Julie Paré

NEOSPORA CANINUM ANTIBODIES IN COWS DURING PREGNANCY AS A PREDICTOR OF CONGENITAL INFECTION AND ABORTION

A cohort study was undertaken on a dairy experiencing endemic Neospora caninum abortions, to characterize dam serologic variations during pregnancy, and to determine if dam N. caninum antibody levels during gestation predicted congenital infection or abortion. Blood samples were collected monthly during pregnancies of 254 cows and precolostrally from 87 of their calves. Antibody levels, as measured by an enzyme-linked immunosorbent assay, indicated 60.6% of cows were seropositive at some time during pregnancy and 87.4% of seropositive cows were seropositive throughout pregnancy. The rate of seroconversion was 8.5/100 cows/yr. The risk of abortion for seropositive cows at the time of pregnancy diagnosis and during gestation was twice that for seronegative cows (P = 0.025, P = 0.006). Calves born to seropositive cows were more likely to be seropositive at birth if the dam had high antibody levels at 240 days of gestation (P = 0.04) and an increase in antibody levels between 90 and 240 days (P = 0.08) than if the respective values of the dam were low or decreasing. Seropositive cows with high antibody levels at 180 and 210 days of gestation were less likely to abort than cows with low antibody levels at those times (P = 0.05, P = 0.03). Results support a causal effect between exposure to N. caninum and abortion, indicate that acquisition of infection during pregnancy is not necessary for congenital infection or abortion to occur, and suggest that maternal immune response influences congenitial infection and abortion.

An Enzyme-Linked Immunosorbent Assay (ELISA) for Serological Diagnosis of Neospora Sp. Infection in Cattle

A kinetic enzyme-linked immunosorbent assay (ELISA) was developed and optimized for detection of antibodies to Neospora sp. in cattle. Sonicated tachyzoites of Neospora sp. isolated from an aborted bovine fetus were used as antigen. Variability in immunoblot patterns among positive sera, and the fact that all life stages of the parasites are unknown, justified use of a multiple-antigen ELISA to allow for maximum sensitivity. Immunoblot analysis revealed negligible cross-reactions between Toxoplasma gondii antigen and Neospora sp. antisera and between Neospora sp. antigen and antisera from various apicomplexan parasites. The maximum positive-to-negative Vmax (average maximum slope of the optical density over time) ratio was obtained using 200 ng/well of sonicated tachyzoite antigen and a 1:200 serum dilution. Using logistic regression to determine the optimal cutoff point between known infected and noninfected cattle, a sample-to-positive control Vmax ratio of 0.45 was found to maximize the percent correct classification, with an estimated sensitivity of 88.6% and specificity of 96.5%. Use of Neospora caninum antigen following the same protocol demonstrated no difference in ELISA interpretation. Comparison with an existing indirect immunofluorescent antibody (IFA) test showed the ELISA to be the more sensitive and specific test for serodiagnosis of Neospora infection in cattle.

Interpretation of an Indirect Fluorescent Antibody Test for Diagnosis of Neospora sp. Infection in Cattle

Neospora is a recently discovered protozoan that may cause bovine protozoal abortion (BPA) in cows and encephalomyelitis in congenitally infected calves. An indirect fluorescent antibody (IFA) test has been described for diagnosing Neospora sp. infection in cattle. We report here results using the IFA test on peritoneal and pleural fluids from aborted fetuses, precolostral calf sera, and selected adult cattle. The IFA test employed was the same as described elsewhere, with the following modifications. Teflon-coated slides were precleaned in absolute ethanol and fixed in an-

Identification of Catalase-Negative, Non-Beta-Hemolytic, Gram-Positive Cocci Isolated from Milk Samples

