Detecção de fatores de virulência em estirpes de Campylobacter spp. isoladas de carcaças de suínos abatidos em frigoríficos / Virulence factors in strains of Campylobacter spp. isolated from the carcasses of swine slaughtered in abattoirs

Isolaram-se estirpes de Campylobacter spp. em amostras de carcaças (n=65), fezes (n=65) e linfonodos mesentéricos (n=65) de suínos abatidos em frigoríficos do estado de São Paulo e detectaram, pela técnica da Multiplex-PCR, a presença do complexo de genes cdt, responsáveis pela expressão do fator de virulência da toxina CDT. Do total de 195 amostras de origem suína, Campylobacter spp. foi isolado de 31 (15,9%), sendo 29 (93,6%) de amostras de suabe retal, 1/65 (3,2%) de suabe de carcaça e um (3,2%) de linfonodo. Vinte e oito estirpes de C. coli foram positivas para a detecção dos genes cdt, e três estirpes de C. jejuni foram negativas para a detecção desses genes. Foi detectada, pela primeira vez no estado de São Paulo, a presença dos genes cdt em 100% das estirpes de Campylobacter coli provenientes de suínos abatidos em frigoríficos.

The purposes of this study were to isolate and identify Campylobacter spp. strains from the carcasses (n=65), feces (n=65) and mesenteric lymph nodes (n=65) of swine slaughtered in abattoirs in the State of Sao Paulo and to detect the presence of the cdt gene complex - responsible for the expression of the virulence factor cytolethal distensive toxin - in these Campylobacter spp. strains through Multiplex-PCR. From 195 samples analyzed, Campylobacter spp. was isolated in 31 (15.9%): 29 (93,6%) samples of rectal swab, 1 (3.2%) carcass swab and 1 (3.2%) lymph node sample. The 28 strains of isolated C. coli were positive for CDT toxin genes and the three strains of isolated C. jejuni were negative for these genes. It was also the first time that the cdt gene cluster was detected in strains isolated from swine in the state of São Paulo. These findings indicate swine as a potential spreading source of virulent strains of Campylobacter coli, either for slaughterhouse staff or consumers of carcasses and sub products.

Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows

This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years.

Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows.

This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years.

Detecção dos genes da toxina citoletal distensiva em estirpes de Campylobacter jejuni isoladas de carcaças de frangos / Detection of cytolethal distending toxin genes in strains of Campylobacter jejuni isolated from broiler carcasses

Foram analisadas 80 amostras de sobrecoxas de frangos de corte resfriados provenientes de feiras livres e hipermercados do município de São Paulo, SP. Treze estirpes de Campylobacter spp. foram isoladas em 10 (12,5 por cento) sobrecoxas, sendo cinco amostras originárias de feiras livres e cinco de hipermercados. Onze estirpes foram identificadas como Campylobacter jejuni e duas como Campylobacter coli. As 11 estirpes foram confirmadas como C. jejuni pela PCR do gene da hipuricase (hip), e destas, quatro (36,4 por cento) apresentaram os três genes (cdtA, cdtB e cdtC) codificantes da toxina citoletal distensiva pela multiplex-PCR, sendo três estirpes provenientes de hipermercados e uma de feira livre. Observou-se a presença de estirpes virulentas de C. jejuni, portadoras do complexo de genes cdt, nas amostras de frango resfriado, não só na linha de abate, mas até o ponto final da cadeia de distribuição, nos dois principais centros de venda a varejo.

Eighty samples of refrigerated broiler thighs purchased in street markets and supermarkets in the city of São Paulo, SP, were analyzed. Thirteen Campylobacter spp. strains were isolated in 10 (12.5 percent) thighs, five of them from street market samples and other five from supermarkets. Eleven strains were identified as Campylobacter jejuni and two of them as Campylobacter coli. The 11 strains were confirmed to be C. jejuni using PCR for hippuricase (hip) gene. From these, multiplex-PCR showed that four (36.4 percent) strains presented the three genes (cdtA, cdtB, and cdtC) encoding cytolethal distending toxin: three strains from supermarket and one from street market samples. These results are important, because they demonstrate the presence of virulent C. jejuni strains in refrigerated broiler thigh samples, not only in the slaughterhouse but in the final point of the distribution chain, at the two most important food retail commercer.

