Claudia Del Fava

Analysis of the long control region of bovine papillomavirus type 1 associated with sarcoids in equine hosts indicates multiple cross-species transmission events and phylogeographical structure.

Papillomaviruses are a family of slowly evolving DNA viruses and their evolution is commonly linked to that of their host species. However, whilst bovine papillomavirus-1 (BPV-1) primarily causes warts in its natural host, the cow, it can also cause locally aggressive and invasive skin tumours in equids, known as sarcoids, and thus provides a rare contemporary example of cross-species transmission of a papillomavirus. Here, we describe the first phylogenetic analysis of BPV-1 in equine sarcoids to our knowledge, allowing us to explore the evolutionary history of BPV-1 and investigate its cross-species association with equids. A phylogenetic analysis of the BPV-1 transcriptional promoter region (the long control region or LCR) was conducted on 15 bovine and 116 equine samples from four continents. Incorporating previous estimates for evolutionary rates in papillomavirus implied that the genetic diversity in the LCR variants was ancient and predated domestication of both equids and cattle. The phylogeny demonstrated geographical segregation into an ancestral group (African, South American and Australian samples), and a more recently derived, largely European clade. Whilst our data are consistent with BPV-1 originating in cattle, we found evidence of multiple, probably relatively recent, cross-species transmission events into horses. We also demonstrated the high prevalence of one particular sequence variant (variant 20), and suggest this may indicate that this variant shows a fitness advantage in equids. Although strong host specificity remains the norm in papillomaviruses, our results demonstrate that exceptions to this rule exist and can become epidemiologically relevant.

Ocorrência de anticorpos anti-Neospora caninum em cães da microrregião da Serra de Botucatu, Estado de São Paulo, Brasil

Neosporosis is a parasitic disease caused by a protozoon Neospora caninum, which is recognized as an important cause of bovine abortion and canine neuropathy. Considering the isolation of N. caninum and the frequent serology in bovines of our region, the objectives of the present trial were to evaluate the occurrence of antibodies anti-N. caninum in dogs of the Botucatu range micro region, in the State of Sao Paulo, and its association with gender, age and origin (urban area--exclusively from the city; rural area--only farms and cottages; and periurban area--access to the urban and rural areas) of the dogs studied. A total of 963 dogs of defined breeds or non-defined breeds, of both genders and different ages, showing no clinical symptoms, were analyzed. Animals were randomly selected during the anti-rabies vaccination campaign in the region, from May to September 1998. Serum samples obtained from the animals were evaluated by means of the Indirect Fluorescent Antibody Test (IFAT) using as antigen N. caninum, standard strain NC-1. A total of 245 animals were reagent (25.4% positive results), with 161 (27.5%) males and 84 (22.3%) females. According to the origin--urban, rural and periurban areas--223 (25.8%), 11(16.9%) and 11(33.3%) dogs were, respectively, reagent to the IFAT. All of the 11 cities in this region presented seropositive dogs with occurrence rates ranging from 53.5 to 8.9%. The lower positive percentage was observed in dogs below 1 year old (16.2%) when compared with animals from 1 to 4 years old and more than 4 years old (28.4% and 28.0% respectively), which did not present any differences between them. Results obtained characterized the seropositivity for N. caninum in dogs from all Municipalities in the Botucatu micro region showing the wide distribution of the agent in the region.

