Colostral transmission of bluetongue virus nucleic acid among newborn dairy calves in California.

There have been substantial recent changes in the global distribution and nature of bluetongue virus (BTV) infection of ungulates, perhaps as a result of climate change. To evaluate the epidemiology of BTV infection in California, an area historically endemic for the virus, we monitored newborn dairy calves at different sites for 1 year for the presence of BTV RNA and virus-specific antibodies. The data confirm both localized, vector-mediated, seasonal transmission of BTV as well as dissemination of BTV and/or viral nucleic acid to newborn calves following ingestion of colostrum.

Likelihood ratio estimation without gold standard: a case study evaluating brucellosis c-ELISA in cattle and water buffalo in Trinidad

The likelihood ratio (LR) is a measure of association that quantifies how many more times likely a particular test result is from an infected animal compared to one that is uninfected. They are ratios of conditional probabilities and cannot be interpreted at the individual animal level without information concerning pretest probabilities. Their usefulness is that they can be used to update the prior belief that the individual has the outcome of interest through a modification of Bayes' theorem. Bayesian analytic techniques can be used for the evaluation of diagnostic tests and estimation of LRs when information concerning a gold standard is not available. As an example, these techniques were applied to the estimation of LRs for a competitive ELISA (c-ELISA) for diagnosis of Brucella abortus infection in cattle and water buffalo in Trinidad.Sera from four herds of cattle (n = 391) and four herds of water buffalo (n = 381) in Trinidad were evaluated for Brucella-specific antibodies using a c-ELISA. On the basis of previous serologic (agglutination) test results in the same animals, iterative simulation modeling was used to classify animals as positive or negative for Brucella infection. LRs were calculated for six categories of the c-ELISA proportion inhibition (PI) results pooled for cattle and water buffalo and yielded the following estimations (95% probability intervals): <0.10 PI, 0.05 (0ùC0.13); 0.10ùC0.249 PI, 0.11 (0.04ùC0.20); 0.25ùC0.349 PI, 0.77 (0.23ùC1.63); 0.35-0.499 PI, 3.22 (1.39ùC6.84); 0.50ùC0.749 PI, 17.9 (6.39ùC77.4); ­í0.75 PI, 423 (129ùC­è). LRs are important for calculation of post-test probabilities and maintaining the quantitative nature of diagnostic test results.

Likelihood ratio estimation without a gold standard: a case study evaluating a brucellosis c-ELISA in cattle and water buffalo of Trinidad.

The likelihood ratio (LR) is a measure of association that quantifies how many more times likely a particular test result is from an infected animal compared to one that is uninfected. They are ratios of conditional probabilities and cannot be interpreted at the individual animal level without information concerning pretest probabilities. Their usefulness is that they can be used to update the prior belief that the individual has the outcome of interest through a modification of Bayes' theorem. Bayesian analytic techniques can be used for the evaluation of diagnostic tests and estimation of LRs when information concerning a gold standard is not available. As an example, these techniques were applied to the estimation of LRs for a competitive ELISA (c-ELISA) for diagnosis of Brucella abortus infection in cattle and water buffalo in Trinidad. Sera from four herds of cattle (n=391) and four herds of water buffalo (n=381) in Trinidad were evaluated for Brucella-specific antibodies using a c-ELISA. On the basis of previous serologic (agglutination) test results in the same animals, iterative simulation modeling was used to classify animals as positive or negative for Brucella infection. LRs were calculated for six categories of the c-ELISA proportion inhibition (PI) results pooled for cattle and water buffalo and yielded the following estimations (95% probability intervals): <0.10 PI, 0.05 (0-0.13); 0.10-0.249 PI, 0.11 (0.04-0.20); 0.25-0.349 PI, 0.77 (0.23-1.63); 0.35-0.499 PI, 3.22 (1.39-6.84); 0.50-0.749 PI, 17.9 (6.39-77.4); > or =0.75 PI, 423 (129-infinity). LRs are important for calculation of post-test probabilities and maintaining the quantitative nature of diagnostic test results.

