Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins.

Clostridium perfringens uses its large arsenal of protein toxins to produce histotoxic, neurologic and intestinal infections in humans and animals. The major toxins involved in diseases are alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), and necrotic B-like (NetB) toxins. CPA is the main virulence factor involved in gas gangrene in humans, whereas its role in animal diseases is limited and controversial. CPB is responsible for necrotizing enteritis and enterotoxemia, mostly in neonatal individuals of many animal species, including humans. ETX is the main toxin involved in enterotoxemia of sheep and goats. ITX has been implicated in cases of enteritis in rabbits and other animal species; however, its specific role in causing disease has not been proved. CPE is responsible for human food-poisoning and non-foodborne C. perfringens-mediated diarrhea. NetB is the cause of necrotic enteritis in chickens. In most cases, host⁻toxin interaction starts on the plasma membrane of target cells via specific receptors, resulting in the activation of intracellular pathways with a variety of effects, commonly including cell death. In general, the molecular mechanisms of cell death associated with C. perfringens toxins involve features of apoptosis, necrosis and/or necroptosis

Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents.

Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. Importance: Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural host intestinal contents. These analyses demonstrated that (i) ETX processing in host intestinal contents occurs in an ordered, stepwise fashion, (ii) processing of prototoxin by host intestinal contents results in higher-molecular-mass material and 3 distinct ~27-kDa ETX species, and (iii) serine proteases, such as trypsin, chymotrypsin, and other proteases, including carboxypeptidases, play a role in the activation of ETX by intestinal contents. These studies provide new insights into the activation and processing of ETX and demonstrate that this process is more complicated than previously appreciated

The VirS/VirR two-component system regulates the anaerobic cytotoxicity, intestinal pathogenicity, and enterotoxemic lethality of Clostridium perfringens type C isolate CN3685.

Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells

Diagnóstico de enfermedades clostridiales de los animales

Las enfermedades clostridiales son producidas por bacterias del género Clostridium, bacilos grampositivos, anaerobios y esporulados. La gran mayoría de los animales domésticos y silvestres son susceptibles a una o más de las enfermedades clostridiales descriptas en este resumen. Las enfermedades clostridiales de los animales se clasifican en entéricas, histotóxicas y neurotóxicas.Trabajo publicado en Cagliada, Maria del Pilar Lilia y Galosi, Cecilia Mónica (comps.). I Congreso de Microbiología Veterinaria. Libro de resúmenes. La Plata: Facultad de Ciencias Veterinarias, 2021.Facultad de Ciencias Veterinaria

Chronic inflammatory lung lesions in rabbits free of known respiratory pathogens

Chronic inflammatory lung lesions were studied in 58 purpose bred, clinically healthy rabbits that were found negative for Bordetella branchiseptica, Pasleurel/a multocida, Toxoplasma gondii, Encephalitozoon cuniculi, and lung worms. The changes found consisted of focal chronic interstitial pneumonia and chronic bronchitis and bronchiolitis, which were frequently associated with perivascular infiltrations of mononuclear leukocytes and vasculitis involving small lung vessels. The etiology of these alterations remains unknown

A study of lung lesions in asymptomatic rabbits naturally infected with B. bronchiseptica

A histological study of lungs performed in 40 rabbits carrying a naturally acquired subclinical B. bronchiseptica infection revealed chronic inflammatory pulmonary lesions in 38 rabbits, B. bronchiseptica was the only agent consistently isolated from lungs. Pasteurella spp. was not isolated from any animal. B. bronchiseptica infected rabbits frequently displayed changes characterized by a simultaneous occurrence of focal chronic interstitial pneumonia, vascular and perivascular infiltration of monocytes and lymphoid cells, and inflammation ofbronchi and bronchioli

First report of caprine abortions due to Chlamydia abortus in Argentina.

Infectious abortions of goats in Argentina are mainly associated with brucellosis and toxoplasmosis. In this paper, we describe an abortion outbreak in goats caused by Chlamydia abortus. Seventy out of 400 goats aborted. Placental smears stained with modified Ziehl-Neelsen stain showed many chlamydia-like bodies within trophoblasts. One stillborn fetus was necropsied and the placenta was examined. No gross lesions were seen in the fetus, but the inter-cotyledonary areas of the placenta were thickened and covered by fibrino-suppurative exudate. The most consistent microscopic finding was found in the placenta and consisted of fibrinoid necrotic vasculitis, with mixed inflammatory infiltration in the tunica media. Immunohistochemistry of the placenta was positive for Chlamydia spp. The results of polymerase chain reaction targeting 23S rRNA gene performed on placenta were positive for Chlamydia spp. An analysis of 417 amplified nucleotide sequences revealed 99% identity to those of C. abortus pm225 (GenBank AJ005617) and pm112 (GenBank AJ005613) isolates. To the best of our knowledge, this is the first report of abortion associated with C. abortus in Argentina

Oral microbiota of Patagonian lizards of genus Diplolaemus (Leiosauridae): fable to facts

