Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan / Detecção molecular de Mycoplasma gallisepticum em diferentes raças de aves de Abbottabad e Rawalpindi, Paquistão

The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Frey’s medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan / Detecção molecular de Mycoplasma gallisepticum em diferentes raças de aves de Abbottabad e Rawalpindi, Paquistão

The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Freys medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.(AU)

O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.(AU)

Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan

Abstract The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Freys medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

Resumo O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan / Detecção molecular de Mycoplasma gallisepticum em diferentes raças de aves de Abbottabad e Rawalpindi, Paquistão

Abstract The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Frey's medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

Resumo O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Isolation of Mycoplasma spp. from Geese with Pneumonia and Identification of Microbial Isolates via Molecular Methods

This study aimed to investigate Mycoplasma species in the lungs of 500 geese with pneumonia from the Kars region (Turkey) via cultural and molecular methods. The samples were cultured on Freys Broth and Agar media. To identify Mycoplasma species a Growth Inhibition Test was used. The identification was continued with species-specific PCR and sequence analysis which provide amplification of the genes dnaX, pcrA, rpoB, and the sequence of the 16S rRNA gene, respectively. In addition, Mycoplasma gallisepticum and Mycoplasma synoviae from pneumonic lung samples were directly analyzed via Multiplex Real-time PCR. As a result, 51 Mycoplasma strains were isolated and 32 were identified as Mycoplasma anatis, 9 as Mycoplasma anseris, 5 as Mycoplasma cloacale and 3 as Mycoplasma anserisalpingitis. Two Mycoplasma isolates that could not be identified were grouped in the same branch as a result of 16S RNA sequencing and their nearest neighbour was found to be Mycoplasma sp. 2045 (GenBankNo.MK615061.1). M. gallisepticum DNA was detected in 3 pneumonic lung samples and M. gallisepticum/M. synoviae DNAs were found simultaneously in 1 sample. While some Mycoplasma species identified in this study consolidated their place as pneumonic agents, some increased their potential to become a pneumonic agent when compared with cases caused by well-recognized Mycoplasma strains. Two isolates were identified as -Mycoplasma spp. as their 16S rRNA gene sequence identity levels scored below the threshold of 98.7% for species demarcation and still need to be defined whether they are possible representatives of a novel Mycoplasma species.(AU)

Isolation of Mycoplasma spp. from Geese with Pneumonia and Identification of Microbial Isolates via Molecular Methods

This study aimed to investigate Mycoplasma species in the lungs of 500 geese with pneumonia from the Kars region (Turkey) via cultural and molecular methods. The samples were cultured on Frey’s Broth and Agar media. To identify Mycoplasma species a Growth Inhibition Test was used. The identification was continued with species-specific PCR and sequence analysis which provide amplification of the genes dnaX, pcrA, rpoB, and the sequence of the 16S rRNA gene, respectively. In addition, Mycoplasma gallisepticum and Mycoplasma synoviae from pneumonic lung samples were directly analyzed via Multiplex Real-time PCR. As a result, 51 Mycoplasma strains were isolated and 32 were identified as Mycoplasma anatis, 9 as Mycoplasma anseris, 5 as Mycoplasma cloacale and 3 as Mycoplasma anserisalpingitis. Two Mycoplasma isolates that could not be identified were grouped in the same branch as a result of 16S RNA sequencing and their nearest neighbour was found to be Mycoplasma sp. 2045 (GenBankNo.MK615061.1). M. gallisepticum DNA was detected in 3 pneumonic lung samples and M. gallisepticum/M. synoviae DNAs were found simultaneously in 1 sample. While some Mycoplasma species identified in this study consolidated their place as pneumonic agents, some increased their potential to become a pneumonic agent when compared with cases caused by well-recognized Mycoplasma strains. Two isolates were identified as -Mycoplasma spp. as their 16S rRNA gene sequence identity levels scored below the threshold of 98.7% for species demarcation and still need to be defined whether they are possible representatives of a novel Mycoplasma species.

