Theilerosis is the most important common vector (tick) born and hemoprotozoan diseases (TBDS), which poses a serious threat to the livestock population in terms of mortality, reduced milk yield and lowered draft power. High humidity and temperature are the main contributing factors. Bovine tropical Theileriosis is a tick-borne infection caused by
T. annulata, an intracellular protozoan parasite. It is a lymphoproliferatve disease causing high mortality and morbidity in cattle.
T. sergenti,
T. buffeli and
T. orientalis are the causative organism for bovine benign theileriosis in cattle. In India, bovine theileriosis was first reported in 1905 by Lingard as reported earlier (
Mohan 1972). Prompt efforts are required to screen theileria infected cattle, interpret systemic dysfunctions or pathological alterations to know and eliminate the etiology properly and so that the treatment can be given for increasing the production and productivity of the cattle. Keeping in view the above facts, the present biochemical, molecular, postmortem and histopathological studies were conducted in the bovine theilerioses at different production and reproduction stages.
Screening of animals was carried out in and around Bhubaneswar during 2021 February to December 2022. Blood samples were collected from suspected theilerioses cattle on the basis of clinical signs with complain of aneroxia, drop in milk yield, non-remitting pyrexia, enlarged superficial lymphnodes, pale mucous membrane of oral mucosa and conjunctiva, nasal discharge and coughing signs suspected, which were referred by the Veterinary Clinician. Patient data regarding age, sex, stage of lactation, parturition and pregnancy history and clinical signs were recorded. A total of 1900 suspected cases were screened by blood smear examination and 950 cases found positive for theileria. In positive cases, there was presence of piroplasms in blood smear with different shapes like dot shape, round, ovoid, irregular cocci or bacilli, rod, stick, comma, fusiform, racquet shaped, signet ring shaped or pear shaped inside RBCs indicating
Theileria species. In few positive cases, Koch’s blue bodies were also found in the lymphocytes of lymphnode smear.
Clinical signs were observed in theileriosis positive cows at different stages of production and reproduction (Table 1). Serum samples from 213 affected cattle were taken and compared with normal ranges of biochemical parameters. These samples were analyzed for serum biochemical parameters like serum glucose, blood urea nitrogen, creatinine, AST, ALT, serum calcium, magnesium and phosphorus. For histopathological examination, formalin fixed tissues were processed for routine paraffin techniques and routine H and E techniques as per the standard paraffin methods, stained and viewed for histopathological interpretation. Out of the collected total number of 200 samples, the 100 samples were found positive in Geimsa stain and were kept at -20°C temperature for doing routine PCR. During the research period, total six numbers of cases were subjected for postmortem examination. The average value we got for different parameters in infected and healthy groups were analyzed by analysis of variance (ANOVA) with post hoc analysis by Duncan’s multiple comparison test using SPSS22 soft ware and expressed as mean ± SE with p<0.05 considered statistically significant.
Serum biochemical analysis
Serum samples were collected from 213 positive cases of cattle in different stages of production and reproduction. The serum glucose values of animals of group 1, 2, 3, 4 and 5 were (78.49±1.14) g/dl, (69.22±1.7) g/dl, (74.11±1.5) g/dl, (70.85±2.3) g/dl and (78.44±1.69) g/dl, respectively. The average value of serum glucose in these groups was 73.79 g/dl. In all the groups the average glucose value was significantly lower than the normal value (45-75 g/dl) as per the reports of
Col and Uslu (2006) and
Acharya et al., (2016) due to utilization of glucose by the parasites.
The BUN values of animals of group 1, 2, 3, 4 and 5 were (25.90±0.67) mg/dl, (28.14±0.79) mg/dl, (28.18±0.70) mg/dl, (29.35±1.08) mg/dl and (22.91±0.69) mg/dl, respectively. The higher values of BUN level might be due to liver and renal damage in the affected animals and increased protein catabolism due to anorexia in theileria positive animals or due to dehydration
(Singh et al., 1991). The creatinine levels of animals of group 1, 2, 3, 4 and 5 were (1.83±0.60) mg/dl, (1.96±0.98) mg/dl, (2.09±0.48) mg/dl, (2.28±0.69) mg/dl and (2.16±0.10) mg/dl, respectively. Statistically, there was significant (p<0.05) difference in the creatinine estimates of Gr-1 and Gr-4. There was increase in average creatinine value in all the groups (
Col and Uslu, 2006).
