Evaluation of the anti-LipL32 monoclonal antibodies potential for use in leptospirosis immunodiagnostic tests.

LipL32 is the major lipoprotein in the membrane of pathogenic leptospira. In this work, we report on the production of monoclonal antibodies (MAbs) against recombinant LipL32 (rLipL32) and on the evaluation of their potential for use as reagents in diagnostic tests for leptospirosis. The MAbs were all of the IgG(2b) isotype and reacted specifically with native LipL32 in pathogenic serovars only. MAbs reacted in the same region of the rLipL32 molecule and their affinity constant was between 5x10(7) M(-1) and 6x10(6) M(-1). These results suggest that although the MAbs cannot be used together, they are well suited for diagnostic tests of leptospirosis based on LipL32 detection.

Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil.

AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.

Atypical American visceral leishmaniasis caused by disseminated Leishmania amazonensis infection presenting with hepatitis and adenopathy.

Leishmania amazonensis is widely recognised as a cause of cutaneous leishmaniasis in Latin America, but it can also disseminate to produce atypical visceral leishmaniasis with hepatitis and lymphadenopathy. The patient, an 8-year-old Brazilian boy, presented with a febrile illness and hepatosplenomegaly, elevated liver enzymes and generalised adenopathy. Serological tests using antigens of L. chagasi, the typical cause of visceral leishmaniasis in Latin America, were inconclusive. Leishmania amazonensis was eventually isolated in a culture of a lymph node. The patient recovered fully after treatment with meglumine antimoniate. As this case illustrates, L. amazonensis produces a spectrum of disease that includes atypical American visceral leishmaniasis with evidence of hepatocellular injury and generalised lymphadenopathy.

DNA inoculation with a plasmid vector carrying the faeG adhesin gene of Escherichia coli K88ab induced immune responses in mice and pigs.

pCB01, an eukaryotic expression vector, was constructed by cloning faeG, the gene that encodes the fimbrial adhesin of Escherichia coli K88ab, in pcDNA3. Mice and swine were inoculated by the intramuscular route with different quantities of plasmid DNA to evaluate its capacity to induce an immune response. The immune response was monitored by ELISA and immunoblotting, using purified fimbriae and whole suspensions of fimbriated bacteria. Mice showed seroconversion 21 days after the inoculation of 8.9 microg and swine after the administration of 1100 microg of plasmid DNA. Seroconversions increased after successive reinoculations. Immunoblotting showed that sera collected 73 days after the first inoculation recognized exclusively a protein of 27 kDa present in purified fimbrial suspensions and in whole suspensions of E. coli K88ab. The immune response elicited by pCB01 was mainly due to IgG2a, while that elicited by a bacterin was due to IgG2b and IgG3. Antibodies were still detected 14 months after the initiation of the immunization.

Influence of equine chorionic gonadotropin on weaning-to-estrus interval and estrus duration in early-weaned, primiparous, female swine.

The weaning-to-estrus interval (WEI) influences the total nonproductive days (NPD) accumulated by the breeding herd and affects herd productivity. Short lactation lengths (LL) are commonly followed by prolonged WEI, which are also associated with short estrus duration (ED). Equine chorionic gonadotropin treatment is a tool that has been used to reduce WEI, especially for low-parity females. The objectives for this study were to evaluate the effect of LL on the association between WEI and ED and to estimate the effects of postweaning eCG administration on WEI and ED for early-weaned females. Two treatments (TREAT) consisting of 750 IU of eCG (n = 96) or control (n = 77) were applied 1 d after weaning to first-parity, weaned females. The study was conducted on a commercial farm having a target LL of 18 d. Estrus detection was conducted three times daily, and estrus duration was determined as the interval between the first and the last positive response to back pressure. Analyses of variance were conducted to estimate the effects of LL and TREAT on WEI and the effects of TREAT and WEI on estrus duration. Mean LL was 17.9+/-1.7 d, mean WEI was 106.6+/-29.2 h, and mean estrus duration was 55.9+/-15.5 h. Even though the frequency of short WEI tended to increase with longer LL, mean WEI was shortest for females weaned after 18 d and longest for those weaned after 20 d (P<.05). The WEI for females receiving eCG (98.7+/-2.7 h) was shorter (P = .0001) than that for control females (121.5+/-3.3 h). The WEI was also affected by a LL x TREAT interaction (P = .0014), indicating that the interval was longer (P<.05) for control females weaned after 17 and 20+ d than for other females. The LL and TREAT did not affect estrus duration (P = .20 and P = .157, respectively). However, estrus duration was reduced as the WEI increased (P = .0001), and it was also influenced by a WEI x TREAT interaction (P = .024). A linear regression model estimated that the association between WEI and estrus duration was stronger in the eCG group than in the control group (R2 = .51 and .15, respectively; both P<.001). In conclusion, the use of eCG postweaning was associated with more precise prediction of estrus duration as a function of the WEI and allows optimization of breeding management in early-weaned, primiparous females.

