Seroprevalence of antibodies to Pestivirus infections in South Australian sheep flocks.

OBJECTIVE: Bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) are of the genus Pestivirus. They are known to cause significant reproductive and production losses, with BVDV acknowledged as a major source of economic loss to the Australian cattle industry. Very little is currently known about the prevalence and effect of pestiviruses in the Australian sheep industry. The present study aimed to examine the seroprevalence and effect of both BVDV and BDV in South Australian sheep flocks. METHODS: In total, 875 breeding ewes on 29 properties were serologically tested by ELISA, AGID and VNT assays for the presence of Pestivirus-specific antibodies. RESULTS: Three (0.34%) individual animals returned serological results suggestive of previous BDV infection. All three positive animals were collected from one property, giving a property level seroprevalence of 3.45% and a within-flock seroprevalence of 10%. CONCLUSION: The results suggested that BDV infection is present, albeit at a very low incidence, in the South Australian sheep flock and BVDV infection appears to be absent. Consequently, pestiviruses are unlikely to impair production in South Australian sheep populations.

Investigation of infectious reproductive pathogens of large ruminants: Are neosporosis, brucellosis, leptospirosis and BVDV of relevance in Lao PDR?

N. caninum, bovine viral diarrhoea virus, Brucella abortus and Leptospira interrogans serovar Hardjo are globally significant reproductive pathogens that cause abortion and reproductive loss in large ruminants. Prevalence information is lacking in Lao People's Democratic Republic (Laos) despite the poor reproductive performance of cattle and buffalo. Serological examination of frozen cattle (n=90) and buffalo (n=61) sera by commercially available enzyme-linked immunosorbent assays provided the first reported screening of some of these pathogens in Laos. Seroprevalence differed amongst these large ruminant species, with N. caninum, BVDV and L. interrogans serovar Hardjo antibodies found in 68.9% (95% CI±11.6), 4.9% (95% CI±5.4) and 3.3% (95% CI±4.5) of buffalo sera, respectively, and in 7.8% (95% CI±5.5), 10.0% (95% CI±6.2) and 22.2% (95% CI±8.6) of cattle sera, respectively. Buffalo sera had a significantly higher seroprevalence of N. caninum compared to cattle (p<0.001) and cattle sera had a significantly higher seroprevalence of L. interrogans serovar Hardjo compared to buffalo (p=0.003). Variability was also observed across provinces for N. caninum in buffalo (p=0.007) and for L. interrogans serovar Hardjo in cattle (p=0.071), suggesting provincial risk factors conducive to pathogen transmission. BVDV and N. caninum seropositivity were negatively associated in buffalo (p=0.018) and cattle (p=0.003). In buffalo, L. interrogans serovar Hardjo and BVDV seropositivity were associated (p=0.035, p=0.039). The identification of antibodies against three major abortifacient pathogens in Laos prompts further research to determine if infection is associated with low reproductive efficiency and the risk factors for infection. This is needed for the development of evidence based prevention strategies for improved large ruminant reproductive management among smallholders in Laos.

Antibodies to bovine viral diarrhoea virus (BVDV) in water buffalo (Bubalus bubalis) and cattle from the Northern Territory of Australia.

BACKGROUND: Farmed and feral water buffaloes (Bubalus bubalis) populations often coexist with cattle in the Northern Territory of Australia, but their level of exposure to bovine viral diarrhoea virus (BVDV) is unknown. METHODS: Water buffalo (n = 245) and cattle (n = 184) serum samples were collected by the NT Government as part of an ongoing disease surveillance scheme at varying intervals between 1993 and 2001. All samples were frozen and stored at -80°C until testing. Water buffalo samples from farming properties were identified as 'farmed' animals and the remaining samples as 'feral' populations. Serum samples were analysed using commercially available ELISAs to test for the presence of BVDV antibodies. RESULTS: Testing of historical water buffalo sera for BVDV antibodies revealed a low level of exposure, with 4.5% (95% CI ± 2.6%) being sero-positive; cattle from the same geographical area and time period had higher levels of exposure at 74.5% (95% CI ± 6.3%). DISCUSSION: This survey showed that water buffalo are susceptible to infection with BVDV. No persistently infected water buffalo were identified in this study.

