Editorial
Zoonotic diseases in companion animals
Companion animals have been important to humans since the first dog joined man, and the positive aspects of
human-animal interaction are well known and have been thoroughly documented.
However, there is a concern relating to this close contact between people and their pets: companion animals carry
the risk of transmitting diseases to humans. This group of diseases is termed zoonoses. Zoonotic diseases are
diseases being common to, shared by or transmitted between human beings and other vertebrate animals.
How do we handle the challenge of zoonotic diseases while maintaining the positive sides of sharing
household with companion animals? Not by diminishing the human-animal interaction, but by increasing the
knowledge on the actual diseases. This includes knowledge on disease recognition and management
as well as prophylactic measures.
Veterinarians need to be able to recognise zoonotic diseases and the effect of such diseases in animals primarily.
But companion animal veterinarians also need knowledge on the effect of these diseases on humans, thus being able
to work in cooperation with human doctors on disease control.
The second online issue of EJCAP, the official journal of FECAVA, is dedicated to the topic of zoonotic diseases.
You will find a collection of articles written by outstanding European experts in their field. It is our goal
that the information gained through these articles will help you in identifying and handle zoonoses in
the best way possible.
Ellen Bjerkås
Scientific Editor of EJCAP
212
ZOONOSES
Companion animals as reservoir
for zoonotic diseases
J. Grøndalen, B. Sævik, H. Sørum
SUMMARY
Companion animals live in close contact with the human population, and the risk of transmitting zoonotic diseases
to humans is therefore significant if the animal itself has been infected. Increased travel activity increases the
possibility of transfer of infection between animal populations, and increased risk for contact with new infectious
agents. An increasing number of people suffer from immunodeficiencies. Environmental- and climatic conditions
cause a change in distribution of vectors in need of special climatic conditions to establish. Exotic species are also to
an increasing extent introduced as family pets, which may contribute to a wider panorama of infections. However, the
traditional zoonotic diseases are still the most important. Vaccination, proper hygiene measurements and knowledge
on preventive measures restrict the risk of transmittance of infections from companion animals. The most significant
risk of companion animals in Norway are mostly related to dog and cat bites or other physical injuries. In total the
benefit and pleasure of this type of animal husbandry is more important than the fear of zoonotic diseases.
may be transferred indirectly from companion animals.
Climatic changes cause a change in distribution of invertebrates
that may act as vectors in transmitting diseases between
species. One example is the cat flea (Ctenocephalides felis) that
at present is slowly spreading from Denmark through Southern
Sweden to Norway.
This paper was first published in the Norwegian
Veterinary Journal, 11/2004.
Introduction
Companion animals are of great significance in today’s society.
37% of Norwegian households own a family pet [1]. Of these,
44% include dogs and 50% cats. Stray dogs are a nonexisting problem in Norway. Cats are to a major extent kept as
companions, although in the cities there may be colonies and
stray cats.
The use of medicines and increased prevalence of diseases
causing immunodeficiency result in humans being more
susceptible to infections, and they may develop more serious
clinical symptoms if diseased.
Increased knowledge on epidemiology and infections reveal
zoonotic significance in more infectious diseases than earlier
anticipated. Thus, we must be open to the increased risk of
transmittance between species.
In Norway, with a population of about 4.5 million inhabitants,
there are about 42,000 horses, 216,000 rabbits, 135,000 captive
birds and 11,000 reptiles kept as pets [1]. These animals normally
live in close contact with humans, and, conditions permitting,
there may be risk of transmittance of zoonotic agents.
This paper describes the most relevant zoonotic diseases that
may be transferred from companion animals. Table 1 presents an
overview of the zoonoses that should be known to veterinarians.
Relevant references may also be found in earlier issues of the
Norwegian Veterinary Journal [2-9].
The regulations for import of dogs and cats from the EU and EFTAcountries were subject to change in 1994, thus facilitating easier
transport of animals between the European countries. Through
this, the risk of animals attracting “new” infectious diseases and
further transmittance to humans is increased. Infections may be
transmitted from animals that suffer from illness, or the animals
can be asymptomatic carriers. Agents dependent on a vector
Dogs and cats
Diseases mainly transferred by saliva through bites and scratches
Many bite wounds caused by dogs or cats develop into
(1) The Norwegian School of Veterinary Science.
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through saliva, and there is highest risk of transmittance from
young adult cats [10]. The most common clinical symptoms in
humans are non-pruritic swelling on the inoculation site and
lymphadenopathy. In some cases fever and general malaise
occur. In rare cases, acute encephalopathy, liver- and spleen
abscesses and pneumopathies may develop [14]. Most infected
cats are asymptomatic carriers, but debilitated individuals may
develop generalised infection. Dogs are believed also to develop
and transfer disease caused by Bartonella spp.
In the period 1998-99 samples were taken from 100 cats in
Mid-Norway. None of the cats were seropositive. This finding
corresponds with the fact that the disease has not been reported
in humans in Norway [15].
Rabies
Rabies is an acute, progressive non-treatable virus encephalitis
[2, 3]. Carnivores and bats are the most important reservoirs.
Transmittance occurs through saliva, most often through bites
from infected animals. The virus follows the nerve pathways to
the central nervous system. After virus replication virus is excreted
through saliva. The incubation time is from one to three months,
however, development of clinical symptoms have occurred days
to years after transmittance of infection. The dog represents
the most significant reservoir for disease in humans. About
35,000 humans are infected every year as a consequence of
dog bites [16]. The infection is maintained through infected fox
herds in arctic, tempered and tropical areas, these representing
the most important reservoir for infection in Eastern Europe.
Hares, rabbits, rodents and birds are considered to have little
significance in transmittance of infection. After 1980 more rabies
cases have been reported in cats than in dogs in the US [17].
However, cats still not represent the most important reservoir
for infections to humans. Because of the long incubation time
and increased global travel activity, rabies may be imported to
areas formerly free of the disease. Clinical symptoms in humans
include fever, anorexia, ataxia, anxiety, altered mental state,
paralysis, coma and death. The patient usually dies at latest
ten days after appearance of clinical symptoms. Rabies has
not been diagnosed in mainland Norway since the beginning
of the nineteenth century, but has been diagnosed sporadically
in polar fox, reindeer and seal in Svalbard, the most recent
cases in 1999 [2, 18]. Vaccine delivered in meat dropped form
aeroplanes as feed for foxes has reduced the rabies problem in
Europe significantly the last 25 years. The disease is at present
more common in Eastern Europe.
Turtles are asymptomatic carriers of salmonella. It has been
reported that reptiles can have a carrier frequency of more than
90%. All reptiles must be considered potential carriers. Here an
information leaflet about zoonoses from the Institute of Protection
from Infections in Sweden.
infections. Cat bites are usually associated with a higher risk of
wound infection than dog bites. Potential pathogen bacteria can
be cultivated from about 90 % of bite wounds caused by a cat
or a dog, and in most cases more than one agent is diagnosed.
The most common isolates include species of Pasteurella,
Streptococcus, Staphylococcus, Neisseria and Moraxella. Other
agents are sporadically isolated [10].
Capnocytophaga canimorsus (former Bacteroides ochraceus,
DF-2) is a gram negative rod considered to be part of the normal
flora of the oral cavity in dogs and cats. The bacteria is rarely
causing disease in humans, however, and if so, a predisposing
factor is usually present, including former splenectomy or other
cause of immunodeficiency [11], caused by an infection after a
dog bite. Fatal outcome has also been reported from Norway
[12].
Pasteurella multocida is also part of the normal flora in most
vertebrates, including the dog and cat. Pasterurella infections
are especially seen after cat bites [10, 13]. Clinical signs may
include cellulitis, lymphangitis and lymphadenopaty, in some
cases in combination with arthritis [10].
Enteric diseases with mainly faecal-oral transmittance
Salmonellosis
The most common sero-variant affecting animals in mainland
Norway are S.typhimurium. Rodents and other wild animals,
including birds, serve as a reservoir for infection [4].
“Cat scratch disease”
The disease is caused by Bartonella henselae (former
Rochalimaea henselae) that is a small, bent gram negative rod.
Other Bartonella spp. may also cause disease in humans. Most
humans that develop “cat scratch disease” are children or young
adults, and the disease is considered to be the most common
cause of chronic lymphadenopathy in this age group [11]. In
some geographic areaes where the cat flea (C.felis) is endemic,
up to 40 % of the healthy cats may suffer from Bartonella
bacteriaemia. Cats with chronic bacteriaemia are contagious
Norwegians are most commonly infected when travelling to
other countries, in this cases the sero-variant S.enteritidis is
the most common agent isolated, mainly after consumption
of contaminated food. The most common sources of infection
both for animals and humans in Norway are contaminated food
and direct faecal-oral transmittance. Dogs have a relatively low
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susceptibility for infection with salmonella bacteria compared to
other animals, like horse and cattle, however, salmonellosis has
recently been diagnosed in dogs infected through contaminated
chewing bones made from slaughter offals. So-called inverse
infection, where animals are being infected through owners
suffering from salmonellosis in not uncommon.
and unkempt fur. Clinical signs may be constantly present or
occur periodically. Giardia spp. as cause of diarrhoea in puppies
and young dogs are occasionally diagnosed. The prevalence of
Giardia spp. and cryptosporidiae seems relatively high among
about 600 dogs included in a study in Norway. The prevalence
in cats in Norway has not been studied (I.S.Hamnes, personal
information, 2004).
Clinical signs in animals vary dependent on number of bacteria
and immune status in the host animal. Gastroenteritis is the
most common manifestation of disease. Bacteriaemia and
enterotoxaemia are usually subclinical signs in the gastrointestinal
manifestation in dogs and cats, however, serious depression,
hypothermia and shock may also be seen with or without
gastrointestinal signs. Animals recovering from infection may
shed bacteria up till six weeks post infection.
Toxoplasmosis is caused by Toxoplasma gondii with cat as
definite host. The cat only plays a minor role for direct transfer
of the protozoan. However, cats shed oocysts through faeces.
These oocysts sporulate within one to five days causing the
cat to act as a source of infection. Thus, new faeces does not
represent a source of infection, but sporulated oocysts may
survive for months to years. Infection to humans through
contaminated sand and soil is considered rare compared to
ingestion of uncooked meat. Infected cats only shed oocysts for
a short period in life. Routine screening of cats for antibodies
against T.gondii is of limited value, but pregnant women are
recommended not to touch cat faeces because of the risk of
infection, which may cause abortion or congenital disease in
the child [18]. Infected cats are most often asymptomatic, but
signs of generalised infection may occur in young or debilitated
cats [17].
Camphylobacteriosis
Camphylobacteriosis may be caused by different species of the
camphylobacter- bacteria in dog and cats. The most common
sub species to cause diarrhoea in humans is Camphylobacter
jejuni sub species jejuni. C.jejuni can cause enteric disease in
the dog. The dog is also considered to be the most important
carrier of C.upsaliensis. In a Norwegian survey from 2000-2001,
24 % of 595 dogs and 18 % of 332 cats tested positive for
Camphylobacter spp. Most of the isolates from both dogs and
cats were C.upsaliensis. The frequency was equal in animals
with and without diarrhoea [19]. This bacteria may also cause
diarrhoea in humans.
Echinococcosis
Echinococcus granulosus and E.multilocularis are the tapeworms
of dogs and foxes, respectively. The parasite has a life cycle that
includes two hosts, and it is the larval stage of the parasite that
causes disease. In predators the tapeworm develops to maturation
and produces eggs that are spread to the environment. When eggs
are ingested by grazing animals, rodents or humans, the larvae are
activated and migrate to different organs where cysts develop.
These cysts can be large and destroy the surrounding tissue. The
cysts can also develop microcysts that spread further. Dogs and
cats are invaded when ingesting either prey with tapeworm,
in earlier years also through slaughter offal from contaminated
reindeer. Today antiparasitic treatment of reindeer is routinely
performed and E.granulosus is almost extinct in Mainland Norway.
However, of ten examined mice (Microtus epiroticus) in Svalbard
in 2002, two were found positive for E.multilocularis [18]. Both
types of tapeworm are found in areas in Europe, however, thus
antiparasitic treatment of dogs immediately before and after
import to Norway is mandatory.
An increased risk for camphylobacteriosis has been found in
humans that keep dogs or cats as pets [20].
Toxocarosis
Toxocara canis and T.cati are the nematodes of the dogs and cats,
respectively, and the most important parasite forming migrating
larvae (larvae migrans) in humans. Adult dogs and cats usually
present with only asymptomatic (“dormant”) infections, while
untreated puppies and kittens may be heavily infested.
In connection with pregnancy in the dam the larvae are
reactivated. This may cause transplacental transmittance to the
foetuses. If the dam and puppies do not undergo antiparasitic
treatment during the first two weeks after birth, eggs are shed
through faeces. In humans ingesting eggs, the eggs will develop
in the intestine. Since humans are not natural hosts, the larvae
will migrate and may cause serious damage in many organs,
including the retina. Luckily, such cases are rare in Norway. Cats
carry a lower significance than dogs for transmitting the parasite
to humans [21]. Antiparasitic treatment with regular intervals,
plus removal of faeces in the environment reduces the risk of
transmittance of the parasite.
Diseases mainly transmitted through direct or indirect
contact with skin or skin lesions
Ringworm
Ringworm (dermatophytosis) is caused by closely related fungi
(dermatophytes) that are only able to infect keratinised tissues
in nails and claws, hair and stratum corneum.
Protozoans
In giardiosis and cryptosporidiosis acute and chronic small
intestinal diarrhoea may develop both in humans and animals.
Wild animals serve as potential reservoirs for dogs and cats,
which are infected through contaminated feed or drinking
water. Most infected dogs and cats do not show signs of
disease, however, some animals do develop diarrhoea, anorexia
The dermatophytes are classified as anthropophilic, zoophilic
and geophilic and have humans, animals and soil as their main
reservoirs. All dermatophytes able to cause ringworm in animals
can also cause disease in humans [9].
The significance of dermatophytes as cause of skin lesions in
dogs and cats is not known, but there are indications that the
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papules and/or erythematous maculae, often in groups in the
contact area(s); arms, chest and abdomen. Gradually, vesiculae
and pustules develop [25].
disease is over diagnosed clinically, especially in dogs. Cats
may be an asymptomatic carrier. However, this is considered
rare in dogs. In a Norwegian study of skin and hair samples
from animals suspected of suffering from ringworm 5% of 780
samples from dogs and 30,8% of 279 samples from cats were
found positive [22].
Ear mites
The ear mite, Otodectes cynotis, can invade the aural canal in cats,
dogs and ferrets and cause otitis externa. Kittens and puppies are
most susceptible for invasion. Transfer to humans with development
of pruritic, papular lesions have been reported [26,27].
The cat is considered the most important reservoir for infection
to humans. Transfer of the fungus occurs either through direct or
indirect contact. In Norway, as in other countries, Microsporum
canis is the dominant agent in ringworm infections in the cat
[22, 23]. Stenwig [22] reported that M.canis represented 83 of
the samples in 86 positive cats. Likewise, E. Christensen (personal
information 2003) reported that 33 of 36 positive samples from
cats were diagnosed as M.canis. Affected cats may show clinical
signs, they may be subclinically infected or they may serve as
vectors. About 50% of humans exposed to M.canis positive cats
will show skin lesions. In households with infected cats at least
one person will be infected in about 70% of the households.
Typical skin lesions in humans are circular, slightly prominent,
crustaceous and erythematous [10].
Cowpox
Cowpox virus causes sporadic infection in cats, and cowpoxinfection has also been reported in cats in Norway [28]. The
natural reservoir is wild rodents. Cats are usually infected
through hunting.
Generalised disease transmitted through direct or indirect
contact
Leptospirosis
Leptospirosis is caused by the bacteria Leptospira interrogans, a
spirochaet that is further subdivided into many serologic variants
(serovars), of which many are able to cause disease [7].
Scabies
The mite Sarcoptes scabei var. canis is most often diagnosed
in dogs. However, it is not species specific and the mite has
been diagnosed in many other animal species [24]. About 24
hours after contact with an affected dog, humans may develop
papulous severely pruritic lesions on the contact area of the skin,
most often the arms and body. Spontaneous remission occurs
after 12-14 days. It has been shown that the mite can survive
on a human being up till six days and may produce eggs in this
time period [25].
In Norway the serovariants Ichterohaemorrhagiae and Canicola
have been connected with disease. The disease has not been
reported in humans in Norway for more than 20 years, Thus,
infection from endemic areas in other countries is considered
the most important risk [6]. Up to the 1950ies leptospirosis was
relatively common in dogs in Norway. In 1947 Strande found
that 6.5% of dogs hospitalised for internal medicine disease
suffered from leptospirosis [29]. During the last years, there have
been single cases of leptospirosis in dogs unrelated to import
[6]. In other countries in Europe and in USA the disease is still
stationary, and increased prevalence may be seen in years with
humid weather. Leptospirosis is probably the most widespread
zoonosis in the world [30].
Fur mites
The fur mites, Cheyletiella yasguri, C.blakei, C.parasitivorax,
cause varying severity of pruritus and dandruff in dogs, cats
and rabbits, however, cats and dogs may also be asymptomatic
carriers. In humans, the mites show a “hit-and-run” behaviour, so
that repeated direct contact with an infested animal is necessary
in order for skin lesions to develop. Typical lesions in humans are
Clinical signs of infection in dogs include generalised infection
that may range from per acute to more chronic development.
Petechiae and bleeding tendency may be seen, as well as
icterus, which is more common in the subacute form. Hepatitis
may lead to intrahepatic cholestasis with grey faeces as one
manifestation.
Rabbits are popular and common pets. Rabbits can be affected by fur
mites and transfer mites to humans through direct contact. Rabbits
can also carry ringworm and can spread the infection to humans
[14]. Tularemia can spread to rabbits through infection via ticks and
fleas from wild rabbits and hares. Photo: Yngvild Wasteson
Horse
Salmonellosis
Salmonellosis in horses presents with enteritis and septicaemia,
with foals and stressed adults being especially susceptible.
S.typhimurium is the most common serovar isolated from horses
in Norway [4].
Clinical signs of salmonellosis in horse may be dramatic,
especially in acute enteritis and may even result in the horse
dying. Large amounts of contagious, soft stool, often gaseous
and excreted under strong pressure represent a significant risk
for transfer of bacteria to other animals and to humans. Isolation
of infected horses and restricted access for humans taking care
of the horses is of utmost importance, and caretakers must be
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of the examined animals. Even if many of these salmonella types
belong to subgroups that are not pathogenic to humans, the
risk of salmonella infection represents a significant problem as
all reptiles must be considered potential carriers of infection
[34]. The bacteria are shed periodically, thus single samples for
diagnosis are of restricted value. The risk of infection is lowest
in terrestrial species, snakes and lizards. These animals are rarely
exposed to their own faeces, which in addition is hard and
rarely excreted due to the slow metabolism. Aquatic turtles and
terrapins represent a greater risk, as the water is contaminated
by faeces. Children are especially exposed to infection. The
animals should not be treated with antibacterial agents, as this
may result in resistant bacteria. Reptiles are not recommended
as pets for children (M.Heggelund, personal communication).
instructed about risk of infection and necessary steps to avoid
transfer of bacteria.
Tuberculosis
Horses are relatively resistant to tuberculosis, but may occasionally
be infected with M.bovis, M.tuberculosis or M.avium [5]. Clinical
signs are unspecific, and may include diarrhoea and respiratory
signs. A case of avian tuberculosis was described in a horse in
the nineties [31].
Ringworm
Ringworm in horses in Norway is most often caused by
Tricophyton equinum or Microsporum equinum [9, 22]. Cases
of ringworm may be diagnosed in riding horses and trotters.
Control of ringworm outbreaks in stables with sports horses
and thus transport of animals between competitions and horse
shows may be a challenge. In stables where horses are in
frequent contact with humans, many of which are young people,
the zoonotic aspect of the disease should be emphasized in the
information given.
Vector borne diseases
Many infections causing disease in animals and humans are
spread through an invertebrate vector. In Norway, the tick
Ixodes ricinus is the most important vector transferring disease
between species [8].
Birds
Borreliosis (Lyme disease)
Borreliosis is caused by the bacteria Borrelia burgdorferi and is
probably the most widespread vector borne zoonotic disease in
Northern Europe [35]. In humans, the disease leads to general
infection, with skin, joint, nervous tissue and heart most often
affected. Among animals, dogs and horses are especially
susceptible. Serology is frequently used for establishing a
diagnosis. A Swedish/Norwegian study of dogs from areas
where I.ricinus is endemic, showed 4% and 27% respectively,
of tested healthy animals to be seropositive [36,37]. Most
seropositive animals do not develop clinical illness, but both
acute and chronic disease with inflammatory signs may develop.
The prevalence among cats has not been studied.
In addition to salmonellosis, psittacosis is the most important
zoonotic disease related to infection from wild or captive
birds. The agent is found in secretion form eyes, nose and
from faeces and transmittance is aerogenic or through direct
contact. Birds with psittacosis are most often asymptomatic,
however, generalised signs of infection may be seen in immune
compromised birds. Respiratory symptoms and headache are
the most common symptoms in humans [32].
Rabbits, rodents and ferrets
Rabbits can be affected by fur mites (Cheyletiella spp.) and
transmit invasion through direct contact – see description for dogs
and cats. Rabbits are also susceptible to ringworm (Tricophyton
mentagrophytes, M.canis and Microsporum gypseum) and may
transfer infection to humans [14]. Tularaemia can be transferred
to rabbits from wild rodents through ticks and fleas. Rodents
can also suffer from ringworm (T.mentagrophytes).
Horses may also suffer from borreliosis. Clinical signs are similar to
what is found in dogs. The diagnosis is most often established in
animals with disease, known exposure to ticks and positive serology.
A Swedish study showed a seroprevalence for B.burgdorferi of
17%. No difference in seroprevalence was found between healthy
hoses and horses with signs of disease [36].
Salmonellosis can be transferred from rodents to humans.
Guinea pigs will most often die if they develop salmonellosis,
while rats and mice may have subclinical infection. The most
important serovariants are Typhimurium and Enteritidis.
Anaplasmosis
Anaplasma phagocytophilum is the present name of bacteria
formerly termed Ehrlichia phagocytophila, E.equi and “Human
granulocytic ehrlichial agent” (HGE). The intracellular bacteria
affect granulocytes in the blood in particular and may be
diagnosed through direct microscopy. “Granulocytic ehrlichiosis”
in dogs was described from Sweden in 1989. Later studies showed
that the dogs had been infected with A.phagocytophilum, the
same type causing disease in humans and horses [38]. Clinical
signs in affected dogs include fever, depression, lameness
plus gastrointestinal and nervous symptoms. Haematology
signs include thrombocytopenia, leukopaenia and anaemia.
Asymptomatic cases are common. Studies have shown a
seroprevalence in Swedish dogs to be 17% and 23% in dogs
from Southern Norway. The significance of anaplasmosis in cats
is not known, however, natural infection has been described.
Leptospirosis can be transferred from rats, usually wild, and
these are also considered a cause of infection in humans,
through contaminated water.
Ferrets may suffer from salmonellosis, camphylobacteriosis and
ringworm.
Reptiles
Turtles are asymptomatic carriers of salmonella. A carrier
frequency of more than 90% has been reported. In a Swedish
study on snakes from 1988, salmonella was diagnosed in 41%
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“Equine granulocytic ehrlichiosis” was described in USA in 1969.
In Sweden the first cases were described in 1990. DNA analysis
showed the bacteria to be closely related to E.phagocytophila
and identical with agents causing human granulocytic ehrlichiosis
[38]. Clinical signs in horses are similar to what is described in
dogs, however, the infection can also be asymptomatic. The
seroprevalence in a Swedish study was 17 %, antibody findings
in healthy and diseased animals being equal [38].
Resistance to antibiotics
Bacterial resistance to antibiotics is an increasing and challenging
problem both in veterinary as well as in human medicine.
Antibiotic resistance can develop through genetic adaptation in
the bacteria. This occurs to a significant degree through genes
causing antibiotic resistance frequently being transferred between
bacteria as mobile genetic elements. Mobile resistance genes
can be used by many bacteria, not necessarily closely related.
This means that widespread use of antibiotics and increase in
resistance genes in veterinary medicine can be of significance for
the increase in bacteria being resistant to antibiotics and causing
disease in humans. These bacteria pick up resistance genes
directly from the animal pathogenic bacteria, or resistance genes
are delivered through the normal flora in animals or humans as a
type of zoonotic problem. Similarly, resistance genes included in
animal pathogenic bacteria may be of human origin.
Tick borne encephalitis (TBE)
TBE is caused by flavivirus and is relatively widespread in Eastern
Europe and the Baltic states. The disease has been known in
humans in Sweden from 1954 with 40-80 new cases diagnosed
annually in endemic areas [39]. The disease was diagnosed for
the first time in humans in Norway in 1997. 5 cases are described
from 1998–2001, all from Trom island in Southern Norway.
Based on this, as serologic study of 317 healthy dogs from this
area was carried out. Antibodies against TBE virus were found
in 16.4% of the dogs [40]. Based on this, it can be stated that
the disease is present in Norway and that dogs may serve as
potential carriers. Clinical signs in diseased dogs include fever,
and progressing CNS signs. In a study of 545 dogs from Austria,
131 had antibodies against TBE virus and about half of these
showed clinical signs of disease [41].
Discussion and conclusion
Companion animals are of benefit to humans. One study showed
that people owning pets visited the doctor less frequently and
had lower blood pressure and blood cholesterol than nonowners [43].
The highest risk in companion animal keeping is connected
to the risk of bites and the risk of transfer of diseases from
animals to humans. There has also been a focus on the risk of
sensibilisation and development of allergy, especially to cats,
but this is controversial. More recent studies have shown that
children in households without a cat or a dog are at greater risk
of developing allergies against these animal species [44].
Leishmaniasis
Leishmaniasis is caused by an intracellular protozoan. In the
Mediterranean area, the dog represents the main reservoir for
infection to humans, the parasite being transmitted through a
sand fly [3]. The disease has a cutaneous and a visceral form,
the latter being caused by Leishmani donovania infantum and
being endemic in areas in the Mediterranean area. The disease
may progress slowly, and it may take years before development
of clinical symptoms, although individual differences exist. In
both humans and animals the visceral form leads to generalised
symptoms of infection, emaciation, muscular atrophy, anaemia,
liver- and kidney failure plus failure of other organs. The disease
is diagnosed in Norway in animals imported from endemic areas
in Italy and Spain. The sand fly does not survive the Norwegian
winters, however, thus there is no risk for the disease to become
endemic. The parasite may also be transferred to humans
through skin contact with infected organs.
The risk of transmitting disease from companion animals to humans
is related to certain factors, including young animals, dense animal
population and poor hygiene, insufficient prophylactic measures,
free-roaming animas eating cadavers, contact with stray animals
and increased travel activity. Small children, pregnant women
and immunodeficient persons form the group at risk, but also
veterinarians, nurses and other staff working with animals with
infections are exposed. Even if the risk of transferring infection
from companion animals is relatively low, the risk of infection can
be reduced further. Owners, and especially those in the group at
risk must be informed about the potential risk and possible routes
of infection, including the life cycles of vectors and agents. This is
a task for both veterinarians and doctors.
Heartworm (Dirofilariasis)
Dirofilaria immitis is the dog heartworm transferred through a
mosquito that demands certain temperature and environmental
conditions for survival. The mosquito has spread north in USA,
but routine antiparasitic treatment of dogs during the summer
months has prevented spreading of the parasite. Single cases of
infection causing abortion have been described in humans. In
Europe, Dirofilaria repens is diagnosed in humans in an increasing
number. Noduse develop in skin, subcutis and internal organs.
This parasite is also transferred through a mosquito. Studies of
dogs from endemic areas in Italy have shown an increase in
number of seropositive dogs. It therefore seems reasonable to
consider a role from the dog as reservoir and in transfer of the
parasite between species [42]. Dogs rarely develop skin nodules,
but the parasite migrates through subcutaneous tissues.
In short, the risk of transfer of infection can be reduced
through
- Good hygiene, in particular hand hygiene
- Removal of faeces in outdoor areas, and regular cleaning of
litter boxes
- Antiparasitic treatment of the animals
- Not letting children play in contaminated areas
- Cover sand boxes when not being used
- Avoid scratches and bites
- Not bringing animals into areas with increased risk of infection/
invasion unless proper prophylactic measures have been taken
However, in total the benefit of having a pet is higher than the
fear of zoonotic disease.
218
Poxviridae
Genus Orthopoxvirus
Cowpox and other
pox virus
Brucella canis
Campylobacter jejuni
C. upsaliensis
Bartonella henselae
Bartonella spp.
Leptospira
interrogans serovar
Ichterohemorrhagiae
and Canicola
Bacillus anthracis
Chlamydophila
psittaci
Chlamydophila felis
Salmonella spp.