Incidence of Mycoplasma gallisepticum and Mycoplasma synoviae in broiler flocks at the Federal District of Brazil and its surrounding areas / Incidência de lotes de frangos afetados por Mycoplasma gallisepticum e Mycoplasma synoviae no Distrito Federal e Entorno, Brasil

Mycoplasma is an important avian pathogen that can cause both respiratory disease and synovitis in birds, resulting in considerable economic losses to the poultry industry worldwide. This study aimed to determine the incidence of Mycoplasma gallisepticum and M. synoviae in broiler flocks at the Federal District and its surrounding regions using polymerase chain reaction (PCR). All slaughtered lots (57 flocks) were analyzed from July to November in one of the two slaughterhouses at the Federal District with Federal inspection services. Approximately 10 samples of broiler tracheae per slaughtered batch were collected from the evisceration line. The results obtained from the accumulated incidence over the study period were 7.02% and 35.09% for M. gallisepticum and M. synoviae, respectively. A greater concentration of flocks affected by M. synoviae was observed during October. The sample design as well as the PCR technique assisted in detecting both agents in the broiler batches in the first epidemiological study of these two agents in the region.(AU)

Os micoplasmas são importantes patógenos aviários, que podem causar doenças respiratórias e sinovites em aves, resultando em consideráveis perdas econômicas para a indústria avícola em todo o mundo. O objetivo deste estudo foi determinar a incidência de Mycoplasma gallisepticum e Mycoplasma synoviae em lotes de frangos na região do Distrito Federal e Entorno, por meio da reação em cadeia de polimerase (PCR). Todos os lotes abatidos (57 lotes) foram analisados durante os meses de julho a novembro, em um dos dois abatedouros frigoríficos do Distrito Federal com Serviço de Inspeção Federal. Na linha de evisceração foram coletadas cerca de 10 amostras de traqueia de frangos de corte por lote abatido. Os resultados obtidos da incidência acumulada no período de estudo foram de 7.02% para M. gallisepticum e 35,09% M. synoviae. Uma maior concentração do número de lotes afetados por M. synoviae foi observada durante o mês de outubro. O desenho amostral, assim como a técnica de PCR, permitiu a detecção de ambos os agentes nos lotes de frangos de corte analisados, sendo este o primeiro estudo epidemiológico desses dois agentes na região de estudo.(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.(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.

Investigation of Mycoplasma spp. in birds of the Rio de Janeiro Zoo by isolation and PCR / Investigação de Mycoplasma spp. em aves do Zoológico do Rio de Janeiro por isolamento e PCR

Brazil is one of the countries with the most abundant avifauna in the world. The confinement of birds associated with close contact with other animals and humans favor the spread of agents of respiratory diseases. Among them, mycoplasmas can cause asymptomatic or apparent disease that manifests in birds by coughing, sneezing, rales, conjunctivitis, ocular and nasal discharge. Several described mycoplasmas cause disease in birds, especially Mycoplasma gallisepticum (MG) and Mycoplasma synoviae(MS). The diagnosis of Mycoplasma spp. can be done by clinical observation and laboratory analysis. Molecular diagnosis by PCR was boosted by its speed, sensitivity, and low cost of agent isolation techniques that take up to 21 days to complete. This study aimed to verify the occurrence of Mycoplasma spp. in birds of the Rio de Janeiro Zoo (Rio Zoo), by isolation and PCR. Of the total 635 birds from the Rio Zoo, 81 were studied for detection of Mycoplasma spp., when taken for routine health assessment exams. These birds belonged to the following orders: Psittaciformes (45), Accipitriformes (18), Galliformes (7), Piciformes (5), Strigiformes (4), Falconiformes (1) and Cariamiformes (1), all individuals already identified by microchip or leg-ring. There was no isolation of mycoplasmas in any of the samples tested, whereas, in the PCR, 62.96% (51/81) were positive, with 1.96% (1/51) identified as MG and 19.61% (10/51) as MS, representing 1.23% (1/81) and 12.34% (10/81) of the total population studied. PCR was shown to be a more effective technique than isolation in the detection of Mycoplasma spp. in birds. It was possible to detect mycoplasmas in birds from Riozoo with no clinical respiratory signs, with higher MS prevalence than MG. The positivities for Mycoplasma spp., MS, and MG were different among the orders studied, being the highest occurrence in birds of prey, followed by Galliformes and Piciformes...(AU)

