ABSTRACT The control of Salmonella in the poultry production chain combined with biosecurity measures is an important tool to maintain and guarantee the sanitary status of Brazilian flocks. The aim of this work was to compare official laboratory data on molecular typification of Salmonella isolates from poultry breeding flocks in different Brazilian states between 2016 and 2018 and identify the production category with the most positive flocks, in light of current legislation. Surveillance data of positive samples from the official Brazilian Salmonella Control Programme sent to Federal Agricultural Defence Laboratory of São Paulo (LFDA-SP) after molecular characterization were analysed. These data were subject to an exploratory study, undergoing a descriptive statistical analysis followed by the use of frequency and non-parametric hypothesis tests. Overall, 49 serovars were detected in poultry broiler-breeder and layer-breeder flocks. Salmonella ser. Heidelberg, Salmonella ser. Anatum, Salmonella ser. Newport, Salmonella ser. Schwarzengrund and Salmonella ser. Mbandaka were the five most common isolated serovars. The data shows that there is an opportunity to improve biosecurity measures in parent breeder flocks. A total of 16 serovars were identified in turkey-breeders. Salmonella ser. Anatum, Salmonella ser. Newport, Salmonella ser. Brandenburg, Salmonella ser. Litchfield, and Salmonella ser. Livingstone were the most common ones. The four official controlled serovars represented a small part of the isolated strains. These data demonstrate the importance of an official program in Brazil for Salmonella surveillance in breeder flocks combined with biosecurity measures.

ABSTRACT Salmonellosis is an important gastrointestinal infection in humans and cause of foodborne outbreaks in the world. In this context, molecular characterization is essential to understand how the strains circulate. The aim of this study was to evaluate the genotypic distribution of S. Heidelberg according to the source of isolation. The genetic relatedness of the S. Heidelberg isolates was determined by pulsed-field gel electrophoresis (PFGE). The most prevalent pulsotypes of cluster A were BRJF6X01.006 (27/95 = 28,42%) related between 1995 and 2011 in broilers, poultry meat and poultry farms, meat product and human, and BRJF6X01.001 (21/95 = 22,10%) related between 2011 and 2017 in wild animals, broilers, poultry meat, poultry farms, meat product, animal feed, and pork meat. The pulsotype BRJF6X01.001 shows a high distribution in the environmental and productive chain. The degree of similarity between pulsotypes BRJF6X01.006 and BRJF6X01.001 is 88%. To ensure the safety of human and animal health, holistic approaches, including surveillance of Salmonella throughout the environment and in the production chain, together with control measures, are critical. As transmission of Salmonella from food producing animals to wildlife and to the environment is considered potential public health problem, information on the survival and persistence of Salmonella in the environment and in potential reservoirs is of considerable importance.

ABSTRACT Enteropatogenic Escherichia coli (EPEC) and shigatoxigenic E. coli (STEC), are generally poultry and poultry product isolate and can cause serious human infections. Many strains may become resistant to various antimicrobials, which can hinder the treatment of bacterial diseases. Organic farming seeks to avoid the selection and frequency of antimicrobial-resistant bacteria. This study aims to verify the resistance of EPEC and STEC from organic and conventional (industrial) broiler isolates to antimicrobials. All isolates were submitted to disk diffusion test with tetracycline, gentamicin, enrofloxacin, ceftriaxone and amoxicillin + clavulanate (TET, GEN, ENO, CTX, AMC) and PCR to detect specific virulence genes for EPEC and STEC. A total of 297 E. coli strains were isolated, 213 from conventional. In organic broiler, 84 strains were isolated. The strains from the conventional broiler isolates were resistant to five antimicrobials tested: TET 48.82% (104/213), ENO 28.17% (60/213), CTX 15.49% (33/213), GEN 14.55% (31/213), and AMC 7.04% (15/213), and 9.86% (21/213) were considered multidrug-resistant. Organic chicken strains were resistant to four of the antimicrobials tested: TET 35.7% (30/84), ENO 9.5% (8/84), CTX 2.4% (2/84), GEN 4.8% (4/84). Of the strains from the organic broiler chicken isolates, only 1.2% (1/84) was considered multidrug-resistant. No EPEC and STEC were found in the organic chicken samples. The multidrug resistance was characterized in 9.52% (2/21) of the EPEC and 4.76% (1/21) of the STEC. The study demonstrated the absence of EPEC and STEC strains in organic broilers and carcasses and a lower frequency of multiresistant strains compared to conventional breeding.