Capnocytophaga
canimorsus (DF-2)
Pasteurella multocida
Mycobacterium spp.
Brucellosis
Campylobacteriosis
“Cat Scratch
Disease”
Leptospirosis
Anthrax
Psittacosis,
ornithosis
Salmonellosis
Capnocytophaga
infection (wound
infection,
septicaemia)
Pasteurellosis
(wound infection,
septicaemia)
Tuberculosis
Bacterioses
Rhabdoviridae
Genus Lyssavirus
Agent
Rabies
Viroses
Disease
219
Uncommon in
Western countries
Occurs, serious
in humans with
immunodeficiency
Occurs, serious
in humans with
immunodeficiency
Rel. common
Occurs
Global
Dog, cat
Dog, cat
Dog, cat, most
mammals, birds,
reptiles
Caged birds, wild
birds Cat (zoonotic
aspect uncertain)
Horse
Dog, rat, rarely cat
and other mammals
Cat, dog
Dog, cat, other
mammals, birds
Most often (aerogenic) Dog, cat, rodents,
infection from humans primates, horse, fish
to animals (”inverse
zoonosis”)
Bites
Bites
Global
Global
Faecal-oral
transmittance
Aerogenic, faecal-oral
transmittance
Aerogenie, alimentary,
contact infected
organs
Global
Global
Global
Contact, urine and
urine contaminated
water, bite
Global
Not uncommon in
Mid- and Southern
Europe and other
parts of the world
Uncommon in the
Western world
Cat bites, cat
scratches, through
fleas and ticks
Faecal-oral
transmittance
Dog
Cat, dog, small
rodents
Contact with infected
skin
Infected organs,
amniotic fluid, urine
All mammals
Affected species
Saliva, usually through
bites
Transmittance
Global,
Global
Not in Norway
Global, endemic
among small
rodents in Norway
Global, except
Australia, Iceland,
Sweden, Mainland
Norway, UK
Geographic
distribution
Rel. common in
certain areas with
fleas and ticks
Rel. common
Uncommon
Uncommon
Uncommon in
Western world
Human aspects
Asymptomatic, granulomas in lungs, gen.
infection, ulcerative dermatitis, meningitis
Normal bacterium in the oral cavity
Normal bacterium in the oral cavity
Asymptomatic, gastroenteritis, septicaemia
Asymptomatic, gen. infection. Conjunctivitis,
upper respiratory tract signs (cat)
More protracted development in horse than in
cattle
Fever, haemorrhage, icterus
Asymptomatic, fever, uveitis, gingivitis, CNS
signs. Dogs: endocarditis, hepatitis
Asymptomatic, gastroenteritis
Asymptomatic, abortion, lymphadenopathy,
orchitis, bacteriaemia, discospondylitis
Skin lesions on head, neck, front legs.
Asymptomatic in small rodents
Progressive CNS signs and death
Clinical signs in animals
EJCAP - Vol. 18 - Issue 3 December 2008
Tab. 1 Zoonoses that can be transferred from companion animals to humans. Under ”affected species” only companion animals are included.
220
Toxoplasma gondii
Toxoplasmosis
Echinococcus
multilocularis
Echinococcosis (Fox
tapeworm)
Toxocara canis
Toxocara cati
Toxocariasis
(human: larvae
migrans)
Fur mite
(cheyletiellosis)
Cheyletiella spp.
Parasittoses – mites and insects
Ancylostoma
spp. Uncinaria
stenocephala
Hookworm
Parasittoses - nematodes
Echinococcus
granulosus
Echinococcosis
(Dog tapeworm)
Parasittoses - tapeworm
Giardia spp.
Cryptosporidium spp.
Giardiosis
Cryptosporidiosis
Parasittoses - protozooans
Sporotrichosis ”rose Sporothrix schenkii
growers disease”
Ringworm
(dermatophytoses)
Hunters esp.
susceptible
Human aspects
Occurs
Uncommon
Uncommon
Uncommon in the
Western world
Uncommon in the
Western world
Uncommon
Rel. common
Rel. common
Uncommon
Rel. common
Microsporum
canis, Tricophyton
mentagrophytes,
Microsporum
equinum, Tricophyton
equinum
Francisella tularensis
Tularaemia
Mycoses
Agent
Disease
Global
Global
Global
Global
Global
Global
Global
Global
Most common
in warm environment
Global
Global
Geographic
distribution
Contact
Faecal-oral
transmittance
Faecal-oral
transmittance
Faecal-oral
transmittance
Faecal-oral
transmittance
Faecal-oral
transmittance
Faecal-oral infection
through contaminated
water
Faecal-oral
transmittance
Contact skin through
scratches and bites,
environmental
pollutant
Contact skin, directly
or indirectly
Direct contact with
infected organs and
bites from infected
animals
Transmittance
Dog, cat, rabbit
Dog, cat
Dog, cat
Dog, cat, rodents
Dog
Cat
Asymptomatic, dandruff, pruritus
Asymptomatic, depression, gastrointestinal and
respiratory signs
Asymptomatic, anaemia, diarrhoea, respiratory
signs, linear skin lesions, pododermatitis
Asymptomatic, organ changes due to pressure
from cysts and granulomas
Asymptomatic, organ changes due to pressure
from cysts and granulomas
Asymptomatic, gen. infection and organ failure
in debilitated humans (rare)
Dog, cat, bird, reptiles Asymptomatic, diarrhoea
Asymptomatic, diarrhoea
Topical cutaneous form, lymphocutaneous form
and multifocal disseminated form
Cat
Cat, transfer from
dog not documented
Skin lesions with varying morphology, also
asymptomatic (mainly cat)
Ulcerativ dermatitis, lymphadenopathy, fever
Clinical signs in animals
Cat, dog, rabbits,
rodents, horse
Hare and other wild
rodents, dog, cat,
other animals
Affected species
Companion animals as reservoir for zoonotic diseases - J. Grøndalen
Global
Ctenocephalides felis, Occurs
Ctenocephalides
canis
Fleas
221
Borrelia burgdorferi
(bacterium)
Anaplasma
phagocytophilum
m.fl (bacterium)
Leishmania spp.
(protozooan)
Dirofilaria repens
Borreliosis
Anaplasmosis
(ehrlichiosis)
Leishmaniosis
Dirofilariosis
(heartworm)
Global
Areas where the
vector is present
Southern Europa
Mediterranean,
Africa, Asia,
South- America
Rel. common in areas Mediterranean,
with sandflies
Africa, Asia,
South- America
Global
Mid-Eastern
Europe, Nordic
countries, Baltic
states
Areas where the
vector is present
Occurs, rel. common
in the Baltic states
Dog, cat
Cat, dog and ferret
Dog (cat and other
mammals)
Affected species
Mosquito bites
Bites from sandflies,
probably also direct
transmittance of
infection
Tick bites Ixodes
ricinus, Rhipicephalus
sanguineus m.fl.
Dog
Dog, cat
Dog, horse
Tick bites Ixodes ricinus Dog, horse
m.fl.
Tick bites Ixodes ricinus Dog
Contact, direct and
indirect
Contact
Contact
Transmittance
Asymptomatic, skin and subcutaneous nodules,
heart symptoms
Cutane form, visceral form, gen. signs of
infection
Asymptomatic, gen. Signs of infections,
polyarthritis
Asymptomatic, fever, polyarthritis
Asymptomatic
Asymptomatic, skin lesions, pruritus
Asymptomatic, otitis externa, pruritus
Papules, pruritus
Clinical signs in animals
The diseases listed below are uncertain with regard to the zoonotic aspect and if they can be transmitted from companion animals to humans.
• Helicobacteriosis, enteric disease (Helicobacter spp.) may possibly be transferred through contact with infected organs via cats
• Anaerobiospirillosis, enteric disease (Anaerobiospirillum spp.) may be transferred from infected cats
• Yersiniosis, (Yersinia pseudotuberculosis) septicaemic form and classical syndrome with organ nodules in guinea pigs and other rodents, rabbits and wild and captive birds may possibly be
transferred via dog and cat. Y. enterocolitica causes enteric form
• Q-fever, infectious syndrome (Coxiella burnetti) may possibly be transferred through contact with infected organs via cat
• Babesiosis, (Babesia spp.) may possibly be transferred via ticks and through bites and scratches from dogs
• ”Rocky Mountain spotted fever”, infectious syndrome(Rickettsia rickettsiae) may possibly be transferred from cats to humans through flea bites
Flaviviridae TBE virus
TBE (tick borne
encephalitis)
Vector borne zoonoses
Global
Occurs
Otodectes cynotis
Global
Ear mite
(otodectosis)
Occurs
Sarcoptes scabiei var.
canis
Geographic
distribution
Scabies
(sarcoptosis)
Human aspects
Agent
Disease
EJCAP - Vol. 18 - Issue 3 December 2008
Companion animals as reservoir for zoonotic diseases - J. Grøndalen
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222
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223
ZOONOSES
Diagnostic tools for the detection
of rabies virus
L. M. McElhinney(1,3), A. R. Fooks(1,3), A. D. Radford(1,2)
SUMMARY
Rabies has the highest case-fatality rate of any currently recognized infectious disease with an estimated 55,000
human deaths per year. The disease is routinely diagnosed in animals based on clinical signs and on epidemiological
grounds in rabies-endemic countries. The use of conventional diagnostic tests including the fluorescent antibody test
(FAT), rabies tissue-culture infection test (RTCIT) and mouse inoculation test (MIT) can now be complemented with
molecular diagnostic tools such as reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridisation
(ISH). Negative diagnostic test results do not exclude the clinical diagnosis as these tests are entirely dependent
on the quality of the sample supplied. Molecular tools and virus typing are becoming more widely used for the
rapid detection and strain identification of rabies viruses from ante-mortem clinical samples. The currently available
diagnostic methodologies for both post-mortem and ante-mortem detection of rabies virus are reviewed.
(Aravan virus, Khujand virus, Irkut virus and West Caucasian Bat
Virus), have recently been proposed as new members of the
Lyssavirus genus [4-6].
This paper was commissioned by FECAVA for
publication in EJCAP.
Rabies virus is largely maintained in two ecologically inter-related
disease cycles; urban and sylvatic (wildlife). Urban rabies, primarily
in dogs and cats, has been eliminated in an increasing number
of developed countries via parenteral vaccination programmes.
However, sylvatic rabies remains widespread throughout parts of
Europe, Africa and North America and is maintained in a variety
of species including the red fox, raccoon-dog, raccoon, skunks
and bats. Urban canine rabies remains widespread throughout
developing countries and human mortality from endemic canine
rabies was calculated to be 55,000 deaths per year with 56%
of the deaths estimated to occur in Asia and 44% in Africa
[7]. The total global cost of rabies prevention is estimated to
exceed $1 billion per year. However, the prevalence and cost of
rabies is believed to be significantly underestimated due to poor
reporting and surveillance [7].
Introduction
Animal rabies is endemic throughout all continents with the
exception of several islands and peninsulas including Australia
and New Zealand, an increasing number of European countries
and Antarctica. Having eliminated rabies, countries that are
striving to maintain their rabies free status, usually do so at
considerable cost and with a continual risk of re-importation
[1,2].
Rabies viruses are members of the Lyssavirus genus, within the
Rhabdoviridae family. The lyssaviruses comprise seven related
virus groups (genotypes 1 - 7). These include, classical rabies virus
(RABV, genotype 1), Lagos bat virus (LBV, genotype 2), Mokola
virus (MOKV, genotype 3), Duvenhage (DUVV, genotype 4),
European bat lyssavirus type 1 (EBLV-1, genotype 5), European
bat lyssavirus type 2 (EBLV-2, genotype 6) and Australian bat
lyssavirus (ABLV, genotype 7). With one exception, (MOKV),
all genotypes have been isolated from bats (Tab. 1) [3]. Four
additional rabies-related viruses, isolated from bats in Eurasia
In Europe, sylvatic rabies (predominantly fox rabies) has been
significantly controlled by the successful implementation of
oral rabies vaccination (ORV) campaigns. The majority of the
western European countries are now free of Classical rabies
(RABV), with reported rabies restricted to relatively rarer bat
(1) National Centre for Zoonosis Research, Neston, South Wirral, CH64 7TE, UK
(2) Department of Veterinary Clinical Sciences, Leahurst Campus, Neston, South Wirral, CH64 7TE, UK
(3) Rabies and Wildlife Zoonoses Group, WHO Collaborating Centre for the Characterisation of Rabies and Rabies-Related Viruses, Veterinary Laboratories
Agency (VLA, Weybridge), New Haw, Addlestone, Surrey, KT15 3NB, UK
Corresponding author: Dr Lorraine McElhinney. Tel.: +44 (0)151 7956058; Fax: +44 (0)151 7946005. E-mail: l.mcelhinney@vla.defra.gsi.gov.uk
224
Diagnostic tools for the detection of rabies virus - L. M. McElhinney, A. R. Fooks, A.D. Radford
Genotype
Phylogroup Species
Abbrev.
(ICTV)
Distribution
Potential vector(s)
1
I
Rabies virus
RABV
World (except
several islands)
carnivores (world) bats (Americas)
2
II
Lagos bat virus
LBV
Africa
frugivorous bats
3
II
Mokola virus
MOKV
Africa
unknown (isolated from shrews)
4
I
Duven-hage virus
DUVV
Africa
insectivorous bats
5
I
European bat lyssavirus 1
EBLV-1
Europe
insectivorous bats (Eptesicus
serotinus)
6
I
European bat lyssavirus 2
EBLV-2
Europe
insectivorous bats (Myotis sp)
7
I
Australian bat lyssavirus
ABLV
Australia
frugivorous/insectivorous bats
Proposed
I
Aravan virus
ARAV
Central Asia
Insectivorous bat (isolated from
Myotis blythi)
Proposed
I
Khujand virus
KHUV
Central Asia
insectivorous bat (isolated from
Myotis mystacinus)
Proposed
I
Irkut virus
IRKV
East Siberia
insectivorous bat (isolated from
Murina leucogaster)
Proposed
II or III
West Caucasian bat virus
WCBV
Caucasus region
insectivorous bat (isolated from
Miniopterus schreibersi)
Tab. 1 Classification of Lyssaviruses.
or imported cases. The most recent European country to be
declared rabies free was Germany (September 2008). Rabies
Bulletin Europe (WHO) reported 9563 cases of rabies in Europe
in 2007 (includes figures for Russian Federation, Ukraine and
Turkey). These cases were associated with red foxes (4515 cases,
47.2%), cats (1411 cases, 14.8%), dogs (1394 cases, 14.6%),
cattle (1288 cases, 13.5%), raccoon-dog (364 cases, 3.8%), bat
(26 cases, 0.3%) and humans (9 cases, 0.1%). Although fox
rabies is the principal vector in Europe as a whole, the raccoondog plays a significant role in the epidemiology of rabies in the
Baltic countries where numbers of infected raccoon-dogs can
exceed that of foxes. The cattle and human cases are generally
considered to represent dead end host spill-over events. Dog and
cat rabies is still prevalent in some Eastern European countries,
Belarus, Ukraine, Russia and Turkey.
to demand serological testing under an EU derogation in place
until June 2010. In addition, treatment for ticks and tapeworm
is required for entry into Ireland, Malta and the United Kingdom,
while Sweden requires tapeworm treatment and not treatment
for ticks. Approximately 546,908 pets have entered the UK
between February 2002 and August 2008 (http://defraweb/
animalh/quarantine/pets/procedures/stats.htm). Commercially
traded eligible animals are transported without quarantine
under the Balai Directive (Council Directive 92/65/EEC of 13 July
1992) as amended (e.g. regulation 998/2003).
Rabies cases in Europe are principally attributed to three of the
Lyssavirus genotypes, namely genotype 1 (RABV, classical rabies)
and to a lesser extent genotypes 5 and 6 (European bat lyssaviruses
type-1 and -2). The 26 insectivorous bat cases reported in 2007
were due to European Bat Lyssaviruses (EBLV-1 or -2). The majority
(>95%) of the reported EBLV-1 cases in European bats have been
identified in one bat species, Eptesicus serotinus (Serotine bat).
The rarer EBLV-2 cases (2%) are associated with Myotis species,
M.daubentonii (Daubenton’s bat) and M.dascyneme (pond bat).
EBLV-1 has been reported in several countries across Europe
(not UK) whereas EBLV-2 has only been confirmed in the UK,
Netherlands, Switzerland and Germany.
In 2000, the UK Government introduced a system whereby
eligible dogs and cats from qualifying countries could enter or
return to the UK without being subjected to quarantine. The UK
Pet Travel Scheme (PETS) comprised of identification, vaccination
and serological testing combined with tick and tapeworm
treatment prior to entry. The scheme was extended to include
further qualifying countries and in 2004 the EU Pet Travel Scheme
succeeded it. The EU PETS allowed for the transportation of
ferrets, a greater list of qualifying countries but no obligation to
confirm seroconversion in the vaccinated animal or administer
treatment for the cestode Echinococcus multilocularis (exotic to
the UK). The UK, Malta, Sweden and Ireland retain the authority
Spill-over of EBLV-1 into sheep has occurred on two separate
occasions in Denmark, into a stone marten in Germany,
domestic cats in France and one confirmed and possibly other
unconfirmed human cases [8]. EBLV-2 has been recorded in two
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Human and animal rabies is usually acquired via the transdermal
inoculation of infected saliva in the bite of a rabid animal but on
rare occasions human rabies can be transmitted via aerosols or
corneal and organ transplantation [9] or via routes other than
bites [10-13]. In the majority of human cases, the exposure to an
infected animal is known and the disease can be prevented by
timely post exposure treatment. The successful administration
of anti-rabies prophylaxis is enhanced by speedy and accurate
diagnosis.
early but significant antibody response (6 days after the onset
of signs) which encouraged the clinicians to attempt a druginduced coma and a cocktail of anti-viral drugs. No virus or
antigen was detected. Her treatment included the induction of
coma with the anaesthetic ketamine and antiviral agents ribavirin
and amantadine. This appeared to be successful and the patient
recovered with only minor residual neurological deficits [17].
Despite a number of attempts to follow the costly procedure
(known as the Milwaukee Protocol), this remarkable success has
not been repeated. It is not clear if the success was due to the
viral strain (assumed to be an American bat strain), development
of paralytic rabies (lack of damaging hydrophobic spasms) or the
immune status of the patient (rare early neutralising antibody
production) [18].
Clinical Signs
Clinical Diagnosis
The long and variable incubation period is a key feature of rabies.
It relates to the time that the neurotropic virus is protected
from immune responses within peripheral cells and nerves. The
incubation period can vary between 15 days to 6 years (average
2–3 months) depending on the strain, species, viral dose and the
proximity of the site of virus entry to the CNS. A short incubation
period would be expected for a deep bite near to the head of
the victim. Most clinically infected species excrete rabies virus
in the saliva. Experimental data suggests that different species
exhibit varying times of virus excretion before the onset of
clinical signs (cats <24hours, dogs<13 days, foxes <29 days post
infection) [14].
Rabies is a notifiable disease in the EU. Clinical signs are not
characteristic, especially early in the course of disease. Clearly
the history may raise the index of suspicion e.g. travel especially
smuggling, contact with infected wildlife including bats, lack
of vaccination. When presented with a suspect case, owners
and veterinarians must contact the competent authority in their
country.
human cases (Finland 1985 and Scotland 2002) but no other
spill-over event has been reported. There has been no evidence
of transmission of EBLV by any other terrestrial species; hence to
date all spill-over events have been dead-end.
Clinical diagnosis is based on the observation of clinical signs, the
first of which usually appear after the variable incubation period
and vary depending upon species. Differential diagnosis can be
difficult due to common sequelae resulting from various diseases
including transmissible spongiform encephalopathies, tetanus,
listeriosis, poisoning, Aujeszky’s disease and other viral nonsuppurative encephalitidies. Paralytic rabies is often mistaken for
Guillain-Barré Syndrome [19, 20]. In addition, secondary infections
can mask the presence of rabies leading to misdiagnosis [21]. A
report by Laothamatas et al. [22] demonstrated that Magnetic
Resonance Imaging (MRI) could not differentiate the two forms
of human rabies (paralytic and furious) as both share a similar MRI
pattern. However, such a pattern in a non-comatose patient with
suspected encephalitis may prove useful in distinguishing rabies
from other viral encephalitidies.
Prophylaxis and treatment
Vaccination is the mainstay of control in at-risk human and
animal populations. In humans, following suspect exposure,
prophylaxis including repeated vaccinations with or without
immunoglobulin therapy is usually administered. In cats and dogs,
there is little evidence to show the effectiveness of such postexposure prophylaxis, and this is not generally recommended.
Animals that are bitten by potentially infected animals should be
immediately reported to the relevant authority.
In the clinical course of rabies, three stages are classically
described; prodromal, excitement (furious) and paralytic (dumb)
(Tab. 2). However, it is important to note that not all stages are
observed in individual cases. The first clinical symptom is usually
neuropathic pain at the site of infection (bite wound) due to viral
replication in dorsal root ganglia and ganglionitis. This is usually
accompanied by non-specific signs typical of a viral encephalitis.
Following the non-specific prodromal phase, either or both the
excitement and/or paralytic forms of the disease may be observed
in a particular species, with disease commonly progressing to the
paralytic form. Cats are more likely to develop furious rabies than
dogs [23]. In some cases, no clinical signs are observed and rabies
virus has been identified as the cause of sudden death.
Without intensive care interventions, death occurs within
1–10 days of neurological signs. In cats and dogs, the clinical
phase can be relatively rapid at 3-4 days [15,16]. Although the
mortality for clinical rabies is believed to be 100%, sporadically
cases of survival are reported. At least five humans that had all
received either pre- or post-exposure prophylaxis are believed to
have recovered after developing clinical signs of rabies [7]. There
is some doubt as to whether the clinical signs in some of these
cases could be attributable to vaccine-induced encephalitis
rather than rabies infection. However, in 2004, an unvaccinated
teenager in Wisconsin, USA, was bitten by a bat, developed
early signs of disease, yet became the first person to recover
from the disease after experimental therapy was initiated.
Rabies biologicals (vaccine or rabies immunoglobulin), usually
administered as post exposure treatment, were not given to
this patient. She developed fever, hypersalivation, parasthesiae
of the bitten hand, progressive cranial nerve paralysis and leg
weakness. Rabies was diagnosed by the detection of a rare
In a recent study, Laothamatas et al. compared furious and
paralytic rabies in dogs using neuroimaging (MRI) and RT-PCR
(RABV RNA and brain cytokine mRNA) [24]. They concluded
that the early stage of furious rabies in dogs is characterized
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Stage
Signs
Prodromal
Pyrexia, altered personality, increased nervousness, loss of fear,
disorientation, loss of appetite, hyperactivity, hypersensitivity to
noise or light, rubbing or biting the site of inoculation (wound),
tremor, dyspnoea.
Excitement (furious)
Increased aggression, abnormal vocalisations (high pitch howl
in canines, characteristic bellowing in ruminants), ‘fly snapping’
at imaginary targets, wandering long distances, hyper sexuality,
head butting (particularly ruminants), staring eyes, partial
paralysis of tongue and lower jaw with drooling of saliva, ‘bone
in throat’ syndrome. Acute colic with abdominal straining
(horses and ruminants).
Paralytic (dumb)
Hind limb paralysis, generalised paralysis, dysphagia, respiratory
arrest, inability to drink or swallow, excessive salivation,
hydrophobia (only in man), convulsions, coma, death.
Tab. 2 Clinical stages of rabies. Clinical progression is variable and although most end in the paralytic phase, some do not show many signs
associated with the excitement phase.
Rabies virus antigen detection
techniques
by severe virus neuroinvasiveness and moderate inflammation
whereas paralytic rabies was associated with a delayed viral
neuroinvasion and a more intense inflammation. However,
during the late stage of infection, little difference was observed
in the brains of furious and paralytic rabid dogs.
In humans, classical signs of brain involvement include
spasms in response to tactile, auditory, visual or olfactory
stimuli (e.g. aerophobia and hydrophobia). Such signs occur
during the clinical phase in almost all rabid patients in whom
excitation (furious rabies) is prominent and are interspersed
with periods of lucidity, agitation, and confusion. However,
excitation is less evident in paralytic rabies. Atypical rabies or
non-classic rabies is increasingly observed and may result in
underreporting.
In the past decade, a diverse number of methods have been
published for the detection and identification of rabies viruses in
clinical specimens. However, the most commonly used diagnostic
test is the fluorescent antibody test (FAT) which detects virus
antigen in the brain using fluorescently labelled anti-rabies
antibodies [26] and is recommended by both WHO and OIE. It
may be used to confirm the presence of rabies virus nucleocapsid
protein in the original sample (brain smear) or sub-passaged
material (see virus isolation below). The FAT gives reliable results
on fresh specimens within a few hours in 95–99% of cases
[25]. However, the sensitivity of the FAT is dependent on the
quality of the specimen, conjugate, equipment and the skills of
the diagnostic staff. The sensitivity of FAT can be affected by
autolysis, lyssavirus species and the accuracy of dissection of the
brain and may be lowered in samples from vaccinated animals.
For direct rabies diagnosis, smears prepared from hippocampus,
cerebellum or medulla oblongata are fixed in high-grade cold
acetone and then stained with a drop of a specific antibody
conjugated to fluorescein isothiocyanate. In the FAT, the specific
aggregates of nucleocapsid are identified by their specific applegreen fluorescence.
There are no gross lesions characteristic for rabies visible at
autopsy. In most animals, microscopic non-specific lesions
suggestive of viral encephalomyelitis with ganglioneuritis may
be observed in the nerve centres, as well as histolymphocytic
cuffs and gliosis. The most significant lesions are usually in the
cervical spinal cord, hypothalamus and pons. The only specific
lesions consist of intracytoplasmic eosinophil inclusions (Negri
bodies) corresponding to the aggregation of developing rabies
virus particles. These oval inclusions, measuring from 4 to 5 µm,
are usually located in Ammon’s horn.
The enzyme-linked immunosorbent assay (ELISA) and rapid
rabies enzyme immunodiagnosis (RREID) methodologies have
been shown to be of benefit for large scale surveillance having
the potential to be automated [27, 28]. These methods detected
the presence of EBLV-1 antigen in a cat in France in 2007 when
conventional FAT failed (H. Bourhy, personal communication).
Consequently, diagnosis can only be confirmed by laboratory
tests preferably conducted post mortem on central nervous
system (CNS) tissue removed from the cranium. Diagnostic
techniques for rabies in animals have been internationally
standardised [25]. The hippocampus, cerebellum and the
medulla oblongata are the recommended tissues of choice.
The head of the suspect animal is generally submitted
for laboratory diagnosis. However, if this is impractical,
for example for large numbers of carcasses, the ‘straw
technique’ may be considered, whereby brain material is
collected by passing a plastic straw through the occipital
foramen [25].
Rabies virus isolation
The OIE recommends the use of a confirmatory virus isolation
test, particularly when FAT results are equivocal and for human
exposure cases.
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rabies infection was confirmed using FAT on subsequent post
mortem brain specimens [32]. All positive PCR products should
be sequenced to confirm the origin of the virus and rule out
possible contamination. Such studies are increasingly reported in
the literature and are giving new insights into the epidemiology
and evolution of this most important virus.
The mouse inoculation test (MIT) involves the intracerebral
inoculation of mice with a clarified supernatant of a homogenate
of brain material (cortex, Ammon’s horn, cerebellum, medulla
oblongata). Clinical signs (positive result) can be observed as soon
as six to eight days for RABV but have been known to take up to
20 days for EBLV-2 [29]. Mice should be observed for a period
of 28 days. This in vivo test is time-consuming, expensive and
involves the use of animals. The OIE recommends that it should
be avoided for routine diagnosis if validated in vitro methods
are established within the laboratory. In vitro virus isolation
tests such as the Rabies Tissues Culture Inoculation Test (RTCIT)
involve the inoculation of the sample into a neuroblastoma cell
line. Positive results are commonly obtained within 2-4 days.
The FAT is then used to confirm the presence of rabies antigen
in both infected mice or cell monolayers. One advantage of the
above in vivo and in vitro assays includes the amplification of the
virus isolate for future typing and analysis.
Several RT-PCR methods have been reported over the past decade
for the detection of lyssaviruses. These methods invariably involve
multiple transfers of nucleic acids between different tubes and
coupled with the high sensitivity of PCR methodologies, any
small amount of contamination will undoubtedly produce falsepositive results. Attempts have been made to adapt RT-PCR to
reduce manipulations thereby reducing contamination risks.
The visualisation of PCR products by gel electrophoresis exposes
facilities and operators to large quantities of amplified material
and thus many adaptations have been directed at replacing
this step. New and improved rapid diagnostic tools for rabies
including PCR ELISA [33] and Taqman technology [34-36] have
recently been developed for diagnostic purposes.