Existe la creencia popular de que los lagartos del género Diplolaemus (Leiosauridae) de Patagonia, Argentina, son capaces de conferir mordeduras venenosas que pueden causar inflamación e incluso la muerte de animales de granja o salvajes. No obstante, no existe evidencia de la existencia de glándulas de veneno en estos lagartos. La inflamación y absceso provocado como resultado de las heridas de mordedura podría ser causado por agentes infecciosos. Este es el primer estudio acerca de la microbiota oral de los lagartos del género Diplolaemus de Argentina. Para este trabajo se analizó un espécimen de D. darwini y tres individuos de Diplolaemus "forma alto patagonica". Las muestras de hisopados de boca de los lagartos fueron incubados aeróbica y anaeróbicamente, algunos de ellos fueron subcultivados en agar de sangre ovina al 5% e incubados nuevamente. Todas las colonias representativas observadas fueron subcultivadas para su purificación y sujetas a tests bioquímicos. La bacteria Clostridium perfigrens fue determinada por medio de la técnica de reacción en cadena de la Polimerasa. Se identificaron cuatro especies de bacterias: Staphylococus warneri, Clostridium bifermentans, Clostridium perfringens y Stomatococcus muscilaginosus. Estas especies son conocidas como agentes etiológicos de numerosas infecciones de humanos y animales. Si bien estas bacterias representan sólo un pequeño número de las posibles bacterias aisladas de la cavidad oral de Diplolaemus, están indicando la presencia de patógenos humanos y animales. <br> Lizards of genus Diplolaemus (Leiosauridae) from Patagonia, Argentina are believed to have "poisonous" bites that can cause inflammation and death of farm and wild animals. However there is no evidence of poisonous glands in these lizards. It is proposed that the inflammation and abscesses resulting from these bite wounds are caused by infectious agent(s). This is the first study of the oral microbiota of Diplolaemus lizards from Argentina. One specimen of D. darwini and three individuals of Diplolaemus "forma alto patagonica" were used for the present work. Swabs were taken from the mouths of the lizards, subcultured onto 5% sheep blood agar plates, incubated at 37 C for 48 hours aerobically and anaerobically. All representative colonies observed were subcultured for purity and subjected to conventional biochemical tests. Isolates identified as Clostridium perfringens were typed by a polymerase chain reaction technique. Four species of bacteria were identified as Staphylococcus warneri, Clostridium perfringens, Clostridium bifermentans and Stomatococcus muscilaginosus. These species are known etiologic agents of a number of human and animal infections. While these bacteria represent only a small number of possible isolates from the oral cavity of Diplolaemus species nevertheless it indicates that human and animal pathogens are present in their oral cavity

Clostridium perfringens epsilon toxin induces blood brain barrier permeability via caveolae-dependent transcytosis and requires expression of MAL.

Clostridium perfringens epsilon toxin (ETX) is responsible for causing the economically devastating disease, enterotoxaemia, in livestock. It is well accepted that ETX causes blood brain barrier (BBB) permeability, however the mechanisms involved in this process are not well understood. Using in vivo and in vitro methods, we determined that ETX causes BBB permeability in mice by increasing caveolae-dependent transcytosis in brain endothelial cells. When mice are intravenously injected with ETX, robust ETX binding is observed in the microvasculature of the central nervous system (CNS) with limited to no binding observed in the vasculature of peripheral organs, indicating that ETX specifically targets CNS endothelial cells. ETX binding to CNS microvasculature is dependent on MAL expression, as ETX binding to CNS microvasculature of MAL-deficient mice was not detected. ETX treatment also induces extravasation of molecular tracers including 376Da fluorescein salt, 60kDA serum albumin, 70kDa dextran, and 155kDA IgG. Importantly, ETX-induced BBB permeability requires expression of both MAL and caveolin-1, as mice deficient in MAL or caveolin-1 did not exhibit ETX-induced BBB permeability. Examination of primary murine brain endothelial cells revealed an increase in caveolae in ETX-treated cells, resulting in dynamin and lipid raft-dependent vacuolation without cell death. ETX-treatment also results in a rapid loss of EEA1 positive early endosomes and accumulation of large, RAB7-positive late endosomes and multivesicular bodies. Based on these results, we hypothesize that ETX binds to MAL on the apical surface of brain endothelial cells, causing recruitment of caveolin-1, triggering caveolae formation and internalization. Internalized caveolae fuse with early endosomes which traffic to late endosomes and multivesicular bodies. We believe that these multivesicular bodies fuse basally, releasing their contents into the brain parenchyma

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 &frac12;, 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.Um total of 110 amostras de Streptococcus suis isoladas de suínos doentes, no Brasil foram sorotipificadas e analisadas para a virulência. Sorotipificação das amostras resultou na seguinte classificação: 42 amostras do sorotipo 2 (38,2%), 10 amostras do sorotipo 14 (9,1%), sete amostras do sorotipo 9 (6,4%), três amostras de cada sorotipo, 7 e 11 (2,7%), duas amostras de cada sorotipo, 1 e 8 (1,8%) e uma amostra de cada um dos sorotipos, &frac12;, 3, 5, 6 e 10 (0,9%). Reações cruzadas entre os sorotipos 1, 14 e 7 foram observadas em 21 amostras (19,1%). Somente 41,9% das amostras foram patogênicas para camundongos