An assessment of caprine tuberculosis prevalence in Lubumbashi slaughterhouse, Democratic Republic of Congo.

Caprine tuberculosis is a major health problem for goats and a major zoonosis of veterinary public health interest. In order to prepare a response to and control of caprine tuberculosis, to evaluate the potential risks to public health, and to assess the prevalence of the disease in Katanga province, Democratic Republic of Congo, 656 goats that were slaughtered at the Kabasele abattoir of Mzee Laurent-Desire Kabila Market in Lubumbashi were subjected to rigorous veterinary inspection during June to August 2012. All goat specimens came from the Kasumbalesa, Kasenga, and Kipushi areas of Katanga province. Consequently, suspected organs presenting signs of tuberculosis were collected and examined using Ziehl-Neelsen stains for diagnosis. Through this investigative inspection in the province, we found an overall prevalence of caprine tuberculosis of 1.68%. Although females showed higher prevalence of caprine tuberculosis (1.07%) compared to males (0.61%), and adults showed higher prevalence (1.22%) than juveniles (0.45%), these comparisons were not statically significant. However, lung and intestine infection by tuberculosis showed significantly higher prevalence of positive cases (1.21 and 0.46%, respectively) (p < 0.05). Goats from Kasumbalesa had the highest prevalence of caprine tuberculosis (1.22%) compared to goats from Kipushi (0.31%) and Kasenga (0.18%). These findings show the risk of caprine tuberculosis in the province for the first time, and we therefore recommend the implementation of strict animal biosecurity and tuberculosis controlling protocols.

Comparative phylogeography of African fruit bats (Chiroptera, Pteropodidae) provide new insights into the outbreak of Ebola virus disease in West Africa, 2014-2016.

Both Ebolavirus and Marburgvirus were detected in several fruit bat species of the family Pteropodidae, suggesting that this taxon plays a key role in the life cycle of filoviruses. After four decades of Zaire Ebolavirus (ZEBOV) outbreaks in Central Africa, the virus was detected for the first time in West Africa in 2014. To better understand the role of fruit bats as potential reservoirs and circulating hosts between Central and West Africa, we examine here the phylogeny and comparative phylogeography of Pteropodidae. Our phylogenetic results confirm the existence of four independent lineages of African fruit bats: the genera Eidolon and Rousettus, and the tribes Epomophorini and Scotonycterini, and indicate that the three species suspected to represent ZEBOV reservoir hosts (Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata) belong to an African clade that diversified rapidly around 8-7 Mya. To test for phylogeographic structure and for recent gene flow from Central to West Africa, we analysed the nucleotide variation of 675 cytochrome b gene (Cytb) sequences, representing eight fruit bat species collected in 48 geographic localities. Within Epomophorina, our mitochondrial data do not support the monophyly of two genera (Epomops and Epomophorus) and four species (Epomophorus gambianus, Epomops franqueti, Epomops buettikoferi, and Micropteropus pusillus). In Epomops, however, we found two geographic haplogroups corresponding to the Congo Basin and Upper Guinea forests, respectively. By contrast, we found no genetic differentiation between Central and West African populations for all species known to make seasonal movements, Eidolon helvum, E. gambianus, H. monstrosus, M. pusillus, Nanonycteris veldkampii, and Rousettus aegyptiacus. Our results suggest that only three fruit bat species were able to disperse directly ZEBOV from the Congo Basin to Upper Guinea: E. helvum, H. monstrosus, and R. aegyptiacus.

The comparative phylogeography of fruit bats of the tribe Scotonycterini (Chiroptera, Pteropodidae) reveals cryptic species diversity related to African Pleistocene forest refugia.

