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Lubembe DM, Odongo DO, Joubert F, Sibeko-Matjila KP. "Limited diversity in the CD8+ antigen-coding loci in Theileria parva parasites from cattle from southern and eastern Africa." Vet Parasitol. 2021;291:109371. Abstract

Theileria parva infections in cattle causes huge economic losses in the affected African countries, directly impacting the livelihood of the poor small-holder farmers. The current immunization protocol using live sporozoites in eastern Africa, is among the control measures designed to limit T. parva infections in cattle. However, the ability of the immune protection induced by this immunization to protect against field parasites has been compromised by the diversity of the parasite involving the schizont antigen genes. Previous studies have reported on the antigenic diversity of T. parva parasites from southern and eastern Africa, however, similar reports on T. parva parasites particularly from cattle from southern Africa remains scanty, due to the self-limiting nature of Corridor disease. Thus, we evaluated the diversity of CD8+ T-cell regions of ten schizont antigen genes in T. parva parasites associated with Corridor disease and East Coast fever (ECF) from southern and eastern Africa respectively. Regions of schizont antigen (TpAg) genes containing the CD8+ T-cell epitopes (CTL determinants) were amplified from genomic DNA extracted from blood of T. parva positive samples, cloned and sequenced. The results revealed limited diversity between the two parasite groups from cattle from southern and eastern Africa, defying the widely accepted notion that antigen-encoding loci in cattle-derived parasites are conserved, while in buffalo-derived parasites, they are extensively variable. This suggests that only a sub-population of parasites is successfully transmitted from buffalo to cattle, resulting in the limited antigenic diversity in Corridor disease parasites. Tp4, Tp5, Tp7 and Tp8 showed limited to absence of diversity in both parasite groups, suggesting the need to further investigate their immunogenic properties for consideration as candidates for a subunit vaccine. Distinct and common variants of Tp2 were detected among the ECF parasites from eastern Africa indicating evidence of parasite mixing following immunization. This study provides additional information on the comparative diversity of TpAg genes in buffalo- and cattle-derived T. parva parasites from cattle from southern and eastern Africa.

Nyabongo L, Kanduma EG, Bishop RP, Machuka E, Njeri A, Bimenyimana AV, Nkundwanayo C, Odongo DO, Pelle R. "Prevalence of tick-transmitted pathogens in cattle reveals that Theileria parva, Babesia bigemina and Anaplasma marginale are endemic in Burundi." Parasit Vectors. 2021;14(1):6. Abstract

Tick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi.

Mulinge E, Odongo D, Magambo J, Njenga SM, Zeyhle E, Mbae C, Kagendo D, Addy F, Ebi D, Wassermann M, Kern P, Romig T. "Diversity of Taenia and Hydatigera (Cestoda: Taeniidae) in domestic dogs in Kenya." Parasitol Res. 2020;119(9):2863-2875. Abstract

Taenia species of domestic dogs can cause cysticercosis and coenurosis in a wide range of intermediate hosts including humans. Most taeniids of dogs are globally distributed, but some wildlife-transmitted species can be specific for certain regions. Generally, little information exists on the species composition and frequency in most regions of the world, which impairs risk assessment and control strategies. This study determined the range of taeniid species in dogs in four widely spaced areas of Kenya by genetic identification of eggs in faeces collected from the environment. Individual taeniid eggs were characterised by nested polymerase chain reaction of NADH dehydrogenase subunit 1 and cytochrome C oxidase 1 genes, restriction fragment length polymorphism and partial sequencing. Overall 79/1621 (4.9%) faecal samples contained eggs of Taenia or Hydatigera (8.0% in Turkana, 4.8% in Isiolo, 3.8% in Maasai Mara and 1.3% in Meru). Taenia hydatigena and T. multiceps were the most frequent, found in 36 and 15 samples, respectively. Other eggs found in the faeces belonged to T. serialis (sensu lato), T. madoquae (the first record in domestic dogs), T. ovis, T. saginata and Hydatigera taeniaeformis. Polymorphism of nad1 sequences revealed 22 and 8 haplotypes of T. hydatigena and T. multiceps, respectively. The results show the involvement of dogs in both domestic and sylvatic transmission cycles. In addition to the species range, this study provides data on the intraspecific diversity of T. hydatigena and T. multiceps in Kenya, which will serve as baseline information for further studies into cysticercosis and coenurosis in livestock and humans in the region.

Mwamuye MM, Odongo D, Kazungu Y, Kindoro F, Gwakisa P, Bishop RP, Nijhof AM, Obara I. "Variant analysis of the sporozoite surface antigen gene reveals that asymptomatic cattle from wildlife-livestock interface areas in northern Tanzania harbour buffalo-derived T. parva." Parasitol Res. 2020;119(11):3817-3828. Abstract

Buffalo-derived Theileria parva can 'break through' the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such 'breakthroughs' have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine 'breakthroughs' have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this.

Bishop RP, Kappmeyer LS, Onzere CK, Odongo DO, Githaka N, Sears KP, Knowles DP, Fry LM. "Equid infective Theileria cluster in distinct 18S rRNA gene clades comprising multiple taxa with unusually broad mammalian host ranges." Parasit Vectors. 2020;13(1):261. Abstract

Equine theileriosis, a tick-transmitted disease caused by the hemoprotozoan parasites Theileria equi and Theileria haneyi, affects equids throughout tropical and subtropical regions of the world. It is a significant regulatory concern in non-endemic countries, where testing for equine theileriosis is required prior to horse import to prevent parasite entry. Within endemic areas, infection causes significant morbidity and mortality, leading to economic losses. No vaccine for equine theileriosis is available, and current drug treatment protocols are inconsistent and associated with significant side effects. Recent work has revealed substantial genetic variability among equine theileriosis organisms, and analysis of ribosomal DNA from affected animals around the world indicates that the organisms can be grouped into five distinct clades. As these diverse parasites are capable of infecting a wide range of both tick and mammalian hosts, movement of different equine Theileria species between endemic countries, and eventually into non-endemic countries, is a significant concern. Furthermore, the substantial genetic variability of these organisms will likely render currently utilized importation diagnostic tests unable to detect all equine Theileria spp. To this end, more complete characterization of these diverse parasites is critical to the continued global control of equine theileriosis. This review discusses current knowledge of equine Theileria spp. in this context, and highlights new opportunities and challenges for workers in this field.

Nanteza A, Obara I, Kasaija P, Mwega E, Kabi F, Salih DA, Njahira M, Joyce Njuguna, Odongo D, Bishop RP, Skilton RA, Ahmed J, Clausen P-H, Lubega GW. "Antigen gene and variable number tandem repeat (VNTR) diversity in Theileria parva parasites from Ankole cattle in south-western Uganda: Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci." Transbound Emerg Dis. 2020;67 Suppl 1:99-107. Abstract

Theileria parva is a tick-transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti-transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south-western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen-encoding genes that are targets of CD8+T-cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south-western Uganda. Cattle-derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved.

