Subclinical Ovine Gammaherpesvirus 2-Related Infections in Free-Ranging Wild Boars (Sus scrofa) from Southern Brazil.

Ovine gammaherpesvirus 2 (OvGHV2), is a Macavirus and the cause of sheep-associated malignant catarrhal fever (SA-MCF), in which sheep are the asymptomatic reservoir hosts. Susceptible mammalian populations infected by OvGHV2 may develop clinical SA-MCF or subclinical infections. All members of the Macavirus genus known to be associated with MCF are collectively referred to as the MCF virus (MCFV) complex. This report describes the occurrence of subclinical OvGHV2-related infections in free-ranging wild boars (Sus scrofa) from southern Brazil. Specific body organs (n = 14) and biological samples (nasal and oral swabs; n = 17) were collected from 24 asymptomatic wild boars from a conservation unit located within the Central-eastern mesoregion of Paraná State. Organs were processed to observe histopathological patterns suggestive of diseases of domestic animals; only pulmonary samples were used in an immunohistochemical assay designed to detect MCFV tissue antigens. Furthermore, all samples were submitted to molecular assays designed to detect the OvGHV2 tegument protein gene. Viral-induced pneumonia was diagnosed in two wild boars; one of these contained OvGHV2 DNA, with MCFV antigens identified in the other. Additionally, MCFV tissue antigens were detected within pulmonary epithelial cells of the lungs with and without pulmonary disease. Collectively, OvGHV2 was detected in 37.5% (9/24) of all wild boars, with detection occurring in the organs of 57.1% (8/14) wild boars and the oral cavity of one animal. These results demonstrated that these wild boars were subclinically infected by OvGHV2, and that infection produced typical pulmonary alterations. In addition, the detection of OvGHV2 within the oral cavity of one wild boar may suggest that this animal may be a potential disseminator of this pathogen to susceptible animal populations, including livestock and wildlife, acting as a possible bridge host for OvGHV2. Furthermore, infection by OvGHV2 probably occurred due to incidental contact with asymptomatic sheep maintained within the surrounding rural areas and not within the conservation units.

Quantification of ovine gammaherpesvirus 2 in clinical cases of cattle with sheep-associated malignant catarrhal fever.

Ovine gammaherpesvirus 2 (OvGHV2) produces sheep-associated malignant catarrhal fever (SA-MCF), a frequently lethal, lymphoproliferative disease that is characterized by widespread vascular lesions. Most studies that evaluated the viral load in tissues of animals with SA-MCF were done in the Northern Hemisphere, with scant information from the Southern part of the globe. This study investigated the viral load of OvGHV2 in the tissues of cattle and an underdeveloped fetus with SA-MCF from three distinct biomes of Brazil. All animals had clinical and histopathological manifestations consistent with SA-MCF. Molecular testing identified the OvGHV2 tegument protein and glycoprotein B genes in the tissues of all animals and the fetus. Viral quantification based on the DNA polymerase gene detected elevated loads of OvGHV2 in tissues with histopathological evidence of SA-MCF and organs with unknown histological data, except for the tissues of the fetus, where the viral load was comparatively reduced. The viral loads detected in multiple organs of cattle from this study with SA-MCF are consistent with those identified in different animal species from the USA and Europe. The detection of a low viral load of OvGHV2 in fetal tissue confirmed transplacental dissemination since elevated viral loads were detected in multiple tissues of the cow with SA-MCF. Furthermore, the elevated viral loads detected in the pulmonary tissues of cattle with interstitial pneumonia indicate that OvGHV2 is an inductor of pulmonary disease in cattle.

Molecular detection of ovine gammaherpesvirus 2 in free ranging wild boars (Sus scrofa) from Southern Brazil.

Ovine gammaherpesvirus 2 (OvGHV2) is a member of Macavirus genus, subfamily Gammaherpesvirinae, family Herpesviridae, and causes sheep associated-malignant catarrhal fever (SA-MCF) in a wide range of ungulates. However, no descriptions of SA-MCF and/or infections due to OvGHV2 were identified in the wild boar (Sus scrofa). This study investigated the occurrence of OvGHV2 in the lungs (n = 44) of asymptomatic, free ranging wild boars captured in several regions of Paraná State, Southern Brazil. A PCR assay targeting the OvGHV2 tegument protein gene amplified OvGHV2 DNA in 4.55% (2/44) of the pulmonary tissues evaluated. Sequence analysis confirmed that the OvGHV2 strains herein identified have 98.4% deduced amino acid (aa) sequence identity with the prototype strain of OvGHV2 and 96.4-100% aa identity with similar strains of OvGHV2 detected in several animal species from diverse countries. These findings confirmed that these two wild boars were infected by OvGHV2, represent the first description of this infection in these animals, and add to the number of pathogens identified in this animal species. Furthermore, these findings contrast earlier descriptions of OvGHV2 in swine since in all previous reports the infected pigs demonstrated clinical manifestations of disease. Consequently, these wild boars from Southern Brazil were subclinically infected or suffered asymptomatic infections by OvGHV2.

