Clinico-pathological studies of cattle naturally infected with Mycobacterium avium subspecies paratuberculosis in Khartoum State , Sudan

Four crossbred cows, 3-5 yearold, naturally infected with Mycobacterium avium subspecies paratuberculosis (MAP) were sacrificed and necropsied. Clinically, they showed profused diarrhoea, emaciation and rough coat with an area of alopecia on the tail. The most prominent macroscopic lesions were thickening, oedema and corrugation of the wall of small and large intestines. The mesenteric lymph nodes were enlarged and oedematous. Microscopically, all cows presented granulomatous enteritis. The inflammatory exudates varied from accumulation of lymphoid cells mixed with some epithelioid macrophages and giant cells to sheets of epithelioid macrophages intermingled with some lymphoid cells. The lymphatics in the submucosa of both the small and large intestines were dilatated and filled with pink homogenous proteinous materials. Acid-fast bacilli (AFB) were demonstrated in the infiltrating epithelioid macrophages and giant cells. Culture of inocula from the small and large intestines and the mesenteric lymph nodes of all animals showed small, round, smooth and glistening colonies 5-7 weeks following incubation in Herrold's Egg Yolk Medium at 37°C.


To cite this article :
Introduction lymphangitis and lymphadenitis associated with the presence of AFB in macrophages (Chiodini et Paratuberculosis is a chronic enteropathy of al., 1984;Kreeger, 1991).Culture of intestinal ruminants caused by Mycobacterium avium subsp.tissues is more sensitive than culture of faecal paratuberculosis (MAP).Organisms ingested by materials.Animals negative in successive faecal neonatal animals are taken up by the Peyer's cultures may become positive by culture from patches and infect intestinal macrophages.In intestinal tissues after slaughter (Whittington and most natural infections it is presumed that the Sergeant, 2001).The aims of this study were to host response eliminates the organisms, but some describe macrosopic and microscopic appearance of the animals remain infected.Adult cows of paratuberculosis in naturally clinical infected develop clinical disease following a long crossbred cows and to draw the correlation subclinical phase (Chiodini et al., 1984; Kreeger, between impression smear and bacterial culture 1991; Clarke, 1997).

the gross lesions of paratuberculosis were
Pathological methods : Necropsies were carried characterized by intestinal thickening and corrugation and the associated lymph nodes were out on four crossbred cows (No118, 119, 229 swollen and oedematous.The main microscopic from Soba area, and 198 from Hilat Kuku area) findings consist of granulomatous enteritis, that presented clinical signs of paratuberculosis.Specimens of the intestinal segments Approximately 0.1 ml of inoculum was transferred to each of two slants of Herrold's medium containing (duodenum, jejunum, ileum, caecum, colon and mycobactin and to one slant of Herrold's medium rectum), mesenteric lymph nodes, brain, heart, without mycobactin.The innoculum was layered liver, kidneys and spleen were fixed in 10% evenly over the surface of the slants.The tubes formalin and embedded in paraffin wax, were allowed to remain in a slant position at 37°C sectioned at 4-5 µm thick and stained with haemafor one week with the screw caps loose.The tubes toxylin and eosin (HαE) and Ziehl-Neelsen (ZN).
were returned to a vertical position when the free

Bacteriological methods
moisture evaporated from the slants.The screw Impression smears : Impression smears were caps were tightened and the tubes were placed prepared from brains, hearts, kidneys, livers, into baskets in an incubator at 37°C for 12 weeks spleens, mesenteric lymph nodes, large and small and observed weekly from the sixth week intestines of the four cows by impression of slides onwards for the presence of MAP (OIE, 2004).against a cut surface of each of the afore-

