Anthony J. Musoke

Identification of a Theileria mutans-specific antigen for use in an antibody and antigen detection ELISA

Summary Purified piroplasms of Theileria mutans were used to immunize BALB/c mice to generate monoclonal antibodies (MoAbs). The MoAbs recognized an antigen of a relative molecular mass of 32 kDa in Western blots. This antigen was also recognized by sera from cattle which had recovered naturally from experimental tick‐transmission or infections induced by the blood stages of T. mutans. The MoAbs did not react, in indirect immunofluorescence or enzyme‐linked immunosorbent assays (ELISA), with the common haemoparasites of cattle, namely, T. parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma congolense, T. vivax or T. brucei. An antigen capture ELISA was established with two of the MoAbs which recognized different epitopes on the 32 kDa molecule. Using this test it was possible to detect circulating antigens or immune complexes in sera collected from cattle during the acute or chronic phases of infection. When the purified 32 kDa protein was used as antigen in a micro‐ELISA to detect circulating antibodies in both experimental and field cattle sera, it was found that the titres of antibodies ranged between 1:20 and 1:10 240. Results of this study indicate that the antigen and immune complex capture assays and the antibody detection ELISA can be complementary in the immunodiagnosis of acute and chronic T. mutans infections. Moreover, the tests are useful in the differential diagnosis of the disease and for epidemiological studies.

Characterization of a polymorphic immunodominant molecule in sporozoites and schizonts of Theileria parva

Summary This study examines several aspects of a polymorphic, immunodominant molecule (P!M) found in the protozoan parasite. Theileria parva. The antigen is present in all T. p. parva stocks examined, and in the related subspecies, T. p. bovis and T. p. lawrencei. It is the predominant antigen recognized by antisera from immune cattle on Western blot analysis of schizont‐infected lymphocytes, and is the only antigen which has been shown to react with anti‐schizont monoclonal antibodies (MoAbs) on Western blots or in immunoprecipitations. The antigen shows polymorphism in both size and expression of antibody epitopes among the different stocks of T. parva. The antigen is present in sporozoites as well as schizonts.

Confirmation and dissection of QTL controlling resistance to malaria in mice

We developed an F11 AIL population from an F1 cross of A/J (susceptible) and C57BL/6J (resistant) mouse strains. One thousand F11 mice were challenged with P.c. chabaudi 54X, and 340 mice selected from the phenotypic extremes for susceptibility and resistance were genotyped for microsatellite markers on Chromosomes (Chrs) 5, 8, and 17. QTL originally detected in backcross and F2 populations were confirmed on the three chromosomes within narrower genomic regions, by maximum likelihood and regression analyses. Each of the previously mapped QTL on Chrs 5 and 17 resolved into two linked QTLs. The distal and proximal QTLs on Chrs 5 and 17, respectively, map to the previously reported QTL.

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4-7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9-10 X 10(6) base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7-8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G + C content than T. parva or the presence of highly reiterated G + C-rich regions.

Different Vaccine Strategies Used to Protect against Theileria annulataa

ABSTRACT: SPAG‐1, a sporozoite surface antigen of T. annulata, has previously been shown to elicit partial protection when used, as an hepatitis B core antigen fusion, to immunize cattle. The objective of this study was to try and improve the protective capacity of this antigen by enlisting different vaccine strategies. Cattle were immunized with SPAG‐1, as a fusion protein with a His6 tag, either incorporated into ISCOMs, with or without the merozoite antigens TAMS 1‐1 and 1‐2, or with RWL as adjuvant three times at monthly intervals. Another group of cattle were immunized with p67, the T. parva sporozoite antigen, in RWL to assess whether any cross‐protection could be induced. The animals were then challenged with an estimated LD50 of T. annulata sporozoites, and their ability to resist the infection was investigated. Serum responses and T‐cell proliferative responses were analyzed throughout the trial. Post‐challenge analyses included lymph node biopsies and blood smears to check for the presence of parasites, routine hematological parameters, and observation for clinical manifestations of the disease. The results of this trial will be discussed.

Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity

Background Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8+ T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8+ T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8+ T-cell epitopes, and to analyse the sequences for evidence of selection. Methodology/Principal Findings Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. Conclusions/Significance The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.

