Isolation and Antimicrobial Sensitivity of Mycoplasma synoviae and Mycoplasma gallisepticum from Vaccinated Hens in Mexico.

Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) strains were isolated at 39.5 °C to rule out temperature-sensitive strains (ts+) and identified using random amplification of polymorphic DNA. Then, their minimum inhibitory concentrations (MIC100) were calculated in isolated strains from broiler breeders and laying hens vaccinated with ts+ MS-H and ts+ MG TS-11 vaccines in Mexico. We sampled 631 lots of hens. A total of 28 of the 123 MS isolates and 12 of the 23 MG isolates were analyzed using random amplification of polymorphic DNA, of which 24 and 3 matched the DNA banding patterns of the MS-H and MG-F strains, respectively. The isolated MS and MG strains were sensitive to tiamulin and tylosin and showed intermediate sensitivity or resistance to lincomycin, florfenicol, erythromycin, enrofloxacin, and curcumin. Although both the MS and MG strains were sensitive to the same antibiotics (MIC100 lower than 1 mg mL-1), the MG strains were 5 to 10 times more sensitive than the MS strains. MS is the most frequently isolated mycoplasma in Mexican poultry production. The MS vaccine used (ts+ MS-H) could reverse its thermosensitivity and therefore could regain its virulence. MS was less sensitive to tiamulin and tylosin compared to MG.

Molecular detection of Mycoplasma synoviae and avian reovirus infection in arthritis and tenosynovitis lesions of broiler and breeder chickens in Santa Catarina State, Brazil.

Infectious arthritis or tenosynovitis in broiler and breeder chickens results in major loss of productivity because of reduced growth and downgrading at processing plants. The most common causative agents of avian infectious arthritis are the bacterium Mycoplasma synoviae and avian reoviruses (ARVs) (family Reoviridae, genus Orthoreovirus). In this study, we evaluated the occurrence of these two pathogens in arthritis or tenosynovitis lesions of broilers and breeder flocks in southern Brazil using molecular detection. Tissue sections from tibiotarsal joints with visible lesions from 719 broilers and 505 breeders were analysed using pathogen-specific polymerase chain reaction (PCR) assays. In breeders, 41.2% (n = 296) of lesions were positive for M. synoviae, 26.4% (n = 190) were positive for ARV, while co-infection was present in 12.2% (n = 88) of the samples. In broilers, 20.8% (n = 105) of lesions were positive for M. synoviae, 11.9% (n = 60) for ARV and 7.7% (n = 39) of these cases were positive for both pathogens. Post-mortem examination revealed lesions with varying degrees of gross pathological severity. Histopathological examination showed intense, diffuse lymphohistiocytic inflammatory infiltrates with heterophil accumulation, primarily in the synovial capsule and digital flexor tendon, in all samples. Improved strategies for early detection and control of these major avian pathogens are highly desirable for preventing the spread of infection and reducing economic losses in the poultry industry.

Detection of Antibodies to Seven Priority Pathogens in Backyard Poultry in Trinidad, West Indies.

Backyard poultry farms in Trinidad and Tobago (T&T) play a vital role in providing food and income for rural communities. There is currently no information on the presence and circulation of pathogens in backyard poultry farms in T&T, and little is known in relation to the potential risks of spread of these pathogens to the commercial poultry sector. In order to address this, serum samples were collected from 41 chickens on five backyard farms taken from selected locations in Trinidad. Samples were tested for antibodies to seven priority pathogens of poultry by enzyme-linked immunosorbent assay (ELISA). Antibodies were detected in 65% (CI 95%: 50-78%) of the sampled birds for Infectious bronchitis virus (IBV), 67.5% (CI 95%: 52-80%) for Infectious bursal disease virus (IBDV), 10% (CI 95%: 4-23%) for Newcastle disease virus (NDV), 0% (CI 95%: 0-0%) for Avian influenza virus (AIV), 0% (CI 95%: 0-0%) for West Nile virus (WNV), 31.7% (CI 95%: 20-47%) for Mycoplasm gallisepticum/synoviae and 0% (CI 95%: 0-0%) for Salmonella enterica serotype Enteritidis. These results reveal the presence and circulation of important pathogens of poultry in selected backyard farms in Trinidad. The results provide important information which should be taken into consideration when assessing the risks of pathogen transmission between commercial and backyard poultry farms, as well as between poultry and wild birds.

