Toxoplasmosis in sheep. Influence of various factors on the antibody contents.

The contents of toxoplasma antibodies were estimated by a micro-modification of the dye test for up to 3 years after the initial stage of infection both in ewes which had aborted and in ewes with normal pregnancies. During the first 2 years the titres in ewes which had aborted were significantly higher than in ewes with normal pregnancies. The investigation indicated that dye test titres ≥ 1/512 usually occur during the first stage of infection, and are mainly found in ewes with clinical toxoplasmosis.The dye test titres in lambs due to antibodies transferred with the colostrum were up to 4 twofold dilutions higher than in their dams during the first 2 days after birth. Later the titres declined, and at the age of about 2 months only 3 of 29 lambs had higher titres than their dams. After the age of about 3 months maternally derived antibodies were not detected.The contents of toxoplasma antibodies in sheep with listeric encephalitis were nearly the same as found by a serological survey of the local sheep population. The examination indicated that the dye test titres in sheep are little influenced by conditions that may affect the defence mechanism.Sheep with haemoglobin type B had significantly higher dye test titres than sheep with the haemoglobin types A and AB when examined less than about 6 months after they had acquired the infection. No association was found between the susceptibility to toxoplasma infection and the haemoglobin type. kw|Keywords|k]toxoplasma infection; k]antibody formation; k]sheep {fn1|This work was supported by grants from The Norwegian Research Council for Science and for the Humanities.}     

Local antibody responses in the bile and faeces of sheep infected with Fasciola hepatica

The effect of infection with the liver fluke Fasciola hepatica on serum, bile and faecal immunoglobulin and antibody levels was studied in Scottish Blackface sheep. In the serum the immunoglobulins showing the most marked increase were IgG1 and IgG2 and their maximal values were reached at 16 weeks after infection. In the bile IgG2 rose to peak values at two weeks and IgG1, IgA and IgM were maximal at four weeks after infection. The levels of faecal IgG and IgA were low after primary infection but after reinfection a rapid increase in IgA concentration was observed within one to two weeks. Haemagglutinating antibody levels against egg antigens, juvenile and adult excretory-secretory antigens and adult fluke somatic antigens were evaluated. In the sera high titres were observed starting from two to four weeks after infection and persisting until 14 to 16 weeks. Bile haemagglutinating antibodies against excretory-secretory antigens showed the highest level at two and four weeks after infection while antibodies against adult somatic antigens reached maximal titres between four and eight weeks. Faecal antibody levels after primary infection were low but increased rapidly within two weeks after reinfection, coinciding with the elevation in faecal IgA concentration. However, there was no reduction in the number of flukes established in reinfected animals.     

Evaluation of ELISA for the detection of Toxoplasma antibodies in swine sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from greater than 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving less than 10 EIU were negative in the other tests, and all those with greater than 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10-70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.     

Neospora caninum in sheep: a herd case report

Neospora caninum was detected by means of PCR in the brain of 4 out of 20 aborted fetuses in a flock of 117 sheep exhibiting a persistent abortion problem, and N. caninum tissue cysts were furthermore found in encephalitic lesions in one of the PCR-positive fetuses. Toxoplasma gondii was detected as aborting agent in another 3 out of 20 fetuses. Antibodies to N. caninum (by indirect fluorescence antibody test (IFAT)) were found in 10.3% of 117 ewes and antibodies for T. gondii were found in 97.4% of 117 ewes. Other organisms associated with abortion were Chlamydia psittaci in three fetuses and Pasteurella multocida in one fetus. This is the first report of N. caninum associated abortion in naturally infected sheep.     

