Donal O’Toole

Proposal of a 2-Tier Histologic Grading System for Canine Cutaneous Mast Cell Tumors to More Accurately Predict Biological Behavior

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples.

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

The Pathology of Malignant Catarrhal Fever, With an Emphasis on Ovine Herpesvirus 2

The enigmatic pathogenesis of malignant catarrhal fever (MCF) involves dysregulated immune responses in susceptible ruminant species. Economically important outbreaks of MCF are due to 2 of the 10 viruses currently comprising the malignant catarrhal fever virus group: ovine herpesvirus 2 (OvHV-2) and alcelaphine herpesvirus 1 (AlHV-1). Attempts to develop effective vaccines for this group of viruses in the 1970s were sufficiently discouraging that they were temporarily abandoned. This review focuses on recent efforts to understand the pathogenesis of MCF, particularly the sheep-associated form of the disease, with the goal of developing rational control methods, including vaccination. The past 2 decades have seen several advances, including recognition of new members of the MCF virus group, better diagnostic assays, induction of disease by a natural route (aerosol), and clearer understanding of OvHV-2’s shedding patterns by domestic sheep. A consistent theme in experimental studies of OvHV-2 in susceptible species is that there are 2 peaks of OvHV-2 gene expression: a preclinical peak involving the respiratory tract and a second in multiple organ systems leading to clinical disease. Latent and lytic gene expression may coexist in tissues during clinical stages in symptomatic animals.

Transmission of ovine herpesvirus 2 among adult sheep

Previous studies from this laboratory have defined the pattern of acquisition of ovine herpesvirus 2 (OHV-2) in lambs under natural flock conditions. This study examined the question of whether OHV-2 could be transmitted between adult sheep. Two potential routes of transmission were examined: (1) direct inoculation of either viable leukocytes or whole blood from OHV-2 positive sheep, and (2) horizontal transmission through natural contact with OHV-2 positive sheep. Two groups of OHV-2 negative adult sheep were inoculated with material from infected sheep, one with 5x10(8) viable peripheral blood leukocytes (PBL), and the other with 100 ml of whole peripheral blood. No PCR signals were detected in any of the three sheep inoculated with the PBL during the 20 weeks following inoculation. In the group of five sheep inoculated with whole blood, two became PCR-positive at 7 and 8 weeks post-inoculation, respectively, and the remaining three sheep maintained their negative status until termination of the experiment at 20 weeks post-inoculation. In two experiments conducted in different flocks, a total of 20 adult sheep were used to examine horizontal transmission by contact; all animals became PCR-positive within 12 months of mixing the uninfected and infected animals. The results of these experiments support two conclusions. First, the susceptibility to OHV-2 is not limited to young lambs; adult sheep remain fully susceptible. Second, the fact that whole blood, but not PBL, from infected sheep was able to transmit the infection to only two of five inoculated sheep suggests that the infection in peripheral blood cells may be largely non-productive.

CD8+/perforin+/WC1- γδ T cells, not CD8+ αβ T cells, infiltrate vasculitis lesions of American bison (Bison bison) with experimental sheep-associated malignant catarrhal fever.

