Condemnation of bovine carcasses due to tuberculosis-gross lesions in Rio Grande do Sul, Brazil: Associated risk factors.

Surveillance of bovine tuberculosis (bTB) lesions in animals at slaughterhouses is useful for controlling and eradicating the disease, besides providing epidemiological information. This study aimed to identify risk factors for bovine tuberculosis (bTB) condemnation in cattle at slaughterhouses in Rio Grande do Sul, Brazil. A logistic regression analysis was conducted using data on bTB-related condemnations. Variables examined included animal origin, number of slaughtered animals, season, inspection level (state or municipality), animal sex, and slaughterhouse location. A total of 297,817 Animal Transport Guides were evaluated, representing the transportation of 3497,521 animals. Among these, 6097 (2.05%) had at least one animal condemned due to bTB lesions. Risk factors for condemnation included larger batch sizes, female animals, slaughterhouses, and animal origin. The higher condemnation frequency in females and regions with dairy farms suggests links to milk production. Variation in condemnation rates by inspection level and slaughterhouse highlights the need for standardized procedures in identifying bTB lesions. Identifying these risk factors enables targeted interventions to enhance disease control and eradication efforts.

An ELISA to Detect Antibodies to Bovine Alphaherpesviruses 1 and 5 and Bubaline Alphaherpesvirus 1 in Cattle Sera.

Bovine alphaherpesvirus 1 (subtypes 1.1, 1.2a, and 1.2b), type 5 (subtypes 5a, 5b, and 5c), and bubaline herpesvirus 1 (BuHV-1) induce highly, though not fully cross-reactive serological responses. Most types and subtypes of these viruses circulate particularly in countries of the southern hemisphere, notably Brazil and Argentina. Therefore, the detection of infected animals is important in defining prevention and control strategies, particularly when flocks are destined for international trade. Identification of infected herds is most often achieved by assays that detect antibodies, such as enzyme immunoassays (ELISAs). However, to date, no ELISA has been evaluated in its capacity to detect antibodies to these alphaherpesviruses. Here, an ELISA was developed to detect antibodies to all currently recognized BoAHV-1, BoAHV-5, and BuAHV-1 types/subtypes, and its sensitivity and specificity were determined. Six hundred bovine sera were screened in serum neutralization tests (SN) against the seven viruses. ELISAs prepared with each of the viruses were compared to SN. Subsequently, a combined assay with multiple antigens LISA was prepared by mixing five viral antigens, chosen for their highest sensitivity in the preparative assays. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity (κ = 0.93; PPV = 95.0%; NPV = 97.3%). The findings reveal that the mAgELISA developed here is highly suitable for the detection of antibodies, comparable in sensitivity and specificity to that of SN when performed with all known types and subtypes of bovine and bubaline alphaherpesviruses.

Molecular survey of porcine respiratory disease complex pathogens in Brazilian wild boars.

Wild boar (Sus scrofa) is an exotic invasive species in Brazil and may be a reservoir for several pathogens, including those related to the porcine respiratory disease complex (PRDC), a critical infectious disease in pig production. The objective of this study was to investigate viral and bacterial pathogens related to PRDC in free-living wild boars from Brazil. Eighty animals were examined in search of genomes of porcine circovirus 2 (PCV2), Torque teno Sus virus 1a (TTSuV1a) and 1b (TTSuV1b), Influenza A virus (IAV), Actinobacillus pleuropneumoniae, Glaesserella parasuis, Pasteurella multocida, and Mycoplasma hyopneumoniae. The results demonstrated that 57.5% (46/80) of the animals had at least one detected pathogen, and 11.3% of them (9/80) were co-infected. TTSuV1a was the most prevalent genome, for which risk factors were associated with increased contact between wild boars and other animals. The other pathogens were detected at much lower frequencies or not detected (M. hyopneumoniae and IAV). An additional IAV serology search identified H1N1pdm09 antibodies in 35.5% (16/45) of the wild boars, bringing concern related to public health. In conclusion, wild boars are infected with pathogens that cause swine diseases, so their eventual contact with domestic pigs might risk animal production in Brazil.

Multidrug-resistant Escherichia coli from free-living pigeons (Columba livia): Insights into antibiotic environmental contamination and detection of resistance genes.

