Fabiana Quoos Mayer

Food safety in raw milk production: risk factors associated to bacterial DNA contamination.

While human illness from milkborne pathogens may be linked to contamination of the product after pasteurization or improper pasteurization, such diseases are usually associated with consumption of raw milk or its by-products. Molecular biology tools were applied to investigate contamination by Listeria monocytogenes, Salmonella spp., some pathogenic strains of Escherichia coli, and Campylobacter jejuni in 548 raw milk samples from 125 dairy farms established in two regions from southern Brazil. Moreover, 15 variables were evaluated for their association with raw milk contamination levels, and the risk factors were determined by multiple regression analysis. Salmonella spp. were more frequently detected, followed by pathogenic E. coli. There was difference in contamination index between the regions, in which risk factors such as temporary cattle confinement, low milk production, low milking machine cleaning frequency, and milk storage area without tile walls were identified. The risk factors were specific to each region studied. Nevertheless, the data can be used to improve milk quality of dairy farms/herds with similar management practices.

How many papillomavirus species can go undetected in papilloma lesions

A co-infection comprising to at least seven papillomavirus (PV) types was detected by next generation sequencing (NGS) of randomly primed rolling circle amplification (RCA) products of a bovine (Bos taurus) papilloma lesion from the Brazilian Amazon region. Six putative new PV types that could not be detected by commonly used PCR protocols were identified. Their overall L1 nucleotide identities were less than 90% compared to described PV species and types. L1 nucleotide BLAST sequence hits showed that each new type was related to Beta, Gamma, Dyokappa, Dyoeta, and Xipapillomavirus, as well as two likely new unclassified genera. Our results show that the employment of NGS is relevant to the detection and characterization of distantly related PV and is of major importance in co-infection studies. This knowledge will help us understand the biology and pathogenesis of PV, as well as contribute to disease control. Moreover, we can also conclude that there are many unknown circulating PVs.

A Novel Chiropteran Circovirus Genome Recovered from a Brazilian Insectivorous Bat Species

ABSTRACT This report describes the identification and characterization of a novel circovirus using metagenomic approaches in respiratory fluid samples from Brazilian free-tailed bats (Tadarida brasiliensis). The genome and deduced protein sequences share low identity with another circovirus recovered in distantly related bats from China.

Faecal virome of healthy chickens reveals a large diversity of the eukaryote viral community, including novel circular ssDNA viruses

This study is focused on the identification of the faecal virome of healthy chickens raised in high-density, export-driven poultry farms in Brazil. Following high-throughput sequencing, a total of 7743 de novo-assembled contigs were constructed and compared with known nucleotide/amino acid sequences from the GenBank database. Analyses with blastx revealed that 279 contigs (4 %) were related to sequences of eukaryotic viruses. Viral genome sequences (total or partial) indicative of members of recognized viral families, including Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, were identified, some of those representing novel genotypes. In addition, a range of circular replication-associated protein encoding DNA viruses were also identified. The characterization of the faecal virome of healthy chickens described here not only provides a description of the viruses encountered in such niche but should also represent a baseline for future studies comparing viral populations in healthy and diseased chicken flocks. Moreover, it may also be relevant for human health, since chickens represent a significant proportion of the animal protein consumed worldwide.

Novel Bovine Papillomavirus Type Discovered by Rolling-Circle Amplification Coupled with Next-Generation Sequencing.

Currently, fifteen bovine papillomavirus (BPV) types have been identified and classified into four genera: Deltapapillomavirus, Epsilonpapillomavirus, Dyoxipapillomavirus, and Xipapillomavirus. Here, the complete genome sequence of a new BPV type (BPV 04AC14) recovered from a papillomatous lesion is reported. The genome is 7,282 bp in length and exhibits the classic genetic organization and motifs of the members of Papillomaviridae. Maximum likelihood phylogenetic analyses revealed that BPV 04AC14 clusters with members of the Xipapillomavirus genus. The nucleotide sequence of the L1 capsid protein of the novel BPV is closely related to its counterpart, BPV3, with which it shares 79% similarity. These findings suggest that this virus is a new BPV type of the Xipapillomavirus genus.

Detection of Mycobacterium tuberculosis and Mycobacterium avium Complexes by Real-Time PCR in Bovine Milk from Brazilian Dairy Farms.

Foodborne diseases are a public health problem worldwide. The consumption of contaminated raw milk has been recognized as a major cause of transmission of bovine tuberculosis to humans. Other mycobacteria that may be present in raw milk and may cause diseases are those belonging to the Mycobacterium avium complex. In this study, molecular biology tools were applied to investigate raw milk contamination with Mycobacterium spp. in family dairy farms from Rio Grande do Sul, southern Brazil. Furthermore, different variables related to the source of the milk, herd characteristics, and management were evaluated for their effect on milk contamination. Five hundred and two samples were analyzed, of which 354 were from the Northwest region (102 farms with samples from 93 bulk tanks and 261 animals) and 148 from the South region of the state (22 farms with samples from 23 bulk tanks and 125 animals). Among them, 10 (1.99%) and 7 (1.39%) were positive for Mycobacterium tuberculosis (9 confirmed as Mycobacterium bovis) and M. avium complexes, respectively. There was no difference in the frequencies of positive samples between the regions or the sample sources. Of the positive samples, 4 were collected from a bulk tank (1 positive for M. avium and 3 for M. tuberculosis). Moreover, 1 sample was positive concomitantly for M. tuberculosis and M. avium complexes. On risk analysis, no variable was associated with raw milk contamination by M. tuberculosis complex species. However, washing the udders of all animals and drying them with paper towels were weakly classified as risk factors for M. avium contamination. Positive samples were obtained from both animals and bulk tanks, which emphasizes the importance of tuberculosis control programs and provides evidence that milk monitoring can be used as a control practice. Moreover, the findings of this study reinforce the need for awareness of the problems of raw milk consumption among the general population.

