Genetic diversity of Bartonella rpoB haplotypes in domestic cats from Chile.

The study aimed to determine the inter and intra-host Bartonella spp. genetic diversity in cats from Chile. 'Seventy-nine cats' blood DNA samples qPCR Bartonella spp. positive were subjected to T-A cloning of Bartonella spp. rpoB partial gene (825 bp), and sequencing by Sanger method. The sequences were submitted to phylogenetic and polymorphism analysis. Thirty-six (45.6%) samples were successfully cloned, generating 118 clones of which 109 showed 99.6%-100% identity with Bartonella henselae whereas 9 showed 99.8-100% identity with Bartonella koehlerae. Haplotype analysis yielded 29 different rpoB-B. henselae haplotypes, one (hap#2) overrepresented in 31 out of 33 cats, and 4 rpoB-B. koehlerae haplotypes, with hap#2 represented in all 3 B. koehlerae infected cats. More than one rpoB -B. henselae and B. koehlerae haplotypes were identified in individual cats, reporting by first time coinfection by different B. henselae/B. koehlerae rpoB variants in cats from Chile.

First Report of Bartonella spp. in Marsupials from Brazil, with a Description of Bartonella harrusi sp. nov. and a New Proposal for the Taxonomic Reclassification of Species of the Genus Bartonella.

The genus Bartonella (Rhizobiales: Bartonellaceae) encompasses facultative intracellular Gram-negative alphaproteobacteria that parasitize mainly erythrocytes and endothelial cells, as well as macrophages, monocytes and dendritic cells. Although they can infect numerous mammal species and arthropod vectors worldwide, reports of Bartonella infections in marsupials are scarce. In fact, such agents have only been detected in marsupials and/or associated ectoparasites in Australia and the United States of America until the present moment. The present study aimed to isolate and characterize molecularly, morphologically and phenotypically Bartonella infecting free-living marsupials sampled in the Brazilian Pantanal, the largest wetland in South America. Two marsupials were captured in December 2018 and six marsupials in February 2019, totaling eight small mammals sampled: five (62.5%) Thylamys macrurus and three (37.5%) Monodelphis domestica. All blood samples were submitted to qPCR for Bartonella spp. based on the nuoG gene, a pre-enrichment liquid culture and a chocolate agar solid culture. Bartonella sp. was isolated from 3 T. macrurus and one M. domestica. One Bartonella isolate obtained from a T. macrurus blood sample (strain 117A) that showed to be closely related to the Bartonella vinsonii complex and Bartonella machadoae was selected for whole genome sequencing using a hybrid approach based on Illumina NovaSeq and Nanopore sequencing platforms. This strain showed a genome of 2.35 Mbp, with an average C + G content of 38.8%, coding for 2013 genes, and a 29 kb plasmid with an average C + G content of 34.5%. In addition, this strain exhibited an average nucleotide identity (ANI) of 85% with Bartonella species belonging to the B. vinsonii group and 91% with B. machadoae. Phylogenomic analysis based on 291 protein coding genes shared by the genomes of 53 Bartonella species positioned this strain closely to B. machadoae. This new isolated species was named Bartonella harrusi sp. nov., which was characterized as having small capnophilic, microaerophilic and aerobic rods with an absence of pili and flagella. In conclusion, the present work describes the biochemical, phenotypic and genomic characteristics of Bartonella harrusi, a new species isolated from the T. macrurus blood samples of the Brazilian Pantanal. Finally, a review of the taxonomic classification of members of the genus Bartonella is proposed, based on the ANI values accessed by whole genome sequencing analyses.

Threat under cats' claws: Molecular detection and risk factors for zoonotic Bartonella species in blood and claw samples from cats in Brazil.