Pesquisa de Mycoplasma em aves da família Psittacidae mantidas em diferentes cativeiros no Brasil Central / Research of Mycoplasma in the family Psittacidae kept in different captivities in Central Brazil

O presente estudo teve como objetivo investigar a presença de Mycoplasma gallisepticum e M. synoviae em diferentes espécies de psitacídeos cativos no Brasil Central. Um total de 300 amostras foram coletadas e corresponderam a 41 espécies de psitacídeos da fauna brasileira, provenientes do CETAS, criadouro comercial e criadouro conservacionista. Quatorze espécies apresentaram amostras positivas para M. gallisepticum destacando a maracanã-verdadeira (Primolius maracana) (01/02, 50%), a arara-canindé (Ara ararauna) (15/48, 33,3%) e a jandaia-verdadeira (Aratinga jandaia) (03/10, 30%). Amostras do CETAS obtiveram total de 21,62% (16/74) de amostras positivas, do criadouro comercial 15,7% (19/121) e do criadouro conservacionista 6,66% (7/105). Apenas três espécies foram positivas para M. synoviae sendo essas, a maracanã-pequena (Primolius maracana) (1/10 - 10%), arara-macao (Ara macao) (1/12, 8,3%) e arara-canindé (Ara ararauna) (2/48, 4,1%). O CETAS obteve 2,7% (2/74) de amostras positivas totais, enquanto o criadouro conservacionista obteve total de 1,9% (2/105) de amostras. Não ocorreram amostras positivas para M. synoviae no criadouro comercial. Os resultados mostraram um considerável número de amostras positivas para M. gallisepticum em espécies da família Psittacidae, indicando que estes animais podem ser uma fonte de infecção silenciosa para outras aves, uma vez que não apresentaram sintomatologia clínica.(AU)

The study aimed to investigate the presence of Mycoplasma gallisepticum and M. synoviae in different species of captive parrots, in Central Brazil. A total of 300 samples were collected from 41 brazilian species of Psittacidae at three captivities: Centro de Triagem de Animais Silvestres (CETAS), a conservation and a commercial captivity. Fourteen species presented positive samples for M. galisepticum, the most affected were blue-winged macaw (Primolius maracana) (01/02, 50%), blue-and-yellow macaw (Ara ararauna) (15/48, 33.3%), and jandaia parakeet (Aratinga jandaia) (03/10, 30%). CETAS facility showed 21.62% (16/74) of positive samples, while the commercial captivity showed 15.7% (19/121), and the conservation captivity 6.66% (7/105). Only three species presented positive samples for M. synoviae: blue-winged macaw (Primolius maracana) (1/10, 10%), scarlet macaw (Ara macao) (1/12, 8.3%) e blue-and-yellow macaw (Ara ararauna) (2/48, 4.1%). CETAS facility showed 2.7% (2/74) of positive samples, while the conservation captivity presented 1.9% (2/105), and no positive samples were found in the commercial captivity. Results showed a considerable number of positive samples for M. galisepticum in species of Psittacidae family, indicating that these animals can be a silent source of infection for other birds, since they did not present clinical symptoms.(AU)

Pesquisa de Mycoplasma em aves da família Psittacidae mantidas em diferentes cativeiros no Brasil Central / Research of Mycoplasma in the family Psittacidae kept in different captivities in Central Brazil

O presente estudo teve como objetivo investigar a presença de Mycoplasma gallisepticum e M. synoviae em diferentes espécies de psitacídeos cativos no Brasil Central. Um total de 300 amostras foram coletadas e corresponderam a 41 espécies de psitacídeos da fauna brasileira, provenientes do CETAS, criadouro comercial e criadouro conservacionista. Quatorze espécies apresentaram amostras positivas para M. gallisepticum destacando a maracanã-verdadeira (Primolius maracana) (01/02, 50%), a arara-canindé (Ara ararauna) (15/48, 33,3%) e a jandaia-verdadeira (Aratinga jandaia) (03/10, 30%). Amostras do CETAS obtiveram total de 21,62% (16/74) de amostras positivas, do criadouro comercial 15,7% (19/121) e do criadouro conservacionista 6,66% (7/105). Apenas três espécies foram positivas para M. synoviae sendo essas, a maracanã-pequena (Primolius maracana) (1/10 - 10%), arara-macao (Ara macao) (1/12, 8,3%) e arara-canindé (Ara ararauna) (2/48, 4,1%). O CETAS obteve 2,7% (2/74) de amostras positivas totais, enquanto o criadouro conservacionista obteve total de 1,9% (2/105) de amostras. Não ocorreram amostras positivas para M. synoviae no criadouro comercial. Os resultados mostraram um considerável número de amostras positivas para M. gallisepticum em espécies da família Psittacidae, indicando que estes animais podem ser uma fonte de infecção silenciosa para outras aves, uma vez que não apresentaram sintomatologia clínica.(AU)

