Vincent N. Tanya

Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

Of Mice, Cattle, and Humans: The Immunology and Treatment of River Blindness

River blindness is a seriously debilitating disease caused by the filarial parasite Onchocerca volvulus, which infects millions in Africa as well as in South and Central America. Research has been hampered by a lack of good animal models, as the parasite can only develop fully in humans and some primates. This review highlights the development of two animal model systems that have allowed significant advances in recent years and hold promise for the future. Experimental findings with Litomosoides sigmodontis in mice and Onchocerca ochengi in cattle are placed in the context of how these models can advance our ability to control the human disease.

Onchocerca ochengi infections in cattle as a model for human onchocerciasis: recent developments.

The bovine parasite Onchocerca ochengi is a nodule-dwelling filarial nematode, closely related to O. volvulus, the causal agent of human River Blindness, and, sharing with it, the same vector. This brief review, based on a presentation at the BSP Autumn Symposium 1999, describes recent work supported by the WHO Drug Development Research Macrofil programme and the Edna McConnell Clark Foundation vaccine development programme, to research the chemotherapy and immunology of onchocerciasis utilising this model system, with experimental infections in Liverpool and field infections in northern Cameroon. In a series of chemotherapeutic trials involving 10 compounds in 20 treatment regimes, the comparability of drug efficacy against O. ochengi with that described against O. volvulus has been demonstrated. Repeated, long-term treatment with oxytetracycline has been shown to be macrofilaricidal and the effect is hypothesized to be related to action on Wolbachia endobacteria, abundant in O. ochengi. Avermectins/milbemycins are not macrofilaricidal (even in high and repeated long-term treatments) but induce sustained abrogation of embryogenesis. In prospective, field exposure experiments with naive calves, prophylactic treatments with ivermectin and moxidectin prevented the development of adult worm infection, raising the possibility that drug-attenuated larval challenge infections may induce immunity. Putatively immune adult cattle exist in endemically exposed populations, and these have been shown to be significantly less susceptible to challenge than age-matched naive controls, whereas radically drug-cured, previously patently-infected cattle were not. Experimental infections with O. ochengi have revealed the kinetics of the immune response in relation to parasite development and demonstrate analogous responses to those reported in O. volvulus infection in humans and chimpanzees. In an immunization experiment with irradiated L3 larvae, cattle were significantly protected against experimental challenge--the first such demonstration of the experimental induction of immunity in a natural Onchocerca host-parasite system. Taken collectively, these studies not only demonstrate the similarity between the host-parasite relationships of O. ochengi in cattle and O. volvulus in humans, but promise to advance options for the control of human onchocerciasis.

Antibiotic Chemotherapy of Onchocerciasis: In a Bovine Model, Killing of Adult Parasites Requires a Sustained Depletion of Endosymbiotic Bacteria (Wolbachia Species)

Development of a drug lethal to adult Onchocerca volvulus (i.e., macrofilaricide) is a research priority for the control of human onchocerciasis. Using bovine O. ochengi infections, we investigated the effects of oxytetracycline administered in a short intensive regimen (SIR; 10 mg/kg daily for 14 days), compared with a prolonged intermittent regimen (PIR; 20 mg/kg monthly for 6 months) or a combination of both (COM), on the viability of adult worms and their endosymbiotic bacteria (Wolbachia species). The long-term treatments eliminated >80% (COM) or >60% (PIR) of adult female worms (P<.001), and the COM regimen effected a sustained depletion of Wolbachia organisms. Conversely, SIR was not macrofilaricidal and only transiently depleted Wolbachia densities, which repopulated worm tissues by 24 weeks after treatment. These results unequivocally demonstrate the macrofilaricidal potential of tetracyclines against Onchocerca infection and suggest that intermittent, protracted administration will be more effective than continuous shorter term treatment.

Molecular Epidemiology of Foot-and-Mouth Disease Viruses in the Adamawa Province of Cameroon

ABSTRACT Foot-and-mouth disease virus (FMDV) causes a highly contagious viral disease of even-toed ungulates and is one of the most important economic diseases of livestock. Most studies of FMDV are done in countries where control measures are being implemented. In contrast, in areas such as sub-Saharan Africa, where FMDV is endemic and new strains are likely to emerge, there are only sporadic submissions to the World Reference Laboratory, Pirbright, United Kingdom. This paper describes the molecular epidemiology of FMDV in the Adamawa province of Cameroon based on a population sample of cattle herds. Serotypes SAT2 and A were isolated in the cross-sectional study. SAT2 isolates were all similar, with phylogenetic distances of <6%, and were most closely related to published sequences of isolates from Eritrea and Saudi Arabia. Serotype A isolates were more variable, with phylogenetic distances of 0 to 11%, and were most closely related to historic isolates from Cameroon. Use of a population-based sample gives a representative sample of virus diversity and will improve our understanding of the evolution of FMDV and its epidemiology. A supplementary study of pigs passing through the railhead collection yard at Ngaoundere detected a serotype O virus. A third pilot longitudinal study monitored viral persistence in three cattle herds over 12 months, and serotype O and A viruses were recovered from a herd 12 months after it was first recorded as being infected with SAT2 virus. The pig type O isolate was not closely related to that recovered from the cattle, suggesting that the pigs had not introduced the O virus into the cattle herds.

