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The objective of this work was to evaluate the reduction of Campylobacter jejuni in chicken meat at 6 and 70°C, after the inoculation of a Brazilian strain. The kinetics of C. jejuni survival showed a 0.26 log (CFU g-1) decrease for each day of exposure at 6°C, and a 1.35 log (CFU g-1) decrease for each 1 log increase at 70°C. Although reduced levels of C. jejuni were found after regular intervals at both temperatures, its incomplete inactivation underlines the need of good hygiene practices for consumers to avoid campylobacteriosis. Resumo: O objetivo deste trabalho foi determinar a redução de Campylobacter jejuni em carne de frango a 6 e 70°C, após inoculação de uma cepa brasileira. A cinética de sobrevivência de C. jejuni mostrou redução de 0,26 log (UFC g-1) a cada dia de exposição a 6°C e redução de 1,35 log (UFC g-1) para cada aumento de 1 log a 70°C. Embora níveis reduzidos de C.jejuni sejam encontrados após intervalos regulares de exposição a ambas as temperaturas, a inativação incompleta ressalta a necessidade de boas práticas de higiene para os consumidores evitarem a campilobacteriose.

The vast capacity for maintenance and dissemination in the environment are major challenges for the control of Salmonella spp. in poultry farms. The aim of this study was to assess environmental contamination by nontyphoidal Salmonella in successive broiler flocks in nine commercial broiler farms integrated with three companies in the south of Brazil, for a twelve-month production period. Recycled broiler litter, feed and swabs from the evaporative cooling system pads were analyzed, and the total enterobacteria count in the litter samples was ascertained. Positive broiler houses were identified in two of the three broiler companies studied, in which nontyphoidal Salmonella were detected for the first time in the first or second flock, and recurred in the recycled litter of subsequent flocks. Feed and evaporative cooling pad swab samples were also positive in at least one of the assessed flocks. The majority of the isolates (87.5%) originating from different flocks, broiler houses and companies that were sampled were identified as S. Heidelberg, with the prevalence of one single genotype. The total enterobacteria levels in the litter diminished as the flocks progressed, but the presence of Salmonella spp. was constant over the course of time, indicating that the litter management procedures were not capable of interrupting the cycle of residual contamination. The predominance of S. Heidelberg highlights its emergence and dissemination in this region, as well as its resistance and maintenance in the environment, and reinforces the need to improve prevention and recycled litter management measures. Made available in DSpace on 2019-11-04T18:11:46Z (GMT). No. of bitstreams: 1 final9057.pdf: 1748574 bytes, checksum: 8d934aa119670b291408db1f1fbc6c90 (MD5) Previous issue date: 2019

Campylobacter is not routinely tested in foodborne disease investigations in Brazil. Here, we studied the occurrence of Campylobacter among other food-related bacteria commonly found in foodborne disease outbreaks reported in Rio Grande do Sul State, Southern Brazil. Seventy-two food samples were analyzed by using culture-based detection methods during the 18-month investigation of 36 foodborne disease outbreaks. The sampled foods from the foodborne disease outbreaks were all negative for Campylobacter. However, at least one of other routinely investigated foodborne-related bacteria was detected in 29.17% (21/72) of the samples. Taken together, these results suggest the need to monitor Campylobacter in foodborne diseases to detect sporadic cases caused by Campylobacter that might go unnoticed in Rio Grande do Sul. Made available in DSpace on 2020-11-18T09:16:29Z (GMT). No. of bitstreams: 1 final9338.pdf: 303006 bytes, checksum: 2023a019be4954fee3b2c31a4bd13aa9 (MD5) Previous issue date: 2020