Armored RNA as virus surrogate in a real-time reverse transcriptase PCR assay proficiency panel.

In recent years testing responsibilities for high-consequence pathogens have been expanded from national reference laboratories into networks of local and regional laboratories in order to support enhanced disease surveillance and to test for surge capacity. This movement of testing of select agents and high-consequence pathogens beyond reference laboratories introduces a critical need for standardized, noninfectious surrogates of disease agents for use as training and proficiency test samples. In this study, reverse transcription-PCR assay RNA targets were developed and packaged as armored RNA for use as a noninfectious, quantifiable synthetic substitute for four high-consequence animal pathogens: classical swine fever virus; foot-and-mouth disease virus; vesicular stomatitis virus, New Jersey serogroup; and vesicular stomatitis virus, Indiana serogroup. Armored RNA spiked into oral swab fluid specimens mimicked virus-positive clinical material through all stages of the reverse transcription-PCR testing process, including RNA recovery by four different commercial extraction procedures, reverse transcription, PCR amplification, and real-time detection at target concentrations consistent with the dynamic ranges of the existing real-time PCR assays. The armored RNA concentrations spiked into the oral swab fluid specimens were stable under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling of proficiency panel samples, including 24 h at 37 degrees C and 2 weeks at temperatures ranging from ambient room temperature to -70 degrees C. The analytic test performance, including the reproducibility over the dynamic range of the assays, indicates that armored RNA can provide a noninfectious, quantifiable, and stable virus surrogate for specific assay training and proficiency test purposes.

The isolation of exotic Newcastle disease (END) virus from nonpoultry avian species associated with the epidemic of END in chickens in southern California: 2002-2003.

During the first 11 months of the 2002-2003 exotic Newcastle disease (END) epidemic in chickens in southern California, a total of 27,688 cloacal and tracheal (oropharyngeal) swab pools and/or tissue pools from 86 different avian species other than chickens and turkeys were submitted for Newcastle disease virus (NDV) isolation and characterization. Fifty-seven specimens (0.23%), representing 12 species of birds and 13 unspecified species, from a total of 24,409 accessions or submissions were positive for NDV. The NDV isolate was characterized as ENDV by real-time reverse transcription-polymerase chain reaction (RT-PCR). Of the 11,486 premises with other avian species, 1599 also had chickens. There were 1900 positive chicken samples from 164 premises, and 56 positive other avian species from 51 premises. Twelve premises had both positive chickens and positive other avian species. All positive other avian species were located on premises either on or within a 1 km radius of known infected premises. In this epidemic, premises with positive other avian species were significantly more likely to have chickens, and were significantly more likely to have positive chickens (OR = 3.7, P < 0.0001).

Risk factors associated with Neospora caninum abortion in Ontario Holstein dairy herds.

The objective of this epidemiological study was to identify risk factors for Neospora caninum-related abortions in Ontario Holstein dairy herds. A total of 88 herds, consisting of 5080 cattle, and utilizing Dairy Herd Improvement (DHI) services, were divided into three groups. Case (n = 30) and first control (n = 31) herds were selected from 1998 and 1999 fetal abortion submissions to the Animal Health Laboratory, University of Guelph, that were histopathologically positive or negative, respectively, for N. caninum. A second control group (n = 27) was selected from multiple sources of herds sampled within the previous 4 years that had a low seroprevalence (<7%) to N. caninum. Between May and December 1999, all available cows on all farms, in parity one or greater, were blood sampled. The sera were then analyzed for antibody to N. caninum using a kinetic ELISA. A survey administered at the time of sampling recorded information on housing, animal species present, manure management, reproduction, biosecurity practices, wildlife observations, peri-parturient cow management, herd disease history and nutrition. Production and other herd parameters were obtained from DHI records. Logistic regression indicated that the following parameters were positively associated with a N. caninum abortion in a herd: the N. caninum herd seroprevalence (OR = 1.1), the total number of dogs on a farm (OR = 2.8), the frequency that dogs were observed defecating in mangers (OR = 2.8), the number of horses on a farm (OR = 3.1), the observed annual rate of retained fetal membranes (OR = 1.2) and the observed annual rate of cows returning to estrus after pregnancy confirmation (OR = 1.2). Factors negatively associated were the frequency of stray cats and wild canids observed on a farm (OR = 0.4 and OR = 0.7, respectively) and the housing of heifers on loafing packs (a housing pen divided into feed manger, scrape alley and bedded pack areas, OR = 0.1).

