Faculty of Veterinary
Medicine, University
of Calgary, Canada
The incidence of Methicillin-Resistant
Staphylococcus aureus in milk samples of
Canadian dairy cows
Eva Bervoets
Supervisors:
Prof. Dr. H.W. Barkema &
Dr. T. van Werven
April – Juni 2008
Contents
1.
2.
3.
4.
5.
6.
7.
8.
9.
Abstract …………………………………………………………………………………….3
Abbreviations ……………………………………….………………………………….3
Introduction ……………………………………………………………………………..4
Materials and methods …………………………………………………………….9
Results …………………………………………………………………………………….11
Discussion ……………………………………………………………………………….11
Conclusions ……………………..……………………………………………………..15
Acknowledgements ………………………………………………………………..15
References ……………………………………………………………………………..16
2
Abstract
Mastitis is the most important disease in the dairy industry. In Canada, 10.3% of the clinical
mastitis cases is caused by Staphylococcus aureus. More than 10% of them were found to be
resistant to penicillin. Methicillin-resistant S. aureus (MRSA) contains the Mec-A gene, which
makes the bacteria resistant to all β-lactam antibiotics. More and more MRSA infections and
carriers are part of a community-associated strain, where originally most of them were
healthcare-associated. Animals, farms and farmers seem to be a source, and are therefore a
possible risk for public health. Worldwide, MRSA was found in pigs, horses, pets, cattle,
chickens and sheep. In Canada it has been reported in pigs, horses, dogs, cats, but not in
dairy cows. The first report of MRSA in dairy cattle was in Belgium, in 1975. More recently,
researchers reported MSRA in dairy cattle in Italy (2006) and in Korea and Hungary (2007). In
this study, milk samples were collected from about 80 farms all over Canada for two years.
Approximately 2000 randomly collected S. aureus samples will be analyzed for MRSA. 550
samples are now done, all of them were not methicillin-resistant.
Abbreviations
CBMRN – Canadian Bovine Mastitis Research Network
BTSCC – Bulk Tank Somatic Cell Count
IRCM – Incidence Rate of Clinical Mastitis
S. aureus – Staphylococcus aureus
MRSA – Methicillin-Resistant Staphylococcus aureus
HA-MRSA – Healthcare Associated Methicillin-Resistant Staphylococcus aureus
CA-MRSA – Community Associated Methicillin-Resistant Staphylococcus aureus
PCR – Polymerase Chain Reaction
3
Introduction
Mastitis
Mastitis is the most common and economically most significant disease that affects the dairy
industry in Canada and worldwide. Economically, milk production is ranked fourth in the
Canadian agricultural sector (Agriculture and Agri-Food Canada). Annual losses due to
mastitis in Canada are estimated at 300 million dollar (www.medvet.umontreal.ca/
reseau_mammite). These losses include the cost of reduced milk production, discarded milk
due to clinical episodes, antibiotic treatment, veterinary fees, therapy, early culling, and cow
replacement cost. Besides economic losses, it has significant effects on the animals and the
farmers working with them. Mastitis causes infringement of animal welfare, a lot of hassle
and therefore decreased pleasure in one’s work.
The incidence rate of clinical mastitis (IRCM) in Canada has, until recently, never been
studied nationwide, but in 2007, Olde Riekerink et al. (2007) found an IRCM of 23 cases per
100 cows per year. Keeping in mind such a high rate, there is sufficient room for
improvement in this field.
What is MRSA?
The Staphylococcus aureus bacterium is a major pathogen in bovine mastitis. In Canada, it
causes 10.3% of the clinical mastitis cases (Olde Riekerink et al., 2007). In Alberta, 4.3% of
mastitis cases were ascribed to this pathogen. Some of these S. aureus bacteria have
become resistant to several antibiotics, mainly penicillins. In the CBMRN study on
antimicrobial resistance, more than 10% of the S. aureus isolates were found to be resistant
to penicillin (table 1).
At first, most of the resistance to penicillin was a result of penicillinases, produced by the
bacteria. Therefore, methicillin which is a β-lactam antibiotic and insensitive to
penicillinases, was widely used to cure S. aureus cases in humans. However, soon after its
introduction in the 1950s the first methicillin-resistant isolates were found in the UK (Jevons,
1961; Barber, 1961). In MRSA, the resistance to methicillin is caused by an altered penicillinbinding protein, which makes it resistant to all β-lactam antimicrobials. The mec-A gene
encodes for this protein and is therefore used to identify MRSA, with a polymerase chain
reaction (PCR). In veterinary medicine methicillin has never been used.
