Bulk-freezing of Theileria parva stabilates for vaccine production.

The freezing and thawing characteristics of different volume samples of freezing medium and Theileria parva stabilates were studied in order to identify suitable conditions for freezing large volumes of stabilates for use in the preparation of vaccines. Conventionally, 1.8 ml aliquots are used but are cumbersome to handle in the field preparation of trivalent vaccines. In this study, 75 ml aliquots of the stabilates were found to exhibit freezing and thawing characteristics similar to those of the 1.8 ml aliquots. Bulk freezing of 2 stabilates yielded sporozoites which remained infective for cattle upon thawing, induced a range of clinical reactions in vaccinated cattle and stimulated the development of immune reactions.

Effects of haemagglutination (lectin) inhibitory sugars on Theileria parva infection in Rhipicephalus appendiculatus.

We have previously described the presence of haemagglutinins in tissues of the tick, Rhipicephalus appendiculatus and determined their sugar specificities by inhibition experiments. In this study, haemagglutination inhibitory sugars are shown to have an effect in vivo on the abundance of Theileria parva infected salivary gland acini in Rhipicephalus appendiculatus. A significant increase (P < 0.05) was observed in T. parva acinar infection rates in the salivary glands of R. appendiculatus fed on ears of rabbits infused with melibiose and raffinose. In contrast, mannose and turanose (non-haemagglutination inhibitory sugars) did not cause elevation of T. parva acinar infection rates. The effect of melibiose in elevating acinar infections was observed when used only during T. parva maturation in the salivary glands but not during parasite pick-up from an infected bovine host. Stabilates produced from ticks with elevated acinar infections did not differ from control stabilates in infectivity to cattle, by comparison of prepatent periods to pyrexia, or parasitosis, or in the severity of reactions.

Viability of Theileria parva trivalent stabilate after thawing and maintenance at 4 degrees C for up to 15 hours.

Two experiments were conducted to determine the effect of prolonged maintenance at 4 degrees C on thawed Theileria parva stabilates. Samples maintained at 4 degrees C for 1, 12, 15 and 18 h were all infective for cattle. Immunized cattle were able to resist potentially lethal challenges 29 days later. These results indicate that the handling of T. parva trivalent stabilates for immunization against East-Coast fever in the field should be much easier than previously envisaged.

Isolation and transmission of Theileria mutans (Chisamba) in Zambia.

Theileria mutans (Chisamba) was isolated from a steer at Chisamba, Central Province, Zambia by inoculation of blood into a susceptible unsplenectomised calf. The parasite was then transmitted on three occasions by nymphs and once by adult Amblyomma variegatum ticks. Macroschizonts, typical for T mutans, were detected in two calves for short periods. The parasite caused varying degrees of anaemia in all experimental calves, whose sera showed high antibody titres to T mutans in the indirect fluorescent antibody test.

Preliminary investigations on isoenzyme variants of lymphoblastoid cell lines infected wih Theileria species.

The electrophoretic mobilities of seven enzymes from eight theileria-infected bovine lymphoblastoid cell lines originating in Kenya and Iran were compared. The isoenzyme patterns of phosphoglucomutase, malate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase were the same for all cell lines infected with any of the three Theileria species. Theileria annulata could be clearly differentiated from T parva and T lawrencei on the basis of three enzymes: glucose phosphate isomerase, glyceraldehyde phosphate dehydrogenase and aldolase. T parva and T lawrencei isoenzyme patterns were alike except for glucose phosphate isomerase, where two sets of isoenzyme mobility were shown which, however, did not separate the two species.

Methods currently used for the control of East Coast fever: their validity and proposals for future control strategies.

Restriction of cattle movements, vector control, treatment and immunization are identified as the main control methods against East Coast fever (ECF). The effectiveness of these methods is very much influenced by cultural practices, economic and political pressures and development of resistance by ticks to acaricides. The proposed strategies for the future include continued restriction of cattle movements, less intensive vector control in enzootic areas so as to maintain enzootic stability, chemotherapy and immunization of the improved cattle. In addition, research efforts to evaluate strategic vector control, develop cheaper drugs, improve the quality and delivery of the stabilate vaccine, and identify and mass produce appropriate immunogenic subunit vaccines should be intensified.

Theileria taurotragi in Zambia.

Theileria sp. (Bwengwa) of low virulence was isolated by feeding R. appendiculatus ticks collected from the field on a susceptible calf and subsequently transmitted between cattle by R. appendiculatus ticks- Theileria sp. (Bwengwa) was shown to be T. taurotragi on parasitological, clinical and serological grounds. T. taurotragi is the fourth Theileria sp. shown to be present in Zambia.

