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Home , Clostridia, Clostridium chauvoei, Clostridium novyi, Clostridium sordellii, Clostridium tetani, Tetanus

IN THE LAB

CLOSTRIDIAL DISEASE IS frequently associated with sudden 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 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 identification depends on suspected . 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 »» perfringens Type A is the intestinal tract of healthy animals. Gram-positive . 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 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 . 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 . 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 testing (FAT) and the potency and effects of alpha of enterotoxaemia cannot be based mouse inoculation) are not currently toxin and 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 . 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 in horses, rabbits, piglets and a range of other species, often after treatment Diseasea Speciesb Definitive diagnosis or hospitalisation. requirements »» Gross lesions will include areas 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) »» C. difficile can be cultured by routine Lambs 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. »» Testing for C. difficile toxins is Clostridium perfringens Necrotic enteritis Poultry Histology 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 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) Tyzzers disease Rabbits Histology or PCR Horses Cats Rodents () Ruminants Clostridium chauvoei Blackleg Cattle Postmortem/Histology This is a disease of ruminants, mainly Malignant oedema Sheep Culture/PCR associated with lameness leading to Malignant oedema Ruminants Postmortem/Histology sudden death, although it can also be Horses Culture/PCR involved in malignant oedema. The 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 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, 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

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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 the diagnosis. from a necrotic focus are consistent »» There are no diagnostic gross or »» C. chauvoei culture is supportive of with the diagnosis. histological lesions in botulism. the diagnosis since it is not a post- »» The bacteria are rarely cultured from mortem contaminant. affected animals. CLOSTRIDIUM »» Most cases are diagnosed on clinical HAEMOLYTICUM (AKA C grounds and by ruling out other CLOSTRIDIUM SEPTICUM, NOVYI TYPE D) causes of death. C PERFRINGENS AND C This species causes intravascular SORDELLII AS PART OF haemolysis in cattle and sheep. Bacteria REFERENCES: MALIGNANT OEDEMA, OR latent in tissues sporulate within anaerobic Cooper BJ, Valentine BA. Chapter 3, Volume GAS liver foci (generally secondary to fluke 1. Muscle and Tendon; Uzal FA, Plattner BL, Hostetter JM. Chapter 1, Volume 2. Alimentary These bacteria cause lesions of oedema migration) and produce a beta toxin. System; Cullen JM, Stalker MJ. Chapter 2, Volume and necrosis within muscle and adjacent »» Grossly, there is a large area 2. Liver and Biliary System. In: Maxie MG (ed). Jubb, tissues of ruminants, horses and pigs, of necrosis found in the liver, Kennedy and Palmer’s Pathology of Domestic Animals, Sixth Edition. Elsevier, St. Louis, MO, USA, 2016 generally after trauma allowing bacteria to peritonitis, anaemia, red/brown be introduced into an anaerobic wound. C. kidneys and ‘port-wine’ urine. Freedman JC, Theoret JR, Wisniewski JA, Uzal FA, Rood JI, McClane BA. Clostridium perfringens septicum also causes abomasitis (braxy). »» Histologically, the liver is as C. type A-E toxin . Research in Microbiology 166 »» Grossly, there will be haemorrhage, novyi, and there is haemoglobinuric (4), 266-279, 2015 oedema and emphysema; this will be nephrosis. Garcia JP, Anderson M, Blanchard P, Mete A, Uzal easier to find in fresh animals. »» Histology and culture of C. FA. The pathology of enterotoxaemia by Clostridium »» Histopathology of affected areas haemolyticum from a necrotic focus perfringens type C in calves. Journal of Veterinary Diagnostic Investigation 25 (3), 438-442, 2013 shows the same changes along with are consistent with the diagnosis. Jones AL, Dagleish MP, Caldow GL. Clostridium necrosis, scant inflammation and perfringens type-D enterotoxaemia in cattle: the scattered Gram-positive bacilli. This diagnostic significance of intestinal epsilon toxin. can be difficult to distinguish from CLOSTRIDIUM TETANI Veterinary Record 177 (15), 390, 2015 putrefaction if animals are not fresh, (TETANUS) Ortega J, Daft B, Assis RA, Kinde H, Anthenill but histology may be suggestive of Tetanus is mainly suspected in horses L, Odani J, Uzal FA. of internal umbilical remnant in foals by Clostridium sordellii. Veterinary the diagnosis. and dogs showing signs such as spastic Pathology 44 (3), 269-275, 2007 » » Bacterial culture from tissues of a paralysis and rigor leading to death. Shrestha A, Uzal FA, McClane BA. Enterotoxic fresh animal is also suggestive of the This is thought to occur after C. tetani Clostridia: Clostridium perfringens enteric diseases. diagnosis, but these species can also living and dying in an anaerobic wound Microbiology Spectrum 6 (5), 2018, doi: 10.1128/ microbiolspec.GPP3-0003-2017 represent post-mortem invasion, and release tetanus toxins. so careful interpretation of culture »» There are no diagnostic gross or Sindern N, Suchodolski JS, Leutenegger CM, Gohari IM, Prescott JF, Proksch A, Mueller RS, results is required. histological lesions in tetanus. Busch K, Unterer S. Prevalence of Clostridium »» The bacteria could theoretically be perfringens netE and netF toxin genes in the faeces of cultured from a contaminated wound. dogs with acute haemorrhagic diarrhoea syndrome. Journal of Veterinary Internal Medicine 33, 100-105, CLOSTRIDIUM NOVYI »» However, most cases are diagnosed 2019 TYPE B on clinical grounds. Uzal FA, Navarro MA, Li J, Freedman JC, This species causes rapid death, mainly in Shrestha A, McClane BA. Comparative pathogenesis sheep. Bacteria latent in tissues sporulate of enteric clostridial in humans and animals. within anaerobic liver foci (generally CLOSTRIDIUM BOTULINUM Anaerobe 53, 11-20, 2018 secondary to fluke migration) and (BOTULISM) Uzal FA, Songer JG. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. produce their own alpha and beta toxins. In New Zealand, botulism is mainly Journal of Veterinary Diagnostic Investigation 20 (3), »» Grossly, there is marked suspected in waterfowl undergoing 253-265, 2008 subcutaneous congestion (‘black warm weather mass mortality and Uzal FA, Songer JG, Prescott JF, Popoff MR. disease’), oedema, cavitary effusions flaccid paralysis. The bacteria live and Clostridial Diseases of Animals. John Wiley & Sons, and haemorrhages, with a yellow die in anaerobic stagnant ponds, and Ames, Iowa USA, 2016 area of necrosis found in the waterfowl ingest their toxins. Other Zachary JF. Chapter 4, Section 1. Mechanisms of Microbial Infections. In: Zachary JF, McGavin MD liver. This may require thorough species may ingest toxins in forage (eds). Pathologic Basis of Veterinary Disease, Fifth dissection to locate. contaminated by carcasses. There are Edition. Elsevier, St. Louis, MO, USA, 2012.

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