Deprez, Piet

Ducatelle, Richard

Goossens, Evy

Haesebrouck, Freddy

Pardon, Bart

Timbermont, Leen

Valgaeren, Bonnie

Van Immerseel, Filip

Verherstraeten, Stefanie

English

Ghent University Academic Bibliography

Downloaded from www.microbiologyresearch.org byIP:  157.193.240.1On: Thu, 08 Oct 2015 10:19:29Haemorrhagic enteritis in newborn calvesassociated with Clostridium perfringens andcolostrum deliveryBonnie R. Valgaeren,1 Bart Pardon,1 Stefanie Verherstraeten,2Evy Goossens,2 Leen Timbermont,2 Freddy Haesebrouck,2Richard Ducatelle,2 Filip Van Immerseel2 and Piet Deprez1CorrespondenceBonnie R. ValgaerenBonnie.Valgaeren@Ugent.Be1Department of Large Animal Internal Medicine, Faculty of Veterinary Medicine, Ghent University,Salisburylaan 133, B-9820 Merelbeke, Belgium2Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine,Ghent University, Salisburylaan 133, B-9820 Merelbeke, BelgiumIntroduction: This report describes 13 cases of fatal haemorrhagic enteritis in newborn calves.Case presentation: Clostridium perfringens type A was isolated both from the intestinalcontents of seven calves and from three visibly worn and soiled bucket teats and stomachtubes used for colostrum administration. After replacement of the colostrum delivery device andintroduction of hygienic measures, no new cases appeared on any of the farms.Conclusion: The results suggested that contamination of colostrum with C. perfringens causedthese cases of neonatal haemorrhagic enteritis.Keywords: Clostridium perfringens; colostrum delivery; haemorrhagic enteritis; hygiene.Received 3 December 2014Accepted 1 August 2015Clostridium perfringens is a spore-forming bacterium, andis divided into five types (A–E), based on the majortoxins produced (Songer, 1996). Type A strains are predo-minantly isolated from calves with clostridial problems,such as haemorrhagic abomasitis in newborn calves andhaemorrhagic enteritis in older calves (Roeder et al.,1988; Songer & Miskimins, 2005; Miyashiro et al., 2009;Valgaeren et al., 2013b). In these older calves, recent exper-imental studies have suggested that a-toxin and perfringo-lysin are the primary toxins in the development ofhaemorrhagic intestinal lesions (Verherstraeten et al., 2013).AlthoughC. perfringens is an anaerobic bacterium, spores sur-vive in the presence of oxygen, explaining its ubiquitous pre-sence in the environment and in faeces (Songer, 1996, 2006;Van Kruiningen et al., 2009). Only one author has suggesteda link between clostridial abomasal disease and insufficienthygiene of colostrum and feeding utensils; however, the pre-sence of C. perfringens strains on these potential carriers hasnot yet been provided (Songer, 2006). This case reportdescribes colostral contamination by the presence ofC. perfringens in colostrum administration utensils.Case reportOver 4 years, 13 cases of haemorrhagic enteritis in new-born calves were observed on six farms (Table 1). Onecase only occurred on a dairy farm in a Holstein Friesian(HF) calf, while multiple cases occurred within a 3-weekperiod on four of the five Belgian Blue (BB) farms. Clinicalsigns started 4–24 h after birth and consisted of anorexia,colic, shock, severe acidosis, apathy, pale mucosae, swollenabdomen, and melena or absence of faeces. Some calveswere hypothermic, while others had fever (range 35.4–39.8 uC). All calves died within 3–30 h after onset ofsigns, despite treatment with penicillin in several cases.A glutaraldehyde coagulation test (Beetson et al., 1985)was performed on sera of six calves from four differentfarms and revealed insufficient immunoglobulin levels(v6 g l21) in all of them. Necropsy, performed on 10 ofthe 13 calves, revealed small intestinal haemorrhagic enter-itis. In four cases, abomasitis was also present, and threeother cases had haemorrhagic typhlocolitis. Histology con-firmed necrohaemorrhagic enteritis, similar to the lesionsseen in enterotoxaemia cases in older calves (Valgaerenet al., 2013a) (Fig. 1). In one calf, histology revealed gasbubbles in the submucosa and lamina propria of themucosa, as well as thrombi in the blood vessels of the sub-mucosa. In seven calves, intestinal content was sampledbetween 10 min and 24 h after death. An overgrowth ofC. perfringens (w108 c.f.u. g21) was consistently presenton Columbia agar (Oxoid) supplemented with 5 % defibri-nated sheep blood. On four farms, all calves had received2–4 l colostrum by stomach tube immediately after birth.In the other farms, calves were bucket-fed 2 l fresh colos-trum from their dam immediately after birth. None ofAbbreviations: BB, Belgian Blue; HF, Holstein Friesian.JMM Case Reports (2015) DOI 10.1099/jmmcr.0.000088G 2015 The Authors. Published by Society for General MicrobiologyThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/). 