Bartonelloses are zoonoses widely dispersed throughout the world caused by bacteria of the genus Bartonella. Domestic cats play an important role in the epidemiology of bartonelloses, since these animals are considered natural hosts of B. henselae, B. koehlerae and B. clarridgeiae. This study aimed to determine the occurrence of Bartonella spp. in domestic cats' blood and claw samples in the southern region of Bahia, northeastern Brazil. Additionally, the main clinical and hematological changes in Bartonella-positive animals were investigated, as well as the risk factors associated with the infection. For this purpose, 188 indoor house domestic cats were clinically evaluated and submitted to claw and blood sample collection. Additionally, data regarding the clinical history of the animals were recorded. Out of 188 cats' blood samples, 20.7% (39/188) were positive in the qPCR for Bartonella spp. based on the nuoG gene. Out of 39 claw samples collected, 23.9% (9/39) were positive for Bartonella spp. The parameters of the blood and claw samples ranged from 1.42 to 395,000 and 4.32 - 108,000 copies/µL of a fragment of Bartonella nuoG gene, respectively. The amplified sequences shared identity ranging from 99% to 100% with the three main cat-related Bartonella species. Higher platelet values (p = 0.0082) were observed in animals positive for Bartonella spp. Young and unsterilized cats with outdoor access were more prone to infection by Bartonella spp. The data reported here demonstrated the occurrence of Bartonella spp. in blood and claw samples from cats in northeastern Brazil showing no significant clinical and hematological disorders.

Bartonella machadoae sp. nov. isolated from wild rodents in the Pantanal wetland.

It has been estimated that 75% of emerging infectious diseases comprise zoonoses, whose majority have free-living animals as reservoirs and are mainly transmitted by arthropod vectors. Although rodents represent important Bartonella reservoirs, there are few studies on the genotypic characterization of Bartonella species commonly found in this taxon and from different Brazilian biomes. Therefore, the present study aimed to investigate the occurrence, isolate and molecularly, morphologically and phenotypically characterize a new Bartonella species infecting free-living rodents sampled in the Brazilian Pantanal, the largest wetland in South America. For this purpose, 129 free-living rodents (79 Thrichomys fosteri, 4 Clyomys laticeps, and Oecomys mamorae) were captured. While blood samples were collected from 57 T. fosteri, 4 C. laticeps and 32 O. mamorae; spleen samples were collected from 22 T. fosteri and 14 O. mamorae. Blood and spleen samples were submitted to a qPCR for Bartonella spp. targeting the nuoG gene, using DNA samples extracted directly from blood/spleen, after passage in pre-enrichment liquid culture, and from colonies obtained from solid culture on chocolate agar. Combining all techniques, occurrence of 24.8% for Bartonella sp. was found among the sampled rodents. One Bartonella isolate (strain 56A) obtained from a T. fosteri's blood sample was closely related to the Bartonella vinsonii complex and selected for Whole Genome Sequencing (WGS) hybrid approach using Illumina NovaSeq and Nanopore sequencing platforms. This strain exhibits a circular 2.7 Mbp genome with an average C+G content of 39% and encoding to 2239 genes. In the phylogenomics based on 291 shared protein-coding genes, this strain was positioned in a unique clade, closely related to Bartonella vinsonii subsp. vinsonii, B. vinsonii subsp. berkhoffii and B. visonii subsp. arupensis. An Average Nucleotide Identity of 85% was found between the obtained isolate and Bartonella species belonging to B. vinsonii complex. These findings supported the separation of this strain, now formally named as Bartonella machadoae sp. nov., from the Bartonella vinsonii complex. In addition, Bartonella machadoae sp. nov. was characterized by capnophilic, microaerophilic and aerobic small rods with absence of pili and flagella. Phylogenetic and distance analyses based on five concatenated molecular markers suggest that Bartonella machadoae may parasite rodents from different Brazilian biomes. In conclusion, we described biochemical, phenotypic and genomic characteristics of Bartonella machadoae nov. sp. isolated from blood samples of T. fosteri rodents from the Brazilian Pantanal.

Molecular Detection of Tick-Borne Agents in Cats from Southeastern and Northern Brazil.

