Human Adenovirus, Mesophilic Bacteria and Fungi in Puppies’ Food Marketed in Bulk in Southern Brazil

Background: The Brazilian domestic canine population are the second largest in the world and their feeding means 0.4% of the Brazilian gross domestic product. For maintaining the quality of the food, the companies use worldwide standards for technical prevention and control of contaminants and biological conservation. The packaging is part of this process, since it provides a barrier between food and environment. However, in Brazil, packagings are often opened in retail stores for bulk marketing. The objectives of this work were to develop a methodology to detect viruses in foods and to analyze the bacterial and fungal contamination in puppies’ food sold in bulk in Ivoti and Estância Velha, cities in Southern Brazil. Materials & Methods & Results: Twenty samples collected between September and October 2016 were analyzed for most probable number of coliforms, Salmonella sp., mesophilic aerobic bacteria and yeast/mold following the regulation of the Brazilian Ministry of Agriculture, Livestock and Food Supply guidelines. They were also tested for Human Mastadenovirus C (HAdV), Canine Mastadenovirus A (CAdV), and Carnivore Protoparvovirus 1 (CPV) genomes. Viral analysis were performed by polymerase chain reaction (PCR) detection. During the collection of the samples hygienic-sanitary conditions, storage of feeds, animals’ access, dog grooming, and veterinary care were considered to evaluate the conditions of each store. A pilot study was carried out using one food sample marketed in bulk and one sample from the original package (closed package) and testing them for bacterial and fungal contamination for standardizing viral detection. Ten grams of food from the original package were mixed with 90 mL of Eagles’ Minimal Essential Medium (E-MEM) in 100 mL sterile bottles. These bottles were kept in room temperature and shaken for 60 min. Subsequently, aliquots were obtained by sequentially diluting the sample (10-2 to 10-4). All final specimens contained 10 mL and each diluted sample was spiked with HAdV-C prototype viral strain (AdV5). A standard solution of HAdV-C was diluted from 3.6x106TCID50mL, (50% tissue culture infective dose) to 3.6x103 TCID50mL, and DNA extraction was performed. Nested-PCR targeting AdV DNApol was performed to detect adenoviruses from different hosts. AdV-positive samples were submitted to nucleotide sequencing and phylogenetic analysis. Specific PCRs were also carried out for CAdV and CPV. Mesophilic aerobic bacteria were detected in all samples and Aspergillus sp. was found in five samples, among which one sample was co-infected with Penicillium sp. One sample was positive for AdV, which was identified as HAdV by sequencing; while coliforms and Salmonella sp. were not detected.Discussion: The presence of fungi with mycotoxigenic potential, such as Aspergillus sp. and Penicillium sp. represents a threat for canines, due to toxins that may persist for a long period even after the fungus is not viable. Moreover, dogs seem to be more susceptible to the effects of the toxins, which is probably because of low glutathione s-transferase activity. Some species of Penicillium sp. genus may produce ocrathoxin A, which nephrotoxic and immunosuppressive effects in dogs are widely reported. Mesophilic bacteria were detected in all samples (at 104 CFU/g) and considered harmless. The detection of human viruses points to the presence of anthropic contamination; on the other hand, ingestion of contaminated feed, even if it is by a heterologous species, turns the dogs into carriers of the virus. In addition, manipulation of those feeds by children who share the same space with dogs can result in gastroenteritis episodes

Epidemiological status of felid herpesvirus type-1 and feline calicivirus infections in Brazil

Feline calicivirus (FCV) and felid herpesvirus type-1 (FeHV-1) are the main infectious agents of domestic and wild felines worldwide. The FCV and FeHV-1 viruses were isolated in Brazil in 1988 and 2012, respectively. Serology surveys were performed among domestic feline in the State of Rio Grande do Sul and among wild felines in central Brazilian States. Felines with acute or chronic infections may become carriers for both viruses and, viral transmission occurs mainly by ocular and nasal secretions. In addition, FCV may be transmitted by oropharyngeal secretion and fomites. The clinical signs commonly observed in cats are fever, sneezing, coughing and nasal and ocular discharge; however, oral lesions are restricted to FCV infection. A systemic syndrome showing hemorrhagic lesions, alopecia, facial edema and jaundice has been associated with FCV. Attenuated as well as inactivated vaccines against FCV and FeHV-1 were developed in the middle 1970s, and they are effective at reducing the presentation/development of the diseases, but they are not capable of eliminating the persistence of FCV and FeHV-1. This article presents a brief review of the main aspects of the FCV and FeHV-1 infections, with an emphasis in the current situation on the domestic feline population from Brazil