Sheep-associated malignant catarrhal fever (SA-MCF) caused by ovine herpesvirus-2 (OvHV-2), a gamma-herpesvirus in the Macavirus genus, is a fatal disease associated with lymphoproliferation, lymphocytic vasculitis, and mucosal ulceration in clinically susceptible species. SA-MCF is an important threat to American bison (Bison bison) due to their high susceptibility to this disease. Currently, the pathogenesis of disease in SA-MCF is poorly understood, and the immunophenotype of lymphocytes that infiltrate the vascular lesions of bison and cattle with SA-MCF has been only partially defined. Previous single-color immunohistochemistry studies have demonstrated that CD8(+) cells and CD4(+) cells predominate within vascular infiltrates in cattle and bison. The CD8(+) cells detected in the vascular lesions of cattle and bison were assumed to be cytotoxic alphabeta T lymphocytes. However, polychromatic immunophenotyping analyses in this study showed that CD8(+)/perforin(+) gammadelta T cells, CD4(+)/perforin(-) alphabeta T cells, and B cells infiltrate vascular lesions in the urinary bladder, kidney, and liver of six bison with experimentally-induced SA-MCF. CD8(+) alphabeta T cells and WC1(+) gammadelta T cell cells were only infrequently and inconsistently identified. This study confirmed our hypothesis that the predominant CD8(+) lymphocytes infiltrating the vascular lesions of bison with SA-MCF are cytotoxic lymphocytes of the innate immune system, not CD8(+) alphabeta T cells. Results of the present study support the previous suggestions that MCF is fundamentally a disease of immune dysregulation.

Ovine herpesvirus 2 infection in American bison: virus and host dynamics in the development of sheep-associated malignant catarrhal fever.

Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus that causes sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease mainly of ruminants. This study was designed to define virus-host dynamics following experimental OvHV-2 infection in bison. A transient peak in viral DNA accompanied by the presence of OvHV-2 ORF25, ORF50 and ORF73 transcripts was observed in lungs only from 9 to 12 days post-inoculation (DPI), suggesting occurrence of viral replication. This initial viral replication was associated with only a subtle increase in transcription of inflammation related genes in lungs and tracheal bronchial lymph nodes, while the level of expression of the majority of immune genes measured remained comparable to uninfected animals. Increasing viral load was observed in the blood and peripheral tissues at 16 and 21 DPI, respectively, indicating systemic viral dissemination. Clinical signs of MCF were observed between 28 and 35 DPI and the severity of lesions increased as disease progressed. Lesion scores were positively correlated with expression levels of ORF25, suggesting a contribution of viral replication in the pathogenesis of SA-MCF. Viral transcripts were observed in all tissues examined from 23 DPI to the end of the experiment at 35 DPI and expression levels of ORF25 were significantly higher in clinically infected animals as compared to pre-clinical stage. The data from this study provide a predictable viral-host interaction time course to test hypotheses concerning disease pathogenesis as well as mitigation of SA-MCF in susceptible species.

Right ventricular hypertrophy with heart failure in Holstein heifers at elevation of 1,600 meters

A syndrome of progressive right-sided heart failure occurred among yearling Holsteins at a heifer-raising facility and 2 dairies on the Colorado Front Range between 2007 and 2011. Most cases were seen at the heifer-raising facility, where the disease ranked second only to pneumonia as a major cause of death in animals aged <1.5 years. The disease resulted in the death or premature sale of 55 animals over the 5-year period. Affected heifers were 4–15 months old when they developed dyspnea, tachycardia, distention and pulsation of jugular veins, lethargy, and weight loss. Clinical progression in most was rapid (2 days to 2 weeks). Ten cattle with typical clinical signs were examined postmortem between 2008 and 2010. Seven developed clinical signs after transportation 57–238 days earlier from Wisconsin (elevation: < 275 m); the remaining 3 animals were born and raised at an altitude of 1,600 m. At necropsy, the 10 cattle had marked hypertrophy of right ventricular myocardium, dilated right atria, right ventricles, and pulmonary trunks, as well as hepatomegaly, ascites, and serous atrophy of fat. The principal histological change in lungs was hypertrophied tunicae adventitia and media of muscular arteries. Hepatic changes were typical of chronic passive congestion. Ultrastructural changes in heart were consistent with uncomplicated hypertrophy of cardiocytes with no evidence of primary cardiomyopathy. The syndrome most likely represents brisket disease due to pulmonary hypertension at the modest elevation of 1,600 m.