Histopathology
Rabies diagnosis based upon the detection of Negri bodies and
histological tests such as Sellers’s or Mann’s tests, are no longer
routinely performed by the majority of diagnostic laboratories,
as they are considered unreliable, particularly for decomposed
material.
A rapid rabies TaqMan assay that distinguishes between
classical rabies virus (genotype 1) and European bat lyssaviruses
1 and 2 (genotypes 5 and 6) has recently been reported [37].
This sensitive and specific assay is performed in a single step in
a closed tube system, thereby dramatically decreasing the risk
of contamination and allows for the genotyping of unknown
isolates. Such real time assays can be applied quantitatively
and the use of an internal control (e.g. ß-actin RNA or 18s
ribosomal RNA) enables the quality of the isolated template to
be assessed, thereby minimising the chance of false negative
results associated with poor sample quality. Real time Taqman
assays have been successfully employed in the rapid diagnosis
and genotype confirmation of EBLV-2 infected Daubenton’s
bats in the UK [38]. The assay was more recently used for the
intra vitam detection and genotyping of a human rabies case in
the UK in July 2005 within five hours of sample receipt [20] and
for the detection of a rabies infected puppy in UK quarantine in
2008 [39].
A protocol suitable for the detection of classical rabies virus
and European bat lyssaviruses type 1 and 2 has more recently
been described [30]. A robust, highly sensitive and specific in
situ hybridisation technique, employing digoxigenin labelled
riboprobes was used for the detection of lyssavirus RNA in
mouse-infected brain tissue. Using this method, both genomic
and messenger RNA were detected. The ability to detect
messenger RNA is indicative of the presence of replicating
virus.
Nucleic acid based methods
Molecular based tools are becoming more widely acceptable
and accessible for the diagnosis of rabies. The use of the reverse
transcriptase polymerase chain reaction (RT-PCR), nested or
hemi-nested RT-PCR and other PCR based techniques are
increasingly reported but are not currently recommended by the
WHO for routine post mortem diagnosis of rabies. However,
in laboratories with strict quality control procedures in place
and demonstrable experience and expertise, these molecular
techniques have been successfully applied for confirmatory
diagnosis and epidemiological surveys. Reverse transcription
(RT)-PCR has been reported to confirm rabies diagnosis intra
vitam in suspect human cases, when conventional diagnostic
methods have failed and post-mortem material is not available
[31]. Rabies virus RNA can be detected in a range of biological
fluids and samples (e.g. saliva, cerebrospinal fluid, tears, skin
biopsies and urine). Owing to the intermittent shedding of virus,
serial samples of fluids such as saliva and urine should be tested
but negative results should not be used to exclude a diagnosis of
rabies. This was demonstrated in 2001 in the UK when multiple
ante mortem saliva specimens taken during the clinical phase of
infection in a Nigerian patient failed to yield rabies viral RNA but
In 2001 Wacharapluesadee and Hemachudha reported the use
of a rapid automated nucleic acid sequence-based amplification
(NASBA) technique which was successfully applied to the saliva
and CSF of four living patients in Thailand with rabies and
detected rabies viral RNA as early as two days after onset of
symptoms [40]. This technique differs from RT-PCR in that the
viral RNA is directly amplified under isothermal conditions and
detected by an automated reader. It is relatively easy to use and
the whole process from extraction to detection can take as little
as four hours using automated equipment. This technique has
also been adapted to investigate rabies virus replication in situ
[41].
Serology
Post-infective rabies antibodies are rarely detectable before
death, allowing antibody detection methods such as the rapid
fluorescent focus inhibition test (RFFIT) or the fluorescent
antibody virus neutralisation (FAVN) test to determine post-
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potential benefits of such methodologies in the hands of trained
staff in appropriately equipped laboratories, both in terms of
diagnosis and molecular epidemiological surveys. As molecular
methodologies are adopted and adapted in increasingly more
laboratories in both developed and developing countries, global
recognition of the importance of quality assurance is essential.
In order to ensure confidence in diagnostic test results, the
OIE published guidelines for quality assurance in veterinary
laboratories (http://www.oie.int/eng/publicat/ouvrages/A_112.
htm) based on the requirements of the internationally recognised
standard ISO/IEC 17025:1999.
vaccinal immunity in vaccinated animals. In previously nonimmunised humans, virus neutralisation assays are sometimes
successful in detecting rabies specific antibodies in the CSF or
serum and thus confirming exposure to a lyssavirus. Neutralising
antibodies, if present in serum tend to appear on average eight
days after clinical signs occur whereas rabies antibodies are
infrequently found in CSF.
Virus neutralisation (VN) assays in cell cultures are more
commonly used to determine levels of immunity in vaccinated
animals (domestic and wildlife). The FAVN and RFFIT are the OIE
prescribed tests for international trade of companion animals. In
both cases, a given quantity of the virus is mixed with different
dilutions of the serum to be tested and the neutralising limit
dilution is determined. Virus escaping neutralisation by the test
antisera is identified by fluorescence. The results are expressed
in international units (IU) calibrated from standard reference
materials available from the OIE Reference Laboratory in AFSSA,
Nancy, France (for animal tests) or from the WHO (for human
tests). Due to the nature of both tests, dedicated biosecure
facilities and highly trained vaccinated staff are required. Enzyme
linked immunosorbent assays (ELISAs) suitable for the detection
of rabies virus-specific antibodies in serum samples from
companion animals and humans have recently been developed
[42-45]. The companion animal ELISAs have been accepted by
the OIE as a screening tool or alternative to the FAVN but await
approval for the EU Pet Travel Scheme. The major advantages of
the ELISA test are that it can be completed in four hours, does
not require the use of live virus and can be performed without
the need for specialised laboratory containment. This contrasts
with the four day turnaround using conventional rabies antibody
virus neutralisation assays.
With respect to serology for international trade, annual proficiency
schemes coordinated by the OIE and Community reference
laboratory AFFSA, Nancy, Malzeville, France, have been in operation
for a number of years. Inter-laboratory ring trials of diagnostic
techniques in European rabies laboratories will also be coordinated
by AFSSA in the near future. Rabies diagnostic proficiency schemes
are in operation in the USA and have highlighted the need for the
standardisation of methodologies. The Centers for Disease Control
and Prevention (CDC) have published a protocol for the FAT on
their website to encourage harmonisation (http://www.cdc.gov/
ncidod/dvrd/rabies/professional/publications/DFA diagnosis/DFA
protocol-b.htm). A report by Rudd et al. emphasised the dramatic
effects that small modifications to the FAT protocol, such as a
change in mountant composition, can have on the specificity or
sensitivity of the test [47].
All laboratories should endeavour to validate their procedures
using the array of virus strains likely to be locally encountered
and not rely on the positive control material (e.g. laboratory
adapted strain) for test validation. Test validation exercises must
be repeated upon the introduction of any modifications to the
test, however minor, and also if the range of lyssaviruses to be
detected is expanded to include, for example, newly isolated
diverging bat strains. As increasing numbers of laboratories
strive to achieve quality assurance standards, the requirement to
participate in inter-laboratory proficiency schemes will facilitate
a more harmonised approach to rabies diagnosis and a greater
confidence in epidemiological data.
Recently, a virus neutralisation based assay has been developed
utilising lentiviral pseudotypes as antigen [46]. The pseudotype
viruses were constructed using G-protein cDNA sequences from
rabies Challenge Virus Standard-11 (CVS-11), European bat
lyssavirus type-1 or -2 (EBLV-1 and -2), cloned and co-expressed
with HIV/MLV gag-pol and GFP/luciferase in human epithelial
cells. The pseudotypes have three major advantages over the
conventional virus neutralisation assays: they can be handled in
biohazard level 2 laboratories, the use of reporter genes such
as green fluorescent protein (GFP), luciferase or β-galactosidase
allows the assay to be used at low cost in laboratories throughout
the world and the pseudotype assay only requires minimal
volumes (<10µl) of sera. The applicability of the pseudotype
viruses is currently being validated in a collaborative study
involving a number of Rabies Reference Laboratories.
Conclusion
Rabies is one of the best known and most feared of viral
infections. It is notifiable and suspect cases must be reported to
competent authorities that are responsible for further diagnosis.
Diagnostic tests based on antigen detection, virus isolation and
histopathology have been the mainstay of diagnosis. However,
these are relatively slow and insensitive, and in some cases require
the use of live animals. There is now an increasing pressure towards
the development and use of modern molecular technologies.
These promise great advances with increased sensitivity providing
for ante-mortem diagnosis. By allowing sequence analysis, they
have also revolutionised our understanding of the evolution and
epidemiology of these viruses, challenging concepts of what
it is to be “rabies free”. Inter-laboratory collaboration and test
standardisation will allow for a better understanding of the global
presence of rabies viruses, and is likely to be necessary to facilitate
their greater control.
Validation of Diagnostic Assays
Rabies is a notifiable disease in most developed countries and
as such, standardised diagnostic procedures are prescribed by
the OIE. Due to the high sensitivity of PCR based methods,
there is concern that appropriate procedures aimed at reducing
the risk of contamination will not be fully adhered to, thereby
generating false positive results. Consequently, the WHO
expert committee agreed that molecular methods should not
be globally recommended [7]. However, they did recognise the
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231
ZOONOSES
Canine leishmaniosis a challenging zoonosis
L. Solano-Gallego(1), G. Baneth(2)
SUMMARY
Leishmania infantum is the causative agent of canine leishmaniosis (CaNL) and human visceral leishmaniasis in the
Mediterranean basin and some other parts of the world. Dogs are the major reservoir for this infection. Surveys
have revealed that dog infection rates reach 70% in some foci in the Mediterranean basin. Canine leishmaniosis is a
good example of a disease in which infection does not equal clinical illness due to the high prevalence of subclinical
infection. Population studies have shown that a low proportion of the canine population develops a severe disease while
another fraction has persistent subclinical infection. Susceptibility to CaNL has been associated with genetic factors
and mutations in several loci. The main clinical signs associated with CaNL are skin lesions, lymphadenomegaly,
splenomegaly, ocular abnormalities, abnormal nails growth (onychogryphosis) and poor body condition. Additional
findings include: epistaxis, renal failure, decreased appetite, polyuria and polydypsia, vomiting, melena and lameness.
The diagnosis of CaNL is performed by microscopic detection of parasites, serology and PCR. Several drugs including
allopurinol, meglumine antimoniate, amphotericin B, and miltefosine are used for treatment of CaNL. However,
although most dogs recover following therapy, complete elimination of the parasite is usually not achieved and
infected dogs may relapse following treatment. Prevention of CaNL includes the use of topical insecticides against
sand flies, such as pyrethroid collars, spot-on formulations and sprays. In addition, new canine vaccines are being
developed and commercially introduced or evaluated in several countries.
the third most important vector-borne disease after malaria and
lymphatic filariasis. Based on clinical signs in humans, the disease
can be divided into cutaneous, mucocutaneous and visceral
forms. An estimated 12 million human cases of leishmaniosis
exist worldwide, with an estimated number of 1.5-2 million
new cases occurring annually: 1-1.5 million cases of cutaneous
leishmaniosis and 500.000 cases of visceral leishmaniosis [5].
Epidemiologically, two different situations occur: (i) The zoonotic
form of visceral leishmaniosis found in the Mediterranean
basin and South America, with the dog as the main source
of L. infantum infection for the female sand fly; and (ii) the
anthroponotic form found in East Africa, Bangladesh, India and
Nepal where L. donovani transmission is passed from person to
person through the sand fly vector [5]. Anthroponotic visceral
leishmaniosis caused by L. donovani is responsible for a large
part of the fatalities due to the visceral disease in people [6].
This paper was commissioned by FECAVA for
publication in EJCAP.
Introduction
Leishmaniosis has a long history of association with mankind.
The disease is known to have been present in Africa and India
since at least the mid-eighteenth century [1]. In 1903, Leishman
and Donovan separately described the protozoan now termed
Leishmania donovani in splenic tissue from human patients in
India [2, 3]. In 1908, Nicolle and Comte in Tunisia reported
infection by Leishmania in dogs for the first time [4].
Leishmania infections caused by different Leishmania species
are present in a variety of biogeographical regions of the Old
and New Worlds. These are responsible for the wide spectrum
of clinical illnesses observed in people. Human leishmaniasis is
Visceral leishmaniosis caused by L. infantum in the Mediterranean
basin was traditionally predominantly a disease of young children
(1) Laia Solano-Gallego. Department of Pathology and Infectious Diseases, Royal Veterinary College, University of London, United Kingdom
(2) Gad Baneth. School of Veterinary Medicine, Hebrew University, P.O. Box 12, Rehovot 76100, Israel.
Corresponding author address: Laia Solano-Gallego. Department of Pathology and Infectious Diseases
Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, United Kingdom, Tel. +44(0)1707666326, Fax. +44(0)1707661464, E-mail: lsolano@rvc.ac.uk
232
Canine leishmaniosis - a challenging zoonosis - L. Solano-Gallego, G. Baneth
Transmission of canine leishmaniosis
and the name of the causative agent of this disease reflects
the predilection to infants. Malnutrition has been recognized
as risk factor for infantile leishmaniosis, and may explain why
this disease is more prevalent among children in poor countries
as compared with affluent ones despite high prevalence rates
in the dog populations [6]. With the appearance of the AIDS
epidemic, adult HIV+ patients are now the predominant risk
group for human visceral leishmaniosis in Southern Europe. The
co-infection of HIV and leishmaniosis reported from more than
33 countries where these infections geographically overlap has
been described as a “deadly gridlock” that does not respond
well to therapy [7, 8].
Female sand flies from the genera Phlebtomus (Old World) or
Lutzomyia (New World) are the principal agents of transmission
of Leishmania in humans and dogs [27]. The activity of the adult
sand flies is crepuscular and nocturnal from early spring to late
autumn in the Mediterranean basin and all year round in South
America [27].
Leishmania is a diphasic parasite that completes its life cycle in
two hosts, a sand fly which harbors the flagellated extracellular
promastigotes and a mammal where the intracellular amastigote
parasite forms develop. Dogs are infected by Leishmania
promastigotes deposited in the skin during the bites of infected
female sand fly vectors. The promastigotes invade host cells
and replicate as intracellular amastigotes. The transmission of
Leishmania metacyclic promastigotes by phlebotomine sandflies
is extensively reviewed elsewhere [28].
CaNL is a major zoonotic disease endemic in more than 70
countries in the world. It is present in regions of southern Europe,
Africa, Asia, South and Central America [9] and has recently
emerged in the USA [10, 11]. CaNL is also an important concern
in non-endemic countries where imported disease constitutes
a veterinary and public health problem [12]. Dogs are the main
reservoir for human visceral leishmaniosis and the disease is
usually fatal if not treated in people and dogs. Phlebotomine
sand flies are the proven vectors of Leishmania infantum, the
causative agent of CaNL in the Old World and for its New World
synonym Leishmania chagasi. The majority of epidemiological
studies about the prevalence and incidence of Leishmania
infection in dogs have been based on serologic surveys. The
seroprevalence in the Mediterranean basin ranges from 10% to
40% depending on the region [13]. Surveys employing other
methods to calculate the prevalence of Leishmania infection
by detecting Leishmania DNA in different tissues [14-16] or by
detecting specific-Leishmania cellular immunity (17-19) have
revealed even higher infection rates approaching 70% in some
foci. It has been estimated based on seroprevalence studies from
Italy, Spain, France and Portugal that 2.5 million dogs in these
countries are infected [20] and L. infantum infection is spreading
north in Europe reaching the foothills of the Alps in Northern
Italy [21]. The number of infected dogs in South America is also
estimated in millions with high infection rates in some areas of
Brazil and Venezuela [22].
An important medical and epidemiological question is whether
dogs found positive to the Leishmania parasite with different
techniques such as serology and/or PCR are also infective to
phlebotomine sand flies. This information would provide the best
indicator of the burden of infectiousness in a dog population in
an endemic area. Seropositive dogs with and without clinical
signs are infectious to sand flies [29-31]. The rate of infected
sand flies increased with the appearance and severity of the
clinical signs [30] and with the decrease in CD4+ T cells counts
[32]. No studies have been published on transmission from
seronegative subclinically infected dogs. Further studies are
needed to ascertain the potential of seronegative subclinically
infected dogs to transmit Leishmania to vector sand flies.
Other less common transmission routes have been reported in
dogs. The transmission of L. infantum through blood products
has been reported in dogs that received blood transfusions from
infected donors in North America [33, 34]. Vertical in-utero
transmission from dam to its offspring has been documented in
one study [35] but disputed by other authors [36]. In addition,
ticks and fleas have been proposed as alternative vectors of
Leishmania transmission but evidence of such transmission is
lacking [37, 38]. Direct transmission without involvement of a
hematophageous vector has been suspected in some cases of
infection in areas where vectors of the disease are apparently
absent [11].
Public health considerations
Transmission of L. infantum from dogs or wildlife animal reservoirs
via sand flies is the main route for human infection. People in
endemic regions share the same habitat and are frequently in
close physical contact with infected dogs [23]. Several studies
have investigated the association between canine and human
leishmaniosis in the same region and examined to what degree
infection in dogs poses a risk for human disease [23]. These
studies reported that (i) increased prevalence in canine population
is associated with increased incidence of human leishmaniosis
[22, 24], (ii) poor socioeconomical conditions are risk factors
for the association between canine and human infections [22]
and (iii) dog density and infected dog ownership are risk factors
for infantile human leishmaniosis [25, 26]. Therefore, effective
control of canine leishmaniosis could lead to a decrease in
human leishmaniosis in endemic regions. Control measures of
CaNL could include effective vaccines, topical insecticides, and
environmental control of sand flies.
Disease versus infection by L. infantum
The classical stages of an infectious disease process include
the initial infection, an incubation period and a clinical disease.
Numerous studies on these stages in relation to Leishmania
have indicated that Leishmania infection and disease are not
always considered synonymous in rodents, humans and dogs.
The concept that all dogs infected with Leishmania infantum will
eventually develop severe clinical leishmaniosis after a variable
incubation period [39, 40] has been disproved. Specific antiLeishmania cellular immunity was demonstrated in apparently
healthy dogs naturally infected with Leishmania [18, 41].
Leishmania infantum infection in dogs can display a broad
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spectrum of immune responses and clinical manifestations from
a clinically healthy condition to a severe clinical disease. This
is similar to findings in human infection where clinical disease
represents one pole and subclinical infection the other pole of
infection [42].
[48, 49] but decreased or absent leishmanial specific lymphocyte
proliferation and delayed type hypersensitivity (DTH) reaction
[41, 50, 51]. Conversely, healthy infected dogs (resistant) produce
specific lymphocyte proliferation, strong DTH reaction, variable
anti-parasite antibody levels [17-19, 41] and lower parasite loads
when compared with sick or susceptible dogs [48, 49].
Leishmania infection in dogs may vary between a subclinical
infection and a severe fatal disease [9]. In dogs, the two opposite
extreme poles of this spectrum are characterized by: (i) protective
immunity that is T cell mediated and (ii) disease susceptibility
that is associated with the production of a marked humoral
non-protective immune response and a reduced or depressed
cell mediated immunity (CMI) [9, 13, 44]. For example, clinical
disease can range from a mild papular dermatitis associated
with specific cellular immunity and low humoral responses [43]
to a severe/fatal disease characterized by renal pathology with
glomerulonephritis due to immune complex deposition mainly
with an extensive humoral response and high parasite loads
[45-47].
Specific immune responses play a major role in susceptibility and
resistance to infection. An experimental model of cutaneous
leishmaniosis in mice infected with L. major has shown that
a susceptible mice strain (BALB/c) which typically develops
a T helper type 2 cellular response will succumb to infection
with secretion of specific cytokines such as IL-4 and IL-10, and
production of a significant antibody response. Another mice
strain (C3H) that responds with a different set of cytokines typical
of a T helper type 1 response such as IFN-γ; IL-2, is resistant to
infection [52]. This general model appears to be only partially
valid for CaNL and human visceral disease where studies of the
immune response have shown mixed Th-1 and Th-2 types of
reaction. An update on canine immune responses in L. infantum
infection is reviewed elsewhere [9, 44].
Population studies in Leishmania-endemic areas have shown
that a low proportion of the canine population develops a
severe disease and another fraction has subclinical infection. The
immune responses mounted by dogs at the time of infection
and thereafter appear to be the most important factor in
determining if they will develop a lasting infection and whether
and when it will progress from a subclinical state into a disease
condition. Animals that are predisposed and will develop severe
disease are considered “susceptible”. Dogs able to control/
restrict the infection and remain constantly subclinical, have
been termed “clinically resistant” [18, 19]. However, subclinical
infection is not necessarily permanent and factors such as
immunosuppressive conditions or concomitant disease could
break the equilibrium and lead to the progression of clinical
disease in dogs as observed in human patients with AIDS and
Leishmania co-infection [7].
Immune-mediated mechanisms are responsible for much of the
pathological findings in CaNL. Circulating immune complexes
[53] and antinuclear antibodies [54, 55] have been detected
in animals with CaNL. Glomerulonephritis associated with the
deposition of immune complexes in the kidneys is a hallmark of
CaNL [45-47]. Vasculitis induced by immune complexes which
activate the complement cascade is an important pathological
mechanism responsible for tissue necrosis and accountable
for dermal, visceral and ocular lesions found in this disease
[56-58].
Dogs with severe disease or progressing toward overt disease
have high antibody levels, high parasite load in numerous tissues
It is not known for certain what mechanisms in dogs are
responsible for protection or susceptibility, nor the way factors
such as age, gender, nutrition, host genetics, coinfections and
concomitant disease, immunosuppressive conditions, cytokine
environment, parasitic burden, nature of Leishmania antigens
or different Leishmania strains, previous infections and way of
Fig. 1 Exfoliative dermatitis, hypotrichosis and alopecia of the face
and pinna in a dog with leishmaniosis.
Fig. 2 Exfoliative dermatitis and alopecia on the ear tips of a dog
with leishmaniosis.
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Canine leishmaniosis - a challenging zoonosis - L. Solano-Gallego, G. Baneth
transmission can affect the polarity of clinical manifestations of
Leishmania infection.
Age seems to be an important factor with a peak in the prevalence
of disease in dogs younger than 3 years or older than 8 year [5961]. Studies have indicated that several dog breeds are apparently
more susceptible to disease. These include the Boxer, Cocker
Spaniel, Rottweiler and German shepherd [62, 63]. Other breeds
that have evolved in endemic areas such as the Ibizian Hound
rarely develop the disease and present a protective predominant
cellular immunity [19]. The Slc11c1 (Solute carrier family 11 member
a1) gene [64] and certain dog major histocompatibility complex
(MHC) class II alleles have been found to be associated with the
susceptibility to CanL [65]. Further studies on specific genes and
whole genome comparisons among resistant and susceptible
breeds might further elucidate the mechanisms responsible for
susceptibility to CaNL.
alopecia which can be generalized or localized over the face,
ears and limbs (Fig. 1 and 2), (ii) ulcerative dermatitis, nodular
dermatitis, (iii) mucocutaneous proliferative dermatitis and (iv)
papular dermatitis [43].
The most common ocular manifestations are anterior
uveitis, blepharitis (exfoliative, ulcerative, or nodular) and
keratoconjunctivitis, either common or sicca [70-72]. About 25%
of dogs with clinical leishmaniosis have ocular and periocular
lesions including keratoconjunctivitis and uveitis [72]. Ocular
consequences of systemic hypertension such as retinal detachment
and/or hemorrhages, retinal arterial tortuosity and hyphema are
present in the disease but not diagnosed frequently [73].
The main clinicopathological findings of canine leishmaniosis
are hyperglobulinemia (polyclonal gammaglobulinemia),
hypoalbuminemia, decreased albumin/globulin ratio, mild to
severe proteinuria, non-regenerative anemia, renal azotemia,
elevated liver enzyme activities, thrombocytopenia and
thrombocytopathy [68-70, 74, 75].
Clinical findings in canine
leishmaniosis
Some degree of renal pathology is present in most dogs with
CaNL and subsequent renal failure due to immune-complex
glomerulonephritis eventually develops and is believed to be the
main cause of death in dogs with CaNL [45-47]. Epistaxis, ocular
abnormalities or renal failure may be the only presenting clinical
findings in CaNL and this disease should be considered among
the differential diagnoses for these conditions in endemic areas
or in dogs that have traveled or were imported from an endemic
region. Marked hyperglobulinemia with no apparent cause
in dogs from endemic regions should also be investigated for
CaNL.
The clinical features of leishmaniosis vary widely as a consequence
of the numerous pathogenic mechanisms of the disease process,
the different organs affected, and because of the diversity of
immune responses of individual hosts, whether humans or other
mammals [66].
The main clinical findings found on physical examination
in classical CaNL include skin lesions (Fig. 1 and 2), local or
generalized lymphadenomegaly, loss of body weight, exercise
intolerance, decreased appetite, lethargy, splenomegaly, polyuria
and polydypsia, ocular lesions, epistaxis, onychogryposis,
lameness, vomiting and diarrhea [67-70].
It is sometimes difficult to define if dogs have clinical disease
due to CaNL because they may be infected with the parasite
without clinical signs and be ill due to other causes, or because
other diseases may present similar clinical signs. In the past,
Skin lesions are the most frequent manifestation of CaNL in dogs
brought for treatment due to the disease. Several dermatological
entities have been described [39]: (i) exfoliative dermatitis with
Tab. 1 Diagnostic techniques used for the diagnosis of canine leishmaniosis.
Diagnostic technique
1. Serology
1. Indirect fluorescent antibody test (IFAT)
2. Enzyme linked immunosorbent assay (ELISA)
3. Direct agglutination test (DAT)
4. Antigen-specific rK39 serology
5. In-house immunochromatographic devices
6. Western blot
2. Cytology
1. Skin touch preparation
3. Histopathology
1. Formalin fixed tissues stained with hematoxylin-eosin
2. Fine needle aspirates from lymph nodes, bone marrow and spleen
2. Immunohistochemistry of tissues using anti-leishmanial antibodies
4. Detection of Leishmania DNA by PCR
1. Conventional PCR targeting the kinetoplast DNA (kDNA) or genomic ribosomal RNA genes
2. Nested PCR
3. Quantitative real-time PCR
5. Parasite culture
1. Culture of aspirates from the bone marrow, spleen or lymph node in a specialized medium
for the cultivation of Leishmania
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EJCAP - Vol. 18 - Issue 3 December 2008
Fig. 3 Touch impression from the skin of the ear in a dog with
leishmaniosis.
Fig. 5 Fine needle aspiration from the prescapular lymph node of a
dog suspected for canine leishmaniosis.
researchers have used the clinical classification of asymptomatic,
oligosymptomatic and polysymptomatic dogs [76] based only
on physical examination. This classification has a limited value
because it does not consider clinicopathological abnormalities
and disregards dogs that have widespread organ dysfunction
without apparent visual manifestations. The authors consider a
sick dog suffering from leishmaniosis if it presents with compatible
clinical signs and/or clinicopathological abnormalities and the
diagnosis is confirmed by specific tests for the infection
and ultrasound can assist in raising the suspicion index for this
disease. Numerous diagnostic techniques have been developed
to help in the diagnosis of canine leishmaniosis (Tab. 1). It is
important to understand the basis of each diagnostic test, the
limitations and the appropriate clinical interpretation.
Leishmania amastigotes can be demonstrated by cytology from
cutaneous lesions (Fig. 3), spleen (Fig. 4), lymph nodes (Fig. 5),
bone marrow [13, 78] and less commonly in other tissues [7981] or body fluids such as joint, cerebrospinal and abdominal
fluids [82-86] stained with Giemsa, or modified Wright’s stain,
or a quick commercial stain. Detection of amastigotes by
cytology is frequently unrewarding due to a low to moderate
number of detectable parasites present even in dogs with a
full blown clinical disease [87, 88]. Leishmania parasites may
also be viewed in histophatologic formalin-fixed, paraffinembedded biopsy sections of the skin or other infected organs.
Leishmania parasites should be suspected in pyogranulomatous,
granulomatous or lymphoplasmacytic inflammations in different
tissues [79, 80, 89-91] or lymph node reactive hyperplasia
[92, 93] on cytological or histological preparations. Definite
identification of parasites within tissue macrophages may be
difficult and an immunohistochemical staining method can be
employed to detect or verify the presence of Leishmania in the
tissue [94].
Diagnosis of canine leishmaniosis
CaNL is a good example of a disease in which infection does
not equal clinical illness due to the high prevalence of subclinical
infection. This makes CaNL a diagnostic challenge for the
veterinary practitioner, clinical pathologist and public health
official in endemic countries as well as non-endemic regions
where imported infection is a concern [23, 77].
