Prevalence and risk factors of bovine trypanosomosis in Kilwa district, Lindi region of southern Tanzania.

African animal trypanosomosis (AAT) and human African trypanosomosis (HAT) are complex chronic, debilitating, emaciating and often fatal diseases of animals and humans, respectively. This cross-sectional study was conducted to determine the prevalence and risk factors associated with bovine trypanosomosis in tsetse-infested Kilwa district, Lindi region, southern Tanzania. Blood samples were collected from 420 cattle randomly selected from 86 herds from ten villages. A maximum of ten herds per village and at most six animals from each herd were selected for sampling. At the same time, a questionnaire was administered. Individual animal samples were analysed using microscopy and pooled sample at herd level were analysed by loop mediated isothermal amplification (LAMP). A herd was considered positive if at least one animal in the herd was positive for AAT. A prevalence of 9.3% (95% CI: 2.9-14.9) was recorded for AAT by microscopy, mainly caused by Trypanosoma congolense (5.8%, 95% CI=0.9-10.7), Trypanosoma brucei species (5.8%, 95%, CI=0.9-10.7) and Trypanosoma vivax (3.5%, 95% CI=0-7.4). Loop mediated isothermal amplification (LAMP) recorded a heard prevalence of 41.9% (95% CI: 30.0-51.4%), mainly caused by T. congolense (30.2%, 95% CI: 20.5-39.9), T. brucei species (25.6%, 95% CI: 16.4-34.8) and T. vivax (20.9%, 95% CI: 12.3-29.7). Most of the cattle herds had mixed infections of these parasites. According to LAMP, Miteja and Matandu villages had the highest AAT herd prevalence of 57% (95% CI: 20.3-93.7) while Mavuji had the lowest prevalence of 14% (95% CI: 0-39.7). Data from the present study suggest that district of origin, grazing in game reserve, water source and form of watering point are risk factors associated with AAT in Kilwa district, southern Tanzania. Continuous surveillance and monitoring of AAT using more sensitive are recommended.

Immunization of young chicks using graded dose of wild strain of Eimeria tenella.

A wild strain of Eimeria tenella was isolated and utilized for immunization studies. Its optimal sporulation was attained at room temperature 24-25 degrees C after 24-48 h. Two groups of chicks were immunized by dosing a graded dose of five oocysts/chick/day for 6 days followed by 50 oocysts/chick/day for 7 days. A third group was not immunized and served as a negative control. Immunized chicks gained mass at the same rate as unimmunized ones, but when challenged with 200,000 oocysts/chick, mass gains declined in the unimmunized group. The growth rate of immunized chicks was not affected by challenge (P > 0.05). Upon challenge, unimmunized chicks produced significantly more oocysts than immunized chicks (P < 0.005). Immunized chicks withstood a challenged with 200,000 oocysts/chick without developing any clinical signs whereas the unimmunized chicks developed typical clinical signs of coccidiosis. Unimmunized chicks had significantly more severe lesions in the caecum than any other group (P > 0.005) and also produced significantly more oocysts than any other group (P > 0.005).

Theileria parva: in vitro studies on the effects of holding temperature, pH and medium on sporozoite infectivity.

The effects of holding temperature, pH and medium on the infectivity of Theileria parva sporozoites were investigated using an in vitro infectivity assay. The sporozoite infectivity lasted for 72 h at a holding temperature of 4 C but for only 24 h at 24 degrees C. Sporozoite infectivity was found to be sensitive to pH variations and sporozoites were most infective between pH 7 and pH 8. There was a significant loss in infectivity at pH 5 and infectivity was almost totally abolished at pH 9. Theileria parva sporozoites are usually held and manipulated in Eagle's minimum essential medium (MEM) with Earles' salts. In this study. Leibovitz-15 supplemented with 15% fetal bovine serum gave a significantly better infectivity than Eagle's MEM (3.8 log units versus 1.0 log units) or any other medium. The importance of proper management of the T. parva sporozoite environment in the laboratory or field is emphasized by the findings in these studies and might also explain some of the failures of vaccination when the pH of the holding medium was allowed to deteriorate.

Comparison of cryoprotectants in the preservation of Theileria parva sporozoites using an in vitro infectivity assay.

