Phylogenetic analyses of Chlamydia psittaci ompA gene sequences from captive blue-fronted Amazon parrots (Amazona aestiva) with hepatic disease in Brazil.

Chlamydia psittaci was detected in 152 (72%) blue-fronted Amazon parrots (Amazona aestiva, parrot from the Psittacidae family) out of a population of 212 that died during 2009-2011 in a wildlife rescue and rehabilitation centre in Minas Gerais, Brazil, following rescue from illegal wildlife trafficking. The macroscopic changes observed in these animals were hepatomegaly with multifocal white foci visible at the serosal surfaces of the liver, and extending into the parenchyma, and splenomegaly. The microscopic lesions observed in the liver included multifocal to coalescing miliary necrosis of hepatocytes with infiltration by heterophils, lymphocytes and plasma cells. In the spleen, loss of the normal architecture and infiltration by macrophages and plasma cells were observed. Stained tissue sections (Gimenez technique) revealed small round clusters suggestive of C. psittaci (reticulate bodies) in the cytoplasm of macrophages from the liver and spleen. Nine sequences of segments of the ompA gene, obtained from different individuals, were randomly selected for sequencing. The phylogenetic analyses showed that all strains clustered with genotype A, which is the most virulent genotype for birds. This genotype is involved in mortality of psittacines, is easily transmitted in captivity and represents a problem for successful rehabilitation. The results indicate the necessity to improve biosecurity in triage and to provide individual personal protection for professionals and caretakers.

Chlamydia psittaci a été détectée chez 152 (72 %) amazones à front bleu (Amazona aestiva, perroquet de la famille des Psittacidés) sur un total de 212 individus rescapés du trafic illégal et décédés en 2009 et 2011 dans un centre de sauvetage et de réhabilitation de la faune sauvage à Minas Gerais (Brésil). Les modifications macroscopiques observées sur ces oiseaux étaient une hépatomégalie avec des foyers blancs multifocaux visibles sur les surfaces séreuses du foie et s'étendant dans le parenchyme, et une splénomégalie. Les lésions microscopiques observées dans le foie comprenaient une nécrose miliaire multifocale à coalescente des hépatocytes avec infiltration d'hétérophiles, de lymphocytes et de plasmocytes. Dans la rate, une perte de l'architecture normale et l'infiltration de macrophages et de plasmocytes ont été observées. La coloration de coupes de tissus (technique de Gimenez) a révélé de petites grappes rondes évoquant C. psittaci (corps réticulés) dans le cytoplasme des macrophages du foie et de la rate. Neuf produits segmentés d'une partie du gène ompA, obtenus de différents individus, ont été sélectionnés de manière aléatoire pour le séquençage. Les analyses phylogénétiques ont montré que toutes les souches se regroupaient dans le génotype A, qui est le plus virulent pour les oiseaux. Ce génotype est responsable de cas de mortalité chez les psittacidés et se transmet facilement en captivité, ce qui représente un risque pour la réussite des opérations de réhabilitation. Au vu de ces résultats, les auteurs soulignent la nécessité d'améliorer la biosécurité lors du tri des animaux dans les centres de soins et de fournir une protection individuelle aux professionnels et aux gardiens.

Se detectó Chlamydia psittaci en 152 (72%) amazonas frentiazules (Amazona aestiva, loro de la familia Psittacidae) de un total de 212 que murieron durante 2009­2011 en un centro de rescate y rehabilitación de fauna silvestre de Minas Gerais, Brasil, tras haber sido rescatadas del tráfico ilegal. Los cambios macroscópicos que se observaron en estos animales fueron hepatomegalia con focos blancos multifocales visibles en las superficies serosas del hígado y que se extendían hacia el parénquima, y esplenomegalia. Las lesiones microscópicas observadas en el hígado consistieron en necrosis miliar multifocal a coalescente de hepatocitos con infiltración de heterófilos, linfocitos y células plasmáticas. En el bazo, se observó pérdida de la arquitectura normal y infiltración de macrófagos y células plasmáticas. Cortes de tejido teñidos (con la técnica de Giménez) revelaron pequeños racimos redondos que sugerían la presencia de C. psittaci (cuerpos reticulados) en el citoplasma de macrófagos del hígado y del bazo. A partir de distintos individuos, se escogieron aleatoriamente nueve segmentos del gen ompA para ser secuenciados. Los análisis filogenéticos mostraron que todas las cepas correspondían al genotipo A, que es el más virulento para las aves. Este genotipo está involucrado en la mortalidad de psitácidas, se transmite fácilmente en cautiverio y supone un riesgo para el éxito de la rehabilitación. Los resultados indican la necesidad de mejorar la bioseguridad en el triaje y de procurar protección personal individual a profesionales y cuidadores.