Silatsa BA, Simo G, Githaka N, Kamga R, Oumarou F, Christian Keambou Tiambo, Machuka E, Domelevo J-B, Odongo D, Bishop R, Kuiate J-R, Njiokou F, Djikeng A, Pelle R. "First detection of Theileria parva in cattle from Cameroon in the absence of the main tick vector Rhipicephalus appendiculatus." Transbound Emerg Dis. 2020;67 Suppl 1:68-78. Abstract

A major risk factor for the spread of livestock diseases and their vectors is the uncontrolled transboundary movement of live animals for trade and grazing. Such movements constrain effective control of tick-transmitted pathogens, including Theileria parva. Only limited studies have been undertaken to identify ticks and tick-borne diseases (TTBDs) affecting cattle in central African countries, including Cameroon. We hereby report the collection of baseline data on the prevalence of T. parva in Cameroon through a countrywide cross-sectional survey, conducted in 2016, involving collection of blood samples from cattle from 63 sites across the five agro-ecological zones (AEZs) of the country. ELISA-based surveillance of infected cattle was performed on 479 randomly selected samples and revealed specific antibodies to T. parva in 22.7% and T. mutans in 41.1% of cattle. Screening of 1,340 representative DNA samples for the presence of T. parva identified 25 (1.86%) positives using a p104 antigen gene-based nested PCR assay. The positives were distributed across agro-ecological zones I, II, III and V. None of the p104 positive cattle exhibited clinical symptoms of East Coast fever (ECF). Using reverse line blot (RLB), 58 (4.3%) and 1,139 (85%) of the samples reacted with the T. parva and T. mutans oligonucleotide probes, respectively. This represents the first report of T. parva from Cameroon. Surprisingly, no Rhipicephalus appendiculatus ticks, the main vector of T. parva, were identified in a parallel study involving comprehensive morphological and molecular survey of tick species present in the country. Only two of the 25 p104 positive cattle were PCR-positive for the CD8+ T-cell target schizont-expressed antigen gene Tp1. Cloning and sequencing of Tp1 amplicons revealed sequence identity with the reference T. parva Muguga. This new finding raises serious concerns of a potential spread of ECF into the central African region.

Bishop RP, Odongo DO, Spooner PR, Morzaria SP, Oura CAL, Skilton RA. "Multilocus genotyping of Theileria parva isolates associated with a live vaccination trial in Kenya provides evidence for transmission of immunizing parasites into local tick and cattle populations." Transbound Emerg Dis. 2020;67 Suppl 1:88-98. Abstract

The live infection and treatment (ITM) vaccination procedure using the trivalent Muguga cocktail is increasingly being used to control East Coast fever, with potential implications for Theileria parva population genetic structure in the field. Transmission of the Kiambu V T. parva component to unvaccinated cattle has previously been described in Uganda. We monitored the T. parva carrier state in vaccinated and control animals on a farm in West Kenya where an ITM stabilate derived from the Kenyan T. parva Marikebuni stock was evaluated for field efficacy. A nested PCR-based Marikebuni-specific marker identified a carrier state in nine of ten vaccinated animals, detectable for a period of two years. We used 22 variable number tandem repeat (VNTR) markers to determine multilocus genotypes (MLGs) of 19 T. parva schizont-infected lymphocyte isolates derived from cattle and field ticks. Two isolates from unimmunized cattle were identical to the Marikebuni vaccination stock. Two cattle isolates were identical to a Muguga cocktail component Kiambu V. Seven isolates from ticks exhibited MLGs that were identical to the Serengeti/Muguga vaccine stocks. Six cattle and two tick-derived stocks exhibited unique MLGs. The data strongly suggest transmission of immunizing genotypes, from Marikebuni vaccine-induced carrier cattle to unimmunized cattle. It is possible that genotypes similar to those in the Muguga cocktail are present in the field in Western Kenya. An alternative hypothesis is that these parasites may have originated from vaccine trial sites in Eastern Uganda. If correct, this suggests that T. parva stocks used for immunization can potentially be disseminated 125 km beyond the immediate vaccination site. Regardless of their origin, the data provide evidence that genotypes similar to those in the Muguga cocktail are circulating in the field in East Africa, alleviating concerns about dissemination of 'alien' T. parva germplasm through live vaccination.

Bishop RP, Odongo D, Ahmed J, Mwamuye M, Fry LM, Knowles DP, Nanteza A, Lubega G, Gwakisa P, Clausen P-H, Obara I. "A review of recent research on Theileria parva: Implications for the infection and treatment vaccination method for control of East Coast fever." Transbound Emerg Dis. 2020;67 Suppl 1:56-67. Abstract

The infection and treatment (ITM) live vaccination method for control of Theileria parva infection in cattle is increasingly being adopted, particularly in Maasai pastoralist systems. Several studies indicate positive impacts on human livelihoods. Importantly, the first detailed protocol for live vaccine production at scale has recently been published. However, quality control and delivery issues constrain vaccination sustainability and deployment. There is evidence that the distribution of T. parva is spreading from endemic areas in East Africa, North into Southern Sudan and West into Cameroon, probably as a result of anthropogenic movement of cattle. It has also recently been demonstrated that in Kenya, T. parva derived from cape buffalo can 'breakthrough' the immunity induced by ITM. However, in Tanzania, breakthrough has not been reported in areas where cattle co-graze with buffalo. It has been confirmed that buffalo in northern Uganda national parks are not infected with T. parva and R. appendiculatus appears to be absent, raising issues regarding vector distribution. Recently, there have been multiple field population genetic studies using variable number tandem repeat (VNTR) sequences and sequencing of antigen genes encoding targets of CD8+ T-cell responses. The VNTR markers generally reveal high levels of diversity. The antigen gene sequences present within the trivalent Muguga cocktail are relatively conserved among cattle transmissible T. parva populations. By contrast, greater genetic diversity is present in antigen genes from T. parva of buffalo origin. There is also evidence from several studies for transmission of components of stocks present within the Muguga cocktail, into field ticks and cattle following induction of a carrier state by immunization. In the short term, this may increase live vaccine effectiveness, through a more homogeneous challenge, but the long-term consequences are unknown.

Nthiwa D, Bett B, Odongo D, Kenya E, Wainaina M, Grazioli S, Foglia E, Brocchi E, Alonso S. "Seroprevalence of foot-and-mouth disease virus in cattle herds raised in Maasai Mara ecosystem in Kenya." Prev Vet Med. 2020;176:104929. Abstract

A cross-sectional study was carried out to determine foot-and-mouth disease (FMD) seroprevalence and identify risk factors of exposure among cattle herds raised in three zones with different types of land use and progressively distant from the Maasai Mara National Reserve (MMNR) boundary. We selected five villages purposively; two in zone 1 (area < 20 km from the MMNR), another two in zone 2 (area between 20-40 km away from the MMNR) and one in zone 3 (area >40 km away from the MMNR). A total of 1170 cattle sera were collected from 390 herds in all the zones and tested for antibodies against the non-structural proteins (NSPs) of FMD virus (FMDV) using two 3ABC-based Enzyme-Linked Immunosorbent Assay ELISA kits. All sera samples were also screened for serotype-specific antibodies using Solid Phase Competitive ELISA (SPCE) kits (IZSLER, Italy). We targeted FMDV serotypes A, O, South African Territory [SAT] 1 and SAT 2, known to be endemic in East Africa including Kenya. Data on putative risk factors for FMD seropositivity in cattle were collected using a questionnaire. The overall apparent animal-level FMD seroprevalence based on the parallel comparison of the two anti-NSPs ELISA kits was 83.8 % (95 % CI; 81.8-85.9), and differed significantly across zones. Zone 1 had a higher seroprevalence than zones 2 and 3 (χ = 116.1, df = 2, p < 0.001). In decreasing order, the overall seroprevalences of FMDV serotypes A, SAT 2, O and SAT 1 were 26.3 % (95 % CI; 23.5-29.2), 21.4 % (95 % CI; 18.8-24.0), 21.2 % (95 % CI; 18.7-23.9) and 13.1 % (95 % CI; 11.1-15.3), respectively. The distribution of these serotypes differed significantly between zones (p < 0.05) except for SAT 2 serotype (χ = 0.90, df = 2, p = 0.639). Both serotypes A and O were more prevalent in zones 1 and 2 than zone 3 while serotype SAT 1, was higher in zone 3 compared to other zones. The results of multivariable analyses identified animal sex (i.e., female), raising of cattle in zones 1 and 2 (areas < 40 km away from the MMNR); mixing of cattle from multiple herds at watering points, and pastoral husbandry practices, as significant predictors of animal-level FMD seropositivity. This study established that FMD seroprevalence declined with distance from the MMNR.

