The Identification Of Theileria Biology Essay

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Theileriosis and heartwater are considered to be the most important tick-borne diseases of livestock in sub-Saharan Africa, resulting in extensive economic losses to farmers in endemic areas. Corridor disease, caused by Theileria parva, is a controlled disease in South Africa. Buffalo are the natural reservoir hosts ofthis parasite, which is transmitted by Rhipicephalus appendiculatus and R. zambeziensis. Buffalo also appear to be the original hosts of two other Theileria species infecting cattle, the relatively benign T. mutans and the apathogenicT. velifera. Theileria buffeli and the uncharacterized Theileria sp. (buffalo) have only been identified in some buffalo populations in South Africa and their vectors remain unknown. Theileria parasites usually occur as mixed infections and the benign and non-pathogenic forms do not have any significant economic importance. However, their presence interferes with the interpretation of results obtained in some diagnostic tests designed to diagnose the pathogenic T. parva in South Africa.

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It has been shown that African buffalo are carriers of Ehrlichia ruminantium, anintracellular rickettsial bacterium. It is the causative agent of heartwater (cowdriosis) and is transmitted by three-host ticks belonging to the genus Amblyomma. Although buffalo show no disease symptoms, they may represent a threat to the livestock industry. It is also likely that, in areas where buffalo populations and both Rhipicephalus and Amblyomma tick species occur, buffalo may be carriers of both Theileria and Ehrlichia species. Little is known about the Ehrlichia spp. infection status of African buffalo.

Life cycle

The life cycle of T. parva consists of different developmental stages spread between the tick carrier and the cattle/buffalo hosts.

It starts of as sporozoites in the salivary glands of ticks where they matured to full size (3). Here after it gets transmitted to the host cells when the tick feeds, the sporozoites infiltrate the host’s lymphocytes where they differentiate to form schizonts (4). These schizonts cause an excess of lymphocytes to be produced in an effort to increase its own numbers. In the lymphocytes, schizonts differentiate to form merozoites at a later stage in the cycle. Merozoites have the ability to invade red blood cells, and from there they change into piroplasms.

Piroplasms are taken with blood that a new tick feeds off and the life cycle continues in the gut of the tick. Here, fertilization and gametogenesis occurs and a zygote forms (5). The zygote then leaves the gut and enters the gut cells, where it transforms into a structure named a single motile kinete. Kinetes travel to the salivary gland where they stay dormant until the tick feeds again, upon which sporozoites are rapidly formed in the gland. In future when the tick feeds again the sporozoites infect the new host and the cycle starts all over again (6).

Life cycle of Theileria parva

Infection during feeding on host

In salivary glands of tick

In lymphocytes

differentiate

Schizont

Sporozoite

Rapid development when feeding is initiated

In salivary glands of tick

New sporozoites

differentiate

Ingut cell of tick

Ingested by tick. Sexual development Gametogenesis and fertilization in tick gut

In erythrocytes

In erythrocytes

differentiate

Motile kinete

Zygote

Piroplasms

Merozoite

Replication only occurs in the lymphocytes, but asexual replication has been found in both lymphocytes and erythrocytes in T. annulata (6).

Vector

Rhipicephalus appendiculatus(Brown ear tick) from the tick family Ixodidae(hard ticks) are usually found in the ears of cattle as its name implies (7). This tick’s main hosts include buffalo, cattle and other large antelopes, but can also be found on other livestock namely sheep and goats. They can be found inthe eastern, central and southeastern parts of Africa.Rhipicephalus appendiculatusis known to be the main vector of T. parva. Sporozoites of T. parva from the salvary glands of adult or nymph ticks are introduced into the susceptible animal during feeding and so enters the host. The parasite is transmitted between hosts through infected ticks.

Diseases

East Coast Fever (ECF)

A disease that is fatal to cattle caused by T. parva parva. The disease is transmitted between cattle through infected ticks. Characteristics include swollen lymph nodes near bite marks and fever. Death may occur in 18-30 days after infected ticks are attached to the animals (8). Susceptibility towards the disease varies between cattle, some will perish but others might recover. Animals that recover from the disease show lower amounts of fat reserves and muscle content.

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It can be controlled through different ways including the use acarcides, pre-immunization against ECF, pasture management and to keeping infected animals apart from those that are healthy.

Corridor disease

In principal, Corridor disease is almost the same as ECF but death in cattle occurs more rapidly, death may occur in just a few days after infection (9). The disease is transmitted from buffaloes to cattle by T. parva lawrencei when they come into contact with each other. Buffaloact as carriers and develop no clinical symptoms of the disease. Corridor disease is fatal to cattle, so much that new infective sporozoites are unable to be produced because the animal dies off to quickly. Infected animals may be cured through the use of chemotherapy. Chemotherapy and treating of animals are not allowed in South Africa, to prevent the formation of other subtypes. But these animals may turn out to become carriers themselves because not all the parasites are always destroyed (10).