Accurate diagnosis of canine leishmaniosis often requires
an integrated approach (clinicopathological diagnosis and
specific laboratory tests). Pertinent clinical history, a thorough
physical examination and several routine diagnostic tests such
as CBC, biochemical profile, urinalysis, serum electrophoresis,
coagulation profile, imaging of the abdomen by radiographs
Various serological methods for the detection of anti-Leishmania
antibodies have been developed. These include the indirect
immunofluorescence assay (IFA), enzyme-linked immunosorbent
assay (ELISA), direct agglutination assays (DAT) and western
blotting [95]. In general, good sensitivities and specificities are
gained with these methods for the diagnosis of clinical CaNL
[23]. High antibody titers are usually associated with disease
and a high parasite density [49, 96] and, for this reason; they
are conclusive of a diagnosis of leishmaniosis. However, the
presence of lower antibody levels is not necessarily indicative of
patent disease and needs to be confirmed by other diagnostic
method such as PCR, cytology or histology [23, 77]. Serological
cross-reactivity with different pathogens is possible with some
serological tests, especially those based on whole parasite
antigen. Cross reactivity has been reported with other species
of Leishmania [97-99], and Trypanosoma cruzi [98].
Fig. 4 Fine needle aspiration
from the spleen of a dog for the
diagnosis of leishmaniosis.
236
Canine leishmaniosis - a challenging zoonosis - L. Solano-Gallego, G. Baneth
First line drugs
Generic name
1. Meglumine antimoniate
2. Allopurinol
3. Combination of meglumine antimoniate and allopurinol
4. Amphotericin B
5. Miltefosine
6. Combination of amphotericin B or miltefosine and allopurinol
Second line drugs
1. Aminosidine
2. Metronidazole and spiramycin
3. Metronidazole and enrofloxacin
4. Marbofloxacin
5. Ketoconazole
6. Pentamidine
Tab. 2 First and second line drugs for the treatment of canine leishmaniosis.
evaluation for dogs [23]. Immunotherapy is also under extensive
investigation in dogs with promising results [116, 117].
Detection of parasite-specific DNA in tissues by PCR allows
sensitive and specific diagnosis. Several different assays with
various target sequences using genomic or kinetoplast DNA
(kDNA) have been developed for CaNL. PCR can be performed on
DNA extracted from tissues, blood or even from histopathologic
specimens. Assays based on kDNA appear to be the most
sensitive for direct detection in infected tissues [23, 100]. Several
tissues have been studied: lymph node [88, 101, 102], bone
marrow aspirates [87, 103, 104], whole blood [101. 105, 106],
buffy coat [104], paraffin-embedded skin biopsies [107], spleen
[108], frozen skin and eye conjunctiva [14, 15], conjunctival
swab [109, 110] and urine samples [111] with varying results.
PCR on bone marrow, lymph node, or skin are most sensitive
and specific for the diagnosis of canine leishmaniosis [15, 87,
101, 107, 112, 113]. PCR on whole blood and urine samples
seem to be less sensitive than the tissues mentioned above [101,
111, 113]. PCR on aspirates of lymph node and bone marrow
have been shown to be more sensitive than stained smears or
parasite culture [87, 88, 101, 114]. Currently, three different
PCR techniques are available and they are listed by the level of
sensitivity from less to more sensitive: conventional PCR, nestedPCR and real-time PCR [23, 100].
Keeping and treating infected dogs presents a dilemma to
owners, veterinarians and public health officials especially in
areas where suitable vectors are found, because of the risk of
transmission to people and pets in the community. Treated dogs
often remain carriers of the disease and commonly experience
clinical relapses. However, it appears that they are less infectious
to sandflies [118, 119]. The use of adequate treatment is
advocated for the control of canine leishmaniasis and to reduce
risks of L. infantum transmission [119]. Owners must receive
a thorough and realistic explanation about the disease, its
zoonotic potential, the prognosis for their dog, and what should
be expected from treatment.
Prevention of canine leishmaniosis
The use of topical insecticides against CaNL [120] in collars
[121] or spot-on formulation has been shown to be effective
in reducing disease transmission [122, 123]. Delthamethrinimpregnated collar significantly reduced the number of dog
sand fly bites under experimental conditions [124, 125] and
decreased infection transmission in field studies [121]. In a study
supported by the WHO in Iran, collaring of dogs in intervention
and control villages significantly reduced the seroconversion rate
in dogs and in children living in the intervention villages [126].
A commercial vaccine against CaNL has recently been approved
in Brazil [127, 128] and several vaccine candidates are under
experimental [129] or field evaluation in Europe [130].
Therapy
Anti-leishmanial treatment often achieves only clinical
improvement in dogs with leishmaniosis and it is frequently
not associated with the elimination of the parasite. The main
drugs used against canine leishmaniosis include the pentavalent
antimony meglumine antimoniate which selectively inhibits
leishmanial glycolysis and fatty acid oxidation and allopurinol
that acts by inhibiting protein translation through interfering
with RNA synthesis (Tab. 2). The combination of antimony
meglumine antimoniate and allopurinol is the most common
treatment protocol used against canine leishmaniosis in Europe
[115]. Amphotericin B, which acts by binding to ergosterol in
the parasite’s cell membrane and altering its permeability is also
used but it is highly nephrotoxic. New drugs are currently under
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241
ZOONOSES
Toxoplasmosis
F. van Knapen,(1) P.A.M. Overgaauw(2)
SUMMARY
This paper is a short introduction to the impact of Toxoplasma gondii infection in humans. Major routes of transmission
occur through tissue cysts infected raw meat and furthermore oocysts shed by (merely) young cats which contaminate
litterboxes, gardens, sandboxes and playgrounds. Clinical symptoms in man are rare, however long-lasting
lymphadenitis and general malaise may happen. Special attention is given to congenital toxoplasmosis in which the
disease burden may be dramatic. The role of dogs and cats in human toxoplasmosis is discussed.
Our housecat (Felix domestica) plays an important role.
In the jejunum (epithelium) Toxoplasma behaves like a real
coccidium: schizogeny and gametogeny are followed by
excretion of oocysts. The oocysts are shedded with the
faeces into the environment, where they have to sporulate to
become infectious (Fig. 3). Sporulation takes at least 2 to 3
days. Sporulated oocysts are very resistant to environmental
conditions and may remain infectious for two years in for
example garden soil [5].
This paper was commissioned by FECAVA for
publication in EJCAP.
Introduction
Toxoplasma gondii is an obligate intracellular protozoan
parasite and part of the Sporozoa family. There are three
known stages of this parasite with different appearances.
Epidemiology
During an active Toxoplasma infection the free parasites
(tachyzoites) replicate by division in the host cell until this cell
is lysed (Fig. 1). Extracellular parasites released by the host
cell lysis actively invade new host cells and cause severe tissue
damage. There is no preference for cell types or organ systems.
The tachyzoites are approximately 10 microns long and 2 to 3
microns wide and have a characteristic moon shape.
The consumption of, tissue cysts infected, undercooked meat
[2], especially from herbivores, is the most common route of
infection in man and carnivorous animals. Herbivores infect
themselves with oocysts from the environment. Significant
variation in frequency of toxoplasmosis infected animals has
been reported from different studies and countries. Especially
farm animals grazing outdoors may become infected, while
production animals housed indoors during their (short) life
are usually not infected with Toxoplasma. Herbivores and
vegetarians can also get infected by the intake of sporulated
oocysts shedded by cats (via soil, hands, vegetables etc.).
A second stage is characterized by tissue- and organ cysts of
10-100 microns in diameter; the cysts have a clear wall in which
hundreds of slim parasites without significant metabolism
are piled up (so called rest phase or latent toxoplasmosis;
(Fig. 2). These cysts remain present for many years and even
stay infectious for a long period after the host has died (for
example in meat).
Cats only shed oocysts once during their lifetime, and only
during a limited period of time (2-3 weeks) when they have
lost their passive immunity and have started to hunt or to eat
raw meat (ingestion of tissue cysts). Most of the spontaneous
excretors found in surveys are younger than 6 months of age.
A third stage of T. gondii is only found in cat species
(Felidae) and more specifically in the genera Felix and Lynx.
(1) Diplomate of the European College of Veterinary Public Health, Certified Parasitologist, Institute for Risk Assessment Sciences
(IRAS), Division Veterinary Public Health, Veterinary Faculty, Utrecht University, Yalelaan 2, NL - 3584 CM Utrecht.
E-mail: f.vanknapen@uu.nl
(2) Certified Veterinary Microbiologist, Certified Parasitologist, RnA Assays, Yalelaan 2, NL - 3584 CM, Utrecht. E-mail:
P.overgaauw@rnassays.com
* Presented by NACAM (The Netherlands)
242
Toxoplasmosis - F. van Knapen, P.A.M. Overgaauw
Fig. 1 Toxoplasma tachyzoites one hour and 24 hours after infecting a macrophage cell line.
(for example in the brain) no inflammatory response is visible
(Fig. 2).
During the excretion period they build up enough immunity to
resist a new infection. There are some exceptions to this, i.e.
primary infection at a very young age, long-term treatment
with corticosteroids, secondary infection with Cystoisospora,
etc. In these cases even mature cats may start shedding oocysts
again [4, 6]. Infection of a non-immunized cat with oocysts
results in a significant antibody response, although no oocysts
will be shedded [10]. In the Netherlands 60% of all one-year
old cats have passed this period, thus they do not longer play
an important role in the epidemiology of toxoplasmosis (Van
Knapen, 1974 and 1993, unpublished data). Not in all countries
seroconversion is seen in the first year of the cat’s life, as this is
also depending on outdoor access, contact with other cats and
the source of feed they obtained [9, 3]. This results in a persistent
contamination of the environment where (young) cats deposit
their faeces. In man it is estimated that 30-60% of all adults
have been infected with Toxoplasma. Depending on the genetic
type, the amount and virulence of the ingested parasites, clinical
symptoms can be expected within 2 to 3 weeks after infection.
The enteroepithelial phase of T. gondii is not usually associated
with enteric disturbance [13]. The intermediate tissue cyst stage,
however, may produce clinical signs in the cat as well as a variety
of other animals, including the dog. The clinical signs are in that
case non-specific and reflect damage to the organ systems
infected, including the central nervous system, musculature,
liver, lungs and the eye.
Since Toxoplasma-parasites show no cell type preference,
the clinical signs are variable. An adult person has enough
immunity to resist an infection and far most infections proceed
without any clinical symptoms. Occasionally parasitaemia is
seen when chronic (latent) infections are reactivated. Special
attention should be paid when a latent infection flares up
again due to immunosuppression. This can be the result of
another (viral) infection, or from a long-term treatment with
for example corticosteroids or cytostatics.
When clinical symptoms occur after an acute, acquired
toxoplasmosis infection, most evident signs are lymphadenitis,
fever and malaise. In rare cases a severe hepatitis, splenitis,
pneumonia, polymyositis or even meningo-encephalitis can
occur.
Fig. 2 Toxoplasma tissue cyst containing numerous bradyzoites
in brain material.
Pathogenesis, pathology and clinical
features in man
During an acute invasion of Toxoplasma-parasites (proliferative
phase, tachyzoites), there is mild to major tissue damage
(necrosis). Histology shows inflammatory infiltrates consisting
of round cells with free parasites and cyst formation on the
borders. Probably due to a combination of cellular and humoral
immune responses, the parasites are forced into a resting
stage (tissue cysts). This latent toxoplasmosis is characterized
by more or less round cysts with a firm wall, around which
243
EJCAP - Vol. 18 - Issue 3 December 2008
The role of dogs and cats in human
toxoplasmosis
It is obvious that the major source of human Toxoplasma
infection is the consumption of raw or improperly cooked
meat [2, 15]. However, oocysts shed by cats may be a source
of soil transmitted disease in man (children) like this is the
source for herbivorous livestock and rodents to become
infected. Indeed Taylor et al. [14] indicated a parallel in
seroprevalence studies in Ireland of specific Toxocara and
Toxoplasma antibodies in children between 4-18 years of age,
suggesting that contaminated soil may have been a common
source. Sporulated oocysts certainly contaminate the direct
environment of man (garden, sandpits) although very little
research is done in this field. Having a cat has never been
associated with a higher risk of getting a Toxoplasma infection.
On the other hand, however, having a dog does in some
studies correlate with an increased risk of having antibodies
to Toxoplasma [7]. Dogs definitely do not play a biological role
in the transmission of Toxoplasma. Soil transmitted infections,
like Toxocara have recently been demonstrated in the fur of
dogs, probably because of dogs behaviour on playing grounds
[11, 12].
Fig. 3 Oocyst of Toxoplasma in faeces.
A special form of toxoplasmosis is congenital toxoplasmosis.
If a woman receives her first exposure to Toxoplasma while
pregnant, the uterus and the unborn foetus(es) can become
infected. In early pregnancy this can lead to severe malformation
of the foetus leading to abortion or to malformations that
are not compatible with life shortly after birth. A majority
of congenital infections however go undiagnosed for years
after birth, before clinical symptoms related to congenital
toxoplasmosis are found (mental retardation, ocular defects)
[8].
If there is indication of soil transmitted infections through
dogs’ fur, it cannot be excluded that the same is true for
toxoplasmosis. Therefore hygiene precautions (e.g. hand
washing) should be taken after contact with dogs as a
preventive measure for toxoplasmosis.
Prevention
Diagnosis in man
Toxoplasmosis in man can be prevented by not eating raw
or undercooked meat (do not even taste it!). Especially
pregnant women should be careful because of the possibility
of transplacental transmission. Furthermore, contact with
sporulated oocysts should be avoided. This can be done
by cleaning the cat litter box daily (within that time no
sporulation has taken place), but far more important is it to
pay extra attention to personal hygiene when gardening (do
not eat or smoke while gardening, wear gloves) and do not
eat unwashed, raw vegetables. Wash hands after contact
with dogs.
Despite the high incidence of infections, clinical toxoplasmosis
is rare. Because the clinical signs of toxoplasmosis resemble
other infectious diseases, laboratory tests are necessary for a
diagnosis. Detection of specific antibodies alone is not enough,
because many individuals (people and animals) already have
an antibody titer. A new infection can be distinguished by the
detection of increasing amounts of antibodies (seroconversion)
of the different isotypes (IgG, IgM, IgA) or by circulating
antigens. The detection of free parasites (tachyzoites) in
combination with clinical symptoms can confirm an infection
(for example in biopsy or abortion material). The detection of
tissue cysts (just like antibodies alone) does not confirm an
active infection. PCR-tests are available for examination of the
foetus, the placenta or amniotic fluid.
Toxoplasmosis in cats can be prevented by feeding them
solely pre-fabricated food or cooked meat and by preventing
cats from hunting and eating prey animals. Small rodents and
birds form an important source of infection in cats, just like
oocysts infected soil (licked from their fur).
Diagnosis in animals
Since clinical symptoms are mostly absent, the only way to
demonstrate toxoplasmosis in animals is by serological testing.
Seropositivity is an indication for past experience and adult
animals of all kinds show moderate to high seroprevalences. In
cats the excretion of oocysts can be detected by examination
of the faeces. Oocysts are only shed during a short period of
time, so this examination is not useful when looking for the
source of clinical toxoplasmosis in a family.
There are no disinfectants effective against Toxoplasma
oocysts. Boiling water or steam kill them immediately.
244
Toxoplasmosis - F. van Knapen, P.A.M. Overgaauw
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gondii oocysts by cats. J Parasitol, 1995, 81: 410-415.
[7] FISHER (S.), REID (R.R.) - Antibodies to Toxoplasma gondii and
contact with animals in the home. The Med. J. of Australia 1973,
1: 1275–1277.
[8] GILBERT (R.E.), STANFORD (M.R.) - Is ocular toxoplasmosis caused
by prenatal or postnatal infection? Br J Ophthalmol, 2000, 84:
224-226.
245
ZOONOSES
Toxoplasmosis – an update
G. Miró,(1) A. Montoya,(2) M. Fisher,(3) I. Fuentes(4)
SUMMARY
Toxoplasma gondii is an obligate intracellular protozoan parasite, with the potential to infect human beings and a broad
spectrum of vertebrate hosts. Toxoplasmosis is an endemic disease with a worldwide distribution. Cats and wild
felidae play a crucial role in the epidemiology of this disease, as they are the only species that shed the environmentally
resistant oocysts in their faeces. Sporulated oocysts from infected cats and bradyzoites from tissue cysts are infectious
to all warm-blooded animals including human beings. Dogs and cats may act as intermediate hosts and could also
suffer clinical toxoplasmosis of variable severity. The majority of cases in immunocompetent people are asymptomatic
or produce only mild symptoms, while in congenitally infected children and immunocompromised people, infection
causes high rates of morbidity and mortality. Due to the potential public health risk of toxoplasmosis, veterinarians
need to be able to identify, control infected and treat sick cats and recommend measures to prevent T. gondii infection
in cats as well as in human beings.
first feline toxoplasmosis case was not reported until 1942,
by Olafson and Monlux. After a brief illness characterized by
anorexia, fever and a cough, the cat died. At necropsy, tumorlike enlargements of the mesenteric lymph nodes, ulceration of
the intestinal mucosa, and multiple nodules in the lungs were
observed. T. gondii was identified in the lymph nodes and lungs
[4].
This paper was commissioned by FECAVA
for publication in EJCAP.
Introduction
Toxoplasmosis is an important parasitic disease caused by the
protozoan Toxoplasma gondii. The parasite was first discovered
by Nicolle and Manceaux in 1908, in the gundi (Ctenodactylus
gundi), a North African rodent. The name Toxoplasma gondii
(“toxon”= arc; “plasma”= form in Greek) is derived from its
crescent shape and from the animal from which was first isolated.
The complete life cycle of T. gondii was not fully described until
1970 [1,2].
Life cycle of Toxoplasma gondii
The complex life cycle of Toxoplasma gondii involves two
phases. The sexual phase takes part in the Felidae family, and
the asexual phase in any warm-blooded animal, i.e. mammals
(including humans) and birds.
There are three infective stages:
– Sporulated oocysts from infected cats. The oocysts are
excreted unsporulated and uninfective when passed in
feces. Sporulation occurs in the environment after 1-5 days
(dependent on temperature and moisture; e.g. 1 day at 2425ºC, 5 days at 15ºC and 21 days at 11ºC).
The first record of canine toxoplasmosis was made by Mello
(1910), who described an acute toxoplasmosis in a dog in
Turin, Italy. The dog presented with fever, anorexia, anemia,
dyspnoea and haemorrhagic diarrhoea. T. gondii was identified
in sanguineous exudates and in nodules in the lungs [3]. The
1) Guadalupe Miró. Animal Health Department, Faculty of Veterinary Medicine / Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda Puerta Hierro s/n, 28040-Madrid (Spain). Tel.: 34-91-3943711. Fax: 34-91-3943908. E-mail: gmiro@vet.ucm.es
2) A Montoya. Animal Health Department, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain.
3) M. Fisher. Shernacre Cottage, Lower Howsell Road, Malvern, Worcestershire WR14 1UX, UK.
4) I. Fuentes. Department of Parasitology, National Center of Microbiology, ISCIII, 28224 Majadahonda, Madrid, Spain.
246
Toxoplasmosis – an update - G. Miró, A. Montoya, M. Fisher, I. Fuentes
Fig. 1 Life cycle of T. gondii (Montoya, 2007 [51]).
time to sporulation depending on environmental temperature
and humidity. Sporulated oocysts are more resistant to the
environmental and chemical conditions than are unsporulated
oocysts; their thermic tolerance range being – 4 to 55ºC. In
liquid medium they can survive for several years [6] (Fig. 1).
– Bradyzoites (“brady”= slow in Greek) from tissue cysts.
– Tachyzoites (“tachos”= speed in Greek), the rapidly dividing
forms that multiplies in any cell of the intermediate host and
non-intestinal epithelial cells of the definitive hosts.
The asexual phase or extra-intestinal cycle occurs in any warmblooded animal after infection by any infectious stage. After
ingestion, sporozoites are free in the intestinal lumen and divide
by an asexual process (endodyogeny). Tachyzoites multiply
rapidly, invading different cells during active infection. Once host
immunity develops the process slows down and during chronic
infection and cysts containing bradyzoites are formed [7].
The sexual phase or entero-epithelial cycle occurs in the
cat’s intestine after ingestion of any infective form. Zoites
penetrate the epithelial cells of the small intestine and initiate
a sequence of numerous generations (termed Types A to E) [5],
whereafter a variable number of schizogonies, microgamonts
and macrogamonts are formed. The microgamonts fertilize
the macrogamonts and oocysts are formed. Unsporulated
oocysts are shed with the feces into the environment, with
The prepatent period is around 18 days in cats fed infectious
oocysts as compared with 3 to 10 days in cats that are fed
tissues cysts [8].
Fig. 2 Epidemiological cycle of T. gondii (Courtesy Dr. M. Mateo).
Epidemiology
Cats and wild felidae play a crucial role in the epidemiology of
T. gondii, because they are the only hosts that can shed oocysts
in the environment (Fig. 2).
All nonfeline hosts are intermediate hosts that harbor tissue
cysts. It is generally assumed that cats probably play a major
role in transmitting the parasite through faecal contamination
of soil, food or water. In studies carried out so far, there is no
toxoplasmosis in areas where there are no cats [9] . However,
Prestud et al. (2007), reported other possible sources of infection
due to T. gondii in the Arctic [10].
Cats and dogs can be infected by T. gondii by different routes:
– Ingestion of water, food, vegetables contaminated with
sporulated oocysts from cats faeces.
– Ingestion of infected tissues with tissue cysts containing
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EJCAP - Vol. 18 - Issue 3 December 2008
Country
Nº
cats
Seropre- Test
valence
(%)
Reference
Pathogenesis
In the majority of acute infections acquired after ingestion of
tissue cysts or sporulated oocysts, the main infection route is
the intestine. Tachyzoites multiply actively, and are disseminating
through the lymphatic system to the different organs where they
produce cell necrosis caused by the intracellular growth of the
parasites. Heavily infected animals may die during this phase.
During this time parasites can be excreted through different
biological fluids (faeces, urine, etc.), but these tachyzoites are
very labile and are easily destroyed. For that reason transmission
to other hosts is very unlikely, even when the animals are
very close together. This fact has been demonstrated under
experimental conditions (e.g. sick mice living in the same cage).
Spain
Madrid
101
54,5
IFAT
Aparicio et al., 1972 [19]
Madrid
69
63,4
IFAT
Alonso et al., 1997 [20]
Madrid
279
30,8
IFAT
Miró et al., 2004 [17]
Barcelona
220
45
MAT
Gauss et al., 2003 [21]
La Rioja
306
33,7
IFAT
Miró et al., 2004 [17]
Germany 267
59,9
DT
Knaus & Fehler, 1989 [22]
Belgium
346
70,2
MAT
Dorny et al., 2002 [23]
Poland
105
52,5
LAT
Czech
republic
2.560 61,3
IFAT
Smielewska & Pacon, 2002
[24]
Svobodová et al., 1998 [25]
Sweden
244
ELISA Uggla et al., 1990 [26]
42
The sub-acute phase of the disease is characterized by the
appearance of IgA antibodies specific for T. gondii enteroepithelial
stages, which eliminate tachyzoite replication in the intestinal
phase but not those localized in the nervous system.
DT: Sabin Feldman Dye Test; IFAT: Indirect Fluorescent Antibody
Test; ELISA: Enzyme-linked Immunosorbent Assay; MAT: Modified
Agglutination Test; LAT: Latex Agglutination Test.
The chronic form starts with tachyzoites beginning to disappear
from visceral tissues and is characterized by persistence of
bradyzoites within cysts. These may be long-lasting; up to 10
months in the dog and 3 years in rats and pigeons, or maybe for
life. This phase is associated with a systemic immune response,
which inhibits tachyzoites proliferation in blood and tissues
(liver, spleen, lungs, etc.) [27].
Tab. 1 Seroprevalence to T. gondii in cats from Europe.
bradyzoites from mammals and birds. This is the most frequent
form of infection.
– Ingestion of infectious oocysts directly from faeces: dogs
ingesting cat litter could serve as mechanical vectors for
transmission to humans as they shed ingested sporulated
oocysts in their stool [11].
– Congential infection: parasitaemia during pregnancy can
cause transfer and spread of tachyzoites to the fetus [12,
13].
Other minor important routes of transmission include tachyzoites
shed in milk and ingestion through sucking [14], and blood
transfusion.
Clinical signs
T. gondii is the most important coccidian of the cat as it is the
cause of one of the most significant zoonoses. However, clinical
signs related to infection are normally not severe in the feline
host, although cats may have diarrhoea alternating with normal
faeces. Oocysts excretion from an infected cat occurs 3-10 days
post infection, and is maintained for a period up to 3 weeks.
Cats can also be asymptomatic during this phase [28].
The reason why infected dogs or cats develop clinical disease is
unknown but may depend on factors such as: age, sex, strain of T.
gondii infection rate, infection acquisition (post-natally acquired
infection is less severe than pre-natally acquired infection), stress,
concomitant illness (retrovirus, FIP, mycoplasmosis, distemper,
leishmaniasis, ehrlichiosis, etc) [29-33] or immunosuppression
(glucocorticoid or cyclosporine therapy) [34-36].
There are numerous studies on the seroprevalence of T. gondii
in felines reported from many countries in Europe, however
these studies are difficult to compare due to the differences in
the diagnostic techniques used, in the size and origin of the
samples, and time of the study conducted (Tab. 1). In all of
these reports the frequency of finding oocysts in faeces is low,
however, probably because oocysts are shed during a short
period of time [15].
Cat
In general, the cats that have a major influence in the epidemiology
of the disease are naive and farm cats. This fact is explained by
the preying habits of this group and their diet that includes wild
birds, rodents and Toxoplasma-infected placentas and stillborn
fetuses [15, 16]. The higher seroprevalence reported in adult
cats indicates that the risk of exposure to T. gondii increases
with age. Sex is not considered to be a determining factor of
infection [17]. Nonetheless, some authors suggest a higher
prevalence in males associated with their territorial habits, as
they have a wider area of operation than females [18].
When infected cats act as intermediate hosts they may suffer a
more or less severe clinical picture (clinical toxoplasmosis) such
as: fever (40 to 41ºC), lethargy, dyspnoea, lymphadenomegaly,
vomiting, diarrhoea, icterus, respiratory tract disease, neurologic
signs (stupor, ataxia, seizures, circling, partial or total blindness,
etc.) (Fig. 3), ocular signs (anterior uveitis, retinochoroiditis etc.)
(Fig. 4). Cats with ocular lesions have a higher seroprevalence
than cats with normal eyes [37]. These animals with toxoplasmosis
have circulating immunocomplexes that may play an important
role in development of ocular lesions, and ocular signs may
occur without polysystemic clinical signs of disease [38].
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Toxoplasmosis – an update - G. Miró, A. Montoya, M. Fisher, I. Fuentes
Fig. 4 Uveitis due to toxoplasmosis in a cat (Courtesy Dr. A. Rodríguez).
Dog
Mild infections are almost always asymptomatic, but in severe
cases clinical signs in dogs include: respiratory disorders (in 50%
of the cases), digestive disorders (in 25%) and neurological
disorders (in 25%). This is common in young dogs with
generalized toxoplasmosis and on some occasions several
clinical signs may occur concurrently (unpublished observation).
Because of this, it is important to consider other diseases,
including distemper and neosporosis as differential diagnosis.
Fig. 3 Adult cat with clinical toxoplasmosis.
All these clinical signs may persist for several days up to several
months. Theoretically any organ may be infected, thus clinical
signs are very variable; even though generalized infections are
quite rare in cats. Central nervous system toxoplasmosis is not
common in cats, with only 7% of histology confirmed cases of
toxoplasmosis showing neurological signs [39].
The onset of clinical toxoplasmosis may be insidious, with clinical
signs such as fever, anorexia, dyspnoea, vomiting, diarrhoea,
seizures and ataxia. In contrast to the frequency of ocular
toxoplasmosis that occurs in cats, there are few cases described
in dogs, but occasional cases appear, with clinical signs being
similar to what is seen in the cat.
The disease is more severe in cats co-infected with retroviruses
(mainly FIV) [29, 30] or feline infectious peritonitis (FIP) [33].
Diagnosis
Epidemiological studies have shown that clinical toxoplasmosis
is more prevalent in stray adult cats or owner cats than have
access to prey. This higher prevalence is correlated with a higher
risk of being infected, but not with age or sex.
Lappin (1990) suggested three criteria for diagnosing clinical
toxoplasmosis: (i) clinical signs of toxoplasmosis, (ii) serological
evidence of recent or active infection, and (iii) response to antiToxoplasma drugs [41].
Likewise, clinical toxoplasmosis is much more severe in kittens
that have acquired the infection by the congenital route where
the disease can be severe and fatal. Clinical illness varies with the
stage of gestation at the time of infection, and some newborn
kittens may even shed oocysts [12]. In these cases, animals may
be stillborn or die within a few hours of birth [40]. The most
typical clinical signs are anorexia, lethargy and sudden death.
