Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations.

The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota--LS--(vaccine strain) and Sao Joao do Meriti--SJM--strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity.

Chlamydophila psittaci infections in hyacinth macaws (Anodorhynchus hyacinthinus) confiscated in Brazil.

The hyacinth macaw (Anodorhynchus hyacinthinus) is the largest species of psittacine birds. It is considered endangered and illegal trade is one of the main factors involved in its decline. In this study, 26 hyacinth macaws maintained under poor husbandry conditions and destined for the illegal trade were confiscated in São Paulo State, Brazil. These birds were evaluated for the presence of antibodies against Chlamydophila psittaci by complement fixation test and C. psittaci DNA by seminested polymerase chain reaction. Results showed that 65.4% of the macaws were positive for at least one test. Birds with subclinical infections can shed chlamydiae intermittently over long periods, contributing to the dissemination of the agent. Global trade is one of the most important drivers of disease emergence. The high percentage of positive samples in this study emphasizes the potential risk that the illegal trade of wild birds represents for both human and animal health.

Prevalence of Salmonella spp. antibodies to Toxoplasma gondii, and Newcastle disease virus in feral pigeons (Columba livia) in the city of Jaboticabal, Brazil.

The rock pigeon (Columba livia) may serve as a reservoir for several pathogenic agents that can be transmitted to poultry, wildlife, domesticated pets, and/or humans via excreta, secretions, or dust from feathers. In addition, ingestion of infected pigeons by wild and domestic animals can also transmit these pathogenic agents. The health status of 126 free-living pigeons in an urban area was evaluated by microbiologic culture for Salmonella and serologic testing for the presence of antibodies for Toxoplasma gondii and for Newcastle disease virus (NDV) from 120 and 109 pigeons, respectively. After drawing blood, the birds were euthanized, and fragments of the liver, spleen, lungs, and gonads, and feces were cultured for Salmonella spp. Salmonella spp. was isolated from 10 birds (7.94%), of which 8 were Salmonella typhimurium, one was Salmonella enterica subsp. enterica serotype 4,12 and one was Salmonella enterica subsp. enterica serotype 4,12,i. Six of 109 pigeons (5.50%) were positive for NDV antibodies when using the hemagglutination inhibition test. Toxoplasma gondii antibodies were detected by immunofluorescence in one of 120 sera tested (0.83%). The results indicate that feral rock pigeons were exposed to NDV and T. gondii, although the exposure was low. In addition, these birds had Salmonella spp. and could disseminate this pathogen in the environment.

Natural host relationships and genetic diversity of rodent-associated hantaviruses in southeastern Brazil.

OBJECTIVE: Hantaviruses are rodent-borne RNA viruses that have caused hantavirus cardiopulmonary syndrome in several Brazilian regions. In the present study, geographical distribution, seroprevalence, natural host range, and phylogenetic relations of rodent-associated hantaviruses collected from seven counties of Southeastern Brazil were evaluated. METHODS: ELISA, RT-PCR and phylogenetic analysis were used in this study. RESULTS: Antibodies to hantavirus were detected in Bolomys lasiurus, Akodon sp. and Oligoryzomys sp., performing an overall seroprevalence of 5.17%. All seropositive rodents were associated with grasslands or woods surrounded by sugar cane fields. Phylogenetic analysis of partial S- and M-segment sequences showed that viral sequences isolated from B. lasiurus specimens clustered with Araraquara virus. However, a sequence from Akodon sp. shared 100% similarity with Argentinian/Chilean viruses based on the partial S-segment amino acid sequence. CONCLUSION: These results indicate that there are associations between rodent reservoirs and hantaviruses in some regions of Southeastern Brazil, and suggest the existence of additional hantavirus genetic diversity and host ecology in these areas.

Experimental infection of Newcastle disease virus in pigeons (Columba livia): humoral antibody response, contact transmission and viral genome shedding.