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.

The pathology of canine mammary candidiasis with embolic dissemination in a dog.

Candidiasis is a fungal disease caused by Candida albicans or other members of the genus Candida. Descriptions of candidiasis are comparatively reduced in veterinary relative to human medicine, with no cases of mammary candidiasis being identified in pet animals. This report presents the cytological, pathological, and molecular findings of mammary candidiasis with embolic dissemination in a postpartum dog. A 1-year-old, female Shih-tzu dog that had recently given birth was admitted to a veterinary teaching hospital in Southern Brazil after repeated episodes of intermittent mammary disease and a neurological syndrome. The dog was euthanized due to worsened clinical status and poor prognosis despite adequate clinical therapy and was submitted for routine post-mortem evaluation to determine the cause of the neurological manifestations. Cytological analysis of purulent mastitis identified intralesional fungal hyphae. Gross evaluation revealed multiple masses within the kidneys, liver, myocardium, pancreas, and brain. Routine histopathology and histochemistry identified fungal nephritis, hepatitis, myocarditis, pancreatitis, and encephalitis associated with intralesional fungal hyphae, frequently with fungal emboli and vasculitis. Pure cultures of C. albicans were obtained from fragments of the masses observed at the myocardium and kidneys, with the typical germ tube of C. albicans being identified by microscopic evaluation. A PCR assay that targeted the ITS1 and 4 generic regions of fungi, amplified the desired amplicon, and direct sequencing confirmed C. albicans. Immunohistochemical and molecular assays designed to identify common infectious disease pathogens of dogs did not confirm the participation of canine distemper virus, canine parvovirus, or canine adenovirus in the target tissues of this dog. These findings suggest that this dog suffered an initial cutaneous lesion, that probably served as portal of entry to the mammary gland, resulting in mammary candidiasis with subsequent embolic dissemination to multiple organs. This report represent the first description of mammary candidiasis in pet animals and probably one of the few pathological descriptions of mammary candidiasis in domestic animals. In this case, the cause of the fungal infection was probably associated with factors intrinsic to abdominal surgery, pregnancy, and the utilization of antibiotics.

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.

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.

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.

Case report: Mixed infection of bovine papillomaviruses associated with squamous papilloma of the upper alimentary tract in a dairy cow.

Bovine papillomavirus (BPV) infection can induce neoplastic lesions in both cutaneous and mucosal epithelia in cattle. This study describes the BPV types associated with proliferative lesions with diverse histopathological features present in the upper alimentary tract of a dairy cow suffering from chronic diarrhea from Midwestern Brazil. At autopsy, warts and plaques composed of multiple spherical nodules were observed in the esophageal mucosa, the areas surrounding and constricting the opening of the cardia, and the rumen pillars. One esophageal papillomatous proliferative lesion and a smooth-surfaced proliferative lesion located at the rumen entrance were evaluated by histopathological and molecular analyses. PCR amplification of partial fragments of the BPV L1 and E1 genes was performed followed by sequencing of the obtained amplicons. Upon histopathological evaluation, the esophageal lesion was classified as a squamous papilloma, whereas the other ruminal proliferative lesion consisted of a fibropapilloma. Direct sequencing of PCR products obtained from ruminal fibropapilloma DNA revealed the presence of BPV2. Sequencing of inserts from selected clones containing partial fragments of the BPV L1 and E1 genes revealed a mixed infection of BPV types 2 and 4 in the esophageal squamous papilloma. The findings reported in our investigation reinforce the association of BPV with benign lesions of the bovine alimentary tract in both single and mixed infections, as previously demonstrated to occur in a buffalo. In addition, this report represents the documentation of the occurrence of massive alimentary papillomatosis associated with BPV types 2 and 4 in cattle raised on lands without infestation by bracken fern in Midwestern Brazil.

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.

Porcine lymphotropic herpesvirus (Gammaherpesvirinae) DNA in free-living wild boars (Sus scrofa Linnaeus, 1758) in Brazil.

