Identification,occurrence and clinical findings of canine hemoplasmas in southern Brazil

Hemoplasmas are ubiquitous pleomorphic and epicellular bacteria detected in erythrocytes in several species. In Brazil, studies on hemoplasmas have not included information on occurrence, clinical signs, and risk factors in dogs. This paper investigates the occurrence of hemoplasmas in dogs, focusing on risk factors and clinical status. Conventional PCR for the four types of canine hemoplasmas was performed in 331 blood samples collected from dogs clinically treated at a teaching veterinary hospital. Of all samples, 17/331 (5.1%) were positive for Mycoplasma haemocanis and 6/331 (1.8%) were positive for a ‘Candidatus Mycoplasma haemominutum-like’ organism. Risk factors included the presence of vectors, old age, dog bite wounds, and neoplastic diseases. In the multivariate analysis, a 4.40 odds ratio in dogs with vector-borne diseases indicated risk for hemoplasmosis. There was correlation between hemoplasma infection and neoplastic disease, suggesting that neoplastic conditions are a risk factor for hemoplasma infection in dogs.     

Molecular evolution and deorphanization of bitter taste receptors in a vampire bat

Bats represent the largest dietary radiation in a single mammalian order, and have become an emerging model group for studying dietary evolution. Taste receptor genes have proven to be molecular signatures of dietary diversification in bats. For example, all 3 extant species of vampire bats have lost many bitter taste receptor genes (Tas2rs) in association with their dietary shift from insectivory to sanguivory. Indeed, only 8 full-length Tas2rs were identified from the high-quality genome of the common vampire bat (Desmodus rotundus). However, it is presently unknown whether these bitter receptors are functional, since the sense of taste is less important in vampire bats, which have an extremely narrow diet and rely on other senses for acquiring food. Here, we applied a molecular evolutionary analysis of Tas2rs in the common vampire bat compared with non-vampire bats. Furthermore, we provided the first attempt to deorphanize all bitter receptors of the vampire bat using a cell-based assay. We found that all Tas2r genes in the vampire bat have a level of selective pressure similar to that in non-vampire bats, suggesting that this species must have retained some bitter taste functions. We demonstrated that 5 of the 8 bitter receptors in the vampire bat can be activated by some bitter compounds, and observed that the vampire bat generally can not detect naturally occurring bitter compounds examined in this study. Our study demonstrates functional retention of bitter taste in vampire bats as suggested by cell-based functional assays, calling for an in-depth study of extra-oral functions of bitter taste receptors.     

Mycoplasma sp. infection in captive Marcgrave's capuchin monkeys (Sapajus flavius)

Three species of hemoplasmas are known to infect monkeys, ‘Candidatus Mycoplasma kahanei’, ‘Candidatus Mycoplasma haemomacaque’ and ‘Candidatus Mycoplasma aoti’. Marcgrave’s capuchin monkey (Sapajus flavius) is a critically endangered neotropical primate species of the Brazilian Atlantic Forest, and thus, sustained efforts are needed to protect the health and well-being of these animals. This study has aimed to screen 12 captive S. flavius from the State of Paraíba, northeastern Brazil, for hemoplasma infection. Packed cell volume (PCV) was measured and a pan-hemoplasma PCR protocol performed. A total of 8/12 (66.6%) monkeys were positive for the 16S rDNA gene of Mycoplasma sp., showing an identity of 99% to Mycoplasma sp. previously detected in Sapajus apella and Sapajus sp. monkeys from the Brazilian Amazon. Further studies should be conducted in order to elucidate whether this potentially novel Mycoplasma sp. is able to cause anemia in Sapajus sp. monkeys from Brazil.     