Cross-sectional observational survey of serum biochemistry values in a population of 69 adult female alpacas (Vicugna pacos) in South Australia.

Blood samples were collected from 69 'healthy' female alpacas aged ≥12 months from 11 properties in South Australia. The 10-90 percentile ranges of the 16/19 analytes measured in this sample population were within the published ranges of four healthy alpaca populations from other geographic locations. Marginal exceptions were glutamate dehydrogenase and bicarbonate. Potassium was notably elevated, probably because of haemolysis of some samples. The sample size was insufficient to provide the appropriate statistical power to define diagnostic references ranges according to international standards. The health status of the sample population of alpacas was presumptive based on a physical examination.

Serological survey for antibodies against bovine viral diarrhoea virus and Neospora caninum in a population of South Australian alpacas (Vicugna pacos).

OBJECTIVE: Bovine viral diarrhoea virus (BVDV) and Neospora caninum may cause clinical disease in alpacas. Both diseases are present in the Australian cattle population. The objective of this study was to perform a serological prevalence survey for BVDV and N. caninum exposure in a regional alpaca population of South Australia. METHODS: Serum samples were taken from 182 alpacas on 10 farms, which had a combined population of 1308 alpacas. Serological analysis for BVD antibodies was performed using a competitive BVDV antibody ELISA kit. Serological analysis for N. caninum was performed using an anti-Neospora ELISA with a protein G conjugate. RESULTS: Of the 182 alpacas sampled, 5 animals located on three properties were positive for BVDV antibodies, constituting a prevalence of 2.7% (95% confidence interval 1-6%). All samples tested negative for N. caninum antibodies. CONCLUSION: There is a low BVDV seroprevalence and N. caninum is currently either absent or present at a very low prevalence in this population of alpacas in South Australia. There is serological evidence for the presence of both organisms in South Australian beef and dairy cattle herds. Appropriate biosecurity protocols to minimise the risk of introduction and exposure should be a high priority to maintain this favourable status.

Survey of farmer knowledge and attitudes to endemic disease management in South Australia, with a focus on bovine viral diarrhoea (bovine pestivirus).

OBJECTIVE: We aimed to establish the attitudes of South Australian cattle farmers towards endemic animal disease prevention and control, with a particular focus on the awareness of and attitudes towards bovine viral diarrhoea (BVD). METHODS: This cross-sectional postal survey involved mailing a questionnaire to all South Australian cattle owners with 35 or more head of cattle. RESULTS: Worms and lice were the most common animal disease concerns. Less than half of responding farmers were adequately vaccinating their herds against clostridial diseases, but 53.0% stated that they utilised quarantine procedures. Less than 20% of respondents had actively taken part in BVD educational opportunities, or had vaccinated or tested their herd for BVD; less than 20% of respondents were actively involved in any systematic control of Johne's disease. Overall, farmers' actual knowledge of BVD was lower than their perceived understanding, although their interest in BVD and its control was high. CONCLUSIONS: Disease prevention measures such as vaccination, quarantine and participation in systematic control schemes were used by a minority of respondents. The results suggest that respondents acknowledge BVD as an important and relevant disease, despite many believing it was not a problem in their herd. Interest in BVD appears to be high and it is likely that an education program would be well received.

Milk as a diagnostic sample for a commercially available ELISA to identify bovine viral diarrhoea (BVD) antibodies in dairy herds.

