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.

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.

Species identity and phylogeny of Paramphistomoidea Fischoeder, 1901 occurring in cattle and sheep in North Cameroon.

Paramphistomidae and Gastrothylacidae are parasitic flatworms occurring in wild and domestic ruminants in different parts of the world especially in Asia and Africa. In Central Africa, few studies have been done using molecular techniques to resolve taxonomical groupings and understand the epizootiology of these parasites. In this study, we molecularly characterized two hundred adult flukes collected from the fore stomachs of cattle and sheep in the Adamawa region of the northern Cameroon. PCR and sequencing of the nuclear ITS-2 of the ribosomal DNA gene and a portion of the mitochondrial cox-1 locus revealed the presence of at least nine species belonging to the genera of Cotylophoron, Calicophorn, Orthocoelium and Carmyerius. In Zebu cattle, we identified Ca. microbothrium, Ca. clavula, Ca. phillerouxi, Co. cotylophorum, Co. fuelleborni, O. scoliocoelium, Car. gregarius, Car. graberi and Car. mancupatus and one yet unknown Paramphistomoidea sp, whereas in sheep, only Ca. microbothrium was found. The present study also strongly suggests cross-hybridization between the two Cotylophoron species coexisting in cattle. These results have implications for the diagnosis and control of rumen flukes in the region and point to the need for accurate species identification to understand parasite distribution and population genetics.

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.

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.

Molecular screening of tsetse flies and cattle reveal different Trypanosoma species including T. grayi and T. theileri in northern Cameroon.

BACKGROUND: African trypanosomes are mainly transmitted through the bite of tsetse flies (Glossina spp.). The present study investigated the occurrence of pathogenic trypanosomes in tsetse flies and cattle in tsetse fly-infested areas of Northern Cameroon. RESULTS: Trypanosomes were identified using nested polymerase chain reaction (PCR) analysis of internal transcribed spacer 1 (ITS1) region, both by size estimation and sequencing of PCR products. Apparent density indices recorded in Gamba and Dodeo were 3.1 and 3.6 tsetse flies per trap and day, respectively. Trypanosoma prevalence infection rate for the tsetse fly gut (40%) and proboscis (19%) were recorded. Among the flies where trypanosomes were detected in the gut, 41.7% were positive for T. congolense and 14.6% for T. brucei ssp., whereas in the proboscis 36% harboured T. congolense and 62% contained T. vivax. T. grayi was highly prevalent in tsetse fly gut (58%). The most common mixed infections were the combination of T. congolense and T. grayi. Trypanosome prevalence rate in cattle blood was 6%. Among these, T. vivax represented 26%, T. congolense 35%, T. brucei ssp. 17% and T. theileri 17% of the infections. Surprisingly, in one case T. grayi was found in cattle. The mean packed cell volume (PCV) of cattle positive for trypanosomes was significantly lower (24.1 ± 5.6%; P < 0.05) than that of cattle in which trypanosomes were not detected (27.1 ± 4.9%). Interestingly, the occurrence of T. theileri or T. grayi DNA in cattle also correlated with low PCV at pathological levels. CONCLUSION: This molecular epidemiological study of Trypanosoma species in Northern Cameroon revealed active foci of trypanosomes in Dodeo and Gamba. These findings are relevant in assessing the status of trypanosomosis in these regions and will serve as a guide for setting the priorities of the government in the control of the disease.

Ongoing Transmission of Onchocerca volvulus after 25 Years of Annual Ivermectin Mass Treatments in the Vina du Nord River Valley, in North Cameroon.

BACKGROUND: Recent reports of transmission interruption of Onchocerca volvulus, the causing agent of river blindness, in former endemic foci in the Americas, and more recently in West and East Africa, raise the question whether elimination of this debilitating disease is underway after long-term treatment of the population at risk with ivermectin. The situation in Central Africa has not yet been clearly assessed. METHODS AND FINDINGS: Entomologic data from two former endemic river basins in North Cameroon were generated over a period of 43 and 48 months to follow-up transmission levels in areas under prolonged ivermectin control. Moreover, epidemiologic parameters of animal-borne Onchocerca spp. transmitted by the same local black fly vectors of the Simulium damnosum complex were recorded and their impact on O. volvulus transmission success evaluated. With mitochondrial DNA markers we unambiguously confirmed the presence of infective O. volvulus larvae in vectors from the Sudan savannah region (mean Annual Transmission Potential 2009-2012: 98, range 47-221), but not from the Adamawa highland region. Transmission rates of O. ochengi, a parasite of Zebu cattle, were high in both foci. CONCLUSIONS/SIGNIFICANCE: The high cattle livestock density in conjunction with the high transmission rates of the bovine filaria O. ochengi prevents the transmission of O. volvulus on the Adamawa plateau, whereas transmission in a former hyperendemic focus was markedly reduced, but not completely interrupted after 25 years of ivermectin control. This study may be helpful to gauge the impact of the presence of animal-filariae for O. volvulus transmission in terms of the growing human and livestock populations in sub-Saharan countries.