Occurrence of hemotropic mycoplasmas in non-human primates (Alouatta caraya,Sapajus nigritus and Callithrix jacchus) of southern Brazil

Hemoplasmas, the erythrocyte-associated mycoplasmas, have been detected in several primates, causing mostly subclinical infection. This study aimed to determine the prevalence of hemoplasma infection in captive and free-ranging monkeys from southern Brazil, as well as factors and hematological abnormalities associated with infection. Blood samples from 40 non-human primates (NHP) were tested for hemoplasmas and coinfections. An overall of 10/40 (25.0%) NHP tested positive for hemoplasmas using PCR-based assays, including 9/14 (64.3%) black howler monkeys (Alouatta caraya) and 1/24 (4.2%) black-horned capuchin (Sapajus nigritus). Infection was not statistically associated with anemia, but wild-born monkeys and male black howler monkeys were more likely to be positive when compared with captive-born animals and female black howler monkeys, respectively. The sequences from the black howler monkey hemoplasma were similar (94% identity) to the squirrel monkey hemoplasma (“Candidatus Mycoplasma kahanei”) and were phylogenetically located in a different cluster when compared to the human hemoplasma (“Candidatus Mycoplasma haemohominis”).     

Hemotropic mycoplasmas in bats captured near human settlements in Nigeria

The presence of DNA of hemotropic mycoplasmas (hemoplasmas) was investigated for the first time in bats in Africa. Blood samples from 90 bats captured within or near human settlements in nine study areas from five states in Nigeria belonging to six genera of the families Pteropodidae, Rhinolophidae, and Molossidae were analyzed using conventional PCR protocol targeting a 391 bp part of the 16S rRNA gene. Of these, 32 samples (35 %) resulted positive. Eight nucleotide sequence types were identified, which were assigned to five genotypes showing between 93–99 % similarity with hemoplasmas from bats and/or rodents from other parts of the world, and/or Candidatus Mycoplasma haemohominis from a human patient. Network analysis showed genetic structure of hemoplasma sequences among bat species, but some sequences were shared among bats of different taxonomic groups and distant study areas. Further characterization of the samples using a protocol targeting ∼1200 bp of the 16S rRNA gene resulted in seven sequences that confirmed the results of the screening protocol. Hemoplasmas in Nigerian bats are prevalent, widely distributed and genetically diverse. The zoonotic risk to local inhabitants should not be neglected, due to the high similarity of some of the retrieved sequences with the human pathogen C. M. haemohominis.     

Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal,an endemic area for bovine trypanosomiasis in South America

Hemotropic mycoplasmas (hemoplasmas) are Gram-negative bacteria that parasitize the erythrocyte surface of a wide variety of mammals. The present study aimed at investigating the occurrence of hemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. Additionally, the objective of this study was to characterize molecularly the genotypes of the found hemoplasmas. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in Corumbá, Nhecolândia sub-region, Mato Grosso do Sul, in Midwest Brazil. Blood samples underwent DNA extraction and standard 16S rRNA gene-based PCR assays for hemoplasmas. The sequences obtained were submitted to phylogenetic inferences, distance analysis, and genotype diversity. The Indirect Enzyme-Linked Immunoabsorbent Assay (iELISA) indicated the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity of said agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive for hemoplasmas in cPCR. The phylogenetic analysis of the obtained sequences confirmed the presence of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii DNA in 0.5% (2/400) and 1.75% (7/400) animals, respectively. Five genotypes of M. wenyonii and one of 'Candidatus M. haemobos' were detected among the sequenced amplicons. The present study showed low molecular occurrence of haemoplasmas in beef cattle sampled in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis. Despite of the conservation of the 16S rRNA gene, there was considerable diversity of hemoplasma genotypes infecting the sampled beef cattle.     

