Canine Circovirus 1 (CaCV-1) and Canine Parvovirus 2 (CPV-2): Recurrent Dual Infections in a Papillon Breeding Colony.

Recurrent outbreaks of sudden death and bloody diarrhea were reported in March 2013 and February 2014 in a breeding colony of Papillon dogs. During the first outbreak, 1 adult dog and 2 eight-month-old puppies died. During the second outbreak, 2 ten-week-old puppies died. One puppy from the first outbreak and 2 puppies from the second outbreak were examined at necropsy. Histologically, all 3 puppies had severe segmental crypt necrosis of the small intestine and marked lymphoid follicle depletion in the spleen and Peyer's patches. Real-time (RT) polymerase chain reaction (PCR) demonstrated abundant canine parvovirus (CPV-2) DNA (Ct<15) in the affected small intestine, and immunohistochemistry detected large amounts of CPV-2 antigen in intestinal crypt epithelium and Kupffer cells but few positive macrophages in lymphoid organs. All puppies had marked sinusoidal histiocytosis and multifocal granulomatous inflammation in mesenteric lymph nodes and spleen, prompting additional RT-PCR testing for canine circovirus 1 (CaCV-1). Very high levels of CaCV-1 DNA (Ct<13) were detected in small intestine, lymph nodes, and spleen. In situ hybridization for CaCV-1 detected rare positive nuclei of regenerating crypt epithelium but abundant amounts of CaCV-1 nucleic acid in the cytoplasm and nuclei of histiocytes in all lymphoid tissues, including granulomatous inflammatory foci and hepatic Kupffer cells. Significant levels of CaCV-1 DNA were detected in blood and serum (Ct as low as 13) but not feces from 3 surviving dogs at 2 months or 1 year after the outbreak, respectively. We hypothesize that CPV-2 infection predisposed dogs to CaCV-1 infection and ultimately resulted in more severe clinical disease.

Beyond H&E: integration of nucleic acid-based analyses into diagnostic pathology.

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets.

Distribution of eastern equine encephalomyelitis viral protein and nucleic acid within central nervous tissue lesions in white-tailed deer (Odocoileus virginianus).

An outbreak of eastern equine encephalomyelitis (EEE) occurred in Michigan free-ranging white-tailed deer (Odocoileus virginianus) during late summer and fall of 2005. Brain tissue from 7 deer with EEE, as confirmed by reverse transcriptase polymerase chain reaction, was studied. Detailed microscopic examination, indirect immunohistochemistry (IHC), and in situ hybridization (ISH) were used to characterize the lesions and distribution of the EEE virus within the brain. The main lesion in all 7 deer was a polioencephalomyelitis with leptomeningitis, which was more prominent within the cerebral cortex, thalamus, hypothalamus, and brainstem. In 3 deer, multifocal microhemorrhages surrounded smaller vessels with or without perivascular cuffing, although vasculitis was not observed. Neuronal necrosis, associated with perineuronal satellitosis and neutrophilic neuronophagia, was most prominent in the thalamus and the brainstem. Positive IHC labeling was mainly observed in the perikaryon, axons, and dendrites of necrotic and intact neurons and, to a much lesser degree, in glial cells, a few neutrophils in the thalamus and the brainstem, and occasionally the cerebral cortex of the 7 deer. There was minimal IHC-based labeling in the cerebellum and hippocampus. ISH labeling was exclusively observed in the cytoplasm of neurons, with a distribution similar to IHC-positive neurons. Neurons positive by IHC and ISH were most prominent in the thalamus and brainstem. The neuropathology of EEE in deer is compared with other species. Based on our findings, EEE has to be considered a differential diagnosis for neurologic disease and meningoencephalitis in white-tailed deer.

Polyomavirus-associated nephritis in 2 horses.

Polyomaviruses produce latent and asymptomatic infections in many species, but productive and lytic infections are rare. In immunocompromised humans, polyomaviruses can cause tubulointerstitial nephritis, demyelination, or meningoencephalitis in the central nervous system and interstitial pneumonia. This report describes 2 Standardbred horses with tubular necrosis and tubulointerstitial nephritis associated with productive equine polyomavirus infection that resembles BK polyomavirus nephropathy in immunocompromised humans.

