Molecular detection of Leishmania (Sauroleishmania) adleri (Trypanosomatida: Trypanosomatidae) in Sergentomyia sp. sand flies (Diptera: Psychodidae) in Mali and Niger.

Phlebotomine sand flies of the genus Sergentomyia are considered to be of minor importance as vectors of Leishmania parasites pathogenic to humans, but are known to transmit lizard parasites of the subgenus Sauroleishmania, including L. (S.) adleri. However, knowledge on the geographic distribution of Sauroleishmania spp. and the infection rates in the vectors is very limited. Therefore, our study aimed (1) to further elucidate the distribution and prevalence of Sauroleishmania spp. in their respective vectors and (2) to assess the potential risk for occasional transmission of Leishmania parasites to international military personnel deployed in camps in Mali and Niger. A total of 1,482 wild-caught sand flies (Sergentomyia spp. and closely related Grassomyia spp.) were screened by real-time PCR for the presence of Leishmania DNA. Thirty-two sand fly pools were tested positive, with six from Mali and 26 from Niger. The DNA of four representative isolates was sequenced. The resulting sequences revealed a homology to L. adleri, which leads to the first report of this species from Mali and Niger to the best of our knowledge. The results suggest that Sergentomyia (Sintonius) clydei might be the natural sand fly vector, while Grassomyia spp. appear to be refractory. No Leishmania sp. pathogenic to humans was detected in these sand flies.

Mosquito survey in Mauritania: Detection of Rift Valley fever virus and dengue virus and the determination of feeding patterns.

In Mauritania, several mosquito-borne viruses have been reported that can cause devastating diseases in animals and humans. However, monitoring data on their occurrence and local distribution are limited. Rift Valley fever virus (RVFV) is an arthropod-borne virus that causes major outbreaks throughout the African continent and the Arabian Peninsula. The first Rift Valley fever (RVF) epidemic in Mauritania occurred in 1987 and since then the country has been affected by recurrent outbreaks of the disease. To gain information on the occurrence of RVFV as well as other mosquito-borne viruses and their vectors in Mauritania, we collected and examined 4,950 mosquitoes, belonging to four genera and 14 species. The mosquitoes were captured during 2018 in the capital Nouakchott and in southern parts of Mauritania. Evidence of RVFV was found in a mosquito pool of female Anopheles pharoensis mosquitoes collected in December on a farm near the Senegal River. At that time, 37.5% of 16 tested Montbéliarde cattle on the farm showed RVFV-specific IgM antibodies. Additionally, we detected IgM antibodies in 10.7% of 28 indigenous cattle that had been sampled on the same farm one month earlier. To obtain information on potential RVFV reservoir hosts, blood meals of captured engorged mosquitoes were analyzed. The mosquitoes mainly fed on humans (urban areas) and cattle (rural areas), but also on small ruminants, donkeys, cats, dogs and straw-colored fruit bats. Results of this study demonstrate the circulation of RVFV in Mauritania and thus the need for further research to investigate the distribution of the virus and its vectors. Furthermore, factors that may contribute to its maintenance should be analyzed more closely. In addition, two mosquito pools containing Aedes aegypti and Culex quinquefasciatus mosquitoes showed evidence of dengue virus (DENV) 2 circulation in the city of Rosso. Further studies are therefore needed to also examine DENV circulation in Mauritania.

Molecular detection of dugbe orthonairovirus in cattle and their infesting ticks (Amblyomma and Rhipicephalus (Boophilus)) in Nigeria.

