UTRECHT UNIVERSITY
Leptospirosis in wildlife
and cats- Pilot study
Research internship
Supervisor New Zealand:
Supervisor the Netherlands:
Student:
Peter Wilson
Ruurd Jorritsma
Mark van de Pol
Abstract
The studies presented in this paper are both studies about the role of Leptospirosis in animals
in the (sub-) urban area. The first part is a pilot study on the prevalence of Leptospirosis in
pest animals in the (sub-) urban area of Palmerston North which. Out of 36 caught animals
(possums, hedgehogs, stoats, mice and rats) 6 were PCR positive (17%). Of the same 36
animals, 1 out of 7 (14%) urine samples were DNA positive and 6 out of 23 (26%) blood
samples were seropositive for Leptospira spp. Three of the seropositive animals were positive
for L. Hardjo, two were positive for L. Ballum, and one sample was positive for both
serovars. The amount of animals though is insufficient to say something about the prevalence
in the different species investigated, but there is already evidence of both L. and L. Ballum in
pest animals living in the (sub)-urban area
The second part of the studies was a pilot study on the prevalence of Leptospirosis in cats. A
total of 180 cat serum samples was tested of which a total of 59 cats (32.7%) were positive
for one or more serovars. L. Ballum was the serovar most found among the positive cats: 56
of the 180 tested cats had antibodies against L. Ballum (31,1%). It is likely that cats get in
contact with leptospirosis due to contact with infected rodents, but to prove that, a more
comprehensive study is needed.
1
General introduction
Leptospirosisis a disease in animals and humans which is distributed worldwide and is
mainly known because of the disease of Weil. Leptospira is an obligate aerobic spirochete
bacteria of which 20 different serotypes and over 300 different serovars are known (Mansell
& Benschop, 2014).
Leptospirosis is a zoonosis, while no human-to-human transmission has ever been reported.
Humans become most often infected with leptospirosis by direct contact with urine of
infected animals or due to indirect contact, for example with water or soil contaminated with
leptospires. The severity of the pathogen depends on the serovar, but also on the health status,
age and immunological competence of the person. Symptoms can be mild: for example an
influenza-like illness, but also very severe and leading to death (Adler & de la Peña
Moctezuma, 2010). Approximately 10% of the severe infections with leptospires develop the
Weil syndrome with symptoms of jaundice, acute kidney failure and pulmonary disease
(Seguro & Andrade, 2013). Transmission is seen in both developing and industrialised
countries and it is known that the incidence of infection in humans is higher in tropical
regions than in regions with a moderate climate. The reported incidence rates are probably an
underestimation (Bharti et al., 2003).
Besides humans, leptospirosis is seen in all different kind of domesticated animals, like cats,
dogs, cattle and swine, but also in wild animals like mice, rats and possums. Symptoms can
vary strongly between species and individuals and include fever, renal and hepatic failure, but
also pulmonary manifestations and reproduction failure. Almost every mammal has been
shown to be able to act as a carrier of leptospires without showing clinical signs. This in
contrast to humans, who suffer more often acute infections and almost never become carriers.
It is also known that some species serve as a particular reservoir for certain leptospirosis
serovars. For example, it is known that mice and the black rat can be carriers of L. Ballum
(Adler & de la Peña Moctezuma, 2010; Mansell & Benschop, 2014). These mice and rats can
harbor the leptospires in their kidneys and shed it into their environment, without showing
clinical signs themselves (Adler & de la Peña Moctezuma, 2010).
2
Leptospirosis in wildlife and domesticated household cats. Pilot study
Introduction and aim of this study
In New Zealand, six serovars of leptospirosis are endemic of which L. Hardjo, L. Pomona and
L. Ballum are the three serovars responsible for the majority of human cases, and L. Ballum
being the serovar most identified in human cases in 2010 (Mansell & Benschop, 2014).
