Presence of antibodies against H5, H7 and H9 influenza A virus in wild birds in the State of São Paulo, Brazil

Although the natural reservoirs of the avian influenza (AI) virus have been extensively studied in many countries, there is a clear lack of information on this subject in South America, particularly in Brazil. The objective of this study was to conduct a serological survey for H5, H7 and H9 antibodies to AI-subtype viruses in wild birds in the state of São Paulo , Brazil. Serum samples were tested using the hemagglutination-inhibition assay. Out of the 31 wild birds sampled between January and December of 2006, seven (22.58%), were seropositive for H5, H7 and H9; four (12.90%) were seropositive for H5 and H7; 13 (41.94%), were seropositive only for H7; three (9.7%), were seropositive only for H9; and four (12.90%) were negative for all three hemagglutinin subtypes. These results indicate that AI viruses belonging to H5, H7 and H9 subtypes circulate among wild birds in the state of São Paulo in the form of either concurrent or consecutive infections. This study contributes to the knowledge of AI epidemiology in Brazil, and stresses the need of further detailed and long-term epidemiological and ecological investigation to determine the current status of this virus.(AU)

Presence of antibodies against H5, H7 and H9 influenza A virus in wild birds in the State of São Paulo, Brazil

Although the natural reservoirs of the avian influenza (AI) virus have been extensively studied in many countries, there is a clear lack of information on this subject in South America, particularly in Brazil. The objective of this study was to conduct a serological survey for H5, H7 and H9 antibodies to AI-subtype viruses in wild birds in the state of São Paulo , Brazil. Serum samples were tested using the hemagglutination-inhibition assay. Out of the 31 wild birds sampled between January and December of 2006, seven (22.58%), were seropositive for H5, H7 and H9; four (12.90%) were seropositive for H5 and H7; 13 (41.94%), were seropositive only for H7; three (9.7%), were seropositive only for H9; and four (12.90%) were negative for all three hemagglutinin subtypes. These results indicate that AI viruses belonging to H5, H7 and H9 subtypes circulate among wild birds in the state of São Paulo in the form of either concurrent or consecutive infections. This study contributes to the knowledge of AI epidemiology in Brazil, and stresses the need of further detailed and long-term epidemiological and ecological investigation to determine the current status of this virus.

Seroprevalence of antibodies to Chlamydophila psittaci in zoo workers in Brazil.

To evaluate the prevalence of antibodies to Chlamydophila psittaci 364 serum samples were collected from veterinarians, biologists, animal scientists, veterinary students, animal keepers and others employees in 20 zoos, and from veterinary practitioners in 10 Brazilian states. Subjects ranged from 15 to 64 years of age, with 268 (74%) males and 96 (26%) females. Chlamydial antibodies were determined by the complement fixation test (CFT) and specific anti-C. psittaci IgG antibodies were determined by the microimmunoflurescence (MIF) test. Complement fixation test showed 23.9% (87/364) and MIF test showed 4.7% (17/364) positive serum samples. Titres ranged from 16 to 256 in both assays, demonstrating evidence of recent or current infection. Although chlamydial antibodies were detected in workers of seventeen zoos, MIF test only detected specific C. psittaci antibodies in seven of them. Previous psittacosis infection was suspected in eight workers of two zoos, five of whom reported having pneumonia, while employed at the zoos. However, diagnosis was not established in any of these cases in the past. Results indicated the occurrence of infection and previous contact of Brazilian zoo workers with C. psittaci, as well as the zoonotic potential of psittacosis in this risk population. Other studies are necessary to evaluate the risk factors of infection in this population. This seroepidemiological survey confirmed the need to adopt preventive measures to control avian chlamydiosis and protect the health of zoo workers in the country.

Neospora caninum infection in birds: experimental infections in chicken and embryonated eggs.

Neospora caninum causes economical impact in cattle-raising farms since it is implicated as the major cause of bovine abortions. Although infection by the parasite has been widely described in mammals, the role of birds in its life-cycle is still obscure. Therefore, this work aimed to evaluate the infection by N. caninum in different chicken models. Experimental infections were conducted in 7-day-old chicks, laying hens and embryonated eggs, where samples were analysed for parasite burden, IgG antibodies and lesions promoted. Chickens demonstrated an asymptomatic infection, although with seroconversion and systemic replication of the parasite. In laying hens, no signs of vertical transmission were observed. However, embryonated eggs inoculated by the allantoic cavity route demonstrated susceptibility to infection, with mortality rates around 50% independent of the inoculum dose. Additionally, dogs became infected after ingestion of different amounts of inoculated eggs, producing either oocysts or specific IgG antibodies. The results herein presented demonstrate that chickens may be intermediate hosts of N. caninum and that embryonated eggs could be a useful model to study the parasite's biology.