Results
mentioned and stained by Ziehl-Neelsen stain.
Clinical signs : Though all four cows had good Preparation of inoculum : Approximately 4 g of appetite, they were emaciated and had profused mucosa from the specimens of intestinal diarrhoea, rough coat with an area of alopecia on segments (duodenum, jejunum, ileum, caecum, the tail.Mandibular oedema was observed in colon and rectum) and mesenteric lymph nodes animal no 198.were placed into a sterile blender jar containing 50 ml of trypsin (2.5%).The mixture was Macroscopic findings : At necropsy, the animals adjusted to neutrality (PH 7) using 4% NaOH and were emaciated and showed serous atrophy of fat stirred for 30 min at room temperature on a magnetic deposits.The most prominent gross lesions were stirrer.The digested mixture was filtered through thickening, oedema and corrugation of the wall of a gauze sheet.The filtrate was centrifuged at the small and large intestines.Mild pathological 2000-3000 g for 30 min.The supernatant fluid changes were observed in the duodenum and was poured off and discarded.The sediment was rectum.These changes were more marked in the resuspended in 20 ml of 0.75% hexadecylpyrilower jejunum and ileum, particularly at the dinium chloride (HPC) and allowed to stand ileocaecal valve where the mucosa was folded undisturbed for 18 hours at room temperature.into transverse ridges that can not be reduced by The particles that settled to the bottom of the tube stretching.Furthermore, animal no 118 showed were used as the inoculum and were removed by a segmented lesions with thickened portions of the pipette without disturbing the supernatant fluid.
small intestine being alternating with normal intestine.In the thickened portions, the mucosa Inoculation of culture media and incubation :   was folded into thick transverse rugae similar to cellular reactions were observed between Brunner's the convolution of cerebral cortical gyrus (Fig. 1).glands and focal aggregates of epithelioid macrophages were encountered only at the tip of Lymph nodes : The mesenteric lymph nodes were villi of animal no 198.enlarged and oedematous especially in animals no Jejunum and ileum : The most prominent 198 and 229 (Fig. 2).No other gross lesions were changes were atrophic villi and desquamation of observed in the four emaciated cows, apart from the lining epithelium.The changes were more congestion of the brain.Ecchymotic haemorrhages marked in animal no 198.The mucosal reaction in the kidney was present only in animal no 198.
was accompanied with intense and diffuse Microscopic findings : Generally, all animals infiltration of mononuclear cells and few langhans presented granulomatous enteritis.Though, both giant cells in the mucosa and submucosa (Fig. 5). the small and large intestines showed pathological However, the epithelioid macrophages effusion changes, the severity of lesions varied considerably was prominent in animal no 118.In this animal from moderate reaction in the proximal parts of the mucosa and submucosa were heavily infiltrated the small intestine, duodenum, to an intense with nests or sheets of epithelioid macrophages severe reaction in the distal part of the small resulting in compression and/or obliteration of intestine, lower jejunum and ileum, and mild mucosal crypts which in some areas were effaced changes in the large intestine.
by the inflammatory cells (Fig. 3).In other parts The inflammatory exudates varied from the epithelioid macrophages form sheets either intermixed or encircled by mononuclear cell and accumulation of lymphoid cells mixed with some few langhans giant cells.In animals no 119, 198 epithelioid macrophages and giant cells (Fig. 3) to and 229 some mucosal glands or crypts were sheets or nests of epithelioid macrophages atrophic forming cyst-like structures filled with intermingled with some lymphoid and giant cells.
tissue debris.The submucosal reaction also varied The lymphatics in the submucosa of both the from heavy infiltration of lymphoid cells to small and large intestines were dilatated and filled moderate or focal areas of lymphoid cells whereas with pink homogenous proteinous materials.
in animal no 118 the predominant cellular reaction Small intestine in the submucosa was sheets of epithelioid Duodenum : It showed mild villous atrophy with macrophages.denudation of lining epithelium, moderate but Acid-fast bacilli were encountered in the diffuse infiltration of mononuclear cells, the jejunum and ileum forming clumps in the majority of which were lymphoid cells, concomitant infiltrating cells (Fig. 6).with some plasma and epithelioid macrophages in the mucosa, submucosa and between the Large intestine : All segments of the large compressed crypts of Lieberkuhn (Fig. 4).Mild intestine showed mild villous atrophy, mild to   moderate mononuclear reaction in the mucosa in Herrold's egg yolk medium (HEYM) revealed and submucosa.Focal collection of lymphoid colonies of MAP 5-7 weeks post incubation.cells were observed superficially or/and the base These colonies were small, colourless, translucent of the mucosa.However, animal no 118 showed and hemispherical.Their margins were round and sheets or nests of epithelioid macrophages in the even, whereas their surfaces were smooth and mucosa and submucosa of the large intestine glistening (table 2).particularly the colon.

