Molecular diagnosis and characterization of Anaplasma marginale and Ehrlichia ruminantium infecting beef cattle of Maputo Province, Mozambique.

BACKGROUND: Members of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique. METHODS: A total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses. RESULTS: Anaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters. CONCLUSION: Cattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified.

Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous).

Piroplasmids and Hepatozoon spp. Are apicomplexan protozoa that may cause disease in several canid species. The present study aimed to expand the knowledge on the diversity of piroplasmids and Hepatozoon in crab-eating foxes (Cerdocyon thous; n = 12) sampled in the Pantanal of Mato Grosso do Sul State, central-western Brazil. PCR assays based on the 18S rRNA were used as screening. Three (25%) and 11 (91.7%) were positive for piroplasmids and Hepatozoon spp., respectively. Co-infection was found in three C. thous. Phylogenetic analyses based on the near-complete 18S rRNA, cox-1 and hsp70 genes evidenced the occurrence of a novel of Babesia spp. (namely Babesia pantanalensis nov. sp.) closely related to Rangelia vitalii and Babesia sp. 'Coco'. This finding was supported by the genetic divergence analysis which showed (i) high divergence, ranging from 4.17 to 5.62% for 18 S rRNA, 6.16% for hps70 and 4.91-9.25% for cox-1 and (ii) the genotype network (which displayed sequences separated from the previously described Piroplasmida species by median vectors and several mutational events). Also, phylogenetic analysis based on the 18S rRNA gene of Hepatozoon spp. positioned the sequences obtained herein in a clade phylogenetically related to Hepatozoon sp. 'Curupira 2', Hepatozoon sp. detected in domestic and wild canids from Uruguay and Hepatozoon americanum. The present study described Babesia pantanalensis nov sp. and Hepatozoon closely related to H. americanum in crab-eating foxes from Brazil. Moreover, the coinfection by piroplasmids and Hepatozoon sp. for the first time in crab-eating foxes strongly suggesting that this wild canid species potentially acts as a bio-accumulate of hemoprotozoan in wild environment.

Bartonella spp. and Typhus Group Rickettsiae among Persons Experiencing Homelessness, São Paulo, Brazil.

Persons experiencing homelessness in São Paulo, Brazil, were seropositive for Bartonella spp. (79/109, 72.5%) and typhus group rickettsiae (40/109, 36.7%). Bartonella quintana DNA was detected in 17.1% (14/82) body louse pools and 0.9% (1/114) blood samples. Clinicians should consider vectorborne agents as potential causes of febrile syndromes in this population.

Bacterial pathogens' screening in Brazilian chigger mites (Trombidiformes: Trombiculidae), with the first report of 'Candidatus Rickettsia colombianensi'-like in avian-associated chiggers.

Chiggers are larval ectoparasites of the Trombiculidae that can transmit pathogens to their hosts. In this study, chiggers collected from birds in Brazil were morphologically identified as Blankaartia sinnamaryi, Eutrombicula batatas, Eutrombicula daemoni, Eutrombicula goeldii, Eutrombicula tinami, and Parasecia gilbertoi. For these specimens, a beginning attempt at molecular identification were also provided, as well as, were genetically screened to detect bacterial pathogens. The species B. sinnamaryi and E. tinami were positive for Rickettsia felis-like and 'Candidatus Rickettsia colombianensi'-like, respectively. For the other agents (Anaplasmataceae, Borrelia spp. and Orientia tsutsugamushi), the tests were negative. This is the first report of 'Ca. R. colombianensi'-like and the second record of R. felis-like in chigger collected on birds from Brazil.

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.

Expanding the universe of Piroplasmids: morphological detection and phylogenetic positioning of putative novel piroplasmids in black-eared opossums (Didelphis aurita) from southeastern Brazil, with description of "South American Marsupialia Group" of Piroplasmida.

