Transplacental Infections Associated with Macavirus in Aborted Bovine Fetuses.

The Macavirus genus, Gammaherpesvirinae subfamily, Herpesviridae family, contains ovine gammaherpesvirus 2 (OvGHV2), the cause of sheep-associated malignant catarrhal fever (SA-MCF). Members of the Macavirus genus associated with the development of malignant catarrhal fever (MCF) in their respective hosts share the 15A antigenic epitope, are conserved within the DNA polymerase gene and are collectively referred to as the malignant catarrhal fever virus (MCFV) complex. The ability of MCFV and/or OvGHV2 to produce abortions in ruminants is currently unknown, with little documentation of infections by these agents in bovine fetuses. This report presents the findings observed due to the detection of OvGHV2 DNA and MCFV tissue antigens in aborted bovine fetuses from southern Brazil. Four aborted bovine fetuses from three farms, located in a geographical region of Paraná State with elevated immunohistochemical (IHC) prevalence of MCFV tissue antigens, with gestational ages varying between 78 to 208 days were investigated. Significant gross and histopathological alterations were not observed in any of these fetuses. An IHC assay using the 15A-monoclonal antibody (15A-MAb), which is based on the 15A antigenic epitope of Macavirus, identified MCFV tissue antigens in multiple organs from two fetuses (#1 and #4); however, positive immunoreactivity to the 15A-MAb IHC assay was not detected in Fetus #2 and #3. Molecular testing amplified OvGHV2 DNA only from the myocardium and lungs of Fetus #1 that had positive intracytoplasmic immunoreactivity to the 15A-MAb IHC assay in these tissues. Furthermore, infections by Leptospira spp. were confirmed by molecular assays in fetuses #1, #3, and #4, while PCR detected Neospora caninum in the myocardium of Fetus #2. Additionally, molecular assays to identify well-known fetopathy agents of cattle, including bovine viral diarrhea virus, bovine alphaherpesvirus 1, Histophilus somni, and Listeria monocytogenes, did not amplify the nucleic acids of these pathogens. PCR assays to identify bovine gammaherpesvirus 6 (BoGHV6), another Macavirus known to infect cattle in Brazil, were unsuccessful. These findings confirmed that the 15A-MAb IHC assay can be efficiently used to detect MCFV antigens in organs of aborted bovine fetuses. The identification of MCFV antigens with the simultaneous detection of OvGHV2 DNA confirmed that Fetus #1 was infected by OvGHV2 and added to the few descriptions of this infection in aborted fetuses of ruminants worldwide. Moreover, the IHC detection of MCFV in multiple organs of Fetus #4, without the molecular detection of OvGHV2 or BoGHV6, may suggest that this fetus was infected by a Macavirus that was not previously diagnosed in cattle herds from Brazil. These findings strongly suggest that OvGHV2 and MCFV can produce transplacental infections in cattle.

Respiratory illness in young and adult cattle caused by bovine viral diarrhea virus subgenotype 2b in singular and mixed bacterial infection in a BVDV-vaccinated dairy herd.

Bovine respiratory disease (BRD) is a common global health problem in dairy cattle. The definitive diagnosis of BRD is complex because its etiology involves several predisposing and determining factors. This report describes the etiology of a BRD outbreak in a dairy herd in the mesoregion of Central Eastern Paraná, which simultaneously affected young (calves and heifers) and adult (cows) Holstein-Friesian cattle. Nine biological samples, consisting of five lung samples from two cows and three suckling calves, and four nasal swab samples from heifers, were used for etiological diagnosis. The nucleic acids extracted from lung fragments and nasal swabs were subjected to PCR and RT-PCR assays for partial amplification of the genes of five viruses [bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoAHV1), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (BPIV-3), and bovine coronavirus (BCoV)] and four bacteria (Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) involved in the etiology of BRD. All nine biological samples from the animals with BRD tested negative for BoAHV1, BRSV, BPIV-3, BCoV, and H. somni. Therefore, the involvement of these microorganisms in the etiology of BRD outbreak can be ruled out. It was possible to identify the presence of BVDV and M. bovis in singular and mixed infections of the lower respiratory tract in cattle. BVDV was also identified in two nasal swabs: one as a single etiological agent and the other in association with two bacteria (P. multocida and M. haemolytica). The phylogenetic analysis conducted in the nucleotide sequence of the 5'UTR region and Npro gene of the BVDV amplicons demonstrated that the BVDV field strains of this BRD outbreak belong to subgenotype 2b. To the best of our knowledge, this is the first report of BVDV-2b involvement in the etiology of BRD in Brazil. Finally, it is necessary to highlight that the cattle were obtained from an open dairy herd with biannual vaccinations for BVDV-1a and - 2a.