Preliminary Evaluation of Enteric Viruses in Bottled Mineral Water Commercialized in Brazil

In Brazil, mineral water is widely consumed and as of yet there have been no studies done in the country that aim to detect enteric viruses in this water source. The aim of this study was to analyze the presence of the human adenovirus (HAdV), the human rotavirus genogroup A (GARV) and the human enterovirus (hEV) in mineral water samples from six different brands that are commercialized in southern Brazil, using molecular techniques and comparing the results with bacterial indicators. Samples of 1.5 L and 500 mL were analyzed for viruses through PCR and total and thermotolerant coliforms. Additionally, heterotrophic bacteria were assayed using a commercial kit. The most prevalent virus was adenovirus (32.5%) followed by rotavirus (25%) and enterovirus (17.5%). Total and thermotolerant coliforms were absent in all samples and only three samples out of the 60 analyzed presented heterotrophic bacteria contamination. We conclude that, following the example taken into consideration regarding the public supply of drinking water, stricter measures for microbiological control should also be applied to mineral water so that this actually becomes a safer alternative

Proteção fetal contra o vírus da diarréia viral bovina (BVDV) em vacas prenhes previamente imunizadas com uma vacina experimental atenuada

Esse artigo relata a avaliação da resposta sorológica e proteção fetal conferida por uma vacina experimental contendo duas amostras atenuadas do vírus da diarréia viral bovina tipos 1 (BVDV-1) e 2 (BVDV-2). Vacas foram imunizadas com a vacina experimental (n=19) e juntamente com controles não-vacinadas (n=18) foram colocadas em cobertura e desafiadas, entre os dias 60 e 90 de gestação, pela inoculação intranasal de quatro amostras heterólogas de BVDV-1 e BVDV-2. A resposta sorológica foi avaliada por testes de soro-neutralização realizados a diferentes intervalos após a vacinação (dias 34, 78 e 138 pós-vacinação [pv]). A proteção fetal foi monitorada por exames ultra-sonográficos e clínicos realizados durante o restante da gestação; e pela pesquisa de vírus e anticorpos no sangue pré-colostral coletado dos fetos abortados e/ou dos bezerros recém nascidos. No dia do desafio (dia 138 pv), todas as vacas vacinadas apresentavam anticorpos neutralizantes em títulos altos contra o BVDV-1 (1.280- >10.240) e, com exceção de uma vaca (título 20), todas apresentavam títulos médios a altos contra o BVDV-2 (80-1.280). O monitoramento da gestação revelou que, dentre as 18 vacas não-vacinadas, apenas três (16,6%) pariram bezerros saudáveis e livres de vírus. As 15 restantes (83,3%) apresentaram indicativos de infecção fetal e/ou falhas reprodutivas. Sete dessas vacas (38,8%) pariram bezerros positivos para o vírus, sendo que cinco eram saudáveis e sobreviveram (27,7%); e dois apresentavam sinais de prematuridade ou fraqueza e morreram três e 15 dias após o nascimento, respectivamente. As oito vacas controle restantes (44,4%) abortaram entre o dia 30 pós-desafio e às proximidades do parto, ou deram à luz bezerros prematuros, inviáveis ou natimortos. Por outro lado, 17 de 19 (89,4%) vacas vacinadas deram à luz bezerros saudáveis e livres de vírus. Uma vaca vacinada abortou 130 dias pós-desafio, mas o produto não pôde ser examinado para a presença de vírus. Outra vaca vacinada pariu um bezerro positivo para o vírus (5,2%). Em resumo, a vacina experimental induziu títulos adequados de anticorpos na maioria dos animais; e a resposta imunológica induzida pela vacinação foi capaz de conferir proteção fetal e prevenir as perdas reprodutivas frente ao desafio com um pool de amostras heterólogas de BVDV. Assim, essa vacina experimental pode representar uma boa alternativa para a redução das perdas reprodutivas associadas com a infecção pelo BVDV