Hathaway et al. (1981) performed a study on the prevalence of leptospirosis in free-living
species in New-Zealand. They found the highest seroprevalence in possums, hedgehogs, ship
rats, Norway rats and the house mouse, varying between 5% (Norway rats) and 56%
(possums). Of the kidney cultures performed in this study, the prevalence varied between 1637%. Possums were mostly infected by L. Balcanica, while of the rodents, L. Ballum was the
isolate most identified. Of the hedgehogs only the serogroep Ballum was determined, but not
the serovar. Four years before that research, Brockie (1977) already isolated L. Ballum in the
ship rat, the Norway rat and the house mouse from dairy farms mainly in the North Island. He
also found L. Copenhageni in the Norway rat. In a study by Hathaway & Blackmore (1981),
the prevalence of L. Ballum in both black and brown rats were respectively 34% and 26%.
There is some old literature about prevalences of leptospirosis in mice, rats, possums and
hedgehogs in New Zealand. Whereas there is an increasing number of humans infected with
L. Ballum (Mansell & Benschop, 2014), the question remains what the role of wildlife at the
moment is in the epidemiology of human leptospirosis in the sub-urban area. Therefore, it is
tried to catch as much animals as possible to collect their kidneys, blood and urine to gain
more information about their infection status. The kidneys and urine will be used to search
for the live leptospires and the blood samples will be used to search for antibodies against
leptospirosis.
The situation for domesticated house cats is a bit different. These animals are common pets in
New Zealand and up to 1.4 million households keep them (MacKay et al., 2011). Different
studies have been done in different countries to the prevalence of leptospiral antibodies in
cats, varying between 4,8 and 35%. There is also evidence that cats are able to shed
leptospires intermittently in their urine for several weeks after experimental infection.
Although there have been some cases reported in the field of cats becoming ill of the bacteria,
it is still thought that cats are resistant against the acute stage of leptospirosis (Rodriquez et
al., 2012). In New Zealand, there has been a serological survey among cats as well. This
study by Shophet (1979) showed the seroprevalence of 11 different serovars among 225 cats
with a microscopic agglutination test (MAT). A total of 20 samples (8,8%) gave positive
titres of which L. Copenhageni, L. Hardjo (both five), L. Ballum and L. Pomona (both four)
were most determined serovars.
While the study of Sophet (1979) is the latest research among cats in New Zealand, it is time
to revisit this study to the infectious status of pet cats. It is still not known what the role of
cats is in the epidemiology of human leptospirosis in New Zealand, despite the fact that they
live in close contact with humans throughout this country. In contrast to the wildlife part of
the study, it is not possible to gain the kidneys of these animals and perform a PCR and/or
culture. Therefore, we try to collect blood samples of domesticated house cats to search for
antibodies, which gives us an indication what serovar these cats come in contact with. It
would also be interesting to compare the Leptospirosis serovars of domesticated house cats
with the wildlife species.
3
Material and methods
In order to catch possums in sub-urban areas, we used a total of ten steel possum cages of
which five were hook- and the other five were plate cages for a period of four weeks. Some
possums were caught by kill traps by the regional council and delivered for research to
leptospirosis. For the collection of rats, we used five wooden, closed stoat cages. All cages
were checked daily.
Possum- and stoat traps were set out on different spots throughout the (sub-) urban part of the
city Palmerston North. The bait used in the possum cages were apples (Cowen, 1987). The
stoat cages were filled with bait containing apple and peanut butter to attract rats (Wilson et
al., 2007).
Once the animals were trapped alive, they were killed by carbondioxide (CO2) gas by putting
the cage in a plastic bag and filling this bag with CO2. After killing, the animals were sexed
and at least 2 ml of blood was collected immediately by heart puncture with a 21G 1 TW
(0.8mm x 25 mm) BD PrecisionGlide™ Needle and a BD 10ml Syringe and put in a 10 ml
BD Vacutainer Serum. If not enough blood could have been collected, the jugular vene was
cut through and blood was caught in the 10 ml BD Vacutainer Serum directly. If possible,
urine was collected also with a 21G 1 TW (0.8mm x 25 mm) BD PrecisionGlide™ Needle
and a BD 10ml Syringe and put in a 10 ml BD Vacutainer®. The kidneys were taken out
aseptically the same day and put in a transparent lock-zip plastic bag. After that, they were
dissected following the protocol in appendix 1 resulting in a 1,5ml Eppendorf® cup with 800
μl dilution for the DNA extraction and one to three samples used for culturing the leptospires
in a Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (appendix 1). Until the 18th
of February 2015, one culture per fresh kidney sample was used and from the 19th of
February 2015 the dilution of three cultures per fresh kidney sample was used, as written in
the appendix. These cultures can be read for 13 weeks before being discarded, because of the
slow growth of this bacteria (Adler & de la Peña Moctezuma, 2010).