The aim of this study was to evaluate the humoral antibody response, the genome viral excretion and the contact transmission of pathogenic chicken origin Newcastle disease virus (NDV) from experimentally infected pigeons (Columba livia) to in-contact pigeon. The antibody response to infection was assessed by the hemagglutination inhibition (HI) test and the genome viral excretion was detected by RT-PCR. Viral strain induced high antibody levels, both in inoculated and in sentinel birds. The pathogenic viral strain for chickens was unable to produce clinical signs of the disease in experimentally infected pigeons, although it induced the humoral antibody response and produced NDV genome shedding. NDV genome was detected intermittently throughout the experimental period, from 5 days post-infection (dpi) to 24 dpi. Therefore, viral genome shedding occurred for 20 days. The viral genome was detected in all birds, between 11 and 13 dpi. Furthermore, the high infectivity of the virus was confirmed, as all non-inoculated sentinel pigeons showed antibody levels as high as those of inoculated birds.

Chlamydophila psittaci in free-living Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil.

Chlamydophila psittaci (C. psittaci) infection was evaluated in 77 free-living nestlings of Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil. Tracheal and cloacal swab samples from 32 wild parrot and 45 macaw nestlings were submitted to semi-nested PCR, while serum samples were submitted to complement fixation test (CFT). Although all 32 Amazon parrot serum samples were negative by CFT, cloacal swabs from two birds were positive for Chlamydophila DNA by semi-nested PCR (6.3%); these positive birds were 32 and 45 days old. In macaws, tracheal and cloacal swabs were positive in 8.9% and 26.7% of the samples, respectively. Complement-fixing antibodies were detected in 4.8% of the macaw nestlings; macaw nestlings with positive findings were between 33 and 88 days old. These results indicate widespread dissemination of this pathogen in the two evaluated psittacine populations. No birds had clinical signs suggestive of chlamydiosis. To the best of our knowledge, this is the first report on C. psittaci in free-living Blue-fronted Amazon parrots and Hyacinth macaws in Brazil.

Experimental infection with Brazilian Newcastle disease virus strain in pigeons and chickens.

This study was designed with the goal of adding as much information as possible about the role of pigeons (Columba livia) and chickens (Gallus gallus) in Newcastle disease virus epidemiology. These species were submitted to direct experimental infection with Newcastle disease virus to evaluate interspecies transmission and virus-host relationships. The results obtained in four experimental models were analyzed by hemagglutination inhibition and reverse transcriptase polymerase chain reaction for detection of virus shedding. These techniques revealed that both avian species, when previously immunized with a low pathogenic Newcastle disease virus strain (LaSota), developed high antibody titers that significantly reduced virus shedding after infection with a highly pathogenic Newcastle disease virus strain (São Joao do Meriti) and that, in chickens, prevent clinical signs. Infected pigeons shed the pathogenic strain, which was not detected in sentinel chickens or control birds. When the presence of Newcastle disease virus was analyzed in tissue samples by RT-PCR, in both species, the virus was most frequently found in the spleen. The vaccination regimen can prevent clinical disease in chickens and reduce viral shedding by chickens or pigeons. Biosecurity measures associated with vaccination programs are crucial to maintain a virulent Newcastle disease virus-free status in industrial poultry in Brazil.

Foot-and-mouth disease in tropical wildlife.

This review of foot-and-mouth disease in cloven-hoofed, free-living animals, describes the disease, the wide range of the hosts, the carrier state, and the interrelationship between disease in domestic livestock and wildlife. This information becomes even more crucial to the development of control strategies when linked to the process of pathogenesis and the epidemiology of the disease.

Evidence of Chlamydophila psittaci infection in captive Amazon parrots in Brazil.

The prevalence of Chlamydophila psittaci (formerly Chlamydia psittaci) infection was assessed in 95 apparently healthy, captive Amazon parrots from three breeder collections in southeastern and west-central Brazil. Cloacal swabs from 95 birds were tested for chlamydial antigen, which was detected by direct immunofluorescence (DIF), and serum samples from 44 of these birds were tested for antibodies to C. psittaci using an enzyme-linked immunosorbent assay. The prevalences of active infection as detected by DIF were 16.7%, 22.2%, and 56.1%, and seroprevalences were 100%, 87.5%, and 60% in flocks A, B, and C, respectively. We can therefore infer that C. psittaci may be widespread in captive parrot populations in Brazil.