BACKGROUND: Suid gammaherpesvirus 3, 4, and 5 (porcine lymphotropic herpesvirus - PLHV-1, -2, and -3) are viruses that infect domestic and feral pigs. OBJECTIVES: This study examined the presence of PLHV DNA in biological samples from free-living wild boars circulating in a Brazilian geographical region with a high density of commercial domestic pigs. METHODS: Lung samples of 50 free-living wild boars were collected by exotic wildlife controller agents between 2017 and 2019 in the state of Paraná, southern Brazil. Lung and spleen fragments were obtained from six fetuses collected by hysterectomy post mortem from a pregnant sow. A polymerase chain reaction (PCR) assay using consensus primers (pan-herpesviruses) was performed to detect PLHV DNA. The samples showing positive results for PLHV DNA were submitted to single-round PCR assays with the specific primers for identifying PLHV-1 (213-S/215-As), PLHV-2 (208-S/212-As), and PLHV-3 (886s/886As). The specificity of the species-specific PCR products was assessed by nucleotide sequencing of the amplicons. RESULTS: Forty-eight (96%) of the 50 lung samples analyzed were positive for PLHV by PCR using pan-herpesvirus primers. In 33 (68.75%) of the positive samples, at least two PLHV species were identified simultaneously. The DNA of PLHV-1, -2, and -3 was found in free-living wild boars of all ages, but not in the fetuses, even though they were from a sow that tested positive for all three viruses. CONCLUSION: These viruses are endemic to the population of feral pigs in the Brazilian region evaluated, as well as in domesticated pigs.

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.

The Participation of a Malignant Catarrhal Fever Virus and Mycoplasma bovis in the Development of Single and Mixed Infections in Beef and Dairy Cattle With Bovine Respiratory Disease.

The bovine respiratory disease (BRD) complex is a multietiological and multifactorial disease associated with a wide range of viral and bacterial pathogens. This study evaluated the contribution of specific infectious disease agents in the development of BRD in cattle from Brazil and determined if a virus within the malignant catarrhal fever virus (MCFV) group and Mycoplasma bovis, acting individually or in conjunction, can be associated with the development of BRD. Formalin-fixed paraffin-embedded pulmonary sections were used in immunohistochemical assays to determine the intralesional presence of six antigens associated with BRD: bovine alphaherpesvirus 1 (BoHV-1), bovine parainfluenza virus 3 (BPIV-3), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), MCFV, and M. bovis. Pneumonia was diagnosed in 82.7% (120/145) of all cattle evaluated. Interstitial pneumonia (60%, 72/120) and suppurative bronchopneumonia (25.8%, 31/120) were the most frequent patterns of pneumonia identified. Intralesional antigens of MCFV (53.3%, 64/120) were the most frequently associated with BRD, followed by M. bovis (47.5%, 57/120), BVDV (42.5%, 51/120), BoHV-1 (28.3%, 34/120), BRSV (24.2%, 29/120), and BPIV-3 (8.3%, 10/120). Additionally, antigens of BVDV, MCFV, and M. bovis were the most frequently identified agents associated with singular and concomitant infections. The MCFV identified during this study is more likely to be ovine gammaherpesvirus 2 (OvHV-2), since OvHV-2 is the only MCFV identified within the geographical region of this study. Interstitial pneumonia with proliferative vascular lesions may be a useful histologic feature to differentiate MCFV-induced pneumonia from other viral pneumonias of cattle. These results demonstrate that MCFV and M. bovis, in single or mixed infections, can produce pneumonia in cattle and should therefore be considered as primary agents in the development of BRD.

Detection and Quantification of the E6 Oncogene in Bovine Papillomavirus Types 2 and 13 From Urinary Bladder Lesions of Cattle.