Effect of equine chorionic gonadotropin on weaning-to-first service interval and litter size of female swine.

We evaluated the effect of PMSG on the weaning-to-first service interval, total litter size and born alive litter size in swine. Four doses of PMSG (0, 500, 750 and 1,000 IU) were administered intramuscularly after weaning to sows at 3 different farms, grouped by parities (1, 2 and 3 or higher) and 2 distinct time periods. The associations among main effects and response variables were assessed by analysis of variance. Polynomial orthogonal terms were used to adjust the estimates of weaning-to-first service interval, total litter size and born alive litter size for the interaction effect of parity and PMSG treatment. The weaning-to-first service interval did not differ across periods and farms (P>0.05), although the interval was shorter (P<0.05) for Parity 3+ sows (4.97 d) than for Parity 1 sows (5.29 d), with no other differences in intervals observed across parities (P>0.05). Time period did not influence litter size (P>0.05), but there were differences in litter size across farms (P<0.05). Both litter size traits were lower for Parity 1 sows than for higher parity sows (P<0.05), but there were no differences in litter size between Parity 2 and 3+ sows (P>0.05). Litter size increased with PMSG dose in both Parities 1 and 2 (P<0.05), but not in Parity 3+ (P>0.05). A significant quadratic effect (P<0.05) of PMSG treatment in weaning-to-first service interval was observed for both Parity 1 and 2 sows, with the shortest intervals occurring with the 750 IU dose for Parity 1 sows. Administration of PMSG after weaning was associated with a shortened weaning-to-first service interval in Parity 1 sows and increased litter size in Parity 1 and 2 sows.

Generation of monoclonal antibodies against surface antigens of Moraxella bovis.

Six hybridoma lines producing monoclonal antibodies (MAbs) against Moraxella bovis were established from fusions between the SP2/0 myeloma cells and BALB/c mice splenocytes. Three antibodies were of the IgG1 isotype, two were IgG2a, and one was IgG2b. The specificity of the antibodies was determined by indirect enzyme-linked immunosorbent assay (ELISA) using whole cells of M. bovis and of other Gram-negative bacteria, and lipopolysaccharide (LPS) from M. bovis JUR2 and E. coli as antigens. Ascitic fluid produced by the six hybridoma lines inhibited hemagglutination by M. bovis GF9. One MAb (35F) reacted specifically with purified M. bovis LPS in the ELISA test. The MAb panel detected heterogeneity among the isolates recovered from different geographical regions.

Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies.

Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.

A fluorescent enzyme immunoassay for Salmonella detection.

A double antibody sandwich immunoassay (EIA) was developed for the detection of Salmonella. The assay utilizes a beta-galactosidase-murine myeloma monoclonal antibody (M467) conjugate prepared with the heterobifunctional coupling reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and uses 4-methyl umbelliferyl beta-D-galactoside as a fluorogenic substrate for the enzyme. The EIA is sensitive and rapid, and compared favorably with the conventional cultural technique in the analysis of 60 feed samples naturally contaminated with Salmonella. Proteins or natural contaminants from the sample did not interfere in the assay.

Enzyme immunoassay: binding of Salmonella antigens to activated microtiter plates.

A heat extract prepared from radiolabeled Salmonella cells was used to determine if covalent binding to activated surface of polystyrene plates would improve antigen retention thus contributing to increase sensitivity in an enzyme immunoassay for Salmonella antigen. The effect of treatment with ethylchloroformate on the retention of antigens passively absorbed to polyvinylchloride and polystyrene plates was also investigated. Chemically modified plates retained more radiolabeled antigens after washing than did untreated plates in which the antigens had been physically adsorbed. However, improvement of assay sensitivity depended on the type of plate used for covalent binding of antigen. N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), was found to be potentially useful for mediation of covalent binding of antigens to activated plates.

Comparacao de meios com sulfito na recuperacao de esporos de Clostridium perfringens tratados termicamente. / Comparision of the recovery capacity of three sulphite media for heat treated Clostridium perfringens spores

Tres meios com sulfito, disponiveis comercialmente, foram comparados quanto a sua capacidade de recuperacao de esporos de Clostridium perfringens tratados e tratados termicamente e quanto a producao de colonias negras. Os meios comparados foram Agar SFP (Shahidi-Fergunson-perfingens), Agar SPS (sulfito - polimixia - sulfadiazina) e Agar TSN (triptona-sulfito neomicina), incubados a 30 graus C/40 hs e 37 graus C/24 hs. As contagens em SFP e SPS nao foram estatisticamente diferentes. As contagens en TSN foram significativamente mais baixas. As contagens obtidas nas duas temperaturas de incubacao nao diferiram estatisticamente. Produziram-se colonias negra caracteristicas em SFP. Em SPS e TSN produziram-se colonias negras e brancas, tanto com esporos tratados como nao tratados termicamente, Progenies de colonias brancas, cultivadas nos tres meios considerados produziram colonias negras