IN THE LAB CLOSTRIDIAL DISEASE IS frequently associated with sudden death syndromes in both production WAS IT and companion animals in New Zealand, but has been notoriously difficult to prove. CLOSTRIDIAL? Over the last 15 years there has been extensive research in this area which has led to fulfillment of Koch’s HOW DO WE postulates in a number of diseases. However there are a lot of suspected diseases which have still not been PROVE IT? proven to occur in veterinary species. There has been a recent surge in the popularity of clostridial toxin testing Michael Hardcastle, of Gribbles Veterinary, Auckland, overseas and in New Zealand. However, the significance of toxin, gene or outlines how to rule in or rule out the most commonly pathogen identification depends on suspected clostridia. clinical signs, gross and histological lesions, clostridial and veterinary species involved and sometimes the quantity of toxin identified. 50 – VetScript April 2019 IMAGERY: ISTOCKPHOTO.COM IN THE LAB For example: this is lacking. It is commonly found in in the lumen, along with many large » Clostridium perfringens Type A is the intestinal tract of healthy animals. Gram-positive bacilli. commonly found in faecal samples of Exceptions to this may occur in pigs, » The previous comments regarding C. normal production and companion where the gene for beta2 has been perfringens culture apply here. animals therefore isolation is frequently found in Type A strains » The beta toxin gene of C. perfringens insignificant on its own. isolated from cases of enteritis, and Type C can be detected by PCR. If » Alpha toxin is produced by all C. in dogs, where C. perfringens Type A found, this is consistent with the perfringens therefore isolation is netE and netF toxin genes have been diagnosis. insignificant on its own. more frequently found in cases of » The gross and histological findings » Up to 20% of normal sheep and cattle haemorrhagic gastroenteritis than in alone may be suggestive of the can be positive for Epsilon toxin healthy dogs. Overseas, it is linked diagnosis. which causes Enterotoxaemia (“Pulpy to ‘yellow lamb disease’, but to my Kidney Disease”) most commonly knowledge that is not seen in New caused by C. perfringens Type D. Zealand. Points to note: CLOSTRIDIUM » C. septicum and C. sordellii are the » Gross lesions will include areas PERFRINGENS TYPE D most commonly isolated post- of haemorrhagic intestine and (ENTEROTOXAEMIA) mortem clostridia and are only sometimes stomach in dogs. This strain is characterised by alpha considered significant in cases of » Histologically, there is and epsilon toxins. It causes disease Malignant Oedema in production haemorrhagic necrosis of the in lambs, kids and occasionally calves, animals in New Zealand. mucosa lined by large Gram-positive mainly presenting as neurological signs Diagnostic testing includes: bacilli. and/or sudden death in well-fed animals » Bacterial identification (Culture, PCR, » C. perfringens can be cultured by – classically, those fed concentrate or IHC, FAT). routine methods, but a positive grain. It is also reported to cause chronic » Toxin testing e.g. ELISA. culture is not diagnostic given enterocolitis and diarrhoea in goats. » Toxin gene testing e.g. PCR, qPCR. that non-pathogenic strains can be » There may be non-specific gross » Histology. commonly found in the intestinal changes including fibrinous » Mouse inoculation. tract. effusions, haemorrhage or oedema. Some of these tests (e.g. » There are PCR tests for some The kidneys autolyse quickly after immunohistochemistry (IHC), C. perfringens toxin genes, but death in ruminants, and a diagnosis fluorescent antibody testing (FAT) and the potency and effects of alpha of enterotoxaemia cannot be based mouse inoculation) are not currently toxin and enterotoxin in domestic solely on ‘pulpy’ kidneys. available in New Zealand. animals are controversial and » The only diagnostic histological Some toxin or IHC tests could be probably limited. lesions of enterotoxaemia are found referred overseas, but these would be » Overall, the gross and histological in the brain, where there can be expensive and have a slow turnaround findings combined with PCR may be regions of perivascular oedema or time. Considering all of this, the suggestive of the diagnosis. malacia after vascular permeability following is an attempt to list how the changes caused by epsilon toxin. most commonly suspected Clostridia This is worse in certain sites, and might be ruled in or out in the New CLOSTRIDIUM so it is best if the entire brain is Zealand context. PERFRINGENS TYPE C submitted for histopathology. This strain is characterised by alpha » Goats may have gross colonic ulcers, and beta toxins. It is found less with histological enterocolitis and