Bovine papillomavirus types 2 and 13 can induce tumors in both the cutaneous and mucosal epithelia of cattle. These viral types are associated with the development of benign cutaneous papillomas and malignant lesions in the urinary bladders of cattle, with the latter being known as bovine enzootic hematuria. Among the viral oncoproteins encoded by Deltapapillomavirus DNA, the E6 oncoprotein has an important role in cell proliferation and might be related to cancer initiation and promotion. The aim of this study was to present a standardized SYBR Green-based quantitative PCR for detection and quantification of the bovine papillomavirus 2 and 13 E6 oncogenes in urinary bladder samples from cattle. Twenty-four urinary bladders from cattle displaying tumors (n = 12) and normal bladder mucosa (n = 12) were tested by quantitative PCR. Of the 12 urinary bladders with tumors, six presented bovine papillomavirus 2 DNA concentrations ranging from 1.05 × 104 to 9.53 × 103 copies/µL, while two had bovine papillomavirus 13 DNA amplified at concentrations of 1.30 × 104 to 1.23 × 104 copies/µL. The healthy bladder mucosa samples were negative for both bovine papillomaviruses. Once the results were confirmed by conventional PCR and direct sequencing, the quantitative PCR assay developed in this study was shown to be a sensitive and specific tool for detecting and quantifying the E6 ORF of bovine papillomavirus 2 and 13 in a variety of clinical samples. Our findings of identification of bovine papillomavirus 2 and 13 DNA in urothelial tumors from cattle suffering from bovine enzootic hematuria agree with data from previous studies, representing the first detection of bovine papillomavirus 13 DNA in malignant bladder lesions of cattle from Brazil.

First detection of canine parvovirus 2b DNA in a crab-eating fox pup (Cerdocyon thous, Linnaeus, 1766).

The crab-eating fox (Cerdocyon thous) is a small wild mammal present in all Brazilian biomes and in some countries of South America. This study aimed to verify the involvement of viral infectious agents in the death of a wild crab-eating fox pup (Cerdocyon thous) in Brazil. The Center for Medicine and Research of Wild Animals of the Universidade Estadual Paulista received a free-living crab-eating fox aged approximately 21 days and apparently healthy. After 13 days, the animal presented anorexia, diarrhea, fever, prostration, and neurological signs progressing to death with an inconclusive diagnosis. In a retrospective study, tissue fragments stored at - 80 °C were used to identify nucleic acids from major canine viruses, such as canine parvovirus-2 (CPV-2), canine adenovirus A types 1 and 2, canid alphaherpesvirus 1, and canine distemper virus. The amplified product with the expected length for CPV-2 was obtained from the heart fragment. After performing nucleotide (nt) sequencing of the amplicon, it was possible to demonstrate that the crab-eating fox strain exhibited high (99.8%) nt identity with the CPV-2b prototype (CPV-39 strain). Additionally, deduced amino acid (aa) sequence analysis showed the GAT codon for the aa Asp (D) at position 426 of the CPV-2 viral protein VP2, which characterizes the subtype 2b. To the best of the authors' knowledge, this report describes the first detection of CPV-2b DNA in tissue fragments from a crab-eating fox.

Porcine lymphotropic herpesvirus DNA detection in multiple organs of pigs in Brazil.

We investigated the porcine lymphotropic herpesvirus (PLHV) DNA presence in multiple organs of pigs. Biological samples (n = 136) included tissue fragments of the central nervous system, heart, kidney, liver, lungs, spleen, urinary bladder, and urine. Sixty-eight (50%) organs were PLHV DNA-positive. None of the urine samples were detected with the virus genome. Although the presence of the PLHV DNA in the urinary bladder and kidney has been detected, it was not possible to show whether urine can be considered an effective route of virus shedding. This study warns to the risk of PLHV zoonotic transmission by xenotransplantation of tissues of porcine origin.

Molecular characterization of Brazilian wild-type strains of bovine respiratory syncytial virus reveals genetic diversity and a putative new subgroup of the virus.

Background: Bovine orthopneumovirus, formerly known as bovine respiratory syncytial virus (BRSV), is frequently associated with bovine respiratory disease (BRD).Aim: To perform the molecular characterization of the G and F proteins of Brazilian wild-type BRSV strains derived from bovine respiratory infections in both beef and dairy cattle.Materials and Methods: Ten BRSV strains derived from a dairy heifer rearing unit (n = 3) in 2011 and steers of three other feedlots (n = 7) in 2014 and 2015 were analyzed. For the BRSV G and F partial gene amplifications, RT-nested-PCR assays were performed with sequencing in both directions with forward and reverse primers used.Results: The G gene-based analysis revealed that two strains were highly similar to the BRSV sequences representative of subgroup III, including the Bayovac vaccine strain. However, the remaining seven Brazilian BRSV strains were diverse when compared with strains representative of the BRSV I to VIII subgroups. The central hydrophobic region of the Brazilian BRSV G gene showed the replacement of conserved cysteines and other residues of importance to antibody reactivity. The deduced F gene amino acid sequences from the Brazilian BRSV strains showed changes that were absent in the representative sequences of the known subgroups. Viral isolation on the nasopharyngeal swab suspensions failed to isolate BRSV.Conclusion: Results suggest that these strains represent a putative new subgroup of BRSV with mutations observed in the immunodominant region of the G protein. However, further studies on these Brazilian BRSV strains should be performed to establish their pathogenic potential.