Zooplankton community structure of two marginal lakes of the River Cuiabá (Mato Grosso, Brazil) with analysis of Rotifera and Cladocera diversity.

In the present study, two small lakes on the margins of the River Cuiabá were analyzed regarding taxonomic composition and population densities of the zooplankton. Diversity was evaluated for two groups, Rotifera and Cladocera; sampling was carried out on two dates: 2 March 1999, in the rainy season, and 25 August 1999, in the dry season. Seventy-nine rotifer taxa, 30 cladoceran taxa, and 6 copepod taxa were found. Comparing the species identified in the present study with those recorded by other authors for several water bodies in Mato Grosso and Mato Grosso do Sul states, it was found that 9 species of Cladocera, 2 of Copepoda, and 14 of Rotifera are new records for the region. The most abundant rotifer species were Keratella cochlearis, Brachionus angularis, Polyarthra vulgaris, and Keratella americana. Moina minuta and Bosminopsis deitersi were dominant among the cladocerans, and Notodiaptomus transitans and N. devoyorum among the copepods. Comparing both lakes, the greatest species richness of both Rotifera and Cladocera was observed in Lake Souza Lima, during the rainy season. This is probably linked to the fact that the littoral region of this lake is densely colonized by macrophytes. The lake also has better environmental conditions since it does not receive domestic sewage inputs, as does Lake Parque Atalaia. The diversity of the Rotifera was markedly low in Lake Parque Atalaia, during the dry season, again perhaps linked domestic sewage input found in this water body.

Zooplankton community structure of two marginal lakes of the River Cuiabá (Mato Grosso, Brazil) with analysis of Rotifera and Cladocera diversity

In the present study, two small lakes on the margins of the River Cuiabá were analyzed regarding taxonomic composition and population densities of the zooplankton. Diversity was evaluated for two groups, Rotifera and Cladocera; sampling was carried out on two dates: 2 March 1999, in the rainy season, and 25 August 1999, in the dry season. Seventy-nine rotifer taxa, 30 cladoceran taxa, and 6 copepod taxa were found. Comparing the species identified in the present study with those recorded by other authors for several water bodies in Mato Grosso and Mato Grosso do Sul states, it was found that 9 species of Cladocera, 2 of Copepoda, and 14 of Rotifera are new records for the region. The most abundant rotifer species were Keratella cochlearis, Brachionus angularis, Polyarthra vulgaris, and Keratella americana. Moina minuta and Bosminopsis deitersi were dominant among the cladocerans, and Notodiaptomus transitans and N. devoyorum among the copepods. Comparing both lakes, the greatest species richness of both Rotifera and Cladocera was observed in Lake Souza Lima, during the rainy season. This is probably linked to the fact that the littoral region of this lake is densely colonized by macrophytes. The lake also has better environmental conditions since it does not receive domestic sewage inputs, as does Lake Parque Atalaia. The diversity of the Rotifera was markedly low in Lake Parque Atalaia, during the dry season, again perhaps linked domestic sewage input found in this water body

Zooplankton community structure of two marginal lakes of the River Cuiabá (Mato Grosso, Brazil) with analysis of Rotifera and Cladocera diversity

In the present study, two small lakes on the margins of the River Cuiabá were analyzed regarding taxonomic composition and population densities of the zooplankton. Diversity was evaluated for two groups, Rotifera and Cladocera; sampling was carried out on two dates: 2 March 1999, in the rainy season, and 25 August 1999, in the dry season. Seventy-nine rotifer taxa, 30 cladoceran taxa, and 6 copepod taxa were found. Comparing the species identified in the present study with those recorded by other authors for several water bodies in Mato Grosso and Mato Grosso do Sul states, it was found that 9 species of Cladocera, 2 of Copepoda, and 14 of Rotifera are new records for the region. The most abundant rotifer species were Keratella cochlearis, Brachionus angularis, Polyarthra vulgaris, and Keratella americana. Moina minuta and Bosminopsis deitersi were dominant among the cladocerans, and Notodiaptomus transitans and N. devoyorum among the copepods. Comparing both lakes, the greatest species richness of both Rotifera and Cladocera was observed in Lake Souza Lima, during the rainy season. This is probably linked to the fact that the littoral region of this lake is densely colonized by macrophytes. The lake also has better environmental conditions since it does not receive domestic sewage inputs, as does Lake Parque Atalaia. The diversity of the Rotifera was markedly low in Lake Parque Atalaia, during the dry season, again perhaps linked domestic sewage input found in this water body.