Outbreak of Bluetongue virus serotype 4 in dairy sheep in Rio de Janeiro, Brazil

In late January 2013, 10 nonpregnant Lacaune dairy ewes raised under extensive husbandry management on a farm in Rio de Janeiro, Brazil, presented with the general clinical signs of lethargy, hyporexia, edema of the face, hyperemia of the exposed parts of the skin, mouth lesions, pyrexia, and lameness. Additionally, 2 pregnant ewes died suddenly after the onset of respiratory signs. The complete blood counts and biochemistry analyses showed neutrophilic leukocytosis with monocytosis and reactive lymphocytes, normocytic normochromic anemia and increased aspartate aminotransferase levels. Postmortem examination revealed erosions on the lingual mucosa, bilateral submandibular ganglia infarctions, yellow foamy fluid accumulation in the trachea and bronchial bifurcation, pulmonary congestion, and edema associated with hemorrhagic lesions on the pulmonary artery and heart. The clinical and pathological findings were suggestive of bluetongue. For a molecular and virological diagnosis, tissue samples were analyzed by Bluetongue virus–specific real-time reverse transcription polymerase chain reaction (qRT-PCR), and viral isolation was performed in embryonated chicken eggs. For viral typing, positive tissue and egg-isolated samples were analyzed by qRT-PCR using primers and probes specific for the structural VP2 gene in genome segment 2 of all 26 serotypes. There are still no contingency plans for responding to an outbreak of bluetongue disease in Brazil, and this episode emphasizes the need for continuing serological and entomological surveillance programs. Additionally, this report describes the isolation of Bluetongue virus serotype 4 in sheep in the Americas.

Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies

Bovine leukemia virus (BLV) was investigated in the central nervous system (CNS) of cattle with neurological syndrome. A total of 269 CNS samples were submitted to nested-PCR (BLV env gene gp51), and the viral genotypes were identified. The nested-PCR was positive in 4.8% (13/269) CNS samples, with 2.7% (2/74) presenting at histological examination lesions of nonpurulent meningoencephalitis (NPME), whereas 5.6% (11/195) not presenting NPME (P > 0.05). No samples presented lymphosarcoma. The PCR products (437 bp) were sequenced and submitted to phylogenetic analysis by neighbor-joining and maximum composite likelihood methods, and genotypes 1, 5, and 6 were detected, corroborating other South American studies. The genotype 6 barely described in Brazil and Argentina was more frequently detected in this study. The identity matrices showed maximum similarity (100%) among some samples of this study and one from Argentina (FJ808582), recovered from GenBank. There was no association among the genotypes and NPME lesions.

Neospora caninum as causative agent of bovine encephalitis in Brazil

For supporting the Brazilian bovine encephalitis surveillance program this study examined the differential diagnosis of Neospora caninum in central nervous system (CNS) by histological analysis (HE staining), immunohistochemistry (IHC), and nested-PCR using a set of primers from the Nc5 region of the genomic DNA and ITS1 region of the ribosomal DNA. A sample of 302 cattle presenting neurological syndrome and negative for rabies, aged 0 to 18 years, from herds in 10 Brazilian states was evaluated for N. caninum from January 2007 to April 2010. All specimens tested negative with IHC and nested-PCR using primers from the ITS1 region of ribosomal DNA, while two positive cases (0.66%) were found using primers from the Nc5 region of genomic DNA: a 20 month-old male and a 72 month-old female, both from São Paulo State. Only the male presented severe multifocal necrotizing encephalitis associated with mononuclear cell infiltration, a pathognomonic lesion caused by parasites of the family Sarcocystidae, and only this case was associated with N. caninum thus representing 0.33% positivity. Future studies should explore the association of IHC and nested-PCR with real-time PCR, a quantitative method that could be standardized for improving the detection of N. caninum in bovine CNS specimens.

Serological response of guinea pigs to oily and aqueous inactivated vaccines containing a Brazilian isolate of the Bovine Viral Diarrhea Virus (BVDV)

Bovine Viral Diarrhea Virus (BVDV) is widespread in cattle in Brazil and research shows its large antigenic variability. Available vaccines are produced with virus strains isolated in other countries and may not be effective. In this study, inactivated vaccines containing the Brazilian BVDV-Ib IBSP11 isolate were developed and tested on 6 groups of 10 guinea pigs (Cavia porcellus). Animals in groups A and C received an aqueous vaccine (aluminum hydroxide); B and D groups received an oily vaccine (Montanide ISA50); Group E positive-control animals were given an imported commercial vaccine with BVDV-Ia Singer; Group F animals were sham vaccinated (negative control). Groups A, B and E received two doses, and Groups C and D, three, every 21 days. Twelve blood samples were taken, at 21-day intervals over 231 days, and evaluated for antibody titer through virus-neutralization (VN), using a homologous strain (IBSP11), and a heterologous strain (BVDV-Ia NADL). Most animals, 42 days following the first dose, seroconverted to both strains and, after the second dose, there was a significant increase of titers in all groups. The oily formulation induced greater response after the third administration. This increase was not observed with the aqueous vaccines, regardless of the virus used in the VN. Antibody decline was more rapid in animals that received aqueous vaccines. The results showed the importance of studying the influence of endemic strains of commercial vaccines, to improve the efficacy of BVD vaccination. Use of the endemic strain in vaccine formulation presented promising results, as well as the use of guinea pigs as a laboratory model.