Molecular Diagnosis of Beak and Feather Disease in Native Brazilian Psittacines

The incidence of the psittacine beak and feather disease virus (BFDV) was investigated in Brazilian native parrots with normal feathering arriving at rescue and triage centers for wild animals (CETAS, IBAMA) in the state of Minas Gerais, Brazil. BFDV DNA was investigated by previously described PCR technique for the partial amplification of BFDV ORF-1 in DNA extracts from blood, cloacal swab or liver of psittacines. Some birds provided more than one sample. Nine species of psittacines were sampled between January 2009 and October 2010. Blood (n=46) or cloacal swab (n=128) samples were obtained from psittacines immediately upon arrival at the triage centers. Liver samples were collected from necropsied birds dead on arrival (n=167). All swab samples were negative, except for one Ara ararauna individual (n=3) which blood presented the BFDV DNA. On the other hand, 11 liver samples were positive for BFDV DNA, with a prevalence of 7.8% in Amazona aestiva (n=140). No BFDV DNA was detected in the liver of Amazona amazonica (n=11), A. vinacea (n=5), A. rhodochorytha (n=4), Anodorhynchus hyacinthinus (n=3), Ara ararauna, (n=3), Aratinga leucophtalma (n=2), Guarouba guarouba (n=1) and Pionus maximiliani (n=1). In most cases, alopecia was not associated with BFDV detection in liver, and liver histopathology was inconclusive. Although all cloacal swab samples were negative, a few psittacines (n=19) that died at CETAS-Belo Horizonte were retested, and 21% were detected as positive in liver. A group of psittacines (n=16) was clinically evaluated, and despite showing feather dystrophy, all birds were negative in the cloacal swabs, except for one, which blood sample was positive (A. ararauna). The obtained sequences of the BFDV strains BH 215 and BH 732 were deposited in the GenBank (JQ649409 and JQ649410). A 98% similarity with strain sequences described in Australia, Japan, and New Zealand was observed. It is possible that these strains arrived in Brazil through the legal and illegal trade of parrots. However, it was not possible to associate BFDV infection with the geographical origin of birds and no local marker was detected. The rates of detection, although similar to other studies, indicate the tendency of a high incidence of the disease, possibly associated with stress, and high bird density and wide transmission in captivity conditions.

Molecular Diagnosis of Beak and Feather Disease in Native Brazilian Psittacines

The incidence of the psittacine beak and feather disease virus (BFDV) was investigated in Brazilian native parrots with normal feathering arriving at rescue and triage centers for wild animals (CETAS, IBAMA) in the state of Minas Gerais, Brazil. BFDV DNA was investigated by previously described PCR technique for the partial amplification of BFDV ORF-1 in DNA extracts from blood, cloacal swab or liver of psittacines. Some birds provided more than one sample. Nine species of psittacines were sampled between January 2009 and October 2010. Blood (n=46) or cloacal swab (n=128) samples were obtained from psittacines immediately upon arrival at the triage centers. Liver samples were collected from necropsied birds dead on arrival (n=167). All swab samples were negative, except for one Ara ararauna individual (n=3) which blood presented the BFDV DNA. On the other hand, 11 liver samples were positive for BFDV DNA, with a prevalence of 7.8% in Amazona aestiva (n=140). No BFDV DNA was detected in the liver of Amazona amazonica (n=11), A. vinacea (n=5), A. rhodochorytha (n=4), Anodorhynchus hyacinthinus (n=3), Ara ararauna, (n=3), Aratinga leucophtalma (n=2), Guarouba guarouba (n=1) and Pionus maximiliani (n=1). In most cases, alopecia was not associated with BFDV detection in liver, and liver histopathology was inconclusive. Although all cloacal swab samples were negative, a few psittacines (n=19) that died at CETAS-Belo Horizonte were retested, and 21% were detected as positive in liver. A group of psittacines (n=16) was clinically evaluated, and despite showing feather dystrophy, all birds were negative in the cloacal swabs, except for one, which blood sample was positive (A. ararauna). The obtained sequences of the BFDV strains BH 215 and BH 732 were deposited in the GenBank (JQ649409 and JQ649410). A 98% similarity with strain sequences described in Australia, Japan, and New Zealand was observed. It is possible that these strains arrived in Brazil through the legal and illegal trade of parrots. However, it was not possible to associate BFDV infection with the geographical origin of birds and no local marker was detected. The rates of detection, although similar to other studies, indicate the tendency of a high incidence of the disease, possibly associated with stress, and high bird density and wide transmission in captivity conditions.(AU)

Occurrence of Aviadenovirus in chickens from the poultry industry of Minas Gerais / Ocorrência de Aviadenovirus em aves da industria avícola de Minas Gerais

The occurrence of Aviadenovirus (FAdV) was investigated in chickens from the poultry industry of Minas Gerais state, Brazil. The investigation was conducted due to the scarcity of recent data in the country and its description in neighboring countries. For this purpose, livers were collected from layer chicks (n=25), older layers (n=25), broilers (n=300), and livers (n=25) and stool (n=25) samples from broiler breeders, representing the major poultry regions of the state. FAdV DNA was demonstrated using a previously described PCR protocol for amplifying part of the hexon gene encoding sequence. FAdV was found in layer chicks (36 percent), widespread (100 percent) in older layers, and with regional differences in broilers (24-86 percent). Although all broiler breeder stools were negative, FAdV DNA was detected in livers (16 percent, 4/25) of stool-negative birds. In order to obtain additional information on the circulation of the infection, livers of subsistence chickens collected from one poultry intensive region, were evaluated (n = 12), with FAdV being detected in all samples. FAdV was found in young and old layers, broilers, broiler breeders and free-range chickens, and results suggest the circulation of FAdV among different types of chickens. The detection in older layer chickens may indicate an extended risk of horizontal transmission in regions of Minas Gerais with mixed activity of egg and meat type chickens and poor biosecurity strategies. The infection in breeders may indicate vertical transmission and the continuous production of infected progenies. The hexon-gene-targeted PCR amplicon sequences aligned with FAdV of species D of Aviadenovirus. Results indicate the necessity for biosecurity, especially for breeders, separating flocks according to origin, age and health status, which will be an advantage regarding any pathogen.(AU)