Even though the epidemiology of tick-borne agents (TBA) in dogs has been extensively investigated around the world, the occurrence, vectors involved, and molecular identity of these agents in cats remains elusive in many regions. Among TBA, Ehrlichia, Anaplasma, Babesia, Cytauxzoon, and Hepatozoon are responsible for diseases with non-specific clinical signs in cats, making essential the use of molecular techniques for accurate diagnosis and proper treatment. The present work aimed to investigate the occurrence and molecular identity of tick-borne agents (Ehrlichia, Anaplasma, Babesia/Theileria, Cytauxzoon, and Hepatozoon) in cats from southeastern (states of São Paulo (SP) and Minas Gerais (MG)) and northern (state of Rondônia (RO)) Brazil. For this purpose, 390 blood samples were collected from domiciled cats in MG (n = 155), SP (n = 151), and RO(n = 84) states, submitted to DNA extraction and PCR assays for Ehrlichia spp. (dsb gene), Anaplasma spp. (rrs gene), piroplasmids (18S rRNA gene), and Hepatozoon spp. (18S rRNA gene), sequencing, and phylogenetic inferences. The overall positivity for Anaplasma spp., Ehrlichia spp., Babesia/Theileria spp., Cytauxzoon spp., and Hepatozoon spp. were 7.4% (12.3% (MG) and 6.6% (SP)), 2% (4.5% (MG) and 0.6% (SP)), 0.7% (0.6% (MG), 0.6% (SP) and 1.2% (RO)), 27.2% (41.9% (MG), 24.5% (SP) and 4.8% (RO), and 0%, respectively. The phylogenetic analysis grouped the obtained sequences with 'Candidatus Anaplasma amazonensis', A. platys, B. vogeli, and Cytauxzoon sp. previously detected in wild felids from Brazil. qPCR specific for E. canis based on the dsb gene confirmed the molecular identity of the detected ehrlichial agent. The present study expanded the list and geographical distribution of hemoparasites in cats. 'Candidatus Anaplasma amazonensis', recently detected in sloths from northern Brazil, was described for the first time in cats. This is the first report of piroplasmids infecting cats in northern Brazil. Coinfection by Cytauxzoon and other TBA (Ehrlichia, Anaplasma, and B. vogeli) reported in the present study raises the need for veterinary practitioners' awareness of cats parasitized by multiple TBA.

Genetic diversity and Multilocus Sequence Typing Analysis of Bartonella henselae in domestic cats from Southeastern Brazil.

Bartonella henselae is the causative agent for the infectious disease Cat Scratch Disease (CSD), which can be fatal. Domestic and wild felines are known to be its main mammal reservoirs. The present study aimed to investigate the occurrence and genetic diversity of Bartonella spp. in cats sampled in São Paulo (SP) and Minas Gerais (MG) States, Southeastern Brazil. Based on a quantitative real-time PCR (qPCR) assay, a Bartonella sp. nuoG gene fragment was detected in 39.9% (122/306) of the blood samples (46/151 cats of SP; 76/155 cats of MG). The blood samples were submitted to a pre-enrichment culture technique that allowed the detection of 12 additional positive samples, which showed to be negative in the qPCR using DNA blood samples as templates. Furthermore, five B. henselae isolates were obtained from qPCR-negative samples for both blood and pre-enrichment culture. Seven out of 24 Ctenocephalides felis fleas were positive for Bartonella spp. in the qPCR assay; 4/7 positive fleas were collected from Bartonella-negative cats. Twenty-three rpoB B. henselae cloned sequences were obtained from nine cats' blood samples, showing the occurrence of 13 different genotypes. Median-joining network and SplitsTree distance analysis showed that the obtained sequences represented distinct B. henselae genotypes when compared to those previously deposited in GenBank. Intra-host diversity was found, since different rpoB genotypes of B. henselae were detected in individual single cats. Bartonella henselae isolates showed two allelic profiles (ST37 in cats from MG state and ST9 in SP state) by MLST (Multilocus Sequence Typing) based on sequencing of eight molecular markers. The present study is the first molecular report of Bartonella sp. in cats from Minas Gerais State. In summary, this body of work showed the occurrence of different B. henselae rpoB genotypes at an intra-reservoir host level. Based on qPCR from blood samples and pre-enrichment liquid culture and isolation, occurrence of 33.1% (50/151) and 56.8% (88/155) for Bartonella sp. was found in cats from SP and MG states, respectively. Two different allelic profiles of B. henselae were found in cats from the states of São Paulo (ST9) and Minas Gerais (ST37), suggesting a clonal evolution of Bartonellae in a certain geographical region.

