Phenotypic and molecular characterisation of Salmonella spp. isolates in healthy poultry.

1. Epidemiological surveillance of Salmonella spp. serves as a primary tool for maintaining the health of poultry flocks. Characterising circulating serotypes is crucial for implementing control and prevention measures. This study conducted phenotypic and molecular characterisation of S. enterica Pullorum, S. enterica Heidelberg, and S. enterica Corvalis isolated from broiler chickens during slaughtering.2. All strains were susceptible to gentamicin, neomycin and norfloxacin. However, resistance rates exceeded 50% for ciprofloxacin and tiamulin, irrespective of the serotype. Approximately 64% of strains were classified as multidrug-resistant, with S. enterica Heidelberg strains exhibiting significantly higher overall resistance. The isolates demonstrated the ability to adhere and produce biofilm at a minimum of three temperatures, with S. enterica Pullorum capable of biofilm production at all temperatures encountered during poultry rearing.3. Each strain possessed between two and seven different virulence-associated genes. Genetic similarity, as indicated by pulsed field gel electrophoresis, exceeded 90% for all three serotypes and strains were classified in the R5 ribotype by PCR, regardless of serotype. Sequencing revealed high similarity among all strains, with homology ranging from 99.61 to 100% and all were classified to a single cluster.4. The results suggested a clonal relationship among the strains, indicating the possible circulation of a unique clonal group of S. enterica Pullorum in the southern region of Brazil.

Sistema de lavagem de carcaças e controle de Salmonella spp. em abatedouros de frangos de corte / Carcass washing system and Salmonella spp. control in poultry slaughterhouses

Avaliaram-se os dados do controle e monitoramento de Salmonella em carcaças de frangos de corte, antes e após a implantação do sistema de lavagem de carcaças. Foram amostradas 2692 carcaças antes da instalação do sistema e 1940 após a instalação, totalizando 4632 amostras em cinco abatedouros sob Inspeção Federal, no sul do Brasil. Anteriormente à instalação dos lavadores, obtiveram-se 156 resultados positivos para Salmonella spp. e, após a instalação, 83 resultados positivos, com diferença significativa (P<0,05/OR 1,4) entre os resultados gerais. Em dois dos cinco abatedouros avaliados, houve redução na positividade para Salmonella spp. nas carcaças amostradas após a instalação do lavador. Entretanto, em três estabelecimentos não houve diferença significativa após a instalação desse sistema. Os resultados sugerem que o aumento da vazão da água está relacionado com a redução da contaminação, enquanto o aumento da pressão de água do sistema de lavagem não foi suficiente para reduzir o patógeno nas carcaças amostradas. Dessa forma, conclui-se que o sistema de lavagem tem vantagens do ponto de vista operacional, ao evitar os desperdícios atribuídos à prática do refile, porém não elimina o risco da presença de Salmonella na carcaça de frango.(AU)

Data of the Control and Monitoring of Salmonella in broiler carcasses were evaluated before and after the implementation of a carcass washing system. A total of 2692 carcasses prior to system installation and 1940 after installation were sampled, totaling 4632 samples in five slaughterhouses under Federal Inspection in southern Brazil. Prior to installation of the washers, 156 carcasses were positive for Salmonella spp. and after installation, 83 carcasses tested positive, with a significant difference (P <0.05/OR 1.4) on the overall results. Two of the five evaluated slaughterhouses showed a prevalence decrease of Salmonella spp. in the sampled carcasses after installing the washer. However, in three establishments, there was no significant difference after installing this system. The results suggest that the increase of the water flow is related to the reduction of the contamination, while the increase of the water pressure of the washing system was not enough to reduce the pathogen in the sampled carcasses. The results show operational results since the washing system avoids the trimming of some carcasses, but doesn't eliminate the risk of Salmonella.(AU)

Salmonella Gallinarum field isolates and its relationship to vaccine strain SG9R.

