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ABSTRACT

Bubaline paratuberculosis is a chronic, debilitating disease that has been implicated in causing reduced milk yield and poor carcass quality in affected animals. The objective of the current study was to depict the histopathological manifestations of paratuberculosis in subclinically infected Nili-Ravi buffaloes. For this purpose a total of 53 slaughtered carcasses were selected on the basis of thickened/ corrugated intestinal mucosae along with edema of the associated lymph nodes. Acid-fast bacilli (AFB) were observed only in 22.64% of the cases on smear method examination. Histological examination of the tissue samples was performed using hematoxylin-eosin and Ziehl-Neelsen staining methods. Heavy mononuclear cells infi ltration was detected in the tissues. The foamy cytoplasm of epithelioid-macrophages was observed to be engorged with AFB in the positive cases. The results of this study demonstrated tissue sections showed much higher positive results (100%) as compared to the smear method (18.68%). Furthermore, the overall histological representation of tissue sections was more defi ned and illustrative than their smears. The

histologically positive cases were also confi rmed by means of ELISA testing. Despite being a time-honored technique, histopathology is an incredibly accurate and reliable protocol for diagnosing bubaline paratuberculosis.

Keywords: Nili-Ravi buffaloes, paratuberculosis, staining, histology, ELISA

INTRODUCTION

Recently the paratuberculosis (PTB) has become one of the most important livestock diseases. It is highly infectious and incurable in nature (Jones et al., 2006) and has been documented in domestic animals throughout the world (Buergelt and Ginn, 2000). Also, it has been observed in zoo and wild ruminants (Ayele et al., 2001; Erume et al., 2001). The causative agent of PTB is an acid fast gram positive bacillus, termed as Mycobacterium avium subsp. paratuberculosis (MAP) (Antognoli et al., 2007). Young animals are believed to be highly susceptible to PTB infection (Collins, 2003). Two distinct forms of PTB have been distinguished in animals as paucibacillary and multibacillary forms

HISTOLOGICAL EVALUATION OF THE GUT OF NILI-RAVI BUFFALOES FOR DIAGNOSING PARATUBERCULOSIS

Arbab Sikandar1*, Muhammad Adil1, Abdur Rahman Ansari1, Amar Nasir2, Touseef Ur Rehman3, Muhammad Raza Hameed4 and Ishtiaq Ahmad5

1Department of Basic Sciences, College of Veterinary and Animal Sciences, Jhang, Punjab, Pakistan, E-mail: arbab.sikandar@uvas.edu.pk 2Department of Clinical Sciences College of Veterinary and Animal Sciences, Jhang, Punjab, Pakistan 3Department of Parasitology, University College of Veterinary and Animal Science, Bahawalpur, Pakistan4Department of Pathobiology, College of Veterinary and Animal Sciences, Jhang, Punjab, Pakistan5Department of Pathology, University of Veterinary and Animal Sciences, Lahore, Pakistani, Pakistan

Original Article

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(Catton, 2002). Crohn’s disease (CD) in humans is also suspected to be caused by MAP (Erume et al., 2001). Subclinically infected animals constitute the most signifi cant source of infection and accurate diagnosis of these animals is critical for the control of PTB. Among large number of laboratory tests, the histology of intestine and mesenteric lymph nodes is the most suitable trial for the diagnosis of PTB (Buergelt and Ginn, 2000; Maxie et al., 2007). Although culture technique is regarded as the gold standard (Huntley et al., 2005) nevertheless, it is a labor intensive technique and requires a prolonged incubation period of up to 16 weeks. The current study was devised to investigate various histological changes in the gut (intestine) and mesenteric lymph nodes and to compare its diagnostic performance with Ziehl-Neelsen (ZN) stained smears and ELISA technique.

MATERIALS AND METHODS

SamplesA total of 53 tissue (intestine and mesenteric

lymphnodes) samples were collected from animals suspected of having PTB for investigation from two abattoirs in the Municipal Area of Jhang, Pakistan. The majority of the animals brought for slaughtering were weak and emaciated. Collection of blood (for serological evaluation) and tissue samples was performed before slaughtering and at postmortem, respectively. The collected samples were kept in sterile vaccutainers and plastic bags, respectively, that were subsequently transferred to a cooling jar containing ice. The tissue samples were conveyed to the histopathology laboratory, College of Veterinary and Animal Sciences, Jhang for further processing. Whereas serological evaluation was conducted at the University Diagnostic

Laboratory (UDL), University of Veterinary and Animal Sciences, Lahore, Pakistan.

