Characterization of ovine gammaherpesvirus 2 in a goat by nanoplate digital PCR and other diagnostic methods.

The Macavirus, ovine gammaherpesvirus 2 (OvGHV2), is the cause of sheep-associated malignant catarrhal fever (SA-MCF). Although SA-MCF occurs in a wide range of mammalian hosts, there are few descriptions of this disease and/or infection in goats. This report describes the findings observed in a goat that was infected by OvGHV2 and adds to the rare description of this infection in this animal species. A 6.5-year-old, female, Anglo Nubian goat, with a neurological syndrome, that was euthanized after severe esophageal obstruction was investigated to determine the cause of the brain disease. Histopathology revealed cerebral cortical edema, hemorrhagic rhombencephalitis, severe hepatic necrosis, and atrophic enteritis. An immunohistochemical (IHC) assay identified intracytoplasmic antigens of a malignant catarrhal fever virus (MCFV) within epithelial cells of the intestine, liver, lungs, and kidneys. A semi-nested PCR assay amplified the partial fragment of the OvGHV2 tegument protein gene from the intestine, confirming that the MCFV identified by IHC was OvGHV2. A qPCR assay that targeted the OvGHV2 polymerase gene revealed an elevated quantification cycle (Cq), while nanoplate-based digital PCR (dPCR) detected low viral copy load within the OvGHV2 DNA. Furthermore, the nucleic acids of several disease pathogens associated with diseases in ruminants were not amplified. However, the exact cause of the neurological syndrome remained obscure since nucleic acids of neurological disease pathogens such as bovine viral diarrhea virus, bovine alphaherpesvirus 1 and 5, Histophilus somni, and OvGHV2 were not detected from the brain. Collectively, the results of the Cq and dPCR confirmed that this goat was infected with a low viral load of OvGHV2, which probably was insufficient to induce the typical histopathological alterations and subsequent clinical manifestations associated with SA-MCF and/or infections by OvGHV2. Therefore, elevated viral loads of OvGHV2 would have been required for the development of histological lesions and/or clinical manifestations of SA-MCF in this goat. Furthermore, the dPCR methodology can be used for the efficient detection and quantification of OvGHV2 DNA in animals with or without clinical and/or histopathological evidence of SA-MCF. Additionally, since previous cases of OvGHV2 infections in goats did not have the typical clinical manifestations of SA-MCF, one wonders if this Macavirus can induce SA-MCF in goats.

Immunohistochemical identification of a malignant catarrhal fever virus in cattle with renal diseases from Paraná state, Southern Brazil: a retrospective epidemiological study.

Malignant catarrhal fever (MCF) is a viral infectious disease caused by specific members of the Macavirus genus that are referred to as the MCF virus (MCFV) complex group. This study determined the prevalence of MCFV-associated infections in cattle within the mesoregions of the state of Paraná, Southern Brazil, by analyzing the histopathologic patterns of renal lesions in association with positive immunoreactivity to intralesional antigens of MCFV. Intracytoplasmic MCFV antigens were identified in 41.7% (48/115) of the kidneys of cattle evaluated. Lymphocytic interstitial nephritis, vascular degeneration, and ballooning degeneration of the renal tubules were the principal histopathological findings associated with positive immunoreactivity to MCFV. The results indicate that MCFV infections are endemic within the state of Paraná and suggest that the kidney can be of diagnostic value in suspected cases of MCF-associated infections in cattle. Furthermore, the utilization of an in situ diagnostic technique resulted in the detection of a greater number of cases of infections by MCFV than previously identified using other diagnostic methods. Additionally, degenerative vascular lesions of the kidney should be considered during the establishment of a histological diagnosis of MCFV-induced infections in cattle in the absence of fibrinoid change or necrotizing vasculitis.

Association of ovine gammaherpesvirus 2 with an outbreak of acute respiratory disease in dairy cattle.

