Nanopore sequencing as a rapid tool for identification and pathotyping of avian influenza A viruses.

We report whole-genome sequencing of influenza A virus (IAV) with 100% diagnostic sensitivity and results available in <24-48 h using amplicon-based nanopore sequencing technology (MinION) on clinical material from wild waterfowl (n = 19), commercial poultry (n = 4), and swine (n = 3). All 8 gene segments of IAV including those from 14 of the 18 recognized hemagglutinin subtypes and 9 of the 11 neuraminidase subtypes were amplified in their entirety at >500× coverage from each of 16 reference virus isolates evaluated. Subgenomic viral sequences obtained in 3 cases using Sanger sequencing as the reference standard were identical to those obtained when sequenced using the MinION approach. An inter-laboratory comparison demonstrated reproducibility when comparing 2 independent laboratories at ≥99.8% across the entirety of the IAV genomes sequenced.

Genomic and antigenic characterization of a cytopathic bovine viral diarrhea virus 1i isolated in the United States.

Bovine viral diarrhea viruses (BVDV) are a common global viral pathogen of ruminants. Considerable genetic variability is found amongst BVDV1 isolates, with at least 21 subgenotypes being described. In the United States, BVDV1a and 1b are the only subgenotypes described to date. Here, the genomic sequence of CA2005, a cytopathic BVDV1, was determined. This virus, isolated in California, did not segregate into either BVDV1a or 1b subgenotypes. BLAST analysis showed CA2005 was most closely related to BVDV1i isolates. CA2005 was also the first cytopathic BVDV1i and one of few non-1a, non-1b cytopathic viruses reported. The genomic sequence was 15,752 nucleotides in length. Cytopathogenicity was conferred by duplication of the NS3 protein with a small ubiquitin B insertion at the border of the NS2/NS3 proteins. Virus neutralization assays using antisera against BVDV1a vaccine viruses revealed variable neutralization, suggesting modified live vaccines may not be totally protective against CA2005 and similar viruses.

Evaluation of Fast Technology Analysis (FTA) Cards as an improved method for specimen collection and shipment targeting viruses associated with Bovine Respiratory Disease Complex.

In order to improve the analytic quality of respiratory specimens collected from cattle for nucleic acid-based diagnosis, a study was undertaken to verify realtime PCR efficiency of specimens collected and stabilized on FTA Cards™, filter paper which is treated chemically. Nucleic acids collected using FTA Cards without the need for a cold-chain or special liquid media handling provided realtime PCR results consistent (96.8% agreement, kappa 0.923 [95% CI=0.89-0.96]) with the same specimens collected using traditional viral transport media and shipped on ice using the U.S. Department of Transportation mandated liquid handling requirements. Nucleic acid stabilization on FTA Cards was evaluated over a temperature range (-27 °C to +46 °C) for up to 14 days to mimic environmental conditions for diagnostic sample handling between collection and processing in a routine veterinary laboratory. No significant difference (P≥0.05) was observed in realtime PCR cycle threshold values over the temperature range and time storage conditions for Bovine Viral Diarrhea virus, Bovine Respiratory Syncytial virus, Bovine Coronavirus, and Bovine Herpesvirus I. The four viruses evaluated in the study are associated with Bovine Respiratory Disease Complex where improvements in ease and reliability of specimen collection and shipping would enhance the diagnostic quality of specimens collected in the field, and ultimately improve diagnostic efficiency.

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows.

Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection.