Western blot analysis of the IgG response of sheep vaccinated with S48 Toxoplasma gondii (Toxovax)

The IgG antibody responses of sheep vaccinated by the subcutaneous injection of live tachyzoites of ‘incomplete’ strain S48 toxoplasma (Toxovax) were analysed by Western blotting. Antibodies corresponding to a range of tachyzoite antigens (13 to 48 kD) were detected, but the response was dominated by antibody recognising a 30 to 32 kD band. Unvaccinated ewes challenged orally with oocysts of the ‘complete’ M3 toxoplasma strain had a more complex IgG response that recognised antigens in six dominant bands of similar intensity as those in sheep vaccinated with S48 tachyzoites and then challenged with M3 oocysts. No differences were detected between the antigenic structures of the S48 tachyzoites and RH strain tachyzoites when the antigens were probed with immune ovine sera. Many of the anitgens of the S48 tachyzoites that were recognised had molecular weights similar to those of antigens that have been identified in other strains of toxoplasma.     

Attempted venereal transmission of Chlamydia psittaci in sheep

Attempts were made to transmit Chlamydia psittaci, the causal agent of enzootic abortion of ewes, in three different ways. Ten ewes were inseminated artificially with freshly collected semen containing 10(5) CELD50 chlamydia. Serological evidence of infection was found two weeks before parturition in nine ewes and the organism was recovered from three of them. By six weeks post partum antibody titres had fallen and were negative in six ewes. Twenty-four hours after service two groups of 10 ewes were infected intravaginally with 10(8) CELD50 and 10(3) CELD50 chlamydia respectively. Positive complement fixation titres were present in the first half of pregnancy in all the ewes in the high dose group but not the low dose group. None of the ewes showed evidence of infection at parturition. Fourteen ewes were served by four rams which had been intravenously infected with 10(8) CELD50 chlamydia four to six days earlier. Following service seroconversion occurred but titres became negative again by late pregnancy. No microbiological evidence of infection was detected in any of the ewes at parturition but complement fixation titres were positive in 12 of 14 ewes sampled six weeks post partum. The 14 ewes were sampled during their pregnancies the next year and none showed any evidence of chlamydial infection. It is concluded that venereal transmission of C psittaci is biologically feasible in sheep, but that under normal systems of flock management in Britain it is unlikely to contribute greatly to the epidemiology of enzootic abortion of ewes.     

Four-layer enzyme immunoassay (EIA) detection of differences in IgG, IgM and IgA antibody response to Aujeszky's disease virus in infected and vaccinated pigs

The use of the four-layer enzyme immunoassay (EIA) for the detection of IgG, IgM and IgA antibodies against Aujeszky's disease virus in blood and oropharyngeal swabs of infected and vaccinated pigs is described. Mean antibody titres obtained using the four-layer EIA were 6.1 and 3829 times higher compared with the indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test, respectively. The VN test detected mainly IgG antibodies, while the IgM antibodies did not react. Using the EIA, the first antiviral antibodies in sera were demonstrated on Days 5-7 after infection or vaccination. Up to the 7th day, demonstrable antibodies were almost exclusively of the IgM class. In infected pigs high titres of IgM antibodies were still detected on Day 18, while in vaccinated animals they were absent by this time. Antibodies of the IgG class appeared in infected pigs sooner (Day 7) than in vaccinated pigs (Day 10) and reached higher mean titres. Antibodies of the IgA class were demonstrable from Day 10 only in samples from infected pigs. Similar antibody dynamics and distribution were detected in oropharyngeal swabs, except that the IgG and IgM titres were roughly 100 times lower than in sera. However, titres of IgA antibodies in oropharyngeal swabs were two times higher than in sera. The greatest differences between both groups of animals were recorded on Day 18; in the infected pigs, IgG, IgM and IgA antibodies were present in sera and oropharyngeal swabs at that time, while in vaccinated pigs only IgG antibodies were demonstrable. The effect of infection and vaccination on the pattern of the immune response as well as the importance of the detection of individual immunoglobulin classes for the specificity of the enzyme immunoassay are discussed.     