Descreve-se a ocorrência de Aviadenovirus (FAdV) na avicultura mineira. Foram amostrados fígados de poedeiras jovens (n=25) e velhas (n=25) e de frangos de corte (n=300). Em matrizes pesadas foram amostrados fígados (n=25) e fezes (n=25). O estudo envolveu as principais regiões avícolas do Estado de Minas Gerais. O DNA de FAdV foi pesquisado por PCR universal, descrito para a amplificação do gene que codifica o hexon de Aviadenovirus, usando FAdV Phelps como referência. Foi demonstrada a presença do DNA de FAdV em 100 por cento (25/25) das poedeiras velhas (78 semanas de idade) e em 36 por cento (9/25) das jovens (18 dias). Em frangos de corte, a detecção variou entre 24 e 86 por cento. Embora as fezes das matrizes tenham sido negativas, foi obtido o amplicon específico em 4/25 dos fígados dessas mesmas aves, indicando menor sensibilidade para detecção nas fezes. Em amostras da avicultura familiar (fígado), colhidas de uma das regiões de avicultura intensificada, foi detectado o genoma de FAdV em 100 por cento das galinhas (n=12). A constatação de alta disseminação de FAdV em aves da avicultura industrial e familiar de Minas Gerais contribui para o entendimento da epidemiologia de Aviadenovirus. As sequências nucleotídicas dos produtos de PCR alinharam com FAdV da espécie D de Aviadenovirus. A demonstração de FAdV em reprodutores indica risco de transmissão vertical e reforça a necessidade de biosseguridade estrita nesses plantéis. A presença de FAdV em diversos setores avícolas, incluindo poedeiras comerciais, frangos de corte, reprodutores e galinhas da avicultura familiar, recomenda a biosseguridade estrita entre as criações de mesmo tipo e de tipos diferentes de aves. A detecção em matrizes pode indicar a continuada geração de progênies infectadas.(AU)

Occurrence of Aviadenovirus in chickens from the poultry industry of Minas Gerais / Ocorrência de Aviadenovirus em aves da industria avícola de Minas Gerais

The occurrence of Aviadenovirus (FAdV) was investigated in chickens from the poultry industry of Minas Gerais state, Brazil. The investigation was conducted due to the scarcity of recent data in the country and its description in neighboring countries. For this purpose, livers were collected from layer chicks (n=25), older layers (n=25), broilers (n=300), and livers (n=25) and stool (n=25) samples from broiler breeders, representing the major poultry regions of the state. FAdV DNA was demonstrated using a previously described PCR protocol for amplifying part of the hexon gene encoding sequence. FAdV was found in layer chicks (36 percent), widespread (100 percent) in older layers, and with regional differences in broilers (24-86 percent). Although all broiler breeder stools were negative, FAdV DNA was detected in livers (16 percent, 4/25) of stool-negative birds. In order to obtain additional information on the circulation of the infection, livers of subsistence chickens collected from one poultry intensive region, were evaluated (n = 12), with FAdV being detected in all samples. FAdV was found in young and old layers, broilers, broiler breeders and free-range chickens, and results suggest the circulation of FAdV among different types of chickens. The detection in older layer chickens may indicate an extended risk of horizontal transmission in regions of Minas Gerais with mixed activity of egg and meat type chickens and poor biosecurity strategies. The infection in breeders may indicate vertical transmission and the continuous production of infected progenies. The hexon-gene-targeted PCR amplicon sequences aligned with FAdV of species D of Aviadenovirus. Results indicate the necessity for biosecurity, especially for breeders, separating flocks according to origin, age and health status, which will be an advantage regarding any pathogen...

Descreve-se a ocorrência de Aviadenovirus (FAdV) na avicultura mineira. Foram amostrados fígados de poedeiras jovens (n=25) e velhas (n=25) e de frangos de corte (n=300). Em matrizes pesadas foram amostrados fígados (n=25) e fezes (n=25). O estudo envolveu as principais regiões avícolas do Estado de Minas Gerais. O DNA de FAdV foi pesquisado por PCR universal, descrito para a amplificação do gene que codifica o hexon de Aviadenovirus, usando FAdV Phelps como referência. Foi demonstrada a presença do DNA de FAdV em 100 por cento (25/25) das poedeiras velhas (78 semanas de idade) e em 36 por cento (9/25) das jovens (18 dias). Em frangos de corte, a detecção variou entre 24 e 86 por cento. Embora as fezes das matrizes tenham sido negativas, foi obtido o amplicon específico em 4/25 dos fígados dessas mesmas aves, indicando menor sensibilidade para detecção nas fezes. Em amostras da avicultura familiar (fígado), colhidas de uma das regiões de avicultura intensificada, foi detectado o genoma de FAdV em 100 por cento das galinhas (n=12). A constatação de alta disseminação de FAdV em aves da avicultura industrial e familiar de Minas Gerais contribui para o entendimento da epidemiologia de Aviadenovirus. As sequências nucleotídicas dos produtos de PCR alinharam com FAdV da espécie D de Aviadenovirus. A demonstração de FAdV em reprodutores indica risco de transmissão vertical e reforça a necessidade de biosseguridade estrita nesses plantéis. A presença de FAdV em diversos setores avícolas, incluindo poedeiras comerciais, frangos de corte, reprodutores e galinhas da avicultura familiar, recomenda a biosseguridade estrita entre as criações de mesmo tipo e de tipos diferentes de aves. A detecção em matrizes pode indicar a continuada geração de progênies infectadas...

