Genetic Analyses and Genome-Wide Association Studies on Pathogen Resistance of Bos taurus and Bos indicus Cattle Breeds in Cameroon.

Autochthonous taurine and later introduced zebu cattle from Cameroon differ considerably in their resistance to endemic pathogens with little to no reports of the underlying genetic make-up. Breed history and habitat variations are reported to contribute significantly to this diversity worldwide, presumably in Cameroon as well, where locations diverge in climate, pasture, and prevalence of infectious agents. In order to investigate the genetic background, the genotypes of 685 individuals of different Cameroonian breeds were analysed by using the BovineSNP50v3 BeadChip. The variance components including heritability were estimated and genome-wide association studies (GWAS) were performed. Phenotypes were obtained by parasitological screening and categorised in Tick-borne pathogens (TBP), gastrointestinal nematodes (GIN), and onchocercosis (ONC). Estimated heritabilities were low for GIN and TBP (0.079 (se = 0.084) and 0.109 (se = 0.103) respectively) and moderate for ONC (0.216 (se = 0.094)). Further than revealing the quantitative nature of the traits, GWAS identified putative trait-associated genomic regions on five chromosomes, including the chromosomes 11 and 18 for GIN, 20 and 24 for TBP, and 12 for ONC. The results imply that breeding for resistant animals in the cattle population from Northern Cameroon might be possible for the studied pathogens; however, further research in this field using larger datasets will be required to improve the resistance towards pathogen infections, propose candidate genes or to infer biological pathways, as well as the genetic structures of African multi-breed populations.

Host specificity and phylogeny of Trichostrongylidae of domestic ruminants in the Guinea savannah of the Adamawa plateau in Cameroon.

Gastro-intestinal tracts were examined from thirteen Gudali zebu cattle, ten goats and ten sheep from the Adamawa highland in Northern Cameroon. A total of 28,325 adult helminths were recovered from the abomasa, small and large intestines. Five trichostrongylid genera were identified by their morphology: Haemonchus, Trichostrongylus and Oesophagostomum were predominant in both cattle and small ruminants, whilst Cooperia was only found in cattle both in the abomasum and small intestines. The molecular species identification and the inference of their phylogenetic relationships was based on the analysis of the hypervariable region I of the small subunit 18S rDNA (SSU) and the Second Internal Transcribed Spacer (ITS-2) of 408 adult trichostrongylid worms, which were PCR-amplified, sequenced, and compared with available database entries. Consistent with earlier findings, the SSU was invariable within the Haemonchus and Trichostrongylus genera, confirming the prior classification based on the morphology of the worms, but the ITS-2 was highly inter- and intraspecifically variable and thus allowed to distinguish individual species and to study the haplotype diversity within the different species. In cattle, we report for the first time in Cameroon co-infection with two species of Haemonchus (H. placei and H. similis), together with two species of Cooperia (C. punctata and C. pectinata) and one species of Trichostrongylus (T. axei). In goats and sheep, we found one highly polymorphic clade of Haemonchus contortus and two Trichostrongylus species (T. axei and T. colubriformis). When compared with other Trichostrongylidae from different regions of the world and wildlife, the analysis of haplotypes did not indicate any host and geographical isolation, but a very high haplotype diversity among H. contortus. These findings illustrate the complexity of trichostrongylid populations in domestic ruminants and suggest grazing overlap between domestic and wildlife hosts.

Whole genome characterization of autochthonous Bos taurus brachyceros and introduced Bos indicus indicus cattle breeds in Cameroon regarding their adaptive phenotypic traits and pathogen resistance.