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.

Serological Profile for Major Respiratory Viruses in Unvaccinated Cows from High-Yielding Dairy Herds.

This study aims to determine the serological profile of high-yielding dairy cows for four main viruses (bovine alphaherpesvirus 1 (BoAHV1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV3), and bovine respiratory syncytial virus (BRSV)) related to bovine respiratory disease (BRD) in cattle herds worldwide. In this survey, 497 blood serum samples were collected from non-vaccinated dairy cows without clinical respiratory signs in 39 herds in the central-eastern mesoregion of Paraná State, South Brazil. The presence of neutralizing antibodies was determined by virus neutralization (VN) tests. VN antibodies against BoAHV1, BVDV, BPIV3, and BRSV were detected in 355 (71.4%), 280 (56.3%), 481 (96.8%), and 315 (63.4%) serum samples, respectively. The frequencies of seropositive herds for BoAHV1, BVDV, BPIV3, and BRSV were 79.5 (n = 31), 82.0 (n = 32), 100 (n = 39), and 84.6% (n = 33), respectively. The frequencies of seropositive cows varied according to the type of herd management and the number of cows in the herd. The detection of VN antibodies in unvaccinated dairy cattle herds demonstrated the endemic circulation of the four viruses in the herds evaluated. For BRD prevention, it is recommended to implement a vaccination program for cows that provides passive immunity in calves and active immunity in cows.

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.

Outbreak of persistently infected heifer calves with bovine viral diarrhea virus subgenotypes 1b and 1d in a BVDV-vaccinated open dairy herd.

Bovine viral diarrhea virus (BVDV) infection has a significant economic impact on beef and dairy industries worldwide. Fetal infection with a non-cytopathic strain may lead to the birth of persistently infected (PI) offspring, which is the main event in the epidemiological chain of BVDV infection. This report describes the birth of 99 BVDV-PI heifer calves within 52 days of birth in a regular BVDV-vaccinated Brazilian dairy cattle herd and the subgenotypes of the infecting field strains. This study was conducted in a high-yielding open dairy cattle herd that frequently acquired heifers from neighboring areas for replacement. The farm monitors the birth of PI calves by screening all calves born using an ELISA (IDEXX) for BVDV antigen detection. All calves aged 1-7 days were evaluated. For positive and suspected results, the ELISA was repeated when the calves were close to one month old. A total of 294 heifer calves were evaluated between February and March 2021. Of these, 99 (33.7 %) had positive ELISA results and were considered PI calves. To evaluate the predominant BVDV species and subgenotypes in this outbreak, whole blood samples were collected from 31 calves born during the study period. All samples were submitted to the RT-PCR assay for the partial amplification of the BVDV 5'-UTR region, and these amplicons were subjected to nucleotide sequencing. Phylogenetic analysis identified BVDV-1b and BVDV-1d in 16 and 13 heifer calves, respectively. In two calves, it was not possible to determine the BVDV-1 subgenotype. Detection of PI animals and monitoring of circulating BVDV subgenotype strains are central to disease control. This study shows that regular BVDV vaccination alone may be insufficient to prevent BVDV infection in high-yielding open dairy cattle herds. Other biosecurity measures must be adopted to avoid the purchase of cattle with acute infections by BVDV or BVDV-PI, which can cause a break in the health profile of the herd and economic losses.

Serological Survey for Three Canine Viruses in Brazilian Wild Carnivores : Antibodies Against Canine Viruses in Wild Carnivores.

We evaluated the presence of antibodies against CaHV-1, CDV, and CPV-2 in serum samples from Brazilian wild carnivore species. Nine maned wolves and six crab-eating foxes were tested for CaHV-1 and CDV by virus neutralization test and CPV-2 by hemagglutination inhibition assay. Antibodies to CaHV-1, CDV, and CPV-2 were detected in serum samples of 1 (6.7%), 5 (33.3%), and 10 (66.7%) wild carnivores, respectively. Two maned wolves and one crab-eating fox were seropositive simultaneously for CDV and CPV-2. Antibodies against all viruses were detected in one crab-eating fox. This is the first report of CaHV-1 antibody detection in crab-eating foxes.

