Pathology of subclinical paratuberculosis in goats of Mahakausal region Jatav Maneesh2, Verma Yamini2,*, Dubey Amita2, Swamy Madhu2, Singh A.P.1,2 2Department of Veterinary Pathology, College of Veterinary Science and AH, Jabalpur, Nanaji Deshmukh Veterinary Science University, Jabalpur, Madhya Pradesh, India 1Center of Animal Biotechnology, College of Veterinary Science and AH, Jabalpur, Nanaji Deshmukh Veterinary Science University, Jabalpur, Madhya Pradesh, India *Corresponding author: dryaminiverma@rediffmail.com
Abstract The present study was carried out to determine the prevalence and the associated enteric pathology of subclinical paratuberculosis in goats of Mahakausal region. Tissue samples including intestine, mesenteric and ileocaecal lymph nodes were collected from 70 goats and examined for presence of acid-fast bacilli in the smears, MAP genome by IS 900 PCR and gross/histopathological changes. The postmortem prevalence of subclinical paratuberculosis was recorded in 7.14% (5/70) goats by Ziehl Neelsen staining of the intestine and lymph node smears and MAP genome was detected in intestine tissue of 4.28% (3/70) goats. Characteristics gross lesions of paratuberculosis were observed as mild to moderate thickening of intestinal wall and focal to generalized mucosal corrugation. Mesenteric lymph nodes were pale, oedematous and enlarged. The microscopic lesions in intestine and mesenteric lymph nodes were characterized by infiltration of macrophages, lymphocytes and epithelioid cells. Histopathologically lesions were classified as paucibacillary and multibacillary based on inflammatory cell infiltration and distribution of acid fast bacilli(AFB) laden epithelioid cells. Top Keywords Goat, Johne's disease paratuberculosis, Pathology, PCR. Top |
INTRODUCTION Paratuberculosis is one of the major economically important diseases of small ruminants andhas a worldwide distribution. It is a serious economic or public health concern1. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a slow growing acid fast bacterium confined to small intestine and draining lymph nodes. The bacteria invade M cells of follicle associated epithelium and subsequently engulfed by intestinal macrophages and they remain for several months to years (subclinical infection) before developing the clinical disease2,3. Clinical symptoms are not visible before the disease gets fully established in the body. The disease is characterized by granulomatous enteritis and associated lymphadenitis2. |
Paratuberculosis has been frequently reported from farm herds and sacrificed/slaughtered goats. Most of losses occurs due to subclinical stage of disease in the form of progressive weight loss, reduced milk yield, increased incidence of mastitis, altered milk constituents, increased somatic cell counts, poor feed conversion, reduced reproductive efficiency and fertility, premature culling, increased susceptibility to other diseases and eventually death4. The bacterium has also been implicated in Crohn‘s disease and is, therefore, now considered a public health concern. Till now, the disease is regarded as incurable5. |
In India, paratuberculosis is endemic in goats, sheep, cattle and buffalo population in almost every state of the country6. Incidences of paratuberculosis in slaughtered buffaloes of Malwa regions of Madhya Pradesh were reported by previous workers8,9. However, perusal of available published literature showed that no systemic study has been conducted in Mahakaushal region to identify the MAP infection from either subclinically, clinically or animals dying from paratuberculosis. Keeping in view above facts, the present investigation was carried out to study the pathology of subclinical paratuberculosis in goats. |
Top MATERIALS AND METHODS Study material A total of 70 goats of both sexes, above 6 month of age and irrespective of breed were included in the present study. During the study period July, 2017 to February 2018, total 8 goats carcasses were received for post-mortem at the Department of Veterinary Pathology CoVSc, Jabalpur and total 62 goats slaughtered for table purpose at various slaughter houses of Jabalpur were subjected to detailed gross morphological examination and lesions were recorded. The representative pieces of small intestine and mesenteric and ileocaecal lymph nodes were collected and transported to laboratory under refrigerated condition (−4°C). Tissue samples from intestine, mesenteric lymph nodes and ileocaecal lymph nodes showing no gross changes (n=6) were also collected from slaughtered goats and were used in the study as control. Individual tissue was divided into three parts, one each for smears preparation, molecular diagnosis and for histopathology. All tissues to be used for DNA extraction were washed thoroughly with sterile distilled water and stored at −20°C. For histopathology, tissues were preserved in 10% neutral buffered formalin. All the samples were properly labeled for identification. Tissue smear examination