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.

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.

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MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil. Key words: Mycobacterium bovis -spoligotyping -MIRU-VNTR typing -bovine tuberculosis -molecular epidemiologyGenotyping of M. bovis from Brazil • Patrícia Martins Parreiras et al. 65While IS6110-RFLP lacks resolution in this species, spoligotyping has been demonstrated to be a fast and cost-effective method for first-line typing (Haddad et al. 2004). The MIRU-VNTR typing procedure has been extensively evaluated in M. tuberculosis and the recent data available about this method on M. bovis strains confirmed its considerable power of discrimination (Sola et al. 2003, Michel et al. 2008, 2010. Smith et al. (2006) inferred that a combination of spoligotyping and VNTR typing resulted in the simplest and most cost-effective method for routine molecular typing and epidemiological tracing of bovine TB in Great Britain.Some previous studies have successfully employed techniques such as IS6110-RFLP and spoligotyping to genotype M. bovis strains isolated from cattle in Brazil (Zanini et al. 2001, Rodriguez et al. 2004), but as far as we know, no data exist on genotyping of M. bovis by combining spoligotyping and MIRU-VNTR typing in our country. We aimed to use spoligotyping and MIRU-VNTR typing to assess the genetic diversity of Brazilian M. bovis isolates. MATERIALS AND METHODS Sampling and conventional procedures -During 2001and 2002, tissue samples from cattle with lesions suggestive of TB were collected immediately after slaughter at the time of inspection in abattoirs. The study samples were collected in abattoirs that followed slaughter inspection procedures from the states of Amazonas (AM), Paraíba (PB), Distrito Federal (DF), Mato Grosso do Sul (MS), São Paulo, Santa Catarina (SC) and Minas Gerais (MG). The sampled specimens from respective carcasses were pooled, put on ice and sent to the Laboratório Nacional Agropecuário de Minas Gerais (Pedro Leopoldo, MG), where they were processed for inoculation in Stonebrink culture medium (Mota 1985). The cultures were incubated at 37ºC and verified for bacterial growth every week for a period of at least two months. Bacterial isolates were submitted to standard procedures for differentiation of certain species of the MTBC, including the evaluation of niacin production, nitrate reductase activity, Tween hydrolysis, catalase activity at 68ºC and resis...

Macrorhabdus ornithogaster in ostrich, rhea, canary, zebra finch, free range chicken, turkey, guinea-fowl, columbina pigeon, toucan, chuckar partridge and experimental infection in chicken, japanese quail and mice [Macrorhabdus ornithogaster em avestruzes, ema, canário, mandarim, galinha, peru, galinha da Angola, pombo doméstico, rolinha, tucano, perdiz ABSTRACTSince 2000, Macrorhabdus ornithogaster "megabacteriosis" has been diagnosed in the avian diseases laboratory in a diversity of avian species and varied spectrum of disease. The disease in some species (chickens, turkeys, guinea fowls) was clinically characterized by emaciation, prostration, loss of appetite, cachexia and death, with a typically chronic course. A more acute disease was observed in finches (canary-Serinus and zebra-Taeniopygia) and budgerigars (Melopsittacus undulatus). The large rod shaped organism, visible from 100 times magnification, with and without staining, could be detected in sick and also in reasonably normal individuals of some species, such as chickens, turkeys, quails and pigeons. In rheas (Rhea americana), ostriches (Struthio camelus), canaries, zebra-finches, guinea-fowl (Numida meleagris) and budgerigars. The disease was severe, causing to up to 100% mortality. The infection could be detected in some species along with other infectious or disease problems, such as endoparasites (helminths, coccidia) and ectoparasitism (order Mallophaga or/and order Acarina). The cultivation of M. ornithogaster was successfully achieved in solid and liquid media, originated from chickens (four isolates), guinea fowl (1 isolate), chuckar partridge (1 isolate) and canary (1 isolate). A very interesting finding at microscopy was motility of M. ornithogaster, as detected both in cultures obtained on agar for pathogenic fungi and passaged into thioglycolate broth, as well as on samples observed in wet preparations from in vivo. Differences in colony aspects were noted among the isolates. Experimental infections were attempted in chicken and japanese quail, using a chicken isolate, allowing the detection of the organism in the proventriculus and liver in apparently normal birds. One chicken isolate was injected intraperitoneally in Balb/c mice and resulted in 100% mortality.

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.

Abstract. Omphalitis and the resulting septicemia contribute to perinatal mortality in several animal species. In foals, the most important causes of omphalitis are Escherichia coli and Streptococcus zooepidemicus. However to date, no information has been published about the role of Clostridium sordellii in these infections. In this paper, we describe 8 cases of perinatal mortality in foals associated with internal umbilical remnant infection by C. sordellii. The foals studied were between 12 and 21 days old at the time of death, and various breeds were represented in the group. Five of the foals were male and 3 were female. The diagnosis was established on the basis of the detection of C. sordellii by 3 methods (culture, fluorescent antibody test, and immunohistochemistry) and on gross and histopathologic findings. All foals had acute peritonitis, and the internal umbilical remnant was thickened by edema, hemorrhage, and fibrosis. A moderate amount of serosanguinous fluid with fibrin strands was present in the pericardial sac and pleural cavity. Histopathologically, the urachus and umbilical arterial walls were thickened by edema and exhibited hemorrhage, fibrin, and leukocytic infiltration. Gram-positive bacterial rods were observed in subepithelial areas of the urachus, the adventicia of umbilical arteries, and interstitium of the internal umbilical remnant. On the basis of these findings, we suggest that C. sordellii should be considered in the differential diagnosis for infections of the internal umbilical remnant in foals.

Abstract. This study was designed to experimentally reproduce enterotoxemia by Clostridium perfringens type D in cattle and to characterize the clinicopathologic findings of this disease. Fourteen 9-month-old calves were inoculated intraduodenally according to the following schedule: group 1 (n 5 4), C. perfringens type D whole culture; group 2 (n 5 3), C. perfringens type D washed cells; group 3 (n 5 5), C. perfringens type D filtered and concentrated supernatant; group 4 (n 5 2), sterile, nontoxic culture medium. In addition, all animals received a 20% starch solution in the abomasum. Ten animals from groups 1 (4/4), 2 (3/3), and 3 (3/5) showed severe respiratory and neurologic signs. Gross findings were observed in these 10 animals and consisted of acute pulmonary edema, excessive protein-rich pericardial fluid, watery contents in the small intestine, and multifocal petechial hemorrhages on the jejunal mucosa. The brain of one animal of group 2 that survived for 8 days showed multifocal, bilateral, and symmetric encephalomalacia in the corpus striatum. The most striking histologic changes consisted of perivascular high protein edema in the brain, and alveolar and interstitial proteinaceous pulmonary edema. The animal that survived for 8 days and that had gross lesions in the corpus striatum showed histologically severe, focal necrosis of this area, cerebellar peduncles, and thalamus. Koch's postulates have been met and these results show that experimental enterotoxemia by C. perfringens type D in cattle has similar clinical and pathologic characteristics to the natural and experimental disease in sheep.