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.

Serological profile of respiratory viruses in unvaccinated steers upon their arrival at Brazilian feedlot facilities.

Bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoAHV1), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza virus 3 (BPIV-3) are involved in bovine respiratory disease. These viruses can infect the respiratory system and cause considerable economic losses to beef and dairy cattle herds. This study aimed to determine the serological profiles of steers for BVDV, BoAHV1, BRSV, and BPIV-3 upon their arrival at Brazilian feedlot facilities. A total of 1,282 serum samples from unvaccinated steers were obtained on the first day of feeding. Samples were collected from 31 beef cattle herds reared in an extensive rearing system in six Brazilian states. Antibodies against BVDV, BoAHV1, BRSV, and BPIV-3 were detected using a virus neutralization test. The steers were distributed in agreement with their age and the Brazilian state of origin. The highest seropositivity was for BoAHV1 and BPIV-3 at 92.1% (1,154/1,253) and 86.6% (1,100/1,270), respectively. The seropositivity of BRSV was 77.1% (959/1,244). BVDV presented a lower rate, at slightly more than 50% (51.8%; 656/1,266). Age was a risk factor for the presence of antibodies against BVDV, BoAHV1, and BPIV-3 but not BRSV. A positive correlation was identified between BoAHV1 and BPIV-3 (P = 0.85) and between BRSV and BPIV-3 (P = 0.47). The high rate of seropositive steers for these four respiratory viruses on the first day of confinement identified in this serological survey provides important epidemiological information on respiratory infections, as the seropositivity of the four main bovine respiratory viruses in Brazilian beef cattle herds in an extensive rearing system.

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.

Presence of antibodies against Mycobacterium avium subspecies paratuberculosis in Brazilian high-producing dairy herds.

This study aimed to determine the presence of antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) in high-producing dairy cows, the presence of the pathogen in the feces, and the risk factors associated with the disease. Blood and fecal samples were collected from 708 dairy cows over 2 years from 54 herds located in five municipalities of Paraná, Brazil. The serum samples were evaluated for the presence of antibodies against MAP using enzyme-linked immunosorbent assay (ELISA). Fecal samples from 100 cows (69 seropositive and 31 seronegative) were assessed using real-time PCR (qPCR) for IS900 of MAP. The herd prevalence of antibodies against MAP was 61.1% (33/54; 95% CI 46.88-74.08), ranging from 12.5 to 80% across the municipalities, and the prevalence in the animals was 9.8% (69/708; 95% CI 7.77-12.15); it ranged from 0 to 87.5% per herd. Only one of the 69 (1.45%) fecal samples from the seropositive cows was positive for the qPCR. The factors associated with the occurrence of paratuberculosis in herds were the use of compost barn system and the type of bed, whereas only the type of bed was associated with the infection of cows. The only risk factor (OR = 2.45; 95% CI 1.03-5.85) associated with the occurrence of paratuberculosis was the introduction of animals purchased from other dairy farms. The prevalence of active infection was low; however, our results demonstrate the presence of MAP in high-producing dairy herds in Paraná state, Brazil.

Infectious Disease Agents Associated with Pulmonary Alterations in Aborted Bovine Fetuses.

This study investigated the occurrence of selected pathogens of bovine respiratory disease in fetal pulmonary tissue of cattle and associated these with patterns of disease. Fetal pulmonary (n = 37) tissues were evaluated by histopathology; immunohistochemical assays identified intralesional antigens of bovine alphaherpesvirus 1 (BoAHV1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and Mycoplasma bovis. Molecular assays were performed to amplify reproductive disease pathogens and bovine gammaherpesvirus 6 (BoGHV6) from 12 lungs. The 2 patterns of pulmonary diseases were interstitial pneumonia (12/37) and suppurative bronchopneumonia (1/37). The frequency of the intralesional antigens identified was BRSV (16.2%; 6/37), BVDV (13.5%; 5/37), BoAHV1 (8.1%; 3/37), M. bovis (5.4%; 2/37), and BPIV-3 (2.7%; 1/37). Interstitial pneumonia was associated with BRSV (n = 3), BoAHV1 (n = 3), and BVDV (n = 2); suppurative bronchopneumonia contained a Gram-positive bacterium and BVDV and BRSV. Reproductive pathogens detected included Leptospira spp., (n = 3), BVDV, Neospora caninum, and Brucella abortus (n = 2). BoGHV6 DNA was identified in the lungs of two fetuses with interstitial pneumonia. These findings suggest that these fetuses were infected transplacentally by several pathogens. The role of some of these pathogens herein identified must be further elucidated in the possible participation of fetal disease.

