Molecular Detection and Characterization of Mycoplasma spp. in Marine Mammals, Brazil.

Mycoplasma spp. are wall-less bacteria able to infect mammals and are classified as hemotropic (hemoplasma) and nonhemotropic. In aquatic mammals, hemoplasma have been reported in California sea lions (Zalophus californianus) and river dolphins (Inia spp.). We investigated Mycoplasma spp. in blood samples of West Indian manatees (Trichechus manatus), pinnipeds (5 species), and marine cetaceans (18 species) that stranded or were undergoing rehabilitation in Brazil during 2002-2022. We detected Mycoplasma in blood of 18/130 (14.8%) cetaceans and 3/18 (16.6%) pinnipeds. All tested manatees were PCR-negative for Mycoplasma. Our findings indicate that >2 different hemoplasma species are circulating in cetaceans. The sequences from pinnipeds were similar to previously described sequences. We also detected a nonhemotropic Mycoplasma in 2 Franciscana dolphins (Pontoporia blainvillei) that might be associated with microscopic lesions. Because certain hemoplasmas can cause disease and death in immunosuppressed mammals, the bacteria could have conservation implications for already endangered aquatic mammals.

Multilayer Networks Assisting to Untangle Direct and Indirect Pathogen Transmission in Bats.

The importance of species that connect the different types of interactions is becoming increasingly recognized, and this role may be related to specific attributes of these species. Multilayer networks have two or more layers, which represent different types of interactions, for example, between different parasites and hosts that are nonetheless connected. The understanding of the ecological relationship between bats, ectoparasites, and vector-borne bacteria could shed some light on the complex transmission cycles of these pathogens. In this study, we investigated a multilayer network in Brazil formed by interactions between bat-bacteria, bat-ectoparasite, and ectoparasite-bacteria, and asked how these interactions overlap considering different groups and transmission modes. The multilayer network was composed of 31 nodes (12 bat species, 14 ectoparasite species, and five bacteria genera) and 334 links, distributed over three layers. The multilayer network has low modularity and shows a core-periphery organization, that is, composed of a few generalist species with many interactions and many specialist species participating in few interactions in the multilayer network. The three layers were needed to accurately describe the multilayer structure, while aggregation leads to loss of information. Our findings also demonstrated that the multilayer network is influenced by a specific set of species that can easily be connected to the behavior, life cycle, and type of existing interactions of these species. Four bat species (Artibeus lituratus, A. planirostris, Phyllostomus discolor, and Platyrrhinus lineatus), one ectoparasite species (Steatonyssus) and three bacteria genera (Ehrlichia, hemotropic Mycoplasma and Neorickettsia) are the most important species for the multilayer network structure. Finally, our study brings an ecological perspective under a multilayer network approach on the interactions between bats, ectoparasites, and pathogens. By using a multilayer approach (different types of interactions), it was possible to better understand these different ecological interactions and how they affect each other, advancing our knowledge on the role of bats and ectoparasites as potential pathogen vectors and reservoirs, as well as the modes of transmission of these pathogens.

Hemotropic Mycoplasma spp. in Aquatic Mammals, Amazon Basin, Brazil.

Hemotropic Mycoplasma spp. (hemoplasmas) are uncultivable bacteria that infect mammals, including humans. We detected a potentially novel hemoplasma species in blood samples from wild river dolphins in the Amazon River Basin, Brazil. Further investigation could determine pathogenicity and zoonotic potential of the detected hemoplasma.

Evaluation of hematological alteration of vector-borne pathogens in cats from Bangkok, Thailand.