O Brasil é um dos países com maior avifauna do mundo. O confinamento de aves associado ao contato próximo a outros animais e seres humanos favorece a disseminação de agentes etiológicos causadores de doenças respiratórias. Dentre eles, os micoplasmas podem causar doença assintomática ou aparente que se manifesta em aves por espirros, estertores, conjuntivite, corrimentos oculares e nasais. São diversos os micoplasmas descritos causadores de doença em aves, com destaque para Mycoplasma gallisepticum (MG) e Mycoplasma synoviae (MS). O diagnóstico de Mycoplasma spp. pode ser feito pela observação clínica e análises laboratoriais. O diagnóstico molecular pela Reação em Cadeia da Polimerase (PCR) ganhou impulso por sua rapidez, sensibilidade e baixo custo em relação às técnicas de isolamento do agente que levam até 21 dias para conclusão do gênero Mycoplasma. Objetivou-se verificar a ocorrência da infecção por Mycoplasma spp. em aves no Zoológico do Rio de Janeiro (Rio Zoo), por isolamento e PCR. Do plantel de 635 aves do Rio Zoo, foram estudadas 81 para detecção de Mycoplasma spp., quando contidas para exames rotineiros de avaliação da condição de saúde. Essas aves eram pertencentes às ordens Psittaciformes (45), Accipitriformes (18), Galliformes (7), Piciformes (5), Strigiformes (4), Falconiformes (1) e Cariamiformes (1), todas já identificadas por microchip ou por anilha. Não houve isolamento de micoplasmas em nenhuma das amostras testadas, enquanto na PCR, 62,96% (51/81) foram positivas, sendo 1,96% (1/51) identificadas como MG e 19,61% (10/51) como MS, representando 1,23% (1/81) e 12,34% (10/81) da população total estudada. A PCR demonstrou ser uma técnica mais efetiva que o isolamento na detecção de Mycoplasma spp. em aves. Foi possível detectar micoplasmas nas aves do Riozoo sem sinal clínico respiratório, tendo MS maior prevalência do que MG...(AU)

Investigation of Mycoplasma spp. in birds of the Rio de Janeiro Zoo by isolation and PCR / Investigação de Mycoplasma spp. em aves do Zoológico do Rio de Janeiro por isolamento e PCR

Brazil is one of the countries with the most abundant avifauna in the world. The confinement of birds associated with close contact with other animals and humans favor the spread of agents of respiratory diseases. Among them, mycoplasmas can cause asymptomatic or apparent disease that manifests in birds by coughing, sneezing, rales, conjunctivitis, ocular and nasal discharge. Several described mycoplasmas cause disease in birds, especially Mycoplasma gallisepticum(MG) andMycoplasma synoviae(MS). The diagnosis ofMycoplasmaspp. can be done by clinical observation and laboratory analysis. Molecular diagnosis by PCR was boosted by its speed, sensitivity, and low cost of agent isolation techniques that take up to 21 days to complete. This study aimed to verify the occurrence ofMycoplasmaspp. in birds of the Rio de Janeiro Zoo (Rio Zoo), by isolation and PCR. Of the total 635 birds from the Rio Zoo, 81 were studied for detection ofMycoplasmaspp., when taken for routine health assessment exams. These birds belonged to the following orders: Psittaciformes (45), Accipitriformes (18), Galliformes (7), Piciformes (5), Strigiformes (4), Falconiformes (1) and Cariamiformes (1), all individuals already identified by microchip or leg-ring. There was no isolation of mycoplasmas in any of the samples tested, whereas, in the PCR, 62.96% (51/81) were positive, with 1.96% (1/51) identified as MG and 19.61% (10/51) as MS, representing 1.23% (1/81) and 12.34% (10/81) of the total population studied. PCR was shown to be a more effective technique than isolation in the detection ofMycoplasmaspp. in birds. It was possible to detect mycoplasmas in birds from Riozoo with no clinical respiratory signs, with higher MS prevalence than MG. The positivities forMycoplasmaspp., MS, and MG were different among the orders studied, being the highest occurrence in birds of prey, followed by Galliformes and Piciformes. The presence of MG and MS in birds of Rio de Janeiro Zoo confirms the circulation of these agents and the need for further studies on the dissemination of mycoplasmas in zoos for the epidemiological analysis of these bacteria in these places.(AU)