An in vitro infectivity assay was used to examine five cryoprotectants for their suitability for preserving Theileria parva sporozoites. All five were capable of preserving T. parva sporozoites through freezing, the optimal concentrations being 7.5% for glycerol, 5% for dimethyl sulphoxide (DMSO), poly (vinylpyrrolidone) (PVP) and poly(ethylene glycol) (PEG), and 2.5% for hydroxyethyl starch (HES). When the five cryoprotectants were compared at their optimal concentrations, using a modification of the standard method of stabilate preparation, glycerol was significantly better than the others (p < 0.05). Measurement of the effects of each cryoprotectant on the osmolality of the media revealed that glycerol and DMSO elevated the osmolality significantly (p < 0.05). Resuscitation of glycerol-preserved sporozoites required the presence of glycerol in the diluent to maintain infectivity. Studies on the effects of equilibration time in glycerol on the infectivity of sporozoites showed that those frozen immediately after mixing (2 min) were as infective as those frozen after 60 min of equilibration.

Application of PCR and DNA probes in the characterisation of trypanosomes in the blood of cattle in farms in Morogoro, Tanzania.

Polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) probes were used to characterise trypanosomes from cattle in Morogoro region of Tanzania. Blood samples collected from 390 beef and dairy cattle in selected farms in Morogoro region were examined for presence of trypanosomes using the buffy coat technique (BCT) and blood smears (BSs). Fifty-two animals were found infected: 40 with Trypanosoma congolense, 10 with T. vivax and two with both T. congolense and T. vivax. DNA extracted from all the parasitologically positive and 62 randomly selected parasitologically negative samples were subjected to PCR amplification using primers specific for different trypanosome species. Using a set of seven specific-pairs of primers on the parasitologically positive samples, we detected only T. congolense, either the Savannah- or the Kilifi-type, as single or mixed infections. With the PCR, trypanosome DNA could be detected in 27 (43%) out of 62 samples that were parasitologically negative. DNA hybridisation using probes specific for Savannah- or Kilifi-types T. congolense, or T. vivax, confirmed the presence of these parasites in cattle kept on some farms in Morogoro region of Tanzania. From these studies, it is clear that there is a need to undertake molecular epidemiological studies to determine the distribution of trypanosome species and subspecies, and to assess the economic impact of these parasites in the productivity of livestock in Tanzania. In particular, it would be desirable to verify the assumed association between the different presentations of trypanosomosis on one hand and genotypes of T. congolense on the other.

Breed-associated resistance to tick infestation in Bos indicus and their crosses with Bos taurus.

The relative resistance to tick infestation of zebu (Bos indicus) in comparison to crossbred (B. indicus x B. taurus) cattle was investigated. B. indicus breeds, all belonging to Tanganyika shorthorn zebu were Meru, Mbullu and Iringa red. Crossbreds were Meru x Friesian and Iringa red x Friesian. Parameters to distinguish between 'tick resistant' and 'tick susceptible' cattle were tick counts on naturally exposed animals, serum complement levels and delayed skin hypersensitivity response to phytohaemagglutinin. Results have shown that pure zebu cattle are less infested with ticks when compared to zebu-taurine crosses under identical field conditions. Zebu cattle also had significantly higher serum complement level than crossbred cattle. While serum complement and tick burden were negatively associated (r = -0.27, P < 0.001), the cutaneous response to phytohaemagglutinin did not vary with tick infestation. The influence of cattle breed on tick infestation and serum complement level is demonstrated.

Use of an in vitro infectivity assay in comparison with histological techniques in the study of Theileria parva sporozoite maturation.

Adult male and female Rhipicephalus appendiculatus ticks infected with Theileria parva (Muguga 3087) were fed on rabbits and the development of infection was monitored daily using light microscopy and an in vitro titration technique able to quantify the infectivity of sporozoite suspensions. The salivary glands stained with methyl green pyronine showed presence of infection in some unfed ticks. The intensity of staining was shown to increase with the number of days the ticks had fed. The in vitro technique, on the other hand, could detect infection only in ticks which had fed for 3-5 days. Feeding of ticks on rabbits for 4 days produced significantly more sporozoites than any other lengths of feeding (P = 0.001). The in vitro assay was also able to demonstrate differences between male and female R. appendiculatus in production of infective sporozoites. Female ticks produced significantly more sporozoites than male ticks (P = 0.002).

Response of Trypanosoma congolense in goats to single and double treatment with diminazene aceturate.

