LEPTOSPIROSIS IN DAIRY CATTLE
“The effectiveness of long-term vaccination of dairy
cattle on New Zealand farms”
A research presented in partial fulfillment of the requirements for the
research internship of Veterinary Medicine of the University of
Utrecht, the Netherlands
Drs. M.B.R. Meenks, 2010
Leptospirosis in dairy cattle; the effectiveness of long-term vaccination of dairy cattle on
New Zealand farms.
A report written by: Drs. M.B.R. Meenks
Supervised by:
P.R. Wilson BVSc, PhD, MACVScProfessor,
Deer Health and Production IVABS, Massey University,
PB 11222Palmerston North, New Zealand
Dr. G.A. Hooijer, Dipl. ECBHM, Univ. hoofddocent, hoofd herkauwergezondheidszorg,
faculteit Diergeneeskunde, Universiteit Utrecht
Yalelaan 7, 3584 CL, Utrecht, The Netherlands
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Preface
During the study of Veterinary Medicine at the University of Utrecht, the Netherlands,
students have to undergo a research internship of three months. This research internship
can take place all over the world, and I have chosen to do this research in Palmerston North,
New Zealand.
The preparations of this research already started back home in Utrecht. After contacting
Peter Wilson about a leptospirosis study in dairy cattle, I decided to take this opportunity
and to do my research at Massey University. With help of Gerrit Hooijer, I wrote a research
proposal and, after I got approval of the faculty of Veterinary Medicine of the University of
Utrecht, I packed my bags and flew 24 hours, to the other side of the world.
In the beginning I really had to get used to the laid back attitude of the people in New
Zealand, but after a while, I found it refreshing!
My research went almost completely according to plan. There were some difficulties to get
over (ordered material which took four weeks to arrive, tests which did not work etc.), but I
learned a great lesson from this: things never go the way you planned, that is also research!
Beside my own research I got the opportunity to help among other studies. I have been to a
sheep slaughterhouse with Marieke to collect samples of the mesenterical lymfenodes of
sheep for her study and for Nicole’s research I have interviewed people about the
correlation between workload and horse feeding. Both Marieke and Nicole study Veterinary
Medicine in Utrecht and did their research internship in New Zealand as well. In the
laboratory I helped Fang with processing her samples. She is doing her PhD about
leptospirosis at Massey University. She learned me a lot about PCR and helped me with my
research by answering my questions and helping me find my way in the laboratory.
For a research of Johne’s disease in deer I have been to the South Island to collect blood,
faeces and lymfenodes of deer. This was a great opportunity to get in contact with a species
we do not have in the Netherlands as a farm animal. It was also a great opportunity to see
something of this beautiful country.
After three months of research internship I am on the verge of a few months to travel and
leave all this behind. I have had a great time at Massey University and I think it was a great
opportunity to see more of the (veterinary) world!
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Table of contents
Preface ........................................................................................................................................3
Table of contents ........................................................................................................................4
Abstract ......................................................................................................................................5
Introduction ................................................................................................................................5
Materials and methods ..............................................................................................................9
Selection of herds and animals ..............................................................................................9
Sample collection ............................................................................................................... 10
Questionnaire...................................................................................................................... 10
Dark-field microscopy ......................................................................................................... 10
Polymerase Chain Reaction................................................................................................. 10
Agarose gel electrophoresis ................................................................................................ 12
Results ..................................................................................................................................... 12
Leptospires in urine............................................................................................................. 12
Vaccination history.............................................................................................................. 14
Herd biosecurity .................................................................................................................. 14
Discussion ................................................................................................................................ 15
Conclusion ............................................................................................................................... 18
Acknowledgements ................................................................................................................. 18
Appendix .................................................................................................................................. 23
Appendix 1 .......................................................................................................................... 23
Appendix 2 .......................................................................................................................... 26
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Abstract
Recently, there has been an observation of high leptospirosis titres in vaccinated dairy
herds. This study was conducted to answer the question whether vaccination totally
eliminates urine shedding of Leptospira in dairy herds or not. The outcome of this study
could act as a model for predicting the possible outcome of vaccination in deer and beef
cattle.
This study was conducted in November 2010 at 17 farms all serviced by Massey University
Farm Services Veterinary Clinic. Ten out of the 17 farms were open farms and bought new
animals. Of each farm urine was collected of 10 cows. A questionnaire was filled in with
questions about the livestock, other animals on the farm, vaccination program against
leptospirosis, the leptospirosis history and movement of cattle. Three different vaccines
were included: Leptavoid®-2, Ultravac® 7-in-1 and Leptoshield®-3. The vaccination protocol
for calves varied from farm to farm. Vaccination of adult cows was between February and
August. The urine was tested by dark-field microscopy and Polymerase Chain Reaction (PCR).
Positive PCR samples where confirmed by agarose gel electrophoresis. One sample was PCR
and agarose gel electrophoresis positive, dark-field microscopy negative and one sample was
dark-field microscopy positive and PCR negative.
In this study, animals on two farms were shedding Leptospira in urine. This may implicate
that vaccination was not fully effective. It is not possible to identify the cause of infection in
this research. Therefore, further research is necessary on these farms. To make clear
conclusions about the effectiviness of vaccination against leptospirosis, further study is
needed.
It is recommanded to sample more farms and take urine and blood samples of more animals
of the positive tested farms, to find out which serovar causes the infections. For optimizing
the vaccination strategy of the different farms, a well conducted vaccination program with a
regular follow-up by the veterinarians is needed.
Introduction
Leptospirosis is an emerging zoonotic disease with a worldwide distribution (Marta et al.,
2009; Subharat et al., 2008). There is a peak incidence of the disease in wet seasons
(Subharat et al., 2008). The infection is caused by Leptospira, gram-negative obligate aerobic
spirochetes. Once leptospires are excreted in the urine, they can survive in moist
environments for months or even years (Marta et al., 2009). Infections spread through direct
contact with blood, urine or kidneys from infected animals or through indirect contact with
5
contaminated water, mud and soil (Subharat et al., 2008 and Marta et al., 2009). Leptospires
invade the host via mucous membranes (Wagenaar et al. 2000; Marta et al., 2009), in
particular the mucosa of the conjunctivae or nasal mucosa. It can also penetrate abraded or
inflamed skin (Moffat, 2007).