4
Table 1: resistance of Staphylococcus aureus to most frequently used antibiotics (n= 207).
Antibiotics used for S. %
No % No % Resistant
aureus treatment:
interpretation MIC
%
Susceptible
Ampicillin
1.45
6.76
0.97
90.82
Ceftiofur
92.75
6.76
0.48
0
Cephalothin
0
6.76
0.48
92.75
Erythromycin
0
6.76
0.48
92.75
Oxacillin
0
6.76
0.48
92.75
Penicillin
0
6.76
10.63
0.48
82.13 *
Penicillin/Novobiocin
0
6.76
0
93.24
Pirlimycin
0
6.76
3.38
89.86
Sulfadimethoxine
0
6.76
0.48
92.75
Tetracyclin
0.48
6.76
1.45
91.30
* after correction.
Relevance of MRSA
MRSA can be found in many places. Initially, the bacterium was found prominently in places
like hospitals and health centers, because of antimicrobial drugs use and susceptible
patients. Traditionally, it was seen as a healthcare associated problem. Now, more and more
infections are community-associated (Layton et al., 1995). The strains of healthcareassociated MRSA differ from the community-associated strains. This implies they came from
different sources which need to be identified.
Public health
Probably the most important reason to look into MSRA in dairy cows is not the cows
themselves, but public health. A lot of people are colonized by S. aureus in the nose or on
5
the skin, without being affected by it. They are so-called carriers. A growing percentage of
these are MRSA (Lu et al., 2005). S. aureus can cause serious diseases in humans and animals
alike, so it is important to be able to treat them. MRSA can spread from human to human,
from animal to human (Loo et al., 2007) and most likely from human to animal too. Because
MRSA infections are so hard to treat and get rid of, researchers try to find the different
sources of and risk factors related to MRSA infections. The focus on animals and farmers as
possible sources of MRSA is increasing.
MRSA positive animal species
Until now, MRSA was found in different species such as swine, cattle (Loo et al., 2007; Wulf
and Voss, 2008), horses (Weese, 2004; Weese et al., 2005), dogs and cats (Weese et al.,
2006), chickens (Lee, 2003) and sheep (Goñi et al., 2004). In several countries studies have
been carried out on milk samples from dairy cows. The first case of MRSA in dairy cattle was
described in 1975 and concerned Belgian dairy herds, this was supposed to be due to human
contamination, since the mastitis was caused by a typical human strain. (Devriese and
Hommez, 1975). More recently Juhasz-Kaszanyitzky et al. (2007) reported a case in Hungary.
On this farm they found MRSA in 27 of the 375 cows and in one of the 12 farm workers. In
Korea, 2.5% (21) of more than 3000 mastitis samples contained MRSA, with 13 of them
carrying the Mec-A gene (Moon et al., 2007). In Italy, MRSA was found in samples of raw
bovine milk and also in cheese made from raw sheep milk (Normanno et al., 2006). In 2008 a
Belgian study was done on five MRSA positive farms. One of these was a Dutch farm. In this
study the farms were previously proven to be MRSA-positive and all cows were tested to
estimate the spread of MRSA strains within the herds. The prevalence varied from 0 to
14.3%. (Vicca et al., 2008).
Pigs were the first food animal species in which MRSA colonization was linked to
transmission to humans. In the Netherlands, 39% of a representative group of slaughter pigs
were MRSA carriers. More than 80% of the pig farms had carrier animals (de Neeling et al.,
2007). In Canada, Khanna et al. (2008) found MRSA colonization in almost 25% of the pigs
and in 20% of the pig farmers.
MRSA colonization also occurs in horses. In a Canadian study of approximately 1000 horses,
4.6% were found to be carrying MRSA, and 13 % of the people working with them, were
proved to be colonized (Weese et al., 2005). All isolates were of the same subtypes, an
uncommon strain in humans. So far, the presence of MRSA has been documented in horses
in Austria (Cunyet al., 2006), the UK (Baptiste et al., 2005), Japan (Anzai et al., 1996) and
Ireland (O’Mahony et al., 2005). Weese et al. (2006) found MRSA carriers in pets as well. All
strains had the same community-associated type. Both animal-to-human and human-toanimal transmission were suspected to have occurred in these cases.
6
Canadian Bovine Mastitis Research Network
The Canadian Bovine Mastitis Research Network (CBMRN) originates from the eastern part
of Canada has subsequently spread throughout the country. It is a network which involves
researchers, dairy producers, industry, government and several private partners. There are
nine universities involved in the research and so are Agriculture and Agri-food Canada, the
Public Health Agency of Canada and many others.