The hypothesis of Pleistocene forest refugia was tested using comparative phylogeography of Scotonycterini, a fruit bat tribe endemic to Africa containing four species: Scotonycteris zenkeri, Casinycteris argynnis, C. campomaanensis, and C. ophiodon. Patterns of genetic structure were assessed using 105 Scotonycterini (including material from three holotypes) collected at 37 localities, and DNA sequences from the mitochondrial cytochrome b gene (1140 nt) and 12 nuclear introns (9641 nt). Phylogenetic trees and molecular dating were inferred by Bayesian methods. Multilocus analyses were performed using supermatrix, SuperTRI, and *BEAST approaches. Mitochondrial analyses reveal strong phylogeographical structure in Scotonycteris, with four divergent haplogroups (4.9-8.7%), from Upper Guinea, Cameroon, western Equatorial Africa, and eastern Democratic Republic of the Congo (DRC). In C. argynnis, we identify two mtDNA haplogroups corresponding to western and eastern Equatorial Africa (1.4-2.1%). In C. ophiodon, the mtDNA haplotypes from Cameroon and Ivory Coast differ by only 1.3%. Nuclear analyses confirm the validity of the recently described C. campomaanensis and indicate that western and eastern populations of C. argynnis are not fully isolated. All mtDNA clusters detected in Scotonycteris are found to be monophyletic based on the nuclear dataset, except in eastern DRC. In the nuclear tree, the clade from western Equatorial Africa is closely related to individuals from eastern DRC, whereas in the mitochondrial tree it appears to be the sister-group of the Cameroon clade. Migrate-n analyses support gene flow from western Equatorial Africa to eastern DRC. Molecular dating indicates that Pleistocene forest refugia have played an important role in shaping the evolution of Scotonycterini, with two phases of allopatric speciation at approximately 2.7 and 1.6 Mya, resulting from isolation in three main forest areas corresponding to Upper Guinea, Cameroon, and Equatorial Africa. Two cryptic species and two subspecies are described herein in the genus Scotonycteris. Female philopatry and male biased dispersal are supported for the smallest taxa, i.e., the three species of Scotonycteris and C. argynnis. The Congo, Ntem, and Sanaga rivers are identified as biogeographic barriers to the dispersal of Scotonycteris during interglacial periods. A greater capacity for long-distance dispersal is inferred for the largest species, C. ophiodon.

Molecular systematics and phylogeography of the tribe Myonycterini (Mammalia, Pteropodidae) inferred from mitochondrial and nuclear markers.

The tribe Myonycterini comprises five fruit bat species of the family Pteropodidae, which are endemic to tropical Africa. Previous studies have produced conflicting results about their interspecific relationships. Here, we performed a comparative phylogeographic analysis based on 148 complete cytochrome b gene sequences from the three species distributed in West Africa and Central Africa (Myonycteris torquata, Lissonycteris angolensis and Megaloglossus woermanni). In addition, we investigated phylogenetic relationships within the tribe Myonycterini, using a matrix including 29 terminal taxa and 7235 nucleotide characters, corresponding to an alignment of two mitochondrial genes and seven nuclear introns. Our phylogenetic analyses confirmed that the genus Megaloglossus belongs to the tribe Myonycterini. Further, the genus Rousettus is paraphyletic, with R. lanosus, sometimes placed in the genus Stenonycteris, being the sister-group of the tribes Myonycterini and Epomophorini. Our phylogeographic results showed that populations of Myonycteris torquata and Megaloglossus woermanni from the Upper Guinea Forest are highly divergent from those of the Congo Basin Forest. Based on our molecular data, we recommended several taxonomic changes. First, Stenonycteris should be recognized as a separate genus from Rousettus and composed of S. lanosus. This genus should be elevated to a new tribe, Stenonycterini, within the subfamily Epomophorinae. This result shows that the evolution of lingual echolocation was more complicated than previously accepted. Second, the genus Lissonycteris is synonymised with Myonycteris. Third, the populations from West Africa formerly included in Myonycteris torquata and Megaloglossus woermanni are now placed in two distinct species, respectively, Myonycteris leptodon and Megaloglossus azagnyi sp. nov. Our molecular dating estimates show that the three phases of taxonomic diversification detected within the tribe Myonycterini can be related to three distinct decreases in tree cover vegetation, at 6.5-6, 2.7-2.5, and 1.8-1.6Ma. Our results suggest that the high nucleotide distance between Ebolavirus Côte d'Ivoire and Ebolavirus Zaire can be correlated with the Plio/Pleistocene divergence between their putative reservoir host species, i.e., Myonycteris leptodon and Myonycteris torquata, respectively.

New STLV-3 strains and a divergent SIVmus strain identified in non-human primate bushmeat in Gabon.