The first case of human leptospirosis in New Zealand was identified in 1951 (Baker and
Lopez, 2004). In human, leptospirosis is characterised by a wide spectrum of symptoms and
clinical signs of disease. The clinical signs vary from a mild influenza-like form to multiple
organ failure resulting in a high mortality rate. Ninety percent of the affected humans
develop a mild form of disease. Because of the wide variety of symptoms, it is difficult to
make an accurate clinical diagnosis (Marta et al., 2009). New Zealand has a relatively high
incidence rate of leptospirosis in the population overall, but particularly in meat workers
where at risk groups have been shown to have up to 9,5% seroprevalence (Benschop et al.,
2009). It is also common in farmers, probably beacause livestock animals play an important
role in the spread of the disease in New Zealand (Subharat et al., 2008).
Leptospira is classified into 18 genomspecies, which are divided into pathogenic, nonpathogenic and opportunistic pathogenic Leptospira. A genomspecies corresponds to a DNA
relatedness group (Brenner et al., 1999). Each genomospecies contains one or more serovars
(Subharat, 2010). In New Zealand there are 8 pathogenic serovars, divided into two
genomspecies (table 1). In this table the maintenance host and the accidental host of the
different serovars in New Zealand are listed. A maintenance population can be defined as a
species population which acts as a continuous reservoir of a specific serovar in a particular
ecosystem, and is characterised by natural transmission of infection between individuals of
the same generation and between consecutive generations (Hathaway, 1981).
A maintenance host for a serovar is a suitable host species to be parasitised by the serovar.
Maintenance host species have a high susceptibility to infection, which means that the
infective dose will be low. Maintenance hosts have a long-term renal infection and, within
the host species, there is a natural transmission of the serovar (Hathaway, 1981). Infection
of a maintenance host causes mild to no disease (Marta et al., 2009). A serovar can also
infect individuals of other species who are not the maintenance host. These accidental host
species are not necessary for the survival of the serovar. The susceptibility to infection is
often low, the renal phase is of limited duration and the transmission within the species is
inefficient (Hathaway, 1981). However, the clinical signs are more severe in accidental hosts
(Marta et al., 2009).
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Tabel 1. Classification of pathogenic Leptospira in New Zealand
Genomspecies
Serovar
Maintainance
Accidental host (s)
host
L. interrogans
Australis
Not available*
Human
Canicola
Not available*
Dog
Copenhageni
Norway rat
Human, cattle,
deer, dog, horse
Pomona
Pig
Human, cattle,
sheep, deer
L. borgpetersenii
Hardjobovis
Cattle, deer
Human, sheep
Tarrasovi
Pig
Human, dog, cattle.
Ballum
Black rat, mouse,
Human, cattle
Balcanica
Hedgehog,
Human, cattle
Possum
Adapted from (Subharat, 2010; Moffat, 2007)
* Isolated from human or dog, but not endemic.
The most common serovar found in cattle in New Zealand is Leptospira borgpetersenii
serovar Hardjobovis, but also Leptospira interrogans serovar Pomona can be found in cattle.
Infection with Leptospira interrogans serovar Copenhageni occurs less frequently and
infection with Leptospirosis borgpetersenii serovar Tarrasovi or serovar Ballum is rare
(Cordes et al., 1982).
Leptospira borgpetersenii serovar Hardjobovis causes a mild infection in cattle. The most
common clinical sign is acute febrile disease with haemoglobinurea (Fain et al., 1999).
Infection is often subclinical, but can cause reproduction failure and production losses (Bolin
and Alt, 2001). Chronic infections result in intermittent urine shedding ( O’Keefe, 2002).
Research suggests that in 1977 over 88 % of the herds of dairy cattle in New Zealand had
antibodies against Leptospira (Cordes et al., 1982). In beef cattle the prevalence of
leptospirosis was even higher: a serological survey in Hawkes Bay (New Zealand) suggested
that of 50 beef herds tested, all herds had positive titres to Leptospira borgpetersenii serovar
Hardjobovis and 64 % of the tested animals had significant titres against Leptospira
(Matthews and Collins-Emerson, 1999). Deer in New Zealand also have a high prevalence of
leptospirosis. It is suggested that 78 % of the tested herds are seropositive for Leptospira
borgpetersenii serovar Hardjobovis , 4% with Leptospira interrogans serovar Pomona, and
7
17% dual infection, with an individual animal seroprevalence of 60,8 % (Ayanegui-Alcérreca
et al., 2010).
To prevent sporadic infection in an accidental host population or to reduce the prevalence in
a maintenance host population, vaccination of animals can be used. In New Zealand, in 1979
a bivalent animal vaccine against Leptospira borgpetersenii serovar Hardjobovis and
Leptospira interrogans serovar Pomona was introduced in dairy cattle, resulting in a drop of
the incidence of human leptosprirosis within a couple of years (Figure 1, Marshall and
Manktelow, 2002; Baker and Lopez, 2004). Nowadays, long-term whole herd vaccination of
dairy cattle is the primary means of controlling leptospirosis in New Zealand (Bolin et al.,
1989a).
Fig. 1 Leptospirosis incidence by year, 1980 – 2003 (Baker and Lopez, 2004)
In April this year, there has been an observation of high titres in 8 dairy herds in North
Otago, prompting the question whether vaccination totally eliminates shedding or not, or
whether these titres were a result of immune response to challenge from an external source
or aberrantly high titres to vaccination.
To diagnose leptospirosis, there are several laboratory diagnostic methods, which can be
divided into those which detect bacteria or their antigens and which detect antibodies
produced in reaction to the bacteria (O’keefe, 2002). The methodologies available for
8
determination of Leptospira shedding in urine and used in this study are dark-field
microscopy, polymerase chain reaction (PCR) and agar gel electrophoresis. For dark-field
microscopy, a positive outcome depends on the observation of intact leptospires (Smith et
al., 1994). Dark-field microscopy will also require a high degree of operator skills. This will
cause a low sensitivity and specificity for dark-field microscopy (Subharat, 2010), which
means that false positive and false negative diagnoses easily can be made (Ahmand et al.,
2005). To be sure that positive samples would not be missed, other tests are used, like a
PCR.
The sensitivity and specifity of the PCR is high (Subharat, 2010; OIE, 2008). To confirm the
PCR outcome, a agarose gel electrophoresis can be used. By using agarose gel
electrophoresis on positive PCR samples, it is possible to see how long the DNA fragment is
which causes the PCR to be positive. In this way you know the melting temperature of the
DNA fragment and the length of the fragment. Comparing both outcomes with a positive
controle, this will give you a great certainty about the chance that a positive tested sample
is indeed positive. Working mechanisms of these techniques can be found in appendix 1.