1. The aim of the present study was to determine if the 9R-strain of the Salmonella Gallinarum live vaccine was responsible for having fowl typhoid outbreaks in chicken flocks from both chicken and turkey breeders as well as to verify the antimicrobial resistance of the isolates from the outbreaks. 2. The triplex polymerase chain reaction, standard antimicrobial test, beta-lactamase genes identification and Ion Torrent PMG whole-genome sequence were used in the field isolates and in the vaccine strain of S. Gallinarum. 3. The 60 tested isolates were not from vaccine origin and manifested high resistance to drugs from macrolide and quinolone groups. Whole-genome sequencing (WGS) and single nucleotide polymorphism analysis on selected isolates for core genes from Salmonella enterica confirmed the wild origin of these isolates and showed two possible sources of S. Gallinarum in the studied outbreaks. 4. S. Gallinarum isolated from fowl typhoid outbreaks in the studied period were not caused by the use of the SG9R live vaccine. The source of strains sequenced was diverse.

Assessment of FTA card employment for Pasteurella multocida DNA transport and detection of virulence-associated genes in strains isolated from fowl cholera in the United States / Avaliação do emprego de cartões FTA para o transporte do DNA de Pasteurella multocida e pesquisa de genes associados à virulência em cepas isoladas de cólera aviária nos Estados Unidos

Fowl Cholera (FC) is a disease caused by Pasteurella multocida. The severity of this disease is partly caused by virulence factors. Genes encoding fimbriae, capsule, sialidases and proteins for iron metabolism may be related to P. multocida's ability to infect the host. Besides to examining DNA for the presence of virulence genes, DNA is essential for the diagnostic and FTA cards are an alternative for genetic material transport. The study aims to evaluate the viability of P. multocida DNA transport using the cards and to detect 14 virulence genes in 27 strains isolated from FC cases in the United States by multiplex-PCR. No growth was observed in any of the FTA cards, which was essential to assess the security. Furthermore, DNA detection was possible in 100% of the samples, independent of the storage period (7 to 35 days) and temperature (4°C and 37°C). ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH and pfhA genes were detected in more than 80% of the samples. FTA cards have proven to be a viable and safe tool for DNA transport of P. multocida. A majority of genes showed a high frequency, which was similar to strains isolated from FC cases.(AU)

Cólera aviária (CA) é uma doença causada pela bactéria Pasteurella multocida e a severidade dos casos é em parte justificada por fatores de virulência. Genes codificando fímbrias, cápsulas, sialidases, dismutases e proteínas do metabolismo férrico podem ser relacionados à capacidade do agente em infectar o hospedeiro. Além da obtenção do DNA para pesquisa de genes de virulência, o material genético é fundamental para o diagnóstico, e os cartões FTA seriam uma alternativa no transporte de microrganismos. Os objetivos da presente pesquisa foram avaliar a viabilidade do transporte de DNA de P. multocida através dos cartões e detectar 14 genes de virulência em 27 cepas isoladas de CA nos Estados Unidos, por meio de multiplex-PCR. Nenhuma das amostras para análise microbiológica da segurança dos cartões apresentou crescimento. Foi possível a detecção do DNA em 100% das amostras, independentemente do tempo de estocagem (sete a 35 dias) e das temperaturas (4°C e 37°C) avaliadas. Genes ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH e pfhA foram detectados em mais de 80% das amostras. Os cartões FTA demonstraram ser uma ferramenta viável e segura para o transporte do DNA de P. multocida. A maioria dos genes apresentou uma alta frequência, compatível com isolados de CA.(AU)

Histomorphometric study of the anterior latissimus dorsi muscle and evaluation of enzymatic markers of broilers affected with dorsal cranial myopathy.

