<div>Background: Equine piroplasmosis is a haemoprtozoan disease of equids and enzootic in Rajasthan prefecture of India. In endemic areas, the subclinical condition is more common, as infected equids recover from the disease and become latently infected. This study aimed to investigate the seroepidemiology and risk factors associated with the endemicity of Theileria equi infection in Rajasthan state.&nbsp; 
 
Methods: A cross-sectional study on the seroprevalence of Theileria equi, was performed. Total of 151 serum samples collected from different areas of Rajasthan. The serum samples were screened by ELISA for assessment of seroprevalence of T. equi infection. 
 
Result: Overall seroprevalence of T. equi was 49.66%. A total of 75 T. equi seropositive horses were detected and one horse was clinically positive for T. equi infection. The clinical signs observed were - fever, haemoglobinuria, mild colic, anaemia and icteric conjunctival mucous membrane. Theileria equi infection is endemic among horses in Rajasthan state and Ajmer district found the most endemic. Haematological observations in T. equi clinically infected and healthy horses were studies. A decreased haemoglobin concentration, packed cell volume, total erythrocytic counts were observed in T. equi infected horse in comparison to healthy horses. This observation showed anaemic condition in T. equi clinically infected equine. An increased concentration of liver enzymes &ndash; AST, ALP, GGT in T. equi infected equine was also recorded, which indicated the liver damage.</div>

<div>Seroprevalence and Risk Factor Associated with Endemicity of Theileria equi Infection in Horses in Rajasthan State, India</div>

<div>Background: Equine piroplasmosis (EP) is a tick-borne disease of horses caused by the intraerytrocytic protozoan parasites. The infected animals remain carriers of these blood parasites for long periods and spread the disease. The introduction of carrier animals into areas where competent tick vectors are prevalent can lead to an epizootic spread of the disease. 
 
Methods: Total 295 blood smears from diseased and healthy horses were examined and 295 serum samples were analyses by cELISA for the presence of antibodies against T. equi and B. caballi whereas 90 DNA samples from seropositive horses were screened by PCR for presence of parasite&rsquo;s DNA.&nbsp; 
 
Result: In present study, 1.35% horses were found positive for T. equi by means of blood smear examination. Using c-ELISA, it was found that 03 (1.02%) horses had antibodies against B. caballi and 182 (61.69%) against T. equi, while none of the sample showed mixed reactions. Ninety (90) seropositivehorses screened for T. equi and B. caballi by PCR method, out of which, only Nine (09) horses werefound positive indicating an overall prevalence rate of T. equi was 10.00% by PCR. None ofthe horses found positive for B. caballi through blood smear examination and PCR method.</div>

<div>Prevalence of Theileria equi and Babesia caballi Infections in Horses in South Gujarat, India</div>

Present study demonstrates the prevalence and risk factors of infection to equines &nbsp;with Theileria species in Punjab, (India). The prevalence by thin blood film examination and Polymerase chain reactions based molecular assay was 4.17 and 33.33%, respectively. PCR targeting 18S rRNA gene of Theileria sp. &nbsp;produced high fidelity 1,100 bp amplification products with 100% sensitivity to blood films. The assessment of various risk factors revealed the prevalence of Theileria sp. to be uniformly distributed in equine population; the age of host animal being apparently the most influential factor for infection (Odds ratio= 1.6154, P= 0.41). The multiple sequence alignment revealed that the primer pair used could perceive the presence of wide range of species of Theileria genus; however the phylogenetic analysis of the custom sequenced amplicons clustered in one node with diverse Theileria equi isolates indicating the evolutionary homology. &nbsp;

Detection of Theileria species infecting equine population in Punjab&nbsp;by 18S rRNA PCR

<div>Background: Clinical theileriosis of small ruminants is tick-borne disease caused by Theileria lestoquardi, Theileria uilenbergi and Theileria luwenshuni. Theileria annulata, the causative agent of bovine tropical theileriosis in cattle, can also infect sheep but does not cause any significant illness. It is one of the economically important diseases. There are no reports of ovine clinical theileriosis from Maharashtra state and there is paucity of information on its epidemiology. This paper reports first confirmed outbreak of ovine theileriosis based on clinical signs, microscopic examination, PCR and sequencing in the Maharashtra State of India.&nbsp; 
 
Methods: Whole blood samples from 22 ailing sheep were collected and subjected to hematological examination. Blood smears stained with Leishman&rsquo;s stain were examined under 100X objective of the microscope. The blood samples from sheep found positive by microscopic method were subjected to PCR detection of 18S rRNA gene of hemoprotozoa and then for nucleotide sequencing and sequence analysis. 
 