Este trabalho se baseia no estudo das comunidades zooplanctônicas de duas lagoas marginais ao rio Cuiabá, próximas às cidades de Cuiabá e Várzea Grande, e teve por objetivo analisar a composição de espécies, a abundância das populações zooplanctônicas e a diversidade dos Rotifera e dos Cladocera. Realizaram-se duas campanhas para amostragens, com coletas efetuadas nos dias 2/3/1999, no período chuvoso, e 25/8/1999, no período seco. Foram identificados no total 115 táxons, distribuídos em 79 táxons de Rotifera, 30 táxons de Cladocera e 6 táxons de Copepoda. Comparando-se as espécies registradas no presente estudo com aquelas já registradas por diferentes autores para os Estados de Mato Grosso e Mato Grosso do Sul, observa-se que 9 espécies de Cladocera, 2 espécies de Copepoda e 14 espécies de Rotifera são novas ocorrências para esses Estados ou região. As espécies mais abundantes foram Keratella cochlearis, K. americana, Brachionus angularis e Polyarthra vulgaris para Rotifera, Moina minuta e Bosminopsis deitersi entre os Cladocera e Notodiaptomus transitans e N. devoyorum entre os Copepoda. Comparando-se ambas as lagoas, observa-se que a maior riqueza de espécies foi registrada para a lagoa Souza Lima durante a estação chuvosa, em relação aos Rotifera e aos Cladocera, o que pode estar relacionado ao fato de a região litorânea dessa lagoa ser densamente colonizada por macrófitas e não receber esgoto doméstico, como observado na lagoa Parque Atalaia. A diversidade de Rotifera foi notadamente baixa na lagoa Parque Atalaia durante a estação seca, o que também pode estar relacionado à entrada de esgoto doméstico nesse corpo d'água.

Extensive antigenic and genetic variation among foot-and-mouth disease type A viruses isolated from the 1994 and 1995 foci in São Paulo, Brazil.

Nine foot-and-mouth disease virus (FMDV) type A isolates recovered from the field FMD foci in São Paulo State, Brazil, during 1994 and 1995 (a period preceding the last reported focus of FMD in 1996 in this state) were compared among themselves and with the reference vaccine strain A(24)Cruzeiro. The techniques used were sandwich ELISA, virus neutralization (VN), polyacrylamide gel electrophoresis (PAGE) of the structural polypeptides and direct sequencing of the VP1-coding region (1D gene). Results of VN were recorded as serological relationships "R" and those from ELISA were expressed as percentage of the homologous reaction "r". ELISA and VN gave comparable results (correlation coefficient, 0.936) allowing assignment of these field viruses to four groups which were distinct from the A(24)Cruzeiro strain. PAGE and 1D nucleotide sequencing were also able to distinguish between these viruses. The high level of genetic and antigenic variation found when comparing the A(24)Cruzeiro vaccine strain and type A strains recovered from the last identified foci of FMD came from a formerly endemic area where vaccination with polyvalent vaccines (O(1)Campos, A(24)Cruzeiro and C(3)Indaial) had been extensively applied. The similarity between the results of the serological and genetic analyses suggest that the antigenic differences found are mainly located in the 1D protein.

Detection of infectious bronchitis virus in experimentally infected chickens by an antigen-competitive ELISA.

An antigen-competitive enzyme-linked immunosorbent assay (Ag-C-ELISA) was developed for the detection of infectious bronchitis virus (IBV) antigens, M41 strain, in tissues from experimentally infected chickens, or in allantoic fluid harvested from inoculated embryonated eggs. The detection limit of IBV in the Ag-C-ELISA was 10(4.1) median embryo infective doses (EID(50))/well. Tracheal and lung samples from chickens vaccinated with 10(2.5) EID(50) of live attenuated infectious bronchitis (H120) vaccine were negative in the direct detection Ag-C-ELISA. The results indicate that the Ag-C-ELISA has the potential to detect IBV, either directly in tissue samples or when combined with the passage of material in embryonated eggs, thereby constituting an alternative method for the diagnosis of IBV.

