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.

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.

Serological exposure of spotted fever group Rickettsia in capybaras (Hydrochoerus hydrochaeris) from urban parks in Campo Grande, Brazilian Midwest.

BACKGROUND: Rickettsia of the spotted fever group (SFG) has been reported in ticks and domestic animals in Campo Grande (CG), Midwest Brazil. METHODS: We searched for Rickettsia in the SFG in capybaras and their ticks in an urban park in the CG. RESULTS: The seropositivity rate was 88.2% (15/17). Although 87.7% of the capybaras sampled showed infestations with Amblyomma sculptum, A. dubitatum, and Amblyomma spp., no molecular results were detected in ticks. CONCLUSIONS: Since Rickettsia from the SFG circulates among capybaras in the urban parks of Campo Grande, this large rodent species should be monitored within the One Health Agenda.

Genetic diversity and lack of molecular evidence for hemoplasma cross-species transmission between wild and synanthropic mammals from Central-Western Brazil.

Globally, hemotropic mycoplasmas (hemoplasmas) comprise an emerging or remerging bacteria group that attaches to red blood cells of several mammal's species and in some cases, causing hemolytic anemia. Herein, we assessed the occurrence, genetic diversity, the factors coupled to mammals infection, and the phylogeographic distribution of hemoplasmas in sylvatic and synanthropic mammals and their associated ectoparasites from Brazil. We collected spleen and/or blood samples from synanthropic rodents (Rattus rattus [N = 39] and Mus musculus [N = 9]), sylvatic rodents (Hydrochoerus hydrochaeris [N = 14]) and opossums (Didelphis albiventris [N = 43]). In addition, ticks (Amblyomma spp. [N = 270] and lice (Polyplax spinulosa [N = 6]) specimens were also sampled. Using a PCR targeting the 16S rRNA region, out of 48 small rodents, 14 capybaras and 43 opossums DNA samples, hemoplasma DNA was found in 25%, 50%, and 32.5% animals, respectively. Besides, we reported hemoplasma DNA in Amblyomma sp. (22.2% [2/9]) and lice (100% [2/2]) pools samples from rats, and one female A. sculptum DNA sample (3% [1/33]) obtained from a capybara. Additionally, and in agreement with ML analysis, the network analyses showed a clear phylogenetic separation among the hemoplasmas genotypes found in the different host species sampled, thus, suggesting the absence of cross-species hemoplasmas transmission between the mammals trapped. Finally, using the NTC network analysis, we reported the same 16S rRNA Mycoplasma genotype circulating in Rattus sampled in Brazil, Hungary, and Japan.

Serological occurrence for tick-borne agents in beef cattle in the Brazilian Pantanal.

This study investigated the seropositivity for five different tick-borne agents, namely Anaplasma marginale, Babesia bovis, Babesia bigemina, Coxiella burnetii, Anaplasma phagocytophilum, and Trypanosoma vivax in beef cattle in the Brazilian Pantanal. The serum samples collected from animals (200 cows; 200 calves) were used in indirect enzyme-linked immunosorbent assays (iELISA) to detect IgG antibodies against A. marginale, B. bovis, B. bigemina, and T. vivax, and Indirect Fluorescent Antibody Test (IFAT) for detecting IgG antibodies against C. burnetii and A. phagocytophilum. No correlation was observed between seropositivity for C. burnetii and A. phagocytophilum with other agents whereas moderate correlation was observed for A. marginalexB. bigemina x B. bovis. Cows were more seropositive for T. vivax whereas calves were more seropositive for B. bovis and B. bigemina. The highest number of seropositive animals by a single agent was observed for T. vivax (15.2%). Co-seropositivity for T. vivax + A. marginale was higher in cows (25.5%) and for T. vivax + B. bovis + B. bigemina + A. marginale was higher in calves (57.5%). The high seropositivity correlation for A. marginale x B. bovis x B. bigemina is probably due to the presence of the tick biological vector, Rhipicephalus microplus, in the studied farms. Common transmission pathways, mediated by hematophagous dipterans and fomites, may explain the high co-seropositivity of cows for A. marginale and T. vivax. Low seropositivity to C. burnetii is probably due to the type of breeding system employed (extensive). Seropositivity for A. phagocytophilum in only one animal suggests the occurrence of a cross-serological reaction with another agent of the genus Anaplasma.

Molecular detection and genetic diversity of Bartonella species in large ruminants and associated ectoparasites from the Brazilian Cerrado.