At necropsy the liver is the most frequently affected organ with
diffuse hepatitis (more than 75% of the liver is destroyed due to
T. gondii infection) followed by gross lesions in the lungs (diffuse
edema and congestion).
Clinical signs, clinico-pathological
findings and complementary
examinations
Encephalitis in newborn kittens has been described, animals may
sleep continuously or present with signs of hyperaesthesia with
atypical crying and incoordination. These clinical signs prevent
feeding and affected kittens can die within few days. Cases of
infertility in queens have also been described.
The main haematological and biochemical abnormalities that
have been reported are non regenerative anaemia, neutrophilic
leucocytosis, lymphocytosis and eosinophilia. Severe leucopenia
could be present in animals during the acute phase of the disease
[42]. Leucocytosis is mainly seen in the recovery phase.
Toxoplasmosis should be suspected in dogs and cats with
anorexia, fever, dyspnoea, abdominal discomfort, hepatitis
with icterus, pancreatitis, anterior uveitis, retinochoroiditis and
central nervous system (CNS) disease manifestations. Thoracic
radiographs and abdominal ultrasonography may aid in the
diagnosis of toxoplasmosis in suspected cases.
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EJCAP - Vol. 18 - Issue 3 December 2008
are excreted by cats during a short period of time (1-2 weeks)
after first infection and because the oocyst size (10x12µm)
makes microscopic analysis difficult (Fig. 5). Moreover, oocysts
shedding is not associated with clinical signs such as diarrohea
[15, 44].
T. gondii oocysts are morphometrically indistinguishable from
oocysts of Hammondia hammondi and Besnoitia sp. Oocysts of
these coccidians can be differentiated by in vitro sporulation and
subsequent mouse inoculation for xenodiagnosis [45].
Serological tests
There are many tests for diagnosing toxoplasmosis: the Sabin
Feldman Dye Test, the indirect fluorescent antibody (IFAT),
enzyme-linked immunosorbent assay (ELISA), agglutination
procedures and Western Blot immunoassay being the most
commonly used tests (Fig. 6).
Fig. 5 Unsporulated oocyst of T. gondii (x 40).
IgM antibodies are detected within 1-2 weeks post-infection
and titers remain high for 12-16 weeks. An IgM titer > 1:64 with
negative IgG indicates active infection. When IgM antibodies
remain persistently it could be associated with reactivation
of a chronic infection after repeated exposure to T. gondii,
glucocorticoid therapy or concomitant infectious diseases (FIV).
Since the IgM titer may remain increased for long periods, the
interpretation of elevated IgM can be difficult [46].
A biochaemical profile characterized by hypoproteinaemia
and hypoalbuminaemia is typical of the acute phase, while
hypergammaglobulinemia has been reported in cats with
chronic toxoplasmosis.
Dogs with hepatic necrosis present with increased serum levels
of alanine aminotransferase (ALT) and alkaline phosphate (ALP),
while cats that develop colangiohepatitis or hepatic lipidosis
show high serum bilirubin levels.
IgG antibodies do not develop until about 2 weeks postinfection and remain at high levels for several years, even for
life. Diagnosis of active toxoplasmosis using IgG test requires
a fourfold or greater increase in IgG antibody titer during 2-3
weeks.
Animals with acute pancreatitis due to T. gondii infection may
show increased serum amylase and lipase activities.
Faecal examination in cats
Detection of T. gondii antibodies in aqueous humor and
cerebrospinal fluid have been used as an aid in the diagnosis of
dogs and cats with toxoplasmic encephalitis or uveitis.
Whilst some serological tests have been developed for the
detection of ocular and CNS toxoplasmosis, they are not
conclusive, nonetheless they maybe helpful in some cases [47,
48].
Most cats with clinical toxoplasmosis will not be excreting
oocysts at the time of presentation [43]. In cats, Toxoplasma
oocysts (10 x 12 μm) can be identified in feces using any of the
standard fecal flotation techniques. However, T. gondii oocysts
in feces have been detected in less than 1% of the cats that have
been examined in numerous studies, because oocysts usually
Fig. 6 Indirect Fluorescent Antibody Test (IFAT) to detect Toxoplasma infection.
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Toxoplasmosis – an update - G. Miró, A. Montoya, M. Fisher, I. Fuentes
T. gondii may also be diagnosed by cultivation techniques such
as isolation in mice or cell cultures, although these methods are
only available in specialized laboratories.
Molecular methods of diagnosis such as polymerase chain
reaction (PCR) from samples including blood, aspirates,
placentas and amniotic fluid are being used in human medicine
to diagnose toxoplasmosis. The PCR can detect 1-10 tachyzoites
in CSF and aqueous humor and as little as 5 tachyzoites in blood
samples [49-51]. Results of PCR testing in veterinary medicine
indicate that it can be an aid in diagnosis, as suggested by the
most recent studies done by Montoya 2006 [52] who identified
T. gondii DNA by nested-PCR and real time PCR from feline brain
and blood samples (Fig. 7).
Treatment
Treatment is recommended in cats and dogs with clinical
toxoplasmosis and works against replication of T. gondii.
However, the available drugs are not completely effective in
killing the parasite (Tab. 2). Clindamycin is the first line drug for
treatment of disseminated toxoplasmosis in both species. Animals
can also be treated with a combination of sulfonamides and
pyrimethamine, although they are contraindicated in pregnant
queens and bitches. Spiramycin can be used in pregnant females
[43].
Fig. 7 Sensitivity determination of B1 direct PCR (A) and nestedPCR (B).
Clinical signs of systemic illness usually begin to resolve within
24-48 hours after institution of therapy.
Bone marrow suppression can occur with the use of
pyrimethamine or trimethoprim sulfonamide combinations and
can be prevented or corrected with the addition of folic acid (5
mg per day) or yeast (100 mg/kg) to the cat’s diet [43].
Lastly, the detection of antibodies to diagnose neonatal
toxoplasmosis is controversial. Dubey et al., 1995, showed that
transplacental transfer of T. gondii does occur in cats but the
frequency is not well known [12], so if the queen is seronegative
and lives indoors the kittens will not have transplacental
transmission. If the queen acquires the infection during
pregnancy, both kittens and queen will present with high IgG
titers.
Treatment of suspected ocular toxoplasmosis includes controlling
anterior uveitis if present with topical anti-inflammatory agents
(prednisolone acetate or dexamethasone) and the administration
of systemic antitoxoplasmic drug [52].
Direct detection of T. gondii
T. gondii oocysts shedding by cats can be suppressed by
coccidiostatics such as toltrazuril, clazuril and sulfonamides
[54].
Cytologic examination of bronchoalveolar washes, aqueous
humour, cerebrospinal fluid (CSF), lymph node, peritoneal
or thoracic fluid aspirates can be useful to detect tachyzoites
of T. gondii. A diagnosis may be established by microscopic
examination of impression smears stained with Giemsa.
Lastly, seropositive cats must be monitored through a routine
serologic test, at least once per year, in order to detect eventual
seroconversion caused by reactivation from a chronic phase, but
specific treatment is not needed in asymptomatic cats.
Tab. 2. Treatment of toxoplasmosis in dogs and cats.
Drug
Route
Dose-rate
Duration
Clindamycin hydrochloride
Per os
10-20 mg/kg BID
4 weeks
Clindamycin phosphate
IM, IV
12.5-25 mg/kg BID
4 weeks
Pyrimethamine plus trimethoprin-sulfonamide
Per os
0.25-0.5 mg/kg BID (pyrimethamine) 4 weeks
30 mg/kg BID (TMP-sulfonamides)
Spiramycin
Per os
8-10 mg /kg BID
10 days
Toltrazuril/Clazuril
Per os
5-10 mg/kg SID
5-10 days
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EJCAP - Vol. 18 - Issue 3 December 2008
Prevention
It is very important to know the T. gondii life cycle and the
epidemiology of the disease to define the control measures in
order to reduce feline infection and environmental contamination
with oocysts. The following measures are recommended:
– Do not feed cats with raw or rare meat. If raw meat is used,
it can be frozen or gamma-ray irradiated to kill tissue cysts
– Change litter boxes daily to avoid oocysts sporulation
– Do not allow cats to bring their prey into the house
– Perform annual fecal and serologic examination of cats at
risk
– Cats should be prevented from entering buildings where
food producing animals are housed or feed is stored
– Animal blood donors should be screened before transfusion
– Do not allow dogs to consume cat feces
– For killing parasites, use chemical disinfection with ammonia
10% for 10 minutes, for the litter trays and all surfaces, or
immerse in boiling soapy water or use steam cleaning
– Control different invertebrates which may act as mechanical
vectors such as: bed bugs, earthworms, cockroaches, etc.
[55, 56].
Fig. 8 Distribution of human toxoplasmosis (Tenter et al., 2000 [61]).
occurs in 10% to 50% of patients with AIDS who are seropositive
to Toxoplasma and have a low CD4+T lymphocyte count. Since
the introduction of highly active antiretroviral therapy in 1996,
the incidence rates have decreased in some regions. However,
toxoplasmosis is a large health problem in many countries [64].
Serological tests are useful in detecting infection, but they
cannot distinguish between reactivated and latent infections
and may give false negative results for immunocompromised
patients. Therefore, PCR detection of active parasites needs to
be performed [65].
Although commercial vaccines are still not available, a candidate
T-263 (bradyzoites of a mutant strain) has been designed
against Toxoplasma infection in cats. Cats developed immunity
without concomitant shedding of infectious oocysts following
oral administration of a vaccine containing T-263 bradyzoites
[57, 58]. The vaccine can only be administered to healthy cats,
and it is not recommended in pregnant females [43].
Congenital toxoplasmosis occurs if the mother is infected for
the first time during gestation. The parasite reaches the foetus
transplacentally and the outcome of the infection depends on
the virulence of the strain, on the woman’s immune response
and the period of pregnancy. Risk of congenital toxoplasmosis
is lower if infection occurs during the first trimester (10%25%) than if it occurs during the second or the third trimester
(60%-90%) [66]. Clinical manifestation of toxoplasmosis
in foetuses and neonates vary from mild disease to severe
signs (abortion, hydrocephalus, chorioretinitis, intracranial
calcification, hepatosplenomegaly). Most infected neonates are
asymptomatic at birth but may present much later with ocular
alteration or mental and psychomotor disorders.
Human toxoplasmosis and public
health considerations
Human toxoplasmosis has a wide geographical distribution and
is estimated to affect around two billion people worldwide [27].
There is a considerable range in seroprevalence (7.5% - 95%)
in different parts of the world and indeed between different
population groups within the same country [59]. In Europe
seroprevalence varies from less than 20% in northern Europe to
more than 60% in southern Europe [60] (Fig. 8).
Toxoplasma gondii is transmitted to humans via a variety
of routes: ingestion of raw or undercooked contaminated
meat; exposure to sporulated oocysts in cat litter or soil, from
unwashed fruit, vegetables, gardening or contaminated water;
or congenital transmission when a primary maternal infection is
passed transplacentally to the foetus. Additionally, toxoplasmosis
may be acquired by organ transplant or laboratory accident [62,
63].
The diagnosis of T. gondii infection is most commonly
established by detecting IgG and IgM antibodies in the blood;
however, these tests cannot estimate the time of infection
precisely enough to properly manage the risk to the foetus of
a maternal infection. A positive IgM test result may reflect an
acute infection; however, Toxoplasma-specific IgM antibodies
may be found for up to several years, and thus may lead to
erroneous interpretation. Recently, it has been suggested that
the combination of a sensitive test for Toxoplasma-specific IgM
antibodies and measurement of the avidity of IgG antibodies for
T. gondii had the highest predictive value with regard to the time
of infection [60]. Diagnostics by PCR is also useful.
Prevention of human toxoplasmosis mainly concerns avoiding
infection. Health education may decrease the incidence during
pregnancy by 60% [67]. Education programs, prenatal and
The majorities of cases in immunocompetent people are
asymptomatic or produce only mild symptoms, but result in
life-long chronic infection with parasites inside tissue cysts.
Reactivation of latent infection may therefore lead to severe and
life-threatening disease in immunocompromised individuals.
Toxoplasmosis is the most prevalent disorder affecting the brain
in HIV-patients, causing toxoplasmic encephalitis (TE). It is the
second most common AIDS-related opportunistic infection and
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Toxoplasmosis – an update - G. Miró, A. Montoya, M. Fisher, I. Fuentes
[13] DUBEY (J.P.), MATTIX (M.E.), LIPSCOMB (T.P.) - Lesions of neonatally
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survey of Toxoplasma gondii, feline immunodeficiency virus and
feline leukaemia virus in urban stray cats in Belgium. Vet Rec,
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cats in epizootiological and clinical aspects. Pol J Vet Sci, 2002,
5:227-230.
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IgG and IgM antibodies specific to Toxoplasma gondii in cats. Vet
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[26] UGGLA (A.), MATTSON (S.), JUNTTI (N.) - Prevalence of antibodies
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screening of neonates to identify and treat congenital infection,
together with animal rearing and production methods designed
to reduce T. gondii contamination of meat, are principal
interventions. Toxoplasma infection in humans may be prevented
by the following measures: cooking meat to a sufficient
temperature to kill Toxoplasma (internal temperature higher
than 67ºC; microwaving does not kill T. gondii cysts); peeling or
thoroughly washing fruits and vegetables before eating; steam
cleaning cooking surfaces and utensils after they have been in
contact with raw meat, poultry or unwashed fruits or vegetables;
pregnant women or immunocompromised individuals should
avoid changing cat litter, but if done, gloves should be used
and the hands should be washed thoroughly afterwards [68].
Children’s sandboxes should be covered to prevent cats from
defecating in them and animal care technicians who clean cats
should wear protective clothing and masks [36].
If all of these recommended measures are put in practice the
risk of toxoplasmosis to human beings decreases dramatically.
Finally, it must be remembered that preventing human exposure
to cats is not the same as preventing exposure to Toxoplasma
gondii oocysts [43]. Oocysts originating from neighbour’s cat
can still be present in vegetables.
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254
ZOONOSES
Echinococcus multilocularis
in veterinary practice in Europe
E. R. Morgan(1)
SUMMARY
The tapeworm Echinococcus multilocularis is relevant to veterinary practice primarily because dogs are important
definitive hosts, shedding eggs that pose a significant zoonotic threat. Dogs can also occasionally become infected
as intermediate hosts and develop severe disease. The major role of the veterinary clinician is to ensure adequate deworming treatment in dogs in endemic areas, and to apply protocols aimed at reducing the risk of introducing the
parasite into new areas through animal movement. In both cases, up to date knowledge of the apparently changing
geographical range and epidemiology is crucial.
Eggs are produced into the faeces, where they are immediately
infective, and can survive for many months in the environment
[1]. Normal summer and winter temperatures in Europe are not
sufficient to render the eggs non-infective. After ingestion by
the intermediate host, usually a rodent, the eggs hatch and
migrate to the tissues. The liver is the most common site for
further development, and cysts are formed, with a germinal
epithelium surrounded by a carbohydrate-rich outer layer which
apparently protects the parasite from the host immune response.
Asexual reproduction produces more immature parasite stages,
known as protoscoleces, into the fluid interior of the cyst [1].
Aggregations of cysts form, with ill-defined boundaries, and
ingestion of an infected intermediate host leads to a variable
number of adult parasites in the gut of the definitive host.
This paper was commissioned by FECAVA for
publication in EJCAP.
Introduction
Echinococcus multilocularis is an important zoonosis in
several parts of the world. In Europe, veterinary practitioners
will find themselves in one of three areas: those endemic for
the parasite, those with recently recognised cases of alveolar
echinococcosis (AE), and those currently free of the parasite, for
which exclusion is a priority. The disease is therefore relevant for
all, especially since its distribution and epidemiology appears to
be undergoing a period of change.
Alveolar echinococcosis (AE)
Infection in the gut of the definitive host is benign, and
dogs can tolerate burdens of many thousands of worms
without apparent ill effect [1]. Cysts in the intermediate host,
however, can be highly pathogenic. Inadvertent infection of
humans leads to alveolar echinococcosis (AE). Unlike its sister
species Echinococcus granulosus, cysts in humans are poorly
circumscribed, and expand by local tissue infiltration and possibly
distant metastasis. The liver is the most common site for the
cysts, and by the time tissue destruction is sufficient to provoke
clinical signs, complete surgical excision is invariably difficult or
impossible [1]. Although advances in diagnosis and treatment
have reduced case fatality from its previous rate of more than
The parasite
Canids, including dogs and foxes, are the usual definitive hosts.
Adult worms are small (2-5mm in length, Fig. 1) and live in the
small intestine, where they attach by embedding their anterior
section in the mucosa. Following infection as the definitive host,
dogs produce eggs after about a month, and continue to do
so for up to 4 months [1]. There is some evidence for partial
acquired immunity, but this is not sufficient to prevent future
infection following re-exposure. The parasite’s biotic potential
is similar in dogs and foxes, but infections in cats produce very
few eggs and cats are therefore not of overall significance in
maintaining parasite populations [2].
(1) Eric R. Morgan. School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK. Tel: +44 117 9287485.
E-mail: eric.morgan@bristol.ac.uk
255
Echinococcus multilocularis in veterinary practice in Europe - E.R. Morgan
by albendazole treatment (daily 10 mg / kg per os). Provided
treatment is maintained, remission periods of up to 3.5 years
have been recorded, with no instances of clinical recurrence in
dogs undergoing chemotherapy after surgery [1].
Clinical cases in non-travelled dogs are an indication that the
parasite is completing its life cycle in the area, and people are
at risk. This should trigger surveillance and re-evaluation of local
recommendations for treatment of dogs.
Current distribution and epidemiology
The approximate current distribution of E. multilocularis in
Europe is shown in Fig. 2. There is some concern that the parasite
is spreading, although sparse objective records especially in the
East, improved diagnostic tests in recent years, and increased
awareness leading to more diagnostic effort, make this difficult
to prove. Cases of autochthonous AE and infection in foxes
have been recorded in several new areas in the past few years.
In some endemic areas, the parasite has colonised urban fox
populations [4], placing increasing numbers of people at risk.
There is evidence for real increases in parasite biomass within
core endemic areas [5] as well as on the edge of the known
range [6], which could be linked to increases in fox populations
as a result of successful control of rabies [5].
Fig. 1 Adult Echinococcus granulosus, which is very similar in
appearance to E. multilocularis. The eggs are typical of taeniids
and morphologically indistinguishable from those of other species
in the group. The eggs (insert) 30-40 microns in diameter, are
typical.
In the natural or sylvatic cycle, foxes act as the definitive host and
a range of rodents as the intermediate hosts. Raccoon dogs are
also competent definitive hosts and may be of epidemiological
importance in northern areas [5]. The most important
intermediate hosts are microtine voles rather than rats or mice,
although other species including wood mice and musk rats can
also be infected. The risk to humans comes from inadvertent
ingestion of eggs from infected faeces, with subsequent cyst
development. Contamination of fingers or food with eggs in
areas frequented by foxes therefore poses a risk of infection, and
people working outdoors and in contact with the soil are more
likely to encounter the parasite. However, since human contact
with dogs is much closer than with foxes, spillover of infection
into domestic dog populations constitutes a great multiplication
of risk. This would occur when dogs ingest rodents or rodent
remains in an area in which foxes are present and the sylvatic
cycle is operating. In this context, the increasing urbanisation of
fox populations in many European cities is of justified concern
[4]. Work in Switzerland suggests that the suburban fringes,
where elevated fox numbers coincide with high densities of
intermediate hosts and green areas frequented by people and
their dogs, are the area of highest risk for transmission. Dogs can
also act as a mechanical carrier of infective eggs, for example
after rolling in fox faeces.
95%, the disease still carries a poor prognosis, and surgery is
followed by life-long parasitostatic anthelmintic chemotherapy
[3]. Although cases in humans are not common, the severity
of disease and the ease with which sources of infection in
companion animals can be eliminated places great responsibility
on the practising veterinarian for protection of public health.
In recent years a number of cases of AE have also been reported
in which domestic animals act as intermediate hosts and develop
cysts, usually in the liver. This has been observed in pigs, several
species of primate, and dogs [1]. In at least one case a dog has
acted as both definitive and intermediate host at the same time.
This raises questions over whether autoinfection can occur, with
rupture of cysts into the intestine or hatching of eggs before
leaving the host. However, the most likely general source of
infection is ingestion of eggs from the environment.
AE in dogs follows a similar course to that in humans, although
cysts develop more quickly [1]. Cranial abdominal mass is the
main presenting sign, and may be accompanied by ascites,
diarrhoea, vomiting, dyspnoea, exercise intolerance and weight
loss. Clinical investigation may reveal signs of liver failure if
the functional capacity of the organ is sufficiently reduced.
Ultrasound examination is suggestive, with the main differential
diagnosis being neoplasia, and exploratory laparotomy reveals
a large polycystic mass in the cranial abdomen with liver
involvement. Cytology on material collected by fine needle
aspirate is unlikely to be helpful, but examination of this material
by antigen-ELISA, if available, is diagnostic. Treatment options
are euthanasia, or surgical resection of the mass. This is rarely
completely achievable due to its locally invasive nature, and might
involve removal of entire liver lobes, with inadvertent rupture
of the cyst leading to major complications. Surgery is followed
Diagnosis and treatment in the definitive host
Eggs are easily recognised on salt flotation of faeces, but cannot
be differentiated from those of other taeniid tapeworm species.
Definitive diagnosis is by identification of the adult parasite using
sedimentation at necropsy. In vivo, expulsion of tapeworms and
enumeration of burdens, for epidemiological research purposes,
can be achieved by arecoline purge. In practice, however,
detection of parasite antigen by ELISA, or DNA in faeces by PCR,
provide accurate diagnosis [1, 4].
256
EJCAP - Vol. 18 - Issue 3 December 2008
parasite control through protection of infection-free status in
non-endemic areas and reduction of infection pressure in the
endemic zone.
Praziquantel is the drug of choice for prophylactic treatment in
the definitive host. However, it has a very short persistence time
with a half-life of only a few hours, so repeated administration
every month is needed to ensure that no egg excretion occurs.
Since this provokes problems of owner compliance, a more
selective approach has been suggested, focusing on dogs that
have the closest contact with humans or the greatest opportunity
to eat rodents [1]. Routine screening by faecal flotation would
also identify infected dogs, although false positives would be
caused by other taeniid infections, and false negatives by low test
sensitivity. Screening also presents a zoonotic hazard to practice
laboratory staff. Routine treatment of all dogs in at-risk areas
is therefore the safest strategy. When recommending worming
regimes to clients, veterinarians in endemic areas should be
wary of combination products targeted to Toxocara canis and
Dirofilaria immitis along with ectoparasites such as fleas, which
do not necessarily have any effect against tapeworms. Diagnosis
of patent infections in dogs should prompt the owners and their
families to seek medical advice, and it is recommended that
they are screened for specific antibodies [1]. The pathogenesis
in humans is chronic and years can go by between infection and
the appearance of clinical signs, with prognosis deteriorating as
the cysts grow. Early diagnosis in people at high risk of infection
is therefore beneficial.
Fig. 2 Approximate geographical distribution of E. multilocularis
in Europe. Dark shading indicates the core endemic area, in which
infection in foxes and dogs is locally common, and light shading
areas in which infection is at a lower level or autocthonous cases of
AE have been recently reported. Data from multiple sources [5, 8,
11, 12 and others]. Larval stages have also been identified in humans
in additional countries in the Balkans and Eastern Europe [11], but
without surveys of definitive and natural intermediate hosts the
endemic status of these countries cannot be confirmed. Regional
distributions within countries are approximate, and records to the
East of the marked range are poor.
Dogs travelling from endemic to uninfected areas could carry
the parasite, leading to establishment in new countries [7].
Consequently, cross-border transport of dogs in some cases
carries a legal requirement for treatment. The protocol is a
compromise between the gut transit time (ensuring that all
eggs are voided before arrival in the destination country) and
the chance of re-infection (which is in theory possible almost
immediately after treatment). Currently, the favoured strategy is
a single dose of praziquantel between 24 and 48 hours before
entry, and there are so far no confirmed reports of introduction
of E. multilocularis by dogs so treated. However, there is political
pressure to weaken requirements for such treatment in the
interests of European harmonisation and free movement, and
given the severity of disease in humans the veterinary profession
has a responsibility to ensure that changes to regulations for
cross-border animal movement takes due account of the risks for
disease transmission. Infection of dogs visiting an endemic area
can further be reduced by avoiding opportunities for ingestion
of rodents or carrion.
Praziquantel is the most effective drug against adult parasites,
and administration at a rate of 5.0 mg / kg body weight per os
or 5.7 mg / kg body weight by intramuscular injection eliminates
the entire worm burden in most cases. A second administration
a day later, followed by copro-antigen or copro-PCR testing
after 4-5 days, provides further security [1].
Prevention of infection in dogs
The veterinary practitioner has an important role in the
prevention of disease and the protection of public health. In
endemic areas, this should focus on identifying dogs at risk
of infection as definitive hosts and preventing egg shedding
by chemoprophylactic treatment, and elimination of burdens
in dogs travelling to non-endemic areas. Outside the endemic
zone, veterinarians should provide appropriate advice to owners
of travelling dogs concerning prevention of infection and correct
application of preventative measures before returning, and
should also be vigilant for cases of AE in animals that might
indicate introduction of the parasite. The veterinary profession
also has an important responsibility to provide public education
concerning the risk of infection and the importance of responsible
pet ownership, and to co-ordinate multi-national strategies for
Prospects for control in endemic areas
In most parts of its range, E. multilocularis is maintained in a
sylvatic cycle, with foxes and voles as the major hosts, although
there is evidence in Asia that dogs can take over as the main
definitive host [1]. Whether or not dogs can maintain the parasite
cycle in the absence of foxes, infection of dogs undoubtedly
leads to increased risk of human disease. In hyper-endemic
areas, the lifetime incidence of E. multilocularis infection in dogs
could be as high as 80% [4]. Routine treatment of dogs with
praziquantel at an appropriate dose and frequency (or, in future,
257
Echinococcus multilocularis in veterinary practice in Europe - E.R. Morgan
References
other proven drugs) and proper disposal of dog faeces should
therefore form the cornerstone of control.
[1]
DEPLAZES (P.), ECKERT (J.) - Veterinary aspects of alveolar
echinococcosis - a zoonosis of public health significance. Vet
Parasitol, 2001, 98: 65-87.
[2] KAPEL (C.M.O.), TORGERSON (P.R.), THOMPSON (R.C.A.),
DEPLAZES (P.) - Reproductive potential of Echinococcus
multilocularis in experimentally infected foxes, dogs, racoon dogs
and cats. Int J Parasitol, 2006, 36: 79-86.
[3] CRAIG (P.) - Echinococcus multilocularis. Curr Opin Infect Dis,
2003, 16: 437-444.
[4] DEPLAZES (P.), HEGGLIN (D.), GLOOR (S.), ROMIG (T.) - Wilderness
in the city: the urbanisation of Echinococcus multilocularis. Trends
Parasitol, 2004, 20: 77-84.
[5] ROMIG (T.), DINKEL (A.), MACKENSTEDT (U.) - The present
situation of echinococcosis in Europe. Parasitol Int., 2006, 55:
187-191.
[6] TAKUMI (K.), DE VRIES (A.), CHU (M.L.), MULDER (J.), TEUNIS
(P.), VAN DER GEESEN (J.) - Evidence for an increasing prevalence
of Echinococcus multilocularis in foxes in The Netherlands. Int J
Parasitol, 2008, 38: 571-578.
[7] GOODFELLOW (M.), SHAW (S.), MORGAN (E.) - Imported disease
of dogs and cats exotic to Ireland: Echinococcus multilocularis.
Irish Vet J, 2006, 59: 14-216.
[8] DEPLAZES, (P.) - Ecology and epidemiology of Echinococcus
multilocularis in Europe. Parassitologia, 2006, 48: 37-39.
[9] MC DONALD (R.A.), DELAHEY (R.J.), CARTER (S.P.), SMITH (G.C.),
CHEESMAN (C.L.) - Perturbing implications of wildlife ecology for
disease control. Trends Ecol Evol. 2008, 23: 53-56.
[10] MORGAN (E.R.), MILNER-GULLAND (E.J.), TORGERSON (P.R.),
MEDLEY (G.F.) - Ruminating on complexity: macroparasites of
wildlife and livestock. Trends Ecol Evol, 2004, 19: 181-188.
[11] KOLAROVA (L.) - Echinococcus multilocularis: new epidemiological
insights in Central and Eastern Europe. Helminthologia, 1999, 36:
193-200.
[12] JENKINS (D.J.), ROMIG (T.), THOMSON (R.C.A.) - Emergence / reemergence of Echinococcus spp. – a global update. Int J Parasitol,
2005, 35: 1205-1219.