The mission of CBMRN is to mobilize national and international scientific and financial
resources to decrease the incidence of mastitis, reduce financial losses and maintain milk
quality trough concerted research, and effective and rapid transfer of results to end-users.
The network has two research themes: the mastitis control theme and the mastitis
monitoring theme. The CBMRN has formed a national cohort of about 80 dairy farms,
representing the Canadian provinces of Alberta, Ontario, Quebec, Nova Scotia, Prince
Edward Island and New Brunswick. The farms were selected to represent the provinces in
terms of housing types (free-stall or tie-stall), bulk tank somatic cell count (BTSCC), in order
to have a cross-section of low, high and average herds in the cohort, breed (at least 80% of
the cows are of the Holstein Friesian breed), a two-times-a-day milking schedule and
participation in Dairy Herd Improvement. All farms in this cohort contribute to the extended
database used in various research projects within the CBMRN. Data collection has started in
November 2006 and will continue until December 2008.
University of Calgary, Faculty of Veterinary Medicine
Until recently, there were four veterinary schools in Canada. Most of them are located in
eastern Canada (fig. 1). Because there is a shortage of veterinarians, especially in large
animals, and the only place in western Canada with a vet school was Saskatoon in
Saskatchewan, it was decided to start a fifth school in Alberta. In September 2008 the
Faculty of Veterinary Medicine of the University of Calgary will start an undergraduate
curriculum. This faculty has a couple of areas of special interest: production animal health,
ecosystem and public health, equine medicine, and investigative medicine. The first 30
students will start their education here in the fall of 2008. This faculty will have between 150
and 200 graduate students.
7
Figure 1: The five Canadian Faculties of Veterinary Medicine.
8
Materials and Methods
In this study, milk samples will be used that have been collected in the CBMRN cohort herds.
Clinical and subclinical mastitis milk samples containing S. aureus will be further analyzed for
the presence of MRSA.
Sample collection:
The CBMRN cohort was formed by selecting farms in six Canadian provinces, the number
and types of farms representing the province. For example, the amount of farms in the
cohort from Quebec was bigger than from the other provinces, because Quebec has the
most dairy farms of all the provinces. In total, about 80 farms contributed to the project. This
number varies, because in these two years some farmers quit the project after a while and
some new farmers joined in. Alberta, where I contributed to the network, has a cohort of
fourteen farms. The university staff and I visited these farms once a month to pick up milk
samples from the farmer, to provide them with sample materials and collect health record
data. Two times a year there was a so called intensive sampling period. The first period was
is spring and the second in the summer. During these periods we visited each farm every
three weeks to take samples during milking. All milk samples were taken aseptically
according to a standard protocol. The farmers were given demonstrations and instructions
on how to do this at the outset of the project.
In intensive sampling periods, the university staff members took milk samples of fifteen
random lactating cows during milking. These were four quarter samples and the same
fifteen cows were sampled three times with a three week interval. Teat condition (teat end)
was also scored during the first and last sampling. The samples got taken back to the
university in coolers and frozen there to -20°C as soon as possible. All the frozen samples
were shipped in coolers with icepacks to the laboratory of the faculty of veterinary medicine
in Saskatoon. This laboratory analysed all the milk samples and the results were sent back.
Apart from the intensive sampling by the staff, farmers were asked to collect milk samples of
all their cows with clinical mastitis, year round. The cows with clinical mastitis had to be
sampled before treatment and two to three weeks and four to five weeks after treatment.
Also, fifteen random cows per year had to be sampled throughout their lactation. The first
sample at three weeks before drying off, the second at drying off, the third at the first day of
the new lactation and the last one at three weeks of lactation.
All four quarters are sampled separately and vials have to be marked with farm number, cow
number, date and quarter. The farmers stored them in the provided boxes, in their freezer
and once a month a university staff member visited the farm to collect the boxes with
samples and provide the farmer with new boxes and vials. At the beginning of the project,
farmers were instructed by the staff on how to take samples.
Number of samples:
Using a confidence level of 95%, an expected prevalence of 1% of MRSA among S. aureus
isolates, and an accepted error of 0.5%, a minimum of 1522 samples will have to be
examined. Twenty randomly selected S. aureus isolates, approximately 10 clinical and 10
9
subclinical isolates, from each CBMRN cohort herd will be screened for evidence of
methicillin/oxacillin resistance. This will result in approximately 2000 S. aureus isolates being
screened from this cohort group. These isolates are already being collected and stored using
a standardized microbiological protocol as determined by CBMRN.