Epizootiologic investigations on a sheep farm with Toxoplasma gondii-induced abortions

During the lambing season of 1983/1984, 8 of 44 purebred Hampshire ewes on a farm in Knoxville, Md had reproductive problems. In at least 4 of these ewes, the problem was attributed to toxoplasmosis. Necrosis and Toxoplasma gondii tachyzoites were found in placental specimens from 3 ewes. Agglutinating antibody to T gondii, at a titer of 1:80, was found in pleural fluids of both fetuses aborted from 1 ewe; this ewe had an antibody titer of 1:6,400 at the time of abortion. In another ewe, the diagnosis was confirmed by the isolation of T gondii from the placenta and 1 of her lambs. Of numerous free-roaming adult cats on the farm, 16 were trapped, euthanatized, and examined for T gondii. Agglutination antibody to T gondii, at titers of 1:4 to 1:64, was found in serum samples from all the cats. Toxoplasma gondii was isolated from the brain and skeletal muscles of 9 of the cats, and from the feces of 1 cat. Blood samples obtained from all 78 sheep on the farm 6 months after the episode of abortion were examined for antibody to T gondii. Agglutinating antibody titers to T gondii were less than 1:16 in 46 sheep, 1:16 in 16, 1:64 in 12, 1:256 in 2, 1:024 in 1, and 1:4,096 in 1. Analyses of serologic data in sheep of various age groups suggested that the Toxoplasma infection was acquired sporadically, probably from feed contaminated with oocysts.     

Serology of a Neospora abortion outbreak on a dairy farm in New Zealand: a case study

AIM: To describe the kinetics of serological titres after an abortion outbreak in April-May 1995 due to Neospora caninum affected 17 dairy cows in a herd of 320. METHODS: Thirty-five cows, that had either aborted, carried mummified calves, were not pregnant or calved normally were: bled several times at regular intervals and the sera tested for Neospora antibodies in the indirect fluorescence antibody test (IFAT). RESULTS: Maximal IFAT titres of up to 1:4000 occurred within 6 weeks of the abortion outbreak, decreased over the next 2 months to < or = 1:200 and remained at this level until the next scheduled bleed a further 2 months later. A rise in titres was subsequently observed in the cows that had aborted or were not pregnant (at the time of the abortions) or had carried mummified foetuses. Seroconversion was also observed in some of the control cows, which had, up until then, remained seronegative. A dog and cat in contact with the cows in the herd investigated were, however, negative in the IFAT. CONCLUSIONS: Maximal serological titres in Neospora abortions are observed within weeks of the abortion event and then quickly return to very low levels. Subsequently, a recrudescence of titres can be observed in infected cows during the next pregnancy, without it being associated with repeat abortions.     

IFAT detection of IgG specific to toxoplasma in thoracic fluids from aborted lambs: evaluation on routine diagnostic submissions

Thoracic fluids from 171 aborted lambs from 55 flocks were examined by the indirect fluorescent antibody test for the presence of IgG specific to Toxoplasma gondii. The technique was shown to be both sensitive and specific for the diagnosis of toxoplasma abortion at a titre of 1/256, when compared with diagnoses made in the flocks and in the small number of individual fetuses in which the pathology of the cotyledons and the serology of the ewe were known.     

Associations between pregnancy outcome and serological response to Neospora caninum among a group of dairy heifers

AIM: To monitor pregnancy in a group of rising 2-year-old dairy heifers on a farm on which abortion due to Neospora caninum was known to occur in previous years. METHODS: A prospective cohort study group of 164 rising 2-year-old heifers was pregnancy-tested and blood-sampled at 4-5-week intervals throughout gestation. Sera were tested for antibodies to N. caninum at 3-4-month intervals, using an enzyme-linked immunosorbent assay (ELISA). When loss of pregnancy was detected, an N. caninum indirect fluorescent antibody test (IFAT) was conducted retrospectively on stored sera collected the month before abortion, the month abortion was detected, and for the following 2 months, from heifers that aborted. All fetal and placental material detected following abortion was subjected to gross post-mortem and histopathological examination. RESULTS: Eleven of 18 (61%) heifers that were seropositive and 4/146 (3%) heifers that were seronegative to N. caninum by ELISA, aborted. The relative risk for abortion among ELISA-positive heifers was 23.6. Abortion occurred predominantly between Days 120 and 152 gestation among the ELISA-positive heifers and throughout gestation among the ELISA-negative heifers. IFAT titres rose around the time of abortion in most of the heifers that were previously seropositive by ELISA, but dropped rapidly again in post-abortion samples. IFAT titres among 4/6 ELISA-positive heifers that did not abort increased, but later in gestation than the time other heifers aborted. IFAT titres remained negative in heifers that aborted that were ELISA negative. CONCLUSIONS: Heifers that were seropositive to N. caninum by ELISA had a much greater risk of abortion than seronegative heifers. Most seropositive heifers showed evidence of a reactivation of infection during pregnancy. High (> or =1:2,000) N. caninum IFAT titres also occurred in non-aborting heifers. CLINICAL RELEVANCE: Culling of replacement heifers seropositive to N. caninum may be a cost-effective strategy for minimising risk of abortion. Pregnancy testing heifers before 5 months gestation may overestimate the number that calve in N. caninum-infected herds, but would assist in documenting the occurrence of abortion. Reliance on a high (>1:2,000) IFAT titre to rule-in N. caninum as a cause of abortion is likely to produce false-positive results.     