Bacterial resistance is a public and one health problem. Free-living birds can be reservoirs of multidrug-resistant bacteria and resistance genes. This study aimed to characterize the antimicrobial resistance of Escherichia coli isolated from free-living urban pigeons (Columba livia) in South Brazil. Ninety-two animals were sampled, and one isolate was obtained from each one. The isolates were characterized, and the antimicrobial resistance profile and beta-lactam and colistin resistance genes were investigated. The isolates were classified as phylogroups B1 (35%), B2 (33%), A (16%) and D (16%), and 14% of the strains had the eae virulence gene. All isolates were resistant to at least one antimicrobial, and 63% of them were multidrug-resistant. Geographical location where the pigeons were captured and presence of the eae gene were associated with multidrug resistance. blaVIM and mcr-1 genes were detected in one and two isolates, respectively. This is the first report of these genes in E. coli of pigeons. The blaVIM -positive isolate was classified as Shiga toxin-producing E. coli, and the isolates with mcr-1 were classified as Enterohaemorrhagic E. coli and Enteropathogenic E. coli, which raise additional concerns related to public health since these are zoonotic pathotypes. The results reveal that pigeons carry multidrug-resistant pathogenic E. coli, which may interest public health. Nonetheless, further studies on whether these animals are sources of contamination for humans must be performed to understand their role in spreading antimicrobial resistance.

A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs.

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named 'Suismacovirus', comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses' genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.

A molecular strategy to optimize bovine tuberculosis post-mortem diagnosis and the exposure to Mycobacterium tuberculosis variant bovis.

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium tuberculosis var. bovis, for which the definitive diagnosis is accomplished by bacterial isolation, which has biosafety issues and requires long time. Thus, diagnostic methods with potential to be faster and more efficient can represent an advance in bTB epidemiological knowledge and decrease exposure to M. tuberculosis var. bovis. This study aimed to validate a molecular test for bTB post-mortem diagnosis, as a strategy to reduce waste in bovine production. A total of 185 tissues from animals of infected herds or with suspected lesions at abattoir were evaluated through bacterial isolation, PCR and histopathology. PCR and histopathology showed sensitivities of 45.1% and 71.2%, respectively, and specificities of 83.3% and 83.0%, respectively, when compared to bacterial isolation. The combination of both tests resulted in enhanced specificity and positive predictive values.Therefore, PCR in conjunction with histopathology may be used as screening, in which concordant results can be considered conclusive, and discordant results may be submitted to bacterial isolation.

Investigation on porcine circovirus type 3 in serum of farrowing sows with stillbirths.

Since its first identification in 2016, porcine circovirus 3 (PCV3) has been detected in healthy and/or diseased swine in many countries worldwide. In a previous study by our group, PCV3 was detected in sera of sows which had at least one stillborn piglet in the last parturition. As such, it became important to investigate if the presence of PCV3 in sows' sera could be associated to the occurrence of stillbirths. With that aim, the frequency of PCV3 infections and viral DNA loads in sows' sera was investigated through a real-time quantitative PCR in 89 serum samples of just farrowed sows with or without stillbirths. PCV3 genomes were identified in most samples, with genome loads ranging between less than 10 to 200,000 copies per mL of serum. No significant differences were observed either in the frequency of infection or PCV3 viral loads in sows with or without stillbirths. Thus, no association could be established between PCV3 infection of sows at farrowing and stillbirths' occurrence.

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 intestinal virome of malabsorption syndrome-affected and unaffected broilers through shotgun metagenomics.

Malabsorption syndrome (MAS) is an economically important disease of young, commercially reared broilers, characterized by growth retardation, defective feather development and diarrheic faeces. Several viruses have been tentatively associated to such syndrome. Here, in order to examine potential associations between enteric viruses and MAS, the faecal viromes of 70 stool samples collected from diseased (n = 35) and healthy (n = 35) chickens from seven flocks were characterized and compared. Following high-throughput sequencing, a total of 8,347,319 paired end reads, with an average of 231 nt, were generated. Through analysis of de novo assembled contigs, 144 contigs > 1000 nt were identified with hits to eukaryotic viral sequences, as determined by GenBank database. A number of known and unknown representatives of Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, as well as novel uncharacterized CRESS-DNA viruses, were identified. However, the distribution of sequence reads of viral genomes identified in diseased or healthy birds revealed no statistically significant differences. These findings indicate no association between the occurrence of MAS and enteric viruses. The viral genomes reported in the present study, including a variety of novel viruses, seem part of the normal intestinal microbiota of chickens.

Pathogenic Leptospira spp. in bats: Molecular investigation in Southern Brazil.