Smear slides were prepared from scraping of intestinal mucosal surface and impression from mesenteric and ileocaecal lymph nodes, air dried, heat fixed and stained with ZN staining (ZN staining kit, Hi-media) for demonstration of acid fast bacilli as per previously described method10. Each slide was examined under microscope using oil immersion objective. Smear exhibiting presence of clumps or short acid fast bacilli was considered positive for MAP, in dispersed form as suspected for MAP, and negative if neither of the two forms observed1. Molecular diagnosis DNA extraction:PCR was used for detection of IS 900 to identify the Mycobacterium avium sub species paratuberculosis. Protocol for DNA extraction and molecular diagnosis from tissue samples was used according to earlier worker Sonawane et al.11 with slight modifications. Polymerase chain reaction Extracted DNA samples were amplified using specific IS 900 (BA5 and BA6) primers BA'5; 5'- CTC GCT ACC AAA CTC CCG A-3' and BA'6; 5'- GAA CTC AGC GCC CAG GAT-3' published by earlier workers11. Briefly, in a volume of 12.5 μl of PCR master mix, 1 μl forward primer (BA5) and 1 μl reverse primer, 9.5 μl nuclease free water and 1 μl of genomic DNA (total volume 25 μl). Total of thirty five cycles were performed in a thermal cycler for complete amplification reaction. Thermal cycling conditions were initial denaturation at 94°C for 5 min (1 cycle), followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, synthesis at 72°C for 1 min and final extension at 72°C for 4 min. The specific amplicon of 314bp product was analyzed by 1.5% agarose ethidium bromide gel electrophoresis and were taken as positives. For every reaction, positive control from genomic DNA extracted from a culture of the MAP and a negative control were used to cross check for any contamination of foreign DNA in reaction component. The amplified product was visualized as a single compact fluorescent band of expected size 314bp under U-V light and documented by gel documentation system (Syngene, Gene genius bio imaging). Histopathology Formalin fixed tissues were processed to obtain paraffin embedded tissue blocks and cut in to 5 μ thick sections and stained by routine staining Haematoxylin and Eosin (H&E) for histopathological examination as per the standard procedure12. Duplicate sections of intestine and lymph nodes were also stained by Ziehl Neelsen method for demonstration of acid fast bacilli as per standard procedure using commercially available staining kit. Special staining such as Masson‘s Trichrome and Mayeb s Mucicarmine were also used for demonstration of connective tissue and mucin respectively12. Top RESULTS All the carcasses exhibited normal healthy to fair body condition with no features of illness. In the present study, 5 (7.14%) out of the 70 goats intestine and mesenteric and ileocaecal lymph nodes smears revealed typical acid fast bacilli either individually or in clumps of 5 to 20 organisms in the cytoplasm of macrophages (Fig. 1). The lymph node smears showed individually or clumps of three or more acid fast bacilli in the cytoplasm of macrophages or outside of the cells (Fig. 2). The intestinal tissues of 70 goats were processed for identification of MAP specific genome by IS 900 PCR and found only three (4.28%) cases positive for MAP genome (Fig. 3). These positive goats were identified as paratuberculosis infected. |
All positive goats belonged to slaughter house, female, above 2 years of age and apparently healthy without any previous clinical history of illness. The associated gross and microscopic pathology in five goats revealed characteristic lesions of paratuberculosis. |
Gross pathology Intestinal tract were distended, ballooned with watery contents and mildly congested (Fig. 4). In small intestine both ileum and jejunum revealed congestion, semisolid mucus mixed content in the lumen. Distal part of small intestine wall revealed mild to severe thickening, hyperemic and transverse corrugated mucosal surface which did not disappear on stretching or pulling (Fig. 5). The mesenteric lymph nodes showed varying degree of enlargement and cut surfaces revealed oedema and congestion. The ileocaecal lymph nodes appeared as carded. On sectioning, cut surfaces revealed oedema, congestion and hemorrhages with gray-white foci in the cortical areas, whose demarcation from medullary region was usually indistinguishable (Fig. 6). Histopathology Microscopically lesions were classified as paucibacillary and multibacillary based on cellular infiltration and distribution of AFB laden epithelioid cells. Paucibacillary lesions were found in two cases. Sections of small intestine revealed mildly congested mucosa with infiltration of lymphocytes predominantly and occasional few scattered AFB laden epithelioid cells observed in the lamina propria. Mild infiltration of mononuclear cells (MNC) around the blood vessels was also observed. Sections of lymph node revealed mild infiltration of MNC and few epithelioid