1.1 INTRODUCTION Animal husbandry plays an important role in Indian agriculture. Indians by large are vegetarian and as such the only source of animal protein is milk and milk products. With the increasing population not only food security but also nutritional security is required. In this context milk is valuable part of our nutrition. The total population of cows in India was approx. 199.1 million and the total buffalo population was 105.3 million (18 th Livestock Census, Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture, 2007). But as per 19 th livestock census (19 th Livestock Census, Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture, 2012) the total cow population reduced to 190.9 million. This shows an overall decline of 3.33% in the total livestock population as compared to 18 th livestock census. Cattle reared in India are low producers. On an average the native cow produces 2-3 litres of milk and buffalo milk is hardly on an average 7 or 8 litre per day, whereas cross bred cows produce 15-30 litres of milk per day. During the last decade the dairy industry has evolved towards intensive milk production system to fulfil the demand of the increasing human population. Hence, large numbers of high yielding exotic cattle (Holstein Friesian) are being imported under various integrated development projects throughout the country. Since 70 s the massive cross breeding programme with the semen of high yielding cows of American and European origin has been taken up. The 20% of cattle population, approximately 39.82 million are cross bred cows. There are many cross bred cows which are producing over 5000 litre of milk per lactation. Thus the milk production has initiated jobs, income and foreign exchange generation. This is likely to pick up. However, cross bred cows are very susceptible to heat stress and blood protozoans whereas, the native cows are somewhat resistant. Cross bred cows are sensitive to hot and humid climate. The impact of heat stress on dairy cows is measured by calculating the Thermal humidity Index (THI). It is used to evaluate the climatic conditions that contribute to heat stress [1, 2]. THI is used for estimation of the magnitude of the heat stress by using ambient temperature and relative humidity [3]. Theileriosis and babesiosis are important blood protozoan diseases in cattle that affect the economy of country. Both diseases are transmitted by ticks [4]. Theileria and Babesia both have same symptoms like high fever and
both are fatal diseases. In babesiosis blood comes out with the urine and hence it is also known as Red water disease. Theileria vaccines are not easily available as they are very costly and these vaccines also require liquid nitrogen for its preservation. Theileriosis is a vector borne disease and transmitted by Hyalomma ticks. Different Theileria species are found all over the world in cattle. Arnald Theiler first reported the Theileria parasite [5]. Theileria can exist in erythrocytes and lymphocytes of their host. The taxonomy of Theileria species are based upon morphology and geographic area [6]. The two known pathogenic species of Theileria in cattle are T. parva and T. annulata [7], which cause East Cost Fever and Tropical Theileriosis respectively in tropical and subtropical countries [8]. The mortality rate due to T. annulata is 70-80% while due to T. parva, 100% mortality rate has been recorded in exotic cattle [9]. In calves high mortality has been observed as compare to adult cattle, however if adult cattle survives after infection it becomes immune [10]. In introduced exotic cattle 30 to 90% mortality rate has been observed due to Theileria but in indigenous breed mortality rate is either 5% or less [11]. The Sahiwal breeds of cattle are highly resistant to ticks, which are responsible for transmission of Theileria [12]. However the cross bred cows suffer from blood protozoans which are also known as tick borne infections. Exotic livestock kept even at low tick challenge are highly susceptible [13, 14]. If introduced exotic cattle are kept in the same herd with indigenous breeds, the chances of parasitic infection increases [9]. Babesia is limited to adult animals whereas Theileria affects all ages of cow and it can be transmitted to new borne calf through mother. The total number of death by theileriosis may be 80-90%, if not treated. Theileria annulata (T. annulata) causes tropical theileriosis (also known as tropical piroplasmosis, Egyptian fever or Mediterranean Coast fever) in cattle with a mortality rate from 10-90% [6]. T. annulata is vectored by Hyalomma ticks, which become infected as a larva or nymph and infection is then passed on by the nymph or adult, respectively [6]. In the first step of life cycle of Theileria, sporozoites present in the salivary gland of tick are transmitted into the host with tick saliva while feeding and these sporozoites infect the lymphocytes. Infected lymphocytes start dividing and schizonts (also known as Koch s Blue Bodies) are formed inside the infected lymphocytes converting into the merozoites. These merozoites come out from lymphocytes and enter into the erythrocytes where they are known as piroplasms [15]. Two forms of piroplasms have been observed in
