Implication of Clostridium perfringens type a in Young Calves

Mahmoud Hamouda¹, Fahad Al–hizab¹, Taha Fouda², Mahmoud Fayez³

*Corresponding author: mhamouda@kfu.edu.sa

ARTICLE CITATION: Hamouda M, Al–hizab F, Fouda T and Fayez M (2014). Implication of clostridium perfringens type A in young calves. Res. j. vet. pract. 2(1): 9 – 12.
Received: 2013–10–30, Revised: 2013–12–14, Accepted: 2013–12–16
The electronic version of this article is the complete one and can be found online at (http://dx.doi.org/10.14737/journal.rjvp/2014/2.1.9.12) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Abstract

Clostridium perfringens produces enteric diseases in cattle, sheep and goats. The microorganism can be a normal inhabitant of the intestine of most animals and human. A total of 8 calves aged between 8 to 11 month suffering from colic, and dark clotted blood in the faeces (melena) proceeded with death. The main postmortem findings were observed as massive hemorrhage and clot formation within the small intestine as well as abomasal ulcerations. Histologically, the abomasal mucosa was sloughed and the intestinal villi appeared necrotic along with a characteristic submucosal oedema. The bacterial culture and toxin detection showed presence of Clostridium perfringens type A. There were no other potential microorganisms or aflatoxins have been identified.

INTRODUCTION

Clostridium perfringens causes food poisoning and fatal enterotoxemia (Yamagishi, 1997; Yoo, 1997). Enteric C. perfringens infections in animals and man called enterotoxemias. There are five types of C. perfringens (A, B, C, D, E), which are identified by the main types of toxins they produce (alpha, beta, iota, epsilon and theta) (Niilo, 1987; Songer, 1996). C. perfringens type A is the most common C. perfringens types. It is part of the normal gut flora in cattle. However, dietary changes or parasitism may produce a favorable growth environment, resulting in overgrowth and production of potent toxins. C. perfringens type A can rapidly produce potent toxins, primarily alpha toxin. Alpha toxin is thought to be associated with a number of potentially deadly gastrointestinal diseases (Hatheway, 1990). Some isolates of C. perfringens type A produce β2–toxin which may contribute, along with α–toxin, to the development of hemorrhagic enteritis in cattle (Jelinski et al., 1995; Bueschel et al., 2003; Abutarbush and Radostits, 2005). C. perfringens type A is also commonly isolated in calves in cases where abomasal ulcers and abomasal haemorrhage are found (Roeder et al., 1987).

History, clinical signs, and gross postmortem findings are useful in establishing a presumptive diagnosis of clostridial enterotoxemia, but confirmation requires laboratory testing. Detection of toxins in intestine is very important to establish a diagnosis. ELISAs are considered one of the most important laboratory technique for C. perfringens toxins detection (Francisco and Glenn, 2008). The purpose of this paper is to present a description of Clostridium perfringens type A in calves that died suddenly with severe intra–luminal hemorrhage in the jejunum and abomasal ulcerations.

MATERIALS AND METHODS

Animals and Samples Collection
A total of 8 calves aged between 8 to 11 month suffering from colic, and dark clotted blood in the faeces (melena) proceeded with death was admitted to the Veterinary Teaching Hospital of the college of Veterinary Medicine and Animal resources, king Faisal, Saudi Arabia. The disease was coincided with the presentation of a new total mixed ration to animals. Blood samples were collected in heparinized tubes for determination of total and differential white blood cells count (Ve.Scan 5HM–ABaxis–USA/2002). Moreover, feedstuffs were analyzed for a total aflatoxin using a slightly modified immunoaffinity method based on Association of Official Analytic Chemists method (AOAC) (Trucksess et al., 1991).

Postmortem and Histopathology
Four cadavers were available to necropsy. Impression smears prepared by scraping intestinal mucosa and the cutting surfaces of mesenteric lymph nodes were stained by Gram’s Method. Specimens of abomasums, small intestine and large intestine, and mesenteric lymph nodes were preserved in 10% neutral buffered formalin.The formalin fixed tissue samples were dehydrated through graded ethanol and embedded in paraffin blocks. Sections of 4–5 um thickness were cut and routinely stained with Haematoxylin and Eosin (HE). The selected sections were stained by Gram and Gomori methylamine silver (GMS) stains to detect bacterial or fungal organisms respectively.

Mycology and Bacteriology
The isolation of A. fumigatus was carried from the trachea, lungs, liver, kidney, brain; small and large intestine.These samples were directly streaked on sabouraud agar plates for culturing and were incubated for 7 days at 37 ° (Darise, 1987). A. fumigatus was identified according to its specific colony characteristics, slides were also prepared for identification of mycelium and hyphial arrangement with lactophenol blue staining method. Additionally, intestinal contents were cultivated in Cooked Meat Broth (CMB) and incubated anaerobically at 37°C for 48 hours. From these cultures, 0.1mL loop aliquot was streaked on 5% sheep blood agar and incubated under anaerobic conditions at 37°C for 24 hours. Colonies showing related characteristics of C. perfringens (aspect, color, and hemolysis) were submitted to Gram stain; colonies corresponding to Gram–positive bacilli were cultivated in CMB. After the incubation period, cultures were submitted to additional tests including catalase, lecithinase and gelatinase production, and glucose, lactose, and skimmed milk fermentation for species identification (Cowan, 1974). Strains identified as C. perfringens were then sub–cultivated in Triptose Yeast Extract Broth (TYB) and incubated under anaerobic conditions. Cultures were then centrifuged at 7.500 rpm for 15 min. at 4°C and cell–free culture supernatants were recovered. After that, 0.3 ml of broth culture supernatant and intestinal contents were injected into white mice (25 – 40 g) via intraperitoneal route and observed for either death or disease signs within three days.

C. Perfringens Toxins ELISA
Intestinal contents and broth cultures supernatant were tested for CPA, CPB, and ETX via a commercial capture ELISA kit (Bio–X, Diagnostics, Belgium), following the manufacturer’s instructions.