Cutaneous lymphomas in European pet rabbits (Oryctolagus cuniculus).

Cutaneous lymphoma is a common skin neoplasm of pet rabbits in Europe but is rarely reported in pet rabbits in North America. These neoplasms have not been previously characterized, nor has the cause for the apparent predilection for cutaneous lymphoma in European pet rabbits compared with North American pet rabbits been investigated. In this retrospective study, the authors morphologically and immunohistochemically characterized 25 cutaneous lymphomas in European pet rabbits according to the World Health Organization classification. Tumors were classified as diffuse large B cell lymphomas, with 14 lymphomas exhibiting a centroblastic/centrocytic subtype and 11 tumors exhibiting a T cell-rich B cell subtype. To investigate a potential viral etiology of these lymphomas, 3 diffuse large B cell and 3 T cell-rich B cell lymphomas were evaluated by polymerase chain reaction for retroviral and herpesviral genes. Neither virus was detected. In contrast to other domestic animals, cutaneous lymphomas in European pet rabbits were highly pleomorphic and frequently contained multinucleated giant cells. Unexpectedly, the second most common subtype was T cell-rich B cell lymphoma, a subtype that is rare in species other than horses. Based on a limited number of samples, there was no support for a viral etiology that would explain the higher incidence of lymphoma in European pet rabbits compared with American pet rabbits. Further investigation into genetic and extrinsic factors associated with the development of these tumors is warranted.

Nested case-control study of feline calicivirus viruria, oral carriage, and serum neutralizing antibodies in cats with idiopathic cystitis.

BACKGROUND: The epidemiology of feline calicivirus (FCV) infection in cats with idiopathic cystitis (FIC) has not been investigated by contemporary molecular biologic methods. OBJECTIVES: To determine the prevalence of and evaluate risk factors for FCV viruria, oral carriage, and virus neutralizing (VN) antibodies in cats with and without FIC. ANIMALS: Cats with nonobstructive FIC (n = 47), obstructive FIC (n = 22), and FCV upper respiratory tract infection (URI; n = 25), and healthy client-owned (n = 18) and colony-housed (n = 24) cats. METHODS: Oropharyngeal secretions and urine were evaluated with a FCV p30 gene-based real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay. Serum VN antibody titers were determined by a modified microtiter assay. Associations of risk factors with log-transformed antibody titers were determined by multivariable generalized linear regression. RESULTS: FCV viruria was detected in 4 (6%) and 3 (12%) cats with FIC and URI, respectively. In 3 FIC cats, viruria was unassociated with detectable oral virus carriage. Oral FCV carriage was detected in 7 (10%) FIC cats. Median antibody titers were significantly higher in cats with obstructive FIC (1 :256), nonobstructive FIC (1:128), and URI (1:512) compared with healthy client-owned (1:16) and colony-housed (1:4) cats (P < .001). Other than disease, multivariate analysis did not identify any other explanatory variables for increased titers in cats with FIC or URI. CONCLUSIONS AND CLINICAL IMPORTANCE: FCV viruria was detected in cats with FIC and URI, however, its etiologic significance is uncertain. Serologic results suggest increased FCV exposure in FIC cats compared with controls. Further investigations are needed to clarify the potential role of FCV in FIC.

Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples.

