The use of Prevotella bryantii 3C5 for modulation of the ruminal environment in an ovine model

Abstract In the Southern Hemisphere, ruminants are mostly raised in grazing systems where animals consume forage and are supplemented with low amounts of concentrates. Concentrates are usually given separately and are rapidly ingested. This practice leads to changing rumen environment conditions during the day, may alter the rumen microbial metabolism and could affect host performance. The native ruminal Prevotella bryantii strain 3C5 was administered every 48 h to wethers under experimental conditions simulating Southern-Hemisphere feeding to evaluate its potential as a rumen fermentation modulator. The inoculum potential was assessed on day 17. The ammonia nitrogen (NH3-N), volatile fatty acids and ruminal pH were monitored on a 24-h basis 19 days after the beginning of the experiment, and the microbial community structure was assessed by pyrosequencing. The administration of P. bryantii modified the fermentation products and daily pH values compared to the control. The NH3-N concentration in the rumen of treated animals was significantly higher than that of the untreated animals. Modification of the ruminal environment and fermentation pathways was achieved without altering the general structure of the microbial community or the potential methane production. P. bryantii 3C5 could be considered in potential probiotic formulations for ruminants in semi-intensive systems.

Isolation and characterization of a novel endo-beta-1, 4-glucanase from a metagenomic library of the black-goat rumen

ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.

População de protozoários ruminais em novilhos zebuínos alimentados com ou sem volumoso / Population of Protozoa in zebu steers fed with or without bulk

This study aimed to quantify and identify the profile of the rumen protozoa population of beef steers fed with or without roughage. Nellore crossbred steers raised in extensive system on lignified tropical pastures with mineral supplementation and steers confined for 60 days only receiving pelletized concentrate and whole corn kernels were evaluated. After slaughter, rumen fluid was collected and one ml aliquots were diluted in nine ml formaldehyde solution at 10%. The counts of small, medium and large protozoa were held in Sedgewick Rafter chambers and identification of genus was possible after staining lugol and optical microscope with a 40X objective. The concentration of rumen protozoa positively correlated with pH ruminal. Cattle fed without roughage had significantly lower rumen protozoa population (P<0.05). Animals fed roughage had higher occurrence of Dasytrichia genus, Charonina, Entodinum, Diplodinium, Ostracodinium and Epidinium while those fed without bulk, the Buetschilia, Isotricha, Eodinium, Polyplastron, Elyplastron, Metadinium and Enoploplastron were the most frequent genus.(AU)

Utilização da técnica in vitro semiautomática de produção de gases na avaliação da fermentação microbiana do ceco de suínos / Use of the semi-automatic in vitro gas production technique for the evaluation of swine cecal microbial fermentation

O presente estudo teve como objetivo avaliar o os padrões de fermentação microbiana do ceco de suínos, utilizando-se um modelo aplicado na técnica in vitro de produção de gases para ruminantes. Três tipos de inóculo foram utilizados: ceco de suínos criados ao ar livre (CSF), ceco de suínos criados confinados (CSC) e líquido ruminal de bovino (LRB). Os substratos utilizados derivaram de uma dieta de suínos à base de farelo de soja e de grãos de milho, atendendo as exigências nutricionais dos suínos. Para composição dos substratos, foram estabelecidos diferentes níveis de substituição da dieta basal pela torta da amêndoa da Acrocomia aculeata (AA), coproduto da produção do biodiesel, sendo: BAS - 100% de dieta basal; M10 - 90% de dieta basal e 10% AA; M20 - 80% de dieta basal e 20% AA e o TF - feno de Tifton 85 (Cynodon spp.). A produção de gases foi avaliada nos tempos zero, três, seis, nove, 12, 16, 24, 48, 72 e 96 horas após a incubação. Foram analisados o potencial máximo de produção de gás (A) e o tempo de colonização (L). O delineamento experimental utilizado foi inteiramente ao acaso, em arranjo fatorial 4 x 3. Os dados de A e L foram submetidos à análise de variância, e as médias comparadas pelo teste de Tukey a 5%. Comparando os inóculos para A (mL/gMS), LRB e CSF apresentaram valores similares, diferindo apenas para TF. O tempo de colonização (L) entre inóculos, o CSC e o CSF apresentaram os menores tempos de colonização quando a ração foi BAS. Com os substratos M10 e M20, o inóculo LRB apresentou o menor tempo, CSF o maior tempo e CSC não diferiu de ambos. Não houve diferença significativa entre os inóculos para o substrato TF. A técnica de produção de gases utilizada para o ceco de suínos apresentou resultados semelhantes aos encontrados para ruminantes. O modelo matemático usado foi adequado para descrever a curva de fermentação no ceco de suínos, mostrando semelhanças entre as microbiotas do ceco e do rúmen.(AU)

