Haemorrhagic bowel syndrom naif by Naif Aljabry

HAEMORRHAGIC BOWEL SYNDROME IN DAIRY CATTLE IN SAUDI ARABIA NAIF AHMAD AL-GABRI ABSTRACT An outbreak of a fatal disease of dairy cows was reported in a dairy farm in AlAhsa province. Clinically, the disease was characterized by sudden death of apparently healthy animals and proceeded with colic or abdominal pain, later dark clotted blood in the feces (melena). The disease outbreaks coincided with the presentation of a new total mixed ration to animals. Mortality rate was about 80%.The main post-mortem findings include 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, and submucosal oedema alongside the small intestine was evident. The bacterial culture of the intestinal ingesta and lesions showed the presence of a large number of Clostridium perfringens. No other potential microorganisms or toxic agents has been identified. INTRODUCTION Hemorrhagic bowel syndrome (HBS) is a devastating disorder affecting high producing dairy cattle. As an important emerging disease, HBS is now estimated to account for > 2% of the deaths of dairy cattle (Kirkpatrick et al, 2001). It is now referred to by a variety of names including HBS, jejunal hemorrhagic syndrome (JHS), acute hemorrhagic enteritis of the small intestine, and dead gut. Maximal milk production is a product of carbohydrate consumption and dry matter intake, both of which could be considered as possible risk factors for HBS (Kirkpatrick et al. 2001). 1

Feeding total mixed ration has been suggested as a risk factor for HBS. The Minnesota survey (Godden et al., 2001) found that 83% of affected herds were fed a total mixed ration. The identification of total mixed ration feeding as a risk factor is supported by the fact that only 38% of the herds in Minnesota at the time of the survey were fed total mixed ration. Several investigators have thought that Clostridium perfringens type A may be a cause of HBS (Dennison et al., 2002 and Godden et al., 2001). More recently, the haemorrhagic condition seen in HBS cows is similar to enteric hemorrhagic diseases caused by A. fumigatus in immuno-suppressed in humans (Forsberg, 2003). HBS is characterized by acute signs of profound depression, decreased milk production, tachycardia, ruminal stasis, abdominal distension, and dark clotted blood in the feces (Tajik et al., 2010). At necropsy, segmental lesions localized to the jejunum are observed (Peek 2005). These areas consist of frank hemorrhage with immediate clotting, forming a functional occlusion of the lumen of the small intestine. There is limited published information describing HBS in dairy cattle and little is known about the etiology, history, physical examination and the role of predisposing factors. The purpose of this paper is to present a description of JHS cases presented to College of veterinary Medicine and Animal Resources King Faisal University.

MATERIALS AND METHODS Animals: A dairy farm contains 500 dairy cows in Al-Ahsa province (Eastern region).

The

age of cows ranged from 3-5 years. The dairy cows were fed a new total mixed ration. There is an extremely high case fatality rate about 80% of affected animals dying within 24 to 36 hours of the onset of clinical signs. Feedstuffs analysis for aflatoxin: The total mixed ration was analyzed for total aflatoxin using a slightly modified immunoaffinity method based on Association of Official Analytic Chemists (AOAC) method (Trucksess et al.1991). Briefly, the whole sample was ground and a 100 g sample was removed for analysis. Methanol:water (80:20) solvent (100 ml) and 5 g NaCl were added to each sample and the mixture was blended at high speed for 3 min. The mixture was then filtered through a fluted filter paper (Whatman 2V, Whatman plc, Middlesex, UK), and the filtrate was diluted (1:4) with water and refiltered through a glass–fiber microfilter paper. Two milliliters of the glass–fiber filtrate was placed on Afla Test immunoaffinity column (VICAM, Water town, MA, USA) and allowed to elute at 1–2 drops/s. The columns were washed two times with 5 ml water, and aflatoxin was eluted from the column with 1 ml high performance liquid chromatography (HPLC)-grade methanol. A bromine developr (1 ml) was added to the methanol extract, and the total aflatoxin concentration was read in a recalibrated VICAM Series-4 fluorometer set at 360 nm excitation and 450 nm emissions Blood samples: Blood was collected from a jugular vein by syringe and transferred into evacuated containers. EDTA was the anticoagulant used for blood for haematological examinations. Most haematological examinations were made using a coulter 3

counter electronic analyzer. These included total white cell count, red cell count, haematocrit, haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) (Vet scan 5 HM-ABaxis-USA).` Serum samples: Biochemical assays were performed on serum from blood collected into plain containers and centrifuged. This samples was stored at-20 until analysis. Determinations made were concentration of total protein, albumin, glucose, blood urea nitrogen (BUN), creatinine, calcium, phosphorous, cholesterol , bilirubin and uric acid and the activities of aspartate aminotransferase (AST), alanine aminotransferase