The collected urine samples were submitted to DNA extraction following the protocol
included in the QIAamp DNA mini kit (QIAGEN, Germany).
The DNA extraction of all the kidney samples was performed with use of the High Pure PCR
Template Preparation Kit (Roche, Mannheim, Germany) following the manufacturer’s
instructions. Only 160 μl out of the total 800 μl of every kidney sample was used. The 640 μl
leftover was put back in the -80°C freezer being held as a back-up sample. This resulted in
final kidney DNA samples of 200 μl each to be used for the PCR.
The wildlife blood samples were within four hours after collection centrifuged in a Heraeus
multifuge 1S-R centrifuge at 3000 rpm for 10 minutes to separate the serum from the red
blood cells. After centrifugation, the blood was stored in the -5°C fridge for a maximum of
10 days.
For the domesticated house cats, we used a total of 180 serum samples from the New Zealand
Veterinary Pathology (NZVP) laboratories. These samples were sent to the NZVP for all
different kind of reasons, both sick and healthy animals. All samples were used, so no
samples were discarded because of hemolysed serum or other reasons.
Of all the collected blood samples, three masterplates were made to use for the Microscopic
Agglutination Test (MAT): one for the wildlife samples and two for the domesticated house
cat samples. The protocol for the preparation of the masterplate can be found in appendix 2.
The MAT was performed as described by Cole et al. (1973). The collected blood samples
4
were investigated for the presence of antibodies against the four most common serovars in
New Zealand: L. interrogans serovar , Pomona and Copenhageni and L. borgpetersenii
serovar Ballum. The cut-off titre of ≥48 was used for differentiation between negative and
positive seroprevalence (Dreyfus, 2013).
Results
After the trapping period of four weeks, a total of seventeen mature possums were collected.
Four of them were caught by a kill trap and thirteen of the possums were caught by live traps.
Of the total of seventeen possums caught dead or alive, eight possums were carrying a
juvenile possum with them. One juvenile possum was caught alone (without the mother) in a
live trap.
Animal
Amount
Possum adult
17 Besides the possums, a total of three stoats, five hedgehogs, one
mouse and one rat were caught dead or alive (table 1). All birds
Possum juvenile
9
in the traps were released immediately. Data on the performed
Hedgehog
5 tests and the location where the different species were caught
Stoat
3 can be found in appendix 4. Figure 1 shows the locations where
Rat
1 one or more animals where caught (green balloon) or the spots
Mouse
1 where no animals were caught (red balloon).
Total
36 We were able to collect kidney samples of all caught animals.
Table 1. Animals caught during
study
Blood samples were collected from 23 animals and a total of 7
urine samples were collected.
Figure 1. Locations in Palmerston North where one or more cages were put down during the collecting period
5
Figure 1. Global distribution of the household cat samples investigated
PCR kidney samples
Of the 36 kidney samples taken during the four weeks of trapping, six animals were positive
in the PCR, which was confirmed using PFGE (figure 3).
Figure 3. Results of positive PCR results for the pulsed-field gel electrophoresis
PCR urine samples
Of the seven urine samples collected, one sample was found positive with the PCR, which
was confirmed during PFGE (figure 3).
6
Kidney cultures
Of the kidneys cultured, two were positive: one hedgehog and one possum. The hedgehog
was positive for L. Ballum and the possum for L. Hardjo.
Microscopic Agglutination Test
Of the 23 wild animals of which blood was collected, six animals (26%) of which five
possums and one hedgehog had one or more positive titres with a cut-off titre of ≥48 (Fang,
2014). One hedgehog was positive for L. Ballum (1:192). One possum was also positive for
L. Ballum, but with a lower titre of 1:48. Another possum was also positive for L. Ballum
with a titre of 1:48, but this animal had also a positive titre of 1:768 for L. Hardjo. Three
possums were positive for L. Hardjo as well with titres of respectively 1:768, 1:1536 and
1:192. There are no positive titres found of the serovars Copenhageni and Pomona (table 2).