This study was conducted to determine whether long-term vaccination eliminates urine
shedding of leptospirosis. The outcome of this study could act as a model for predicting the
possible outcome of vaccination in deer and beef cattle, which have been shown to have
high herd and individual animal seroprevalence (Matthews and Collins-Emerson, 1999;
Ayanegui-Alcérreca et al., 2010).
Materials and methods
Selection of herds and animals
Seventeen commercial seasonal dairy herds in the vicinity of Massey University, clients of
the Massey University Farm Services Veterinary Clinic, were selected upon advice of the
senior veterinarian, with due regard to the following criteria: the selected herds had
undergone long-term vaccination with a bivalent animal vaccine against Leptospira
borgpetersonii serovar Hardjobovis and Leptospira interrogans serovar Pomona, according
to clinic records. In every herd 10 lactating cows were opportunistically selected for urine
sampling as they urinated naturally.
9
Sample collection
Urine collection took place during morning or evening milking. Mid-flow urine samples were
taken of spontaneous discharged urine or urination was induced by ventral vulval
stimulation. Urine samples were collected in a 60 ml plastic collector which was individually
labeled with an individual cow identifier, and immediately stored on ice. The age of the cows
was retrieved from the data management program of the farm. The urine samples were
transported to the laboratory immediately after collection, where they were stored at a
temperature of 4 o C. The samples were analysed in the laboratory within 24 hours.
Questionnaire
Every farmer contributing to this research, was interviewed and a questionnaire was filled in.
In this questionnaire the following subjects were questioned: the livestock, other animals on
the farm, vaccination program against leptospirosis, the leptospirosis history and movement
of cattle. The questionnaire used can be found in appendix 2.
Dark-field microscopy
For this research a quantity of 1.5 ml urine was centrifuged at 6000 runs-per-minute (rpm)
for 1 minute. The pellet was re-suspended with 1.4 ml urine and centrifuged again at 6000
rpm for 1 minute. Twenty microliters of the pellet were pipetted on a microscope slide and
covered with a coverglass. This preparation was used to look at by dark-field microscopy
using an Olympus BH2 microscope. The preparation was scanned for the presence of
Leptospira.
Polymerase Chain Reaction
The PCR technique used in this research has been developed in the Hopkirk Research
Institute. The DNA Gyrase Subunit B gene (gyrB) is described as a successfully used target
gene. The gyrB gene used for PCR can amplify a 504 bp product from eight pathogenic
Leptospira species, including Leptospira interrogans and Leptospira borgpetersenii. The
melting point of the gyrB PCR product lies between 83,4 oC and 84,8 oC (Slack et al., 2006).
The real time PCR technique that was used for this study is described by Subharat, 2010. The
sensitivity and specificity of PCR for this target gene in deer urine was tested as respectively
96.7% and 100%. As fluorescence double stranded DNA specific intercalating dye, CYTO9
was used (Subharat, 2010).
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DNA extraction from urine samples was undertaken using QIAamp DNA Mini kit (QIAGEN®),
following the indications of manufacturer: twenty µl Proteinase K was pipetteted in a 1.5 ml
microcentrifuge tube, which was labeled with the sample ID. 200 µl urine sample was added
to the microcentrifuge tube and also 200 µl Buffer AL was added to the sample. This was
mixed by pulse-vortexing for 15 seconds and incubated at 56 oC for 10 minutes. After
incubating 200 µl ethanol (97 %) was added to the sample. The mixture was carefully applied
to the QIAamp spin column, which was placed in a 2 ml collection tube. After centrifuging at
6000 X g for 1 minute, the QIAamp spin column was placed in a clean 2 ml collection tube.
500 µl Buffer AW1 was added to the QIAamp spin column and the sample was centrifuged at
6000 X g for 1 minute. The QIAamp spin column was placed in a clean 2 ml collection tube
and 500 µl Buffer AW2 was added. This was centrifuged at full speed (14.000 rpm) for 3
minutes and after placing the QIAamp spin column in a new 2 ml collection tube, it was
centrifuged again at full speed for 1 minute.
The QIAamp spin column was placed in a clean 1,5 ml microcentrifuge tube and 200 µl
Buffer AE was added. This was incubated at room temperature for 5 minutes, and then
centrifuged at 6000 x g for 1 minute (QIAamp® DNA Mini and Blood Mini Handbook, 2010).
After DNA extraction samples were stored at – 20 oC.
For the PCR reaction a PCR tube was prepared for each sample and for the two positive and
one negative control sample. Each tube was labeled with the sample number.
For each tube there was a reaction medium prepared, containing: 1 x PCR buffer, 1,5 mM of
magnesium chloride (MgCl2), 200 µM of dNTPs, 5 pmol of Forward Primer 2For (5’TGAGCCAAGAAGAAACAAGCTACA-3’),
5
pmol
of
Reverse
Primer
504Rev
(5’-
MATGGTTCCRCTTTCCGAAGA-3’), 1 unit of Taq Polymerase(New England Biolabs®), 1,5 µM of
SYT09 (Invitrogen®) and double distilled water (ddH2O) to make up a final volume of 23 µl.
The reaction medium was added into each sample tube and in the positive and negative
control tubes. 2 µl of sample DNA extract was added to each sample tube and 2 µl of double
distilled water was added to the negative control tube. 2 positive controls were used with
field isolates of serovar Hardjobovis and serovar Pomona.
The Roto-Gene Q RT-PCR machine was programmed, started with the initial denaturation at
95 oC for 10 minutes, followed by 40 cycles of: denaturation at 95 oC for 10 seconds,
annealing at 63 oC for 20 seconds and extension at 72 oC for 10 seconds.
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Fluorescence readings took place at the end of each extension cycle in the F1 channel. The
PCR product was heated from 78oC to 95oC and the fluorescence change was monitored
every 0,2 oC to form a melting curve.
The melting temperature of the samples was compared with the melting temperature of the
positive controls, to assess the positive samples (Subharat, 2010).
Agarose gel electrophoresis
Confirmation of PCR positive samples was determined by performing an agarose gel
electrophoresis of the PCR product. 1,5 % agarose gel was used. It was stained with ethidium
bromide and visualised on UV light source (Subharat, 2010).
The protocol used for this electrophoresis was made by Subharat (personal communication).
A weight of 1,5 grams of agarose was added to 100 ml of Tris Borate EDTA Buffer (TBE) and
swirled gently. The agarose was melted by heating it in a microwave and cooled down to 50 55 oC. The comb was placed in the slots on the end of the gel tray and the agarose was
poured into the tray. It was led to cool and form a solid gel. After removing the comb, the gel
tray was placed into the electrophoresis chamber and the TBE Buffer solution was poured in
the chamber as well until it covers the gel.