Neonatal diarrhea and rotavirus A infection in beef and dairy calves, Brazil, 2006-2015 / Diarreia neonatal e infecção por rotavírus A em bezerros de corte e leite, Brasil, 2006-2015

Calf diarrhea causes substantial economic losses in the cattle industry worldwide. Bovine rotavirus A (RVA) is the main viral agent that leads to enteric infection and diarrhea outbreaks in calves throughout the world. The aim of this retrospective (2006-2015) study was to determine the frequency of RVA detection in diarrheic fecal samples from beef and dairy calves from the three main cattle-producing regions of Brazil. Diarrheic fecal samples (n=1,498) of 124 beef and 56 dairy cattle herds from the Midwest, South, and Southeast geographical regions of Brazil were evaluated using the silver-stained polyacrylamide gel electrophoresis (ss-PAGE) technique. RVA double stranded-RNA was identified by the ss-PAGE technique in 410 (27.4%) fecal samples. The frequency of positive samples found in beef calves (31.9%; 328/1,027) was higher than the frequency found in diarrheic fecal samples from dairy calves (17.4%; 82/471). RVA infection was identified in calves from the three Brazilian geographical regions analyzed. However, the frequency of positive diarrheic calves in the Midwest region (39.4%), predominantly beef calves, was higher than in the South (19.4%) and Southeast (17.6%) regions. The temporal distribution of RVA-infected calves evaluated by two five-year periods (2006-2010, 24.5%; 2011-2015, 28.8%) demonstrated a very similar frequency of RVA in both periods. Considering the wide regional and temporal scope of this study, it can be concluded that RVA remains an important etiology of neonatal diarrhea in calves of Brazilian cattle herds.(AU)

A diarreia neonatal ocasiona perdas econômicas importantes na pecuária bovina em todo o mundo. Rotavírus A (RVA) é o principal agente etiológico viral de infecções entéricas e surtos de diarreia em bezerros de rebanhos de corte e leite. O objetivo deste estudo retrospectivo (2006-2015) foi determinar a frequência de detecção de RVA em amostras de fezes diarreicas de bezerros de corte e leite das três principais regiões produtoras de bovinos do Brasil. Amostras de fezes diarreicas (n=1.498) de 124 rebanhos bovinos de corte e 56 rebanhos bovinos de leite das regiões Centro-Oeste, Sul e Sudeste do Brasil foram avaliadas utilizando a técnica de eletroforese em gel de poliacrilamida (EGPA). O genoma segmentado de RVA foi identificado pela técnica de EGPA em 410 (27,4%) amostras de fezes. A frequência de amostras positivas encontrada em bezerros de rebanhos de corte (31,9%; 328/1.027) foi maior que a frequência identificada em amostras de fezes diarreicas de bezerros de rebanhos leiteiros (17,4%; 82/471). A infecção por RVA foi identificada em bezerros das três regiões geográficas brasileiras analisadas. No entanto, a frequência de bezerros com diarreia positivos para RVA na região Centro-Oeste (39,4%), predominantemente de bezerros de rebanhos de corte, foi maior que nas regiões Sul (19,4%) e Sudeste (17,6%). A distribuição temporal dos bezerros infectados com RVA avaliados por dois períodos de cinco anos (2006-2010, 24,5%; 2011-2015, 28,8%) demonstrou uma frequência muito semelhante em ambos os períodos. Considerando a amplitude regional e temporal deste estudo, pode-se concluir que RVA continua sendo uma importante etiologia de diarreia neonatal em bezerros de rebanhos bovinos brasileiros.(AU)

Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential / Pseudomonas spp. e outros micro-organismos psicrotróficos em queijos Minas Frescal inspecionados e não inspecionados: potencial proteolítico, lipolítico e produção de AprX