This study aimed to evaluate the microbial fermentation patterns of the pig ceca using the technique of in vitro gas production for ruminants. Three types of inoculums were used: swine ceca raised in a free range system (CSF), swine ceca raised in a conventional confined system (CSC) and bovine rumen fluid (LRB). The substrates used were derived from pig's diet with soybean meal and corn grits, according to the nutritional requirements of swine. The substrates were composed by different set levels of substitution of basal diet by meal almond Acrocomia aculeata (AA), as: BAS - 100% of basal diet; M10 - 90% of basal diet and 10% AA (M10); M20 - 80% of basal diet and 20% AA (M20) and TF - Tifton 85 hay (Cynodon spp.).The gas production was evaluated at times 0, 3, 6, 9, 12, 16, 24, 48, 72 and 96 hours after incubation. The study analyzed the maximum potential of gas production (A) and lag time (L). The experimental design was completely randomized in a 4 x 3 factorial arrangement. The A and L data were submitted to analysis of variance and the averages compared by Tukey 5% test. Comparing the inoculum for A (ml / gDM), LRB, and CS, they showed similar values, differing only for TF. The lag time (L) between inoculum, the CSC and CSF had the lowest colonization times when the meal was BAS. With M10 and M20 substrates, the LRB inoculum showed the lowest time, CSF had the longest time and CSC did not differ from both. There was no significant difference between the inoculum for TF substrate. The gas production technique used for the ceca of pigs showed similar results to those for ruminants. The mathematical model used was adequate to describe the fermentation curve in the ceca of pigs, showing similarities between the microbiota of the ceca and the rumen.(AU)

Efeito da inclusão da torta de macaúba na população de protozoários do rúmen de vacas leiteiras / The effect of macauba meal inclusion in the ruminal protozoa population of dairy cows

Avaliaram-se os efeitos da inclusão da torta de macaúba (TM) na dieta sobre a concentração e o perfil da população de protozoários ciliados do rúmen. Foram utilizadas oito vacas Holandesas, dispostas em dois quadrados latinos 4x4 e alimentadas com diferentes níveis de inclusão da TM (0, 100, 200 e 300g kg-1 de matéria seca na dieta total). Para quantificação e identificação dos protozoários, amostras de 1mL do líquido ruminal de cada animal foram diluídas em 9mL de solução de formaldeído a 10%. Após diluições decimais sucessivas em solução salina, os protozoários foram quantificados em câmara de Sedgewick. A identificação foi realizada sob a luz da microscopia óptica, utilizando-se a objetiva de 40x para caracterizar o mínimo de 100 indivíduos por amostra. As concentrações de protozoários pequenos (P<0,01), médios (P=0,05), grandes (P=0,01) e o número total (P<0,01) apresentaram comportamento linear decrescente com o aumento dos níveis de inclusão da TM nas dietas. Foram encontradas características morfológicas típicas de 16 gêneros de protozoários ciliados, sendo os gêneros Entodinium e Eodinium os mais frequentes no líquido ruminal dos animais avaliados. O perfil da população de protozoários ciliados sofreu pequenas alterações com a inclusão da TM, sendo observada alteração na participação percentual dos gêneros Charonina (P = 0,03) e Polyplastron (P<0,01). A utilização da TM na dieta de bovinos promove redução na concentração e modificação no perfil da população de protozoários ciliados do rúmen.