Currently, five Bartonella species and an expanding number of Candidatus Bartonella species have globally been reported in ruminants. Likewise, different Bartonella genotypes were identified. However, studies relating to ruminant-associated Bartonella in Brazil are scarce. The current study aimed to assess the prevalence and genetic diversity of Bartonella in cattle, buffaloes and associated ectoparasites in Brazil. For this purpose, EDTA-blood samples from 75 cattle and 101 buffaloes were sampled. Additionally, 128 Rhipicephalus microplus and one Amblyomma sculptum ticks collected from cattle, and 197 R. microplus, one A. sculptum and 170 lice (Haematopinus tuberculatus) collected from buffaloes were included. Bartonella DNA was initially screened through an HRM real-time PCR assay targeting the 16S-23S internal transcribed spacer (ITS), and the positive samples were submitted to an additional HRM assay targeting the ssrA gene. The HRM-positive amplicons were sequenced, and the nucleotide identity was assessed by BLASTn. Bartonella spp.-positive DNA samples were analysed by conventional PCR assays targeting the gltA and rpoB genes, and then, the samples were cloned. Finally, the phylogenetic positioning and the genetic diversity of clones were assessed. Overall, 21 of 75 (28%) cattle blood samples and 13 of 126 (10.3%) associated ticks were positive for Bartonella bovis. Out of 101 buffaloes, 95 lice and 188 tick DNA samples, one (1%) buffalo and four (4.2%) lice were positive for Bartonella spp. Conversely, none of the ticks obtained from buffaloes were positive for Bartonella. The Bartonella sequences from buffaloes showed identity ranging from 100% (ITS and gltA) to 94% (ssrA) with B. bovis. In contrast, the Bartonella DNA sequences from lice were identical (100%) to uncultured Bartonella sp. detected in cattle tail louse (Haematopinus quadripertusus) from Israel in all amplified genes. The present study demonstrates the prevalence of new B. bovis genotypes and a cattle lice-associated Bartonella species in large ruminants and their ectoparasites from Brazil. These findings shed light on the distribution and genetic diversity of ruminant- and ectoparasite-related Bartonella in Brazil.

Low occurrence of Bartonella in synanthropic mammals and associated ectoparasites in peri-urban areas from Central-Western and Southern Brazil.

Worldwide, Bartonella species are known to infect a wide range of mammalian and arthropod hosts, including humans. The current study aimed to investigate the prevalence of Bartonella spp. in synanthropic mammals captured in peri-urban areas from Central-Western and Southern Brazil and their ectoparasites. For this aim, 160 mammals belonging to four species, and 218 associated arthropods were sampled. DNA was extracted and subjected to different Bartonella screening assays. Additionally, blood samples from 48 small rodents were submitted to liquid BAPGM culture followed by qPCR assay and solid culture. Two out of 55 Rattus captured in Santa Catarina state were PCR-positive for Bartonella when targeting the nuoG, 16S, and ITS loci. Sequences showed high homology with Bartonella coopersplainsensis. Conversely, all 48 small rodents, 14 capybaras and 43 opossum DNA samples from animals trapped in Mato Grosso do Sul were Bartonella negative in the HRM real time PCR assays targeting the ITS locus and gltA gene. Additionally, all mammal-associated ectoparasites showed negativity results based on HRM real time PCR assays. The present study showed, for the first time, the occurrence of B. coopersplainsensis in Brazil, shedding some light on the distribution of rats-related Bartonella in South America. In addition, the majority of rodents and marsupials were negative for Bartonella spp. Since B. coopersplainsensis reservoirs - Rattus spp. - are widely dispersed around the globe, their zoonotic potential should be further investigated.

Detection of Brucella spp. in dogs at Pantanal wetlands.

Canine brucellosis is an infectious disease that produces reproductive disease in both males and females. Although Brucella canis is more common, the infection by Brucella abortus is more frequent in dogs sharing habitats with livestock and wild animals. We decided to investigate the role of dogs in the maintenance of Brucella spp. in the Pantanal wetland. Serum and whole blood samples were collected from 167 dogs. To detect antibodies against B. abortus and B. canis, buffered acidified plate antigen (BAPA) and agar gel immunodiffusion (AGID) tests were performed. To detect Brucella spp., B. abortus and B. canis DNA, PCR was performed using the bcsp31, BruAb2_0168, and BR00953 genes, respectively. To confirm the PCR results, three bcsp31 PCR products were sequenced and compared with sequences deposited in GenBank. The seropositivity rates of 7.8% and 9% were observed for the AGID and BAPA tests, respectively. Positivity rates of 45.5% and 10.8% were observed when testing bcsp31 and BruAb2_0168, respectively, while there was no positivity for BR00953. The sequenced products had 110 base pairs that aligned with 100% identity to B. abortus, B. canis, and B. suis. Considering our results, dogs may be acting as maintenance hosts of Brucella spp. in the Pantanal region.

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.