This study investigated the cause of an outbreak of an acute respiratory disease syndrome followed by episodes of diarrhea in a dairy cattle herd from Southern Brazil. Deep nasal swabs (DNS) from asymptomatic calves, calves with pulmonary discomfort, and diarrheic calves after episodes of respiratory distress were used in molecular assays designed to detect the principal pathogens associated with bovine respiratory disease (BRD). Fecal samples were used for the molecular detection of bovine enteric disease agents. Pulmonary tissues from three calves and a cow that died were evaluated by molecular assays to identify 11 agents associated with the development of BRD. The intestinal and pulmonary fragments of one calf and the cow revealed atrophic enteritis and interstitial pneumonia by histopathology, respectively. Immunohistochemistry (IHC) identified intralesional antigens of a malignant catarrhal fever virus, genus Macavirus, within epithelial cells of the lungs and intestines. Molecular assays amplified ovine gammaherpesvirus 2 (OvGHV2) from most of the DNS, and the pulmonary and intestinal fragments from the animals that died, confirming that the Macavirus identified by IHC was OvGHV2. Concomitant pulmonary infections of OvGHV2 with bovine gammaherpesvirus 6 and bovine coronavirus were identified. Additionally, bovine viral diarrhea virus 1b and Aichivirus B were detected in the fecal samples. These findings demonstrated that OvGHV2, a Macavirus, was the disease agent most frequently (81.2%; 13/16) associated with singular pulmonary infections during this outbreak of BRD, suggesting that this virus may be another potential agent of respiratory disease of cattle.

Histophilus somni disease conditions with simultaneous infections by ovine gammaherpesvirus 2 in cattle herds from Southern Brazil.

This report investigated the cause of cattle mortality in two farms in Southern Brazil. The tissues of one animal from each farm (animals #1 and #2) respectively were used in pathological and molecular investigations to determine the possible cause of death. The principal pathological findings observed in animal #1 were pulmonary, myocardial, and encephalitic hemorrhages with vasculitis, and lymphoplasmacytic interstitial pneumonia with proliferative vascular lesions (PVL). The main pathological findings observed in animal #2 were purulent bronchopneumonia, hemorrhagic myocarditis, and lymphoplasmacytic interstitial pneumonia with PVL. An immunohistochemical assay detected intralesional antigens of a malignant catarrhal fever virus (MCFV) from multiple tissues of animal #2 while PCR confirmed that the MCFV amplified was ovine gammaherpesvirus 2 (OvGHV2), genus Macavirus, subfamily Gammaherpesvirinae; OvGHV2 was also amplified from multiple tissues of animal #1. Furthermore, PCR assays amplified Histophilus somni DNA from multiple fragments of both animals. However, the nucleic acids of Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, bovine respiratory syncytial virus, bovine alphaherpesvirus virus 1 and 5, bovine coronavirus, and bovine parainfluenza virus 3 were not amplified from any of the tissues analyzed, suggesting that these pathogens did not participate in the development of the lesions herein described. These findings demonstrated that both animals were concomitantly infected by H. somni and OvGHV2 and developed the septicemic and encephalitic manifestations of H. somni. Furthermore, the interstitial pneumonia observed in cow #2 was more likely associated with infection by OvGHV2.

Possible Association of Bovine Gammaherpesvirus 6 with Pulmonary Disease in a Cow.

Bovine gammaherpesvirus 6 (BoGHV6), previously known as bovine lymphotropic virus, is a member of the Macavirus genus, subfamily Gammaherpesvirinae. Other members of the genus Macavirus include viruses that produce malignant catarrhal fever (MCF) in mammalian hosts, collectively referred to as the MCF virus (MCFV) complex, and the porcine lymphotropic herpesvirus (PLHV). However, the current role of BoGHV6 in the development of diseases and/or disease syndromes remains uncertain and controversial. This paper investigated the participation of BoGHV6 in the development of pulmonary disease in a cow with interstitial pneumonia by histopathology and molecular testing. Tissue antigens of common viral agents of respiratory diseases and Mycoplasma bovis were not identified by immunohistochemistry. Additionally, molecular assays designed to amplify common bacterial and viral pathogens of pulmonary disease did not amplify the nucleic acids of these agents. However, a pan-PCR assay amplified the DNA of the herpesvirus polymerase gene, while the specific BoGHV6 nested-PCR assay amplified the partial fragment of the BoGHV6 polymerase gene derived from the pulmonary tissue with interstitial pneumonia. Phylogenetic analysis revealed that the BoGHV6 strain herein identified had 99.8% nucleotide (nt) sequence identity with reference strains of BoGHV6, but only 72.2-73.5% and 67.9-68.6% nt identity with reference strains of MCFV and PLHV, respectively. Consequently, these results suggest that BoGHV6 was associated with the pulmonary disease observed in this cow.