Neotropical primary bat cell lines show restricted dengue virus replication

Dengue is the most widespread arboviral disease affecting humans. Bats are recognized carriers of emerging viral zoonoses and have been proposed as dengue reservoirs, since RNA/NS1 and/or antiviral antibodies have been detected. Yet, experimental inoculation of Artibeus bats failed to show virus replication. This conflicting results prevent drawing further conclusions of whether bats sustain dengue infection. To test bat cellular permissivity to dengue infection, we established primary bat embryonic cells from diverse organs and tissues of Artibeus jamaicensis, Molossus sinaloae, and Desmodus rotundus. We observed a limited serotype-, organ-, and bat species- specific dengue susceptibility. Only some Molossus-derived primary cells sustained poorly initial Dengue serotype-1 replication, though it was latter absent. To elucidate if Molossus bats may play a role in dengue replication, ecological or in vivo experiments must be performed. Taken together our results show that Dengue did not replicate efficiently in cell lines derived from Neotropical bat species.     

Widespread infection with hemotropic mycoplasmas in bats in Spain,including a hemoplasma closely related to “Candidatus Mycoplasma hemohominis”

Molecular analyses of blood samples revealed infection with hemoplasmas in 97% of 31 cave bats captured in three caves in North-Eastern Spain. The characterization of 1250 bp of the 16S rRNA gene in 29 of the positive bats identified two different groups of sequences. Twenty-two Schreibers’ bats (Miniopterus schreibersii) and one long-eared bat (Myotis capaccinii) shared one group, composed of seven closely related sequences. These sequences showed an identity of about 97% with “Candidatus Mycoplasma hemohominis” and the phylogenetic branch including bat and human sequences showed a 100% bootstrap value, supporting a close phylogenetic relationship between these hemoplasmas. The second group, representing a potentially novel species, was composed of a single sequence shared by six Schreibers’ bats that had 91% identity with the recently reported hemoplasma from little brown bats in North America. Large bat aggregations in roosting caves probably benefits intra and inter-species transmission explaining the high observed prevalence.     

Diversity and molecular characterization of novel hemoplasmas infecting wild rodents from different Brazilian biomes

Although hemoplasma infection in domestic animals has been well documented, little is known about the prevalence and genetic diversity of these bacteria in wild rodents. The present work aimed to investigate the occurrence of hemotrophic mycoplasmas in wild rodents from five Brazilian biomes, assessing the 16S rRNA phylogenetic position of hemoplasma species by molecular approach. Spleen tissues were obtained from 500 rodents, comprising 52 different rodent species trapped between 2000 and 2011. DNA samples were submitted to previously described PCR protocols for amplifying Mycoplasma spp. based on 16S rRNA, followed by sequencing and phylogenetic inferences. Among 457 rodent spleen samples showing absence of inhibitors, 100 (21.9%) were PCR positive to Mycoplasma spp. The occurrence of hemotropic mycoplasmas among all sampled rodents was demonstrated in all five biomes and ranged from 9.3% (7/75) to 26.2% (38/145). The Blastn analysis showed that amplified sequences had a percentage of identity ranging from 86 to 99% with other murine hemoplasmas. The ML phylogenetic analysis of 16S rRNA gene of 24 positive randomly selected samples showed the presence of ten distinct groups, all clustering within the Mycoplasma haemofelis. The phylogenetic assessment suggests the circulation of novel hemoplasma species in rodents from different biomes in Brazil.     

Genetic diversity and hematological and biochemical alterations in Alouatta primates naturally infected with hemoplasmas in Brazil

