Screening wild and semi-free ranging great apes for putative sexually transmitted diseases: Evidence of Trichomonadidae infections.

Sexually transmitted diseases (STDs) can persist endemically, are known to cause sterility and infant mortality in humans, and could have similar impacts in wildlife populations. African apes (i.e., chimpanzees, bonobos, and to a lesser extent gorillas) show multi-male mating behavior that could offer opportunities for STD transmission, yet little is known about the prevalence and impact of STDs in this endangered primate group. We used serology and PCR-based detection methods to screen biological samples from wild and orphaned eastern chimpanzees and gorillas (N = 172 individuals, including adults, and juveniles) for four classes of pathogens that either commonly cause human STDs or were previously detected in captive apes: trichomonads, Chlamydia spp., Treponema pallidum (syphilis and yaws), and papillomaviruses. Based on results from prior modeling and comparative research, we expected STD prevalence to be highest in females versus males and in sexually mature versus immature individuals. All samples were negative for Chlamydia, Treponema pallidum, and papillomaviruses; however, a high percentage of wild chimpanzee urine and fecal samples showed evidence of trichomonads (protozoa). Analysis revealed that females were more likely than males to have positive urine-but not fecal-samples; however, there was no evidence of age (sexual maturity) differences in infection status. Sequence analysis of chimpanzee trichomonad samples revealed a close relationship to previously described trichomonads within the genus Tetratrichomonas. Phylogenetic comparisons to archived sequences from multiple vertebrate hosts suggests that many of the chimpanzee parasites from our study are likely transmitted via fecal-oral contact, but the transmission of some Tetratrichomonas sequence-types remains unknown and could include sexual contact. Our work emphasizes that only a fraction of infectious agents affecting wild apes are presently known to science, and that further work on great ape STDs could offer insights for the management of endangered great apes and for understanding human STD origins.

EXPERIMENTAL CHALLENGE STUDY OF FV3-LIKE RANAVIRUS INFECTION IN PREVIOUSLY FV3-LIKE RANAVIRUS INFECTED EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) TO ASSESS INFECTION AND SURVIVAL.

The Maryland Zoo in Baltimore experienced an outbreak of Frog virus-3 (FV3)-like ranavirus during the summer of 2011, during which 14 of 27 (52%) of its captive eastern box turtles (Terrapene carolina carolina) survived. To assess survival, immunity, and viral shedding, an experimental challenge study was performed in which the surviving, previously infected turtles were reinfected with the outbreak strain of FV3-like ranavirus. Seven turtles were inoculated with virus intramuscularly and four control turtles received saline intramuscularly. The turtles were monitored for 8 wk with blood and oral swabs collected for quantitative polymerase chain reaction (qPCR). During that time, one of seven (14%) inoculated turtles and none of the controls (0%) died; there was no significant difference in survival. Clinical signs of the inoculated turtles, except for the turtle that died, were mild compared to the original outbreak. Quantitative PCR for FV3-like ranavirus on blood and oral swabs was positive for all inoculated turtles and negative for all controls. The turtle that died had intracytoplasmic inclusion bodies in multiple organs. Three inoculated and two control turtles were euthanized at the end of the study. No inclusion bodies were present in any of the organs. Quantitative PCR detected FV3-like ranavirus in the spleen of a control turtle, which suggested persistence of the virus. The surviving five turtles were qPCR-negative for FV3-like ranavirus from blood and oral swabs after brumation. Quantitative PCR for Terrapene herpesvirus 1 found no association between ranavirus infection and herpesvirus loads. In conclusion, previously infected eastern box turtles can be reinfected with the same strain of FV3-like ranavirus and show mild to no clinical signs but can shed the virus from the oral cavity.

Projecting the impact of an ebola virus outbreak on endangered mountain gorillas.

Ebola virus is highly lethal for great apes. Estimated mortality rates up to 98% have reduced the global gorilla population by approximately one-third. As mountain gorillas (Gorilla beringei beringei) are endangered, with just over 1000 individuals remaining in the world, an outbreak could decimate the population. Simulation modeling was used to evaluate the potential impact of an Ebola virus outbreak on the mountain gorilla population of the Virunga Massif. Findings indicate that estimated contact rates among gorilla groups are high enough to allow rapid spread of Ebola, with less than 20% of the population projected to survive at 100 days post-infection of just one gorilla. Despite increasing survival with vaccination, no modeled vaccination strategy prevented widespread infection. However, the model projected that survival rates greater than 50% could be achieved by vaccinating at least half the habituated gorillas within 3 weeks of the first infectious individual.

Ghost admixture in eastern gorillas.

