Experimental infection of Artibeus lituratus bats and no detection of Zika virus in neotropical bats from French Guiana, Peru, and Costa Rica suggests a limited role of bats in Zika transmission.

Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested to play an important role in the Zika virus (ZIKV) transmission cycle. However, the exact role of these animals as reservoirs for flaviviruses is still controversial. To further expand our understanding of the role of bats in the ZIKV transmission cycle in Latin America, we carried out an experimental infection in wild-caught Artibeus lituratus bats and sampled several free-living neotropical bats across three countries of the region. Experimental ZIKV infection was performed in wild-caught adult bats (4 females and 5 males). The most relevant findings were hemorrhages in the bladder, stomach and patagium. Significant histological findings included inflammatory infiltrate consisting of a predominance of neutrophils and lymphocytes, in addition to degeneration in the reproductive tract of males and females. This suggests that bat reproduction might be at some level affected by ZIKV. Leukopenia was also observed in some inoculated animals. Hemorrhages, genital alterations, and leukopenia are suggested to be caused by ZIKV; however, since these were wild-caught bats, we cannot exclude other agents. Detection of ZIKV by qPCR was observed at low concentrations in only two urine samples in two inoculated animals. All other animals and tissues tested were negative. Finally, no virus-neutralizing antibodies were found in any animal. To determine ZIKV infection in nature, the blood of a total of 2056 bats was sampled for ZIKV detection by qPCR. Most of the sampled individuals belonged to the genus Pteronotus sp. (23%), followed by the species Carollia sp. (17%), Anoura sp. (14%), and Molossus sp. (13.7%). No sample of any tested species was positive for ZIKV by qPCR. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and may not have an important role in ZIKV transmission dynamics.

Natural co-infection of divergent hepatitis B and C virus homologues in carnivores.

In humans, co-infection of hepatitis B and C viruses (HBV, HCV) is common and aggravates disease outcome. Infection-mediated disease aggravation is poorly understood, partly due to lack of suitable animal models. Carnivores are understudied for hepatitis virus homologues. We investigated Mexican carnivores (ringtails, Bassariscus astutus) for HBV and HCV homologues. Three out of eight animals were infected with a divergent HBV termed ringtail HBV (RtHBV) at high viral loads of 5 × 109 -1.4 × 1010 copies/ml serum. Two of the RtHBV-infected animals were co-infected with a divergent hepacivirus termed ringtail hepacivirus (RtHV) at 4 × 106 -7.5 × 107 copies/ml in strain-specific qRT-PCR assays. Immunofluorescence assays relying on HBV core and RtHV NS3/4a proteins indicated that none of the animals had detectable hepadnavirus core-specific antibodies, whereas one RtHV-infected animal had concomitant RtHV-specific antibodies at 1:800 end-point titre. RtHBV and RtHV complete genomes showed typical HBV and HCV structure and length. All RtHBV genomes were identical, whereas RtHV genomes showed four amino acid substitutions located predominantly in the E1/E2-encoding genomic regions. Both RtHBV (>28% genomic nucleotide sequence distance) and RtHV (>30% partial NS3/NS5B amino acid sequence distance) formed new species within their virus families. Evolutionary analyses showed that RtHBV grouped with HBV homologues from different laurasiatherian hosts (carnivores, bats, and ungulates), whereas RtHV grouped predominantly with rodent-borne viruses. Ancestral state reconstructions showed that RtHV, but not RtHBV, likely emerged via a non-recent host switch involving rodent-borne hepacivirus ancestors. Conserved hepatitis virus infection patterns in naturally infected ringtails indicate that carnivores may be promising animal models to understand HBV/HCV co-infection.

Detection of Bartonella species, including Candidatus Bartonella ovis sp. nov, in ruminants from Mexico and lack of evidence of Bartonella DNA in saliva of common vampire bats (Desmodus rotundus) predating on them.

Bartonella spp. have been identified in many bat species worldwide, including the zoonotic species, Candidatus Bartonella mayotimonensis. The common vampire bat (Desmodus rotundus) preys preferentially on livestock in Latin America and is frequently infected with Bartonella spp. To determine the potential role of D. rotundus in transmitting Bartonella to livestock, common vampire bats and bat-bitten domestic ruminants from Mexico were tested for Bartonella infection by blood culture or conventional PCR. Furthermore, to explore the possibility of bite transmission during blood feeding, saliva swabs from 35 D. rotundus known to be either Bartonella bacteremic (N = 17) or blood culture negative (N = 18) were tested by PCR to detect the presence of Bartonella DNA. Twenty (17.1%) of 117 sheep and 16 (34.8%) of 46 cattle were Bartonella bacteremic by PCR testing. However, none of them were infected with Bartonella strains previously isolated from vampire bats and none of the 35 D. rotundus saliva swabs tested were PCR positive for Bartonella. All but two animals among those which were Bartonella culture and/or PCR positive, were infected with either B. bovis (cattle) or B. melophagi (sheep). Two sheep were infected by a possible new species, Candidatus Bartonella ovis, being phylogenetically closer to B. bovis than B. melophagi. This study does not support the role of D. rotundus as a reservoir of Bartonella species infecting livestock, which could be transmitted via bite and blood feeding and therefore suggest limited risk of zoonotic transmission of Bartonella from common vampire bats to humans.