Mycoplasma spp. and Bartonella spp. are Gram-negative bacteria transmitted by arthropod vectors that infect red blood cells of several mammal species. This study investigated the occurrence and genetic diversity of hemoplasmas and Bartonella spp. in 68 howler monkeys kept in captivity in São Paulo, a southeastern state in Brazil. In addition, possible hematological, biochemical and electrophoretic changes of serum proteins associated with the occurrence of hemoplasmas and Bartonella spp. in captive primates were also investigated. The cPCR results showed that all sampled howler monkeys were negative for Bartonella spp. based on the gltA gene. The cPCR results indicated that 18 (26.47%) non-human primates (NHP) were positive for hemoplasmas based on the 16S rRNA gene. Monocyte and lymphocyte counts were higher in hemoplasma-positive howlers (P < 0.05). Platelet counts decreased in nonhuman primates (NHP) positive for hemoplasmas (P < 0.05). The results from the blood serum proteinogram and biochemistry analyses were not significantly different between NHPs positive and negative for hemotrophic mycoplasmas. Phylogenetic analysis using Bayesian Inference (BI) based on the 16S rRNA gene positioned the obtained sequences close to ‘Candidatus Mycoplasma kahanei’. The analysis of sequence diversity of the 16S rRNA gene showed that 5 different genotypes are circulating in NHP in Brazil and in the world; besides, a clear separation between the sequences of hemoplasmas that infect NHP of the Sapajus and Alouatta genus in Brazil was found, probably corresponding to two different species. The pathogenic potential of this hemoplasma species in NHP should be further investigated.     

Microscopic and molecular identification of hemotropic mycoplasmas in South American coatis (Nasua nasua)

Hemoplasmas were detected in two apparently healthy captive South American coatis (Nasua nasua) from southern Brazil during an investigation for vector-borne pathogens. Blood was subjected to packed cell volume (PCV) determination, a commercial real-time PCR panel for the detection of Anaplasma spp., Babesia spp., Bartonella spp., Hepatozoon spp., Leishmania spp., Mycoplasma haemofelis, ‘Candidatus Mycoplasma turicensis’, ‘Candidatus Mycoplasma haemominutum’, Neorickettsia risticii, Rickettsia rickettsii and Leptospira spp., and a pan-hemoplasma conventional PCR assay. PCV was normal, but both coatis tested positive for hemoplasmas and negative for all the remaining pathogens tested. Using different techniques for microscopy (light, confocal or SEM), structures compatible with hemoplasmas were identified. Sequencing of the 16S rRNA gene identified an organism resembling Mycoplasma haemofelis and another hemotropic Mycoplasma sp., with a sequence identity of 96.8% to a Mycoplasma sp. previously detected in capybaras.     

Detection of a novel hemoplasma based on 16S rRNA gene DNA in captive and free-ranging capybaras (Hydrochaeris hydrochaeris)

Two different species of hemoplasmas, Mycoplasma coccoides and M. haemomuris, are known to infect small rodents such as mice and rats. However, there are no previous reports of hemoplasma infection in capybara (Hydrochaeris hydrochaeris). The aim of our study was to determine whether these hemoplasmas might infect capybaras from Southern Brazil. Blood samples from 31 animals: 10 captive and 21 free-ranging capybaras were collected and packed cell volume and total plasma protein were measured. DNA was extracted and PCR assays for M. coccoides and M. haemomuris were performed. Using the M. coccoides-PCR assay 64% of the capybaras were positive, 80% free-ranging and 30% from captive animals. The prevalence of infection between the groups was significantly different (p = 0.001). Sequencing of the nearly entire 16S rRNA gene from the positive samples suggested a novel hemoplasma isolate with identity of 92% with M. coccoides and 86% with M. haemomuris. All capybara samples were negative for M. haemomuris infection. DNA of a housekeeping gene was successfully amplified from all samples. This is the first evidence of a hemoplasma infection in capybaras.     

Occurrence and molecular characterization of Bartonella spp. and hemoplasmas in neotropical primates from Brazilian Amazon