The present study aimed to investigate the frequency of pathogenic Leptospira spp. in Brazilian bats and to determine possible risk factors associated to it. Ninety two bats of 12 species were evaluated. Whole genomic DNA from kidneys was extracted and real-time PCR specific to pathogenic Leptospira spp. was applied. Association between the frequency of specimens positive for Leptospira spp. and sex, age, bat species or family, season of collection, geographic localization and feeding habits was evaluated. The results showed that 39.13% of analyzed bats were found positive for Leptospira spp. Nine bat species had at least one positive result. There was no association among the evaluated variables and frequency of pathogenic Leptospira spp. Although the limitations due to lack of Leptospira spp. isolation, leptospiral carriage was demonstrated in bats of different species from southern Brazil, which reinforces the need for surveillance of infectious agents in wild animals.

Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis / A PCR em tempo real de swab nasal não é adequada para o diagnóstico in vivo de tuberculose bovina

Bovine tuberculosis (bTB) is a zoonosis causing economic losses and public health risks in many countries. The disease diagnosis in live animals is performed by intradermal tuberculin test, which is based on delayed hypersensitivity reactions. As tuberculosis has complex immune response, this test has limitations in sensitivity and specificity. This study sought to test an alternative approach for in vivo diagnosis of bovine tuberculosis, based on real-time polymerase chain reaction (PCR). DNA samples, extracted from nasal swabs of live cows, were used for SYBR® Green real-time PCR, which is able to differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. Statistical analysis was performed to compare the results of tuberculin test, the in vivo gold standard bTB diagnosis method, with real-time PCR, thereby determining the specificity and sensitivity of molecular method. Cervical comparative test (CCT) was performed in 238 animals, of which 193 had suitable DNA from nasal swabs for molecular analysis, as indicated by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and were included in the study. In total, 25 (10.5%) of the animals were CCT reactive, of which none was positive in the molecular test. Of the 168 CCT negative animals, four were positive for M. tuberculosis complex at real time PCR from nasal swabs. The comparison of these results generated values of sensitivity and specificity of 0% and 97.6%, respectively; moreover, low coefficients of agreement and correlation (-0.029 and -0.049, respectively) between the results obtained with both tests were also observed. This study showed that real-time PCR from nasal swabs is not suitable for in vivo diagnosis of bovine tuberculosis; thus tuberculin skin test is still the best option for this purpose.(AU)

A tuberculose bovina (bTB) é uma zoonose que causa perdas econômicas e riscos à saúde pública em muitos países. O diagnóstico da doença em animais vivos é realizado pelo teste intradérmico da tuberculina, que é baseado em reações de hipersensibilidade tardia. Como a tuberculose tem resposta imunológica complexa, este teste tem limitações em termos de sensibilidade e especificidade. Este estudo procurou desenvolver uma abordagem alternativa para o diagnóstico in vivo da tuberculose bovina, com base na reação em cadeia da polimerase (PCR) em tempo real. As amostras de DNA, extraídas de suabes nasais de vacas vivas, foram usadas para PCR em tempo real com SYBR® Green, capaz de diferenciar os complexos Mycobacterium tuberculosis e Mycobacterium avium. A análise estatística foi realizada para comparar os resultados de teste de tuberculina, padrão ouro para o diagnóstico in vivo da bTB, com PCR em tempo real, determinando-se assim a especificidade e sensibilidade do método molecular. O teste cervical comparativo (TCC) foi realizado em 238 animais, dos quais 193 tiveram DNA dos suabes nasais adequados para análise molecular, como indicado pela amplificação do gene gliceraldeído-3-fosfato-desidrogenase (GAPDH), e foram incluídos no estudo. No total, 25 (10,5%) animais foram reativos no TCC, dos quais nenhum foi positivo no teste molecular. Dos 168 animais negativos no TCC, quatro foram positivos para o complexo M. tuberculosis na PCR em tempo real a partir dos suabes nasais. A comparação destes resultados gerou valores de sensibilidade e especificidade de 0% e 97,6%, respectivamente; além disso, baixos coeficientes de concordância e correlação (-0,029 e -0,049, respectivamente) entre os resultados obtidos com ambos os testes também foram observados. Este estudo mostrou que a PCR em tempo real a partir de suabes nasais não é adequada para o diagnóstico in vivo da tuberculose bovina; portanto, o teste da tuberculina ainda é a melhor opção para este fim.(AU)

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.

Genome sequence of bubaline alphaherpesvirus 1 (BuHV1) isolated in Australia in 1972.