Dorsal cranial myopathy (DCM), which affects the anterior latissimus dorsi (ALD) muscles of commercial broilers, is of unknown etiology, and it represents up to 6% of the partial condemnations in Brazilian slaughterhouses. This study was performed to achieve histomorphometric characterizations of the ALD muscles from male Cobb 500 broilers slaughtered at either 35 d or 42 d and to evaluate the effects of DCM on the enzymatic markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH) and on uric acid and creatinine metabolites. Blood samples (1.5 to 3 mL) and ALD muscle fragments were collected from each carcass, all of which were processed in a commercial inline processing system. For each age, twelve macroscopically normal animals and twelve animals found to exhibit DCM were randomly selected for histomorphometric evaluation and analysis of serologic profiles. Microscopic evaluations demonstrated that the muscle fibers of those with DCM exhibited a strong presence of multifocal regenerative myodegeneration as well as a substitution of muscle tissue with connective tissue (P < 0.001) through fibrosis, thus characterizing the chronicity and hardness of the affected muscle. It is suggested that DCM is a localized muscle lesion because the detected serum levels of CK (P < 0.001), AST (P < 0.001), ALT (P = 0.01), and LDH (P < 0.001) enzymes were strongly associated with the group affected by DCM. Additional studies are needed to gain an understanding of this myopathy because it is an emerging problem in the poultry industry. In addition, it is related to DCM lesions in fast-growing broilers with the greatest slaughter weights.

Atividade antimicrobiana do óleo essencial de rizomas de açafrão (Curcuma longa L. ) e gengibre (Zingiber officinale Roscoe) frente a salmonelas entéricas isoladas de frango resfriado / Antimicrobial activity of essential oil from Curcuma longa and Zingiber officinale rhizomes against enteric Salmonella isolated from chicken

Objetivou-se com este trabalho identificar, quantificar os constituintes, e avaliar a atividade antibacteriana dos óleos essenciais extraídos de rizomas de açafrão (Curcuma longa L.) e gengibre (Zingiber officinale Roscoe) cultivados nas condições de Manaus/AM frente a 14 salmonelas entéricas isoladas de frango resfriado. A extração dos óleos essenciais foi realizada utilizando-se aparelho tipo Clevenger e a composição determinada por Cromatografia Gasosa acoplada a Espectrometria de Massas (CG-MS). A atividade antibacteriana foi realizada com o emprego de técnica de microdiluição em caldo. O óleo essencial de gengibre se mostrou expressivamente mais eficiente do que o óleo de açafrão, tanto em termos de ação bacteriostática (concentração inibitória mínima de 2500 a 5000 µg.mL-1) quanto bactericida (concentração bactericida mínima de 5000 a 10000 µg.mL-1) observando-se variação apenas em duas as amostras em termos de resistência a ação bactericida deste óleo. Assim, o óleo essencial de gengibre, representa uma alternativa para o controle de Salmonella enterica, entretanto, demais estudos abordando o sinergismo com alimentos são indicados.

The objective of this work was to identify, quantify constituents and evaluate the antibacterial activity of essential oils from rhizomes of turmeric (Curcuma longa L.) and ginger (Zingiber officinale Roscoe) grown under conditions of Manaus/AM front of enteric salmonella isolated from chilled poultry. The extraction of essential oils was performed using the Clevenger type apparatus and composition determined by Gas Chromatography coupled to Mass Spectrometry (GC-MS). The antibacterial activity was performed with the use of microdilution broth. The essential oil of ginger proved significantly more efficient than tumeric oil, both in terms of bacteriostatic action (minimum inhibitory concentration 2500-5000 mg µg mL-1) and bactericidal (minimum bactericidal concentration 5000-10000 mg µg mL-1) observing changes in only two samples in terms of resistance to bactericidal activity of this oil. Thus, the essential oil of ginger, is an alternative for the control of Salmonella enterica, however, other studies addressing the synergism with food are indicated.

Individual and combined effects of Salmonella typhimurium lipopolysaccharide and fumonisin B1 in broiler chickens.