erythrocytes; either they are slender comma shaped or spherical ovoid form [15]. The infective stage is piroplasm stage which is taken by ticks while taking a blood meal from an infected host. After attachment of infected ticks, the first clinical sign of theileriosis appears within 7 to 15 days in cattle. The body temperature increases to 42 C [16, 17]. Anaemia develops in the infected animal followed by loss in health condition. Other clinical signs observed in animals due to theileriosis are lacrimation, corneal opacity, nasal discharge, terminal dyspnea, diarrhoea and sometimes haemoglobinuria is also seen. Cattle infected with T. annulata are generally diagnosed by examining Giemsa s-stained lymph node biopsy smears and blood smears for the detection of macroschizonts and piroplasms respectively [18]. The morphology of piroplasms of Theileria species is more or less similar so it becomes difficult to differentiate them on blood slides by microscopic examination [19]. Serological tests like indirect immunofluorescent antibody test (IFA) are used for detecting antibodies present in the blood of the infected animal [19]. However, IFA test is not successful to detect all infected cattle especially carrier cattle. It also has a drawback i.e. cross-reactivity within closely related Theileria species, which limits the specificity of serological tests [20]. Furthermore in carrier animals the antibodies disappear after a period but Theileria piroplasms remain present in low numbers. Therefore, carrier animals are not diagnosed by this serological test and they are still capable of infecting ticks [21]. The 18S small subunit ribosomal RNA gene is a highly conserved region in the nuclear genome. The small subunit ribosomal RNA gene (SSUrRNA) has high levels of conservation so it is useful for phylogenetic analysis [22]. The small subunit ribosomal RNA gene of T. annulata was compared to SSUrRNA gene sequence of Apicomplexans, Dinoflagellates and Ciliates and a close phylogenetic relationship was observed between Apicomplexa and Dinoflagellata [23]. To differentiate six Theileria species, primers have been designed for small subunit ribosomal RNA gene and it is useful for identification of Theileria infected animals as well as carrier animals [24]. Through microscope only 50% animals can be diagnosed [25]. However PCR based diagnostic test can detect the positive clinical and sub clinical cases. If diagnosed positive, the drug Buparvaquone if given in time can save the animal. But Buparvaquone doesn t give 100% effective results as it is effective only if animal is treated in early stage of infection. In the severe stage of disease it failed to cure the animal [26]. The parasite has the detrimental effect on
the cows, it causes high mortality and the animals that survive have irreversible loss of milk production and reproduction. In carrier animals piroplasms persist in their blood at very low number but they are sufficient to infect the ticks and are not diagnosed easily by the routinely used diagnostic techniques [25]. In the sub clinical cases, the parasite is in the lymph gland and it reduces the production and upset the reproduction. The PCR test once settled and equipment is available, costly animals can be saved by using this technique and increase production and reproduction. Nonimmune cattle that are imported from the places where Theileria is prevalent are susceptible to disease. Theileria infection is a major issue for the livestock development programmes in many places. Cattle of European origin like Holstein Friesian (Bos taurus) are very much susceptible to Theileria, thus it is a big issue for cattle farming [27]. Seasonal activity of Rhiphicephalus appendiculatus was studied in Zimbabwe and Rhodesia and it was found that the adults are highly active in rainy season and larval and nymphal are highly active in dry season [28]. They also found that activity was regulated by influence of temperature, humidity and dry length. Theileria is usually thought as disease of warmer climate and has not been reported in Uttarakhand to the best of our knowledge. Earlier studies suggested that this disease did not occur in the Himalayan regions [29]. It was thought that in Uttarakhand haemoprotozoans were not prevalent. As no sensitive diagnostic method for detection of clinical cases or carrier animals was in use and also no cost effective treatment