OBJECTIVE: The aims of this study were to evaluate a commercially available ELISA for the detection of bovine viral diarrhoea virus (BVDV)-specific antibodies in individual milk compared with individual serum samples, and in bulk milk samples compared with within-herd antibody prevalence and bulk milk quantitative reverse transcription polymerase chain reaction (qRT-PCR) results. METHODS: Paired individual serum and individual milk samples were collected from 125 lactating cows and tested by ELISA; 96 bulk milk samples were also tested. Within-herd antibody prevalence was calculated based on milk ELISA results for 25 individual cows in each herd. Additionally, 167 bulk milk samples were tested for BVDV-specific antibodies by ELISA and for the presence of BVDV by qRT-PCR to establish the correlation between antibody result and virus presence. RESULTS: Good agreement was observed between individual milk and serum results (Kappa = 0.865). The ELISA was observed to detect BVDV-specific antibodies in individual milk samples with a relative sensitivity of 96.6% and specificity of 89.2%. The bulk milk samples revealed a strong (r(2) = 0.95) relationship between the ELISA result and the within-herd antibody prevalence. The proportion of herds that tested positive by bulk milk qRT-PCR increased as the bulk milk antibody S/P ratio increased. CONCLUSION: Commercially available ELISA testing of individual and bulk milk samples is an appropriate alternative to serum testing with good test performance in these samples. Determining a threshold for the detection of herds containing active BVD infection by testing bulk milk is a novel use for an antibody ELISA kit and provides more practical, relevant test results.

Bovine viral diarrhoea virus ('pestivirus') in Australia: to control or not to control?

BACKGROUND: Acute infection with bovine viral diarrhoea virus (BVDV) usually causes only mild clinical disease in cattle, but infection of animals of breeding age can result in immune suppression (resulting in an increased incidence and severity of secondary disease) and decreased reproductive performance. If infection occurs during pregnancy, the virus may cross the placenta and either cause abortion, establish immunotolerance and persistent infection (PI) in the fetus or cause congenital deformities. These outcomes depend on the stage of pregnancy at the time of infection. INTERNATIONAL PERSPECTIVE: BVDV is recognised as a disease of significant financial impact in a number of countries. As a result, national and regional BVDV control programs are now in place in several regions around the world. In Europe, these programs largely rely on the identification and removal of the PI animals, whereas vaccination has tended to be the chosen method of control in the United States. BVD IN AUSTRALIA: BVDV is endemic in Australian cattle populations, with more than 80% of herds surveyed showing some level of exposure to the pathogen. The cost to the national industry is estimated to be AUD57.9 million annually. This review identifies and discusses the challenges to BVDV control in Australia, including farmer attitudes, herd size, sheep as a potential reservoir host and diagnostic capabilities. We conclude that systematic BVDV control in Australia is, or soon will be, an option; however, detailed cost-benefit analyses will need to be undertaken.

Neospora caninum serostatus is affected by age and species variables in cohabiting water buffaloes and beef cattle.

The aim of this study was to investigate how Neospora caninum serostatus may be affected by variables such as host species (water buffaloes or cattle) and age in animals cohabiting in the same ranch. A convenience cross-sectional study was performed on four ranches in the Northeast of Argentina, where water buffalo are cohabitating with beef cattle. Blood samples were collected from 1350 female water buffaloes (Bubalus bubalis) and 880 female beef cattle (Bos taurus and Bos indicus crossbreeds) from four ranches. Calving and weaning percentages at herd level for each ranch were also recorded. N. caninum antibody levels were measured by an indirect fluorescent antibody test (IFAT) (reciprocal antibody titers ≥ 100). Serological results were classified into 2 categories (0: negative; 1: positive). A logistic regression model was used to describe the relationship between N. caninum serostatus and specie (water buffalo or cattle), age or ranch and their interactions. Likelihood ratio tests were used to assess the significance of the model and their terms. Odds ratios were estimated and 95% profile likelihood (LR) and Wald confidence intervals (CI) obtained. Overall, specific antibody titers were found in 43.3% (584/1350) of water buffaloes and 28.6% (252/880) of cattle. Seropositive water buffaloes and cattle were observed on all ranches. Age was statistically significant (p=0.01) with an overall estimate of logit (log odds) of age of 0.03 for both species. This indicates that for every one year increase in age, the expected change in log odds of being seropositive increased by 0.03. On three of four ranches a water buffalo was 4.48, 1.54 and 2.25 times more likely to be seropositive than cattle for animals of the same age. The N. caninum serostatus was affected by age in the first place, but also by species on at least three of the four ranches. Calving and weaning percentages were higher in water buffaloes than in beef cattle (p<0.05). Even though the low pathogenicity that N. caninum seems to have in water buffaloes, this study reinforces the importance of this specie as maintenance of the disease.