Health assessment of raptors in triage in Belo Horizonte, MG, Brazil

Falconiformes (n=82), Strigiformes (n=84) and Cathartiformes (n=14) at a triage center (CETAS-Belo Horizonte, IBAMA, Brazil) were examined between 2008 and 2010 . No bird was reactive at hemagglutination-inhibition (HI) for antibodies against Mycoplasma gallisepticum (Mg). Two Caracara plancus (2/68) had HI titers (16-32) against Newcastle disease virus. No Chlamydophila psittaci DNA was detected in the liver (PCR; n=95). Blood smears (Giemsa; n=89) and spleen fragments (PCR; n=82) were 13.5% and 8.5% positive, respectively, for Haemoproteus only. Necropsy of Cathartiformes (n=10), Falconiformes (n=42) and Strigiformes (n=57) showed that trauma injuries were the main cause (63.3%) of admission and death, being fractures (38.5%) of the thoracic limbs (57.1%) the most frequent. Nematode (12.8%), cestode (1.8%), trematode (0.9%), and acanthocephalan (2.7%) parasite infections were relevant. Mites (Acari) were the most frequent (17.4%) external parasites, particularly Ornithonyssus sylviarum in Asio clamator and Amblyomma cajennense in Tyto alba. Chewing lice (10.1%) and Pseudolynchia spp. (9.2%) were also found. Histomonas spp. (6.4%) was found in the ceca of Bubo virginianus, Athene cunicularia, Tyto alba, and Asio clamator, but not in Falconiformes or Cathartiformes. Trichomonas spp. (oral cavity, pharynx and upper esophagus; 9.1%) was detected in Falconiformes and Strigiformes, but not in Cathartiformes. Trichomonas spp. were found in A. cunicularia, Asio clamator, Glaucidium brasilianum and Tyto alba (Strigiformes), and in Rupornis magnirostris, Milvago chimachima, Falco femoralis, Falco sparverius and Caracara plancus (Falconiformes). Coccidia (9.1%) (Sarcocystis spp., 6.4%) and mycosis were observed in most Tyto alba (70%). The evaluated Orders may not pose risks for commercial poultry production. Habitat loss and urban adaptation may be increasingly affecting raptors.(AU)

Health assessment of raptors in triage in Belo Horizonte, MG, Brazil

Falconiformes (n=82), Strigiformes (n=84) and Cathartiformes (n=14) at a triage center (CETAS-Belo Horizonte, IBAMA, Brazil) were examined between 2008 and 2010 . No bird was reactive at hemagglutination-inhibition (HI) for antibodies against Mycoplasma gallisepticum (Mg). Two Caracara plancus (2/68) had HI titers (16-32) against Newcastle disease virus. No Chlamydophila psittaci DNA was detected in the liver (PCR; n=95). Blood smears (Giemsa; n=89) and spleen fragments (PCR; n=82) were 13.5% and 8.5% positive, respectively, for Haemoproteus only. Necropsy of Cathartiformes (n=10), Falconiformes (n=42) and Strigiformes (n=57) showed that trauma injuries were the main cause (63.3%) of admission and death, being fractures (38.5%) of the thoracic limbs (57.1%) the most frequent. Nematode (12.8%), cestode (1.8%), trematode (0.9%), and acanthocephalan (2.7%) parasite infections were relevant. Mites (Acari) were the most frequent (17.4%) external parasites, particularly Ornithonyssus sylviarum in Asio clamator and Amblyomma cajennense in Tyto alba. Chewing lice (10.1%) and Pseudolynchia spp. (9.2%) were also found. Histomonas spp. (6.4%) was found in the ceca of Bubo virginianus, Athene cunicularia, Tyto alba, and Asio clamator, but not in Falconiformes or Cathartiformes. Trichomonas spp. (oral cavity, pharynx and upper esophagus; 9.1%) was detected in Falconiformes and Strigiformes, but not in Cathartiformes. Trichomonas spp. were found in A. cunicularia, Asio clamator, Glaucidium brasilianum and Tyto alba (Strigiformes), and in Rupornis magnirostris, Milvago chimachima, Falco femoralis, Falco sparverius and Caracara plancus (Falconiformes). Coccidia (9.1%) (Sarcocystis spp., 6.4%) and mycosis were observed in most Tyto alba (70%). The evaluated Orders may not pose risks for commercial poultry production. Habitat loss and urban adaptation may be increasingly affecting raptors.