cells in the cortical region. Multibacillary lesions were observed in three goats. Sections of small intestine revealed short club shaped and atrophied villi, epithelial lining of the tip of villi showed degenerative and necrotic changes and were desquamated in various extents (Fig. 7). Diffuse infiltration of lymphocytes, polymorphs and epithelioid cells were observed in mucosa. Lamina propria and submucosa was oedematous and infiltrated predominantly with lymphocytes and epithelioid cells (Fig. 8). Plasma cells and polymorphs were observed in less numbers. Dilatation of crypts, lacteals and intestinal glands with mucin were also observed in Mayer‘s Mucicarmine stained sections (Fig. 9). The serosa was oedematous and infiltrated with inflammatory cells. On Ziehl Neelsen staining, small intestine tissue sections revealed intracellular acid fast bacilli laden epithelioid cells scattered in lamina propria and submucosa (Fig. 10). Mesenteric and ileocaecal lymph nodes revealed thickened capsule and trabeculae with varying degree of fibrous connective tissue proliferation observed by Masson‘s Trichrome stain and infiltration of mononuclear cells and epithelioid cells in the cortical region. Few epithelioid macrophages were also seen scattered in the subcapsular, cortical and paracortical areas. Duplicate tissue sections stained with Ziehl Neelsen staining revealed AFB laden epithelioid cells in cortex and the medullary region of mesenteric and ileocaecal lymph nodes (Fig. 11). Top DISCUSSION The results of the present study confirm the presence of MAP infection in goats of Mahakausal region. The goats included in the present study were exhibited normal healthy to fair body condition with no features of illness. Similar findings were also reported by earlier workers13,14. Tissue smear revealed presence of typical acid fast bacilli in the cytoplasm of epithelioid cells was also reported by earlier researcher15,16. Low prevalence rate of Johne‘s disease observed in this study supported the findings reported by earlier workers17,18. However, the higher prevalence rate has alsobeen reported by other workers14,15,19. |
In current work we found only three (4.28%) goat intestines positive for MAP genome by PCR and these were identified as paratuberculosis infected and showed multibatillary lesions. The low prevalence by PCR found in the present study was in accordance with the observations of earlier worker14. However, higher prevalence rates were reported ranging from 14.8% to 36.6% by tissue PCR by earlier workers15,16. The low prevalence rate in the present study was not surprising in view of subclinical paratuberculosis infection as the samples were collected from apparently healthy goats or due to less number of bacteria present in tissues, i.e., below the detection limit (230 bacilli/g); these observations are in congruence with the earlier report11. The present findings are in accordance with the findings of earlier workers20 as they also reported that the tissue PCR targeting IS 900 has been found highly sensitive in detection of multibacillary cases as compared to paucibadllarycases. However, PCR is an accurate and reliable method for detecting the MAP genome21,22. |
Typical gross and microscopic pathology of paratuberculosis was observed in tissue sections of goats that were from slaughter house, above 2 years of age females, and apparently healthy without any previous clinical history of illness. Because of the chronic nature of disease the clinical signs appear late even though infections are present or in early stage of the disease, therefore goats appeared apparently healthy. Previous workers stated that in many cases clinical signs of disease are missed until the infection reaches significant prevalence and the clinical signs may not be present a linear relationship with the findings at necropsy23. |
Gross lesions of thickened and corrugated intestinal mucosa observed in present study are in accordance with the findings reported in goats and sheep by earlier workers10,24,25. The thickening of intestine wall with transverse mucosal corrugation observed were due to excessive infiltration and accumulation of mononuclear inflammatory cells including epithelioid macrophages in the submucosa as evidenced from histopathology. Similar findings were also reported by previous researchers16, whocalso opined that inflammatory cells infiltrates were responsible for much of the increased thickness in the mucosa and submucosa and for the villous atrophy. |
In present study, gross lesions observed only in the distal portion of the small intestine and could not be seen in the duodenum, colon and rectum. Similar observations were reported by others26,27 but contrasted with the findings of earlier report28 as they observed extension of lesions up to duodenum anteriorly and colon posteriorly. |
