Susy Carman

Severe acute bovine viral diarrhea in Ontario, 1993-1995

In 1993, noncytopathic bovine viral diarrhea virus (BVDV) strains with enhanced virulence caused unprecedented outbreaks of severe acute bovine viral diarrhea (BVD) in dairy, beef, and veal herds in Ontario (Canada). Fever, pneumonia, diarrhea, and sudden death occurred in all age groups of cattle. Abortions often occurred in pregnant animals. Gross lesions in the alimentary tract were similar to those associated with mucosal disease, especially in animals >6 months of age. Cattle of all age groups had microscopic lesions in the alimentary tract similar to those seen with mucosal disease. The epidemic peaked in the summer of 1993, with 15% of all bovine accessions from diseased cattle presented to the diagnostic laboratory being associated with BVDV. The virus strains involved in the outbreak were analyzed using monoclonal and polyclonal antibodies and the polymerase chain reaction. The virus isolates from these outbreaks of severe disease were determined to be type 2 BVDV. Type 2 BVDV has been present in Ontario at least since 1981 without causing widespread outbreaks of severe acute BVD, which suggests that type 2 designation in itself does not imply enhanced virulence. Cattle properly vaccinated with type 1 BVDV vaccines appear to be protected from clinical disease.

Differences in virulence among porcine circovirus type 2 isolates are unrelated to cluster type 2a or 2b and prior infection provides heterologous protection

Porcine circovirus type 2 (PCV2) is divided into two genetic clusters designated PCV2a and PCV2b. The objectives of this study were to determine whether isolates from different clusters vary in virulence and to determine whether infection with PCV2a isolates induces protective immunity against subsequent infection with a recent PCV2b isolate. One-hundred and thirteen conventional specific-pathogen-free (SPF) pigs were assigned randomly to treatment groups and rooms: pigs inoculated with PCV2a cluster isolates (ISU-40895 or ISU-4838), pigs inoculated with PCV2b cluster isolates (NC-16845 or Can-17639) and uninoculated pigs. Necropsies were performed at 16 or 51 days post-inoculation (p.i.). There were no significant differences in PCV2-associated lymphoid lesions between PCV2a and PCV2b clusters; however, within the same cluster, significant differences were found between isolates: ISU-4838- and Can-17639-inoculated pigs had significantly (P<0.05) less severe lesions compared with ISU-40895- and NC-16845-inoculated pigs. To evaluate cross-protection, six pigs within each group were challenged at 35 days p.i. with an isolate from the heterologous cluster and were necropsied 51 days p.i. The severity of PCV2-associated lesions was reduced in pigs with prior exposure to an isolate from the heterologous cluster in comparison with singly inoculated pigs. Results indicate that the virulence of PCV2a and PCV2b isolates is not different in the conventional SPF pig model; however, the virulence of isolates within the same cluster differs. Increased virulence as reported to be associated with PCV2b isolates in the field was not observed under the conditions of this study. Moreover, cross-protection between PCV2a and PCV2b exists.

Diseases and Pathogens Associated with Mortality in Ontario Beef Feedlots

This study determined the prevalence of diseases and pathogens associated with mortality or severe morbidity in 72 Ontario beef feedlots in calves that died or were euthanized within 60 days after arrival. Routine pathologic and microbiologic investigations, as well as immunohistochemical staining for detection of bovine viral diarrhea virus (BVDV) antigen, were performed on 99 calves that died or were euthanized within 60 days after arrival. Major disease conditions identified included fibrinosuppurative bronchopneumonia (49%), caseonecrotic bronchopneumonia or arthritis (or both) caused by Mycoplasma bovis (36%), viral respiratory disease (19%), BVDV-related diseases (21%), Histophilus somni myocarditis (8%), ruminal bloat (2%), and miscellaneous diseases (8%). Viral infections identified were BVDV (35%), bovine respiratory syncytial virus (9%), bovine herpesvirus-1 (6%), parainfluenza-3 virus (3%), and bovine coronavirus (2%). Bacteria isolated from the lungs included M. bovis (82%), Mycoplasma arginini (72%), Ureaplasma diversum (25%), Mannheimia haemolytica (27%), Pasteurella multocida (19%), H. somni (14%), and Arcanobacterium pyogenes (19%). Pneumonia was the most frequent cause of mortality of beef calves during the first 2 months after arrival in feedlots, representing 69% of total deaths. The prevalence of caseonecrotic bronchopneumonia caused by M. bovis was similar to that of fibrinosuppurative bronchopneumonia, and together, these diseases were the most common causes of pneumonia and death. M. bovis pneumonia and polyarthritis has emerged as an important cause of mortality in Ontario beef feedlots.