The effects of meal macaúba (MM) inclusion on the concentration and profile of the ciliate protozoa of the bovine rumen were evaluated. Eight Holstein cows were used during second lactation, with average 480 kg of body weight, arranged in two 4x4 Latin squares simultaneously and fed four diets with different concentrations of the MM (0, 100, 200 and 300 g kg-1). For identification and quantification of protozoa, samples of ruminal fluid from each animal with 1 ml were diluted in 9mL of 10% formaldehyde. After consecutive decimal dilutions in saline solution, protozoa were assessed in a Sedgewick chamber. The identification was carried out under the optical light microscope using a 40x objective to characterize at least 100 individuals per sample. The concentrations of small (P<0.01), medium (P=0.05), large (P= 0.01), and the total number (P<0.01) of protozoa decreased linearly with the increasing MM inclusion. Morphological characteristics typical of 16 genera of ciliates were found, being the Entodinium spp. and Eodinium spp. the most frequent in the rumen fluid of animals evaluated. The profile of the population of ciliated protozoa suffer minor changes with the addition of MM, with a change in the percentage share of Charonina spp. (P = 0.03) and Polyplastron spp. (P <0.01). The use of the MM in the cattle diet promotes reduction in the concentration and change in the profile of the population of rumen ciliates protozoa.

Bastonetes Gram-negativos aeróbios e anaeróbios facultativos no fluido ruminal de bovinos de corte alimentados em pastagem lignificada e em novilhos com acidose ruminal / Aerobes and anaerobe facultative Gram-negative rod-shaped bacteria in the ruminal fluid of beef cattle fed lignified pasture and steers with ruminal acidosis

Objetivou-se analisar a população de bastonetes Gram negativos aeróbios e anaeróbios facultativas no suco ruminal bovinos zebuínos de diferentes categorias, alimentados em pastagem tropical, e de novilhos alimentados com alto teor de grão e sem volumosos. Foram coletados fluido ruminal de 32 vacas, 50 novilhos e 50 bezerros alimentadas em pastagem de Brachiaria spp. e de 20 novilhos com acidose ruminal. Após diluições decimais, amostras foram inoculadas em placas contendo ágar MacConkey a 39°C. Para a identificação dos gêneros mais frequentes foram utilizadas provas bioquímicas. A concentração dessas bactérias não diferiu no ambiente ruminal de vacas, novilhos e bezerros de corte alimentados com pastagem tropical lignificada. Os gêneros mais frequentemente identificados para esses animais foram Escherichia, Enterobacter e Klebsiella. Novilhos alimentados sem volumoso e com acidose apresentaram maior taxa de detecção e maior população dessas bactérias no ambiente ruminal (>6 log/ml) quando comparados aos novilhos alimentados somente em pastagem. A espécie Escherichia coli foi predominante entre as bactérias isoladas do fluido ruminal de novilhos alimentados com dieta com alta concentração de grãos e com acidose (p<0,01). Constatou-se que em bovinos de corte, criados em pastagem tropical lignificada, a população desses microrganismos é baixa no ambiente ruminal e com maior diversidade de gêneros bacterianos. Entretanto em novilhos confinados e alimentos sem volumoso, apresentando acidose ruminal subaguda, ocorre desequilíbrio populacional com aumento da população de E. coli...

This study aimed to analyze the population of Gram negative bacteria, rod-shaped aerobic and facultative anaerobes, in ruminal fluid of health Zebu cattle of different categories fed in tropical pasture and steers fed high levels of grain and without bulky. Rumen fluid from 32 cows, 50 steers and 50 calves fed on Brachiaria spp. and 20 steers with ruminal acidosis were collected. After decimal dilutions, the samples were inoculated on petri dishes with agar MacConkey at 39°C. Biochemical tests were used to identify the most common genera these bacteria. The concentration of these bacteria did not differ in the rumen of cows, calves and calves fed lignified tropical pasture and the most frequently identified genera for these animals were Escherichia, Enterobacter and Klebsiella. However, steers fed without forage and with acidosis showed a higher detection rate and larger population of these bacteria in the rumen (>6 log/ml) compared to steers fed only pasture. The Escherichia coli species was predominant among theses bacteria isolated from the rumen fluid of steers with acidosis (p<0.01). In beef zebu cattle raised on pasture lignified, the population of these microorganisms in the rumen is low showing greater diversity of genera. However in confined zebu steers fed without forage and with sub acute ruminal acidosis occur disequilibrium with increased E. coli population...