Discussion
Lymph nodes : The mesenteric lymph nodes were The clinical signs and macro-and microscopic enlarged, oedematous and hyperplastic with findings observed in this study are similar to infiltration of mononuclear cells and sparse giant those described in MAP-infected cattle (Buergelt cells in subcapsular and medullary sinuses.The et al., 1978;Chiodini et al., 1984; Whitlock and infiltrating cells were mainly lymphoid, some Buergelt, 1996Radostits et al., 2000).plasma and epithelioid macrophages.In animals In the present study emaciation and rough no.118 and 229, the epithelioid macrophages coat associated with alopecia were observed in all were found in nests throughout the lymph nodes four crossbred cows.Mandibular oedema was (Fig. 7).The medullary sinuses and lymphatics only observed in animal no 198 which may be were filled with oedematous fluid.AFB were attributed to protein malabsorption and loss as a demonstrated in all sections and imprints of the result of necrosis and destruction of intestinal mesenteric lymph nodes of all four examined mucosa of small intestine, particularly the ileum cows (Fig. 8).
and jejunum where the ingesta are completely digested and absorption.This is similar to finding Impression smears : Brains, hearts, kidneys, of Stehman (1990) and Radostits et al. (2000).livers and spleens were negative AFB.The results The profused diarrhoea observed presented of mesenteric lymph nodes, large and small by all four crossbred cows is the cardinal signs of intestines are summarized in Table -1.paratuberculosis.The faeces were soft and thin, Bacterial culture of organs : Decontaminated like thick pea soup, homogeneous and inoffensive.innocula prepared from tissue of small and large The faeces were neither blood nor mucous tinged.intestines and mesenteric lymph nodes incorporated These findings were compatible with the    The mucosa and submucosa in animal no by chronic diarrhoea.The chronic nature of 118 were heavily infiltrated with nests or sheets paratuberculosis is usually sufficient to differentiate of epithelioid macrophages resulting in compression it from other common enteritis of cattle such as and/or obliteration of mucosal crypts.This is in salmonellosis, coccidiosis and gastrointestinal agreement with Radostits et al. (2000).helminthosis which are usually acute; the latter In the inflammatory exudation, in the four two disease occur principally in young animals animals is comprised of mononuclear cells and are distinguishable on faecal examination.mainly lymphoid cells concomitant with Secondary copper deficiency (chronic molybdenum granulation tissue.This indicate that the most poisoning) is likely to be confused with paratuimportant immuno mechanism operating in case berculosis in cattle, but is usually a problem of MAP-infected cows is cellular than humoral.affecting large number of animals that responds

Animal number
The mesenteric lymph nodes were enlarged, well to the supplementation of copper (Radostits oedematous and hyperplastic with infiltration of et al., 2000).mononuclear cells and sparse giant cells in the In the present study, the macro-and subcapsular and medullary sinuses.These cells microscpic findings are similar to those described were mainly lymphoid, some plasma and by others investigators (Jubb and Kennedy, 1970; epithelioid macrophages.The medullary sinuses Radostits et al., 2000).The severity of these and lymphatics were distended with oedematous changes in the lower small intestine and at fluid.This is in agreement with the findings of ileocaecal valve may be attributed to the fact that Jubb and Kennedy (1970) and Radostits et al. these are predilection sites and the Peyer's (2000) but it is in contrast with the findings of patches are well developed and play an important Payne and Rankin (1961) who found that giant role in mucosal immunity.cells occurred less frequently in the intestines Serous atrophy of fat deposits, congestion than in the associated lymph nodes.
Results presented in tables 1 and 2 indicate of the brain in the four crossbred cows and that there is poor correlation between impression ecchymotic haemorrhages in the kidneys that smear and bacterial culture of organs for the observed only in animal no 198.The first finding detection of MAP in duodenum, caecum, rectum was observed previously (Buergelt et al., 1978).and mesenteric lymph nodes but it is high for Congestion of brain and ecchymotic haemorrhages detection of MAP in jejunum, ileum and in the kidneys were not previously described in ileocaecal valve.This indicates that the jejunum, MAP-infected cattle.
ileum and ileocaecal valve are the predilection Similar to the results of the the current sites of choice for diagnosis of paratuberculosis investigation, the granulomatous inflammatory in cattle for various tests (impression smear, cells in the mucosa and submucosa of the small bacterial culture and microscopic apperance).It intestine and paracortex and sinuses of the is worth mentioning that, in view of the small mesenteric lymph nodes together with presence

Fig 1 :
Fig 1: Ileum of animal No 229.Note, the mucosa was folded into thick transverse rugae.

Fig 2 :
Fig 2: Note, swollen of mesenteric lymph node of animal No 198.

Fig 5 :
Fig 5: Jejunum of animal No 198.Note, intense and diffuse effusion of lymphoid cells and one langhans giant cell in the submucosa and mucosa.(H&EX10).

Fig 7 :
Fig 7: Lymph node of animal No 229.Note, sheet of epithelioid macrophages in the cortical area.H&EX40.

Fig 8 :
Fig 8: Imprint of lymph node of animal No 198.Note, clumps of acid-fast bacilli intra and extracellular.ZNX40.

Table 1 : Results of impression smears of large, small intestines and mesenteric lymph nodes from Crossbred cows in Khartoum State during 2006-2007.
Clinico-pathological studies of cattle naturally infected withMycobacterium avium subspecies paratuberculosis observations of other investigators (Buergelt et of intracellular AFB were reported by other al.,1978;Whitlock and Buergelt, 1996).Moreover, investigators (Buergelt et al., 1978; Pembertson, in adult cattle the clinical disease should be 1979).differentiated from other diseases characterized