Detection of bovine gammaherpesvirus 6 in tissues of aborted fetuses from dairy cows concomitantly infected by Histophilus somni.

Bovine gammaherpesvirus 6 (BoGHV6), formerly known as bovine lymphotropic virus, is a member of the Macavirus genus, subfamily Gammaherpesvirinae, family Herpesviridae, that was initially associated with proliferative diseases in cattle. While the Macavirus genus contains agents, including alcelaphine gammaherpesvirus 1 (AlGHV1), ovine gammaherpesvirus 2 (OvGHV2), and caprine gammaherpesvirus-2 (CpGHV2), known to cause malignant catarrhal fever (MCF), and are collectively referred to as MCF virus (MCFV) group of organisms, diseases and/or clinical syndromes have not been associated with BoGHV6 and porcine lymphotropic herpesvirus (PLHV). This report investigated the occurrence of BoGHV6 in tissues of aborted dairy fetuses known to be infected by Histophilus somni to identify possible disease patterns associated with infection by this Macavirus. A nested-PCR (nPCR) assay was used to amplify the BoGHV6 polymerase gene from multiple tissues of 13 fetuses and the cow of one of these which were derived from seven dairy herds located in three geographical regions of Brazil. Direct sequencing confirmed the results of the nPCR assays. Additionally, all fetal tissues were previously investigated for the presence of H. somni, Listeria monocytogenes, Neospora caninum, Brucella abortus, Leptospira spp., bovine alphaherpesvirus 1, and bovine viral diarrhea virus (BVDV) by PCR and/or RT-PCR assays. The nPCR assay amplified BoGHV6 DNA from fetuses of most dairy herds (85.7%; 6/7) investigated, resulting in the amplification of BoGHV6 from 76.9% (10/13) of all fetuses evaluated from two geographical and important cattle-producing regions of Brazil. Furthermore, only BoGHV6 was identified in the spleen (n = 3), myocardium, and kidney (n = 2) of five fetuses, and BoGHV6 was the only agent associated with myocarditis in one of these. Nevertheless, dual, triple, and quadruple infections (including BVDV, B. abortus, and N. caninum) were identified in fetuses that were concomitantly infected by H. somni. Phylogenetic analysis revealed that the strain herein identified has 100% nucleotide (nt) sequence identity with wild type strains of BoGHV6 circulating in ruminants from Brazil and 99.8% nt identity with the reference strain of BoGHV6 but was 72.2-73.3% and 67.4-68.2% different from members of the MCFV group and PLHV, respectively. These results demonstrated that 76.9% of the fetuses evaluated were infected by BoGHV6, most likely via vertical infection resulting in transplacental transmission. Considering that most fetuses were concomitantly infected by BoGHV6 and H. somni the real impact of this viral infection cannot be efficiently determined. However, since BoGHV6 was the only pathogen identified in the myocardium of one fetus with myocarditis by histopathology, the possible participation of this Macavirus in the etiopathogenesis of the myocardial disease observed in this fetus cannot be ignored or discarded. However, the mere amplification of BoGHV6 DNA from the myocardium is not enough to establish a definite association between cause and effect, since in situ evaluations and experimental studies would be needed to confirm this agent in the etiopathogenesis of fetal diseases and/or abortions in cattle. Consequently, additional studies are needed to determine the exact role, if any, of BoGHV6 in the development of fetal disease, and possibly fetal mortality.

Ovine gammaherpesvirus 2 infections in cattle without typical manifestations of sheep-associated malignant catarrhal fever and concomitantly infected with bovine coronavirus.