ABSTRACT The present study aimed to investigate the occurrence of Mycoplasma synoviae (MS), M. gallisepticum (MG), Ornitobacterium rhinotracheale (OR), Avibacterium paragallinarum (AP), Pasteurella multocida (PM) and Infectious Bronchitis Virus (IBV) in laying hens with respiratory clinical signs in two phases of production. 140 tracheal swabs and 140 blood samples were collected from laying hens in the rearing and production phases, the chickens belonged to six farms (A-F) located in the state of São Paulo, Brazil. The samples were analyzed by PCR for MG, MS, OR, AP, PM and IBV and by ELISA for MG and MS. The highest frequencies observed by PCR were for MS at farms B and C with 95 and 100% positivity, followed by MG at farms D and E with 35% and 65%, IBV with 35% at farm F and ORT with 15% at farm A. All flocks were positive for MG and MS in serology. Although MG and IBV have been detected, this can be explained by the vaccination protocols, since live attenuated vaccines are widely used for immunization against these pathogens. It was also possible to detect OR and AP thorugh PCR in some flocks. The occurrence of several etiological agents that cause respiratory diseases in laying hens was confirmed by PCR and serology, with MS being the most prevalent and being present in all farms studied.

ABSTRACT The present study aimed to investigate, by culture and PCR, the occurrence of Mollicutes, Mycoplasma gallisepticum and Mycoplasma synoviae in free-living Muscovy-ducks (Cairina moschata) from the Rio Zoo, RJ, Brazil. Tracheal swabs were obtained from 82 asymptomatic ducks and the samples were submitted to culture of mycoplasmas and PCR for identification of Mollicutes Class, Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). Samples were also analyzed directly by PCR, without prior culture, for Mollicutes, MG and MS. Eighteen (18/82) Muscovy-ducks were positive for Mollicutes by culture, all isolates were confirmed as Mollicutes and seven were identified as MG. Of the samples analyzed directly by PCR, without prior culture, 17,1% (14/82) was positive for Mollicutes, being 35,7% (5/14) identified as MG and 21,4% (3/14) as MS. The occurrence of Mollicutes class bacteria was detected in Muscovy-ducks. MG and MS were identified in these animals suggesting the circulation of these agents in the Rio de Janeiro Zoo and may present a risk for the health status of the other birds.

ABSTRACT The extensive use of antimicrobial agents has contributed to the emergence of antimicrobial resistance and multidrug resistance (MDR) in Salmonella, an important zoonotic pathogen that causes outbreaks and sporadic cases of gastroenteritis in humans. The study aimed to investigate the antimicrobial resistance profile of Salmonella strains isolated from poultry in Brazil. A total of 230 Salmonella strains, isolated from cloacal swabs (n=56) and broiler carcasses swabs (n=174) before and after chilling from slaughterhouses under Federal Inspection Service within the period 2012-2017, were analyzed. Serotyping and antibiotic susceptibility testing were performed on all the isolates. Serotyping results showed that 41% of the strains were Salmonella Heidelberg, 29% S. Minnesota, 12% S. Saintpaul, 6.5% S. Enteritidis, 3.9% S. Anatum, 2.2% S. Cerro, 2.2% S. Senftenberg, 1.7% S. Newport, 0.4% S. Ealing, 0.4% S. O:4,5 and 0.4% S. O:9,12. MDR rates of the isolates were 67.4%. S. Heidelberg 89.5%, S. Minnesota 51.5%, S. Saintpaul 82.1%, S. Anatum 66.7%, S. Cerro 60%, S. Senftenberg 40%. Out of the 230 strains, 41.3% presented resistance to Penicillins + beta-lactamase inhibitor, Penicillin, 1st and 2nd Generation Cephalosporin, 3rd and 4th Generation Cephalosporin, Tetracycline and Sulfonamide. Salmonella Heidelberg, S. Saintpaul, S. Anatum, S. Cerro, S. Senftenberg and S. Minnesota were isolated after chilling tank highlighting a food safety concern for the industry of poultry and poultry products indicating a risk to collective health. The high prevalence of MDR nontyphoidal Salmonella obtained in this study limit the options available to treat infectious disease in humans and animals.