An outbreak of chlamydiosis in captive blue-fronted Amazon parrots (Amazona aestiva) in Brazil.

Fifty-eight blue-fronted Amazon parrot (Amazona aestiva) nestlings, recovered from the illegal trade, became ill at a wildlife rehabilitation center in São Paulo State, Brazil. Clinical signs observed were nonspecific, and the mortality rate was 96.5% despite initial treatment with norfloxacin. Postmortem examinations were performed on 10 birds. Liver and spleen smears showed structures suggestive of Chlamydophila psittaci in four cases. Diagnosis was confirmed by seminested polymerase chain reaction on tissue samples. Other birds from the same location showed no clinical signs of the disease, although high complement fixation titers to C. psittaci were found in 10 adult psittacines. All birds in the facility were treated with doxycycline. The two surviving nestlings did not recover after two doxycycline treatments and were euthanatized. The high mortality rate observed in this outbreak was attributed to poor conditions of husbandry and delays in the diagnosis and treatment of the disease. After diagnosis, improved control measures for chlamydiosis were instituted.

Detection of hemoplasma and Bartonella species and co-infection with retroviruses in cats subjected to a spaying/neutering program in Jaboticabal, SP, Brazil.

Hemotrophic mycoplasmas and Bartonella species are important pathogens that circulate between cats and invertebrate hosts, occasionally causing diseases in humans. Nevertheless, there are few reports on occurrences of these agents in cats in Brazil. The present study aimed to detect the presence of hemoplasma and Bartonella DNA by means of PCR and sequencing. FIV antigens and anti-FeLV antibodies, were studied by using a commercial kit on blood and serum samples, respectively, among 46 cats that were sampled during a spaying/neutering campaign conducted in Jaboticabal, SP. Three (6.5%) cats were positive for hemoplasmas: two (4.3%) for 'Candidatus M. haemominutum' and one (2.2%) for both M. haemofelis and 'Candidatus M. turicensis'. One of the two 'Candidatus M. haemominutum'-infected cats was also positive for FeLV antigens and showed antibodies for FIV. Two cats (4.3%) were positive for B. henselae. One of them was also positive for FeLV antigens. Eight cats (17.4%) were positive for FeLV, and just one (2.2%) showed anti-FIV antibodies. Bartonella species and hemoplasmas associated with infection due to retroviruses can circulate among apparently healthy cats.

Experimental infection with Brazilian Newcastle disease virus strain in pigeons and chickens

Abstract This study was designed with the goal of adding as much information as possible about the role of pigeons (Columba livia) and chickens (Gallus gallus) in Newcastle disease virus epidemiology. These species were submitted to direct experimental infection with Newcastle disease virus to evaluate interspecies transmission and virus-host relationships. The results obtained in four experimental models were analyzed by hemagglutination inhibition and reverse transcriptase polymerase chain reaction for detection of virus shedding. These techniques revealed that both avian species, when previously immunized with a low pathogenic Newcastle disease virus strain (LaSota), developed high antibody titers that significantly reduced virus shedding after infection with a highly pathogenic Newcastle disease virus strain (São Joao do Meriti) and that, in chickens, prevent clinical signs. Infected pigeons shed the pathogenic strain, which was not detected in sentinel chickens or control birds. When the presence of Newcastle disease virus was analyzed in tissue samples by RT-PCR, in both species, the virus was most frequently found in the spleen. The vaccination regimen can prevent clinical disease in chickens and reduce viral shedding by chickens or pigeons. Biosecurity measures associated with vaccination programs are crucial to maintain a virulent Newcastle disease virus-free status in industrial poultry in Brazil.