Molecular Detection of Bartonella Species in Blood-Feeding Bat Flies from Mexico.

Bartonellae are emerging blood-borne bacteria that have been recovered from a wide range of mammalian species and arthropod vectors around the world. Bats are now recognized as a potential wildlife reservoir for a diverse number of Bartonella species, including the zoonotic Candidatus B. mayotimonensis. These bat-borne Bartonella species have also been detected in the obligate ectoparasites of bats, such as blood-feeding flies, which could transmit these bacteria within bat populations. To better understand this potential for transmission, we investigated the relatedness between Bartonella detected or isolated from bat hosts sampled in Mexico and their ectoparasites. Bartonella spp. were identified in bat flies collected on two bat species, with the highest prevalence in Trichobius parasiticus and Strebla wiedemanni collected from common vampire bats (Desmodus rotundus). When comparing Bartonella sequences from a fragment of the citrate synthase gene (gltA), vector-associated strains were diverse and generally close to, but distinct from, those recovered from their bacteremic bat hosts in Mexico. Complete Bartonella sequence concordance was observed in only one bat-vector pair. The diversity of Bartonella strains in bat flies reflects the frequent host switch by bat flies, as they usually do not live permanently on their bat host. It may also suggest a possible endosymbiotic relationship with these vectors for some of the Bartonella species carried by bat flies, whereas others could have a mammalian host.

Addition of C3d-P28 adjuvant to a rabies DNA vaccine encoding the G5 linear epitope enhances the humoral immune response and confers protection.

Rabies DNA vaccines based on full-length glycoprotein (G) induce virus neutralizing antibody (VNA) responses and protect against the virus challenge. Although conformational epitopes of G are the main target of VNAs, some studies have shown that a polypeptide linear epitope G5 is also able to induce VNAs. However, a G5 DNA vaccine has not been explored. While multiple doses of DNA vaccines are required in order to confer a protective immune response, this could be overcome by the inclusion of C3d-P28, a molecular adjuvant is know to improve the antibody response in several anti-viral vaccine models. To induce and enhance the immune response against rabies in mice, we evaluated two DNA vaccines based on the linear epitope G5 of Rabies Virus (RABV) glycoprotein (pVaxG5 vaccine) and another vaccine consisting of G5 fused to the molecular adjuvant C3d-P28 (pVaxF1 vaccine). VNA responses were measured in mice immunized with both vaccines. The VNA levels from the group immunized with pVaxG5 decreased gradually, while those from the group vaccinated with pVaxF1 remained high throughout the experimental study. After challenge with 22 LD50 of the Challenge Virus Strain (CVS), the survival rate of mice immunized with pVaxG5 and pVaxF1 was increased by 27% and 50% respectively, in comparison to the PBS group. Furthermore, the in vitro proliferation of anti-rabies specific spleen CD4+ and CD8+ T cells from mice immunized with pVaxF1 was observed. Collectively, these results suggest that the linear G5 epitope is a potential candidate vaccine. Furthermore, the addition of a C3d-P28 adjuvant contributed to enhanced protection, the sustained production of VNAs, and a specific T-cell proliferative response.

Detection of Dengue Virus in Bat Flies (Diptera: Streblidae) of Common Vampire Bats, Desmodus rotundus, in Progreso, Hidalgo, Mexico.

Blood-feeding arthropods play a major role in the transmission of several flaviviruses, which represent an important problem for human health. Currently, dengue is one of the most important arboviral emerging diseases worldwide. Furthermore, some previous studies have reported the presence of viral nucleic acids and antibodies against dengue virus (DENV) in wild animals. Our knowledge of the role played by wildlife reservoirs in the sylvatic transmission and maintenance of DENV remains limited. Our objective was to screen blood-feeding ectoparasites (bat flies) and their common vampire bat (Desmodus rotundus) hosts, for flaviviruses in Hidalgo, Mexico. We detected Flavivirus sequences in 38 pools of ectoparasites (Diptera: Streblidae, Strebla wiedemanni and Trichobius parasiticus) and 8 tissue samples of D. rotundus by RT-PCR and semi-nested PCR using FlaviPF1S, FlaviPR2bis, and FlaviPF3S primers specific for NS5, a gene highly conserved among flaviviruses. Phylogenetic inference analysis performed using the maximum likelihood algorithm implemented in PhyML showed that six sequences clustered with DENV (bootstrap value = 53.5%). Although this study supports other reports of DENV detection in bats and arthropods other than Aedes mosquitoes, the role of these ectoparasitic flies and of hematophagous bats in the epidemiology of DENV still warrants further investigation.

