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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code OZ0404

2. Project title

Bovine neosporosis: the evaluation of zoonotic risk and the development of evidence - based control strategies

3. Contractororganisation(s)

Veterinary ParasitologyLiverpool School of Tropical MedicinePembroke PlaceLiverpoolL3 5QA

54. Total Defra project costs £ 242,267

5. Project: start date................ 01 November 2002

end date................. 31 December 2005

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Neospora caninum is a newly recognised protozoan parasite of dogs and cattle, first described in 1988. Now recognised as one of the major infectious cause of abortion in dairy cattle, it is known that cattle can be infected transplacentally or by ingestion of oocysts excreted by dogs. However, very little is known about the frequency and intensity of oocyst shedding by dogs or the infectivity and clinical consequences of oocyst infection in cattle. The importance of environmental infection from dogs to cattle, is a critical question since it has profound implications for control. Environmental contamination with N. caninum oocysts also has potential implications for humans, Given its relationship to the ubiquitous and common zoonotic protozoan Toxoplasma gondii, and the fact that N. caninum will infect primates and grow in human cells in vitro, there is concern to determine if N. caninum is infecting humans with, as yet, unidentified consequences. Ingestion of oocysts, as well as with parasite stages in undercooked beef, are the two major potential routes of human infection.

This project has addressed these key questions about N. caninum and has successfully achieved its major objectives to determine the zoonotic potential of N. caninum and to elucidate the significance of oocyst infection for cattle.

In collaboration with the Health Protection Agency, the most comprehensive serological survey of a human population yet undertaken was conducted using over 3200 samples randomly drawn from anonymised specimens accumulated by the Public Health Laboratories services (PHLS) and representing A major and logistically challenging experiment was conducted to determine the effect of N. caninum oocysts infection on pregnant cattle. Whilst it has been presumed that oocyst infection might cause the abortion ‘storms’ (epidemic outbreaks) characteristic of N. caninum, until this project began almost no oocyst infections of pregnant cattle had been conducted and abortion had not been provoked. This is due to the substantial logistical problems in conducting such experiments because of difficulties of conducting dog infection (to get oocysts) and the extremely low numbers of oocysts produced. Accordingly, we collaborated with Professor Milt McAllister of the University of Illinois who had first demonstrated that dogs are a definitive host of N. caninum. Using 18 cows in three groups with synchronised oestrus and artificial insemination, transplacental infection was confirmed consistently (4/5 calves infected) when cows were infected at 210 days gestation, but not at 70 days (0/6 calves infected) and in only 1/6 pregnancies at 120 days, in which case the foetus was aborted. This experiment demonstrated that oocyst infection could cause transplacental infection in mid to late pregnancy; that it could cause abortion – albeit infrequently; and that those effects could be produced by relatively low numbers of oocysts (estimated oocyst dose between 127 – 40,000 oocysts; because oocyst concentrations were too low to count viable dose was estimated by bioassay). Because it is now realised that chronic maternal infections recrudescing in pregnancy are an important feature of N. caninum in cows, we exploited the unique opportunity of having oocyst-infected cows, to investigate whether such post-natal infection led to chronic infection which could infect subsequent pregnancies. In a second

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experiment, seven cows shown by immunological responses to have been infected with N. caninum oocysts in their first pregnancy, were rebred. There was no evidence of spontaneous infection in their calves.

The cattle experiments revealed the possible effects of oocyst consumption by cows. A third part of the project investigated the natural production of oocysts by dogs. In a cross-sectional survey of faecal samples from foxhounds, farm dogs and pet dogs, N. caninum oocysts were found in only 1/261 foxhounds, and 0/69 farm dogs and 0/105 pet dogs. Morphologically similar oocysts of Hammondia heynorni were found in 13 foxhounds, one farm dog, and one pet dog and were identified by species-specific PCR. In the N. caninum positive foxhound sample there were <100 oocysts /g faeces.

In the fourth major part of the project the possible relationship of infections in dogs and cattle was investigated on 96 Cheshire dairy farms using a questionnaire for risk factors, estimates of herd prevalence from bulk-milk tank assays for N. caninum-specific antibody and determination of farm dog antibody status by N. caninum-specific IFAT, analysed by logistic regression and multivariate analysis. No significant (at p<0.05) risk factors were identified that would suggest that high herd prevalence (>6%) was associated with transmission from dogs to cattle, but a number of identified risk factors suggested dogs acquire infections from cattle.

In conclusion – in the largest study yet conducted, there was no evidence of exposure to N. caninum in humans this is consistent with very low levels of environmental contamination likely by N. caninum based

on the cross-sectional faecal survey. It also indicates that there is negligible risk from consumption of meat.

low numbers of oocysts were experimentally shown to cause abortion in 1/6 cows when infection was in mid-pregnancy, and to congenitally infect 4/5 calves when infection was in late-pregnancy – but chronic infections which infected a subsequent pregnancy were not established.

in the first study of risk factors on British dairy farms, no factors were identified suggesting higher herd prevalence than normal was associated with transmission from dogs. However, there was an association between seropositivity in dogs and herd seroprevalence. Without indicating whether dogs are infected cattle or cattle infected from dogs, this result nonetheless indicates that farmers should be advised to minimise contact between dogs and cattle, to prevent access of dogs to calving membranes, and to prevent dog fouling of cattle feed.

Overall whilst this project confirmed that oocysts can infect pregnant cows and cause abortion or congenital infection the likelihood of this occurring appears low based on epidemiological risk factors and the prevalence and intensity of oocyst shedding. Control of bovine neosporosis in dairy herds should focus on identifying positive cows and removing them from the replacement breeding programme. The study suggests that oocyst infection may be most significant in causing congenital infection in heifer calves. If congenitally infected female calves are retained they may go on to endogenously infect all their progeny with an associated incidence of sporadic or endemic abortion. This possibility requires to be proven.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

CONTENTS

People involved in the Project

Glossary and abbreviations

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1.0. Scientific Objectives2.1. Objective 1: To determine the zoonotic potential of N.caninum 2.1.1. Introduction2.1.2. Materials and methods2.1.2.1. Populations tested2.1.2.2. Ethical Approval2.1.2.3. Testing of sera2.1.3. Results2.1.3.1. Samples tested2.1.3.2. Inhibition ELISA2.1.3.3. IFAT2.1.3.4. T. gondii serology2.1.4. Discussion2.2. Objective 02a: An intervention study to determine the effectiveness of embryo transfer to eliminate congenital infection.2.2.1. Introduction2.2.2. Materials and methods2.2.2.1. Supply and collection of embryos2.2.2.2. Sample processing2.2.2.3. Testing embryo DNA and bovine serum2.2.3. Results2.2.4 Discussion2.2.5. Future possible work2.3. Objective 02bi: Primary objective: To determine the outcome of challenging pregnant heifers with sporulated N.caninum oocysts at different stages of pregnancy. Secondary objective: To determine whether infection of cattle with oocysts during pregnancy can result in endogenous transplacental infection occurring in a subsequent pregnancy. 2.3.1. Introduction 2.3.2. Material and methods2.3.2.1. Production and supply of N.caninum oocysts2.3.2.2. N.caninum oocyst infection of cattle.2.3.2.3. Gerbil bioassay2.3.2.4. Immunological responses in cattle.2.3.2.5. Necropsy of aborted foetuses and neonatal calves2.3.2.6. Rebreeding of cattle2.3.3. Results2.3.3.1. Gerbil bioassay2.3.3.2. Infection of heifers2.3.3.3. Rebred cows.2.3.4. Discussion2.4. Objective 02bii: To determine the prevalence and intensity of natural oocyst shedding in certain canid populations2.4.1. Introduction2.4.2. Materials and methods2.4.2.1. Collection of faecal samples2.4.2.2. Screening of samples2.4.2.3. Concentration of oocyst and DNA extraction2.4.2.4. PCRs to confirm identity of oocysts.2.4.2.5. Bioassay of oocysts and in-vitro culture.2.4.3. Results2.4.3.1. Screening of samples2.4.3.2. Bioassay and in-vitro culture.2.4.3.3. Questionnaire2.4.4. Discussion2.4.5 Future work2.5. Objective 02c: To determine the potential risk factors for Neospora- associated infection in cattle and dogs.2.5.1. Introduction2.5.2. Materials and methods.2.5.2.1. Populations studied2.5.2.2. Variables studied2.5.2.3. Analysis of samples2.5.2.4. Data management and analysis2.5.3. Results2.5.4. Discussion

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Appendix 1Appendix 2

People involved in the Project

Veterinary Parasitology, LTSM and Faculty of Veterinary Science, University of Liverpool

Principal Investigator Professor Alexander Trees

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Co-Investigator Dr Diana J.L. Williams

Research Associate Ms Catherine McCann

Other contributors Dr Jane Hodgkinson; Dr John McGarry; Dr Sarah Felstead

Collaborators

Department of Veterinary Clinical Science and Animal Husbandry, Faculty of Veterinary Science, University of Liverpool:

Dr Robert F SmithDr Peter J Cripps

Department of Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool:

Dr Anja Kipar

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois, USA:

Associate Professor M. McAllisterDr Pita Gondim

Health Protection Agency, Centre for Infections, Immunisation Department, 61 Colindale Avenue, London NW9 5HT:

Dr Richard PebodyDr Andrew Vyse

Preston Microbiology Services, Royal Preston Hospital, Preston, PR2 9HT:

Dr Louise Hesketh

Public Health Laboratories Farm Cohort Steering Group:

Dr Daniel Thomas and Dr Roland Salmon, NPHS Communicable Disease Surveillance Centre, Cardiff, CF14 3QXDr Sue Kench, Hereford Public Health Laboratory , County Hospital, Hereford.

Members of the Masters of Foxhounds Association

Glossary and abbreviations

BVDV Bovine Virus Diarrhoea virus

CI confidence interval

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DNA deoxyribonucleic acid

ELISA enzyme-linked immunosorbent assay

ET embryo transfer

FITC fluorescein isothiocyanate

g gravitational field (centrifuging)

h hour

HPA Health Protection Agency

IBR Infectious Bovine Rhinotracheitis

IFAT Indirect Fluorescence Antibody Test

IFNγ interferon gamma

IgG immunoglobulin G

IPX immunoperoxidase

L litres

m minutes

NBF neutral buffered formalin

nd not done

NHS National Health Service

OR Odds ratio

P probability

PAP peroxidase anti-peroxidase

PBS phosphate buffered saline

pc post challenge

PCR polymerase chain reaction

PHLS Public Health Laboratories

PI percentage inhibition

PP percent positivity

SI stimulation index

TPI transplacental infection

VLA Veterinary Laboratories Agency

1.0. Scientific Objectives

1. Objective 01: To determine the zoonotic potential of N.caninum

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2. Objective 02a: An intervention study to determine the effectiveness of embryo transfer to eliminate congenital

infection

3. Objective 02bi: Primary objective: to determine the outcome of challenging pregnant heifers with sporulated

N.caninum oocysts at different stages of pregnancy.

Secondary objective: To determine whether infection of cattle with oocysts during pregnancy can result in

endogenous transplacental infection occurring in a subsequent pregnancy.

4. Objective 02bii: To determine the prevalence and intensity of natural oocyst shedding in certain canid

populations.

5. Objective 02c: To determine potential risk factors for Neospora-associated infection in cattle and dogs.

2.1. Objective 01: To determine the zoonotic potential of N.caninum

2.1.1. Introduction

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The newly recognised protozoan parasite Neospora caninum has recently emerged as a major disease of cattle and dogs worldwide (1). It is closely related to the common zoonotic parasite Toxoplasma gondii, can be cultured in vitro in human and primate cells (2) and can infect primates (3, 4). For all these reasons there is concern that N.caninum may be zoonotic. People may become infected with T.gondii by ingestion of oocysts from soil, water or cat litter contaminated with cat faeces. Oral transmission may also occur by ingestion of raw or poorly cooked meat containing tissue cysts. It is hypothesized that N.caninum has the potential to infect human beings by similar routes to T.gondii. Other possible modes of transmission that may affect persons working with animals (farmers and veterinary surgeons) are through drinking raw milk or exposure to infective organisms from cattle placentas. Six percent of cattle in Britain are infected with N.caninum (5); these infections are chronic and persist lifelong in the animals. It has been shown that dogs fed meat from N.caninum infected animals may shed N.caninum oocysts thus it is plausible that people may become infected by consuming raw or inadequately cooked meat or oocysts contaminating the environment.

To date N.caninum infection has not been associated with any clinical disease in humans. A study of human sera from blood donors in the USA provided immunological evidence of human exposure to N.caninum. IFAT antibodies were reported at (1:100) in 69 of 1,029 sera. However all sera were negative at 1:200 (6). Such evidence was not found in sera of 247 blood donors and agricultural workers in Northern Ireland tested by an IFAT (7). In a study of 76 women who had had repeated abortions in Denmark, no N.caninum antibodies were detected by ELISA, IFAT and Western Blot (8).

In order to determine the zoonotic potential of N.caninum, evidence of human exposure to N.caninum was sought by examining two populations: a cross-sectional sample of the English population (for which a sample size was calculated to detect a prevalence of 2.42 % with a precision of 0.5%), and a putative high-risk group, a cohort of farm workers - the subject of an historic Public Health Laboratories (PHLS) zoonotic diseases study. A high throughput, inhibition ELISA was used as a screening test and an IFAT was used to examine putative positives. In order to determine whether there may be any association between infection with N.caninum and T.gondii, the sera of all individuals in which percent inhibition values of ≥ 20% were measured in the inhibition ELISA were examined for antibodies to T.gondii.

