Alex A. Ardans

Detection of Serum Antibody Responses in Cattle with Natural or Experimental Neospora Infections

Parasite-specific antibody responses were detected using an indirect fluorescent antibody (IFA) test in cattle that were naturally or experimentally infected with Neospora parasites. The test was developed using Neospora tachyzoites isolated from an aborted bovine fetus and grown in bovine cell cultures (isolate BPA1). In all cases, infections were confirmed by the identification of Neospora tachyzoites and/or bradyzoite cysts in fetal or calf tissues using an immunoperoxidase test procedure. Fifty-five naturally infected cows that aborted Neospora-infected fetuses had titers of 320-5,120 at the time of abortion. The titer of 6 cows that were serologically monitored over a prolonged period decreased to 160–640 within 150 days after they aborted infected fetuses. Two of the cows showed an increase in their Neospora titers during their subsequent pregnancy, and they gave birth to congenitally infected calves that had precolostral titers of 10,240-20,480. Postcolostral titers of these calves and of 4 other calves with congenital Neospora infections were all 25,120, whereas calves with no detectable parasites had titers ≤ 160. Two pregnant heifers that were experimentally infected with the BPA1 isolate at approximately 120 days gestation seroconverted to Neospora antigens within 9 days and developed peak titers of 5,120 and 20,480 within 32 days of infection. The fetus taken by caesarian section 32 days postinfection from 1 heifer and the full-term calf born to the other had Neospora titers of 640 and 10,240, respectively. Nine cows that aborted uninfected fetuses and 61 adult cattle maintained under pasture or feedlot conditions, where risk of exposure to Neospora was considered to be low, had titers ≤ 320. Some of the feedlot cattle tested had serologic reactivity that was restricted to antigens at the apical end of both Neospora and Toxoplasma gondii tachyzoites. This type of reactivity, which may result from serologic cross-reactivity between conserved apical complex antigens of closely related sporozoan parasites, differed from the whole parasite fluorescence that was observed with sera from Neospora-infected animals. The significance of these results and the potential application of the IFA test for the diagnosis of Neospora infections in cattle are discussed.

Experimental reproduction of bovine fetal Neospora infection and death with a bovine Neospora isolate

Studies were conducted to determine the pathogenic potential of the recently isolated bovine Neospora protozoa (BPA-1) for the bovine fetus. Cows chosen for study had Neospora titers < 160 using an indirect immunofluorescent antibody (IFA) test. Four experimental groups were studied. In group 1, 2 fetuses were inoculated in utero at 118 days gestation with culture-derived Neospora tachyzoites. A pregnant control cow was housed in the same pen, observed daily and screened serologically for evidence of exposure to Neospora. In group 2, 2 cows were infected with Neospora tachyzoites at 138 or 161 days gestation, and 1 control cow was given uninfected cell culture suspension simultaneously at 154 days gestation. Groups 3 (85 days gestation) and 4 (120 days gestation) each consisted of 2 cows infected with Neospora tachyzoites and 1 control cow given uninfected material at the same stage of gestation. Dead fetuses were surgically removed from the infected cows in group 1 on postinfection day (PID) 17. The histopathology was compatible with protozoal fetal infection, and protozoa were identified by immunohistochemistry. Viable fetuses were removed surgically from cows in group 2 on PID 28-30. The histopathology was compatible with protozoal fetal infection, protozoa were identified by immunoperoxidase techniques, and Neospora tachyzoites were reisolated in vitro from tissues of the 2 infected fetuses. In groups 3 and 4, the control fetus and 1 infected fetus were removed surgically between PID 26 and PID 33. The remaining infected cows were observed until fetal death or abortion occurred. In group 3, the fetus that was surgically removed from 1 infected cow had no pathologic abnormalities, and parasites were not found (PID 26). The second fetus in group 3 died in utero, and expulsion of a mummified fetus was induced on PID 67. Brain histopathology was compatible with protozoal infection, and parasites were identified by immunoperoxidase techniques. The fetus that was surgically removed (PID 32) from 1 infected cow in group 4 had lesions compatible with protozoal infection, and Neospora tachyzoites were reisolated in vitro from fetal tissues. The second infected cow in group 4 produced a full-term live calf that had a precolostral Neospora titer of 20,480. Clinically, this calf had depressed conscious proprioception in all limbs. Very mild lesions were found in the central nervous system, but protozoa were not found in the tissues. The results demonstrate that the bovine Neospora protozoa can be transplacentally transmitted, resulting in fetal infection and death, and mimics the naturally occurring fetal disease.