Influência das condições ambientais na transmissão da leptospirose entre criações de ovinos e bovinos da região de Sorocaba, SP / The influence of environment conditions on the transmission of leptospirosis between sheep and cattle herds of Sorocaba region, SP, Brazil

Leptospiras excretadas pela urina podem sobreviver por longos períodos em águas de superfície e solos, na dependência do pH e teor de umidade e de matéria orgânica. Investigou-se a influência do meio ambiente na transmissão da leptospirose em dois rebanhos exclusivos de ovinos (A e C) e dois de ovinos consorciados com bovinos (F e H) da região de Sorocaba, SP, no período de dezembro de 2007 a setembro de 2008. Foram examinadas amostras de soro pela reação de soroaglutinação microscópica; de urina, água e solo pelo cultivo para leptospiras e urina de ovinos pela PCR. Condições edafoclimáticas, pH das águas de superfície e solo, granulometria e permeabilidade do solo foram analisadas. Todos os rebanhos apresentaram pelo menos um animal sororeagente para Leptospira spp. Apenas a PCR de um pool de urina de ovinos (H) foi positiva. Leptospira spp. foi isolada do lago de F. O pH das águas de superfície variou entre 6,0-7,0; e nos solos entre 4,5 e 6,8. Os índices de matéria orgânica em A, C e H variaram de 24 a 35 g/dm3, e 63 g/dm3 em F. A composição do solo de A e F mostrou-se franco-argiloarenosa, C argilosa e H franco-siltosa; como texturas mistas são capazes de manter a umidade, principalmente devido a argila. Diante da presença de animais sororeatores e portanto da circulação de Leptospira spp. nos rebanhos, conclui-se que o ciclo de transmissão é dependente da interação sinérgica e antagônica de muitas variáveis; onde o pastejo num habitat com alto teor de umidade parece ser limitante.

Leptospires excreted by urine are able to survive for long periods in surface water and soil depending on the pH, humidity and organic matter presence. This paper reported the influence of environment conditions on the transmission of leptospirosis in two sheep-only farms (A and C) and two cattle-sheep farms (F and H) from December 2007 to September 2008. Serum samples were examined by microscopic agglutination test; urine, surface water and soil samples were cultured for leptospires, and ovine urine pools were analyzed by PCR. Regional edaphoclimatic conditions, pH of surface water and soil, granulometry and permeability of soil were analyzed. All herds presented at least one reactor to Leptospira spp. Only the PCR of an ovine urine pool of herd H was positive and Leptospira spp. was isolated from the F lake. The pH of water samples ranged from 6.0 to 7.0; while in soil it was around from 4.5 to 6.8. Soil organic matter were 24 to 35 g/dm3 in A, C e H, and 63 g/dm3 in F. Soil samples of A and F showed loamy-clay texture; C had clay soil, and H loamy-silt soil; as mixed compositions are able to maintain the humidity, mainly where clay is present. As the presence of reactors in all herds indicated the contact with Leptospira spp., it was concluded that the cycle of transmission is dependent on the synergistic and antagonistic interaction of many variables; but the close contact of animals grazing in a high humidity habitat seems to be limiting.

Prevalência de anticorpos contra os agentes da maedi-visna e clamidofilose em ovinos no município de Uberlândia, MG / Prevalence of antibodies against the agents of maedi-visna and chlamydophilosis in sheep from Uberlândia county, MG, Brazil

Objetivou-se com este estudo realizar o primeiro inquérito soro-epidemiológico para o vírus da maedi-visna e Chlamydophila spp. em 12 rebanhos de ovinos do Município de Uberlândia, MG. Foram utilizadas 334 amostras de soro sanguíneo de ovinos e aplicou-se um inquérito epidemiológico a cada propriedade. Os testes realizados para a pesquisa de anticorpos contra o vírus da maedi-visna e Chlamydophila spp. foram imunodifusão em gel de ágar (IDGA) e reação de fixação do complemento (RFC), respectivamente. Não foram detectados ovinos reagentes para maedi-visna. Verificou-se uma prevalência de 3,3% para Chlamydophila spp., com títulos variando de 32 a 64. Não houve diferença estatística significativa (p > 0,05) para os fatores de risco analisados. Ressalta-se a importância dos sistemas de vigilância epidemiológica para atuar no controle dessas infecções, evitando a introdução do vírus da maedi-visna e uma maior propagação da Chlamydophila spp. neste município.