The study aimed to investigate the presence of Mycoplasma gallisepticum and M. synoviae in different species of captive parrots, in Central Brazil. A total of 300 samples were collected from 41 brazilian species of Psittacidae at three captivities: Centro de Triagem de Animais Silvestres (CETAS), a conservation and a commercial captivity. Fourteen species presented positive samples for M. galisepticum, the most affected were blue-winged macaw (Primolius maracana) (01/02, 50%), blue-and-yellow macaw (Ara ararauna) (15/48, 33.3%), and jandaia parakeet (Aratinga jandaia) (03/10, 30%). CETAS facility showed 21.62% (16/74) of positive samples, while the commercial captivity showed 15.7% (19/121), and the conservation captivity 6.66% (7/105). Only three species presented positive samples for M. synoviae: blue-winged macaw (Primolius maracana) (1/10, 10%), scarlet macaw (Ara macao) (1/12, 8.3%) e blue-and-yellow macaw (Ara ararauna) (2/48, 4.1%). CETAS facility showed 2.7% (2/74) of positive samples, while the conservation captivity presented 1.9% (2/105), and no positive samples were found in the commercial captivity. Results showed a considerable number of positive samples for M. galisepticum in species of Psittacidae family, indicating that these animals can be a silent source of infection for other birds, since they did not present clinical symptoms.(AU)

Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine / Avaliação de uma PCR multiplex para detecção e diferenciação de Mycoplasma synoviae, Mycoplasma gallisepticum e Mycoplasma gallisepticum cepa F vacinal

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD) of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR) demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%), and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks.(AU)

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) são micoplasmas que causam infecção de maior preocupação para a indústria avícola. MG é a bactéria responsável pela infecção, comumente designada, como doença crônica respiratória (DCR) de galinhas e sinusite infecciosa de perus. MS é responsável por infecções subclínicas do trato respiratório superior e tenosinovite ou bursite em galinha e perus. A reação da PCR multiplex foi padronizada para detectar simultaneamente MS, MG cepa de campo e MG-F cepa vacinal. A PCR genérica para detecção de qualquer espécie de Mycoplasma foi realizada e comparada a PCR multiplex e a PCR com primers específicos. O total de 129 amostras de suabes de traqueia foi coletado de reprodutoras pesadas, poedeiras e frangos em sete diferentes empresas avícolas e então foram examinados por PCR multiplex. O sistema da PCR multiplex demonstrou ser muito rápido, sensível e específico. Então, os resultados mostraram uma alta prevalência de MS nos lotes examinados ( 27,9%), e indica que MS é um patógeno recorrente nos lotes de aves comerciais brasileiro.(AU)

Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine / Avaliação de uma PCR multiplex para detecção e diferenciação de Mycoplasma synoviae, Mycoplasma gallisepticum e Mycoplasma gallisepticum cepa F vacinal

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD) of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR) demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%), and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks...

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) são micoplasmas que causam infecção de maior preocupação para a indústria avícola. MG é a bactéria responsável pela infecção, comumente designada, como doença crônica respiratória (DCR) de galinhas e sinusite infecciosa de perus. MS é responsável por infecções subclínicas do trato respiratório superior e tenosinovite ou bursite em galinha e perus. A reação da PCR multiplex foi padronizada para detectar simultaneamente MS, MG cepa de campo e MG-F cepa vacinal. A PCR genérica para detecção de qualquer espécie de Mycoplasma foi realizada e comparada a PCR multiplex e a PCR com primers específicos. O total de 129 amostras de suabes de traqueia foi coletado de reprodutoras pesadas, poedeiras e frangos em sete diferentes empresas avícolas e então foram examinados por PCR multiplex. O sistema da PCR multiplex demonstrou ser muito rápido, sensível e específico. Então, os resultados mostraram uma alta prevalência de MS nos lotes examinados ( 27,9%), e indica que MS é um patógeno recorrente nos lotes de aves comerciais brasileiro...

Seroprevalence of Salmonella and Mycoplasma in commercial broilers, backyard chickens, and spent hens in the region of Triângulo Mineiro, State of Minas Gerais, Brazil

Avian salmonellosis and mycoplasmosis are infectious diseases that, in addition of causing lack of flock uniformity, represent a hazard to human health. The objective of the present study was to evaluate the seroprevalence of mycoplasmosis and salmonellosis in commercial broilers, backyard chickens, and spent hens slaughtered at a processing plant with local health inspection in Uberlândia, MG, Brazil. A total of 210 samples were randomly collected at the time of bleeding. Samples were submitted to rapid plate serum agglutination test (RSA) for the classification of Salmonella pullorum, Salmonella gallinarum, Mycoplasma gallisepticum and Mycoplasma synoviae. In order to increase result specificity, mycoplasmosis-positive samples were submitted to hemagglutination inhibition test (HI). No samples presented detectable antibodies against Salmonella pullorum or Salmonella gallinarum in the RSA test. Only Mycoplasma synoviae was detected in 14% of the backyard chickens and 0.74% in commercial broilers, whereas no antibodies were detected in spent hens. The seroprevalence rates found in the present study emphasize the need of keeping chicken flocks free from disease using effective biosafety systems.