Molecular characterization of contaminating infectious anemia virus of chickens in live commercial vaccines produced in the 1990s.

The presence of infectious chicken anemia virus (CAV) was detected in a previous study by nested-PCR as a contaminant in seven commercial vaccines, produced in the 1990s by three different manufacturers, prepared against the most relevant virus etiologies. In order to phylogenetically characterize the genome and compare it to CAV isolates from Brazil and other parts of the world, sequences of approximately 675 bp of the gene encoding the hypervariable region of VP1 protein of three CAV vaccine contaminant strains were studied. The CAV genome in contaminated vaccines showed high similarity (> 98.9%) with the Brazilian BR91/99 and Argentinian ArgA001028 (> 99%) strains. However, the comparison with the Cuxhaven-1 vaccine strain showed a lower identity of between 96.8% and 97.7%, and comparing it with the CAV26P4 vaccine strain showed an identity between 97.2% and 98.2%; both are available in Brazil. Such differences might be relevant for the highly conserved CAV genome. CAV contaminants were positioned in the same genetic group (clusters) with the Brazilian strain BR91/99 and Argentinian strain ArgA001028. Results indicated that the contamination of live vaccines by CAV may have influenced CAV epidemiology in the Brazilian and Argentinian poultry industry.

The occurrence of orthoreovirus, rotavirus and chicken anemia virus in chickens of the poultry industry in Minas Gerais, Brazil / Ocorrência de orthoreovirus, rotavirus e vírus da anemia das galinhas em frangos de corte da indústria avícola de Minas Gerais

Fifty-four fecal samples taken from broiler chickens from 1 to 45 days of age, and of pullets from 10 to 13 weeks of age, original from eight different poultry regions in the state of Minas Gerais, Brazil, were collected from March 2008 to January 2010 for avian Orthoreovirus (ARV) and avian Rotavirus (AvRV) analyses. For the assay of ARV, RNA was immediately extracted (Trizolâ) and transcribed into cDNA for assaying in a nested-PCR with ARV-specific primers. For AvRV, polyacrylamide gel electrophoresis (PAGE) was performed with RNA extracts obtained by phenol-chloroform extraction. CAV was additionally investigated through a nested-PCR of thymus and spleen. Results found 5.55% positive for ARV and 9.25% for AvRV. Also, CAV and ARV genomes were detected in co-infection, in a highly prostrated and claudicating chicken flock. No ARV or AvRV infections were detected in pullets. Material of a clinically affected flock was inoculated into SPF embryos, resulting in embryonic hemorrhage, whitish foci in the chorio-allantoic membrane and death. Sequencing of ARV amplicons and isolate cDNA grouped local strains with the ARV S1133 strain, historically used in live vaccines, suggesting the continued circulation of this vaccine virus strain in intensive poultry regions. Detection rates for ARV and AvRV, as well as the presence of CAV, were additionally indicative of failing biosecurity strategies for the intensive poultry regions examined.

Avaliou-se a ocorrência de Orthoreovirus (ARV) e Rotavirus (AvRV) aviários na avicultura industrial de Minas Gerais. Foram colhidas cinquenta e quatro amostras de fezes de frangos de corte entre um e 45 dias e de frangas de postura de 10 a 13 semanas de idade. Para análise de ARV, o RNA foi imediatamente extraído (Trizol), transcrito em cDNA e avaliado em uma PCR com oligonucleotídeos iniciadores específicos para ARV. Para a investigação de AvRV, os extratos de RNA foram obtidos por fenol-clorofórmio e submetidos à eletroforese em gel de poliacrilamida. Todas as amostras foram também avaliadas para o DNA do vírus da anemia das galinhas (CAV) em uma nested-PCR específica. Em frangos de corte, a positividade encontrada para ARV foi de 5,55% e para AvRV de 9,25%. CAV foi detectado em coinfecção em um plantel com refugagem, claudicação e prostração. Nenhuma amostra de poedeiras foi positiva para ARV ou AvRV. Material de plantel com sinais clínicos foi purificado e inoculado em ovos SPF embrionados, sendo obtidas lesões hemorrágicas e focos brancos na membrana cório-alantóide. O sequenciamento dos produtos de PCR e de embrião agrupou os isolados de ARV com a estirpe S1133, historicamente usada como vacina viva. Os resultados sugerem a continuada circulação da infecção por estirpes assemelhadas a ARV S1133 nas regiões de avicultura industrial. Os índices de detecção de ARV, AvRV e CAV indicam que a intensificação nas regiões produtoras tem resultado em falhas de biosseguridade.