Titre
1:48
1:96
1:192
1:384
1:768
1:1536
Total
Hardjo
0
0
1
0
2
1
4
Ballum
2
0
1
0
0
0
3
Copenhageni
0
0
0
0
0
0
0
Pomona
0
0
0
0
0
0
0
Table 2. Titre results of the Microscopic Agglutination Test of the wildlife animals caught during the study
Of the 180 household cat serum samples investigated, 59 samples (32,7%) were positive for
one or more leptospirosis serovars on the MAT (table 3). Two cats (1,1%) had a positive titre
of L. Hardjo with both a titre of 1:48. One cat had a positive titre of L. Pomona of 1:384
(0,6%). Eight cats (4,4%) had a positive titre of L. Copenhageni of which five cats had a titre
of 1:48, two of 1:96 and one of 1:768. The serovar Ballum was with 56 positive MAT results
the serovar most common in the cats investigated (31,1%). The titres varied between 1:48 up
to 1:3072. The global results can be found in table 3, the more comprehensive results can be
found in appendix 5.
Titre
1:48
1:96
1:192
1:384
1:768
1:1536
1:3072
Total
Hardjo
2
0
0
0
0
0
0
2
Ballum
18
18
7
6
4
2
1
56
Pomona
0
0
0
1
0
0
0
1
Copenhageni
5
2
0
0
1
0
0
8
Table 3. Titre results of the Microscopic Agglutination Test performed on cats from namely the Northern Island
Of the 180 cats, six cats were positive for two serovars of which five were positive for the
combination of L. Ballum and L. Copenhageni and one cat was positive for L. Ballum and L.
Pomona.
7
Of the 180 cats, one cat was positive for three serovar, namely L. Hardjo, L. Ballum and L.
Copenhageni.
Wildlife combinations
As can be seen in table 4, there are animals with more than one positive result for different
tests. Of the six positive MAT results, there were three animals with just a positive MAT and
the other three animals also had a positive PCR result. One of those three animals even had a
positive culture on top of that. The other positive culture was combined with a positive
kidney and urine PCR, but without a positive MAT. There are two animals with just a
positive kidney PCR. The serovar involved in these individuals couldn’t be determined.
Combinations found
# of animals serovars
Only positive MAT
Positive MAT+ positive kidney PCR
Positive MAT+ positive kidney PCR + positive culture
Positive kidney PCR
Positive kidney PCR + positive urine PCR+ positive culture
3
2
1
2
1
2x L. Ballum 1x L. Hardjo
2x L. Hardjo
1x L. Hardjo
2x unknown
1x L. Ballum
Table 4. Overview of the combinations of positive test results found in the wildlife study
8
Conclusion and discussion
In this study there have been a total of three weeks of trying to catch rats around people’s
houses. Only two hedgehogs (of which one was released by the householder) were caught
during 60 trapnights. So this trapping at individuals’ houses barely provided any animals for
receiving data. It is more useful to put the traps down at places with high rat densities, as seen
in the study of Wilson et al. (2007), where 31 rats in 100 trapnights were caught. It also can
be that the wooden stoat cages are not good for capturing rats. Wilson et al. (2007) found that
more rats are caught in cage traps than in Elliot traps. Elliot traps are closed traps and look
more like the stoat traps than the cage traps do.
Wildlife
The MAT performed on the wildlife serum samples collected, shows that some animals
captured for this research have been in contact with L. Ballum and some with L. Hardjo. L.
Hardjo is antigenetically indistinguishable from L. Balcanica using MAT without further
testing (Hathaway et al., 1978a). Because previous research showed that L. Balcanica is often
present in possums, instead of L. Hardjo, it is likely that this is the case here as well, but this
was not possible to perform in our laboratory (Hathaway et al., 1981). Hathaway et al. (1981)
also found most positive serum samples in possums for both the Ballum and Hebdomadis
serogroup (where both L. Hardjo and L. Balcanica belong to). They also found the highest
seroprevalence of L. Ballum in hedgehogs, of which also the only positive hedgehog out of
five was positive for in this research. That none of the animals have been found positive for
L. Copenhageni and L. Pomona suggests that these serovars play a minor role in wildlife
epidemiology in the sub-urban area. But more research should be done to the real prevalences
of leptospirosis in the five species investigated in this study in which the results of this study
can be used or added. The good thing is that a lot of tests in individual animals are combined
and that most of these animals were relatively fresh, which makes investigation easier and
more reliable: DNA and antibodies are not degraded yet and for the culture, live leptospires
are needed.