was available for theileriosis, many of diseased animals were left untreated. Dehradun district of Uttarakhand is located at the foothills of Himalayas. Dehradun is situated between latitudes 29 58' N and 31 2'N and longitudes 77 34' E and 78 18'E. The district is bordered by hills, the Himalayas in the north and the Shivalik Hills to the south. The district is also bordered by the rivers, Ganges to the east and Yamuna to the west. The physical geography of Dehradun district varies from Himalayan Mountains to Plains. The spring here spans from March to mid April. Summer follows the spring season from mid April to June end. Monsoon starts after June and ends in October. By November the winter starts. Transportation of animals occurs here mainly from Haryana, Punjab. In Dehradun district particularly in Dehradun city the cost of milk is more than the cost of diesel, whereas
the fodder production is scanty, not much green are available, and cattle are kept on bhusa. There is a great paucity of land. So there is no space for grazing calves. As such the high producing animals producing over 30 litre of milk per day are kept for making good profit. There is no room for low producers. These high producing animals are mainly crosses of Holstein, which are prone to heat stress and blood protozoans like Theileria. Figure 1.1: Map of Dehradun showing Border States and black arrows show locations of sampling (Source: www.slbcuttarakhand.org.in/images/dehradun.jpg)
Uttarakhand is a relatively new state and many cross bred cattle were introduced to boost the milk yield of the state. As Cross-bred cattle of Holsteins are high milk producers so they are introduced in Uttarakhand, especially in Dehradun region from the neighbour states like Haryana, Punjab etc. where these diseases are prominent and many of these animals may be carriers. These carrier animals are the source of infection. Infection is usually transmitted by ticks that migrate from carrier animals to non-infected animals. Despite all efforts, the milk yield of dairy in Uttarakhand is comparatively low. The problem of heat stress in dairy cattle has recently increased due to anticipated increase in environmental temperature by global warming. Heat stress is very important stress factor and it cannot be indicated only in terms of temperature. When high humidity level coincides with high temperature, it results in oppressive and stressful conditions. The THI is measured by combining temperature and humidity both and has been used to quantify heat stress in dairy animals. Temperature humidity index (THI) was originally developed for humans and extended to cattle. High yielding cross bred cows are more sensitive to hot and humid environment than low yielding cows while the native cattle are well adapted to these environmental conditions. The higher susceptibility of high yielding dairy cattle to heat stress could be explained by the substantial increase in metabolic heat output these cows are experiencing due to the high milk production, which may exacerbate the problem of heat stress in high yielding dairy cows [30]. The exotic Holstein crosses are uncomfortable above the temperature of 25 C. When Heat Stress Index for Cattle is 71 or below, cattle are in the zone of comfort i.e. 72 is optimum, below this animals are in zone of comfort. Values ranging from 72-80 indicate Mild Stress and from 81-90 indicate Medium Stress. When Heat Stress Index values goes above 90 it indicates Severe Stress. Heat stress makes many physiological as well as behavioural changes in animal s body like less feed intake, decline in milk production, reduce growth,
reduction in reproduction activity, increase in respiratory rate and rise in body temperature. Hormones in plasma are potential indicator of the physiological status of a cow. Environmental temperature, especially high temperature, depresses the thyroid function. In heat stressed animals, 25 % lower concentrations of T3 and T4 in blood plasma were observed. It takes several days for T3 and T4 level to return at normal range. The readjustment in thyroid response is chronic and as a result, summer thyroid activity remains low with decreased metabolic rate, reduced feed intake and growth and low milk production under heat stress [31]. Figure 1.2: Thermal Humidity Index chart showing different levels of heat stress at different temperature and relative humidity (Source: www.extension.umn.edu) This study was aimed to assess the occurrence of Theileria in district Dehradun of Uttarakhand using sensitive molecular diagnostic technique polymerase chain reaction (PCR) based diagnostic tool and optimization of thermal humidity index (THI). This DNA technology monitors the presence of parasites, which otherwise are not visible by microscopic examination and in preclinical cases.