1 µl of loading dye was placed onto a square of Parafilm and 5 µl of PCR product was
pipetted onto the spot of loading dye and the mixture was slowly pipetted up and down to
mix it well. This mixture was pipetted into a well of the gel. In one of the wells 5 µl of DNA
ladder (100bp) was pipetted.
The lit was connected to the electrophoresis chamber and the positive and negative leads
were connected. The power supply was adjusted to a voltage of 80 volts. After 60 minutes
the power supply was turned off and the gel was carefully lifted out of the tray. The gel was
placed into ethidium bromide staining tray for 10 – 20 minutes. After removing the gel from
the ethidium bromide staining tray it was placed on the UV light source and photographed.
Results
Leptospires in urine
There was evidence of leptospirosis in two different herds. One animal was PCR positive
(Farm ID 3), and there was a dark-field positive animal (Farm ID 11). Results are summarised
in table 2.
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Table 2. Results of dark-field microscopy and PCR on urine of dairy cattle, summarised per
farm.
Farm Sampling Number of Number of
Dark-field
PCR positive
ID
date
dairy cows cows sampled microscopy
positive
1
3 nov
270
10
0
0
2
9 nov
430
10
0
0
3
9 nov
200
9
0
1
4
10 nov
46
10
0
0
5
10 nov
53
10
0
0
6
11 nov
410
10
0
0
7
12 nov
276
10
0
0
8
12 nov
290
10
0
0
9
16 nov
490
10
0
0
10
16 nov
321
10
0
0
11
17 nov
135
10
1
0
12
17 nov
160
10
0
0
13
18 nov
766
10
0
0
14
18 nov
445
10
0
0
15
19 nov
244
10
0
0
16
25 nov
658
10
0
0
17
26 nov
350
10
0
0
The PCR positive sample was confirmed by agarose gel electrophoresis, as seen in figure 2.
Fig. 2 Agarose gel electrophoresis results
L = DNA ladder, Lane 1: positive control serovar Hardjobovis, lane 2: positive control serovar
Pomona, lane 3: positive PCR sample. ( lane 4: sample from other research)
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Vaccination history
The questionnaire data showed that there were 3 different vaccines used against
leptospirosis (table 3). Leptavoid®-2 (Intervet) is used in 4 farms and is used for
immunization against the serovars Hardjobovis and Pomona. Ultravac® 7-in-1(Pfizer) is used
in 9 farms and is a combination of a 5-way clostridial vaccine containing antigens to serovar
Hardjobovis and serovar Pomona. Leptoshield®-3 (Pfizer) is used in 5 farms and is a vaccine
which contains the serovars Hardjobovis, Pomona and Copenhageni.
The vaccination protocol for calves varied from farm to farm, starting from 4 weeks to one
year of age, with a second injection 3 weeks to one year later. The time of vaccination for
adult cows was between February and August, with 7/17 herds vaccinated in March.
Table 3. Summary of the use of vaccines on study farms.
Farm Vaccine used
Time of year
ID
vaccination of
adult cows
1
Leptavoid 2
March
2
Leptavoid 2
March
3
Leptavoid 2
March
4
7-in-1
March
5
7-in-1
March
6
7-in-1
February
7
Leptoshield 3
May
8
Leptoshield 3
May
9
7-in-1 calves, Leptavoid 2
April
cows
10
Unknown
March
11
7-in-1
August
12
7-in-1 calves, cows unknown
May
13
Leptoshield 3
May
14
7-in-1 calves, leptoshield 3
April
cows
15
7-in-1 calves, leptoshield 3
May
cows
16
Unknown
March
17
7-in-1
June
Age at calf Time-interval
vaccination between 1st – 2nd
vaccination
12 wk
6 wk
6m
1 year
6m
1 year
6 wk
4 wk
6 wk
4 wk
5m
6 wk
1 year
6 wk
1 year
6 wk
6 wk
5 wk
8 wk
1 year
4 wk
8 wk
12 wk
5 wk
6 wk
3 wk
6 months
6 wk
6 wk
4 wk
10 wk
10 wk
4 wk
6 wk
Herd biosecurity
The questionnaire shows that ten of the 17 farms were open and bought new animals. Two
of those 10 farms did not know anything about the leptospirosis status of the new animals.
Of the farms who claimed that new animals where vaccinated, three farms tested new
animals on leptospirosis and two farms only asked whether the animals were vaccinated
against leptospirosis. One farm knew animals where being vaccinated, because the manager
14
knows the vaccination protocol of the other farm and one farm asked for vaccination
certificates. There was one farm who thought new bought animals where being vaccinated,
but did not verify this. None of the open farms treated new animals with antibiotics. Only
one farm put new animals in quarantine for 48 hours.
Discussion
Farm ID 3 had a PCR positive, but dark-field microscopy negative sample. The PCR positive
was confirmed by agar gel electrophoresis, which suggests that dark-field microscopy gave a
false negative result.
Visualization of Leptospira by dark-field microscopy suffers from poor sensitivity and
specificity and requires a high degree of operator skills (O’Keefe, 2002; Subharat, 2010).
Visualization of leptospires requires approximately 104 or 105 leptospires/ml for one cel per
field necessary in urine (Ahmad et al., 2005; Subharat, 2010). If there are less leptospires in
the urine, this may give a false negative result. Also the
intermittently shedding of
Leptospira in urine (O’keefe, 2002) can be the reason for false negative results from darkfield microscopy.
A positive result by dark-field microscopy depends on the observation of intact leptospires
(Smith et al., 1994). Research shows that the optimum pH for survival of leptospires is
between 7.2 and 7.6 (Subharat, 2010). In this study, there was no transport medium used for
buffering. The urea in the urine might be converted into ammonia, which causes a raise in
pH. A raise in pH may result in a reduced survival of leptospires. A reduced, or no survival of
leptospires results in false negative dark-field microscopy results, yet the DNA form dead
organisms can be detected by PCR.
Farm ID 11 has a dark-field microscopy positive, but PCR negative sample. The dark-field
microscopy positive was confirmed by two other researchers who had a lot of experience
with dark-field microscopy and leptospirosis. This indicates that the PCR may have given a
false negative result. Indeed, the positive control which was used was positive after PCR, so
false negative PCR results may be due to the absence of DNA after DNA extraction and may
not be due to errors in the PCR process itself.
Errors in the DNA extraction process may result in the absence of leptospiral DNA in positive
samples. All the other samples processed on the same day were all negative as well, but on
other days, there was a positive sample. This suggest that errors were not due to failing of
15
the method for DNA extraction. The errors in the DNA extraction of Farm ID 11 might be due
to human error.