Low occurrence of equine herpesvirus 1 (EHV-1) as cause of abortion and perinatal mortality in Brazil

Arq. Inst. Biol., v.85, 1-7, e0852017, 2018 RESUMO: O herpes-vírus equino tipo 1 (HVE-1) é um importante agente patogênico causador de aborto, doença neonatal, distúrbios respiratórios e síndrome neurológica em populações de equinos em todo o mundo. Para avaliar a ocorrência do HVE-1 como agente causal de abortamento e mortalidade perinatal no Brasil, foram examinadas amostras de 105 fetos equinos abortados, natimortos e potros de até 1 mês de idade, utilizando as técnicas de isolamento viral, imuno-histoquímica (IHQ), histopatologia e reação em cadeia da polimerase aninhada (nested-PCR). Dois fetos foram positivos na análise de PCR, e um deles apresentou corpúsculos de inclusão viral eosinofílicos e sincícios no epitélio brônquico, porém foi negativo na análise de isolamento viral. O outro feto não apresentou lesões histológicas características de infecção herpética, mas foi positivo na análise de isolamento viral. Nenhuma amostra apresentou resultado positivo pela análise de IHQ. Os resultados demonstraram baixa ocorrência de HVE-1 na população estudada e que o uso de diferentes técnicas diagnósticas aumenta a probabilidade de um diagnóstico preciso para o HVE-1.

Bovine Leukosis Virus and Antibodies against Bovine Herpesvirus Type-1 and Bovine Viral Diarrhea Virus and in Buffaloes of the State of Pará, Brazil

Background: Viral diseases affecting reproduction cause economic losses in cattle, as reproductive failure interrupts the production cycle, thus reducing herd productivity. Buffaloes are susceptible to most of the diseases that affect cattle. Some of the viral diseases of reproductive importance are infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD), and bovine leukosis (BL). The objective of this study was to determine the seroprevalence of IBR virus (bovine herpes virus-1, BoHV-1), BVD virus (BVDV), and BL virus (BLV) in female buffaloes living in wetland areas or plains in the state of Para, as well as the seroprevalence of BoHV-1, BVDV, and BLV in females of different age groups. Materials, Methods & Results: It were used 225 crossbred buffaloes from 4 buffalo exclusive farms. The buffaloes reared on farms A (n = 50) and B (n = 89) were kept in wetland areas on Marajo Island (n = 139), Para. On farms C (n = 30) and D (n = 56) buffaloes were kept in plains (n = 86) in a northeastern mesoregion of Para. Animals were categorized into age groups: I: 0-3 years (n = 34), II: 3-6 years (n = 58), III: 7-9 years (n = 55), and IV: over 9 years (n = 78). Blood samples were collected and sera were sent for processing at the Bovine Virus Laboratory, Biological Institute of Sao Paulo, Brazil. The presence of IBRV and BVDV was determined by neutralization and BLV antibodies by immunodiffusion in agar gel. Statistical analysis was performed using the Ȥ2 test at a significance level of 5%. Among the seroprevalences of IBRV, BVDV and BLV antibodies, the difference in prevalence for BVD [P < 0.01] was observed between wetland areas and plains of Para, varying from 12.4% to 96.0% and 0.0% to 13.3%, respectively. While levels of IBRV remained high on farms A (79.6%), B (86.5%), C (83.3%) and D (89.1%) [P = 0.60] and BLV was negative in all the animals. BVDV was similar for all the age groups [P = 0.60], while IBRV was more prevalent in animals over 6 years of age with group I: (76.5%), II (74.5%), III (92.3%) and IV (91.3%) [P = 0.01]. It was not possible verify the influence of age in prevalence for BL. Discussion: The prevalence obtained for the BoHV-1 on farms was high, with little variation between 79.6% and 89.1%. The high prevalence of BoHV-1 suggests the free circulation of the virus in the state of Para. Notably, the animals in this study had not been vaccinated, so the determined viral antibody titers were independent of vaccination. BVDV results showed high variation between 0.0% and 96.0%. The highest prevalence in wetland areas may be related to the breeding environment, because of a possible horizontal contamination. The lack of BLV antibodies prevalence can be attributed to the fact that all the evaluated animals were kept in an extensive breeding system where they had little direct contact. The prevalence of IBR was higher in the older animals. Variation in prevalence with age was not observed for BVD. It was concluded that the prevalence for IBR and BVD were high indicating that the etiological agents are circulating in Para, with IBR having a higher prevalence in older animals. Breeding buffalo in wetland areas may favor horizontal transmission of BVDV because facilitate contact of healthy animals with water contaminated by virus carriers animal secretion and the absence of seropositive animals at the BLV does not necessarily indicate that buffaloes are resistant to the virus.