2.1.2. Material and methods

2.1.2.1. Populations tested

a. Health Protection Agency (HPA) Seroepidemiology Programme (formally the PHLS Serological

Surveillance Programme)

The HPA maintains a collection of anonymised residues of specimens, submitted for microbiological or biochemical testing to Public Health Laboratories in England and Wales (9). This is currently the best serum collection available that represents the general population of England and Wales. With the assistance of Dr Andrew Vyse of the HPA a sample size estimate of 3,430 was calculated to detect an overall prevalence of 2.42% (95% CI, 1.93, 2.92) with a precision of 0.50%. in adults (aged 20-69 years). The samples were selected from English regions using stratified random sampling techniques from the sera collected in the year 2000. The prevalence of N.caninum was expected to increase from 1% in those aged 20-29 years to 5% in those aged 60 -69 years (Table 1). These estimates were calculated considering the results of Tranas et al. (6) who found an overall prevalence of 6.7% in blood donors in the USA and an assumption that prevalence might increase with age (as is the case for T. gondii).

Table 1: Sample Size estimates for HPA cohort

Age Assumed Target Lower Upper Sample Size No IgG +ve

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group prevalence precision Confidence Interval

Confidence Interval

expected

20-29 1% 0.50 % 0.50 % 1.50 % 1,520.64 15.206430-39 2% 1.00 % 1.00 % 3.00 % 752.64 15.052840-49 3% 1.50 % 1.50 % 4.50 % 496.64 14.899250-59 4% 2.00 % 2.00 % 6.00 % 368.64 14.745660-69 5% 2.50 % 2.50 % 7.50 % 291.84 14.592Overall20-69 2.42% 0.50% 1.93% 2.92% 3430.40 88.9344

b. PHLS Farm Cohort

The PHLS Farm Cohort was recruited in 1991 in order to provide a representative population based sample that could be used to perform seroprevalence studies on zoonotic diseases and also collect data on on-farm risk factors for acquiring zoonoses (10,11). Initially 606 persons, comprising farmers, farm workers and family members, were recruited into the cohort. Recruitment having been carried out in 8 local government areas in the catchment areas of Hereford, Norwich and Preston Public Health laboratories. Catherine McCann (Research Assistant) made a presentation to the PHLS Farm Cohort Steering Group in March 2005 on our proposed use of the sera. Following agreement to test the sera, serum samples, collected during the 1995 annual sampling round, were made available.

2.1.2.2. Ethical Approval

Ethical approval to test the HPA sera for evidence of N.caninum and T.gondii infection was granted by the NHS Liverpool Paediatric Research Ethics Committee on 17 June 2005. Formal ethical approval for examination of the PHLS Farmworkers Cohort for evidence of N.caninum infection was given by the Liverpool School of Tropical Medicine Research Ethics Committee on 6 May 2005.

2.1.2.3. Testing of sera

No serological assay has been validated for N.caninum antibodies in humans. Faced with this problem, for the high throughput screening of the large number of sera needed to detect the low prevalence expected, we applied an inhibition ELISA developed in our laboratory (12). This has previously been validated in cattle and dogs. In the inhibition ELISA, the binding of rabbit polyclonal antiserum to N.caninum tachyzoites is inhibited by antibodies in the test sera. Anti-N.caninum IgG from a rabbit that had been infected with N.caninum was conjugated with horse-radish peroxidase to produce the polyclonal anti-serum. The test uses whole tachyzoite- coated plates as developed for a conventional ELISA for bovine use (the Mastazyme ELISA, Mast Diagnostic, Bootle, UK ), (13,14). The inhibition ELISA method was as described previously (12) but using human sera diluted 1:10 in PBS, pH 7.2 containing Tween 20 (PBS/Tween). Two bovine sera with known high or low percent positivity (PP) values as defined by the Mastazyme ELISA were used as positive controls on each plate. The negative control sera used was from a cow that had consistently tested negative for N.caninum antibodies by the Mastazyme ELISA. In the absence of a human positive control, we used a primate serum sample kindly donated by Dr Brad Barr, University of California, Davis. This sample was from a rhesus macaque (Macaca mulata) experimentally infected with N.caninum as previously described. (3).

The optical density (OD) was read at 450nm. Percent inhibition (PI) values were calculated using the formula:

100 – [(Test OD/negative control OD) X 100]

Without specific validation, a cut-off of 20% inhibition was chosen to indicate putative positives. This cut-off has been used for previous comparison of the inhibition ELISA with the conventional Mastazyme ELISA in bovine sera (12). All samples with inhibition ≥20% were subsequently tested at a dilution of 1:50, using an IFAT. Positive controls used in the IFAT were bovine and primate N.caninum positive sera. The negative control used was bovine N.caninum negative serum. Monoclonal anti-human IgG FITC conjugate (Sigma) was used in the assay for the human and primate samples and anti-bovine IgG FITC (Sigma) conjugate for the bovine controls. All the samples from the HPA cohort with inhibition ≥20% in the inhibition ELISA were also tested for antibodies to T.gondii using Toxoscreen (bioMerieux®), a direct agglutination test, according to the manufacturers’ directions at a dilution of 1:40. The T.gondii serostatus of PHLS cohort members were obtained from source. For the HPA samples, the inhibition ELISA results were categorised according to % inhibition and a chi squared test was performed to determine whether there was any association between the T. gondii serostatus and inhibition ELISA PI for those samples that were putative positives.

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For the HPA sera inhibition ELISA results, the distribution of the data was examined firstly by plotting % inhibition with the data aggregated into bands of 10%. The plots were repeated after logging the data and putting it into bins (equal width reactivity categories based on log 10 % inhibition). The distribution of log10 inhibition was also examined according to sex, age and submitting laboratory. A similar analysis was done for the PHLS farm cohort data.

2.1.3. Results

2.1.3.1. Samples tested

518 samples from the PHLS Farm Cohort and 3232 serum samples from the HPA Seroepidemiology serum bank were screened for N.caninum antibodies. The latter number was less than planned because inadequate serum was available from nearly 200 of the selected samples. 1342 (41.52%) of the HPA samples were from male subjects, 1889 (58.45%) were from females, the gender of one subject was unknown. The distribution of the subjects tested by age and submitting laboratory are given in Tables 2 and 3 respectively.

Table 2: Distribution by age of samples tested from HPA cohort

Age Group

Estimate before study Actual numbers tested Difference (%)Frequency Percentage Frequency Percentage

20 – 29

1520.64 44.34 1480 45.79 40.64 (97.33 %)

30 - 39

752.64 21.94 674 20.85 78.64 (89.55%)

40 – 49

496.64 14.48 467 14.45 29.64 (94.03%)

50 – 59

368.64 10.75 349 10.8019.64 (94.67%)

60 - 69

291.84 8.51 262 8.11 29.84 (89.78%)

Total 3430.40 100 3232 100 197.4 (94.22%)

Table 3: Distribution of HPA subjects tested by region

Submitting Laboratory

No. of samples (%) English Region No. of samples (%)

Preston 23 (0.71 %)

North 993 (30.72%)Leeds 366 (11.32 %)Manchester 350 (10.83 %)Liverpool 254 (7.86 %)Birmingham 94 (2.91 %) Midlands 94 (2.91 %)Cambridge 521 (16.12 %) South & SE

529 (16.37%)London 8 (0.25 %)Dorchester 1107 (34.25 %)

SW 1616 (50%)Exeter 125 (3.87 %)Portsmouth 101 (3.13 %)Bristol 283 (8.76 %)TOTAL 3232 (100%) 3232 (100%)

2.1.3.2. Inhibition ELISA

For the bovine positive controls, mean percentage inhibition for the high positive control was 79.76% ((5% CI 78.94, 80.58) and for the medium positive control 74.64% (95% CI 73.55, 75.42).The primate positive sera had a PI of 63%. Of the test samples, 691 (21.38%) of the HPA samples (Table 4) and 29 (5.56%) of the PHLS samples (Table 5) produced percent inhibition of ≥ 20% in the inhibition ELISA test.

Table 4: Results of inhibition ELISA for N.caninum for HPA cohort

HPA N.caninum Inhibition ELISA: Percentage Inhibition N.caninum

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(samples tested

IFAT No. +ve @ ≥ 1/50/ No tested

<20 ≥20 <30 ≥30 <40 ≥40 < 50 ≥ 50

3232 2541 350 201 98 42 0/691Toxoplasma gondii Direct agglutination test

Result - ve +ve - ve +ve - ve +ve - ve +veNumbers 297 53 173 28 89 9 40 2Percent positive

15.14 13.93 9.18 4.76

To determine the true significance of the inhibition percentage data, the frequency distribution of actual percentage inhibitions was plotted. For the HPA samples, this plot showed a single positively skewed distribution. However, after logging the data, the plot showed a very clear single log normal distribution, with a mean close to 0% inhibition, i.e. similar to the negative control used. This is shown in Figure 1 in which the data are plotted according to age. There was no association with age, sex or submitting laboratory (results not shown). The distribution plot indicated that all samples conformed to a single population with no evidence of a positively exposed population.

Table 5: Results of serological tests for N.caninum for PHLS Farm Cohort

PHLS Farm Cohort (samples tested)

N.caninum Inhibition ELISA: Percentage Inhibition N.caninum IFAT No. +ve @ ≥ 1/50 / No tested

<20 ≥20 <30 ≥30 <40 ≥40 < 50 ≥ 50

Norwich (196) 195 1 O 0 0 0/1Hereford (163) 145 11 14 3 0 0/28

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Preston (159) 159 0 0 0 0 0/0

When the inhibition data for the PHLS Farm Cohort was also plotted as a frequency distribution it produced a single normal distribution distributed about a mean close to 0% inhibition similar to Fig. 1 above (data not shown).

2.1.3.3. IFAT

The primate and bovine positive controls consistently (test repeated 10 times) gave a positive fluorescence at 1/50. When samples with a PI ≥ 20% in the inhibition ELISA were tested using the IFAT, all failed to give positive fluorescence at 1/50 (see Tables 4 and 5.)

2.1.3.4. T.gondii serology

Five of the 29, 17.24% (95% CI 3.5, 30.99) PHLS and 92 of the 691, 13.31% (95% CI 10.78, 15.84) HPA, inhibition ELISA positive samples were positive for T.gondii No association was found between T.gondii serostatus and inhibition ELISA result for those samples that were putatively positive for N.caninum (Chi squared = 5.19, p=0.158) .

2.1.4. Discussion

We sought evidence of human exposure to N.caninum infection by specific antibody detection in two populations – a cross sectional English population and a cohort of farm workers, the latter considered to be a putative high risk group. An inhibition ELISA, was used to screen samples and putative positives were further examined with an IFAT. The inhibition ELISA, which was developed as an appropriate test to examine serum of multiple species for N.caninum antibodies, has not been validated in humans so a nominal “cut-off” was adopted which has been used for bovine sera. Using this cut-off of 20 percent inhibition, 21.38% of the HPA samples and 5.56% of the PHLS Farm Cohort samples screened as putative positives. However, when the actual inhibition results were plotted in a frequency distribution curve (Fig. 1), the data for the HPA samples aggregated into a single distribution and there was no evidence that the samples were distributed discretely into those with antibody to Neospora and those without. The same result was obtained with the inhibition results from the PHLS Farm Cohort. In previous studies, a similar analysis of Varicella zoster (VZV) specific IgG using sera from the same collection showed an aggregation of the data into two clear distributions of results relating to an uninfected population and a previously infected population that is age associated (15). For N. caninum, the quantitative results of the inhibition ELISA distribute normally around a mean that is very close to the % inhibition of the negative controls. This provides strong evidence for there being no Neospora-specific IgG in any of the samples and that the distribution observed describes a single population of non-exposed individuals. Moreover, no association was observed with age, further evidence that the data set is from a single large population of negative results. A small proportion of the samples gave a result ≥ 20% inhibition, but there was no evidence that this could be due to cross-reactivity with T. gondii antibodies. It is most likely that they are forming the right hand tail of what is clearly a normal distribution. Lastly, confirmatory testing with the IFAT of all samples with more than 20% inhibition in the inhibition ELISA, failed to detect any specific fluorescence indicative of true positives. It is therefore concluded that there is no evidence of exposure to N. caninum in this population.

The sera used from the HPA epidemiology collection are not a random sample of the population, they are derived from residual blood samples obtained for diagnostic and screening tests. Selection bias for such residual sera is likely to be limited because the provision of free access to health care for all by the NHS. Each submitting laboratory offers a comprehensive diagnostic service thus substantial differences for the reason sera are submitted are unlikely. The collection may therefore be considered to approximate the general population of England in terms of its exposure to N.caninum and T.gondii.

We have performed the largest and most thorough N.caninum antibody prevalence study to date of human sera and have detected no evidence of human exposure. In addition we have tested serum samples from a cohort of individuals who may be considered to be at high risk of N.caninum infection, assuming that any infection in humans would occur via similar routes to human infections of T.gondii, with negative results. These results indicate that the risk to human in the UK of N. caninum infection is negligible. The results of the only study to date which has reported seropositivity in humans to N. caninum (6) need to be treated with great caution. That study utilised an IFAT and no frequency distributions were presented. The low titre responses observed may have indicated the extremes within a negative population.

2.2. Objective 02a: An intervention study to determine the effectiveness of embryo transfer to eliminate congenital infection

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This objective was not met in full because the commercial company who supplied the embryos and ova ceased

its embryo transfer programme 4 months after they started to supply us with samples.

