Caracterização do viroma de soro de suínos, equinos e bovinos

Os suínos, equinos e bovinos possuem uma histórica importância econômica e social. A criação de suínos de exposição para participar em eventos agrícolas é uma atividade relevante na região Centro-Oeste dos Estados Unidos. Os equinos são utilizados para o lazer, esporte, equoterapia e força de tração no trabalho rural. Os bovinos apresentam um papel essencial na indústria de carne e leite. Além disso, equinos e bovinos são fontes de soro, um produto biológico utilizado na pesquisa e para à fabricação de vacinas. Dessa forma, o conhecimento do status sanitário de suínos, equinos e bovinos é um ponto estratégico para à epidemiologia molecular de agentes virais que causam prejuízos econômicos à saúde animal. O conhecimento limitado da comunidade viral presente no soro desses animais dificulta o esclarecimento das possíveis causas de doenças de etiologia infecciosa. O emprego do sequenciamento de alto desempenho (high-throughput sequencing, HTS) tem permitido a detecção de agentes virais em amostras biológicas de muitas espécies animais. Porém, os soros de suínos de exposição, e lotes de soros equinos e bovinos comerciais de diferentes países ainda carecem de investigação da presença de vírus adventícios através da metagenômica viral. Nesse sentido, o presente estudo visa ampliar o conhecimento sobre a diversidade do viroma do soros desses animais utilizando o HTS. No Capítulo 1, foram analisadas 400 amostras de soros de suínos, divididas em dois pools de 200 amostras cada. Foram obtidas sequências de genomas das famílias Arteriviridae, Circoviridae, Flaviviridae, Herpesviridae, Parvoviridae e Retroviridae. Vinte e três espécies virais foram detectadas, incluindo o primeiro bufavírus suíno nos Estados Unidos. Assim como, o vírus da síndrome respiratória e reprodutiva suína, pestivírus atípico de suíno e circovírus suíno. Além disso, 17 genomas completos foram recuperados e os dois primeiros bocaparvovirus ungulado 3 dos Estados Unidos. No Capítulo 2, foram investigados cinco lotes de soros comerciais de equinos com origem na Nova Zelândia (três lotes), Brasil e dos Estados Unidos (um lote cada). Nestes soros foram detectadas sequências genomicas das famílias Flaviviridae, Herpesviridae e Parvoviridae. Particularmente, hepacivírus e pegivírus equinos foram os mais frequentes. No Capítulo 3, foram sequenciados sete lotes de soros comerciais de bovinos com origem na Nova Zelândia (dois lotes), México (um lote) e dos Estados Unidos (quatro lotes). Os soros de bovinos apresentaram genomas das famílias Adenoviridae, Flaviviridae, Parvoviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae e vírus circulares DNA fita simples codificantes de replicase (circular rep-encoding single-stranded, CRESS) (Genomoviridae, Circoviridae e Smacoviridae). Doze genomas completos de parvovírus bovino 2 e 3, bosavírus, hokovírus bovino e poliomavírus bovino 1 foram recuperados. Além disso, foram detectadas sequências relacionadas à crAssphage, um bacteriófago associado a contaminação fecal humana. Portanto, o estudo ilustra os agentes virais presentes nos soros de suínos, equinos e bovinos, buscando contribuir para o conhecimento do viroma presente nos soros desses animias e auxiliar no esclarecimento de possíveis causas de doenças de etiologia infecciosa.Pigs, horses, and cattle have historical economic and social importance. Raising show pigs to participate in agricultural events is a major activity in the Midwest region of the United States. Horses are used for leisure, sport, hippotherapy and traction force in rural work. Cattle play an essential role in the meat and milk industry. In addition, horses and cattle are sources of serum, a biological product used in research and for the manufacture of vaccines. Thus, knowledge of the health status of pigs, horses and cattle is a strategic point for the molecular epidemiology of viral agents that cause economic damage to animal health. The limited knowledge of the viral community present in the serum of these animals makes it difficult to clarify the possible causes of diseases of infectious etiology. The use of high-throughput sequencing (HTS) has allowed the detection of viral agents in biological samples of many animal species. However, the sera from show pigs, and batches of commercial equine and bovine sera from different countries still need to be investigated for the presence of adventitious viruses through viral metagenomics. In this sense, the present study aims to expand the knowledge about the diversity of the sera virome of these animals using the HTS. In Chapter 1, 400 swine serum samples were analyzed, divided into two pools of 200 samples each. Genome sequences were obtained from the Arteriviridae, Circoviridae, Flaviviridae, Herpesviridae, Parvoviridae and Retroviridae families. Twenty-three viral species were detected, including the first porcine bufavirus in the United States. As well as the porcine reproductive and respiratory syndrome virus, porcine atypical pestivirus and porcine circovirus. In addition, 17 complete genomes were retrieved and the first two ungulate bocaparvovirus 3 from the United States. In Chapter 2, five batches of commercial equine sera originating in New Zealand (three batches), Brazil and the United States (one batch each) were investigated. In these sera, genomic sequences of the Flaviviridae, Herpesviridae and Parvoviridae families were detected. In particular, equine hepacivirus and pegivirus were the most frequent. In Chapter 3, seven batches of commercial bovine sera from New Zealand (two batches), Mexico (one batch) and the United States (four batches) were sequenced. Bovine sera showed genomes of the Adenoviridae, Flaviviridae, Parvoviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae and circular Rep-encoding single-stranded (CRESS) DNA virus (Genomoviridae, Circoviridae e Smacoviridae). Twelve complete genomes of bovine parvovirus 2 and 3, bosavirus, bovine hokovirus and bovine polyomavirus 1 were recovered. Furthermore, sequences related to crAssphage, a bacteriophage associated with human fecal contamination, were detected. Therefore, the study illustrates the viral agents present in the pigs, horses, and cattle sera, seeking to contribute to the knowledge of the virome present in these animals sera and to help clarify possible causes of infectious etiology diseases