Lutta HO, Odongo D, Mather A, Perez-Casal J, Potter A, Gerdts V, Berberov EM, Prysliak T, Martina Kyallo, Kipronoh A, Olum M, Pelle R, Naessens J. "Baseline analysis of Mycoplasma mycoides subsp. mycoides antigens as targets for a DIVA assay for use with a subunit vaccine for contagious bovine pleuropneumonia." BMC Vet Res. 2020;16(1):236. Abstract

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia in cattle. A prototype subunit vaccine is being developed, however, there is currently no diagnostic test that can differentiate between infected cattle and those vaccinated with the prototype subunit vaccine. This study characterized Mmm proteins to identify potential antigens for use in differentiating infected from vaccinated animals.

Obara I, Githaka N, Nijhof A, Krücken J, Nanteza A, Odongo D, Lubembe D, Atimnedi P, Mijele D, Njeri A, Mwaura S, Owido G, Ahmed J, Clausen PH, Bishop RP. "The Rhipicephalus appendiculatus tick vector of Theileria parva is absent from cape buffalo (Syncerus caffer) populations and associated ecosystems in northern Uganda." Parasitol Res. 2020;119(7):2363-2367. Abstract

Rhipicephalus appendiculatus is the major tick vector of Theileria parva, an apicomplexan protozoan parasite that causes the most economically important and lethal disease of cattle in East and central Africa. The African cape buffalo (Syncerus caffer) is the major wildlife host of T. parva from southern Uganda and Kenya to southern Africa. We show herein that R. appendiculatus appears to be absent from the two largest national parks in northern Uganda. Syncerus caffer is common in both of these national parks, specifically Murchison falls (MFNP) and Kidepo Valley (KVNP). We re-confirmed the previously reported absence of T. parva in buffalo sampled in the two northern parks based on RLB data using a nested PCR based on the T. parva p104 gene. By contrast, T. parva-infected R. appendiculatus ticks and parasite-infected buffalo were present in Lake Mburo (LMNP) in South central Uganda. This suggests that the distribution of R. appendiculatus, which is predicted to include the higher rainfall regions of northern Uganda, may be limited by additional, as yet unknown factors.

Mwamuye MM, Obara I, Elati K, Odongo D, Bakheit MA, Jongejan F, Nijhof AM. "Unique Mitochondrial Single Nucleotide Polymorphisms Demonstrate Resolution Potential to Discriminate Vaccine and Buffalo-Derived Strains." Life (Basel). 2020;10(12). Abstract

Distinct pathogenic and epidemiological features underlie different strains resulting in different clinical manifestations of East Coast Fever and Corridor Disease in susceptible cattle. Unclear delineation of these strains limits the control of these diseases in endemic areas. Hence, an accurate characterization of strains can improve the treatment and prevention approaches as well as investigate their origin. Here, we describe a set of single nucleotide polymorphisms (SNPs) based on 13 near-complete mitogenomes of strains originating from East and Southern Africa, including the live vaccine stock strains. We identified 11 SNPs that are non-preferentially distributed within the coding and non-coding regions, all of which are synonymous except for two within the gene of buffalo-derived strains. Our analysis ascertains haplotype-specific mutations that segregate the different vaccine and the buffalo-derived strains except Muguga and Serengeti-transformed strains suggesting a shared lineage between the latter two vaccine strains. Phylogenetic analyses including the mitogenomes of other species: , , and , with the latter two sequenced in this study for the first time, were congruent with nuclear-encoded genes. Importantly, we describe seven haplotypes characterized by synonymous SNPs and parsimony-informative characters with the other three transforming species mitogenomes. We anticipate that tracking mitochondrial haplotypes from this study will provide insight into the parasite's epidemiological dynamics and underpin current control efforts.

Lubembe DM, Odongo DO, Salih DA, Sibeko-Matjila KP. "Microsatellite and minisatellite genotyping of Theileria parva population from southern Africa reveals possible discriminatory allele profiles with parasites from eastern Africa." Ticks Tick Borne Dis. 2020;11(6):101539. Abstract

The control of Theileria parva, a protozoan parasite that threatens almost 50% of the cattle population in Africa, is still a challenge in many affected countries. Theileria parva field parasites from eastern Africa, and parasites comprising the current live T. parva vaccine widely deployed in the same region have been reported to be genotypically diverse. However, similar reports on T. parva parasites from southern Africa are limited, especially in Corridor disease designated areas. Establishing the extent of genetic exchange in T. parva populations is necessary for effective control of the parasite infection. Twelve polymorphic microsatellite and minisatellite loci were targeted for genotypic and population genetics analysis of T. parva parasites from South Africa, Mozambique, Kenya and Uganda using genomic DNA prepared from cattle and buffalo blood samples. The results revealed genotypic similarities among parasites from the two regions of Africa, with possible distinguishing allelic profiles on three loci (MS8, MS19 and MS33) for parasites associated with Corridor disease in South Africa, and East Coast fever in eastern Africa. Individual populations were in linkage equilibrium (VL) was observed. Genetic divergence was observed to be more within (AMOVA = 74%) than between (AMOVA = 26%) populations. Principal coordinate analysis showed clustering that separated buffalo-derived from cattle-derived T. parva parasites, although parasites from cattle showed a close genetic relationship. The results also demonstrated geographic sub-structuring of T. parva parasites based on the disease syndromes caused in cattle in the two regions of Africa. These findings provide additional information on the genotypic diversity of T. parva parasites from South Africa, and reveal possible differences based on three loci (MS8, MS19 and MS33) and similarities between buffalo-derived T. parva parasites from southern and eastern Africa.

Mukolwe LD, Odongo DO, Byaruhanga C, Snyman LP, Sibeko-Matjila KP. "Analysis of p67 allelic sequences reveals a subtype of allele type 1 unique to buffalo-derived Theileria parva parasites from southern Africa." PLoS One. 2020;15(6):e0231434. Abstract

East Coast fever (ECF) and Corridor disease (CD) caused by cattle- and buffalo-derived T. parva respectively are the most economically important tick-borne diseases of cattle in the affected African countries. The p67 gene has been evaluated as a recombinant subunit vaccine against ECF, and for discrimination of T. parva parasites causing ECF and Corridor disease. The p67 allele type 1 was first identified in cattle-derived T. parva parasites from East Africa, where parasites possessing this allele type have been associated with ECF. Subsequent characterization of buffalo-derived T. parva parasites from South Africa where ECF was eradicated, revealed the presence of a similar allele type, raising concerns as to whether or not allele type 1 from parasites from the two regions is identical. A 900 bp central fragment of the gene encoding p67 was PCR amplified from T. parva DNA extracted from blood collected from cattle and buffalo in South Africa, Mozambique, Kenya, Tanzania and Uganda, followed by DNA sequence analysis. Four p67 allele types previously described were identified. A subtype of p67 allele type 1 was identified in parasites from clinical cases of CD and buffalo from southern Africa. Notably, p67 allele type 1 sequences from parasites associated with ECF in East Africa and CD in Kenya were identical. Analysis of two p67 B-cell epitopes (TpM12 and AR22.7) revealed amino acid substitutions in allele type 1 from buffalo-derived T. parva parasites from southern Africa. However, both epitopes were conserved in allele type 1 from cattle- and buffalo-derived T. parva parasites from East Africa. These findings reveal detection of a subtype of p67 allele type 1 associated with T. parva parasites transmissible from buffalo to cattle in southern Africa.