2.2.1. Introduction

It is hypothesised that vertical transmission of N.caninum infection occurs pre-natally across the placenta post-implantation. Embryo transfer (ET) should thus prevent vertical transmission and eliminate infection from a given cattle germ line if embryos or ova are proven to be free of infection and are transferred into non-infected recipients. This could allow elimination of congenital infection from germ lines of high genetic merit.Three papers have recently been published describing small in vivo studies on the effectiveness of ET to eliminate vertical transmission of N.caninum in cattle. (17, 18, 19). In the largest of these studies, Baillargeon et al., (17) compared the results of transferring embryos from N.caninum seropositive and seronegative donors into seronegative recipients and embryos from seronegative donors into seropositive recipients. All calves born from embryos transferred into seronegative recipient cows were N.caninum seronegative; whilst 5 out of 6 calves born from the embryos transferred into the N.caninum seropositive cows were born N.caninum seropositive. In the light of the results of these papers a proposal to remove the in vivo study was discussed and agreed at the review of DEFRA’s non-food borne zoonosis research programme in May 2004. An alternative in vitro study was proposed and approved at the meeting. The aim of this study was to show that N.caninum infection can be eliminated from a given bovine germ line by ET. The specific objective was to examine unfertilised ova and embryos obtained from cows of known N.caninum antibody serostatus for the presence of N.caninum DNA by N.caninum - specific PCR.

2.2.2. Materials and method

2.2.2.1. Supply and collection of embryos

A commercial cattle breeding company agreed to supply us with unfertilised ova and grade 3 embryos that were not suitable for embryo transfer so that we could test them for evidence of N.caninum infection. Most of the embryos were derived from maiden cows that are brought into the Embryo Transfer Farm from other farms owned by the company. A few donors were older cows. Standard methods were used to produce the embryos, that is, the donors underwent super ovulation and artificial insemination prior to non-surgical embryo collection. The embryos were evaluated, categorised, washed and treated with trypsin according to standards set by the International Embryo Transfer Society. Visual inspection, to ensure integrity of the zona pellucida, was performed before and after the washing and trypsin treatment procedure. Grade 3 embryos and unfertilised ova were put in an eppendorf tube suspended in ViGroTM Holding PLus (Bioniche Animal Health) medium and sent to our laboratory together with a clotted blood sample from each donor cow. Information on the age of most of the donors was supplied.

2.2.2.2. Sample processing

Upon receipt of the blood samples, they were centrifuged and the serum removed and stored at -20oC prior to testing for N.caninum specific antibodies. DNA was extracted from the embryos and ova using a DNeasy kit (Qiagen, Crawley, UK). The DNA was eluted in 100µl of Buffer AE supplied with the kit.

2.2.2.3. Testing embryo DNA and bovine serum

To test for successful DNA extraction, all DNA samples were subject to PCR using primers that recognise the bovine 1.715 satellite DNA (SAT) (20).DNA extracted from the embryo and ova samples was then tested using a nested N.caninum specific PCR that amplifies the internal transcribed (ITS1) region as described by Uggla et al., (21), using primer pairs F5.8b/F6 and PN3/PN4). The N.caninum PCR was performed in duplicate for all samples. To establish the sensitivity of this PCR, DNA was extracted from a known quantity of N.caninum (Isolate NC-Liverpool) tachyzoites and PCR performed on serial 10-fold dilutions from 10-1 – 10-8. In addition, to test for any inhibition of the PCR by the embryos or medium, parallel PCRs were performed using serially diluted N.caninum DNA samples and equivalent DNA samples spiked with DNA extracted from bovine embryos/ova.

Sera from donor cows was tested for N.caninum antibodies using a commercial ELISA (Mastazyme, Mast Diagnostics, Bootle, UK) as previously described (14). The results were calculated as the optical density (OD) expressed as a percentage of a high positive control (percent positivity PP) (14). A cut off of PP ≥ 20 was used to classify cows as positive, PP ≥ 15 < 20 as inconclusive and PP < 15 as negative.

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2.2.3. Results

Embryos or ova were submitted from 36 cows, 25 animals were less than 18 months of age, 9 older than 18 months, age of 7 cows was not supplied. Information on whether a sample came from ova, embryos or both was not available. The N.caninum specific PCR was shown to be able to detect the DNA equivalent of that extracted from 1 x 10-1 N.caninum tachyzoites. The sensitivity was not reduced when DNA extracted from tachyzoites was spiked with DNA from embryos/ova. The results of the PCRs according to donor cow N.caninum serostatus are given in Table 6. All DNA extractions from ova/embryos were successful as shown by the positive PCR result for the bovine SAT region. All embryo/ova samples were negative for N.caninum DNA. One donor cow was N.caninum seropositive, 28 were seronegative and for 7 cows the ELISA result was inconclusive.

Due to the small sample size no statistical analysis was performed.

Table 6: Results of PCR tests of embryo/ova DNA for bovine SAT region and N.caninum according to N.caninum serostatus of donor as determined by ELISA

ELISA PP value Number of cows SAT PCR (no positive/no. tested)

N.caninum PCR (no positive/no tested)

< 15 (negative) 28 28/28 0/28≥15<20 (inconclusive) 7 7/7 0/7≥ 20 (positive) 1 1/1 0/1

2.2.4. DISCUSSION

Insufficient samples were obtained to test the hypothesis that ova are uninfected irrespective of the infection status of the dam.

2.2.5. FUTURE POSSIBLE WORK

It has been possible to develop a protocol that could be implemented in a large scale study to determine whether ova and embryos from cows that are N.caninum seropositive are free of N.caninum infection. It would be important, in such a study that information should be collected (if possible) about the infection status of the bulls used as sires if the DNA tested is derived from a bovine embryo as opposed to an ovum, since it has recently been shown that N. caninum DNA may be present in semen from seropositive bulls (22).

2.3. Objective 02bi: Primary objective: To determine the outcome of challenging pregnant cows with sporulated N.caninum oocysts at different stages of pregnancy. Secondary objective: To determine whether infection of cattle with oocysts during pregnancy can result in endogenous transplacental infection occurring in a subsequent pregnancy.

2.3.1. Introduction

Transplacental infection (TPI) is a major route of transmission of N. caninum to cattle and may result in either foetopathy or the birth of congenitally infected calves. Recently, two types of TPI have been defined, namely endogenous and exogenous TPI (23). Endogenous TPI refers to foetal infection occurring as a result of a recrudescence of maternal infection during pregnancy in dams which, probably (not proven), were themselves infected congenitally. Exogenous TPI occurs as a result of a de novo infection of the dam when pregnant by oocyst ingestion. Dogs and coyotes are definitive hosts of N.caninum and produce oocysts. (24, 25,). It is postulated that abortion storms may result from a point source of infection such as may occur when cattle ingest feed or water contaminated with oocysts. When this project began, although calves had been infected with oocysts (26), only one small experiment had previously been reported in which pregnant cows were so challenged (27). That experiment used three cows and a single time of challenge and none of the infections caused abortion or transplacental infection. Accordingly, to better determine the pathogenic potential of oocysts challenge in pregnancy, the current study was designed to compare the effects of challenge at three different times in pregnancy. Furthermore in view of the emerging significance of endogenous TPI (23), we extended the experiment by rebreeding seven cows to determine if they were able to endogenously infect their foetuses as a result of oocyst infection in the previous pregnancy . In order to establish the dose of viable oocysts used in the experiment we performed bioassays in gerbils (Meriones unguiculatus). Gerbils have been shown to be highly susceptible to oral infection with N.caninum oocysts with evidence of infection being shown following the administration of one oocyst (28, 27).

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2.3.2. Materials and methods

Cattle and gerbil N.caninum oocyst infections were carried out in accordance with the Animals (Scientific Procedures) Act, 1986 under licence from the Home Office (Project License #PP40/23/63).

2.3.2.1.Production and supply of N.caninum oocysts

N.caninum oocysts were produced in March 2003 at the University of Illinois, by Dr Milt McAllister, from a hound pup fed mixed tissue from 4 calves that had been inoculated intravenously with N.caninum (Isolate NC- Liverpool) tachyzoites. Oocysts shed by the pup were collected, purified and sporulated as previously described (29). Approximately 792,000 sporulated oocysts, suspended in 2 litres (L) of dilute sulphuric acid, in 4 separate bottles, were stored at 4oC prior to being shipped to Liverpool.

2.3.2.2. N.caninum oocyst infection of cattle

Twenty-one Freisian-Holstein maiden cows were purchased from local farms and housed at the University of Liverpool’s Animal Husbandry Farm where they were loose housed on straw bedding. Serological tests for evidence of infection with common abortifacient agents (N.caninum, Bovine Viral Diarrhoea (BVD), Infectious Bovine Rhinotracheitis (IBR) and Leptospira hardjo) were performed by the Veterinary Laboratory Agency and gave negative results. N.caninum-specific ELISAs (Mastazyme) were also performed on sera from each cow on two occasions, prior to N.caninum oocyst infection, to confirm that the cattle had not previously been exposed to N.caninum. The cattle were vaccinated for BVD (Bovidec, Novartis Animal Health UK Ltd., Hertfordshire, England ) and also given a prophylactic 3-day course of streptomycin at 25mg/kg (Streptocare, Animalcare, Dunnington, York, England) because several animals came from herds in which other cattle were seropositive for L. hardjo.

Oestrus was synchronised by means of a progesterone releasing device (PRID, Ceva Animal Health Ltd., Chesham, Buckinghamshire, England) and the cows were artificially inseminated. Pregnancy was confirmed by transrectal ultrasonography 35 days later. The oocysts were stored at 4oC upon arrival in Liverpool. The four batches of oocysts were mixed and all doses drawn from a single pool. Doses of a nominal 40,000 oocysts, based on counts provided by McAllister, were prepared shortly before they were administered to the cows. The appropriate volume of oocysts suspended in 2% sulphuric acid was reduced to 20ml by centrifugation and made up to 1L of water for dosing. No attempt was made to count oocysts because the numbers available and their density was so low. Cows were orally dosed with the oocysts and immediately afterwards were dosed with a further 1L of water. Eighteen animals were challenged with the oocysts between January and April 2004 in three groups of 6 animals. Group 1 were challenged on Day 70 of pregnancy, Group 2 on Day 120 and Group 3 on Day 210. At the time when each group of animals was challenged, one control animal was dosed with 1L of water containing 20mls of 2% sulphuric acid. For 48 h after dosing with oocysts, animals were isolated from the control animal and kept on sawdust which was later collected and incinerated. The control animal was then kept with the challenged cattle and acted as a sentinel for adventitious infection. Foetal viability was assessed three times a week by transrectal ultrasonography for the first month post-challenge and thereafter weekly. If a foetus was found to be non-viable as indicated by the absence of a heart beat, it was checked again after 24 hours to confirm foetal death. One cow in Group 2 was found to have a non-viable foetus 33 days after the challenge and was injected with an anti-progestative (Alizin, Virbac Ltd. England) to induce expulsion of the foetus. Pre-colostral blood samples were collected from all calves after birth.

2.3.2.3. Gerbil bioassay

Bioassay was performed in gerbils in December 2003, prior to the first group of cattle being challenged, and at the same time as each challenge. Doses of 100, 101, 102 and 103 oocysts were prepared by measuring the appropriate volumes of sulphuric acid containing the oocysts and centrifuging (1,500g, 15min), the supernatant was removed and the oocysts washed by resuspending in PBS and centrifuging. The washing was repeated two more times before each dose was finally prepared in 300µl PBS. Two control gerbils in each group were dosed with PBS alone. Blood samples were taken prior to infection and at weekly intervals from the tail vein and tested for antibody to N. caninum using an inhibition ELISA (12). Samples that were confirmed positive were also tested by IFAT as previously described (30), using tachyzoites of the Nc-Liverpool strain of N.caninum at a cut-off value of 1:50. The conjugate used was an anti-murine IgG FITC (Sigma). The gerbils were euthanased after 28 – 42 days. At post mortem brain samples were taken under sterile conditions for parasite isolation and for PCR. Isolation of N.caninum from brain tissue was attempted by in-vitro culture as described by Barber et al., (31) and cultures were examined every 2 days for the presence of tachyzoites. DNA was extracted and purified from brain and heart tissue with a DNeasy kit (Qiagen, Crawley, UK). Replicate PCR reactions were carried out using a modification of the nested PCR reaction of Uggla et al., (21). The PCR reactions were performed with Biotaq DNA

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Polymerase (1.25 IU per reaction). The positive control was N.caninum tachyzoite DNA and the negative control ultra-pure water. PCR products were analysed on 2% agarose gels stained with ethidium bromide and visualised under ultraviolet light. The proportion of oocysts that was viable was estimated from the proportion of gerbils infected in each titration group as shown by seroconversion and it was assumed that one or more viable oocysts would always cause infection and that no oocysts were lost in washing the inoculum dose.

2.3.2.4. Immunological responses in cattle.

Blood samples were collected one week prior to oocyst challenge and thereafter weekly until calving, by jugular venepuncture. Serum was tested for N. caninum antibody using a commercial ELISA (Mastazyme, Mast Diagnostics, Bootle,UK) as previously described (14). The results were calculated as the optical density (OD) expressed as a percentage of a high positive control (percent positivity PP) (14). Calf pre-colostral serum samples were also tested using this ELISA. A cut off of PP ≥ 20 was used to classify cows and calves as seropositive.

From the day of oocyst challenge and weekly post challenge (pc) for 8 weeks and thereafter monthly, blood samples were collected into heparinised vacutainers. N.caninum antigen-specific PBMC proliferation was determined as previously described (32) but using OptiprepTM (density 1.320g/ml; Axishield, Norway) and expressed as a Stimulation Index (SI). For IFN-γ assays, duplicate cell cultures were set up and supernatants were harvested after 3 days, centrifuged at 10,000g for 5 min and stored at -20 oC (32. Williams et al, 2000). IFN- levels were subsequently measured in the culture supernatants using the BOVIGAM IFN-γ test kit (CSL, Australia). The levels of IFN- in test samples were quantified using a standard curve, derived from a series of dilutions of a recombinant bovine standard (0.95ng/ml; Ciba Geigy, Switzerland).

