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.

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.