Little is known about the prevalence and genetic diversity of Bartonella spp. and hemoplasmas in nonhuman primates (NHP). The present study aimed to investigate the occurrence of and assess the phylogenetic position of Bartonella spp. and hemoplasma species infecting neotropical NHP from Brazilian Amazon. From 2009 to 2013, a total of 98 blood samples from NHP belonging to the Family Cebidae were collected in the island of São Luís, state of Maranhão, of which 87 NHP were from Wild Animal Screening Center (CETAS) in the municipality of São Luís, and 11 (9 Sapajus sp. and 2 Saimiri sciureus) were from NHP caught in the Sítio Aguahy Private Reserve. DNA samples were screened by previously described PCR protocols for amplifying Bartonella spp. and Mycoplasma spp. based on nuoG, gltA and 16S rRNA genes, respectively. Purified amplicons were submitted to sequencing and phylogenetic analysis. Bacteremia with one or more Bartonella spp. was not detected in NHP. Conversely, 35 NHP were PCR positive to Mycoplasma spp. The Blastn analysis of seven positive randomly selected sequencing products showed percentage of identity ranging from 95% to 99% with other primates hemoplasmas. The Maximum Likelihood phylogenetic analysis based on a 1510 bp of 16S rRNA gene showed the presence of two distinct clusters, positioned within Mycoplasma haemofelis and Mycoplasma suis groups. The phylogenetic assessment suggests the presence of a novel hemoplasma species in NHP from the Brazilian Amazon.     

Extended‐spectrum beta‐lactamase‐producing Escherichia coli in common vampire bats Desmodus rotundus and livestock in Peru

Antibiotic resistance mediated by bacterial production of extended‐spectrum beta‐lactamase (ESBL) is a global threat to public health. ESBL resistance is most commonly hospital‐acquired; however, infections acquired outside of hospital settings have raised concerns over the role of livestock and wildlife in the zoonotic spread of ESBL‐producing bacteria. Only limited data are available on the circulation of ESBL‐producing bacteria in animals. Here, we report ESBL‐producing Escherichia coli in wild common vampire bats Desmodus rotundus and livestock near Lima, Peru. Molecular analyses revealed that most of this resistance resulted from the expression of bla_CTX‐M‐15 genes carried by plasmids, which are disseminating worldwide in hospital settings and have also been observed in healthy children of Peru. Multilocus sequence typing showed a diverse pool of E. coli strains carrying this resistance that were not always host species‐specific, suggesting sharing of strains between species or infection from a common source. This study shows widespread ESBL resistance in wild and domestic animals, supporting animal communities as a potential source of resistance. Future work is needed to elucidate the role of bats in the dissemination of antibiotic‐resistant strains of public health importance and to understand the origin of the observed resistance.     

Real-time PCR-based prevalence study, infection follow-up and molecular characterization of canine hemotropic mycoplasmas

Hemotropic mycoplasmas (hemoplasmas) have been reported in several mammalian species including dogs. Infections may lead to hemolytic anemia, but investigations in the dog had been hampered by the lack of adequate diagnostic methods. Only recently sensitive PCR-based assays were reported for the two canine hemoplasmas, Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum. By applying these assays, 15.4% of 460 dogs from the south of France tested hemoplasma positive. It was hypothesized that this high prevalence may be associated with the presence of Rhipicephalus sanguineus, a proposed vector for canine hemoplasmas. To address this hypothesis and expand the PCR-based knowledge on canine hemoplasmosis, we investigated dogs in a climatic zone that does not allow for the permanent establishment of R. sanguineus. Blood samples were collected throughout a year from 889 dogs in Switzerland: 1.2% of the dogs tested real-time PCR positive. The infection status was not significantly associated with anemia, age or gender. Phylogenetic analyses of Candidatus M. haematoparvum and M. haemocanis isolates revealed > or =99.8% identity to published sequences. All samples collected from three infected dogs throughout a follow-up period of < or =13 months tested PCR positive. Interestingly, the majority of the infected dogs either had been imported from or had visited regions where R. sanguineus is indigenous. Thus, canine hemoplasma prevalence was found to be low in a country with a climate incompatible with frequent occurrence of R. sanguineus. Nonetheless, veterinarians may expect hemoplasma infections in dogs with a travel history and/or after potential tick vector exposure.     