Detection and quantification of antibodies to Newcastle disease virus in ostrich and rhea sera using a liquid phase blocking enzyme-linked immunosorbent assay.

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method. The cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement (kappa = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Antibody response to Newcastle disease vaccination in a flock of young partridges (Rhynchotus rufescens).

Ten young partridges (Rhynchotus rufescens) were vaccinated with the lentogenic strain of Newcastle disease virus. Another eight unvaccinated birds were kept in close contact with the treated flock. Antibodies levels were measured over the course of 3 mo in all birds using the hemagglutination inhibition (HI) test and the liquid-phase blocking enzyme-linked immunosorbent assay (LPB-ELISA). The LPB-ELISA was standardized, and the results were compared with those obtained with the HI test. Antibodies increased after 23 days postvaccination in 16 birds with no side effects as determined by both the HI test and the LPB-ELISA.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Babesia bovis in cattle.

A method for the isolation of Babesia bovis merozoites from infected erythrocytes (Machado et al., 1994) and an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-B. bovis antibodies were developed. This ELISA utilizes a soluble, alkali-digested B. bovis antigen. Sera from calves experimentally infected with B. bovis were screened by this technique from day 9 to day 233 postinfection (PI). Maximum titers were reached between days 29 and 149 PI. Sera from calves (n = 62), heifers (n = 38) and cows (n = 49), raised in tick-infested areas of São Paulo State, showed higher antibody levels in heifers and cows. A higher percentage of negative sera (19.4%) was found among calves. Sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and immunoblotting have identified proteins of similar molecular mass in the two species. Sera from calves experimentally infected with B. bovis reacted with homologous antigens at the level of 95, 66 and 23 kDa. The same serum reacted with the 23 kDa band of heterologous antigen. Sera from calves experimentally infected with B. bigemina recognized 82, 66, 58, 36 and the 23 kDa polypeptides of homologous and heterologous antigens. The experimental ELISA described may prove to be a practical serological test for bovine Babesia infection with the choice of specific test antigen for B. bovis and B. bigemina.

Liquid-phase blocking sandwich enzyme-linked immunosorbent assay for detection of antibodies against foot-and-mouth disease virus in water buffalo sera.

OBJECTIVE: To develop and apply the liquid-phase blocking sandwich ELISA (BLOCKING-ELISA) for the quantification of antibodies against foot-and-mouth disease virus (FMDV) strains O1 Campos, A24 Cruzeiro, and C3 Indaial. DESIGN--Antibody quantification. SAMPLE POPULATION: 158 water buffalo from various premises of Sao Paulo State-Brazil. The sera were collected either from systemically vaccinated or nonvaccinated animals. PROCEDURE: The basic reagents of BLOCKING-ELISA (capture and detector antibodies, virus antigens, and conjugate) were prepared and the reaction was optimized and standardized to quantify water buffalo antibodies against FMDV. An alternative procedure based on mathematical interpolation was adopted to estimate more precisely the antibody 50% competition titers in the BLOCKING-ELISA. These titers were compared with the virus-neutralization test (VNT) titers to determine the correlation between these techniques. The percentages of agreement, cutoff points, and reproducibility also were determined. RESULTS: The antibody titers obtained in the BLOCKING-ELISA had high positive correlation coefficients with VNT, reaching values of 0.90 for O1 Campos and C3 Indaial, and 0.82 for the A24 Cruzeiro (P < 0.0005). The cutoff points obtained by use of the copositivity and conegativity curves allowed determination of high levels of agreement between BLOCKING-ELISA and VNT antibody titers against the 3 FMDV strains analyzed. CONCLUSIONS: The results characterized by high correlation coefficients, levels of agreement, and reproducibility indicate that the BLOCKING-ELISA may replace the conventional VNT for detection and quantification of antibodies from water buffalo sera to FMDV.

Rapid coagglutination test for the detection and typing of foot and mouth disease virus.