Screening through ZN smears techniqueThe thickened portion of intestine having

prominent mucosal corrugations was used for the preparation of a few hard pressed mucosal impression smears. The ZN staining method proposed by Cappuccino and Sherman (2008) was adopted for staining of intestinal impression smears (ISs). Acid-fast bacilli (AFB) were observed as reddish rod-shaped structures under the microscope. Those samples in which the ISs were ZN stain negative were all discarded and all those that showed AFB were further processed for histopathological examination following Bancroft and Cook (2007). ZN staining of the tissue samples was performed using the method proposed by Huntley et al. (2005). Serum samples of the ZN positive animals were subjected to an indirect ELISA test. The indirect immunoenzymatic technique was executed using the diagnostic kit of anti-MAP antibodies in bovine sera as per instructions of the manufacturers (SerelisaTM M.Para TB Ab Mono Indirect, Cat. No. ASPTB3 (2 Plates), Symbiotic Europe SAS, 2, rue Alexander Fleming, F-69367 Lyon, Cedex 07, France). Data thus collected for ZN acid-fast staining (smears as well as tissue sections) and the ELISA test were analyzed by applying descriptive statistical methods (Zar, 2003).

RESULTS AND DISCUSSION

A total of 53 samples obtained from Nili-Ravi buffaloes suspected of having PTB were tested. All the specimens illustrated relatively thickened and extremely corrugated mucosae more around the distal ileum; however, the gross pathological

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changes were evident in smear samples that were positive for AFB. The diverse elevated corrugations of the thickest parts were prominent enough to retain their appearance even after fi rm stretching. However, mesenteric lymph nodes of only nine animals were swollen and enlarged.

3.1 Histopathological examination of tissue sections Overall 22.64% (12/53) mucosal smears from intestines were found to be positive for ZN stain. Taking them as the confi rmed PTB cases, further investigation of their impression smears and tissues sections was conducted. Intestine and mesenteric lymph nodes were the organs of study. Histological fi ndings refl ected that all (100%) of the (12/12) intestinal sections were ZN stain positive. It is, therefore, clear that tissue section is a relatively superior technique for the diagnosis of PTB as compared to the smear method. A signifi cantly greater number of AFB was observed in intestinal tissue sections as compared to intestinal ISs. Likewise, the number of positive detected samples was also higher through histological sections of mesenteric lymph nodes contrary to its ISs. Histopathological studies revealed that the epithelial cells lining the intestinal villi had mostly sloughed-off in the posterior small intestine, and the mucosal lamina propria was fully distended with granulomatous infi ltrations. The predominant cells of the infi ltrate were the epithelioid macrophages having foamy cytoplasm and round to oval, eccentric nuclei (Figure 1). The intestinal walls were thickened up to twice normal owing to increase in the size of mucosa and submucosa. Fibrous connective cells along with lymphocytes, plasma cells and eosinophils were extensively infi ltrated in the mucosa and submucosa. Crypts of Lieberkuhn had become shallow, slit-like, atrophied, reduced in

numbers and were separated from each other due to cellular infi ltration. Exfoliated cells were observed in the opening of the crypts. A few infi ltrations by mononuclear cells in the tunica muscularis and even in serosa were also evident. While using ZN stain, the cytoplasm of epithelioid macrophages was stuffed with acid-fast microorganisms. The AFB appeared red to pink inside macrophages (Figure 2). Large collections of laden macrophages were observed in the mucosa and submucosa. Multinucleated giant cells were also noticeable. Examination of all (n=09) oedematous lymph node samples revealed that their covering fi brous connective tissue capsules were very thick. There were variable sized areas containing shapeless, acellular brown caseous material surrounded by thin fi brous membrane. All the histologically positive cases were also confi rmed by the indirect ELISA test. The study was aimed to detect PTB in Nili-Ravi buffaloes via traditional methods i.e. ZN staining, histopathology and indirect ELISA. Emphasis was laid on the histological examination of intestine and mesenteric lymph nodes. Grossly observable gut lesions in small domestic ruminants are often mild (Radostits et al., 2007), but in the recent study the intestines were about two to three times thicker than normal and presented conspicuous transverse corrugations the same as reported by Sikandar et al. (2012). The thickness and corrugations occurred as a result of cellular infi ltration in the mucosa and submucosa as described by Ayele et al. (2001; Jones et al. (2006); Sivakumar et al. (2006); Khan et al. (2010); and Sikandar et al. (2013). Intestinal tissue contained multinucleated giant cells. However, our results do not comply with those of Tafti and Rashidi (2000), who described giant cells inside mesenteric lymph nodes in goats. This study highlighted the

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Figure 1. Mucosa of the small intestine. Epithelioid macrophages having foamy cytoplasm and eccentric nuclei are seen in the lamina propria. H&E (400X).

Figure 2. Mucosa of the large intestine. Macrophages laden with rose red acid-fast bacilli. Crypts are present wide apart from each other in upper portion of mucosa of the cecum. ZN (100X).

