Chapter I

Introduction

Livestock is one of the most potential sub-sector of agriculture in Bangladesh which plays an indispensable role to promote human health and national economy of the country. Livestock not only assist to upgrade the financial condition but also made a substantial contribution to human nutrition.

Livestock is an integral part of farming system which has a better contribution to enhance the economy of Bangladesh. large ruminants (Cattle and Buffalo) and small ruminants (sheep and goat) constitute the major portion of livestock. The present population of livestock is 22.9 million cattle, 1.2 million Buffalo, 21.5 million goat and 8.78 million sheep (DLS, 2006) 

The total contribution of livestock sub-sector to Gross Domestic Product (GDP) in Bangladesh is approximately 7.23%, livestock in agricultural production 17.325% (Economic review, 2007). It also generates 13% of foreign currency and provides 20% fulltime employment and 50% partial employment of rural population (Alam, 1993). 

The climatic condition of Bangladesh favors the incidence of different diseases. Among parasitic diseases, tick born diseases are considered a major obstacle in the health and product performance of cattle. Ticks comprise a burning veterinary problem because they transmit diseases, induce paralysis or toxicosis and cause physical damage to livestock. (Rajput, 2005). Despite good genetic potentiality, most dairy animals in Bangladesh have considerably low quantity of milk due to lack of disease control.

The hot and humid weather of Bangladesh favors the growth and multiplication of arthropods vector like tick, flies, mosquitoes that are responsible for transmission of different causative agents form diseased animal to healthy animal resulting different diseases including Babesiosis, Anaplamosis, Theileriosis etc. Babesiosis, Anaplamosis and Theileriosis  are distributed world wide, have got a serious economic impact due to obvious reason of death, decreased production and lowered working efficiency and acting as a serious economic challenge, particularly in developing countries (Uilenberg, 1955). These diseases not  only acts as serious constrains in the productivity of animal but also involve in zoonoses (such as Babesia divergens, Babesia microti) (Sparagano, 1999). Prevalence of blood protozoa such as Babesia bigemina, Babesia divergens, Theileria annulata, Theileria mutans and blood rickettsia such as                Anaplasma marginale, Anaplasma centrale has been reported in animals of Bangladesh (Ahmed, 1976; Samad and Gautam, 1984). 

Babesiosis is of economic importance in many countries, 900 million cattle could be at risk (Montenegro- James and James, 1998). Costs are from death and abortion in clinical cases, loss of production in sick and recovered animals, and cost associated with preventive measures such as tick control. The diseases constrain efficient production and in developing countries limit the introduction of susceptible cattle breeds with superior genetics (Blood (1968). Samad (1989) reported the occurrence of sub-clinical Anaplasmosis infection in cattle was 5.93% in Bangladesh.

Therefore, epidemiological study should be undertaken to investigate present status of blood parasites in cattle in Bangladesh at upazila or thana level.. Considering the above mentioned facts, the present study was conducted with following objectives:

•	To measure the prevalence of haemoprotozoal infection at Noakhali Sadar Upazila, Noakhali.
•	To identify the different risk factors associated with the haemoprotozoal infections.      

Chapter II
Review of literature
Babesiosis

Babesiosis is a tick-borne disease of cattle, sheep, goat and swine caused  which is characterized by fever, erythrocyte destruction resulting anemia, anorexia, haemoglobinuria in certain instances icterus occasionally central nervous system involvement and death in severe cases (Daniel, 1985).

Babesia sp caused Texas fever, an acute febrile condition of cattle that occurred in the southern part of USA (Simth and Kilborne ,1893).

Babesiosis is caused by intra-erythocytic protozoan parasites of the genus Babesia. The disease, which is transmitted by ticks, affects a wide range of domestic and wild animals and occasionally humans (Marck Veterinay Manual, 9th edition).

Etiology of Babesiosis in cattle: 

Babesiosis in cattle is caused by Babesia bovis (includes, Babesia argentiana, Babesia berbera), Babesia bigemina, Babesia major, Babesia divergens, Babesia ovata (1), babesia ovata oshimensis (n.var) (2) (Blood et al., 1968).

Two important species in cattle causes Babesiosis namely Babesia bigemina and              Babesia bovis (Marck Veterinay Manual, 9th edition).

Babesia sp. has been described in all of the domestic animals and ticks were the only vectors and in cattle, it is caused by, Babesia argentiana, Babesia berbera, Babesia bigemina,     Babesia bovis, Babesia divergens, Babesia major  (Ristic, 1981).

there has been much confusion in referring to the various tick fever diseases. thus, in the literature of the world there is a tendency to call the organisms as Babesia argentina and Babesia bigemina (Hungerford, 1962).

the causes of Babesiosis in cattle was Babesia bigemina, Babesia bovis, Babesia major    (Hugan et al., 1960).

There were six species of Babesia have been identified from cattle. these are                    Babesia argentiana, Babesia bigemina, Babesis bovis, Babesia divergens and  Babesia majo. (Soulsby, 1968).								

Babesiosis in cattle is primarily caused by 4 Babesia spp. The most important species are Babesia bovis and Babesia bigemina, Babesia divergens and Babesia major (Daniel, 1985).

Epidemiology of Babesiosis:

Geographical occurrence:

Babesiosis frequently range over wide areas exposed to different climatic condition and the combination of large numbers of animal with variation in the density of  vector population pre  disposes to the occurrence of  severe outbreak of diseases (Kreier, 1993).

the distribution of the causative protozoa is governed by the geographical and seasonal distribution of the insect vector transmits them. In general terms, Babesia bigemina and Babesia bovis are infections of the tropics and sub tropics. Babesia major and Babesia divergens occurs in South America, the west Indies, Austrilia and Africa; Babesia agrentina in the tropics including  South and Central America, Austrilia, Asia and Southern Europe. Babesia divergens occurs in North-West Europe, Spain, are the principal cause of Babesiosis in the United Kingdom. Babeisa bovis occurs in Europe, South America and Africa. Babesia berbera in Mdeiterranean Europe and North Africa; Babesia major in the United Kingdom and Europe (Blood et al., 1968).

Babesia bigemina and Babesia bovis, both the agents occur in Europe, Africa, Austrilia, Asia and Central South America (Ristic, 1968).