1Downloaded from www.microbiologyresearch.org byIP:  157.193.240.1On: Thu, 08 Oct 2015 10:19:29the farmers disinfected the tube or bucket teats betweencalves. On several farms, teats and stomach tubes were vis-ibly contaminated with milk residues. On farms 2, 3 and 6,swabs were taken from the stomach tubes and on farm 4,swabs were taken from the bucket teat. C. perfringens wasisolated from the feeding utensils on three out of thefour farms. Forty-three strains from the abomasal andintestinal contents of seven calves on six farms and 12strains from the tubes and teats were typed by multiplexPCR (Gholamiandekhordi et al., 2006) and were identifiedas type A C. perfringens. Only two strains from the intesti-nal content of one calf were positive for the b2-toxin gene.Randomly amplified polymorphic DNA analysis (Leflon-Guibout et al., 1997) showed that the investigated isolateswere all different strains. Apart from the tubes and teats,two colostrum samples were tested from farm 4, ofwhich one was negative and the other was positive forC. perfringens. Herdsmen were advised to replace thestomach tubes and bucket teats, to clean and disinfectwith 3 % hydrogen peroxide between calves, and toavoid contamination of colostrum during collection andstorage (in a refrigerator). No new cases occurred on anyof the six farms after the implementation of theseprecautions.DiscussionThis report demonstrated that C. perfringens strains can bedetected in colostrum and utensils used for colostrumdelivery. Ingested clostridial spores can become vegetativein the abomasum and intestine. The high abomasal pHand the lack of a competitive microbiota can lead to clos-tridial overgrowth and toxin production (Songer, 2006;Luka´s et al., 2007). Colostrum has a trypsin-inhibitoryactivity, which can protect both a-toxin and perfringolysinTable 1. Cases of haemorrhagic enteritis in neonatal calvesCase Farm TimeperiodBreed Means ofadministrationof colostrumNecropsyresultsCulture ofteatCulture ofintestinalcontent1 1 October 2011 BB Stomach tube – – –2 1 October 2011 BB Stomach tube Haemorrhagic enteritis – –3 1 October 2011 BB Stomach tube Necrohaemorrhagic abomasitisand enteritis– One strain:type A, b224 1 November 2011 BB Stomach tube Haemorrhagic enterotyphlocolitis – One strain:type A, b225 2 July 2012 BB Stomach tube Haemorrhagic enteritis – –6 2 July 2012 BB Stomach tube Necrohaemorrhagic enteritis Negative Four strains:type A, b227 3 May 2013 HF Stomach tube Necrohaemorrhagic abomasitisand enteritisFive strains:type A, b22Five strains:type A, b228 4 August 2013 BB Bucket – – –9 4 August 2013 BB Bucket – – –10 4 August 2013 BB Bucket Haemorrhagic enteritis Five strains:type A, b22Five strains:type A, b2211 5 January 2014 BB Bucket Segmental haemorrhagic enteritis – –12 5 January 2014 BB Bucket Necrohaemorrhagic abomasitis,haemorrhagic ruminitis andhaemorrhagic enterotyphlocolitis– Five strains:type A, b2-13 6 June 2014 BB Stomach tube Necrohaemorrhagic abomasitisand enteritisTwo strains:type A, b2220 strains: type A,b2-2 strains:type A, b2+Fig. 1. Small intestine of a case of haemorrhagic enterotyphloco-litis (case 4). Haematoxylin and eosin staining. In the luminal halfof the mucosa, there is necrosis, infiltration with neutrophils,hyperaemia and haemorrhage.B. R. Valgaeren and others2 JMM Case ReportsDownloaded from www.microbiologyresearch.org byIP:  157.193.240.1On: Thu, 08 Oct 2015 10:19:29against enzymic degradation, possibly facilitating thedevelopment of haemorrhagic intestinal lesions (Quigleyet al., 1995; Uzal et al., 2010; Diab et al., 