ABSTRACT This study was undertaken in an effort to improve the identification scheme of catalase-negative, non-beta-hemolytic, gram-positive cocci isolated from milk samples obtained from cows. First, the sensitivity and specificity of the identification procedure currently in use in our laboratory were compared to the results obtained with API 20 STREP strips which were set as the gold standard. Second, a number of other identification tests, which could contribute to increase the sensitivity and specificity of the identification procedure of these microorganisms, were evaluated and selected. The data have shown that there is a necessity to review the identification procedure. Some modifications are suggested to laboratories doing milk sample analyses. A standardized procedure, using the CAMP test, esculin and sodium hippurate hydrolysis, the presence of the enzymes pyrolidonyl arylaminase and leucine aminopeptidase, and acid production from 1% inulin and raffinose broth, would not only improve the results of the identification process of gram-positive cocci isolated from milk samples but also ensure greater uniformity of the epidemiological data.

Virulence factors in Escherichia coli isolated from the blood of bacteremic neonatal calves

Twenty-five Escherichia coli isolates from the blood of critically ill bacteremic calves sampled in two separate studies on a calf-rearing farm housing over 15,000 calves, in the San Joaquin Valley, California were studied. Isolates were characterized for O serogroups and for pathotypes as determined by the presence of specific virulence factors including heat-labile enterotoxin (LT), heat-stable enterotoxins a and b (STa, STb), verotoxins 1 and 2 (VT1, VT2), cytotoxic necrotizing factor (CNF), aerobactin, intimin Eae and P, F17 and CS31A fimbrial adhesins, and resistance to bactericidal effects of serum. These isolates constituted a heterogeneous group. However, isolates were mostly aerobactin positive and often resistant to the bactericidal effects of serum. Isolates of pathotypes O78 (n=6), O119:CS31a (n=3), and P positive but O non-typeable (n=3) were associated with a high mortality rate. The remaining isolates belonged to diverse pathotypes, often possessing the adhesins P, F17, CS31A and Eae but belonging to O serogroups other than O78 and O119, and were less frequently associated with mortality. Although no virulence factor common to all isolates was identified, the capacity to use iron by the presence of aerobactin which is important to the capture of iron was a predominant factor. Moreover, certain pathotypes appear to be associated with primary colisepticemia whereas other pathotypes may cause a bacteremia without necessarily leading to septicemia.

Rare detection of Neospora caninum in placentas from seropositive dams giving birth to full-term calves

Neospora caninum is thought to be transmitted to cattle by dogs, the only known definitive host. Although aborted fetuses seem the most likely source of infective material for dogs, placentas from seropositive dams appear also as a potential source of infective material. The objective of the study was to evaluate the presence of N. caninum organisms in placentas of full-term calves born to seropositive cows. Sixteen placentas, 11 from Neospora-seropositive cows, were examined histologically and by immunohistochemistry and polymerase chain reaction (PCR) assay for the presence of N. caninum. Mild placentitis was observed in all placentas. Neospora caninum was not identified by immunohistochemistry, but placentas from 2 seropositive dams were positive for N. caninum by PCR. These results suggest that placentas of full-term calves from seropositive cows may be a potential source of N. caninum for dogs, but the incidence of this mode of transmission is likely to be low.

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum.

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.

Evaluation of a PCR assay on overgrown environmental samples cultured for Mycobacterium avium subsp. paratuberculosis

Culture of Mycobacterium avium subsp. paratuberculosis (MAP) is the definitive antemortem test method for paratuberculosis. Microbial overgrowth is a challenge for MAP culture, as it complicates, delays, and increases the cost of the process. Additionally, herd status determination is impeded when noninterpretable (NI) results are obtained. The performance of PCR is comparable to fecal culture, thus it may be a complementary detection tool to classify NI samples. Our study aimed to determine if MAP DNA can be identified by PCR performed on NI environmental samples and to evaluate the performance of PCR before and after the culture of these samples in liquid media. A total of 154 environmental samples (62 NI, 62 negative, and 30 positive) were analyzed by PCR before being incubated in an automated system. Growth was confirmed by acid-fast bacilli stain and then the same PCR method was again applied on incubated samples, regardless of culture and stain results. Change in MAP DNA after incubation was assessed by converting the PCR quantification cycle (Cq) values into fold change using the 2−ΔCq method (ΔCq = Cq after culture − Cq before culture). A total of 1.6% (standard error [SE] = 1.6) of the NI environmental samples had detectable MAP DNA. The PCR had a significantly better performance when applied after culture than before culture (p = 0.004). After culture, a 66-fold change (SE = 17.1) in MAP DNA was observed on average. Performing a PCR on NI samples improves MAP culturing. The PCR method used in our study is a reliable and consistent method to classify NI environmental samples.