BACKGROUND: Human retroviral infections such as Human Immunodeficiency Virus (HIV) or Human T-cell Lymphotropic Virus (HTLV) are the result of simian zoonotic transmissions through handling and butchering of Non-Human Primates (NHP) or by close contact with pet animals. Recent studies on retroviral infections in NHP bushmeat allowed for the identification of numerous Simian Immunodeficiency Viruses (SIV) and Simian T-cell Lymphotropic Viruses (STLV) to which humans are exposed. Nevertheless, today, data on simian retroviruses at the primate/hunter interface remain scarce. We conducted a pilot study on 63 blood and/or tissues samples derived from NHP bushmeat seized by the competent authorities in different locations across the country. RESULTS: SIV and STLV were detected by antibodies to HIV and HTLV antigens, and PCRs were performed on samples with an HIV or/and HTLV-like or indeterminate profile. Fourteen percent of the samples cross-reacted with HIV antigens and 44% with HTLV antigens. We reported STLV-1 infections in five of the seven species tested. STLV-3 infections, including a new STLV-3 subtype, STLV-1 and -3 co-infections, and triple SIV, STLV-1, STLV-3 infections were observed in red-capped mangabeys (C.torquatus). We confirmed SIV infections by PCR and sequence analyses in mandrills, red-capped mangabeys and showed that mustached monkeys in Gabon are infected with a new SIV strain basal to the SIVgsn/mus/mon lineage that did not fall into the previously described SIVmus lineages reported from the corresponding species in Cameroon. The same monkey (sub)species can thus be carrier of, at least, three distinct SIVs. Overall, the minimal prevalence observed for both STLV and SIV natural infections were 26.9% and 11.1% respectively. CONCLUSIONS: Overall, these data, obtained from a restricted sampling, highlight the need for further studies on simian retroviruses in sub-Saharan Africa to better understand their evolutionary history and to document SIV strains to which humans are exposed. We also show that within one species, a high genetic diversity may exist for SIVs and STLVs and observe a high genetic diversity in the SIVgsn/mon/mus lineage, ancestor of HIV-1/SIVcpz/SIVgor.

Risk factors for Zaire ebolavirus--specific IgG in rural Gabonese populations.

BACKGROUND: In Gabon, several Ebolavirus outbreaks have occurred exclusively in the northeastern region. We conducted a large serosurvey to identify areas and populations at risk and potential demographic, clinical, and behavioral risk factors. METHODS: Blood samples and clinical and sociodemographic data were collected from 4349 adults and 362 children in a random sample of 220 villages in the 9 provinces of Gabon. An enzyme-linked immunosorbent assay was used to detect Zaire ebolavirus (ZEBOV)-specific IgG, and thin blood smears were used to detect parasites. Logistic regression was implemented using Stata software (Stata), and a probability level of <.05 was considered to be statistically significant. RESULTS: The prevalence of ZEBOV-specific IgG was 15.3% overall, increasing to 32.4% (P< .001) in forest areas. No sociodemographic risk factors were found, but the antibody prevalence increased linearly up to 20 years of age. Chronic arthralgia and amicrofilaremia were the only factors associated with ZEBOV seropositivity. CONCLUSIONS: These findings confirm the endemicity of ZEBOV in Gabon and its link to the ecosystem. Human antibody positivity would appear to be to the result of exposure to contaminated fruits.

African monkeys are infected by Plasmodium falciparum nonhuman primate-specific strains.

Recent molecular exploration of the Plasmodium species circulating in great apes in Africa has revealed the existence of a large and previously unknown diversity of Plasmodium. For instance, gorillas were found to be infected by parasites closely related to Plasmodium falciparum, suggesting that the human malignant malaria agent may have arisen after a transfer from gorillas. Although this scenario is likely in light of the data collected in great apes, it remained to be ascertained whether P. falciparum-related parasites may infect other nonhuman primates in Africa. Using molecular tools, we here explore the diversity of Plasmodium species infecting monkeys in Central Africa. In addition to previously described Hepatocystis and Plasmodium species (Plasmodium gonderi and Plasmodium sp DAJ-2004), we have found one African monkey to be infected by a P. falciparum-related parasite. Examination of the nuclear and mitochondrial genomes of this parasite reveals that it is specific of nonhuman primates, indicating that P. falciparum-related pathogens can naturally circulate in some monkey populations in Africa. We also show that at least two distinct genetic entities of P. falciparum infect nonhuman primates and humans, respectively. Our discoveries bring into question the proposed gorilla origin of human P. falciparum.