Archaic admixture has had a substantial impact on human evolution with multiple events across different clades, including from extinct hominins such as Neanderthals and Denisovans into modern humans. In great apes, archaic admixture has been identified in chimpanzees and bonobos but the possibility of such events has not been explored in other species. Here, we address this question using high-coverage whole-genome sequences from all four extant gorilla subspecies, including six newly sequenced eastern gorillas from previously unsampled geographic regions. Using approximate Bayesian computation with neural networks to model the demographic history of gorillas, we find a signature of admixture from an archaic 'ghost' lineage into the common ancestor of eastern gorillas but not western gorillas. We infer that up to 3% of the genome of these individuals is introgressed from an archaic lineage that diverged more than 3 million years ago from the common ancestor of all extant gorillas. This introgression event took place before the split of mountain and eastern lowland gorillas, probably more than 40 thousand years ago and may have influenced perception of bitter taste in eastern gorillas. When comparing the introgression landscapes of gorillas, humans and bonobos, we find a consistent depletion of introgressed fragments on the X chromosome across these species. However, depletion in protein-coding content is not detectable in eastern gorillas, possibly as a consequence of stronger genetic drift in this species.

Novel adenoviruses in wild primates: a high level of genetic diversity and evidence of zoonotic transmissions.

Adenoviruses (AdVs) broadly infect vertebrate hosts, including a variety of nonhuman primates (NHPs). In the present study, we identified AdVs in NHPs living in their natural habitats, and through the combination of phylogenetic analyses and information on the habitats and epidemiological settings, we detected possible horizontal transmission events between NHPs and humans. Wild NHPs were analyzed with a pan-primate AdV-specific PCR using a degenerate nested primer set that targets the highly conserved adenovirus DNA polymerase gene. A plethora of novel AdV sequences were identified, representing at least 45 distinct AdVs. From the AdV-positive individuals, 29 nearly complete hexon genes were amplified and, based on phylogenetic analysis, tentatively allocated to all known human AdV species (Human adenovirus A to Human adenovirus G [HAdV-A to -G]) as well as to the only simian AdV species (Simian adenovirus A [SAdV-A]). Interestingly, five of the AdVs detected in great apes grouped into the HAdV-A, HAdV-D, HAdV-F, or SAdV-A clade. Furthermore, we report the first detection of AdVs in New World monkeys, clustering at the base of the primate AdV evolutionary tree. Most notably, six chimpanzee AdVs of species HAdV-A to HAdV-F revealed a remarkably close relationship to human AdVs, possibly indicating recent interspecies transmission events.

Coronaviruses Detected in Bats in Close Contact with Humans in Rwanda.

Bats living in close contact with people in Rwanda were tested for evidence of infection with viruses of zoonotic potential. Mucosal swabs from 503 bats representing 17 species were sampled from 2010 to 2014 and screened by consensus PCR for 11 viral families. Samples were negative for all viral families except coronaviruses, which were detected in 27 bats belonging to eight species. Known coronaviruses detected included the betacorona viruses: Kenya bat coronaviruses, Eidolon bat coronavirus, and Bat coronavirus HKU9, as well as an alphacoronavirus, Chaerephon Bat coronavirus. Novel coronaviruses included two betacorona viruses clustering with SARS-CoV, a 2d coronavirus, and an alphacoronavirus.

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage.

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.

Occupational health and gorilla conservation in Rwanda.

The design and implementation of an employee health program for people who work with mountain gorillas in Rwanda is described. This program aims to improve worker health and to reduce human-to-gorilla transmission of infectious disease. The program covered approximately 111 workers, generally healthy men and women 25-45 years old, including essentially all people in Rwanda who have regular contact with gorillas. Initial assessment included a questionnaire, medical examination, and local tests. U.S. laboratory facilities were utilized to confirm some results and for serologic testing for zoonotic (simian) viruses. Initial interventions included STD/HIV prevention health education, tetanus immunization, and anthelminthic treatment. Local physicians continue to provide health services, including follow-up testing and treatment. Mountain Gorilla Veterinary Project (MGVP) veterinarians assist in planning and implementing continuing program components in collaboration with local health authorities and the other employing organizations.

Prevalence of antibodies to alphaviruses and flaviviruses in free-ranging game animals and nonhuman primates in the greater Congo basin.