O Brasil é um dos países com maior avifauna do mundo. O confinamento de aves associado ao contato próximo a outros animais e seres humanos favorece a disseminação de agentes etiológicos causadores de doenças respiratórias. Dentre eles, os micoplasmas podem causar doença assintomática ou aparente que se manifesta em aves por espirros, estertores, conjuntivite, corrimentos oculares e nasais. São diversos os micoplasmas descritos causadores de doença em aves, com destaque para Mycoplasma gallisepticum (MG) e Mycoplasma synoviae (MS). O diagnóstico de Mycoplasma spp. pode ser feito pela observação clínica e análises laboratoriais. O diagnóstico molecular pela Reação em Cadeia da Polimerase (PCR) ganhou impulso por sua rapidez, sensibilidade e baixo custo em relação às técnicas de isolamento do agente que levam até 21 dias para conclusão do gênero Mycoplasma. Objetivou-se verificar a ocorrência da infecção por Mycoplasma spp. em aves no Zoológico do Rio de Janeiro (Rio Zoo), por isolamento e PCR. Do plantel de 635 aves do Rio Zoo, foram estudadas 81 para detecção de Mycoplasma spp., quando contidas para exames rotineiros de avaliação da condição de saúde. Essas aves eram pertencentes às ordens Psittaciformes (45), Accipitriformes (18), Galliformes (7), Piciformes (5), Strigiformes (4), Falconiformes (1) e Cariamiformes (1), todas já identificadas por microchip ou por anilha. Não houve isolamento de micoplasmas em nenhuma das amostras testadas, enquanto na PCR, 62,96% (51/81) foram positivas, sendo 1,96% (1/51) identificadas como MG e 19,61% (10/51) como MS, representando 1,23% (1/81) e 12,34% (10/81) da população total estudada. A PCR demonstrou ser uma técnica mais efetiva que o isolamento na detecção de Mycoplasma spp. em aves. Foi possível detectar micoplasmas nas aves do Riozoo sem sinal clínico respiratório, tendo MS maior prevalência do que MG. As positividades para Mycoplasma spp., MG e MS foram diferentes entre as ordens de aves estudadas, sendo a maior ocorrência nas aves de rapina, seguida dos Galliformes e dos Piciformes. A presença de MG e MS nas aves do Rio de Janeiro Zoo confirma a circulação destes agentes e a necessidade de mais estudos sobre a disseminação de micoplasmas em zoológicos para análise epidemiológica dessas bactérias nesse local.(AU)

Comparison of Mycoplasma gallisepticum Infection in Different Samples and Ages of Chicken Breeder Flocks

This study aimed to compare method-based and newly developed sample-based methods for Mycoplasma gallisepticum (MG) detection in different samples of breeder flocks suffering from respiratory disease problems by using culture, real-time PCR (rPCR) and ELISA from chicks and embryonated eggs. Overall, 450 samples of 19-day-old chicken embryos trachea, 450 samples of 8-day-old chicken tracheal swabs and 900 blood samples of 20-, 27-, 34-, 40- and 46-week-old breeder chickens from 5 flocks were sampled for 26 weeks, and were all tested for MG by culture, MG-rPCR and MG-ELISA. Culturing assays and rPCR were applied to 450 mixture samples from 19-day-old chicken embryos trachea and 450 tracheal swab samples (each pooled into groups of 3) from 8-day-old chicks from the same flocks. Also, 900 blood samples from the same 5 breeder flocks suffering from respiratory disease problems were tested by MG-ELISA. In individual sample-based analyses, 55 (18.3%) of the 300 pooled swab samples were positive for MG using culture methods, and 106 (35.3%) of the same samples were found positive by rPCR (sensitivity, specificity). The ELISAs indicated that 252 (28%) of the 900 breeding blood samples were MG seropositive. Using age-based analyses, the most positive period was 46 weeks, followed by 40 weeks, 34 weeks, 27 weeks and at least 20 weeks, in order of decreasing seropositivity. When comparing the culture and rPCR results of the two different sampling methods, chicken embryos trachea yielded more positive results than did tracheal swabs from the same flocks. In conclusion, rPCR is a highly specific, sensitive and reliable method for MG identification.(AU)

Comparison of Mycoplasma gallisepticum Infection in Different Samples and Ages of Chicken Breeder Flocks