Validation and evaluation of a commercially available ELISA for the detection of antibodies specific to bovine viral diarrhoea virus (bovine pestivirus).

OBJECTIVE: To compare the performance of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies specific to bovine viral diarrhoea virus (BVDV) with a virus neutralisation test (VNT) and agarose gel immunodiffusion (AGID) test. DESIGN: A total of 125 cattle serum samples were tested by a commercially available ELISA for antibodies specific to BVDV and by a VNT as the reference standard. A comparison between AGID and ELISA for detection of BVDV antibodies was also carried out, using 1182 serum samples from unvaccinated South Australian cattle. METHODS: Two-graph receiver operating characteristics (TG-ROC) analysis was used to confirm that the manufacturer's recommended cut-off value for the ELISA was appropriate. Two-by-two tables were constructed to analyse the concordance of serological results among the three assays. McNemar tests were used to assess the agreement among serological tests. RESULTS AND CONCLUSIONS: Using the manufacturer's cut-off threshold, supported by TG-ROC analysis, the ELISA's sensitivity and specificity were calculated to be 96.7% and 97.1%, respectively, compared with the VNT. Compared with AGID, ELISA with specific BVDV antibodies may be more sensitive and detect 5.8% more samples than AGID. McNemar test also showed a significant difference (P < 0.001) between AGID and ELISA.

Treating Cryptosporidium parvum infection in calves.

The present study evaluated the therapeutic efficacy of azithromycin, co-trimoxazole and kalvangi (Nigella sativa, also known as Black Cumin) against Cryptosporidium parvum infection in calves under field conditions. The experimental calves were treated with azithromycin (group A) at 1500 mg/calf/day, co-trimoxazole (group B) at 30 mg Kg-1 and kalvangi seeds powder (group C) at 750 mg Kg-1 BW orally for 7 days. Calves in the group D were naturally infected with C. parvum , untreated animals (positive control) while the calves in the group E were uninfected negative control animals. A significant decrease (p < 0.05) in oocyst counts for calves in groups A, B and C was observed compared to group D. When the oocyst counts amongst the treatment groups A, B and C were compared, a significant decrease (p < 0.05) was observed in group A. On day 21 post-treatment, the efficacy of azithromycin, co-trimoxazole and kalvangi in calves was 88.2% (95% C.I. ± 15.4), 45% (95% C.I. ± 21.8) and 27.8% (95% C.I. ± 20.7), respectively. This study confirmed previous reports of azithromycin efficacy against C. parvum infection, but found co-trimoxazole and kalvangi to be ineffective for this infection under these treatment regimens.

Prevalence of Neospora caninum antibodies in sheep and goats in Pakistan.

The purpose of the present study was to obtain seroepidemiological information on the Neospora caninum infection status of sheep and goats in different areas of Punjab Province and Azad Kashmir (Pakistan). A cross-sectional study, with the use of a competitive ELISA, showed an overall 27.7% (35 of 128) (95% confidence interval [CI] ± 7.7%) and 8.6% (13 of 142) (95% CI ± 4.6%) seroprevalence of N. caninum antibodies in sheep and goats, respectively. The difference in seroprevalence between sheep and goat populations was statistically significant (P < 0.05). The highest prevalence (37.4% ± 13.2%) was recorded in the tailless breed of sheep.

Use of molecular and milk production information for the cost-effective diagnosis of bovine viral diarrhoea infection in New Zealand dairy cattle.