There are different vaccines used on the tested farms. The Leptavoid®-2 vaccine is claimed
by the manufacuturer to give immunisation for at least 12 months against infection with
Leptospirosis borgpetersenii serovar Hardjo and Leptospirosis interrogans serovar Pomona.
Inactivated cultures were used to produce the vaccine. Calves can be vaccinated from three
months of age, with a second dose 4 to 6 weeks later. An annual booster has to be given
before the period of great risk, which is autumn till the end of spring. This will also ensure
maternal antibody protection for newborn calves (Intervet/Schering-Plough, 2010).
Leptoshield®-3 is claimed by the manufacturer to provide immunization against Leptospirosis
borgpetersenii serovar Hardjo and Leptospirosis interrogans serovar Pomona and serovar
Copenhageni. Inactivated cultures are used in this vaccine. Vaccination can start in calves at
4 weeks of age and it is claimed to be efficacious in the present of maternal antibodies. A
second dose must be given 4 to 6 weeks later and an annual booster must be given before
the season of greatest risk (Pfizer, 2008a).
Ultravac® 7-in-1 is claimed by the manufacturer to provide immunization against
Leptospirosis borgpetersenii serovar Hardjobovis and Leptospirosis interrogans
serovar
Pomona. Inactivated cultures are used. It is claimed to work in the presence of maternal
antibodies and vaccination can start in calves at 4 weeks of age. A second dose is necessary 4
to 6 weeks later. An annual booster must be given (Pfizer, 2008b).
Of Ultravac® 7-in-1 and Leptoshield®-3 it is claimed that vaccination still works in the
presence of maternal antibodies.
This is supported by Palit et al. (1991), but calves
vaccinated at 4 weeks of age showed the smallest rise in post-vaccination antibody titres,
compared with calves vaccinated at an older age (Palit et al., 1991).
All vaccines contain inactivated leptospirosis cultures. Inactivated vaccines will give a lower
immunerespons compared with attenuated vaccines. This is because inactivated vaccines
are not able to multiply in the animal and there is only local lymfenode activation (IAP
Committee on Immunisation, 2010). To get a higher immune response, a booster vaccination
is necessary (Wang et al., 2007).
The vaccination strategy for immunisation of calves is summarised in table 3. The three
vaccines used on farms in this study all need a booster vaccination after 4 to 6 weeks (Pfizer,
2008a; Pfizer 2008b; Intervet/Schering-Plough, 2010). As seen in table 3, farm ID 2, 3 and 14
16
have a longer vaccination interval between the first and second vaccination. This might not
give the desired protection against leptospirosis. It is suggested that single time vaccination
only results in immunity for a short period of time (Bolin et al., 1989b).
Both farms with positive animals vaccinate the herd with a bivalent leptospirosis vaccine
against the serovars Hardjobovis and Pomona. Cattle can be a accidental host for other
serovars, like the serovars Copenhageni, Tarassovi or Ballum as stated in table 1. Infection of
cattle with serovar Copenhageni is less common, infection with Tarassovi or Ballum is rare.
(Cordes, 1982). Both the vaccine Leptavoid®-2 and Ultravac® 7-in-1 do not provide
protection against these serovars. Both farms ID 3 and ID 11 are being vaccinated with one
of these vaccines. Because the PCR and dark-field microscopy can not determine between
diverent serovars (Smith et al., 1994; Slack et al., 2006; OIE, 2008), there will be also a
positive test result when there is shedding of serovar Copenhageni or serovars Tarassovi or
Ballum. To determine which serovar caused infection other tests has to be used, like culture
or serology. As a serology test, a Microscopic Aglutination Test (MAT) can be used. This test
has a high sensitivity and specificity and can identify the different serovars (Subharat, 2010).
The MAT is the standard serological test for leptospirosis (Ahmad et al., 2005; OIE, 2008).
All vaccines suggest vaccination before the period of greatest risk for leptospirosis, which is
from the autumn till the end of spring. So it is recommended to vaccinate around March.
Farm ID 11 vaccinates the animals in August. This is in the middle of the period of great risk.
It is possible that the protection derived from vaccination is lower just before vaccination, so
the animals of farm ID 11 might not be enough protected against leptospirosis in the
beginning of the period of greatest risk.
The herd biosecurity of some of the farms, including farm ID 3, is not optimal, which is
showed by the questionnaire. Buying new animals is a risk for introducing leptospirosis in
the herd, as there is no certainty about the leptospirosis status of new bought animals.
Farm ID 3, which tested positive by PCR, is an open farm, but claimed to test new bought
animals. Farm ID 11, tested positive by dark-field microscopy, is a closed farm. After
informing the veterinarians about the vaccination strategy on different farms, they said that
only one farm tests new bought animals against leptospirosis. From the questionnaire we
can see that three farms claimed to test new animals. It is possible that Farm ID 3 did not
test it’s animals. This means that there is no certainty about the leptospirosis status of new
17
bought animals on this farm. Most farms only bought new bulls. Not only shedding of
leptospires in urine, but also infected semen can be a source of disease (Faine et al. 1999).
The positive results are only single samples, and even while false positives are not common,
the results may be false. It is recommended to take more samples and to take urine and
blood samples as well.
Conclusion
Based on this research there is evidence that animals on 2 farms are shedding Leptospira in
urine. This may implicate that vaccination is not fully effective. It is not possible to identify
the cause of infection in this research, but it is showed that the vaccination program of the
two positive tested farms is not optimal, which might result in a leptospirosis infection. Risk
factors on these farms are a too long time interval between first and second vaccination in
calves (farm ID 3), buying animals with unknown leptospirosis status (farm ID 3), vaccination
in the wrong time of year (farm ID 11) or infection with a serovar which is not included in the
used vaccination (both farm ID 3 and farm ID 11).
To make further conclusions about the effectiviness of vaccination against leptospirosis,
further research is necessary. It is recommended to sample more farms and to go back to
the positive tested farms and sample more animals. For sampling on the positive farms, it is
recommended to take blood and urine samples from more animals. Furthermore, urine
collection is necessary for prevalence studies if shedding Leptospira. Blood samples can be
used for serology by using MAT, to find out which serovar causes infection.
The questionnaire showed that the vaccination strategy is not always according to the
guidelines of the manufacturer. For optimising the vaccination strategy, a follow-up for
these farms by the veterinarians is recommended.