Household cats
As can be seen in table 3 and appendix 5, antibodies against the serovar Ballum are by far
most found among the household cats investigated. L. Hardjo, L. Pomona and to a lesser
extend L. Copenhageni play a minor role in this species.
Rodriguez et al. (2014) found that outdoor access and hunting lifestyles are significant risk
factors for seropositivity in cats. It is known that the serovar most common in black rats and
mice is L. Ballum (Mansell & Benschop, 2014) and that is exactly the same serovar most
found among cats in this study. It is likely that cats get in contact with leptospirosis due to
contact with infected rodents, although there is no information available of these cats about
their hunting lifestyle or outdoor access.
When we compare the results of the household cats to the study of Sophet (1979), there is a
big difference in the percentage of total cats positive, but also of cats with positive antibodies
against L. Ballum. Although his study is not for 100% comparable with this study (he used a
cut-off value of ≥24 and with two positive serovars in the same cat, he only marked the
highest titre as being positive), a difference of incidence for L. Ballum of 1,77% versus
31,1% is a big difference and suggests a higher presence of L. Ballum in the environment of
humans. This is in correlation with the fact that in 2010, L. Ballum was the serovar most
identified among human cases (Mansell & Benschop, 2014).
9
There is not much information available of the cats investigated. It is known that antibodies
against leptospirosis are more common in outdoor cats and in known hunters (Rodriguez et
al., 2014) but all information available of the cats used in this investigation was (often) a
short history. There was also no urine available to investigate for live leptospires, which
could provide more information about the shedding status of the individual. The study of
Rodriguez et al., (2014) is therefore more comprehensively, because there was more
information available about the cats due to a questionnaire for the owner. For a follow-up of
this research, it would be useful to combine urine samples with blood samples to gain a more
complete view of the shedding status of the cats. Also, the questionnaire for owners used in
the study of Rodriguez et al., (2014) about the behavior of their cat would be useful to gain
more information about the behavior of infected and non-infected cats to find any risk
factors.
The cut-off titre for the MAT of ≥48, which has been used by both Dreyfus (2013) and Fang
(2013) is a bit arbitrary. A human case will be confirmed for example when the person has a
titre of 400 or higher (World Health Organisation, 2011). Often, a cut-off value of 100 and
higher is considered indicative for infection (Fang, 2013). On the other hand it has been seen
that cattle in New Zealand which were actively shedding, sometimes did have lower MAT
titres (in this case of L. Hardjo) than ≥100 (Mackintosh et al. 1980; Ellis et al. 1986) so it’s
not clear what the ideal cut-off titre has to be. That is why is chosen for the same cut-off titre
as Dreyfus (2013) and Fang (2014).
Another tool for confirmation of an infection with leptospirosis is a fourfold or greater rise of
the MAT titre (Fang, 2013). In that case, it is possible to distinguish between animals with a
low titre. The animals with a low titre in the acute phase will show an incline of the MAT
titre, while the animals with a low titre and a past infection will not show a rise and might
even show a decline. With only a single titre available of the 23 wildlife animals and 180 cats
used for this research, nothing can be said about the phase of infection of the animals with
lower titres. So for a follow up study, it would be useful to collect paired titres when possible
because that provides more information.