BACKGROUND: African indigenous taurine cattle display unique adaptive traits shaped by husbandry management, regional climate and exposure to endemic pathogens. They are less productive with respect to milk and meat production which has been associated with amongst others, small size, traditional beliefs, husbandry practices, limited feed resources, disease burden and lack of sustained breeding for trait improvement. This resulted in the severe dwindling of their population size rendering them vulnerable to extinction. The Namchi taurine cattle breed is referred to as [Namchi (Doayo)] and shows resistance traits against trypanosome infection and exposure to tick infestation. Nonetheless, the historically later introduced Zebu cattle are the main cattle breeds in Africa today, even though they suffer more from locally prevailing pathogens. By using a whole genome sequencing approach, we sequenced with high depth for the first time the genomes of five cattle breeds from Cameroon in order to provide a valuable genetic resource for future African cattle breeding: the Namchi, an endangered trypano-tolerant taurine breed, the Kapsiki, an indigenous trypano-susceptible taurine breed, and three Zebu (Bos indicus indicus) breeds: Ngaoundere Gudali, White Fulani and Red Fulani. RESULTS: Approximately 167 Gigabases of raw sequencing data were generated for each breed and mapped to the cattle reference genomes ARS-UCD1.2 and UMD3.1.The coverage was 103 to 140-fold when aligning the reads to ARS-UCD1.2 with an average mapping rate of ~ 99%, and 22 to 30-fold when aligning the reads to UMD3.1 with an average mapping rate of ~ 64%. The single nucleotide polymorphisms (SNPs) obtained from analysis using the genome ARS-UCD1.2 were compared with reference genomes of European Bos taurus Holstein, the Asian Bos indicus Brahman, and the African trypanotolerant N'Dama breeds. A total of ~ 100 million (M) SNPs were identified and 7.7 M of those were breed-specific. An approximately 11.1 M constituted of small insertions and deletions. By using only breed-specific non-synonymous variants we identified genes as genetic signatures and associated Gene Ontology (GO) terms that could explain certain cattle-breed specific phenotypes such as increased tolerance against trypanosome parasites in the Namchi breed and heat tolerance in the Kapsiki breed. Phylogenetic analysis grouped, except for Namchi, the Bos taurus breeds Kapsiki, N'Dama and Holstein together while the B. indicus breeds White and Red Fulani, Gudali and Brahman clustered separately. The deviating result for Namchi indicates a hybrid status of the selected animal with a recent introgression of Zebu genes into its genome. CONCLUSIONS: The findings provide the first comprehensive set of genome-wide variant data of the most important Cameroonian cattle breeds. The genomic data shall constitute a foundation for breed amelioration whilst exploiting the heritable traits and support conservation efforts for the endangered local cattle breeds.

Widespread co-endemicity of Trypanosoma species infecting cattle in the Sudano-Sahelian and Guinea Savannah zones of Cameroon.

BACKGROUND: African animal trypanosomosis remains the major constraint of livestock production and livelihood of pastoral communities in Cameroon. Despite several decades of vector and parasite control efforts, it has not been eradicated. Alternative and sustainable control strategies require a sound knowledge of the local species, strains and vectors. In the Sudano-Sahelian and Guinea Savannah of Cameroon the prevalence and genetic diversity of trypanosomes infecting cattle was investigated by microscopy of cattle blood buffy coat and molecular methods using generic primers targeting parts of the internal transcribed spacer 1 (ITS-1) and encoded glycosomal glyceraldehyde 3-phosphate dehydrogenase-gene (gGAPDH). RESULTS: A total of 1176 randomly chosen cattle from five divisions in the Sudano-Sahelian and Guinea Savannah of Cameroon were examined. The overall prevalence of trypanosomes by microscopy was 5.9% (56/953) in contrast to 53.2% (626/1176) when molecular tools were used. This indicated a limited sensitivity of microscopy in subclinical infections with frequently low parasitemia. Three trypanosome species were identified by light microscopy: T. vivax (2.3%), T. brucei (3.7%) and T. congolense (3.0%), whereas five were identified by PCR, namely T. grayi/T. theileri (30.8%), T. vivax (17.7%), T. brucei (14.5%) and T. congolense (5.1%). Unexpected cases of T. grayi (n = 4) and T. theileri (n = 26) were confirmed by sequencing. Phylogenetic analysis of the gGAPDH revealed the presence of T. vivax, clade A and T. vivax clade C, which were co-endemic in the Faro et Deo division. T. grayi/T. theileri were the predominant species infecting cattle in tsetse free areas. In contrast, T. vivax, T. brucei and T. congolense were more abundant in areas where the Glossina-vectors were present. CONCLUSIONS: The abundance of pathogenic trypanosomes in tsetse infested areas is alarming and even more, the occurrence of T. vivax, T. brucei, T. congolense, T. theileri and T. grayi in tsetse-free areas implies that tsetse control alone is not sufficient to control trypanosomosis in livestock. To implement control measures that reduce the risk of spread in tsetse free areas, close monitoring using molecular tools and a thorough search for alternative vectors of trypanosomes is recommended.