Acknowledgements
First of all, I would like to thank my supervisors: professor Peter Wilson for having me here
and for the help and guidance he gave me during my research, and Gerrit Hooijer, for his
support and enthousiasm about my research and his help and advice with writing my report.
I would like to thank the other supervisors from the leptospirosis group at Massey
University: Dr. Julie Collins-Emerson for her help in the laboratory and Cord Heuer for his
18
help with the sample size calculation. I would like to thank Jenny Watson for helping me
selecting the herds for sampling and for helping me getting in contact with the farmers.
I like to thank Nevill Haack for helping me to get the supplies I needed for sampling.
My thanks also go to Marieke Verra, who helped me with sampling. Without her help I
wouldn’t have as many samples I have now. I would like to thank Fang Fang for her patience,
explanation and help in the laboratory. Without her help I wouldn’t know how to process my
samples. My thanks goes to Emilie Vallee who helped me with my questionnaire, report and
processing some of my samples.
I would like to thank all the people participating in my research, even for just a small part of
my research, like Leslie, Marrit and all the people in the Hopkirk laboratory who helped me,
and answered my questions.
A special thanks goes to the farmers who participated in this research. Without them there
where no samples to process and there wouldn’t be a research at all.
19
References
1. Ahmad SN, Shah S, Ahmad FMH. Laboratory diagnosis of Leptospirosis. J Postgrad Med,
51, 3, 195-200, 2005
2. Ayanegui-Alcérreca MA, Wilson PR, Mackintosh CG, Collins-Emerson JM, Heuer C,
Midwinter AC, Castillo-Alcala F. Regional seroprevalence of Leptospirosis in deer farms
in New Zealand. New Zealand Veterinary Journal 58(4), 184 – 189, 2010
3. Baker M, Lopez, L. The changing epidemiology of human Leptospirosis in New Zealand.
Proc. 34th annual seminar, society of sheep & beef cattle veterinarians NZVA, 27-34, 2004
4. Benschop J, Heuer C, Jaros P, Collins-Emerson J, Midwinter A, Wilson P. Seroprevalence of Leptospirosis in workers at a New Zealand slaughterhouse. The New
Zealand Medical Journal 122, 1307, 2009
5. Bolin CA, Alt DP. Use of a monovalent leptospiral vaccine to prevent renal colonization
and urinary shedding in cattle exposed to leptospira borgpetersenii serovar hardjo.
American Journal of Veterinary Research,62, 995-1000, 2001
6. Bolin CA, Thiermann B, Handsaker AL, et al. Effect of vaccination with a pentavalent
leptospiral vaccine on leptospira interrogans serovar hardjo type hardjo-bovis infection
of pregant cattle. American Journal of Veterinary Research, 50, 161-165, 1989a
7. Bolin CA, Thiermann B, Handsaker AL, et al. Effect of vaccination with a pentavalent
leptospiral vaccine on leptospira interrogans serovar hardjo type hardjo-bovis infection
of cattle. American Journal of Veterinary Research, 50, 2004-2008, 1989b
8. Brenner DJ, Kaufmann AF, Sulzer KR, Steigerwalt AG, Rogers FC, Weyant RS. Further
determination of DNA relatedness between serogroups and serovars in the family
Leptospiraceae with a proposal for Leptospira alexandri sp. nov. and for new Leptospira
genomspecies. International Journal of Bacteriology, 49,839-858, 1999
9. Cordes DO, Carter ME, Townsend KG, Lewis SF, Holland JTS. Leptospirosis: I. clinical
infestigation of the infection in dairy cattle in the Waikato district of New Zealand. New
Zealand Veterinary Journal, 30, 122-124, 1982
10. Eys GJJM, Gravenkamp C, Gerritsen MJ, Quint W, Cornelissen MTE, Ter Scheggert J,
Terpstra WJ. Detection of leptospires in urine by polymerase chain reaction. Journal of
clinical Microbiology, 10, 2258-2262, 1989
11. Faine S, Adler B, Bolin C, Perolat P. Leptospira and Leptospirosis. MediSci, Melbourne,
Australia, 2nd edition. 1999
20
12. Intervet/Schering-Plough
Animal
Health,
Leptavoid
2,
October
2010,
http://www.intervet.co.nz/binaries/Leptavoid_2_Label_tcm_90-190279.pdf, consulted:
16 december 2010
13. Hathaway SC. Leptospirosis in New Zealand: an ecological view. New Zealand
Veterinarian Journal, 29, 109-112, 1981
14. IAP Committee on Immunisation, Guidebook of Immunization, chap. 2: Basic
vaccinology, 2010, http://iapcoi.com/pdf/chapter02basicvaccinology.pdf, consulted: 20
December 2010
15. Matthews M, Collins-Emerson J. Leptospirosis in beef herds. Vetscript, april, 4, 1999
16. Marshall RB, Manktelow BW. Fifty years of Leptospirosis research in New Zealand: a
perspective. New Zealand Veterinary Journal, 50, 61-63, 2002
17. Marta A. Zoonosis update, Leptospirosis. Journal of the American Veterinary Medical
Association, 4, 472-478, 2009
18. Moffat JR. Leptospirosis immunity.
Proceedings of the Society of Dairy Cattle
Veterinarians of the NZVA, 2007
19. OIE, Terrestrial Manual. 6th Edition, chapter 2.1.9., 2008 OIE, Paris, France
20. O’Keefe JS. A brief review on the laboratory diagnosis of Leptospirosis. New Zealand
Veterinary Journal 50, 9-13, 2002
21. Palit A, Middleton H, Sheers J, Basilone C. The influence of maternal antibody and age
of calves on effective vaccination against Leptospira interrogans serovar hardjo,
Australian Veterinary Journal, 68, 299-303, 1991
22. Pfizer
Animal
Health
New
Zealand,
Leptoshield®
3,
2008a,
http://www.pfizeranimalhealth.co.nz/sites/pfizeranimalhealth/Pages/Leptoshield3.aspx
?Species=Dairy, consulted: 16 December 2010
23. Pfizer
Animal
Health
New
Zealand,
Ultravac®
7-in-1,
2008b,
http://www.pfizeranimalhealth.co.nz/sites/pfizeranimalhealth/Pages/Ultravac7in1.aspx
?Species=Dairy, consulted: 16 december 2010
24. QIAamp® DNA Mini and Blood Mini Handbook. Third edition, QIAGEN®, april 2010
25. Smith CR, Ketterer PJ, McGowan MR, Corney BG. A review of laboratory techniques and
their use in the diagnosis of Leptospira interrogans serovar hardjo infection in cattle.