A retrospective PCR investigation of avian Orthoreovirus, chicken infectious anemia and fowl Aviadenovirus genomes contamination in commercial poultry vaccines in Brazil / Estudo retrospectivo de vacinas avícolas vivas por PCR para os vírus da anemia das galinhas, Aviadenovirus aviário e Orthoreovirus aviário

Vacinas avícolas vivas comerciais produzidas entre 1991 e 2005 foram examinadas para a presença de genomas dos vírus da anemia infecciosa das galinhas (Gyrovirus CAV), da hepatite por corpúsculo de inclusão (Aviadenovirus FAdV) e da artrite viral/síndrome da má absorção (Orthoreovirus aviário ARV). Vinte e seis partidas de vacinas vivas liofilizadas de oito fabricantes com lacre original foram examinadas. As extrações de DNA e PCR de CAV e FAdV, e de RNA e RT-PCR para ARV, foram descritas previamente. Contaminações triplas de ARV, CAV e FAdV foram detectadas em vacinas de mesmo fabricante, produzidas em 1991 e 1992 contra a doença de Newcastle (DN), e para a encefalomielite aviária, produzida em 1994. ARV e CAV em co-infecção foram encontrados em vacinas contra a doença de Marek liofilizadas produzidas em 1996 por dois fabricantes diferentes. Genoma de ARV foi detectado em vacinas contra a bronquite infecciosa de setembro e dezembro de 1998, doença infecciosa bursal, de dezembro de 1998 e DN de janeiro de 1998. Três dos oito fabricantes apresentaram vacinas com contaminação e cinco nunca apresentaram vacinas contaminadas. Nenhuma vacina produzida a partir de 2001 apresentou contaminação. Cogita-se um papel epidemiológico para vacinas vivas, como fonte de infecção para ARV, CAV e FAdV e, potencialmente determinante da atual alta disseminação destes.(AU)

A retrospective PCR investigation of avian Orthoreovirus, chicken infectious anemia and fowl Aviadenovirus genomes contamination in commercial poultry vaccines in Brazil / Estudo retrospectivo de vacinas avícolas vivas por PCR para os vírus da anemia das galinhas, Aviadenovirus aviário e Orthoreovirus aviário

Vacinas avícolas vivas comerciais produzidas entre 1991 e 2005 foram examinadas para a presença de genomas dos vírus da anemia infecciosa das galinhas (Gyrovirus CAV), da hepatite por corpúsculo de inclusão (Aviadenovirus FAdV) e da artrite viral/síndrome da má absorção (Orthoreovirus aviário ARV). Vinte e seis partidas de vacinas vivas liofilizadas de oito fabricantes com lacre original foram examinadas. As extrações de DNA e PCR de CAV e FAdV, e de RNA e RT-PCR para ARV, foram descritas previamente. Contaminações triplas de ARV, CAV e FAdV foram detectadas em vacinas de mesmo fabricante, produzidas em 1991 e 1992 contra a doença de Newcastle (DN), e para a encefalomielite aviária, produzida em 1994. ARV e CAV em co-infecção foram encontrados em vacinas contra a doença de Marek liofilizadas produzidas em 1996 por dois fabricantes diferentes. Genoma de ARV foi detectado em vacinas contra a bronquite infecciosa de setembro e dezembro de 1998, doença infecciosa bursal, de dezembro de 1998 e DN de janeiro de 1998. Três dos oito fabricantes apresentaram vacinas com contaminação e cinco nunca apresentaram vacinas contaminadas. Nenhuma vacina produzida a partir de 2001 apresentou contaminação. Cogita-se um papel epidemiológico para vacinas vivas, como fonte de infecção para ARV, CAV e FAdV e, potencialmente determinante da atual alta disseminação destes.

The occurrence of Orthoreovirus, Rotavirus and chicken anemia virus in chickens of the poultry industry in Minas Gerais, Brazil / Ocorrência de Orthoreovirus, Rotavirus e vírus da anemia das galinhas em frangos de corte da indústria avícola de Minas Gerais

Fifty-four fecal samples taken from broiler chickens from 1 to 45 days of age, and of pullets from 10 to 13 weeks of age, original from eight different poultry regions in the state of Minas Gerais, Brazil, were collected from March 2008 to January 2010 for avian Orthoreovirus (ARV) and avian Rotavirus (AvRV) analyses. For the assay of ARV, RNA was immediately extracted (Trizolâ) and transcribed into cDNA for assaying in a nested-PCR with ARV-specific primers. For AvRV, polyacrylamide gel electrophoresis (PAGE) was performed with RNA extracts obtained by phenol-chloroform extraction. CAV was additionally investigated through a nested-PCR of thymus and spleen. Results found 5.55% positive for ARV and 9.25% for AvRV. Also, CAV and ARV genomes were detected in co-infection, in a highly prostrated and claudicating chicken flock. No ARV or AvRV infections were detected in pullets. Material of a clinically affected flock was inoculated into SPF embryos, resulting in embryonic hemorrhage, whitish foci in the chorio-allantoic membrane and death. Sequencing of ARV amplicons and isolate cDNA grouped local strains with the ARV S1133 strain, historically used in live vaccines, suggesting the continued circulation of this vaccine virus strain in intensive poultry regions. Detection rates for ARV and AvRV, as well as the presence of CAV, were additionally indicative of failing biosecurity strategies for the intensive poultry regions examined.(AU)