Évaluation des tests ELISA réalisés sur des échantillons de lait et de sérum pour la détection de la néosporose et de la leucose chez les vaches laitières en lactation. Des échantillons de sérum et de lait provenant de 1229 vaches dans 22 fermes laitières de l'Ontario ont été testés individuellement pour déceler des anticorps particuliers au virus de la leucose bovine (VLB) et de Neospora caninum à l'aide d'un test ELISA. Les anticorps contre le VLB étaient présents dans 361 échantillons de sérum (29,4 %) et 369 échantillons de lait (30,0 %). En comparant les 2 tests, la concordance était quasiment parfaite (k = 0,86; IC de 95 % = de 0,83 à 0,90) et les proportions d'échantillons positifs n'étaient pas significativement différentes (P = 0,56). Les deux tests ont identifié les même 3 troupeaux comme étant libres du virus de la leucose bovine. Des anticorps contre N. caninum ont été détectés dans 138 échantillons de sérum (11,2 %) et 111 échantillons de lait (9,0 %). La concordance entre les 2 tests était modérée (k = 0,52; IC de 95 % = de 0,43 à 0,59). Quatre troupeaux étaient libres de néosporose lors du test pour le sérum, tandis que 10 troupeaux étaient négatifs lors du test pour le lait. Le test ELISA sur les échantillons de lait facilite le prélèvement d'échantillons pour déclarer les troupeaux comme étant libre du VLB; le test ELISA du lait pour N. caninum était moins fiable pour prédire l'infection au niveau du troupeau.(Traduit par Isabelle Vallières).

Identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus 229E.

In 2007, a novel coronavirus associated with an acute respiratory disease in alpacas (Alpaca Coronavirus, ACoV) was isolated. Full-length genomic sequencing of the ACoV demonstrated the genome to be consistent with other Alphacoronaviruses. A putative additional open-reading frame was identified between the nucleocapsid gene and 3'UTR. The ACoV was genetically most similar to the common human coronavirus (HCoV) 229E with 92.2% nucleotide identity over the entire genome. A comparison of spike gene sequences from ACoV and from HCoV-229E isolates recovered over a span of five decades showed the ACoV to be most similar to viruses isolated in the 1960's to early 1980's. The true origin of the ACoV is unknown, however a common ancestor between the ACoV and HCoV-229E appears to have existed prior to the 1960's, suggesting virus transmission, either as a zoonosis or anthroponosis, has occurred between alpacas and humans.

Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

Effect of changes in testing parameters on the cost-effectiveness of two pooled test methods to classify infection status of animals in a herd.

Monte Carlo simulation was used to determine optimal fecal pool sizes for identification of all Mycobacterium avium subsp. paratuberculosis (MAP)-infected cows in a dairy herd. Two pooling protocols were compared: a halving protocol involving a single retest of negative pools followed by halving of positive pools and a simple protocol involving single retest of negative pools but no halving of positive pools. For both protocols, all component samples in positive pools were then tested individually. In the simulations, the distributions of number of tests required to classify all individuals in an infected herd were generated for various combinations of prevalence (0.01, 0.05 and 0.1), herd size (300, 1000 and 3000), pool size (5, 10, 20 and 50) and test sensitivity (0.5-0.9). Test specificity was fixed at 1.0 because fecal culture for MAP yields no or rare false-positive results. Optimal performance was determined primarily on the basis of a comparison of the distributions of numbers of tests needed to detect MAP-infected cows using the Mann-Whitney U test statistic. Optimal pool size was independent of both herd size and test characteristics, regardless of protocol. When sensitivity was the same for each pool size, pool sizes of 20 and 10 performed best for both protocols for prevalences of 0.01 and 0.1, respectively, while for prevalences of 0.05, pool sizes of 10 and 20 were optimal for the simple and halving protocols, respectively. When sensitivity decreased with increasing pool size, the results changed for prevalences of 0.05 and 0.1 with pool sizes of 50 being optimal especially at a prevalence of 0.1. Overall, the halving protocol was more cost effective than the simple protocol especially at higher prevalences. For detection of MAP using fecal culture, we recommend use of the halving protocol and pool sizes of 10 or 20 when the prevalence is suspected to range from 0.01 to 0.1 and there is no expected loss of sensitivity with increasing pool size. If loss in sensitivity is expected and the prevalence is thought to be between 0.05 and 0.1, the halving protocol and a pool size of 50 is recommended. Our findings are broadly applicable to other infectious diseases under comparable testing conditions.

An outbreak of late-term abortions, premature births, and congenital deformities associated with a bovine viral diarrhea virus 1 subtype b that induces thrombocytopenia.