Differentiating between inflammatory bowel disease (IBD) and small intestinal lymphoma in cats is often difficult, especially when only endoscopic biopsy specimens are available for evaluation. However, a correct diagnosis is imperative for proper treatment and prognosis. A retrospective study was performed using surgical and endoscopic intestinal biopsy specimens from 63 cats with a history of chronic diarrhea or vomiting or weight loss. A diagnosis of lymphoma or inflammation was based on microscopic examination of hematoxylin and eosin (HE)-stained sections alone, HE-stained sections plus results of immunohistochemical labeling (IHC) for CD3e and CD79a, and HE staining, immunophenotyping, and polymerase chain reaction (PCR) results for B and/or T cell clonality. In addition, various histomorphologic parameters were evaluated for significant differences between lymphoma and IBD using Fisher's exact test. The sensitivity and specificity of each parameter in the diagnosis of lymphoma were also determined. Results of Bayesian statistical analysis demonstrated that combining histologic evaluation of small intestinal biopsy specimens with immunophenotyping and analysis of clonality of lymphoid infiltrates results in more accurate differentiation of neoplastic versus inflammatory lymphocytes. Important histologic features that differentiated intestinal lymphoma from IBD included lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. Based on the results of this study, a stepwise diagnostic algorithm that first uses histologic assessment, followed by immunophenotyping and then PCR to determine clonality of the lymphocytes, was developed to more accurately differentiate between intestinal lymphoma and IBD.

Detection of group C rotavirus in juvenile ferrets (Mustela putorius furo) with diarrhea by reverse transcription polymerase chain reaction: sequencing and analysis of the complete coding region of the VP6 gene.

Nine juvenile ferrets (Mustela putorius furo) with a history of diarrhea were severely dehydrated and had distended abdomens and thin-walled small intestines that contained gas and fluid. Histologically, small intestines exhibited acute superficial atrophic enteritis. Transmission electron microscopy of the small intestine showed rotavirus-like particles within apical vacuoles. Reverse transcription polymerase chain reaction (RT-PCR) was negative for group A rotavirus. A group C rotavirus-specific RT-PCR assay was developed using consensus primers designed from the alignment of VP6 gene sequences of porcine, bovine, and human strains. A 182-bp product of the VP6 gene was sequenced and showed significant similarity to group C rotavirus VP6 sequences. This strain was designated "Ferret Rota C-MSU." The entire coding sequence of VP6 was determined and compared with other rotaviruses. Ferret Rota C-MSU virus was found to be most closely related to Shintoku group C rotavirus. This is the first definitive identification of a group C rotavirus in ferrets, based upon RT-PCR, sequencing, and genetic analysis.

Clinico-pathologic features of fatal disease attributed to new variants of endotheliotropic herpesviruses in two Asian elephants (Elephas maximus).

The first herpesviruses described in association with serious elephant disease were referred to as endotheliotropic herpesviruses (EEHV) because of their ability to infect capillary endothelial cells and cause potentially fatal disease. Two related viruses, EEHV1 and EEHV2, have been described based on genetic composition. This report describes the similarities and differences in clinicopathologic features of 2 cases of fatal endotheliotropic herpesvirus infections in Asian elephants caused by a previously unrecognized virus within the betaherpesvirus subfamily. EEHV3 is markedly divergent from the 2 previously studied fatal probosciviruses, based on polymerase chain reaction sequence analysis of 2 segments of the viral genome. In addition to ascites, widespread visceral edema, petechiae, and capillary damage previously reported, important findings with EEHV3 infection were the presence of grossly visible renal medullary hemorrhage, a tropism for larger veins and arteries in various tissues, relatively high density of renal herpetic inclusions, and involvement of the retinal vessels. These findings indicate a less selective organ tropism, and this may confer a higher degree of virulence for EEHV3.

Clinicopathologic features of a systemic coronavirus-associated disease resembling feline infectious peritonitis in the domestic ferret (Mustela putorius).

From 2002 to 2007, 23 ferrets from Europe and the United States were diagnosed with systemic pyogranulomatous inflammation resembling feline infectious peritonitis (FIP). The average age at the time of diagnosis was 11 months. The disease was progressive in all cases, and average duration of clinical illness was 67 days. Common clinical findings were anorexia, weight loss, diarrhea, and large, palpable intra-abdominal masses; less frequent findings included hind limb paresis, central nervous system signs, vomiting, and dyspnea. Frequent hematologic findings were mild anemia, thrombocytopenia, and hypergammaglobulinemia. Grossly, whitish nodules were found in numerous tissues, most frequently the mesenteric adipose tissue and lymph nodes, visceral peritoneum, liver, kidneys, spleen, and lungs. One ferret had a serous abdominal effusion. Microscopically, pyogranulomatous inflammation involved especially the visceral peritoneum, mesenteric adipose tissue, liver, lungs, kidneys, lymph nodes, spleen, pancreas, adrenal glands, and/or blood vessels. Immunohistochemically, all cases were positive for coronavirus antigen using monoclonal antibody FIPV3-70. Electron microscopic examination of inflammatory lesions identified particles with coronavirus morphology in the cytoplasm of macrophages. Partial sequencing of the coronavirus spike gene obtained from frozen tissue indicates that the virus is related to ferret enteric coronavirus.