Detection of Chlamydophila felis and Feline Herpesvirus Type-1 in non-domestic felids in Brazil / Detecção de Chlamydophila felis e Herpesvirus felino tipo 1 em felídeo não doméstico no Brasil

Little is known about the occurrence of feline upper respiratory tract disease agents, namely Feline Herpesvirus type 1 (FHV-1) and Chlamydophila felis, and co-infection of these agents with Feline Immunodeficiency virus (FIV) and Feline Leukemia Virus (FeLV) in non-domestic felids in Brazil. Between 2009 and 2010, 72 conjunctival swab and serum samples were collected from eight non-domestic felid species (Leopardus pardalis, Leopardus tigrinus, Panthera leo, Panthera tigris, Puma concolor, Puma yagouaroundi, Oncifelis colocolo, and Panthera onca) maintained in captivity in Brazilian zoos. DNA extracted from conjunctival swabs were used in PCR assays for the detection of Chlamydophila sp, FHV-1, and retrovirus DNA, respectively. Antibodies to FIV and FeLV antigen were detected in non-domestic felid serum samples using a commercial ELISA kit. Antibodies to FIV were found only in five (6.9%) felids. No sampled non-domestic felid was positive for FeLV antigen detection. One (1.3%) out of 72 non-domestic felid conjunctival swab samples was positive for Chlamydophilasp. and Feline Herpesvirus-1 in PCR. This felid was an ocelot and was negative for FIV and FeLV. The results of this survey showed the occurrence of co-infection with C. felis and FHV-1 in an ocelot (Leopardus pardalis) in Brazil...

Poucos trabalhos descrevem a ocorrência dos agentes do complexo respiratório felino, Herpesvírus Felino tipo 1 (FHV-1) e Chlamydophila felis, e a coinfecção com o vírus da imunodeficiência felina (FIV) e leucemia viral felina (FeLV) em felinos não domésticos no Brasil. Entre 2009 e 2010, 72 amostras de swab de conjuntiva e de soro foram coletados de oito espécies de felinos não domésticos (Leopardus pardalis, Leopardus tigrinus, Panthera leo, Panthera tigris, Puma concolor, Puma yagouaroundi, Oncifelis colocolo, and Panthera onca) mantidos em cativeiro em zoológicos brasileiros. O DNA foi extraído das amostras de swab de conjuntiva para detecção de Chlamydophila sp e FHV-1 pela PCR. Anticorpos para FIV e antígeno para FeLV foram determinados pelo kit comercial de ELISA. Anticorpos para FIV foram detectados em cinco felídeos (6,9%). Nenhuma amostra foi positiva para a presença de antígeno de FeLV. Um (1,3%) dos 72 felinos não domésticos apresentou fragmentos de DNA de Chlamydophila sp e FHV-1 pela PCR. Este felino era uma jaguatirica que não apresentou anticorpos para FIV e nem antígeno para FelV. Estes resultados demonstram a ocorrência de coinfecção de C. felis e FHV-1 em uma jaguatirica (Leopardus pardalis) no Brasil...

Detection of hemoplasma and Bartonella species and co-infection with retroviruses in cats subjected to a spaying/neutering program in Jaboticabal, SP, Brazil / Detecção de hemoplasmas e Bartonella sp. e co-infecção com retrovírus em gatos submetidos a um programa de castração/esterilização em Jaboticabal, SP, Brasil

Hemotrophic mycoplasmas and Bartonella species are important pathogens that circulate between cats and invertebrate hosts, occasionally causing diseases in humans. Nevertheless, there are few reports on occurrences of these agents in cats in Brazil. The present study aimed to detect the presence of hemoplasma and Bartonella DNA by means of PCR and sequencing. FIV antigens and anti-FeLV antibodies, were studied by using a commercial kit on blood and serum samples, respectively, among 46 cats that were sampled during a spaying/neutering campaign conducted in Jaboticabal, SP. Three (6.5%) cats were positive for hemoplasmas: two (4.3%) for 'Candidatus M. haemominutum' and one (2.2%) for both M. haemofelis and 'Candidatus M. turicensis'. One of the two 'Candidatus M. haemominutum'-infected cats was also positive for FeLV antigens and showed antibodies for FIV. Two cats (4.3%) were positive for B. henselae. One of them was also positive for FeLV antigens. Eight cats (17.4%) were positive for FeLV, and just one (2.2%) showed anti-FIV antibodies. Bartonella species and hemoplasmas associated with infection due to retroviruses can circulate among apparently healthy cats.