Evaluation of brucellosis RB51 vaccine for domestic water buffalo (Bubalus bubalis) in Trinidad.

Thirty-two young domestic water buffalo (Bubalus bubalis) were obtained from a brucellosis-free farm to determine effectiveness of RB51 vaccination for prevention of Brucella infection under natural-exposure conditions in Trinidad. Study animals (20 males and 12 females 5-20 months old) were assigned to vaccination or control groups, using a block randomization design ensuring equal sex distributions between groups. The vaccination group received commercially available RB51 at the recommended calfhood dose of (1.0-3.4)x10(10) colony-forming units (CFU) and controls received 2ml sterile saline. Vaccination did not result in positive serologic results as measured by four traditional agglutination tests: standard tube agglutination test (STAT), standard plate agglutination test (SPAT), buffered plate agglutination test (BPAT), and card agglutination. Study animals were maintained in a brucellosis-positive herd in southern Trinidad with an estimated 56% prevalence to allow for natural exposure to B. abortus, which was evaluated using STAT, SPAT, BPAT, and card tests. Animals were sampled seven times over 2 years and were classified as positive if they had persistent agglutination titers or had Brucella isolated from specimens collected at completion of the study. Five of the original 32 study animals were lost to follow-up during the field trial. Six of the 14 (43%) vaccinated animals completing the study were classified as positive for Brucella infection-as were two of the 13 (15%) control animals (P=0.21). Isolates from four vaccinates and one control were confirmed as B. abortus biovar 1.

Genetic diversity of avian infectious bronchitis virus California variants isolated between 1988 and 2001 based on the S1 subunit of the spike glycoprotein.

Twenty-nine isolates of avian infectious bronchitis virus (IBV) recovered from commercial chicken flocks in California between 1988 and 2001 and identified as California variants by serotype and direct automated cycle sequencing of the IBV spike glycoprotein S1 subunit, were further characterized phylogenetically and by nucleotide sequence comparison. California variants were grouped according to production type of chicken, by comparison with public access sequence databases (NCBI GenBank and EMBL), or based on phylogenetic analysis. Fisher's Exact test was used to compare mutations per year, purifying and positive selection, predictive antigenicity, and a > or = 6 bp deletion between California variant groups.A high number of mutations at the nucleotide level ( p = 0.013) and a > or = 6 bp deletion in the nucleotide sequence ( p = 0.006) was significantly associated with broiler-type chickens. However, 88% of significant comparisons at the amino acid level such as purifying and positive selection were seen in layer-type chickens. A pronounced predictive antigenicity in the HVR2 region was also associated with layer-type chickens ( p = 0.001). The study indicates that IBV in California is in a phase of slow evolution with different evolutionary patterns being associated with the production type of chicken.

Descriptive epidemiology of postnatal bovine viral diarrhea virus infection in intensively managed dairy heifers.

OBJECTIVE: To evaluate risk of bovine viral diarrhea virus (BVDV) infection between birth and 9 months of age for dairy replacement heifers raised under typical dry-lot management conditions. DESIGN: Longitudinal observational study. ANIMALS: 446 calves. PROCEDURE: Calves were randomly selected from 2 dairies that used killed and modified-live BVDV vaccines. Repeated serologic and BVDV polymerase chain reaction assays were used to estimate risk of BVDV infection in calves of various ages (1 to 60 days; 61 to 100 days; 101 days to 9 months) and to estimate overall infection rate by 9 months of age. RESULTS: Risk of BVDV infection increased with age (maximum risk, 150 to 260 days). Proportion of calves infected with BVDV by 9 months of age was higher for dairy A (0.665), compared with dairy B (0.357). Percentage infected with BVDV type I did not differ between dairy A (18.2%) and dairy B (15.2%), whereas percentage infected with BVDV type II for dairy A (50%) was twice that for dairy B (21%). Between 210 and 220 days of age, infection with BVDV regardless of type was > 1.3%/d on dairy A and 0.5%/d on dairy B. CONCLUSIONS AND CLINICAL RELEVANCE: Under dry-lot conditions, a considerable amount of BVDV infection may occur before 9 months of age. Risk of infection increases with age. Although dairies may appear to have similar management practices, there can be considerably different risks of BVDV infection among dairies.

