Hemato-biochemical studies on paratuberculosis in caprine

Introduction

Paratuberculosis, also known as Johne's disease is a chronic, fatal intestinal mycobacterial infection caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease was first described by Johne and Frothington in 1895 and first reported in sheep in Bosnia in 1908 (Bruère and West, 1993) [4] . Clinically manifested by irreversible wasting, therapy resistant diarrhoea and death from cachexia in ruminants and is widely spread world over in domestic and wild ruminant population (Sweeney, 1996 and Windsor et al., 2014) [21,25] . Johne's disease has shattering effects on the livestock sector in terms of production and economic losses and also has serious impacts on food safety, associated with Crohne's disease in humans (Ott et al., 1999 and Singh et al., 2008) [11,17] . The disease is found in both ovine and caprine, having worldwide distribution and has been designated as multispecies disease in OIE listed disease (Okwumabua et al., 2010) [10] . Johne's disease causes major problems for the livestock sector, leading to reduced production and large economic losses (Ott et al., 1999) [11] . It also raises public health concerns because of its possible link with Crohn's disease in humans (Singh et al., 2008) [17] .

The disease affects both sheep and goats, is widespread globally, and is listed by the OIE as a multispecies disease (Okwumabua et al., 2010; OIE, 2021) [10,9] . Animals usually become infected with Mycobacterium avium subsp. paratuberculosis (MAP) by eating or drinking contaminated material. After entering the intestine, the bacteria move to the lymphatic system and settle in M cells located above Peyer's patches in the ileum. Paratuberculosis

Materials and Methods

Study area

The Jaipur is located in the semi-arid region of Rajasthan, India, at 26°55′ N latitude and 75°49′ E longitude. The area is characterized by hot summers, low annual rainfall, and relatively mild winters.

Animals

In present study, a total of 400 goat animals aged above 1 year, without discrimination the sex and breed were selected from different flocks located in and around Jaipur, Rajasthan. All the 400 animals were screened by using single intradermal (SID) and ELISA tests and the animals that showed positive reaction either in tests were included in Group-I. For comparison of haemato-biochemical parameters, 20 apparently healthy animals aged above 1 year without discrimination the sex and breed, were taken as Group-II or control animals.

Single intradermal skin test

The purified protein derivatives (PPD) were procured from Biological Product Division (B.P. Division), Indian Veterinary Research Institute (IVRI), Izatnagar. The PPD was stored at 4 °C before and after use. The test was performed according to Ritchie (1953) [12] . After complete restraint of animal, an area of 1 square inch will be prepared for SIDT exactly 4 inches below crest of neck was shaved using shaving blade under aseptic conditions. The thickness of skin fold was measured in millimeters using digital vernier calipers. Tuberculin syringe of 1 ml (40 Units) capacity was used for the administration of 0.1 ml (4 Units) Johnin PPD intradermally. The correction of the injection was confirmed by palpation of a small pea-like swelling at the site. After 72 hours, the thickness at the injection sites was measured again and interpreted. The result was interpreted as 3 mm-negative, swelling between 3 to 4 mmdoubtful and swelling of 4 mm and above-positive.

Enzyme linked immuno-sorbent assay

A total of 400 serum samples were tested for antibodies against Mycobacterium avium subsp. Paratuberculosis (MAP). ELISA was carried out by using Mycobacterium paratuberculosis (MAP) Test Kit AsurDxTM (Biostone Animal Health, USA). The test was performed as per manufacturer"s protocol.

Hematological examinations

The blood samples were subjected for estimation of the haematological parameters viz. haemoglobin (Hb), packed cell volume (PCV), total erythrocyte count (TEC), total leucocyte count (TLC), differential leucocyte count (DLC). These parameters were analysed as per standard haematological methods cited by Schalm's veterinary haematology (Jain, 1986) [6] .

Biochemical estimations

Biochemical analysis of serum was carried out to estimate serum total protein, albumin, globulin, serum total bilirubin, serum creatinine, blood urea nitrogen (BUN) and the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). All the above biochemical parameters were estimated by using the -Turbo Chem. 100‖ (Automatic blood Biochemistry Analyzer), in the Department of Veterinary Physiology and Biochemistry of Post Graduated Institute of Veterinary Education and Research (PGIVER), Jaipur. The principles, reagents required, procedure, calculation and precautions used for each of them were followed as per operator's manual.

Comparative statistical analysis

The compared using standard formulas given for mean, standard error, t-test as per the procedures explained by Snedecor and Cochran (2004) [18] .

Results and Discussion

Out of 400 goats a total of 27 (6.75%) were found positive tuberculin reactors and 163 (40.75%) showed positive reaction by ELISA test and 25 (6.25%) animals showed positive reaction in both tests. These 163 goats that show positive reaction in either test, were subjected to further process for haematological and biochemical investigation.