Pasteurella (P.) multocida is the causative agent of pneumonic pasteurellosis in swine, which is commonly associated with the final stages of enzootic pneumonia or porcine respiratory disease complex. Although this syndrome is one of the most common and important diseases of pigs, data on antimicrobial susceptibility of P. multocida isolates are uncommon in Brazil. Therefore, the present study was carried out to determine and to compare antimicrobial susceptibility profile of Brazilian P. multocida isolated from pigs with lesions of pneumonia or pleuritis during two-time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were used to determine the MIC of amoxicillin, enrofloxacin, florfenicol and tetracycline by microbroth dilution. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Multi-drug resistance (MDR) to amoxicillin/florfenicol/tetracycline was observed in 6% of recent isolates. There was a significant increase (p˂0.05) in resistance for amoxicillin and enrofloxacin in recent isolates compared with historic isolates (3.8% and 18%, respectively), most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. The results of this study showed an increase of isolates resistant to important drugs used in treatment of P. multocida infections in pigs, demonstrating the need for the implementation of rational use of antimicrobials in Brazilian swine industry. RESUMO: Pasteurella (P.) multocida é o agente da pasteurelose pneumônica em suínos, a qual é comumente associada com o estágio final da pneumonia enzoótica suína ou complexo das doenças respiratórias dos suínos. Apesar de ser uma das doenças mais comuns e importantes na suinocultura, dados sobre suscetibilidade antimicrobiana de isolados de P. multocida são raros no Brasil. Dessa forma, o presente estudo foi realizado para determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. Isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) foram usados para determinar a concentração inibitória mínima (CIM) de amoxicilina, enrofloxacina, florfenicol e tetraciclina através do teste de microdiluição em caldo. Florfenicol apresentou o menor nível de resistência para ambos os isolados históricos e contemporâneos (0% e 6%, respectivamente), enquanto que tetraciclina apresentou o maior nível de resistência (20.5% e 34%, respectivamente). Resistência a múltiplos antimicrobianos (amoxicilina, florfenicol e tetraciclina) foi observada em 6% dos isolados recentes. Foi observado aumento significativo (p˂0.05) na resistência a amoxicilina e enrofloxacina em isolados recentes comparado com isolados históricos (3.8% e 18%, respectivamente), provavelmente devido à pressão de seleção de antimicrobianos usados no tratamento e prevenção de infecções causadas por P. multocida. Os resultados deste trabalho demostraram o aumento de isolados resistentes a importantes drogas utilizadas no tratamento de infecções causadas por P. multocida em suínos, evidenciando a necessidade da implementação do uso racional de antimicrobianos na suinocultura brasileira.

The microbiological risk of recycled litter depends on the efficacy of the management system applied to inactivate residual microorganisms and preserve the health of the successive broiler flock. This study aimed to assess the viability and infectivity of the Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV) and Salmonella Heidelberg in recycled litter exposed to different treatments. The litter was contaminated with microorganisms and submitted to the treatments (T): T1: shallow fermentation; T2: quicklime (calcium oxide); T3: shallow fermentation followed by addition of quicklime; T4: no treatment. Sentinel chicks housed on the treated litter showed that T1 and T3 inactivated residual IBDV. Analysis of the litter subjected to T1 also showed reduced levels of total enterobacteria. T2 was not able to reduce the microorganisms assessed and its association with T1 (T3) failed to enhance the effect of the treatment. NDV did not survive in the broiler litter, regardless of the treatment applied, and it was also not detected in the sentinel chicks. S. Heidelberg remained viable in the litter submitted to all studied treatments, being isolated from the sentinel chicks of all the experimental groups. The antimicrobial activity of T1 and T3 was associated to higher ammonia contents in the broiler litter. The results indicate that the shallow fermentation treatment is efficient for controlling residual IBDV and total enterobacteria in the recycled litter.

Made available in DSpace on 2020-12-12T01:14:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Empresa Brasileira de Pesquisa Agropecuária Infectious bursal disease (IBD) is an immunosuppressive viral disease of chickens, associated with severe economic losses and major threats to poultry production worldwide. Disease prevention programs rely on unequivocal identification of the pathogen, as well as vaccination programs. This study developed a sensitive, one-step, real-time, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay using a hydrolysis probe system for infectious bursal disease virus (IBDV, VP1 gene) detection and quantification, which was compared to other routinely used diagnostic methods. The assay successfully detected IBD reference viruses and field isolates. The absence of cross-reactivity was detected with negative samples or with other avian viruses in the analytical specificity test. The detection limit of this assay was 70 RNA copies. RT-qPCR was more sensitive in the detection of serially diluted IBDV isolates compared to virus isolation. For clinical samples, the sensitivity and specificity values of RT-qPCR compared to enzyme-linked immunosorbent assay (ELISA) were 97.5% and 100%, respectively, and compared to histopathology, these values were 100% and 93.94%, respectively. RT-qPCR can provide a simple and reliable assay for IBDV surveillance programs and for evaluation of control strategies. Embrapa Suínos E Aves, BR 153, Km 110 Embrapa Pecuária Sudeste, Rodovia Washington Luiz, Km 234, Fazenda Canchim Departamento de Zootecnia Universidade Estadual Paulista - UNESP Departamento de Zootecnia Universidade Estadual Paulista - UNESP Empresa Brasileira de Pesquisa Agropecuária: 0313100900