Conclusion: Samples from 14 out of 22 sheep were found positive for piroplasms of Theileria spp by light microscopy. All positive samples were further confirmed by PCR detection of 18S rRNA gene of hemoprotozoa. PCR amplification yielded expected product of 1750 bp for all samples. BLAST and phylogenetic analysis of one sample revealed high sequence homology with T. luwenshuni reported from India and other countries. Characteristic clinical signs like fever, progressive anaemia, laboured breathing, lymphadenopathy, debility and non-responsiveness to antibiotic therapy were recorded. The animals responded to specific treatment against theileriosis. It is the first ever confirmed report of ovine theileriosis in Maharashtra state of India and hence reported.</div>

Investigations on First Confirmed Outbreak of Ovine Theileriosis (Theileria luwenshuni) from Maharashtra State, India

<div>Background: Theileria annulata is predominant and of utmost economical importance tick borne pathogen of bovines in the region which is routinely diagnosed based on the microscopic examination of Romanowsky stained thin blood smears. Present study was intended to evaluate and analyze the detection efficacy of a commercial polymerase chain reaction kit based assay in comparison to conventional PCR assay and classical microscopy for detection of T. annulata from blood samples of bovines from Punjab state. 
 
Methods: In this comparative study 360 bovine blood samples from various districts of agro-climatic zones in Punjab were first screened for T. annulata by Giemsa-stained thin blood smear (GSTBS) examination. The same panel of blood samples was tested for T. annulata by a commercial PCR kit Bovi-TheiDX Theileria annulata (Genext Genomics) (PCR1) and established conventional PCR assay targeting merozoite piroplasm surface antigen (Tams1) gene of T. annulata (PCR2). 
 
Result:&nbsp;Out of 360 samples screened, positivity of&nbsp;T. annulata by GSTBS was found to be 12.5% (45/360) with a sensitivity of 37.20% and specificity of 96.00% when compared with commercial kit (PCR1), the difference was statistically significant (p&lt;.0001). The detection prevalence by PCR1 29.70% (107/360) and PCR2 assay 33.90% (122/360) showed significant (p&lt;.0001) difference. The conventional PCR targeting the Tams1 gene (PCR2) was found to be more sensitive (84.6%) (p&lt;0.001) than PCR1 (69.7%).</div>

Comparative Performance and Evaluation of Two Molecular Assays and Conventional Detection of Theileria annulata in Bovines

<div style="text-align:justify">Background: Theileriosis is a haemoprotozoan illness that poses severe economic loss to dairy farmers due to its negative impact on mortality and productivity, especially in tropical and subtropical areas of the world. 
 
Methods:&nbsp;The present study was conducted for a period of one year (June 2020 to July 2021) during which 55 blood samples of cattle showing clinical signs suggestive of theileriosis were examined and quantified by using multiplex real time PCR for Theileria orientalis and Theileria annulata using Taq-Man probe. 
 
Result: Prevalence of theileriosis in three Theileria-endemic districts of Odisha due to Theileria annulata and Theileria orientalis were 26.11 per cent (11/42) and 57.14 per cent (24/42), respectively. Mixed infection were reported in 16.6 percent (7/42) cases. Parasite load for T. annulata in clinically affected cases ranged from 2.85&times;103 to 1.51&times;105 parasites per ml of blood while for T. orientalis,&nbsp;it ranged from 1.94&times;104&nbsp;to 5.88&times;105&nbsp;parasites per ml of blood. Ten cattle positive for T. orientalis were administered two doses of buparvaquone (Butalex) @2.5 mg/kg b.w. at an interval of 48 hours after which degree of parasitemia were quantified ten days post treatment. The parasitic load in blood ten days post treatment decreased significantly from 4.04&times;104 to 2.21&times;102 per ml of blood.</div>

RT PCR Assay to Determine Efficacy of Buparvaquone in Reducing Degree of Parasitemia of Theileria orientalis

Background: Bovines are domesticated ungulates mainly raised as livestock for milk, meat and leather and as draught animals. A slaughterhouse study helps to assess the disease status of herds and contains valuable information about the incidence and epidemiology of animal diseases. Haemoprotozoan diseases are one of the major problems which adversely affect the health and productivity of cattle. The diverse climatic zones of India are highly conducive to the survival and propagation of vectors and vector-borne pathogens. However, there is no data on proper slaughterhouse study of bovines in Mizoram even in India for determination of the prevalence of haemoprotozoan infections. 
 
Methods: The blood samples were collected in an EDTA vials. The blood smear was prepared, stained with Giemsa stain and examined for the presence of haemoprotozoan. The DNA was isolated from positive samples by use of a DNeasy Blood and Tissue Kit. Then PCR was performed for all the isolated DNA collected from positive samples. After confirmation through PCR, the representative samples for Anaplasma sp. and Theileria sp. were sent for sequencing. Then the sequencing results were annotated using BLAST, ClustalW multiple alignment program of MEGA9 software. The phylogenetic tree was generated by using the Neighbour-Joining method, keeping bootstrap consensus from 1000 replicates.&nbsp; 
 
Result: After the blood smear examination, PCR was performed for confirmation. Molecular detection of Anaplasma marginale, Theileria orientalis was done. Molecular characterization of 2 isolates from Anaplasma marginale and Theileria orientalis was performed by sequencing. The sequence analysis of rpoB gene of isolates of Anaplasma revealed both the isolates were placed in the same clade in the phylogenetic tree. The phylogenetic analysis of Thileria isolates revealed that isolate Miz-86 was closely placed with the Type-N3 clade. Whereas the other isolate i.e. Miz-87, belonged to the Type-5 clade.