Mean PBMC proliferative responses and IFN- responses were calculated for the three sentinel animals as the mean of the weekly values obtained for the 8 weeks following oocyst challenge of the infected heifers.

2.3.2.5 Necropsy of aborted foetuses and neonatal calves

Calves were euthanased within 14 days of birth by intravenous injection of 20% (w/v) pentobarbitone sodium (Euthatal, Merial Animal Health, UK). Samples of brain, heart, kidney, lung, skeletal muscle (from front and hind limbs) and, when available placental tissue, were taken from each aborted foetus and calf, and stored at -20oC for PCR or fixed in 10% neutral buffered formalin (NBF). In vitro culture of parasites from calf brain and heart tissues was done using the method of Barber et al., (31). The cultures were examined for evidence of tachyzoites every 2 days, until 8 weeks post inoculation. For each calf and foetus, six 50mg samples from different regions of the brain were pooled, and homogenised in liquid nitrogen. DNA extraction and purification was performed on six 50mg samples extracted from the pooled tissues using a DNeasy kit (Qiagen, Crawley, UK). DNA preparations were also made from 50mg samples of heart tissues from each calf and foetus and from other tissues from aborted foetuses. Replicate PCRs were performed on DNA from the brain samples and other tissues using the nested PCR reaction of Uggla et al., (21) as for the gerbil samples.

Immunohistology for the demonstration of N.caninum was performed on neonatal calves and the aborted fetuses using peroxidase anti-peroxidase (PAP) method. Briefly, after deparaffination and blocking of endogenous peroxidase, sections were incubated with a rabbit anti-N.caninum antiserum at 4C for 15-18 h. They were then washed and incubated with swine anti-rabbit IgG and rabbit PAP complex and the enzyme substrate diaminobenzidin. As a negative control, consecutive slides were incubated with tris-buffered saline. An N.caninum-infected cell pellet served as positive control.

2.3.2.6. Rebreeding of cattle

Seven cows were selected for rebreeding according to the immunological results and the outcome of the pregnancy during which they were given oocysts Five cows had a N.caninum infected calf or foetus, one cow had an undiagnosed abortion, and one cow had a persistently high antibody response from three weeks post-infection. Animals were rebred by artificial insemination following oestrous synchronisation as before. Three uninfected control animals were co-housed but not rebred. Foetal viability and N. caninum-specific antibody responses were monitored as before. Precolostral blood samples were collected from all calves at birth and tested for N. caninum-specific antibodies as before. Calves were euthansed and subject to post mortem as previously. N. caninum-specific PCR’s were performed on DNA extracted from calf brain and heart tissue. Oocyst infected cows were euthansed within two weeks of calving, and brain removed for analysis by PCR. For each cow, forty 50mg samples from different areas of the brain were pooled and homogenised in liquid nitrogen. Ten samples of 50mg were removed from each pool and DNA extraction and purification performed as before. All DNA samples were subject to PCR using the primers of Uggla et al., (21).

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2.3.3. Results

2.3.3.1. Gerbil bioassay

N.caninum infection was confirmed in 10 of the gerbils dosed with oocysts (Table 7). The minimum nominal dose that resulted in infection was 100 oocysts. DNA was detected by PCR in eight of the gerbils that were positive by serology but N.caninum was not isolated in vitro from any of the gerbils. After preliminary examination of the data the results from challenging gerbils with 1000 oocysts (4/4 infected) were ignored because it seemed likely that more than one viable oocyst would have been present in each challenge. For dosages of 100 or less oocysts it was assumed that not more than one viable oocyst would be present in the challenge dose. The remaining results (from 30 gerbils with a dose of between 1 and 100 oocysts) were pooled: they showed that from 1892 oocysts there were 6 infections. From this we calculated that the proportion of viable oocysts was 1/315.3 = 0.003171, with 95% Exact Confidence limits being 0.001165, 0.006890. When multiplied up to 40,000 this meant that each cow received an estimated 127 viable oocysts (95% Confidence Limits, 47 – 276). There was no evidence that the viability of oocysts decreased during the 5 month period that the bioassays were carried out.

Table 7: Results of bioassay of N.caninum oocysts given to gerbils, throughout the duration of the experiment, according to number of gerbils that seroconverted in each dosage group (0 – 103 oocysts)

Date infected Number of oocysts0 100 101 102 103

Dec 2003 0/2 0/2 0/2 1/2 ndJan 2004 0/2 0/2 0/2 0/2 ndFeb 2004 0/2 nd 0/2 1/2 2/2Apr 2004 0/2 nd 0/2 4/12 2/2

nd = not done

2.3.3.2. Infection of cows

All 18 cows showed evidence of systemic infection as shown by a N.caninum - specific PBMC proliferative response of a SI of at least 15. Fourteen cows showed an N.caninum -specific IgG response; the mean IgG response for each group is shown in Figure 2. An IFN- response of at least 3ng/ml was detected in 17 cows. The mean PBMC proliferation and IFN-γ responses are shown in Figures 3 and 4 respectively. No evidence of N.caninum infection was found in the three sentinel cows or their calves (one cow failed to become pregnant). IgG responses are shown in Figure 2. The mean PBMC proliferative response was an SI of 6 (Figure 3) and the mean IFN- response in the controls was 0.6ng/ml (Figure 4)

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The outcome of the N.caninum oocyst challenge of pregnant cows is summarised in Table 8. All cows infected at Day 70 of pregnancy gave birth to live uninfected calves at term. Four cows dosed at Day 120 also gave birth to live uninfected calves at term and a fifth calf born to this group was stillborn but uninfected. The sixth cow dosed at Day 120 aborted 33 days post challenge (pc) and the foetus was shown to be infected with N.caninum as shown by a positive PCR of brain and cotyledon tissue. In addition parasites were identified within skeletal muscle by immunohistology. One cow in the group dosed at Day 210 aborted 22 days pc but the cause of the abortion was not established, however this cow did have an N.caninum - specific antibody response. Four cows in this group gave birth to live congenitally infected calves which had positive precolostral N.caninum – specific antibodies and in one N.caninum DNA was detected. The sixth cow, which did not have an N.caninum – specific IgG response, gave birth to a live uninfected calf.

Table 8 : Results of oocyst infection of first gestation cows at different stages of pregnancy

Cows CalvesGroup Time in

gestation(days)

Nc – specific antibodies(PP≥20 )

IFN – γ response(> 1ng/ml)

PBMC proliferation(SI > 15)

Abortion Parasite DNA (foetus)

Precolostral Nc – specific antibodies

Parasite DNA

1 70 4/6 5/6 6/6 0/6 0/6 0/62 120 5/6 6/6 6/6 1/6 1/1 0/5 * 0/5 *3 210 5/6 6/6 6/6 1/6 0/1 4/5 1/5

Footnote: * includes one stillborn calf.

2.3.3.3. Rebred cows

The results (outcome of pregnancy and immune response) for their first pregnancy when challenged with oocysts are summarised in Table 9. In their second pregnancy all 7 animals gave birth to live uninfected calves at term. All PCR analyses for N.caninum DNA in the cow brains and calf brain and heart tissue were negative. There was no evidence of recrudescence of N.caninum infection in the cows as shown by the N.caninum - specific IgG responses recorded throughout pregnancy (Figure 5). Only two cows were N.caninum seropositive at calving and 5 cows had a PP value at calving that was less than the PP value at the start of pregnancy (Table 10). PP values for 6 of the animals showed some fluctuations during pregnancy however the maximum increase seen over a two week period was approximately 10 PP. The ELISA PP values of the 3 control cows stayed at about 10 PP throughout the experiment.

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Table 9: Cows selected for rebreeding: first pregnancy outcome and immunological responses following oocyst challenge.

Cow ID#

Day pregnancy oocyst challenge

Pregnancy outcome

Calf/foetus infected with N.caninum

Maximum ELISA PP

Maximum PBMC Proliferation (SI)

Maximum IFN – γ response (ng/ml)

165 210 Live calf Yes 63 235 14.0142 210 Live calf Yes 40 15 21.896 210 Live calf Yes 44 283 3.3187 210 Live calf Yes 68 178 5.084 120 Abortion Yes 78 58 3.6139 210 Abortion No 24 115 11.595 70 Live calf No 65 67 1.4

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Table 10: Rebred cows: N.caninum - specific antibody responses during second pregnancy and pregnancy outcome.

Cow ID #

Interval (weeks) between oocyst challenge and AI for rebreeding

ELISA PP at AI

ELISA PP at 20 weeks in gestation

ELISA PP at calving

Pregnancy outcome

Calf infected with N.caninum (precolostral serology & PCR)

Cow infected with N.caninum (PCR)

165 15 60 25 19 Live calf No No142 15 48 36 31 Live calf No No96 15 61 17 16 Live calf No No187 15 29 26 21 Live calf No No84 29 32 26 17 Live calf No No139 18 5 10 8 Live calf No No95 15 18 21 18 Live calf No No

2.3.4. Discussion

In the first part of this experiment, 18 maiden cows challenged with a nominal dose of 40,000 N.caninum oocysts showed evidence of systemic infection as demonstrated by an N.caninum - specific PBMC proliferative response. In addition, 14 cows became seropositive as shown by an N.caninum-specific ELISA PP value of ≥ 20 and 17 cows had an IFN- response. All cows had at least two immunological indicators of exposure, namely antigen-specific PBMC proliferation together with an antigen-specific antibody response and/or an antigen-specific IFN- response. Exogenous TPI occurred in 5 of the animals but not in any of the cows infected at 70 days. One animal infected at 120 days had an N.caninum-associated abortion and 4 cows infected at 210 days gave birth to congenitally infected calves. These results concur well with, and extend results of, previous experiments when cows have been given an exogenous N.caninum infection. Trees et al., (27) infected 3 cows with 600 N.caninum oocysts at Day 70 and all gave birth to uninfected calves at term. In a more recent study in the US concurrent with this one (33), 19 pregnant cows were given nominal doses between 1,500 – 115,000 oocysts between Days 70 – 176 of pregnancy. One cow infected at Day 120 with 41,000 oocysts aborted and 6 cows gave birth to congenitally infected calves with the risk of TPI increasing the later the time of oocyst challenge. In our experiment the timing of oocyst dose also appeared to influence outcome, with abortion associated with mid-term challenge and viable infected calves born only to cows in the late challenge group. These results are similar to those following intravenous tachyzoite challenge (32). One major difference from the results of Williams et al (32) is the failure of oocyst challenge at 70 days of gestation to cause abortion, something also observed by Gondim et al (33).

The maiden cows were challenged with a nominal dose of 40,000 oocysts, but the results of the bioassay in gerbils suggested that a minimum of 0.3% of the oocysts were viable, giving a minimum dose of 127 viable oocysts. Several assumptions have been made in calculating this dose. Firstly that it takes only one oocyst to infect a gerbil – based on previous studies this is a well founded assumption (28, 27); secondly, that the initial oocyst count was accurate; and thirdly that there was no loss of oocysts when the H2SO4 was removed by washing with PBS prior to dosing the gerbils. In fact, it is probable that substantial numbers of oocysts were lost in the centrifugation and re-suspension steps involved in preparing the gerbil dose. From previous experience with coccidial oocysts, the principal investigator is aware such losses can be considerable. It was precisely for this reason that, for cows, oocysts were not washed but instead the preservative H2SO4 was diluted in excess water and given in a large volume of oral innoculum. Thus, whilst loss of oocysts would have occurred for gerbils it was minimised for cows; hence it is probable that the cattle dose substantially exceeded 127 oocysts although it was less than 40,000. Whilst immune responses are not linearly related to oocyst dose, the responses in this experiment are less than those described following a dose of 600 oocysts (confirmed by bioassay)at 70 days gestation (27) – which is consistent with the estimated dose above. In the only other study involving oocyst dosing of cattle by Gondim et al (33) bioassays in gerbils were not conducted. Loss of viability of the oocysts may have been due to the prolonged storage prior to dosing the cows or while the oocysts were being shipped to England, but this was unavoidable. There is a major logistic problem in conducting oocysts experiments in cattle with N. caninum in that a number of dog infections need to be conducted in order to accumulate an adequate number of oocysts but, storage involves loss of viability. Thus it is important to conduct in vivo bioassays at the time of oral dosing of cattle.

There was no evidence that these cows carried a chronic infection which was transmissible in their subsequent pregnancy. None of the calves born in the second pregnancy was infected. Moreover, none of the

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cows showed an acute rise in antibody level in the second pregnancy. Such an antibody “surge” is characteristic of naturally-infected, persistently infected cows in which endogenous TPI occurs (34). Finally, despite comprehensive DNA sampling of the cows’ brains after the second pregnancy, it was not possible to detect N.caninum DNA in them by PCR. Thus, there is no evidence from this experiment, that a post-natal infection with oocysts, which itself is able to cause exogenous TPI, is able to establish a chronic maternal infection which may lead to endogenous TPI. This has important implications in the epidemiology of bovine neosporosis

In summary, we have demonstrated that exogenous TPI may occur in cattle as a result of the administration of N.caninum oocysts during pregnancy, that the timing of the challenge is crucial to the outcome and that exogenous TPI can occur following ingestion of relatively small numbers of viable oocysts. However, abortion was only induced in one cow. By rebreeding seven cows which had immune responses to the original challenge, and five of which had been systemically infected (they infected their foetuses), we have been unable to demonstrate endogenous TPI in a subsequent pregnancy. This suggests that cows post-natally infected with oocysts are unlikely to infect their subsequent progeny. Given that they are likely to be able to resist exogenous TPI (based on the results of Williams et al, 2003 and Williams et al, unpublished observations), in field situations, consideration should be given to retaining such cows in the herd.