Seroprevalence of antibodies against equine arteritis virus in horses residing in the United States and imported horses.

OBJECTIVE: To compare seroprevalence of antibodies against equine arteritis virus (EAV) in horses residing in the United States with that of imported horses. DESIGN: Serologic survey. SAMPLE POPULATION: Serum samples from 364 horses on 44 equine operations in California and 226 horses imported from various countries. PROCEDURE: Serum samples were collected from each imported horse and from up to 20 horses on each operation. For resident horses, the number of sampled horses on each operation was determined on the basis of the number of horses on the operation. Samples were tested for antibodies against EAV by use of a serum neutralization test. RESULTS: 1.9% of resident horses and 18.6% of imported horses were seropositive to EAV, including 16.1% of imported stallions. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that the EAV seroprevalence of horses residing in California is considerably lower than that of imported horses, including imported stallions.

Effect of calfhood vaccination on transmission of bovine viral diarrhea virus under typical drylot dairy conditions.

OBJECTIVE: To estimate transmission of bovine viral diarrhea virus (BVDV) and crude morbidity and mortality ratios in BVDV-vaccinated and unvaccinated dairy heifer calves managed under typical dairy drylot conditions. DESIGN: Randomized clinical trial. ANIMALS: 106 female Holstein calves. PROCEDURE: Seroconversion rates for BVDV types I and II and proportional morbidity and mortality ratios were compared between calves given a killed BVDV type-I vaccine at 15 days of age and a modified-live BVDV type-I vaccine at 40 to 45 days of age (n = 53) and calves given no BVDV vaccines (53). Sera were collected at 45-day intervals as calves moved from individual hutches to corrals holding increasingly larger numbers of calves. Seroconversion was used as evidence of exposure to BVDV. RESULTS: Crude proportional morbidity (0.16) and mortality (0.17) ratios for control calves did not differ significantly from those of vaccinated calves (0.28 and 0.12, respectively). The proportion of control calves that seroconverted to BVDV type I through 9 months of age (0.629) was significantly higher than that of vaccinated calves that seroconverted, unrelated to vaccination, during the same period (0.536). Estimated overall protective effect of vaccination against BVDV type I through 4 to 9 months of age was 48%. The proportion of control calves that seroconverted to BVDV type II (0.356) was not different from that of vaccinated calves (0.470). CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest that calfhood vaccination may be an appropriate strategy to help reduce short-term transmission of some but not necessarily all strains of BVDV.

Pooled-sample testing as a herd-screening tool for detection of bovine viral diarrhea virus persistently infected cattle.

The study was conducted to develop methodology for least-cost strategies for using polymerase chain reaction (PCR)/probe testing of pooled blood samples to identify animals in a herd persistently infected with bovine viral diarrhea virus (BVDV). Cost was estimated for 5 protocols using Monte Carlo simulations for herd prevalences of BVDV persistent infection (BVDV-PI) ranging from 0.5% to 3%, assuming a cost for a PCR/probe test of $20. The protocol associated with the least cost per cow involved an initial testing of pools followed by repooling and testing of positive pools. For a herd prevalence of 1%, the least cost per cow was $2.64 (95% prediction interval = $1.72, $3.68), where pool sizes for the initial and repooled testing were 20 and 5 blood samples per pool, respectively. Optimization of the least cost for pooled-sample testing depended on how well a presumed prevalence of BVDV-PI approximated the true prevalence of BVDV infection in the herd. As prevalence increased beyond 3%, the least cost increased, thereby diminishing the competitive benefit of pooled testing. The protocols presented for sample pooling have general application to screening or surveillance using a sensitive diagnostic test to detect very low prevalence diseases or pathogens in flocks or herds.