Submucosal region of intestine showed congestion, infiltration of inflammatory cells and dilated crypts and lacteals. The serosa was oedematous and infiltrated predominantly with lymphocytes. Polymorphs and plasma cells were also observed in the mucosa. The findings are in accordance with the findings of previous worker11,24 they also observed dilated and distended cryptal glands and filled with inflammatory cells and mucin. The infiltration of inflammatory cells in the early stage were correlated with the strong cell mediated immune response and increased mucus production which could be due to natural exposure of intestinal mucosa to other antigenic stimulants (intestinal parasite and protozoa) as apparent by Mucicarmine stain. In the present observation AFB laden epithelioid cells were found in the submucosa and mucosal region of intestine by ZN staining suggested a strong cellular immune response that might have restricted the replication of bacteria. These findings were supported by observations of previous workers24,25,28. |
In the present study, mesenteric lymph nodes showed thickening of capsule and trabeculae with fibrous connective tissue proliferation as apparent by Masson‘s trichrome stainand infiltration of macrophages and epithelioid cells. Similar findings were observed by other workers25, who also recorded thickening of capsule due to fibrous connective tissue proliferation. However, contrary to our findings, some researchers did not report fibrosis in the Johne‘s disease infected mesenteric lymph nodes in goats10. The sections of mesenteric lymph nodes showed few or scarce AFB laden epithelioid cells were scattered in the cortical area. The presence of scarce AFB organisms in the mesenteric lymph nodes was in accordance with the findings of earlier workers29, who also found that in subclinical cases of disease AFB were rarely detected from mesenteric lymph nodes. |
Mesenteric and ileocaecal lymph nodes revealed accumulation of lymphocytes, and epithelioid macrophages with the formation of granuloma in the cortex and medulla and in and around the lymphatics and blood vessels. The lesion around the blood vessels, suggested the possibility of hematogenous and lymphatic route of MAP infection. Similar findings were also reported by earlier workers10,29. Under field condition, disease caused by single pathogen is very rare and occurrence of mixed infections cannot be ruled out in animals. |
In conclusion, low prevalence rate of subclinical paratuberculosis were observed in apparently healthy goats in the Mahakaousal region of Madhya Pradesh, which stood at 7.14% and 4.28% on the basis of Ziehl Neelsen staining and PCR, respectively. The gross and microscopic pathological lesions observed were typical of paratuberculosis. |
Top ACKNOWLEDGEMENTS The present work is part of post-graduate thesis research. The facilities and funds provided by Head of the Department and the Dean of the College are thankfully acknowledged. We are thankful to the Dr B.N. Tripathi, Director, ICAR-NRCE, Hissar, Dr R.V.S. Pawaiya, Principle Scientist, ICAR-CIRG, Makhdoom, Dr G.G. Sonawane, Senior Scientist, ICAR-CSWRI, Avikanagar for providing necessary help and guidance to carry out this study. |
Top Figures | Fig. 1.: SmallIntestine scraping showing pink coloured, rod shaped acid fast bacilli (arrow). ZN x1000
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| | Fig. 2.: Impression smear of mesenteric lymph node showing pink coloured, rod shaped, acid fast bacilli (arrow). ZN x1000
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| | Fig. 3.: PCR amplification of 314bp of IS900 gene for identification of MAP in tissue samples. (M: 100bp DNA ladder, L1: Positive control DNA extracted from MAP culture obtained from CSWRI, Avikanagar, L2, L5, L7: Positive DNA sample, L8: Negative control)
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| | Fig. 4.: Goat found positive for paratuberculosis infection showing congested and ballooned intestine
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| | Fig. 5.: Photograph of Johne‘s disease infected goat intestine showing thickened wall and corrugated mucosa
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| | Fig. 6.: Mesenteric and ileocaecal lymph nodes showing congestion and gray-white foci on cortex region (arrows).
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| | Fig. 7.: Section of intestine showing flattened fused villi and MNC infiltration. H&E ×40
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| | Fig. 8.: Section of intestine showing mild infiltration of lymphocytes, polymorphs and epithelioid macrophages with foamy cytoplasm in mucosa. H&E ×400
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| | Fig. 9.: Sections of intestine showing dilated crypts with mucin (arrow). Mayer‘s Mucicarminex400
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| | Fig. 10.: Section of small intestine showing acid fast bacilli (arrows). ZN x1000
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| | Fig. 11.: Mesenteric lymph node showing acid fastbacilli (arrow). ZN x1000.
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