We will also passively monitor for MRSA mastitis isolates by requesting all provincial as well
as large private veterinary diagnostic labs to submit S. aureus milk isolates that are
suspicious of being methicillin/oxacillin resistant for a 2-year duration. These labs will submit
S. aureus isolates that are resistant on their sensitivity panel for oxacillin/cloxacillin.
Detection and confirmation of MRSA:
The S. aureus isolates will be cultured on blood agar plates to confirm that the cultures are
pure and that the colonies are of typical morphology. Individual colonies from these plates
will be tested with catalase and coagulase to confirm a sample as a coagulase-positive
staphylococcus species. All confirmed coagulase-positive staphylococci will also be screened
for methicillin/oxacillin resistance using the Denim Blue chromogenic MRSA culture media
(Bio-Rad). All MRSA positive isolates will be confirmed using a Pastaurex-Plus latex
agglutination test (Oxoid) to confirm that they are S. aureus and a mecA real-time PCR
(Paule, et al. 2005) assay will confirm the presence of the methicillin resistance gene. All
MRSA isolates will have their MIC’s determined for the common antimicrobials used in
bovine mastitis treatment. SensititreTM Mastitis MIC plates are used to do this.
10
Results
Of all the samples taken since the beginning of this project up until now, 550 that were
analyzed with S. aureus, have been checked for MRSA. So far none of them proved to be
positive. We are very happy to be able to report that as for now Canadian dairy cows seems
to be free of MRSA. Of course this is only yet one third of the required amount of samples
we have to run to get a 95% confidence level, but it is a good sign. We can only be sure if all
2000 S. aureus samples are analyzed. This will take another six months to a year and thus by
the summer of 2009 the final results from this study should be ready.
Discussion
No MRSA have been found in the S. aureus samples up until now. There are several possible
explanations for this. In the first place we will not find it, if it is just not there. Secondly the
number of samples might be too small. Now the number has been calculated with a 95%
confidence level and this could be too low for finding MRSA in dairy. Furthermore, the tests
used for detection of MRSA in S. aureus cultures might cause false-negative results.
Besides, the study has not been completed yet. Only 550 of the required 2000 samples have
been analyzed. This is the first study on MRSA in the Canadian dairy industry, so there are no
other data to compare with. As already indicated above, MRSA has, however, been reported
in isolates from milk samples in several other countries like Belgium (Devriese and Hommez,
1975), Korea (Moon et al., 2007), Italy (Normanno et al., 2006) and Hungary (JuhaszKaszanyitzky et al., 2007). Furthermore, in Canada, MRSA has been found in pigs (Khanna et
al., 2008), horses (Weese, 2004) and pets (Weese et al., 2006). Since MRSA is found in so
many other animal species and in dairy cows in other countries, it would be highly
remarkable, despite our findings so far, if MRSA would not to be found in dairy cows in
Canada. Of course we would be glad to be able to declare Canadian dairy MRSA-free, but we
would also have to consider why we did not find it. It seems very likely that MRSA is there,
so we have to consider what kind of risk factors and possible sources of infection might play
in role in MRSA infection and colonization.
From other studies we know that infections can spread between animals and between
human and animals, both ways. Risk factors for infection or colonization have not been
determined in official studies yet, but it seems just logical that having several species of
animals together on one farm must be a major one. We know that MRSA colonization occurs
in pigs, horses, dogs, cats, sheep and chickens, all of which are found on farms quite often.
One of the possible reasons for not finding MRSA in dairy cows could be that in Canada
farms are usually not mixed. Khanna et al. (2008) found 45% of MRSA colonization in pig
farms in Ontario, Canada. Hence pigs are so far considered to be the group of farm animals
11
with the most MRSA. In the Belgian study mentioned before (Vicca et al., 2008), the MRSA
strains found on five farms were all of the spa type t011, the type that is most prevalent in
pigs in Belgium. Two of these farms were also pig farms. A mixed pig-dairy farm operation
seems therefore at high risk to spread contamination trough the animals. Canada however,
has very few mixed dairy-pig farms.
Horses, however, are found more often on dairy farms. In a study in Ontario, Canada and
New York state, 4.7% of the horses were MRSA carriers. Their main site of colonization is in
the nasal passage (Weese, 2004). If nose to nose animal contact with cows happens,
transmission could take place. Anyhow, this has not been studied yet. Also, horses and dogs
used for farm work are usually not taken of the farm, unlike show horses and companion
dogs, so they are not at risk to pick up MRSA somewhere else and contaminate the animals
back on the farm.