Seroprevalence of Salmonella and Mycoplasma in commercial broilers, backyard chickens, and spent hens in the region of Triângulo Mineiro, State of Minas Gerais, Brazil

Avian salmonellosis and mycoplasmosis are infectious diseases that, in addition of causing lack of flock uniformity, represent a hazard to human health. The objective of the present study was to evaluate the seroprevalence of mycoplasmosis and salmonellosis in commercial broilers, backyard chickens, and spent hens slaughtered at a processing plant with local health inspection in Uberlândia, MG, Brazil. A total of 210 samples were randomly collected at the time of bleeding. Samples were submitted to rapid plate serum agglutination test (RSA) for the classification of Salmonella pullorum, Salmonella gallinarum, Mycoplasma gallisepticum and Mycoplasma synoviae. In order to increase result specificity, mycoplasmosis-positive samples were submitted to hemagglutination inhibition test (HI). No samples presented detectable antibodies against Salmonella pullorum or Salmonella gallinarum in the RSA test. Only Mycoplasma synoviae was detected in 14% of the backyard chickens and 0.74% in commercial broilers, whereas no antibodies were detected in spent hens. The seroprevalence rates found in the present study emphasize the need of keeping chicken flocks free from disease using effective biosafety systems.(AU)

Occurrence of Mycoplasma synoviae on commercial poultry farms of Pernambuco, Brazil / Ocorrência de Mycoplasma synoviae em granjas comerciais da avicultura de Pernambuco

The state of Pernambuco is the largest producer of eggs in the North and Northeast of Brazil and second one in the broiler production. Mycoplasmas are important avian pathogens, which cause respiratory and joint diseases that result in large economic losses. The aim of the present study was to investigate the occurrence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in broilers and commercial laying hens in the state of Pernambuco, Brazil. Tracheal fragments were analyzed from 55 healthy broilers, 35 broilers with respiratory signs and 30 commercial laying hens with respiratory signs, from 24 commercial poultry farms, each sample was composed of a pool of five birds. The bacteriological exam, PCR and nested PCR were used for the detection of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). All samples were negative in bacteriological isolation. In the PCR analyses, seven samples from birds with respiratory signs were positive for MS and one was positive for MG, the latter of which was confirmed as the MG-F vaccine strain. The occurrence of MS in chickens with respiratory signs may indicate inadequate sanitary management on poultry farms, favoring the propagation of mycoplasmosis.(AU)

O estado de Pernambuco é o maior produtor de ovos da região Norte e Nordeste e ocupa a segunda posição na produção de frangos de corte. Os micoplasmas são importantes patógenos aviários que causam doenças respiratórias e sinovite que resultam em grandes perdas econômicas. Objetivou-se pesquisar a ocorrência de Mycoplasma gallisepticum (MG) e Mycoplasma synoviae (MS) em frangos de corte e poedeiras comerciais no Estado de Pernambuco, Brasil. Foram colhidos fragmentos de traquéia de 55 frangos de corte sadios, 35 com sinais respiratórios e de 30 poedeiras comerciais também com sinais respiratórios, provenientes de 24 granjas, cada amostra foi composta por um "pool" de cinco aves. Para detecção de Mycoplasma gallisepticum (MG) e Mycoplasma synoviae (MS) foram utilizados o exame bacteriológico, PCR e Nested-PCR. Todas as amostras apresentaram resultados negativos no exame bacteriológico. Na PCR, sete amostras foram positivas para MS e uma para MG em amostras de aves com sinais respiratórios, sendo a amostra positiva para MG confirmada como cepa vacinal MG-F. A ocorrência de MS em aves com sinais clínicos respiratórios pode indicar ausência de barreiras sanitárias adequadas em granjas de frangos de corte e de poedeira comercial, favorecendo a sua propagação.(AU)

Occurrence of Mycoplasma synoviae on commercial poultry farms of Pernambuco, Brazil / Ocorrência de Mycoplasma synoviae em granjas comerciais da avicultura de Pernambuco

The state of Pernambuco is the largest producer of eggs in the North and Northeast of Brazil and second one in the broiler production. Mycoplasmas are important avian pathogens, which cause respiratory and joint diseases that result in large economic losses. The aim of the present study was to investigate the occurrence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in broilers and commercial laying hens in the state of Pernambuco, Brazil. Tracheal fragments were analyzed from 55 healthy broilers, 35 broilers with respiratory signs and 30 commercial laying hens with respiratory signs, from 24 commercial poultry farms, each sample was composed of a pool of five birds. The bacteriological exam, PCR and nested PCR were used for the detection of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). All samples were negative in bacteriological isolation. In the PCR analyses, seven samples from birds with respiratory signs were positive for MS and one was positive for MG, the latter of which was confirmed as the MG-F vaccine strain. The occurrence of MS in chickens with respiratory signs may indicate inadequate sanitary management on poultry farms, favoring the propagation of mycoplasmosis.