Salmonella Gallinarum biovar Gallinarum (SG) is a host-specific avian pathogen causing fowl typhoid (FT), a notifiable disease that causes septicemia in poultry and significant economic losses. In the state of Santa Catarina, Brazil, several cases of FT were reported from 2012 to 2016. However, strains of these outbreaks have not yet been characterized, and the genotypic characterization of isolates is essential for epidemiological surveillance and outbreak investigations. The objective of this work was to study the genetic diversity of SG isolated from the FT outbreaks, aiming to identify similarity or dissimilarity among strains isolated from the reported foci. For this study, 56 SG strains were submitted to pulsed-field gel electrophoresis (PFGE). Through the PFGE analysis, a prevalent genotypic profile (57.1%) of SG was identified, suggesting the occurrence of an endemic clone, whose dissemination is possibly linked to the transport of infected birds across the regions. Fifteen other genotypic profiles were also obtained, evidencing the genetic variability of circulating strains and multiple contamination sources.

Glässer’s disease (GD) is an important infectious disease of swine caused byHaemophilus (Glaesserella) parasuis. Vaccination with inactivated whole cell vaccines is the major approach for prevention ofH. parasuisinfection worldwide, but the immunity induced is predominantly against the specific polysaccharide capsule. As a consequence, the available vaccines may not induce adequate protection against the field strains, when the capsules present in the vaccine strains are different from those in strains isolated from the farms. Therefore, it is crucial to mapH. parasuisserovars associated with regional outbreaks so that appropriate bacterin vaccines can be developed and distributed for prevention of infection. In this study, 459H. parasuisfield strains isolated from different Glässer’s disease outbreaks that occurred in 10 different Brazilian States were analyzed for serotype using PCR-based approaches. Surprisingly, non-typeable (NT) strains were the second most prevalent group of field strains and along with serovars 4, 5 and 1 comprised more than 70% of the isolates. A PCR-based approach designed to amplify the entire polysaccharide capsule locus revealed 9 different band patterns in the NT strains, and 75% of the NT strains belonged to three clusters, suggesting that a number of new serovars are responsible for a substantial proportion of disease. These results indicate that commercially available vaccines in Brazil do not cover the most prevalentH. parasuisserovars associated with GD.

Resumo: Para avaliação dos aspectos patológicos e microbiológicos de casos clínicos de doenças respiratórias em suínos de terminação foram analisados 75 suínos doentes oriundos de 36 lotes. Suínos que apresentavam sinais clínicos respiratórios evidentes foram necropsiados para avaliação macroscópica e colheita de amostras para análise histopatológica e microbiológica. Foram realizados testes de isolamento bacteriano para as principais bactérias do sistema respiratório dos suínos, PCR para Mycoplasma hyorhinis, imuno-histoquímica para Influenza A, Circovirus suíno tipo 2 e Mycoplasma hyopneumoniae. A sensibilidade antimicrobiana de 24 amostras de Pasteurella multocida tipo A foi avaliada por testes de concentração inibitória mínima para os principais antimicrobianos utilizados em suinocultura. Mycoplasma hyopneumoniae e Pasteurella multocida tipo A foram os agentes infecciosos mais prevalentes. Broncopneumonia supurativa e pleurite foram as principais lesões respiratórias encontradas. Pasteurella multocida tipo A, quando presente, aumentou a extensão das lesões pulmonares. Todas as amostras de Pasteurella multocida testadas foram sensíveis aos antimicrobianos Doxiciclina, Enrofloxacina e Tilmicosina. Em 58% das amostras foi identificado mais de um agente infeccioso, evidenciando a alta prevalência da associação de agentes nas doenças respiratórias de suínos em terminação.