The average values in Gr-1, Gr-2, Gr-3, Gr-4 and Gr-5 were (121.98±0.75) IU/L, (142.83±0.14) IU/L, (164.19±3.51) IU/L, (157.22±5.5) IU/L and (107.66±2.3) IU/L, respectively. The AST estimate in Gr-5 was found to be significantly (p<0.05) lower than Gr-2, Gr-3 and Gr-4. The average values in Gr-1, Gr-2, Gr-3, Gr-4 and Gr-5 were (28.55±0.96) IU/L, (33.66±1.96) IU/L, (41.34±0.83) IU/L, (37.05±1.55) IU/L and (26.68±0.71) IU/L, respectively. The ALT estimate in Gr-3 was found to be significantly (p<0.05) higher than Gr-1, Gr-2 and Gr-5. Recorded increase in the enzyme level indicated muscle trauma due to recumbent position of animals
(Selim et al., 2022) and debility caused by the depressed liver function.
The average values in Gr-1, Gr-2, Gr-3, Gr-4 and Gr-5 were (7.99±022) mg/dl, (7.04±0.22) mg/dl, (7.39±0.12) mg/dl, (7.49±0.18) mg/dl and (7.66±0.13) mg/dl, respectively. But the estimated calcium value was lower than the normal range, which might be attributed to the hypoalbuminaemia in the affected animals (
Ellah, 2015). The average values in Gr-1, Gr-2, Gr-3, Gr-4 and Gr-5 were (0.30±0.008) mmol/l, (0.28±0.009) mmol/l, (0.27±0.006) mmol/l, (0.25±0.007) mmol/l and (0.33±0.011) mmol/l, respectively. The serum Mg value was higher in Gr-5 than all other groups and there is statistically significant (p<0.05) difference of serum Mg value of Gr-5 with Gr-2, Gr-3 and Gr-4.
The average values of serum phosphorus in Gr-1, Gr-2, Gr-3, Gr-4 and Gr-5 were (0.41±0.011) mmol/l, (0.42±0.006) mmol/l, (0.37±0.009) mmol/l, (0.40±0.008) mmol/l and (0.42±0.006) mmol/l, respectively. The serum phosphorus estimate in Gr-2 and Gr-5 were significantly (p<0.05) different with Gr-3. The serum Ca, Mg and P level in all the groups were observed lower than the normal range, which might be due to the physiological stress in pregnancy and lactating stage leading to metabolic disturbances along with systemic dysfunction (
Col and Uslu, 2006).
Molecular diagnosis through PCR
The DNA was extracted from 200 number of blood samples. Out of them 120 were clinically positive and 80 samples were suspected cases (Fig 1). Polymerase chain reaction for 768bp fragment for Tams1 gene and 776bp fragment for MPSP gene was done and specific fragments were amplified in both primers using standard protocol. Then the PCR samples were sequenced by Sanger’s sequencer and nucleotide sequences were obtained by FASTA format (Fig 2). The nucleotide sequences were interpreted using Finch TV Software. The Forward and Reverse sequences aligned to obtain 768 bp and 776 bp (Fig 3 and 4). The nucleotide sequences were submitted in GenBank and the accession numbers (OQ230445 and OQ230446) were obtained. The identification of
T. annulata and
T. orientalis from phylogenetic tree was based on the reports of
Selim et al., (2022).
Total eight closely related nucleotide sequences from different places and two regions of Odisha were received from GenBank. Analysis of Tams 1 gene partial nucleotide sequence of
Theileria annulata (OQ230445) showed similarity of 98%, 100%, 100%, 95% with MW12309, Khorda isolate, MF346012, MN818858 Puri isolate and
Theileria annulata isolate OD2MF346013, respectively. Higher genetic divergence was found in between Khorda isolate and Puri isolate. The lower genetic divergence was found in Odisha and Jammu isolate (Fig 5). Phylogenetic relationship was estimated based on the partial nucleotide sequences (768 bp) of merozoite piroplasm surface antigen of
Theileria annulata with available other variants in GenBank.