Dugbe orthonairovirus (DUGV), a tick-borne zoonotic arbovirus, was first isolated in 1964 in Nigeria. For over four decades, no active surveillance was conducted to monitor the spread and genetic variation of DUGV. This study detected and genetically characterized DUGV circulating in cattle and their infesting ticks (Amblyomma and Rhipicephalus (Boophilus)) in Kwara State, North-Central Nigeria. Blood and or ticks were collected from 1051 cattle at 31 sampling sites (abattoirs and farms) across 10 local government areas of the State. DUGV detection was carried out by RT-qPCR, and positive samples sequenced and phylogenetically analysed. A total of 11824 ticks, mostly A. variegatum (36.0%) and R. (B.) microplus (63.9%), were obtained with mean tick burden of 12 ticks/cattle. Thirty-four (32 A. variegatum and two R. (B.) microplus) of 4644 examined ticks were DUGV-positive, whereas all of the cattle sera tested negative for DUGV genome. Whole genome sequence (S, M and L segments) and phylogenetic analyses indicate that the positive samples shared up to 99.88% nucleotide identity with and clustered around the Nigerian DUGV prototype strain IbAr 1792. Hence, DUGV with high similarity to the previously characterised strain has been detected in Nigeria. To our knowledge, this is the first report of DUGV in North-Central Nigeria and the most recent information after its last surveillance in 1974.

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.

Galectins from Onchocerca ochengi and O. volvulus and their immune recognition by Wistar rats, Gudali zebu cattle and human hosts.

BACKGROUND: During the last two decades research on animal filarial parasites, especially Onchocerca ochengi, infecting cattle in savanna areas of Africa revealed that O. ochengi as an animal model has biological features that are similar to those of O. volvulus, the aetiological agent of human onchocerciasis. There is, however, a paucity of biochemical, immunological and pathological data for O. ochengi. Galectins can be generated by parasites and their hosts. They are multifunctional molecules affecting the interaction between filarial parasites and their mammalian hosts including immune responses. This study characterized O. ochengi galectin, verified its immunologenicity and established its immune reactivity and that of Onchocerca volvulus galectin. RESULTS: The phylogenetic analysis showed the high degree of identity between the identified O. ochengi and the O. volvulus galectin-1 (ß-galactoside-binding protein-1) consisting only in one exchange of alanine for serine. O. ochengi galectin induced IgG antibodies during 28 days after immunization of Wistar rats. IgG from O. ochengi-infected cattle and O. volvulus-infected humans cross-reacted with the corresponding galectins. Under the applied experimental conditions in a cell proliferation test, O. ochengi galectin failed to significantly stimulate peripheral blood mononuclear cells (PBMCs) from O. ochengi-infected cattle, regardless of their parasite load. CONCLUSION: An O. ochengi galectin gene was identified and the recombinantly expressed protein was immunogenic. IgG from Onchocerca-infected humans and cattle showed similar cross-reaction with both respective galectins. The present findings reflect the phylogenetic relationship between the two parasites and endorse the appropriateness of the cattle O. ochengi model for O. volvulus infection research.

Epidemiological investigation of Crimean-Congo haemorrhagic fever virus infection among the one-humped camels (Camelus dromedarius) in southern Tunisia.

Crimean-Congo haemorrhagic fever is a viral tick-borne zoonotic disease caused by a Nairovirus, Crimean-Congo haemorrhagic fever virus (CCHFV). The present survey aimed to determine the exposure of one-humped camels (Camelus dromedarius) from southern Tunisia to CCHFV. A total of 273 sera from extensively reared camels were collected from Tataouine district, Tunisia, and tested by CCHFV-specific enzyme linked immunosorbent assays. By combining the results of three serological tests, the overall seroprevalence of CCHFV was estimated as 89.7% (245/273). No viral RNA was detected from camel sera using quantitative real-time PCR (RT-qPCR). A total of 165 ticks were collected from camels and tested with RT-qPCR, and only one Hyalomma impeltatum tick was positive for virus RNA.

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.

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.

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.

Reproductive biology of Onchocerca ochengi, a nodule forming filarial nematode in zebu cattle.