Micoplasmas hemotróficos e espécies de Bartonella são importantes patógenos que circulam entre gatos e hospedeiros invertebrados, causando ocasionalmente doenças no homem. Apesar disto, poucos são os estudos acerca da ocorrência destes agentes entre gatos no Brasil. O presente estudo objetivou detectar o DNA de hemoplasmas e Bartonella sp. pela PCR e sequenciamento. Antígeno de FIV e anticorpos anti-FeLV foram estudados utilizando um "kit" comercial, em amostras de sangue e soro, respectivamente, de 46 gatos amostrados em uma campanha de castração em Jaboticabal, SP. Três gatos (6,5%) foram positivos para hemoplasmas: dois (4,3%) para 'Candidatus M. haemominutum' e um (2,2%) para M. haemofelis and 'Candidatus M. turicensis'. Um dos gatos positivos para 'Candidatus M. haemominutum' mostrou-se também positivo na detecção de antígeno de FeLV e de anticorpos para FIV. Dois (4,3%) gatos mostraram-se positivos para B. henselae, sendo que um deles também se mostrou positivo para antígeno de FeLV. Oito gatos (17,4%) foram positivos para FeLV, e apenas um gato mostrou anticorpos anti-FIV. Bartonella sp. e hemoplasmas associados à infecção por retrovírus podem circular entre gatos aparentemente saudáveis.

Aplicaçäo de uma técnica de ELISA ("Enzyme-linked immunosorbent assay") no diagnóstico direto do vírus da febre aftosa / Use of a technic ELISA-Enzyme-linked immunosorbent assay in the direct diagnosis of fost-and-mouth disease virus

O método do "Sandwich indireto" do ELISA ("Enzyme-Linked Immunosorbent Assay"), foi nesse trabalho empregado com vantagens para detecçäo e tipificaçäo direta dos sorotipos O1, A24, AVenceslau e C3 do vírus da febre aftosa presentes em suspensöes antigênicas brutas provenientes de cultura celular, do epitélio de coxim plantar de cobaias, de carcaça de camundongos e de epitélio lingual bovino, infectados com essas estirpes virais. Os títulos obtidos no ELISA foram sempre superiores aos da reaçäo de fixaçäo de complemento, apresentando uma sensibilidade 3,8 a 80 vezes maior do que esta última reaçäo. Quanto à especificidade do ensaio imunoenzimático foi observado o desenvolvimento de reaçöes heterotípicas cruzadas principalmente da estirpe O1 com o anti-soro anti-C3, da estirpe A24 com o anti-O1, e anti-C3, da estirpe AVenceslau com anti-O1 e da estirpe C3 com o anti-O1, o que näo ocorreu na reaçäo de fixaçäo de complemento. No entanto, isto näo impediu que as 4 estirpes virais estudadas (O1, A24, AVenceslau e C3) fossem classificadas pelo ELISA em seus respectivos sorotipos

Leucocyte migration inhibition test (LMI) for the in vitro detection of cellular immunity in Newcastle disease

O teste de inibiçäo da migraçäo de leucócitos (IML), descrito neste trabalho, mostrou-se apropriado para avaliaçäo da imunidade anti-Newcastle em aves. O teste de inibiçäo da hemaglutinaçäo (HI) foi usado como parâmetro de comparaçäo para medida da resposta imune humoral. Os resultados mostraram näo haver correspondência entre as imunidades celular e humoral

Relation between the hemaglutination inhibition test and the indirect ELISA in the sorologic monitoring of laying hens submitted to different systems of vaccination against newcastle disease

A relaçäo entre os testes de ELISA indireto (EI) e de inibiçäo de hemaglutinaçäo (HI) foi estudada em 240 soros de poedeiras submetidas a diferentes sistemas de vacinaçäo contra a Doença de Newcastle. Uma elevada correlaçäo foi obtida entre as duas provas sorológicas, embora tenha sido observada uma ampla variaçäo de absorvância no EI para soros do mesmo título de HI