Control in foxes or in rodents has been considered [8]. Culling
foxes is unlikely to be a realistic option, not only because of
logistical and ethical problems, but also because the ensuing
disruption could be counterproductive. Disturbance of stable
family groups with defended territories is likely to lead to
increased dispersal, especially by young foxes, which carry the
highest worm burdens. This could lead both to geographical
spread of infection and increased opportunity for contact
with humans and domestic animals. Experiences with badger
culling to control bovine tuberculosis in the UK should prompt
caution when considering wildlife culling as a means of
disease control, especially in territorial species [9]. In practice,
a sustained decrease in fox populations might reduce the
force of infection, but given the high reproductive potential
of foxes is unlikely to be achievable by conventional means. A
more successful strategy has employed treatment, with five- to
ten-fold reductions in the prevalence of E. multilocularis in fox
populations given praziquantel-laced baits over a 2 year period
[4]. Treatment of urban and suburban foxes in this way could
directly reduce zoonotic risk, as well as the chances of spillover
infections in domestic dogs. However, such treatment should
be strategic and take into account the population dynamics of
foxes and the parasite, and the spatial and dynamic complexities
of transmission [10].
Conclusions
The severity of zoonotic disease caused by E. multilocularis
compels the practising veterinary surgeon to take serious
responsibility for control of infection in dogs. This can be
achieved by routine anthelmintic treatment in endemic areas,
and strategic treatments in dogs moving from endemic to
uninfected countries. Veterinary efforts to control this parasite
will benefit from awareness among practitioners of its life cycle,
epidemiology and diagnosis, risk factors for human and canine
infection, and the presentation and diagnosis of disease in
animals acting as intermediate hosts. They should also be aware
of the apparent changes in epidemiology in terms of urban
fox populations and expansion of the known geographical
distribution.
258
ZOONOSES
Toxocarosis, an important zoonosis
P.A.M. Overgaauw(1) F. van Knapen(2)
SUMMARY
This paper reviews the current knowledge about an important zoonotic dog and cat parasite, Toxocara. A good
understanding of the epidemiology is required so that effective prevention of infection in man, dogs and cats can
be possible. Education of the dog and cat owner will be significant in prevention. In this review, the epidemiology,
clinical symptoms, diagnosis, prevention and control in the dog, the cat and the human will be discussed. Uniform
guidelines for deworming dogs and cats will be given.
Toxocara infected paratenic hosts by dogs or cats, larvae will be
released and in most cases develop directly into adult worms
in the intestinal tract. This may explain the higher Toxocara
prevalence rate in adult cats, which catch prey animals more
often than dogs. In the pregnant bitch, ‘dormant’ somatic larvae
are reactivated and migrate in the bitch across the placenta to
infect the fetuses. New-born puppies and kittens also acquire
infection through ingestion of larvae in the milk [49, 59, 60].
This paper was commissioned by FECAVA for
publication in EJCAP.
Introduction
Toxocarosis of dogs and cats
Toxocara canis and Toxocara cati are roundworms of dogs
and cats and the reported infection rates in Western Europe
vary from 3.5% to 17% for T. canis in dogs and 8% to 76%
for T. cati in cats [8, 15, 22, 35, 39, 41, 52]. The prevalence of
patent Toxocara infections is highest in young dogs and cats
and much less common in adult animals. Toxocara infection
follows ingestion of embryonated Toxocara eggs or larvae in
a paratenic host. Migration of larvae can lead to (overt) clinical
disease (toxocarosis) in the (paratenic) host.
Environment
Toxocara eggs are unembryonated and not infectious when
passed into the environment in the faeces of dogs and cats.
Within a period of 3 weeks to several months, depending on soil
type and climatic conditions such as temperature and humidity,
eggs will develop to an infectious stage that can survive for
at least one year under optimal circumstances. Studies from
all over the world demonstrated high rates (10-30%) of soil
contamination with Toxocara eggs in backyards, sandpits,
parks, playgrounds, lake beaches, and other public places [37].
In a survey in the Netherlands, the presence of T. canis eggs in
public parks was comparable with reports from other European
cities, but most of the investigated sand-boxes were polluted
with T. cati eggs [27].
Epidemiology
Infection of the dog and cat
Adult worms in the intestinal tract of infected dogs and cats
shed large numbers of eggs into the environment (Fig. 2) via
the faeces where they embryonate (Fig. 3) and maybe ingested
by natural hosts as well as paratenic hosts. In the intestine the
larvae hatch (Fig. 3) and migrate throughout the body via blood
vessels. This is called visceral larva migrans or VLM (Fig. 4 and 5).
In young animals larvae migrate from the lungs up the trachea
and after swallowing, mature in the intestinal tract. In paratenic
hosts and most adult dogs and cats that have some degree
of acquired immunity, the larvae undergo somatic migration
and remain as somatic larvae in the tissues. After predation of
Infection routes
Tracheal migration
After young dogs ingest infective Toxocara eggs, larvae migrate
through the liver, the vascular system and the lungs to the
trachea. After swallowing, they complete their development in
the stomach and small intestine. Eggs first appear in the faeces
4 to 5 weeks post-infection [49]. Depending on previous
exposure to infection, the migratory pathway and deworming
(1) Certified Veterinary Microbiologist, Certified Parasitologist, RnA Assays, Yalelaan 2, NL - 3584 CM, Utrecht. E-mail: P.overgaauw@rnassays.com
(2) Diplomate of the European College of Veterinary Public Health, Certified Parasitologist, Institute for Risk Assessment Sciences (IRAS), Department of
Veterinary Public Health, Veterinary Faculty, Utrecht University, Yalelaan 2, NL - 3584 CM Utrecht. E-mail: f.vanknapen@uu.nl
* Presented by NACAM (The Netherlands)
259
Toxocarosis, an important zoonosis - P.A.M. Overgaauw, F. van Knapen
Fig. 1 Toxocara egg 400x (Photo
Felix Yaya).
bitch during pregnancy. Within hours of birth, the larvae that
were present in the liver of the neonate, migrate to the lungs
and undergo a tracheal migration. Adult worms can be found at
two weeks of age and large numbers of eggs may be passed in
the faeces after a minimum period of 16 days [32].
Transmammary transmission
After activation, somatic Toxocara larvae in dogs and cats will also
be transmitted via the colostrum and the milk (transmammary
transmission, lactogenic or milk-borne infection). Following
ingestion by the offspring, the larvae undergo development
without tracheal migration. Larvae are found to pass in the
bitch’s milk for at least 38 days after parturition [72]. This route is
less important than intra-uterine transmission in the puppy, but
it is the primary mode of infection in the kitten. Kittens infected
by lactogenic transmission will show faecal egg excretion after
7 weeks.
[12], in pups of one to two months, the probability that newly
hatched T. canis larvae will develop into adult ascarids may
fall to a lower level, while the probability of somatic migration
progressively increases. This is called age resistance and the
mechanism operates partly within the lungs. Age resistance
seems to be low in older cats. An explanation is not known
[69].
Infection of the dam by the offspring
Infection of the lactating bitch will occur mainly by ingestion
of immature fourth-stage larvae from vomit or faeces from the
puppies. Larvae develop to adults without a tracheal migration.
Toxocara eggs shed in the faeces of puppies or kittens can be
ingested by the mother, where they pass through the digestive
tract, causing a false-positive diagnosis of Toxocara infection
upon faecal examination. There is no development of intestinal
infection with T. canis from somatic larvae at other times during
gestation and the bitch is not at higher risk of Toxocara infection
during metoestrus [44].
Although the prevalence of T. canis is highest in young dogs,
a certain proportion of the adult canine population can also
be infected [12, 35, 43, 53], mainly by ingestion of infective
Toxocara eggs from contaminated soil.
Somatic migration
After ingestion of infective Toxocara eggs, larvae will migrate
actively by penetration of the tissues and invasion of all parts
of the body. Gradually somatic larvae accumulate in the tissues,
persisting for long periods in a manner similar to that seen in
paratenic hosts. Larvae of T. cati prefer to migrate to the muscles,
while T. canis larvae were more commonly found in the central
nervous system.
Transplacental migration
Nearly 100% of puppies are infected by somatic larvae in utero
from day 42 of the gestation [33]. This so called transplacental
migration or intra-uterine infection is the most important
mode of transmission in dogs. In cats, prenatal infection via
the placenta does not occur. The larvae in pregnant bitches are
probably reactivated by the changing hormonal status of the
Transmission through paratenic hosts
Paratenesis is the mode of infection of some larval nematodes
like Toxocara, ensuring its continuing survival by its distribution
in prey species [21]. This route of infection exists because of
the development of somatic larvae in paratenic hosts, including
vertebrates such as rodents and birds or invertebrates such as
earthworms and insects (e.g. flies). After ingestion of an infected
paratenic host, the larvae develop directly in the intestine. Cats
catch and eat more prey animals than dogs and this may be the
explanation why higher infection rates are found.
Fig. 2 Embryonating Toxocara egg 400x (Photo RVC).
Fig. 3 Hatching Toxocara larvae 400x (Photo RVC).
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EJCAP - Vol. 18 - Issue 3 December 2008
Fig. 4 Migrating Toxocara larvae in gut wall (Photo RVC).
Fig. 5 Migrating Toxocara larvae in tissue (Photo RVC).
Clinical symptoms
and remain infective for years. Since no practical methods exist
for reducing environmental egg burdens, prevention of initial
contamination of the environment is most important. This can
be achieved by taking measures such as eliminating patent
infections in dogs and cats, preventing defecation by pets in
public areas (Fig. 8), taking hygienic precautions and education
of the public [19]. Household garden soil was found to be a
potentially greater source of Toxocara infection than soil in public
green areas [25]. A decrease in contamination can be achieved
by methods including: restriction of uncontrolled dogs and cats,
cleaning up faeces from soil and on pavements by dog owners,
preventing access of dogs and cats to public places (especially
children’s playgrounds) and by use of strategic anthelmintic
treatment of dogs and cats with emphasis on worming puppies,
kittens, nursing bitches and queens.
The clinical symptoms depend on the age of the animal and on
the number, location and stage of development of the worms.
Toxocara infection is highest in puppies and kittens up to 6
months of age. After birth, puppies can suffer from pneumonia
associated with the tracheal migration and die within 2 to 3
days. At an age of 2 to 3 weeks, puppies can show emaciation
and digestive disturbances, caused by mature worms in the
stomach and intestine (Fig. 6 and 7). Diarrhoea, constipation,
vomiting, coughing and nasal discharge can be found at clinical
examination. Distension of the abdomen (‘potbelly’) can occur,
probably as result of gas formation caused by dysbacteriosis
[41].
Anthelmintic treatment strategy
The most serious and concentrated sources of infection are found
in bitches nursing a litter and puppies aged between 3 weeks
and 6 months. A major aim of long-term prophylactic treatment
programmes is to suppress T. canis egg-output throughout
the whole of puppyhood using a multidose schedule. Puppies
should be treated with appropriate anthelmintics at the age of 2
weeks and because milk transmission occurs continuously for at
least 5 weeks post partum, repeated treatments are necessary
[3]. Larvae that reach the intestine need at least 2 weeks to
mature and start passing eggs, therefore the treatment should
be repeated every 14 days. The treatment schedule therefore
requires deworming at 4, 6 and 8 weeks of age and then
monthly until 6 months of age. Because prenatal infection
does not occur in kittens, fortnightly treatment can begin at
3 weeks of age. Nursing bitches and queens should be treated
concurrently with their offspring since they may develop patent
infections along with their young.
Kittens are older when worms are maturing (adult from day
28 and egg producing from day 49 after birth) and tracheal
migration with related symptoms does not occur. Therefore,
kittens have a better chance to grow and in the meantime
develop better bodily condition before problems may be seen.
For this reason clinical symptoms similar to those in puppies are
usually inapparent.
Diagnosis
Patent Toxocara infection in dogs and cats can be tentatively
diagnosed from the medical history, particularly the use or
otherwise of an appropriate anthelmintic schedule, and the
clinical symptoms. Confirmation of the diagnosis can be obtained
by finding dark brown coloured eggs with thick pitted shells in
faecal samples (Fig. 1). The direct faecal smear technique is not a
sensitive test and generally should never be used for recovering
eggs from faecal samples. Examination of faeces by a floatation
technique is a useful method for detecting helminths [7].
Control in older dogs and cats can be achieved by periodic
treatments of dogs and cats with anthelmintics, or by treatments
prescribed based on the results of periodic diagnostic faecal
examinations. Annual or twice annual treatments have been shown
not to have a significant impact on preventing patent infection
within a population, so a treatment frequency of at least 4 times
Control measures
There are two reasons for Toxocara control: to prevent human
infection and to reduce the risk of infection to pets. Toxocara
eggs are very resistant to adverse environmental conditions
261
Toxocarosis, an important zoonosis - P.A.M. Overgaauw, F. van Knapen
owners should routinely collect and safely dispose of their pets’
faeces, especially from children’s play areas and finally advice for
children not to play in potentially contaminated environments.
Although veterinarians should be the most appropriate sources
of information for their clients regarding the dangers and the
control of toxocarosis, surveys have demonstrated that client
education on this issue is lacking [23, 42].
per year is proposed as a general recommendation. Anthelmintics
at the recommended doses are not highly effective against inhibited
somatic larvae [11] and treatment of bitches before mating and two
weeks before the anticipated whelping date has no useful effect on
prenatal transmission [10, 14]. Therefore it is generally not advised
to deworm pregnant dogs and cats.
In the past, uniform guidelines for the control and treatment
of parasites in pet animals were developed and published by
CAPC in the US (www.capcvet.org) and recently by ESCCAP
in Europe (www.esccap.org). The guidelines give overviews of
worms, their significance and suggest rational control measures
for the most important species in order to prevent animal and/
or human infection.
Human toxocarosis
Infection routes
Toxocarosis is a public health problem. Man acts as an unnatural
host in which Toxocara larvae will not develop but will migrate
and survive for a long time. The mode of transmission to humans
is by oral ingestion of infective Toxocara eggs from contaminated
soil (sapro-zoonosis), from unwashed hands or consumption of
raw vegetables [19]. Some infections may occur from ingestion
of larvae in under-cooked organ and muscle tissue of infected
paratenic hosts such as chickens, cattle and sheep [2, 38, 55,
61, 62].
Hygiene
For dogs and cats, hygiene can be achieved by removing the
faeces and by thorough cleaning of kennels. A 20% solution
of commercial bleach can be used. This does not kill the eggs,
but removes their sticky outer protein coat. These decorticated
eggs are then easier to remove from inaccessible areas. Any
worms vomited or passed in faeces should be destroyed and
predation and scavenging on carcasses by dogs and cats should
be prevented.
Recent studies indicate the fur of as an important source of
Toxocara eggs for infection after direct contact [1, 54, 71]. Direct
contact with dogs and cats that harbour a patent Toxocara
infection is usually not considered a risk, since the eggs need
to mature several weeks before they are infective [41, 45, 46].
Moreover, Toxocara eggs are very sticky and therefore difficult
to remove from the coat of a dog or cat and this makes ingestion
of a sufficient number of eggs unlikely. A low percentage of
these eggs were embryonated in the studies and even in the
worst case scenario of highly contaminated fur, it is necessary to
ingest several grams of heavily contaminated hair to get infected
[48].
Education
Dog and cat owners can help to avoid contamination of the
environment with Toxocara eggs and the exposure of other
persons to unnecessary risks of Toxocara infections. Proper
information about this zoonosis and the social concept of
responsible pet ownership is required. Pet owners should be
advised about deworming schemes, effective anthelmintics
and to prevent their animals from defecating on children’s
playgrounds [57].
Information disseminated to pet owners should include a
description of T. canis and T. cati and how they affect their
hosts; clearing of prenatal and transmammary transmission; how
Toxocara can be transmitted to and produce damage in humans;
how prevention can most effectively be achieved; how pet
The importance of Toxocara cati in human toxocarosis
The role of T. cati as a zoonotic parasite is not always clearly
recognised. Despite the fact that differentiation between
T. canis and T. cati infections is not performed in surveys, the
majority of reported human cases of toxocarosis in the past have
been associated with T. canis and not with T. cati [13]. The greater
Fig. 6 Patent Toxocara infection resulting in death of 6 months
old Border Collie (Photo Paul Overgaauw).
Fig. 7 T. canis adult worms in intestine (Photo RVC).
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EJCAP - Vol. 18 - Issue 3 December 2008
Fig. 8 Defecating cat in sandpit (Photo Frans van Knapen).
Fig. 9 Granulomatous retinal lesion in human eye due to ocular
larva migrans (Photo RVC).
abundance of T. canis eggs in park soil and playgrounds might
be considered the reason for this, but sand-boxes and garden
soil (Fig. 8) are more often polluted with T. cati eggs [27]. The
large number of common antigenic fractions shared between
T. canis and T. cati and the similarity in the mode of infection
are indications that there is no difference in zoonotic risk.
Furthermore, in Islamic countries, dogs are avoided for religious
reasons, while cats are favoured pets. The seroprevalence of
human toxocarosis in these countries is considerable as well
[58]. The role of T. cati in human toxocarosis should therefore
not be underestimated [40, 58].
serum IgE concentration, the presence of allergen-specific IgE
and eosinophilia is established. The occurrence of asthma or
recurrent bronchitis and hospitalization due to asthma were
significantly related to seroprevalence, while eczema tended to
be more frequent. It was concluded that allergic phenomena in
children who are predisposed to asthma, are more frequently
manifested after Toxocara infection [4]. Toxocara is not a
causative agent but contributes to the development of atopic
diseases and the allergic manifestation of asthma [51].
General clinical symptoms often include malaise, fever, abdominal
complaints (vague upper abdominal discomfort attributed to
hepatomegaly), wheezing or coughing [17]. Toxocara infection
should be considered in the differential diagnosis of any child
with a persistent and unexplained eosinophilia or recurrent
abdominal pain. Chronic ‘idiopathic’ urticaria, chronic pruritus,
and miscellaneous eczema in adults and children are found
strongly associated with toxocarosis [16, 50].
Infection risk to children
Children are more frequently infected than adults and VLM with
more severe clinical symptoms is mainly found in children of 1
to 3 years of age. This can be explained because young children
play and have closer contact with potentially contaminated soil
in yards and sand-pits. In addition, children may often put their
fingers into their mouth and sometimes eat dirt.
Severe clinical symptoms are reported including life-threatening
pneumonia after massive infection [28], eosinophilic meningoencephalitis in children [36], and thrombosis of the aorta [67].
Clinical symptoms
Visceral larva migrans
After ingestion of infective Toxocara eggs by a human, Toxocara
larvae hatch in the stomach and migrate into the mucosa of
the upper small intestine and disperse throughout the body via
blood and lymphatic vessels. A more marked, inflammatory,
immune response is called visceral larva migrans syndrome or
VLM. This multisystem invasion can be associated with varied,
non-specific clinical symptoms as a result of the host’s immune
response.
Ocular larva migrans
Migrating Toxocara larvae can induce granulomatous retinal
lesions, which are characterised by complaints of loss of visual
acuity, squint and ‘seeing lights’ (Fig. 9). This is called Ocular
Larva Migrans syndrome (OLM) [6, 63]. In a minority of cases,
total blindness of one or both eyes can result. The mean age of
patients with OLM is 8 years, but it is diagnosed in adults as well
[31, 56]. Ocular larva migrans is usually caused by no more than
a single larva.
VLM is mainly diagnosed in children between 1 to 7 years
of age (mean age 2 years) and is characterised by persistent
eosinophilia, leukocytosis, an elevated GT γ level and
hypergamma-globulinaemia [56].
Covert toxocarosis
A third clinical syndrome, called ‘covert toxocariasis’ (CT),
was found in patients with a vague complex of non-specific
clinical symptoms, which do not fall within the categories of
VLM or OLM. Symptoms such as hepatomegaly, cough, sleep
disturbances, abdominal pain, headaches and behavioural
changes have been associated with raised Toxocara antibodies
Eosinophilia is seen more often in children than in adults [68]
and a relationship between Toxocara seroprevalence and the
incidence of chronic airway disorder (asthma), elevation of
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Toxocarosis, an important zoonosis - P.A.M. Overgaauw, F. van Knapen
[64]. The diagnosis ‘Idiopathic Abdominal Pain of Childhood’ is
usually made in children.
To increase the awareness of potential zoonotic hazards,
particularly amongst pet owners, veterinary practitioners,
general practitioners and public health agencies should
provide sufficient information and advice for appropriate
measures to be taken to minimize the risk of infection.
Several reports, however, have indicated a significant lack of
knowledge within the professions [23, 42, 47]. All authors
concluded that continuing education with emphasis on the
zoonotic risks is still strongly recommended.
Cerebral toxocarosis
There are some indications that larval involvement in the human
brain may have subtle public health implications, such as changes
to cognitive function in children [26]. A large study to determine
OLM in 120,000 Irish schoolchildren, ranging in age from 3 to
19 years, revealed a strong association between having had a
convulsion and ocular toxocarosis [20].
Recommendations include the following: be careful when in
contact with young dogs and cats; wash hands before eating
and after contact with animals; deworm dogs and cats regularly
especially puppies and kittens; prevent children from eating
earth and from playing on areas soiled with animal faeces;
remove pet faeces; keep children’s nails clipped.
Diagnosis
Direct diagnosis of Toxocara infection is not easy because
patients do not excrete parasite material such as eggs or larvae.
Serodiagnostic techniques (ELISA) are the most reliable tools to
detect antibodies and circulating antigens [58]. Anti-Toxocara
antibodies measured by ELISA were found to persist for up
to 2.8 years in infected adults and their presence alone does
not distinguish between current and past infections. It should
therefore be accompanied by other laboratory tests for a blood
eosinophil count and total serum IgE [34]. Seroprevalence
for Toxocara in some reports varied between 4.6 to 7.3% in
children in the USA [24], 2.5% in Germany to 83% for children
in the Caribbean [65]. In the Netherlands the prevalence was
found to be 19% on average: between 4% and 15% in people
younger than 30 years and 30% in adults older than 45 years
[5]. Regularly re-infection of adults is probably the cause of the
higher prevalence. Titres fall gradually over a period of about
three years but should be considered as a balance between the
fading memory of the immune system and its stimulation by
continuing ingestion of viable ova or reactivation of dormant
larvae. A review of cases of toxocarosis (VLM and OLM) from all
over the world revealed that more than half of the patients were
less than three years old, one fifth were adults and 60% were
males [9]. For OLM, serum antibodies are not diagnostic but the
presence of intraocular antibodies appears more promising as a
diagnostic aid [63].
Treatment of patients
Patients with severe Toxocara infections can be treated with
systemic acting and larvicidal anthelmintics [34]. Clinicians,
however, should balance the risk of therapy with the severity of
the disease, because treatment can lead to severe hypersensitivity
reactions caused by dying larvae. Especially in OLM cases the
anthelmintic dose should be increased gradually over a period
of days and accompanied by the concomitant administration of
steroids [34].
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264
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266
ZOONOSES
Update on avian chlamydiosis and
its public health significance
D. Vanrompay (1)
SUMMARY
The present review gives updated information on Chlamydophila psittaci strain classification, epidemiology,
transmission, clinical disease, new diagnostic tests, public health significance, present legislation on psittacosis and
recommendations for prevention and control of the disease.
At present, analysis of the MOMP gene encoding the
outer membrane protein A (ompA) is more often used
to characterize avian Cp. psittaci strains as molecular
techniques can differentiate Cp. psittaci strains more
precisely and serovar-specific monoclonal antibodies are
not commercially available restricting their use by veterinary
clinical laboratories. Analysis of the ompA gene by
sequencing, genotyping real-time PCR [10] or genotyping
micro array [33] allows for the detection of genotypes A to
F as well as the recently discovered genotype E/B [11]. These
genotypes correspond to MOMP serovars A to F. Monoclonal
antibodies cannot distinguish genotype E/B from B and E.
Genotype E/B was mainly isolated from ducks.
This paper was commissioned by FECAVA for
publication in EJCAP.
Cp. psittaci strain classification
In 1999, the family Chlamydiaceae was reclassified into two
genera and nine species [7]. The genus Chlamydia (C) includes C.
trachomatis (human), C. suis (swine), and C. muridarum (mouse,
hamster). The genus Chlamydophila (Cp) includes Cp. psittaci
(avian), Cp. felis (cats), Cp. abortus (sheep, goats, cattle), Cp.
caviae (guinea pigs), Cp. pneumoniae (human) and Cp. pecorum
(ruminants).
Epidemiology
All known avian strains belong to the species Chlamydophila
psittaci, which includes six avian serovars A through F, and two
mammalian isolates WC and M56 isolated from epizootics in
cattle and muskrats, respectively. Serotyping of avian strains
is performed by a micro-immunofluorescence test using
monoclonal antibodies against serovar-specific epitopes of
the major outer membrane protein (MOMP) [1, 42]. The avian
serovars are relatively host-specific. Serovars A and B are usually
associated with psittacine birds and pigeons, respectively.
Serovar C has primarily been isolated from ducks and geese,
and serovar D mainly from turkeys. Serovar F was isolated from
a psittacine bird and from turkeys. The host range of serovar
E is the most diverse of the strains: it has been isolated from
pigeons, ratites, ducks, turkeys and occasionally from humans.
All serovars should be considered to be readily transmissible to
humans.
The psittacosis outbreaks of 1929-1930 and 1930-1938 were
attributed to psittacine birds. However, in the next years it
became clear that Cp. psittaci infections were not limited to
psittacine birds, but could affect other bird species. In 1939, the
bacterium was isolated from two South African racing pigeons
and soon, additional pigeon isolates were obtained, this time
in California. At that time, psittacosis in two New York citizens
could be attributed to contact with infected feral pigeons.
In 1942, serological evidence showed ducks and turkeys to
be frequently infected and within the next 3 years, human
infections due to contact with infected ducks were reported in
California and New York. In the early 1950s, Cp. psittaci could be
isolated from turkeys and from humans in contact with infected
turkeys, during severe respiratory outbreaks in the US turkey
industry. The incidence of severe Cp. psittaci epidemics in US
poultry declined during the 1960s. However, avian chlamydiosis
(1) Daisy Vanrompay. Ghent University, Faculty of Bioscience Engineering, Department of Molecular Biotechnology, Coupure Links 653, 9000 Ghent,
Belgium. Phone: +32 09 2645972. Fax: +32 09 2646219. E-mail: Daisy.Vanrompay@ugent.ne
267
EJCAP - Vol. 18 - Issue 3 December 2008
herons, egrets, pigeons, blackbirds, grackles, house sparrows,
and killdeer, all of which freely intermingle with domestic birds.
Seagulls and egrets could be asymptomatic carriers of highly
virulent strains of Cp. psittaci excreting a high number of
bacteria.
Transmission
Cp. psittaci is excreted in faeces and nasal discharges. Faecal
shedding occurs intermittently and can be activated by stress
caused by nutritional deficiencies, prolonged transport,
overcrowding, chilling, breeding, egg-laying, treatment or
handling. During natural infection, bacterial excretion periods
vary according to strain virulence, infection dose and host
immune status. However, shedding may occur for several
months. Transmission of chlamydiae is mainly by inhalation
of contaminated material and sometimes by ingestion. Large
numbers of Cp. psittaci can be found in respiratory tract exudate
and faecal material from infected birds. In turkeys, the lateral
nasal glands become infected early and remain infected for
more than 60 days. Choanal/oropharyngeal swabs are more
consistent for isolation of the agent than faecal swabs, especially
during the early stages of infection.
Turkey experimentally infected with the 84/55 genotype A strain.
Note the thickened abdominal airsac totally covered with fibrin
cloths (arrow).
remained a continuing threat to both birds and humans. During
the 1980s, chlamydiosis was reported again in US turkeys and in
the 1990s also in the European turkeys [14, 30, 43]. Nowadays,
Cp. psittaci is nearly endemic in the Belgian turkey industry [40,
46] and probably in other European countries as well. However,
devastating, explosive outbreaks like those in ‘ancient days’ are
rare and respiratory signs with no or low mortality characterize
outbreaks nowadays. More recently, an increase in the number
of chlamydiosis outbreaks in ducks and in people in contact with
infected ducks has been reported [2, 21]. In the past, human
infections associated with outbreaks in ducks had already been
described [2].
Avian species, including domestic poultry sharing aquatic or moist
soil habitats with wild infected aquatic birds may become infected
via contaminated water. Granivorous birds like pigeons, doves,
pheasants and house sparrows may become infected by dust
inhalation in faecal contaminated barnyards and grain storage sites.
The consumption of infected carcasses may transmit Cp. psittaci to
host species that are predators or scavengers of other birds.