2.4. Objective 02bii: To determine the prevalence and intensity of natural oocyst shedding in certain canid populations

2.4.1. Introduction

Whilst the experimental infection of dogs has been shown to lead to the excretion of N.caninum oocysts (24, 36, 37), there are few reports of the natural excretion of oocysts in dogs and only two recent faecal surveys of dog populations ( 38, 39). The experimental infections have led to the excretion of relatively low numbers of oocysts (e.g. 103 – 106 per patency compared to Toxoplasma gondii in cats of 107 – 108) and raise important questions about the possible role of canine oocysts in the epidemiology of bovine N.caninum infections and abortion. This element of the project sought to determine oocyst excretion prevalence and intensity in natural canid populations.

Examination of canid faeces for low numbers of small oocysts (10 -12 μm) is made more difficult because the morphologically similar Hammondia heydorni also occurs in dogs. Its taxonomic relationship to N.caninum has been the subject of bitter controversy (40), although the current consensus is that it is a distinct parasite (41), not associated with disease in cattle. Accordingly, in addition to semi-quantitative faecal floatation as a screening method, bioassay and PCR identification were employed in this study.

2.4.2.Material and methods

Gerbil and mice N.caninum infections were carried out in accordance with the Animals (Scientific Procedures) Act, 1986 under licence from the Home Office (Project License #PP40/23/63).

2.4.2.1. Collection of faecal samples

Farm dogs: Samples were collected from dogs on dairy farms in Cheshire (see Section 2.5 - Objective 02c). Approximately one week prior to a farm visit to take blood samples (see later), sterile plastic containers were sent to the farms during October and November 2003 for farmers to collect faecal samples. The samples were stored refrigerated. Foxhounds: With the assistance of the Master of Foxhounds Association and their website, twelve foxhound kennels in England and Wales were visited between January and December 2003. At each visit, fresh faecal samples were collected as they were passed so that the identity, age and sex of each hound for each sample was known. Information was collected by questionnaire on the size of the pack, and its health, husbandry, and feeding practices (Appendix 1).

Pet Dogs: Faecal samples submitted by the University of Liverpool’s Small Animal Hospital to the Veterinary Parasitology Laboratory for routine parasitological examination during 2003 and 2004 were examined for oocysts resembling those of N.caninum. The samples were collected from pet dogs that were referred to the Small Animal Hospital for a variety of conditions including gastrointestinal disorders.

2.4.2.2. Screening of samples

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Faecal samples were screened for the presence of oocysts that morphologically resembled N.caninum and H. heydorni, by microscopy after centrifugal floatation in saturated sodium chloride of 1g samples using a semi-quantitative cover slip method (42).

2.4.2.3. Concentration of oocysts and DNA extraction

For faeces in which oocysts had been observed, oocysts were extracted and concentrated from 10 g of the sample as previously described (42), with the final pellet of oocysts being resuspended in phosphate buffered saline (PBS). DNA was subsequently isolated and purified using the DNeasy® tissue kit (Qiagen, Crawley, Sussex, England), according to the manufacturer’s instructions. The DNA was eluted in 100µl of Buffer AE supplied with the kit and stored at -20oC. For samples that were initially PCR negative, further DNA extractions were made from oocysts, using a QIAamp® DNA Stool Mini Kit. (Qiagen, Crawley, Sussex, England). This kit employs a reagent “InhibitEX”, which absorbs DNA-damaging compounds and PCR inhibitors present in faeces.

2.4.2.4. PCRs to confirm the identity of the oocysts

DNA was examined by PCR using the apicomplexan generic primers COC-1 and COC-2 (43), the N.caninum –specific nested PCR of Uggla et al., (21), and the H.heydorni-specific primers of Šlapeta et al., (38). All PCRs were carried out in duplicate. The positive controls were N.caninum DNA extracted from tachyzoites of NC-Liverpool and H.heydorni DNA extracted from oocysts of H.heydorni Giessen – 1999 (AY 189897, 39), kindly supplied by Dr. G. Schares. The negative control for each reaction was ultra-pure water.

2.4.2.6. Bioassay of oocysts and in-vitro culture

Samples in which more than 10 oocysts / g were found were subject to bioassay in gerbils. From 10g of faeces, oocysts were concentrated (as above) and mixed with 2% potassium dichromate and left at room temperature in a Petri dish for 4 days to sporulate. Once sporulation was confirmed by visual examination the oocysts were washed three times in PBS. For one sample (#79) the number of sporulated oocysts / ml of suspension was estimated by counting the oocysts present in 10µl. Three groups of two gerbils were infected with 101, 102 and 103 oocysts suspended in 0.3ml PBS by gavage. For two other samples (#238 and #188), oocysts could not be counted prior to infecting the gerbils; each sample was used to infect two gerbils, two sentinel gerbils were dosed with PBS alone.

For in-vitro culture of oocysts, following sporulation of oocysts, the oocysts were washed and pelleted by centrifugation and the outer layer of the oocyst wall removed by resuspending the pellet in 500µl 2% w/v sodium hypochlorite solution in 16.5% w/v sodium chloride (Milton’s fluid) and leaving at room temperature for 30 min. The suspension was centrifuged (700g, 10 min) and the oocyst-rich supernatant added to 5 times its original volume of sterile PBS. It was then centrifuged (1,600g , 10 min), the supernatant removed and the pellet resuspended in 100µl PBS. The suspension was vortexed to disrupt the sporocysts, then 400-500µl excystation fluid, (0.25% trypsin and 5% sodium taurocholate in PBS ,pH 7.3), was added to the supernatant and it was incubated at 37oC for 30 minutes. The suspension was then centrifuged (1,600 g, 10 min) and the supernatant removed. The pellet was resuspended in Roswell Park Memorial Institute (RPMI) medium with 10% horse serum and 1% penicillin/streptomycin and cultured in a 25cm3 flask containing a mono-layer of Vero cells. (The medium in the flask was changed after 24 hours and the cells passaged onto new Vero cells 11 days later). After tachyzoites had been observed in culture, two BALBc mice were infected with 1 x 104 tachyzoites, one week after they had been immunosuppressed by the injection of 0.05ml (40mg/ml) of methylprednisolone (Depo-medrone –V).

For gerbils and mice, blood samples were taken prior to infection and at weekly intervals from the tail vein and tested for the presence of Neospora -specific antibody using an inhibition ELISA (12) and IFAT, using tachyzoites of N.caninum NC-Liverpool as antigen, at a cut off value of 1:50. The positive controls used were serum from a gerbil that had been infected with 1,000 N.caninum (Isolate NC-Liverpool) oocysts and serum from a mouse that had been infected with 105 N.caninum NC-Liverpool tachyzoites. An anti-mouse IgG FITC (Sigma) was used as conjugate. The mice and gerbils were euthanased at 28 – 35 days post-infection. At post mortem brain, heart, liver and lung samples were taken for parasite isolation and PCR. DNA was extracted and purified from 25mg of tissue with a DNeasy® tissue kit, as for the oocysts. Replicate PCRs were carried out to test the DNA for N.caninum and H.heydorni as above. In vitro parasite isolation of gerbil and mice tissues was done according to the method of Barber et al., (31). The cultures were examined every 2 days for the presence of tachyzoites.

2.4.3. Results

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2.4.3.1. Screening of samples

Faecal samples were collected and examined from 261 foxhounds, 69 farm dogs and 105 pet dogs (Table 10). On microscopic examination, 15 samples had oocysts that resembled N.caninum /H.heydorni. Thirteen of these samples were from foxhounds; there was one positive sample each from the farm dogs and pet dogs groups. The oocysts ranged in size from 10.4 – 13.1 µm x 10.4 – 13.8 µm. DNA extracted from nine of the samples was positive in the PCR using primers COC1 /COC2 that replicates apicomplexan SSU-rDNA. All 15 of the oocyst samples were positive using the JS4/JS5 primers thus confirming their identity as H.heydorni. However one of these samples (#188) was also positive using the N.caninum specific primers suggesting that the sample was a mixed infection of N.caninum and H.heydorni (Table 11).

2.4.3.2. Bioassay and in-vitro culture

None of the gerbil infections with putative N.caninum oocysts induced an N.caninum specific immune response as shown by negative results in the inhibition ELISA and IFATs performed. Tissues of gerbils inoculated with oocysts from samples #188 and #238 were negative in all PCRs. Tissues from gerbils infected with oocyst sample #79 were negative in N.caninum - specific PCRs and positive in H.heydorni - PCRs. Results of in-vitro culture of oocyst from sample #79 and mouse bioassay were inconclusive.

Table 10: Results of coprological screening of faecal samples from 3 canid groups for oocysts resembling N.caninum / H.heydorni

Canid Group

Numbers sampled/Numbers on premises sampled

No. with oocysts resembling N.caninum /H.heydorni

Positive Apicomplexan PCR

Positive H.heydorni PCR

Positive N.caninum PCR

Foxhounds 261/1332 13 (4.98%) 9 13 1

Farm Dogs 69/149 1(1.45%) 0 1 0Pet Dogs 105 randomly

selected from submitted samples

1(0.95%) 0 1 0

Table 11: Results of tests performed to confirm identity of oocysts resembling N.caninum / H.heydorni

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Sample #

Canid Group

Oocyst/g Apicomplexan PCR

N.caninum PCR

H.heydorni PCR

PCR Gerbil Tissues

Serology Gerbil

79 Foxhound >1,500 + ve - ve + ve -ve N.caninum +ve

H.heydorni

-ve N.caninum

81 Foxhound <10 + ve - ve + ve nd nd115 Foxhound <10 - ve - ve + ve nd nd118 Foxhound <10 + ve - ve + ve nd nd124 Foxhound <10 + ve - ve + ve nd nd130* Foxhound <10 + ve - ve + ve nd nd238 Foxhound ~32 + ve - ve + ve -ve N.caninum

-ve H.heydorni-ve

N.caninum41 Foxhound <10 - ve - ve + ve nd nd49 Foxhound <10 - ve - ve + ve nd nd51 Foxhound <10 + ve - ve + ve nd nd143 Foxhound <10 - ve - ve + ve nd nd144 Foxhound <10 - ve - ve + ve nd nd188 Foxhound ~100 + ve + ve + ve -ve N.caninum

-ve H.heydorni-ve

N.caninumWD 104 Farm Dog <10 - ve - ve + ve nd ndP 177 Pet Dog <10 - ve - ve + ve nd nd

* Sample taken from same dog as sample #79 one week laternd = not done

2.4.3.2. Questionnaire

Questionnaires were completed at the 12 foxhound kennels visited. All kennels fed the foxhounds with bovine carcasses and 5 kennels also fed ovine carcasses. All kennels were registered under the Animal By-Products Order 1999 and adhered to current Specified Risk Material (SRM) controls. None of the kennels reported symptoms of neural disease in puppies or had had clinical neosporosis diagnosed.

2.4.4. Discussion

In this study only one faecal sample containing putative oocysts was shown to contain oocysts of N.caninum by PCR and this appeared to be a mixed infection with H.heydorni. There has only been one previous report of N.caninum oocysts in a naturally infected dog in the UK (42). In each case, N.caninum oocysts had been shed by foxhounds. All foxhound kennels visited reported that foxhounds were fed bovine tissues, however the prevalence of shedding in foxhounds appears to be very low. These results concur well with other studies which found a very low prevalence of N.caninum oocyst shedding. In Germany, 24,089 canine faecal samples were examined, oocysts of 9 - 14µm were found in 47 samples however following gerbil bioassay only 7 induced an N.caninum specific antibody response (39). Whilst the objective of that study had been to generate a crude estimate of N.caninum shedding in dogs in Germany the authors stated that the samples were more likely to have come from cities and that farm dogs were under represented in the study. The dogs were not randomly chosen and 70% of the samples had been submitted from dogs with diarrhoea. In the Czech Republic, 3155 faecal samples from 2240 dogs, most of which were German Shepherds, were screened for the presence of N.caninum /H.heydorni oocysts, five were positive but these were all later shown to be oocysts of H.heydorni (38).

In conclusion in this study involving 3 canid populations, only one faecal sample was positive for low number of N.caninum oocysts,. This suggests that the prevalence and intensity of N.caninum shedding by canids in England is low. An association was found between herd seroprevalence and seropositivity in dogs. It is not possible to conclude in which direction infection predominantly flows from this association, but given the findings we have reported earlier, with respect to oocyst infection causing transplacental infection in cattle farmers should be advised to minimise contact between dogs and cattle. All calving membranes should be properly disposed of and attempts made to prevent dogs defecating in either cattle feeding areas or stored feed.

In this study gerbils fed oocysts, that were later identified as H.heydorni oocysts, were successfully infected as shown by positive PCR amplification of DNA extracted from heart and brain tissues by H.heydorni-specific primers.

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2.4.5. Further Work

In this study cross sectional sampling of several canid populations was performed to estimate prevalence of N.caninum oocyst shedding. This method would not indicate if animals sampled had ever excreted N.caninum oocysts. To better assess the risk of N.caninum oocyst shedding by any canid group that has access to potentially infected intermediate host material, such as farm dogs and foxhounds, it would be necessary to perform an intensive study, with regular (2 – 3 times weekly) faecal sampling from the time of first exposure to potentially infective material.

2.5. Objective 02c: To determine the potential risk factors for Neospora associated infection in cattle and dogs.

2.5.1. Introduction

No study of on-farm risk factors associated with N.caninum infection has been carried out in the UK. In order to better inform management practice to reduce the prevalence of infection and the incidence of N.caninum –associated abortion, a cross-sectional study was carried out in Cheshire dairy farms. This study was conducted in conjunction with a parallel investigation by S Felstead in which a bulk-milk tank ELISA was developed to determine within-herd N.caninum prevalence in these dairy herds.