Bovine viral diarrhea virus 1 (BVDV-1) subtype b was isolated from premature Holstein calves from a dairy herd that experienced an outbreak of premature births, late-term abortions, brachygnathism, growth retardation, malformations of the brain and cranium, and rare extracranial skeletal malformations in calves born to first-calf heifers. Experimental inoculation of 3 colostrum-deprived calves aged 2-4 months old with this BVDV isolate resulted in thrombocytopenia, lymphopenia, and leukopenia. Outbreaks of brachygnathism are rarely associated with BVDV, and thrombocytopenia is rarely associated with BVDV-1 strains.

Evaluation of the Western immunoblot as a detection method for Brucella abortus exposure in elk.

Brucella abortus has been an important wildlife disease issue for most of the last century, especially because wildlife species are considered to be important disease reservoirs for cattle. Diagnostic uncertainty, caused in part by cross-reactions of antibodies to environmental pathogens such as Yersinia enterocolitica O:9 on standard Brucella serology, has exacerbated the challenges of managing the disease and has highlighted the need for test validation in wildlife species. The western immunoblot was evaluated for use in detecting B. abortus exposure in elk (Cervus elaphus) and for ruling out exposure to cross-reacting bacteria. Samples collected from 2003 to 2006, including 54 female and immature elk from four different elk herds, were tested using standard Brucella serologic methods (card, rapid automated presumptive [RAP], and rivanol tests), as well as the western immunoblot. Samples (n=28) from animals known to be naturally infected with B. abortus biovar 1 served as positive controls. For presumed negative samples, sera (n=26) were collected from two elk herds in which negative serologic tests, and the absence of clinical signs of disease such as abortions, supported Brucella-negative classification. In addition to these study samples, serologic data from 12 tule elk (Cervus elaphus nannodes) were provided from the California Department of Fish and Game in order to illustrate a field application of the western blot. The western immunoblot had the highest sensitivity (1.0; % 0.899-1.0) and specificity (1.0; 0.891-1.0) among all tests used in the study. The Kappa statistic for agreement between the western blot and the card, rivanol, and RAP tests were 0.701, 0.808, and 0.921, respectively, showing good to excellent agreement with the standard diagnostic tests currently in use. Although the western immunoblot is more expensive and time intensive than other tests, in this limited study, it was shown to be reliable for establishing and confirming B. abortus disease status in elk. In addition to this study, subsequent applications of the western blot assay have been successful in detecting Yersinia sp. exposure in elk after their antibodies cross-reacted on standard Brucella serology.

Characteristics of a very virulent infectious bursal disease virus from California.

An outbreak of infectious bursal disease (IBD) in two California layer flocks resulted in the isolation of two infectious bursal disease viruses designated rA and rB. Increased mortality plus gross and histopathology in the layer flocks suggested rA and rB could be very virulent infectious bursal disease virus (vvIBDV). Preliminary studies indicated that high mortality resulted when bursa homogenates from the layer farms were used to inoculate specific-pathogen-free (SPF) chicks. In addition, rA and rB contained VP2 amino acid sequences typically seen in vvIBDV. Molecular and in vivo studies were conducted to more thoroughly identify and characterize the rA and rB viruses. Nucleotide sequence analysis demonstrated that rA and rB had identical sequences across the hypervariable VP2 (hvVP2) and segment B regions examined, and their amino acid sequences in the hvVP2 region were identical to the vvwIBDV type strains UK 661, OKYM, and Harbin. Furthermore, the genome segment B nucleotide sequences examined for rA and rB were a 98.1% match with vvIBDV and only an 88.0% match with classic IBDV strains. Phylogenetic analysis placed the rA and rB viruses with other vvIBDV and confirmed these viruses were close genetic descendants of vvIBDV seen around the world. Pathogenicity studies in 4-wk-old SPF chicks demonstrated that at a high dose (105.5 50% egg infective dose [EID50]) and a low dose (102.0 EID50) of rA and rB, mortality ranged from 91% to 100%. A pathogenic classic virus, standard challenge (STC), at similar doses did not cause mortality in the SPF chicks. In addition, mortality occurred in three out of four SPF birds exposed by direct contact to rA and rB inoculated chicks. Serum from convalescent birds inoculated with rA had high titers to IBDV but were negative for antibodies to infectious bronchitis virus, avian influenza virus, chicken anemia virus, Newcastle disease virus, Mycoplasma gallisepticum, and Mycoplasma synoviae. Virus isolation attempts on the rA and rB bursa homogenate inocula also indicated that no contaminating microorganisms contributed to the high mortality and pathology observed in the SPF chicks. In one experiment, broilers with maternal immunity to IBDV were protected from infection and disease when they were challenged with 10(2) EID50 and 10(5) EID50 of the STC virus. When challenged with 10(2) EID50 of the rA virus, the maternally immune broilers were protected from disease but not infection as evidenced by a positive reverse transcription-polymerase chain reaction (RT-PCR) assay for the virus. When the broilers were challenged with 10(5) EID50 of the rA virus, they had typical gross and histopathologic signs of IBD but no mortality by 7 days postinoculation. It was concluded that the rA and rB viruses meet the genotypic and phenotypic characteristics of a vvIBDV.