Vaccines against Theileria parva.

Abstract: Bovine theileriosis caused by Theileria parva continues to be a major economic problem in many parts of Eastern, Southern, and Central Africa. Due to the unsustainable nature of the present control method‐using toxic acaricides to kill ticks‐alternative control methods are being sought. Live vaccines are being used in many countries in the region. These vaccines are based on the infective sporozoite stage of the parasite. Sporozoites are inoculated in cattle with simultaneous administration of a long‐acting formulation of oxytetracycline. These vaccines are poorly adopted in the region, mainly because of problems associated with the use of live parasites. An experimental recombinant vaccine based on a sporozoite surface antigen (p67) has been developed. Immunization with this antigen induces neutralizing antibodies and, under laboratory conditions, this technique protects approximately 70% of the immunized cattle to a defined needle challenge. The efficacy of the vaccine is currently being evaluated under field challenge in Kenya. Since a vaccine based on a single antigen may not be sustainable under field conditions, a search for schizont antigens that induce protective cell‐mediated immune responses continues. It is expected that the ultimate vaccine against theileriosis will incorporate a mixture of several antigens derived from both sporozoite and schizont stages, contributing to robust immunity.

Evaluation of recombinant sporozoite antigen SPAG-1 as a vaccine candidate against Theileria annulata by the use of different delivery systems

Summary The major sporozoite surface antigen of Theileria annulata (SPAG‐1) is a candidate for inclusion in a subunit vaccine. In this paper we summarize the results of 4 vaccination experiments using recombinant SPAG‐1 expressed in different systems and presented in different adjuvants. The antigen has been presented as either a C terminal 108 amino acid peptide (called SR1) expressed as both β‐galactosidase and hepatitis B core antigen fusions or as a full‐length form expressed as a GST fusion with an N terminal His6 tag. We used different adjuvants, namely Freunds, saponin, ISCOMs and a proprietary adjuvant supplied by SmithKline Beecham, which we call SKBA. The data point to the conclusion that SPAG‐1 can elicit partial protection and is therefore suitable for inclusion in an eventual multicomponent subunit vaccine.

Identification of Babesia bigemina infected erythrocyte surface antigens containing epitopes conserved among strains

The presence of previously uncharacterized antigens (new antigens) on the surface of intact erythrocytes infected with three strains of Babesia bigemina from Kenya and one each from Puerto Rico, Mexico, St. Croix, and Texcoco‐Mexico was demonstrated by indirect immunofluorescent antibody (IFA) reactions. These antigens were not strain specific because antibodies in bovine immune serum to either the Mexico or Kenya isolates reacted with all seven strains tested. Homologous and heterologous immune serum antibodies bound a maximum of 83% and 55%, respectively, of intact erythrocytes infected with the Kenya‐Ngong strain but not uninfected erythrocytes. Both sera caused agglutination of only infected erythrocytes. Antibodies eluted from the surface of glutaraldehyde (0.25%) fixed infected erythrocytes had IFA reaction patterns among strains similar to those of immune sera before elation. Eluted antibodies were used to determine if these antigens were protein and encoded by B. bigemina. Eluted antibodies bound seven parasite‐encoded proteins of 240, 220, 66, 62, 58, 52 and 38 kDa in an erythrocyte surfacespecific immunoprecipitation reaction of 35‐methionine labelled proteins. It was concluded that the surface of B. bigemina infected erythrocytes had parasite‐encoded proteins and that these proteins had surface exposed epitopes that were conserved among the seven strains examined which were from two continents.

Sequence and expression of a 90-kilodalton heat-shock protein family member of Theileria parva.

A Theileria parva specific full-length cDNA clone, T7, which encodes a protein with more than 60% homology to heat shock protein 90 (hsp90) of other organisms, has been identified. T7 appears to be a single copy gene. The gene is expressed as a protein of 87 kDa in both the sporozoite and schizont stages of T. parva. The protein was not found in the piroplasm stage, although the corresponding transcript was detected, suggesting post-transcriptional regulation of the gene. In the schizont stage the T7 protein is upregulated upon heat shock and localized in the cytoplasm.