The aim of this research was to evaluate the individual and combined effects of Salmonella typhimurium lipopolysaccharide (sLPS) and fumonisin B(1) (FB) on performance, relative weight of liver, biological parameters, and histological evaluation of several tissues from four hundred thirty-two 1-d-old male broiler chickens divided into 9 treatments according to the dose of FB (0, 100, or 200 mg/kg, from d 1 to d 28) and sLPS (0, 250, or 500 µg/application per bird, every other day, from d 15 to 27) administered. At the end of the experiment (28 d), significant effects caused by sLPS, FB, and the interaction of sLPS × FB were observed on several parameters. Histopathological evaluations showed significant lesions in liver and kidney caused by sLPS, FB, and their association. According to these results, both sLPS and FB (isolated or in association) cause significant effects on performance and biological parameters of broilers at 28 d of age.

Downgrading of heavy broiler chicken carcasses due to myodegeneration of the anterior latissimus dorsi: pathologic and epidemiologic studies.

Recently, in some Brazilian poultry companies, a dorsal cranial muscular lesion has been increasingly detected in broilers, causing heavy economic losses due to carcass downgrading. The observed gross lesions located in the anterior latissimus dorsi (ALD) muscle are characterized by yellowish discoloration of the skin and swelling on the dorsal cranial region of that muscle. When the ALD muscle is cut, subcutaneous edema, muscular superficial hemorrhage, pallor, adherence, and increased thickness and density are observed. Microscopically, findings indicate degenerative and polyphasic features, variation in fiber size and splitting, presence of hyaline, necrotic and regenerative myofibers, extensive fibrosis, and adipose tissue with lymphohistiocytic infiltration in all ALD muscles affected. The etiology of the lesion is unknown, and no detailed report was found in literature. The highest frequency of carcass downgrading due to this lesion was found in the heaviest and the oldest males of high-yield broiler strains (P < 0.01). This study is the first to describe the pathologic and some epidemiologic aspects of this new myopathy.

Inoculation of newly hatched broiler chicks with two Brazilian isolates of Salmonella Heidelberg strains with different virulence gene profiles, antimicrobial resistance, and pulsed field gel electrophoresis patterns to intestinal changes evaluation.

Salmonella Heidelberg is one of the 3 most frequently isolated serovars from human Salmonella cases in Canada, and the fourth most commonly reported Salmonella serovar in human foodborne disease cases in the United States. Since 1962, Salmonella Heidelberg has been isolated and reported in poultry and poultry products in Brazil. The poultry industry has focused efforts on reducing salmonellae incidence in live production in an effort to reduce Salmonella in the processing plant. A better understanding of the initial infection in chicks could provide approaches to control Salmonella contamination. The objective of the present study was to evaluate 2 Salmonella Heidelberg strains that differed in the presence of virulence genes invA, agfA, and lpfA; antimicrobial resistance profiles; and epidemiologic profiles on aspects of pathogenicity and intestinal morphology. Newly hatched broiler chicks were inoculated with 2 strains (SH23 and SH35) of Salmonella Heidelberg and cecal morphometry, histopathology, electron microscopy, and bacterial counts in the liver and cecum were assessed. The SH23 and SH35 strains resulted in different changes in villi height and crypt depth and inflammatory cell infiltration in the cecum. The SH35 group had higher liver and cecum bacterial cell counts when compared with SH23 strains.

Resistência antimicrobiana em Salmonella Enteritidis isoladas de amostras clínicas e ambientais de frangos de corte e matrizes pesadas / Antimicrobial resistance in Salmonella Enteritidis isolated from clinical and environmental broiler chickens and breeders broiler

The antimicrobial resistance of Salmonella Enteritidis strains isolated from clinical and environmental poultry samples in the Southern Brazil during the years of 1999, 2000 and 2001 was evaluated. Among the 79 isolated samples, 64 (81 percent) were resistant to at least one of the antimicrobial agents tested, showing 22 different resistance patterns. Tetracycline showed the highest percentage (64,5 percent) of resistance among the antimicrobial agents used. Resistance to drugs at different levels was found as the following: ampicillin (1.2 percent), kanamycin (1.2 percent), ciprofloxacin (2.5 percent), enrofloxacin (8.8 percent), gentamicin (21.5 percent), streptomycin (20.2 percent), nitrofurantoin (26.6 percent), and nalidixic acid (30.4 percent). None of the S. Enteritidis strains were resistant to chloramphenicol, norfloxacin, and polimycin B. Among the 64 S. Enteritidis strains that showed resistance, 43 (67.2 percent) were resistant to two or more antimicrobial agents. Twenty-one (32.8 percent) strains were resistant to only one of the antimicrobial agents, 14 to tetracycline, three to nalidixic acid, three to nitrofurantoin, and one to gentamycin. These antimicrobial resistance levels suggest a high occurrence of tetracycline resistant S. Enteritidis strains and resistance to two or more antimicrobial agents.