Neospora caninum infection in a clinically healthy calf: parasitological study and serological follow-up

In this study a case of congenital infection in a clinically healthy calf is reported. The mother showed high antibody levels (IFAT) at 230 days of gestation (IgG titres > or = 1:1600, IgM titres > or = 1:320) and the parasite was isolated from placental cotyledonary villi at calving. The IgM values are indicative of a recent infection in the third trimester of gestation. The calf was monitored serologically for IgM and IgG from birth until slaughtering, at 8 months of age. IgM titre showed a peak at birth, while IgG peak was observed at 40-60 days of age. Parasitic isolation was obtained by biological tests using Swiss mice or VERO cell cultures inoculated with brain and spinal cord tissues. The parasitic presence in the calf was also evidenced in the myocardium with immunohistochemical method. The results are very important because they demonstrate that the period of gestation in which the cow becomes infected is an important factor in the pathogenesis of N. caninum induced abortion: in fact, the acquisition of infection in the third trimester of gestation allowed the foetus to develop a sufficient grade of immunocompetency to limit parasite multiplication with the result of a calf born clinically healthy.     

Neospora caninum Infection in a Clinically Healthy Calf: Parasitological Study and Serological Follow‐up

In this study a case of congenital infection in a clinically healthy calf is reported. The mother showed high antibody levels (IFAT) at 230 days of gestation (IgG titres 1:1600, IgM titres 1:320) and the parasite was isolated from placental cotyledonary villi at calving. The IgM values are indicative of a recent infection in the third trimester of gestation. The calf was monitored serologically for IgM and IgG from birth until slaughtering, at 8 months of age. IgM titre showed a peak at birth, while IgG peak was observed at 40–60 days of age. Parasitic isolation was obtained by biological tests using Swiss mice or VERO cell cultures inoculated with brain and spinal cord tissues. The parasitic presence in the calf was also evidenced in the myocardium with immunohistochemical method. The results are very important because they demonstrate that the period of gestation in which the cow becomes infected is an important factor in the pathogenesis of N. caninum induced abortion: in fact, the acquisition of infection in the third trimester of gestation allowed the foetus to develop a sufficient grade of immunocompetency to limit parasite multiplication with the result of a calf born clinically healthy.     

Systemic and local immune responses of gnotobiotic calves to respiratory infection with Mycoplasma bovis

Gnotobiotic calves were inoculated by the intratracheal route with Mycoplasma bovis and the specific antibody response in sera and tracheo-bronchial washings examined by radioimmunoassay. In sera an IgM response which reached a peak two weeks post infection was followed by IgG1 and IgG2 antibody responses. Low levels of IgA antibody were detected in sera three and four weeks after infection. The predominant antibody in tracheo-bronchial washings 2 weeks after infection was IgA. Four weeks after infection IgG1 antibody predominated, but IgG2 and IgA antibodies were also present. Cells containing Ig were present in the cellular accumulations around the necrotic zones produced by M. bovis in the lung parenchyma two and four weeks after infection. IgG1 containing cells predominated in these cellular infiltrates. IgG2 producing cells were the next in frequency. It is concluded that the lung lesion caused by M. bovis is partly due to the host's immune response, presumably contributing to the control of the infection, and that the cells infiltrating the lung are a major source of the local and systemic IgG antibody that is detected after infection. IgA staining cells were observed in the submucosa of tissues from nasal cavity and trachea. These cells are probably the source of IgA in tracheo-bronchial washings and sera since IgA-producing cells were not a predominant component of the lesion in the lung parenchyma.     