Treatment of theileriosis with parvaquone in Zambia.

Parvaquone was used to treat 126 cattle with theileriosis. Theileria species schizonts were present in their lymph node biopsy smears and the majority of the animals had clinical signs of theileriosis. One hundred and fifteen treated and one untreated cattle survived the infection while 11 treated and 12 untreated animals died of the disease. Despite serological evidence of a parasite challenge during the subsequent rainy season, recovered cattle did not develop clinical signs of theileriosis but untreated cattle in the area continued to die from the disease. An intermittent low piroplasm parasitaemia (less than 1 per cent) was observed in recovered cattle for up to 14 months after detailed monitoring of cattle in the trial; this could be evidence for a carrier status for the Theileria species or strains involved.

Isolation of Theileria parva (SAO Hill) and Theileria parva (West Kilimanjaro) and their cross-immunity with Theileria parva (Kasoba).

Two Theileria parva stocks were isolated from control cattle during East Cost fever (ECF) field immunization trials at SAO Hill and West Kilimanjaro in the southern and northern parts of Tanzania respectively. Both parasite stocks caused severe clinical ECF which required antitheilerial treatment for 3 of the 5 experimentally infected cattle. Cattle recovering from infection with the two T. parva stocks did not develop a fever and only 1 of 4 animals developed scanty schizont parasitosis for one day during a challenge with T. parva (Kasoba) from northern Malawi. In contrast, both control cattle developed fever and schizonts, and one required antitheilerial treatment to survive.

The fine structural relationship between Theileria schizonts and infected bovine lymphoblasts from cultures.

The fine structure of Theileria-lymphoblast relationship was studied using cultured bovine lymphoblastoid cells infected with Theileria parva, T lawrencei, or T. annulata. The major findings of this study were: (1) the presence of a very active Golgi complex with the associated annulate lamelae; (b) the presence of cytoplasmic microtubules which joined the parasites and host cell centriole during lymphoblast mitosis; and (c) the absence of morphological evidence to suggest that the host cell developed a reaction to the parasitic presence. The significance of these findings is discussed.

Ecology and phenology of ticks in Zambia: seasonal dynamics on cattle.

A study of the seasonality and infestation rates of ticks was carried out in 11 cattle herds in different ecological habitats in Zambia between 1980 and 1982. Wherever possible supplementary data were obtained from opportunistic collections from cattle and other hosts. Analysis of over 1000 tick collections from cattle indicated that infestation rates of the most important species, Amblyomma variegatum and Rhipicephalus appendiculatus vary in different ecological habitats: (i) In Western Province, infestations are much lower than elsewhere; (ii) in Central and Southern Provinces, moderate to high infestations occur; and (iii) in Eastern Province, R. appendiculatus numbers are generally low and A. variegatum numbers are moderate. These two species, however, have similar life cycles throughout their range with one generation per year. Larvae occur mainly from March to May, nymphae from May to September, and adults of A. variegatum from October to December and of R. appendiculatus from December to April. Boophilus decoloratus appears to have two to four generations per year but is uncommon during the rainy season. In some areas in central Zambia Rhipicephalus compositus adults are seasonally common in September-October whereas Rhipicephalus evertsi is more or less ubiquitous. Low to moderate infestations of Hyalomma truncatum and Hyalomma rufipes occur in most areas. At least 14 other less common or rare species of Rhipicephalus, Amblyomma, Haemaphysalis and Ixodes were taken infrequently from cattle. These and other host-specific species were also collected from dogs, sheep, various wildlife hosts and the environment. Infestation rates, seasonality and host-relationships of tick species are discussed in relation to their ecology. Relevant biosystematic and disease relationships are reviewed briefly. The baseline data derived from this study are adequate for integrated analysis with those from other ecological and economic investigations to formulate tick control strategies.

Epidemiology of bovine babesiosis and anaplasmosis in Zambia.

The serological prevalence of bovine babesiosis and anaplasmosis in the traditional farming sector of six provinces of Zambia was determined using the indirect fluorescent antibody test (IFAT) for babesiosis and the card agglutination test (CAT) for anaplasmosis. Antibodies to Babesia bigemina occurred throughout the country whereas the prevalence of B. bovis followed the distribution of its tick vector Boophilus microplus which is limited to the north-eastern part of the country. Low numbers of B. bovis serologically positive cattle were demonstrated in central and southern Province. Anaplasma spp. occurred throughout Zambia but the overall percentages of positive sera were low ranging between 14.7% and 38.6% using the CAT. Two hundred sera were retested for anaplasmosis using an enzyme-linked immunosorbent assay (ELISA). Sero-prevalence rates were 1.5 to 2.3-fold greater with the ELISA than with the card agglutination test.