Amzati GS, Djikeng A, Odongo DO, Nimpaye H, Sibeko KP, Muhigwa J-BB, Madder M, Kirschvink N, Marcotty T. "Genetic and antigenic variation of the bovine tick-borne pathogen Theileria parva in the Great Lakes region of Central Africa." Parasit Vectors. 2019;12(1):588. Abstract

Theileria parva causes East Coast fever (ECF), one of the most economically important tick-borne diseases of cattle in sub-Saharan Africa. A live immunisation approach using the infection and treatment method (ITM) provides a strong long-term strain-restricted immunity. However, it typically induces a tick-transmissible carrier state in cattle and may lead to spread of antigenically distinct parasites. Thus, understanding the genetic composition of T. parva is needed prior to the use of the ITM vaccine in new areas. This study examined the sequence diversity and the evolutionary and biogeographical dynamics of T. parva within the African Great Lakes region to better understand the epidemiology of ECF and to assure vaccine safety. Genetic analyses were performed using sequences of two antigen-coding genes, Tp1 and Tp2, generated among 119 T. parva samples collected from cattle in four agro-ecological zones of DRC and Burundi.

Omondi WP, Owino EA, Odongo D, Mwangangi JM, Torto B, Tchouassi DP. "Differential response to plant- and human-derived odorants in field surveillance of the dengue vector, Aedes aegypti." Acta Trop. 2019;200:105163. Abstract

Linalool oxide (LO) and hexanoic acid (HA) represent plant- and human-derived odorants, respectively, previously found as attractants for the dengue vector Aedes aegypti. Here, we investigated if a blend of both compounds can improve captures of this mosquito species in field trials in two dengue endemic sites, Kilifi and Busia Counties in Kenya. Ae. aegypti captures were significantly higher in Kilifi than Busia (χ = 170.63, P < 0.0001) and varied by treatments (χ = 151.19, P = 0.002). We found that CO-baited BG Sentinel traps combined with a blend of both odorants decreased Ae. aegypti captures about 2- to 4-fold compared to captures with the individual compounds (LO or HA) used as positive controls. This was the case for all blends of LO and HA, irrespective of the doses tested. Our findings indicate that combining plant- and human-derived odors may elicit a masking effect in trapping Ae. aegypti. These results partly corroborate previous findings for malaria mosquitoes which showed that combining lures from both host sources either decreases or increases trap catches depending on the dose. Further investigations in the usefulness of combining plant and animal odorants in mosquito trapping are therefore necessary.

Hassan S, Skilton RA, Pelle R, Odongo D, Bishop RP, Ahmed J, Seitzer U, Bakheit M, HASSAN SM, El Hussein AM. "Assessment of the prevalence of Theileria lestoquardi in sheep from the Sudan using serological and molecular methods." Prev Vet Med. 2019;169:104697. Abstract

Malignant theileriosis of sheep and goats caused by Theileria lestoquardi is considered to be among the most important tick borne diseases in the Sudan. Information on the prevalence of the disease in different parts of the Sudan is limited. The purpose of this study was to estimate the prevalence of the disease in five states of the Sudan using molecular and serological assays. A total of 393 blood and serum samples from clinically asymptomatic sheep were analysed using nested reverse line blot (nRLB) and loop mediated isothermal amplification (LAMP), as well as an enzyme-linked immunosorbent assay (ELISA). The results indicated a sero-prevalence of 33.8% while RLB and LAMP assays revealed molecular prevalences of 29.5 and 22.6% respectively. The prevalence of Theileria lestoquardi varied significantly according to the geographical origin of the infected animals, whereas age and gender did not have a significant effect. RLB data indicated that T. lestoquardi usually occurred as a co-infection with the non-pathogenic Theileria ovis. Using RLB as a gold standard, a sensitivity of 68.1% and a specificity of 96.4% were recorded for LAMP and a sensitivity of 75.9% and a specificity of 83.8% for ELISA. The Kappa coefficient between nRLB and LAMP indicated a significant level of agreement (0.692), but only moderate concordance (0.572) between nRLB and ELISA. The results of the present study confirm and extend earlier findings regarding the widespread of T. lestoquardi infections in sheep in the Sudan. The data provide evidence that should enable the veterinary authorities to deploy appropriate control measures.

Nthiwa D, Alonso S, Odongo D, Kenya E, Bett B. "Zoonotic Pathogen Seroprevalence in Cattle in a Wildlife-Livestock Interface, Kenya." Ecohealth. 2019;16(4):712-725. Abstract

A cross-sectional study was conducted to determine the seroprevalence of Brucella spp. and Leptospira spp. and risk factors of exposure in cattle in three zones with varying land use types and wildlife-livestock interactions. Five villages were selected purposively; two in areas with intensive livestock-wildlife interactions (zone 1), another two in areas with moderate livestock-wildlife interactions (zone 2) and one in areas where wildlife-livestock interactions are rarer (zone 3). Sera samples were collected from 1170 cattle belonging to 390 herds in all the zones and tested for antibodies against Brucella abortus and Leptospira interrogans serovar hardjo using ELISA kits. Data on putative risk factors for seropositivity of these pathogens in cattle were collected using a questionnaire. The overall apparent animal-level seroprevalence of brucellosis and leptospirosis was, respectively, 36.9% (95% CI 34.1-39.8) and 23.5% (95% CI 21.1-26.0). Brucella spp. seroprevalence was higher in zone 1 than in zones 2 and 3 (χ = 25.1, df = 2, P < 0.001). Zones 1 and 2 had significantly higher Leptospira spp. seroprevalence than zone 3 (χ = 7.0, df = 2, P = 0.029). Results of multivariable analyses identified animal sex (female) and zones (high interface area) as significant predictors (P < 0.05) of animal-level seropositivity of Brucella spp. For Leptospira spp., important predictors of animal-level seropositivity were animal sex (female), zones (moderate interface area) and herds utilizing a communal grazing reserve. The seroprevalences of Brucella spp. and Leptospira spp. in cattle were higher in areas with moderate to high wildlife-livestock interactions than those with rare interactions.

E Mulinge, SM Njenga OD, Magambo J. "Molecular identification of zoonotic hookworms in dogs from four counties of Kenya." Journal of helminthology. 2019:1-8.
Daniel Nthiwa, Silvia Alonso DO, Eucharia Kenya BB. "A participatory epidemiological study of major cattle diseases amongst Maasai pastoralists living in wildlife-livestock interfaces in Maasai Mara, Kenya." Tropical animal health and production. 2019:1-7.
Nyangacha RM, Oyieke F, Erastus Muniu, Stanley Chasia MO. "Spatial distribution, prevalence and potential risk factors of Tungiasis in Vihiga County, Kenya." PLoS neglected tropical diseases. 2019;13(3):e0007244.
Bishop RP, Thomas T Dolan, Rosemary B Dolan RSPSAD. "THEILERIOSIS IN MOUNTAIN BONGO REPATRIATED TO KENYA: A CLINCAL AND MOLECULAR INVESTIGATION." Journal of Zoo and Wildlife Medicine. 2019;50(2):342-349.
Knowles DP, Kappmeyer LS, Haney D, Herndon DR, Fry LM, Munro JB, Sears K, Ueti MW, Wise LN, Silva M, Schneider DA, Grause J, White SN, Tretina K, Bishop RP, Odongo DO, Pelzel-McCluskey AM, Scoles GA, Mealey RH, Silva JC. "Discovery of a novel species, Theileria haneyi n. sp., infective to equids, highlights exceptional genomic diversity within the genus Theileria: implications for apicomplexan parasite surveillance." Int J Parasitol. 2018;48(9-10):679-690. Abstract

A novel apicomplexan parasite was serendipitously discovered in horses at the United States - Mexico border. Phylogenetic analysis based on 18S rDNA showed the erythrocyte-infective parasite to be related to, but distinct from, Theileria spp. in Africa, the most similar taxa being Theileria spp. from waterbuck and mountain zebra. The degree of sequence variability observed at the 18S rDNA locus also suggests the likely existence of additional cryptic species. Among described species, the genome of this novel equid Theileria parasite is most similar to that of Theileria equi, also a pathogen of horses. The estimated divergence time between the new Theileria sp. and T. equi, based on genomic sequence data, is greater than 33 million years. Average protein sequence divergence between them, at 23%, is greater than that of Theileria parva and Theileria annulata proteins, which is 18%. The latter two represent highly virulent Theileria spp. of domestic cattle, as well as of African and Asian wild buffalo, respectively, which differ markedly in pathology, host cell tropism, tick vector and geographical distribution. The extent of genome-wide sequence divergence, as well as significant morphological differences, relative to T. equi justify the classification of Theileria sp. as a new taxon. Despite the overall genomic divergence, the nine member equi merozoite antigen (EMA) superfamily, previously found as a multigene family only in T. equi, is also present in the novel parasite. Practically, significant sequence divergence in antigenic loci resulted in this undescribed Theileria sp. not being detectable using currently available diagnostic tests. Discovery of this novel species infective to equids highlights exceptional diversity within the genus Theileria, a finding with serious implications for apicomplexan parasite surveillance.