Occurrence and characterization of Campylobacter in the Brazilian production and processing of broilers.

Twenty-two commercial broiler flocks and their carcasses, totaling 546 samples (450 collected from a poultry farm and 96 from a slaughterhouse), were surveyed for the presence of Campylobacter. The positive results for Campylobacter among the analyzed samples were homogeneous, yielding 81.8% for cecal droppings, 80.9% for feces, and 80.4% for cloacal swabs. Pre-enrichment and direct plating showed that 77.85% and 81.8% of cloacal swabs, respectively, were positive for Campylobacter compared to 99.0% and 97.9% of carcasses testing positive with the pre-enrichment and direct plating methods. The Campylobacter count averaged 7.0 log10 colony-forming units (CFU)/g in cecal droppings, 5.15 log10 CFU/carcass after defeathering, and 4.24 log10 CFU/carcass after chilling. The samples were identified by the API Campy system as Campylobacter jejuni subsp. jejuni (68.8%), Campylobacter coli (8.3%), Campylobacter jejuni subsp. doylei (6.3%), Campylobacter upsaliensis (4.2%), and Campylobacter fetus subsp. fetus (2.1%). The analyzed broiler flocks were positive for Campylobacter in 81.8% of the cases, thus characterizing the occurrence of this pathogen in a broiler-producing region in southern Brazil. These results highlight the importance of programs targeted at the reduction of Campylobacter in poultry products, in order to minimize the risks for consumers.

Effects of probiotics and maternal vaccination on Salmonella enteritidis infection in broiler chicks.

The effects of probiotics and maternal vaccination with an inactivated Salmonella Enteritidis (SE) vaccine on day-old chicks challenged with SE were evaluated. A 2 X 3 factorial arrangement was used (with or without probiotics; breeders nonvaccinated, vaccinated intramuscularly, or vaccinated intraperitoneally). Three trials were conducted in isolation cabinets and SE challenge was different between trials. The number of SE organisms per chick and the time interval between housing and introduction of seeder birds (hereafter called challenge) were 1.6 X 10(8) and 1 hr (Trial I), 1.8 X 10(6) and 12 hr (Trial II), and 1.2 X 10(4) and 24 hr (Trial III). SE recovery was assessed in ceca and liver at 3, 5, and 7 days postchallenge, and the number of colony-forming units (CFU) in ceca was evaluated at 5 and 7 days postchallenge. The number of SE (log CFU) in the ceca reduced 0.56 log (from 7.59 to 7.03) and 1.45 log (7.62 to 6.17) because of the treatment with probiotics in Trials II and III, respectively. The greater reduction in Trial III indicates the importance of the early use of probiotics on the prevention of SE infection. Treatment with probiotics resulted in a smaller number of SE-positive livers after 5 days postchallenge on Trial III. Although there was no significant effect of maternal vaccination on the number of SE CFU in the ceca, a significant effect of maternal vaccination on the SE CFU was observed in the liver, but not in the ceca at 5 days after challenge.

Use of artificial neural networks to estimate production variables of broilers breeders in the production phase.