Diminazene aceturate is one of a limited number of compounds currently marketed for treatment of trypanosomiasis in cattle, sheep and goats. The pharmacokinetics of the compound in goats suggest that double treatment with diminazene aceturate might enhance the compound's therapeutic activity. A study was therefore conducted in goats using two clones of Trypanosoma congolense, IL 3274 and IL 1180, which were previously shown to be resistant and sensitive, respectively, to single treatment with diminazene aceturate. The results indicated that, as compared to single treatment, double treatment with diminazene aceturate at a dose of 7.2 mg kg-1 bodyweight, at either eight or 24 hour intervals, did not greatly enhance the therapeutic activity of the drug. Furthermore, treatment with the same drug dose eliminated infections with T congolense IL 3274 when treatment was administered 24 hours after infected Glossina morsitans centralis had fed, but failed to do so if treatment was delayed until after goats were detected to be parasitaemic. This suggests that failure of T congolense IL 3274 to respond to treatment with diminazene may not be due to drug resistance per se.

Use of antigens of cultured Trypanosoma brucei in tests for bovine trypanosomiasis.

MicroELISA tests for the detection of trypanosomal antibodies in cattle infected with Trypanosoma brucei, T. vivax and T. congolense were carried out using antigens prepared from culture forms and bloodstream forms of T. brucei. Antigens prepared from culture forms gave similar microELISA values to those obtained with bloodstream form antigens and statistical analysis of the results obtained with both antigen preparations and sera from the infected animals showed significant high positive correlation. It would thus be possible to use culture from antigens as an alternative to bloodstream form antigens in diagnostic tests for trypanosomiasis, with the advantage that culture form antigens are more easily prepared free from extraneous protein.

Studies on Theileriidae (Sporozoa) in Tanzania. X. A large-scale field trial on immunization against cattle Theileriosis.

50 cattle immunized by an infection and treatment method with 3 strains of Theileria parva (Mugaga, Kiambu 5 and Serengeti transformed), together with 19 controls, were exposed to natural tick infestation in Tanzania, in a site where a continuous influx of infected ticks from different regions of the country may be expected to occur. Exposure lasted for 2 months, monitoring continued for 3 more weeks after exposure ended. All 19 controls contracted East Coast Fever and died. 40 of the 50 immunized animals survived the whole period of monitoring; the other 10 died of accidents (2), heartwater (3) and unidentified causes (5); none died of ECF. It is recommended that this method of immunization be routinely applied to protect valuable animals at risk. An attempt to immunize against pathogenic Theileria mutans, by injecting blood containing piroplasms of a strain of low pathogenicity, showed that this method may protect against high parasitaemias caused by natural tick-borne infections and may be useful in eliminating the confusing factor of T. mutans in future trials on East Coast Fever.

Studies on Theileriidae (Sporozoa) in Tanzania. VI. Second field trial on immunization against cattle theileriosis.

Nine cattle, immunized by an infection and treatment method with 3 strains of Theileria parva (Muguga, Kiambu 5 and Serengeti transformed) and nine controls, were exposed to natural tick infestation in Tanzania. All controls contracted fatal East Coast Fever within 2 months of exposure. All immunized animals survived the period of exposure (over 2 months), but later one died of ECF, one of heart-water, one of an undetermined cause (not a theileriosis), and one disappeared from the herd. The 5 remaining animals survived for over 3 months after exposure ended. It is thought that this method of immunization is of value in protecting valuable animals at risk. An attempt to immunize against pathogenic Theilereia mutans, by injecting blood containing an apathogenic strain of this parasite, gave inconclusive results as no pathogenic strains were encountered during the trial.

Studies on Theileriidae (Sporozoa) in Tanzania. IV. A field trial on immunization against East Coast fever (Theileria parva infection of cattle).

Seven cattle, immunized by an infection and treatment method with 3 strains of Theileria parva (Muguga, Kiambu 5 and Serengeti transformed), and five controls were exposed to natural tick infestation in Tanzania, at a site where 2 precontrols had previously contracted fatal East Coast fever within 3 weeks. All controls became infected with fatal ECF within a month of exposure. One immunized animal survived, two died from a Theileria mutans infection and one from a Babesia bigemina infection. The cause of death of the other three animals could not be ascertained, but East Coast fever could not be incriminated. Immunized cattle took longer to succumb than the controls, and serological results showed that all immunized animals became infected with T. parva during exposure, without showing symptoms of ECF.