Atividade in vitro de plantas condimentares (Rosmarinus officinalis L., Lippia graveolens HBK e Thymus vulgaris L.) contra o calicivírus felino

<p>O calicivírus felino (FCV) é um importante patógeno de gatos que causa lesões ulcerativas orais e infecções respiratórias. O vírus tem sido utilizado como modelo experimental para avaliação de agente antivirais contra norovírus (NoVs). Nesse estudo, investigou-se a ação dos óleos essenciais de alecrim (Rosmarinus officinalis L.), orégano mexicano (Lippia graveolens HBK.) e tomilho (Thymus vulgaris L.) frente ao FCV, in vitro. A toxicidade celular foi testada pelo método de MTT e os ensaios antivirais pelo teste de redução de placas. Três protocolos foram aplicados: a) diferentes concentrações não tóxicas dos óleos essenciais (CNTOE) foram incubadas com o vírus por 1 hora antes da inoculação (ensaio virucida); b) CNTOE foram adicionadas às células CRFK e incubadas por 1 hora antes da adsorção viral (ensaio de pré-tratamento); c) CNTOE foram adicionadas às células após a inoculação do FCV e mantidas por 18 horas (ensaio de pós-tratamento). A CC50 para os óleos de alecrim, orégano mexicano e tomilho foram: 1300,21 μg mL-1; 435,92 μg mL-1 e 675,34 μg mL-1; respectivamente. O óleo essencial de tomilho apresentou índice de seletividade [IS=CC50/CI50] de 8,57 para o ensaio de pré-tratamento e 6,2 no ensaio virucida. O óleo de alecrim mostrou atividade antiviral no ensaio virucida (IS=6,54) e de pós-tratamento (IS=6,86). O orégano mexicano apresentou IS de 5,75 no ensaio virucida e 5,59 no de pós-tratamento. Conclui-se que os óleos essenciais de tomilho e alecrim apresentaram atividade frente ao FCV em diferentes momentos da infecção viral.</p><p><br />Palavras-chave: Norovírus. FCV. óleos essenciais. Citotoxicidade. MTT. ensaio de placa.</p><p> </p><p>ABSTRACT</p><p><br />In vitro activity of plants used as condiments (Rosmarinus officinalis L., Lippia graveolens HBK e Thymus vulgaris L.) against the feline calicivirus</p><p><br />The feline calicivirus (FCV) is an important pathogen of feline causing oral ulcerative lesions and respiratory disease. This virus has been used as a model to evaluate antiviral compounds against Norovirus (NoVs). In this study, the essential oils of rosemary (Rosmarinus officinalis L.), mexican oregano (Lippia graveolens HBK) and thyme (Thymus vulgaris L.) were examined for their activity towards FCV, in vitro. The cytotoxicity was determined by the MTT test and the antiviral assays were performed by the plaque reduction test. Three protocols were applied: a) different non-toxic concentrations of the essential oils (NTCEO) were incubated with the virus for 1 hour before viral inoculation (virucidal assay); b) NTCEO were added to CRFK cells and incubated for 1 hour before viral adsorption (pre-treatment assay); c) NTCEO were added to cells after virus inoculation and maintained for 18 hours (post-treatment assay). The cytotoxic concentration at 50% (CC50) for the essential oils of rosemary, mexican oregano, and thyme were: 1300.21 μg mL-1; 435.92 μg mL-1 and 675.34 μg mL-1; respectively. The essential oil of thyme showed a selectivity index (IS=CC50/CI50) of 8.57 at the cell pre-treatment assay and 6.2 at the virucidal assay. The essential oil of rosemary showed antiviral activity at the virucidal assay (IS=6.54) and, also, at the posttreatment assay (IS=6.86). The mexican oregano showed an IS of 5.75 at the virucidal assay and 5.59 at the post-treatment. Therefore, it can be concluded that the essential oils of thyme and rosemary show antiviral activity against FCV in different times of the infection.</p><p><br />Keywords: Norovirus. FCV. Essential oils. Cytotoxicity. MTT. Plaque assay.</p