Mode of infection/Transmission: 
Ticks are the natural vectors of Babesiosis; the causative parasites persist as pass through part of their life cycle in the vertebrate host. Both Babesia bovis and Babesia bigemina pass part of their life cycle in the Boophilus microplus & Boophilus annulatus and Boophilus microplus are the major vectors of Babesiosis. Ixodes ricinus is the common carrier of Babesia divergens in UK (Blood et al., 1968).

Babesia bovis and Babesia bigemina  both are  world wide  distributed including large parts of Africa, Southern Europe, Southern Asia, Austrilia and Central and South America as well as many islands in the Caribean and South pacific. Their distribution corresponds to the occurrence of their 1 hostic tick vectors Boophilus microplus, Boophilus decoloratus and               Boophilus annulatus  (Daniel, 1985).

Babesia organisms are transmitted by tick only and mechanical transmission does not occur. Distribution of Babesia bovis and Babesia bigemina corresponds to the occurrence of their 1 host-tick vectors Boohilus microplus, Boophilus decoloratus and Boophilus annulatus. Babesia divergens, transmitted by 3 host-tick  Ixodes recinus and Babesia divergens is transmitted by 3 host tick Haemaphysalis punctata. Depending on the Babesia spp. and tick species, larvae, nymphae, or adults of the next generation transmit the infection (Daniel, 1985).

Under natural condition, the Babesia species are transmitted by ticks which were first demonstrated by Smith and Kilborne (1893). sexual reproduction occurred with               Babesia bigemia in the tick Boophilus annulatus (Dennish, 1932).

development and transmission of Babesia spp. in tick is either by transovarian transmission (Babesia bigemina) or by state to stage transmission (Babesia bovis). The main vectors of Babesia bigemina and Babesia bovis are 1-host Boophilas spp. Ticks, in which transmission occurs transovarially (Soulsby, 1968).

Babesia bigemina transmitted by Boophilus microplus. (Reik,1964).

Certain developmental forms in the stomach of the arthropod have been followed, but nothing further is known except that some from of the parasite is carried from the adult female tick through her eggs to the larval form, which when feeding upon the blood of a bovine, introduces the infection into the host (Hagan et at., 1961).

Risk factors:
Host factors:

All ages of cattle are equally susceptible to Babesia bigemina but Zebu (Bos indicus) and Afrikaner cattle have a higher resistance to Babesis bovis  (Blood et  al., 1968).

European (Bos taurus) sanga, Zebu (Bos indicus) breeds are all susceptible to Babesiosis but Bos indicus and their cross with Bos taurus often possess appreciable resistance. (Daniel, 1985) 
Age:
There is a variation in susceptibility to the infection calves under 2 months of age from previously unexposed cows are highly susceptible to the infection and its effect, whereas the offspring of immune mother are resistant. After the age of 2 months, all calves developed a natural, nonspecific, innate resistance that persist for at least a further 4 to 6 months and is not depended on immune status of the cow. (Daniel, 1985)
It is frequently stated that there is an inverse age resistance to Babesia infection in that young animals are less susceptible to Babesiosis than other animals  (Urquhart et al., 1996).

Calves and foals from infected dams receive antibodies via the colostrum and this passive immunity persist form 3-4months after birth. The greatest infection rate is in animal in the 6-12 months of age group and infection is uncommon in animal over 5years of age. Animal under 1 year of age are infected predominantly with Babesia bigemina and those over 2 years of age by Babesia bovis. (Blood et al., 1968). 

Environmental factors:
There is a seasonal variation in the prevalence of clinical Babesiosis. The greatest incidence occurring soon after the peck of the tick population. Of the climatic factors, air, temperature is the most important because of its effect on tick activity, higher temperature increases it, humidity and rainfall have little effect and even with temperature the effect is limited once a threshold of 7 – 100C  minimum temperature is exceed (Blood et al., 1968).

In endemic areas, where there are many infected ticks, the immunity of the host is maintained at a high level through repeated challenge and overt disease is rare. In contrast, where there are few ticks or when they are confined to limited areas, the immune status of the population is low, and the young animal receive low little, if any, colostral protection (Urquhart et al.,1996).
.
Other factors:
In housed cattle, the level of antibodies in the patient are at their lowest when the cattle comes out of the barn in the spring and gradually increases as they are exposed to vectors ticks. In enzootic areas, the animals most commonly affected by clinical diseases are susceptible cattle introduced for breeding purposes, for slaughter, or in transit, other stress like parturition, starvation or concurrent disease. Break down of immunity are likely to occur if there is a superimposed infection with different parasites especially Anaplasma marginale                (Blood et al., 1968).

Endemic (enzootic) stability is achieved in area where all calves  are frequently exposed to the parasite while they are still protected by colostral and innate immunity (i.e. first 6 to 9 months of age) and endemic instability occurs if some animal fail to become infected for prolong period after birth. Various factors such as changes in climatic condition and frequency of acaricidal treatment can influence the tick population (Daniel, 1985).

Diagnosis:
diagnosis can be done by clinical signs, the history, and the geographic regions are often suggestive of Babesiosis Therefore, microscopic demonstration of the parasite is necessary to confirm the diagnosis. Babesia bovis is a “small” Babesia measuring up to 2µm in diameter. Single organisms are rounded, oval, or irregular in shape; paired form is pyriform or club-shaped with an obtuse angle between the paired merozoits. Large forms of Babesia bovis are, however, not uncommon (Howard, 1985). 

diagnosis of existing babesiosis in clinically affected animals of all species depends on the demonstration of protozoa in a Giemsa-stained smear. venous blood may give a false –negative in Babesia bovis infcctions. in Babesia bigemina infection, protozoa are numerous in peripheral capillaries.  As the Babesia bovis are found infrequently in the thin blood smear and this difficulty can be largely overcome by the use of thick blood smear (Blood et al., 1968).

diagnosis of Babesiosis is based on the clinical signs and confirmed by the detection of parasites in the peripheral blood. Both thick and thin blood smears may be employed, being stained by one of the Romanowsky stains; however, the organism may not be apparent, and it may be necessary to examine a number of smears to establish their presence. In the cerebral form, examination of cerebral capillaries is necessary  (Soulsby, 1968). 

Examination of stained blood films shows the organism to be within red cells, almost always singly or as pairs, often arranged at a characteristic angle with their narrow ends opposed. Typically they are pyriform, but may be rounded, elongated or cigar shaped. Conventionally, the various species are grouped into the small Babesia whose bodies are 1.0-2.5µm long and Large Babesia which are 2.5-5.0 µm long.  Babesia bigemina, a large Babesia whose piroplam measures 4.5X2.0 µm and Babesia bovis, a small Babesia, whose piroplam measures 2.0X1.5 µm (Urquhart et al., 1996).