Animal trypanosomosis in clinically healthy cattle of north Cameroon: epidemiological implications.

BACKGROUND: The control of animal trypanosomosis consists, amongst other things, of the punctual treatment of new cases, primarily diagnosed by pastoralists on the basis of clinical signs. This practice suggests that many apparently healthy infected animals are left untreated. In this study animal trypanosomosis in clinically healthy zebu cattle was evaluated, the distribution of the vectors established and the epidemiological implications discussed. METHODS: In 2014 two cross-sectional surveys were carried out in the Cambeef ranch. A total of 866 blood samples were collected from cattle in different sites: 549 in the dry season and 317 in the rainy season. The blood samples were subjected to parasitological examination using the buffy coat method and to PCV determination. An entomological survey on animal trypanosomosis vectors was undertaken during tsetse flies caught were identified and the mid-gut of each living non-teneral tsetse fly was examined for infections using a microscope. RESULTS: An overall trypanosomosis prevalence of 9% was found in the cattle examined. There were significantly (P < 0.05) more trypanosome infected cattle in the dry season than the rainy season. Trypanosome-infected cattle had significantly (P < 0.05) lower Body Condition Scores (BCS) and Packed Cell Volumes (PCV) in the dry season than in the rainy season. Anemia was positively correlated with trypanosome infection. The likelihood for an animal to be parasitologically free of trypanosome infection was at least three times as high in the Gudali breed as compared with the white and red Fulani breeds. Species of trypanosomes identified were Trypanosoma vivax (73.23%), Trypanosoma congolense (15.49%) and Trypanosoma brucei (11.27%). A total of 390 tsetse flies and 103 tabanids were trapped. Two species of tsetse flies were identified: Glossina tachinoides (33.59%) and G. morsitans submorsitans (41%). Nine of the 194 non-teneral flies were infected with trypanosomes. CONCLUSION: Carriers of trypanosomes are present amongst apparently healthy cattle in the study site. Attempts to successfully reduce the population of reservoir trypanosomes within herds and control the disease will need to consider mass screening once every year and this should be associated with drug sensitivity tests.

Isolation, identification and functional profile of excretory-secretory peptides from Onchocerca ochengi.

Parasitic helminths excrete or secrete a variety of functional molecules into the internal milieu of their mammalian hosts and arthropod vectors which reveal distinct immunomodulatory and other biological activities. We identified and initially characterized the low molecular weight peptide composition of the secretome from the filarial parasite Onchocerca ochengi. A total of 85 peptides were purified by liquid chromatography and further characterized by mass spectrometry. 72 of these peptides were derived from already described Onchocerca proteins and 13 peptide sequences are included in the sequence of uncharacterized proteins. Three peptides, similar to host defense peptides, revealed antibacterial activity. The present analysis confirms the putative involvement of low molecular weight compounds in the parasite-host cross-talk.

Molecular evidence of 'Siisa form', a new genotype related to Onchocerca ochengi in cattle from North Cameroon.

Onchocerca ochengi, a filarial nematode parasite from African Zebu cattle is considered to be the closest relative of Onchocerca volvulus, the causative agent of river blindness. Both Onchocerca species share the vector, black flies of the Simulium damnosum complex. Correct identification of their infective third-stage larvae in man-biting vectors is crucial to distinguish the transmission of human or animal parasites. In order to identify different closely related Onchocerca species we surveyed the sequences from the three mitochondrial loci 12S rRNA, 16S rRNA and coxI in both adult worms isolated from Onchocerca-induced nodules in cattle and infective third stage larvae isolated from vector flies from North Cameroon. Two distinct groups of mitochondrial haplotypes were found in cattle as well as in flies. One of them has been formerly mentioned in the literature as Onchocerca sp. 'Siisa', a filaria isolated from the vector S. damnosum sensu lato in Uganda with hitherto unknown host. Both variants are found sympatric, also in the same nodule of the animal host and in the vector. In the flies we also found the mitochondrial haplotype that had been described for O. volvulus which is about equally different from the two previously mentioned ones as they are from each other. These results suggest a higher genetic diversification of Onchocerca ochengi than previously reported.

Livestock pathology in the central African region: some epidemiological considerations and control strategies.

Disease consistently features among the major constraints of livestock production in the central African region, orchestrating important economic losses. This article reviews livestock diseases of economic importance, including foot-and-mouth disease, trypanosomosis and dermatophilosis in cattle, peste des petits ruminants and gastrointestinal helminthosis in sheep and goats, and Newcastle disease in poultry. Some aspects of epidemiology such as pathogen identification, prevalence and risk factors are examined in the light of research findings in the region. Control tools such as vaccines, chemotherapeutic or prophylactic agents, and protocols developed for their efficient use are also reviewed. Constraints to the effective use of these tools have been identified as mostly due to institutional insufficiencies and measures for improvement have been proposed. These include the promotion of private professional veterinary services endowed with greater responsibility in animal health care, creation and promotion of community-based animal health care units in areas of marginal professional coverage, and adoption of a regional approach to the control of diseases of economic importance.