Transmission of disease between animals of the same species can occur when new animals
are bought from another farm and introduced into the herd. This is quite common practice
for Canadian dairy producers: approximately half the farms (48 of 90) in our national cohort
is closed and half is open. This entails a great risk to introduce new diseases in a free herd.
But, for MRSA, this might not (yet) be a very important risk factor since there are no or very
few positive herds. Still, from an epidemiological point of view, it would be better to have
closed farms.
Lee (2003) suggested that transmission between cows and humans can occur, because he
found the same milk-associated MRSA types in milk samples and in humans, but he could
not prove the transmission between them, let alone prove whether it would be the animals
infecting the humans or the other way around. If humans are indeed able to transfer their
MRSA colonization to animals, then the people working on a farm are a risk factor as well,
especially if these people are in close contact with possible CA-MRSA or HA-MRSA sources,
like other colonized people or health centers.
Like in most contagious diseases, humans could act as a possible vector for MRSA. Especially
if they visit different farms regularly, like vets, inseminators, food companies etc. Humans
carry MRSA mainly on the skin and in the nose. When they work in close contact with the
cows, they could transfer them. And maybe even their equipment, clothes, vehicles and
other things brought to the farms could act as a vector.
In their Canadian mastitis study Olde Riekerink et al. (2007) found a relation between the
type of mastitis pathogens and housing types. Housing types are also area-related in Canada.
For example, in Quebec 80% of the dairy farms have tie-stalls and in Alberta 70% are freestalls. In Ontario and the Atlantic provinces the proportion of free-stalls and tie-stalls are
almost the same (Fig. 2). In tie-stall barns, cows have a higher chance to have mastitis
caused by contagious pathogens. In cows housed in tie-stalls, an IRCM of 4.04 cases of
clinical mastitis per 100 cow-years was found for S. aureus (Olde Riekerink et al., 2007). In
12
free-stalls S. aureus IRCM was only 1.62 per 110 cow-years. If there is more S. aureus, there
is also a bigger chance that some of them are methicillin-resistant. In regions like Quebec,
with mostly tie-stall barns, the chance of finding MRSA should thus be higher.
Figure 2: Distribution of barn types over the four regions in Canada. (Olde Riekerink et al., 2007)
Methicillin-resistance can also be found in coagulase-negative staphylococci (CNS), due to
the same Mec-A gene. It was suggested by Juhasz-Kaszanyitzy et al. (2007) that methicillinsusceptible S. aureus (MSSA) could pick up the Mec-A gene from the CNS and become
Methicillin resistant. In another CBMRN project CNS has been screened for the Mec-A gene,
but like in S. aureus, nothing has been found yet.
In pigs, the use of standard antimicrobials, for example in group medication, seems to be a
risk factor for finding MRSA-positive animals on pig farms (Van Duijkeren et al., 2008). In
dairy cows group medication is not as common as in pigs, but some standard antimicrobial
treatment is used on most dairy farms. Dry cow treatment is probably the most important
one. This standard use of antimicrobials could also be a risk factor for MRSA on dairy farms,
since standard treatments in all the animals select for antimicrobial resistant bacteria.
In the CBMRN cohort of 84 farms, almost 60% of the farms dry of 65% or more of their cows
with intra-mammary antibiotics. This 65% (or more) of the cows is probably an
underestimation, since these numbers result from collecting and counting empty drug
packages on the farms and such method is very likely to miss some of them. Olde Riekerink
et al. (2007) found that in Canada about 61% of the dairy farms dry of all their cows with an
antibiotic treatment. This probably means that all the farms counted with an over 65%
antibiotic dry cow treatment, actually do 100%.
13
Conclusions
MRSA has so far not been found on Canadian dairy farms. It was found, however, in pigs,
horses and pets in Canada. And it has been found in dairy in other countries like Belgium,
Italy, Hungary and Korea. The fact that all the samples were negative might mean there is
simply no MRSA in dairy cows in Canada, but also that the amount of samples run so far is
too small, or that specific risk factors should be taken into consideration when selecting
farms. There seem to be quite a few risk factors at play. In order to determine these risk
factors and their importance, more research needs to be done.
Acknowledgements
I would like to thank everybody that made this project possible for me. First of all, Herman
Barkema for giving me the opportunity to come here, my co-workers and co-students; Anke,
Andrea, Vineet, Niccole, James and Amanda, my parents and of course Ruud.
14
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