The growing proximity of wildlife to large urban niches arouses greater interest in understanding wild reservoirs in the epidemiology of diseases of importance to animal and human health. The aim of the present study was to investigate the presence of piroplasmids in opossums rescued from the metropolitan region of Rio de Janeiro state, Brazil. Blood and bone marrow samples were collected from 15 Didelphis aurita and subjected to DNA extraction and PCR using primers for the 18S rRNA, cox1, cox3, and hsp70 genes of piroplasmids. Clinical and hematological evaluation of the animals was also performed. Five (33.3%) of the 15 opossums tested positive for piroplasms in the nested PCR based on the 18S rRNA, and in two animals, it was possible to observe intra-erythrocytic structures compatible with merozoites. One of the positive animals showed clinical signs of infection such as jaundice, fever, and apathy. Anemia, low level of plasma protein, leukocytosis, and regenerative erythrocyte signs were observed in positive animals. Phylogenetic analysis based on both 18S rRNA and cox-3 genes demonstrated that the piroplasmids detected in D. aurita formed a unique sub-clade, albeit related to piroplasmids previously detected in Didelphis albiventris and associated ticks from Brazil. This study proposes the novel Piroplasmida Clade, namely "South American Marsupialia Group," and reinforces the need for new clinical-epidemiological surveys to understand the dynamics of these infections in didelphids in Brazil.

Molecular detection of piroplasmids in mammals from the Superorder Xenarthra in Brazil.

Xenarthra mammals can be found from southern North America to southern South America, including all Brazilian biomes. Although it has been shown that Xenarthra mammals can play a role as reservoirs for several zoonotic agents, few studies investigate the diversity of piroplasmids (Apicomplexa: Piroplasmida) in this group of mammals. Taking into account that piroplasmids can cause disease in animals and humans, understanding the prevalence and diversity of piroplasmids in Xenarthra mammals would contribute to conservation efforts for this group of animals as well as to infer risk areas for transmission of emergent zoonosis. The present study aimed to investigate the occurrence and molecular identity of piroplasmids in free-living mammals of the Superorder Xenarthra from four Brazilian states (Mato Grosso do Sul, São Paulo, Rondônia, and Pará). For this, DNA was extracted from blood or spleen samples from 455 animals. A nested PCR based on the 18S rRNA gene was used as screening for piroplasmids. Of the 455 samples analyzed, 25 (5.5%) were positive. Additionally, PCR assays based on 18S rRNA near-complete, cox-1, cox-3, hsp70, cytB, ß-tubulin genes and the ITS-1 intergenic region were performed. Five out of 25 positive samples also tested positive for ITS-1-based PCR. The phylogenetic analysis positioned three 18S rRNA sequences detected in Priodontes maximus into the same clade of Babesia sp. detected in marsupials (Didelphis albiventris, Didelphis marsupialis, and Monodelphis domestica) and Amblyomma dubitatum collected from opossums and coatis in Brazil. On the other hand, the 18S rRNA sequence obtained from Dasypus novemcinctus was closely related to a Theileria sp. sequence previously detected in armadillos from Mato Grosso State, grouping in a subclade within the Theileria sensu stricto clade. In the phylogenetic analysis based on the ITS-1 region, the sequences obtained from Myrmecophaga tridactyla and Tamandua tetradactyla were placed into a single clade, apart from the other piroplasmid clades. The present study demonstrated the molecular occurrence of Piroplasmida in anteaters and Babesia sp. and Theileria sp. in armadillos from Brazil.

A bottom-up proteomics workflow for a system containing multiple organisms.

RATIONALE: Discovery proteomics has been popularized to be essential in the investigator's biological toolbox. Many biological problems involve the interplay of multiple organisms. Herein, a bottom-up proteomics workflow was developed to study a system containing multiple organisms to promote a thorough understanding of how each interacts with the others. METHODS: A label-free quantification proteomics workflow was developed with nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). This protocol describes a bottom-up proteomics workflow used to study differential protein expression in the context of fleas (Ctenocephalides felis felis) experimentally infected by the bacterium Bartonella henselae, the etiological agent of Cat Scratch Disease (CSD). RESULTS: Step-by-step instructions are provided for protein extraction, protein cleanup, total protein measurement, nanoLC-MS/MS data acquisition, and data analysis using Proteome Discoverer software. Comprehensive and exhaustive details are included to promote the adoption of this proteomics workflow in other laboratories. CONCLUSION: A proteomics protocol is detailed for a system containing multiple proteomes from different taxonomic lineages using CSD (cats bitten by fleas infected with Bartonella henselae) as a model. The operating protocol can be readily applied to other label-free proteomics work involving multiple proteomes from taxonomically distinct organisms.

Hepatozoon parasites (Apicomplexa: Hepatozoidae) in fish Hoplias aimara (Characiformes, Erythrinidae) from the Eastern Amazon, Brazil.