Babesia bigemina is a large piroplam, 4-5 µm in length by about 2 µm wide, round forms 2-3 µm in diameter. The organism is characterized by pear shaped and lie in pairs forming an acute angle in the RBC, round , oval or irregularly shaped forms may occur, depending on the stage of the development of the parasites. A small  piroplam, 2.4 µm by 1.5 µm and slightly larger than Babesia divergens. In contrast to Babesia divergens , there are usually no divergent form lying superficially in the RBC, and vacuolated signet ring forms are particularly common        (Soulsby, 1968)

Babesia bigemina is larger, around 4 to 5 X 2 to 3 µm, and can extend the full width of an erythrocyte. Single forms of Babesia bigemina are elongated or amoeboid in shape. Paired forms are piriform with an acute angle between the meroziotes. Babesia divergens is a piriform, paired or club-shaped organism, smaller than Babesia bovis and is around 1.5 to 2.0 X 0.4µm.it frequently occurs on the margin of red blood cells. Babesia major is very smaller to but slightly smaller than Babesia bigemina (Howard, 1985).

Serological test:
A variety of serologic tests have been used to demonstrate the presence of antibodies of   Babesia spp. The most commonly used include the Complement Fixation Test, indirect Fluorescent antibody, indirect hemagglutination, radioimmunoassay, and Enzyme Linked Immunosorbent Assay (ELISA) tests. Of these, indirect Fluorescent antibody is the most popular.

Immunodiagnostic tests are increasingly used to detect infection, especially in the sub-clinical situation when organisms are not demonstrable in the blood. The provision of antigen free from host protein contamination presents problems, but Godman and Bukovsky (1975) prepared a soluble antigen from parasite-stroma suspensions Babesia bigemina for the use of immunodiagnostic tests. The other diagnostic tests are available including Complement Fixation Test (Mahoney,1962), the indirect Fluorescent antibody test (FAT) (Ross and Lohr, 1968) .

The most –used in bovine Babesiosis is a Complement Fixation Test. Other tests include a passive agglutination test, an indirect Fluorescent antibody test, an Enzyme- Linked Immunoabsorbent Assay (ELISA). A microplate enzyme  immunoassay (EIA), a Latex agglutination test, a capillary agglutination, a slide agglutination  and a card agglutination test. All of the tests have good reputations, with the EIA being probably the most sensitive. The most recently introduced tests are an ELISA suing a recombinant Babesia bovis antigen. PCR and a DNA probe, which can detect specific parasitemias at very low levels of infcction. the DNA probe has the added advantages of being able to detect protozoa in necropsy specimens and in tick tissues. The PCRs are most useful because of their high sensitivity, which makes them identical for the detection of carrier animals(Howard , 1985).

Examination using a DNA probe may be a satisfactory solution where those facilities are available. Diagnosis of past present infection is adequately demonstrated by any one of a wide range of serological tests. Because of the difficulty in finding protozoa in smears in animal during the sub-clinical stages of the diseases, especially in surveillance studies of the detection of the infection in herds or areas, much attention has been directed to serological tests. There are now well established, but none of them enjoys a completely satisfactory reputation            (Blood et. al.; 1968)
 
Necropsy findings: 
In acute cases of Babesiosis in all species, in which patients die after a brief illness and during an anemia crisis, the typical lesions are jaundice, thin watery blood, pale tissues, and enlargement of the spleen which has a soft, pulpy consistency, and gross enlargement and dark brown discoloration of the liver. The gall bladder is distended with thick, granular bile, the kidneys are enlarged and dark, and the bladder contains red brown urine. Ecchymotic hemorrhages are present under the epicardium and enocardium, and the percardial sac contains and increased quantity of blood stained fluid. Characteristic In sub-acute or chronic cases of fairly long duration, the carcass is emaciated but haemogobinuria is absent; the other changes observed in acuter cases are present but less pronounced (Blood et al., 1968).

Post-mortem  changes associated with Babesiosis include watery blood, a pale carcass, icterus, splenomegaly, hepatomegaly (yellow brown in color). Swollen dark-coloured kidneys, partly congested lungs, lung edema (more common  in Babesia bigemina infection), dark red urine due to hemoglobinura (more pronounced in  B. bigemina infection). In case of cerebral Babesiosis, the gray matter of the cerebrum and cerebellum is congested and has a characteristically cherry pink color. In sub-acute cases, the carcass is pale and icteric with varying degrees of swelling and congestion of the internal organs (Howard, 1985).   

Differential Diagnosis:
Theileriasis is very similar clinically and the differentiation can be done only by absent of coffee color urine and presence of petechial haemorrhage in vaginal mucous membrane                (Blood et al., 1968).

Post parturient haemoglobinuria does not require the presence of vectors, occurs only in most recently calved cows in full milk and on low phosphorous diets. (Blood et al., 1968).

Bacterial Haemoglobinuria characterized by a necrotic infarct under the diaphragmatic surface on the liver in cattle grazing lush pasture. (Blood et al., 1968).

SMCO poisoning occurs only in cattle grazing crops of rape or other Brassica spp. (Blood et al., 1968)

Leptospirosis occurs only in this form of the diseases in calves kept in unsanitary conditions which are wet underfoot. Diagnosis of this disease depends on isolation of the leptospires. (Blood et al., 1968).

Anaplamosis:
Bovine anaplasmosis is an infectious vector borne disease of cattle caused by obligate inta-erythrocytic rickettsia like organism of the genus anaplasma. The disease is generally characterized by fever, inappitence, a drop in milk yield, progressive anemia, icterus and constipation (Wilelm, 1985)

The disease is characterized by fever, severe anemia, jaundice, brownish urine, loss of appetite, dullness or depression, rapid deterioration of physical condition, muscular tremors, constipation, yellowing of mucous membrane and labored breathing (Bram, 1983).

Anlaplasmosis, formerly known as Gall sickness, traditionally refers to a disease of ruminants caused by obligate inta-erythocytic bacteria of the order Rickettsiales, family Analplasmataceae, genus Anaplasma. Cattle, sheep, goat, buffalo, and some wild ruminants can be infected with the erythrocytic anaplasma. (Marck Veterinary Manual, 9th edition)

Etiology of Anaplasmosis:
Anaplasma marginale is the causative agent in cattle and wild ruminants.   Anaplasma centrale is closely related to Anaplasma marginale and causes mild Anaplasmosis in cattle             (Blood et al., 1968).