O estado de Pernambuco é o maior produtor de ovos da região Norte e Nordeste e ocupa a segunda posição na produção de frangos de corte. Os micoplasmas são importantes patógenos aviários que causam doenças respiratórias e sinovite que resultam em grandes perdas econômicas. Objetivou-se pesquisar a ocorrência de Mycoplasma gallisepticum (MG) e Mycoplasma synoviae (MS) em frangos de corte e poedeiras comerciais no Estado de Pernambuco, Brasil. Foram colhidos fragmentos de traquéia de 55 frangos de corte sadios, 35 com sinais respiratórios e de 30 poedeiras comerciais também com sinais respiratórios, provenientes de 24 granjas, cada amostra foi composta por um "pool" de cinco aves. Para detecção de Mycoplasma gallisepticum (MG) e Mycoplasma synoviae (MS) foram utilizados o exame bacteriológico, PCR e Nested-PCR. Todas as amostras apresentaram resultados negativos no exame bacteriológico. Na PCR, sete amostras foram positivas para MS e uma para MG em amostras de aves com sinais respiratórios, sendo a amostra positiva para MG confirmada como cepa vacinal MG-F. A ocorrência de MS em aves com sinais clínicos respiratórios pode indicar ausência de barreiras sanitárias adequadas em granjas de frangos de corte e de poedeira comercial, favorecendo a sua propagação.

The effect of eggshell apex abnormalities on table egg quality during storage in 2 seasons of the year.

Mycoplasma synoviae infection of hens has been associated with problems of eggshell quality called eggshell apex abnormalities (EAA). Little is known about the quality of EAA eggs from a commercial point of view, especially during their storage. The study aimed to examine the differences between EAA and normal eggs during storage under controlled conditions in 2 seasons, summer and winter, by comparing internal and external quality parameters. In a conventional egg production farm with white laying hens of varying ages in the city of Bastos, state of São Paulo, Brazil, 232 eggs were used in the summer season and 400 eggs in the winter season. Half of the eggs had EAA, and the other half were considered normal eggs for each season. The eggs were analyzed at 2, 7, 14, 21, and 28 d after being laid and stored from 24.6 to 25.8°C in summer and from 24 to 25°C in winter. There was no difference (P > 0.05) in the average egg weight between EAA and normal eggs at any studied time point, but in both seasons, the weight loss in EAA eggs was higher than in normal eggs. The losses in Haugh unit scores from the first to the last measurements were approximately 40% regardless of egg type or season of production. In comparing eggshell thickness, only the apices of the EAA eggs were thinner (P < 0.0001) than normal eggs in the summer, but in the winter, the EAA egg apices (P < 0.0001) and sides (P = 0.03) were both thinner. The presence of EAA did not affect the eggshell weight (P > 0.05) or eggshell percentage (P > 0.05). The eggshell strength of the EAA eggs was lower (P < 0.0001) than normal eggs in both the summer (16.57%) and winter (19.86%). The presence of EAA did not affect the internal quality of the egg, but was related to a greater loss of external quality and weight during storage.

A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks.

Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.

A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.

A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.(AU)

Detecção do vírus da Influenza Aviária, Paramyxovirus tipo 1 (vírus da Doença de Newcastle), Mycoplasma gallisepticum e Mycoplasma synoviae em aves silvestres e domésticas próximas às granjas avícolas comerciais nas regiões de Mogi das Cruzes e Louveira do Estado de São Paulo / Detection of Influenzavirus, Paramyxovirus I, Mycoplasma gallisepticum and Mycoplasma synoviae in free-ranging birds and backyard chicken around poultry farms in Mogi das Cruzes and Louveira, São Paulo state

Objetivou-se, neste trabalho, detectar o vírus da Influenza aviária, Paramyxovirus tipo 1 (doença de Newcastle), Mycoplasma gallisepticum e Mycoplasma synoviae, respectivamente pelas técnicas de RT-PCR e PCR, em aves domésticas e aves em vida livre próximas às granjas avícolas nas cidades de Mogi das Cruzes e Louveira do Estado de São Paulo. As aves silvestres foram capturadas, anilhadas, submetidas à avaliação de estado geral e à coleta de suabes de orofaringe e cloaca. As aves de subsistência ou fundo de quintal seguiram o mesmo protocolo com a exceção do anilhamento, e tiveram amostras de sangue coletadas para a pesquisa de anticorpos contra o vírus da Doença de Newcastle, Mycoplasma gallisepticum e Mycoplasma synoviae pela técnica de ELISA indireto. Foram considerados os aspectos da biodiversidade entre as espécies silvestres capturadas e a biossegurança nas granjas. As aves silvestres apresentaram resultados negativos nesta pesquisa, no entanto, Mycoplasma gallisepticum e Mycoplasma synoviae foram detectados pela técnica da PCR nas aves de subsistência, assim como apresentaram títulos de anticorpos para os agentes acima citados e para o Paramyxovirus tipo I. Duas granjas não possuíam medidas de biosseguridade adequadas permitindo o contato de animais de vida livre com as aves de fundo de quintal e com as aves de produção, o que pode facilitar a disseminação de patógenos de interesse para a saúde pública e para a avicultura comercial