Australian Veterinary Journal, 71, 9, 290-294, 1994
26. Slack AT, Symonds ML, Dohnt, MF SMythe LD. Identification of pathogenic leptospira
species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B
encoding gene. BMC Microbiology, 6, 95, 2006
21
27. Subharat S, Wilson PR, Heuer C, Collins-Emerson JM. Leptospirosis: A Massey University
research update. In: A Deer Course for Veterinarians. Proc. Deer Branch NZVA 25, 115120, 2008
28. Subharat S. Epidemiology, diagnosis and vaccination control of Leptospirosis in farmed
deer in New Zealand, PhD in Veterinary Clinical Science thesis, Massey University,
Palmerston North, New Zealand, 271pp. 2010
29. Wagenaar J, Zuerner RL, Alt D, Bolin CA. Comparison of polymerase chain reaction
asseys with bacteriologic culture, immunofluorescence, and nucleic acid hybridization
for detection of Leptospira borgpetersenii serovar hardjo in urine of cattle. AJVR, 61, 3,
316 – 320, 2000
30. Wang Z, Jin L, Wegrzyn A. Leptospirosis vaccines. Microbial cell factories 6, 39, 2010
22
Appendix
Appendix 1
Dark-field microscopy
Particles which are too thin to be seen by transmission microscopy can be visualised by
observing the reflections form their surface using dark-field microscopy. These reflections
are magnified microscopically. To avoid distortion and loss of light, it is necessary to use a
thin homogenous preparation of organisms. A dense preparation is unsuitable because it
destroys contrast. Slides and coverslips must be optically clean (Fain et al., 1999).
Visualisation of leptospira by dark-field microscopy suffers from poor sensitivity and
specificity. It will also require a high degree of operator skills (Subharat, 2010).
To be visible under dark-field microscopy, there are approximately 104 or 105 leptospires/ml
for one cel per field necessary in urine (Ahmad et al., 2005; Subharat, 2010). A positive result
by dark-field microscopy depends on the observation of intact leptospires (Smith et al.,
1994).
Polymerase Chain Reaction
Polymerase Chain reaction is a reaction which is used to make large numbers of copies of a
specific DNA fragment. The DNA sample is unwound by heating the sample. This will break
the hydrogen bonds between the base pairs. Each DNA strand is used as a template. For
synthesis a primer must be used. This is a short DNA sequence that is complementary to the
template sequence. For annealing of the primers, the temperature must be lowered. The
annealing temperature varies depending on the primers. This temperature is important to
ensure a high specificity in the reaction (McPherson and Møller, 2006).
DNA polymerase adds nucleotides to the free 3’-OH end of the primers. For DNA polymerase
activity the temperature is adjusted to an optimal, normally 72 oC. The next step in the PCR
process is another heating step to denaturise the new formed DNA strand. Each DNA strand
can act as a template for the next round of DNA synthesis. In every reaction cycle the
amount of DNA will be doubled (McPherson and Møller, 2006).
Magnesium is one of the most critical components in the PCR. The magnesium concentration
can affect the efficiency and specificity of the reaction. Taq DNA polymerase activity
depends on the presence of magnesium. The dNTP concentration affects the concentration
of free Mg2+. It is important that the four dNTPs are present in equimolar concentrations for
a successful PCR (McPherson and Møller, 2006).
23
To design a primer you have to know the DNA sequence of the template. A primer should be
16 – 30 nucleotides long and contain approximately a equal number of each nucleotide. At
least the first three nucleotides of the 3’-end of the primer should be perfectly matched to
the template (McPherson and Møller, 2006).
The amplified sequence of DNA in the PCR process is called the amplicon (Dorak, 2006).
Real-time Polymerase Chain Reaction is the continuous collection of fluorescent signal from
a polymerase chain reaction (Dorak, 2006).
A real-time PCR contains a fluorescent reporter in the form of a fluorescent DNA-binding dye
or as a fluorescent oligonucleotide primer. There is only a fluorescence signal when
associated with the product amplicon. The increase in recorded fluorescence signal during
amplification is in direct proportion to the amount of amplification product in the reaction
(McPherson and Møller, 2006).
Every piece of double stranded DNA has a melting point. This is the temperature at which
50% of the DNA is single stranded. When DNA binding dyes are used, the melting point (Tm)
is detected when there is a sudden decrease in fluorescence when heated (Dorak, 2006).
After amplification of the product, a meting curve analysis can be performed. If there is
amplification of only the product, a single and well-defined peak should be observed at the
melting temperature of this amplicon. This demonstrates that the fluorescence is a direct
measure of specific product accumulation. When series of peaks are observed, there is
nonspecific product formation (McPherson and Møller, 2006).
Agar gel electrophoresis
Nucleic acids are negatively charged molecules, which, when put in an electric field, migrate
under the influence of the electric field towards the positive electrode. Different sizes of
nucleic acid move at different rates. This provides a basis for their separation.
For the migration of nucleic acids, the presence of charged ions is essential. For this, a gel is
used. The gel entraps a buffer in a solid matrix, what results in series of pores through which
the molecules must pass. There are two gels used for analysis of molecules: polyacrylamide
and agarose. Agarose is melted in a electrophoresis buffer and forms a gel after cooling. The
concentration of agarose used determines the size of the pores (Martin, 1996).
Polyacrylamide is mostly used to separate proteins, Agarose is mainly used to separate
larger molecules, such as nucleic acids (Hames, 1998). Agarose gels have larger pores than
polyacrylamide gels.
24
There are different techniques to detect nucleic acids. One way for detecting is binding of
fluorescent dyes. Fluorescence dyes have a high fluorescence when bound to nucleic acids.
Ethidium bromide is the most used dye. After binding, it absorbs UV light and makes the
nucleic acids visible (Martin, 1996).
References:
1. Dorak MT. (2006) Real-time PCR. 1st edition. Taylor & Francis Group, pp. 1 - 8
2. Faine S, Adler B, Bolin C, Perolat P. Leptospira and Leptospirosis. MediSci,
Melbourne, Australia, 2nd edition. 1999
3. McPherson Mj, Møller SG. 2006 PCR 2nd edition. Taylor & Francis Group, pp. 1 - 19
4. Hames BD. (1998) Gel electrophoresis of proteins. 3th edition. Oxford University
Press, New York ,p. 1
5. Martin R. (1996) Gel electrophoresis: Nucleic Acids. 1st edition. BIOS Scientific
Publishers Limited, Oxford, United Kingdom,pp. 1 – 12, 49 - 54
6. Smith CR, Ketterer PJ, McGowan MR, Corney BG. A review of laboratory techniques
and their use in the diagnosis of Leptospira interrogans serovar hardjo infection in
cattle. Australian Veterinary Journal, 71, 9, 290-294, 1994
7. Subharat S. Epidemiology, diagnosis and vaccination control of Leptospirosis in
farmed deer in New Zealand, PhD in Veterinary Clinical Science thesis, Massey
University, Palmerston North, New Zealand, 271pp. 2010
25
Appendix 2
Leptospirosis in cattle
‘The effectiviness of long-term vaccination of dairy cattle on New Zealand farms.’