2012). From the43 strains isolated from the abomasal and intestinal con-tent of the calves, only two were positive for atypical b2-toxin, and none was positive for consensus b2-toxin. Con-sensus b2-toxin has been suggested as to make an import-ant contribution to haemorrhagic enteritis in older calves(Lebrun et al., 2007). However, recent studies have shownthat any C. perfringens strain can induce necrohaemorrhagiclesions in calves and that a-toxin and perfringolysin areprobably the predominant virulence factors (Valgaerenet al., 2013a; Verherstraeten et al., 2013). Thus, similar tothe situation in older calves, b2-toxin does not seem to beof importance in neonatal haemorrhagic enteritis. Thereare comparable numbers of BB and HF calves in Belgium.However, only one case was seen in a HF calf, in contrastto the 12 cases seen in BB calves. On the BB farms, multiplecases occurred in a short period of time, while the HF casewas solitary. This suggests that BB calves are predisposedto haemorrhagic enteritis, as has already been confirmedin the veal industry (Pardon et al., 2012). The reasons forthis predilection are not clear, and might be genetic, man-agement related, feed related or some combination ofthese. The IgG content of the delivered colostrum was notknown. Normally in BB calves, 2 l colostrum from theirdams should be sufficient to provide 200 g immunoglobu-lins, but the five examined calves were hypogammaglobuli-naemic (Hoflack et al., 2004). It is possible that the diseasedescribed here decreased absorption. The problem wascompletely resolved after replacement and systematic clean-ing and disinfection (e.g. using hydrogen peroxide) of thetubes and teats used for colostrum feeding (Venczel et al.,1997). The present observations suggest that the uptake ofC. perfringens from the use of contaminated teats or tubesis a cause of abomasitis and haemorrhagic enteritis. Thestrains isolated from the feeding utensils were not identicalto those isolated from the intestines of diseased calves. Therequired number of strains that should be examined inorder to be 95 % confident that all strains present havebeen covered is not known. Possibly, and probably, toofew strains were tested. Further work is necessary to elabor-ate the means of transfer of clostridial strains to neonates.In conclusion, the present report demonstrated C. perfringensin colostrum and utensils used for colostrum delivery,suggesting a possible source of infection for neonatal haemor-rhagic enteritis. More work on a larger number of strains iso-lated from the intestines of cases and from colostrum deliverydevices is necessary to definitively confirm the current obser-vations. Experimental infection of newborn calves throughadministration of C. perfringens-contaminated colostrum isnecessary to definitively confirm this route of infection.Based on the observations in these cases, to avoid haemorrha-gic enteritis in newborns, we suggest that attention should bepaid to hygienic administration of colostrum and proper dis-infection of colostrum tubes and teats.AcknowledgementsThe authors acknowledge the help of the supporting staff from thelaboratories of pathology, bacteriology and internal medicine, aswell as the role of the farmers and field veterinarians in providingus with the calves, used teats and tubes, and case information.ReferencesBeetson, S. A., Hilbert, B. J. & Mills, J. N. (1985). The use of theglutaraldehyde coagulation test for detection of hypo-gammaglobulinaemia in neonatal foals. Aust Vet J 62, 279–281.Diab, S. S., Kinde, H., Moore, J., Shahriar, M. F., Odani, J., Anthenill,L., Songer, G. & Uzal, F. A. (2012). Pathology of Clostridiumperfringens type C enterotoxemia in horses. Vet Pathol 49, 255–263.Gholamiandekhordi, A. R., Ducatelle, R., Heyndrickx, M.,Haesebrouck, F. & Van Immerseel, F. (2006). Molecular andphenotypical characterization of Clostridium perfringens isolates frompoultry flocks with different disease status. Vet Microbiol 113, 143–152.Hoflack, G., Laureyns, J., Dewulf, J., Opsomer, G. & de Kruif, A.