The aim of this study was to carry out the first investigation into the serological prevalence of maedi-visna virus and Chlamydophila spp. on 12 sheep breeding farms in Uberlândia County, MG, Brazil. A total of 334 blood serum samples were used and an epidemiological survey was completed by each farm. The tests to detect maedi-visna and Chlamydophila spp. antibodies were an agar gel immunodiffusion (AGID) and a complement fixation test (CFT), respectively. None of the sheep were reactive to maedi-visna. The detection of antibodies against Chlamydophila spp. was 3.3%, with titers varying from 32 to 64. There was no statistically significant difference (p > 0.05) in regard to the risk factors analyzed. The importance of epidemiological surveillance systems to aid in the control of these infections is emphasized, in order to avoid the introduction of maedi-visna virus and a wider spread of Chlamydophila spp. in this county.

Use of pcr-rflp of thefla a gene for detection and subtyping of Campylobacter jejuni strains Potentially related to Guillain-barré syndrome, isolated from humans and animals

The objectives of the present study were the subtyping of Campylobacter jejuni subsp. jejuni strains obtained from humans and different animal species using PCR-RFLP, and the detection, by means of the same technique, of strains related to serotype PEN O19:LIO 7, the main C. jejuni serotype linked to Guillain-Barré Syndrome (GBS). Seventy C. jejuni strains isolated from human feces (n=33), primates (n=15), dogs (n=5), swine (n=2), bovines (n=1), abortion material from goats (n=2) and poultry carcasses (n=12), all collected in the state of São Paulo, were subtyped by means of PCR-RFLP of fla A gene, using restriction endonucleases Hae III, Afa I and Mbo I. Seven subtypes were observed when using the enzyme Hae III; eight when using Mbo I; and seven when using Afa I. The combination of the three endonucleases led to 16 fla-RFLP subtypes, from which ten subtypes shared strains of human and animal origin. From these, seven subtypes were observed in human and broiler strains. In eight subtypes, the other animal species shared patterns with human strains. It was inferred that, besides broilers, swine, goats, dogs and primates may be sources of infection for human in São Paulo. PCR-RFLP is a highly discriminatory technique that may be applied to molecular epidemiology studies of samples from different origins. Besides, the study also enabled the detection of two human strains and two primate strains related to serotype PEN O19: LIO 7.

Use of pcr-rflp of the fla a gene for detection and subtyping of Campylobacter jejuni strains Potentially related to Guillain-barré syndrome, isolated from humans and animals.

The objectives of the present study were the subtyping of Campylobacter jejuni subsp. jejuni strains obtained from humans and different animal species using PCR-RFLP, and the detection, by means of the same technique, of strains related to serotype PEN O19:LIO 7, the main C. jejuni serotype linked to Guillain-Barré Syndrome (GBS). Seventy C. jejuni strains isolated from human feces (n=33), primates (n=15), dogs (n=5), swine (n=2), bovines (n=1), abortion material from goats (n=2) and poultry carcasses (n=12), all collected in the state of São Paulo, were subtyped by means of PCR-RFLP of fla A gene, using restriction endonucleases Hae III, Afa I and Mbo I. Seven subtypes were observed when using the enzyme Hae III; eight when using Mbo I; and seven when using Afa I. The combination of the three endonucleases led to 16 fla-RFLP subtypes, from which ten subtypes shared strains of human and animal origin. From these, seven subtypes were observed in human and broiler strains. In eight subtypes, the other animal species shared patterns with human strains. It was inferred that, besides broilers, swine, goats, dogs and primates may be sources of infection for human in São Paulo. PCR-RFLP is a highly discriminatory technique that may be applied to molecular epidemiology studies of samples from different origins. Besides, the study also enabled the detection of two human strains and two primate strains related to serotype PEN O19: LIO 7.