The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to microbial contamination and clandestine production and commercialization can pose a risk to consumer health. The storage of this fresh product under refrigeration, although more appropriate, may favor the growth of spoilage psychrotrophic bacteria. The objective of this study was to quantify and compare Pseudomonas spp. and other psychrotrophic bacteria in inspected and non-inspected MFC samples, evaluate their lipolytic and proteolytic activities and their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of the proteolytic and lipolytic potential of the isolates, and identification of potential producers of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts were 1.07 (±2.18) × 109CFU/g in the inspected samples and 4.5 (±5.86) × 108CFU/g in the non-inspected, with no significant difference (p=0.37). The average score of Pseudomonas spp. was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC samples, respectively, with no significant difference (p=0.1). Pseudomonas spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively. Collectively, 694 psychrotrophic strains and 47Pseudomonas spp. were isolated, of which 59.9% and 68.1% were simultaneously proteolytic and lipolytic, respectively. Of the 470 psychrotrophs isolated from inspected and 224 from non-inspected cheese samples, 5.74% and 2.23% contained aprX, respectively, while 100 and 86.96% of the Pseudomonas spp. isolates in inspected and non-inspected cheese samples contained the gene. The production potential of AprX did not, however, determine the proteolytic activity on plates of these isolates under the conditions evaluated in this study. Of total, 65.63% of the psychrotrophs that contained aprX gene were confirmed as Pseudomonas spp., using genus-specific PCR. Phylogenetic analysis of the 16S rRNA gene of the other psychrotrophs that were potential producers of AprX identified them as Serratia spp. (n=7), Raoultella ornithinolytica (n=1), and Acinetobacter schindleri (n=1) in the inspected samples and Psychrobacter sanguinis (n=1) and Leuconostoc mesenteroides (n=1) in the non-inspected samples. The production conditions of Brazilian MFC of these samples, while meeting the legal determinations, are not sufficient to control Pseudomonas and other spoilage-related psychrotrophs. Thus, stricter hygienic measures are required during the formal production of this type of cheese.(AU)

O mais consumido no Brasil, o queijo Minas Frescal (QMF) é altamente suscetível à contaminação microbiana e a produção e comercialização clandestina podem representar um risco para a saúde do consumidor. O armazenamento deste produto fresco sob refrigeração, embora mais apropriado, pode favorecer a multiplicação de bactérias psicrotróficas deteriorantes. O objetivo deste estudo foi quantificar e comparar Pseudomonas spp. e outras bactérias psicrotróficas em amostras de QMF inspecionadas e não inspecionadas, avaliar o potencial lipolítico, proteolítico e de produção de metaloprotease alcalina. Vinte amostras de QMF foram avaliadas: 10 inspecionadas e 10 não inspecionadas. Foram avaliadas as contagens de bactérias psicrotróficas e Pseudomonas spp., o potencial proteolítico e lipolítico dos isolados e a identificação de potenciais produtores de metaloprotease alcalina (AprX). A média total das contagens de bactérias psicrotróficas foi de 1,07 (±2,18) × 109UFC/g nas amostras inspecionadas e 4,5 (±5,86) × 108UFC/g nas não inspecionadas, sem diferença significativa (p=0,37). A média de Pseudomonasspp. foi de 6,86 (±18,6) × 105 e 2,08 (±3,65) × 106UFC/g para as amostras QMF inspecionadas e não-inspecionadas, respectivamente, sem diferença significativa (p=0,1). Pseudomonas spp. representaram 0,06% e 0,004% de bactérias psicrotróficas encontradas em amostras QMF inspecionadas e não-inspecionadas, respectivamente. Das amostras inspecionadas e não inspecionadas, foram isoladas 694 colônias psicrotróficas e 47 Pseudomonasspp., dos quais 59,9% e 68,1% foram simultaneamente proteolíticos e lipolíticos, respectivamente. Dos 470 isolados de psicrotróficos das amostras de queijo inspecionados e dos 224 isolados das não inspecionadas, 5,74% e 2,23% continham o gene aprX, respectivamente, enquanto 100 e 86,96% das Pseudomonasspp. isoladas em amostras de queijo inspecionadas e não inspecionadas continham o potencial de expressão de AprX. Esse potencial, no entanto, não determinou a atividade proteolítica em placas desses isolados nas condições avaliadas neste estudo. Do total, 65,63% dos psicrotróficos que continham o gene aprX foram confirmados como Pseudomonasspp., utilizando PCR gênero-específico. A análise filogenética do gene 16S rRNA dos outros psicrotróficos que foram produtores potenciais de AprX os identificou como Serratia spp. (n=7), Raoultella ornithinolytica (n=1) e Acinetobacter schindleri (n=1) nas amostras inspecionadas e Psychrobacter sanguinis (n=1) e Leuconostoc mesenteroides (n=1) nas amostras não inspecionadas. As condições de produção do QMF dessas amostras, atendendo às determinações legais, não são suficientes para controlar a Pseudomonas e outros psicrotróficos relacionados à deterioração. Assim, medidas higiênicas mais rígidas são necessárias durante a produção formal deste tipo de queijo.(AU)