Rift Valley fever virus seroprevalence in human rural populations of Gabon.

BACKGROUND: Rift Valley fever (RVF) is a mosquito-borne viral zoonosis caused by a phlebovirus and transmitted by Aedes mosquitoes. Humans can also be infected through direct contact with blood (aerosols) or tissues (placenta, stillborn) of infected animals. Although severe clinical cases can be observed, infection with RVF virus (RVFV) in humans is, in most cases, asymptomatic or causes a febrile illness without serious symptoms. In small ruminants RVFV mainly causes abortion and neonatal death. The distribution of RVFV has been well documented in many African countries, particularly in the north (Egypt, Sudan), east (Kenya, Tanzania, Somalia), west (Senegal, Mauritania) and south (South Africa), but also in the Indian Ocean (Madagascar, Mayotte) and the Arabian Peninsula. In contrast, the prevalence of RVFV has rarely been investigated in central African countries. METHODOLOGY/PRINCIPAL FINDINGS: We therefore conducted a large serological survey of rural populations in Gabon, involving 4,323 individuals from 212 randomly selected villages (10.3% of all Gabonese villages). RVFV-specific IgG was found in a total of 145 individuals (3.3%) suggesting the wide circulation of Rift Valley fever virus in Gabon. The seroprevalence was significantly higher in the lakes region than in forest and savannas zones, with respective rates of 8.3%, 2.9% and 2.2%. In the lakes region, RVFV-specific IgG was significantly more prevalent in males than in females (respectively 12.8% and 3.8%) and the seroprevalence increased gradually with age in males but not in females. CONCLUSIONS/SIGNIFICANCE: Although RVFV was suggested to circulate at a relatively high level in Gabon, no outbreaks or even isolated cases have been documented in the country. The higher prevalence in the lakes region is likely to be driven by specific ecologic conditions favorable to certain mosquito vector species. Males may be more at risk of infection than females because they spend more time farming and hunting outside the villages, where they may be more exposed to mosquito bites and infected animals. Further investigations are needed to determine the putative sylvan cycle of RVFV, including the mosquito species and the reservoir role of wild animals in the viral maintenance cycle.

First serological evidence of West Nile virus in human rural populations of Gabon.

To investigate West Nile virus (WNV) circulation in rural populations in Gabon, we undertook a large serological survey focusing on human rural populations, using two different ELISA assays. A sample was considered positive when it reacted in both tests. A total of 2320 villagers from 115 villages were interviewed and sampled. Surprisingly, the WNV-specific IgG prevalence was high overall (27.2%) and varied according to the ecosystem: 23.7% in forested regions, 21.8% in savanna, and 64.9% in the lakes region. The WNV-specific IgG prevalence rate was 30% in males and 24.6% in females, and increased with age. Although serological cross-reactions between flaviviruses are likely and may be frequent, these findings strongly suggest that WNV is widespread in Gabon. The difference in WNV prevalence among ecosystems suggests preferential circulation in the lakes region. The linear increase with age suggests continuous exposure of Gabonese populations to WNV. Further investigations are needed to determine the WNV cycle and transmission patterns in Gabon.

Large serological survey showing cocirculation of Ebola and Marburg viruses in Gabonese bat populations, and a high seroprevalence of both viruses in Rousettus aegyptiacus.