Prevalência de listeria spp em amostras de carne de frango obtidas em abatedouros do Estado de São Paulo / Prevalence of listeria spp in samples of chicken meat obtained in s slaughtherhouses of the State of São Paulo

Listeria monocytogenes está envolvida em surtos humanos relacionados a alimentos, embora, no Brasil a doença em humanos seja pouco relatada. Enfoque renovado foi dado a esta bactéria após surtos de doenças de origem alimentar (DOA) ocorridos na América do Norte e Europa durante os anos de 1980 e 1990. Listeria spp é freqüentemente isolada de carnes cruas, incluindo as de frango, como resultado de ampla contaminação cruzada em plantas industriais. A carne de frango é parte integrante da dieta dos brasileiros como fonte de proteína animal, assim, é importante que se conheça a prevalência deste agente neste tipo de alimento. Para tanto, foram examinadas 74 (setenta e quatro) amostras de carne de frango (coxa, sobrecoxa, peito, frango à passarinho e inteiro) utilizando-se métodos de isolamento de Listeria spp com meios de enriquecimento e seletivos, e reação em cadeia de polimerase (PCR), para confirmação dos testes bioquímicos. Em apenas uma das amostras foi detectada a presença de L. monocytogenes. Aventa-se, para o baixo índice de listérias encontrado, a ação antimicrobiana determinada pelo uso de descontaminantes nos tanques de resfriamento dos abatedouros.

Listeria monocytogenes is responsible for food borne disease, although in Brazil there is little data about this agent. Renewed emphasis has been given to this bacterium after the North American and European outbreaks during the 1980s and 1990s. Listeria spp is usually isolated from raw meat, including chicken, due to cross contamination in industrial plants. Chicken meat plays an important role in the diet of Brazilian people as an animal protein source. This work was aimed at investigating this bacterium' s prevalence in different cuts of chicken sampled at slaughterhouses. To achieve this, 74 chicken samples (drumsticks, thighs, breasts, entire chickens cut-up and whole) were cultivated in Listeria spp enrichment and selective culture media, and submitted to polymerase chain reaction to confirm the biochemical tests. Just one sample was positive for L. monocytogenes. As one possible explanation for the low level of listeria found, the authors point to the antiimicrobial action of disinfectant products used in the chilled tanks of slaughther houses.

Prevalência de listeria spp em amostras de carne de frango obtidas em abatedouros do Estado de São Paulo / Prevalence of listeria spp in samples of chicken meat obtained in s slaughtherhouses of the State of São Paulo

Listeria monocytogenes está envolvida em surtos humanos relacionados a alimentos, embora, no Brasil a doença em humanos seja pouco relatada. Enfoque renovado foi dado a esta bactéria após surtos de doenças de origem alimentar (DOA) ocorridos na América do Norte e Europa durante os anos de 1980 e 1990. Listeria spp é freqüentemente isolada de carnes cruas, incluindo as de frango, como resultado de ampla contaminação cruzada em plantas industriais. A carne de frango é parte integrante da dieta dos brasileiros como fonte de proteína animal, assim, é importante que se conheça a prevalência deste agente neste tipo de alimento. Para tanto, foram examinadas 74 (setenta e quatro) amostras de carne de frango (coxa, sobrecoxa, peito, frango à passarinho e inteiro) utilizando-se métodos de isolamento de Listeria spp com meios de enriquecimento e seletivos, e reação em cadeia de polimerase (PCR), para confirmação dos testes bioquímicos. Em apenas uma das amostras foi detectada a presença de L. monocytogenes. Aventa-se, para o baixo índice de listérias encontrado, a ação antimicrobiana determinada pelo uso de descontaminantes nos tanques de resfriamento dos abatedouros.

Polygenic control of quantitative antibody responsiveness: restrictions of the multispecific effect related to the selection antigen.