The aim of this study is to detect avian influenza virus, Newcastle disease virus (Paramyxovirus I), Mycoplasma gallisepticum and Mycoplasma synoviae in backyard chicken and wildlife birds around commercial poultry farms using RT-PCR and PCR. The birds were captured with mist nets, identified with alluminium leg rings, subjected to the assessment of clinical conditions and samples were collected by oral and cloacal swabs. The same was done with backyard chicken without the identification with leg rings. Blood samples were collected from backyard chicken and tested for antibodies against Mycoplasma gallisepticum, Mycoplasma synoviae and Paramyxovirus I by indirect ELISA test. This study was conducted in Mogi das Cruzes and Louveira, São Paulo state, where the commercial poultry is considered an activity of great importance. The results were negative to wild birds, but we could detect Mycoplasma gallisepticum and Mycoplasma synoviae by PCR and antibodies titles for Mycoplasma gallisepticum, Mycoplasma synoviae and Newcastle disease in backyard chickens.Two farms didn´t have appropriate biosecurity measures, allowing intense contact with free-living birds, backyard chicken and poultry facilitating spread of pathogens with concern to human health and poultry farms

DETECCIÓN Y DIFERENCIACIÓN DE Mycoplasma gallisepticum Y Mycoplasma synoviae MEDIANTE LA TÉCNICA DE PCR A PARTIR DE HISOPOS TRAQUEALES DE AVES CON SÍNTOMAS RESPIRATORIOS / Detection and Differentiation of Mycoplasma gallisepticum and Mycoplasma synoviaeby PCR from Tracheal Swabs from Birds with Respiratory Symptoms

Los micoplasmas son importantes patógenos en las aves por ser responsables de cuadros respiratorios que ocasionan grandes pérdidas económicas a la industria avícola alrededor del mundo. Existen principalmente dos especies de micoplasmas como causantes de enfermedad en aves comerciales, el Mycoplasma gallisepticum(MG) y el Mycoplasma synoviae(MS). Teniendo en cuenta su importancia y la necesidad de conocer y diferenciar las diferentes especies de micoplasmas presentes en las explotaciones avícolas, se tomaron 91 muestras de hisopos traqueales de aves con síntomas respiratorios, provenientes de igual número de granjas de pollo de engorde, ponedoras comerciales y reproductoras pesadas ubicadas en los departamentos de Cundinamarca y Boyacá, Colombia, y se determinó la presencia de MG y MS por la técnica de PCR. La prevalencia determinada fue de 39,6 % para MG y 47,3 % para MS, encontrándose diferencias estadísticamente significativas cuando se comparó la positividad a MG y MS y el tipo de explotación (p < 0,05), siendo mayor la presentación en ponedoras y reproductoras que en explotaciones de pollo de engorde. Se encontraron diferencias cuando se compararon los resultados en diferentes grupos etáreos, siendo mayor el porcentaje de positividad para MG y MS en las aves con edades entre 20 y 60 semanas tanto en ponedoras comerciales como en reproductoras, mientras que en el grupo de pollo de engorde se encontró una mayor positividad para MG en aves de cinco semanas y para MS en aves de dos semanas.

Mycoplasmas are worldwide pathogens that affect the poultry industry causing respiratory illness which cause a negative economic impact. Two mycoplasmas species are the most important in the commercial poultry: Mycoplasma gallisepticum(MG) and Mycoplasma synoviae(MS). By its importance and necessity to know and differentiate between mycoplasmas species in locals poultry houses this study used the PCR technique like a diagnosis tool, using tracheal swabs from bird with respiratory symptoms. A total of 91 samples from broilers, layers and breeders farms located in the departments of Cundinamarca and Boyacá was processed. The punctual prevalence founded in this study was 39.6 % for MG and 47.3 % for MS. Statistical differences for type of production and positive samples for MG y MS (p < 0.05) were founded, a bigger number of positive samples from layers and breeder in comparison to broilers was found. In the same way, the positive samples for the layers and breeder from the age group between 20 and 60 weeks was greater, while for the broilers group most of the positive samples were from five weeks old birds for MG and two weeks old birds for MS.