There are many Anaplasma species parasites but Anaplasma marginale and Anaplasma centrale are the most important species (TFRC, 1996). Bovine anaplasmosis is usually caused by Anaplasma marginale (Bram, 1975; Kocan et al., 2000) and A. centrale can induce a moderate degree of anaemia, with clinical outbreaks in the field being extremely rare                         

Three spccies of the causative organisms occur in cattle Anaplasma marginale,           Anaplasma centrale, and Anaplasma caudatum. Anaplama centrale, which enjoys a far more limited natural distribution than does Anaplasma marginale, is less pathogenic and responsible for a relatively mild form of the disease in cattle. However, Anaplasma centrale  may cause severe clinical reaction is susceptible adult cattle and should not be disregarded  (Wilelm, 1985).

the three Anaplasma  species Anaplasma marginale is the most pathogenic for cattle. Anaplasma centrale  causes a relatively mild form of Bovine Anaplasmosis in Africa. Clinical bovine Aaplasmosis is usually caused by Anaplasma marginale. Cattle are also infected with Anaplasma centrale, which generally reusts in mild disease (Ristic , 1968).

Anaplasma marginale,  a small spherical body located in the stroma of erythocyte and Anaplasma centrale, a relatively non-pathogenic species located centrally in the erythrocytes are responsible for anaplasmosis in cattle (Fraser,1991) 

Epidemiology of Anaplamosis:
Geographical occurrence:
Anaplasmosis in cattle is common on all six continents, being present in South Africa, Austrilia, Asia, the former USSR, South America and the united states  (Blood et al., 1968).

Anaplasmosis caused by Anaplasma marginale has been reported from most tropical and subtropical parts of the world. It occurs in Europe bordering on the Mediterranean, the Middle and far east, South America, the Caribbean, the Soviet Union, Austrilia, North America, and large parts of the African Continents (Wilhelm, 1985).

Bovine Anaplasmosis in India is an endemic disease and sub clinical infection and mild clinical cases occur in indigenous cattle. At times the animals show clinical diseases under stress of certain intercurrent diseases, inclement weather, pregnancy and lactation. The exotic and to lesser extent crossbred animals are fully susceptible. The disease causes direct losses due to prolonged period of convalescence, low productivity and mortality (Gautam, et al., 1982).

Anaplasmosis is transmitted b y a diverse group of biological and mechanical vectors. Infection in cattle is endemic in tropical and subtropical areas that support large population of these vectors. Infection occurs sporadically in temperate climate areas (Blood et al., 1968).

the occurrence of sub-clinical anaplasmosis in cattle was 5.93% in Bangladesh. Anaplamosis occurs in tropical and subtropical areas throughout the world and is a major constraint to the cattle production in many countries. (Samad et al., 1980) 

Mode of infection/Transmission:
At least 20 tick species have been shown experimentally to be capable of transmitting Analplasmosis. These include argasid ticks (Ornithodorus ssp. And Argas persicus) and a number of 1 host, 2 host and 3 host Isodid tick species of which Boophilus sp., Dermacentor spp. and Rhipicephaus spp. are regarded as the  most important. Trans-stadial (larva to nymph or nymph to adult) and intrastadial transmission (e.g. tranfer of male ticks both hosts) appear to be the usual mode of transmission employed by both 1-host and multi host ticks. Mechanical transmission of Anaplamosis by hemaophagous arthopods, blood  sucking flies as horse flies (Tabanus spp.), stable flies (Stomoxys spp) is considered important in the maintenance of infection, even in the absence of tick vectors. Flies of the genera Musca, Chrysops, and Siphora and eye gants (Hippelates spp.) have also  reported that mechanical transmission can also take place by iatrogenic means, through the unsanitary use of instruments employed for vaccination, dehorning, castration, and tattooing that become contaminated with fresh infected blood (Wilhelm, 1985)

Anaplasma is one of the most important parasites transmitted by at least 20 ticks species, including Argas persicus, Ornithodoros lahorensis, Boophilus annulatus, B. decoloratus, B. microplus, Dermacentor albipictus, D. andersoni, D. occidentalis, D. variabilis, Hyalomma excavatum, Ixodes ricinus, Rhipicephalus bursa, R. sanguineus and R. simus (Marchette and Stiller, 1982) but mostly Boophilus microplus causing Anaplasmosis (TFRC, 1996). Various other biting arthropods have been implicated as mechanical vectors. Experimental transmission has been demonstrated with a number of species of Tabanus (horseflies), and with mosquitoes of the genus Psorophora (Ristic, 1968).

experimental and epizootiological evidence incriminates horse flies (Tabanus sp) as the most significant insect vector of Anaplamosis. Transmission by flies is affected by direct transfer of blood from infected to susceptible cattle and must take place within a few minutes after feeding on an infected animals.  (Friedhoff and Ristic ,1996) 

It is reported that spread from animal occurs chiefly by insect vectors. A variety of arthropods may act as vectors  but significant natural vectors are ticks in the family Ixodidae and flies in the family Tabanidae. Of ticks, the one-host Boophilus spp. are major importance in tropical and subtropical regions and three-host Dermacentor spp. of major importance in the western US. (Blood et al., 1968)

Numerous species of tick vectors (Boophilus, Dermacentor, Rhipicephalus, Ixodes, Hyaloma and Ornithodoros) can transmit Anaplasma spp. After feeding on an infected  animal, intrastadial or trans-stadial transmission may occur. Transplacental transmission has been reported and is usually associated with acute infection of the dam in the second or third trimester of generation. Anaplasmosis may  also be spread through the use of contaminated needles or dehorning or other surgical instruments (Marck Veterinary Manual, 9th edition).

Transmission by blood sucking flies is well recognized and Tabanids deer flies, stable flies and mosquitoes are the insects chiefly concerned. Direct transfer of infected blood must take place for insect transmission and this must occur within a few minutes after feeding on an infected animal. Mechanical transmission of Anaplasmosis is wel know and major and minor operation operations in cattle husbandry such as dehorning, castration, vaccination, blood sampling etc. may be resposisble for the transmission of Anaplasmosis both in  and out of season         (Soulsby, 1986)

Risk factors:
Susceptible host
Bos indicus, Bos taurus and their crosses have equal susceptibility to infection and show the same age susceptibility, but under field condition Bos indicus are not as commonly affected, probably because of their relative resistance to heavy tick infestation. Breeds with black or red coat color have a higher risk of infection than those with white coats in regions where biting flies are the insect vectors. Dairy breeds be at greater risk for iatrogenic transmission          (Blood et al.,1968). 

