Prevalence of theileriosis in cross-bred cattle : its detection through blood smear examination and polymerase chain reaction in Dehradun district , Uttarakhand , India

Aim: Theileriosis is a major threat to dairy and related industries. Theileria is a protozoan parasite that causes theileriosis, a fatal disease. The aim of the present study was to assess the prevalence of theileriosis in crossbred cattle belonging to Dehradun district, Uttarakhand, India. Materials and Methods: We screened 301 blood samples each month from apparently normal/healthy crossbred cattle from various locations of Dehradun district. Samples were tested using Giemsa's staining technique and specific PCR test. Result: Microscopic examination of blood smears revealed 27.2% (82) overall prevalence of theileriosis. The highest prevalence was found in rainy season with a prevalence rate 45.4%. By Theileria genus specific PCR test, 98 samples (32.5 %) were found to be positive for theileriosis. Conclusion: Our study suggests that theileriosis is spreading to even the temperate region in Uttarakhand and immediate screening norms/policies are needed to reduce the extent of spread. There is a need for further investigation using molecular technique.


Introduction
crossbred cattle has been reported in 2009 [3].In Northern Kerala 16 % positive cases of theileriosis has Haemoprotozoan diseases cause devastating been reported in crossbred cattle [4].37% cattle losses to the livestock industry and thus pose major reported positive for the haemoprotozoan infection in constraints to the dairy industry throughout the world.
Kaira and Anand Districts of Gujrat [5].Vahora et al [5] In this regard, ticks are mostly related to initiation of also reported that the higher incidence of Theileria many diseases.The hot and humid climate is highly during the monsoon season.A case of tropical favourable for the development and survival of ticks.In theileriosis was also reported from West Bengal in particular, ticks spread Theileria which pose a serious 2012 [6].An outbreak of theileriosis in cattle has been challenge to the exotic crossbred cattle population.
reported from Punjab with 4.86% mortality rate [7].Theileria annulata and Theileria parva are considered The present study was aimed to assess the spread to be the most pathogenic species of Theileria.Tropical of Theileria in Uttarakhand, this paper highlights the theileriosis is one of the most prevalent diseases of problem of theileriosis and its season wise prevalence cattle caused by T. annulata [1] and is transmitted rate in crossbred cattle in Dehradun district of through Ixodid tick of genus Hyalomma.Theileriosis Uttarakhand, India.has serious economic impact in view of mortality and reduced milk yield.Most of the haemoprotozoan

parasites are tick borne and are of great economic
The study was conducted in the Dehradun district importance in Asia and have always been formidable of Uttarakhand, India.Geographic location of barriers to the survival of crossbred cattle in India.In Dehradun is in between 29°58' and 31°2'30" North India the annual loss reported due to tropical land 77°34'45"and 78°18'30"East.The climate of theileriosis is approximately US$ 800 million [2].In Dehradun is moderate due to its location at the foot of India theileriosis has been reported from geographical the Himalayas.The clinical manifestation of regions such as Punjab, Haryana, and Gujarat.In theileriosis recorded in cross bred cattle during the Bangalore north, the occurrence of T. annulata among summer and monsoon season are high temperature, swelling of lymph nodes, weakness, increased respiration and nasal discharge.
Institutional Ethics Committee, Graphic Era Univer-negative control were obtained from clinical case and sity, Dehradun.
healthy cattle diagnosed respectively at Indian Veterinary Research Institute (IVRI), Bareilly, India Collection of samples: A total number of 301 crossbred (from the lab in Division of Parasitology).Positive cattle were screened for haemoprotozoan parasite from control and negative control was run along with the different areas of Dehradun (Raipur, Subhashnagar, other samples.The amplification products were Ajabpur and Selaqui region) Uttarakhand, India subjected to electrophoresis on 1% agarose gel.The (Fig. -1).Blood samples from ear veins of examined length of amplified product was estimated using a base animals were collected for detection of Theileria pair standard (1Kb, Merck) and the amplified products piroplasms microscopically after staining with were visualized with an U.V. transilluminator.Giemsa's stain.Approximately 3ml blood sample was