Postnatal Neospora caninum transmission and transient serologic responses in two dairies.

Cattle on two typically managed drylot dairies were serologically monitored from birth through year 1 to year 4 of life to characterise congenital and postnatal Neospora caninum transmission. Of the 456 calves enrolled, 284 were classified as N. caninum negative and 172 were classified as N. caninum positive. Ninety-six percent of congenitally infected calves were seropositive for all samples tested. Seven (4%) of the 172 congenitally infected animals had a period that persisted for 9 to 18 months when they were seronegative; however, all returned to seropositive status by 25 months of age. In N. caninum-negative calves, colostral antibody decayed by 128 days, with an estimated half-life of 19.6 +/- 5.2 days. Of the 284 calves classified as negative, 18% had sporadic, isolated responses to N. caninum, typically between 29 and 35 months of age, without subsequent seroconversion or infection. During the study, 17 animals seroconverted and remained seropositive throughout the follow-up. Thirteen of the seroconversions occurred in the neonatal period; however, in nine of 10 where dam status was available, the dam was N. caninum positive, suggesting late gestation congenital infection rather than postnatal infection. Seroconversion was detected in an additional four animals, between 13 and 22 months of age. The estimate of postnatal infection rate was less than 1% per year despite a high N. caninum seroprevalence in the herds, and the presence of potential definitive and intermediate hosts on the dairy throughout the study. The extremely low rate of postnatal infection, as well as the lifelong persistence of congenital infection, emphasises the importance of congenital transmission in maintaining N. caninum infection in dairies.

Infertility and abortion among first-lactation dairy cows seropositive or seronegative for Leptospira interrogans serovar hardjo.

OBJECTIVE: To estimate the extent to which exposure to Leptospira hardjo before or at the time of first parturition was associated with infertility and abortion during the first lactation among dairy cows that had not been vaccinated for > or = 12 months. ANIMALS: 207 first-lactation cows from a herd of 2,000 lactating cows. PROCEDURE: Cows were tested for antibodies to L hardjo within 40 days after calving. Time from calving to first breeding, time from calving to conception, number of breedings per conception, and risk of abortion were compared between cows seropositive for L hardjo and cows that were seronegative. RESULTS: For the 9 (4.3%) cows that were seropositive for L hardjo, median time from calving to conception (132.6 days) was significantly longer than time for seronegative cows (95.4 days). Cows that were seropositive were twice as likely (relative risk, 2.07) to fail to conceive as seronegative cows. Mean number of breedings required per conception for seropositive cows (3.4) was significantly higher than that for seronegative cows (2.1). The proportion of seropositive cows that aborted was not significantly different from the proportion of seronegative cows that aborted. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of nonvaccinated dairy cows to L hardjo can be associated with a subsequent reduction in fertility, as indicated by a greater time from calving to conception and higher number of breedings required per conception. The efficacy of leptospiral vaccines should be assessed to determine whether vaccination will minimize herd infertility associated with L hardjo infection.

Seroprevalence of Neospora caninum and abortion in dairy cows in northern Spain.

The seroprevalence of Neospora caninum infection was estimated from a sample of 889 cattle from 43 dairy herds in three counties in the Asturias region of Spain. The true prevalence of infection was estimated to be 30.6 per cent (95 per cent confidence interval (CI) 27.6 to 33.6). Seropositivity was associated with abortion during the previous year (odds ratio (OR)=3.31, P<0.001) and was slightly higher among purchased cattle (37.6 per cent), than among cattle raised on the farm (29.1 per cent) (P=0.078). Seropositive cows were more likely than seronegative cows to have had a seropositive dam (OR=2.3, P=0.011), suggesting that congenital transmission contributed to about 56 per cent of the infections. Herds with a true seroprevalence above 10 per cent had more dogs on the farm, than herds with a lower prevalence (P=0.032). The ORS relating abortion to seropositivity in individual herds ranged from 0.7 to 19, indicating that some herds experienced few abortions caused by N. caninum, while others experienced more abortions due to the organism. Overall, 38.7 per cent of the abortions were estimated to have been attributable to N. caninum.