2.5.2. Materials and method

2.5.2.1. Populations studied

One hundred and twenty of the 160 dairy farms that used the veterinary services of a single veterinary practice, had volunteered to participate in a prospective study two years previously (S. Felstead, 2005, PhD thesis). Ninety six of the farms that had submitted bulk milk samples at least once every three months were included in this study. The farmers were sent a questionnaire (Appendix 2) which was conducted by telephone or face to face. They were also sent a container to collect faecal samples from their dogs (see Objective 02bii). A visit was made to the farms during October and November 2003 during which blood samples, to be used to test the dogs for N.caninum antibodies, were taken from farm dogs at the request of farmers, by the Veterinary Research Assistant (S. Felstead) working in association with the farmers’ veterinary practice.

2.5.2.2. Variables studied

The questionnaire, comprising 57 mostly closed questions with a total of 155 variables, collected data on farm demography, husbandry practices such as disposal of placentas and dead foetuses, housing nutrition, the presence of other animals on farms, fertility and production parameters. These are summarised in Table 12.

2.5.2.3. Analysis of samples

Blood samples were collected by venipuncture from the brachial vein into serum gel microtubes, incubated overnight and the serum was removed after centrifugation and stored at -20oC. The serum was tested for the presence of N.caninum - specific IgG antibodies using the indirect fluorescent antibody test (IFAT) as described by Trees et al., (30). All sera were tested at dilutions of 1:50, 1:200 and 1:800 in PBS. For cattle, herd prevalences were kindly provided by S. Felstead. They were estimated using a commercial N.caninum - specific ELISA (Mastazyme, Mast Diagnostics, Bootle, UK) used on herd bulk milk tank samples using methods developed by S Felstead (PhD thesis, 2005) to calculate the percentage N.caninum positive cows in each herd for each year that bulk samples had been collected.

Table 12: Details of the information collected by questionnaire that was used to determine risk factors for N.caninum infection in cows and farm dogs.

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General Farm Characteristics: Farm type including other enterprises; Number of cows in milk; average annual milk yield; breed; source and type of cattle purchased; number of family lines; abortifacant diseases diagnosed/vaccinated against; percentage culled annually and reason for culling; management changes; action taken when cattle are diagnosed as N.caninum seropositive.Fertility and calving: Number of calves born each month; calving intervals and other fertility indices; frequency of pregnancy diagnosis, if practised; use of artificial insemination /natural service/sweeper bull/ embryo transfer; calving accommodation; disposal of afterbirths/dead foetuses& calves.Feed management & Housing: Components of ration during housing and grazing period for milking and dry cows and heifers; whether ration is mixed; whether mouldy silage is fed to cattle; frequency of cleaning feeding troughs and disposal of leftover feed; where cattle are housed during housing period; zero grazing; strip grazing; buffer feeding; location of cattle away from farm during year; drinking water sources; over wintering of sheep; public access to farmland.Other species: Frequency that dogs, cats, foxes and badger have been seen on farmland and in farm buildings/yards; main species of birds in buildings/yards; types of feeds stored where any other animals could enter; number and age of farm dogs; introduction of new dogs on farm; bitches whelped on farm and details of puppies born and any illness; Areas on farm that dogs have been seen with cattle; dogs seen eating afterbirths and /or foetuses/dead calves; hound packs cross farm land.

2.5.2.4. Data management and analysis

Each questionnaire was checked for completeness, errors and inconsistencies. Corrections were made either at the time of the farm visit of later during subsequent telephone calls. The answers were coded for data entry and entered into date files created in Epi-info software programme (Centre for Disease Control and Prevention, Atlanta, USA).

The statistical programme STATA (Stata Corporation, Texas, USA) was used to analyse the data from the questionnaire together with the results of the dog serology and herd N.caninum prevalence rates. Farms that did not have a dog or whose dog(s) was/were not tested were excluded from further analysis. The mean annual prevalence of N.caninum seropositivity in each herd was calculated from the annual prevalence for 2002 and 2003. A cut off value of 6% was chosen in order to categorise herds as seropositive or seronegative. In order to study the possible association between environmental characteristics (explanatory variables) and N.caninum infection in farm dogs and N.caninum infection in cows(outcome variables), univariate analysis of single risk factors was performed and results expressed as odds ratio (OR) and a 95% confidence interval. In order to further investigate the association between farms with a seropositive dog and the seroprevalence rate in the cows all variables with an OR that was significant at P≤ 0.10 were tested for an association with herd seroprevalence. Those variables found to be associated with herd seroprevalence were selected for further analysis using logistical non- conditional regression techniques for multivariate analysis. A final model was produced using all variables found to be significant confounders of the association between farms with a seropositive dog and farms with a herd seroprevalence > 6%.

2.5.3. Results

Questionnaires were completed for 92 farms, a compliance rate of 96%. Blood samples were collected from 120 dogs on 68 farms. Eighteen dogs as reported in the questionnaire responses, present on 11 farms were not sampled, because a farm visit was not made or the dogs were not around or were too vicious to be sampled; 13 farms reported no dogs present. Fifty-one (42.5%, 95% CI 33.7 – 51.3) dogs were positive by IFAT at 1:50 or greater. There was no association between seropositivity and sex. Dogs older than 2 years were more likely to be positive than those aged 2 years or less, OR 3.68, p=0.029 (95% CI 1.15 – 11.82). Thirty-five farms (51.47%) had at least one positive dog (95% CI 39.59 – 63 .34). The mean herd seroprevalence for the 68 herds whose dogs were sampled was 8.72% (range: 0 -33.23%) for 2002 and 6.72% (range: 0 -30.27) for 2003. The reduction in mean prevalence between the two years was not significant (t = 1.49, p>0.1). The distribution of farms with seropositive dogs, seronegative dogs and number of dogs in relation to herd seroprevalence is given in Table 13. Farms that had 2 or more dogs compared to one or no dogs were at a greater risk of having a herd seroprevalence greater than 6% (OR 2.07, 95% CI 0.90 – 4.27, p=0.089). This risk was much reduced when only the farms who had dogs that were tested were included in the analysis (OR 1.88, 95% CI 0.67 – 5.27, p = 0.231) when farms with 1 dog were compared to farms with 2 or more dogs.

In the logistic regression, a farm having a N.caninum seropositive dog was found to be a positive predictor of the N.caninum seroprevalence of the herd being greater than 6% (OR 4.36, 95% CI 1.58 – 12.06, p=

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0.004). No other variables were found to be significant predictors of a herd seroprevalence greater than 6% at p≤ 0.05. Factors found to be positive predictors of herd seroprevalence greater than 6% at p ≤0.10 were farm dogs eat foetuses/dead calves, milking cows fed straights during housing season, afterbirths not disposed of and dead calves/foetuses buried. Negative predictors at p≤ 0.10 were seeing dogs around buildings/yards, calving in individual pens during the grazing season and foxes seen frequently on the land (Crude odds ratios are given in Table 14). Variables found to be positive predictors at p≤ 0.05 of a farm having a N.caninum positive dog were dogs seen in cattle feeding and bedding area, dogs frequently seen in cattle loafing area, maize silage stored where dogs and other animals could get access, farm dogs eat foetuses/ dead calves and afterbirths, mouldy silage fed to pregnant cows, two or more dogs present on farm and pigeons one of three commonest bird species found on farm. Wholecrop stored where dogs and other animals could get access and heifers fed concentrates during the grazing season were marginally significant (P≤ 0.10) as predictors of a seropositive dog. Calving in individual pens in housing period and swallows one of three commonest species of birds found on farm were found to act as protective factors against farms having an N.caninum seropositive dog (Table 15). Five variables were found to act as confounders to the association between herd seroprevalence of >6% farms having a N.caninum positive dog (Table 16). A final model including these variables resulted in an adjusted OR of 3.47 (95% CI 1.03 – 11.64, p=0.043).

Table 13: Distribution of farms by Neospora caninum seropositive dogs and numbers of dogs according to herd seroprevalence for N.caninum

Herd Seroprevalence

Number of farms

Farms with seronegative dogs only

Farms with ≥ 1 seropositive dogs

Farms not tested with ≥ 1 dog

Farms with no dogs

Farms with 1 dog only

Farms with ≥2 dogs

0 – 1.25% 23 11 5 3 4 7 121.26 – 6.0% 25 11 6 4 4 11 106.1 – 12% 23 8 10 2 3 7 1312.1 – 32% 21 3 14 2 2 4 15Totals 92 33 35 11 13 29 50

Table 14: Factors found to be predictors of the herd N.caninum seroprevalence being greater than 6%

Variable Odds ratio 95% CI P-valuePositive associationsAt least on farm dog positive for N.caninum by IFAT

4.36 1.58 – 12.06 0.004

Farm dogs eat foetuses/dead calves 3.87 0.96 – 15.63 0.058Milking cows fed straights during housing season 2.52 0.91 – 6.94 0.074Afterbirths not disposed of 2.31 0.87 – 6.10 0.092Dead calves/foetuses buried 2.48 0.85 – 7.25 0.098Negative associationsSeeing dogs around buildings/yard 0.40 0.15 – 1.08 0.069Calving in individual pens during grazing season 0.25 0.061 – 0.102 0.054Foxes seen frequently on land a 0.34 0.096 – 1.24 0.10

a frequently compared to occasionally

Table 15: Factors found to be predictors of a farm having at least one dog with a positive titre for N.caninum by IFAT

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Variable Odds ratio 95% CI P-valuePositive associationsMouldy silage fed to pregnant cows 5.37 1.06 – 27.07 0.042Farm dogs eat afterbirths 4.8 1.64 – 14.06 0.004Farm dogs eat foetuses/dead calves 6.88 1.39 – 34.03 0.0182 or more dogs on farm 4.55 1.49 – 13.84 0.008Pigeons one of 3 main bird species seen on farm 3.76 1.2 – 11.56 0.021Maize silage stored where other animals have access* 9.86 1.07 – 90.65 0.043Wholecrop stored where other animals have access* 6.25 0.84 – 46.57 0.074Heifers fed concentrates in grazing season 8.17 0.94 – 71.17 0.057N.caninum seroprevalence in herd >6% 4.36 1.58 – 12.06 0.004Dogs seen in cattle feeding area 5.71 1.64 – 19.92 0.006Dogs seen in cattle bedding area 12.75 1.51 – 107.40 0.019Dogs seen frequently seen in cattle loafing area a 4.05 1.42 – 11.52 0.009Negative associationsCalving in individual pen during housing period 0.29 0.11 – 0.79 0.016Swallows one of 3 main bird species seen on farm 0.40 0.15 – 1.08 0.071

* “other animals” includes all other animals seen in yard/buildingsa frequently compared to occasionally

Table 16: Odds ratio for herds with seroprevalence ≤ 6% v herds with seroprevalence > 6% farms having a N.caninum positive dogs adjusted for confounders

Adjustment Odds ratio

95% CI p-value P-value for likelihood ratio test for significance of confounder

None 4.36 1.58 – 12.06 0.004Farm dogs eat afterbirths

5.09 1.65 – 15.64 0.005 0.0024

Farm dogs eat foetuses/dead calves

3.45 1.19 – 9.94 0.022 0.0174

Dogs seen in cattle feeding area

4.15 1.41 – 12.22 0.010 0.0071

Dogs seen in cattle bedding area

3.96 1.36- 11.56 0.012 0.0076

Dogs seen frequently seen in cattle loafing area a

3.56 1.24 – 10.25 0.019 0.0349

a frequently compared to occasionally

2.5.4. Discussion

A number of studies, carried out in different cattle populations and using different study designs, have concluded that farm dogs may be associated with increased N.caninum seroprevalence and N.caninum associated abortion in cattle (44, 45, 46, 47, 48, 49, 50, 51). In this study, the first to be carried out on UK dairy farms, a highly significant association was found between herds with a N.caninum seroprevalence of >6% and the presence of at least one N.caninum seropositive dog. Following adjustment for several confounding factors the odds ratio was reduced but remained significant. Data on possible risk factors for on-farm N.caninum horizontal transmission were collected by means of a comprehensive questionnaire. No significant (at p≤ 0.05), risk factors were identified that would suggest that seroprevalence >6% was associated with transmission of N.caninum from dogs to cattle. Whilst several risk factors - farm dogs eat foetuses/dead calves, milking cows fed straights during housing season, afterbirths not disposed of and dead foetuses/calves buried - were identified at p≤0.10, none of these was related to possible oocyst excretion e.g. dogs in cattle feeding or bedding areas. Foxes seen frequently on land was found to be a protective factor (p≤0.10) for herd seroprevalence at >6%, and this may be due to the scavenging activities of foxes who to date have not been shown to be definitive hosts of N.caninum.

A number of risk factors were found to be predictors of a farm having at least one N.caninum seropositive dog and importantly several of these are directly related to farm dogs being in contact with cattle. Significantly (P≤0.05) associated with farms having a seropositive dogs were dogs seen in cattle bedding, feeding and loafing

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areas and farm dogs seen eating afterbirths and foetuses/dead calves. These predictors suggest that dogs are becoming infected by exposure to N.caninum infected material. Calving in individual pens during the housing period was found to be a significant protective factor against a farm having a N.caninum seropositive dogs. The explanation for the other factors found to be predictors of farms having an N.caninum seropositive dog however is less straight forward. The highly significant finding that a farm having two or more dogs were more likely to have a seropositive dogs than one with only one dog may be because farmers are more inclined to keep single dogs chained up and which would thus limit their contact with cattle compared to the situation on farms with more than one dog, when dogs are more likely to move around freely. This theory was suggested by Otranto et al., (52) but no observations pertaining to restraint of farm dogs were made in this study.