Protein A containing Staphylococcus aureus was used to develop a coagglutination (COA) test for the detection and typing of foot and mouth disease virus (FMDV) O, A and C serotypes in infected cells and tissues. Different batches and amounts of guinea pig anti-FMDV sera were assessed to optimize the preparation of COA conjugates. The sensitivity and specificity of the COA Test for the detection of FMDV O, A and C serotypes and heterologous viruses was also characterized. Comparison between the COA Test and complement fixation test for the detection and typing of FMDV obtained from extracts of tongue epithelial tissues from infected cattle revealed high agreement in the results and indicated a potential application of the COA Test for the direct diagnosis of viruses.

Efficiency test for foot and mouth disease vaccine. II. Relationship between the C index in Guinea pigs and the mouse protection index

O exame da eficiência de seis vacinas antiaftosa, realizado com os testes "Indice Proteçäo Camundongos" e "Indice C" utilizando 15 ou mais cobaias por diluiçäo de vírus na titulaçäo, demonstrou a presença de boa correlaçäo e alta significância entre os resultados das duas provas. O valor do Indice Proteçäo Camundongos foi aproximadamente o dobro do valor do Indice C. Assim, o Indice Proteçäo Camundongos pode ser recomendado como teste de eficiência de vacians antiaftosa, principalmente quando se necessita de um grande número de provas, devido a seu baixo custo e facilidade de execuçäo, por usar exclusivamente camundongos

Eficciency test for foot and mouth disease vaccines. I. Reduction in C index variation by increasing the number of guinea pigs (Cavia cavia)

Duas vacinas foram submetidas a seis repetiçöes cada, à prova de eficiência, "Indice C", usando-se para titulaçäo, quatro cobaias por diluiçäo de vírus. Os valores encontrados, quando transformados em qualidade de vacina, demonstraram que uma mesma vacina poderia ser enquadrada como sendo "reprovada regular", "aprovada boa" ou "aprovada muito boa", indicando que a variaçäo dos resultados, dependendo do caso, pode indefinir a classificaçäo do imunógeno. Para verificar se tais variaçöes säo devidas ao pequeno número de cobaias, uma vacina foi submetida a seis repetiçöes, usando-se cinco cobaias por diluiçäo do vírus na prova "Indice C". Os arranjos 2 a 2, 3 a 3, 4 a 4, 5 a 5, realizados a partir dos resultados correspondentes a todas as combinaçöes possíveis quando säo usadas 5, 10, 15, 20, 25 ou 30 cobaias por diluiçäo viral, demonstraram que a variaçäo de mais ou menos 0,5 logaritmo com 95 por cento de segurança, corresponde a 15 cobaias

A pro-inflammatory effect of foot and mouth disease virus on immune and non immune guinea pigs

A estirpe O 1campos do vírus da febre aftosa (VFA) usada como agente indutor do teste de pleurisia foi capaz de desencadear um efeito pró-inflamatório em cobaias normais e imunes. A atividade pró-inflamatória do VFA, detectada em dois intervalos de pleurisia (24 e 48 horas) foi demonstrada, somente por quimiotaxia de leucócitos mononucleares (MN), no primeiro intervalo e por efeito edematogênico, migraçäo de MN e polimorfonucleares (PMN), no último intervalo de reaçäo. Os perfis de reaçäo inflamatória induzida pelo VFA em cobaias imunes (imunizadas com vacinas oleosas anti-VFAO 1 campos), aos 7 e aos 30 dias pós-vacinaçäo (PV) apresentaram intensidades mais baixas do que as observadas em cobaias normais, embora nas cobaias com 7 dias de vacinaçäo a quimiotaxia de leucócitos totais e de PMN tenha sido similar àquela encontrada nos animais normais, no intervalo de 48 horas de reaçäo. Ademais, nas cobaias com 30 dias PV, o VFA induziu um aumento significante no volume de exsudato e na infiltraçäo de MN, no intervalo de 24 horas, sendo que os valores de todos os parâmetros do exsudato inflamatório caíram a níveis normais, no segundo intervalo de reaçäo. Nas cobaias imunes foi observada uma associaçäo negativa entre o aumento no título de anticorpos soro-neutralizantes, de 7 para 30 dias PV e as intensidades dos parâmetros inflamatórios pleurais. O teste de pleurisia revelou-se um procedimento adequado para avaliar a atividade pró-inflamatória do VFA