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.

Congenital Neurological Disease Associated With HoBi-like Pestivirus Infection in a Newborn Dairy Calf From Brazil.

HoBi-like pestivirus (HoBiPeV) has been reported in several biological samples from cattle worldwide, but there are no descriptions of this virus associated with neurological symptoms. This report described the first occurrence of neurological disease associated with HoBiPeV in a newborn dairy calf. A mixed-breed Holstein calf had severe neurological symptoms at birth and died at 21 days old. The tissue fragments (central nervous system (CNS), myocardium, liver, kidney, lung, intestine, and spleen) were submitted to reverse transcription (RT)-PCR assay for the partial 5'-untranslated region (5'UTR) and N-terminal autoprotease (Npro) gene of the pestivirus genome, and the CNS tissue fragments were submitted to histopathological and immunohistochemical evaluation. The RT-PCR assay indicated that the kidney, CNS, and intestinal tissue fragments were positive for the pestivirus 5'UTR, and the CNS and intestinal tissue fragments were positive for the pestivirus Npro gene. Amplicons with high DNA quantification in the 5'UTR (CNS-cerebral cortex) and Npro (CNS-cerebral cortex and intestine) RT-PCR assays were sequenced. The nucleotide (nt) sequence and phylogenetic analysis of the 5'UTR strain exhibited 93.6 to 99.4%, 85%, 89.4 to 89.9%, 85.1%, and 90.5 to 91.5% nt identity with HoBiPeV strains from clades a, b, c, d, and e, respectively. The Npro amplicons showed 99.7% nt identity to each other and 90.4 to 96.5%, 85.1 to 85.3%, 79.2 to 79.7%, and 85.8 to 86.5% nt identity with HoBiPeV strains from clades a, c, d, and e, respectively. A histopathology revealed neuronal necrosis at the cerebrum, cerebellum, and brain stem. An immunohistochemical assay designed to identify antigens of bovine viral diarrhea virus revealed positive intracytoplasmic immunoreactivity within neurons at the cerebral cortex, cerebrum, cerebellum, and spinal cord. Thus, this report provides information about the first identification of HoBiPeV in tissues of the CNS in a newborn dairy calf with neurological symptoms.

Leptospira spp. in Naturally Infected Dairy Cow from a Brazilian Border Region.

Leptospirosis is an important infectious disease, which can generate large economic losses, especially in the dairy herd. The pathogen that causes this disease may have its entry in Brazilian herds facilitated by the existence of a large extension of land borders. Therefore, the objective of this work was to investigate the presence of DNA and antibodies against Leptospira spp. in samples of vaginal mucus and serum from naturally infected bovine females from small rural dairy farms in a border region. Blood and vaginal mucus samples were collected from 70 Holstein cows, from small rural dairy farms between October 2017 and June 2018. The inclusion criteria for dairy cattle of any breed were aged over 2 years, not vaccinated against leptospirosis, and presenting a history of any reproductive problem such as abortion, stillbirth, repetition of heat, absence of heat, and lack of conception. Blood was collected by puncturing the coccygeal vein; for the collection of vaginal mucus, it was necessary to use a tampon with an applicator. For the detection of anti-Leptospira spp. antibodies, the sera were submitted to microscopic agglutination test (MAT) and, for DNA detection, the vaginal mucus was submitted to the PCR technique. Among the 70 cows, 42.86% had reagents in MAT and the most likely serovar was Wolffi (43.47%). In 74.28% of the vaginal mucus samples, it was possible to amplify the Leptospira spp. DNA. The results of this work show the presence of Leptospira spp. antibodies and DNA in samples of serum and vaginal mucus from naturally infected bovine females from small rural dairy farms in a border region (Brazil × Paraguay). These results demonstrate the importance of considering bovine females as potential vaginal carriers of Leptospira spp. Thus, it highlights the importance of further studies to better understanding of this issue, in addition to carrying out molecular and serological tests, to monitor the infection and further characterize epidemiological studies of leptospirosis in herds from regions that face this international frontier challenge.

Gender effects on pork quality and calpain-1 and calpastatin gene expression in male pig muscle.