1.2 STATEMENT AND GENESIS OF THE PROBLEM: - The Graphic Era University in Dehradun district maintains a herd of 70 cattle and supplies milk. It is also a source of vermicompost. In 2010 some cows fell sick, few cows were treated with antibiotic and three cows died. The blood examination at IVRI and at this lab revealed that these animals were suffering from theileriosis which mainly attacks high yielding cross bred cows but not the native cows. Six cows added from Rajasthan were diagnosed carriers hence affecting the cow population. Microscopic examination of blood smear did not reveal all positive cases as were as the carrier cases. So the molecular diagnostic tool, polymerase chain reaction test was applied to diagnose the true status of the disease. Further the same was applied to different herds in the Dehradun district. To increase the milk production in Uttarakhand many dairy farms have imported high yielding exotic cattle. However milk production usually declined in most of the dairies. Some cows fell sick and some even died. Hence this study was planned to investigate the possible reason for decrease in milk production focussing on blood protozoans and heat stress. 1.3 OBJECTIVES: - The aim of the study was early diagnosis of theileria by polymerase chain reaction in crossbred cows in Dehradun, Uttarakhand. The study has been focused on Uttarakhand with the following objectives- 1. To check/reduce the mortality in cows by diagnosis of blood protozoans. 2. To check the carrier animals (preclinical cases of Theileria). 3. To know the species of Theileria.
4. To find out correlation between Theileria, Thermal Humidity Index (THI) and milk production. 5. To study hormone profile to monitor the degree of stress. 1.4 ORGANIZATION OF THESIS The thesis has been presented in 7 chapters: Chapter 1 deals with the introduction and motivation of the research problem. Livestock production is very important for the improvement in agriculture of India. Indians by large are vegetarian and as such the only source of animal protein is milk and milk products. Cattle reared in India are low milk producers. Hence large numbers of high yielding exotic cattle are being imported throughout the country. There are many cross bred cows which are producing over 5000 litres of milk in 365 days per lactation. But cross bred cows are very susceptible to heat stress and blood protozoans, which may cause 90 % mortality and also lowered milk production. The native cows usually do not suffer from Theileria infection. Haemoprotozoans pose a serious challenge to the exotic cross bred cattle population. Tropical theileriosis is a disease found in cattle, caused by T. annulata and is transmitted through Ixodid ticks of genus Hyalomma. This disease has not been reported from the Himalayan regions as the temperature is not favourable for the survival of ticks. Earlier it was thought that Uttarakhand was free from bloodborne parasites and their vectors. As no sensitive diagnostic method is available and in use for detection of clinical cases and carrier animals of Theileria and also no effective treatment within reach of small dairy holders is available for theileriosis, thus resulting in maximum cases left untreated. Carriers are the source of infection and aid in spread of the disease. Hence this study was aimed at assessment of extent of spread of this disease in district Dehradun of Uttarakhand. The study also aimed at monitoring the heat stress through Thermal Humidity Index (THI) and monitoring the blood protozoan by microscopic examination and by Polymerase Chain Reaction (PCR) which is a recent DNA Technology. This
DNA technology monitors the presence of parasites which is not visible by routine microscopic examination and in preclinical cases. This critical review on Theileria parasite is presented in chapter 2. Theileria is intracellular protozoan parasite. The Theileria parasite was first reported by Arnald Theiler. Theileria are small round, ovoid, irregular or bacilliform shaped parasites. T. annulata was first described in 1904 in Transcaucasian cattle [32]. It usually infects exotic and cross bred cattle. This chapter reviews history, epidemiology, etiology, symptoms, diagnosis and control of Theileria. It also includes reports of theileriosis from different regions of India. Ticks act as biological vectors for the transmission of Theileria species. Rhipicephalus appendiculatus and Hyalomma ticks are the most important vectors for the transmission of T. parva and T. annulata