Ana Carolina Diniz Matos

Bovine vaccinia, a systemic infection: Evidence of fecal shedding, viremia and detection in lymphoid organs

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that affects dairy cattle and milkers, causing economic losses and impacting animal and human health. Based on the clinical presentation, BV appears to be a localized disease, with lesions restricted to the skin of affected individuals. However, there are no studies on the pathogenesis of the disease in cows to determine if there is a systemic spread of the virus and if there are different ways of VACV shedding. The objective of this work was to study if there is a systemic spread of VACV in experimentally infected cows and to study the kinetics of VACV circulation in the blood and shedding in the feces of these animals. To this end, eight crossbred lactating cows were used. Three teats of each cow were inoculated with the GP2V strain of VACV. All animals were monitored daily, and blood and fecal samples were collected for 67 days post-infection (dpi). After this period, four of these previously infected cows were immunosuppressed using dexamethasone. Viral DNA was continuously detected and quantified in the blood and feces of these animals in an intermittent way, even after the resolution of the lesions. At slaughter, tissues were collected, and viral DNA was detected and quantified in the mesenteric and retromammary lymph nodes, ileum, spleen and liver. The detection of VACV DNA in the feces for a longer period (67 dpi) and in the lymphatic organs provides new evidence about VACV elimination and suggests that BV could be a systemic infection with a chronic course and viral shedding through the feces.

Clinical, hematological and biochemical parameters of dairy cows experimentally infected with Vaccinia virus

Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an important zoonosis that affects dairy cattle. There are many aspects of the disease that remain unknown, and aiming to answer some of these questions, the clinical, hematological, and biochemical parameters of VACV experimentally infected cows were evaluated. In the first part of the study, lactating cows were infected with VACV-GP2 strain. In the second part, animals previously infected with VACV-GP2 were divided into two treatment groups: Group 1, immunosuppressed cows; and Group 2, re-infected cows. In this study, BV could be experimentally reproduced, with similar lesions as observed in natural infections. Moreover, a short incubation period and local lymphadenopathy were also observed. VACV could be detected by PCR and isolated from scabs taken from teat lesions of all inoculated and re-inoculated animals. Lymphocytosis and neutrophilia were observed in all animals from the first part of the experiment, and lymphopenia and relative neutrophilia were observed in the immunosuppressed animals. Detection of viral DNA in oral mucosa lesions suggests that viral reactivation might occur in immunosuppressed animals. Moreover, clinical disease with teat lesions may occur in previously VACV-infected cows under the experimental conditions of the present study.

Detection of Vaccinia virus in blood and faeces of experimentally infected cows.

Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus (VACV), affects dairy cattle and milkers, causing economic, veterinary and human health impacts. Despite such impacts, there are no experimental studies about the pathogenesis of BV in cows to assess whether there is a systemic spread of the virus and whether there are different ways of VACV shedding. Trying to answer some of these questions, a study was proposed using experimental inoculation of VACV in cows. All experimentally infected cows developed lesions compatible with VACV infection in cattle. Two of the six animals presented VACV DNA in blood and faecal samples, starting at the 2nd and the 3rd day post-infection (d.p.i.), respectively, and lasting until the 36th d.p.i., in an intermittent way. This study provides new evidence that VACV can be detected in blood and faeces of infected cows, suggesting that BV could be a systemic disease, and also bringing new information about the epidemiology and pathogenesis of BV.

Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model.

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 107 PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20th and 30th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit without exhibiting any clinical signs.

Short communication: Survival of Vaccinia virus in inoculated cheeses during 60-day ripening

Bovine vaccinia is a neglected zoonosis caused by Vaccinia virus (VACV) and has a major economic and public health effect in Brazil. Previous studies showed infectious VACV particles in milk from either experimentally or naturally infected cows and in fresh cheeses prepared with experimentally contaminated milk. Ripening is a process that leads to major changes in the physical and chemical characteristics of cheese, reducing contamination by spoilage, pathogenic microorganisms, or both. However, it is not known if VACV infectious particles persist after the ripening process. To investigate this issue, viral infectivity at different ripening times was studied in cheeses manufactured with milk experimentally contaminated with VACV strain Guarani P2 (GP2). Cheeses were analyzed at 1, 7, 14, 21, 45, and 60 d of ripening at 25°C. Viral DNA was quantified by real-time PCR, and VACV isolation and titration were performed in Vero cells. The whole experiment was repeated 4 times. Analysis of the mean viral DNA quantification and infectivity indicated a reduction of approximately 2 logs along the ripening process; however, infectious viral particles (1.7 × 102 pfu/mL) could still be recovered at d 60 of ripening. These findings indicate that the ripening process reduces VACV infectivity, but it was not able to inactivate completely the viral particles after 60 d.