Experimental inoculation of pregnant swine with type 1 bovine viral diarrhoea virus.

The ability of bovine viral diarrhoea virus type 1 (BVDV-1) to induce transplacental infection in pigs was evaluated. Control pigs (n = 4) were sham-inoculated while infected pigs (n = 4) were intranasally inoculated with BVDV-1 on day 65 of gestation. Blood samples were tested throughout the study for BVDV and antibody to BVDV. On day 110 of gestation, a Caesarean section was performed. Serum was obtained for virus isolation and antibody determination from all piglets, and all experimental animals were killed. Tissues were collected for virus isolation and histopathology. Bovine viral diarrhoea virus was isolated on days 5 and 7 after infection and seroconversion was demonstrated in all infected gilts; however, BVDV was only isolated from one fetus from an infected pig. Viraemia and seroconversion were demonstrated in the pregnant gilts; however, transplacental infection at day 65 of gestation in the pig was not consistently demonstrated.

Relationship between degree of viremia and disease manifestation in calves with experimentally induced bovine viral diarrhea virus infection.

OBJECTIVE: To compare degree of viremia and disease manifestations in calves with type-I and -II bovine viral diarrhea virus (BVDV) infection. ANIMALS: 16 calves. PROCEDURE: Colostrum-deprived calves obtained immediately after birth were assigned to 1 control and 3 treatment groups (4 calves/group). Calves in treatment groups were inoculated (day 0) by intranasal instillation of 10(7) median tissue culture infective dose BVDV 890 (type II), BVDV 7937 (type II), or BVDV TGAN (type I). Blood cell counts and virus isolation from serum and leukocytes were performed daily, whereas degree of viremia was determined immediately before and 4, 6, 8, and 12 days after inoculation. Calves were euthanatized on day 12, and pathologic, virologic, and immunohistochemical examinations were performed. RESULTS: Type-II BVDV 890 induced the highest degree of viremia, and type-I BVDV TGAN induced the lowest. Virus was isolated more frequently and for a longer duration in calves inoculated with BVDV 890. A parallel relationship between degree of viremia and rectal temperature and an inverse relationship between degree of viremia and blood cell counts was observed. Pathologic and immunohistochemical examinations revealed more pronounced lesions and more extensive distribution of viral antigen in calves inoculated with type-II BVDV. CONCLUSIONS AND CLINICAL RELEVANCE: Degree of viremia induced during BVDV infection is associated with severity of clinical disease. Isolates of BVDV that induce a high degree of viremia may be more capable of inducing clinical signs of disease. Strategies (eg, vaccination) that reduce viremia may control clinical signs of acute infection with BVDV.

Platelet aggregation responses and virus isolation from platelets in calves experimentally infected with type I or type II bovine viral diarrhea virus.