Among the differences observed between the various high (H) and low (L) antibody responder lines of mice resulting from distinct bidirectional selective breedings, one of the most puzzling is the variation in the "multispecific effect," i.e., in the modification of antibody responses to antigens unrelated to those used during the selection. The best examples are the H and L lines of selection IV, selected on the basis of responses to somatic antigen of Salmonella which do not differ in their antibody responses to sheep erythrocytes (SE). However, a wide range of variability is observed in the responses of (HIV X LIV)F2 hybrids to this antigen, and it was therefore hypothesized that distinct groups of genes might regulate antibody responses to SE and the somatic antigen. Indeed, a new selection (IV-A) for anti-SE responsiveness started from these (HIV X LIV)F2 successfully produced a high and a low anti-SE responder line. The results of selection IV-A and the variance analysis of (HIV-A X LIV-A)F2 hybrids are reported. They are roughly similar to those in selection I, also carried out for anti-SE responsiveness. In vivo attempts to identify the major regulatory mechanism which contributes to the interline difference indicate that the efficiency of macrophage accessory function has been modified in selection IV-A, as was observed in selection I, whereas this function did not differ in HIV and LIV lines. Probably in relation to the involvement of macrophage function there is a notable increase of the multispecific effect in selection IV-A when compared with selection IV. The results of selection IV-A demonstrate that responsiveness to heterologous erythrocytes and to somatic antigen of Salmonella are under separate polygenic control operating through distinct regulatory mechanisms. The choice of the selection antigen and immunization procedure is of major importance for defining the gene interaction operating in each selective breeding experiment and the extent of its multispecific effect.

Genetic regulation of multispecific antibody responsiveness: improvement of "high" and "low" characters.

The five selections carried out in the mouse for high or low antibody responsiveness to various multideterminant immunogens were successful. In all cases the large interline difference was shown to result from the additive effects of several independently segregating loci (polygenic regulation). However, important peculiarities were demonstrated in these original selections concerning either the cellular mechanisms operating or the effect of the selected genes on antibody responses to antigens unrelated with those used for the selection (multi-specific effect). In an attempt to improve and generalize the effect of selection, the 5 high and the 5 low lines were inter-crossed to obtain populations with a balanced proportion of the 5 genomes. These two populations were then submitted to selective breedings in which the phenotypic character was the weighted responses to pluri-antigen immunization. The data obtained in 16 consecutive generations of two selective breedings (general-primary, GP and general-secondary, GS, responses) carried out from these populations are reported. The genetic parameters of the response to GP and GS selections are compared with those obtained in the original selections. The final result of both GP and GS selections demonstrate a marked improvement of the high and low antibody production traits, both quantitatively (interline divergence) and qualitatively (multi-specific effect). The success of GP and GS selections agrees with the concept that distinct groups of genes are preferentially affected by selection according to the nature of the selection antigen and the immunization procedure.

Effect of silica on the genetic regulation of antibody responsiveness.

The high (H) and low (L) antibody responder lines of mice produced by selective breeding are characterized by different modifications in immunocompetent cell potentialities, according to the immunization procedure used for the selection process. In selections I and II, the difference in antibody responsiveness between H and L lines was clearly shown to depend mainly on macrophage function: the more rapid catabolism of antigens in L mice was the main cause of the low antibody production. In contrast, up to now, no difference has been observed between H and L mice of selections III and IV in terms of the macrophage accessory role. The administration of silica particles has a well known impairment effect on macrophage activity. Therefore, the effect of silica injection on the kinetics of antibody responses to selection antigens was compared in H and L mice of the four selections. Silica was given either intravenously or locally in one hind footpad 6 or 24 h before immunization by the same route. Silica treatment consistently improved antibody responsiveness in the L mice of selections I and II, but had no effect in the L mice of selections III and IV. The antibody responses of the H lines of the four selections were not substantially modified by silica injections. Therefore, the silica treatment reduced the interline difference in antibody responses in selections I and II only, by interfering with the expression of the genetic modification of macrophage activity. However, a similar effect was not obtained with other substances known to affect macrophages, including dextran sulphate or carrageenan. The results reported here are in agreement with the above-mentioned statement that the genetic modification of macrophage function plays a major role in the interline difference in selections I and II and is not involved in selections III and IV.