Detection and Antimicrobial Resistance Profile of Enteropathogenic (EPEC) and Shigatoxigenic Escherichia coli (STEC) in Conventional and Organic Broiler Chickens

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.(AU)

Genetic variability of Mycoplasma synoviae detected in commercial layers in southeastern Brazil / Variabilidade genética de Mycoplasma synoviae detectado em poedeiras comerciais do sudeste do Brasil

Este estudo teve como objetivo caracterizar genótipos de M. synoviae circulantes em poedeiras comerciais, na Região Sudeste do Brasil. Para estabelecer a relação evolutiva entre as cepas de MS, foi analisada a sequência genética do gene vlhA de 11 cepas de MS identificadas no Rio de Janeiro e em São Paulo, de 10 outras cepas de MS identificadas no Brasil e cujas sequências foram depositadas no banco de dados GenBank, e da cepa vacinal MSH. O método da máxima verossimilhança e o modelo de Kimura com dois parâmetros foram utilizados para comparar as cepas. As sequências obtidas foram depositadas no Genbank, sob os números de acesso OP279775 a OP279785. Foi possível verificar a presença de diferentes cepas circulantes no Brasil, com alta similaridade entre as cepas do Rio de Janeiro pela análise do gene vlhA. As duas cepas paulistas detectadas no presente estudo possuem o baixo percentual (68%) de similaridade, demonstrando a variabilidade das cepas dessa localidade.

Detection of Mycoplasma Synoviae and Other Pathogens in Laying Hens with Respiratory Signs in the Rearing and Production Phases

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.(AU)

Detection of Mycoplasma Synoviae and Other Pathogens in Laying Hens with Respiratory Signs in the Rearing and Production Phases

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.

Proposal of an iELISA for Mycoplasma bovis diagnosis in dairy cattle and associated risk factors / [Proposta de um iELISA para diagnóstico de Mycoplasma bovis em bovinos leiteiros e fatores de risco associados]

Mycoplasma bovis is a highly contagious agent associated with several pathologies in cattle. The detection of reactive antibodies to M. bovis by Indirect Enzyme-Linked Immunosorbent Assay (iELISA) identifies if there was an exposure to the microorganism. The current study aimed to optimize an iELISA from M. bovis total cell antigen, applying it to bovine serum samples, and to evaluate risk factors. Serum samples were obtained from 400 cows from 17 herds from Southeast Brazil. In the optimization of iELISA, the following was established: 2 µg/mL of antigen, sera dilution 1:300, and conjugate dilution 1:15000. The frequency was 62.3% (249/400) of reactive animals and 100% (17/17) of reactive herds. Risk factors were: herds with more than 100 animals (OR= 3.1; CI= 95%); Holstein breed (OR= 72.5; CI= 95%); cows (OR= 29.7; CI= 95%); intensive breeding system (OR= 3.3; CI= 95%); associated small ruminant production (OR= 4.4; CI= 95%); milk production above 500L (OR= 2.9; CI= 95%); no quarantine (OR= 1.5; CI= 95%); mechanical milking (OR= 5.5; CI= 95%) and cases of mastitis (OR= 5.5; CI= 95%). The proposed iELISA was able to detect antibodies reactive to M. bovis in bovine serum. Knowledge of these risk factors can assist in the implementation of prophylactic measures.(AU)

Mycoplasma bovis é um agente altamente contagioso relacionado a várias patologias em bovinos. A detecção de anticorpos reativos a M. bovis por Ensaio de Imunoadsorção Enzimática Indireto (iELISA) identifica se houve exposição ao microrganismo. O presente estudo teve como objetivo otimizar um iELISA de antígeno celular total de M. bovis, aplicando-o a amostras de soro bovino, bem como avaliar fatores de risco. Amostras de soro foram obtidas de 400 vacas de 17 rebanhos da Região Sudeste do Brasil. Na otimização do iELISA foram obtidos: 2µg/mL de antígeno, diluição dos soros 1:300 e do conjugado 1:15000. A frequência de animais reativos foi de 62,3% (249/400) e de 100% (17/17) para os rebanhos. Os fatores de risco foram: rebanhos com mais de 100 animais (OR= 3,1; IC= 95%); raça Holandesa (OR= 72,5;IC= 95%); vacas (OR= 29,7;IC= 95%); sistema intensivo (OR= 3,3; C= 95%); produção de pequenos ruminantes (OR= 4,4;IC=95%); produção de leite acima de 500L (OR= 2,9;IC= 95%); sem quarentena (OR= 1,5;IC= 95%); ordenha mecânica (OR= 5,5;IC= 95%) e casos de mastite (OR= 5,5;IC= 95%). O iELISA proposto foi capaz de detectar anticorpos reativos a M. bovis no soro bovino. O conhecimento desses fatores de risco pode auxiliar na implementação de medidas profiláticas.(AU)