Frequency of antibodies and risk factors associated with Toxoplasma gondii infection in backyard pig production in the city of Mossoró, state of Rio Grande do Norte, Brazil / Frequência de anticorpos e fatores de risco associados à infecção por Toxoplasma gondii em criações de suínos em Mossoró, Rio Grande do Norte

Abstract Toxoplasmosis is an important zoonosis for pregnant women and immunosuppressed people. The pig population also becomes infected by this pathogen, and undercooked or raw meat is an important source of infection for humans. The aims of the present study were to evaluate the rate of exposure of pigs to T. gondii in the municipality of Mossoró, Rio Grande do Norte and seek to identify associations with possible risk factors. Blood samples were collected from 412 pigs and were analyzed using the immunofluorescence assay. Among these 412 serum samples, 40.7% were seropositive for T. gondii. The IgG antibody titers were 64 (56 specimens), 128 (32), 256 (37), 512 (23), 1024 (14), 2048 (5) and 4046 (1). Seropositivity for T. gondii was found to be related (p-value < 0.05) to the following factors: female gender, semi-confined rearing system, use of well water, dewormed animals, presence of cats, goats, sheep, mice and vultures on the farm and carcasses left on the ground. In contrast, seropositivity was not related (p-value < 0.05) to the age of the pigs, type of facility or feeding with human food remains. Preventive measures need to be adopted on the farms studied here, with the aim of decreasing the animals' intake of sporulated oocysts.

Resumo A toxoplasmose é uma importante zoonose para mulheres grávidas e pessoas imunossuprimidas. Os suínos também são infectados por este patógeno; e a carne crua ou malcozida é uma importante fonte de infecção para o ser humano. O objetivo do presente trabalho foi avaliar a taxa de exposição suína à T. gondii no município de Mossoró, Rio Grande do Norte e identificar associações com possíveis fatores de risco. Amostras de sangue foram coletadas em tubos sem anticoagulante e o soro foi separado de 412 porcos e analisadas pelo ensaio de imunofluorescência. Dentre as 412 amostras de soro, 40,7% foram positivas para T. gondii. Os títulos para o anticorpo IgG foram 64 (56 amostras); 128 (32); 256 (37); 512 (23); 1024 (14); 2048 (5) e 4046 (1). A soropotividade para T. gondii foi relacionada (p-valor < 0,05) aos seguintes fatores: gênero feminino, sistema de criação de semi-confinamento, uso de água de poço, animais vermifugados, presença de gatos, cabras, ovelhas, ratos e urubus na fazenda e carcaças deixadas à céu aberto. Em contrapartida, a soropositividade não foi relacionada (p-valor < 0,05) à idade dos suínos, tipo de instalações ou alimentação com restos de comida humana. As adoções de medidas preventivas são necessárias na propriedade estudada, objetivando diminuir a ingestão de oocistos esporulados pelos animais.

Frequency of antibodies and risk factors associated with Toxoplasma gondii infection in backyard pig production in the city of Mossoró, state of Rio Grande do Norte, Brazil.

Toxoplasmosis is an important zoonosis for pregnant women and immunosuppressed people. The pig population also becomes infected by this pathogen, and undercooked or raw meat is an important source of infection for humans. The aims of the present study were to evaluate the rate of exposure of pigs to T. gondii in the municipality of Mossoró, Rio Grande do Norte and seek to identify associations with possible risk factors. Blood samples were collected from 412 pigs and were analyzed using the immunofluorescence assay. Among these 412 serum samples, 40.7% were seropositive for T. gondii. The IgG antibody titers were 64 (56 specimens), 128 (32), 256 (37), 512 (23), 1024 (14), 2048 (5) and 4046 (1). Seropositivity for T. gondii was found to be related (p-value < 0.05) to the following factors: female gender, semi-confined rearing system, use of well water, dewormed animals, presence of cats, goats, sheep, mice and vultures on the farm and carcasses left on the ground. In contrast, seropositivity was not related (p-value < 0.05) to the age of the pigs, type of facility or feeding with human food remains. Preventive measures need to be adopted on the farms studied here, with the aim of decreasing the animals' intake of sporulated oocysts.