Detection and Molecular Characterization of Adenoviruses in Captive and Free-Roaming African Green Monkeys (Chlorocebus sabaeus): Evidence for Possible Recombination and Cross-Species Transmission.

In the present study, 31 samples (12 fecal, 9 nasal and 10 rectal swabs) from 28/92 (30.43%, 10 captive and 18 free-roaming African green monkeys (AGMs, Chlorocebus sabaeus)) apparently healthy AGMs in the Caribbean Island of St. Kitts tested positive for adenoviruses (AdVs) by DNA-dependent DNA polymerase (pol)-, or hexon-based screening PCR assays. Based on analysis of partial deduced amino acid sequences of Pol- and hexon- of nine AGM AdVs, at least two AdV genetic variants (group-I: seven AdVs with a Simian mastadenovirus-F (SAdV-F)/SAdV-18-like Pol and hexon, and group-II: two AdVs with a SAdV-F/SAdV-18-like Pol and a Human mastadenovirus-F (HAdV-F)/HAdV-40-like hexon) were identified, which was corroborated by analysis of the nearly complete putative Pol, complete hexon, and partial penton base sequences of a representative group-I (strain KNA-08975), and -II (KNA-S6) AdV. SAdV-F-like AdVs were reported for the first time in free-roaming non-human primates (NHPs) and after ~six decades from captive NHPs. Molecular characterization of KNA-S6 (and the other group-II AdV) indicated possible recombination and cross-species transmission events involving SAdV-F-like and HAdV-F-like viruses, corroborating the hypothesis that the evolutionary pathways of HAdVs and SAdVs are intermingled, complicated by recombination and inter-species transmission events, especially between related AdV species, such as HAdV-F and SAdV-F. To our knowledge, this is the first report on detection and molecular characterization of AdVs in AGMs.

Case report: Control of intestinal nematodes in captive Chlorocebus sabaeus.

There are limited data on the efficacy of antiparasitic treatments and husbandry methods to control nematode infections in captive populations of African green monkeys (AGMs), Chlorocebus sabaeus. In faecal egg count (FEC) tests, 10 of the 11 (91%) adult male AGMs captured from the large feral population on the island of St Kitts had evidence of nematode infections, mostly Capillaria (8/11, 73%), Trichuris trichiura (7/11, 64%) and strongylid species (7/11, 64%) specifically (hookworm and Trichostrongylus, 50/50), but also Strongyloides fuelleborni (1/11, 9%). When kept in individual cages with cleaning and feeding regimens to prevent reinfections and treated concurrently with ivermectin (300 µg/kg, given subcutaneously) and albendazole (10 mg/kg, given orally) daily for 3 days, 60% (6/10) of the AGMs were negative at a follow-up FEC at 3 months and by FEC and necropsy at the end of the study 5-8 months later. One monkey appeared to have been reinfected with T. trichiura after being negative by FEC at 3 months post-treatment. Four AGMs were positive for T. trichiura at the 3 month FEC follow-up but were negative at the end of the study after one further treatment regimen. Although initially being cleared of Capillaria following treatment, three AGMs were found to be infected at the end of the study. The ivermectin and albendazole treatment regimen coupled with good husbandry practices to prevent reinfections effectively controlled nematode infections in captive AGMs.

Trichuris trichiura egg extract proteome reveals potential diagnostic targets and immunomodulators.

Embryonated eggs are the infectious developmental stage of Trichuris trichiura and are the primary stimulus for the immune system of the definitive host. The intestinal-dwelling T. trichiura affects an estimated 465 million people worldwide with an estimated global burden of disease of 640 000 DALYs (Disability Adjusted Life Years). In Latin America and the Caribbean, trichuriasis is the most prevalent soil transmitted helminthiasis in the region (12.3%; 95% CI). The adverse health consequences impair childhood school performance and reduce school attendance resulting in lower future wage-earning capacity. The accumulation of the long-term effects translates into poverty promoting sequelae and a cycle of impoverishment. Each infective T. trichiura egg carries the antigens needed to face the immune system with a wide variety of proteins present in the shell, larvae's surface, and the accompanying fluid that contains their excretions/secretions. We used a proteomic approach with tandem mass spectrometry to investigate the proteome of soluble non-embryonated egg extracts of T. trichiura obtained from naturally infected African green monkeys (Chlorocebus sabaeus). A total of 231 proteins were identified, 168 of them with known molecular functions. The proteome revealed common proteins families which are known to play roles in energy and metabolism; the cytoskeleton, muscle and motility; proteolysis; signaling; the stress response and detoxification; transcription and translation; and lipid binding and transport. In addition to the study of the T. trichiura non-embryonated egg proteome, the antigenic profile of the T. trichiura non-embryonated egg and female soluble proteins against serum antibodies from C. sabaeus naturally infected with trichuriasis was investigated. We used an immunoproteomic approach by Western blot and tandem mass spectrometry from the corresponding SDS-PAGE gels. Vitellogenin N and VWD and DUF1943 domain containing protein, poly-cysteine and histidine tailed protein isoform 2, heat shock protein 70, glyceraldehyde-3-phosphate dehydrogenase, actin, and enolase, were among the potential immunoactive proteins. To our knowledge, this is the first study on the T. trichiura non-embryonated egg proteome as a novel source of information on potential targets for immunodiagnostics and immunomodulators from a neglected tropical disease. This initial list of T. trichiura non-embryonated egg proteins (proteome and antigenic profile) can be used in future research on the immunobiology and pathogenesis of human trichuriasis and the treatment of human intestinal immune-related diseases.

Should monkeys wash their hands and feet: A pilot-study on sources of zoonotic parasite exposure.