ABSTRACT The present study investigated the frequency, level of contamination and serotyping of Salmonella strains isolated from broiler flocks in different processing sites and the fulfillment of a Performance Objective (PO) in frozen chicken breasts, as a risk assessment to measure the efficacy of prevention and control programs applied to reduce the risk of Salmonella spp. in raw poultry meat that contribute to reach food safety and public health goals. From 1,800 samples of cloacal swabs, carcasses before and after immersion chilling and frozen breasts derived from 20 broiler flocks slaughtered at two processing plants located in the mid-west and southern regions of Brazil, 278 samples were positive for Salmonella spp. by polymerase chain reaction (PCR) automated BAX System (DUPONT QUALICOM, USA), and 118 were enumerated by miniaturized most probable number technique. 122 Salmonella spp. strains were serotyped at the National Reference Laboratory of Cholera and Enteric Diseases of Oswaldo Cruz Institute Foundation (FIOCRUZ), showing a dominance of Salmonella Minnesota in every processing steps of the slaughterhouse located in the Brazilian mid-west region. Only 1 lot failed to reach the expected result for the Performance Objective (PO), using a maximum of 10% positivity acceptance for Salmonella spp. in frozen chicken breasts. Qualitative and quantitative results combined may be considered an effective tool to evaluate the effect of prevention and control programs for Salmonella spp. on the safety of the final product.

ABSTRACT The objective of this study was to identify the species and characterize the genetic relationships among mycoplasma isolates from commercial layer hen flocks using 16S-23S rDNA intergenic spacer region (IGSR) sequencing. Twenty-one isolates were obtained from samples collected from commercial layer flocks in four Brazilian states: São Paulo, Minas Gerais, Rio de Janeiro and Espírito Santo. The isolates were recovered from the São Paulo, Rio de Janeiro and Espírito Santo states. Eleven isolates were originated from tracheal swabs, five from shell gland swabs and five from ovary fragment collection. The 16S-23S rDNA IGSR of isolates were amplified by PCR, and the obtained products were subsequently sequenced. The consensus of each isolate was compared to the available sequences using Nucleotide BLAST® to determine the mycoplasma species. A phylogenetic analysis of the Mycoplasma gallisepticum (MG) sequences was performed. Pairwise analyses showed homologies of 99% to 100% with the previously characterized sequences listed in GenBank®. Four Mycoplasma gallinaceum were isolated from three flocks and seven M. pullorum isolates were obtained from a single flock. The other 10 isolates were all identified as MG and were obtained from four flocks. The 16S-23S rDNA IGSR sequencing was a good method to identify Mycoplasma species isolated from field samples, providing fast and reliable results at relatively low costs. The results were also satisfactory for the single-locus sequence typing of MG isolates.

ABSTRACT The present study investigated the effectiveness of a single Salmonella prevention and control program applied in two different processing plants, located in the states of Mato Grosso do Sul (plant A) and Santa Catarina (Plant B), belonging to the same company, and identified Salmonella strain subtypes isolated from broilers, carcasses before and after chilling, and frozen chicken breasts. The Salmonella prevention and control program was 90% effective in plant A and 100% in plant B, considering a level of 10% positive samples per frozen chicken breast batch acceptable. A total of 128 strains were serotyped, being 10 from drag swabs, 31 from cloacal swabs, 83 from carcasses, and 4 from frozen chicken breasts. After serotyping analysis, 30 strains isolated at different processing steps and drag swabs, and three Salmonella Minnesota strains isolated in 2012 in plant A, were genotyped by PFGE. In plant A, the most frequently strain isolated was Salmonella Minnesota (90.35%), followed by Salmonella Newport (8.77%), and in Plant B, Salmonella Senftenberg (80%). Salmonella Minnesota strains were differentiated by PFGE into 19 pulsotypes distributed in three clusters. The phenotypic identification by serotyping of four strains diverged from their PFGE genotypic results. Most Salmonella Minnesota strains genotyped in plant A and the strains isolated from environmental samples in 2012 in the same broiler processing plant belong to a single cluster, confirming the dominance and persistence of this clone over time.