Avaliou-se a ocorrência de Orthoreovirus (ARV) e Rotavirus (AvRV) aviários na avicultura industrial de Minas Gerais. Foram colhidas cinquenta e quatro amostras de fezes de frangos de corte entre um e 45 dias e de frangas de postura de 10 a 13 semanas de idade. Para análise de ARV, o RNA foi imediatamente extraído (Trizol), transcrito em cDNA e avaliado em uma PCR com oligonucleotídeos iniciadores específicos para ARV. Para a investigação de AvRV, os extratos de RNA foram obtidos por fenol-clorofórmio e submetidos à eletroforese em gel de poliacrilamida. Todas as amostras foram também avaliadas para o DNA do vírus da anemia das galinhas (CAV) em uma nested-PCR específica. Em frangos de corte, a positividade encontrada para ARV foi de 5,55% e para AvRV de 9,25%. CAV foi detectado em coinfecção em um plantel com refugagem, claudicação e prostração. Nenhuma amostra de poedeiras foi positiva para ARV ou AvRV. Material de plantel com sinais clínicos foi purificado e inoculado em ovos SPF embrionados, sendo obtidas lesões hemorrágicas e focos brancos na membrana cório-alantóide. O sequenciamento dos produtos de PCR e de embrião agrupou os isolados de ARV com a estirpe S1133, historicamente usada como vacina viva. Os resultados sugerem a continuada circulação da infecção por estirpes assemelhadas a ARV S1133 nas regiões de avicultura industrial. Os índices de detecção de ARV, AvRV e CAV indicam que a intensificação nas regiões produtoras tem resultado em falhas de biosseguridade.(AU)

Ostrich (Struthio camelus) gastric diseases in Minas Gerais, Brazil, from 1997 to 2009 / Doenças gástricas de avestruzes em Minas Gerais

Descrevem-se as doenças gástricas de avestruzes diagnosticadas em um Laboratório de Doenças das Aves, Belo Horizonte-MG, entre 1997 e 2009. As afecções gástricas corresponderam a 46,2% de todos os casos de doença, com quadros que incluíram impactação (83,3%), infecções e parasitoses (16,7%). As impactações foram causadas por material não alimentar diversificado e as infecções e parasitoses incluíram o fungo Macrorhabdus ornithogaster (megabacteria) e o nematódeo Libyostrongylus douglassii.(AU)

Ostrich (Struthio camelus) gastric diseases in Minas Gerais, Brazil, from 1997 to 2009 / Doenças gástricas de avestruzes em Minas Gerais

Descrevem-se as doenças gástricas de avestruzes diagnosticadas em um Laboratório de Doenças das Aves, Belo Horizonte-MG, entre 1997 e 2009. As afecções gástricas corresponderam a 46,2% de todos os casos de doença, com quadros que incluíram impactação (83,3%), infecções e parasitoses (16,7%). As impactações foram causadas por material não alimentar diversificado e as infecções e parasitoses incluíram o fungo Macrorhabdus ornithogaster (megabacteria) e o nematódeo Libyostrongylus douglassii.

Purificação de IgG de codorna a partir de soro e gema de ovos / Purification of Japanese quail IgG from serum and egg yolk

The purification of Japanese quail IgG from serum was performed using caprilic acid and ammonium sulfate, and from egg yolk using PEG-6000 and ethanol. After confirming the purification and the concentration of IgG, the yolk samples had twice the amount of protein compared to serum samples. The IgG extracts were analyzed by SDS-PAGE and western blot showing similar results as the ones of chicken IgG used as the standard. So, these methodologies can be used for purifying quail serum or egg yolk IgG, which would enable the development of diagnostic assays.(AU)

ELISA indireto para detecção de IgG antivírus da doença de Newcastle em soro de codorna / Indirect ELISA for the detection of IgG specific to Newcastle disease virus in quail serum

An indirect ELISA for the detection of japanese quail IgG specific to Newcastle disease virus (NDV) was developed. The secondary anti-quail IgG was produced in Balb/c mice, by inoculating Freund's complete adjuvant emulsified japanese quail-IgG extract. The purification of IgG was achieved using the caprilic acid method. The ELISA was compared to the haemagglutination-inhibition (HI) test for antibodies to NDV. ELISA cut-off point was established through TG-ROC analysis. Total correlation was observed between the ELISA and the HI, being the ELISA efficient in the identification of positive and negative sera, with high sensitivity and specificity (100 percent). These results validate the use of the indirect ELISA as an alternative for the detection of NDV-specific IgG in japanese quail sera, with the advantage of high sensitivity and automation(AU)

ELISA indireto para detecção de IgG antivírus da doença de Newcastle em soro de codorna / Indirect ELISA for the detection of IgG specific to Newcastle disease virus in quail serum

An indirect ELISA for the detection of japanese quail IgG specific to Newcastle disease virus (NDV) was developed. The secondary anti-quail IgG was produced in Balb/c mice, by inoculating Freund's complete adjuvant emulsified japanese quail-IgG extract. The purification of IgG was achieved using the caprilic acid method. The ELISA was compared to the haemagglutination-inhibition (HI) test for antibodies to NDV. ELISA cut-off point was established through TG-ROC analysis. Total correlation was observed between the ELISA and the HI, being the ELISA efficient in the identification of positive and negative sera, with high sensitivity and specificity (100 percent). These results validate the use of the indirect ELISA as an alternative for the detection of NDV-specific IgG in japanese quail sera, with the advantage of high sensitivity and automation

Apoptose e expressão de VP2 e GAPDH na infecção precoce pelo vírus da doença infecciosa da bursa de Fabricius em pintos SPF / Apoptosis and expression of VP2 and GADPH in an experimental infectious bursal disease in SPF chicks