CLOSTRIDIUM often than Type A in the intestinal sometimes necrosis with Gram- PERFRINGENS TYPE A tract of healthy animals, and causes positive bacilli. This strain is mainly characterised necrohaemorrhagic enteritis in mainly » The previous comments regarding C. by production of alpha toxin, neonatal calves, lambs, foals and pigs perfringens culture apply here. although it sometimes also produces due to the effects of beta toxin in the » PCR for the epsilon toxin gene is not beta2, enterotoxin and a range of absence of trypsin digestion. currently available in New Zealand. other toxins. It is considered to » Gross lesions will include areas of » Glycosuria can be seen in cause necrotic enteritis in poultry, red/black small intestine. other conditions and is not and has also been associated with » Histologically, there is mucosal pathognomonic. gastroenteritis in a range of other to transmural necrosis with » However, classical histological species – although, absolute proof of thrombosis, haemorrhage or fibrin findings are diagnostic. VetScript April 2019 – 51 IN THE LAB CLOSTRIDIUM DIFFICILE This produces two toxins (A and B) and causes enteritis and/or colitis in horses, rabbits, piglets and a range of other species, often after antibiotic treatment Bacteria Diseasea Speciesb Definitive diagnosis or hospitalisation. requirements » Gross lesions will include areas Clostridium perfringens Haemorrhagic enteritis Dogs Clinical signs of congested and haemorrhagic Type A Histology intestine. Toxin (NetF) » Histologically, there is mucosal Enteritis/enterocolitis Pigs Clinical signs Horses Histology necrosis with thrombosis, Clostridium perfringens Lamb Dysentery Sheep Histology haemorrhage, fibrin and neutrophils Type B Toxin (CPB and ETX) in the lumen, along with short Clostridium perfringens Neonatal Necrotising Horses Histology Gram-positive bacilli. Type C Enteritis Pigs Toxin (CPB) Lambs » C. difficile can be cultured by routine Calves methods, but a positive culture Clostridium perfringens Pulpy Kidney Disease Sheep Histology (brain) is not diagnostic given that non- Type D Goats pathogenic strains can be found in Cattle Histology (brain) the intestinal tract. Clostridium perfringens Necrotic enteritis Poultry Histology » Testing for C. difficile toxins is Type G Toxin (NetB) available by referral to a medical Clostridium difficile Enterocolitis Horses Histology laboratory, but these tests are not Pigs Toxin (A + B) validated in animals. Rabbits Rodents » The gross and histological findings Clostridium novyi Type B Blacks disease Ruminants Postmortem/Histology may be suggestive of the diagnosis. Horses Culture/PCR » Positive PCR for the toxin genes Clostridium novyi Bacillary Ruminants Postmortem/Histology of C. difficile is consistent with the Type D (Clostridium haemoglobinuria Culture/PCR diagnosis. haemolyticum) Clostridium piliforme Tyzzers disease Rabbits Histology or PCR Horses Cats CLOSTRIDIUM CHAUVOEI Rodents (BLACKLEG) Ruminants Clostridium chauvoei Blackleg Cattle Postmortem/Histology This is a disease of ruminants, mainly Malignant oedema Sheep Culture/PCR associated with lameness leading to Clostridium septicum Malignant oedema Ruminants Postmortem/Histology sudden death, although it can also be Horses Culture/PCR involved in malignant oedema. The Clostridium sordellii Malignant oedema Ruminants Postmortem/Histology bacterium cause lesions of necrosis Foal omphalophlebitis Horses Culture/PCR Postparturient Sheep within cardiac and skeletal muscles; metritis these are classically ascribed to trauma Clostridium tetani Tetanus Ruminants Clinical history creating anaerobic conditions favourable Horses Gram stains of wounds Pigs to the growth of resident spores. Dogs » Grossly, there will be haemorrhagic, Clostridium botulinum Botulism Avian Clinical history crepitant and oedematous areas with Horses a ‘rancid butter’ odour in muscle Ruminants bellies. These will be easier to find Clostridium spiroforme Enteric disease Rabbits Histology Culture in ‘fresh’ animals, may be small, and so may require extensive dissection to locate. a Clinical diseases in bold font are confirmed in Veterinary Species (Fulfil Koch's postulates). b Species in bold font are most commonly affected by this disease » Histopathology of affected areas shows haemorrhage, oedema, necrosis, inflammation and scattered Gram-positive bacilli. This can 52 – VetScript April 2019 IN THE LAB be difficult to distinguish from » Histologically, bacteria are found at also reports of C. botulinum colonisation putrefaction if animals are not fresh. the margin of the necrotic focus. of the intestinal tract or anaerobic » The gross and histological findings » Histology and culture of C. novyi wounds (toxicoinfectious botulism). may be suggestive of