This study aimed to compare method-based and newly developed sample-based methods for Mycoplasma gallisepticum (MG) detection in different samples of breeder flocks suffering from respiratory disease problems by using culture, real-time PCR (rPCR) and ELISA from chicks and embryonated eggs. Overall, 450 samples of 19-day-old chicken embryos trachea, 450 samples of 8-day-old chicken tracheal swabs and 900 blood samples of 20-, 27-, 34-, 40- and 46-week-old breeder chickens from 5 flocks were sampled for 26 weeks, and were all tested for MG by culture, MG-rPCR and MG-ELISA. Culturing assays and rPCR were applied to 450 mixture samples from 19-day-old chicken embryos trachea and 450 tracheal swab samples (each pooled into groups of 3) from 8-day-old chicks from the same flocks. Also, 900 blood samples from the same 5 breeder flocks suffering from respiratory disease problems were tested by MG-ELISA. In individual sample-based analyses, 55 (18.3%) of the 300 pooled swab samples were positive for MG using culture methods, and 106 (35.3%) of the same samples were found positive by rPCR (sensitivity, specificity). The ELISAs indicated that 252 (28%) of the 900 breeding blood samples were MG seropositive. Using age-based analyses, the most positive period was 46 weeks, followed by 40 weeks, 34 weeks, 27 weeks and at least 20 weeks, in order of decreasing seropositivity. When comparing the culture and rPCR results of the two different sampling methods, chicken embryos trachea yielded more positive results than did tracheal swabs from the same flocks. In conclusion, rPCR is a highly specific, sensitive and reliable method for MG identification.

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)

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.

Investigation of Mycoplasma spp. in birds of the Rio de Janeiro Zoo by isolation and PCR

ABSTRACT: Brazil is one of the countries with the most abundant avifauna in the world. The confinement of birds associated with close contact with other animals and humans favor the spread of agents of respiratory diseases. Among them, mycoplasmas can cause asymptomatic or apparent disease that manifests in birds by coughing, sneezing, rales, conjunctivitis, ocular and nasal discharge. Several described mycoplasmas cause disease in birds, especially Mycoplasma gallisepticum(MG) andMycoplasma synoviae(MS). The diagnosis ofMycoplasmaspp. can be done by clinical observation and laboratory analysis. Molecular diagnosis by PCR was boosted by its speed, sensitivity, and low cost of agent isolation techniques that take up to 21 days to complete. This study aimed to verify the occurrence ofMycoplasmaspp. in birds of the Rio de Janeiro Zoo (Rio Zoo), by isolation and PCR. Of the total 635 birds from the Rio Zoo, 81 were studied for detection ofMycoplasmaspp., when taken for routine health assessment exams. These birds belonged to the following orders: Psittaciformes (45), Accipitriformes (18), Galliformes (7), Piciformes (5), Strigiformes (4), Falconiformes (1) and Cariamiformes (1), all individuals already identified by microchip or leg-ring. There was no isolation of mycoplasmas in any of the samples tested, whereas, in the PCR, 62.96% (51/81) were positive, with 1.96% (1/51) identified as MG and 19.61% (10/51) as MS, representing 1.23% (1/81) and 12.34% (10/81) of the total population studied. PCR was shown to be a more effective technique than isolation in the detection ofMycoplasmaspp. in birds. It was possible to detect mycoplasmas in birds from Riozoo with no clinical respiratory signs, with higher MS prevalence than MG. The positivities forMycoplasmaspp., MS, and MG were different among the orders studied, being the highest occurrence in birds of prey, followed by Galliformes and Piciformes. The presence of MG and MS in birds of Rio de Janeiro Zoo confirms the circulation of these agents and the need for further studies on the dissemination of mycoplasmas in zoos for the epidemiological analysis of these bacteria in these places.