Sheep-associated malignant catarrhal fever (SA-MCF) is a severe, frequently fatal, lymphoproliferative disease that affects a wide variety of ruminants and is caused by ovine gammaherpesvirus 2 (OvHV-2), a member of the MCF virus (MCFV) complex. The typical clinical manifestations of SA-MCF are well known and easily recognized by veterinarians, resulting in clinical diagnosis of MCF when characteristic clinical signs are present. This article describes the findings observed in cattle infected with OvHV-2 but without typical clinical manifestations of SA-MCF. Three calves with episodes of diarrhea before death and a yearling that died suddenly were investigated. Gross alterations were not suggestive of SA-MCF. Histopathology revealed a combination of proliferating vascular lesions (PVLs) and necrotizing vasculitis in three animals (two calves and the yearling); with PVLs being identified only at the carotid rete mirabile of two calves infected with OvHV-2. Additional significant histopathologic lesions included atrophic enteritis, portal lymphocytic hepatitis, interstitial pneumonia, suppurative bacterial bronchopneumonia, and pulmonary hemorrhage. An immunohistochemical assay designed to identify only antigens of MCFV revealed, positive, intralesional, intracytoplasmic immunoreactivity within epithelial cells of multiple tissues of all animals with PVLs. PCR assays amplified OvHV-2 DNA from multiple tissues of the animals that contained MCFV proteins, confirming the MCFV identified as OvHV-2. Additionally, bovine coronavirus (BCoV) nucleic acids were amplified from tissues of all animals, including the animal not infected by OvHV-2. Collectively, these findings confirmed the participation of OvHV-2 in the development of the disease patterns observed in these animals that were concomitantly infected by BCoV and provide additional confirmation that cattle can be subclinically infected with OvHV-2. Consequently, the real occurrence of OvHV-2-related disease may be more elevated than reported, since asymptomatic or subclinically infected animals are not likely to be investigated for OvHV-2. Furthermore, PVLs should be included as possible histologic indicators of OvHV-2-related diseases in ruminants.

Canine distemper virus and canine adenovirus type-2 infections in neotropical otters (Lontra longicaudis) from Southern Brazil.

All descriptions of infectious diseases affecting otters were published in the Northern Hemisphere, with no occurrence identified in neotropical otters (Lontra longicaudis). Consequently, a retrospective histopathological study using archival tissue samples from six free-living neotropical otters was done to investigate the possible occurrence of disease patterns associated with common viral infectious disease agents of the domestic dogs. Immunohistochemical (IHC) assays were designed to identify intralesional tissue antigens of canine distemper virus (CDV), and canine adenovirus-1 (CAdV-1) and canine adenovirus-2 (CAdV-2). The most frequent histopathological patterns diagnosed were interstitial pneumonia (83.33%; 6/5) and hepatocellular vacuolar degeneration (50%; 3/6). IHC identified intralesional intracytoplasmic immunoreactivity to CDV antigens in all otters evaluated, with positive immunolabeling occurring within epithelial cells of the lungs, stomach, kidneys, and liver, and skin. Intracytoplasmic CAdV-2 antigens were identified within epithelial cells of the peribronchial glands in four otters with interstitial pneumonia. These findings resulted in singular and simultaneous infections in these neotropical otters, represented the first report of concomitant infections by CDV and CAdV-2 in free-living neotropical otters from the Southern Hemisphere, and suggested that this mammalian species is susceptible to infections by viral disease agents common to the domestic dogs and may develop similar histopathologic disease patterns.

Ovine gammaherpesvirus-2 infection associated with chronic interstitial pneumonia in a sheep.