The findings of the variable pigeons positively associated, and swallows negatively associated, with a farm having at least one dog with a N.caninum positive titre are difficult to interpret especially as the method used to find out this information in the questionnaire was rather subjective – farmers were asked to identify the three most common species of bird found on the farm. Bartels et al., (53) found the presence and number of poultry on farms to be linked with N.caninum associated epidemics, suggesting that they could act as vectors of infection or as intermediate hosts for infection in dogs.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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1. Dubey, J. P., Carpenter J. L., Speer, C. A., Topper, M. J., Uggla, A. (1988). Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med. Assoc. 192, 1269-1285.

2. Dubey, J. P., Lindsay, D. S. (1993) Neosporosis. Parasitol. Today. 9, 452-458.

3. Barr, B. C. Conrad P. A. Sverlow K. W. Tarantal A. F. Hendrickx A. G. (1994). Experimental fetal and transplacental Neospora infection in the nonhuman primate. Lab. Invest. 71, 236-242.

4. Ho, M. S, Barr, B.C., Tarantal, A.F., Lai, L.T., Hendrickx, A.G., Marsh, A.E., Sverlow, K.W., Packham, A.E., Conrad, P. A. (1997). Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization. J. Clin. Microbiol. 35, 1740-1745.

5. Davidson, H.C., Otter, A., Trees, A.J. (1999). Significance of Neospora caninum in British dairy cattle determined by estimation of seroprevalence in normally calving and aborting cattle. Int. J. Parasitolol. 29, 1189 – 1194.

6. Tranas, J., Heinzen, R. A. Weiss, L. M., McAllister, M. M. (1999). Serological evidence of human infection with the protozoan Neospora caninum. Clin. Diagn. Lab. Immunol. 6, 765-767.

7. Graham, D.A., Calvert, V., Whyte, M., Marks, J. (1999). Absence of serological evidence for human Neospora caninum infection. Vet. Rec. 144, 672-673.

8. Peterson, E., Lebech, M., Jensen, L., Lind, P., Rask , M., Bagger , P., Björkman, C., Uggla, A. (1999). Neospora caninum infection and repeated abortions in humans. Emerg. Infect. Dis. 5, 278-280.

9. Osborne, K., Gay, N., Hesketh, L., Morgan-Capner, P., Miller, E. (2000). Ten years of serological surveillance in England and Wales: methods, results, implications and action. Int. J. Epidemiol. 29, 362-368.

10. Thomas, D. R., Salmon, R. L., Kench, S. M., Meadows, D., Coleman T. J., Morgan-Capner, P. Morgan, K. L. (1994). Zoonotic illness: determining risks and measures of effects: association between current animal exposure and a history of illness in a well characterised rural population. J. Epidemiol. and Community Health 48, 151-155.

11. Chalmers, R M., Thomas, D. Rh., Sillis, M, et al. (1998). Coxiella burnetti in farmworkers and their families. In Thrusfield MV Goodal, EA, eds. Proceedings of the Society for Veterinary Epidemiology and Preventive Medicine, 25- 27 March 1998,128 -138.

12. McGarry, J. W., Guy, F., Trees, A. J., Williams, D. J. L. (2000). Validation and application of an inhibition ELISA to detect serum antibodies to Neospora caninum in different host species. Int J. Parasitol. 30, 880-884.

13. Williams, D. J. L., McGarry, J., Guy, F., Barber, J., Trees , A. J. (1997). Novel ELISA for detection of Neospora-specific antibodies in cattle. Vet.Rec. 140, 328-331.

14. Williams, D. J. L., Davison, H. C., Helmick, B., McGarry, J., Guy, F., Otter, A., Trees, A. J. (1999). Evaluation of a commercial ELISA for detecting serum antibody to Neospora caninum in cattle. Vet. Rec. 145, 571-575.

15. Vyse, A., Gay, N.J., Hesketh, L.M., Morgan-Capner, P., Miller, E. (2004). Seroprevalence of antibody to varicella zoster virus in England and Wales in children and young adults. Epidemiol. Infect. 132, 1129 – 1134.

16. Dubey, J.P. (2003). Review of Neospora caninum and neosporosis in animals. Korean J. Parasitol. 41, 1-16.

17. Baillargeon, P., Fecteau, G., Paré J., Lamothe, P., Sauvé, R. (2001). Evaluation of the embryo transfer procedure proposed by the International Embryo Transfer Society as a method of controlling vertical transmission of Neospora caninum in cattle. J. Am. Vet. Med. Assoc. 218, 1803-1806.

18. Campero, C. M., Moore, D. P., Lagomarsino, H., Odeon, A. C., Castro, M., Visca, H. (2003). Serological status and abortion rate in progeny obtained by natural service or embryo transfer from Neospora caninum-seropositive cows. J. Vet. Med. B. Infect. Dis. 50, 458-460.

19. Landmann, J. K., Jillella, D., O'Donoghue, P. J., McGowan, M. R. (2002).Confirmation of the prevention of vertical transmission of Neospora caninum in cattle by the use of embryo transfer. Aust. Vet. J. 80, 502-503.

20. Mara, L., Pilichi, S., Sanna, A., Accardo, C., Chessa, B., Chessa, F. , Dattena, M., Bomboi, G., Cappai, P.

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(2004). Sexing of in vitro produced ovine embryos by duplex PCR. Mol. Reprod. Dev. 69, 35-42.

21. Uggla, A., Stenlund, S., Holmdahl, O. J. M., Jakubek, E. B., Thebo, P., Kindahl, H., Björkman, C. (1998). Oral Neospora caninum inoculation of neonatal calves. Int. J. Parasitol. 28, 1467-1472.

22. Ortega-Mora, L. M., Ferre, I., del-Pozo, I., Caetano-da-Silva, A., Collantes-Fernandez, E., Regidor-Cerrillo, J., Ugarte-Garagalza, C., Aduriz, G. (2003). Detection of Neospora caninum in semen of bulls. Vet. Parasitol. 117, 301-308.

23. Trees, A. J., Williams, D. J. L. (2005). Endogenous and exogenous transplacental infection in Neospora caninum and Toxoplasma gondii. Trends Parasitol. 21, 558-561.

24. McAllister, M. M., Dubey, J. P., Lindsay, D. S., Jolley, W. R., Wills, R. A., McGuire, A. M. (1998). Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473-1478.

25. Gondim, L. F., McAllister, M. M., Pitt, W. C., Zemlicka, D. E. (2004). Coyotes (Canis latrans) are definitive hosts of Neospora caninum. Int. J. Parasitol. 34, 159-161.

26. de Marez, T., Liddell, S., Dubey, J. P., Jenkins, M. C., Gasbarre, L. (1999). Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses. Int. J. Parasitol. 29, 1647-1657.

27. Trees, A. J., McAllister, M. M., Guy, C. S., McGarry, J. W., Smith, R. F. , Williams, D. J. L. (2002). Neospora caninum: oocyst challenge of pregnant cows. Vet. Parasitol. 109, 147-154.

28. Dubey, J. P., Lindsay D. S. (2000). Gerbils (Meriones unguiculatus) are highly susceptible to oral infection with Neospora caninum oocysts. Parasitol. Res. 86,165-168.

29. Gondim, L. F., Gao, L. , McAllister, M. M. (2002). Improved production of Neospora caninum oocysts, cyclical oral transmission between dogs and cattle, and in vitro isolation from oocysts. J. Parasitol. 88, 1159-1163.

30. Trees, A. J., Guy, F., Tennant, B. J., Balfour,A. H. Dubey J. P. (1993). Prevalence of antibodies to Neospora caninum in a population of urban dogs in England. Vet. Rec. 132, 125-126.

31. Barber, J. S., Holmdahl, O. J., Owen, M. R., Guy, F., Uggla, A. Trees, A. J. (1995). Characterization of the first European isolate of Neospora caninum (Dubey, Carpenter, Speer, Topper and Uggla). Parasitol. 111, 563-568.

32.Williams, D. J. L., Guy, C. S. , McGarry, J. W., Guy, F. , Tasker, L., Smith, R. F., MacEachern, K., Cripps, P. J., Kelly, D. F., Trees, A. J. (2000). Neospora caninum- associated abortion in cattle: the time of experimentally -induced parasitaemia during gestation determines foetal survival. Parasitol. 121, 347-358.

33. Gondim, L. F., McAllister, M. M. Anderson-Sprecher, R. C., Bjorkman, C., Lock ,T. F., Firkins, L. D., Gao, L., Fischer, W. R. (2004). Transplacental transmission and abortion in cows administered Neospora caninum oocysts. J. Parasitol. 90, 1394-1400.

34. Guy, C. S., Williams, D. J. L., Kelly, D. F., McGarry, J. W. Guy, F. Björkman, C. , Smith, R. F., Trees, A. J. (2001). Neospora caninum in persistently infected cows: spontaneous transplacental infection is associated with an acute increase in maternal antibody. Vet.Rec. 149, 443-449.

36. Lindsay, D. S., Dubey, J. P., Duncan, R. B. (1999). Confirmation that the dog is a definitive host for Neospora caninum. Vet Parasitol. 82, 327-333.

37. Schares, G., Heydorn, A. O., Cuppers, A. , Mehlhorn, H., Geue, L., Peters, M., Conraths, F. J. (2002). In contrast to dogs, red foxes (Vulpes vulpes) did not shed Neospora caninum upon feeding of intermediate host tissues. Parasitol. Res. 88, 44-52.

38. Šlapeta, J. R., Koudela, B., Votýpka, J., Modrý, D., Horejs, R., Lukes, J. (2002). Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum. Vet. J. 163, 147-154.

39. Schares, G., Pantchez, N., Barutzki, D., Heydorn, A. O., Bauer, C., Conraths, F. J. (2005). Oocysts of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and Hammondia hammondia in faeces collected from dogs in Germany. Int. J. Parasitol. 35, 1525-1537.

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40. Mehlorn, H., Heydorn, A. O. (2000). Neospora caninum: Is it really different from Hammondia heydorni or is it a strain of Toxoplasma gondii? An opinion. Parasitol. Res. 86, 169-178.

41. Dubey, J. P., Hill, D. E., Lindsay, D. S., Jenkins, M. C., Uggla, A., Speer, C. A. (2002). Neospora caninum and Hammondia heydorni are separate species/organisms. Trends Parasitol. 18, 66-69.

42. McGarry, J. W., Stockton, C. M., Williams, D. J. L., Trees, A. J. (2003). Protracted shedding of oocysts of Neospora caninum by a naturally infected foxhound. J. Parasitol. 89, 628-630.

43. Ho, M. S., Y. Barr, B. C., Marsh, A. E., Anderson, M. L., Rowe, J. D., Tarantal, A. F., Hendrickx, A. G., Sverlow, K., Dubey, J. P., Conrad, P. A. (1996). Identification of Bovine Neospora parasites by PCR amplification and specific small-subunit rRNA sequence probe hybridisation. J. Clin. Microbiol. 34, 1203-1208.

44. Paré, J., Fecteau, G., Fortin, M., Marsolais, G. (1998). Seroepidemiology study, of Neospora caninum in dairy herds. J. Am. Vet. Med. Assoc. 213, 1595-1598.

45. Bartels, C. J. M., Wouda, W., Schukken, Y. H. (1999). Risk factors for Neospora caninum associated abortion storms in dairy herds in the Netherlands (1995 - 1997). Theriogenology 52, 247-257.

46. Wouda, W., Bartels, C. J. M., Moen, A. R. (1999). Characterisation of Neospora caninum-associated abortion storms in dairy herds in the Netherlands (1995 - 1997). Theriogenology 52, 233-245.

47. Mainar-Jaime, R. C., Thumond, M. C., Berzal-Herranz, B., Hietala, S. K. (1999). Seroprevalence of Neospora caninum and abortion in dairy cows in Northern Spain. Vet. Res. 145, 72-75.

48.Moore, D.P., Campero, C.M., Odeon, A.C., Posso, M.A., Cano, D., Leunda, M.R., Basso, W., Venturini, M.C., Spath, E. (2002). Seroepidemiology of beef and dairy herds and fetal study of Neospora caninum in Argentina. Vet. Parasitol. 107, 303-316.

49. Sanchez, G.F., Morales, S.E , MartÍnez, M.J., Trigo, J.F. (2003). Determination and correlation of anti-Neospora caninum antibodies in dogs and cattle from Mexico. Can. J. of Vet. Res. 67, 142-145.

50. Guimarães, J, S,. Souza, S.L.P., Bergamaschi, D.P., Gennari, S.M. (2004). Prevalence of Neospora caninum antibodies and factors associated with their presence in dairy cattle of the north of Parana state. Brazil. Vet. Parasitol. 124, 1-8.

51.Hobson, J. C., Duffield, T. F., Kelton, D., Lissemore, K., Hietala, S. K., Leslie, K. E., McEwen, B., Peregrine, A. S. (2005). Risk factors associated with Neospora caninum abortion in Ontario Holstein dairy herds. Vet. Parasitol. 127, 177 – 88.

52. Otranto, D., Llazari, A., Testini, G., Traversa, D., Frangipane di Regalbono, A., Badan, M., Capelli, G. (2003). Seroprevalence and associated risk factors of neosporosis in beef and dairy cattle in Italy. Vet. Parasitol. 118, 7-18.

53. Bartels, C.J.M., Wouda, W., Schukken, Y.H. (1999). Risk factors for Neospora caninum associated abortion storms in dairy herds in the Netherlands (1995 – 1997). Theriogenology. 52, 247 – 257.