The probiotic properties of Enterococcus faecium strains isolated from buffalo milk : food matrix studies

Buffalo milk has been increasingly explored in the production of dairy foods. Given the diversity of lactic acid bacteria present in this raw material, the functional potential of this type of milk should be studied. This study aims to assess the probiotic potential of two strains of Enterococcus faecium isolated from buffalo milk. We evaluated E. faecium M7AN7-1 and E. faecium M7AN10 for sustained cell viability under different conditions of conservation. We also assessed adhesion and cell viability in a simulated gastrointestinal transit test. E. faecium M7AN10 was selected for microencapsulation in sodium alginate and testing in the food matrix. The isolates maintained cell viability after refrigeration, freezing, and freeze-drying. E. faecium M7AN10 showed higher concentrations of viable cells than E. faecium M7AN7-1 after 180 min of contact with simulated gastric fluid, reaching 7.19 ± 0.59 Log10 CFU mL-1. We also observed sustained cell viability after exposure to simulated intestinal fluid. After encapsulation, E. faecium M7AN10 was tested in the food matrix of UHT milk. Cell count viability was maintained and its release was sustained in this medium for 30 days. The results of the in vitro assessment demonstrated that E. faecium M7AN10 remained functionally active under these experimental conditions. Similarly, they showed that it is a bacterium capable of sustaining viability in this type of food and after transit in a simulated gastrointestinal tract. Based on the data, we suggest this isolate may be a probiotic bacterial candidate for in vivo behavioral assessment

Virome characterization in commercial bovine serum batches : a potentially needed testing strategy for biological products