Studies that investigate the expression of genes related to the tenderness of meat from entire and immunocastrated male pigs have not yet been performed. The objective of this study was to investigate the association between gender (entire male and immunocastrated) and the meat quality of pigs, as well as to quantify the expression of calpain-1 and the calpastatin gene. Regarding carcass measurements and meat quality, boars presented lower values of muscle depth (P = 0.028), subcutaneous fat thickness (P = 0.046), L* value (P = 0.004) and cook loss (P = 0.008) than the immunocastrated pigs. The boars presented greater calpain-1 gene expression (P = 0.006) and lower calpastatin gene expression (P = 0.003) than immunocastrated pigs. This study shows that combined with other factors the gene expression can contribute to a tender meat from boars due to their higher calpain-1 expression and lower calpastatin expression than those of immunocastrated male pigs.

IMMUNOHISTOCHEMICAL AND MOLECULAR EVIDENCE OF PUTATIVE NEORICKETTSIA INFECTION IN COATIS ( NASUA NASUA) FROM SOUTHERN BRAZIL.

The pathologic, molecular, and immunohistochemical findings associated with Neorickettsia helminthoeca are described in coatis ( Nasua nasua). Tissue sections (small intestine, lungs, kidney, liver, and spleen) of coatis ( n = 3) that died at the Bela Vista Biological Refuge, Foz do Iguaçu, Paraná, southern Brazil were routinely processed from histopathology. Selected formalin-fixed paraffin-embedded (FFPE) tissue sections of the small intestine, lungs, and spleen were used in an immunohistochemical (IHC) assay designed to identify the antigens of N. helminthoeca. Additionally, FFPE tissue sections of the small intestine were used to demonstrate antigens of canine parvovirus-2 (CPV-2) by IHC. Histopathology revealed chronic enteritis in all coatis. Parasitic enteritis was diagnosed in two coatis; one of these contained examples of a trematode within the lumen of the small intestine and the ovum of a trematode encysted in the intestinal mucosa. Other significant pathologic findings included interstitial pneumonia ( n = 2) and pyogranulomatous splenitis ( n = 1). Positive immunolabeling for N. helminthoeca was identified within macrophages of the small intestine and reticuloendothelial cells within the germinal centers of the spleen of all coatis; the intestinal trematode was N. helminthoeca IHC-positive. All pulmonary sections revealed negative immunolabeling for N. helminthoeca. Furthermore, the antigens of CPV-2 were not identified in the intestine of any coati. These findings indicate that these coatis were infected by N. helminthoeca, but since clinical and gross pathological findings were not recorded, it is uncertain if this pathogen produced clinical disease in this canid host; therefore, coatis may be asymptomatic or dead-end hosts for this organism.

Bovine viral diarrhea virus subgenotype 1b in water buffalos (Bubalus bubalis) from Brazil.

Serological studies have characterized the presence of the bovine viral diarrhea virus (BVDV) infection in water buffalo herds worldwide. However, the molecular characterization of BVDV strains circulating in this animal species is uncommon. The aim of this study was to identify young water buffalo with acute infection and characterize the subgenotype of the infecting wild-type BVDV strain. Two dairy water buffalo herds from Northeastern Brazil were selected based on the results of virus neutralization test which showed high titers of anti-BVDV antibodies. To identify viremic animals, the BVDV RNA was assessed by RT-PCR assay in serum samples from 44 asymptomatic young water buffalos, where 31 serum samples from herd A and 13 from herd B. Amplicons with 288 bp of BVDV 5'UTR region were obtained in 7 (15.9%) serum samples (herd A, n = 5; herd B, n = 2). One good-quality amplicon from each herd was selected for nucleotide sequencing. The phylogenetic analysis demonstrated that the two BVDV wild-type strains clustered with BVDV strains of the subgenotype 1b. This study identified for the first time the active infection by BVDV subgenotype 1b in two dairy water buffalo herds from Brazil. These results highlight the importance of that, as well as in cattle herds, also in water buffalo herds prophylaxis measures to control BVDV infection should be intensified, mainly because these species clearly coexist in buffalo farms within Brazil.

Virus neutralization technique as a tool to evaluate the virological profile for bovine viral diarrhea virus infection in dairy water buffalo (Bubalus bubalis) herds.