Appendix 1

University of Liverpool

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Investigation of Excretion of oocysts of Neospora caninum in the faeces of foxhounds

Questionnaire number

Date

SECTION A: General

1. Name of Kennel -------------------------------------------------------------------

2. Address of Kennel -------------------------------------------------------------------

-------------------------------------------------------------------

3. Name of respondent -------------------------------------------------------------------

4.Contact telephone number -------------------------------------------------------------------

5. How many adult dogs are kept at kennel?

(Write in numbers in boxes provided)i. Male

ii. Female

iii. Total

6. a. Are puppies bred at kennels(1 = Yes, 0 = No)

If No, go to question 76.b. Are puppies fostered out?(1 = Yes, 0 = No)

If No, go to question 76c. How old are puppies when they are fostered out?

6d. How old are puppies when they return to the kennel?

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Months

Months

6e. How long is it since puppies were bred at the kennels?(Write in number of months)

7.When was the last time a new dog was brought into the kennels?(Write in approximate date)

8. Do the foxhounds have regular contact with cattle?(1 = Yes, 0 = No)

9a. How often are the hounds exercised?

i. Twice daily

ii. Once a day

iii. Every other day

iv. Weekly

iv. Other (Specify below)

9b. Where are they exercised?

-------------------------------------------------------------------------------------------------------

SECTION B: Housing

10. Which of the following apply to the method whereby the hounds are housed? Tick appropriate box

i. Individual

ii. Group Housing, mixed sex

iii. Group Housing, separation of sexes

11. What type of bedding is used for the hounds?Tick appropriate box

i. None

ii. Straw

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iii. Wood shavings


12. How often are kennels cleaned out?Tick appropriate box

i. Daily

ii. Weekly

iii. Twice a day


SECTION C: Feeding

13. a Are the hounds fed any or all of the following ?

Tick appropriate boxesi. Dry ration

ii. Bovine carcasses

iii. Ovine carcasses


If bovine carcasses are fed answer Question 14 If ovine carcasses are fed, answer Question 15

If neither bovine or ovine carcasses are fed, go to question 21

14. Bovine carcasses

14.a Are stillborn carcasses carcasses fed?(1 = Yes, 0 = No)

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14.b. If yes, Which of the following are removed from the carcasses before feeding?Tick appropriate box

i. Thymus glands

ii. Intestines

iii. Head

iv. Tonsils

v. Tongue

vi. Spleen

vii. Spinal cord

viii. Vertebral column

14.c Are carcasses less than 6 months old fed?(1 = Yes, 0 = No)

14.d. If yes, Which of the following are removed from the carcasses before feeding?Tick appropriate box

i. Thymus glands

ii. Intestines

iii. Head

iv. Tonsils

v. Tongue

vi. Spleen

vii. Spinal cord


14e. Are carcasses older than six months old fed?(1 = Yes, 0 = No)

14f. If yes, which of the following are removed from carcasses over 6 months of age before feedingTick appropriate box

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i. Thymus glands

ii. Intestines

iii. Head

v. Tonsils

vi. Tongue

vii. Spleen

viii. Spinal cord

ix. Vertebral Column

15. Ovine carcasses

15a Are stillborn carcasses and /or carcasses less than 12 months old fed?(1 = Yes, 0 = No)

15b. If yes, which of the following are removed from carcasses under 12 months of age before feeding?Tick appropriate box

i. Thymus glands

ii. Intestines

iii. Head

iv. Tonsils

v. Tongue

vi. Spleen

vii. Spinal cord


15c. Are carcasses older than twelve months old fed?(1 = Yes, 0 = No)

15d.. If yes, which of the following are removed from carcasses over 12 months of age before feedingTick appropriate box

i. Thymus glands

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ii. Intestines

iii. Head

iv. Tonsil

v. Tongue

vi. Spleen

vii. Spinal cord


16. Are carcasses fed fresh?

(1 = Yes, 0 = No)

17. How many carcasses are fed on average everyday?Write number in box

18. Are uneaten carcasses removed?(1 = Yes, 0 = No)

19. What do you do with carcase remains?Tick appropriate box

i. Incinerate on site

ii. Incinerate off site

iii. Bury


20. At what age are puppies first fed bovine or ovine carcasses?

-------------------------------------------------------------------------------------------------------

SECTION D: Health

21a Have there been any puppies with signs of nervous disease, such as paralysis or lameness, or any puppies diagnosed with clinical neosporosis, .at the kennels? (1 = Yes, 0 = No)

. If Yes, Please answer the following questions, If No, Move to question 22.

21b When was neosporosis diagnosed?

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Write month and year in box

21c. What are the names of the dams that had puppies with neosporosis, or neonatal paralysis/paresis or lameness?

21d. Please give any other information about the dams below:

22. How often do you give the hounds deworming medicines?Tick appropriate box

i. Every month

ii. Every two months

v. Every six months

vi. Every year

23. What deworming medicines do you use?Write names in box below

24. What is the age of the oldest dog?

Please write any other information that may be relevant to this study below:

APPENDIX 2

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Section 1: Farm GeneralSection 1: Farm General

Q1: What is the farm type? Dairy only go to Q3(please tick) Mixed enterprise go to Q2

Mixed enterprise can include non-animal enterprise too.

Q2: If a mixed enterprise, please specify other enterprise(s):________________

Q3: What was the approximate number 2001______ 2002______2003______ of cows in milk in the following years?

Q4: What is the average annual milk yield of an individual cow in your herd? litres

Q5: What is the main breed of your dairy herd?

Holstein-Friesian Jersey Ayrshire Guernsey Friesian Other

Q6: What percentage of the animals on your farm are homebred?

Q7: If you purchase animals, which of the following do you buy? (tick all applicable)a) open heifers b) in calf heifers c) fresh calved heifers d) fresh calved cows e) cows in milk

Q8: From where do you purchase these animals? (tick all applicable)a) Direct from farm b) Dealer c) Market d) Farm dispersals e) Other (please state)

Q9: How many family lines does your herd consist of? (tick only one)a) <5 b) 5 – 10 c) 11 – 15 d) 16 – 20 e) >20

Q10: Have any of the following diseases been diagnosed on the farm in the last 2 years in the adult cows (>15 months). ( tick all applicable)BVDV Lepto Salmonella Campylobacter IBR

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Other (please state)

Q11: Do you vaccinate adult cows for any of the following diseases? (tick all applicable)IBR, BVDV, Lepto, Salmonella

Other (please state)

Q12: What percentage of the herd is replaced each year? %

Q13:Please indicate the two most common reasons for culling over the last two years?a) Abortion or empty at drying off Indicate primary and b) Failure to conceive secondary reasons for c) Lameness cullingd) Downer cow e) Mastitis or High SCC f) Other

Q14: Have there been any recent (in the last 2 years) management changes to:a) cow numbers b) feed regime c) pasture d)housing

Briefly describe changes

Section 2: Fertility and CalvingsSection 2: Fertility and Calvings

Q15: Please give the number of all calves born in each month (include those from first calved heifers) Count twins as one calving

2002

Jan. Feb. Mar Apr May June July Aug. Sept Oct. Nov. Dec.

2003 (predicted Oct- Dec)

Jan Feb Mar Apr May June July Aug Sept Oct Nov Dec

Q16: Using your fertility records (NMR or other) please complete the following:

Rolling 12 month

Rolling 3 month

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What is the calving to conception?

days days

What is the conception to 1st

service?% %

What is the number serves/conception?

Q17: What proportion of the herd have calving interval of (percentage or actual figures):a) <344d

b) 344 – 385d

b) 386 – 425d

c) >425d

Q18: Do you have the cows routinely PD’d?No go to Q19Yes How often are the PD visits?

At what stage(s) of pregnancy?Ie what stages do you present to the vet/scanner

Q19: What percentage of the herd are served by AI? %

Q20: Do you use a sweeper bull? No Yes which group of cows?

Q21: Where do calvings occur? (please indicate all applicable)

Cubicles Straw Yard

Individual Pens

Separate Pastureland

Pasture not separated

Housing Period

Grazing Period

Q22: Disposal of afterbirths (please tick all applicable)a) Buried b) Incinerated c) Slurry lagoon d) No Action e) Other (please state)

Q23: Disposal of dead calves and foetuses? (please tick all applicable)a)Knackerman b) Hunt Kennels c)Incinerated d)Bury e)Slurry lagoon

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f)No Action g) Other (please state)

May mention that this has changed but question is about the last 2 years

Q24: Do you use embryo transfer? Yes go to Q25No go to Q26

Q25: Do you test the recipients for Neospora ? Yes No

Section 3: Feed Management and HousingSection 3: Feed Management and Housing

Q26: Please indicate the components of each ration fed during the housing periods of 2001 to 2003.

When asking for the milking cow diet first asked if the same ration is fed to all milkers or is there a separate high and low yielders ration. Similarly for weaned heifers, prior to bulling or bulling/pregnant onwards.

Ration ComponentMilking CowHigh Low

Dry Cow HeifersNon-breed Breeding

Fresh GrassGrass SilageMaize SilageWholecropHayStrawComplete Concentrate FeedHome Mixed StraightsOther (please state)

Q27: Is the ration mixed/partially mixed?

Q29: During the housing period where are the following groups of cattle housed?

Milking CowsHigh Low

Dry Cows Pregnant Heifers

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Straw YardsCubicles

Q30: Please indicate if the cubicles have the following: (please tick if present)a) automatic scrapers b) slatted floors

Q31: Has mouldy silage been fed to pregnant cows? Yes No

Q32: How often are the feeding troughs emptied? (please tick one only)a) Every feed b) Once a day c) Once a week d) Never e) Other (please state)

Q33: What happens to the leftovers?

Q34: Is the herd zero-grazed? Yes Go to Q35No Go to Q36

Q35: If the herd is zero-grazed, are the cows at pasturea) Never? b) Only in dry period? c) Exercise paddocks milking or dry period? d) Exercise paddocks milking period only? d) Other Go to Q41

Q36: Do you use strip grazing?Never Occasionally Frequently

Q37: During the grazing period do you buffer feed? No Go to Q41This isn’t about parlour conc. Yes Go to Q38

Q38: Please indicate the components of each ration fed during the grazing periods of 2001 to 2003.

Ration Component Milking CowHigh Low

Dry CowHeifers

Non-breed BreedingGrass Silage

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Maize SilageWholecropHayStrawComplete Concentrate FeedHome Mixed StraightsOther (please state)

Q39: Is the ration mixed/partially mixed?

Q40: Please indicate if buffer feeding is carried out Buildings/Yard Pasture

Q41: Are cattle located away from the holding during the year Yes go to Q42No go to Q43

Q42: Please complete the following table:

Housed At Pasture Grid RefIf possible

Dry CowsDairy

Heifers

Q43: Please indicate whether the following water sources are supplied to cattle:

Mains Spring River Borehole Stream Pond OtherHousing period

Grazing period

Q44: Do you over winter sheep on grazing land or silage fields Yes No

Q45: Is there public access to grassland (grazing or silage fields) ie footpathsYes No

Section 4: Other SpeciesSection 4: Other Species

For Q46 and Q47 dogs and cats are those belonging to the general public or strays.

Q46: How often have these animals been or are found on the farmland in the last 2 years? (Please circle the appropriate answer)

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Dogs: Never Occasionally Frequently Cats: Never Occasionally Frequently Foxes: Never Occasionally Frequently Badgers: Never Occasionally Frequently

Q47: How often have these animals been found in the farm buildings/yard in the last 2 years?(Please circle the appropriate answer)

Dogs: Never Occasionally Frequently Cats: Never Occasionally Frequently Foxes: Never Occasionally Frequently Badgers: Never Occasionally Frequently Birds: Never Occasionally Frequently

Q48: What are the main species of birds in buildings/yards? (please tick all applicable)

Crows Pigeons Starlings Poultry Swallows

Other no more than 3 and indicate order

Q49: Which feeds are stored in areas that could have been entered by animals in the last 2 years? (Please circle the appropriate answer)Ask which would be the main species involved for each food source

SpeciesGrass silage: Never Occasionally Frequently Maize Silage: Never Occasionally Frequently Wholecrop: Never Occasionally Frequently Hay: Never Occasionally Frequently Straw: Never Occasionally Frequently Calf Feed: Never Occasionally Frequently Adult Concentrate Feed: Never Occasionally Frequently Straights: Never Occasionally Frequently

Q50: Please indicate the number of farm dogs or frequent visiting dogs in each category?

At present2 years ago

Puppies, less than 6 months

Dogs 6 – 12 months

Dogs more than 12 months

Q51: Please complete the following table for new dogs when introduced onto the farm:

Introduced Dog Date Age Origin

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Q52: Have there been any pregnant dogs on the farm in the last 2 years?Yes go to Q43 No go to Q44

Q53: Please complete the following information for each pregnant bitch/pregnancy:

Pregnant Bitch

Date No. of puppies

Number of puppies with hind limb lameness/ inco-ordination

Where housed

With puppies?

Contact with

pregnant cows

Q54: Have the farm dogs and/or the regular visiting dogs been seen in cattle:(Please circle the appropriate answer)

a)Loafing areas Never Occasionally Frequently b)Bedding areas Never Occasionally Frequently c)Feeding area Never Occasionally Frequently d)Calving area Never Occasionally Frequently e)Pasture Never Occasionally Frequently f)Pasture Feeding TroughsNever Occasionally Frequently

Q55: Do your dogs eat: Afterbirths Yes No Foetuses/dead calves? Yes No

Q56: Have hound packs hunted over any part of your land in the last 2 years? Yes No

Q57:Have you taken any action after a cow being diagnosed as Neospora positive? (please tick all applicable)Try to get an indication of numbers for each category.

a) Culled cow b) Retained cow and bred to beef c) Culled family line d) No action

SID 5 (2/05) of 50

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