Altered platelet function has been reported in calves experimentally infected with type II bovine viral diarrhea virus (BVDV). The purpose of the present study was to further evaluate the ability of BVDV isolates to alter platelet function and to examine for the presence of a virus-platelet interaction during BVDV infection. Colostrum-deprived Holstein calves were obtained immediately after birth, housed in isolation, and assigned to 1 of 4 groups (1 control and 3 treatment groups). Control calves (n = 4) were sham inoculated, while calves in the infected groups (n = 4 for each group) were inoculated by intranasal instillation with 10(7) TCID50 of either BVDV 890 (type II), BVDV 7937 (type II), or BVDV TGAN (type I). Whole blood was collected prior to inoculation (day 0) and on days 4, 6, 8, 10, and 12 after inoculation for platelet function testing by optical aggregometry by using adenosine diphosphate and platelet activating factor. The maximum percentage aggregation and the slope of the aggregation curve decreased over time in BVDV-infected calves; however, statistically significant differences (Freidman repeated measures ANOVA on ranks, P < 0.05) were only observed in calves infected with the type II BVDV isolates. Bovine viral diarrhea virus was not isolated from control calves, but was isolated from all calves infected with both type II BVDV isolates from days 4 through 12 after inoculation. In calves infected with type I BVDV, virus was isolated from 1 of 4 calves on days 4 and 12 after inoculation and from all calves on days 6 and 8 after inoculation. Altered platelet function was observed in calves infected with both type II BVDV isolates, but was not observed in calves infected with type I BVDV. Altered platelet function may be important as a difference in virulence between type I and type II BVDV infection.

Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan.

The gammaherpesvirus bovine herpesvirus-4 (BHV-4) has been isolated from a wide variety of animals, including lions and domestic cats. Although BHV-4 antibodies have been detected in normal cats and cats with urinary disorders, the epidemiology and pathogenic role of BHV-4 in cats is unknown. The purpose of this study was to determine the prevalence of BHV-4 antibodies and viral nucleic acid in a population of free-roaming cats. Plasma and peripheral blood leukocyte samples were collected from 52 male and 52 female free-roaming cats impounded at a regional animal control facility in Central Michigan. Plasma concentrations of BHV-4 antibodies were measured with an indirect fluorescent antibody test. Peripheral blood leukocyte DNA was isolated, and a 2-stage polymerase chain reaction with heminested primers delineating a conserved portion of the BHV-4 glycoprotein B gene homologue was used to amplify BHV-4-specific DNA sequences. BHV-4 antibodies were detected in 38 (73%) male and 23 (44%) female cats. Seropositive cats were significantly more likely to be male than female (odds ratio = 3.22; P = .007). Cell-associated viremia was detected in 17 (33%) male and 11 (21%) female cats. Of the 61 seropositive cats, 23 (38%) had a detectable viremia; only 5 (12%) seronegative cats had detectable viremia. Seropositive cats were significantly more likely to be viremic than seronegative cats (OR = 4.30: P = .009). Our results suggest that BHV-4 infection may be more widespread in certain cat populations than previously reported. Furthermore, many cats seropositive for BHV-4 antibodies have a concurrent cell-associated viremia.

Effects of formalin on bacterial growth in mink feed, feed consumption and reproductive performance of adult mink, and growth of mink kits.

Feed that is typically used on commercial mink ranches is an ideal environment for bacterial growth because of the raw animal by-products used as ingredients. Recently, formaldehyde was approved for use as an antimicrobial agent in poultry feed. Experiments in our laboratory were carried out to investigate the effects of incorporating different concentrations of formalin into the feed of mink on the growth of gram-negative and gram-positive bacteria. Feed containing 0, 550 or 1100 ppm formalin was kept refrigerated for up to 7 d and the number of colony forming units of gram-negative and gram-positive bacteria derived from the feed was determined each day. Colony forming units in the formalin-treated feed were significantly fewer than colony forming units in untreated feed. In the second trial, feed containing the same concentrations of formalin was maintained at 30 C for 24 h and cultured bacterial colonies were counted at 0, 12 or 24 h of feed incubation. Both concentrations of formalin were effective in significantly reducing the number of colony forming units. A feed consumption trial determined if mink (Mustela vison) preferred formalin-treated feed to non-treated feed kept refrigerated for up to 7 d. Consumption of feed treated with 1100 ppm formalin was significantly lower than consumption of the non-treated feed on d 1, 2, 4 and 5, but body weight was not affected. A long-term feeding trial determined the effects of formalin on mink reproduction, early growth of offspring and quality of fur. Mink were fed formalin at concentrations of 0, 550 or 1100 ppm for approximately 140 d beginning 1 mo prior to mating until kits were weaned at 6 w of age. Mating success was not affected by consumption of formalin-treated diets, but kit survival at birth was adversely affected in mink consuming 1100 ppm formalin. Hemoglobin concentration, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin were significantly decreased in 6-w-old kits, but there were no significant differences in any of these parameters between the kits exposed to 0 and 550 ppm formalin. In a second phase, some kits and their dams were continued on their respective dietary treatments from weaning through pelting (approximately 220 and 320 d, respectively). At pelting, hematocrits and hemoglobin concentrations for the kits fed 1100 ppm formalin were significantly less compared to the control and 550 ppm formalin groups. There were no significant differences in body weights among female kits or adult female mink. The body weights of male kits in the 1100 ppm formalin group became significantly less than the body weights of male kits in the control and 550 ppm formalin groups as the trial progressed. The quality of fur was highest for mink in the control group and lowest for mink in the 1100 ppm formalin group. While dietary 1100 and 550 ppm formalin were effective in suppressing bacterial growth in the feed of mink, the deleterious effects of 1100 ppm formalin on kit survival, hematologic parameters, body weight, and quality of fur preclude formalin use at this concentration.