Salih DA, Mwacharo JM, Pelle R, Njahira MN, Odongo DO, Mbole-Kariuki MN, Marcellino WL, Malak AK, Kiara H, El Hussein ARM, Bishop RP, Skilton RA. "Genetic diversity and population structure of Theileria parva in South Sudan." Ticks Tick Borne Dis. 2018;9(4):806-813. Abstract

Theileria parva is a parasitic protozoan that causes East Coast fever (ECF), an economically important disease of cattle in eastern, central and southern Africa. In South Sudan, ECF is considered a major constraint for livestock development in regions where the disease is endemic. To obtain insights into the dynamics of T. parva in South Sudan, population genetic analysis was performed. Out of the 751 samples included in this study, 178 blood samples were positive for T. parva by species-specific PCR, were collected from cattle from four regions in South Sudan (Bor = 62; Juba = 45; Kajo keji = 41 and Yei = 30) were genotyped using 14 microsatellite markers spanning the four chromosomes. The T. parva Muguga strain was included in the study as a reference. Linkage disequilibrium was evident when populations from the four regions were treated as a single entity, but, when populations were analyzed separately, linkage disequilibrium was observed in Bor, Juba and Kajo keji. Juba region had a higher multiplicity of infection than the other three regions. Principal components analysis revealed a degree of sub-structure between isolates from each region, suggesting that populations are partially distinct, with genetic exchange and gene flow being limited between parasites in the four geographically separated populations studied. Panmixia was observed within individual populations. Overall T. parva population genetic analyses of four populations in South Sudan exhibited a low level of genetic exchange between the populations, but a high level of genetic diversity within each population.

Erastus Mulinge, Japhet Magambo DOSNEZCM, Dorothy Kagendo, Francis Addy DEMWPKTR. "Molecular characterization of Echinococcus species in dogs from four regions of Kenya." Veterinary parasitology. 2018;255:49-57.
Lutta HO, Mather A, Maina TW, Odongo DO, Ndiwa NN, Wesonga HO, Naessens J. "Preliminary Findings of Lipoprotein B in Detecting Cattle Chronically Infected with Contagious Bovine Pleuropneumonia." Journal of Veterinary Science & Medical Diagnosis . 2018;7:2.
Odongo DO, Tiampati CM, Mulinge E, Mbae CK, Bishop RP, Zeyhle E, Magambo J, Wasserman M, Kern P, Romig T. "Prevalence and genotyping of Echinococcus granulosus in sheep in Narok County, Kenya." Parasitology research. 2018;117(7):2065-2073.
Nyangacha RM, Odongo D, Oyieke F, Ochwoto M, Korir R, Ngetich RK, Nginya G, Makwaga O, Bii C, Mwitari P, Tolo F. "Secondary bacterial infections and antibiotic resistance among tungiasis patients in Western, Kenya." PLoS Negl Trop Dis. 2017;11(9):e0005901. Abstract

Tungiasis or jigger infestation is a parasitic disease caused by the female sand flea Tunga penetrans. Secondary infection of the lesions caused by this flea is common in endemic communities. This study sought to shed light on the bacterial pathogens causing secondary infections in tungiasis lesions and their susceptibility profiles to commonly prescribed antibiotics. Participants were recruited with the help of Community Health Workers. Swabs were taken from lesions which showed signs of secondary infection. Identification of suspected bacteria colonies was done by colony morphology, Gram staining, and biochemical tests. The Kirby Bauer disc diffusion test was used to determine the drug susceptibility profiles. Out of 37 participants, from whom swabs were collected, specimen were positive in 29 and 8 had no growth. From these, 10 different strains of bacteria were isolated. Two were Gram positive bacteria and they were, Staphylococcus epidermidis (38.3%) and Staphylococcus aureus (21.3%). Eight were Gram negative namely Enterobacter cloacae (8.5%), Proteus species (8.5%), Klebsiellla species (6.4%), Aeromonas sobria (4.3%), Citrobacter species (4.3%), Proteus mirabillis(4.3%), Enterobacter amnigenus (2.1%) and Klebsiella pneumoniae (2.1%). The methicillin resistant S. aureus (MRSA) isolated were also resistant to clindamycin, kanamycin, erythromycin, nalidixic acid, trimethorprim sulfamethoxazole and tetracycline. All the Gram negative and Gram positive bacteria isolates were sensitive to gentamicin and norfloxacin drugs. Results from this study confirms the presence of resistant bacteria in tungiasis lesions hence highlighting the significance of secondary infection of the lesions in endemic communties. This therefore suggests that antimicrobial susceptibility testing may be considered to guide in identification of appropriate antibiotics and treatment therapy among tungiasis patients.

Osebe T, Mbaria J, Yole D, Odongo DO, Nderitu J, Ochanda H. "Bioactivity and toxicity of Bridelia micrantha, Chenopodium ambrosoides and Ocimum americanum plant extracts." International Journal of Basic & Clinical Pharmacology. 2017;6:5-11.
Lutta HO, Wesonga HO, Odongo DO, Thiaucourt F, Naessens J. "Inoculation of Mycoplasma mycoides by endotracheal intubation produces a milder disease than by contact transmission." Bulletin of Animal Health and Production Africa. 2017;65:477-484.
Mwamuye MM, Kariuki E, D O, Kabii J, Odongo D, Masiga D, Villinger J. "Novel Rickettsia and emergent tick-borne pathogens: A molecular survey of ticks and tick-borne pathogens in Shimba Hills National Reserve, Kenya." Ticks and Tick-borne Diseases. 2017;8(2): 208-218.
Olds CL, Mwaura S, Odongo DO, Scoles GA, Bishop R, Daubenberger C. "Induction of humoral immune response to multiple recombinant Rhipicephalus appendiculatus antigens and their effect on tick feeding success and pathogen transmission." Parasit Vectors. 2016;9(1):484. Abstract

Rhipicephalus appendiculatus is the primary vector of Theileria parva, the etiological agent of East Coast fever (ECF), a devastating disease of cattle in sub-Saharan Africa. We hypothesized that a vaccine targeting tick proteins that are involved in attachment and feeding might affect feeding success and possibly reduce tick-borne transmission of T. parva. Here we report the evaluation of a multivalent vaccine cocktail of tick antigens for their ability to reduce R. appendiculatus feeding success and possibly reduce tick-transmission of T. parva in a natural host-tick-parasite challenge model.

Norling M, Bishop RP, Pelle R, Qi W, Henson S, Drábek EF, Tretina K, Odongo D, Mwaura S, Njoroge T, Bongcam-Rudloff E, Daubenberger CA, Silva JC. "The genomes of three stocks comprising the most widely utilized live sporozoite Theileria parva vaccine exhibit very different degrees and patterns of sequence divergence." BMC Genomics. 2015;16:729. Abstract

There are no commercially available vaccines against human protozoan parasitic diseases, despite the success of vaccination-induced long-term protection against infectious diseases. East Coast fever, caused by the protist Theileria parva, kills one million cattle each year in sub-Saharan Africa, and contributes significantly to hunger and poverty in the region. A highly effective, live, multi-isolate vaccine against T. parva exists, but its component isolates have not been characterized. Here we sequence and compare the three component T. parva stocks within this vaccine, the Muguga Cocktail, namely Muguga, Kiambu5 and Serengeti-transformed, aiming to identify genomic features that contribute to vaccine efficacy.

