Conducive climatic conditions favour the tick population and increase the risk of tick-borne diseases in tropical countries like India. Extensive grazing practices further enhance the risk among sheep and goats. Several workers have reported theileriosis in small ruminants based on microscopic examination of blood smears from many countries (Altay et al. 2007; Fatima et al. 2015; Osman et al. 2017) and a few from India (Anumol et al. 2011; Tayo et al. 2011; Velusamy et al. 2015; Sahu et al. 2016; Das 2017; Begam et al. 2019 and Jayaram et al. 2019). Reports indicated the microscopy-based prevalence of theileriosis was as high as 20% in sheep and 68% in goats (Jayaram et al. 2019; Shruthi et al. 2018). However, reports on species identification are scarce in India. Hence, data on prevalent pathogenic species are less. Six different species have been reported from sheep and goat viz. T. lestoquardi, T. uilenbergi, T. luwenshuni, T. ovis, T. separata and T. recondite, among which T. lestoquardi, T. uilenbergi and T. luwenshuni have been described as highly pathogenic (Friedhoff, 1997; Ahmed et al. 2006; Yin et al. 2008). Recently, Nagaraj et al. (2019) reported prevalence of T. ovis, T. luwenshuni and T. lestoquardi as 75.28%, 32.58% and 7.86%, respectively in apparently healthy goats in Kerala, India indicating carrier status of infection. There are no documented reports on clinical disease due to T. lestoquardi from India.
In the present study, the overall morbidity rate, cumulative mortality and case fatality rate was around 27.95%, 17.46%, 62.5%, respectively. High morbidity and mortality rates in this report could be attributed to the more deaths in Farm A which may be due to pregnancy stress (Osman et al. 2017). Morbidity, mortality and CFR were more in sheep in comparison to goats in the present study. Moreover, T. Lestoquardi infection has been reported to cause heavy loss with high morbidity and mortality in sheep (Ghali and Hussein 1995; Tageldin et al. 1992) and low susceptibility of goats to the pathogen (Brown et al. 1998). However, in Sudan, Taha et al. (2011) reported high mortality of 72.7% in goats suffering with malignant ovine theileriosis due to T. lestoquardi. A previous study by Al‑Hamidhi et al. (2021) indicated that the lone infection of T. lestoquardi (pathogenic) resulted in higher lethalities in comparison to coinfection with T. ovis (non-pathogenic) which might be due to possible competitive interaction between the parasites in mixed-parasite infection. It reflected that T. ovis guards against the pathogenicity of T. lestoquardi infection.
Clinical presentation of high fever, anorexia, lymphadenopathy, anaemia with pale mucus membrane and weakness in affected animals was consistent in all the farms. Similar clinical observations were made in small ruminants by Taha et al. (2011). The affected animals were anaemic with Low haemoglobin, TEC, PCV values and high TLC count. Nazifi et al. (2012) and El Imam et al. (2015) also reported anaemia and low PCV due to erythrocyte destruction in sheep infected with T. lestoquardi, whereas marked leucopenia was reported in contrast to our findings. ME detected intraerythrocytic piroplasms which were pleomorphic under microscopy. Mamatha et al. (2017) and Dhaygude et al. (2020) also observed pleomorphism in piroplasms. The predominance of piroplasms over Koch blue bodies may be due to the late stage of the disease (Dhaygude et al. 2020).
At necropsy, some affected sheep showed enlargement of lymph nodes, enlarged and icteric liver, mucosal ulceration of abomasum and lung oedema. Histopathological findings of focal necrotic foci in the liver, interstitial oedema of the lung, enteritis with mononuclear infiltration and lymphadenopathy were in agreement with gross pathology. Tageldin et al. (2005) also reported similar necropsy and histopathological findings in affected sheep.
All the samples which were positive by ME were found positive by PCR for Theileria spp. targeting 18s rRNA gene. In order to differentiate the species of Theileria organism, species-specific PCR was used which confirmed the presence of T. lestoquardi in all the episodes while mixed infection T. lestoquardi with T. ovis in two outbreaks (farms C and D). Other Theileria species infective to small ruminants viz. T. luwenshuni and T. uilenbergi were not detected. Sequence analysis of PCR products further confirmed and revealed 100% homology with T. lestoquardi and T. ovis. This is the first confirmed report of outbreaks of malignant ovine theileriosis in the Haryana state of India which caused high morbidity, mortality and case fatality among sheep and goats. Most of the previous studies in India concluded that small ruminant theileriosis was due to T. hirci, merely based on microscopic examination of blood smears without any species level differentiation studies (Rao et al. 1991; Sasmal et al. 1982; Gautham et al. 1975).
The phylogenetic analysis of partial sequences of the small subunit of ribosomal RNA revealed that the identified sequences of T. ovis and T. lestoquardi shared the closest evolutionary relationship with the Asian isolates. The occurrence of theileriosis caused by T. hirci in sheep and goats in India has been documented in previous reports (Ramanujachari and Alwar 1954; Rao et al. 1991; Sasmal et al. 1982). This study confirms the conclusions which were drawn in previous findings based on microscopy.
During the study. H. anatolicum anatolicum was identified as tick in affected animals. However, further research is required to study the possibility of other ticks in the transmission of T. lestoquardi and T. ovis.
Despite the apparent clinical presentation of theileriosis in affected animals viz. anaemia, fever, superficial lymph node enlargement and unresponsive to antibiotic therapy, the disease went undiagnosed by the field veterinarian because theileriosis in small ruminants was not considered while making a differential diagnosis. The present investigation underscores the requirement of systemic studies to know the prevalence and vectors. In conclusion, this study improves our understanding of theileriosis disease in small ruminants.