Phylogeography of msp4 genotypes of Anaplasma marginale in beef cattle from the Brazilian Pantanal.

The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1ß gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.

Genetic diversity of Anaplasma marginale in beef cattle in the Brazilian Pantanal.

There are few studies on the genetic diversity of Anaplasma marginale in Brazilian cattle herds, especially about beef cattle. The objective of this study was to evaluate the genetic diversity of A. marginale, based on the msp1α gene in Bos taurus indicus sampled from the Brazilian Pantanal. Aliquots of blood with and without EDTA were taken from 400 cattle (200 cows and 200 calves) across five extensive farms. The samples were submitted to the indirect immunoenzymatic assay (iELISA), quantitative real-time PCR (qPCR) for the msp1ß gene and to the semi-nested (sn) PCR for the msp1α gene. Positive samples were sequenced by the Sanger method and subjected to diversity analysis using the RepeatAnalyser software. The percentage of positive animals by iELISA, qPCR and (sn) PCR was 72.2% (289/400), 56.7% (227/400) and 23% (52/227), respectively. Cows (154/200) showed to be significantly more seropositive than calves (135/200). In qPCR, the number of calves and average quantification value (138/200; 1.3 × 106) A. marginale msp1α copies per µL proved to be higher when compared to that found for the cows (89/200; 3.9 × 104). The microsatellite analysis of the 26 sequences obtained from the msp1α gene revealed the presence of E (77%), C (15.4%) and B (7.7%) genotypes. Fourteen A. marginale strains were identified in the studied region, with eight that have never before been described in the literature (τ-10-13-13-18; τ-27-18; EV8-EV8-17; α-ß-ß-ß-100; EV7-11-10-15; τ-11-11-27-18; τ-11-10-15; τ-27-13-18). Beef cattle are highly exposed to A. marginale in the Brazilian Pantanal. Moreover, a high genetic diversity of A. marginale, with eight new strains, was found in the studied region. While cows may act as chronic carriers, perpetuating the pathogen within the herd, male beef calves sold to other regions may disperse these strains.

Maintenance of Trypanosoma cruzi, T. evansi and Leishmania spp. by domestic dogs and wild mammals in a rural settlement in Brazil-Bolivian border.

Domestic dogs are considered reservoirs hosts for several vector-borne parasites. This study aimed to evaluate the role of domestic dogs as hosts for Trypanosoma cruzi, Trypanosoma evansi and Leishmania spp. in single and co-infections in the Urucum settlement, near the Brazil-Bolivian border. Additionally, we evaluated the involvement of wild mammals' in the maintenance of these parasites in the study area. Blood samples of dogs (n = 62) and six species of wild mammals (n = 36) were collected in July and August of 2015. The infections were assessed using parasitological, serological and molecular tests. Clinical examination of dogs was performed and their feeding habits were noted. Overall, 87% (54/62) of sampled dogs were positive for at least one trypanosomatid species, in single (n = 9) and co-infections (n = 45). We found that 76% of dogs were positive for T. cruzi, four of them displayed high parasitemias demonstrated by hemoculture, including one strain types TcI, two TcIII and one TcIII/TcV. Around 73% (45/62) of dogs were positive to T. evansi, three with high parasitemias as seen by positive microhematocrit centrifuge technique. Of dogs sampled, 50% (31/62) were positive for Leishmania spp. by PCR or serology. We found a positive influence of (i) T. evansi on mucous pallor, (ii) co-infection by T. cruzi and Leishmania with onychogryphosis, and (iii) all parasites to skin lesions of sampled dogs. Finally, feeding on wild mammals had a positive influence in the Leishmania spp. infection in dogs. We found that 28% (5/18) coati Nasua nasua was co-infected for all three trypanosamatids, demonstrating that it might play a key role in maintenance of these parasites. Our results showed the importance of Urucum region as a hotspot for T. cruzi, T. evansi and Leishmania spp. and demonstrated that dogs can be considered as incidental hosts.

Serological exposure of spotted fever group Rickettsia in capybaras (Hydrochoerus hydrochaeris) from urban parks in Campo Grande, Brazilian Midwest

ABSTRACT Background: Rickettsia of the spotted fever group (SFG) has been reported in ticks and domestic animals in Campo Grande (CG), Midwest Brazil. Methods: We searched for Rickettsia in the SFG in capybaras and their ticks in an urban park in the CG. Results: The seropositivity rate was 88.2% (15/17). Although 87.7% of the capybaras sampled showed infestations with Amblyomma sculptum, A. dubitatum, and Amblyomma spp., no molecular results were detected in ticks. Conclusions: Since Rickettsia from the SFG circulates among capybaras in the urban parks of Campo Grande, this large rodent species should be monitored within the One Health Agenda.