Obara I, Ulrike S, Musoke T, Spooner PR, Jabbar A, Odongo D, Kemp S, Silva JC, Bishop RP. "Molecular evolution of a central region containing B cell epitopes in the gene encoding the p67 sporozoite antigen within a field population of Theileria parva." Parasitol Res. 2015;114(5):1729-37. Abstract

Protective immunity induced by the infective sporozoite stage of Theileria parva indicates a potential role for antibodies directed against conserved serologically reactive regions of the major sporozoite surface antigen p67 in vaccination to control the parasite. We have examined the allelic variation and determined the extent of B cell epitope polymorphism of the gene encoding p67 among field isolates originating from cattle exposed to infected ticks in the Marula area of the rift valley in central Kenya where the African cape buffalo (Syncerus caffer) and cattle co-graze. In the first of two closely juxtaposed epitope sequences in the central region of the p67 protein, an in-frame deletion of a seven-amino acid segment results in a truncation that was observed in parasites derived from cattle that co-grazed with buffalo. In contrast, the variation in the second epitope was primarily due to nonsynonymous substitutions, resulting in relatively low overall amino acid conservation in this segment of the protein. We also observed polymorphism in the region of the protein adjacent to the two defined epitopes, but this was not sufficient to provide statistically significant evidence for positive selection. The data indicates that B cell epitopes previously identified within the p67 gene are polymorphic within the Marula field isolates. Given the complete sequence identity of the p67 gene in all previously characterized T. parva isolates that are transmissible between cattle by ticks, the diversity observed in p67 from the Marula isolates in combination with the clinical reaction of the infected cattle is consistent with them originating from ticks that had acquired T. parva from buffalo.

Bishop RP, Hemmink JD, Morrison WI, Weir W, Toye PG, Sitt T, Spooner PR, Musoke AJ, Skilton RA, Odongo DO. "The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes." Int J Parasitol Parasites Wildl. 2015;4(3):333-42. Abstract

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. 'Deep 454 pyrosequencing' of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva.

Nthiwa DM, Odongo DO, Ochanda H, Khamadi S, Gichimu BM. "Trypanosoma Infection Rates in Glossina Species in Mtito Andei Division, Makueni County, Kenya." J Parasitol Res. 2015;2015:607432. Abstract

African Animal Trypanosomiasis (AAT) transmitted cyclically by tsetse fly (Glossina spp.) is a major obstacle to livestock production in the tropical parts of Africa. The objective of this study was to determine the infection rates of trypanosomes in Glossina species in Mtito Andei Division, Makueni County, Kenya. Tsetse fly species, G. longipennis and G. pallidipes, were trapped and DNA was isolated from their dissected internal organs (proboscis, salivary glands, and midguts). The DNA was then subjected to a nested PCR assay using internal transcribed spacer primers and individual trypanosome species were identified following agarose gel electrophoresis. Out of the 117 flies trapped in the area 39 (33.3%) were teneral while 78 (67%) were nonteneral. G. pallidipes constituted the largest percentage of 58% while G. longipennis were 42%. The overall trypanosomes infection rate in all nonteneral Glossina spp. was 11.53% with G. longipennis recording the highest infection rate of 23.08% while G. pallidipes had an infection rate of 5.77%. T. vivax was the most infectious (10.26%) compared to T. congolense (1.28%). Mean apparent densities were strongly positively correlated with infection rates (r = 0.95) confirming the importance of this parameter as an indicator of AAT transmission risk.

Githaka N, Konnai S, Bishop R, Odongo D, Lekolool I, Kariuki E, Gakuya F, Kamau L, Isezaki M, Murata S, Ohashi K. "Identification and sequence characterization of novel Theileria genotypes from the waterbuck (Kobus defassa) in a Theileria parva-endemic area in Kenya." Vet Parasitol. 2014;202(3-4):180-93. Abstract

Waterbuck (Kobus defassa), an ungulate species endemic to the Eastern African savannah, is suspected of being a wildlife reservoir for tick-transmitted parasites infective to livestock. Waterbuck is infested by large numbers of Rhipicephalus appendiculatus, the tick vector for Theileria parva, and previous data suggests that the species may be a source of T. parva transmission to cattle. In the present study, a total of 86 cattle and 26 waterbuck blood samples were obtained from Marula, a site in Kenya endemic for East Coast fever (ECF) where the primary wildlife reservoir of T. parva the Cape buffalo (Syncerus caffer) is also common. To investigate for the presence of cattle-infective Theileria parasites, DNA specimens extracted from the blood samples were subjected to two diagnostic assays; a nested PCR based on the p104 gene that is specific for T. parva, and a reverse line blot (RLB) incorporating 13 oligonucleotide probes including all of the Theileria spp. so far described from livestock and wildlife in Kenya. Neither assay provided evidence of T. parva or Theileria sp. (buffalo) infection in the waterbuck DNA samples. By contrast, majority of the cattle samples (67.4%) were positive for T. parva using a nested PCR assay. The RLB assay, including a generic probe for the genus Theileria, indicated that 25/26 (96%) of the waterbuck samples were positive for Theileria, while none of the 11 Theileria species-specific probes hybridized with the waterbuck-derived PCR products. Phylogenetic analysis of 18S ribosomal RNA (18S rRNA) and internal transcribed spacer (ITS) sequences within the RLB-positive waterbuck samples revealed the occurrence of three Theileria genotypes of unknown identity designated A, B and C. Group A clustered with Theileria equi, a pathogenic Theileria species and a causative agent of equine piroplasmosis in domestic equids. However, DNA from this group failed to hybridize with the T. equi oligonucleotide present on the RLB filter probe, suggesting the occurrence of novel taxa in these animals. This was confirmed by DNA sequencing that revealed heterogeneity between the waterbuck isolates and previously reported T. equi genotypes. Group B parasites clustered closely with Theileria luwenshuni, a highly pathogenic parasite of sheep and goats reported from China. Group C was closely related to Theileria ovis, an apparently benign parasite of sheep. Together, these findings provided no evidence that waterbuck plays a role in the transmission of T. parva. However, novel Theileria genotypes detected in this bovid species may be of veterinary importance.

Cassandra Olds, Mwaura S, Crowder D, Odongo D, van Oers M, Owen J, Bishop R, Daubenberger C. "Immunization of cattle with Ra86 impedes Rhipicephalus appendiculatus nymphal-to-adult molting." Ticks Tick Borne Dis. 2012;3(3):170-8. Abstract

Commercial vaccines based on the tick gut protein Bm86 have been successful in controlling the one-host tick Rhipicephalus (Boophilus) microplus and provide heterologous protection against certain other non-target ixodid tick species. This cross protection, however, does not extend to the three-host tick R. appendiculatus, the vector of the protozoan parasite Theileria parva. When transmitted to cattle, T. parva causes the often fatal disease East Coast fever. Here, we used insect cell-expressed recombinant versions of the R. appendiculatus homologs of Bm86, named Ra86, to vaccinate cattle. We measured multiple fitness characteristics for ticks that were fed on cattle Ra86-vaccinated or unvaccinated. The Ra86 vaccination of cattle significantly decreased the molting success of nymphal ticks to the adult stage. Modeling simulations based on our empirical data suggest that repeated vaccinations using Ra86 could reduce tick populations over successive generations. Vaccination with Ra86 could thus form a component of integrated control strategies for R. appendiculatus leading to a reduction in use of environmentally damaging acaricides.