In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses

A Neospora sp. was isolated from the brains of two aborted bovine foetuses and grown continuously in vitro in bovine cell cultures. A comparison of the antigenic reactivity of in vitro cultivated tachyzoites with polyclonal antisera to Neospora caninum, Hammondia hammondi or Toxoplasma gondii revealed that the bovine protozoal isolates were similar to N. caninum and antigenically distinct from T. gondii. Tachyzoites of both bovine isolates had similar ultrastructural features, including an apical polar ring, conoid, electron-dense rhoptries and micronemes. The orientation of the micronemes, presence of micropores and a large number of electron-dense granules in the posterior portion of the bovine isolate tachyzoites differed from previous descriptions of N. caninum in vivo. Tachyzoites of the bovine isolates were ultrastructurally more similar to in vitro cultivated N. caninum tachyzoites than to tachyzoites of T. gondii or H. hammondi. The antigenic and ultrastructural similarities between N. caninum and the protozoal parasites isolated from aborted bovine foetuses in this study support the proposition that these parasites belong to the genus Neospora.

Salmonella enteritidis, phage type 4 infection in a commercial layer flock in southern California: bacteriologic and epidemiologic findings.

Salmonella enteritidis, phage type 4 (SE PT4), was isolated from five of six 27-wk-old layer chickens submitted for necropsy from a flock of 43,000. Bacteriologic and epidemiologic investigations on the ranch revealed that five of the eight flocks (n = 176,000) were infected. The prevalence of SE PT4 in randomly selected healthy birds ranged from 1.7% (in caged birds) to 50% (in free-range birds) and prevalence in culled birds (kept on dirt floor houses) ranged from 14% to 42%. The estimated overall prevalence of group D Salmonella in eggs contaminated with group D Salmonella was 2.28 per 10,000. The estimated prevalence of group D Salmonella in eggs from caged birds in three infected houses ranged from 1.5 to 4.1 per 10,000, whereas in two houses of free-range birds, prevalence was 14.9 to 19.1 per 10,000. Three of the eight flocks on the ranch remained negative for Salmonella between May 1994 and December 1995 or until removed from the ranch. Salmonella enteritidis PT4 was also isolated from 12.5% (6 of 48) of mice; 57% (four of seven) of cats; and two of two skunks tested. Environmental drag swabs and well water samples yielded multiple serotypes of Salmonella (23/180 and 5/14, respectively) but not S. enteritidis.

Cumulative racing-speed exercise distance cluster as a risk factor for fatal musculoskeletal injury in Thoroughbred racehorses in California

