Clostridium perfringens is an anaerobic, spore forming, Gram positive bacteria found widely distributed in the soil and the digestive tract of many domestic animals. On the basis of the exotoxins they produce, six types (A, B, C, D, E and F) have been identified. Among these, the types B, C and D are found commonly associated with disease in domestic animals. Clostridium perfringens normally inhabits the digestive tract in small numbers without causing any disease. If any toxin is produced, even in small quantities and it can passes through the animal without causing problems. If an animal is exposed to sudden increase in carbohydrates, such as a heavy feeding of milk, lush pastures or supplementary concentrates, resident bacteria may multiply rapidly and produce large amounts of toxin. These toxins may damage the intestines, facilitating the absorption of toxins into the bloodstream. The end result of this intoxication is usually rapid death due to enterotoxaemia¹.

Cyanides are found in the plants, fumigants, fertilizers, pesticides, rodenticides and a variety of cyanide compounds are encountered in industries such as electroplating, metal cleaning, gold extraction and plastics industry. The toxicity can result from the accidental exposure or malicious use but in the case of animals, the most frequent cause of poisoning is ingestion of plants containing cyanogenic glycosides. HCN is rapidly absorbed from the gastrointestinal tract following ingestion and from lungs through inhalation. The onset of symptoms depends upon the quantity ingested or inhaled. Inhalation of airborne concentrations in excess of 100 ppm can be fatal within an hour and concentrations above 300 ppm are generally fatal within several minutes².

In rabbitsthere is paucity of the data on HCN toxicity³. Very few studies are known that too on experimental HCN toxicity by inhalation route in rabbits⁴,⁵.

In another subchronic study, New Zealand white rabbits (6 per group) were administered with potassium cyanide in their diet over a period of 40 week experiment⁶. The average cyanide intake was 36.5 mg/day. The cyanide-exposed group exhibited higher feed consumption with reduced weight gain and focal necrosis was noted in the liver and kidneys. However, to best of our knowledge, there are no reports of spontaneous/field cases of HCN toxicity in rabbits due to feeding of green fodder. In present communication, an outbreak of highmortality was investigated in a closed colony of New Zealand White laboratory rabbits to find out its causes so that such incidences may be prevented in future.

A total of 97 New Zealand White rabbits of Laboratory Animal Research Section, ICAR- IVRIwhich suddenly died in the second week of November, 2013 were received for necropsy examination and diseasediagnosis. Mortality continued for 5–6 days. All the animals were systematically examined andsamples were collected.for aerobic and anaerobic microbiological examination, visceral organs (lung, liver and spleen) and caecal contents were collected aseptically For toxicological examination, stomach content, feed mixture and green fodder (maize leaves) were collected. Hind quarter fecal contents and intestinal scrapings were collected for coccidial oocysts examination.Brain, lungs, heart, stomach, spleen, small and large intestines, kidneys and adrenal glands were collected in 10 per cent formalin for histopathological examination.

The intestinal contents were cultured in Robertson‘s cooked meat medium (HiMedia labs) at 37°C under anaerobic conditions for 48 hrs. Culture was then examined by Gram staining, and sub-cultured on anaerobic blood agar under anaerobic conditions. DNA from bacterial culture was then isolated by boiling for 5 minutes followed by snap chilling. Bacterial DNA was then subjected to multiplex PCR for typing Clostridium perfringens as described elsewhere⁷. Amplified PCR products were examined by agarose gel electrophoresis. Detection of HCN in maize fodder and stomach contents of rabbits was done by a qualitative method-Picrate Paper test⁸,⁹. A change in colour of picrate paper strip from yellow to brick red indicated the presence of HCN in the sample. Tissue specimens collected in 10% formalinfor histopathology were processed routinely and 5 mm thick sections were obtained and stained with Haematoxylin and Eosin. Duplicate sections of caecum were stained with Gram‘s Good Pasteur method for demonstration of bacteria.

Clostridial enteropathyis a major cause of morbidity and mortality in growing rabbits in rabbitries. The feeding of high energy and low fiber diets to weanlings is one of the most important predisposing factors of enterotoxaemia. The clinical signs are often lethargy, rough coat and perineal region covered with greenish brown fecal material and death within 48 hours. Clostridiumspiroforme is the major pathogen reported inthis disease which is an anaerobicnon-motile, sporeforming, Gram‘s-positive bacteria capable of producing potent enterotoxins. Conditions that can alter the normal gut flora and favor the colonization and proliferation of Clostridialspiroforme include weaning, abrupt diet change, parturition and lactation, illness, antibiotic administration. Clostridium perfringens also causes enterotoxaemia-like condition in young rabbits rarely that results in bleeding enteritis in the blind sack and edema Clostridium difficile and Clostridium cocleatum has also been reported in rabbits¹,¹⁰.

Affected rabbits are often in good condition with sufficient body fat but there may be evidence of dehydration, fecal soiling of the perineum and death within 48 hrs. In the present case also, animals showed sudden mortality in a span of 3 days. The major necropsy findings in the present outbreak were soiling of hind quarter, haemorrhagic typhlitisand pulmonary congestion or haemorrhages. The most characteristic findings of Clostridial infection are petechial and ecchymotic haemorrhages on the caecal serosal surface¹⁰. This may also be evident on the proximal colon, appendix, and distal ileum. The caecum wasdistended with gas and contains fluid to mucoid dark brown feces, mucosal haemorrhages with occasional ulcers or pseudo-membranes with thickened and edematous submucosa¹,¹⁰.

Histopathologically, caecum showed pronounced submucosal edema, haemorrhages, splenic haemorrhages with necrosis and pulmonary edema. Gram‘s Good Pasteur staining in tissue sections revealed presence of numerous bacilli in different parts of caecum. These lesions were suggestive of Clostridial enterotoxaemia. In Clostridial enteropathy, diffuse necrosis or focal erosions/ulcers of the stomach and intestinal mucosahave been reported¹,¹⁰. Submucosa and lamina propriaexhibits inflammatory cell infiltration, mainly neutrophils, and ulcers are frequently overlaid by an exudate layer composed of cell debris, inflammatory cells and proteinaceous fluid¹,¹⁰. In caecum the most consistent changes are submucosal edema and serosal haemorrhages. There may be evidence of epithelial cell proliferation in areas of intact mucosa, e.g., increased numbers of mitoses and lengthening of crypts. Mucosal attenuation is also reported1.In the present study, intestinal contents were found alpha toxin positive by PCR indicating Clostridium perfringens infection.

HCN toxin was also demonstrated in green maize fodder, ballies and stomach content. Pulmonary edema was seen in some cases which is indicative of HCN toxicity. Although there is no information available on the presence of cyanogenic glycoside hydrolyzing enzyme in rabbit stomach, it is quite possible that the bacterial enzymatic degradation and/change in the gastric pH could have induced liberation of HCN which ultimately resulted in toxicity and mortalities in rabbits.

In conclusion, it may be stated that the present outbreak and high mortality in rabbits was caused due to combined effects of Clostridial and HCN toxins.

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