1. Although the poultry industry uses state-of-the-art equipment and up-to-date services, in Brazil it generally makes decisions involving all its production variables based on purely subjective criteria. This paper reports the use of artificial neural networks to estimate performance in production birds belonging to a South Brazilian poultry farm. 2. Recorded data from 22 broiler production breeder flocks were obtained, from April, 1998 to December, 1999, which corresponded to 689 data lines of weekly recordings. 3. These data were processed by artificial neural networks using the software NeuroShell 2 version 4.0 (Ward Systems Group). The artificial neural network models generated were compared and selected based on their largest determination coefficient (R2), lowest Mean Squared Error (MSE), as well as on a uniform scatter in the residual plots. The authors conclude that it is possible to explain the performance variables of production birds, with the use of artificial neural networks. 4. The method allows the decisions made by the technical staff to be based on objective, scientific criteria, allows simulations of the consequences related to these decisions, and reports the contribution of each variable to the variables under study.

Polymerase chain reaction (PCR) for the detection of Salmonella in artificially inoculated chicken meat.

The aim of this study was to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in artificially contaminated chicken meat. Tests were performed with different dilutions of Salmonella Typhimurium or Salmonella Enteritidis cells (10(-7), 10(-8) or 10(-9) CFU/mL) inoculated in chicken meat samples, in order to establish the limits of detection, incubation times (0, 6, 8 and 24 hours of pre-enrichment in PBW 1%) and three DNA extraction protocols (phenol-chloroform, thermal treatment and thermal treatment and Sephaglass). The assay was able to detect until 10(-9) CFU/mL of initial dilution of Salmonella cells inoculated in chicken meat, which allows detection of Salmonella within 48 hours, including 24 hours of pre-enrichment and using the phenol-chloroform DNA extraction protocol. As the results are obtained in a shorter time period than that of microbiological culture, this procedure will be useful in the methodology for detection of Salmonella in chicken.

Diagnosis of skin lesions in condemned or downgraded broiler carcasses - a microscopic and macroscopic study.

Eight hundred skin samples from broiler carcasses condemned or downgraded for skin lesions were collected at five processing plants. Histologically, 45.25% were cellulitis, 19.00% were atypical fowlpox, 3.25% were dermal squamous cell carcinoma (DSCC), 17.00% were non-specific dermatitis, 0.5% were focal haemorrhages and 0.25% were haemangiomas. Of the samples, 14.75% were histologically normal. Macroscopically, increased thickening of the skin was a frequent finding occurring in all the skin diseases, as well as skin discoloration, dark nodules in feather follicles and crusts. Skin scratches were associated with all of the diseases, except DSCC. Crater-like ulcers occurred in DSCC and fowlpox. Cellulitis was more frequent on the abdomen, fowlpox on the dorsum, DSCC in thighs, and non-specific dermatitis on the dorsum, thighs, legs and breast. It was concluded that macroscopic changes are not specific and examinations at slaughterhouses do not allow classification of broiler skin diseases. Histological examination appears to be an important tool to provide more accurate data.

Squamous cell carcinoma-like and pox lesions occurring simultaneously in chorioallantoic membranes of chicken embryos inoculated with materials from squamous cell carcinoma and pox lesions in broiler chickens.

The finding of closely associated squamous cell carcinoma (SCC)-like lesions and pox lesions in chorioallantoic membranes (CAMs) inoculated with skin and palate samples taken from broilers is described. The samples were obtained from two broilers coming from different flocks that were not vaccinated against fowl pox. Both birds presented skin lesions, which were diagnosed in one bird as fowl pox, and in the other as SCC. After inoculation of CAMs with fresh tissues from both birds, histologic examination revealed, in all CAMs, lesions that were characteristic of fowl pox together with lesions consistent with those seen in the skin of broilers affected with SCC. This finding was unexpected and may shed some light on the etiology of SCC.

Relationship between shell structure and movement of Salmonella enteritidis across the eggshell wall.

1. Ultrastructural analyses of the eggshells of one strain of commercial layers indicated that the cuticular layer is rarely present as an even covering over the shell surface and the paired shell membranes are invariably pitted with holes larger than bacterial dimensions. 2. The above conditions pertain irrespective of the age of the laying bird. 3. Bacterial penetration of the eggshell is independent of pore numbers. 4. In the absence of a functional cuticle and with the shell membranes removed, bacteria are checked in their movement by structural modifications in the mammillary layer.