Comparison of type I and type II bovine viral diarrhea virus infection in swine.

Some isolates of type II bovine viral diarrhea virus (BVDV) are capable of causing severe clinical disease in cattle. Bovine viral diarrhea virus infection has been reported in pigs, but the ability of these more virulent isolates of type II BVDV to induce severe clinical disease in pigs is unknown. It was our objective to compare clinical, virologic, and pathologic findings between type I and type II BVDV infection in pigs. Noninfected control and BVDV-infected 2-month-old pigs were used. A noncytopathic type I and a noncytopathic type II BVDV isolate were chosen for evaluation in feeder age swine based upon preliminary in vitro and in vivo experiments. A dose titration study was performed using 4 groups of 4 pigs for each viral isolate. The groups were inoculated intranasally with either sham (control), 10(3), 10(5), or 10(7) TCID50 of virus. The pigs were examined daily and clinical findings were recorded. Antemortem and postmortem samples were collected for virus isolation. Neither the type I nor type II BVDV isolates resulted in clinical signs of disease in pigs. Bovine viral diarrhea virus was isolated from antemortem and postmortem samples from groups of pigs receiving the 10(5) and the 10(7) TCID50 dose of the type I BVDV isolate. In contrast, BVDV was only isolated from postmortem samples in the group of pigs receiving the 10(7) TCID50 dose of the type II BVDV isolate. Type I BVDV was able to establish infection in pigs at lower doses by intranasal instillation than type II BVDV. Infection of pigs with a type II isolate of BVDV known to cause severe disease in calves did not result in clinically apparent disease in pigs.

Experimental model of type II bovine viral diarrhea virus-induced thrombocytopenia in neonatal calves.

Thrombocytopenia has been associated with type II bovine viral diarrhea virus (BVDV) infection in immunocompetent cattle, but the mechanism is unknown. The purpose of the present study was to develop and characterize a model of type II BVDV-induced thrombocytopenia. Colostrum-deprived Holstein calves were obtained immediately after birth, given a BVDV-negative and BVDV antibody-negative plasma transfusion, housed in an isolation facility, and randomly assigned to either control (n = 4) or infected (n = 5) groups. Infected calves were inoculated by intranasal instillation on day 3 of age with 10(7) TCID50 of the prototype type II isolate, BVDV 890, whereas control calves were sham inoculated. Blood counts and virus isolations from serum, white blood cells, and platelets were performed daily until day 12 after infection, at which time all experimental calves were euthanatized, and pathologic, virologic, and immunohistochemical examinations were performed. On physical examination, the control calves remained normal, but the infected calves developed pyrexia and diarrhea characteristic of type II BVDV infection. The platelet count decreased in all infected calves, and a statistically significant difference in the platelet count between control and infected calves was observed on days 7-12 after infection. In addition, the mean platelet volume and white blood cell counts also decreased. Examination of the bone marrow from the infected calves revealed immunohistochemical staining for BVDV antigen in megakaryocytes and evidence of concurrent megakaryocyte necrosis and hyperplasia.