Malignant Catarrhal Fever in Brazilian cattle presenting with neurological syndrome

Malignant Catarrhal Fever (MCF) was investigated in the central nervous system of cattle with neurological syndrome. Two-hundred-ninety samples were analyzed by histology, and molecular methods to detect ovine herpesvirus type 2 (OvHV-2) were optimized and validated. The qualitative polymerase chain reaction (qualitative PCR) analytical sensitivity was 101 DNA copies/μL and found 4.8% (14/290) positive for OvHV-2. The quantitative polymerase chain reaction (qPCR) analytical sensitivity was 100 DNA copy/μL and 5.9% (17/290) positivity, with 47.1% (8/17) of the positive samples presenting histological evidence of non-purulent meningo-encephalitis. The qualitative PCR products (422 bp of the ORF75 region) were sequenced and submitted to phylogenetic analysis. Identity matrices showed 100% similarity in OvHV-2 samples obtained in this study and those recovered from GenBank, corroborating other studies.

Demodex phylloides infection in swine reared in a peri-urban family farm located on the outskirts of the Metropolitan Region of São Paulo, Brazil.

This paper reports the occurrence of porcine demodicosis caused by the mite Demodex phylloides in hogs reared in a peri-urban family farm located in Francisco Morato, a municipality of the Metropolitan Region of São Paulo, capital city of the state of São Paulo, Brazil. In a parcel of forty Landrace - Large White cross hogs, approximately four months old, four animals presented severe skin lesions in the form of small nodules over their entire body, especially in the periocular region, snout, lower abdomen and flanks. Two hogs had to be euthanized for animal welfare reasons, which enabled post-mortem examination. Skin scrapings revealed eggs, larvae, nymphs and adults of D. phylloides. Purulent subcutaneous nodules with intense parasitic folliculitis and intense perifollicular inflammatory reaction were present. Enterobacteria and coagulase-positive Staphylococcus spp. were isolated from the skin pustules. Necropsy revealed milk spotted liver, enteritis and enlargement of mesenteric lymph nodes. Protozoa (Eimeria spp. and Balantidium sp.), helminth eggs (Ascaris suum, Trichuris suis and strongyles) and Brachyspira spp. were found in faeces. Staphylococcus spp. and enterobacteria were isolated from internal organs. All remaining hogs were treated with ivermectin at a daily oral dose of 0.45g/kg of feed, during seven days. Fifteen days after treatment, remission of symptoms was observed in the surviving animals with demodicosis; absence of mites was confirmed by skin scraping examinations. The hogs were reared under poor environmental, nutritional and sanitary conditions, resulting in multimorbidity and immunosuppression. Severe clinical porcine demodicosis was triggered when the animals were castrated. Family pig farmers had been suffering economic losses due to the stunted growth of the herd. In addition to that, the lesions found on the skin and in the internal organs would result in condemnation of meat and viscera for human consumption. As part of a Public Policies Project, farm facilities were renovated with governmental aid, while family farmers received training. Good management practices and biosecurity measures were introduced in the herd. Educative policies and financial support were important to guide family pig farmers towards better husbandry practices, allowing them to raise healthy hogs in compliance with market demands.