As can be seen in appendix 4, two possums and one hedgehog are positive for both the MAT
and the PCR on the kidneys. These MAT titres vary between 1:192 and 1:1536 and in all
cases they were positive MAT results for L. Hardjo. The PCR method is a really sensitive and
specific method, but the disadvantage is that it is not possible to differentiate between
different serovars (O’Keefe, 2002). While these MAT results correspond with a positive PCR
result, it is likely that the PCR tested positive on L. Hardjo. The one possum that was positive
for MAT, kidney PCR and kidney culture showed the presence of L. Hardjo in the culture and
antibodies against this same serovar, so these test results match with each other. That is why
the combination of tests is relevant. With only the PCR, it is known that leptospires are
present, but it is unknown what serovar is involved. With only the MAT, it is known that the
individual has been in contact with a certain serovar, but nothing can be said about the actual
infection status of that individual. If animals are positive for both PCR and MAT, the serovar
can be determined and it is known that that animal is actually shedding the bacteria. That the
combination of tests performed provides more information is also seen in the case of
Hedgehog 44. The serovar, L. Ballum, has been determined based on the positive culture,
despite of the absence of a positive MAT. Besides this positive culture, this hedgehog was
positive on the PCR for both the urine and kidney. The explanation of the fact that three PCR
positive animals didn’t have a corresponding MAT titre is that it can be that the infection is
too acute and that no antibodies against leptospires are produced yet. Fang (2014) did
research to the comparison of both urine and kidney PCR results and the seroprevalence of
10
the same individuals in cows and sheep. He found out that approximately 40% of the
seropositive animals had a positive PCR for both urine and kidney and that approximately 3%
of the seronegative animals had a positive PCR result for their urine or kidney. It is possible
that three animals caught in this study were in the acute phase of infection, but this would be
a coincidence.
Another explanation for a negative MAT result, in combination with a positive PCR result is
that the MAT is not specific enough, but Adler & de la Peña Moctezuma (2010) state that the
MAT is a highly specific and sensitive method, so this theory is unlikely as well. Perhaps, the
study of Fang (2014) in sheep and cows cannot be extrapolated to wildlife.
In short: a high prevalence of L. Ballum has been found in domesticated house cats. In
wildlife, both L. Ballum and L. Hardjo have been isolated. The majority of human cases are
caused by L. Ballum, L. Hardjo and L. Pomona (Mansell & Benschop, 2014). Although it is
still unclear what the exact role of both domesticated house cats and wildlife is in the
epidemiology of humans, the information obtained by this research is indicative that both
groups might play a role in the epidemiology of at least L. Ballum and L. Hardjo.
11
Appendix 1. Protocol for kidney dissection
1. Weight the kidneys together at the weighting scale and write down the weight of the
kidneys together.
2. Turn the fume cupboard on and take the kidneys to the fume cupboard.
3. Take for each kidney sample one (and from the 19th of February three) bottle(s)
containing the Ellinghausen-McCullough-Johnson-Harris (EMJH) medium out of the
fridge and leave it at room temperature for at least 30 minutes.
4. Undo the kidney of its capsule with your forceps.
5. Take a total amount of 10 grams (estimated) of the kidneys and drench it with Ethanol
70% above a funnel and flask. If the kidneys weight less than 10 grams, then take
both entire kidneys.
6. Flame the kidneys with the Bunsen burner for a few seconds until the entire surface is
egally grey/brown.
7. Put the kidneys in a Stomacher® bag and add an amount of 5 ml per gram kidney,
with a maximum of 50 ml 0,01M Phosphate Buffer Saline (PBS).
8. Put the Stomacher® bag in the Colworth stomacher 400 (AJ Seward Ltd, London,
United Kingdom) and wait for 1 minute. If the total amount of kidneys is <2 grams,
crush the kidneys for at least 1 minute manually with a blunt object until they are well
crushed.
9. Take the Stomacher bag out of the Colworth stomacher 400 and see if the kidneys are
well crushed.
10. If they are not well crushed enough, repeat step 7 & 8.
11. Take an 1,5 ml Eppendorf cup (Raylab, Auckland, New Zealand) for each sample and
write the sample number on top.
12. Put 800 μl of fluid out of the bag into the destinated Eppendorf cup using a Gilson
Pipetman P200.
13. Put the 1,5 ml Eppendorf cup in the -20°C freezer
14. Label each EMJH medium with Mark, species, animal number, date and dilution.
15. Put 100 μl of fluid out of the bag into an EMJH medium (dilution 0) using a Gilson
Pipetman P200.
16. From the 19th of February: take 100 μl of fluid out of the EMJH medium dilution 0
and put it in a new EMJH medium (dilution -1) and repeat this step one more time for
the dilution -2.