BACKGROUND: Cats can be carriers of infected arthropods and be infected with several vector-borne pathogens (VBPs) but there is limited knowledge about their pathogenic role in cats. This study aimed to assess the prevalence of some feline vector-borne agents by molecular technique and to characterize the hematological findings associated with these infections in a cat population from Bangkok Thailand. RESULTS: PCR was positive with at least one pathogen in 237 out of 372 subjects (63.7%), with prevalence of 39.5% (147/372) for Babesia spp., 36.9% (137/372) for hemoplasmas and 3.2% (12/372) for Hepatozoon spp. The cats older than 1 year were at significantly greater risk for VBPs infection (P = 0.001; OR = 1.43; 95% CI: 1.12 - 1.81) and hemoplasmas infection (χ2 = 10.8, df = 1; P < 0.0001; OR = 2.45; 95% CI: 1.49 - 4.01). A significant association between hematological findings and hemoplasma infection were identified in the present study. Besides, VBPs infection revealed more frequent in male cats (χ2= 6.38, df = 1, P = 0.01). Macrocytic hypochromic type of anemia was observed in cats infested with blood-sucking arthropods compared to the non-infested cats presented. CONCLUSIONS: The current study confirmed that Babesia, Hepatozoon and hemoplasmas had infected semi-domesticated cats in Bangkok, Thailand, with Babesia and hemoplasmas being dominant in prevalence. Some hematological findings were significantly associated with cats infected with vector-borne pathogens in this study including leukocyte count and platelets count that may help support veterinary technicians in diagnosis and appropriate treatment. Campaigns of VBPs monitoring in Bangkok emphasizing on the investigation of vectors and possible routes of the infection in animals should be conducted to prevent the transmission of the pathogens.

Prevalence and phylogeny of Chlamydiae and hemotropic mycoplasma species in captive and free-living bats.

BACKGROUND: Bats are hosts for a variety of microorganisms, however, little is known about the presence of Chlamydiales and hemotropic mycoplasmas. This study investigated 475 captive and free-living bats from Switzerland, Germany, and Costa Rica for Chlamydiales and hemotropic mycoplasmas by PCR to determine the prevalence and phylogeny of these organisms. RESULTS: Screening for Chlamydiales resulted in a total prevalence of 31.4%. Positive samples originated from captive and free-living bats from all three countries. Sequencing of 15 samples allowed the detection of two phylogenetically distinct groups. These groups share sequence identities to Chlamydiaceae, and to Chlamydia-like organisms including Rhabdochlamydiaceae and unclassified Chlamydiales from environmental samples, respectively. PCR analysis for the presence of hemotropic mycoplasmas resulted in a total prevalence of 0.7%, comprising free-living bats from Germany and Costa Rica. Phylogenetic analysis revealed three sequences related to other unidentified mycoplasmas found in vampire bats and Chilean bats. CONCLUSIONS: Bats can harbor Chlamydiales and hemotropic mycoplasmas and the newly described sequences in this study indicate that the diversity of these bacteria in bats is much larger than previously thought. Both, Chlamydiales and hemotropic mycoplasmas are not restricted to certain bat species or countries and captive and free-living bats can be colonized. In conclusion, bats represent another potential host or vector for novel, previously unidentified, Chlamydiales and hemotropic mycoplasmas.

Prevalence, Genotype Richness, and Coinfection Patterns of Hemotropic Mycoplasmas in Raccoons (Procyon lotor) on Environmentally Protected and Urbanized Barrier Islands.