Molecular identification and prevalence of tick-borne pathogens in zebu and taurine cattle in North Cameroon.

BACKGROUND: Public interest for tick-borne pathogens in cattle livestock is rising due to their veterinary and zoonotic importance. Consequently, correct identification of these potential pathogens is crucial to estimate the level of exposition, the risk and the detrimental impact on livestock and the human population. RESULTS: Conventional PCR with generic primers was used to identify groups of tick-borne pathogens in cattle breeds from northern Cameroon. The overall prevalence in 1260 blood samples was 89.1%, with 993 (78.8%) positive for Theileria/Babesia spp., 959 (76.1%) for Anaplasma/Ehrlichia spp., 225 (17.9%) for Borrelia spp., and 180 (14.3%) for Rickettsia spp. Sanger sequencing of a subset of positively-tested samples revealed the presence of Theileria mutans (92.2%, 130/141), T. velifera (16.3%, 23/141), Anaplasma centrale (10.9%, 15/137), A. marginale (30.7%, 42/137), A. platys (51.1%, 70/137), Anaplasma sp. 'Hadesa' (10.9%, 15/137), Ehrlichia ruminantium (0.7%, 1/137), E. canis (0.7%, 1/137), Borrelia theileri (91.3%, 42/46), Rickettsia africae (59.4%, 19/32) and R. felis (12.5%, 4/32). A high level of both intra- and inter-generic co-infections (76.0%) was observed. To the best of our knowledge, B. theileri, T. mutans, T. velifera, A. platys, Anaplasma sp. 'Hadesa', R. felis and E. canis are reported for the first time in cattle from Cameroon, and for R. felis it is the first discovery in the cattle host. Babesia spp. were not detected by sequencing. The highest number of still identifiable species co-infections was up to four pathogens per genus group. Multifactorial analyses revealed a significant association of infection with Borrelia theileri and anemia. Whereas animals of older age had a higher risk of infection, the Gudali cattle had a lower risk compared to the other local breeds. CONCLUSION: Co-infections of tick-borne pathogens with an overall high prevalence were found in all five study sites, and were more likely to occur than single infections. Fulani, Namchi and Kapsiki were the most infected breed in general; however, with regions as significant risk factor. A better-adapted approach for tick-borne pathogen identification in co-infected samples is a requirement for epidemiological investigations and tailored control measures.

Development of a Low-Density DNA Microarray for Detecting Tick-Borne Bacterial and Piroplasmid Pathogens in African Cattle.

In Africa, pathogens transmitted by ticks are of major concern in livestock production and human health. Despite noticeable improvements particularly of molecular screening methods, their widespread availability and the detection of multiple infections remain challenging. Hence, we developed a universally accessible and robust tool for the detection of bacterial pathogens and piroplasmid parasites of cattle. A low-cost and low-density chip DNA microarray kit (LCD-Array) was designed and tested towards its specificity and sensitivity for five genera causing tick-borne diseases. The blood samples used for this study were collected from cattle in Northern Cameroon. Altogether, 12 species of the genera Anaplasma, Ehrlichia, Rickettsia and Theileria, and their corresponding genus-wide probes including Babesia were tested on a single LCD-Array. The detection limit of plasmid controls by PCR ranged from 1 to 75 copies per µL depending on the species. All sequenced species hybridized on the LCD-Array. As expected, PCR, agarose gel electrophoresis and Sanger sequencing found significantly less pathogens than the LCD-Array (p < 0.001). Theileria and Rickettsia had lower detection limits than Anaplasma and Ehrlichia. The parallel identification of some of the most detrimental tick-borne pathogens of livestock, and the possible implementation in small molecular-diagnostic laboratories with limited capacities makes the LCD-Array an appealing asset.