Theileria orientalis partial MPSP gene (776 bp) found in this research with GenBank accession number OQ230446 showed 80% similarity with MW672060 Khorda isolate and 75% with JX648207 isolate of Wayanad (Fig 6). There was 57% cases
T. annulata as found by PCR assays, which was similar to the findings of
Acharya et al., (2016) and
George et al., (2015) and 20.5% cases
T. orientalis infection, which indicated the occurrence of tropical theileriosis. There was 9.9% of concurrent infection of both the organisms. Hence, the early detection is required to provide proper treatment to reduce the economic loss.
The melting curves of two primers for Tams 1 and MPSP showed single peak, which was suggestive of complete dissociation of primers and specificity of qPCR reactions. The cq values is cycle number above which fluorescence level becomes higher than the back ground fluorescence so that it can be detected. For our experiment, the Cq value for Tams 1 gene was almost 15 cycles and for MPSP gene it was almost 31 cycles. So, Tams 1 gene (
Theileria annulata) can be identified earlier than MPSP gene (
Theileria orientalis) in qPCR. Alternatively, the relative abundance of
Theileria annulata was higher than
Theileria orientalis (Fig 7). To the best of our knowledge, this is nearly a new phylogenetic study of
Theileria orientalis and the occurrence of concurrent infection is also a new finding. Both PCR and qPCR are accurate techniques for the detection of this concurrent infection in bovine theileriosis. From molecular analysis, we found 174 positive cases from 200 samples out of which 114 cows found positive for
T. annulata (57%) and 41 cows (20.5%) were positive for
T. orientalis and 19 cows (9.9%) cows were positive for concurrent infection.
Gross pathological changes
Systemic post-mortem examination of six dead animals was carried out during the study. Some of the animals were weak, debilitated with atrophy of the muscles. There was distended abdomen and soiling of hind quarter due to diarrhea in two cases.
Skeletal muscles were pale in 3 cases. Skeletal muscles were yellowish tinged with atrophy of muscles and prominent ribs in 2 cases. Subcutaneous surface revealed yellowish discoloration indicating Jaundice. Gelatinization of subcutaneous fat and paleness of muscle was observed in 2 cases. In few cases, abdominal cavity revealed presence of straw-colored fluid
(Acharya et al., 2016). Liver was slightly enlarged with congestion in few cases. Hepatic surface was congested with pale patches and distended gallbladder in two cases
(Sahoo et al., 1991). Liver was firm and jaundiced with yellowish discoloration of the cut edges. In one case, the liver was pale with haemorrhagic patches on the surface of liver and with adjacent pale patches. Lungs were congested and edematous slightly and pleura was wrinkled in few cases. Pale with haemorrhagic patches was evident in two cases. In two cases, lungs were icteric with intense yellowish discoloration of the surface, parenchyma, bronchi and bronchioles with scanty yellowish tinged of exudates (
Sastry and Rao, 2001). Pleura was thickened. In some cases, there was cardiac dilation with gelatinization of epicardial fat. In two cases heart was dilated with yellow discoloration of epicardial surface. Spleen surface was pale and yellow in two cases. Cut edges of the spleen revealed reddish yellow discoloration with oozing of reddish yellow fluid (
Sastry and Rao, 2001). In few cases, intestine was slightly distended with yellowish discoloration of serosa. Intestinal wall was thickened with catarrhal exudates in the lumen. There were haemorrhagic and ulcerative patches in the intestinal mucosa. In some cases, cortical surface of kidney was pale with pale medullary area, gelatinization of renal fat. Abomasal mucosa was pale with punched out ulcers.
Histopathology
Liver showed necrosis of hepatocytes with infiltration of neutrophils and mononuclear cells. In some cases, marked sinusoidal congestion and necrosis of hepatocytes were observed
(Sahoo et al., 1991 and
Acharya et al., 2016). The disruption of myocardial fibres and haemorrhages were found. Kidney showed the necrosis of tubular epithelial lining cells and focal infiltration of mononuclear cells. Some cases lumen of tubule filled with necrotic debris and also necrosis in glomerular tuft. Lungs were showing edema in alveolar space filled with edematous fluid and interstitial congestion. There was also necrosis and disruption of pulmonary alveoli with marked congestion, edema and haemorrhage. In some cases, congestion, edema and haemorrhages were found.
There was depletion of lymphoid follicles of lymphnodes. Marked congestion and haemorrhage of lymphoid follicle were noted. There was depletion of lymphocytes with lymphoid follicles. Abomasum showed the presence of fibrinous material on the surface of mucosa and congestion of mucosa with focal infiltration of inflammatory cells.