Although Anaplamosis is essentially a disease of cattle, potentially affecting animals of any age, breed, or sex, Anaplasma spp. are known to also infect a wide range of North American and African wild ruminant species. The importance of free living wild ruminants as reservoir hosts of Anaplasma spp. pathogenic to cattle has not been fully investigated (Wilhelm, 1985).
 
age of infection:
All cattle are susceptible to infection but age at infection is a major determinant of the severity of clinical disease. Young calves are susceptible to infection but seldom show clinical signs. Infection between six months and three years of age has increasing risk of clinical illness and animals infected after 3 years of age are commonly affected by a per acute fatal form of the disease (Blood et al., 1968).

Cattle of all ages are affected by Anaplasmosis. Calves born to immune or non-immune dams an innate resistance protecting them from severe clinical disease. The non specific resistance is probably greatest at the age of 6 months and tends to diminish with increasing age of the animal. Calves up to age of 9 month or even 1 year usually show no clinical signs or develop only  a mild form of the disease, whereas animal between 1 year  and 2 years of age may develop acute, but rarely fatal, disease symptom. Acute and occasionally fatal infections occur in cattle up to 3 years of age; in cattle older than 3 years, the disease is often per-acute and frequently fatal (Wilhelm, 1985)

Nutritional status
Clinical disease is less severe in cattle on a low plane of nutrition. Exposure of infected, clinically normal animals to devitalizing environmental influences, particularly shortage of feed, and the presence of other diseases, may result in the development of acute introduced into outbreaks among them are not uncommon 2-3 weeks after entry (Blood et al.,1968).

Season
In temperate climates a seasonal occurrence of disease occurs in association with seasonal occurrence of the insect vectors. Winter outbreaks are likely associated with iatrogenic transmission or possibly the winter tick (Blood et al., 1968). 

Diagnosis:
Tentative diagnosis can be made on the basis of geographical region, history, clinical signs, and post mortem lesions. A definite diagnosis of anaplasmosis depends on the demonstration of Anaplasma bodies in erythrocytes of clinically affected animals. In Giemsa-stained blood smear, the organisms appear as homogenous, dense, deep purple, more or less round bodies 0.3 to 1.0m in diameter. In  Anaplasma marginale infection, approximately 80 to 90 percent of parasites are situated it or near the periphery of RBC (Wilhelm, 1985).

During the acute stage of Anaplasmosis, the diagnosis is made on the basis of clinical symptoms, hematological changes and microscopic examination of stained peripheral blood films for intraerythrocytic inclusion bodies. Giemsa staning is the oldest and most frequently used method. Other staining methods include toluidine blue and acridine orange. The latter method an ultraviolet microscope for visualization the marginal anaplama bodies. In contrast to the ease with which acute form of anaplasmosis are recognized.(Ristic, 1981).

Anaplasma organism can be diagnosed by the blood smear prepared from the peripheral blood. Diff-Quick staining of blood smears is as accurate as Giemsa in the detection of Anaplama spp. and can be cmpleted in 15 seconds as compared to nearly an hour for Giemsa                     (Blood et al., 1968). 

Various soluble and corpuscular antigens extracted form the blood of infected animals has been used for serologic diagnosis of Anaplasmosis. Currently used tests are compliment fixation (CF), Capillary Tube Agglutination (CTA) and card agglutination (CD) (Ristic, 1981).

Anaplasma organism can be diagnosed by the blood smear prepared from the peripheral blood. Diff-Quick staining of blood smears is as accurate as Giemsa in the detection of Anaplasma spp. and can be completed in 15 seconds as compared to nearly an hour for Giemsa                   (Blood et al., 1968). 

The complement fixation test is the standard test for the detection of carrier animals. It is satisfactory for use in cattle, goat and sheep but antibody titer is highest during he active phase of the disease. A rapid card agglutination test, which tests serum or plasma for antibodies against Anaplasma marginale,  is cheap and quick, and sufficiently accurate to be used as a herd test. Other serological tests like a capillary tube agglutination test, indirect fluorescent antibody test, a dot ELISA  are also used for the detection of Anaplasma spp. Nucleic probe analysis can be used to detect low level of parasitema. (Blood et al., 1968)

It was reported that  a simple test that seems to have some value the diagnosis of anlaplasmosis. The blood is allowed to clot a little clear serum is obtained. Two drops are added to 2ml of distilled water in a tube. The serum of normal animals does not cloud the water, that of affected with Anaplasmosis causes an immediate clouding and after that tube stands overnight, a white precipitate covers the bottom. Animals affected with acute Anaplasmosis and recently recovered carriers give this reaction. The test depends on the precipitation of eugloin, which appears to be present in increased amount in this disease  (Boynton and woods, 1937). 

Post-mortem findings:
The most obvious findings are emaciation, pallor of the tissues, and thin watery blood. There is mild jaundice and the liver is enlarged and deep orange in colour.The kidneys are congested and there may be myocardial hemorrhages. The spleen is enlarged with soft pulp. The bone marrow cavity may be redden by increased hematopoietic tissue in acute case but there may be serous atrophy of marrow fat in chronic case (Blood et al., 1968). 

Gross pathological changes are typical of anemia in which erythrocytes are removed by the reticuloendothelial system. The prominent changes are icterus mucous membrane, enlarged spleen, and obstructed gall bladder. Petechial haemorrhage may be observed on the epicadium and pericardium, and the heart is usually pale and flabby. The liver may be mottled yellow or brown, hepatic and mediastinal lymph nodes are brown, moderately and moist on section (Ristic, 1981)

The tissue appears anemic or icteric in advanced cases and blood appears thin and watery. The colour of the mucous membrane range form pale to yellow. Emaciation, dehydration, and serous atrophy of fatty tissues may also be present. The spleen and liver is enlarged liver appears as yellowish brown color. The gall bladder appears enlarged and distended with thick yellow green bile. The colon and rectum contain dry and hard faces covered in a variable amount of thick mucus, degenerative changes found in kidneys  (Wilhelm, 1985).