Multiplexed molecular assay for rapid exclusion of foot-and-mouth disease.

A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV "look-alike" diagnostic assay panel contains 5 PCR and 12 reverse transcriptase PCR (RT-PCR) signatures for a total of 17 simultaneous PCR amplifications for 7 diseases plus incorporating 4 internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV "look-alike" viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

Prevalence of Neospora caninum and persistent infection with bovine viral diarrhea virus in dairy-breed steers in a feedlot.

OBJECTIVE: To determine the prevalence and effect of Neospora caninum infection and persistent infection (PI) with bovine viral diarrhea virus (BVDV) on weight gain, morbidity, and mortality rate in dairy-breed steer calves located on a feedlot in California. DESIGN: Prospective cohort observational study. ANIMALS: 900 dairy-breed steer calves in 2 pens. PROCEDURES: The 3- to 4-month-old calves were evaluated for serum antibodies against N caninum and infection with BVDV at entry to the feedlot. Five months later, sera were again analyzed for anti-N caninum antibodies; calves that were determined to have BVDV infection initially were retested to evaluate PI status. Average daily gain, morbidity, and deaths were recorded for all calves. RESULTS: Among 900 calves, prevalence of N caninum infection was 16.7% (95% confidence interval, 14.3% to 19.3%); prevalence of BVDV-associated PI was 0.2% (95% confidence interval, 0.03% to 0.9%). Morbidity rate and time to first illness were not significantly different between calves that were seropositive or seronegative for N caninum. At the second sample collection, weight and average daily gain of calves that were seropositive for N caninum was less than that of seronegative steers in 1 pen, whereas these measures did not differ between groups in the other pen. Statistical power was insufficient to evaluate the effect of BVDV PI on any outcome measurement. CONCLUSIONS AND CLINICAL RELEVANCE: Although N caninum serostatus had no significant effect on morbidity rate, some seropositive calves had reduced growth, compared with seronegative calves, 5 months after entry to the feedlot.

Neospora caninum and Leptospira serovar serostatus in dairy cattle in Ontario.

No significant association existed between Neospora caninum titer and serostatus to Leptospira serovar hardjo, icterohaemorrhagiae, or pomona in cattle on 78 dairy herds in Ontario. Leptospira titer increased with parity. Amongst herds not vaccinated against Leptospira, the proportions of herds with > or = 1 animal seropositive to serovar hardjo, icterohaemorrhagiae, or pomona were 45%, 42%, and 58%, respectively.

Avian influenza A virus subtype H5N2 in a red-lored Amazon parrot.