Vinte e nove pintos SPF de um dia foram inoculados com o vírus da doença infecciosa da bursa de Fabricius (VDIB) para avaliar a ocorrência precoce de apoptose e a expressão da proteína viral 2 (VP2) e da enzima gliceraldeído fosfato dehidrogenase (GAPDH). Os animais foram distribuídos em cinco grupos: 1-controle; e 2 a 5- com 24, 48, 72 e 96 horas pós-inoculação, respectivamente. Fragmentos da bursa de Fabricius foram colhidos para processamento histológico e extração de RNA. Lâminas coradas em HE e TUNEL (marcação in situ da fragmentação do genoma com transferase terminal de deoxinucleotídeo) foram utilizadas na morfometria do índice apoptótico. Amostras de mRNA foram testadas para a expressão dos genes VP2 e GAPDH utilizando-se transcrição reversa e RT-PCR. Utilizou-se um kit SYBR GREEN PCR, e a reação foi desenvolvida em ABI Prism 7000 SDS. Os índices apoptóticos cresceram progressivamente indicando uma relação na atrofia bursal causada pelo VDIB. Paralelamente, os resultados da PCR em tempo real demonstraram queda da carga viral nas células linfóides da bursa nos diferentes intervalos de tempo do experimento. Esses resultados sugerem um papel protetor da apoptose na diminuição da replicação viral.(AU)

Twenty-nine SPF 1-day-old chicks were inoculated with infectious bursal disease virus (IBDV) to evaluate early apoptosis and the expression of viral protein 2 (VP2) and glyceraldehyde-3-phosphate dehydrogenease (GAPDH). Five groups were formed: G1-control -and G2 to G5, - 24, 48, 72 and 96 hours post inoculation, respectively. Half of each BF was fixed and processed by routine techniques. To quantify apoptosis, 5µm-thick sections were stained with HE and submitted to TUNEL (terminal transferase UDP nick end labeling) technique. mRNA was extracted from pooled samples of 3 animals/group and used for the expression of VP2 and GADPH genes using the reverse transcription and real-time polymerase chain reaction (RT-PCR). A SYBR GREEN PCR kit was used and the reaction was carried out in an ABI Prism 7000 SDS. Apoptotic indexes progressively increased indicating a role of IBDV in inducing hypotrophy of the BF. Also, it was showed that as long as apoptosis increased, viral protein expression decreased, which suggests that apoptosis plays a role as a defense mechanism against viral replication.(AU)

Apoptose e expressão de VP2 e GAPDH na infecção precoce pelo vírus da doença infecciosa da bursa de Fabricius em pintos SPF / Apoptosis and expression of VP2 and GADPH in an experimental infectious bursal disease in SPF chicks

Vinte e nove pintos SPF de um dia foram inoculados com o vírus da doença infecciosa da bursa de Fabricius (VDIB) para avaliar a ocorrência precoce de apoptose e a expressão da proteína viral 2 (VP2) e da enzima gliceraldeído fosfato dehidrogenase (GAPDH). Os animais foram distribuídos em cinco grupos: 1-controle; e 2 a 5- com 24, 48, 72 e 96 horas pós-inoculação, respectivamente. Fragmentos da bursa de Fabricius foram colhidos para processamento histológico e extração de RNA. Lâminas coradas em HE e TUNEL (marcação in situ da fragmentação do genoma com transferase terminal de deoxinucleotídeo) foram utilizadas na morfometria do índice apoptótico. Amostras de mRNA foram testadas para a expressão dos genes VP2 e GAPDH utilizando-se transcrição reversa e RT-PCR. Utilizou-se um kit SYBR GREEN PCR, e a reação foi desenvolvida em ABI Prism 7000 SDS. Os índices apoptóticos cresceram progressivamente indicando uma relação na atrofia bursal causada pelo VDIB. Paralelamente, os resultados da PCR em tempo real demonstraram queda da carga viral nas células linfóides da bursa nos diferentes intervalos de tempo do experimento. Esses resultados sugerem um papel protetor da apoptose na diminuição da replicação viral.

Twenty-nine SPF 1-day-old chicks were inoculated with infectious bursal disease virus (IBDV) to evaluate early apoptosis and the expression of viral protein 2 (VP2) and glyceraldehyde-3-phosphate dehydrogenease (GAPDH). Five groups were formed: G1-control -and G2 to G5, - 24, 48, 72 and 96 hours post inoculation, respectively. Half of each BF was fixed and processed by routine techniques. To quantify apoptosis, 5µm-thick sections were stained with HE and submitted to TUNEL (terminal transferase UDP nick end labeling) technique. mRNA was extracted from pooled samples of 3 animals/group and used for the expression of VP2 and GADPH genes using the reverse transcription and real-time polymerase chain reaction (RT-PCR). A SYBR GREEN PCR kit was used and the reaction was carried out in an ABI Prism 7000 SDS. Apoptotic indexes progressively increased indicating a role of IBDV in inducing hypotrophy of the BF. Also, it was showed that as long as apoptosis increased, viral protein expression decreased, which suggests that apoptosis plays a role as a defense mechanism against viral replication.