BACKGROUND: Ebola and Marburg viruses cause highly lethal hemorrhagic fevers in humans. Recently, bats of multiple species have been identified as possible natural hosts of Zaire ebolavirus (ZEBOV) in Gabon and Republic of Congo, and also of marburgvirus (MARV) in Gabon and Democratic Republic of Congo. METHODS: We tested 2147 bats belonging to at least nine species sampled between 2003 and 2008 in three regions of Gabon and in the Ebola epidemic region of north Congo for IgG antibodies specific for ZEBOV and MARV. RESULTS: Overall, IgG antibodies to ZEBOV and MARV were found in 4% and 1% of bats, respectively. ZEBOV-specific antibodies were found in six bat species (Epomops franqueti, Hypsignathus monstrosus, Myonycteris torquata, Micropteropus pusillus, Mops condylurus and Rousettus aegyptiacus), while MARV-specific antibodies were only found in Rousettus aegyptiacus and Hypsignathus monstrosus. The prevalence of MARV-specific IgG was significantly higher in R. aegyptiacus members captured inside caves than elsewhere. No significant difference in prevalence was found according to age or gender. A higher prevalence of ZEBOV-specific IgG was found in pregnant females than in non pregnant females. CONCLUSION: These findings confirm that ZEBOV and MARV co-circulate in Gabon, the only country where bats infected by each virus have been found. IgG antibodies to both viruses were detected only in Rousettus aegyptiacus, suggesting that this bat species may be involved in the natural cycle of both Marburg and Ebola viruses. The presence of MARV in Gabon indicates a potential risk for a first human outbreak. Disease surveillance should be enhanced in areas near caves.

Concurrent chikungunya and dengue virus infections during simultaneous outbreaks, Gabon, 2007.

An outbreak of febrile illness occurred in Gabon in 2007, with 20,000 suspected cases. Chikungunya or dengue-2 virus infections were identified in 321 patients; 8 patients had documented co-infections. Aedes albopictus was identified as the principal vector for the transmission of both viruses.

Human Ebola outbreak resulting from direct exposure to fruit bats in Luebo, Democratic Republic of Congo, 2007.

Twelve years after the Kikwit Ebola outbreak in 1995, Ebola virus reemerged in the Occidental Kasaï province of the Democratic Republic of Congo (DRC) between May and November 2007, affecting more than 260 humans and causing 186 deaths. During this latter outbreak we conducted several epidemiological investigations to identify the underlying ecological conditions and animal sources. Qualitative social and environmental data were collected through interviews with villagers and by direct observation. The local populations reported no unusual morbidity or mortality among wild or domestic animals, but they described a massive annual fruit bat migration toward the southeast, up the Lulua River. Migrating bats settled in the outbreak area for several weeks, between April and May, nestling in the numerous fruit trees in Ndongo and Koumelele islands as well as in palm trees of a largely abandoned plantation. They were massively hunted by villagers, for whom they represented a major source of protein. By tracing back the initial human-human transmission events, we were able to show that, in May, the putative first human victim bought freshly killed bats from hunters to eat. We were able to reconstruct the likely initial human-human transmission events that preceded the outbreak. This study provides the most likely sequence of events linking a human Ebola outbreak to exposure to fruit bats, a putative virus reservoir. These findings support the suspected role of bats in the natural cycle of Ebola virus and indicate that the massive seasonal fruit bat migrations should be taken into account in operational Ebola risk maps and seasonal alerts in the DRC.

Isolates of Zaire ebolavirus from wild apes reveal genetic lineage and recombinants.

Over the last 30 years, Zaire ebolavirus (ZEBOV), a virus highly pathogenic for humans and wild apes, has emerged repeatedly in Central Africa. Thus far, only a few virus isolates have been characterized genetically, all belonging to a single genetic lineage and originating exclusively from infected human patients. Here, we describe the first ZEBOV sequences isolated from great ape carcasses in the Gabon/Congo region that belong to a previously unrecognized genetic lineage. According to our estimates, this lineage, which we also encountered in the two most recent human outbreaks in the Republic of the Congo in 2003 and 2005, diverged from the previously known viruses around the time of the first documented human outbreak in 1976. These results suggest that virus spillover from the reservoir has occurred more than once, as predicted by the multiple emergence hypothesis. However, the young age of both ZEBOV lineages and the spatial and temporal sequence of outbreaks remain at odds with the idea that the virus simply emerged from a long-established and widespread reservoir population. Based on data from two ZEBOV genes, we also demonstrate, within the family Filoviridae, recombination between the two lineages. According to our estimates, this event took place between 1996 and 2001 and gave rise to a group of recombinant viruses that were responsible for a series of outbreaks in 2001-2003. The potential for recombination adds an additional level of complexity to unraveling and potentially controlling the emergence of ZEBOV in humans and wildlife species.

Marburg virus infection detected in a common African bat.