Raccoons (Procyon lotor) are successful urban adapters and hosts to a number of zoonotic and nonzoonotic pathogens, yet little is known about their hemoplasma infections and how prevalence varies across habitat types. This study identifies hemotropic Mycoplasma species infection in raccoons from urban and undisturbed habitats and compares hemoplasma infection in sympatric urban cats (Felis catus) from the same geographic region. We collected blood from raccoons (n = 95) on an urban coastal island (n = 37) and an undisturbed coastal island (n = 58) and from sympatric urban cats (n = 39) in Georgia, USA. Based on 16S rRNA gene amplification, 62.1% (59/95) of raccoons and 17.9% (7/39) of feral cats were positive for hemoplasma. There was a greater percentage of hemoplasma-infected raccoons on the undisturbed island (79.3% [46/58]) than on the urban island (35.1% [13/37]; χ2 = 16.9, df = 1, P = 0.00004). Sequencing of the full-length 16S rRNA gene amplicons revealed six hemoplasma genotypes in raccoons, including five novel genotypes that were distinct from three known hemoplasma species identified in the sympatric cats. In addition, the hemoplasma genotypes detected in raccoons were not identified in sympatric cats or vice versa. Although all six hemoplasma genotypes were found in raccoons from urban and undisturbed islands, coinfection patterns differed between sites and among individuals, with the proportion of coinfected raccoons being greater in the undisturbed site. This study shows that raccoons are hosts for several novel hemoplasmas and that habitat type influences infection patterns.IMPORTANCE This study provides information about novel hemoplasmas identified in raccoons (Procyon lotor), which can be used for assessments of the prevalence of these hemoplasmas in raccoon populations and for future studies on the potential pathogenic impacts of these hemoplasmas on raccoon health. Raccoons from the undisturbed habitat had a higher prevalence of hemoplasma infection than urban raccoons. There does not appear to be cross-species transmission of hemotropic mycoplasmas between urban raccoons and feral cats. Raccoons appear to be hosts for several novel hemoplasmas, and habitat type influences infection patterns.

Novel hemotropic mycoplasmas are widespread and genetically diverse in vampire bats.

Bats (Order: Chiroptera) have been widely studied as reservoir hosts for viruses of concern for human and animal health. However, whether bats are equally competent hosts of non-viral pathogens such as bacteria remains an important open question. Here, we surveyed blood and saliva samples of vampire bats from Peru and Belize for hemotropic Mycoplasma spp. (hemoplasmas), bacteria that can cause inapparent infection or anemia in hosts. 16S rRNA gene amplification of blood showed 67% (150/223) of common vampire bats (Desmodus rotundus) were infected by hemoplasmas. Sequencing of the 16S rRNA gene amplicons revealed three novel genotypes that were phylogenetically related but not identical to hemoplasmas described from other (non-vampire) bat species, rodents, humans, and non-human primates. Hemoplasma prevalence in vampire bats was highest in non-reproductive and young individuals, did not differ by country, and was relatively stable over time (i.e., endemic). Metagenomics from pooled D. rotundus saliva from Peru detected non-hemotropic Mycoplasma species and hemoplasma genotypes phylogenetically similar to those identified in blood, providing indirect evidence for potential direct transmission of hemoplasmas through biting or social contacts. This study demonstrates vampire bats host several novel hemoplasmas and sheds light on risk factors for infection and basic transmission routes. Given the high frequency of direct contacts that arise when vampire bats feed on humans, domestic animals, and wildlife, the potential of these bacteria to be transmitted between species should be investigated in future work.

Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and 'Candidatus Mycoplasma haemovis') in sheep and goats in China.

BACKGROUND: Hemotropic mycoplasmas (hemoplasmas) are emerging zoonotic pathogens with a worldwide distribution that can cause mild to severe hemolytic anemia, icterus, ill-thrift, infertility, and poor weight gain. However, understanding of the molecular epidemiology of hemoplasmas (Mycoplasma ovis and 'Candidatus Mycoplasma haemovis') is limited in sheep and goats, and the hemoplasma strain/species/variant 'Candidatus M. haemovis' was poorly studied throughout the world and had never been detected in China until now. Thus, the aim of the present study was to determine the molecular prevalence of hemoplasmas, including M. ovis and 'Candidatus M. haemovis' in sheep and goats from seven provinces and one autonomous region of China. METHODS: A total of 1364 blood samples were collected from sheep and goats in seven provinces and one autonomous region of China. All blood samples were tested for hemoplasmas (M. ovis and 'Candidatus M. haemovis') by nested PCR amplification based on 16S rRNA gene. Positive specimens underwent nucleotide sequencing and phylogenetic analysis. RESULTS: Overall, 610 specimens (44.7%, 610/1364) were shown to be hemoplasmas (M. ovis and 'Candidatus M. haemovis') -positive by nested PCR amplification based on 16S rRNA gene. The prevalence in goats was 44.1% (379/860), and 45.8% (231/504) in sheep, while that in grazing small ruminants was 54.4% (396/728) and 33.6% (214/636) in house feeding small ruminants. Sequencing of the nearly complete 16S rRNA gene was successful for the 103 randomly selected positive specimens from different farms in different sampling sites of China. Among them, analysis of the 16S rRNA gene sequences identified M. ovis (n = 56) and 'Candidatus M. haemovis' (n = 47). Two (KU983740 and KU983746) of the four novel genotypes obtained in this study were closely related to M. ovis, while the other two genotypes (KU983748 and KU983749) had high identity with 'Candidatus M. haemovis'. Remarkably, the genotype (KU983740) of M. ovis in sheep and goats in this study fell in a clade with two human hemoplasmas from USA (KF313922 and GU230144) and shared 99.8%-99.9% with them. CONCLUSIONS: In this study, 'Candidatus M. haemovis' was first detected in Chinese sheep and goats and hemoplasmas (M. ovis and 'Candidatus M. haemovis') are highly prevalent, and widely distributed in China.