CASE DESCRIPTION: A 3-month-old red-lored Amazon parrot (Amazona autumnalis autumnalis) was evaluated for severe lethargy. CLINICAL FINDINGS: Avian influenza virus hemagglutinin subtype H5N2 with low pathogenicity was characterized by virus isolation, real-time reverse transcriptase PCR assay, chicken intravenous pathogenicity index, and reference sera. The virus was also determined to be closely related to a virus lineage that had been reported only in Mexico and Central America. TREATMENT AND OUTCOME: The chick was admitted to the hospital and placed in quarantine. Supportive care treatment was administered. Although detection of H5 avian influenza virus in birds in the United States typically results in euthanasia of infected birds, an alternative strategy with strict quarantine measures and repeated diagnostic testing was used. The chick recovered from the initial clinical signs after 4 days and was released from quarantine 9 weeks after initial evaluation after 2 consecutive negative virus isolation and real-time reverse transcriptase PCR assay results. CLINICAL RELEVANCE: To the authors' knowledge, this is the first report of H5N2 avian influenza A virus isolated from a psittacine bird and represents the first introduction of this virus into the United States, most likely by illegal importation of psittacine birds. Avian influenza A virus should be considered as a differential diagnosis for clinical signs of gastrointestinal tract disease in psittacine birds, especially in birds with an unknown history of origin. Although infection with avian influenza virus subtype H5 is reportable, destruction of birds is not always required.

High-throughput real-time RT-PCR assay to detect the exotic Newcastle Disease Virus during the California 2002--2003 outbreak.

During the 2002--2003 Exotic Newcastle Disease (END) outbreak in Southern California, a high-throughput real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) system was developed to respond to the large diagnostic and surveillance sample workload. A 96-well RNA extraction method, using magnetic bead technology, combined with a 96-well RRT-PCR assay, allowed 1 technician to process and test more than 400 samples per day. A 3-technician team could complete testing on approximately 1,900 samples per day. The diagnostic sensitivity of the high-throughput RRT-PCR assay was 0.9967 (95% CI 0.9937-0.9997) based on 926 virus isolation confirmed positive samples. Diagnostic specificity using an initial 434 virus isolation confirmed negative samples was 100%. A diagnostic specificity of 0.9999 (95% CI 0.9999, >0.9999) was subsequently calculated on the basis of 2 false-positive results among 65,343 surveillance samples collected after the final END-positive case was confirmed in May 2003. Assay performance over 500 replicates, including reproducibility of the combined extraction and RRT-PCR amplification steps yielded a standard deviation of 0.70 RRT-PCR cycle thresholds (Ct) and a standard deviation of 0.59 Ct for the RRT-PCR steps alone. The high-throughput RRT-PCR developed for END contributed significantly to the 2002--2003 END control effort, reducing the predicted timeline for eradication from 3 years to just 11 months, primarily because of the large number of samples that could be rapidly tested. The 96-well approach described for high-throughput END RRT-PCR could be adapted to other rapid, high-volume testing needs, as required for potential foreign animal disease responses or intensive surveillance efforts.

Environmental air sampling to detect exotic Newcastle disease virus in two California commercial poultry flocks.

The 2002--2003 Exotic Newcastle Disease (END) outbreak in Southern California poultry provided an opportunity to evaluate environmental air sampling as an efficient and cost-effective means of sampling flocks for detection of a circulating virus. Exotic Newcastle Disease virus was detected by real-time reverse transcriptase PCR from air samples collected using a wetted-wall cyclone-style air sampler placed within 2 m of birds in 2 commercial flocks suspected of being naturally exposed to END virus during the outbreak. Exotic Newcastle Disease virus was detected after 2 hours of air sampling the poultry-house environments of the 2 naturally infected flocks.

A multilaboratory evaluation of a commercial enzyme-linked immunosorbent assay test for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in cattle.

Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturer's directions. The S/ P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA results with kit lot being a primary contributor. Similar data for other ELISA tests for antibodies to M. avium subsp. paratuberculosis or other antigens also should be developed.

Evaluation of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds.