Transmission of Cp. psittaci in the nest is possible. In many
species, such as Columbiformes, cormorants, egrets, and herons,
transmission from parent to young may occur through feeding,
by regurgitation, while contamination of the nesting site with
infective exudates or faeces may be important in other species
such as snow geese, gulls and shorebirds. Cp. psittaci can be
transmitted from bird to bird by blood-sucking ectoparasites
The list of avian species in which Cp. psittaci infections occur has
rapidly expanded. Today, Cp. psittaci has been demonstrated
in about 465 bird species comprising 30 different bird orders
[19]. The highest infection rates are found in psittacine birds
(Psittacidae) and pigeons (Columbiformes). The seroprevalence
in psittacine birds ranges between 16 and 81% and a mortality
rate of 50% or even higher is not unusual [6, 28]. Psittacidae
are the main chlamydial reservoirs, especially under captive
conditions, and nowadays these birds are extremely popular
pets [4, 44].
Hematoxylin and eosin staining of the lung of a turkey
experimentally infected with the Texas Turkey genotype D strain.
Note the congestion and the infiltration of lymphocytes (arrow A)
and the dilated parabronchi (arrow B).
Data on the seropositivity of racing pigeons range from 35.9
to 60%. Thirty-eight studies on the seroprevalence of Cp.
psittaci in feral pigeons conducted from 1966 to 2005, revealed
a seropositivity ranging from 12.5 to 95.6% [12, 20, 25, 27,
37]. More recent studies, conducted in feral pigeons in Italy,
Bosnia and Herzegovina, and FYROM revealed a seropositivity
of 48.5%, 26.5% and 19.2%, respectively [3, 17, 29]. Freeliving pigeons are distributed in urban and rural areas worldwide
and are in close contact with humans in most outdoor public
places.
Birds mostly living on sea shores and other waters, like geese,
ducks, gulls and penguins are more frequently infected than
hens, pheasants and quails [19]. Common reservoirs of Cp.
psittaci include wild and feral birds such as seagulls, ducks,
268
Update on avian chlamydiosis and its public health significance - D. Vanrompay
For isolation the following samples can be collected: pharyngeal/
choanal slit swabs, lung tissue, thickened exudate-coated air sacs
and free exudates, thickened pericardial tissues and exudates,
sections of enlarged spleen and liver, intestinal mucosa at sites
of hyperemia, colon contents, conjunctival and nasal discharges,
whole blood and peritoneal exudates. In live birds, pharyngeal/
choanal slit swabs are preferred rather than faeces or cloacal
swabs, as faecal shedding is intermittent. In dead birds, samples
of the respiratory tract are most suitable.
such as lice, mites and flies or, less commonly, through bites
or wounds. Transmission of Cp. psittaci by arthropod vectors
would be facilitated in the nest environment.
Vertical transmission has been demonstrated in turkeys,
chickens, ducks, parakeets, seagulls and snow geese, although
the occurrence appears to be fairly low. However, it could serve
as a method of introducing chlamydophila into a poultry flock.
Cp. psittaci can be introduced into susceptible pet birds and
poultry through the wild bird population. Contaminated
feed or equipment can also be a source of infection, and
feed should therefore be protected from wild birds. Careful
cleaning of equipment is extremely important as Cp. psittaci can
survive in faeces and bedding for up to thirty days. Cleaning
and disinfection with most detergents and disinfectants will
inactivate Cp. psittaci, as this Gram-negative bacterium has a
high lipid content.
The same tissues can be collected for molecular diagnosis. The
preservation of DNA from degradation is critically important.
DeGraves et al., [5] suggest all specimens for PCR analysis should
be collected in a DNA stabilization reagent. Such reagents are
commercially available; for instance the RNA/DNA Stabilization
Reagent for Blood/Bone Marrow® from Roche Applied Science.
This reagent is based on the denaturation of proteins in a
concentrated solution of guanidinium isothiocyanate and another
agent, for the inactivation of ribonuclease during RNA isolations.
However, it also works perfectly well for DNA stabilization.
Clinical disease
Polymerase chain reaction
Molecular methods, particularly the polymerase chain reaction
(PCR), permit rapid detection and identification of Chlamydophila
psittaci from clinical specimens. Two different genomic target
regions are usually used for amplification, namely the ribosomal
RNA gene region [7, 23, 24] and the gene encoding the MOMP
antigen designated ompA [15].
Depending on the chlamydial strain and the avian host, chlamydiae
cause pericarditis, air sacculitis (Fig. 1), pneumonia (Fig. 2), lateral
nasal adenitis, peritonitis, hepatitis, and splenitis. Generalized
infections result in fever, anorexia, lethargy, diarrhoea, and
occasionally shock and death. Chlamydiosis is a very common
chronic infection of psittacine birds. Infections cause conjunctivitis,
enteritis, air sacculitis, pneumonitis, and hepatosplenomegaly.
Droppings are often green to yellow-green. Many of the birds
become chronically infected but show no clinical signs until
stressed. These birds often shed chlamydiae intermittently and
serve as a source of infection for humans and other birds.
In some cases, concentration of target organisms by physical
means increases the assays’ sensitivity, but it is important to
ascertain the enrichment effect [5]. Such methods include
immunomagnetic, centrifugal, or filter concentration. Several
in-house-made and commercial DNA extraction methods
have been described [16, 31, 47]. Alternatively, commercial
DNA extraction kits can be used for the extraction of
chlamydial DNA. In our hands, the QIAamp® DNA Mini Kit
performed well for pharyngeal swabs while the High Pure
PCR Template Preparation Kit performed well for cloacal
swabs and faeces.
Recent developments in diagnosis
Sample collection and storage of specimens
The laboratory diagnosis of avian chlamydiosis includes
isolation or identification of the organism from the host
preferably by recently developed molecular techniques.
Because Cp. psittaci requires living cells to multiply, isolation
requires inoculation of cell cultures or specific pathogen free
embryonated chicken eggs in a biosafety level 3 laboratory,
as the infection can be aerogenically transmitted to humans.
Specimens should be collected aseptically, as contaminant
bacteria may interfere with isolation. A special transport
medium [35] should be used consisting of a sterile sucrose–
phosphate–glutamate (SPG) buffer (sucrose, 74.6 g/liter;
KH2PO 4, 0.512 g/liter; K 2HPO 4, 1.237 g/liter; and L-glutamic
acid, 0.721 g/liter), supplemented with foetal calf serum
(10%), vancomycin and streptomycin (100 mg/ml), and
nystatin and gentamicin (50 mg/ml). The medium also serves
for freezing swabs and tissues at -80°C in order to preserve
chlamydial viability. The stability of chlamydiae during
storage depends upon the material in which it is contained.
Chlamydiae in tissue specimens or yolk-sac suspension can be
preserved indefinitely by storage at -80°C [2]. Swabs should
be frozen in transport medium. At 4° C, the organism can
survive with gradual loss of infectivity for 30 days or longer
when in heavily infected tissues or in transport medium.
Recently, two highly sensitive nested PCR assays have been
developed to detect Chlamydophila psittaci in avian samples
[31, 41]. The method of Sachse and Hotzel [31] is based on a
first amplification generating a genus-specific ompA product
(576-597 bp) followed by a second amplification using one
species-specific and one genus-specific primer generating a
Chlamydophila psittaci-specific amplicon (389-404 bp). PCR
results are visualized by agarose gel electrophoresis using
ethidium bromide containing gels. The sensitivity of the nested
enzyme immunoassay (PCR-EIA) was established at 1 to 0.1
infection forming unit (IFU). Van Loock et al., [41] developed a
nested PCR-EIA. The fluorescein–biotin labeled PCR products
were immobilized on streptavidin-coated microtitre plates
and detected with anti-fluorescein peroxidase conjugate
and a colorimetric substrate, although detection by use of a
fluorimeter is also possible using this method. The sensitivity
of the nested PCR-EIA was established at 0.1 infection-forming
units (IFU). Specificity was 100%. An internal inhibition control
269
EJCAP - Vol. 18 - Issue 3 December 2008
through the use of commercially available bird seed impregnated
with chlortetracycline (CTC) (0.5 mg CTC/g of seed). For large
psittacine birds, feed containing 1% CTC (10 mg per g diet)
is recommended. CTC medicated diets are not available in
every country. A medicated mash diet for psittacine birds
can be prepared by cooking 2 pounds of rice, 2 pounds of
hen scratch feed and 3 pints of water for 15 minutes, adding
CTC to the cooled mash. Pigeons can be fed with uncooked
hen feed supplemented with CTC (5000 ppm). To reduce
interference with absorption the calcium content of the diet has
to be reduced to <0.7%. Doxycycline is the drug of choice for
administration by mouth (25 to 50 mg/kg bw) and parenteral
(intramuscular) treatment (75-100 mg/kg bw) every 5-7 days for
the first 4 weeks and subsequently every 5 days for the duration
of the treatment (45-day period).
was included to rule out the presence of inhibitors of DNA
amplification.
A number of real-time PCR tests have been developed. Real-time
PCR systems have the advantage that their sensitivity approaches
that of the nested PCR systems, and they require no additional
pipetting or handling of the PCR product following the initial
set-up of the PCR mix. This reduces both the time needed for
the test and the incidence of contamination. Recently a SYBR
Green-based real-time PCR was developed targeting the rDNA
ribosomal spacer of Chlamydophila psittaci [10]. The test could
detect 10rDNA copies/µL DNA extract. Geens et al., [10] also
developed a genotyping real-time PCR which detects all known
ompA Chlamydophila psittaci genotypes.
Micro arrays
Micro arrays can be coupled with PCR where they serve as a
set of parallel dot-blots to enhance product detection and
identification of bacterial isolates. Sachse et al., [32] developed
a DNA micro array-based detection and identification method
for Chlamydia and Chlamydophila spp. The test was developed
using the ArrayTube platform (CLONDIAG®chip technologies).
Unique species-specific hybridization patterns were obtained for
all nine species of the family Chlamydiaceae. The assay proved
suitable for species identification of chlamydial cell cultures and
showed a potential for direct detection of these bacteria from
tissue samples. Recently, an ompA-based Cp. psittaci genotyping
micro array has been developed [33].
Public health significance
Cp. psittaci can infect humans and should be handled
carefully under conditions of bio-containment. Humans most
often become infected by inhalation when urine, respiratory
secretions or dried faeces of infected birds is dispersed in the air
as very fine droplets or dust particles. Other sources of exposure
include mouth-to-beak contact, a bite from an infected bird or
handling the plumage and tissues of infected birds. Therefore,
post-mortem examinations of infected birds and handling of
cultures should be done in laminar flow hoods or with proper
protective equipment. Human infection can result from transient
exposures. The disease is of public health significance because
of the popularity of psittacine birds as pets and the increased
placement of these birds in child-care facilities and in homes
for the aged. Not only direct contact with birds poses risk of
psittacosis but also the rural environment and activities such as
gardening that may expose individuals to the infectious agent
[8, 38]. Person-to-person transmission of psittacosis is possible
but it is believed to be rare [18].
Serology
Despite the introduction of very sensitive and specific tests
like PCR, the idea of serological diagnosis still lingers in the
minds of several veterinary clinicians. However, serology
alone is not particularly useful for diagnosis of a patent
disease in birds because of the high seroprevalence of this
infection in asymptomatic birds and the long-term (up to
several months) persistence of anti-chlamydial antibodies. In
most bird species, there is a high background rate of antichlamydial antibodies, and until we have more information
on the disease pattern in certain bird species in relation to
direct identification of the bacteria and serology, we are
unable to comment on the real significance of antibody
titres obtained. Thus, to determine if a single bird is
infected, serology should always be used in conjunction with
bacterial antigen or gene detection or paired sera should
be examined. The latter can not be applied for immediate
clinical evaluation. Treatment with antibiotics may delay
and/or diminish the antibody response. Serology is very
helpful in epidemiological research. Recently, a sensitive
and specific recombinant ELISA has been developed based
on the detection of antibodies against the chlamydial major
outer membrane protein [46].
The incubation period in humans is usually 5–14 days or longer.
Human infections vary from asymptomatic to severe systemic
disease with interstitial pneumonia and occasionally encephalitis.
The disease is rarely fatal in properly treated patients; therefore,
early diagnosis is important. Symptoms usually include headache,
chills, malaise and myalgia, with or without signs of respiratory
involvement. Pulmonary involvement is common. Several severe
psittacosis (pneumonia) cases have recently been documented
[13, 26, 36] and Cp. psittaci has been associated with ocular
lymphoma [9, 48]. However, many psittacosis cases in humans
are presented with either mild respiratory symptoms or as
asymptomatic infections [16, 44]. The public health significance
of these infections is unknown.
Diagnosis is usually established through testing paired sera or only
one serum sample by use of the micro-immunofluorescence (MIF)
test which is more sensitive and specific than the complement
fixation (CF) test. However, the MIF test presents cross-reactivity
with other chlamydial species. As in birds, culture or the more
sensitive molecular methods can be used to specifically detect Cp.
psittaci. Tetracyclines are the drugs of choice for therapy of human
psittacosis. Doxycycline or tetracycline is usually administered (not
Treatment
Tetracyclines are the drugs of choice and can be administered
orally (medicated feed or by mouth if the bird does not accept
the medicated diet) or parenterally (injections). Parakeets,
budgerigars, finches, canaries and rice birds can be treated
270
Update on avian chlamydiosis and its public health significance - D. Vanrompay
in pregnant women and children < 9 years), where erythromycin
constitutes a second choice. The duration of treatment depends
on the drug. Tetracyclines should be given for at least 14 days. In
most countries, psittacosis is a notifiable disease.
during quarantine it is suspected or confirmed that psittaciformes
are infected with Cp. psittaci, all birds of the consignment must
be treated by a method approved by the competent authority
and the quarantine must be prolonged for at least two months
following the last recorded case”. Thus, these requirements did
not involve bird species other than poultry nor were there any
rules for the management of psittacosis within the EU. Therefore,
Decisions 2000/666/EC and 2005/760/EC were repealed and a
new Commission Regulation, EC no 318/2007 has been in place
since 1 July 2007 after being published in the Official Journal of
the EU. This regulation lays down the animal health conditions
for imports of certain birds from third countries and parts thereof
into the EU. This Regulation also lays down the quarantine
conditions. For instance: 1) approved quarantine facilities and
centres; 2) direct transport of birds to quarantine stations; 3)
attestation by the importers or their agents; 4) quarantine for at
least 30 days; 5) examination, sampling and testing to be carried
out by an official veterinarian; 6) actions in case of chlamydiosis
suspicion, which are treatment of all birds and prolonged
quarantine for at least two months following the date of the last
recorded case. Importantly, the Regulation allows for imports
of birds only from approved breeding establishments. Thus,
for birds other than poultry, only birds bred in captivity with an
individual identification number and accompanied by an animal
health certificate are allowed for importation into the EU.
Prevention and control
Vaccine development
Recently, attention has been turned to DNA vaccination and
recombinant adenoviral vaccines as a protective means against
chlamydial infections [45, 49]. DNA vaccination has been more
successful at inducing protective immune responses in turkeys
infected with Cp. psittaci [45], than in sheep or mouse models
infected with Cp. abortus [22]. Importantly, DNA vaccination can be
performed in the presence of maternal antibodies [39]. However,
additional research is needed to further enhance the immunogenicity
of DNA vaccines and to lower the vaccination costs.
There are currently no vaccines available against avian chlamydiosis
although research on MOMP-based DNA vaccination in SPF
turkeys has been very promising. DNA vaccination significantly
reduces clinical signs and chlamydial excretion, though it did not
give no full protection. Until an effective vaccine is produced,
risk reduction strategies and treatment will have to be applied.
Worldwide, psittacosis prevention and control recommendations
and legislation are mainly focused on the decrease of human
morbidity. Recommended control measures: 1) maintenance of
accurate records by the seller of all bird transactions. Records
should mention when the birds are sold, the name, address and
phone number of the customer, date of purchase, species and
band number if applicable; 2) Birds with signs compatible with
chlamydiosis should not be purchased or sold; 3) Before adding
new birds to a group, the birds should be quarantined and
observed for a 14-30 day period and tested or prophylactically
treated; 4) accurate preventive husbandary (prevent feathers,
food and other materials moving from one cage to another, daily
cleaning, sufficient exhaust ventilation to prevent accumulation
of aerosols); 5) special husbandary during infection (isolation,
non-dusty litter, keep circulation of feathers and dust to a
minimum by frequent wet-mopping with disinfectants, cleaning
and disinfection); 6) disinfection (1:1000 dilution of quaternary
ammonium compounds, 70% isopropyl alcohol, 1% Lysol, 1:100
dilution of household bleach or chlorophenols can be used); 7)
personal protection (HEPA mask in high risk situations).
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273
ZOONOSES
The role of Bartonella spp. in
veterinary and human medicine
with special emphasis on
pathogenicity mechanisms
V. A. J. Kempf(1), F. Krämer(2)
SUMMARY
Bartonella spp. are important pathogens in human and veterinary medicine. Currently, B. henselae is considered to
be the most relevant zoonotic Bartonella species responsible for cat scratch disease (CSD), bacillary angiomatosis and
peliosis hepatis. Besides B. henselae, several other species have been isolated from cats, dogs and humans suffering
from a variety of clinical manifestations ranging from mild infections to severe disease. The analysis of the underlying
pathogenicity mechanisms will result in better understanding of the diseases and consequent vector control might
allow the prevention of such Bartonella-infections.
Bartonella infections of cats, dogs and humans
Infectious diseases caused by Bartonella have been described
for more than 1,000 years. Historically, infections with B.
bacilliformis (which is endemic in South America) are known
since the dynasty of the Inca. B. quintana was even detected in
4,000-year-old human tissue [15]. However, only 18 years ago,
B. henselae, the most important human pathogenic species,
was discovered by David Relman who identified this pathogen
in 1990 by molecular techniques [41].
This paper was commissioned by FECAVA for
publication in EJCAP.
Introduction
Bartonellosis is considered an emerging zoonosis with increasing
frequency. While cats often remain asymptomatic carriers
for Bartonella spp., dogs may develop clinical signs that are
similar to those observed in humans [21]. Due to their way of
transmission in cats and dogs via fleas and presumably also
ticks [12], bartonellosis is counted into the field of canine vector
borne diseases (CVBD).
Cats have been confirmed as a reservoir for B. henselae. Besides this
species, cats are also the main reservoir for B. clarridgeiae and are
presumably the reservoir of B. koehlerae which has been isolated
from the blood of cats in the USA [16] and was also detected in
cat fleas in France [46]. Mostly, cats with serological, cultural or
PCR-based evidence of an exposure to a certain Bartonella spp.
show no particular signs of infection. Nevertheless, B. henselae
infections of cats have been associated with a variety of clinical
manifestations, including, e.g., uveitis [31, 32].
The ability to cause vasculoproliferative disorders or
intraerythrocytic bacteraemia is a unique feature of the genus
Bartonella. The pathogens are present in a broad spectrum
of mammals including cats, dogs, ruminants and rodents
which might either suffer from these infections [25] or serve
as asymptomatic reservoir hosts for zoonotic infections [13].
Elucidating the course of Bartonella infections in animals,
arthropods and humans will help to prevent such infections
in future; vector control in cats and dogs might also prevent
transmission to humans as well as disease in the pets
themselves.
The most prevalent Bartonella species in dogs, B. vinsonii subsp.
berkhoffii, was initially isolated from a dog with endocarditis and
intermittent epistaxis in 1993 [5, 30]. Meanwhile, this pathogen
has also been associated with cardiac arrythmias, myocarditis,
granulomatous rhinitis, anterior uveitis, and choroiditis [4, 5, 36,
38, 39]. B. henselae [17, 19, 23, 29, 47], B. clarridgeiae [10,
(1) Volkhard AJ Kempf. University Hospital, Institute for Medical Microbiology and Hygiene, Elfriede-Aulhorn-Str. 6, D-72076 Tuebingen, Germany
E-mail: volkhard.kempf@med.uni-tuebingen.de
(2) Friederike Krämer. University of Veterinary Medicine Hannover, Institute for Parasitology, Bünteweg 17, D-30559 Hannover, Germany
274
The role of Bartonella spp. in veterinary and human medicine - V. A. J. Kempf & F. Krämer
23], B. washoensis [11], B. elizabethae [3] and B. quintana [25]
have also been associated with pathology and clinical signs
in dogs, including endocarditis, hepatic disease and sudden
death. Furthermore, B. henselae has been implicated in peliosis
hepatis [29], granulomatous hepatis [23], and granulomatous
sialodenitis [47]. Interestingly, B. henselae and B. quintana were
also detected in the blood or lymph nodes of dogs suffering
from lymphoma [19].
Today, the clinically most important humanpathogenic species
are B. henselae, B. quintana and B. bacilliformis. A synopsis of
Bartonella spp., their reservoir hosts, vectors and the spectrum
of human diseases are given in Tab. 1.
Cat scratch disease (B. henselae, B. clarridgeiae)
Cat scratch disease (CSD) is caused by B. henselae (less
frequently by B. clarridgeiae). The pathogens are transmitted
by bites or scratches of infected cats (Fig. 1). Usually, 2–3
weeks after infection, a unilateral lymphadenitis in the lymph
Fig. 1 Inoculation site (forearm) of a patient suffering from a
B. henselae-caused CSD.
Tab. 1 Bartonella spp.: reservoirs, vectors, human diseases (modified table, taken from ref. 13).
Bartonella spp.
Reservoir
Vector
human
sandfly
Human diseases
Human-specific spp.:
B. bacilliformis
Carrion’s disease, Oroya fever, verruga peruana
46
8
B. quintana
human
body louse (cat flea , ticks )
trench fever, endocarditis, bacillary angiomatosis
B. rochalimae
unknown
unknown
bacteraemia, fever20
B. clarridgeiae
cat
cat flea
cat scratch disease
B. elizabethae
rat
unknown
endocarditis, neuroretinitis
B. grahamii
mouse, vole
unknown
neuroretinitis
B. henselae
cat
cat flea (ticks?)
cat scratch disease, bacillary angiomatosis,
endocarditis, neuroretinitis, bacteraemia
B. koehlerae
cat
unknown
endocarditis
B. vinsonii subsp. arupensis
mouse
tick
bacteraemia, fever, endocarditis (?)
B. vinsonii subsp. berkhoffii
dog
tick
endocarditis
B. washoensis
ground squirrel
unknown
myocarditis, endocarditis (?)
B. alsatica
rabbit
unknown
unknown
B. birtlesii
mouse
unknown
unknown
B. bovis (= B. weissii)
cattle, cat
unknown
unknown
B. capreoli
roe deer
unknown
unknown
B. chomelii
cattle
unknown
unknown
B. doshiae
vole
unknown
unknown
B. peromysci
deer, mouse
unknown
unknown
B. phoceensis
rat
unknown
unknown
B. rattimassiliensis
rat
unknown
unknown
B. schoenbuchensis
roe deer
unknown
unknown
B. talpae
vole
unknown
unknown
B. taylorii
mouse, vole
unknown
unknown
B. tribocorum
rat
unknown
unknown
B. vinsonii subsp. vinsonii
vole
unknown
unknown
Zoonotic spp.:
Animal-specific spp.:
275
EJCAP - Vol. 18 - Issue 3 December 2008
Fig. 2 Typical histology of CSD in a lymph node of a 52-year-old
patient showing granulomatous inflammation and central necrosis.
Fig. 3 Bacillary angiomatosis in a 37-year-old AIDS patient located
at the chest. Newly appeared vessel formations can be noted.
draining region near the site of the scratch or bite occurs (Fig. 2).
In ~10 % of CSD, infected lymph nodes may form a fistula through
the skin draining pus. CSD is a common cause of chronic lymph
node swelling in children, and these patients might also suffer
from fever, headache or splenomegaly. In rare cases, oculoglandular involvement (Parinaud’s syndrome), encephalopathy,
neuroretinitis or osteomyelitis can occur. Usually, the disease is
self-limiting and patients do not require antibiotic treatment.
Serological testing is the best evaluated method for the
laboratory diagnosis of CSD. Relapsing or systemic infections
might be treated with macrolides (e.g., erythromycin).
lobulated proliferations of mainly capillary-sized vessels and are
understood as the result of a chronic infection with B. henselae or
B. quintana. The pathogen is detectable in the vasculoproliferative
lesions, and bacterial eradication by antibiotic treatment results in
a complete regression of the angiomatous tumors.
Bloodstream infections: Trench fever (B. quintana)
B. quintana, the agent of “Trench fever”, caused large epidemics
in Europe during the First and Second World Wars. Trench
fever reemerged at the end of the last century with increasing
frequency. B. quintana bloodstream infections were reported
in homeless people or patients with chronic alcoholism [7, 52].
The disease is characterized by a sudden onset and subsequent
periodical feverish relapses (“five-day fever”) accompanied by an
intraerythrocytic bacteraemia. The presence of B. quintana in the
bone marrow (erythroblasts) has been described [45]. Humans
are the only known natural reservoir host for B. quintana; body
lice transmit the pathogen from patient to patient. Interestingly,
the pathogen has recently been detected in cat fleas [46]
and ticks [8]. B. quintana has also been detected in two dogs
suffering from endocarditis [25].
Cats have been identified as the primary reservoir for B. henselae,
and the domesticated species is most often associated with the
transmission of the infection to humans (mainly children) by
scratches or bites [3]. Between cats, the organism is transmitted
by the cat flea Ctenocephalides felis [9]. Recent studies
have shown a strong correlation between the prevalence of
B. henselae in cats and fleas from these cats [33].
Dogs are also suggested to represent a reservoir for B. henselae
as the pathogen was found to be the causative agent in a case of
human osteomyelitis following a dog scratch [28]. Furthermore,
a dog owner suffering from lymphadenopathy (seroreactive for
B. henselae) was reported and B. henselae DNA was amplified
from gingival swaps of the dog [53]. Currently, four Bartonella
species have been detected in dog saliva [18]. From these
findings, a transmission of Bartonella from dogs to humans
cannot be excluded and it might be assumed that vector control
(flea and tick) reduces the risk of transmission to dogs (e.g., via
ticks [12]) and, thus, possibly from dogs to humans.
Recently, B. henselae and B. vinsonii subsp. berkhoffii were
detected in the peripheral blood of immunocompetent patients
suffering from chronic neurological symptoms including ataxia,
seizures and tremor [6].
Fig. 4 Detection of
anti-B. henselae
antibodies by IFA
using cell culturederived antigen.
Bacteria-induced neoangiogenesis: bacillary
angiomatosis, peliosis hepatis (B. henselae, B. quintana)
The ability to induce tumorous vasculoproliferative disorders is a
unique feature of pathogens of the genus Bartonella. In humans,
mainly immunosuppressed patients (e.g. AIDS patients) suffer
from the vasculoproliferative disorders bacillary angiomatosis
(cutaneous manifestations) (Fig. 3) or peliosis hepatis (hepatic
manifestations). Both diseases are histologically characterized as
276
The role of Bartonella spp. in veterinary and human medicine - V. A. J. Kempf & F. Krämer
B. henselae infections: Diagnostic procedures
Concerning the zoonotic importance of Bartonella infections in
cats and dogs, as well as the general relevance of Bartonella
infections in humans, obviously, B. henselae represents the most
important pathogenic Bartonella species.
include the Bartonella adhesin A (BadA) of B. henselae [43] and
the variably expressed outer membrane proteins (Vomps) of
B. quintana [54].
Intraerythrocytic bacteraemia caused by Bartonella spp.
Several Bartonella species cause a long-lasting intraerythrocytic
bacteraemia in their mammalian reservoir host. This obviously
enables the transmission of the pathogens via blood-sucking
arthropods (e.g. fleas, body lice). Furthermore, a persistent
bacteraemia with B. henselae in asymptomatic cats (and possibly
in dogs) represents an important factor facilitating the spread of
microorganism.
A variety of methods for the laboratory identification of
B. henselae infections have been employed including histological
examination, isolation and culture or molecular and serological
approaches. Nevertheless, the detection of the causative
agent of CSD by histology or PCR (e.g., via amplification of the
16S-rDNA) requires invasive procedures (biopsy, fine-needle
aspiration). Unfortunately, cultivation of the slowly growing
and fastidious pathogens from patient specimen is normally not
successful. Therefore, the most widely used diagnostic method
is the serological testing for B. henselae antibodies. Here, an
indirect immunofluorescence assay using whole cell antigen
from B. henselae co-cultivated with Vero cells is used (Fig. 4).
Most of the knowledge of intraerythrocytic Bartonella infections
was gained using a B. tribocorum rat-infection model which
mimics Trench fever [48]. Here, the pathogens are rapidly cleared
from the circulating blood after an intravenous infection. Five
days later, the bacteria start to be periodically released from their
unknown niche into the bloodstream. B. tribocorum persists
several weeks in the circulating blood in an immunoprivileged
intracellular niche (erythrocyte). This haematotropic infection
strategy is probably a specific adaptation of the pathogen to
the transmission by blood-sucking arthropod vectors and is
shared by many Bartonella species. From all Bartonellae, only
B. bacilliformis causes a massive haemolysis of infected
erythrocytes often resulting in a fatal haemolytic anaemia
[13]. Using human haematopoietic stem cells, it could be
demonstrated that Bartonella spp. are able to infect and to
persist in erythroid-differentiating stem cells suggesting that
haematopoietic stem cells might serve as a potential primary
niche in Bartonella infections [35].