Evaluation of North American antibody detection tests for diagnosis of brucellosis in goats.

The sensitivities and specificities of 17 antibody detection tests for brucellosis in goats were estimated. Tests evaluated included the U.S. Department of Agriculture (USDA) card test with 8% cell concentration (8%Card), USDA rapid automated presumptive test (RAP), Mexican rose bengal plate tests with 8 and 3% cell concentrations (8%RB and 3%RB), French rose bengal plate test with 4.5% cell concentration (4.5%RB), USDA standard plate test (SPT), USDA buffered acidified plate agglutination test (BAPA), USDA and Mexican rivanol tests (URIV and MRIV), USDA standard tube tests with Brucella abortus and Brucella melitensis antigens (SATA and SATM), serum enzyme-linked immunosorbent assay (ELISA), USDA cold-fixation complement fixation tests with B. abortus and B. melitensis antigens (CFA and CFM), USDA and Mexican milk ring tests (UBRT and MBRT), and a milk ELISA. Test sensitivity was evaluated by using two groups of 10 goats experimentally infected with B. melitensis or B. abortus and monitored for 24 weeks. Specificity was evaluated by using 200 brucellosis-free nonvaccinated goats from 10 California herds. The 3%RB was considered a good screening test because of high sensitivity at week 24 postinfection (90%), ease of performance, and low cost. The cold-fixation CFA and CFM had 100% specificity in the field study and were considered appropriate confirmatory tests. The milk ELISA was significantly more sensitive (P < 0.05) than the UBRT and significantly more specific (P < 0.05) than the MBRT. The milk ELISA also had the advantage of objectivity and ease of interpretation.

Risk factors for brucellosis seropositivity of goat herds in the Mexicali Valley of Baja California, Mexico.

A case-control study was conducted in the Mexicali Valley to identify risk factors for goat-herd seropositivity for Brucella melitensis. Nineteen case herds (> or = 2 positive results with the 8% rose bengal plate test (RBT)) and 55 control herds (zero positive results in RBT), matched for herdsize and geographic location, were enrolled. Conditional logistic regression was used to construct a multivariable model of the odds of seropositivity using variables assessed in a questionnaire administered to goat ranchers. The final model for herd seropositivity included increased risk from importation of goats from other Mexican states, the presence of La Mancha breed does, and the presence of does born outside the herd. Increasing herdsize was also highly significant (p < 0.01). In addition, a significant (p < 0.05) positive association was found between the presence of seropositive dogs (as assessed by RBT) and seropositive goats on the same ranch.

Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle.

OBJECTIVES: To estimate the extent to which abortion risk in dairy cattle during subsequent pregnancies was associated with congenitally-acquired Neospora caninum infection and previous abortions. ANIMALS: 468 Holstein cattle. PROCEDURE: Newborn heifer calves were tested for evidence of congenital infection attributable to N caninum and examined repeatedly until the completion of their second lactation for serologic status and evidence of abortion. RESULTS: Compared with noninfected cows, congenitally infected cows had a 7.4-fold higher risk of abortion during their initial pregnancy and a 1.7-fold higher risk of aborting the first pregnancy during their first lactation. During the first pregnancy of their second lactation, congenitally infected cows that had aborted previously had a 5.6-fold higher risk of abortion, compared with cows that had not previously aborted and that were seronegative. The fetal risk period for N caninum-associated death began sooner and extended later during the initial pregnancy compared with subsequent pregnancies. CONCLUSION: Congenitally acquired N caninum infection can cause a substantial number of abortions during the initial pregnancy of heifers, with abortion risk attributable to N caninum decreasing in subsequent pregnancies, possibly because of selective culling. Subsequent abortions can be expected in congenitally infected cows that have aborted previously.