Geographically widespread and novel hemotropic mycoplasmas and bartonellae in Mexican free-tailed bats and sympatric North American bat species.

Bacterial pathogens remain poorly characterized in bats, especially in North America. We describe novel (and in some cases panmictic) hemoplasmas (12.9% positivity) and bartonellae (16.7% positivity) across three colonies of Mexican free-tailed bats (Tadarida brasiliensis), a partially migratory species that can seasonally travel hundreds of kilometers. Molecular analyses identified three novel Candidatus hemoplasma species most similar to another novel Candidatus species in Neotropical molossid bats. We also detected novel hemoplasmas in sympatric cave myotis (Myotis velifer) and pallid bats (Antrozous pallidus), with sequences in the latter 96.5% related to C. Mycoplasma haemohominis. We identified eight Bartonella genotypes, including those in cave myotis, with 96.7% similarity to C. Bartonella mayotimonensis. We also detected Bartonella rochalimae in migratory Tadarida brasiliensis, representing the first report of this human pathogen in bats. The seasonality and diversity of these bacteria observed here suggest that additional longitudinal, genomic, and immunological studies in bats are warranted.

Molecular survey of hemoplasmas and Coxiella burnetii in vampire bats from northern Brazil.

In addition to zoonotic viral pathogens, bats can also harbor bacterial pathogens, including hemoplasmas (hemotropic mycoplasmas) and Coxiella burnetii. The present study aimed to investigate, using molecular techniques, the presence of hemoplasmas and C. burnetii in spleen samples from vampire bats in northern Brazil. For this purpose, between 2017 and 2019, spleen samples were collected from Desmodus rotundus (n = 228) and Diaemus youngii (n = 1) captured in the states of Pará (n = 207), Amazonas (n = 1), Roraima (n = 18) and Amapá (n = 3). DNA samples extracted from the bat spleen and positive in PCR for the endogenous gapdh gene were subjected to conventional PCR assays for the 16S rRNA, 23S rRNA and RNAse P genes from hemoplasmas and to qPCR based on the IS1111 gene element for C. burnetii. All spleen samples from vampire bats were negative in the qPCR for C. burnetii. Hemoplasmas were detected in 10 % (23/229) of spleen samples using a PCR based on the 16S rRNA gene. Of these, 21.73 % (5/23) were positive for the 23S rRNA gene and none for the RNAseP gene. The seven hemoplasma 16S rRNA sequences obtained were closely related to sequences previously identified in vampire bats from Belize, Peru and Brazil. The 23S rRNA sequence obtained revealed genetic proximity to hemoplasmas from non-hematophagous bats from Brazil and Belize. The analysis revealed different circulating genotypes among Brazilian vampire bats, in addition to a trend towards genera-specific hemoplasma genotypes. The present study contributes to the knowledge of the wide diversity of hemoplasmas in vampire bats.