(ALT),

gamma

glutamyltransferase

(GGT),

lactate

dehydrogenase (LDH), creatine kinase (CK) and alkaline phosphatase (AP).These parameter was tested by coulter ELLEPC (Italy 2003). Post-mortem examination and histopathology: Four cadavers were available to do a post-mortem examination. Impression smears prepared by scraping intestinal mucosa and cut 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 Haematoxyline and Eosin (H&E). The selected sections were stained by Gram’s and Gomori methylamine silver (GMS) stains to detect bacterial and fungal organisms respectively. Bacteriologic culture: Intestinal contents samples were cultivated in Cooked Meat Broth (CMB) and incubated anaerobically at 37°C for 48 hours. From these cultures, 0.1mL loop 4

aliquot was streaked on 5% sheep blood agar and incubated in anaerobic conditions at 37°C for 24 hours. Colonies showing related characteristics of C. perfringens form, aspect, color, and hemolysis were submitted to Gram stain; colonies corresponding to Gram-positive bacilli were cultivated in CMB in the same above conditions. After the incubation period, cultures were submitted to additional tests including catalase, lecithinase and gelatinase production, and glucose, lactose, and skimmed milk fermentation used to species identification. Interpretation was performed according to Cowan (1974). Strains identified as C. perfringens were then sub cultivated in Triptose Yeast Extract Broth (TYB) and further incubated in anaerobic conditions. Cultures were then centrifuged at 7.500 rpm for 15 minutes at 4°C and cell-free culture supernatants were recovered to be used for toxin detection and identification. Mycotic culture: At necropsy swabs taken from intestinal mucosa were used to inoculate Sabouraud's glucose agar plates containing chloramphenicol and actidione, and were incubated at 37°C for 24 h and then at 25°C for 3 days. Characteristics of fungal growth were noted from the plates, mounted in cotton blue-lactophenol and examined microscopically (Scott 1990). Toxin detection: White mice (25 - 40 g) were injected via intraperitoneal route with

0.3 ml of

broth culture supernatant and intestinal contents and then observed over a period of three days for either death or disease signs. After that, Intestinal contents and broth cultures supernatant were determined by mouse neutralization assay and an indirect ELISA commercial kit (Bio-X Diagnostics, Belgium) according to the manufacturer’s instructions. 5

RESULTS Clinical signs: Affected cows are often found dead (sudden death) within 24 to 36 hours of the onset of clinical signs. Alternatively, they found recumbent and semiconscious, or still standing. Also, there was anorexia, a severe drop in milk production, signs of colic and dark clotted blood in the feces (melena) (fig.3a). Bacteriologic and Mycotic growth: C. perfringens was isolated from the intestinal contents of cows. All strains were identified as C. perfringens based on colonial morphology , haemolysis on blood agar , Gram stain and biochemical characterization including production of catalase, lecithinase and gelatinase, and fermentation of glucose, lactose and tumultuous fermentation of skim milk. No evidence for mycotic growth in all intestinal samples. Toxin detection: Toxin was detected in intestinal contents and broth culture supernatants, where all mice were die within 72 hours after injection.Toxin was typed by using mouse neutralization assay and indirect ELISA assay. The results revealed that intestinal contents and broth culture supernatants have C. perfringens Alpha toxin. Beta and epsilon toxins were not detected in all examined samples. Hence, it would be properly C. perfringens type A. Clinical Pathology and chemistry findings: The most constant haematological finding in most cases was an often profound neutrophilia. No other noticeable changes in blood or serum samples.

Necropsy findings: The abomasum of most cases revealed multiple ulcers alongside the abomasal folds (fig.3b).The small intestine may contain either blackish blood or a large solid blood clot that obstructs the lumen (fig.3c) as well as the mucosal surface of intestine appeared black in colour (fig.3d). This was associated with congested and haemorrhagic mesenteric lymph nodes. Microscopic lesion: The abomasal mucosa was denuded in more than one part and appeared as depressed area (fig.4a). The villi of jejnum and ileum appeared necrotic and moderately infiltrated by inflammatory cells (fig.4b). The submucosa of ileum revealed a cluster of glands herniate into peyer's patches which called colitis cystica profunda ((fig.4c). The salient lesion in the small intestine is the presence of a characteristic submucosal oedema (fig.4d).