Mycobacterium bovis infection in a collared peccary (Tayassu tajacu): insights on tuberculosis wild reservoirs.

Bovine tuberculosis is a zoonotic disease caused mainly nfection with Mycobacterium bovis (Michel et al., 2010). disease represents a risk to human health and is ponsible for high economic losses to the cattle industry, n in developed countries (Parra et al., 2008). The main y of tuberculosis control and eradication is based on a tem of diagnosis and slaughter of infected animals arez et al., 2012), but this practice is hampered by the stence of wild reservoirs such as skunks, badgers, and r, among others (Taylor et al., 2007). In this report we ntified M. bovis as the causative agent of death of a ared peccary (Tayassu tajacu) in southern Brazil. An adult collared peccary (CP) from a conservation eding located in southern Brazil died after showing a onic condition characterized by respiratory distress and gressive wasting. The CP was kept with other 20 from ame specie in an area of approximately 2000 m, which s delimited by a wire screen and covered by vegetation, provided with no roof. When the animal presented ical disease, it was kept in a covered stall. Additional mals in the farm included cattle, white-lipped peccs, deer, capybaras, agouti, coatis, pacas and birds. mals kept close to the collared peccaries were cattle, us and capybaras. The source of water consumed by st of the animals in the farm was the same and itional six collared peccaries had died previously with similar clinical disease; however, no laboratory testing had been done to identify those ailments. At necropsy, CP lungs showed caseous lesions, which were sampled for isolation of Mycobacterium using the Petroff method for decontamination as described by De Kantor et al. (1998b). Next, the sample was inoculated on Löwenstein-Jensen and Stonebrink media and incubated at 37 8C for primary isolation (Corner, 1994). After 4 weeks, colonies with characteristic growth patterns of Mycobacterium were detected. A culture smear was stained with Ziehl–Neelsen technique (De Kantor et al., 1998a) and revealed acid-fast bacilli (Fig. 1A). The identity of M. bovis was confirmed from a bacterial loop through molecular analysis as described hereafter. A lung sample was fixed in neutral-buffered formalin for 48 h, and processed following standard procedures for histopathology. The analysis evidenced granulomatous pneumonia with prominent caseous necrosis and islands of mineralization. There were epithelioid macrophages and multinucleated giant cells surrounding necrosis, mixed with lymphocytes clusters (Fig. 1B). DNA was extracted from lungs as described by Singh et al. (2000) and quantified at Nanodrop 1000 (Thermo Scientific, USA). About 200 ng were used as template. First, a screening PCR, as described by Gómez-Laguna et al. (2010), differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. The sample was positive for M. tuberculosis complex. Then, primers (Forward: BoF: 50 CCTTCCGCACACCGTTCAG 30 and

Genotypic and antimicrobial characterization of pathogenic bacteria at different stages of cattle slaughtering in southern Brazil

Meat can be contaminated in different stages of the slaughtering process and the identification of these stages is the starting point to implement adequate control measures. The objectives of this study were to assess the presence of pathogenic microorganisms in cattle carcasses, to identify the most important contamination points of the slaughtering process, and to evaluate the possible risk factors related to them in a cattle slaughterhouse. To this aim, 108 cattle carcasses were sampled at three stages of the slaughtering process: Point 1 (hides after bleeding); Point 2 (carcasses after hide removal); and Point 3 (carcasses immediately after division). Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Livingstone were isolated from the carcasses. Phenotypic and genotypic characterization indicated that there was cross-contamination among animals, since bacteria with identical genotypic and phenotypic profiles were isolated from different animals at the same sampling day. Furthermore, this is the first report about the isolation of E. coli O157:H7 in a bovine slaughterhouse from southern Brazil.

Full-Genome Sequence of a Reassortant H1N2 Influenza A Virus Isolated from Pigs in Brazil

ABSTRACT In this study, the full-genome sequence of a reassortant H1N2 swine influenza virus is reported. The isolate has the hemagglutinin (HA) and neuraminidase (NA) genes from human lineage (H1-δ cluster and N2), and the internal genes (polymerase basic 1 [PB1], polymerase basic 2 [PB2], polymerase acidic [PA], nucleoprotein [NP], matrix [M], and nonstructural [NS]) are derived from human 2009 pandemic H1N1 (H1N1pdm09) virus.

Genomic characterization of a bovine viral diarrhea virus subtype 1i in Brazil

Bovine viral diarrhea virus 1 (BVDV-1) belongs to the genus Pestivirus within the family Flaviviridae. Based on the 5’ untranslated region (UTR) sequence, BVDV-1 can be divided into at least 17 subtypes (1a though 1q). BVDV-1i is an uncommon subtype that has been reported in the United Kingdom and Uruguay. Here, we report the complete genome sequence of the first subtype 1i BVDV-1 (strain ACM/BR/2016) isolated from cattle in southern Brazil. The genome is 12,231 nt in length and contains a single ORF that encodes a polyprotein of 3,896 amino acids, flanked by 5’ and 3’UTRs of 325 and 220 nt, respectively. Phylogenetic inferences based on the whole genome, the 5’UTR, and the Npro region showed that strain ACM/BR/2016 is closely related to previously characterized BVDV-1i members. Its 5’UTR shares the highest nucleotide identity (90.5%) with BVDV-1i strains from United Kingdom, and its Npro is most closely related to that of a Uruguayan strain (90.6%). To the best of our knowledge, this is the first BVDV-1i strain from which the whole genome has been completely sequenced and characterized. The complete genome of a BVDV-1i will help future studies on pestivirus evolution and heterogeneity.