A worm's best friend: recruitment of neutrophils by Wolbachia confounds eosinophil degranulation against the filarial nematode Onchocerca ochengi

Onchocerca ochengi, a filarial parasite of cattle, represents the closest relative of the human pathogen, Onchocerca volvulus. Both species harbour Wolbachia endosymbionts and are remarkable in that adult female worms remain viable but sessile for many years while surrounded by host cells and antibodies. The basis of the symbiosis between filariae and Wolbachia is thought to be metabolic, although a role for Wolbachia in immune evasion has received little attention. Neutrophils are attracted to Wolbachia, but following antibiotic chemotherapy they are replaced by eosinophils that degranulate on the worm cuticle. However, it is unclear whether the eosinophils are involved in parasite killing or if they are attracted secondarily to dying worms. In this study, cattle infected with Onchocerca ochengi received adulticidal regimens of oxytetracycline or melarsomine. In contrast to oxytetracycline, melarsomine did not directly affect Wolbachia viability. Eosinophil degranulation increased significantly only in the oxytetracycline group; whereas nodular gene expression of bovine neutrophilic chemokines was lowest in this group. Moreover, intense eosinophil degranulation was initially associated with worm vitality, not degeneration. Taken together, these data offer strong support for the hypothesis that Wolbachia confers longevity on O. ochengi through a defensive mutualism, which diverts a potentially lethal effector cell response.

Extracellular Onchocerca-derived small RNAs in host nodules and blood.

BackgroundmicroRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans.MethodsSmall RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum.ResultsA total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis.ConclusionsWe have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.

Foot and mouth disease and livestock husbandry practices in the Adamawa Province of Cameroon.

Foot and mouth disease (FMD) is a highly contagious viral disease of even-toed ungulates and is endemic in most of the tropics. A cross-sectional study using a stratified, two-stage random sample design was undertaken in the Adamawa Province of Cameroon. The objectives were to measure the reported herd-level prevalence of FMD and a range of husbandry practices important for its transmission. The owner-reported prevalence for the previous 12 months was 57.9% (50.4–65.4%), although there was a significant variation across the Province. During the previous dry season, 46.5% (38.6–54.4%) of herds had gone on transhumance. Herds had high numbers of contacts with other herds while on transhumance (98.6%), at pasture (95.8%) and at night (74.4%), with medians of 7–10, 4–6 and 1–3 daily contacts, respectively. The high level of endemic FMD and potential for disease spread presents a significant challenge for control and eradication. Locally sustainable methods need to be developed upon which larger regional control programmes could be built in the future.

Serological Patterns of Brucellosis, Leptospirosis and Q Fever in Bos indicus Cattle in Cameroon

Brucellosis, leptospirosis and Q fever are important infections of livestock causing a range of clinical conditions including abortions and reduced fertility. In addition, they are all important zoonotic infections infecting those who work with livestock and those who consume livestock related products such as milk, producing non-specific symptoms including fever, that are often misdiagnosed and that can lead to severe chronic disease. This study used banked sera from the Adamawa Region of Cameroon to investigate the seroprevalences and distributions of seropositive animals and herds. A classical statistical and a multi-level prevalence modelling approach were compared. The unbiased estimates were 20% of herds were seropositive for Brucella spp. compared to 95% for Leptospira spp. and 68% for Q fever. The within-herd seroprevalences were 16%, 35% and 39% respectively. There was statistical evidence of clustering of seropositive brucellosis and Q fever herds. The modelling approach has the major advantage that estimates of seroprevalence can be adjusted for the sensitivity and specificity of the diagnostic test used and the multi-level structure of the sampling. The study found a low seroprevalence of brucellosis in the Adamawa Region compared to a high proportion of leptospirosis and Q fever seropositive herds. This represents a high risk to the human population as well as potentially having a major impact on animal health and productivity in the region.

Palaeosymbiosis revealed by genomic fossils of Wolbachia in a strongyloidean nematode.

Wolbachia are common endosymbionts of terrestrial arthropods, and are also found in nematodes: the animal-parasitic filaria, and the plant-parasite Radopholus similis. Lateral transfer of Wolbachia DNA to the host genome is common. We generated a draft genome sequence for the strongyloidean nematode parasite Dictyocaulus viviparus, the cattle lungworm. In the assembly, we identified nearly 1 Mb of sequence with similarity to Wolbachia. The fragments were unlikely to derive from a live Wolbachia infection: most were short, and the genes were disabled through inactivating mutations. Many fragments were co-assembled with definitively nematode-derived sequence. We found limited evidence of expression of the Wolbachia-derived genes. The D. viviparus Wolbachia genes were most similar to filarial strains and strains from the host-promiscuous clade F. We conclude that D. viviparus was infected by Wolbachia in the past, and that clade F-like symbionts may have been the source of filarial Wolbachia infections.