OBJECTIVE: To evaluate sensitivity of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis (MAP) in large dairy herds and assess the use of the method for estimation of MAP prevalence. ANIMALS: 1,740 lactating cows from 29 dairy herds in California. PROCEDURE: Serum from each cow was tested by use of a commercial ELISA kit. Individual fecal samples were cultured and used to create pooled fecal samples (10 randomly selected fecal samples/pool; 6 pooled samples/herd). Sensitivity of MAP detection was compared between Herrold's egg yolk (HEY) agar and a new liquid culture method. Bayesian methods were used to estimate true prevalence of MAP-infected cows and herd sensitivity. RESULTS: Estimated sensitivity for pooled fecal samples among all herds was 0.69 (25 culture-positive pools/36 pools that were MAP positive). Sensitivity increased as the number of culture-positive samples in a pool increased. The HEY agar method detected more infected cows than the liquid culture method but had lower sensitivity for pooled fecal samples. Prevalence of MAP-infected cows was estimated to be 4% (95% probability interval, 2% to 6%) on the basis of culture of pooled fecal samples. Herd-level sensitivity estimate ranged from 90% to 100% and was dependent on prevalence in the population and the sensitivity for culture of pooled fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Use of pooled fecal samples from 10 cows was a cost-effective tool for herd screening and may provide a good estimate of the percentage of MAP-infected cows in dairy herds with a low prevalence of MAP.

Udder health in dairy cattle infected with Neospora caninum.

Blood samples were collected from 3449 cows on 57 representative Ontario dairy herds during the summer of 1998 and analysed for antibody to Neospora caninum using an ELISA. Forty-eight herds (2742 cattle) contained at least one N. caninum-seropositive animal. Two composite milk samples were collected from all cattle: the first on the day of blood collection and the second 68 to 365 days later. All milk samples were submitted for bacteriological culture. Ontario Dairy Herd Improvement Corporation (DHI) data were available for 3162 cattle in the 57 herds at the time of bleeding. Furthermore, complete DHI data were available for 1658 cattle that were culled between 12 and 24 months following blood collection. Using a standardised ELISA sample-to-positive (S/P) cut-off of > or = 0.45, the corrected seroprevalence was 8.2% overall and 10.1% within seropositive herds. At blood collection the odds of N. caninum-seropositive cows having a high linear score (> or = 4.0; equivalent to a somatic cell count > or = 200,000 cells/ml) was 27% less than for seronegative animals. Similarly, at the time of culling, the odds of having a high linear score was 22% less in N. caninum-seropositive cattle. Overall, linear score was lower in N. caninum-seropositive cattle at culling. After controlling for herd, parity, days in milk, and the interval between collection of milk samples, the odds of N. caninum-seropositive cattle testing positive for an environmental pathogen (i.e. environmental Streptococcus species and coliforms) on the second milk sample was 56% less than for seronegative animals. The odds were 83% less at a higher ELISA S/P cut-off of > or = 0.70. Finally, the odds of N. caninum-seropositive cattle developing a new infection with a major pathogen (environmental or contagious) were 60% less than seronegative cows using the higher ELISA S/P cut-off.

Effect of bovine viral diarrhea virus infection on fertility of dairy heifers.

A prospective field study in heifers from birth to first breeding was undertaken on two commercial dairies to assess the effect of bovine viral diarrhea virus (BVDV) congenital and post-natal infection (PNI) on fertility. A high BVDV Type 2 antibody titer (1:4096) at 10 months of age was associated with 32 more days to conceive, compared with a low titer (1:128). Conversely, infection with BVDV by 5-6 months of age and high BVDV Type 2 titers 1 month before conception or breeding was associated with improved fertility. Heifers with evidence of congenital BVDV infection had lower fertility than non-infected heifers (15-42 days longer time-to-first AI), which depended on BVDV Type 2 titers at 10 months of age. Neospora caninum infection was associated with additional services per conception (SPC) and Leptospira interrogans infection was associated with a delay in the time-to-first breeding. It appears that under field conditions, the effect of subclinical BVDV infection on subsequent heifer fertility may be due to a complex of interrelationships among multiple BVDV infections that depend on the type and timing of infection relative to reproductive development and events.