The disease may be controlled through the continuous monitoring off cattle and buffalo herds to keep them apart. This is the simplest way to prevent infection and also it does not give the parasite the chance to build up a resistance or mutate to overcome a treatment of some sort. Testing animals frequently for infection also plays an important role

January disease (Zimbabwean theileriosis)

This disease is a derivative of ECF transmitted by T. parva bovis(11).The main differences are the parasite causing infection and that this disease is seasonal, only occurring in December and March hence the name. January disease is also carried byR. appendiculatus, its activity corresponds to the seasonality of the disease. January disease circulates in Zimbabwe and has not been observed in South Africa (11).

Diagnosis

The identification of the disease can be achieved in different ways including conventional methods: examining blood smears, serological methods: Indirect immunofluorescent antibody test (IFAS) and Enzyme-linked immunosorbent assay (ELISA) and lastly molecular methods: Real-time PCR and Reverse line blot (RLB)hybridization asaay. Buffalo in South Africa must be tested for: foot-and-mouth and Corridor disease, bovine brucellosis and tuberculosis because these illnesses can be transmitted to cattle.

Conventional methods

The use of microscopes to examine lymph and blood smears for the presenceof T. parva can be used for early identification. In smears parasites can be identified by morphological characteristics and by the use of certain staining methods (12). This diagnostic procedure has a few limitations including that piroplasms and schizonts from T. parva are very difficult to distinguish from over Theileria species, if they should occur in the same blood/lymph sample (6). One last limitation is that parasites in a carrier are at such a low level that microscope examination does not detect it.

Serological methods

The IFA test was originally used to detect T. parva antibodies but in present times schizont antigens are used (13,14). The method, although good in many ways, has many limitations which include complications when it comes to standardization, cross-reactivity causing many different immune responses and epitopes (6). When examining carriers there is a chance that antibodies in the animal have diminished but the parasite could still be present - a negative test can, therefore, still deliver an infective state. Indirect immunofluorescent antibody tests are not sufficient enough on their own in identifying disease presence in carriers (15,16).

Enzyme-linked immunosorbent assays use recombinant polymorphic immunodominant molecules (PIM) to detect T. parva antibodies (17). ELISA tests are more sensitive and specific than IFA. Despite this superiority ELISA still has limitations close to that of IFA, the parasite may have been removed from the animal but the test still is positive because ELISA does not test for the parasite but for the presence of an antigen or antibody (16,18).

Molecular methods

Because of the limits of conventional and serological methods, researchers developed molecular diagnostics for the identification of T. parva. These molecular techniques include conventional PCR assays, PCR-based hybridization and RFLP, real-time PCR assays and RLB. We shall concentrate on real-time PCRs and RLB because these techniques will be used in the project.

Real-time PCR’s detection is one of the best diagnostic tools researchers have up to now based on its sensitivity. It has the ability to distinguish between different species in a sample through the use of fluorescent hybridization probes and a melting curve analysis. It not only detects the parasite DNA (19). Up until a few years ago real-time PCR has not been used as a diagnostic test in the identification process of T. parva, recent tests have shown that real-time is far better than its predecessors, the conventional PCR. By using fluorophores real-time can detect more than one species at a time.

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Reverse line blot hybridization uses multiple probes to simultaneous detect different disease agents in one sample (20). It can differentiate between parasites in ticks, blood and tissue samples. RLB makes use of PCR amplification and hybridization to deliver a much higher level of sensitivity than normal PCR assays. Its used to identify different species from Babesia, Theileria, Ehrlichia and Anaplasma families. Also, in the detection process chemiluminescence is used instead of radioactivity, making it more user friendly (21). This method is a cost-effective and quicker way to identify multiple parasites.

Importance in South Africa

East coast fever was first sited in South Africa in the early 1900’s (6,22). Through painstaking work of dipping cattle and killing off of infected ones the disease was completely removed from South Africa in 1960 (23). Till now ECF has not been observed in R.S.A. because of strict government regulations of import and export of cattle.

Following the eradication of ECF a new problem posed itself in 1953 in the form of a new disease (24). Theileria parva was discovered in a buffalo host from where it was able to be transmitted to cattle and kill them off. The buffalo acts as a carrier of the parasite and when they share common pastures with cattle the parasite can be transmitted via ticks to these cattle. The disease was named Corridor disease in accordance to where it was found originally on a corridor of land between two game reserves (24).

The disease is controlled in South Africa by keeping buffalo and cattle apart to prevent any transmission of the parasite at all.