Vector-borne and zoonotic pathogens have comprised a significant proportion of the emerging infectious diseases in humans in recent decades. The role of many wildlife species as reservoirs for arthropod-borne viral pathogens is poorly understood. We investigated the exposure history of various African wildlife species from the Congo basin to mosquito-borne flaviviruses and alphaviruses by testing archived serum samples. Sera from 24 African forest buffalo (Syncerus caffer nanus), 34 African elephants (Loxodonta africana), 40 duikers (Cephalophus and Philantomba spp.), 25 mandrills (Mandrillus sphinx), 32 mountain gorillas (Gorilla beringei beringei), five Grauer's gorillas (Gorilla beringei graueri), two L'Hoest's monkeys (Cercopithecus lhoesti), two golden monkeys (Cercopithecus kandti), and three chimpanzees (Pan troglodytes) sampled between 1991 and 2009 were tested for antibodies against chikungunya virus (CHIKV), o'nyong-nyong virus (ONNV), West Nile virus (WNV), dengue 2 virus (DENV-2), and yellow fever virus (YFV) by plaque reduction neutralization test. Specific neutralizing antibodies against ONNV were found in African forest buffalo in the Democratic Republic of the Congo (DRC) and Gabon, duikers in the DRC, and mandrills in Gabon, providing novel evidence of enzootic circulation of ONNV in these countries. African forest buffalo in the DRC and Gabon also demonstrated evidence of exposure to CHIKV, WNV, and DENV-2, while mandrills in Gabon were antibody positive for CHIKV, DENV-2, WNV, and YFV. All of the elephants tested had a strong neutralizing antibody response to WNV. We also document results from a survey of gorillas for arboviruses, of which 4/32 (13%) had antibody to an alphavirus or flavivirus. Overall, our results demonstrate a high prevalence of neutralizing antibodies against multiple arboviruses in wildlife in equatorial Africa.

Extreme conservation leads to recovery of the Virunga mountain gorillas.

As wildlife populations are declining, conservationists are under increasing pressure to measure the effectiveness of different management strategies. Conventional conservation measures such as law enforcement and community development projects are typically designed to minimize negative human influences upon a species and its ecosystem. In contrast, we define "extreme" conservation as efforts targeted to deliberately increase positive human influences, including veterinary care and close monitoring of individual animals. Here we compare the impact of both conservation approaches upon the population growth rate of the critically endangered Virunga mountain gorillas (Gorilla beringei beringei), which increased by 50% since their nadir in 1981, from approximately 250 to nearly 400 gorillas. Using demographic data from 1967-2008, we show an annual decline of 0.7%±0.059% for unhabituated gorillas that received intensive levels of conventional conservation approaches, versus an increase 4.1%±0.088% for habituated gorillas that also received extreme conservation measures. Each group of habituated gorillas is now continuously guarded by a separate team of field staff during daylight hours and receives veterinary treatment for snares, respiratory disease, and other life-threatening conditions. These results suggest that conventional conservation efforts prevented a severe decline of the overall population, but additional extreme measures were needed to achieve positive growth. Demographic stochasticity and socioecological factors had minimal impact on variability in the growth rates. Veterinary interventions could account for up to 40% of the difference in growth rates between habituated versus unhabituated gorillas, with the remaining difference likely arising from greater protection against poachers. Thus, by increasing protection and facilitating veterinary treatment, the daily monitoring of each habituated group contributed to most of the difference in growth rates. Our results argue for wider consideration of extreme measures and offer a startling view of the enormous resources that may be needed to conserve some endangered species.

Measuring the effects of an ever-changing environment on malaria control.

The effectiveness of malaria control measures depends not only on the potency of the control measures themselves but also upon the influence of variables associated with the environment. Environmental variables have the capacity either to enhance or to impair the desired outcome. An optimal outcome in the field, which is ultimately the real goal of vaccine research, will result from prior knowledge of both the potency of the control measures and the role of environmental variables. Here we describe both the potential effectiveness of control measures and the problems associated with testing in an area of endemicity. We placed canaries with different immunologic backgrounds (e.g., naïve to malaria infection, vaccinated naïve, and immune) directly into an area where avian malaria, Plasmodium relictum, is endemic. In our study setting, canaries that are naïve to malaria infection routinely suffer approximately 50% mortality during their first period of exposure to the disease. In comparison, birds vaccinated and boosted with a DNA vaccine plasmid encoding the circumsporozoite protein of P. relictum exhibited a moderate degree of protection against natural infection (P < 0.01). In the second year we followed the fate of all surviving birds with no further manipulation. The vaccinated birds from the first year were no longer statistically distinguishable for protection against malaria from cages of naïve birds. During this period, 36% of vaccinated birds died of malaria. We postulate that the vaccine-induced protective immune responses prevented the acquisition of natural immunity similar to that concurrently acquired by birds in a neighboring cage. These results indicate that dominant environmental parameters associated with malaria deaths can be addressed before their application to a less malleable human system.