Risk factors associated with Mycobacterium avium subsp. paratuberculosis herd status in Québec dairy herds

Paratuberculosis is a chronic and contagious enteric disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Control of paratuberculosis is justified given the associated economic losses and the potential role of MAP in Crohns disease in humans. Management practices that limit exposure of susceptible animals to MAP are more effective at reducing disease prevalence than testing and culling infected cows. The objective of this retrospective case-control study was to study the association between management practices and MAP status in dairy herds in Québec, Canada. A total of 26 case herds (MAP had been isolated from at least 1 environmental sample in each herd) and 91 control herds (no clinical cases of paratuberculosis and negative on 2 consecutive yearly environmental samplings) were selected among herds enrolled in the Québec Voluntary Paratuberculosis Control Program. A risk assessment questionnaire, completed at enrolment, was available for the selected herds. Culture of MAP was achieved using liquid media and the BACTEC 960 detection system. Multivariable logistic regression was used to evaluate the association between selected risk factors and MAP herd status. Herd size (OR = 1.17; 95% CI: 1.02-1.33) and proportion of cows purchased per year in the last 5 years (OR = 5.44; 95% CI: 1.23-23.98) were significantly associated with a positive MAP herd status. The management risk factors identified in the present study are in accord with previous studies. Management practices aiming to prevent the introduction of new animals into the herd and to reduce the contact of newborn calves with adult animals or their feces are key elements to minimize MAP introduction and transmission into a herd. These elements should be prioritized in control programs.

Estimating diagnostic accuracy of fecal culture in liquid media for the detection of Mycobacterium avium subsp. paratuberculosis infections in Québec dairy cows: A latent class model

A latent class model fit within a Bayesian framework was used to estimate the sensitivity and specificity of individual fecal culture (IFC) in liquid medium (Para TB culture liquid medium and BACTEC MGIT 960 system) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in Québec dairy cows. As a secondary objective, the within-herd paratuberculosis prevalence was estimated. A dataset including 21 commercial Québec dairy herds participating in previous research projects was retrospectively analyzed. In total, 1386 adult cows on which both IFC and serum-ELISA were available were included. The selected latent class model assumed conditional dependence between the tests. Non-informative priors for IFC accuracy and paratuberculosis prevalence were used while informative priors, obtained from the literature, were used for serum-ELISA accuracy. The WinBUGS statistical freeware was used to obtain posterior estimates (medians and 95% Bayesian credibility intervals (95% BCI)) for each parameter. The sensitivity and specificity estimates for IFC were 34.4% (95% BCI: 20.3-66.1) and 99.5% (95% BCI: 98.6-100), respectively. Sensitivity and specificity for serum-ELISA were 27.3% (95% BCI: 18.1-38.3) and 97.4% (95% BCI: 96.6-98.0). Median paratuberculosis within herd prevalence was estimated to be 0.3% (0-3.3). In conclusion, a higher sensitivity of IFC compared to serum-ELISA was observed both in the unconditional and conditional dependent models. Since the sensitivity of both IFC and serum-ELISA was relatively low, conditional dependence between the tests is more likely in the true disease positive animals. We hypothesize that conditional dependence arises because an unmeasured covariate influences the performance of both tests among disease positive animals causing both tests to incorrectly misclassify the animal as negative. One limitation of this study was the very low within herd prevalence of the participant herds.