Ronnie Antunes de Assis

Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia.

Outbreak of clostridial myocarditis in calves

This paper describes the epidemiological and pathological features of an outbreak of clostridial myocarditis in calves due to Clostridium chauvoei. Four of seven two-month-old Hereford calves died in the course of a week. Their gross postmortem lesions were similar and consisted of irregular dark red areas of myocardial necrosis through the full thickness of the atrial and ventricular myocardium. No lesions were observed in skeletal muscle. The heart muscle had extensive multifocal areas of acute coagulative necrosis. The diagnosis was confirmed by a fluorescent antibody technique on tissue smears, by a streptavidin-biotin technique on formalin-fixed, paraffin-embedded tissues, and by a PCR technique specific for the 16S rRNA of C chauvoei.

Presença de anticorpos da classe IgM de Leptospira interrogans em animais silvestres do Estado do Tocantins, 2002

Four hundred and twenty-seven serum samples of wild animals were tested against 18 serovars of Leptospira interrogans. Of 286 samples of Cebus apella, 46 (16.1%) were positive for the serovars pomona, brasiliensis, mini, swajizak, grippotyphosa, sarmin, fluminense, autumnalis, hebdomadis, guaratuba, javanica and icterohaemorrhagiae. Of 82 samples of Alouatta caraya, 2 (2.4%) were positive for the serovars mangus and fluminense. Of 31 samples of Nasua nasua, 4 (12.9%) were positive for the serovars fluminense and javanica, and of 10 samples of Cerdocyon thous, 2 (20 %) were positive for the serovars fluminense and brasiliensis. Seven samples of Dasyprocta sp, 6 of Tamandua tetradactyla and 5 of Euphractus sexcintus did not present reactivity.

PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues

The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.

Avaliação da resposta de antitoxinas beta e épsilon de Clostridium perfringens induzidas em bovinos e coelhos por seis vacinas comerciais no Brasil

The antibody response against beta and epsilon toxins of Clostridium perfringens was evaluated in cattle vaccinated with six commercial clostridial vaccines. Groups of six calves (6-7 months old) were vaccinated with each vaccine (T2 to T7), on days 0 and 42, with the recommended dose and route. Saline solution (T1) and a standard toxoid vaccine (T8) were used as negative and positive controls, respectively. On days 0, 42 and 56 post-vaccination (PV), blood samples were collected for antibody titration. Vaccines, as well as controls, were also injected into 8 rabbits each, on days 0 and 21, using half of the dose recommended for cattle. Rabbits were bled on day 35. Rabbit sera were tested as a pool per vaccine and cattle sera were tested individually against beta and epsilon toxins, by the serum neutralization test in mice. The vaccine T2 had antibody titers of 22.6 and 5.6 IU/ml and T4 had 11.2 and 7.0 IU/ml against beta and epsilon toxins in rabbits, respectively. The standard toxoid had a titer of 45.2 IU/ml against both toxins. In cattle, mean antibody titers against beta toxin on days 42 and 56 PV induced by T2 (1.15 and 8.0 IU/ml) were similar to those induced by the standard toxoid (2.02 and 10.3 IU/ml). T4 titers (0.73 and 4.54 IU/ml), were lower (P<0.05) than those obtained with the standard toxoid, but similar to T2 titers. On day 42, the standard toxoid had a mean titer (0.97 IU/ml) against epsilon toxin significantly higher (P<0.05) than that obtained with T4 (0.15 IU/ml) but similar to T2 mean titer (0.42 IU/ml). On day 56, T2 resulted in a significantly higher titer (4.27 IU/ml) (P<0.05) than that induced by T4 (0.68 IU/ml), but similar to the standard control (4.98 IU/ml). The other vaccines, as well as the saline solution, did not elicit antibody responses, neither in rabbits nor in cattle. T2 induced antibody titers against C. perfringens beta and epsilon toxins similar to those induced by the standard toxoid and higher than those induced by T4.

Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

Serotyping and evaluation of the virulence in mice of Streptococcus suis strains isolated from diseased pigs

A total of 110 strains of Streptococcus suis, isolated from diseased pigs in Brazil were serotyped and analyzed for virulence. Serotyping of the strains resulted in the following classification: 42 strains of serotype 2 (38.2%), 10 strains of serotype 14 (9.1%), seven strains of serotype 9 (6.4%), three strains each of serotype 7 and 11 (2.7%), two strains each of serotype 1 and 8 (1.8%) and one strain each of serotypes 1/2, 3, 5, 6 and 10 (0.9%). Cross reactions among serotypes 1, 14 and 7 were observed in 21 strains (19.1%). Only 41.9% of the strains were lethal for mice using the pathogenicity test.

Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil.

The objective of this study was to detect C. difficile A/B toxins and to isolate strains of C. perfringens and C. difficile from diarrheic and non-diarrheic dogs in Brazil. Stool samples were collected from 57 dogs, 35 of which were apparently healthy, and 22 of which were diarrheic. C. difficile A/B toxins were detected by ELISA, and C. perfringens and C. difficile were identified by multiplex PCR. C. difficile A/B toxins were detected in 21 samples (36.8%). Of these, 16 (76.2%) were from diarrheic dogs, and five (23.8%) were from non-diarrheic dogs. Twelve C. difficile strains (21.1%) were isolated, of which ten were A+B+ and two were A−B−. All non-toxigenic strains were isolated from non-diarrheic animals. The binary toxin gene cdtB was found in one strain, which was A+B+ and was derived from a non-diarrheic dog. C. perfringens strains were isolated from 40 samples (70.2%). Of these, 18 (45%) were from the diarrheic group, and 22 (55%) belonged to the non-diarrheic group. All isolates were classified as C. perfringens type A and there was an association between the detection of the cpe gene and the presence of diarrhea. Interestingly, ten strains (25%) were positive for the presence of the cpb2 gene. The high rate of detection of the A/B toxins in non-diarrheic dogs suggests the occurrence of subclinical disease in dogs or carriage of its toxins without disease. More studies are needed to elucidate the epidemiology of C. difficile and C. perfringens in dogs and to better our understanding of C. difficile as a zoonotic agent. This is the first study to report the binary toxin gene in C. difficile strains isolated from dogs in Brazil.

Detection of enterotoxin A and cytotoxin B, and isolation of Clostridium difficile in piglets in Minas Gerais, Brazil.

Clostridium difficile has emerged as a major cause of neonatal colitis in piglets, displacing classic bacterial pathogens. However, there is no information regarding the distribution of this microorganism in pig farms in Brazil. In the present study, the presence of toxins A/B and of C. difficile strains in stool samples from 60 diarrheic or non-diarrheic newborn piglets (one to seven days old), from 15 different farms, was studied. The presence of toxins A/B was detected by ELISA and PCR was used to identify toxin A, toxin B and binary toxin gene in each isolated strain. C. difficile A/B toxins were detected in ten samples (16.7%). Of these, seven were from diarrheic and three were from non-diarrheic piglets. C. difficile was recovered from 12 out of 60 (20%) fecal samples. Of those, three strains were non-toxigenic (A-B-) and nine were toxigenic. Of the nine toxigenic strains, four were A+B+ strains and five were A-B+ strains. The presence of binary toxin observed in the present study was much higher (50%) than in previously reported studies. All three non-toxigenic strains were isolated from otherwise healthy piglets. The results suggest the occurrence of neonatal diarrhea by C. difficile in farms in Brazil.

Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques

Transmission of Mycobacterium bovis from cattle to humans has been reported and can cause tuberculosis (Tb) and a problem in certain risk populations. Therefore, knowledge of resistance of M. bovis towards antibiotics used for therapy of human Tb could help avoiding cure delay and treatment cost increase when dealing with drug resistant organisms. We therefore evaluated the susceptibility of M. bovis isolates towards streptomycin, isoniazide, rifampicin, ethambutol, and ethionamide, the first line antibiotics for human Tb. Therefore, 185 clinical samples from cattle with clinical signs of tuberculosis were processed and submitted to culturing and bacterial isolates to identification and drug susceptibility testing using the proportion method. Among 89 mycobacterial strains, 65 were identified as M. bovis and none were resistant to any of the antibiotics used. Confirmation of present results by future studies, enrolling a large number of isolates and designed to properly represent Brazilian regions, may favor the idea of using isoniazide preventive therapy as part of a Tb control strategy in special situations. Also, nucleic acids from bacterial isolates were submitted to rifoligotyping, a recently described reverse hybridization assay for detection of mutations causing resistance towards rifampicin. Concordance between the conventional and the molecular test was 100%, demonstrating the use of such methodology for rapid evaluation of drug susceptibility in M. bovis.