Leptospirosis is an important disease of cattle and other livestock, which can also cause
illness in humans. New Zealand has a relative high incidence of leptospirosis, especially in
meat workers and farmers. Animals play an important role in the spread of the disease.
For more than 20 years, vaccination of dairy cattle has been widespread. Within a couple of
years after the start of vaccination the human and animal incidence of leptospirosis
dropped.
Since then there’s never been research which evaluates just how effective vaccination has
been. Does long-term vaccination prevent shedding of the bacteria in urine? This is
something we would like to find out.
1. General information
1.1.
Property name:_________________________________________________
1.2.
Contact person:_________________________________________________
1.3.
Postal address:_________________________________________________
1.4.
Farm address (if different):________________________________________
1.5.
District:_______________________________________________________
1.6.
Phone (home):_________________________________________________
1.7.
Phone (business, if different):______________________________________
1.8.
Fax:__________________________________________________________
1.9.
Mobile:________________________________________________________
1.10. Email:_________________________________________________________
1.11. Farm area:________________________ ha
1.12. Date of visit:____________________________________________________
26
2. Livestock
2.1. Breed:_________________________________________________________
Please fill in the next table:
Age group
Numbers
2.2. Calves (0-12 months) on this farm
2.3. Calves (0-12 months) on other farms
(away for rearing)
2.4. Heifers (12 months – 1st calf) on this
farm
2.5. Heifers (12 months – 1st calf) on
other farms (away for rearing after
weening)
2.6. Cows (24+ months)
2.7. Bulls (24+ months)
2.8. Do you send calves away for rearing? (circle the appropriate answer)
Yes
/
No
2.9. Do you send heifers away for rearing after weening? (circle the appropriate
answer)
Yes
2.10.
/
No
Please estimate the numbers of workers in regular contact with livestock on
the farm, including farmer, family and employed
worker:____________________
3. Other animals
3.1. Do you have other animals on your farm? (circle the appropriate answer)
Yes /
no
3.2. If yes, state (wich animals, how many):_______________________________
______________________________________________________________
27
4. Vaccination
4.1. Dairy cattle
4.1.1. Are the animals vaccinated for leptospirosis? (circle the appropriate
answer)
Yes
/
No
If yes, please answer the following questions:
4.1.2. What year did the whole-herd vaccination strategy for leptospirosis
started on this farm?
□ 0 – 5 years ago
□ 5 – 10 years ago
□ 10 – 15 years ago
□ 15 – 20 years ago
□ more than 20 years ago
(□ Manager isn’t sure, he didn’t worked here when it started.)
4.1.3. What vaccine is used for vaccination for leptospirosis?
______________________________________________________
4.1.4. What time of year (month) are they vaccinated against leptospirosis?
_________________________________________________________
4.1.5. Who vaccinates the animals? (please tick the appropriate box/boxes)
□ Farmer
□ Farm technician
□ Vet
□ Vet technician
□ Other (please specify)
4.2. Calves
4.2.1. At what age do calves get their first vaccination for leptospirosis?
________________________________________________________
4.2.2. Do the calves get a second injection in the first year of vaccination?
(circle the appropriate answer)
Yes
/
No
28
4.2.3. If yes, please give time interval between the two injections:
_______________________________________________________
4.2.4. Are calves vaccinated at the same time with the rest of the herd to
synchronize them? (circle the appropriate answer)
Yes
/
No
4.2.5. Do bull calves also get vaccinated before they leave the farm to go to
a beeffarm? (circle the appropriate answer)
Yes
/
No
4.3. Heifer and cows
4.3.1.
How often a year are they vaccinated against leptospirosis?
_________________________________________________________
4.3.2. When was the last vaccination?
_________________________________________________________
4.3.3. If you send heifers away for rearing, are the heifers vaccinated before
returning at the farm? (circle the appropriate answer)
Yes
/
No
4.4. Bulls
4.4.1. How often a year are they vaccinated against leptospirosis?
________________________________________________________
4.4.2. What time of year are they vaccinated?
________________________________________________________
4.5. Other animals
4.5.1. Are the other animals vaccinated against leptospirosis? (circle the
appropriate answer)
Yes
/
No
4.5.2. If yes, please specify (which animals, what vaccine, how often):
________________________________________________________
________________________________________________________
________________________________________________________
29
5.
Leptospirosis history
5.1. Are there any cases of leptospirosis (any species) known in the past 5 years?
(circle the appropriate answer)
Yes
/
No
5.2. If yes, please fill in the next table:
Animal
Year
Number
Briefly describe the
of cases
cases
Confirmed by:
□ Vet
□ Lab
□ Not confirmed
□ Vet
□ Lab
□ Not confirmed
Animal
Year
Number
Briefly describe the
of cases
cases
Confirmed by:
□ Vet
□ Lab
□ Not confirmed
□ Vet
□ Lab
□ Not confirmed
6.
Movement of cattle
6.1. Type of farm: (circle the appropriate answer)
Open /
Closed
6.2. If the type of farm is open, please answer the following questions:
6.2.1. What’s the frequency of introduction of new animals?
_____________________________________ Animals/year
6.2.2. What’s the origin of the new animals?
6.2.2.1. other farm
Yes
/
No
30
6.2.2.2. Livestock market
Yes
/
No
6.2.2.3. Cattle trader
Yes
/
No
6.2.2.4. Other origin
Yes
/
No
6.2.2.5. If yes, please
specify:__________________________________________________
________________________________________________________
6.2.3. When was the last
introduction?_____________________________________________
________________________________________________________
6.2.4. What is the leptospirosis status of the new introduced animals?
□ Vaccinated against leptospirosis
□ Not vaccinated against Leptospirosis
□ Unknown status
6.2.5. How do you verify?
________________________________________________________
6.2.6. Are new animals treated with antibiotics? (circle the appropriate
answer)
Yes
/
No
6.2.7. Are new animals held in quarantine? (circle the appropriate answer)
Yes
/
No
6.2.8. If yes: for how long?
________________________________________________________
31