(2004). Colostrum quality and quantity in Belgian Blue cows andthe subsequent maternal immunity. In Poster Abstracts of the 23rdWorld Buiatrics Congress, Quebec City, Canada.Lebrun, M., File´e, P., Mousset, B., Desmecht, D., Galleni, M., Mainil,J. G. & Linden, A. (2007). The expression of Clostridium perfringensconsensus (2 toxin is associated with bovine enterotoxaemiasyndrome. Vet Microbiol 120, 151–157.Leflon-Guibout, V., Pons, J. L., Heym, B. & Nicolas-Chanoine, M. H.(1997). Typing of Clostridium perfringens strains by use of randomamplified polymorphic DNA (RAPD) system in comparison withzymotyping. Anaerobe 3, 245–250.Luka´s, F., Koppova´, I., Kudrna, V. & Kopecny´, J. (2007). Postnataldevelopment of bacterial population in the gastrointestinal tract ofcalves. Folia Microbiol (Praha) 52, 99–104.Miyashiro, S., Baldassi, L. & Nassar, A. F. C. (2009). Genotyping ofClostridium perfringens associated with sudden death in cattle.J Venom Anim Toxins Incl Trop Dis 15, 491–497.Pardon, B., De Bleecker, K., Hostens, M., Callens, J., Dewulf, J. &Deprez, P. (2012). Longitudinal study on morbidity and mortalityin white veal calves in Belgium. BMC Vet Res 8, 26.Quigley, J. D. III, Martin, K. R. & Dowlen, H. H. (1995). Concentrationsof trypsin inhibitor and immunoglobulins in colostrum of Jerseycows. J Dairy Sci 78, 1573–1577.Roeder, B. L., Chengappa, M. M., Nagaraja, T. G., Avery, T. B. &Kennedy, G. A. (1988). Experimental induction of abdominaltympany, abomasitis, and abomasal ulceration by intraruminalinoculation of Clostridium perfringens type A in neonatal calves. AmJ Vet Res 49, 201–207.Songer, J. G. (1996). Clostridial enteric diseases of domestic animals.Clin Microbiol Rev 9, 216–234.Songer, J. G. (2006). Bovine enteritis and enterotoxaemia.In Clostridia in Medical, Veterinary and Food Microbiology,Diagnosis and Typing, pp. 51–57. Edited by J. Mainil, C. Duchesnes,P. E. Granum, M. G. Menozzi, M. Peck, S. Pelkonen, M. Popoff,E. Stackebrandt & R. Titball. Brussels, Belgium: EuropeanCommision, Directorate-General for Research, DirectorateE- Biotechnology, Agriculture and Food.Songer, J. G. & Miskimins, D. W. (2005). Clostridial abomasitis incalves: case report and review of the literature. Anaerobe 11,290–294.Haemorrhagic enteritis associated with C. perfringenshttp://jmmcr.sgmjournals.org 3Downloaded from www.microbiologyresearch.org byIP:  157.193.240.1On: Thu, 08 Oct 2015 10:19:29Uzal, F. A., Vidal, J. E., McClane, B. A. & Gurjar, A. A. (2010).Clostridium perfringens toxins involved in mammalian veterinarydiseases. Open Toxinology J 2, 24–42.Valgaeren, B., Pardon, B., Goossens, E., Verherstraeten, S.,Schauvliege, S., Timbermont, L., Ducatelle, R., Deprez, P. & VanImmerseel, F. (2013a). Lesion development in a new intestinalloop model indicates the involvement of a shared Clostridiumperfringens virulence factor in haemorrhagic enteritis in calves.J Comp Pathol 149, 103–112.Valgaeren, B. R., Pardon, B., Verherstraeten, S., Goossens, E.,Timbermont, L., Haesebrouck, F., Ducatelle, R., Deprez, P. R. & VanImmerseel, F. (2013b). Intestinal clostridial counts have no diagnosticvalue in the diagnosis of enterotoxaemia in veal calves. Vet Rec 172, 237.Van Kruiningen, H. J., Nyaoke, C. A., Sidor, I. F., Fabis, J. J., Hinckley,L. S. & Lindell, K. A. (2009). Clostridial abomasal disease inConnecticut dairy calves. Can Vet J 50, 857–860.Venczel, L. V., Arrowood, M., Hurd, M. & Sobsey, M. D.(1997). Inactivation of Cryptosporidium parvum oocysts andClostridium perfringens spores by a mixed-oxidant disinfectantand by free chlorine. Appl Environ Microbiol 63, 1598–1601.Verherstraeten, S., Goossens, E., Valgaeren, B., Pardon, B.,Timbermont, L., Vermeulen, K., Schauvliege, S., Haesebrouck, F.,Ducatelle, R. & other authors (2013). The synergisticnecrohemorrhagic action of Clostridium perfringens perfringolysin andalpha toxin in the bovine intestine and against bovine endothelialcells. Vet Res 44, 45.B. R. Valgaeren and others4 JMM Case Reports

Haemorrhagic enteritis in newborn calves associated with Clostridium perfringens and colostrum delivery

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Haemorrhagic enteritis in newborn calves associated with Clostridium perfringens and colostrum delivery

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