On the possibility of bovine Onchocerca species infecting Simulium damnosum s.l. in the forest zone of Sierra Leone. II. Biting densities and filarial infections in Simulium spp. and Culicoides spp.

Previous studies of bovine Onchocerca spp. in cattle in Sierra Leone indicated that only O. gutturosa was transmitted in the forest zone at high intensity. To determine its vector(s) and the extent to which Onchocerca-like infections in Simulium damnosum were likely to be of bovine origin, three lines of investigation were pursued. Firstly, a study was made of the biting flies attacking an ox bait animal over a 14-month period at Njala University Campus, near Bo. Secondly, attempts were made to infect the dominant local forest cytospecies of S. damnosum s.l. with O. gutturosa by feeding them on an infected ox under a bed-net. Thirdly, S. damnosum s.l. were infected by intra-thoracic injection of O. gutturosa microfilariae (mff). In 113 collections made at dawn and dusk at weekly intervals from the ox bait, 624 simuliids and 7740 Culicoides spp. were collected. Almost all the simuliids were S. damnosum s.l. which, on the basis of iso-enzyme examination and knowledge of local breeding sites, were identified as S. soubrense 'B'. Although this cytospecies fed readily on the ox at ventral sites where O. gutturosa mff occurred and the bed-net experiments showed that 16.1% of engorged S. soubrense 'B' ingested an average of 3.3 O. gutturosa mff each, no development occurred. The refractoriness of S. damnosum s.l. to O. gutturosa was confirmed by the intra-thoracic injection experiments. The predominant Culicoides spp. attacking the ox bait were C. krameri, C. trifasciellus and C. fulvithorax, with smaller numbers of C. schultzei. In 5803 dissected Culicoides spp., natural infections of Onchocerca-like larvae, presumed to be O. gutturosa, were found in 0.3% of C. fulvithorax, 0.1% of C. trifasciellus and 0.06% of C. krameri. It was concluded that, in the forest zone of Sierra Leone, S. damnosum s.l. is not a vector of O. gutturosa and all Onchocerca-like larvae in S. damnosum are likely to be O. volvulus, while the natural vectors of O. gutturosa are the Culicoides species C. fulvithorax, C. trifasciellus and C. krameri.

Eimeria tenella: local antibodies and interactions with the sporozoite surface.

Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10(3) oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10(5) oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and immunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results.(ABSTRACT TRUNCATED AT 250 WORDS)

Class-specific circulating antibodies in infections with Eimeria tenella.

Using a three-layer immunofluorescent test, class-specific, parasite-specific circulating antibody responses to Eimeria tenella were investigated following oocyst infection, drug-truncated oocyst infection and the injection of non-living antigens. Following all three means of antigenic stimulation, IgG, IgM and IgA antibodies were detected. The response to drug-truncated infections was dose-dependent. The sequence of appearance of antibody was IgM, IgA, IgG, whilst the relative quantities were IgG greater than or equal to IgM greater than IgA.

The maintenance and survival of Theileria annulata in colonies of Hyalomma anatolicum anatolicum.

Procedures are described for the colonization of Hyalomma anatolicum anatolicum ticks using gerbils, rabbits and cattle as hosts. On rabbits H. a. anatolicum undergoes a two-host cycle but methods are described for obtaining either unfed or engorged nymphs. Data are given on the life cycle timings and the numbers and timings of tick feeding regimes. The infection of H. a. anatolicum with two strains of Theileria annulata is described and methods given for monitoring the development and survival of T. annulata in H. a. anatolicum. Data are presented indicating the optimum maintenance conditions for T. annulata in H. a. anatolicum. Piroplasm parasitaemias in cattle greater than 2% gave high infection rates in adult ticks if engorged nymphs were moulted at 28 degrees C for 28 days. The Theileria will survive for ten months in such adults stored at 12 degrees C after moult, and when fed the ticks will produce maximum numbers of sporozoites on the third day of feeding, whatever their age. The moult of engorged nymphs is retarded at 18 degrees C but the Theileria in such ticks will develop normally when the ticks are moulted at 28 degrees C after four months storage at 18 degrees C.

The maturation of Theileria annulata in Hyalomma anatolicum anatolicum stimulated by incubation or feeding to produce sporozoites.

Adult Hyalomma anatolicum anatolicum ticks infected with Theileria annulata (Hissar strain) were incubated at 36 degrees C or fed on rabbits. Tick salivary glands were stained whole with methyl green pyronin or ground up and deposited on microscope slides and stained with Giemsa's solution. Separate batches of ticks from both treatments were ground up, centrifuged and filtered to produce sporozoite suspensions. The suspensions were examined as deposits on microscope slides stained with Giemsa's solution. The Theileria in the salivary glands of the fed ticks matured more completely and rapidly than in the incubated ticks. The peak numbers of sporozoites from the fed ticks was greater by at least tenfold than the peak from the incubated ticks. This peak was on the third day of feeding or on the fourth day of incubation. It was confirmed that fed ticks will be more suitable for sporozoite production for infection of cattle and production of stabilates.

Artificial infection of the tick Rhipicephalus appendiculatus with Theileria parva.

Methods for infecting Rhipicephalus appendiculatus ticks with Theileria parva by injection and by artificial feeding were confirmed and compared. The injection method proved simpler and at best as effective and suggested improvements are described.

Rapid quantitative assessment of Theileria infection in ticks.

A simplified method for methyl green pyronin staining is described for Theileria parva and T. annulata in whole salivary glands of Rhipicephalus appendiculatus and Hyalomma anatolicum subspecies respectively. The stain gives results comparable with Feulgen staining and can be used after the ticks have been in cold storage for 3 days. There is considerable variability in the rate and intensity of infection of these ticks with theilerial parasites and it is concluded that the method permits large samples (60 ticks per person per day) to be examined to overcome this variability when assessing infection quantitatively.