Características clínicas e anatomo-histopatologicas da infecção experimental mista por Orthoreovirus aviario e Mycoplasma synoviae em frangos de corte / Clinical and histologic lesions of mixed infection with Avian orthoreovirus and Mycoplasma synoviae in broilers

A artrite infecciosa em frangos de corte representa um problema sanitário e econômico de grande impacto, provocando perdas de produtividade e nos processos de produção e industrialização. Os principais agentes etiológicos associados aos casos de artrites e tenossinovites infecciosas em aves são Mycoplasma synoviae (MS) e Orthoreovirus aviario (ARV). Esse trabalho propôs investigar as alterações anatomohistopatológicas causadas pela infecção experimental concomitante por Mycoplasma synoviae e Orthoreovirus aviario em frangos de corte e confirmar a presença dos agentes através das técnicas de PCR e imuno-luorescência indireta (RIFI). Para tal foram utilizados 16 frangos de corte, alojados em cama, com fornecimento de ração e água ad libitum. A infecção experimental foi realizada utilizando amostras atenuadas de MS e de ARV. Clinicamente as aves inoculadas apresentaram apatia e edemaciação da região da articulação tíbiotársica. Após 30 dias procedeu-se a eutanásia e a necropsia das aves. Na análise histopatológica constatou-se o efeito da infecção mista com MS e ARV sobre os diferentes órgãos/tecidos. Todos os animais apresentaram quadro de artrite e tenossinovite caracterizado pela presença de infiltrado inflamatório linfohistiocitário difuso, com acúmulo de heterófilos na cápsula articular/membrana sinovial e tendão flexor digital. Além disso, foi possível observar infiltrado inflamatório na traquéia, nos pulmões e sacos aéreos, no fígado, baço, pericárdio e proventrículo. A utilização da RIFI foi necessária para visualizar a presença de ambos os agentes nas articulações, identificando a presença de antígenos do ARV e do MS. A técnica de PCR constatou positividade do MS na traquéia, pulmões/sacos aéreos, cápsula articular/membrana sinovial e liquido sinovial. Já para o ARV a PCR foi positiva em amostras de fígado, baço, cápsula articular/membrana sinovial e tendão flexor digital. Com base nas lesões observadas e nos dados da literatura, sugere-se a ação concomitante por MS e ARV nos diferentes tecidos.

Infectious arthritis in broiler represents an economic and health problem resulting in severe losses due to retarded growth and down grading at slaughterhouse. The most common agents associated with cases of infectious arthritis in poultry are Mycoplasma synoviae and Avian orthoreovirus. This study proposed to investigate the histopathological changes caused by mixed infection with Mycoplasma synoviae and Avian orthoreovirus in broilers and confirm the presence of the agents through PCR and immunofluorescence assay (IFA). We used 16 broiler chickens, housed in bed, with supply of food and water ad libitum. Ten-day-old broilers were infected with Mycoplasma synoviae and Avian orthoreovirus. Clinically, they showed lethargy and swelling of the hock joint. After 30 days, we proceeded to their euthanasia and necropsy. Histological lesions were observed due to the mixed infection with Mycoplasma synoviae and Avian orthoreovirus in different tissues. The histopathology of the joints was characterized by infiltration of heterophil leucocytes in the synovial membrane and the digital flexor tendon. The inflammatory process was also found in trachea, lungs, air sac, liver, spleen, pericardium and proventriculus. The use of IFA was necessary to verify the presence of both agents in the hock joints, identifying the antigens of Mycoplasma synoviae and Avian orthoreovirus. The presence of M. synoviae was detected by PCR in trachea, lung, air sacs, synovial membrane and synovial fluid. Avian orthoreovirus was detected with PCR in liver, spleen, synovial membrane and digital flexor tendon. In conclusion, this investigation suggests that a synergistic relationship exists between Mycoplasma synoviae and Avian orthoreovirus.

Características clínicas e anatomo-histopatologicas da infecção experimental mista por Orthoreovirus aviario e Mycoplasma synoviae em frangos de corte / Clinical and histologic lesions of mixed infection with Avian orthoreovirus and Mycoplasma synoviae in broilers

A artrite infecciosa em frangos de corte representa um problema sanitário e econômico de grande impacto, provocando perdas de produtividade e nos processos de produção e industrialização. Os principais agentes etiológicos associados aos casos de artrites e tenossinovites infecciosas em aves são Mycoplasma synoviae (MS) e Orthoreovirus aviario (ARV). Esse trabalho propôs investigar as alterações anatomohistopatológicas causadas pela infecção experimental concomitante por Mycoplasma synoviae e Orthoreovirus aviario em frangos de corte e confirmar a presença dos agentes através das técnicas de PCR e imuno-luorescência indireta (RIFI). Para tal foram utilizados 16 frangos de corte, alojados em cama, com fornecimento de ração e água ad libitum. A infecção experimental foi realizada utilizando amostras atenuadas de MS e de ARV. Clinicamente as aves inoculadas apresentaram apatia e edemaciação da região da articulação tíbiotársica. Após 30 dias procedeu-se a eutanásia e a necropsia das aves. Na análise histopatológica constatou-se o efeito da infecção mista com MS e ARV sobre os diferentes órgãos/tecidos. Todos os animais apresentaram quadro de artrite e tenossinovite caracterizado pela presença de infiltrado inflamatório linfohistiocitário difuso, com acúmulo de heterófilos na cápsula articular/membrana sinovial e tendão flexor digital. Além disso, foi possível observar infiltrado inflamatório na traquéia, nos pulmões e sacos aéreos, no fígado, baço, pericárdio e proventrículo. A utilização da RIFI foi necessária para visualizar a presença de ambos os agentes nas articulações, identificando a presença de antígenos do ARV e do MS. A técnica de PCR constatou positividade do MS na traquéia, pulmões/sacos aéreos, cápsula articular/membrana sinovial e liquido sinovial. Já para o ARV a PCR foi positiva em amostras de fígado, baço, cápsula articular/membrana sinovial e tendão flexor digital. Com base nas lesões observadas e nos dados da literatura, sugere-se a ação concomitante por MS e ARV nos diferentes tecidos.(AU)