Detection and characterization of hemotropic Mycoplasmas in Iberian wolves (Canis lupus signatus) of Cantabria, Spain.

Hemoplasmas (hemotropic mycoplasmas) are uncultivable wall-less bacteria able to infect mammalian erythrocytes. Hemoplasmas can cause anemia, especially in immunocompromised hosts, predisposing to secondary infections and even leading to death. Between 2017 and 2023, spleen samples of 131 wild Iberian wolves (Canis lupus signatus) of Cantabria (Spain) were screened for Mycoplasma spp. using a real-time PCR able to amplify a 360 bp fragment of the 16S rRNA gene and confirmed by direct Sanger sequencing. Additional conventional PCRs were performed to screen for coinfections by different Mycoplasma species and to discriminate between Mycoplasma haemocanis/haemofelis (Mhc/Mhf). Overall, 24/131 (18.3%) animals were PCR-positive. Biological and environmental factors potentially promoting hemoplasma infection in this species were analyzed. Two different hemoplasma species were detected: Mhc/Mhf (18/131; 13.7%) and Candidatus Mycoplasma haematoparvum (CMhp) (3/131; 2.3%), each with one nucleotide sequence type (ntST); three other sequences were not classified. No Mhc/Mhf and CMhp coinfection were observed. The 12 Mhc/Mhf suitable for ribonuclease P RNA sequencing were confirmed as Mhc. Mhc ntST was 100% identical to a Mhc sequence previously obtained in domestic dogs (Canis lupus familiaris), and in wild Iberian wolves of northwestern Spain (Asturias and Galicia) at a similar prevalence to the one found herein, suggesting a high Mhc genetic homogeneity in this wild population. CMhp ntST was 100% identical to CMhp sequences from domestic dogs. To our knowledge, this is the first description of CMhp in the Iberian wolf. The high genetic similarity observed in Mhc and CMhp sequences, as well as their high similarity with domestic dog sequences, suggest its recent introduction, a high level of intraspecific transmission within the wild wolf population, and likely, interspecific transmission between wolves and domestic dogs.

Molecular detection of blood-borne agents in vampire bats from Brazil, with the first molecular evidence of Neorickettsia sp. in Desmodus rotundus and Diphylla ecaudata.

Bats (Mammalia, Chiroptera) represent the second largest group of mammals. Due to their ability to fly and adapt and colonize different niches, bats act as reservoirs of several potentially zoonotic pathogens. In this context, the present work aimed to investigate, using molecular techniques, the occurrence of blood-borne agents (Anaplasmataceae, Coxiella burnetii, hemoplasmas, hemosporidians and piroplasmids) in 198 vampire bats sampled in different regions of Brazil and belonging to the species Desmodus rotundus (n = 159), Diphylla ecaudata (n = 31) and Diaemus youngii (n = 8). All vampire bats liver samples were negative in PCR assays for Ehrlichia spp., Anaplasma spp., piroplasmids, hemosporidians and Coxiella burnetii. However, Neorickettsia sp. was detected in liver samples of 1.51% (3/198) through nested PCR based on the 16S rRNA gene in D. rotundus and D. ecaudata. This is the first study to report Neorickettsia sp. in vampire bats. Hemoplasmas were detected in 6.06% (12/198) of the liver samples using a PCR based on the 16S rRNA gene. The two 16S rRNA sequences obtained from hemoplasmas were closely related to sequences previously identified in vampire and non-hematophagous bats from Belize, Peru and Brazil. The genotypic analysis identified a high diversity of bat-associated hemoplasma genotypes from different regions of the world, emphasizing the need for studies on this subject, in order to better understand the mechanisms of co-evolution between this group of bacteria and their vertebrate hosts. The role of neotropical bat-associated Neorickettsia sp. and bats from Brazil in the biological cycle of such agent warrant further investigation.

Viability of erythrocytes in canine packed red blood cells stored in CPDA-1 is related to the presence of Mycoplasma haemocanis.