Naturally Occurring Mycoplasma Bovis—Associated Pneumonia and Polyarthritis in Feedlot Beef Calves

Mycoplasma bovis is perceived as an emerging cause of mortality in feedlot beef cattle. This study examined the lesions and infectious agents in naturally occurring M. bovis–associated bronchopneumonia and arthritis and the relationship of this condition with bovine viral diarrhea virus (BVDV) infection. Standardized pathologic, immunohistochemical, and microbiologic investigations were conducted on 99 calves that died or were euthanized within 60 days after arrival in 72 feedlots. Cranioventral bronchopneumonia with multiple foci of caseous necrosis was identified in 54 of 99 calves, including 30 with concurrent fibrinosuppurative bronchopneumonia typical of pneumonic pasteurellosis. Mycoplasma bovis was consistently identified in these lesions by culture and immunohistochemistry, but also commonly in healthy lungs and those with pneumonia of other causes. Focal lesions of coagulation necrosis, typical of pneumonic pasteurellosis, were often infected with both Mannheimia haemolytica and M. bovis. Arthritis was present in 25 of 54 (46%) calves with M. bovis pneumonia, and all calves with arthritis had pneumonia. BVDV infection was more common in calves with lesions of bacterial pneumonia than in those dying of other causes, but BVDV infection was not more common in calves with caseonecrotic bronchopneumonia than those with fibrinosuppurative bronchopneumonia. Retrospective analysis identified cases of M. bovis pneumonia in the early 1980s that had milder lesions than the current cases. The findings suggest that, in at least some calves, M. bovis induces caseonecrotic bronchopneumonia within the lesions of pneumonic pasteurellosis.

Bovine viral diarrhea virus in alpaca: abortion and persistent infection

An alpaca herd in eastern Ontario experienced vague signs of illness, including anorexia and lethargy in 9 animals, 2.5 months after the addition of a chronically ill cria and his dam to the farm. Subsequently 2 alpaca had early pregnancy loss; one aborted at 5.5 months gestation and the other at 7 months gestation. Seventeen were found to have serum antibody to bovine viral diarrhea virus (BVDV), with highest titers to BVDV type 1. The fetus that was aborted at 5.5 months gestation, 3 months after the clinical outbreak, was found to be positive for BVDV on immunohistochemical staining, and noncytopathic BVDV type 1b was isolated. Of the 13 cria born alive that season, a single male underweight alpaca cria, born 9 months after the clinical illnesses, was infected with BVDV type 1b. The cria was positive for BVDV at birth, at 3 and 26 days of age and continued to be positive for noncytopathic BVDV using virus isolation, nested reverse transcription PCR, antigen detection ELISA, and immunohistochemical staining until euthanasia at 46 days of age. The cria remained serum antibody negative to both BVDV type 1 and type 2. A diagnosis of persistent infection was made. This is the first report describing persistent infection with BVDV in an alpaca cria.

Restriction fragment length polymorphism of porcine reproductive and respiratory syndrome viruses recovered from Ontario farms, 1998-2000.

From January 1998 to July 2000, 2,456 clinical samples, including lung, tonsil, lymph node, and serum, from 760 cases submitted to the Animal Health Laboratory, Ontario, Canada, were tested for porcine reproductive and respiratory syndrome viruses (PRRSV) using reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR product restriction fragment length polymorphism (RFLP) analysis. A total of 516 samples from 284 cases were RT-PCR positive for the PRRSV open reading frame (ORF) 7 sequence. The RT-PCR RFLP typing assay was performed using 2 different sets of primers, amplifying 716 or 933 base pairs of ORF 4, 5, and 6 of PRRSV. Samples from 254 cases were typeable, yielding 34 different RFLP types. Of these, 164 cases had 32 different RFLP types of field or intermediate strains, 86 had a pattern similar to a commercial PRRSV vaccine or VR 2332 strain of the virus, 4 had a RFLP type shared by another commercial vaccine and a field strain. In 4 cases, 2 different RFLP types were identified from tissues from different pigs that were submitted at the same time from the same farm. Of the 195 farms that submitted PRRSV PCR-positive samples, 48 submitted samples on more than 1 occasion during the specified time frame. In 23 of those 48 farms, RFLP patterns of PRRSV differed between submissions, whereas in the other 25 farms, the RFLP pattern remained unchanged. There were 34 different PRRSV patterns identified from 236 cases using the primer set amplifying 716 base pairs of PRRSV. There were 18 cases, consisting of 9 different patterns, typeable only by using the primers amplifying a 933-base pair fragment of the virus.