Bovine serum has been widely used as a universal supplement in culture media and other applications, including the manufacture of biological products and the production of synthetic meat. Currently, commercial bovine serum is tested for possible viral contaminants following regional guidelines. Regulatory agencies’ established tests focused on detecting selected animal origin viruses and are based on virus isolation, immunofluorescence, and hemadsorption assays. However, these tests may fail to detect new or emerging viruses in biological products. High-throughput sequencing is a powerful option since no prior knowledge of the viral targets is required. In the present study, we evaluate the virome of seven commercial batches of bovine serum from Mexico (one batch), New Zealand (two batches), and the United States (four batches) using a specific preparation and enrichment method for pooled samples and sequencing using an Illumina platform. A variety of circular replicase-encoding single-stranded (CRESS) DNA families (Genomoviridae, Circoviridae, and Smacoviridae) was identified. Additionally, CrAssphage, a recently discovered group of bacteriophage correlated with fecal contamination, was identified in 85% of the tested batches. Furthermore, sequences representing viral families with single-stranded DNA (Parvoviridae), double-stranded DNA (Polyomaviridae and Adenoviridae), single-stranded RNA (Flaviviridae, Picornaviridae, and Retroviridae), and double-stranded RNA (Reoviridae) were identified. These results support that high-throughput sequencing associated with viral enrichment is a robust tool and should be considered an additional layer of safety when testing pooled biologicals to detect viral contaminants overlooked by the current testing protocols

Complete genome sequences of two bovine alphaherpesvirus 5 subtype C strains from southeast Brazil

Bovine alphaherpesvirus 5 causes meningoencephalitis in cattle, belongs to the Herpesviridae family, and can be divided into subtypes a, b, and c. Limited information is available about subtype c. Here, we report the complete genome sequences of two strains, P160/96, and ISO97/45, isolated from cattle in southeast Brazil

Viral DNA genomes in sera of farrowing sows with or without stillbirths

A study was conducted to investigate the serum virome of sows with and without stillbirths after farrowing. Sera from sows with at least one stillbirth or with normal litters were collected immediately after farrowing. Viral DNA was extracted from serum pools and submitted to high throughput sequencing. No differences in the proportion of virus-related reads were found in both groups (p > 0.05). A variety of viral DNA genomes were identified, mostly representative of three viral families: Anelloviridae, Circoviridae and Smacoviridae. Besides, a number of novel unclassified circular Rep-encoding single stranded DNA (CRESS DNA) viruses were also identified. These findings suggest that the presence of such viral genomes in sows’ sera bears no correlation with stillbirths’ occurrence; it seems likely that these constitute part of the normal serum microbiome of sows at farrowing

The complete genome sequence of bovine herpesvirus 5 subtype c

O herpesvírus bovino tipo 5 (BoHV-5) é classificado na ordem Herpesvirales, família Herpesviridae, subfamília Alphaherpesvirinae, gênero Varicellovirus. O BoHV-5 é um agente importante de encefalites ou meningoencefalites em bovinos. Até o presente três subtipos de BoHV-5 são reconhecidos (BoHV-5a, b & c), para que tais correlações possam ser investigadas como maior profundidade, a disponibilidade das sequências genômicas dos diferentes subtipos é de particular importância. Até o presente, somente uma sequência completa de BoHV-5a (SV507/99) encontra-se disponível. No presente estudo é apresentada a sequência genômica completa de uma amostra de BoHV-5 subtipo c, denominada P160/96. O genoma viral foi obtido através de sequenciamento de alto desempenho. As sequências obtidas foram analisadas com o software Geneious (versão 9.1.4). O genoma contém 137.740 pb, com 99% de similaridade em nível de nucleotídeos com a amostra SV507/99. Embora semelhante à sequência de genoma de BoHV-5 já publicada, existe um número de substituições de nucleotídeos e deleções/inserções ao longo do genoma, muitas das quais afetam as sequências de codificação. O genoma da amostra P160/96 apresenta um sítio adicional de clivagem para BstEII, localizado na região repetida interna (IR) da sequência, que revela o perfil de restrição enzimática característico de amostras do subtipo c. Este é o segundo genoma de BoHV-5 até o presente publicado, sendo o primeiro do subtipo c. Espera-se que esta sequência venha representar uma contribuição significativa para uma maior compreensão da biologia dos herpesvírus bovinos, especialmente na investigação de potenciais diferenças na patogenicidade de distintos subtipos de BoHV-5. Esse conhecimento poderá trazer subsídios adicionais para a definição de estratégias de controle ou erradicação mais apropriadas, contribuindo assim para a melhoria a saúde dos rebanhos bovinos.Bovine herpesvirus type 5 (BoHV-5) is classified in the order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. BoHV-5 is an important agent of encephalitis or meningoencephalitis in cattle. For such correlations to be further investigated, the availability of the genomic sequences of the different subtypes is of particular importance. To date, only one complete sequence of BoHV-5 subtype a (SV507/99) is available. In the present study the complete genomic sequence of BoHV-5 subtype c, named P160/96, is presented. The viral genome was determined by high-throughput sequencing. The obtained sequence was analyzed with the Geneious software (version 9.1.4). The P160/96 genome has 137.740 bp, with 99% similarity at the nucleotide level with SV507/99. Although similar to the published BoHV-5 genome sequence, there are a number of nucleotide substitutions and deletions/insertions throughout the genome, many of which affect the coding sequences. The P160/96 genome has the additional BstEII cleavage site located in the internal repeat region (IR), which gives rise to the reveals the characteristic enzyme restriction profile of subtype c samples. This is the second published BoHV-5 genome and the, first of subtype c. It is expected that this sequence will represent a significant contribution to a better understanding of bovine herpesvirus biology, especially in the investigation of potential differences in pathogenicity of distinct BoHV-5 subtypes. This is expected to provide additional information for the definition of appropriate control or eradication strategies, thus contributing to the improvement of cattle health