Abstract The present report aims at describing the occurrence of Marek's Disease (MD) in a free range poultry farm based on macroscopic and microscopic lesions. For this purpose, seven free-range chickens were evaluated by gross and microscopic examination. Tumor lesions were observed in the liver and ovary, in addition of enlarged kidney and spleen, skin thickening and whitish lesions in streak form in the breast muscle. Fragments of the affected organs were collected and placed in 10% formalin for processing by the usual histopathology techniques, and stained with hematoxylin-eosin. Under microscopic evaluation, we observed pleomorphic populations of neoplastic lymphoreticular cells with pyknotic nuclei in the same organs, all compatible with MD. The macroscopic and microscopic lesions presented in this study were consistent with MD, meaning that the MD virus is present in the rearing environment of the studied chicken farm.

A farm with 3,000 free-range hens between 24 and 65 weeks of age was investigated. These hens were separated in small flocks of 400 to 700 birds, presenting 10 to 23% egg production reduction. Twenty serum samples were collected during the period of drop in egg production and three weeks later for the investigation of Mycoplasma synoviae (MS), M. gallisepticum (MG) and Infectious Bronchitis Virus (IBV) antibodies using ELISA. At the time of the second collection, egg production had resumed to normal levels; however, with 10.23% of the eggs showed eggshell abnormalities limited to the apex. Eggshell strength was significantly different between normal and those with eggshell apex abnormalities, but not other egg-quality parameters. ELISA tests showed that MS and IBV titers increased during the evaluated period. MS infection was confirmed by culture and by PCR of tracheal swabs. All samples were negative for MG by ELISA and PCR. Further studies with larger samples to ensure the occurrence of this disease in industrial layer flocks in Brazil are under way.

Avian mycoplasmas occur in a variety of bird species. The most important mycoplasmas for chickens and turkeys are Mycoplasma gallisepticum (MG), M. synoviae (MS), and M. meleagridis. Besides, M. iowe (MI) is an emerging pathogen in turkeys, but of little concern for chickens. Mycoplasmas are bacteria that lack cell wall and belong to the class Mollicutes. Although they have been considered extracellular agents, scientists admit nowadays that some of them are obligatory intracellular microorganisms, whereas all other mycoplasmas are considered facultative intracellular organisms. Their pathogenic mechanism for disease include adherence to host target cells, mediation of apoptosis, innocent bystander damage to host cell due to intimate membrane contact, molecular (antigen) mimicry that may lead to tolerance, and mitotic effect for B and/or T lymphocytes, which could lead to suppressed T-cell function and/or production of cytotoxic T cell, besides mycoplasma by-products, such as hydrogen peroxide and superoxide radicals. Moreover, mycoplasma ability to stimulate macrophages, monocytes, T-helper cells and NK cells, results in the production of substances, such as tumor necrosing factor (TNF-alpha), interleukin (IL-1, 2, 6) and interferon (<FONT FACE=Symbol>a, b, g</FONT>). The major clinical signs seen in avian mycoplasmosis are coughing, sneezing, snicks, respiratory rales, ocular and nasal discharge, decreased feed intake and egg production, increased mortality, poor hatchability, and, primarily in turkeys, swelling of the infraorbital sinus(es). Nevertheless, chronic and unapparent infections are most common and more threatening. Mycoplasmas are transmitted horizontally, from bird to bird, and vertically, from dam to offspring through the eggs. Losses attributed to mycoplasmosis, mainly MG and MS infections, result from decreased egg production and egg quality, poor hatchability (high rate of embryonic mortality and culling of day-old birds), poor feed efficiency, increase in mortality and carcass condemnations, besides medication costs. Mycoplasmas are diagnosed by serologic tests, culture and PCR and are sensitive to antimicrobials whose action sites are other than the bacterial cell wall, such as tetracyclines, macrolides, quinolones and tiamulin. However, mycoplasma control is more efficiently achieved by acquisition of birds free of MG, MS, MM and/or MI, vaccination of layers, and monitoring of breeder flocks, followed by elimination of the infected flocks that are detected.