Molecular Characterisation of Anaplasma marginale and Theileria orientalis in Slaughtered Bovines of Aizawl District, Mizoram

Background: Theileria Spp. is the causative agent of the economically significant hemoprotozoan disease known as theileriosis in cattle. In dairy animals, it results in death and decreased productivity. This study aimed to clarify the effects of Theileria annulata (T. annulata) infection on the histological and haemato-biochemical parameters in infected cattle. 
&nbsp; 
Methods: Between December 2022 and May 2023, 200 cattle were randomly selected from the slaughterhouse and blood samples were collected from them to assess the hemato-biochemical parameters and screen for T. annulata infection using a Giemsa-stained blood smear, furthermore, the histological profile of the spleen, lymph nodes and liver. 
&nbsp; 
Result: T. annulata was detected in 26.5% of the cattle based on blood smear microscopy. The haematological analysis showed a notable increase in lymphocytes and a significant drop in platelets, haemoglobin, total leukocyte count and total erythrocyte count. In cattle infected with T. annulata, biochemical examination showed a significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), high density lipoprotein (HDL) and triglyceride, also increase in bilirubin. Histological examination revealed necrotic degenerations of splenic nodules, oedema, parenchymal necrotic degeneration, increased intercellular spaces, depletion of lymphocytes, increased sinuses in lymph nodes and focal necrosis of hepatocytes with infiltration of inflammatory cells and vacuolar degeneration of hepatocytes.

Impact of Theileria annulata on Hemato-biochemical and Histological Parameters in Cattle

Background: Theileriosis is a tick-borne disease that has a maximum impact on the health and economy of developing countries. A number of clinical manifestations have been found in animals affected with Theileria annulata. A similar trend has been observed in the therapeutic management of theileriosis, especially in cattle. Resistance to available treatment options is one of the significant concerns with theileriosis. 
 
Methods: The present study evaluated the in-vitro activity of moxifloxacin, the fluoroquinolone compound, against T. annulata in 96 well culture plates using resazurin dye. The histopaque method was used for the separation of infected and healthy PBMCs (Polymorph Blood Mononuclear Cells) for growth inhibition assay of T. annulata and cytotoxicity assay measurement, respectively. The hemolytic assay was also evaluated against normal bovine RBCs (red blood cells). The 50% inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50) values were measured and analyzed using a correlation and regression method. 
 
Result: The IC50&nbsp;value of moxifloxacin was observed at 5,559 &micro;M against in-vitro T. annulata. The CC50&nbsp;value of cytotoxicity on PMBCs was found to be 3,984 &micro;M. However, the HC50&nbsp;value of hemolytic assay on bovine red blood cells was observed at 2,59,202 &micro;M. The moxifloxacin showed a wide safety margin on red blood cells. Moxifloxacin can be further investigated in-vivo to determine its actual effect on T. annulata.

In-vitro Activity of Moxifloxacin against Theileria annulata - A Preliminary Study

<div>Background: This study investigated the in vitro activities of naphthoquinones and artemisinin contained in the extracts obtained from Arnebia densiflora and Artemisia annua plants against peripheral blood mononuclear cells infected with Theileria annulata schizonts. For this purpose, the n-hexane extract of A. densiflora and the petroleum ether and methanol extracts of A. annua were obtained. 
 
Methods: Non-infected and infected cells were cultivated and 2&times;106 cells were seeded in each well in 0.5 ml of RPMI 1640 medium. The n-hexane extract of A. densiflora was tested six times at concentrations of 125, 62.5, 15.625, 7.8125 and 3.9062 mg/ml. A. annua petroleum ether and methanol extracts were also tested six times at 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, and 1.9531 mg/ml concentrations on PBMCs. A Mann-Whitney U test was used to determine differences between control and experimental groups. 
 
Result: The A. densiflora extract killed both non-infected and infected cells at significant levels (P&lt;0.05) compared to the control group at all tested concentrations. A. annua methanol extract had no adverse effect on non-infected cells and the effect on infected cells was insufficient, with the only observed significant effect at a concentration of 125 &micro;g/ml. The A. annua petroleum ether extract had no adverse effect on non-infected cells and killed infected cells at significant levels (P&lt;0.05) at concentrations of 15.625 &micro;g/ml and above at 24 hours. At 48 hours, all infected cells were killed at a concentration of 62.5 &micro;g/ml. In conclusion, A. annua petroleum ether extract can be used in future in vivo studies for Theileriosis at these effective concentrations.</div>

In vitro Effect of Arnebia densiflora and Artemisia annua Plant Extracts against&nbsp;Theileria annulata Schizonts

Seroprevalence and Risk Factor Associated with Endemicity of Theileria equi Infection in Horses in Rajasthan State, India

Prevalence of Theileria equi and Babesia caballi Infections in Horses in South Gujarat, India

Detection of Theileria species infecting equine population in Punjab by 18S rRNA PCR