Serological evidence shows that the bovine viral diarrhea virus (BVDV) infections are present in Brazilian dairy and beef water buffalo herds. As few reports describe the BVDV infection profile the aim of this study was to evaluate the dynamics of BVDV circulation in Brazilian dairy water buffalo herds through analysis of the seropositivity rate and the titer range of anti-BVDV neutralizing antibodies in a group of animals that are considered sentinels. Blood samples (n = 305) were obtained from unvaccinated, asymptomatic young water buffalos from four dairy herds randomly identified as A (n = 106), B (n = 62), C (n = 119), and D (n = 18). The detection and titration of anti-BVDV neutralizing antibodies were evaluated by the virus neutralization test according to the World Organization for Animal Health. Analysis of the results revealed two distinct epidemiological conditions. The first is represented by herds A and C where high rates of seropositive animals (A = 39.6%; C = 51.3%) and high and very variable antibodies titers suggested active BVDV infection. The other condition is represented by herds B and D with low rates of seropositive animals (B = 8.1%; D = 11.1%) and low and little variable antibodies titers suggesting an epidemiological condition of infection stability. Some variables were observed in herds with a distinct BVDV infection profile. Herds with active infection were big, open herds, and had more management practices. In contrast, the herds with infection stability were small, closed herds with few management practices. These results highlight the importance of evaluation, monitoring, and control of BVDV infection also in dairy water buffalo herds.

Serological and molecular investigation of viral agents in freeliving jaguars of the Pantanal wetlands, state of Mato Grosso, Brazil / Investigação sorológica e molecular de agentes virais em onças-pintadas de vida livre no Pantanal de Mato Grosso, Brasil

This study investigates the exposure of free-living jaguars from two federal protected areas in the Pantanal of Mato Grosso, Brazil, to a variety viral agents. These viral agents, particularly causing zoonotic diseases, were analyzed using serological and molecular methods. None of the jaguars was positive by RT-PCR for the molecular detection of avian influenza and West Nile Fever (WNF). Only one animal was serologically positive for Eastern Equine Encephalitis (EEE) by virus neutralization test in VERO cell cultures, representing the first reported case of jaguar exposure to EEE virus. However, all the animals were negative for Western Equine Encephalitis (WEE) virus and Venezuelan Equine Encephalitis (VEE) virus. Eleven jaguars were tested by two tests for the detection of antibodies against rabies virus (Simplified Fluorescent Inhibition Microtest ­ SFIMT and Rapid Fluorescent Focus Inhibition Test ­ RFFIT), resulting in five positive animals, two animals in each test and one in both serological tests. Furthermore, three out of 14 samples subjected to the neutralization test were positive for antibodies against canine distemper virus (CDV), and 15 out of 17 samples subjected to the hemagglutination-inhibition test (HI) were positive for antibodies against canine parvovirus (CPV). In view of the findings of this study, it is unlikely that the viruses examined here represent a threat to the jaguar populations in this region.(AU)

Este estudo investigou a exposição de onças-pintadas de vida livre a agentes virais selecionados em duas unidades de conservação federais no Pantanal de Mato Grosso, Brasil. Para a análise desses agentes virais, a maioria de caráter zoonótico, foram utilizados métodos sorológicos e moleculares. Nenhuma das onze onças-pintadas examinadas foi positiva na técnica de real-time RT-PCR para a detecção molecular dos agentes da Influenza aviária e Febre do Nilo Ocidental (WNF). Somente um animal foi positivo sorologicamente para a o vírus da Encefalite Equina do Leste (EEE) pela Microtécnica de vírus neutralização em culturas de células VERO, sendo este o primeiro relato da exposição de onças-pintadas. Todos os animais examinados s foram negativos para o vírus da Encefalite Equina do Oeste (WEE) e Venezuelana (VEE). Amostras de soro colhidas de 11 onças-pintadas foram submetidas a adois testes distintos para a detecção de anticorpos contra o vírus da raiva (Teste Rápido de Inibição de Foco de Fluorescência ­ RFFIT e Microteste Simplificado de Inibição da Fluorescência - SFIMT), resultando em cinco animais positivos, dos quase dois positivos para cada teste e um positivo quando submetido aos dois testes sorológicos. Além disso, três das 14 amostras submetidas a técnica de soroneutralização foram positivas para a pesquisa de anticorpos contra o vírus da cinomose (CDV) e 15 amostras positivas das 17 analisadas para a pesquisa de anticorpos contra o parvovírus canino (CPV) foram identificadas pela técnica de Inibição da Hemaglutinação (HI). De acordo com os resultados deste estudo, é pouco provável que os agentes virais aqui analisados representem ameaça à população de onçaspintadas nesta região.(AU)