Theileri0sis
Etiology:

Theileriosis are those tick-borne protozoan diseases caused by Theileria spp. in cattle, sheep and goats as well as in wild and captive ungulates. The diseases are characterized by fever and lymphoproliferative disorders which may associate with leukopenia and/or anemia. Theileria parva and Theileria anulata cause East Coast fever (ECF) and tropical Theileriosis (Mediterranean Coast fever) respectively.  Theileria orientalis responsible for oriental Theileriosis/Benign Theileriosis, a mild disease characterized by moderate to severe anaemia in heavily parasitized cattle (Blood et al., 1968). 

Theileriasis are a group of tick borne diseases caused by Theileria spp. the most important species are Theileria parva and Theileria anulata which cause widespread death in cattle in tropical and sub-tropical areas of the world (Merck Veterinary Manual, 9th edition.).

The disease caused by several species of Theileria which is transmitted by ticks             (Urquhart et al. 1996)

Theileriosis is caused by protozoan parasites of the genus Theileria, among food animals, the disease occurs only in ruminants. In cattle, 2 highly pathogenic species, Theileria parva is the cause of East Coast Fever in East and Central Africa. Theileria annulata causes tropical Theileriasis, which is widespread through Mediterranean basin. Theileria orientalis (Theileria buffeti)  is generally benign species found in Mediterranean basin, the middle east, Asia and Austrilia may cause disease in imported cattle (Wilhelm, 1985)

Theileria causes the disease East Coast Fever or Bovine Theileriosis which is responsible or  high mortality among susceptible and imported stock. The zebu (Bos indica) in  endemic areas has a high natural resistance to Theileria parva; however animals imported into endemic areas are highly susceptible. Theileria anulata, the organism produces a highly fatal diseases of cattle known as Tropical Theileriasis  (Toit, 1918).

Epidemiology
Geographical occurrence
Within an enzootic area, Theileriosis is primarily a problem introduced non adapted animals, in which it may cause heavy mortality. In local, adapted animal, infection is widespread in young stock. But a degree of innate resistance usually limits mortality to a low level. East Coast Fever caused by Theileria parva mostly occurred in East & Central Africa. Theileria anulata  causes tropical Theileriosis, which is widespread through the Mediterranean basin, the middle East, and Asia (lawerence, 1985)

Theileria spp are found through the world. East Coast fever is mainly distributed in East and Central Africa, Tropical Theileriasis is distributed in Mediterranean countries, indo-china, Oreintal Theileriasis is found in Asia and Benign Theileriosis is cosmopolitan                    (Blood et al., 1968). 

Mode of infection / Transmission:
Theileria parva sporozoites are infected into cattle by infected ticks, Rhipicephalaus appendiculatus  during feeding (Merck Veterinary Manual, 9th edition.).

Theileria parva is transmitted by Rhipicephalaus appendiculatus  and Theileria annulata by Hyalomma anatolicus during feeding. Other species like Theileria buffeli (Theileria orientalis). Theileria mutans are transmitted by Haemophysalis longicornis / Hyalomma punctata and Amblyomma sp. (Blood et al., 1968). 

All species of Theileria  are transmitted by multihostic ticks. Transmission by the tick in all species occurs form one stage to the next and not transovarially from one generation to the next. When a larva or nymphal tick feeds on an infected animal, it ingests parasitized erythrocytes.The principal vectors for the transmission of Theileria parva is Rhipicephalaus spp.,especially Rhipicephalaus appendiculatus, Theileria anulata  by Hyalomma spp. especially Hyalomma d. detritum (Mediterranean basin) and Hyalomma a. anatolicum (Asia and North Africa). Other species like Theileria mutans and Theileria orientalis carried by Amblyomma spp. and Haemaphysais spp. respectively (lawerence, 1985).

Risk factors
The most important risk factors related to the presence of the brown oat tick in a given area and teh levelof tick burden per animal. At low infestation rates, an average of five ticks per head (two to three ticks per ear\0 will sustain endemicity; ne to four per head will invite epidemicity; while an average of less than one can allow sporadic out breaks (lawerence, 1985).

Environmental factors
Areas that are too high or too cold or dry will not allow the tick to undergo more than one life cycle in a year there by reducing the period f transmission of theilerial parasites by the nymhs or adults.ever, round, oval, comma and ring-shaped forms may also occur (lawerence, 1985).

Diagnosis:
Clinical signs: 
In sick animals, macroschizonts are readily detected in biopsy smears of lymphnodes and in dead animals in impression smears of lymphnodes and spleen. In advanced cases, Giemsa stained blood semars show piroplasm in the red cells, up to 80% of which may be parasitized. (Soulsby, 1986)

 In  erythrocytes the piroplasm are predominantly  rod-shaped and up to 2.0µm long and 1.0 µm  wide. Round, oval and ring shaped forms also occur. With the Giemsa stain, the cytoplasm of each is bule with a red chromatin dot at one end (Blood et al., 1968). 

Diagnosis is based on the demonstration of parasites in the red blood cells or in smears of material obtained from lymphnodes or spleen. Theileria parva in red blood cells are mainly rod shaped, 1.5-2 µm by 0.5-1 µm. However, rounded, oval, comma and ring shaped forms may occur. In case of Theileria annulata, the proplasm in the RBC are more or less in distinguishable form those of T. parva but commonly occur as round, oval or ring shaped (0.5 – 1.5 µm) forms. (Soulsby,1986).
Post mortem findings:
Post mortem findings in case of consists of markedly enlarged spleen and liver, there are infacts in the kidnes, the lungs are usually oedematous, and the lymphnodes may be sollowen. (Soulsby, 1986)

 Chapter III

Materials and Method
Study area:

The study was carried out at different farms at Sadar upazila in Noakhali district. Different farms were selected randomly form different villages. the costal area was important for different parasitic  disease as the climatic condition and geographical location of this Upazila supports the incidence of such diseases. 

Study period:

The present study was undertaken for a period of 60 days, starting form November to December’2009.

Target samples:

Twelve (12) medium scale (20-25 cattle) commercial dairy farms were selected for collecting blood samples. A total of 150 blood smears were collected randomly form different farms from different villages (n=12).
Survey design:

A preset questionnaire was used to record information like age, breed, sex, size of the farm, types of the farm, body condition score (BCS), presence of tick, or other arthropods and vaccination. The cattle (Local X  Sahiwal and Local X Holestin Friesian) were grouped into different age (≤1 yr., 1.1 -2.5yr., 2.6-5yr., and 5 to above) groups  to determine the influence of age and breed susceptibility in blood protozoal infections. The minimum age of the animal was 20 days and the maximum was 108 months.