DISCUSSION

Haemorrhagic bowel syndrome (HBS), a deadly digestive tract disease, has been reported with increasing frequency in adult dairy cows. Results of the present study confirm the previous reports that HBS is acute intestinal disorder of adult cattle characterized by intraluminal haemorrhage and obstruction of small intestine. Although various names have been coined for this syndrome, the term HBS was chosen for the present study as being the most descriptive. It was noteworthy in this study to find out colitis cystica profunda. This lesion may be a sequel to local damage to the muscularis mucosa or it may be represent herniation into the space left by an involuated peyer's patches (Jubb et al., 1993). 7

Neutrophilia and typical gross lesions at necropsy, and bacteriology as wells toxin detection supported the diagnosis (Ceci et al., 2006 and Muskens et al., 2007). A final diagnosis of type A infections should not be based solely on toxin detection and should be accompanied by pathological as well as microbiological findings (Francisco et al., 2008). Neutrophilia with an accompanying increase in segmented neutrophils count may be attributable to the release of inflammatory cytokines leading to subsequent release of neutrophils from the bone marrow. Alternatively, high neutrophils count may be attributable, at least in part, to endogenous steroid release associated to the stress of the disease (Carlson 1990 and Morris and large 1990). Colstridium perfringens type A is ubiquitous in the digestive tract of cattle and a hypothesis for the aetiology of C.perfringens overgrowth is the overflow of finely ground carbohydrates from forestomach (Ewoldt and Anderson 2005). This situation arises in association with the same factors which lead to subacute ruminal acidosis due to feeding excess amounts of rapidly fermented carbohydrates or insufficient effective fiber (Godden 2003). Another hypothesis for aetiology of HBS is poorly fermented ensiled feeds which may lead to accumulate the harmful mold or bacteria and their toxins. The occurrence of HBS with feeding poor quality silage has been reported by Kirkpatrick and Timms (2004). Although, there was not mycotic growth in this study. More recently, the mold Aspergillus fumigatus has been associated with HBS. A. fumigatus is also ubiquitous in the digestive tract of cattle. Some authors believe that rumen acidosis, metabolic disease and stress or an immune suppression, creates an environment for A. fumigatus to gain entrance into the blood in high number (Simon et al., 2005). 8

In conclusion, the cause of the disease is not known yet, but C. perfringens type A and more recently Aspergillus fumigatus have been implicated in

the

disease syndrome. Nevertheless, the ubiquitous nature of both of these organisms makes its significance in HBS difficult to ascertain. There are suggested possible risk factors for HBS, include a high amount of fermentable carbohydrate in the diet, the level of dry matter intake, the level of milk production, feeding a total mixed ration, lactation number, herd size, season, and the presence of the causative organism in the feedstuff (Godden et al., 2001). However, more studies and research need to be done to illuminate the disease process.

Fig. (3) A- Dead cow. Note bloody stool (melena) (arrow). BAbomasum showing multiple ulcers (arrows). C- Small intestine showing distension with blackish blood (arrow). D. Ileum showing blackish mucosa (arrow).

Ileum showing black colour of mucosa (arrow). D-

Fig. (4) A- Abomasum. Note sloughed mucosa (arrow). B- Jejunum showing necrotic mucosa (arrow). C- Ileum showing colitis cystic profunda (arrows). D. Jejunum showing submucosal oedema (arrow).

ACKNOWLEDGMENTS

I would like to thank Prof. Dr. Mahmoud hamouda for helping in preparation and writing this article. Department of Pathology College of Veterinary Medicine and Animal Resources. 11

REFERENCES 1- Carlson, C.P. (1990): Clinical chemistry tests. In: Smith, B.P.,ed. Large animal internal medicine. Philadelphia: Cv Mosby Co; 386-418. 2- Ceci, L., Paradies, P.,Sasanelli,M., Caprarils,D.D.,Guarda,F.,Capucchio,M.T., and Carelli, G. (2006): Haemorrhagic bowel syndrome in dairy cattle: possible role of clostridium perfringens type A in the disease complex. J. Vet. Med., 53 (A):518523. 3- Cowan, S.T. (1974): Cown and Steel's manual for the identification of medical bacteria. 2nd ed. Great Britain: Cambridge University Press; p.238. 4- Dennison, A., D. VanMetre, R. Callan, P. Dinsmore, G. Mason, and R. Ellis. (2002): Hemorrhagic bowel syndrome in dairy cattle: 22 cases (1997-2000). J. Am. Vet. Med. Assoc. 331:686-689. 5- Ewoldt, J.M. and Anderson, D.E. (2005): Determination of the effect of single abomasal or jejunal inoculation of Clostridium perfringens type A in dairy cows. Can. Vet. J., 46:821-824. 6- Forsberg, N. (2003): New ﬁndings on jejunal hemorrhagic syndrome. Hoard’s Dairyman 148(8):311, April issue. 7- Francisco, A, Uzal and Glenn Songer, J. (2008): Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. J Vet Diagn Invest 20:253–265. 8- Godden, S., R. Frank, and T. Ames. (2001): Survey of Minnesota dairy veterinarians on the occurrence of and potential risk factors for jejunal hemorrhage syndrome in adult dairy cows. The Bovine Practitioner 35:97-103. 9- Gooden, S. (2003): Jejunal hemorrhage syndrome in adult dairy cattle. In: 6th western dairy management conference proceeding. Reno,Nevada. P: 179. 10-Jubb, K.V.F., Kennedy, P.C. and Palmer, N. (1993): Pathology of Domestic Animals, Fourth ed, Academic Press, San Diego. 12