An increase in veterinary and farmer interest in bovine viral diarrhoea (BVD) in New Zealand over recent years led to requests for cost-effective identification of BVD virus (BVDV) infected herds and individuals. This study was undertaken to determine if the use of real-time reverse transcriptase polymerase chain reaction (RT-PCR) technology and dairy cow production data could identify persistently infected (PI) animals in milking herds. Milk samples were collected from the vats of dairy herds and tested for the presence of BVDV by RT-PCR till four herds were found containing PI animals. Individual serum samples were then collected from every cow in the herd and tested by both RT-PCR and antigen capture enzyme-linked immunosorbent assays (ACE) to identify the PI animals. Individual animal testing found 1/223, 1/130, 2/800 and 1/275 PI's respectively in the four herds. Based on these results a maximum pool size of 400 cows contributing to the bulk tank milk was selected. After removal of the PI from the herds, further bulk milk samples were shown to be BVDV negative by RT-PCR. All the PI animals identified by this method were found in the lowest producing 10-20% of herd. This approach of targeted testing of dairy herds using PCR technology, in conjunction with animal production information, markedly reduced the cost of diagnostic testing for BVDV in dairy herds in New Zealand. Questionnaire follow-up on 81 BVDV-positive herds (15% of those tested) indicated the stratification approach identified milking PIs successfully over 90% of the time and reduced the number of individual tests to 12% of the milking herd.

Genetic diversity amongst isolates of Neospora caninum, and the development of a multiplex assay for the detection of distinct strains.

Infection with Neospora caninum is regarded as a significant cause of abortion in cattle. Despite the economic impact of this infection, relatively little is known about the biology of this parasite. In this study, mini and microsatellite DNAs were detected in the genome of N. caninum and eight loci were identified that each contained repetitive DNA which was polymorphic among different isolates of this parasite. A multiplex PCR assay was developed for the detection of genetic variation within N. caninum based on length polymorphism associated with three different repetitive markers. The utility of the multiplex PCR was demonstrated in that it was able to distinguish amongst strains of N. caninum used as either vaccine or challenge strains in animal vaccination experiments and that it could genotype N. caninum associated with naturally acquired infections of animals. The multiplex PCR is simple, rapid, informative and sensitive and should provide a valuable tool for further studies on the epidemiology of N. caninum in different host species.

Does control of bovine viral diarrhoea infection make economic sense?

AIM: To provide an economic analysis of the costs of control or eradication of bovine viral diarrhoea (BVD) against the estimated costs of the disease. METHODS: A decision-tree approach was adapted to an analysis of the costs of bovine viral diarrhoea virus (BVDV) infection and that of three main control options (vaccination, test-and-cull, and increased biosecurity) and their combinations, to the dairy industry in New Zealand. The model was based on an average herd of 322 milking cows. Endemic, epidemic and sporadic effects of BVDV infection were modelled in the herd, to derive an estimate of costs. RESULTS: The cost of BVDV infection to an infected average-sized dairy herd in New Zealand was estimated to be NZ $11,334 (or NZ $35.19 per cow) per annum, and NZ $48,311 over 10 years. Based on these calculations, the estimate of the annual cost of BVDV infection to the dairy industry in New Zealand was in excess of NZ $23 million per annum. While all of the control options required financial input, the rate of return compared with the cost of BVD, when viewed over a 10-year term, was as high as 123%. CONCLUSIONS: All control options offered considerable savings compared with the cost of BVD infection, and control is economically favourable. Uncertainty over the likely efficacy of the control options under field conditions in New Zealand would not allow a firm choice of one option over another at this stage, and more work on determining the efficacy of those control options in New Zealand is needed.

Evaluation of an enzyme-linked immunosorbent assay for the serological diagnosis of Neospora caninum infection in sheep and determination of the apparent prevalence of infection in New Zealand.

Neospora caninum has recently been shown to be a cause of abortions of sheep in New Zealand. A commercially available enzyme-linked immunosorbent assay (ELISA) was validated for use in sheep with sera from experimentally infected sheep. A cut-off threshold was established that demonstrated sero-conversion between 7 and 14 days post-infection. Higher inocula led to earlier sero-conversion. This ELISA was applied to 640 sera collected from rams across New Zealand and 0.625% (+/-0.61%) (4/640) were shown to be serologically positive. The four positive sera were also demonstrated to be positive by indirect fluorescent antibody test (IFAT). The ELISA evaluated here lends itself more readily to large-scale investigations than IFAT. The low background of N. caninum infection in the New Zealand sheep population suggests that N. caninum abortions could be more easily diagnosed by serological means than in populations with higher background sero-prevalence.