Hemotropic mycoplasmas are associated with subclinical disease in dogs and should be identified in blood donors. The objective was to investigate the presence and effect of M. haemocanis in units of packed red blood cells (pRBC) during storage. Canine donors (n = 10) were screened for M. haemocanis by quantitative real-time PCR. pRBCs were obtained from 5 hemoplasma negative dogs and 5 hemoplasma positive dogs. Each pRBC was aliquoted into two 100 mL transfer bags and stored at 4 °C. M. haemocanis loads and biochemical variables (pH, bicarbonate, potassium, sodium, chlorite, glucose, lactate, ammonia, PCV, and % hemolysis) were evaluated on days 1, 7, 18, and 29. M. haemocanis loads increased in pRBC from day 1-29 of storage. Glucose decreased and lactate increase faster in pRBC with M. haemocanis. This study contributes to understand hemoplasma metabolism and reinforces that dog donors should be tested for hemoplasmas.

High prevalence and genetic diversity of hemoplasmas in bats and bat ectoparasites from China.

Hemoplasmas can cause severe hemolytic anemia in humans. To explore the genetic diversity and the potential transmission routes of hemoplasmas among bat population, bats and bat-ectoparasites including bat-flies, bat-mites, and bat-ticks were collected in Eastern and Central China from 2015 to 2021, and tested with PCR for hemoplasmas 16S rRNA gene. Based on 16S rRNA PCR, 18.0% (103/572) adult bats were positive for hemoplasmas, but none of 11 fetuses from hemoplasmas-positive pregnant bats was positive for hemoplasmas. These results indicated that adult bats had a high prevalence of hemoplasma, but vertical transmission of hemoplasmas did not occurr in the bats. Based on the 16S rRNA gene PCR, the minimum infection rate of bat-ectoparasite for hemoplasmas was 4.0% (27/676), suggesting that bat-ectoparasite also had a high prevalence for hemoplasmas. Phylogenetic analysis revealed that bat hemoplasmas from this study clustered into 4 genotypes (I-IV). Genotype I clustered together with hemoplasmas identified in bats from America. Genotype II shared high similarity with a human-pathogenic hemoplasma Candidatus Mycoplasma haemohominis. Genotype III and IV were unique, representing 2 new hemoplasma genotypes. Only genotype I was identified in both bats and all bat-ectoparasites including bat-flies, bat-mites, and bat-ticks. In conclusion, bats and bat-ectoparasites from China harbored abundant genetically diverse hemoplasmas including potential human-pathogenic hemoplasmas, indicating bats and bat-ectoparasites may play important roles in the maintenance and transmission of hemoplasmas in the natural foci.

Molecular survey and phylogenetic analysis of Bartonella sp., Coxiella sp., and hemoplamas in pudu (Pudu puda) from Chile: first report of Bartonella henselae in a wild ungulate species.

Introduction: Recent evidence shows a high diversity of infectious agents in wildlife that represent a threat to human, domestic, and wild animal health. In Chile, wild populations of the most common cervid species, pudu (Pudu puda), have been reported as hosts for novel pathogens such as Mycoplasma ovis-like and a novel ecotype of Anaplasma phagocytophilum. A better understanding of the epidemiology of this group and other intracellular bacteria that might have cervids as hosts would enlighten their population relevance. This study aimed to determine the occurrence and genetic diversity of Bartonella spp., hemotropic mycoplasmas, and Coxiella burnetii in pudus from Chile. Methods: The DNA was extracted from the blood samples of 69 wild free-ranging and 30 captive pudus from Chile. A combination of real-time (nouG gene for Bartonella and IS1111 element for C. burnetii) and conventional PCR (16S rRNA for hemotropic Mycoplasma spp. and rpoB, gltA, and ITS for Bartonella spp.) was used for pathogen screening and molecular characterization. Results: DNA of Bartonella spp. was detected in 10.1% [95% CI (5.2-18.2%)] samples, hemotropic Mycoplasma spp. in 1.7% [95% CI (0.08-10.1%)], and C. burnetii in 1.0% [95% CI (0.05-6.3%)] samples. Two sequenced samples were identified as Mycoplasma ovis-like, and one free-ranging pudu was positive for C. burnetii. While one captive and two free-ranging pudus were positive for Bartonella henselae, one wild pudu was co-positive for B. henselae and Bartonella sp., similar to Bartonellae identified in ruminants. Discussion: To the best of our knowledge, this is the first report of B. henselae in wild ungulate species, and C. burnetii and Bartonella spp. in wild ungulate species in South America. Further research will be necessary to evaluate the potential role of pudu as reservoirs of infection and identify the sources for disease transmission among humans and wild and domestic animals.