respectively. Female ticks are more responsible for the transmission of Theileria parasite than male ticks. Many cows are apparently healthy but they are carrier of Theileria. Carrier means they harbour parasite and can transfer disease in cows which are not carriers. The carrier animals have developed antibodies and these animals do not suffer from Theileria again but piroplasms remain in their blood in very low number so such carrier animals can t be diagnosed by microscopic examination. However the production of milk and reproduction of such animal is affected. Carrier animals are having low numbers of infected erythrocytes infect ticks and play major role in transmission of the disease through ticks. Therefore, by identifying the carrier animals the spread of infection can be controlled to some extent. However, detection of carrier animals is not easy by microscopy as they have very low number of infected erythrocytes, and also to differentiate pathogenic species from the non-pathogenic species of Theileria is very difficult by microscopic examination. Although serological tests can also be used as effective diagnostic tools by detecting antibodies but these tests are also not 100 % accurate due to cross-reactivity of antibodies of other species [33]. In carrier animals antibodies disappear with time but piroplasms may reside in very low number, therefore these animals show negative serological tests and can still be able to transmit the disease by ticks. Several molecular diagnostic procedures like PCR were developed for the identification of these parasites [34]. If animal suffering from tropical theileriosis are treated with antiparasitic drugs, Theileria annulata does get removed from
lymph nodes but remains in blood in very low number [26]. As DNA sequence of the parasite is known and PCR is available, parasites can be detected within blood samples of the infected animal. PCR has become the sensitive and accurate diagnostic technique for the detection of T. annulata [35]. PCR enables us to detect parasitic infection with clinical or without clinical signs [21]. Exotic cattle are also susceptible to heat stress which is measured by thermal humidity index. This chapter discusses the heat stress, factors responsible for heat stress, effect of heat stress on cattle milk yield and thyroid hormone. In chapter 3 Theileria has been monitored by using microscopic examination technique. This chapter documents the detection of Theileria in cross bred cattle by microscopic examination. On the basis of information collected from different areas of Dehradun district that cows are dying and not responding to antibiotic and usual treatment, the animals were screened for blood protozoan i.e. Theileria which was not prevalent in Garhwal region of Uttarakhand. Blood sampling was done from different locations of Dehradun district. Blood sampling procedure was approved by Institutional Animal Ethical Committee (IAEC). Screening of the animals was done by blood smears examination. The blood smears were examined microscopically using Giemsa stain to detect the occurrence of Theileria. For knowing probable months of the occurrence of Theileria the study was based on season-wise screening of the animals. As per the preliminary survey, in different places of Dehradun like Raipur, Subhashnagar etc. some cases of theileriosis were diagnosed and some carrier animals were established. In this survey 20 positive cases for Theileria out of 58 animals were observed. In the experimental study, 301 cattle were screened for the prevalence of Theileria and 82 (27.2%) were found positive for Theileria by microscopic examination. Highest prevalence was found in the rainy season with prevalence rate of 45.4% followed by summer with 27.4%. In spring season and winter season the prevalence rate was around 9% and 8.8% respectively. Chapter 4 describes the monitoring of theileriosis and carrier cases by using the polymerase chain reaction (PCR) test. The clinical and subclinical cases or carrier cases were subjected to molecular diagnostic tool i.e. polymerase chain reaction. All the samples were examined by PCR test using Theileria specific primer set