Neosporosis and hammondiosis in dogs.

The dog is a definitive host of the protozoan parasite Neospora caninum, and in many parts of the world, infection is relatively common as determined by serology. Reported seroprevalences usually range from 0 to 20 per cent, however, reports of clinically affected dogs are infrequent. Affected dogs are generally less than six months old and predominantly have signs of an ascending hindleg paralysis, with the associated lesions of polyradiculoneuritis and granulomatous polymyositis. Although any organ may be affected, infections are more common in the central nervous system, muscles, lungs and skin. Ante-mortem diagnosis is difficult but serology and cytology can aid diagnosis. The diagnosis can be confirmed by histology, immunohistochemistry, the use of molecular techniques on biopsy material, or on post-mortem examination. Neospora caninum oocysts are rarely found in faeces and must be differentiated from oocysts of related coccidians such as Hammondia heydorni and Toxoplasma gondii. Hammondia heydorni can cause diarrrhoea in immunosuppressed dogs. Neosporosis should be suspected in young pups with an ascending paralysis of the hindlegs. Treatment with clindamycin and potentiated sulphonamides may be useful in cases where muscular atrophy and fibrosis are absent. Feeding of raw meat is a potential risk factor for infection of dogs and should be discouraged.

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle.

AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA.

Immunization of cattle with live tachyzoites of Neospora caninum confers protection against fetal death.

Neospora caninum is an obligate intracellular protozoan parasite that causes abortion in cattle. It is normally found as a latent infection controlled by a T-helper-cell type 1 response involving CD4(+) cytotoxic T cells and gamma interferon. Cattle may be infected by two different routes: transplacentally as a result of activation of the latent infection in the mother causing congenital infection or abortion and by ingestion of oocysts, which, if it occurs during gestation, can also result in abortion. Here, for the first time, we establish proof that live vaccination protects against fetal death, whereas immunization using whole-tachyzoite lysate in different adjuvants fails to protect against fetal death. Strong antibody responses were induced in all the vaccinated groups, and the quality and magnitude of these responses were similar in the live- and the lysate-vaccinated groups. In contrast, only the group immunized with live tachyzoites had strong cellular and gamma interferon responses prior to challenge, and these responses correlated with protection against fetopathy. These results suggest that a cellular immune response may be important in the mechanisms involved in protection against N. caninum-associated abortions.

Prevalence of Neospora caninum infection in Australian (NSW) dairy cattle estimated by a newly validated ELISA for milk.

AIM: To determine the performance characteristics of an Institut Pourquier (IP) enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Neospora caninum in bovine milk and subsequent determination of the prevalence of N. caninum infection in New South Wales (NSW) dairy cattle. METHODS: Matching serum and milk samples from 93 cattle were assayed in two commercially available ELISAs for the detection of anti-N. caninum antibodies. Serum test results of one ELISA (IDEXX) were used to determine the N. caninum infection status of the cattle. Optimised cut-off values for the IP ELISA using milk samples were determined by two-graph receiver operating characteristic (TG-ROC) analysis and then applied to a representative sample of 398 milk samples from dairy herds around NSW. RESULTS: When this ELISA was applied to a representative collection of 398 milk samples from dairy cattle across NSW it demonstrated a 21.1% prevalence of N. caninum infection in those cattle. From the TG-ROC analysis an IP ELISA protocol was derived which suggested a cut-off threshold that would allow milk testing with 97% sensitivity and specificity, respectively, relative to serum testing. CONCLUSIONS: The prevalence of N. caninum in NSW dairy cattle was higher than previously believed. When used on individual milk samples this ELISA demonstrated high sensitivity and specificity and so could be used to accurately identify N. caninum infection. TG-ROC analysis of the IP ELISA optimised the protocol and prescribed cut-off values enabling the ELISA to be used for the screening of N. caninum antibodies in the milk of dairy cattle.