This study reports the occurrence of parasites belonging to the Hepatozoon genus in fish Hoplias aimara from the Eastern Amazon. Fish (n = 54) were sampled from the Falsino River, located in the Amapá National Forest (FLONA), in the state of Amapá, northern Brazil. Fresh liver preparations were examined in the field between a slide and a coverslip under a light microscope. Cysts containing Hepatozoon cystozoites were observed in the liver of 5 (9%) out of 54 H. aimara individuals. The cysts were ovoid (mean dimensions 10.28 × 9.8 µm), presenting up to four elongated cystozoites (mean dimensions 11.04 × 1.68 µm), containing 1 to 4 residual bodies of different sizes. A single liver sample containing cysts was submitted to DNA extraction and PCR analyses based on a fragment of the 18S rRNA gene. The sequencing revealed a 465 bp fragment exhibiting 99% query coverage, 0.0 E-value, and 98.7% identity with Hepatozoon caimani (MF322538 and MF322539), detected in caimans (Caiman yacare) from Brazil. This is the first report of the occurrence of cysts containing Hepatozoon cystozoites in free-living fishes.

Piroplasmid infection is not associated with clinicopathological and laboratory abnormalities in cats from Midwestern Brazil.

Feline piroplasmids include the genera Babesia spp., Cytauxzoon spp., and Theileria spp. In Brazil, there are few reports regarding these hemoprotozoans; however, clinicopathological and molecular data are scarce. This study aimed to characterize the clinical relevance of these parasites through hematological, biochemical, and molecular approaches. For this purpose, 166 cats from Brasilia, Federal District, Midwestern Brazil, were screened using a quantitative polymerase chain reaction (qPCR) for piroplasmids based on the LSU4 mitochondrial gene, which resulted in an overall prevalence of 36/166 (21.7%). Twelve of 166 samples (7.2%) were positive for C. felis, while 19/166 (11.4%) were positive for Babesia vogeli. No samples tested positive for Theileria spp. Babesia vogeli and Cytauxzoon spp. LSU4 sequences showed identities of 97-100% and 99.3%, respectively, to US isolates. The hematological and biochemical findings did not differ significantly between the cats that tested positive and negative for piroplasmids. Although the lack of abnormalities in clinical and laboratory parameters does not eliminate the possibility that these cats were sick and recovered, it may suggest that the Brazilian strain of Cytauxzoon spp. is not as pathogenic as that from the USA, despite the high molecular identity with North American isolates.

Sleeping with the enemy: case reports of Ornithonyssus bursa (Berlese, 1888) (Mesostigmata: Macronyssidae) causing human dermatitis in Brazil.

Ornithonyssus bursa, known as the "tropical fowl mite," is a hematophagous mite of domestic and wild birds, which occasionally bites humans. Accidental bites on humans occur mainly when abandoned bird nests are close to homes or when people are handling parasitized birds. In the present study, we describe five case reports of bites on humans and new records of localities for this species. Based on the material examined, we provide morphological and molecular characterizations for this species herein.

Relationships between vector-borne parasites and free-living mammals at the Brazilian Pantanal.

In nature, parasitic infections must be addressed as complex systems involving parasite-host relationships on a temporal and spatial scale. Since the parasites cover a great biological diversity, we can expect that wildlife are exposed simultaneously to different parasites. In this sense, the objective of this work was to determine the relationships between free-living mammals and their associated hemoparasites in the Brazilian Pantanal. We used the data published during 2017 and 2018 by de Sousa et al. regarding the detection of vector-borne pathogens (VBP), namely Anaplasma, Babesia, Bartonella, Cytauxzoon, Ehrlichia, Hepatozoon, Mycoplasma, and Theileria, in nine species of free-living mammals belonging to orders Carnivora, Rodentia, and Didelphimorphia. We assume as infected an individual positive on any of parasitological, molecular, and/or serological tests. We observed a strong association between the wild felid Leopardus pardalis with Cytauxzoon, the wild canid Cerdocyon thous with Hepatozoon, the small rodent Thrichomys fosteri with Bartonella, and the procyonid Nasua nasua with Mycoplasma and Theileria. Therefore, N. nasua, C. thous, T. fosteri, and the small rodent Oecomys mamorae can be considered key species for the maintenance of selected VBP in the Pantanal region, because they showed a high number of single and coinfections. Together, our results highlighted the importance of coinfection as a common phenomenon in nature.

First molecular detection of piroplasmids in non-hematophagous bats from Brazil, with evidence of putative novel species.