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validity of a thorough histological assessment of the intestine and mesenteric lymph nodes for the identifi cation of PTB in one of the most important dairy animals. The impression smear method used alone to diagnose PTB is not reliable unless verifi ed by the histopathological method. No doubt the histological method is a relatively old and time consuming approach, but it is one of the most economical techniques and provides high specifi city and accuracy as compared to other conventionally used methods. It can be regarded as a routine diagnostic technique for PTB infection in buffaloes. Further study for the identifi cation and differentiation of MAP is required, which is not probable using staining techniques. According to Ayele et al. (2001) and Shiratsuchi et al. (2008) MAP has been suspected to be linked to Crohn’s disease (human chronic granulomatous ileo-colitis), and the existing means are inadequate to avert the access of MAP infected tissues to the food supply (Antognoli et al., 2007). Suffi cient hygienic and public health measures should be undertaken to overcome this problem. Owing to the availability of limited information, it is not regarded as a priority disease for control in Pakistan. It can be concluded from this study that PTB is prevalent in Pakistan and precise diagnosis is possible using histological and serological techniques.

ACKNOWLEDGEMENTS

The authors would like to thank Prof. Dr. Ashiq Hussain Cheema for his endless support and guidance.

REFERENCES

Antognoli, M.C., F.B. Garry, H.L. Hirst, J.E. Lombard, M.M. Dennis., D.H. Gould, and M.D. Salman. 2007. Characterization of Mycobacterium avium subspecies paratuberculosis disseminated infection in dairy cattle and its association with antemortem test results. J. Vet. Microbiol., 10: 1016 - 1025.

Ayele, W.Y., M. Machackova and I. Pavlik. 2001. The transmission and impact of paratuberculosis infection in domestic and wild ruminants. Vet. Med. - Czech., (7-8): 205-224.

Bancroft, J.D., C.H. Cook and R.W. Stirling. 2007. Manual of Hisological Techniques and their Diagnostic Application. Churchill Livingstone, London. p. 17-34.

Buergelt, C.D. and P.E. Ginn. 2000. The histopathologic diagnosis of subclinical Johne‘s disease in North American Bison (Bison bison). Vet Microbiol., 77(3-4): 325-331.

Cappuccino, J.G. and N. Sherman. 2008. Microbiology: A Laboratory Manual, 7th ed. Dorling Kindersley, India. p. 77-79.

Catton, B.A. 2003. Paucibacillary paratuberculosis in a goat. Can. Vet. J., 43: 787-788.

Collins, M.T. 2003. Paratuberculosis: Review of present knowledge. Acta Vet. Scand., 44(3-4): 217-221.

Erume, J., J. Spergser and R. Rosengarten. 2001. Rapid detection of Mycobacterium avium subsp. paratuberculosis from cattle and zoo animals by nested PCR. Afr. Health Sci., 1(2): 83-89.

Huntley, J. F.J., R.H. Whitlock, J.P. Bannantine and J.R. Stabel. 2005. Comparison of diagnostic

Buffalo Bulletin (December 2014) Vol.33 No.4

475

detection methods for Mycobacterium avium subsp. paratuberculosis in North American Bison. Vet. Pathol., 42(1): 42-51.

Jones, T.C., R.D. Hunt and N.W. King. 2006. Veterinary Pathology, 6th ed. Blackwell Publishing, USA. p. 498-501.

Khan, F.A., Z.I. Chaudhry, M.I. Ali, S. Khan, N. Mumtaz and I. Ahmad. 2010. Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples of cattle and buffaloes. Trop. Anim. Health Pro., 42: 633-638.

Maxie, M.G., K.V.F. Jubb, P.C. Kennedy. and N.C. Palmer. 2007. Pathology of Domestic Animals, 5th ed. Vol. 2. Saunders Elsevier, London. p. 222-225.

Radostits, O.M., C.C. Gay, K.W. Hinchcliff and P.D. Constable. 2007. Veterinary Medicine: A textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats, 10th ed. Saunders Elsevier, London. p. 1017-1041.

Shiratsuchi, H., J. Ellner and J.M.D. Basson. 2008. Extracellular-regulated kinase activation regulates replication of Mycobacterium avium intracellularly in primary human monocytes. Cell Tissue Res., 332(2); 237-244.

Sikandar, A., A.H. Cheema, M. Adil, M. Younus, H. Zaneb, M.A. Zaman, M.Y. Tipu and S. Masood. 2013. Ovine paratuberculosis-A histopathological study from Pakistan. J. Anim. Plant Sci., 23(3): 749-753.

Sikandar, A., A.H. Cheema, M. Younus., A. Aslam., M.A. Zaman. and T. Rehman. 2012. Histopathological and serological studies on paratuberculosis in cattle and buffaloes. Pak. Vet. J., 32(4): 547-551.

Sivakumar, P., B.N. Tripathi, N. Singh and A.K. Sharma. 2006. Pathology of naturally

occurring paratuberculosis in water buffaloes (Bubalus bubalis). Vet. Pathol., 43: 455-462.

Tafti, A.K. and K. Rashidi. 2000. The pathology of goat paratuberculosis: Gross and histopathological lesions in the intestines and mesenteric lymph nodes. J. Vet. Med., 47: 487-495.

Zar, J.H. 2003. Biostatistical Analysis, 4th ed. Pearson Education, Singapore.