17. Put the EMJH medium in the 38°C
18. Discard the Stomacher® bags.
12
Appendix 2. Protocol for preparation of a masterplate
1. Centrifuge all the samples in numbered vacutainers at 3000rpm for 10min in a
Heraeus multifuge 1S-R centrifuge.
2. Determine in which well of the Greiner Bio One microplate (REF 655161) you want
to put which sample (from vacutainers) and write this down on the Masterplate-paper.
3. Fill the wells for the vacutainer samples with 150μl sterile saline with a Gilson
Pipetman L Multi 20 μl-300 μl pipet.
4. Take 30μl of the serum/supernatant from the vacutainer with a Gilson Pipetman
Classic™ P200 pipet and put it in the right well with the saline.
5. Take the rest of the serum/supernatant with a Gilson Pipetman Classic™ P200 pipet
and put it in a backup sample tube.
6. Write the sample number on the lid of the backup sample tube, and on the tube itself:
Mark, sample number, date.
7. Put Parafilm “M” (Pechiney Plastic Packaging, Chicago, IL, USA) on top of the
Greiner Bio One microplate (REF 655161) and put the lid on the microplate.
8. Put the Greiner Bio One microplate (REF 655161) in a zip-lock bag and close the ziplock bag.
9. Put the zip-lock bag in the -20°C freezer.
10. Put the backup sample tubes from step 13 in a backup sample box.
11. Name the backup sample box: Mark 1 Lepto 2015. Write down which backup
samples are in which backup sample box.
12. Put the backup sample box in the freezer (-80°C).
13. Discard the used and empty vacutainers.
13
Appendix 3. Total list of trap dates, locations, type of cages and animals caught
11-Feb
12-Feb
13-Feb
6 Plate cage 7 Plate cage
4 Plate cage 5 Plate cage
3 Hook cage
1 Hook cage 2 Hook cage
108 Atawhai rd 108 Atawhai rd 108 Atawhai rd 108 Atawhai rd 7 Fernwood rd 118 Clifton cr 42 The Strand
P
x
x
x
x
x
P
P
x
x
x***
x
P
P
C
x
x***
x
x
x
P
x
x
B
x
x
13 Stoat trap
11 Stoat trap 12 Stoat trap
10 Stoat trap
Stoat trap
8 Plate cage/ kill 9trap
66 Pahiatua st 50A Lockhart av 50A Lockhart av 50A Lockhart av 50A Lockhart av 50A Lockhart av
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
P (KT)
x
x
x
x
x
x (KT)
8 Plate cage
6 Plate cage 7 Plate cage
4 Plate cage 5 Plate cage
3 Hook cage
1 Hook cage 2 Hook cage
108 Atawhai rd 108 Atawhai rd 108 Atawhai rd 108 Atawhai rd 108 Atawhai rd 108 Atawhai rd108 Atawhai rd 66 Pahiatua st
H
x
x
x
H
x
P
P
H
*
x
P
x
x
x
x
x
x
x**
x
B
x
x
13 Stoat trap
11 Stoat trap 12 Stoat trap
10 Stoat trap
9 Stoat trap
50A Lockhart av 50A Lockhart av 50A Lockhart av 50A Lockhart av 50A Lockhart av
x
x
x
x
H*
x
x
x
x
H
x
x
x
x
x
17-Feb
18-Feb
19-Feb
20-Feb
12 Hook cage
156 Aokautere drive
x
x
x
x***
8 Kill Trap
66 Pahiatua st
x
x
x
x
11 Plate cage
9 la Lena gr
x
x
x
x
6 Plate cage 7 Plate cage
4 Hook cage 5 Plate cage
3 Hook cage
1 Hook cage 2 Hook cage
108 Atawhai rd Acacia Birch 3c Acacia Birch 3c Acacia Birch 3c 7 Fernwood rd 118 Clifton cr 42 The Strand
x
x
P
x
x
x
x
B
x
x
x
x
x
x
P
x
P
x
x
x
x
B
x
x
x
x
x
x
10 Plate cage