Bartonella Infection in Hematophagous, Insectivorous, and Phytophagous Bat Populations of Central Mexico and the Yucatan Peninsula.

Although emerging nonviral pathogens remain relatively understudied in bat populations, there is an increasing focus on identifying bat-associated bartonellae around the world. Many novel Bartonella strains have been described from both bats and their arthropod ectoparasites, including Bartonella mayotimonensis, a zoonotic agent of human endocarditis. This cross-sectional study was designed to describe novel Bartonella strains isolated from bats sampled in Mexico and evaluate factors potentially associated with infection. A total of 238 bats belonging to seven genera were captured in five states of Central Mexico and the Yucatan Peninsula. Animals were screened by bacterial culture from whole blood and/or polymerase chain reaction of DNA extracted from heart tissue or blood. Bartonella spp. were isolated or detected in 54 (22.7%) bats, consisting of 41 (38%) hematophagous, 10 (16.4%) insectivorous, and three (4.3%) phytophagous individuals. This study also identified Balantiopteryx plicata as another possible bat reservoir of Bartonella. Univariate and multivariate logistic regression models suggested that Bartonella infection was positively associated with blood-feeding diet and ectoparasite burden. Phylogenetic analysis identified a number of genetic variants across hematophagous, phytophagous, and insectivorous bats that are unique from described bat-borne Bartonella species. However, these strains were closely related to those bartonellae previously identified in bat species from Latin America.

Experimental infection of Artibeus intermedius with a vampire bat rabies virus.

Experimental infection of Artibeus intermedius, the great fruit-eating bat, was performed with vampire bat rabies isolates. Bats (n=35) were captured in the wild and quarantined prior to experimental infection. No rabies antibodies were detected by rapid fluorescent focus inhibition test (RFFIT) prior to infection. Three doses of rabies virus (RV) and three different routes of infection were used. One out of 35 bats died without showing any clinical signs at day 14 and was positive for rabies. None of the 34 other bats showed clinical signs for rabies, but high antibody titers were detected post-inoculation, suggesting either innate immune response to the vampire bat rabies virus or possible pre-exposure to RV and inoculation leading to a booster effect. Rabies virus was detected by hemi-nested RT-PCR (hnRT-PCR) in the brain (n=3), stomach (n=1) of bats that were negative by immunofluorescence and that survived rabies infection. The bat that died on day 14 was positive by hnRT-PCR on the brain, heart and liver. These results suggest that either previous non-lethal exposure to RV or natural low susceptibility to vampire bat viruses somehow protected Artibeus intermedius from clinical rabies infection leading to a marginal lethality effect on this bats species population in the wild.

Identification of Bartonella Species Isolated from Rodents from Yucatan, Mexico, and Isolation of Bartonella vinsonii subsp. yucatanensis subsp. nov.

Bartonella species are highly endemic among wild rodents in many parts of the world. Blood and/or blood clot cultures from 38 rodents, including 27 Yucatan deer mouse (Peromyscus yucatanicus), 7 Gaumer's spiny pocket mouse (Heteromys gaumeri), 2 black rats (Rattus rattus) and 2 big-eared climbing rats (Ototylomys phyllotis) captured near Merida, Yucatan, Mexico, led to the isolation in 3-4 days of small gram-negative bacilli, which were identified as Bartonella spp. based on colony morphology. DNA extraction and PCR testing were also performed from heart samples of 35 of these 38 rodents. Overall, Bartonella spp. were isolated from the blood/blood clots of 22 (58%) rodents. All Bartonella-positive rodents were Yucatán deer mice from San José Pituch. Sequencing of a fragment of the gltA gene showed that all but one rodent isolates were closest to B. vinsonii subsp. vinsonii and one isolate was intermediate between B. vinsonii subsp. berkhoffii and B. vinsonii subsp. arupensis. Further analysis of concatenated housekeeping genes (gltA, ftsZ, rpoB, and 16S rRNA) suggests that this outlier isolate is a new subspecies within the B. vinsonii genogroup, for which we proposed the name B. vinsonii subsp. yucatanensis.