Abstract Thoroughbred racehorses which suffered a fatal musculoskeletal injury (FMI) while racing or race training at a California racetrack during 9 months of 1991 were studied to determine the importance of intensive, high-speed exercise schedules prior to injury. Seventy-seven horses which sustained an FMI while racing and 45 horses which sustained an FMI while race training were successfully matched by race or timed workout session with one control horse and included in the analyses. Race and timed workout (racing-speed exercise) histories were obtained for the case and control horses. Two-month cumulative, racing-speed cutoff distances were calculated from the control horse sample by two methods. Median racing-speed exercise frequencies and distances of the control horses were used to estimate age-specific (2, 3, 4 and ≥ 5 years), 2-month cumulative, racing-speed distances (Method 1). For the second method, the last race or timed workout for each control horse occurring just prior to, or on the date of injury for the matched case horse was identified. Cumulative racing-speed distances 2 months prior to these exercise events were determined for each control horse and used to estimate median age-specific (2, 3, 4 and ≥ 5 years), 2-month cumulative racing-speed distances (Method 2). The cumulative cutoff distances estimated from both methods were used to classify each matched pair according to the presence or absence of a 2-month cumulative, racing-speed distance which exceeded the age-appropriate cutoff distance (exercise distance cluster) within 6 months prior to injury. Manlel-Haenszel matched-pair odds ratios and 95% confidence limits were calculated separately for the racing and race-training fatal injuries. The relative risk for racing FMI was significantly greater for those horses which ran 2-month, cumulative racing and timed workout distances in excess of the cutoff values determined with Method 1 (relative risk (RR) = 3.0, 95% confidence interval (CI) = 1.2, 7.6) and Method 2 (RR = 7.2, 95% CI = 2.6, 20.6). The relative risk for race-training FMI was significantly greater for those horses which ran 2-month, cumulative racing and timed workout distances in excess of the cutoff values determined with Method 2 (RR = 3.4, 95% CI =1.0, 13.2).

Longitudinal Monitoring of Two Commercial Layer Flocks and Their Environments for Salmonella enterica Serovar Enteritidis and Other Salmonellae

Abstract Between August 20, 2001, and September 17, 2002, 1429 samples including drag swabs, egg belt or egg rollout swabs, fan-blade swabs, rodent organ and intestinal pools, beetle (Alphitobius diaperinus) pools, housefly (Musca domestica) pools, chicken organ and intestinal pools, and egg pools were obtained for Salmonella culture from two flocks from two different commercial layer ranches. The two ranches were purposefully selected for the study based on their previous status of Salmonella Enteritidis isolation using environmental drag swabs in cooperation with practicing veterinarians. Salmonella sp. was isolated from 337 out of 979 (34.42%) non-egg samples. No Salmonella was isolated from 450 egg pools collected from either ranch. S. enteritidis was isolated from samples obtained from ranch 1 from manure drag swabs, 4/284 (1.4%); rodent organs, 1/24 (4.2%); and housefly pool cultures 1/21 (4.8%). Salmonella Enteritidis was isolated from ranch 2 from mouse organ and intestinal pool samples, 1/24 (4.2%). Salmonella group B was isolated from all sample types except the insects. There was a statistically significant difference in isolation rates among seven serogroups of Salmonella: groups B, C1, C2, D, E, K, and untypeable (Pearson chi-square 18.96, P = 0.002). Overall, statistically significant differences were observed with respect to Salmonella isolation among the types of samples taken (Pearson chi-square 118.54, P < 0.0001). Intensive monitoring for Salmonella Enteritidis can be used to optimize a Salmonella reduction program for an individual poultry biosecurity unit.

SEWAGE EFFLUENT : LIKELY SOURCE OF SALMONELLA ENTERITIDIS, PHAGE TYPE 4 INFECTION IN A COMMERCIAL CHICKEN LAYER FLOCK IN SOUTHERN CALIFORNIA

Following the diagnosis of Salmonella enteritidis, phage type 4, infection in a commercial layer flock in southern California, effluent from a nearby sewer treatment plant was investigated as a potential source of infection. Between July 1994 and March 1995, 68 Salmonella isolations, comprising 27 serotypes, were made from the inflow (raw sewage) and effluent (treated sewage). Thirty-nine of 68 (57%) isolations yielded six serotypes, which consisted of S. enteritidis 12% (8/68), S. cerro 10% (7/68), S. typhimurium 7.4% (5/68), S. tennessee 7.4% (5/68), S. give 7.4% (5/68), S. mbandaka 7.4% (5/68), and S. panama 6% (4/68). The remaining 43% (29/68) isolations were represented by 21 serotypes. Seventeen S. enteritidis isolates originating from the effluent (creek water), resident feral animals (rodents, stray cats, skunks), and chickens (organs, eggs) of the affected flock were subjected to plasmid profile and restriction endonuclease analysis. Twelve of the 17 isolates had identical plasmid profile and restriction digestion patterns. Two of 17 isolates showed similar patterns but both differed from the rest; and 1 of 17 did not yield plasmids. Two other isolates were found to be different from each other and from the rest of the group.