Isotypic antibody responses in cattle infected with Haemophilus somnus

Bovine antibody responses to Haemophilus somnus were compared on the basis of clinical and bacteriological findings. Serum IgG1 and IgM antibody titres were significantly increased in clinically normal cattle that were bacteriologically positive for H somnus from the nasal or vaginal mucosae compared with clinically normal, negative cows. IgG2 titres did not differ significantly between these two groups. However, IgG2 antibody was significantly higher in animals with H somnus disease (pneumonia or abortion) than in clinically normal cattle (whether bacteriologically positive or negative), while IgG1 and IgM titres did not differ between diseased and bacteriologically positive, clinically normal cattle. These antibody trends were duplicated in experimental H somnus abortion or pneumonia, with the greatest response occurring within the IgG2 subclass. Cattle vaccinated systemically with killed whole H somnus produced a predominant IgG2 response with minimal IgG1 and IgM responses. These results demonstrate that IgG2 antibody is consistently elevated in H somnus disease, and suggest that this response may be useful in discriminating diseased from asymptomatic cattle.     

Preliminary evaluation of diagnostic tests using horses experimentally infected with trypanosoma evansi

Seven surra negative horses were intravenously inoculated with 3 x 10(6)Trypanosoma evansi parasites derived from a camel. One horse was maintained as an uninfected negative control. Three antigen and three antibody detection tests were evaluated for diagnosis of infection in horses. The microhaematocrit centrifugation test (MHCT) was the most sensitive, first detecting parasites between one and three days (x 2.4) post infection (p.i.). The antigen (ag)-ELISA detected antigen between three and ten days (x 6.6) p.i. The latex agglutination test (LAT) first gave positive results on day 3 (x 3.0) p.i. Following the treatment of horses with trypanocidal drugs, the MCHT and the mouse inoculation test (MIT) became negative. Antigen levels using LAT declined and reached pre-infection levels in five out of six horses during the period of observation (92-279 days). Antigen levels using the ag-ELISA declined as well but did not reach pre-infection levels in any of the six horses.Three antibody detection techniques, ab-ELISA, card agglutination test (CATT), and immunofluorescent antibody test (IFAT) detected antibodies in the blood of all seven infected horses but not in the uninfected control. However, the ab-ELISA did not discriminate clearly between sera from infected and uninfected horses because unacceptably high ELISA background readings were detected in 15% of the surra negative horses shipped to the UAE from the UK. The ELISA antibody increased above pre-infection levels in the six horses experimentally infected, but not in one horse. In this horse the ELISA antibody level exceeded the cut-off level only after the reoccurrence of the T. evansi infection. The IFAT detected antibodies 15.7 days p.i. in all infected horses.     

Serum antibody response and Cryptosporidium parvum oocyst antigens recognized by sera from naturally infected sheep.