Bovine herpesvirus-1: Evaluation of genetic diversity of subtypes derived from field strains of varied clinical syndromes and their relationship to vaccine strains

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle. Control programs in North America incorporate vaccination with modified live viral (MLV) or killed (KV) vaccine. BoHV-1 strains are isolated from diseased animals or fetuses after vaccination. There are markers for differentiating MLV from field strains using whole-genome sequencing and analysis identifying single nucleotide polymorphisms (SNPs). Using multiple primer sets and sequencing of products permits association of BoHV-1 isolates with vaccines. To determine association between vaccine virus and strains isolated from clinical cases following vaccination, we analyzed 12 BoHV-1 isolates from animals with various clinical syndromes; 9 corresponded to BoHV-1.1 respiratory group. The remaining three corresponded to BoHV-1.2b, typically found in genital tracts of cattle. Four BoHV-1 isolates were identical to a vaccine strain; three were from post-vaccination abortion episodes with typical herpetic lesions whose dams had received MLV vaccine during pregnancy, and one from a heifer given a related MLV vaccine; Sequences of two respiratory isolates perfectly matched mutations characterizing RLB106 strain, a temperature sensitive mutant used in intranasal and parenteral vaccines. The last three respiratory strains clearly appeared related to a group of MLV vaccines. Previously the MLV vaccines were grouped into four groups based on SNPs patterns. In contrast with above-mentioned isolates that closely matched SNP patterns of their respective MLV vaccine virus, these 3 strains both lacked some and possessed a number of additional mutations compared to a group of MLV vaccine viral genome. Finding BoHV-1.2b in respiratory cases indicates focus should be given BoHV-1.2b as an emerging virus or a virus not recognized nor fully characterized in BRD.

Experimental nebulization of American bison (Bison bison) with low doses of ovine herpesvirus 2 from sheep nasal secretions

Malignant catarrhal fever (MCF), caused by ovine herpesvirus 2 (OvHV-2), is an important cause of mortality in ranched American bison and domestic cattle in North America. Previous studies showed that bison can be infected by intranasal nebulization with sheep nasal secretions containing OvHV-2 and provided preliminary information on viral doses required for infection and disease progression. The goals of this study were to establish optimal minimal infectious and minimal lethal doses of OvHV-2 by the intranasal route in bison, evaluate the influence of dose on incubation period and other clinical parameters and determine if bison seropositive for antibody against MCF-group viruses are resistant to developing MCF after intranasal challenge. In this study, the minimal infectious dose and minimal lethal dose overlap, suggesting that experimental production of subclinically infected bison is impractical. Dose is inversely related to both incubation period and the period between nebulization and first detection of >1000 OvHV-2 DNA copies/500 ng total DNA in peripheral blood leukocytes. Interestingly, all of the bison seropositive for anti-MCF-group viral antibody prior to inoculation died of MCF after nebulization. We conclude that previous exposure to an MCF-group virus does not necessarily provide resistance to OvHV-2-induced MCF in bison.

Canine distemper outbreak in pet store puppies linked to a high-volume dog breeder

Canine distemper is uncommon in the pet trade in the United States, in large part due to effective vaccines against Canine distemper virus (CDV). This is a report of CDV affecting 24 young dogs of multiple breeds shortly after sale by 2 pet stores in Wyoming during August–October 2010. Cases were diagnosed over 37 days. Diagnosis was established by a combination of fluorescent antibody staining, reverse transcription polymerase chain reaction, negative stain electron microscopy, and necropsy with histopathology. Viral hemagglutinin gene sequences were analyzed from 2 affected dogs and were identical (GenBank accession no. JF283477). Sequences were distinct from those in a contemporaneous unrelated case of CDV in a Wyoming dog (JF283476) that had no contact with the pet store dogs. The breeding property from which the puppies originated was quarantined by the Kansas Animal Health Department. Puppies intended for sale were tested for CDV. Canine distemper was diagnosed on site in November 2010. At that point 1,466 dogs were euthanized to eliminate dispersal of the disease through commercial channels. The investigation underscores the risks inherent in large-scale dog breeding when vaccination and biosecurity practices are suboptimal.