Results
collected from jugular vein into tubes containing EDTA and stored at -20°C for DNA extraction.All the The study was conducted for 10 months from sampling was done as per ethical standards by a March to December 2012 to identify the seasonal Veterinarian.
prevalence of Theileria in Dehradun district.From a total number of 301 cattle analysed by Giemsa's stained Microscopic examination (ME): Thin blood smears were prepared and fixed with methanol for 5 minutes.
blood smear (Fig. -2), 9.0% cows were found positive These were then stained with 5% Giemsa's solution and for Theileria as per blood smear in spring season, were examined under microscope after 30 minutes.
19.6% were found positive in summer season, 45.4% Blood smears were carefully examined for Theileria were found positive in rainy season and 8.8% were under the oil immersion lens (100 x magnification).
found positive in winter season (Table -1).Highest Even the presence of single piroplasms was considered numbers of positive cases were obtained in rainy positive for Theileria.
season which corresponds to months between July and October (Fig.

DNA extraction and polymerase chain reaction (PCR):
in previous reports (3, 10).Microscopic examination of DNA extraction was performed according to the blood smears revealed 27.2% overall prevalence of standard Phenol-Chloroform protocol [8] and integrity theileriosis. of extracted DNA was checked by agarose gel PCR reactions amplifying a band of 1098bp were electrophoresis.This purified DNA was used as considered positive for the infection (Fig. -4).Besides template for the PCR.The Theileria genus specific 82 positive samples that were identified using staining primers for SSU rRNA gene used in the present study procedure, 16 other samples were also detected as were 989(F) AGTTTCTGACCTATCAG and 990(R) positive for the disease using PCR.These cattle were TTGCCTTAAACTTCCTTG (9).Amplification was categorized as "carrier cattle".Thus, Theileria genus performed in a thermal cycler (Eppendorf AG,5333) specific PCR could detect 98 samples (32.5%) as under the following conditions: 94°C for 3 min (initial positive.Along all the PCR a theileria positive sample denaturation) followed by 25 cycles of 94°C, 30 sec (obtained/confirmed from IVRI, Bareilly) was run.(denaturation), 52°C, 30 sec (annealing), 72°C 1.5 min Positive control sample always showed the requisite (extension) and a final extension of 72°C for 5 min.A positive control sample representing Theileria sp. and band of size 1098 bp.The present study is focused on blood protozoan The demand for milk is increasing in Dehradun, disease theileriosis and its prevalence as well as capital of Uttarakhand, India.Eventually, dairy with identifying the "carriers".This study was carried out Holstein cows has become a big enterprise but for ten months (March to December) and the cases theileriosis and babesiosis have extensive prevalence positive for theileriosis showed that there was a high and high mortality rates due to these cause huge economic prevalence of these haemoprotozoans, especially in the losses, as in several countries [11].Theileriosis is a rainy season, in Dehradun district.Age, sex, breed, tick fatal parasitic disease which is seasonal -starts from the density and season also affect the prevalence rate [14, month of May, and its occurrence increases gradually 15].With the availability of sequenced parasite genes till rainy season.The highest abundance of the ticks and PCR, it is possible to detect parasites within blood was reported in the month of July [12] whereas samples [16].PCR has allowed the development of Hyalomma sp. of ticks is most abundant in June [13].
sensitive and specific diagnostic assays for Theileria Carrier cattle (infected but not showing symptoms) are [16].It is also reflected from our study that the entire usually the chronic carriers of piroplasms.They positively stained samples were confirmed positive by become the source of infection when carrier cattle are PCR but an additional 16 samples were also detected as reared together with healthy cattle.Ticks from carrier positive ("carriers").As Dehradun is a hilly region, it cattle act as vectors and transmit the disease to healthy was previously thought that the occurrence of these cows (cross bred cows, like Holstein Friesian, are more

Figure- 2 .
Figure-2.Microscopic Examination of Giemsa stained blood smear showing piroplasm of Theileria parasite (arrow shows the piroplasm stage of Theileria in erythrocyte)

Table - 1
. Season-wise incidence of Theileria detected in crossbred cattle during March-December 2012