Marburg and Ebola viruses can cause large hemorrhagic fever (HF) outbreaks with high case fatality (80-90%) in human and great apes. Identification of the natural reservoir of these viruses is one of the most important topics in this field and a fundamental key to understanding their natural history. Despite the discovery of this virus family almost 40 years ago, the search for the natural reservoir of these lethal pathogens remains an enigma despite numerous ecological studies. Here, we report the discovery of Marburg virus in a common species of fruit bat (Rousettus aegyptiacus) in Gabon as shown by finding virus-specific RNA and IgG antibody in individual bats. These Marburg virus positive bats represent the first naturally infected non-primate animals identified. Furthermore, this is the first report of Marburg virus being present in this area of Africa, thus extending the known range of the virus. These data imply that more areas are at risk for MHF outbreaks than previously realized and correspond well with a recently published report in which three species of fruit bats were demonstrated to be likely reservoirs for Ebola virus.

Ngoye virus: a novel evolutionary lineage within the genus Flavivirus.

By using degenerate primers deduced from conserved patterns in the flavivirus polymerase gene, a novel RNA virus was discovered in Rhipicephalus ticks sampled from members of the family Bovidae in Senegal. It was named Ngoye virus (NGOV) after the location from which it was isolated. Viral particles could be observed by electron microscopy, but isolation in vertebrate or invertebrate cell lines or by intracerebral infection of newborn mice remained unsuccessful. This is atypical of recognized arboviruses. The characterization of 4176 nt of the non-structural genes revealed that NGOV is a novel flavivirus species. It forms a distinct phylogenetic lineage related distantly to previously identified members of the genus Flavivirus. Analysis of genetic data suggested that the processing of the NGOV polyprotein and the organization of its replication complex are similar to those of flaviviruses. Together with other recent data, these findings suggest that a large number of viruses related distantly to 'classical' arthropod-borne flaviviruses remain to be discovered.

Molecular characterization of a novel simian immunodeficiency virus lineage (SIVtal) from northern talapoins (Miopithecus ogouensis).

Simian immunodeficiency viruses (SIVs) are found in an extensive number of African primates, and humans continue to be exposed to these viruses by hunting and handling of primate bushmeat and following occupational exposures to captive nonhuman primates. Here, we report the molecular characterization of a new SIV lineage, SIVtal, from wild-caught and captive talapoin monkeys (Miopithecus ogouensis) from Cameroon and U.S. zoos, respectively. Phylogenetic tree analyses of a small fragment in the pol gene indicated that all SIVtal strains clustered together forming a single species-specific lineage. Full-length sequence analysis for two strains, SIVtal-00CM266 and SIVtal-01CM8023, from wild-caught animals in Cameroon confirmed that SIVtal was distinct from all primate lentiviruses isolated so far and represents a new SIV lineage. Phylogenetic analyses in different viral genes showed a significant clustering of the SIVtal lineage with the Cercopithecus-specific SIVs. In addition, SIVtal and Cercopithecus-specific SIVs share functional motifs in Gag and Env that distinguish them from other primate lentiviruses. Like SIVsyk and SIVdeb, a vpu gene homologue was also absent in SIVtal. Although northern talapoins belong to the Miopithecus genus, their SIVs belong to the Cercopithecus SIV lineage, suggesting evolution from a common ancestor or cross-species transmission between both primate genera.

Fruit bats as reservoirs of Ebola virus.

The first recorded human outbreak of Ebola virus was in 1976, but the wild reservoir of this virus is still unknown. Here we test for Ebola in more than a thousand small vertebrates that were collected during Ebola outbreaks in humans and great apes between 2001 and 2003 in Gabon and the Republic of the Congo. We find evidence of asymptomatic infection by Ebola virus in three species of fruit bat, indicating that these animals may be acting as a reservoir for this deadly virus.

The natural history of Ebola virus in Africa.

Several countries spanning the equatorial forest regions of Africa have had outbreaks of Ebola hemorrhagic fever over the last three decades. This article is an overview of the many published investigations of how Ebola virus circulates in its natural environment, focusing on the viral reservoir, susceptible animal species, environmental conditions favoring inter-species transmission, and how the infection is transmitted to humans. Major breakthroughs have been made in recent years but many outstanding questions must be dealt with if we are to prevent human outbreaks by interfering with the viral life cycle.