Onchocerca - infected cattle produce strong antibody responses to excretory-secretory proteins released from adult male Onchocerca ochengi worms.

BACKGROUND: The front line molecules from filarial worms and other nematodes or helminthes are their Excretory-Secretory (ES) products. Their interaction with the host cells, proteins and immune system accounts for the skin and eye pathology or hyposensitivity observed in human onchocerciasis. ES products and adult worms' crude extracts from Onchocerca ochengi, a filarial nematode that infects the African zebu cattle, were utilized in the present study as a model for studying Onchocerca volvulus that causes river blindness in man. METHODS: The ES products were generated from adult male and female worms in vitro and analyzed with poly acrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) using sera from Onchocerca-infected cattle and humans. The cattle sera were collected from a herd that had been exposed for six years to natural transmission of Onchocerca spp. The expressed reactivity was evaluated and differences analyzed statistically using Kruskal-Wallis rank and Chi-square tests. RESULTS: The gel electrophoretic analyses of 156 ES products from O. ochengi female and male worms and of two somatic extracts from three females and 25 males revealed differences in the protein pattern showing pronounced bands at 15, 30-50 and 75 kDa for male ES proteins and 15, 25 and 40-75 kDa for somatic extracts, respectively and less than 100 kDa for female worms. Proteins in the ES products and somatic extracts from female and male Onchocerca ochengi worms were recognized by IgG in sera from both Onchocerca-exposed cattle and humans. Bovine serum antibodies reacted more strongly with proteins in the somatic extracts than with those in the ES products. Interestingly, the reaction was higher with male ES products than with ES products from female worms, suggesting that the males which migrate from one nodule to another are more exposed to the host immune system than the females which remain encapsulated in intradermal nodules. CONCLUSIONS: This study demonstrates that O. ochengi ES products and, in particular, extracts from male filariae may represent a good source of immunogenic proteins and potential vaccine candidates.

Full mitochondrial and nuclear genome comparison confirms that Onchocerca sp. "Siisa" is Onchocerca ochengi.

Onchocerca ochengi is a nodule-forming filarial nematode parasite of cattle. It is the closest known relative of the human parasite Onchocerca volvulus, with which it shares the black fly vector Simulium damnosum. Onchocerca sp. "Siisa" was described in black flies and in cattle and, based on limited mitochondrial sequence information, appeared to be about equally phylogenetically distant from O. ochengi and O. volvulus. Based on molecular genetic markers and apparent interbreeding, we later proposed that O. sp. "Siisa" belongs to the species O. ochengi. However, we did not demonstrate directly that the hybrids were fertile, and we were still unable to resolve the phylogenetic relationship of O. ochengi, O. sp. "Siisa," and O. volvulus, leaving some concerns with the conclusion mentioned above. Here, we present fully assembled, manually curated mitochondrial genomes of O. ochengi and O. sp. "Siisa," and we compare multiple individuals of these two taxa with respect to their whole mitochondrial and nuclear genomes. Based on the mitochondrial genomes, O. ochengi and O. sp. "Siisa" are phylogenetically much closer to each other than to O. volvulus. The differences between them are well within the range of what is expected for within-species variation. The nuclear genome comparison provided no indication of genetic separation of O. ochengi and O. sp. "Siisa." From this, in combination with the earlier literature, we conclude that O. ochengi and O. sp. "Siisa" should be considered one species.