Infectious arthritis in broiler represents an economic and health problem resulting in severe losses due to retarded growth and down grading at slaughterhouse. The most common agents associated with cases of infectious arthritis in poultry are Mycoplasma synoviae and Avian orthoreovirus. This study proposed to investigate the histopathological changes caused by mixed infection with Mycoplasma synoviae and Avian orthoreovirus in broilers and confirm the presence of the agents through PCR and immunofluorescence assay (IFA). We used 16 broiler chickens, housed in bed, with supply of food and water ad libitum. Ten-day-old broilers were infected with Mycoplasma synoviae and Avian orthoreovirus. Clinically, they showed lethargy and swelling of the hock joint. After 30 days, we proceeded to their euthanasia and necropsy. Histological lesions were observed due to the mixed infection with Mycoplasma synoviae and Avian orthoreovirus in different tissues. The histopathology of the joints was characterized by infiltration of heterophil leucocytes in the synovial membrane and the digital flexor tendon. The inflammatory process was also found in trachea, lungs, air sac, liver, spleen, pericardium and proventriculus. The use of IFA was necessary to verify the presence of both agents in the hock joints, identifying the antigens of Mycoplasma synoviae and Avian orthoreovirus. The presence of M. synoviae was detected by PCR in trachea, lung, air sacs, synovial membrane and synovial fluid. Avian orthoreovirus was detected with PCR in liver, spleen, synovial membrane and digital flexor tendon. In conclusion, this investigation suggests that a synergistic relationship exists between Mycoplasma synoviae and Avian orthoreovirus.(AU)

Monitoria sorológica da micoplasmose em plantéis de aves reprodutoras no brasil através do teste de soroaglutinação rápida

ABSTRACT This study was conducted to evaluate the serological responses of 952 flocks of breeder chickens with ages between 6 and 65 weeks, from several areas of the country during the years of 2003 and 2004. A total of 139,096 and 121,818 serum samples were analyzed by rapid serum agglutination test for Mycoplasma gallisepticum and Mycoplasma synoviae, respectively. The sera were inactivated at 56º C, for 30min and processed by the rapid serum agglutination test with commercial antigens. Similar parts of antigen and serum were added, the solution was mixed and after 2min the presence or not of clots was verified, indicating the antigenantibody formation. Initially crude serum was used, and later serum diluted in the proportion of 1:10 in saline 0.85% of NaCl. Of the analyzed samples, 1.58% (1920) resulted positive for the tested antigen in the dilution 1:10 of M. synoviae and negative for M. gallisepticum. Considering these results, the presence of MS in these flocks can be suspected, indicating that rapid serum agglutination test can be used as a control, to facilitate the laboratorial procedures followed, leading to other serological tests and isolation actions and the identification of agents with greater precision.

RESUMO O estudo foi conduzido com o objetivo de avaliar sorologicamente 952 lotes de galinhas reprodutoras com idade entre 6 e 65 semanas, de diversas regiões do País durante os anos de 2003 e 2004. Foram analisadas 139.096 e 121.818 amostras de soro através da prova de soroaglutinação rápida para Mycoplasma gallisepticum e Mycoplasma synoviae, respectivamente. Os soros foram inativados a 56º C, por 30min e processados à prova de soroaglutinação rápida com antígenos comerciais. Adicionou-se partes iguais de antígeno e soro, homogenizando a mistura e após 2min verificou-se a presença ou não de grumos, indicando a formação de reação antígeno-anticorpo. Inicialmente utilizou-se soro bruto, e posteriormente soro diluído na proporção de 1:10 em salina 0,85% de NaCl. Das amostras analisadas, 1,58% (1920) apresentaram resultado positivo na diluição 1:10 ao antígeno testado de MS e negativas ao antígeno testado de M. gallisepticum. Considerandose esses resultados pode-se suspeitar da presença de M. synoviae nesses plantéis, indicando que a prova de soroaglutinação rápida pode ser utilizada preliminarmente como monitoria, facilitando a conduta laboratorial a ser seguida, direcionando outros testes sorológicos e ações de isolamento e a identificação dos agentes com maior precisão.