Use of green fluorescent protein to monitor Lactobacillus plantarum in the gastrointestinal tract of goats

The experiment aimed to specifically monitor the passage of lactobacilli in vivo after oral administration. The green fluorescent protein (GFP) gene was cloned downstream from the constitutive p32 promoter from L. lactis subsp. cremoris Wg2. The recombinant expression vector, pLEM415-gfp-p32, was electroporated into Lactobacillus plantarum (L. plantarum) isolated from goat. Green fluorescent protein (GFP) was successfully expressed in L. plantarum. After 2 h post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, bacteria concentration initially decreased, reached the minimum at 42 h post-oral administration and then increased. However, this concentration decreased constantly in the duodenum. This result indicated that L. plantarum could colonize in the rumen but not in the duodenum.

Historical review of clinical vaccine studies at Oswaldo Cruz Institute and Oswaldo Cruz Foundation - technological development issues

This paper presents, from the perspective of technological development and production, the results of an investigation examining 61 clinical studies with vaccines conducted in Brazil between 1938-2013, with the participation of the Oswaldo Cruz Institute (IOC) and the Oswaldo Cruz Foundation (Fiocruz). These studies have been identified and reviewed according to criteria, such as the kind of vaccine (viral, bacterial, parasitic), their rationale, design and methodological strategies. The results indicate that IOC and Fiocruz have accumulated along this time significant knowledge and experience for the performance of studies in all clinical phases and are prepared for the development of new vaccines products and processes. We recommend national policy strategies to overcome existing regulatory and financing constraints.

Técnicas de quantificação da síntese microbiana no rúmen: uma revisão / Quantification techniques of the protein microbial synthesis in rumen: a review / Técnicas de cuantificación de síntesis microbiana en rumen: una revisión

Quantificar a síntese de proteína microbiana em animais ruminantes é de grande importância para avaliar a qualidade nutritiva das dietas. A determinação dessa síntese se obtém por meio da utilização de marcadores microbianos, osquais são classificados em internos (subprodutos dos microrganismos ruminais) e externos (produtos administrados aos animais que são incorporados pelos microrganismos) e técnicas indiretas. Embora tenham ocorrido avanços ainda não se tem o marcador ideal. O objetivo desta revisão é detalhar a cada uma das vantagens e desvantagens das técnicas para estimar a síntese de proteína microbiana no rúmen e discutir os resultados obtidos utilizando cadatécnica.

The quantification of the microbial protein synthesis in the ruminants is of great importance to assess the nutritional quality of the diets. The determination of microbial synthesis is achieved through the use of microbial markers, which are classified: internal (by-products of rumen microorganisms) and external (substances administeredto animals that are incorporated by rumen microorganisms). Although some advances has occurred lately anideal marker still need to be developed. So the objective of this review is to detail each of the techniques forestimating microbial protein synthesis in the rumen, and discuss the results obtained using each technique.

La cuantificación de la síntesis de proteína microbiana en los rumiantes es de gran importancia para evaluar lacalidad nutricional de las dietas. La determinación de la síntesis microbiana se logra a través del uso de marcadoresmicrobianos que se clasifican como: Internos (subproductos de los organismos de la panza) y externos (sustancias administradas a los animales que son incorporadas por los organismos de la panza). Aunque últimamente ha habido algunos avances, aún es necesario desarrollar un marcador ideal. Así que el objetivo de esta revisión es detallarcada una de las técnicas usadas para estimar la síntesis de proteína microbiana en la panza y discutir los resultados obtenidos al usar cada técnica.