The spread of type 2 porcine reproductive and respiratory syndrome virus (prrsv) in North America: A phylogeographic approach

The emergence and spread of Type 2 Porcine Reproductive and Respiratory Syndrome virus (Type 2 PRRSV) in North America is heavily influenced by the multiple site production system used in the hog industry. However, it is unclear how anthropogenic factors such has this have shaped the current spatial distribution of PRRSV genotypes. We employed Bayesian phylogeographic analyses of 7040 ORF5 sequences to reveal the recent geographical spread of Type 2 PRRSV in North America. The directions and intensities in our inferred virus traffic network closely mirror the hog transportation. Most notably, we reveal multiple viral introductions from Canada into the United States causing a major shift in virus genetic composition in the Midwest USA that went unnoticed by the regular surveillance and field epidemiological studies. Overall, these findings provide important insights into the dynamics of Type 2 PRRSV evolution and spread that will facilitate programs for control and prevention.

A prospective study of sheep and goat abortion using real-time polymerase chain reaction and cut point estimation shows Coxiella burnetii and Chlamydophila abortus infection concurrently with other major pathogens

From 2009 to 2011, 163 sheep and 96 goat abortion submissions were received at the Animal Health Laboratory, University of Guelph, Ontario, Canada, for gross and histologic examination, as well as real-time polymerase chain reaction (PCR) testing for Chlamydophila abortus and/or Coxiella burnetii. Additional testing included immunohistochemistry for Toxoplasma gondii and Chlamydophila spp., routine bacterial culture and selective culture for Campylobacter spp., examination of modified acid-fast–stained placenta smears, enzyme-linked immunosorbent assay testing for Chlamydophila spp., and virus isolation. The final diagnosis made for each case by individual pathologists, based on gross and histologic lesions, as well as ancillary testing, was used as a standard to determine the significance of C. abortus and C. burnetii infection. Coxiella burnetii was identified by real-time PCR in 113 of 163 (69.0%) and 72 of 96 (75%) sheep and goat abortion submissions, respectively, but was considered to be significant in causing abortion in only 11 of 113 (10%) sheep and 15 out of 72 (21%) goat submissions that tested positive. Chlamydophila abortus was identified by real-time PCR in 42 of 162 (26%) and 54 of 92 (59%) sheep and goat submissions, respectively, but was considered the cause of the abortion in 16 of 42 (38%) sheep and 34 of 54 (63%) goat submissions that tested positive. Optimal sensitivity and specificity cut points for the real-time PCR copy number for C. abortus and C. burnetii were determined using the final pathology diagnosis as the reference test.

Interlaboratory comparison of Porcine circovirus-2 indirect immunofluorescent antibody test and enzyme-linked immunosorbent assay results on experimentally infected pigs

A blinded interlaboratory assessment of the diagnostic agreement and accuracy of serologic tests for routine detection of antibodies against Porcine circovirus-2 (PCV-2), including indirect fluorescent antibody tests (IFATs) and enzyme-linked immunosorbent assays (ELISAs) was conducted in 7 North American laboratories. Serum samples were collected weekly, on trial days 0, 7, 14, 21, 28, 35, 42, and 49, from the following groups of animals: 1) negative controls (n = 7), 2) PCV-2a (n = 8), 3) PCV-2b (n = 8), 4) PCV-1 (n = 8), 5) PCV-2 vaccine A (n = 8; Ingelvac® CircoFLEX™), 6) PCV-2 vaccine B (n = 8; Circumvent® PCV2), and 7) PCV-2 vaccine C (n = 8; Suvaxyn® PCV2 One Dose). Results from each laboratory were analyzed by kappa and receiver operating characteristic (ROC) analysis. Kappa analysis indicated that, by trial day 49, IFATs had almost perfect agreement, in-house ELISAs had fair to almost perfect agreement, and commercially available anti–PCV-2 immunoglobulin G ELISAs (I or S) had moderate to substantial agreement. From trial days 14–49, the area under the ROC curve for the 2 laboratories that offered IFATs, the 4 laboratories that offered in-house ELISAs, and the 3 laboratories that used commercially available ELISAs ranged from 0.94 to 1.00, 0.72 to 1.00, and 0.95 to 1.00, respectively. However, test sensitivities varied based on laboratory-specific cutoffs that were used to dichotomize test results.

Comparison of nucleic and amino acid sequences and phylogenetic analysis of the Gs protein of various equine arteritis virus isolates.

The genetic variation in equine arteritis virus (EAV) GS protein encoding gene was investigated. Nucleic and deduced amino acid sequences from eight different EAV isolates (one European, two American and five Canadian isolates) were compared with those of the Bucyrus reference strain. Nucleotide and amino acid sequence identities between these isolates and the Bucyrus reference strain ranged from 92.3 to 96.4%, and 93.2 to 95.5%, respectively. However, phylogenetic tree analysis and estimation of genetic distances based on the GS protein encoding gene sequences showed that the European prototype Vienna strain, the American 87AR-A1 isolate and all other North American EAV isolates could be classified into three genetically divergent groups. Our results showed that the GS protein-encoding gene can be subjected on the basis of phylogenetic analysis to genetic variation, as previously shown for the other three EAV structural protein (M, N and GL)-encoding genes.