The response of specific serum immunoglobulins (IgG, IgM and IgA) and the major antigens of Cryptosporidium parvum recognized by these isotypes were investigated by using enzyme-linked immunosorbent assay and immunoblot techniques in lambs and ewes naturally infected throughout an outbreak of cryptosporidiosis. Serum samples were collected from 20 lambs the first day they showed diarrhoea (D1), and Days 11 and 22, in addition to single serum samples from 17 of their dams. Serum anti-C. parvum IgG, IgM and/or IgA antibodies were detected in lambs as early as Day 1. Levels of IgM antibodies remained steady from D1 to D11 and increased at D22, whereas the IgG response decreased from D1 to D11 and subsequently increased. In contrast, IgA antibodies rapidly fell from D1 and all lambs were seronegative at D11 and D22. The highest levels of specific antibodies were detected in sera from ewes. In fact, all ewes were seropositives for IgM and IgA isotypes and most (16/17) showed positive levels of IgG. Four protein fractions (37-39, 42-48, 51-57 and 60-69 kDa) were the most frequently recognized by IgG and IgM from lamb sera. A low molecular weight fraction (12-14 kDa) reacting with IgG and IgA in most lamb sera was scarcely recognized by IgM and three broad bands were frequently recognized by IgA antibodies (23-25, 51-57 and 90-95 kDa). The recognition pattern of 23-25 kDa peptides by IgA from lamb sera clearly increased with the age. Peptides of 42-48, 51-57, 60-69 and 71-78 kDa were most frequently recognized by IgG and IgM from ewe sera. In relation to IgA antibodies from ewe sera, a frequent immunoreactivity was found with proteins in the intervals between 12 and 22 kDa as well as between 32 and 34 kDa and practically all sera reacted with fractions from 42 to 95 kDa.     

Passive immunisation of neonatal lambs against infection with enteropathogenic Escherichia coli via colostrum of ewes immunised with crude and purified K99 pili

Lambs sucking ewes immunised four to five weeks before parturition with crude preparations of K99 and purified K99 pili of single subunit composition were protected against challenge infection with heterologous enteropathogenic Escherichia coli strains. In contrast, the majority of lambs sucking sham-immunised ewes suffered severe diarrhoea and dehydration, followed by death in nearly half of the affected lambs. Protection was related to the presence of antibody in the colostral whey and lamb sera. K99-specific antibody activity in the colostral whey was found to be confined to IgM and IgG (IgG1 and IgG2) but not to the IgA class.     

Associations between pregnancy outcome and serological response to Neospora caninum among a group of dairy heifers

AIM: To monitor pregnancy in a group of rising 2-year-old dairy heifers on a farm on which abortion due to Neospora caninum was known to occur in previous years.

METHODS: A prospective cohort study group of 164 rising 2-year-old heifers was pregnancy-tested and blood-sampled at 4–5-week intervals throughout gestation. Sera were tested for antibodies to N. caninum at 3–4-month intervals, using an enzyme-linked immunosorbent assay (ELISA). When loss of pregnancy was detected, an N. caninum indirect fluorescent antibody test (IFAT) was conducted retrospectively on stored sera collected the month before abortion, the month abortion was detected, and for the following 2 months, from heifers that aborted. All fetal and placental material detected following abortion was subjected to gross post-mortem and histopathological examination.

RESULTS: Eleven of 18 (61%) heifers that were seropositive and 4/146 (3%) heifers that were seronegative to N. caninum by ELISA, aborted. The relative risk for abortion among ELISApositive heifers was 23.6. Abortion occurred predominantly between Days 120 and 152 gestation among the ELISA-positive heifers and throughout gestation among the ELISA-negative heifers. IFAT titres rose around the time of abortion in most of the heifers that were previously seropositive by ELISA, but dropped rapidly again in post-abortion samples. IFAT titres among 4/6 ELISA-positive heifers that did not abort increased, but later in gestation than the time other heifers aborted. IFAT titres remained negative in heifers that aborted that were ELISAnegative.

CONCLUSIONS: Heifers that were seropositive to N. caninum by ELISA had a much greater risk of abortion than seronegative heifers. Most seropositive heifers showed evidence of a reactivation of infection during pregnancy. High (≥1:2,000) N. caninum IFAT titres also occurred in non-aborting heifers.

CLINICAL RELEVANCE: Culling of replacement heifers seropositive to N. caninum may be a cost-effective strategy for minimising risk of abortion. Pregnancy testing heifers before 5 months gestation may overestimate the number that calve in N. caninum-infected herds, but would assist in documenting the occurrence of abortion. Reliance on a high (>1:2,000) IFAT titre to rule-in N. caninum as a cause of abortion is likely to produce false-positive results.