Bubaline alphaherpesvirus 1 (BuHV1) is a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. To date, no full genome sequence of BuHV has been published. Here, we report the complete genome sequence of bubaline alphaherpesvirus 1 (BuHV1) strain b6 (BuHV1-b6), isolated from a water buffalo (Bubalus bubalis) in 1972 in Australia. The virus was multiplied in MDBK cells, and the DNA was extracted and subjected to high-throughput sequencing. The reads were aligned and combined into a single genome sequence, with bovine alphaherpesvirus 5 (BoHV5) strain SV507/99 (accession number NC005261) as a reference. The BuHV1-b6 genome is a linear double-stranded DNA molecule, 137,452 bp long, with a GC content of 76.8%. The genome consists of two unique sequences: a long, or UL, sequence (103,818 bp) and a short, or US, sequence (9,586 bp), with the latter being flanked by inverted IR and TR elements of 12,024 bp each. The arrangement is typical of herpesvirus genomes of the D-type. The overall sequence has a 92.2% similarity at the nucleotide level to the reference BoHV5 strain. Our report provides a significant landmark in the history of herpesviruses, represented by the genome sequence of this 44-year-old virus isolate.

Chicken parvovirus viral loads in cloacal swabs from malabsorption syndrome-affected and healthy broilers.

Chicken parvovirus (ChPV) has been associated with malabsorption syndrome (MAS) in broilers. However, the participation of this virus in such syndrome is unclear, since it may be detected in diseased and healthy chickens. In the course of these studies, it was argued whether ChPV genome loads might be correlated to the occurrence of MAS. To check such a hypothesis, a SYBR green-based quantitative polymerase chain reaction was developed to detect and quantify ChPV genomes. Cloacal swabs from 68 broilers with MAS and 59 from healthy animals were collected from different poultry farms. Genomes of ChPV were detected in all samples, regardless of their health status. However, viral genome loads in MAS-affected broilers were significantly higher (1 × 105 genome copies per 100 ng DNA) than in healthy animals (1.3 × 103 GC/100 ng DNA). These findings indicate that there is an association between high ChPV genome loads and the occurrence of MAS in broilers.

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.

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.

Detection of human adenovirus, rotavirus and enterovirus in water samples collected on dairy farms from Tenente Portela, Northwest of Rio Grande do Sul, Brazil

Viral gastroenteritis and other waterborne diseases are a major concern for health in Brazil. A number of studies were conducted about the presence of viruses on water samples from Brazilian areas. However, the knowledge about the occurrence of viral contamination of drinking water sources in rural settings of the country is insufficient. On the present work, 15 samples from 5 dairy farms located at the municipality of Tenente Portela were collected and analysed for the presence of human adenoviruses (HAdV), as well as human enteroviruses (EV) and rotaviruses (RV). HAdV was present on 66.66% of the water samples, and have been found in all samples from artesian wells and springs, which are used as sources of drinking water for the individuals inhabiting those farms. EV and RV found only in one sample each. The detection rates of HAdV on the water from these dairy farms are alarming and point towards a situation of elevated environmental contamination by fecal microorganisms of human origin and poor basic sanitation conditions.

Detection of human adenovirus, rotavirus and enterovirus in water samples collected on dairy farms from Tenente Portela, Northwest of Rio Grande do Sul, Brazil.

Viral gastroenteritis and other waterborne diseases are a major concern for health in Brazil. A number of studies were conducted about the presence of viruses on water samples from Brazilian areas. However, the knowledge about the occurrence of viral contamination of drinking water sources in rural settings of the country is insufficient. On the present work, 15 samples from 5 dairy farms located at the municipality of Tenente Portela were collected and analysed for the presence of human adenoviruses (HAdV), as well as human enteroviruses (EV) and rotaviruses (RV). HAdV was present on 66.66% of the water samples, and have been found in all samples from artesian wells and springs, which are used as sources of drinking water for the individuals inhabiting those farms. EV and RV found only in one sample each. The detection rates of HAdV on the water from these dairy farms are alarming and point towards a situation of elevated environmental contamination by fecal microorganisms of human origin and poor basic sanitation conditions.

Management, breeding, and health records from a captive colony of pekin robins (Leiothrix lutea), 2001 - 2010.

Pekin robins (Leiothrix lutea) were once the most widely kept softbills in captivity. As a result of the Convention on the International Trade of Endangered Species of Fauna and Flora (CITES-1997), the worldwide trade of wild-caught pekin robins has been prohibited due to the depletion of native populations of this species. In Brazil, as in other countries, pekin robins imported prior to the enactment of the CITES have disappeared from aviaries because the end of the birds' natural life span has passed, and only very few captive-bred pekin robins now exist. While captive propagation fails to address the primary causes of wild bird population decline, it might help the recovery of populations of this species. This article presents records made over a 10-yr period of a captive colony of pekin robins. Emphasis is placed on the management of the flock, the ailments affecting the birds, and the findings associated with bird losses. The main causes of bird losses included rearing management failures and age-related disorders.