[ effects of saccharomyces cerevisiae and lactic acid producing bacteria on digestibility, rumen and blood parameters of sheep]

Various probiotic products have been used in ruminant nutrition over the past decade. Some of them had positive effects on animal production. Present study was conducted to investigate the effects of Saccharomyces cerevisiae, Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium, Bacillus subtilis mixture on digestibility, rumen and blood parameters of 4 fistulated varamini sheeps. Animals fed with 4 diets during 4 periods using a 2x2 factorial arrangement in a change-over design. First factor included two sources of forages [alfalfa hay and corn silage] and second factor included two levels of above probiotic mixture [0 and 5 gr/day/head]. Digestibility of diets were measured by acid insoluble ash as marker. Blood samples were taken at 0 and 4 hours after feeding via jugular vein to determine concentration of CO2, pH, LDH and Glucose. Also ruminal liquor samples were taken at 0 and 4 hours after feeding to determine rumen pH and concentration of N-NH3 and volatile fatty acids. The results showed that digestibility of acid detergent fiber, neutral detergent fiber, organic matter and dry matter of diets were not significantly different among diets. However, digestibility of crude protein and ether extract in diets containing corn silage were lower and higher than alfalfa hay, respectively. There was no significant difference on blood metabolites of sheep fed various diets. Total volatile fatty acids in sheep fed microbial mixture were more than control group. Diets containing alfalfa hay had higher propionate and acetate concentrations than diets containing corn silage. Ruminal ammonia concentration decreased in sheep fed diets containing microbial mixture [P<0.05]. We concluded that present microbial mixture improved ruminal fermentation but could not significantly affect digestibility and blood metabolites of sheep

Fungos anaeróbios do rúmen de bovinos e caprinos de corte criados em pastagens tropicais / Ruminal anaerobic fungi of beef cattle and beef goats raised on tropical pastures

The presence of anaerobic fungi structures was evaluated in ruminal juice of beef goats and beef cattle raised in the North of Minas Gerais, Brazil. The strains were collected from 18 Anglo-Nubian crossbred male goats and 23 Nellore crossbred steers during the dry period of the year. Physical-chemical characteristics of the juice were evaluated and direct examination with KOH digestion was performed for anaerobic fungi detection. Structures of these fungi were detected in samples of 14 (77.8 percent) goats and 17 (73.9 percent) steers. The monocentric fungi frequency (56.5 percent) was significantly higher in cattle than polycentric fungi frequency (26.1 percent). This study is the first report of anaerobic ruminal fungi in these ruminants in Brazil and showed high prevalence of theses microorganisms in the ruminal ecosystem of both animals.

Frações dos compostos nitrogenados associados à parede celular em forragens tropicais / Fractions of cell wall nitrogenous compounds in tropical forages

The total and undegradable fractions of nitrogenous compounds associated to cell wall organic matrix in some tropical forages were evaluated. Samples of corn silage, elephantgrass silage and sugarcane were used. Neutral detergent insoluble nitrogenous compounds were divided in three different fractions associated with: hemicellulose (HN), cellulose (CN), and lignin (LN). The size of the different fractions varied among feeds. A portion of acid detergent insoluble nitrogen, which is the sum of CN and LN, was potentially degradable in the rumen. A portion of HN was not degradable in the rumen. The heterogeneous dimensions of degradable portion of all nitrogenous compounds among feeds can compromise the estimates of cell wall undegradable nitrogen by simple chemical approaches in tropical forages. The LN overestimated the lignin contents and the correction for those compounds on lignin has been suggested.

In vitro degradation of coast-cross (cynodon dactylon) by rumen microorganisms associated with Saccharomyces cerevisiae or humicola sp

Ruminal degradation of the dry matter and neural detergent figer of coast-cross (cynodon dactylon) forage, when previously inoculated with Saccharomyces cerevisiae or Humicolla sp., was estimated by in vitro incubation. The potential dry matter degradability was incresead in the presence of S. cerevisiae, whereas the incubation with Humicola sp. resulted in improvement of the neutral detergent fiber degradability. The rate of dry matter digestion was enhanced to above 15 per center with the inoculation with S. cerevisiae. The inoculation with S. cerevisiae or Humicola sp. increased the rate of digestion of the neutral detergent fiber in relation to the control treatment.