Identification of bluetongue virus serotypes 1, 4, and 17 co-infections in sheep flocks during outbreaks in Brazil

Bluetongue (BT) is a vector-borne viral disease caused by the Bluetongue virus (BTV), an Orbivirus from the Reoviridae family, affecting domestic and wild ruminants. BTV circulation in Brazil was first reported in 1978, and several serological surveys indicate that the virus is widespread, although with varied prevalence. In 2014, BT outbreaks affected sheep flocks in Rio Grande do Sul state, causing significant mortality (18.4%; 91/495) in BTV-infected sheep. In total, seven farms were monitored, and one or two sheep from each farm that died due to clinical signs of BT were necropsied. Apathy, pyrexia, anorexia, tachycardia, respiratory, and digestive disorders were noted. Additionally, an abortion was recorded in one of the monitored farms. The main gross lesions observed were pulmonary edema, anterior-ventral pulmonary consolidation, muscular necrosis in the esophagus and in the ventral serratus muscle, and hemorrhagic lesions in the heart. The blood and tissue samples were tested for BTV RNA detection by RT-qPCR targeting the segment 10. Positive samples were used for viral isolation. The isolated BTVs were typed by conventional RT-PCR targeting the segment 2 of the 26 BTV serotypes, followed by sequencing analysis. BTV-1, BTV-4 and BTV-17 were identified in the analyzed samples. Double or triple BTV co-infections with these serotypes were detected. We report the occurrence of BT outbreaks related to BTV-1, BTV-4 and BTV-17 infections and co-infections causing clinical signs in sheep flocks in Southern Brazil, with significant mortality and lethality rates.

West Nile virus associated with equid encephalitis in Brazil, 2018

Mosquito-borne arboviruses are a major public health concern worldwide and are responsible for emerging and re-emerging diseases. Taken together, the arboviruses have a strong impact on public health and are the most common causes of equine encephalitis. In-depth diagnostic investigation of equine viral encephalitis is of utmost importance for the epidemiological surveillance and control of this disease. Regarding neurological disorders in equids, in April-May 2018, at least 12 cases of equid mortality with acute neurological signs were reported in six farms from Espirito Santo state, Brazil. To investigate the aetiological agent of this neurological disease outbreak, central nervous system (CNS) fragments from two horses and two donkeys were submitted for virologic diagnosis. Rabies, equine herpesvirus-1, and arbovirus-associated encephalomyelitis were investigated using differential diagnosis techniques. West Nile virus (WNV) was detected by nested RT-PCR in CNS fragments from each of the four animals in the study and confirmed by nucleotide sequencing. This is the first case of neurological disease in equids confirmed to be associated with WNV infection in Brazil. This finding unveils a new and urgent field of research and the need to understand the epidemiological and clinical characteristics of the disease and the risk to public health.

Serological Evidence of Orthopoxvirus Circulation Among Equids, Southeast Brazil

Since 1999 Vaccinia virus (VACV) outbreaks involving bovines and humans have been reported in Brazil; this zoonosis is known as Bovine Vaccinia (BV) and is mainly an occupational disease of milkers. It was only in 2008 (and then again in 2011 and 2014) however, that VACV was found causing natural infections in Brazilian equids. These reports involved only equids, no infected humans or bovines were identified, and the sources of infections remain unknown up to date. The peculiarities of Equine Vaccinia outbreaks (e.g., absence of human infection), the frequently shared environments, and fomites by equids and bovines in Brazilian farms and the remaining gaps in BV epidemiology incited a question over OPV serological status of equids in Brazil. For this report, sera from 621 equids - representing different species, ages, sexes and locations of origin within Minas Gerais State, southeast Brazil – were examined for the presence of anti-Orthopoxvirus (OPV) antibodies. Only 74 of these were sampled during an Equine Vaccinia outbreak, meaning some of these specific animals presented typical lesions of OPV infections. The majority of sera, however, were sampled from animals without typical signs of OPV infection and during the absence of reported Bovine or Equine Vaccinia outbreaks. Results suggest the circulation of VACV among equids of southeast Brazil even prior to the time of the first VACV outbreak in 2008. There is a correlation of OPVs outbreaks among bovines and equids although many gaps remain to our understanding of its nature. The data obtained may even be carefully associated to recent discussion over OPVs history. Moreover, data is available to improve the knowledge and instigate new researches regarding OPVs circulation in Brazil and worldwide.