Prevalência e estudo genético de Mycoplasma gallisepticum e M. synoviae em poedeiras comerciais, na região centro-oeste do estado de São Paulo, Brasil / [Prevalence and genetic study of Mycoplasma gallisepticum and M. synoviae strains from commercial laying hens in the Midwest region of Sao Paulo state, Brazil]

O objetivo deste trabalho foi estudar a prevalência de MG e MS e a filogenia das cepas circulantes, comparando-as com outras já descritas em poedeiras comerciais no Brasil. Foram coletados 140 suabes traqueais de poedeiras comerciais com sinais respiratórios em seis granjas da região centro-oeste de São Paulo. As amostras foram avaliadas por PCR, com posterior sequenciamento e análise filogenética das cepas identificadas. Das 140 amostras, 16,4% foram positivas para MG e 68,6% para MS. Houve diferença significativa nas frequências de MG e MS por granja, segundo o teste G de independência (P<0,05). Todas as cepas identificadas de MG e MS de granjas distintas apresentaram similaridade tanto pela lipoproteína para MG quanto pela região 16s rRNA para MS. Neste estudo, foi possível observar altas prevalências dos agentes estudados, sendo a de MS maior que a de MG. Foi detectada infecção mista por MG e MS em 11,4% das amostras e sabe-se que esses micoplasmas podem agir de forma sinérgica, agravando o quadro respiratório. As cepas circulantes identificadas, pela análise das regiões gênicas da lipoproteína para MG e 16S rRNA para MS, são similares em todas as granjas estudadas.(AU)

The aim of this study was to evaluate the prevalence of MG and MS and the phylogeny of the circulating strains, comparing them with others already described in commercial laying hens from Brazil. A total of 140 tracheal swabs were collected from commercial laying hens with respiratory signs in six farms from the western region of São Paulo state. The samples were analyzed by PCR with subsequent sequencing and phylogenetic analysis of the identified strains. From the 140 samples, 68.6% were positive for MS and 16.4% for MG. There was a significant difference in the frequencies of MG and MS per farm according to G Test of independence (P<0.05). All strains identified as MG and MS from distinct farms presented similarity both by lipoprotein to MG and by 16s rRNA region to MS. In this study, it was possible to observe a high prevalence of MS compared to MG. Mixed MG and MS infection was detected in 11.4% of the samples. These mycoplasmas may act synergistically, worsening the respiratory signs. The circulating strains identified by analysis of the lipoprotein for MG and 16S rRNA for MS are similar on all poultry farms studied.(AU)

Occurrence of Avian Mycoplasmas in Free-Living Muscovy-Ducks (Cairina Moschata)

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.(AU)

Prevalência e estudo genético de Mycoplasma gallisepticum e M. synoviae em poedeiras comerciais, na região centro-oeste do estado de São Paulo, Brasil / [Prevalence and genetic study of Mycoplasma gallisepticum and M. synoviae strains from commercial laying hens in the Midwest region of Sao Paulo state, Brazil]

O objetivo deste trabalho foi estudar a prevalência de MG e MS e a filogenia das cepas circulantes, comparando-as com outras já descritas em poedeiras comerciais no Brasil. Foram coletados 140 suabes traqueais de poedeiras comerciais com sinais respiratórios em seis granjas da região centro-oeste de São Paulo. As amostras foram avaliadas por PCR, com posterior sequenciamento e análise filogenética das cepas identificadas. Das 140 amostras, 16,4% foram positivas para MG e 68,6% para MS. Houve diferença significativa nas frequências de MG e MS por granja, segundo o teste G de independência (P<0,05). Todas as cepas identificadas de MG e MS de granjas distintas apresentaram similaridade tanto pela lipoproteína para MG quanto pela região 16s rRNA para MS. Neste estudo, foi possível observar altas prevalências dos agentes estudados, sendo a de MS maior que a de MG. Foi detectada infecção mista por MG e MS em 11,4% das amostras e sabe-se que esses micoplasmas podem agir de forma sinérgica, agravando o quadro respiratório. As cepas circulantes identificadas, pela análise das regiões gênicas da lipoproteína para MG e 16S rRNA para MS, são similares em todas as granjas estudadas.(AU)