9 la Lena gr
x
P
x
H*
24-Feb
25-Feb
26-Feb
27-Feb
4. Stoat trap 5. Stoat trap
1. Stoat trap 2. Stoat trap 3. Stoat trap
5 Newland crt 5 Newland crt 5 Newland crt 5 Newland crt 5 Newland crt
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
9 Kill Trap
122 Atawhai rd
x
x
P
x
27-Feb
2-Mar
3-Mar
4-Mar
5-Mar
x= empty
P= Possum
H= Hedgehog
H*= Hedgehog released by householder
St= Stoat
B= Bird
KT= Kill Trap
*= cage disappeared
x**= cage sabotaged with flour
x***= cage closed, bait gone, no animal
14
Appendix 4. Comprehensive list of animals caught, tests performed and test results
15
Appendix 5. Comprehensive results of positive (≥48) MAT titres cats
Hardjo
Ballum
Pomona
Copenhageni
Age
Gender
District
P15000804
1:96
15Y
C
Whanganui
P15000819
1:384
16Y
F
South Taranaki
P15000842
1:96
6Y
S
Wellington City
P15000850
1:96
16Y
M
Gisborne
P15000897
1:1536
16Y
S
Wellington City
P15000898
1:48
15Y
C
Whanganui
P15000919
1:768
9Y
C
Central Hawkes Bay
P15000928
1:96
13Y
C
Kapiti Coast
P15000943
1:384
15Y
M
South Taranaki
P15000954
1:48
12Y
S
Tasman
1:3072
16Y
C
Wellington City
P15001055
1:1536
14Y
C
Porirua City
P15001059
1:96
12Y
C
Porirua City
P15001070
1:96
16Y
S
Porirua City
P15001072
1:48
14Y
S
Porirua City
P15001104
1:96
13Y
C
New Plymouth
P15001202
1:48
15Y
U
Kapiti Coast
P15001205
1:48
14Y
C
Porirua City
P15001220
1:768
1:96
9Y
C
Wellington City
P15001226
1:48
1:48
9Y
S
Wellington City
P15001234
1:48
18Y
S
Palmerston North
P15001238
1:48
13Y
C
Horowhenua
P15001265
1:768
15Y
F
Manawatu
P15001288
1:192
14Y
M
Hastings
1:24
13Y
S
Porirua City
P15001302
1:192
8Y
F
Wellington City
P15001305
1:48
14Y
S
Kapiti Coast
P15001353
1:768
13Y
F
Lower Hutt City
P15001359
1:384
P15001366
1:96
P15000989
P15001299
1:24
1:48
1:96
14Y
C
Marlborough
1:48
UN
S
Horowhenua
P15001369
1:48
16Y
F
Gisborne
P15001376
1:48
4Y
S
Porirua City
P15001404
1:192
5Y
C
Palmerston North
P15001416
1:192
3M
C
Whanganui
P15001426
1:96
11Y
S
Wellington City
P15001433
1:192
UN
U
Wellington City
P15001436
1:192
15Y
C
Wellington City
P15001444
1:96
4Y
M
South Taranaki
P15001450
1:96
13Y
S
Wellington City
P15001519
1:96
9Y
C
Palmerston North
P15001538
1:384
13Y
M
Hastings
P15001539
1:48
16Y
F
Kapiti Coast
1:24
1:384
1:24
16
P15001550
1:96
P15001554
13Y
M
Porirua City
1:48
17Y
F
Wellington City
P15001566
1:48
9Y
M
Hastings
P15001612
1:48
13Y
C
Wellington City
P15001615
1:48
15Y
C
Kapiti Coast
P15001624
1:384
14Y
S
Porirua City
P15001628
1:96
1:24
17Y
C
Wellington City
P15001695
1:48
1:24
13Y
F
Manawatu
P15001723
1:96
10Y
C
Wellington City
P15001728
1:48
18Y
S
Central Hawkes Bay
P15001795
1:96
9Y
F
Manawatu
P15001813
1:384
10Y
S
Wellington City
P15001817
1:48
11Y
C
Kapiti Coast
P15001819
1:48
13Y
S
Wellington City
P15001821
1:192
10Y
C
Wellington City
P15001823
1:96
1:768
14Y
C
Wellington City
1:96
1:48
5Y
M
South Taranaki
P15001829
M=
F=
C=
S=
U=
1:48
1:24
Male
Female
Male castrated
Female speyed
Unknown
17
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