Osebe T, Odongo D, Bishop R. "Molecular cloning, sequencing and recombinant expression of a putative tick protective antigen from three ixodid ticks." African Journal of Biotechnology. 2012;Vol. 11(84):15072-15081.
Malak, A.K., Mpoke L, Banak, J., Muriuki S, Skilton R, Odongo D, Kiara H. "Prevalence of livestock diseases and their impact on livelihoods in Central Equatoria State, southern Sudan ." Preventive Veterinary Medicine. 2012;104:216-223.
Kamau L, Skilton RA, Odongo DO, Mwaura S, Githaka N, Kanduma E, Obura M, Kabiru E, Orago A, Musoke A, Bishop RP. "Differential transcription of two highly divergent gut-expressed Bm86 antigen gene homologues in the tick Rhipicephalus appendiculatus (Acari: Ixodida)." Insect Mol. Biol.. 2011;20(1):105-14. Abstract

The transcriptional control of gene expression is not well documented in the Arthropoda. We describe transcriptional analysis of two exceptionally divergent homologues (Ra86) of the Bm86 gut antigen from Rhipicephalus appendiculatus. Bm86 forms the basis of a commercial vaccine for the control of Rhipicephalus (Boophilus) microplus. The R. appendiculatus Ra86 proteins contain 654 and 693 amino acids, with only 80% amino acid sequence identity. Reverse-transcription PCR of gut cDNA showed transcription of only one genotype in individual female ticks. PCR amplification of 3' untranslated sequences from genomic DNA indicated that both variants could be encoded within a single genome. When both variants were present, one of the two Ra86 genotypes was transcriptionally dominant.

Pellé R, Graham SP, Njahira MN, Osaso J, Saya RM, Odongo DO, Toye PG, Spooner PR, Musoke AJ, Mwangi DM, Taracha E, Morrison IW, Weir W, Silva JC, Bishop RP. "Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity." PLoS ONE . 2011;29(6(4)):e19015.
Odongo DO, Sunter JD, Kiara HK, Skilton RA, Bishop RP. "A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals." Parasitol. Res.. 2010;106(2):357-65. Abstract

Theileria parva causes East Coast fever, an economically important disease of cattle in sub-Saharan Africa. We describe a nested polymerase chain reaction (nPCR) assay for the detection of T. parva DNA in cattle blood spotted onto filter paper using primers derived from the T. parva-specific 104-kDa antigen (p104) gene. The sensitivity of this assay was compared to a previously described p104-based PCR and also the reverse line blot (RLB) technique, using serial dilutions of blood from a calf with known T. parva piroplasm parasitaemia. The relative sensitivities of the three assays were 0.4, 1.4 and 4 parasites/microl corresponding to blood parasitaemias of 9.2 x 10(-6)%, 2.8 x 10(-5)% and 8.3 x 10(-5)%, respectively. The three assays were applied to samples from two calves infected with the T. parva Muguga stock. Parasite DNA was consistently detectable by the two p104 PCR assays until 48 and 82 days post-infection, respectively, and thereafter sporadically. RLB detected parasite DNA in the two infected calves until days 43 and 45. Field samples from 151 Kenyan cattle exhibited 37.7% positivity for T. parva by regular p104 PCR and 42.3% positivity using p104 nPCR. Among 169 cattle blood samples from Southern Sudan, 36% were positive for T. parva using nPCR. The nPCR assay represents a highly sensitive tool for detection and monitoring of asymptomatic carrier state infections of T. parva in the blood of cattle.

Odongo DO, Ueti MW, Mwaura SN, Knowles DP, Bishop RP, Scoles GA. "Quantification of Theileria parva in Rhipicephalus appendiculatus (Acari: Ixodidae) confirms differences in infection between selected tick strains." J. Med. Entomol.. 2009;46(4):888-94. Abstract

Theileria parva is the etiologic agent of East Coast fever, an economically important disease of cattle in sub-Saharan Africa. This protozoan parasite is biologically transmitted by Rhipicephalus appendiculatus (Neumann) (Acari: Ixodidae). An understanding of the vector-parasite interaction may aid the development of improved methods for controlling transmission. We developed quantitative polymerase chain reaction (qPCR) and nested PCR (nPCR) assays targeting the T. parva-specific p104 gene to study T. parva pathogenesis in two strains of R. appendiculatus that had previously been selected to be relatively more (Kiambu) or less (Muguga) susceptible to infection. Nymphs from both strains were fed simultaneously to repletion on acutely infected calves. Nymphs from the Kiambu strain showed significantly higher engorgement weights compared with Muguga strain nymphs. Immediately after engorgement qPCR confirmed that nymphal Kiambu ticks had significantly higher parasite loads at repletion than Muguga nymphs. By 12 d postengorgement, parasites were below quantifiable levels but could be detected by nPCR in 83-87% (Muguga and Kiambu, respectively) of nymphs. After the molt, adult feeding on naïve cattle stimulated parasite replication in the salivary glands. PCR detected significantly more infected ticks than microscopy, and there was a significant difference between the two tick strains both in the proportion of ticks that develop salivary gland infections, and in the number of parasites within infected salivary glands. These data confirm that although both tick strains were competent vectors, Kiambu is both a significantly more susceptible and a more efficient host for T. parva than Muguga. The mechanisms that contribute to the levels of susceptibility and efficiency are unknown; however, this study lays the groundwork for a comparison of the transcriptome of these tick strains, the next step toward discovering the genes involved in the tick-parasite interaction.

Kopp N, Diaz D, Amacker M, Odongo DO, Beier K, Nitsch C, Bishop RP, Daubenberger CA. "Identification of a synthetic peptide inducing cross-reactive antibodies binding to Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus BM86 homologues." Vaccine. 2009;28(1):261-9. Abstract

The BM86 antigen, originally identified in Rhipicephalus (Boophilus) microplus, is the basis of the only commercialized anti-tick vaccine. The long-term goal of our study is to improve BM86 based vaccines by induction of high levels of tick gut binding antibodies that are also cross-reactive with a range of BM86 homologues expressed in other important tick species. Here we have used a BD86 derived synthetic peptide, BD86-3, to raise a series of mouse monoclonal antibodies. One of these mAbs, named 12.1, recognized BM86 homologues in immuno-histochemical analyses in four out of five tick species including R. (B.) microplus, Rhipicephalus (Boophilus) decoloratus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus. Our results indicate that broadly cross-reactive tick gut binding antibodies can be induced after immunization with a synthetic peptide derived from the protein BD86.

Odongo D, Kamau L, Skilton R, Mwaura S, Nitsch C, Musoke A, Taracha E, Daubenberger C, Bishop R. "Vaccination of cattle with TickGARD induces cross-reactive antibodies binding to conserved linear peptides of Bm86 homologues in Boophilus decoloratus." Vaccine. 2007;25(7):1287-96. Abstract

Vaccines based on recombinant Bm86 gut antigen from Boophilus microplus are a useful component of integrated control strategies against B. microplus infestations of cattle. The capacity of such vaccines to control heterologous infestations by two African tick species was investigated. The mean weight of engorged female ticks and mean egg mass per tick were significantly reduced in B. decoloratus infestations, but there was no effect of the vaccine against adult Rhipicephalus appendiculatus. We cloned, sequenced and expressed two Bm86 homologues (Bd86) from B. decoloratus. Amino acid sequence identity between Bd86 homologues (Bd86-1 and Bd86-2) and Bm86 was 86% and 85%, respectively, compared to 93% identity between the variants. Native Bd86 protein in B. decoloratus tick mid-gut sections and recombinant Bd86-1 reacted strongly with sera from TickGARD vaccinated cattle. TickGARD can therefore protect against a heterologous tick species with multiple antigen sequences. Epitope mapping using sera from TickGARD-vaccinated cattle identified two linear peptides conserved between the Bd86 homologues and Bm86. These epitopes represent candidate synthetic peptide vaccines for control of Boophilus spp. and the pathogens transmitted by these tick vectors.

