Haematological examination
Microscopic examination of 22 sheep blood smears revealed 14 samples positive for piroplasms of
Theileria species. Ring shaped piroplasms were mostly observed inside erythrocytes. However, some rod, coma and round shaped organisms were also recorded (Fig 1). Similar kind of pleomorphism in morphology of piroplasms has also been reported by
Yin et al., (2011) and
Mamatha et al., (2017). Koch blue bodies (KBBs) in lymphocytes were noted in the buffy coat smears of only 3 (13.63%) cases. As it is described in the earlier literature, the piroplasms are abundant in later stage of the diseases and in the present study late presentation of cases for diagnosis would have resulted in passing of sporozoite/schizont (acute) stage and that could be reason for getting piroplasm stage predominant over KBBs.
@figure1
The animals which were tested positive for theileriosis revealed anaemia of varying degree. The values of Hb (g/dl), PCV (%) and TEC (x 10
6/μl) ranged between 4.0 to 8.4, 10.0 to 28.0 and 1.9 to 4.6 respectively, indicating anaemia and findings are in accordance to several earlier reports
(Shruti et al., 2017; Zangana and Naqid, 2011).
Percentage of parasitemia was assessed by counting number of infected cells per 1000 RBCs and was found to be 41 to 68%, which indicated very severe infection.
Nagaraj et al., (2019) reported 1.0- 1.8% level of parasitemia in clinical and 0.1-0.9% in carrier animals. Parasitemia of 3.2-3.7% has been recorded in goats from China
(Guo et al., 2002). However, the level of parasitemia recorded in present investigation found very high. This could be attributed to the stage of the disease at which the blood smears were made because, high parasitemia will be seen in acute/clinical stage whereas, low parasitemia is characteristic feature of carrier or chronic stage of the disease
(Yin et al., 2008).
Molecular characterization of Theileria species
The blood samples from all positive cases were subjected to DNA extraction and subsequent PCR to detect 18S rRNA gene of hemoprotozoa. All 14 samples which were positive for theileriosis by microscopic examination were also tested positive by PCR (Fig 2). The PCR product was purified and subjected to sequencing. The sequence thereby obtained was subjected to BLAST analysis and phylogenetic analysis to construct phylogenetic tree (Fig 3).
@figure2
@figure3
The sequence revealed 99.53% homology with
T. luwenshuni reported from china (Gene Bank Accession No. JF719832.1 and JF719833.1) and 99.46% homology with
T. luwenshuni sequence from China (Gene Bank Accession No.JX469527.1 and JX469528.1). BLAST analysis revealed 94.99%, 95.54%, 95.82% and 97.19% sequence homology with
T. ovis (Gene Bank Accession No MK216569.1),
T. annulata (Gene Bank Accession No. KT736498.1),
T. lestoquardi (Gene Bank Accession No MG 564224.1) and
Theileria buffeli (Gene Bank Accession No EF126184.1) respectively, reported from India indicating that it is distantly related to all the above species and has a close sequence homology with
T. luwenshuni.
Reports from India, based on the results of blood smear examination, indicated that theileriosis was prevalent amongst small ruminants in different states including Karnataka, Kerala, Arunachal Pradesh, Tamil Nadu, Madhya Pradesh and Tripura
(Harish et al., 2006; Anumol et al., 2011; Tayo et al., 2011; Velusamy et al., 2015; Sahu et al., 2016; Das, 2017). However, there were no reports on molecular characterization of Theileria species involved in causation of this disease in sheep from Maharashtra State of India. So, it is first ever, confirmed report of
Theileria luwenshuni infection in sheep in Maharashtra state (India). Some older reports have shown
T. lestoquardi (earlier called
T. hirci) and
T. ovis infection in small ruminants in Tamil Nadu state of India.
Mamatha et al., (2017) reported first confirmed case of
Theileria luwenshuni infection in from sheep in Karnataka state of India. Recently,
Nagaraj et al., (2019) reported 75.28%, 32.58% and 7.86% goats positive for
T. ovis, T. luwenshuni and
T. lestoquardi respectively in Kerala state of India.
T. luwenshuni has been reported to be as pathogenic as
T. lestoquardi (Altay
et al., 2005;
Li et al., 2014).
Clinical signs
The morbidity was estimated 16.20% and mortality was around 8.30% during the period of outbreak. Substantial variation has been reported earlier by several authors with respect to morbidity and mortality in small ruminant theileriosis.
Durrani et al., (2011) reported 30% morbidity in a sheep flock in Pakistan which is in agreement with present findings. During the year 2015-16 and 2016-17 many flocks of sheep and goats in Karnataka
State of India showed huge mortalities ranging between 20 to 60% (IAHandVB Annual reports, 2016-17). On the contrary as per report of CFSHP (2019), the morbidity in
Theileria lestoquardi infection could be as high as 100%.
Taha et al., (2011) reported high mortality of 72.7% in goats suffering with malignant ovine theileriosis.
The ailing sheep showed fever (³104°F), anorexia, lymphadenopathy, progressive anaemia, debility, labored breathing, weekness, pale mucous membranes, non-responsiveness to antibiotic and other symptomatic therapy; increased respiration rate and dyspnoea in terminal stage as salient clinical signs in the animals examined. In 3 of the 22 animals examined, yellowish discoloration of visible mucous membrane was also noticed
. Similar clinical signs were reported in small ruminants by
Durrani et al., 2011; Taha et al., 2011; Mamatha et al., 2017 and CFSPH, 2019).
Response to treatment
Based on the microscopic diagnosis, the ailing animals were treated with Inj. Buparvaquone @ 3.5 mg /kg BW, injected intramuscularly (single dose); combination of meloxicam and paracetamol @ 0.5 mg /kg BW intramuscularly for three days and injection vitamin B- complex @ 3ml, i/m, per adult sheep for three days. The response to the treatment was swift and fast recovery was noted. In the present investigation Buparvaquone was found to be effective in the treatment of clinical theileriosis in sheep. Three effective drugs
viz., parvaquone, buparvaquone and halofuginone lactate are reportedly used in the treatment of theileriosis worldwide. Buparvaquone, the second-generation hydroxy naphthoquinone has been reported to be more effective
(Kumar et al., 2018; Ngumi et al., 1994). Earlier studies also reported efficacy of buparvaquone in successful treatment of clinical theileriosis in Sheep
(Mamatha et al., 2017).
Based on clinical signs, microscopic examination, PCR, sequence analysis as well as response to specific treatment; the outbreak was confirmed as theileriosis caused by
Theileria luwenshuni infection. It is the first ever confirmed report of ovine theileriosis in Maharashtra state of India. Considering the confirmed cases of ovine theileriosis in western Maharashtra for the first time, the field veterinarians are recommended to consider theileriosis as one of the differentials in sheep showing clinical signs of fever, progressive anaemia, lymphadenopathy, debility and non-responsiveness to antibiotic therapy. The present investigation and tropical climate in this region warrants systemic surveillance studies so as to know prevalence and to develop accurate and rapid diagnostics and effective vaccine.