Human exposure to zoonotic parasites via contaminated soil is a much studied area. Less research has been performed on exposure via contact with animals and surfaces such as picnic tables with which infected animals might be in contact. On St. Kitts, wild African green monkeys (AGM; Chlorocebus aethiops sabaeus), which are known to have zoonotic parasites, roam freely in areas with outdoor dining facilities and are used in the tourist industry. In this study, the hands and feet of eight AGM and picnic tables where AGM were known to walk on were examined for parasitic organisms. Six of the AGM had parasitic organisms on their hands and/or feet. Trichuris spp. eggs, hookworm larvae and eggs and pinworm eggs were recovered from the tables. The results of this pilot study highlight other potential means of transfer of zoonotic parasites and suggest that surfaces without obvious fecal material can be contaminated with zoonotic parasites.

Molecular species identification of Trichuris trichiura in African green monkey on St. Kitts, West Indies.

The population of African green monkeys (AGM, Chlorocebus aethiops sabaeus) on St. Kitts, West Indies is believed to be as large as or greater than the human population. Interactions with humans are frequent and the pathogens carried by AGM, such as Trichuris spp., may pose a risk to humans. The objectives of this study were to assess the use of molecular methods for diagnosing Trichuris spp. in AGM and compare its DNA sequences to those of Trichuris spp. found in other non-human primates and humans. Fecal samples were collected from trapped and individually housed AGM between January and December 2015 and analysed using fecal flotation with Sheather's sugar flotation solution and PCR amplification and DNA sequencing of 18S rRNA and ITS2 fragments. Phylogenetic analysis was performed. 91% (81/89) and 55.4% (31/56) were Trichuris spp. positive by fecal flotation and PCR, respectively. Both AGM-NADH1 gene and T. trichiura-18S rRNA gene showed no variations in sequence and were 100% identical to corresponding sequences deposited in GenBank. Nevertheless Trichuris ITS2 showed some diversities among 12 sequences, which was <5%. Phylogenetic analysis of ITS2 put Trichuris spp. in Kittitian AGM into the same clades of T. trichiura found in human and other non-human primates in many other geographical regions. These data confirm that AGM are reservoirs for T. trichiura in humans. We suggest a one health approach to curtail enteric parasitic infections in human populations in the insular country.

Detection of picobirnaviruses in vervet monkeys (Chlorocebus sabaeus): Molecular characterization of complete genomic segment-2.

During 2014-2015, 270 fecal samples were collected from non-diarrheic, captive and wild African green monkeys (AGMs) on the island of St. Kitts, Caribbean region. By RNA-PAGE, picobirnaviruses (PBVs) were detected in sixteen captive AGMs. By RT-PCR and sequencing of partial gene segment-2, PBVs in 15 of these 16 samples were assigned to genogroup-I. The full-length nucleotide (nt) sequence of gene segment-2 of one of the genogroup-I PBV strains, strain PBV/African green monkey/KNA/016593/2015, was obtained using a non-specific primer-based amplification method with modifications. Gene segment-2 of strain 016593 was 1707bp long, and encoded a putative RNA-dependent RNA polymerase (RdRp) of 538aa. Furthermore, the nearly complete gene segment-2 sequences of three other AGM PBV strains were determined using primers designed from gene segment-2 sequence of 016593. The gene segment-2 of the 4 AGM PBV strains were almost identical to each other, and exhibited a high degree of genetic diversity (maximum nt and deduced aa sequence identities of 66.4% and 65.3%, respectively) with those of PBVs from other host species. The 5'- and 3'- (except for one mismatch) end nt sequences and the three domains of RdRps were retained in the AGM PBV strains. To our knowledge, this is the first report on detection, and molecular characterization of complete gene segment-2 of PBVs in vervet monkeys. PBVs were detected for the first time from the Caribbean region.

Whole genome analysis provides evidence for porcine-to-simian interspecies transmission of rotavirus-A.

We report here whole genome analysis of a porcine rotavirus-A (RVA) strain RVA/Pig-wt/KNA/ET8B/2015/G5P[13] detected in a diarrheic piglet, and nearly whole genome (except for VP4 gene) analysis of a simian RVA strain RVA/Simian-wt/KNA/08979/2015/G5P[X] detected in a non-diarrheic African green monkey (AGM) on the island of St. Kitts, Caribbean region. Strain ET8B exhibited a G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 genotype constellation that was identical to those of Brazilian porcine RVA G5P[13] strains RVA/Pig-wt/BRA/ROTA01/2013/G5P[13] and RVA/Pig-wt/BRA/ROTA07/2013/G5P[13], the only porcine G5P[13] RVAs that have been analyzed for the whole genome so far. Phylogenetically, all the 11 gene segments of ET8B were closely related to those of porcine and porcine-like human RVAs within the respective genotypes. Although the porcine G5P[13] RVAs exhibited identical genotype constellations, ET8B did not appear to share common evolutionary pathways with the Brazilian porcine G5P[13] RVAs. Interestingly, the VP2, VP3, VP6, VP7, and NSP1-NSP5 genes of simian RVA strain 08979 were closely related to those of porcine and porcine-like human RVA strains, exhibiting 99%-100% nucleotide sequence identities to cognate genes of co-circulating porcine RVA strain ET8B. On the other hand, the VP1 of 08979 appeared to be genetically divergent from porcine and human RVAs within the R1 genotype, and its exact origin could not be ascertained. Taken together, these observations suggested that simian strain 08979 might have been derived from interspecies transmission events involving transmission of ET8B-like RVAs from pigs to AGMs. In St. Kitts, AGMs often stray from the wild into livestock farms. Therefore, it may be possible that the AGM acquired the infection from a pig farm on the island. To our knowledge, this is the first report on detection of porcine-like RVAs in monkeys. Also, the present study is the first to report whole genomic analysis of a porcine RVA strain from the Caribbean region.