In Brazil, since 2011 clinical cases of salmonellosis has been increasing substantially. Nevertheless, few information is available about the antimicrobial profile, distribution, serotypes and genetic relationship among the strains. The objectives of this study were: to identify the Salmonella serotypes, to characterize the in vitro antimicrobial resistance profiles and to determine the genetic relationship of clinical isolates in Brazil. During 2016, clinical isolates of Salmonella (111) from nine States were sent to Embrapa Swine and Poultry for complementary analysis. First, isolates were serotyped by Kauffmann White Scheme. In parallel, the strains were tested against fifteen antimicrobials by disk diffusion method and genotyping was performed by Pulsed Field Gel Electrophoresis (PFGE) using the XbaI restriction enzyme. As expected, the main serovars found were Typhimurium and Choleraesuis. Four strains showed resistance to only one antimicrobial and 76.5% (85/111) were considered multiresistant. The highest level of resistance was found against to tetracycline. More than 80% of the strains were susceptible to fosfomycin, lincomycin/spectinomycin and norfloxacin. It was possible to identify one major Choleraesuis clonal group present in different Brazilian States. Further, several small clonal groups were obtained for Typhimurium. In conclusion, clinical salmonellosis caused by Typhimurium and Choleraesuis is endemic in pig production areas and the majority of the strains are multi-resistant

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.

Background: Pasteurella multocida serotypes A and D are commonly associated with pneumonia and pleuritis in pigs. Different phenotypic techniques, such as hyaluronidase and acriflavine tests, and genotyping techniques, such as PCR, are used to distinguish between these serotypes. The objective of this study was to compare the capsular identification methods of type A and type D P. multocida isolated from pigs using both phenotypic (hyaluronidase and acriflavine tests) and genotypic (multiplex PCR) techniques.Materials, Methods & Results: A total of 44 lyophilized P. multocida isolates, obtained between 1981 and 1997 from pig farms at Rio Grande do Sul State, Brazil, were analyzed. The isolates were reactivated in Brain Heart Infusion (BHI) broth and cultured in BHI broth and blood agar supplemented with 5% sheep blood. Colony identity was further confirmed by evaluating colony morphology in blood agar and confirming the absence of growth on MacConkey agar. Bacteria in Tryptone Soy Agar (TSA) were used for the Triple Sugar Iron (TSI), Sulfide-Indole-Motility (SIM), and nitrate, glucose, lactose, sucrose and mannitol fermentation tests. For hyaluronidase test, P. multocida colonies were streaked transversally across the entire plate, approximately 3-5mm apart, in order to observe their lines of growth. Following this, a hyaluronidase producing strain of Staphylococcus aureus was heavily streaked at right angles to the P. multocida lines and the plates were incubated at 37°C for 24 h. Type A isolates were then identified as those with smaller colonies in the region adjacent to the Staphylococcus aureus streak (negative satellitism). For acriflavine test, the isolates were inoculated into tubes containing 2 mL of BHI, incubated at 37°C for 18-24 h, centrifuged (500 g for 15 min) and 1.5 mL of the supernatant was discarded. A 1:1000 solution of acriflavine neutral (0.5 mL) was then added to the residual broth containing bacteria and kept at room temperature. Solutions of acriflavine were freshly prepared each week and stored protected from light. Type D strains were identified by the appearance of a heavy flocculent precipitate within 5 min. DNA extraction by heat shock was performed prior to multiplex PCR for the detection of capsular genes hyaD-hyaC (capsular typing A) and dcbF (capsular typing D). Test of symmetry and a weighted kappa coefficient were used to evaluate correlations and to assess agreement of the results between the identification methods, respectively. Phenotypic tests showed that two isolates were type D (4.55%), 40 were type A (90.9%) and two (4.55%) were untypable isolates (4.55%) while PCR showed that 38 isolates were type A (86.36%) and six were type D (13.64%). The correlation analysis between the phenotypic and genotypic tests showed that 90.9% of the strains were identified as belonging to the same serotype by both tests and the weighted kappa coefficient (K = 0.633) indicates a substantial agreement between the two tests.Discussion: There was a disagreement between the phenotypic and genotypic results in four of the isolates (9.09%). The phenotypically untypable isolates were classified as type D by multiplex PCR. Nonetheless, we conclude that PCR testing is a more reliable method to differentiate between P. multocida serotypes A and D.

