Hemoparasitic diseases have a significant negative impact on global animal health and productivity, particularly vector-borne diseases which pose a substantial threat to the financial stability of impoverished farmers. These diseases adversely affect the well-being, reproductive capabilities, and growth of domestic animals, resulting in increased sickness and death rates within the livestock industry, especially in coastal regions like Odisha. Despite considerable research on blood protozoan diseases in cattle in India, investigations into similar infections in small ruminants are still in their infancy. To tackle the recent rise in infections and mortality among Kendrapada sheep, leading to widespread loss of productivity, this study aims to examine various hemoparasitic parasites affecting Kendrapada sheep in Kendrapara district. The research involves identification using hematological, biochemical, and molecular techniques.
In the present study with 148 sheep from four blocks (Derabish, Aul, Kendrapara, and Marshaghai) between June to November 2023, 13% (19/148) were positive for Theileria piroplasm, while the rest were negative. Blood smear examination using Giemsa stain revealed various shapes of Theileria piroplasm. Hematological assessment and serum extraction were conducted on 51 samples suspected of infection which further subjected to biochemical analysis. The prevalence rates among different age groups were 17.9% (Group I), 19% (Group II), and 13.33% (Group III). Female sheep exhibited a higher prevalence rate (19%) compared to males (15.9%). Prevalence rates varied across different blocks, with the highest rate observed in Aul (27.90%). Semi-intensive rearing practices showed a prevalence of 69%, while stall-fed systems had a prevalence of 31%. The prevalence of theileriosis varied based on socio-economic status, with daily laborers showing the highest prevalence (38%). The aforementioned research findings align with analogous investigations conducted by Nangru et al., (2023) and Ismaeil et al., (2023). Similarly, in regions with tropical climates like Tamil Nadu, a study conducted by Jayalakshmi et al., (2022), indicated that 70% of examined sheep blood smears tested positive for Theileria spp. The Shujaabad area, district of Multan, Punjab, Pakistan, observed a prevalence rate of 3.7% (Riaz and Tasawar 2017). Moreover, the southern districts of Pakistan reported prevalence rates of 27.5%, 13% and 19.5% in Dera Ismail Khan, Tank and Bannu, respectively (Ullah et al. 2018).
Upon reviewing the history and clinical manifestations observed during blood collection, suspicion arose regarding Theileria infection. Typical symptoms encompassed persistent fever, loss of appetite, weight reduction, fatigue, pallor of mucous membranes, coughing, diarrhea, swelling of lymph nodes, and edema of the brisket. These clinical indicators played a pivotal role in the diagnostic assessment of Theileria infection. Sheep were divided into three age groups: Group I (< 1 year), Group II (1–4 years), and Group III (> 4 years). The above studies were consistent with similar studies by El Imam et al., (2016) and Nasreen et al., (2020) indicate varying age-related prevalence patterns of Theileria infection. Further, Osman et al., (2017) found the highest prevalence in sheep aged > 2 years in Nyala, South Darfur state, Sudan, while no piroplasms were detected in younger age groups. In district Multan, Pakistan, Riaz and Tasawar (2017) reported higher commonness rate of theileriosis in the age group of > 2 years for both sheep and goats compared to the age groups of 1 year and 1–2 years. The prevalence rate of theileriosis was found lesser 15.9% (11/69) in male sheep than in 19% (15/79) female sheep. The overall prevalence rate varied among the four blocks: Group I (Derabish) had a rate of 5.88% (3/51 samples), Group II (Aul) had a rate of 27.90% (12/43 samples), Group III (Kendrapara) had a rate of 16.12% (5/ 31 samples) and Group IV had a rate of 19.35% (6/31 samples). Among the four blocks, Aul had the highest prevalence rate of theileriosis. These results align with the findings reported by (Jayalakshmi et al. 2022) where they reported higher rate of prevalence of 14% in small ruminants in Western parts of Tamil Nadu out of Tropical climate of Tamil Nadu. A higher incidence of Theileria piroplasms in sheep and goats was mentioned from Khyber Pukhtoon Khwa compared to those from Punjab province in Pakistan and many other workers like (Kho et al. 2017; Nasreen et al. 2020; Ullah et al. 2021; Mohsin et al. 2022).
Average hematological values were calculated and compared among these groups. A significant difference (P < 0.05) was observed in the average PCV values, but no statistically substantial difference (P > 0.05) was detected in other hematological parameters. Similarly, no significant difference was observed between various hematological parameters among male and female Theileria-positive sheep. Comparisons of average hematological parameters in Theileria-positive sheep from the four blocks of Kendrapara district revealed a significant difference (P < 0.05) in PCV and Eosinophil count, while no statistically substantial difference (P > 0.05) was found in other parameters, including Hb, TLC, TEC, Neutrophils, Lymphocytes, Basophils, Monocytes, MCV, and MCHC. A reduction in the levels of Hb and TEC was noted, accompanied by PCV values falling below the expected range, aligning with the findings documented by Ayaz et al., (2023), Riaz and Tasawar (2017), and Jayalakshmi et al., (2022).
Blood smears, carefully prepared and stained with acridine orange, underwent thorough examination under a high-powered fluorescence microscope at a magnification of 1000X. This meticulous process unveiled the striking green luminescence emitted by the DNA of piroplasms nestled within the red blood cells, presenting a stark contrast against the dark background. Following this methodical analysis, the presence of these distinctive green-colored piroplasms was conclusively identified in 22 samples. It's worth noting that this identification was carried out using the rigorous procedure outlined by Al-Badrani and Hammed, (2022) and Omer et al., (2011) ensuring consistency and reliability in the findings.
Further, the biochemical analysis of 26 serum samples that tested positive was conducted to ascertain the levels of various parameters, including serum glucose, total protein, albumin, globulin, and urea, with the aim of comprehensively understanding the metabolic alterations in affected sheep. Average values were 52.85 ± 2.39 mg/dl for serum glucose, 5.49 ± 0.22 g/dl for total protein, 2.79 ± 0.08 g/dl for albumin, 4.96 ± 0.08 g/dl for globulin, 21.61 ± 0.44 mg/dl for urea, and 0.57 ± 0.02 for the A/G ratio. These findings partially coincide with the findings reported by Ullah et al., (2018) for total protein and urea but differ in terms of albumin, globulin and A/G ratio. Decrease in total serum protein was reported from normal value (Riaz and Tasawar 2017; Ayaz et al. 2023).
Out of 148 blood samples, 19 were positive for Theileria in blood smear examination. All positive cases in blood smear examination were confirmed by PCR. Interestingly, PCR detected Theileria species in 7 samples initially deemed negative by blood smear examination, resulting in a total of 26 positive cases. This highlights PCR's 6% higher sensitivity compared to traditional blood smear examination, making it effective even in subclinical infections.Several researchers made similar observations like Mamatha et al., (2017), Dhaygude et al., (2020), Moudgil et al., (2023), Nangru et al., (2022, 2023). The identified Theileria species bears a striking resemblance to T. luwenshuni, which is consistent with the recognized patterns of host pathology observed in sheep and goats. Within species, the 18S small subunit rRNA gene displays considerable variability, yet remains relatively conserved across members of the same genus. However, the lack of extensive data poses challenges in making deeper insights into relationships within the Theileria group. Despite this, it's noteworthy that intra-specific variation appears to be limited, typically manifesting as a handful of nucleotide substitutions in the 18S rRNA gene.