Piroplasmida is an order of the phylum Apicomplexa that comprises the Babesia, Cytauxzoon, and Theileria genera. These hemoparasites infect vertebrate blood cells and may cause serious diseases in animals and humans. Even though previous studies have shown that bats are infected by different species of piroplasmids, the occurrence and diversity of these hemoparasites have not been investigated in this group of mammals in Brazil. Therefore, the present work aimed to investigate the occurrence and assess the phylogenetic placement of piroplasmids infecting bats sampled in a peri-urban area from Central-Western Brazil. Seventeen (12.6%) out of 135 animals were positive by nested PCR assay for the detection of Babesia/Theileria targeting the 18S rRNA gene. Eleven sequences of the 17 positive samples could be analyzed and showed an identity of 91.8-100% with Theileria bicornis, Babesia vogeli, a Babesia sp. identified in a small rodent (Thrichomys pachyurus) from the Brazilian Pantanal and a Babesia sp. identified in a dog from Thailand as assessed by nBLAST. A phylogenetic tree was constructed from an alignment of 1399 bp length using analyzed and known piroplasmid 18S rRNA sequences. In this tree, piroplasmid 18S rRNA sequences detected in three specimens of Phyllostomus discolor (Piroplasmid n. sp., P. discolor) were placed as a sister taxon to Theileria sensu stricto (Clade V) and Babesia sensu stricto (Clade VI). An additional phylogenetic tree was generated from a shorter alignment of 524 bp length including analyzed piroplasmid 18S rRNA sequences of bat species Artibeus planirostris and A. lituratus (Piroplasmid sp., Artibeus spp.). The two 18S rRNA sequences detected in Artibeus spp. (Piroplasmid n. sp., Artibeus spp.) were placed within Babesia sensu stricto (Clade VI) into a strongly supported clade (bootstrap: 100) that included Babesia vogeli. The two 18S rRNA sequences of Piroplasmid sp., Artibeus spp. showed a single and a two-nucleotide differences, respectively, with respect to B. vogeli in a 709 pb length alignment. For the first time, the present study shows the occurrence of putative new piroplasmid species in non-hematophagous bats from Brazil.

Molecular detection of piroplasmids in synanthropic rodents, marsupials, and associated ticks from Brazil, with phylogenetic inference of a putative novel Babesia sp. from white-eared opossum (Didelphis albiventris).

The order Piroplasmida encompasses tick-borne pathogens of veterinary and medical importance positioned in two main families: Babesiidae and Theileriidae. Even though previous studies carried out in Brazil recorded the occurrence of piroplasmid species circulating in small mammals, 18S RNA gene sequences were only partially sequenced, preventing the assessment of their phylogenetic positioning. The current study aimed to detect and characterize, using morphological, molecular, and bioinformatic approaches, piroplasmids from wild mammals and associated ticks sampled in Central-Western Brazil. Out of 67 Didelphis albiventris sampled, 22 (16.4%) were positive for piroplasmids by PCR. In contrast, none of the 48 small rodents and 14 capybaras (Hydrochoerus hydrochaeris) was PCR-positive. Four Amblyomma dubitatum ticks-one from Rattus rattus, one from H. hydrochaeris, and two from D. albiventris-out of 114 Amblyomma spp. DNA samples were positive for piroplasmids by PCR. The phylogenetic inference performed using the near-complete 18S rRNA gene positioned the putative novel piroplasmid species detected in D. albiventris and associated A. dubitatum ticks near to Babesia sensu lato clade (Western group-cluster III) and distant from the Australian marsupial-associated piroplasms. Phylogenetic inferences based on two additional molecular markers, namely hsp-70 and cox-1, supported the near-complete 18S rRNA gene phylogenetic inference. Finally, the partial 18S rRNA gene sequences detected in ticks from rodents (R. rattus and H. hydrochaeris) showed 97.2-99.4% identity with the Piroplasmida previously detected in a capybara from Brazil, raising evidence that a still uncharacterized piroplasmid species has been identified in the capybara, the largest rodent species from South America.

Tick-borne zoonotic agents infecting horses from an urban area in Midwestern Brazil: epidemiological and hematological features.