Tentativas de defaunaçäo de ovinos / Attempts at sheep defaunation

Foram testados três métodos de defaunaçäo: 1- Dioctyl sulfosuccinato de sódio (DSS) aplicado através de fístula ruminal em diferentes dosagens com e sem jejum prévio; 2- Lavagem do rúmen com soluçäo de formol a 0,15% e 3- Isolamento após o nascimento. Amostras de conteúdo ruminal foram coletadas para pesquisa de protozoários. O DSS e a lavagem do rúmen com formol causaram alta taxa de mortalidade ou näo foram capazes de eliminar os protozoários ruminais. O isolamento após o nascimento näo evitou a contaminaçäo do rúmen por diferentes gêneros de protozoários. Nenhum dos métodos testados foi capaz de produzir animais defaunados

Avaliaçäo dos protozoários ciliados no rúmen de búfalos (Bubalus bubalis L) e bovinos (Bos indicus L) em regime de confinamento / Rumen ciliate protozoa in buffalo (Bubalus bubalis, L) and zebu cattle (Bos indicus, L) kept in a feedlot

Num experimento de engorda em confinamento, foram utilizado nove bubalinos e nove zebuínos, todos machos castrados, de 18 a 24 meses de idade e peso médio de 340 kg, mantidos em baias individuais, consumindo dieta de 2,0 kg de soja crua moída, 2,0 kg de espigas de milho desintegradas (gräo, palha e sabugo) e silagem de sorgo à vontade (controlando-se o consumo diário). Ao final do período de confinamento, todos os animais foram abatidos e o conteúdo ruminal de cada indivíduo foi homogeneizado, sendo colhidas amostras de 30 a 40 ml de líquido ruminal e, desta, retirou-se alíquota de 10 ml em tubo de ensaio com 20 ml de formaldeído diluído em água destilada a 1:2, para fixaçäo dos protozoários ciliados. As contagens de protozoários foram feitas em 100 campos e os resultados permitiram as seguintes conclusöes: 1) a dieta favoreceu maior concentraçäo de protozoários ciliados/ml de líquido ruminal dos zebuínos que no de bubalinos, apesar destes apresentarem maior número de gêneros; 2) os protozoários ciliados digestores de fibra foram detectados em maior quantidade nos zebuínos que nos bubalinos; 3) o gênero Entodinium spp representou mais de 80 por cento do total dos protozoários ciliados identificados nos zebuínos e nos bubalinos

Isolamento de bactérias do gênero Clostridium e detecçäo de toxina botulínica a partir de materiais obtidos de bovinos com suspeita clínica de botulismo / Isolation of genus Clostridium bacterias and detection of botulinum toxin from samples obtained of bovine with clinic cases of botulism

Foram analisadas amostras de fragmentos de fígado, conteúdo ruminal, fezes e soro sangüíneo provenientes de 68 casos de bovinos com diagnóstico clínico de botulismo. O objetivo foi a confirmaçäo laboratorial de botulismo, face ao número crescente de casos clínicos suspeitos em nosso meio. Os materiais eram oriundos de bovinos dos Estados de Säo Paulo, Mato Grosso, Mato Grosso do Sul, Goiás, Minas Gerais, Pará e Bahia e foram coletados através de necrópsia realizada por veterinários de campo e enviados à Seçäo de Bacteriologia Animal do Instituto Biológico de Säo Paulo da Secretaria da Agricultura e Abastecimento. Foi realizada a extraçäo de toxina botulínica e sua classificaçäo sorológica, bem como o isolamento e identificaçäo de bactérias do gênero Clostridium. A toxina botulínica foi detectada em apenas 2 (2,9%) dos casos, sendo 1 (1,5%) no fígado e classificada no tipo C e em outro (1,5%) no soro sangüíneo e conteúdo ruminal, mas näo pertencente aos tipos C e D. Nesses 2 casos näo foram isoladas cepas de Cl. botulinum. Pelo exame bacteriológico, 30 (44,1%) dos casos estudados foram positivos para o isolamento de cepas do gênero Clostridium, dos quais foram isoladas 23 diferentes espécies do gënero. De 19 (63,3%) dos 30 casos positivos foram obtidas cepas de Clostridium, isoladamente ou em associaçäo, identificadas como espécies classicamente consideradas como agentes patogênicos para bovinos, incluindo o isolamento de Cl. botulinum, embora de apenas um caso. Tais fatos demonstram a necessidade de maiores estudos sobre o assunto que possibilitem posiçöes mais conclusivas sobre a real situaçäo desta doença