Molecular detection and characterization of vector-borne agents in common opossums (Didelphis marsupialis) from northeastern Brazil.

Opossums are synanthropic marsupials able to interchange among wild, peri­urban and urban environments, playing an epidemiologically important role as hosts for emerging pathogens and ectoparasites of relevance in public health. The present study aimed to detect and molecularly characterize vector-borne agents in a population of common opossums (Didelphis marsupialis) from the Island of São Luís do Maranhão, northeastern Brazil. Of the 45 animals analyzed, one (2.22%) was positive in the nested PCR assay based on the 18S rRNA gene of piroplasmids. The obtained sequence was phylogenetically positioned in a clade containing sequences of Babesia sp. previously detected in Didelphis aurita, Didelphis albiventris and associated ticks from Brazil. Eight (17.77%) samples were positive in PCR for Ehrlichia spp. based on the dsb gene; four samples were sequenced and positioned into a new clade, sister to E. minasensis and Ehrlichia sp. clade detected in Superorder Xenarthra mammals. No samples tested positive in the screening PCR assays based on the 16S rRNA gene of Anaplasma spp. Two samples were positive in the qPCR for Bartonella spp. based on the nuoG gene. Seven animals (15.56%) were positive in the nPCR based on the 16S rRNA gene of hemoplasmas. Of these, three were positive in a PCR based on the 23S rRNA gene. The phylogenies based on both 16S rRNA and 23S rRNA genes corroborated to each other and positioned the sequences in the same clade of hemoplasmas previously detected in D. aurita and D. albiventris sampled in Brazil. Finally, three (6.66%) animals were positive in the PCR for Hepatozoon spp.; the obtained 18S rRNA sequence was positioned into the H. felis clade.The present study showed, for the first time, the circulation of piroplasmids, Hepatozoon spp., Ehrlichia spp., hemoplasmas and Bartonella spp. in D. marsupialis sampled in northeastern Brazil, with description of putative novel genotypes of Ehrlichia and Hepatozoon and copositivity by different vector-borne agents. The present work consolidates the "South American Marsupialia" piroplasmid clade, adding one more genotype of Babesia sp. to this clade.

An Update of Bovine Hemoplasmas Based on Phylogenetic and Genomics Analysis.

Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos' are bacteria that have been described as significant hemoplasmas that infect cattle worldwide. Currently, three bovine hemoplasma genomes are known. This work aimed to describe the main genomic characteristics and the evolutionary relationships between hemoplasmas, and provide a list of epitopes predicted by immunoinformatics as diagnostic candidates for bovine hemoplasmosis. Thus far, there is no vaccine to prevent this disease that economically impacts cattle production worldwide. Additionally, there is a lack of vaccines against bovine hemoplasmosis. In this work, we performed a genomic characterization of hemoplasmas, including two Mexican strains reported in bovines in the last few years. The generated information is a new scenario about the phylogeny of hemoplasmas. Also, we show genomic features among hemoplasmas that strengthen their characteristic genome plasticity of intracellular lifestyles. Finally, the elucidation of antigenic proteins in Mexican strains represents an opportunity to develop molecular detection methods and diagnoses.