The seroprevalence of anti-B. henselae antibodies in cats ranges
from 0% in Norway [2] over 35% in Alabama, Maryland and
Texas [33] and 71% in Spain [50], up to 93% in North Carolina
[37], depending on the region, the method of examination and
the type of cats examined (e.g., feral/stray or pet cats).
In dogs the seroprevalence of anti-B. henselae antibodies has
been reported between 2% in Brazil [14] and 3% in the UK
[1] over 5% in southern Ontario and Quebec [22] and 17% in
Spain [51], up to 27% in south-eastern USA [49] and 28% in
Italy [40].
The seroprevalence of anti-B. henselae antibodies in humans is
between 5-30% and, therefore, much higher compared with the
seroprevalence of antibodies against, e.g. Borrelia burgdorferi
(causing Lyme disease) and many other pathogens. 13% of
cervical lymph node swellings are caused by Bartonella spp.
[42]. In the United States, the incidence of Bartonella infections
was 9.3 per 100,000 inhabitants in the year 1993 [24].
Vasculoproliferative diseases caused by Bartonella spp.
B. henselae, B. quintana and B. bacilliformis are able to cause
vasculoproliferative disorders. This feature is especially reported
from humans; however, less known, also dogs can develop such
vasculoproliferations (e.g. peliosis hepatis [29]).
Mechanisms leading to this neoangiogenic lesions are best
investigated for B. henselae. Currently, it is speculated that
Bartonella species may cause vasculoproliferations by at least
three different mechanisms which may act synergistically:
(i) triggering the proliferation of endothelial cells directly, (ii)
inhibition of endothelial cell apoptosis (via the VirB/D4 T4SS),
and (iii) angiogenic reprogramming of infected host cells. This
bacterially induced angiogenic gene program is regulated via
the activation of “hypoxia-inducible factor (HIF)-1” (which
represents the key transcription factor involved in the induction
of angiogenesis) and subsequent secretion of angiogenic
cytokines (e.g., vascular endothelial growth factor, VEGF), all
known to be crucially involved in neoangiogenic processes. Both,
HIF-1 activation and VEGF secretion are induced after infection
of cultured host cells with B. henselae in vitro and where also
demonstrated in patient’s bacillary angiomatosis tissue lesions
in vivo.
Pathogenicity of Bartonella spp.
Two aspects are in the focus of Bartonella research: the ability
of the bacteria to induce vessel formation (“neoangiogenesis”)
and their ability to cause a long-lasting bacteraemia, both
being highly interesting phenomena, especially of interest
for developing new strategies of inducing therapeutic vessel
growth, possibly influencing angiogenesis research. Strategies of
how bacteria cause chronic intraerythrocytic infections are also
fascinating as the underlying mechanisms are largely unknown.
However, Bartonella research is hampered by the fact that the
bacteria are slow growing and no sufficient liquid medium is
available. With the recently described “BAPGM”-medium [34]
or our new liquid medium [44] new promising tools for liquid
cultivation of Bartonella spp. now exist.
Mainly two pathogenicity factors of Bartonella spp. have been
investigated in the past: (i) the so-called “VirB/D4 type-4
secretion system” (T4SS), a molecular nanomachine injecting
Bartonella effector proteins (Beps) into human host cells and
(ii) the so-called “trimeric autotransporter adhesins” which
Based on our own previous work, we favour a “two-step”pathogenicity model suggesting that host-cell-derived
vasculoproliferative factors play a crucial role in the pathogenesis
277
EJCAP - Vol. 18 - Issue 3 December 2008
of neoangiogenesis in Bartonella infections. The resulting
proliferation of endothelia might be understood as bacteriumtriggered promotion of its own niche: the endothelial cell. These
proliferating host cells promote persistence and growth of B.
henselae by supplying the bacterium with yet unknown, possibly
proteinacous compounds [27]. It is tempting to speculate that
by affecting host-cell metabolism, B. henselae creates its own
endothelial habitat [26].
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39:3548-54.
[11] CHOMEL (B.B.), WEY (A.C.), KASTEN (R.W.) - Isolation of
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formation. Nat Rev Microbiol, 2005, 3:621-31.
[14] DINIZ (P.P.), MAGGI (R.G.), SCHWARTZ (D.S.), CADENAS (M.B.),
BRADLEY (J.M.), HEGARTY (B.), et al - Canine bartonellosis:
serological and molecular prevalence in Brazil and evidence of coinfection with Bartonella henselae and Bartonella vinsonii subsp.
berkhoffii. Vet Res, 2007, 38:697-710.
[15] DRANCOURT (M.), TRAN-HUNG (L.), COURTIN (J.), LUMLEY (H.),
RAOULT (D.) - Bartonella quintana in a 4000-year-old human
tooth. J Infect Dis, 2005, 191:607-11.
[16] DROZ (S.), CHI (B.), HORN (E.), STEIGERWALT (A.G.), WHITNEY
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combined approach for the enhanced detection and isolation
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[18] DUNCAN (A.W.), MAGGI (R.G.), BREITSCHWERDT (E.B.) Bartonella DNA in dog saliva. Emerg Infect Dis, 2007, 13:194850.
[19] DUNCAN (A.W.), MARR (H.S.), BIRKENHEUER (A.J.), MAGGI
(R.G.), WILLIAMS (L.E.), CORREA (M.T), et al - Bartonella DNA in
the blood and lymph nodes of Golden Retrievers with lymphoma
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(E.T.), MATHEW (S.S.), et al - Bacteremia, fever, and splenomegaly
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2007, 356:2381-7.
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Conclusion
Bartonella spp. are important pathogens in human and
veterinary medicine. B. henselae is currently considered to
be the most relevant zoonotic pathogenic Bartonella species
responsible for CSD, bacillary angiomatosis and other diseases.
Besides B. henselae, several other Bartonella species have been
isolated from cats, dogs and humans with a variety of clinical
manifestations, from symptomless to severe disease. The ability
to cause vasculoproliferative disorders and intraerythrocytic
bacteraemia are unique features of the genus Bartonella.
Obviously, the VirB/D4 T4SS and BadA are key virulence factors
of B. henselae responsible for host-cell infection, inhibition of
endothelial cell apoptosis and induction of angiogenic gene
programming. Elucidating the course of Bartonella infections
in animals, arthropods and humans will help to understand
and thus possibly prevent such infections in future. Taking into
account the current knowledge about Bartonella infections, their
zoonotic nature and the transmission risk by arthropods, it can be
assumed that vector control strategies reduce the ectoparasite –
host interactions thus limiting pathogen transmission.
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279
ZOONOSES
Meticillin-resistant staphylococci in
companion animals
T. Nuttall(1), N. Williams(2), R. Saunders(1), S. Dawson(2)
SUMMARY
Meticillin resistant (MR) Staphylococcus aureus (MRSA), S. intermedius/pseudintermedius, S. schleiferi coagulans, S. schleiferi
schleiferi and other coagulase negative staphylococci have been associated with infections and colonisation in many
animal species. Dogs and cats are usually colonised with the most prevalent human MRSA isolates, whereas other
animal species are colonised with clone types unique to those species. MR staphylococci are resistant to β-lactams,
cefalosporins, fluoroquinolones and clindamycin. They are frequently sensitive to tetracyclines, sufonamides,
fucidic acid and mupirocin, but multi-drug resistant S. intermedius and S. pseudintermedius have been identified. The
prevalence in healthy animals appears to be low. Risk factors for infection include repeated courses of broad spectrum
antibacterials, surgery, joint infections, non-healing wounds and catheterisation. Most infected animals survive,
provided that the primary condition can be managed. It is likely that there is two-way exchange of MR staphylococci
between humans and animals. Animals are therefore at risk of colonisation from contact with veterinary staff and
premises, and colonised animals may act as reservoirs for colonisation of in-contact humans. True zoonotic infections
remain rare and are associated with defined risk factors. Antibacterial use guidelines may limit the development and
spread of multi-drug resistant bacteria. Strict hygienic precautions will help prevent contamination and dissemination
in veterinary clinics.
MRSA in humans
This paper was commissioned by FECAVA
MRSA is of little concern to healthy people. Approximately 2030% of people are colonised with meticillin sensitive S. aureus
(MSSA) but probably less than 1% carry MRSA. The carriage
rate in healthcare workers, however, may be 5-10%. Veterinary
personnel have also been shown to have higher carriage rates
than the general population [1-4] .
for publication in EJCAP.
Introduction
MRSA (meticillin resistant Staphylococcus aureus) and other
meticillin resistant (MR) staphylococci are major medical and
veterinary health care concerns. MRSA and other meticillin
resistant staphylococcal species have been isolated from dogs,
cats, rabbits, birds, horses and farm animals. Animals could be at
risk of colonisation or infection in veterinary premises and/or act
as reservoirs for colonisation or infection of in-contact humans.
Colonisation in this context refers to the non-pathogenic
temporary (also referred to as ‘contamination’) or persistent (or
‘carriage’) presence of MR staphylococci at mucosal surfaces or
on the skin. The aim of this review is to give veterinary clinicians
and nurses or technicians an overview of the current state of
MR staphylococci in companion animals, and an update on the
practical and clinical aspects of controlling these organisms.
There has been a dramatic increase in MRSA infections in recent
years, especially in patients who have skin or mucosal barrier
defects, are immuno-compromised, undergo extensive surgery
or are long term in-patients. MRSA clones can be differentiated
by several techniques including pulsed field gel electrophoresis
(PFGE), multi-locus sequence typing (MLST) and antibiotic
resistance patterns. In the UK, the majority are hospital acquired
(HA) epidemic (E) strains 15 and 16 [5]. EMRSA-15 is resistant to
-lactams, fluoroquinolones and macrolides, whilst EMRSA-16
can be resistant to further antimicrobials. EMRSA-15 is also
prevalent elsewhere in Europe. Based on blood culture isolates,
the prevalence of MRSA infections varies greatly across Europe,
1) Department of Veterinary Clinical Science University of Liverpool Faculty of Veterinary Science, Leahurst Campus, Neston, Cheshire, UK.
2) Department of Veterinary Pathology, National Centre for Zoonosis Research, Leahurst Campus, Neston, Cheshire, UK.
Corresponding author: Dr Tim Nuttall. The University of Liverpool Small Animal Teaching Hospital, Leahurst Campus, Neston, Cheshire, CH64 7TE,
UK. Tel. - +44 151 795 6216. Email. timn@liv.ac.uk
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Meticillin-resistant Staphylococci in companion animals - T. Nuttall, N. Williams, R. Saunders, S. Dawson
Canada and the USA (Weese personal communication). The PVL
gene has also been detected in 50% of US MRSA isolates from
animals [31].
The majority of isolates from horses, rabbits and other animals
in the UK, Ireland, Austria, Germany, Japan, US and Canada,
in contrast, are largely restricted to these animal species and
are only rarely found in the human population [14, 16, 20, 23,
32, 33]. Equine isolates are predominantly identical or closely
related to Canadian MRSA-5 (USA500), which is rare in the
general human population but has been isolated from in-contact
humans [32, 34].
Fig. 1 70% alcohol
hand rub worn on
a nurse’s uniform.
All our staff and
students wear these for
quick and easy hand
disinfection between
patients. Organic
debris must be washed
off with a detergent
though.
An MRSA type identified as sequence type (ST) 398 by multilocus sequence typing has come to prominence because of its
association with pig farming, farmers and veterinarians in the
Netherlands and Germany [21, 35, 36]. ST398 has also been
isolated from horses in Germany and Canada, and neonatal
humans in Scotland.
from <1% in Scandinavia and the Netherlands to >40% in
southern and western Europe [6].
MR S. intermedius/pseudintermedius
MR has also been seen in other staphylococci more commonly
associated with animals, particularly S. intermedius [37-39],
although it is now thought that most, if not all, canine isolates
are actually S. pseudintermedius [40-42]. In a recent Japanese
study, 17/18 MR staphylococcal isolates from dogs were
S. pseudintermedius [41]. In Germany, 12 identical or
closely related S. intermedius strains, resistant to at least five
antimicrobial classes including penicillins, cefalosporins and
fluoroquinolones, were isolated from skin and ear infections in
11 dogs and one cat [43].
Community acquired (CA) clones are genetically distinct from HAMRSA clones [7]. CA-MRSA are usually isolated from individuals
without healthcare contact or other risk factors. Infections
are rare and sporadic but can be severe and life-threatening.
Antibiotic resistance is less marked but CA-MRSA infections are
often associated with Panton Valentine Leucocidin (PVL). PVL
can result in extensive tissue necrosis and severe disease [8]. The
designations HA and CA do not necessarily refer to the source
of colonisation or infection: HA-MRSA can be acquired in the
community [9] and CA-MRSA clones have been associated with
hospital acquired infections [10].
Other MR staphylococci in animals
Other MR staphylococci isolated from animals include the
coagulase positive species (CPS) S. schleiferi subsp. coagulans,
particularly in the US [37-39], and the coagulase negative
staphylococci (CNS) S. schleiferi subsp. schleiferi, S. haemolyticus,
S. vitulinus, S. sciuri, S. epidermidis, and S. warneri [44]. CNS
area usually regarded as non-pathogenic, commensal species,
as they lack many of the virulence factors associated with CPS.
CNS, particularly S. schleiferi subsp. schleiferi, have nevertheless
been isolated from pyoderma and wound infections in animals
[38, 39, 45, 46], although it can be difficult to determine their
importance in mixed infections. Commensal CNS will also be
exposed to antibacterials, and could act as reservoirs of mobile
genetic elements encoding for antimicrobial resistance (see
below). Further research on the clinical relevance of CNS and
the impact of antibacterials on commensal bacteria is therefore
warranted.
MR staphylococci in animals
MR staphylococci have been isolated from healthy and diseased
individuals in many animal species [11-21]. The epidemiology
varies between staphylococcal isolates, animal species and
geographic location, reflecting differences in staphylococcal
species, animal-human interaction, antimicrobial use, and
species susceptibility.
MRSA in companion animals
MRSA has been the most frequent MR staphylococcal species
isolated from animals in the UK, Ireland and Canada [14-16, 19,
22, 23].
British, Irish and German small animal isolates are mostly identical
or closely related to EMRSA-15 [14, 16, 23-25]. EMRSA-16 has
also been isolated from dogs in the UK, but is less common [26].
In contrast, canine isolates unrelated to human clones have been
occasionally identified in the UK, the Netherlands and Germany
[20, 27, 28].
Origins and dissemination of MR
staphylococci
Several studies have associated identical or very closely related
MR staphylococcal isolates with particular veterinary premises
in the UK, Denmark, Austria, Germany, Ireland, Japan and
Canada [22, 23, 33, 35, 41, 43, 47, 48]. The genetic diversity
of MR S. pseudintermedius and intermedius isolates suggests
that colonisation can be acquired either in veterinary clinics or
In the US and Canada the predominant human strain, Canadian
epidemic MRSA-2, (USA100) is most common in animals [15,
29, 30]. More recently PVL-positive Canadian epidemic MRSA10 (USA-300) has been isolated from small animals in both
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Fig. 2 Fully
waterproof and
washable keyboard for
use in clinical areas.
in the community [41, 43], but the similarity of isolates from the
staff and patients in particular premises suggests dissemination
in veterinary clinics occurs. A US study of 27 veterinary teaching
hospitals, however, could not associate specific clones with
individual hospitals and concluded that colonisation was
probably community acquired [49].
will be ineffective. Third line drugs are those very important to
humans and animals (e.g. fluoroquinlones, anti-Pseudomonas
penicillins, ceftazidime, imipenem etc.). They should only be
used where culture indicates they are necessary.
In one study the adoption of antibacterial use guidelines lead to
a significant fall in the use of second and third line drugs with
first line drugs accounting for approximately 90% of treatment
[53].
Antibacterial use and the emergence of MR
staphylococci
It is likely that antibacterials played a role in the emergence of
MR staphylococci in animals. One study concluded that the
prevalence of MR-CNS in companion animals reflected national
and local patterns of antimicrobial use [47]. A significantly
higher prevalence of resistance to two or more antibacterials
was reported in S. intermedius isolates from dogs and domestic
pigeons compared to those from wild birds [50]. Prior use of
antibacterials is also a risk factor for MRSA infection in humans
and horses [18, 51], and recent studies by the authors and others
have shown that courses of broad-spectrum antibacterials are a
risk factor for MRSA infection in dogs.
The prevalence of MR staphylococci in
companion animals
Colonisation in healthy animals
It is likely that the colonisation rate in healthy animals is low.
One Slovenian study did not isolate any MR-CPS from 200
healthy dogs [46]. A UK study failed to isolate MRSA from dogs
sampled in the community over a two month period [16], and
only 1/255 dogs was positive for MRSA in another [54].
Rational and responsible use of antibacterials
Measures to minimise the development and spread of
antimicrobial resistant bacteria should be adopted [52]. It is
beyond the scope of this article to discuss these in detail, but
issues to address include:
Colonisation and infection in sick animals
The colonisation rate in non-healthy animals seems to be much
higher, although not all reports distinguish colonisation and
infection. One UK study found that MRSA was carried by up
to 10% of dogs referred to a small animal veterinary hospital
without MRSA infections [24], although these had received
treatment including antibacterials prior to referral. MRSA was
also isolated from approximately 3% of clinical submissions to a
UK veterinary diagnostic laboratory [25]. In Ireland, MRSA was
recovered from 25 animals (14 dogs, eight horses, one cat, one
rabbit and a seal) and from 10 in-contact veterinary personnel
[14]. Of 869 submissions from a German small animal hospital
over a 20 month period, 3% (approximately 0.05% of the total
caseload) were MRSA [20]. Similar proportions of samples were
from dogs (3%) and cats (2.7%), with MRSA also isolated from a
bird, rabbit, guinea pig, turtle and bat. In the authors’ institution
MR-CPS infections comprised 0.04% of all cases and 0.5% of all
bacterial infections from 2004-2007. Twelve MR S. intermedius
isolates identified in one report accounted for 23% of all S.
intermedius submissions from a German dermatology referral
clinic during an 18 month period [43]. MR S. pseudintermedius,
S. aureus and S. schleiferi coagulans were isolated from 2.1%,
0.5% and 0.5% respectively of 193 dogs referred to a Canadian
Veterinary Teaching Hospital [55].
– Ensuring that the patient has a bacterial infection
– Ensuring that the infection warrants systemic or topical
antibacterial therapy
– Selecting the most appropriate drug
– Using an efficacious dose for an adequate treatment period
– Maximising compliance
It can be helpful to divide antibacterials into first, second and third
line drugs, although this will depend on the patient, target tissue
and type of bacteria. First line drugs include older antibacterials
and/or narrow spectrum drugs such as simple penicillins,
tetracyclines, and sulfonamides. These are no less potent than
other antibacterials in the appropriate circumstances. Second
line drugs include newer, broad spectrum products that are
more important for humans and animals and/or more prone to
resistance (e.g. broad spectrum β-lactamase resistant penicillins,
cefalosporins and macrolides). These should be used where
culture or good empirical evidence indicates that first line drugs
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Meticillin-resistant Staphylococci in companion animals - T. Nuttall, N. Williams, R. Saunders, S. Dawson
MR staphylococcal infections in
animals
There is probably little risk to healthy animals but vulnerable
pets could include immunocompromised animals, long-term inpatients, and animals with open wounds, implants and major
surgery. A wide variety of skin, soft-tissue and other infections
have been reported [13, 24, 43]. In the authors’ clinic MRSA
infections are significantly more likely to be associated with
post-operative infections and non-healing wounds compared
to meticillin sensitive CPS infections, whereas primary skin, ear
and soft-tissue infections were less common. In US reports, MR
S. intermedius was more likely to be associated with superficial
and first-line infections, whereas MR S. schleiferi were more
common in recurrent infections following antimicrobial therapy,
and MRSA were associated with opportunistic soft-tissue
infections and/or septicaemia [30, 38, 39]. One retrospective
study, comparing infection with MRSA versus MSSA, showed the
presence of a urinary catheter, joint infection and prior treatment
with fluoroquinolones to be significant risk factors [59].
Fig. 3 Tape-strip impression smear from a staphylococcal
pyoderma. Note the degenerate neutrophils with intra- and
extracellular cocci. Diff-Quik stain, magnification x 400.
A prevalence study at a UK referral hospital isolated MRSA from
6% of staff and 6% of dogs (2/3 had MRSA infections) but
no cats [16, 56]. A single day prevalence study at another UK
referral hospital found that 18% of staff, 9% of dogs, 10%
of environmental samples and again no cats were colonised by
MRSA [24]. In Japan, MR-CPS were isolated from 15% of staff,
46% of canine in-patients and 19% of canine out-patients [41];
2/3 isolates from staff were MRSA, but only one of the isolates
from the dogs was MRSA and the remaining 17 were MR
S. pseudintermedius.
There is very little data on risk factors for colonisation and
infection in dogs and cats, but the following are currently
considered to be at risk:
– Patients from known MRSA positive households or that belong
to owners with recent or frequent healthcare contact.
– Patients with non-healing wounds.
– Patients with non-antibiotic responsive infections where
previous cytology and/or culture indicates that staphylococci
are involved.
– Nosocomial or secondary infections, especially in at-risk
patients.
In contrast, US studies have identified MR staphylococci at much
higher frequency. They have been isolated from 15% of healthy
cats and 38% of dogs with recurrent pyoderma; MR rates
for S. aureus was 35%, 17% for S. intermedius and 40% for
S. schleiferi [37]. More recently, however, the reported prevalence
has been lower with MR staphylococci isolated from 15% dogs
with inflammatory skin disease (MR in 17% of S. aureus, 8%
of S. intermedius, 20% of S. schleiferi schleiferi and 20% of
S. schleiferi coagulans) and 5% of healthy dogs (MR in 0% of
S. aureus, 3% of S. intermedius, 50% of S. schleiferi coagulans)
[57]. In another report, 20% of 1,772 staphylococci were MR.
These comprised 15.6% of the S. intermedius, 46.6% of the
S. schleiferi and 23.5% of the S. aureus isolates [58]. A six month
surveillance study of 27 veterinary teaching hospitals identified
65 S. aureus isolates, of which 13% were MRSA [49].
Detection of MR staphylococcal infections
Any Staphylococcus with unusual (e.g. clavulanate potentiated
amoxicillin, cefalosporin or fluoroquinolone) antibacterial
resistance is suspicious, and resistance to oxacillin and
identification of the mecA gene is definitive. It is important to
use a reputable laboratory as some techniques overestimate
resistance and not using enrichment may miss 8-13% of MR
isolates (M. Rich, personal communication). Further genetic
typing to identify the strain is important in epidemiology. Swabs
should be taken from clinical lesions and implants as appropriate
in each case. Swabs should be taken from both the nose and
perineum, as single swabs miss 29-56% of dogs colonised with
CPS (J. Fazakerley, accepted for publication).
Nosocomial and community acquired colonisation
Very few studies have differentiated nosocomial colonisation
from animals colonised at admission. One study found that
the rate of community-associated colonisation in horses (27
per 1,000 admissions) was comparable to that of nosocomial
colonisation (23 per 1,000 admissions) [18], although horses
colonized at admission were significantly more likely to develop
clinical MRSA infections. A survey of three large UK equine
veterinary hospitals, in contrast, could not detect any MRSA on
admission or discharge [56].
Treatment
MR staphylococci are usually resistant to all penicillins,
cefalosporins and fluoroquinolones [60]. Most MRSA isolated
from animals also appear to have inducible clindamycin
resistance [25]. Tetracyclines, trimethoprim-sulfonamides,
fucidic acid and mupirocin are often effective. Recently, however,
S. pseudintermedius and S.intermedius isolates resistant to five
or more classes of antibiotics including -lactams, cefalosporins,
fluoroquinlones, macrolides, trimethoprim-sulfonamides, fucidic
acid and mupirocin have been reported [41, 43].
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EJCAP - Vol. 18 - Issue 3 December 2008
Antibiotics should be chosen following bacterial culture and
sensitivity tests. These are, however, less reliable in predicting
the response to topical antibiotics as direct application can
achieve local concentrations in excess of the minimum inhibitory
concentration break points established for systemic therapy. For
example, of 11 multi-drug resistant S. intermedius in one study,
seven responded to a combination of systemic and topical
antibiotics, and four to topical therapy only [43].
– Before surgery, it may be possible to decontaminate the
patient (see above).
– Covering wounds with impermeable dressings and preventing
licking (using collars etc.) may help reduce wound infection.
Routine measures to prevent the spread of MRSA (and
other infectious diseases)
1. Hand washing and disinfection of surfaces and equipment
between patients. Alcohol gel pouches on uniforms and
kennels are a visual cue and can be quickly used immediately
after handling an animal. Remember that alcohol will not be
effective in the presence of organic matter - this must be
removed by hand washing with a detergent.
2. Avoid materials at hand touch sites that cannot be cleaned use washable keyboards or keyboard covers etc. Alcohol wipes
are effective and easy to use on equipment and keyboards,
but soiled surfaces must be cleaned with a detergent.
3. Cover wounds or skin lesions with waterproof dressings.
4. High standards of aseptic technique: minimise theatre
staff; sterile gowns, gloves, hats and masks; sterilisation of
equipment; and single patient use.
5. High standards of cleaning: clean cages and bedding at
least once daily, and thoroughly clean and disinfect between
patients. Antimicrobial impregnated materials have not been
shown to prevent colonisation in clinical settings. The authors
recently found that no difference in bacterial contamination
of standard and tolnaftate/triclosan impregnated veterinary
bedding material.
6. Segregation of all waste and contaminated material.
7. Ensure that all staff understand and adhere to infection
control.
It is most important to identify the underlying cause. Further
treatment depends on the nature of the primary problem (e.g.
removing implants and or the use of gentamicin impregnated
beads, collagen sponges, activated silver dressings etc.) [13]. The
majority of animals with MR staphylococcal infections survive,
and in about half of the fatal cases, death is attributable to the
underlying disease. In the authors’ clinic the mortality rate with
MR staphylococcal infections is comparable to that in meticillin
sensitive CPS infections.
Discharged patients and decolonisation
Patients should be discharged as soon as they are clinically fit.
If the animal remains colonised potential risks and precautions
must be discussed with the owner. Decolonisation of humans
often involves repeated cycles of intranasal fucidic acid or
mupirocin and chlorhexidine washes. Treatment clears 91-99%
of patients but re-colonisation rates are up to 26%. Experience
suggests that dogs require longer courses of treatment of 2-3
weeks. At present, non-antibiotic decolonisation methods are
preferred. Hygiene, cleanliness and removal from the source are
most important and effective.
Preventing contamination with MR
staphylococci in veterinary clinics
Monitoring and surveillance
This is a very controversial and no figures for acceptable
microbiological levels in medical or veterinary premises have
been established. If surveillance is used, it is essential that it is
conducted following advice, is done with a reputable laboratory
and that the policy has clear aims.
Admission
Admit known or suspected cases directly into a consultation
room to avoid contamination and contagion in the waiting
room. To minimise contact and contamination procedures
should be scheduled for the end of the day, discharging wounds
covered with an impermeable dressing and trolleys used to
move patients. Surfaces and equipment should be cleaned and
disinfected before they are used again.
Fig. 4 MRSA wound infection.
Hospitalisation
– Isolate MRSA patients as far as possible from other patients.
– Use disposable gloves, gowns, hats and mask. Long hair
should be tied back and sleeves rolled up to the elbow.
Eye protection should be worn if there is a risk of splashing
or aerosols. Overshoes, however, may actually increase
contamination (presumably from having to balance and
handle shoes).
– Strict washing of the hands and forearms after handling the
patient.
– Equipment should be used with the affected patient only and
then disposed of or disinfected.
– Bathing every 2-3 days with an antibacterial wash may
reduce mucosal and cutaneous carriage, but may be not
be possible with all individuals or species, increases staff or
owner contact and could increase environmental aerosols.
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Meticillin-resistant Staphylococci in companion animals - T. Nuttall, N. Williams, R. Saunders, S. Dawson
Routine environmental screening could be used to monitor
cleanliness – of 82-91% hospital surfaces judged to visually clean
only 30-45% were microbiologically clean. MRSA contamination
rates have declined where cleaners have been trained to think
about microbiological cleanliness. One Canadian study isolated
MRSA from 10% of samples (including tables, floors, telephones,
keyboards, taps, kennels, stethoscopes and otoscopes) [61].
skin infection in three and nasal colonisation in 10 veterinary
staff treating a foal with Canadian epidemic MRSA-5 [70].
Transmission occurred despite barrier nursing precautions and
resulted in infection in healthy individuals without risk factors
for opportunistic infection.
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with consent, confidentiality, stigmatisation and further action.
Screening can also miss transiently contaminated staff, which
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Transmission of MRSA between
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