11- Kirkpatrick, M.A., L.L. Timms, K.W. Kersting, and J.M. Kinyon. (2001): Case report: jejunal hemorrhage syndrome of dairy cattle. The Bovine Practitioner 35:104-116. 12- Kirkpatrick, M.and Timms, L.(2004): Jejunal hemorrhage syndrome of dairy cattle. 2004 mid-south ruminant nutrition conference proceeding. Arlington, Texas. PP: 47-59. 13- Morris, D.D. and Large S.M. (1990): Alterations in the leukogram. In: Smith, B.P.,ed. Large animal internal medicine. Philadelphia: Cv Mosby Co; 425-434. 14- Muskens, J; Veldhorst, GJ; Snoep, JJ and Vos, J. (2007): High mortality in

herd with signs of jejunal hemorrhage syndrome. Tijdschr.Diergeneeskd. 132: 116119.Nordlund, KV; Garrett, EF and Oetzel, GR. 15- Peek, SF. (2005): Jejunal Hemorrhage Syndrome. Preconvention Seminar 7: Dairy herd Problem Investigation Strategies. AABP 38th Annual Conference. 16-Scott, B.M (1990): Natwal Pohonr, AOAC. Washington DC, p 1191 Sheridan JJ (1981) Vet. Res. Commun 5:1 Oxford, p 301. 17- Simon, F.Peek. And Sheila, McGuirk. (2005): Jejunal Hemorrhage Syndrome. Preconvention Seminar 7: Dairy herd Problem Investigation Strategies. AABP 38th Annual Conference. September 19-21. 18-Tajik, J., G.R. Mohammadi, M. Radand A. Barati. (2010): Hemorrhagic bowel syndrome in dairy cattle in Iran: a case report. Iranian Journal of Veterinary Research, Shiraz University. 11: 180-183. 19-Trucksess, M.W., Stack, M.E., Nesheim, S., Page, S.W., Albert, R.H.,Hansen, T.J., et al., (1991): Immunoaffinity column coupled with solution fluorometry or liquid chromatography post-column derivatization for determination of aflatoxins in corn, peanuts, peanut butter: collaborative study. J Assoc. Off. Anal. Chem. 74 (1), 81. 13

‫متالزمة القولون النزفية في األبقار الحلوب في المملكة العربية السعودية‬ ‫الملخص العربي‬

‫تم تسجل تقرير عن تفشي مرض قاتل لألبقار الحلوب في مزرعة ألبان في محافظة األحساء‪.‬‬ ‫االعراض السريريه لهذا المرض تميزت بالموت المفاجئ للحيوانات السليمة ظاهريا مع مغص‬ ‫أو ألم في البطن‪ ،‬وفي مراحل متقدمة من المرض ظهرالبراز بة دم متخثر (‪ .)melena‬تزامن‬ ‫تفشي هذا المراض مع اعطاء اعالف جديده للحيوانات‪ .‬وكان معدل الوفيات حوالي ‪،٪08‬‬ ‫العالمات الرئيسية بعد الوفاة شملت نزيف هائل وتكوين تجلط داخل األمعاء الدقيقة وكذلك‬ ‫تقرحات المنفحه‪ .‬الفحص الميكروسكوبي أظهر انفصال الغشاء المخاطي للمنفحه وتنخر‬ ‫الزغابات المعوية وظهور وذمة تحت الغشاء المخاطي لألمعاء الدقيقة إلى جانب ذالك زرعت‬ ‫البكتيريا المعوية من محتوى االمعاء واالماكن المصابة وأكدت وجود عدد كبير من بكتيريا‬ ‫‪ .clostridium perfreinges‬لم يتم التعرف على كائنات حية دقيقة أخرى محتملة أو مواد‬ ‫سامة‪.‬‬

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