Examination of animals and sample collection:

The animal was examined especially on the basis some parameters like rectal temperature, visible mucous membrane, body conformation score, , lymphnode palpation, presence of ticks, urine colour Single Blood smears from each animal were collected form ear vein by puncturing with sterile needle. The slides were touched to the coming out blood and then spread by another slide. The slides were air dried and fixed by 100% methyl alcohol for 2 min.

Staining and examination of blood samples:

The prepared blood smears were stained with the Giemsa stain (working solution) for 25 to 30 minutes. After rinsing with water water of the stained blood smears, they were air dried and examined under microscope (10X100x) with immersion oil for the identification of blood parasites as described by Soulsby (1982). 

	

	

Fig 1: blood smears preparation technique (Ref:httpwww.funsci.comfun3_enbloodblood_07.gif)

Measuring the prevalence:
The proportional prevalence of hemoprotozoal infections in cattle was estimated by the following formula:
The proportional prevalence of hemoprotozoal infections =

No. of individual having a disease in a particular point of time X 100

No. of individual in the populatin at risk at the  point of  time

(Thrusfielld, 1995)

Analysis of the data:

The procured data through questionnaire and record  of blood smears examination were tabulated, sorted, and coded by using Office 2003 service pack and data were analyzed by STATA version 9.0 ( STATA  Corporation, College Station, Texas, USA). Descriptive univariable (Chi-square) and univariable logistic regression was carried out using STATA software. The result of responded answers for each coded category or continuous variables were expressed as percentage of 95% confidence level. In both univariate analysis (MANTEL HANZEL and logistic regression) the difference between  In this analysis, categorical variable were  measured using Chi-square tests and the test for significance for an association was done and expressed with calculated ‘P’ value.

Chapter IV
Result and Discussion

Prevalence study: 
In the study the proportional prevalence of haemoprotozoal infections in cattle was 16% of 24 positive samples where Babesiosis was 9.34%.  Among 9.34%, Babesia divergens contributed 3.34. %, Babesia bigemina contributed 3.34% and Bebesia sp. (species not identified) caused 2.66%. The proportional prevalence Anaplama marginale and Theileriosis in cattle was 1.33% and 5.33%, respectively..  Samad 1989) reported the occurrence of sub-clinical Anaplasmosis infection in cattle was 5.93% in Bangladesh  which was higher than the observation  of this study. This variation might be due to season, area, resistance of the animal and tick activities.

Factors influencing the occurrence of the diseases:
Age:

The occurrence of heamoprotozoan infections was more in heifers which was 29.17% at the age of 2.6 to 5 years 9 (P>0.728)  whereas, the lowest percentage was recorded in adult cow (5%) at the age of 5.1 to 9 years which also indicated that disease prevalence was lower in adult cows than calf or heifer. Among different heamoprotozoan infections, the occurrence of Babesiosis was higher in calf (≤1year) and heifer (2.6 to 5 years) which were 20.83% in both stages. The disease frequency was less in adult (0.83%) at 5.1 to 9 years of age which indicated that disease prevalence was lower in adult cows. This observation was consistent according to the observation of Blood (1968). Theileriasis occurred in heifers at the age of 1.1 to 5 years which was 8.33%  and higher prevalence of Anaplasmosis (4.17%) was rescored in calf (≤1yr.)  than other stages.  

Breed: 

The prevalence of heamoprotozoan infection was higher in Local X HF cross bred (66.67%) than Local X S cross bred cattle (33.33%) where P>0.326, although the variation was not statistically significant.. The occurrence of Babesiosis in both cross bred cattle was 29.16%. the prevalence was higher in Local X HF cattle which were 8.3% in Anaplasmosis and 29.16% in Theileriosis..

Nutritional status: 
among different hemoprotozoan infections, the prevalence of Babesiosis and Theileriosis was 16.66% and 22.22%, respectively where BCS was 3,which indicated that the prevalence of different hemoprotozoan infection was highest in healthy cattle (BCS 2.75 -3) which was recorded 27.78% - 38.89%. Rodostits et al. (2000) reported that blood parasitic diseases are less severe in cattle on a low plane of  nutrition and observation of this study was consistent with it. 

Table 1: Prevalence and measures of association between intra-variables in different blood protozoal infections in cattle. 

variables		Positive to Giemsa staining	
	
	Frequency
(N)	Babesia sp.
(%)	Anaplasma sp. 
(%)	Theileria sp. 
 (%)	Total positive	Percentage
(%)

	Chi-square 
value	P
Value	

Breed	
L X HF 

L X S
	
112

38	
7 (29.16)

7 (29.16)	
2 (8.34)

0 (0)	
7 (29.16)

1 (4.17)	
16

8	
66.67

33.33	

0.9667	

0.326
	
Age	
< 1yer.

1.1-2.5yr.

2.6 -5yr. 

5.1 to- 9yr 
	
38

39

47

26
	
5 (20.83)

3 (12.50)

5 (20.83)

1(0.835)	
1 (4.17)

0 (0)

0 (0)

1 (0.83)	
0 (0)

2 (8.33)

2 (8.33)

4 (3.34)	
6

5

7

6	
25.00

20.83

29.17

5.00	1.3063	0.728	
BCS	
2

2.25

2.5

2.75

3

10

18

23

22

26

1 (1.56)

1 (5.56)

2 (11.11)

2 (11.11)

3 (16.66)

0 (0)

0 (0)

0 (0)

1 (5.56)

0 (0)

0 (0)

2 (11.12)

0 (0)

2 (11.11)

4 (22.22)
	
1

3

2

5

7	
1.56

16.67

11.11

27.78

38.89
	3.5101	
0.476
	
L XS = Local X Sahiwal, L X HF = Local X Holstein Friesian, yr. = year CS = Body Condition Score. 
 Table 2: The strength of association between hemoprotozoal infections with other variables

variables	Odd ratio	P value	95% Confidence interval	
	Breed	L X S

L X HF 
	1

1.6	

0.328	

.6234337 - 4.106291	
	Age	> 1yer.

1.1	- 2.5yr.

2.6 - 5 yr.