Vaccine genotype and route of administration affect pseudorabies field virus latency load after challenge.

The influence of vaccine genotype and route of administration on the efficacy of pseudorabies virus (PRV) vaccines against virulent PRV challenge was evaluated in a controlled experiment using five genotypically distinct modified live vaccines (MLVs) for PRV. Several of these MLVs share deletions in specific genes, however, each has its deletion in a different locus within that gene. Pigs were vaccinated with each vaccine, either via the intramuscular or intranasal route, and subsequently challenged with a highly virulent PRV field strain. During a 2-week period following challenge with virulent PRV, each of the vaccine strains used in this study was evaluated for its effectiveness in the reduction of clinical signs, prevention of growth retardation and virulent virus shedding. One month after challenge, tissues were collected and analyzed for virulent PRV latency load by a recently developed method for the electrochemiluminescent quantitation of latent herpesvirus DNA in animal tissues after PCR amplification. It was determined that all vaccination protocols provided protection against clinical signs resulting from field virus challenge and reduced both field virus shedding and latency load after field virus challenge. Our results indicated that vaccine efficacy was significantly influenced by the modified live vaccine strain and route of administration. Compared to unvaccinated pigs, vaccination reduced field virus latency load in trigeminal ganglia, but significant differences were found between vaccines and routes of administration. We conclude that vaccine genotype plays a role in the effectiveness of PRV MLVs.

Neutralizing antibodies to type 1 and 2 bovine viral diarrhea viruses: detection by inhibition of viral cytopathology and infectivity by immunoperoxidase assay.

Neutralizing antibodies to type 1 and 2 bovine viral diarrhea virus (BVDV) strains were measured by a microtiter virus neutralization test (MVNT) in cell culture. Antibodies (neutralizing) were detected by inhibition of viral infectivity, by the absence of viral cytopathology for cytopathic strains, or by immunoperoxidase staining for noncytopathic strains. The immunoperoxidase-stained monolayers could be detected without the aid of light microscopy. Twenty BVDV strains were used as challenge viruses in the in vitro MVNT, including 14 type 1 and 6 type 2 strains. Representative noncytopathic and cytopathic strains of both types were used. Positive control serum samples available for diagnostic testing contained both type 1 and type 2 BVDV antibodies. There did not appear to be major differences in antibody titers among the respective type strains, regardless of biotype (cytopathic or noncytopathic). In a study with sera from calves receiving a modified live virus or inactivated BVDV vaccine, the calves receiving type 1 strains responded with higher antibody titers to type 1 strains than to type 2 strains.

Recent developments in latency and recombination of Aujeszky's disease (pseudorabies) virus.

Latency is a characteristic and fascinating part of the biology of alphaherpesvirinae, including ADV. Tissue explanation, blot hybridization, in situ hybridization and more recently PCR are the experimental methods used to demonstrate that latent infections consistently occur in ganglionic neurons and, at a lower level, in tonsillar and possibly other cells. In vivo reactivation of ADV, resulting in shedding of virulent ADV, has been demonstrated experimentally following administration of high doses of corticosteriods. To determine the influence of vaccination with currently used gene deleted vaccines on field virus latency load, it is essential to use quantitative latency detection methods. We have developed chemiluminescence-based quantitative PCR assays specific for gG and gE, and are currently using these to determine field virus latency loads in tissues of pigs vaccinated with one of several gene deleted vaccines. Recombination between ADV strains has been demonstrated both in vitro and in vivo and has raised concerns about the generation of gene deleted virulent ADV strains. Recent studies in a mouse model have shown that high concentrations of both strains have to be present at the same anatomical site for recombination to take place. This led to the conclusion that ongoing ADV eradication programs, based upon the use of gene deleted vaccines and differential serological testing, are not likely to be threatened by recombination between virulent ADV and gene deleted vaccine strains.