989/990 [989(F) AGTTTCTGACCTATCAG and 990(R) TTGCCTTAAACTTCCTTG] for the SSU rrna gene. Identification of Theileria species was made on the basis of polymerase chain reaction based test. PCR positive samples were further diagnosed by using T. annulata specific primer set N516/N517 [N516 (F)-GTAACCTTTAAAAACGT3 and N517 (R) GTTACGAACATGGGTTT]. This is a step forward in saving exotic cattle and making it possible for maintaining exotic cattle for increasing milk production. Thereby it is a thrust area for generation of employment and economy for masses. By using PCR besides 82 microscopically positive samples, 16 other samples were also detected as positive for theileriosis. These cattle were categorized as carrier cattle. So Theileria genus specific PCR could detect 98 samples (32.5%) as positive. Hence PCR technique is more sensitive and accurate. Chapter 5 discusses the effect of heat stress in cross bred dairy cows and its correlation with milk production. To measure the heat stress, the daily temperature and humidity variation for three years were continuously monitored. With the help of these two parameters we calculated the thermal humidity index (THI). To assess the effect of heat stress on milk production 40 cattle were observed. These cattle were divided into two groups i.e. high yielding cattle which were imported from other states and low yielding cattle which were maintained here for the last two years. The daily milk production of all the cattle was measured to verify the effect of heat stress on milk yield. As hormones are the good indicator of stress, we monitored thyroid hormones (T3 and T4) to assess the effect of heat stress on hormone concentration in serum of high yielding cattle by using the enzyme linked immuno-sorbent assay (ELISA) test kits and then correlated it with the thermal humidity index by plotting a graph. The THI rose above 72 in the month of July, till October and in November it decreased. In high yielding cows, once the milk production declined it did not rise. Even after the THI declined there was loss of 8-9 litres of milk per cow everyday and this loss was irreversible. The low yielding cows, producing less than 8-10 litre of milk, were not much affected by increase in thermal humidity index or stress level. As THI increases, milk yield decreases. This decrease in milk production can range from 40-44%. The thyroid hormones level was also observed to be decreased in the summer season when the thermal
humidity index was in stress zone i.e. above 80. When THI value was 72 or below 72, the thyroid hormone levels either increased or remained in normal range. Chapter 6 deals with the general discussion part of the entire study. Theileriosis is a fatal parasitic disease which is seasonal usually starts from the month of May and its occurrence increases gradually till rainy season. The highest prevalence of Theileria was found in the months from August to October as the highest abundance of the ticks was reported in the months of June-July. Carrier cattle (infected but not showing symptoms) are usually the chronic carriers of piroplasms. They become the source of infection when carrier cattle are reared together with healthy cattle. Ticks from carrier cattle act as vectors and transmit the disease to healthy cows (cross bred cows, like Holstein Friesian, are more susceptible to infection). Thermal humidity index was also found high in the months from August to October i.e. above 80. This also causes distress in the animals and lowered the milk production especially in the high yielding cows. However the low milk producers were not much affected by increase in thermal humidity index or stress level. In this connection the thyroid hormone levels were monitored which were found very high during winter months and low in summer months. Low thyroid hormone level in the summer months also shows the degree of heat stress in animals. Chapter 7 concludes the study and recommends the future prospects of the study. This is one of the first reports documenting spread of Theileria in Himalayan region of Garhwal, Uttarakhand. It was concluded that there is outbreak of theileriosis in Dehradun district. Diagnosis of Theileria is done by polymerase chain reaction test. This technique is very sensitive and detects carrier animals. Highest prevalence of Theileria was observed in rainy season when the thermal humidity index was above 80. As the disease is transmitted from the carrier animals introduced from Theileria endemic areas of the country, before their entry they must be quarantined. The high milk producing animals were highly affected by heat stress. Decrease in thyroid hormone level also showed that the animals are affected by heat stress. This chapter also discusses the future prospects of the study. Theileriosis in Dehradun is spreading at a faster rate which is to be tackled immediately.