Onchocerca ochengi is a nodule-forming filarial nematode parasite of cattle in tropical Africa and closely related to the human pathogen Onchocerca volvulus. The adult worms reside in intradermal nodules. While females are sedentary, males may move between nodules. The first stage larvae (microfilariae) disperse in the skin of the host waiting to be taken up by the intermediate host. The density of microfilariae in the skin is largely independent of the number of adult worms present indicating some form of density dependent control. Recently, Onchocerca sp. Siisa, a form of Onchocerca distinguishable from O. ochengi by mitochondrial DNA sequences but not by morphology, was described to occur in cattle. This raised the question if Onchocerca sp. Siisa represents a different mitochondrial clade of O. ochengi or a new species. In order to study the reproductive biology and to understand this self-control of the off-spring population we systematically analyzed all Onchocerca nodules from the skin of one zebu cow and we examined a sample of microfilariae from a skin biopsy. We identified 87 O. ochengi females and 146 males. 56 (64.4%) of the females contained developing embryos. In order to assign the progeny to their respective parents we determined the genotypes at six nuclear and two mitochondrial molecular genetic markers in the adult worms, in a fraction of the progeny present in the uteri of the females and in the skin microfilariae. The 121 skin microfilariae we analyzed originated from at least 17 different mothers, which contributed rather differently to the total. Forty-five larvae (37.2%) were the progeny of a single female. Of the adult worms 16.7% were of the type Onchocerca sp. Siisa. These worms appeared to interbreed freely with the rest of the O. ochengi population and therefore belong to the same species.

Comparative analysis of macrophage migration inhibitory factors (MIFs) from the parasitic nematode Onchocerca volvulus and the free-living nematode Caenorhabditis elegans.

The macrophage migration inhibitory factors (MIFs) from the filarial parasite Onchocerca volvulus (OvMIF) were compared to the MIFs from the free-living nematode Caenorhabditis elegans (CeMIF) with respect to molecular, biochemical and immunological properties. Except for CeMIF-4, all other MIFs demonstrated tautomerase activity. Surprisingly, OvMIF-1 displayed oxidoreductase activity. The strongest immunostaining for OvMIF-1 was observed in the outer cellular covering of the adult worm body, the syncytial hypodermis; moderate immunostaining was observed in the uterine wall. The generation of a strong humoral immune response towards OvMIF-1 and reduced reactivity to OvMIF-2 was indicated by high IgG levels in patients infected with O. volvulus and cows infected with the closely related Onchocerca ochengi, both MIFs revealing identical amino acid sequences. Using Litomosoides sigmodontis-infected mice, a laboratory model for filarial infection, MIFs derived from the tissue-dwelling O. volvulus, the rodent gut-dwelling Strongyloides ratti and from free-living C. elegans were recognized, suggesting that L. sigmodontis MIF-specific IgM and IgG1 were produced during L. sigmodontis infection of mice and cross-reacted with all MIF proteins tested. Thus, MIF apparently functions as a target of B cell response during nematode infection, but in the natural Onchocerca-specific human and bovine infection, the induced antibodies can discriminate between MIFs derived from parasitic or free-living nematodes.

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.

Single worm genotyping demonstrates that Onchocerca ochengi females simultaneously produce progeny sired by different males.

Onchocerca ochengi is a filarial nematode parasite of African cattle and most closely related to Onchocerca volvulus, the causing agent of river blindness. O. ochengi females induce the formation of a nodule in the dermis of the host, in which they remain sedentary in very close association with the host's tissue. Males, which do not adhere to the host's tissue, are also found within the nodules at an average number of about one male per nodule. Young O. ochengi females tend to avoid the immediate proximity of existing nodules. Therefore, O. ochengi nodules are dispersed in the ventral inguinal skin at considerable distances from each other. It has been speculated that males avoid the risk of leaving a female once they have found one and remain in the nodule as territorial males rendering the reproductive strategy of O. ochengi essentially monogamous. We developed a protocol that allows reliable PCR amplification of single copy loci from different developmental stages of O. ochengi including embryos and microfilariae. From 32 O. ochengi nodules, we genotyped the female worms and the 67 adult male worms, found in these nodules, together with a fraction of the progeny from within the uteri of females. In 18 of 32 gravid females progeny derived from multiple males were found. In five nodules, the males isolated from the same nodule as the female were not sufficient to explain the genotypes of the entire progeny. We conclude that frequently O. ochengi females simultaneously produce progeny sired by different males and that most but not all males are still present in the nodule when their offspring is ready to hatch.