RESUMO: O Brasil é um dos países com maior avifauna do mundo. O confinamento de aves associado ao contato próximo a outros animais e seres humanos favorece a disseminação de agentes etiológicos causadores de doenças respiratórias. Dentre eles, os micoplasmas podem causar doença assintomática ou aparente que se manifesta em aves por espirros, estertores, conjuntivite, corrimentos oculares e nasais. São diversos os micoplasmas descritos causadores de doença em aves, com destaque para Mycoplasma gallisepticum (MG) e Mycoplasma synoviae (MS). O diagnóstico de Mycoplasma spp. pode ser feito pela observação clínica e análises laboratoriais. O diagnóstico molecular pela Reação em Cadeia da Polimerase (PCR) ganhou impulso por sua rapidez, sensibilidade e baixo custo em relação às técnicas de isolamento do agente que levam até 21 dias para conclusão do gênero Mycoplasma. Objetivou-se verificar a ocorrência da infecção por Mycoplasma spp. em aves no Zoológico do Rio de Janeiro (Rio Zoo), por isolamento e PCR. Do plantel de 635 aves do Rio Zoo, foram estudadas 81 para detecção de Mycoplasma spp., quando contidas para exames rotineiros de avaliação da condição de saúde. Essas aves eram pertencentes às ordens Psittaciformes (45), Accipitriformes (18), Galliformes (7), Piciformes (5), Strigiformes (4), Falconiformes (1) e Cariamiformes (1), todas já identificadas por microchip ou por anilha. Não houve isolamento de micoplasmas em nenhuma das amostras testadas, enquanto na PCR, 62,96% (51/81) foram positivas, sendo 1,96% (1/51) identificadas como MG e 19,61% (10/51) como MS, representando 1,23% (1/81) e 12,34% (10/81) da população total estudada. A PCR demonstrou ser uma técnica mais efetiva que o isolamento na detecção de Mycoplasma spp. em aves. Foi possível detectar micoplasmas nas aves do Riozoo sem sinal clínico respiratório, tendo MS maior prevalência do que MG. As positividades para Mycoplasma spp., MG e MS foram diferentes entre as ordens de aves estudadas, sendo a maior ocorrência nas aves de rapina, seguida dos Galliformes e dos Piciformes. A presença de MG e MS nas aves do Rio de Janeiro Zoo confirma a circulação destes agentes e a necessidade de mais estudos sobre a disseminação de micoplasmas em zoológicos para análise epidemiológica dessas bactérias nesse local.

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)

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)

Circulation of Major Respiratory Pathogens in Backyard Poultry and their Association with Clinical Disease and Biosecurity

Raising backyard birds is a common practice in Brazil, mainly in the countryside or suburban areas. However, the level of respiratory pathogens in these animals is unknown. We sampled two hundred chickens from 19 backyard flocks near commercial poultry farms and performed ELISA to Infectious Bronchitis Virus, avian Metapneumovirus, Mycoplasma synoviae and Mycoplasma gallisepticum. We evaluated the association between the predictive ability of ELISA and Hemagglutination-inhibition (HI)by comparing results from eight flocks positive to Mycoplasma gallisepticum on ELISA. Besides, we assessed essential biosecurity measures in the properties (multiple species birds, rodent control, hygienic conditions, and water quality for the bird`s consumption). We could access the vaccination program only on four properties; in three of them, the birds were supposedly vaccinated for IBV. Overall the properties had a poor score for the biosecurity measures, and the seroprevalence in backyard poultry flocks for IBV, a MPV, MS, and MG were respectively 87.5% (14/16), 89.5% (17/19), 100 (19/19) and MG 84.21% (16/19). We found low specificity and predictive value between ELISA and HI in MG analysis and a positive correlation between the presence of clinical symptoms and mean MG titers. Backyard chicken are pathogens reservoirs and pose a risk for the commercial poultry farms in the region, and further efforts of the governmental entities and private sector of poultry production should consider these information to avoid future economic losses.(AU)

Circulation of Major Respiratory Pathogens in Backyard Poultry and their Association with Clinical Disease and Biosecurity

Raising backyard birds is a common practice in Brazil, mainly in the countryside or suburban areas. However, the level of respiratory pathogens in these animals is unknown. We sampled two hundred chickens from 19 backyard flocks near commercial poultry farms and performed ELISA to Infectious Bronchitis Virus, avian Metapneumovirus, Mycoplasma synoviae and Mycoplasma gallisepticum. We evaluated the association between the predictive ability of ELISA and Hemagglutination-inhibition (HI)by comparing results from eight flocks positive to Mycoplasma gallisepticum on ELISA. Besides, we assessed essential biosecurity measures in the properties (multiple species birds, rodent control, hygienic conditions, and water quality for the bird`s consumption). We could access the vaccination program only on four properties; in three of them, the birds were supposedly vaccinated for IBV. Overall the properties had a poor score for the biosecurity measures, and the seroprevalence in backyard poultry flocks for IBV, a MPV, MS, and MG were respectively 87.5% (14/16), 89.5% (17/19), 100 (19/19) and MG 84.21% (16/19). We found low specificity and predictive value between ELISA and HI in MG analysis and a positive correlation between the presence of clinical symptoms and mean MG titers. Backyard chicken are pathogens reservoirs and pose a risk for the commercial poultry farms in the region, and further efforts of the governmental entities and private sector of poultry production should consider these information to avoid future economic losses.