The aim of this study was to evaluate the prevalence of MG and MS and the phylogeny of the circulating strains, comparing them with others already described in commercial laying hens from Brazil. A total of 140 tracheal swabs were collected from commercial laying hens with respiratory signs in six farms from the western region of São Paulo state. The samples were analyzed by PCR with subsequent sequencing and phylogenetic analysis of the identified strains. From the 140 samples, 68.6% were positive for MS and 16.4% for MG. There was a significant difference in the frequencies of MG and MS per farm according to G Test of independence (P<0.05). All strains identified as MG and MS from distinct farms presented similarity both by lipoprotein to MG and by 16s rRNA region to MS. In this study, it was possible to observe a high prevalence of MS compared to MG. Mixed MG and MS infection was detected in 11.4% of the samples. These mycoplasmas may act synergistically, worsening the respiratory signs. The circulating strains identified by analysis of the lipoprotein for MG and 16S rRNA for MS are similar on all poultry farms studied.(AU)

Occurrence of Avian Mycoplasmas in Free-Living Muscovy-Ducks (Cairina Moschata)

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.

Detecção de resíduos de enrofloxacina por ensaio imunoenzimático e cromatografia líquida acoplada à espectrometria de massas em ovos comerciais de galinha após tratamento / Investigation of enrofloxacin residues through enzyme-linked immunosorbent assay and liquid cromatography-tandem mass spectrometry in eggs from laying hens after treatment

A enrofloxacina é um dos antimicrobianos mais utilizados na avicultura industrial, e a deposição de resíduos em produtos avícolas, como os ovos, são de grande importância para a saúde pública. Na legislação brasileira não existe padronização do período de carência para o seu uso na produção avícola e não há Limite Máximo de Resíduo (LMR) fixado para enrofloxacina em ovos. Neste estudo, foi utilizado o kit de ELISA comercial (Bioo Scientific(r)) e a LC-MS/MS na pesquisa de enrofloxacina em ovos de 30 galinhas tratadas previamente via água de bebida, com 10mg/kg de enrofloxacina, durante cinco dias. Seis ovos foram coletados diariamente e analisados durante o tratamento e após a sua suspensão, durante 15 dias. A deposição de resíduos obteve níveis máximos no quinto dia de tratamento das aves, declinando gradativamente até não ser detectada a partir do nono dia de suspensão do tratamento. Considerando como base o LMR de 100µg/kg fixado pelo Brasil para tecidos comestíveis de aves e pela União Europeia para músculo, gordura e pele, após seis dias de suspensão do tratamento, os níveis de resíduos foram inferiores a esse limite, tendo como médias 37,43µg/kg na LC-MS/MS e 14,731µg/kg no ELISA. Dentro das condições deste estudo, um período de carência de seis dias seria mais adequado para utilização dos ovos para consumo humano. Foram detectados valores de resíduos nos ovos menores no ELISA em relação à LC-MS/MS para a mesma amostra, mas os dois métodos apresentaram concordância estatística entre si. A LC-MS/MS é o teste recomendado pela legislação brasileira para a análise de resíduos em alimentos; entretanto, pelos resultados obtidos, o kit de ELISA utilizado também pode ser aplicado na detecção de resíduos de enrofloxacina em ovos, com as vantagens de rapidez e simplicidade.(AU)