Orthohepadnavirus infection in a neotropical bat (Platyrrhinus lineatus)

Hepatitis B virus (HBV) is the prototype of the Orthohepadnavirus genus and represents an important cause of chronic hepatitis, liver cirrhosis, and hepatic cancer in humans worldwide. To verify the occurrence and genetic variability of orthohepadnavirus among neotropical bats, we tested 81 liver samples of New World bats from São Paulo State, Southeastern Brazil, collected during 2012. PCR, sequencing, and phylogenetic analysis of Surface/Polymerase and Core viral genes confirmed the occurrence of the first isolate of bat orthohepadnavirus detected in South America. These results may contribute to subsequent studies of the origin, variability, host species, and evolution of bat orthohepadnaviruses in South America.     

Mycoplasma ovis in captive cervids: prevalence, molecular characterization and phylogeny

Hemotrophic mycoplasmas (hemoplasmas) are bacteria that attach to red blood cells of mammals, leading to acute and/or subclinical disease in infected animals. It has been suggested that Mycoplasma ovis, a hemoplasma that infects sheep and goats worldwide, may also infect deer. The aim of this study was to evaluate whether South American deer are infected with M. ovis. EDTA-anticoagulated blood samples from a herd of 32 captive South American deer were collected. DNA extraction of blood samples was performed followed by PCR amplification of the 16S and 23S rRNA genes, and sequencing of products. Using M. ovis PCR, 27/31 (87%) were positive, including 21/22 Mazama nana; 2/3 Mazama americana and 4/6 Blastocerus dichotomus. Sequencing of the nearly entire 16S rRNA gene of 26/27 positive samples showed 98.2-98.8% identity to M. ovis of sheep (GenBank, AF338268) and 98.6-99.4% identity to M. ovis-like of a fawn (FJ824847); the 23S rRNA gene from one of these isolates and the fawn's had 97.6% identity. The remaining isolate had just 94.9% identity to the 16S rRNA gene of M. ovis and only 89.4% identity to the 23S rRNA gene of the fawn's M. ovis. This is the first report of M. ovis in captive South American deer, revealing a high prevalence of hemoplasma infection in these animals.     

Isolation of rabies virus from bats in Bolivia

Summary Rabies virus was isolated from apparently normal blood-sucking(Desmodus rotundus) and insectivorous(Artibeus planirostris) bats caught in Bolivia. The virus was identified by immunofluorescence, biological and sero-neutralisation tests.

---

Aislamiento Del Virus Rábico De Murciélagos en Bolivia

Resumen Se aisló el virus rábico de vampiros (Desmodus rotundus) y murciélagos insectivoros (Artibeus planirostris) aparentemente sanos. El virus se identificó por inmunofluorescencia y pruebas biológicas y de seroneutralización.

---

Isolement Du Virus Rabique Chez Des Chauves-souris, En Bolivie

Résumé Du virus rabique a été isolé chez des chauves-souris hémo (Desmodus rotundus) et insectivores (Artibeus planirostris) apparemment normales, capturées en Bolivie. Le virus a été identifié par immunofluorescence et des tests biologiques et de séro-neutralisation.

     

Haematophagous bats in Brazil, their role in rabies transmission, impact on public health, livestock industry and alternatives to an indiscriminate reduction of bat population

Haematophagous bats exist only in Latin America, from México to the Northern provinces of Argentina. They are represented by three species, Desmodus rotundus, Diphylla ecaudata and Diaemus youngii. While two species feed only on blood of wild birds, one species, D. rotundus, causes losses feeding on livestock and could be a vector for rabies virus. The cases in which humans were bitten by the bat have increased in Brazil. Bats became a target of control activities by farming communities and local governments. Indiscriminate actions such as poisoning bats and destroying their roosts put the lives of other bat species, which are extremely important for the ecologic balance, at risk. The vaccination of exposed livestock against rabies, which would protect the endangered livestock, is not regularly carried out. The importance and current status of D. rotundus in the transmission of rabies in Brazil, the Public Health aspects, the importance for the livestock industry are shown and the consequences of reducing bat population are discussed. Alternatives to an indiscriminate bat-population reduction in the control of rabies are proposed.