Sheep Associated-Malignant Catarrhal Fever (SA-MCF) is severe, frequently lethal, lymphoproliferative disease predominantly of ruminants, that is caused by ovine gammaherpesvirus-2 (OvHV-2), a member of the MCF virus (MCFV) complex. However, SA-MCF in sheep is a rare entity with few demonstrations of natural diseases worldwide. This report documents the clinical, radiographical, pathological, immunohistochemical, and molecular findings of SA-MCF in a sheep. A 4-year-old, female, mixed-breed sheep with progressive emaciation for at least one month was humanely euthanized due to poor prognosis. Clinically, the animal had tachypnea, ruminal hypomotility, productive coughing with bilateral muffling sounds during pulmonary auscultation. Radiographical evaluation revealed alveolar opacity of the cranioventral pulmonary region. Grossly, there were distinct rib impressions on the pleural surface of the lungs, suggestive of interstitial pneumonia. Histopathologic evaluation of the lungs revealed several disease patterns including 1) chronic interstitial pneumonia with vasculitis and proliferating vascular lesions, and thrombosis; 2) pulmonary abscesses associated with embolic dissemination of Corynebacterium pseudotuberculosis from superficial lymph node due to caseous lymphadenitis, CLA; 3) granulomatous pneumonia associated with pulmonary nematodes; and 4) chronic pleuritis, probably due to caseous lymphadenitis. Additional significant histologic findings included widespread lymphocytic vasculitis and proliferating vascular lesions in multiple tissues, atrophic enteritis, segmental degeneration of myocardial fibers with lymphocytic pericarditis, lymphocytic interstitial nephritis, and non-suppurative encephalitis. An immunohistochemistry (IHC) assay, based on the monoclonal antibody 15A (MAb-15A), that is specific to all MCFV known to cause MCF, revealed positive, intracytoplasmic, intralesional immunoreactivity, predominantly within bronchial and bronchiolar epithelial cells of the lungs and cryptal epithelial cells of the small intestine, followed by the renal tubular epithelium, cardiomyocytes, and with patchy immunolabelling within neurons of the cerebral cortex. Molecular testing done to detect a wide range of bacterial and viral agents of ruminant diseases, only amplified OvHV-2 DNA from fresh tissue fragments of the lungs, kidney, liver, spleen, and cerebrum. Direct sequencing confirmed that the PCR amplicon derived from the pulmonary fragments had 99.2-99.7% nucleotide sequence identity with OvHV-2 reference strains and strains of OvHV-2 from Brazil. The clinical, radiographical, gross, histopathologic, IHC, and molecular findings in the lungs are consistent with chronic interstitial pneumonia associated with infection by OvHV-2. Furthermore, the non-detection of other viral agents associated with pulmonary diseases in ruminants suggest that OvHV-2 was directly associated with the development of chronic pneumonia in this sheep. Additionally, the dental alterations, CLA, and the pulmonary nematode may have contributed towards the reduced immunological statue of the animal and facilitated the occurrence of SA-MCF. These findings may indicate that OvHV-2 may be a major participant in the pathogenesis of pulmonary disease of sheep under special conditions. Moreover, the proliferating vascular lesions identified in multiple tissues are additional evidence of chronic manifestations of OvHV-2 infections as described in chronic SA-MCF of cattle, while the widespread vasculitis is consistent with SA-MCF. Additionally, the IHC findings using the MAb-15A confirmed that this diagnostic approach is efficient to identify intralesional antigens of OvHV-2.

Neorickettsia helminthoeca associated lymphoid, enteric, and pulmonary lesions in dogs from Southern Brazil: An immunohistochemical study.

Neorickettsia helminthoeca (NH), the agent of salmon poisoning disease or canine neorickettiosis (CN), is a bacterial endosymbiont of the nematode Nanophyetus salmincola, and infections are spreading among specific fish-eating mammalians. This article describes the pathologic and immunohistochemical findings associated with spontaneous NH-induced infections in dogs from Southern Brazil. The principal pathologic findings were hypertrophy of Peyer patches and lymphadenopathy with lymphocytic proliferation, chronic interstitial pneumonia, and chronic enteritis associated with positive intralesional immunoreactivity to antigens of NH within macrophages and histiocytes. Positive immunoreactivity against canine parvovirus-2 (CPV-2) or/and canine distemper virus was not detected in the evaluated intestinal segments or in the samples from the cerebellum and lungs, respectively, from the dogs evaluated. These findings demonstrated that NH was involved in the enteric, pulmonary, and lymphoid lesions herein described, and provide additional information to confirm the occurrence of this bacterial endosymbiont within this geographical location. It is proposed that chronic pneumonia should be considered as a pathologic manifestation of NH-induced infections. Additionally, our results show that the occurrences of CN seem to be underdiagnosed in Southern Brazil due to the confusion with the incidence of CPV-2.