Neutrophil phagocytic and serum opsonic response of the foal to Corynebacterium equi

This study was undertaken to examine the neutrophil response to Corynebacterium (Rhodococcus) equi, and to assess the possibility of neutrophil immaturity or malfunction in predisposition to C. equi pneumonia in foals. Neutrophil phagocytosis of Corynebacterium (Rhodococcus) equi was studied in foals from birth to 6 months of age. Chemiluminescence (CL) and bactericidal assays were used to assay the phagocytic response of peripheral blood neutrophils to C. equi in vitro. Results of in vitro bactericidal and CL assays indicate that foal neutrophils are able to ingest and kill C. equi, however are significantly more efficient in the presence of opsonization with specific antibody, and less importantly complement. Neutrophil CL was significantly decreased (p greater than .05) or eliminated by antibody adsorption, heat-inactivation, or removal of serum from the assay. The ability of the neutrophil to kill C. equi, as measured by in vitro bactericidal assay, was greater than 90% killing by 6 hours, in the presence of C. equi antiserum. Bactericidal activity was reduced to less than 40% killing when C. equi adsorbed serum was used as the opsonin source. As CL results indicated complement involvement in the opsonization of C. equi, the temporal development of hemolytic and conglutinating complement was measured in normal and C. equi infected foals. Neither defects nor age-related suppression of neutrophil function or complement activity were detected in C. equi affected foals, suggesting that these are not pathogenic mechanisms involved in foal pneumonia.

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera.

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.

Pathologic and bacteriologic findings in 27-week-old commercial laying hens experimentally infected with Salmonella enteritidis, phage type 4.

Two strains of 27-wk-old commercial laying chickens (strain A, brown-egg-laying type and strain B, white-egg-laying type) were inoculated either orally (PO) or intravenously (IV) with a field isolate of Salmonella enteritidis phage type 4. Chickens were sequentially necropsied at regular intervals throughout the 17-wk observation period. Gross and microscopic lesions were most evident between 1 and 14 days postinoculation (DPI). Gross lesions consisted of enlarged livers with white foci, enlarged and mottled white spleens, fibrinous exudate in the peritoneum, and atretic, misshapen ovarian follicles. Microscopic lesions included multifocal coagulative necrosis of hepatocytes and inflammation, fibrinous exudation in vascular sinuses of the spleen, and fibrinosuppurative inflammation of the peritoneum and ovarian follicles. The proportion of reproductive organ infections (ovary and oviduct) in the IV group, 83% (20/24, P = 0.007; 50% and 33% for strains A and strain B birds, respectively), was higher than that of the PO group, 46% (11/24; 29% and 17% for strains A and B, respectively), for the first 16 days of observation postinoculation. The proportion of fecal shedding for the IV group of birds was significantly (P = 0.009) lower, 29% (7/24; 33% and 25% respectively for strain A and strain B birds, respectively), than the PO group, 67% (16/24; 75% and 58% for strain A and strain B birds, respectively). Three (2.6%) of 234 egg pools were culture-positive for group D Salmonella from strain A chickens (1 of 119 pools from the IV group and 2 of 115 pools from the PO group of birds). Chickens infected with the field strain of S. enteritidis phage type 4 harbored the organism in tissues only for a brief time, most clearing within 8 DPI and nearly all within 16 DPI. Overall the percentage of culture-positive birds did not differ significantly (P > 0.05) between birds with and without lesions, but isolation of S. enteritidis tended to be more frequent when lesions were evident. This experiment also demonstrated that brown-egg-laying-type chickens were more susceptible than white-egg-laying-type chickens to S. enteritidis phage type 4 isolated from California based on gross and microscopic lesions and bacteriologic findings.