The emergence of tick-borne diseases has been reported as a serious problem in public health worldwide and many aspects of its epidemiology and effects on the health of its hosts are unclear. We aimed to perform an epidemiological study of tick-borne zoonotic Rickettsia, Borrelia, and Anaplasmataceae in horses from Midwestern Brazil. We also evaluated whether Borrelia spp. and Anaplasmataceae may be associated with hematological disorders in the sampled animals. Blood and serum samples as well as ticks were collected from 262 horses. Serum samples were used to perform serological tests, and hematological analyses were made using whole blood. Furthermore, DNA extracted from whole blood and ticks was used for molecular tests. Campo Grande is enzootic for tick-borne studied bacteria, since we found an overall exposure of 59.9% of the sampled horses, 28.7% of them presented co-exposure. Seropositivity rates of 20.6% for Borrelia spp., 25.6% for Rickettsia spp., and 31.6% for Anaplasmataceae were found in the sampled horses. Considering both molecular and serological tests for Borrelia spp., the infection rate was 48.0% (126/262). None of the tested horses showed molecular positivity for Anaplasma phagocytophilum. The horses sampled displayed 7.2% of parasitism by ixodid ticks in single and coinfestations. We did not find DNA of any studied bacteria in the sampled ticks. Positive horses for Borrelia spp. and Anaplasmataceae agents displayed leukopenia, monocytopenia, and lymphopenia. Together, our results suggest that horses may play a role as sentinel host for zoonotic bacteria and Borrelia spp. and Anaplasmataceae agents can impair the health of horses.

New records and genetic diversity of Mycoplasma ovis in free-ranging deer in Brazil.

Cervids represent a mammal group which plays an important role in the maintenance of ecological balance. Recent studies have highlighted the role of these species as reservoirs for several arthropods-borne pathogens. Globally, hemotropic mycoplasmas (haemoplasmas) are emerging or remerging bacteria that attach to red blood cells of several mammals species causing hemolytic anaemia. Therefore, the aim of this study was to investigate the occurrence and assess the phylogenetic positioning of Mycoplasma ovis in free-ranging deer from Brazil. Using a polymerase chain reaction targeting the 16S rRNA region, 18 (40%) out of 45 sampled deer were positive to M. ovis. Among the nine sequences analysed, four distinct genotypes were identified. The sequences detected in the present study were closely related to sequences previously identified in deer from Brazil and the USA. On the other hand, the Neighbour-Net network analysis showed that the human-associated M. ovis genotypes were related to genotypes detected in sheep and goats. The present study shows, for the first time, the occurrence of M. ovis in Mazama gouazoubira and Mazama bororo deer species, expanding the knowledge on the hosts harbouring this haemoplasma species. Once several deer species have your population in decline, additional studies are needed to evaluate the pathogenicity of M. ovis among deer populations around the world and assess its potential as reservoir hosts to human infections.

Molecular detection of Apicomplexan hemoparasites in anurans from Brazil.

Amphibians are among the most threatened vertebrate groups in the world, and the main causes include climate change, habitat destruction, and emerging diseases. Herein, we investigated the occurrence and characterized molecularly Apicomplexa in anurans from southeastern Brazil. Forty individuals from seven anuran species were sampled in São Paulo state. In the molecular analyses, one Leptodactylus latrans and one Rhinella diptycha were positive in PCR assays for species of Hepatozoon. Two L. latrans were also positive for coccidian infections (Lankesterella sp. and an unidentified coccidian species). Phylogenetic analysis based on 18S rDNA clustered the sequences detected in anurans from the present study with Hepatozoon spp. detected in reptiles and other anurans from Brazil, albeit they were separate from Hepatozoon haplotypes detected in frogs from Africa and North America. Our study showed, for the first time, the molecular detection of Lankesterella sp. and another coccidian in L. latrans. Additionally, co-infection by different species of Hepatozoon haplotypes and an unidentified coccidian in anurans from Brazil was documented.

Genetic diversity of Babesia bovis in beef cattle in a large wetland in Brazil.