Evaluation of Ultraviolet Type C Radiation in Inactivating Relevant Veterinary Viruses on Experimentally Contaminated Surfaces

Many swine farms employ UVC treatment in employees’ personal belongings and small tools entering farms as part of the biosecurity protocol to decrease the risk of pathogen introduction into the operation. However, the UVC efficacy in some veterinary viruses is not fully evaluated. This study evaluated the efficacy of ultraviolet type C (UVC) radiation in inactivating seven relevant veterinary viruses: Swine Poxvirus (SwPV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Epidemic Diarrhea Virus (PEDV), Swine Influenza Virus (SIV), Bovine Viral Diarrhea Virus (BVDV), Porcine Parvovirus (PPV), and Senecavirus A (SVA). The experimentally contaminated materials included polystyrene and filter paper. The samples were exposed to UVC for 5 min (total dose of 360 mJ/cm2). The UVC treatment caused a decrease over 4 log10 in SwPV titer on the polystyrene surface, whereas it consistently reduced about 5 log10 in PPV and SVA samples. No viable virus was recovered from PRRSV, PEDV, SIV, and BVDV samples. In filter paper, conversely, the efficacy was reduced. This study provides essential information on the inactivation effectiveness of a specific dose of UVC on important veterinary viruses, further supporting the rational application and strategic guidance for UVC radiation use to disinfect materials

Prolonged Viability of Senecavirus A in Exposed House Flies (Musca domestica)

House flies (Musca domestica) are often present in swine farms worldwide. These flies utilize animal secretions and waste as a food source. House flies may harbor and transport microbes and pathogens acting as mechanical vectors for diseases. Senecavirus A (SVA) infection in pigs occurs via oronasal route, and animals shed high virus titers to the environment. Additionally, SVA possesses increased environmental resistance. Due to these reasons, we investigated the tenacity of SVA in house flies. Five groups of flies, each composed of ten females and ten males, were exposed to SVA, titer of 109.3 tissue culture infectious dose (TCID50/mL). Groups of male and female flies were collected at 0, 6, 12, 24, and 48 h post-exposure. For comparison purposes, groups of flies were exposed to Swinepox virus (SwPV). Infectious SVA was identified in all tested groups. Successful isolation of SVA demonstrated the titers varied between 106.8 and 102.8 TCID50/mL in female groups and varied from 105.85 to 103.8 TCID50/mL in male groups. In contrast, infectious SwPV was only detected in the female group at 6 h. The significant SVA infectious titer for prolonged periods of time, up to 48 h, indicates a potential role of flies in SVA transmission