Odongo DO, Oura CAL, Spooner PR, Kiara H, Mburu D, Hanotte OH, Bishop RP. "Linkage disequilibrium between alleles at highly polymorphic mini- and micro-satellite loci of Theileria parva isolated from cattle in three regions of Kenya." Int. J. Parasitol.. 2006;36(8):937-46. Abstract

Theileria parva schizont-infected lymphocyte culture isolates from western, central and coastal Kenya were analysed for size polymorphism at 30 T. parva-specific variable number tandem repeat (VNTR) loci using a panel of mini- and micro-satellite markers. The mean number of alleles ranged from 3 to 11 at individual loci and 183 distinct alleles were observed in total, indicating high genetic diversity within the T. parva gene pool in Kenyan cattle. The frequency distribution of the length variation of specific alleles among isolates ranged from normal to markedly discontinuous. Genetic relationships between isolates were analysed using standard indices of genetic distance. Genetic distances and dendrograms derived from these using neighbour-joining algorithms did not indicate significant clustering on a geographical basis. Analysis of molecular variance demonstrated that the genetic variation between individual isolates was 72%, but only 2.3% when isolates from different regions were pooled. Both these observations suggest minimal genetic sub-structuring relative to geographical origin. Linkage disequilibrium was observed between pairs of loci within populations, as in certain Ugandan T. parva populations. A novel observation was that disequilibrium was also detected between alleles at three individual pairs of VNTR loci when isolates from the three regional meta-populations were pooled for analysis.

Musoke A, Rowlands J, Nene V, Nyanjui J, Katende J, Spooner P, Mwaura S, Odongo D, Nkonge C, Mbogo S, Bishop R, Morzaria S. "Subunit vaccine based on the p67 major surface protein of Theileria parva sporozoites reduces severity of infection derived from field tick challenge." Vaccine. 2005;23(23):3084-95. Abstract

Two recombinant vaccines against Theileriaparva, based on a near full-length version of the sporozoite surface antigen p67 (p67(635)), or an 80 amino acid C-terminal section (p67C), were evaluated by exposure of immunized cattle to natural tick challenge in two sites at the Kenya Coast and one in Central Kenya. Vaccination reduced severe ECF by 47% at the coast and by 52% in central Kenya from an average incidence of 0.53+/-0.07 (S.E.) in 50 non-immunised controls to an average of 0.27+/-0.05 in 83 immunised animals. The reduction in severe East Coast fever was similar to that observed in laboratory experiments with p67(635) and p67C. The p67 coding sequence from thirteen T. parva field isolates including seven from vaccinated cattle that were not protected, was 100% identical to the gene on which the recombinant vaccine is based, suggesting a predominantly homologous p67 antigenic challenge. The same parasite isolates were however genetically heterogeneous at several loci other than p67.

Bishop R, Obura M, Odongo D, Odenyo A. "Specific PCR assay for a tannin-tolerant selenomonas ruminantium isolate, derived from helicase coding sequences." Appl. Environ. Microbiol.. 2004;70(5):3180-2. Abstract

Sequences from a tannin-tolerant Selenomonas ruminantium isolate (EAT2) that hydrolyzes gallic acid were identified. Two exhibited identity to helicases with a wide phylogenetic distribution. PCR amplification by using primers from one helicase gene detected 2000 to 5000 EAT2 genome equivalents but did not amplify total gastrointestinal microbial DNA of nine other ungulate species.

Nelson KE, Zinder SH, Hance I, Burr P, Odongo D, Wasawo D, Odenyo A, Bishop R. "Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche." Environ. Microbiol.. 2003;5(11):1212-20. Abstract

At present, there is little information on the phylogenetic diversity of microbial species that inhabit the gastrointestinal tracts of wildlife. To increase understanding in this area, we initiated a characterization of the bacterial diversity in the digestive tracts of three wild African ruminant species namely eland (Taurotragus oryx), Thompson's gazelle (Gazella rufifrons) and Grant's gazelle (Gazella granti), together with a domesticated ruminant species, zebu cattle (Bos indicus), and a non-ruminant species, zebra (Equus quagga). Bacterial diversity was analysed by PCR amplification, sequencing and phylogenetic analysis of 16S ribosomal DNA (rDNA) sequences. A total of 252 full-length 16S rDNA sequences averaging 1,500 base pairs (bp) in length, and an additional 27 partial sequences were obtained and subject to phylogenetic analysis. Using a 98% criterion for similarity, all except for one of the sequences were derived from distinct phylotypes. At least 24 distinct operational taxonomic units (OTU's) could be identified, with the majority of these sequences representing hitherto uncharacterized species and genera. The sequences were generally affiliated with four major bacterial phyla, the majority being members of the Firmicutes (low G+C Gram-positives) related to the genera Clostridium and Ruminococcus. By contrast, with earlier studies using 16S rDNA sequences to assess biodiversity in Bos taurus dairy cattle, Gram-negative bacteria in the Bacteroidales (Prevotella-Bacteroides group) were poorly represented. The lack of redundancy in the 16S rDNA dataset from the five African ungulate species, and the presence of novel sequences not previously described from the gastrointestinal tract of any animal species, highlights the level of diversity that exists in these ecosystems and raises the question as to the functional role of these species in the gastrointestinal tract.

Oura CAL, Odongo DO, Lubega GW, Spooner PR, Tait A, Bishop RP. "A panel of microsatellite and minisatellite markers for the characterisation of field isolates of Theileria parva." Int. J. Parasitol.. 2003;33(14):1641-53. Abstract

Mini- and microsatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis of parasites. Herein we describe the identification of a panel of 11 polymorphic microsatellites and 49 polymorphic minisatellites of the protozoan haemoparasite Theileria parva. The PCR products were run on high resolution Spreadex gels on which the alleles were identified and sized. The sequences of the mini- and microsatellites were distributed across the four chromosomes with 16 on chromosome 1, 12 on chromosome 2, 14 on chromosome 3 and 18 on chromosome 4. The primers from the 60 sequences were tested against all the Theileria species that co-infect cattle in East and Southern Africa and were found to be specific for T. parva. In order to demonstrate the utility of these markers, we characterised eight tissue culture isolates of T. parva isolated from cattle in widely separated regions of Eastern and Southern Africa (one from Zambia, one from Uganda, two from Zimbabwe, four from Kenya) and one Kenyan tissue culture isolate from Cape buffalo (Syncerus caffer). The numbers of alleles per locus range from three to eight indicating a high level of diversity between these geographically distinct isolates. We also analysed five isolates from cattle on a single farm at Kakuzi in the central highlands of Kenya and identified a range of one to four alleles per locus. Four of the Kakuzi isolates represented distinct multilocus genotypes while two exhibited identical multilocus genotypes. This indicates a high level of diversity in a single population of T. parva. Cluster analysis of multilocus genotypes from the 14 isolates (using a neighbour joining algorithm) revealed that genetic similarity between isolates was not obviously related to their geographical origin.

Odongo DO, Irungu LW. "A simple method for storing mosquito blood meals for human DNA profiling." Insect Science and its Application. 2002;22 (2 ):155-158.
Bishop R, Asefa G, Jamnadass R, Odongo D, Osuji P. "Characterization of Tannin-tolerant Bacterial Isolates from East African Ruminants." Anaerobe. 2001;7: 5-15.
Book Chapter
Bishop RP, Odongo DO, Mann J, Pearson TW, Sugimoto C, Haines LR, Glass E, Jensen K, Seitzer U, Ahmed JS, Graham SP, de Villiers EP. "Theileria.". In: Genome Mapping and Genomics in Animal-Associated Microbes. Berlin Heidelberg, Germany: Springer-Verlag; 2009.
Bishop R, Geysen D, Skilton R, Odongo D, Nene V, Allsopp B, Mbogo S, Spooner P, Morzaria S. "Genomic Polymorphism, Sexual Recombination and Molecular Epidemiology of Theileria parva.". In: Theileria. The Netherlands: Kluwer Academic Press; 2002.

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