Babesia bovis is the etiological agent of bovine babesiosis, a disease transmitted by Rhipicephalus microplus, which affects cattle herds in tropical and subtropical regions of the world, causing significant economic losses due to decreasing meat and milk yield. This study used molecular techniques to determine the occurrence and genetic diversity of B. bovis, based on the genes encoding the spherical body protein (sbp-2) and the merozoite surface antigens (MSAs) genes, in a herd of 400 Nellore (Bos indicus) sampled from beef cattle farms in the Pantanal region, state of Mato Grosso do Sul, Midwestern Brazil. The results of the nested PCR assays based on the sbp-2 gene indicated that 18 (4.5%) calves were positive for B. bovis; out of them, while 77.7% (14/18) were positive for the B. bovis msa-2b fragment, 66.6% (12/18) were positive for the msa-2c fragment. The phylogenetic analysis based on the maximum likelihood method using 14 sequences from msa-2b clones and 13 sequences from msa-2c clones indicated that the sequences detected in this study are clearly distributed in different cladograms. These findings corroborated the diversity analysis of the same sequences, which revealed the presence of 14 and 11 haplotypes of the msa-2b and msa-2c genes, respectively. Furthermore, the entropy analyses of amino acid sequences revealed 78 and 44 high entropy peaks with values ranging from 0.25 to 1.53 and from 0.27 to 1.09 for MSA-2B and MSA-2C, respectively. Therefore, the results indicate a low molecular occurrence of B. bovis in beef cattle sampled in the Brazilian Pantanal. Despite this, a high degree of genetic diversity was found in the analyzed B. bovis population, with possibly different haplotypes coexisting in the same animal and/or in the same studied herd.

Pathological and molecular characterization of avian malaria in captive Magellanic penguins (Spheniscus magellanicus) in South America.

Avian malaria is a mosquito-borne disease that affects multiple avian species and is caused by protozoans of the genus Plasmodium. An avian malaria infection caused by Plasmodium sp. in Magellanic penguins (Spheniscus magellanicus) with high mortality is described in a zoo in Southern Brazil. Clinically, three birds presented signs of inappetence, anorexia, pale mucosa, dyspnea, and opisthotonus, with death in a clinical course of 5-8 h. At the necropsy, all birds exhibited pale mucosa, marked splenomegaly and hepatomegaly, in addition to moderate leptomeningeal blood vessels ingurgitation in the brain. Microscopically, multiple exoerythrocytic meronts were observed in the cytoplasm of endothelial cells in the spleen, liver, heart, lungs, brain, kidneys, and pancreas. The spleen had a multifocal perivascular inflammatory infiltrate of lymphocytes, plasma cells, and macrophages, which also exhibited hemosiderosis and erythrophagocytosis. The liver had a multifocal periportal inflammatory infiltrate of lymphocytes, macrophages, and plasma cells, in addition to marked hemosiderosis in the hepatic sinusoids. Fragments of spleen, liver, brain, skeletal muscle, and lung were tested by the polymerase chain reaction technique for the detection of a fragment of the cytochrome B gene from haemosporidians, which resulted positive for Plasmodium spp. After sequencing, the samples were phylogenetically associated to Plasmodium sp. detected in Turdus albicollis (KU562808) in Brazil and matched to the lineage TURALB01 previously detected in T. albicollis. Avian malaria infections caused by Plasmodium sp. of lineage TURALB01 may occur in S. magellanicus with high mortality, and, thus, it is essential to detect and characterize the agent involved to obtain the differential diagnosis of the condition.

Genetic Diversity of Bartonella spp. in Wild Mammals and Ectoparasites in Brazilian Pantanal.

The present work aimed to investigate the genetic diversity of Bartonella in mammals and ectoparasites in Pantanal wetland, Brazil. For this purpose, 31 Nasua nasua, 78 Cerdocyon thous, 7 Leopardus pardalis, 110 wild rodents, 30 marsupials, and 42 dogs were sampled. DNA samples were submitted to a quantitative real-time PCR assay (qPCR). Positive samples in qPCR were submitted to conventional PCR assays targeting other five protein-coding genes. Thirty-five wild rodents and three Polygenis (P.) bohlsi bohlsi flea pools showed positive results in qPCR for Bartonella spp. Thirty-seven out of 38 positive samples in qPCR were also positive in cPCR assays based on ftsZ gene, nine in nuoG-cPCR, and six in gltA-cPCR. Concatenated phylogenetic analyses showed that two main genotypes circulate in rodents and ectoparasites in the studied region. While one of them was closely related to Bartonella spp. previously detected in Cricetidae rodents from North America and Brazil, the other one was related to Bartonella alsatica, Bartonella pachyuromydis, Bartonella birtlesii, Bartonella acomydis, Bartonella silvatica, and Bartonella callosciuri. These results showed that at least two Bartonella genotypes circulate among wild rodents. Additionally, the present study suggests that Polygenis (P.) bohlsi bohlsi fleas could act as possible Bartonella vectors among rodents in Pantanal wetland, Brazil.