Enrofloxacin is one of the most used antibiotics in the poultry industry and the deposition of residues in poultry products, such as eggs, are of great concern to public health. In Brazilian law there is no standard withdrawal period for enrofloxacin in eggs and there is no Maximum Residue Limit (MRL) established for this antimicrobial in eggs. In this study, (Bioo Scientific(r)) commercial ELISA kit and LC-MS/MS were used to investigate enrofloxacin in eggs of 30 hens pretreated via drinking water at 10mg/kg of enrofloxacin for five days. Six eggs were collected daily and analyzed during treatment and after the end of treatment, for 15 days. Residues obtained maximum levels on the fifth day of treatment, declined gradually and were no longer detected from the ninth day to the end of treatment. Based on the MRL of 100mg/kg established for edible tissues of poultry by Brazillian law and for muscle, fat and skin, by the European Union, after six days of treatment withdrawal, the residue levels were below that limit, with the average of 37.43mg/kg in LC-MS/MS and 14.731mg/kg in ELISA. Within the conditions of this study, a withdrawal period of six days would be more appropriate to use the eggs for human consumption. The values obtained by ELISA for residues in eggs were lower than those obtained in LC-MS/MS for the same sample, however both methods showed statistical agreement. LC-MS/MS is the recommended method by Brazilian legislation for analysis of residues in food, however, according to the results the ELISA kit used can also be applied to the detection of enrofloxacin residues in eggs, with the advantages of speed and simplicity.(AU)

Eggshell apex abnormalities in a free-range hen farm with mycoplasma synoviae and infectious bronchitis virus in Rio de Janeiro state, Brazil

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.

Eggshell apex abnormalities in a free-range hen farm with mycoplasma synoviae and infectious bronchitis virus in Rio de Janeiro state, Brazil

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.(AU)

PCR for the detection of the gene fel a of escherichia coli in broiler condemned for airsacculitis by the Brazilian Federal Sanitary Inspection / PCR na detecção de gene Fel A de Escherichia Coli em frangos de corte condenados por aerossaculite pela Inspeção Sanitária Federal

Collibacillosis is considered one of the major diseases of the modern poultry industry, due to the significant losses it causes. Escherichia coli contributes not only to the disease itself, by causing weight loss of the birds, but also to the increase in carcasses condemnation during slaughter and processing. Detection of virulence factors in E. coli strains of the APEC pathotype contributes to the characterization and pathogenicity of this agent. PCR techniques have been very helpful in the search for genes that encode those virulence factors. This study aimed to detect the gene Fel A of E. coli by PCR and relate its positivity to low weight in broiler flocks with airsacculitis as diagnosed by the health inspection service. The study involved 40 flocks of broilers slaughtered in a single poultry slaughterhouse, under Federal Sanitary Inspection, located in the state of Rio Grande do Sul, Brazil. Three broilers were randomly selected to obtain one "pool" of three tracheas for each PCR. DNA was extracted using phenol-chloroform and amplified using a pair of primers specific to gene Fel A of E. coli. Of the 40 flocks analyzed by PCR, 35% (14/40) were positive for the gene Fel A. PCR was an effective technique for the detection of gene Fel A in broiler flocks. There was a relationship between the presence of the gene Fel A, weight loss, and increase of the airsacculitis rate.(AU)

A colibacilose é considerada uma das principais doenças da indústria avícola moderna, devido aos grandes prejuízos econômicos causados. A Escherichia coli contribui não só para a doença em si, levando à perda de peso das aves, bem como para o aumento da taxa condenação de carcaças durante o abate e processamento. A detecção de fatores de virulência de cepas de E. coli do patotipo APEC colabora para a caracterização de sua patogenicidade e as técnicas de PCR têm sido muito úteis na pesquisa de genes que os codificam. Este estudo objetivou diagnosticar E. coli pela detecção o gene Fel A por PCR e relacionar a positividade para este agente com o baixo peso em frangos de corte provenientes de lotes condenados por aerossaculite. Foram estudados 40 lotes de frangos de corte abatidos em um matadouro avícola sob Inspeção Sanitária Federal, localizado no Estado do Rio Grande do Sul. Foram colhidos aleatoriamente 3 frangos e obtidos "pools" de três traqueias em cada um deles para PCR. O DNA foi extraído pelo método de fenol-clorofórmio e amplificado com pares de "primers" específicos para gene Fel A de E. coli. Dos 40 lotes analisados pela PCR, 35% (14/40) foram positivos para o gene Fel A. A PCR foi eficaz para a detecção do gene Fel A em lotes de frangos de corte e houve relação entre a presença do gene Fel A, a queda de peso e aumento na taxa de aerossaculite.(AU)