Characteristics of tick, Rhipicephalus appendiculatus, glands distinguished by surface lectin binding.

Incubation of fluorescein-conjugated and biotinylated lectins with Rhipicephalus appendiculatus salivary glands revealed differences between the basal laminae of the haemocoelic surfaces of the three acinal types. There were some similarities in the lectin binding characteristics of the surfaces of type II and type III acini when Con A, PNA and TVA were applied, indicating the presence of galactose, mannose and glucose moieties on the acinal surfaces. The binding of BPA, HPA and HAA lectins, specific for galactosyl and glycosyl ligands, which occurred only on the surface of type III acini, was moderate to intense. The remaining galactose-reactive lectins (GMA, DBA and VVA) also bound only to type III acini and the level of binding was weak to moderate. Although the relationship between the haemocoelic surface of the R. appendiculatus salivary gland acini and Theileria kinetes is not known, the consistent differences in the surface carbohydrate composition of the acini confirm the existence of differences in the basic physiology of the acini which may correlate with the specific susceptibility of the type III acinus to Theileria parva infection.

Theileria parva (Kasoba): isolation and challenge of cattle recovered from infection with other Theileria parva stocks.

A pathogenic Theileria stock was isolated from control cattle during an East Coast Fever (ECF) field immunization trial at Kasoba near Karonga town in northern Malawi. A stabilate of this stock caused severe fevers and prolonged parasitosis in Theileria parva naive cattle, resulting in the death of 5 out of 12 cattle despite treatment. In contrast, this parasite stock caused mild to moderate reactions in 17 cattle immunized with the trivalent T. parva stabilate except in 3 animals which developed severe reactions, and one of them died. Another time, cattle immunized with buffalo-derived Theileria parva (Serengeti transformed) resisted a potentially fatal challenge, with only mild to moderate reactions being recorded. The parasite stock was morphologically and serologically indistinguishable from Theileria parva (Muguga); it was virulent and could cause mortality, particularly in T. parva naive cattle. The parasite stock was designated Theileria parva (Kasoba).

Integrated tick and tick-borne disease control trials in crossbred dairy cattle in Malawi.

Crossbred dairy heifers on a farm in an East Coast fever (ECF) endemic area in Malawi were immunised against Theileria parva, Anaplasma spp., Babesia bigemina, Babesia bovis and Cowdria ruminantium. They were treated at infrequent intervals with chlorfenvinphos to limit infestation with adult ticks, without providing complete tick control. In one trial, which tested a threshold dipping regimen, 20 heifers were dipped only once in 6 months to control a flush of Boophilus microplus. Unimmunised controls showed serological evidence of exposure to T. parva and B. bigemina, and one died of ECF, but there were no incidents of tick-borne disease in the immunised group. In a second trial, which tested a strategic dipping regimen, 107 animals were dipped 9 times over a 6 month period. Despite heavy challenge by B. bovis and moderate challenge by B. bigemina and Anaplasma spp. demonstrated serologically, there was only a single clinical case of babesiosis. The observations provide encouragement for the introduction of integrated tick and tick-borne disease control programmes in improved cattle in ECF endemic areas.

Challenge of Theileria parva (Boleni) immunised cattle with selected east African Theileria stocks.

Theileria parva (Boleni) stock from Zimbabwe was used to immunise 24 susceptible Friesian calves by infection and treatment with oxytetracycline. Twenty-eight days after immunisation the animals in groups of 6 plus 2 susceptible controls were subjected to challenge: 3 groups with heterologous stocks and one group with the immunising stock. Theileria parva (Boleni) gave good protection against homologous challenge, the T. parva (Muguga, Kenya) and buffalo-derived T. parva (Serengeti transformed, Tanzania) parasite stocks. It did not protect against the T. parva (Kasoba, Malawi) stock and 3 out of 6 immunised cattle died and the remaining 3 had to be treated with parvaquone. In a second experiment, the 6 T. parva (Boleni) immunised animals which had received homologous challenge, together with the 2 controls which had recovered without treatment from T. parva (Boleni) infection, were challenged with the T. parva (Kasoba) stock. Four out of 6 of the immunised animals resisted the challenge with mild to moderate reactions. The other 2 animals had severe reactions and one died. The 2 control animals which recovered from T. parva (Boleni) infection resisted the T. parva (Kasoba) challenge and both had mild reactions. It is suggested that oxytetracycline used in the first experiment may have interfered with the expression of the full protective capacity against the virulent T. parva (Kasoba) stock. Further studies on the use of the T. parva (Boleni) stock without oxytetracycline treatment could identify a more broadly immunising effect and a more economical vaccination method.