In order to understand better the pathological aspects and spread of Pasteurella multocida type A as the primary cause of pneumonia in pigs, was made an experiment with intranasal inoculation of different concentrations of inocula [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml], using two pigs per group. The pigs were obtained from a high health status herd. Pigs were monitored clinically for 4 days and subsequently necropsied. All pigs had clinical signs and lesions associated with respiratory disease. Dyspnoea and hyperthermia were the main clinical signs observed. Suppurative cranioventral bronchopneumonia, in some cases associated with necrosuppurative pleuropneumonia, fibrinous pericarditis and pleuritic, were the most frequent types of lesion found. The disease evolved with septicaemia, characterized by septic infarctions in the liver and spleen, with the detection of P. multocida type A. In this study, P. multocida type A strain #11246 was the primary agent of fibrinous pleuritis and suppurative cranioventral bronchopneumonia, pericarditis and septicaemia in the pigs. All concentrations of inoculum used (105-108 CFU/ml) were able to produce clinical and pathological changes of pneumonia, pleuritis, pericarditis and septicemia in challenged animals. Resumo: Para entender melhor os aspectos patológicos e disseminação de Pasteurella multocida tipo A como causa primária de pneumonia em suínos foi realizado um experimento com inoculação intranasal de diferentes concentrações de inóculos [Grupo (G1): 108 Unidades Formadoras de Colônias (UFC)/ml; G2: 107 UFC/ml; G3: 106 UFC/ml e G4: 105 UFC/ml], usando dois suínos por grupo. Esses suínos foram obtidos de um rebanho de alto status sanitário. Os animais foram monitorados clinicamente por quarto dias e subsequentemente necropsiados. Todos os suínos apresentaram sinais clínicos e lesões associadas com doença respiratória. Dispneia e hipertermia foram os principais sinais clínicos observados. Broncopneumonia cranioventral supurativa, em alguns casos associados com pleuropneumonia necrossupurativa, pleurites e pericardite fibrinosa foram mais frequentes. A doença evoluiu com septicemia, caracterizada por infartos sépticos no fígado e baço, com detecção de P. multocida. Neste estudo, P. multocida tipo A isolado 11246 foi agente primário de pleurite fibrinosa e broncopneumonia cranioventral supurativa, pericardite fibrinosa e septicemia em suínos. Todas as concentrações de inóculo utilizado (105-108 UFC/ml) foram capazes de produzir sinais clínicos e patológicos de alterações de pneumonia, pleurites, pericardites e septicemia nos animais.

Brazil is the largest exporter of chicken meat and poultry farming is one of the most important productive segments, despite major losses due to the bacterium Escherichia coli, which is also a zoonotic microorganism. The objetive of this study was to isolate E. coli and to evaluate its transmissibility potential from the field to chicken meat using the Pulsed Field Gel Electrophoresis (PFGE) technique. Environmental samples (poultry litter, soil and water) were collected from broiler farms located in the South of Brazil where the majority of the Brazilian poultry production occurs. In addition, chicken meat (gizzard, heart, drumette and tulip) samples were collected from local supermarkets. As results, 47.36% of the samples were positives for E. coli. Furthermore, 10 pairs of clones of E. coli were found always in the same substrate (two waterwater pairs; three soil-soil pairs and five meat-meat pairs) using PFGE. These findings suggest that certain strains of E. coli may have habitat preferences, making the transfer from one substrate type to another more difficult to occur. Moreover, since no clones were found between environmental samples and chicken meat, it is possible to imply a low risk of E. coli transmissibility throughout the chicken meat production chain. Made available in DSpace on 2021-08-07T02:04:35Z (GMT). No. of bitstreams: 1 final9680.pdf: 1894617 bytes, checksum: c2155ad0836e2fe3191df0b1572d67eb (MD5) Previous issue date: 2021

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Uma análise preliminar da rotina laboratorial da Embrapa Suínos e Aves entre os anos de 2010 a 2014 mostrou que foram isoladas 1.141 cepas de Campylobacter de material avícola, compreendendo as espécies C. Jejuni e C. Coli.