'Candidatus Mycoplasma Haemoalbiventris' and Tick-Borne Pathogens in Black-Eared Opossum (Didelphis aurita) from Southeastern Brazil.

The black-eared opossum (Didelphis aurita) is a South American synanthropic marsupial. The presence of opossums in domestic spaces is relevant in the One-Health context since they are hosts of pathogens and ectoparasites that may affect the health of domestic animals and humans. In this study, we aim to determine the occurrence of hemoplasmas and selected tick-borne pathogens in free-ranging black-eared opossums, along with their molecular characterization, hematological and biochemical evaluation and factors associated with infection, in the municipality of Viçosa, State of Minas Gerais, southeastern Brazil. Thirty black-eared opossums were trapped between March 2021 and June 2022. Ectoparasites were collected. Hematological and biochemical analyses were performed. DNA from EDTA-blood samples were analyzed by PCR and qPCR assays. By molecular analyses, 'Candidatus Mycoplasma haemoalbiventris' was the most prevalent hemoparasite (73.3%), followed by Hepatozoon sp. (22.2%). Significant differences were observed in the number of platelets, and in the concentration of protein and globulins in the animals infected by 'Ca. M. haemoalbiventris' when compared with the negative group. This is the first report of 'Ca. M. haemoalbiventris' infection in D. aurita.

Molecular detection and genotype diversity of hemoplasmas in non-hematophagous bats and associated ectoparasites sampled in peri-urban areas from Brazil.

Hemoplasmas have already been detected in bats in the United States of America, Spain, Australia, Chile, Brazil, Peru, Belize, Nigeria, Costa Rica, Germany, Switzerland and New Caledonia. The recent detection of hemoplasmas closely related to Mycoplasma haematohominis, an agent causing disease in humans, emphasizes the need for additional studies on the diversity of hemoplasmas in bats. The present work aimed to investigate the occurrence and assess the phylogenetic positioning and genetic diversity of hemoplasmas in bats and associated ectoparasites sampled in central-western Brazil. Overall, 43% (58/135) sampled bats and 1.56% (1/64) bat flies (Megistopoda aranea) were positive for hemoplasmas, however, twenty-four and two hemoplasma sequences were obtained from PCR assays targeting 16S and 23S rRNA genes, respectively, since the majority of the obtained amplicons showed faint bands in agarose gel electrophoresis. The obtained 16S rRNA sequences showed to be broadly distributed along the phylogenetic tree, albeit positioned within the 'Haemofelis group' and clustering with other bat-associated hemoplasmas. Twelve 16S rRNA hemoplasma genotypes were found among the 24 obtained sequences. When compared to other bat-related hemoplasmas sequences retrieved from the Genbank, 52 genotypes were found. The two 23S rRNA sequences obtained were positioned as a sister clade to "Candidatus Mycoplasma haematohydrochaerus", M. haemofelis and M. haemocanis. High genetic diversity was found among 16S rRNA hemoplasma sequences detected in non-hematophagous bats from central-western Brazil and previously detected in other regions of the world. Even though the genotype analysis showed that hemoplasmas from the same genus tend to group together, the results from the unipartite and bipartite analyses did not robustly support the hypothesis. Further studies addressing the specificity of hemoplasma genotypes according to bat species and genera should be performed.

Novel Rickettsia spp. in two common overwintering North American songbirds.

American robins and dark-eyed juncos migrate across North America and have been found to be competent hosts for some bacterial and viral pathogens, but their contributions to arthropod-borne diseases more broadly remain poorly characterized. Here, we sampled robins and juncos in multiple sites across North America for arthropod-borne bacterial pathogens of public health significance. We identified two novel Rickettsia spp. in one wintering migrant per bird species related to bellii, transitional, and spotted rickettsiae fever groups. Stable isotope analyses of feathers suggested spring migration of these common songbirds could disperse these novel rickettsiae hundreds-to-thousands of kilometers to host breeding grounds. Further work is needed to characterize zoonotic potential of these rickettsiae and host reservoir competence.