Haematological parameters

The Mean ± SE values of hematological parameters of caprine paratuberculosis positive animals (Group-I) and apparently healthy animals (Group-II) are presented in Table 1. The mean values of Hb, PCV and TEC were significantly (p<0.01) decreased in positive animals as compare to healthy animals. These findings were in agreement with the study conducted by Weiss and Goodnough, (2005) [24] ; Senturk et al. (2009) [15] ; Tharwat et al. (2012) [23] ; Suman et al. (2016) [20] and Nabi et al. (2024) [8] . Sravani and Ganesan (2025) [19] reported significant anaemia and lower levels of Hb, probably as a result of in-appetence leading to poor body condition of animals. On contrary, Senturk et al. (2009) [15] , Bhardwaj et al. (2011) [3] , El-Deeb et al. (2014) [5] and Nabi et al. (2024) [8] who observed decrease erythrocyte count and PCV in affected animals with PTB. The mean values of total leucocyte count did not change significantly in the affected group as compare to healthy animals group. On statistical analysis there was no significant change in total leucocyte count of affected goats as compared to healthy control animals. The findings of present investigation are agreement with Nabi et al. (2024) [8] who also reported non-significant changes in total leucocyte count.

The mean values of neutrophil were significantly (p<0.05) increased in positive animals as compare to healthy animals. The increased neutrophil count in the affected group could be attributed to the nature of neutrophils which are one of the most abundant and important mediators of innate immunity and are professional phagocytes which mount the acute inflammatory response and act as the first line of defense against invading pathogens Segal, (2005) [14] ; El-Deeb et al. (2014) [5] and Nabi et al. (2024) [8] . The mean values of lymphocyte were significantly (p<0.05) decreased in positive animals as compare to healthy animals. According to Sharma et al. (2020) [16] and Nabi et al. (2024) [8] a decreased levels of lymphocytes in MAPpositive animals and mentioned chronic infections are known to induce 'immune exhaustion'. The mean values of monocyte, eosinophil and basophil revealed non-significant changes in positive animals as compare to healthy animals.

Biochemical Parameters

The Mean ± SE values of biochemical parameters of positive animals (Group-I) and apparently healthy animals (Group-II) are presented in Table 2. The mean values of serum total protein, albumin and globulin were significant (p>0.05) decrease in positive animals as compare to healthy animals. The findings are in agreement with Bhardwaj et al. (2011) [3] ; El-Deeb et al. (2014) [5] ; (Balikci and Gurdogan 2015) [2] and Nabi et al. (2024) [8] who observed lower value of total protein in affected group as compared to healthy group. The hypoproteinemia might be attributed to decrease of albumin in serum. Mishra et al. (2024) [7] studied decrease in serum total protein in PTB affected goats that could be attributed to dehydration caused by recurrent diarrhea. There was a significant decrease in serum albumin levels, which might be attributed to albumin leakage via injured tissues, resulting in a damaging granulomatous inflammatory response that finally leads to intestinal malabsorption and protein losing enteropathy (Sweeney et al., 2012; Balikci and Gurdogan, 2015) [22,2] . Hypoglobulinemia could all be attributed to the malabsorption diarrhea. Other factors such as reduced appetite and emaciation as the disease progressed may have contributed to changes in these serum parameters. The decrease level of globulin in PTB affected goats could be attributed to dehydration caused by recurrent diarrhoea (Schroeder et al., 2001) [13] .

The mean values of ALT, AST and ALP were significant (p>0.05) increase in positive animals as compare to healthy animals. The finding of study is in collaboration with finding of El-Deeb et al. (2014) [5] and Mishra et al. (2024). Significant increase in ALT concentrations were indicators of certain systemic affections, indicating liver damage caused by chronic infections of acid-fast bacilli (Balikci and Gurdogan, 2015) [2] . A significant increase in aspartate aminotransferase (AST) concentrations serves as an indicator of systemic involvement, reflecting hepatic damage associated with chronic infections caused by MAP Balikci and Gurdogan (2015) [2] . Increased activities of ALP can be related to muscle cell damage as a result of weight loss and anorexia in Mycobacterium infection Askar et al. (2018) [1] . The mean values of BUN and creatinine were revealed non-significant (p>0.05) in positive animals as compare to healthy animals. These findings are similar with Balikci and Gurdogan (2015) [2] . The mean values of total bilirubin were significant (p>0.05) increase in positive animals as compare to healthy animals. These findings in accordance with Balikci and Gurdogan (2015) [2] . El-Deeb et al. (2014) [5] reported hepatic involvement in animals infected with Mycobacterium avium subspecies paratuberculosis (MAP), characterized by a significant increase in serum bilirubin levels.

Conclusion

The study revealed that caprine paratuberculosis-positive goats exhibited significant haematological and biochemical alterations compared to healthy animals. Affected goats showed marked reductions in hemoglobin, PCV, TEC, total protein, albumin, and globulin, alongside increased neutrophils, liver enzymes (ALT, AST, ALP), and total bilirubin, while TLC, monocytes, eosinophils, basophils, BUN, and creatinine remained non-significant. These findings indicate anemia, hypoproteinemia, hepatic dysfunction, and immune alterations in MAP-infected goats, consistent with earlier studies, suggesting systemic involvement and metabolic disruption associated with chronic infection.

Acknowledgement

AcknowledgementsAuthors are thankful to Dean, PGIVER, Jaipur and Principal investigator "Centre for Diagnosis, Surveillance & Response of Zoonotic Diseases (CDSRZ)" for financial support and provided facilities.

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