5.1 to- 9 yr.
	1

1.79   

1.94

.6    	

0.465     

0.569     

0.710     

.4529467 - 5.651879

.3852572 - 4.053775

.2177992 - 2.824381	
	BCS	2

2.25

2.5

2.75

3	1

1.8

.86

2.67

3.32	

0.633

0.905

0.406

0.294     

.1617759 - 20.02771

.0686715 - 10.69868

.2669817 - 26.24495

.352864 - 1.15778	

L XS = Local X Sahiwal, L X HF = Local X Holstein Friesian, yr. = year, B CS = Body Condition Score.

The odd ration of heifer and cow were 1.50 (P=0.559) and 1.51 (P=0.42) times , respectively, to have susceptibility with blood protozoal infections than calf.

Local X HF cross bred cattle was 1.6 (P=0.328) times more susceptible to hemoprotozoan infections than Local X S cross bred.

Animals of age between 5.1-9 years was 0.6 (P=0.710 ) times protective against blood protozoal infections than the animal of age group below 1 year. The odd ratio of animal of age group       1.1-2.5years was 1.79 (P=0.465) times and age group 2.6-5years was 1.94 (0.569) times more susceptible to the heaemoprotozon infections than animal of age group below 1 year.

The odd ratio indicated that animal having BCS 2.5 was 0.86 (P=0.90) times more protective than animal having BCS 2.0. on the other hand, animal having BCS 2.75 was 2.67 (P=0.465) times and animal having BCS 3 was 3.32 (P=0.465) times more susceptible to the occurring of the diseases than animal having BCS 2.0.

 

Fig 2: Babesia  bigemina. in thin blood smear (10X100 magnification)

Fig 3: Anaplasma marginale in thin blood smear (10X100 magnification)

	

Fig 4: Theileria in  thin blood smear (10X100 magnification)

Limitations of the study

Duration of the study period:
The study period was too short to perform properly.

Recall bias: 
Farmers were not cooperative and friendly. In many cases, they were uncooperative to allow for collecting of the blood samples of their animals. In some cases, interview was not taken from th right person who involved with the rearing of the animal.
Chapter V

Conclusions

From this study, it might be concluded that prevalence of different hemoprotozoan infections  were present at Noakhali Sadar area, Noakhali at significant level where highest prevalence was recorded in Babesiosis and lowest for analasmosis. It is also significant that L X HF cattle were more infected than L X S. The condition might be more aggravated during summer seasons when tick activities were more. Genetic potentiality as well as production of dairy animals was hampered with various diseases including different haemoprotozal infections. So, epidemiological survey necessary to detect the various blood protozoal infections which was the cause of death, decreased production, lowered working efficiency and also acting as a serious economic challenge for developing country like Bangladesh.

Chapter VI
Recommendation
Form the study, it may be recommended that the result of the study would be more authentic if:
•	It was conducted for a longer period of time.
•	population size would be more.
•	At least two set of data would be taken at two different times of the year. 
•	Previous authentic record of the prevalence of the blood parasitic diseases would be found

Chapter VII

References

Alam,J. (1993). Livestock the sector for more investment in Bangladesh. Asian Livestock, 18:77-81

Bangladesh Bureau of Statistics, (1989). Report on the Bangladesh Livestock Survey 1983-84. Ministry of Planning, Govt. of the peoples’ Republic of Bangladesh.

Blood,D.C., and Henderson, J.A. (1968). A text book of disease of cattle, sheep, pigs, goats and horse. 4th edn. Baillere Tindall Publication, London. pp. 1261-64,1289-94,1324-29,

Bram R.A.,(1983). Tick-borne livestock diseases and their vectors. 1. The global problem. World Anim Rev 6:1–5.

Bram RA., (1983) Tick-borne livestock diseases and their vectors: the global problem. Ticks and tick-born diseases, FAO Animal Production and Health Paper. World Anim Rev, Rome. 1983;36:7–11.

Gautam,O.P, and Banerjee, D.P. (1982). Anaplama in cattle with reference of the disease in India. Indian Journal of Paraistology, 6(2): 169-181.

Hagan,W.A., and Bruner,D.W.(1961). The infectious diseases of domestic animals, 4th edn. Comstock Publishing Association, Ithaca, Newyork. pp. 652-686

Howard, J. L., (1985-2003).Current Veterinary Therapy-3, Food Animal Practices, Texas A&M Vet. Med. Diagnostic Lab., Amarillo, Texas, USA. pp. 584-86,588-91,627-28. 

Hungerford,T.G. (1962).Disease of Livestock, 5th edn. Angus and Robertson Ltd.,Sydney. pp. 201-207

Julish,P.K., (1966). Parasitic protozoa, Vol.IV. Academic press, INC, London, Ltd. Pp.115-120,242-243,
Mahoney, D.F. (1994). The development of control method for tick fevers of cattle in Austrilia. Aust. Vet. J., 71(9), 283-289 

Ristic M. Anaplasmosis. In: Weinman D, Ristic M, editors.,(1960). Infectious Blood Diseases of Man and Animals. Vol. 11. New York, USA: Academic Press; 1968. pp. 473–542.

Ristic,M. (1981). Diseases of Cattle in the Tropics. Martinus Nijhoff Publishers, The Hague. Vol 6,pp.327-344,443-468.

Roadostits, O.M, Blood, D.C and Gay, G.C. (2000). Veterinary Medicine. 9th end. Baillere Tindall Publication, London. pp.1261-1264,1298-94,1324-29.

Samad,M.A., Bashar,S.A., Shahidullah,M., and Ahmed,M.U. (1989). Prevalence of hemoprotozoan parasites in the cattle of Bangladesh. Indian Vet.Med.J.,13:50-51

Soulsby,E.J.L. (1986). Helminths, Arthropods and Protozoa of Domesticated Animals,7th edn. Baillere Tindall, London.pp:707-717, 729-735.

Sparagano,O.,(1999). Molecular diagnosis of Theileria and Babesia species. J. vet. Parasitol, 13 (2): 83-92

Thrusfielld,M.,(1995). Veterinary Epidemiology. Blackwell Science Ltd., a Blackwell Publishing company.,pp.39-41

Talukder,M.H. and Karim,M.J. (2001).Subclinical Anaplasma infection in Crossbred cattle in Bangladesh. Bangladesh Vet. J., 35 (3-4): 159-160.

The Merck Veterinary Manual., (1997). Professional Hand book of Merck and Co.;INC,pp.18-22,30-33.

Uilenberg,G.,(1955). International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health. Vet. Parasitol.;57:19–41. 

URQUHART,G.M.,(1996). Veterinary Parasitology.Blackwell Science Ltd.2nd edn.pp.242-249.