Serological occurrence for tick-borne agents in beef cattle in the Brazilian Pantanal / Ocorrência sorológica para agentes transmitidos por carrapatos em bovinos de corte no Pantanal Brasileiro

Abstract This study investigated the seropositivity for five different tick-borne agents, namely Anaplasma marginale, Babesia bovis, Babesia bigemina, Coxiella burnetii, Anaplasma phagocytophilum, and Trypanosoma vivax in beef cattle in the Brazilian Pantanal. The serum samples collected from animals (200 cows; 200 calves) were used in indirect enzyme-linked immunosorbent assays (iELISA) to detect IgG antibodies against A. marginale, B. bovis, B. bigemina, and T. vivax, and Indirect Fluorescent Antibody Test (IFAT) for detecting IgG antibodies against C. burnetii and A. phagocytophilum. No correlation was observed between seropositivity for C. burnetii and A. phagocytophilum with other agents whereas moderate correlation was observed for A. marginalexB. bigemina x B. bovis. Cows were more seropositive for T. vivax whereas calves were more seropositive for B. bovis and B. bigemina. The highest number of seropositive animals by a single agent was observed for T. vivax (15.2%). Co-seropositivity for T. vivax + A. marginale was higher in cows (25.5%) and for T. vivax + B. bovis + B. bigemina + A. marginale was higher in calves (57.5%). The high seropositivity correlation for A. marginale x B. bovis x B. bigemina is probably due to the presence of the tick biological vector, Rhipicephalus microplus, in the studied farms. Common transmission pathways, mediated by hematophagous dipterans and fomites, may explain the high co-seropositivity of cows for A. marginale and T. vivax. Low seropositivity to C. burnetii is probably due to the type of breeding system employed (extensive). Seropositivity for A. phagocytophilum in only one animal suggests the occurrence of a cross-serological reaction with another agent of the genus Anaplasma.

Resumo Este estudo teve como objetivo determinar a co-soropositividade para agentes transmitidos por carrapatos, como Anaplasma marginale, Babesia bovis, Babesia bigemina, Coxiella burnetii, Anaplasma phagocytophilum, e Trypanosoma vivax em bovinos de corte do Pantanal Brasileiro. Amostras de soro foram colhidas de 400 animais (200 vacas; 200 bezerros) e submetidas a Ensaios Imunoenzimáticos Indiretos (iELISA) para detecção de anticorpos IgG anti- A. marginale, anti- B. bovis, anti- B. bigemina e anti- T. vivax, e à Reação de Imunofluorescência Indireta (RIFI) para detecção de anticorpos IgG anti -C. burnetii e anti- A. phagocytophilum. Ausência de correlação foi vista entre os animais soropositivos para C. burnetii e A. phagocytophilum com os outros agentes e correlação moderada ocorreu entre A. marginale x B. bigemina x B. bovis. Vacas foram mais soropositivas que bezerros para T. vivax e bezerros mais soropositivos que vacas para B. bovis e B. bigemina. Maior número de animais soropositivos para um único agente foi visto para T. vivax (15,2%). Vacas demonstraram maior co-soropositividade para T. vivax + A. marginale (25,5%) e bezerros para T. vivax + B. bovis + B. bigemina + A. marginale (57,5%). A alta correlação entre a soropositividade para A. marginale x B. bovis x B. bigemina é provavelmente devida à presença do vetor biológico, o carrapato Rhipicephalus microplus, nas fazendas estudadas. As vias de transmissão comuns, mediadas por dípteros hematófagos e fômites, podem explicar a alta co-soropositividade das vacas para A. marginale e T. vivax. A baixa soropositividade para C. burnetii é provavelmente devida ao tipo de sistema de criação empregado (extenso). A soropositividade para A. phagocytophilum em apenas um animal sugere a ocorrência de reação sorológica cruzada com outro agente do gênero Anaplasma.

Serological detection of Toxoplasma gondii, Leishmania infantum and Neospora caninum in cats from an area endemic for leishmaniasis in Brazil.

An investigation was made into the occurrence of antibodies to Toxoplasma gondii, Leishmania infantum and Neospora caninum in 151 domestic cats, based on the indirect fluorescent antibody test (IFAT). Serum samples were collected from 151 domestic cats (65 free-roaming and 86 domiciled cats; 55 males and 96 females) in Campo Grande, Mato Grosso do Sul, Brazil between January and April 2013. IgG antibodies to T. gondii, L. infantum and N. caninum were found, respectively, in 49 (32.5%), 34 (22.5%) and 10 (6.6%) sampled cats. A positive correlation was found between T. gondii and N. caninum, T. gondii and L. infantum, and N. caninum and L. infantum (p <0.05) infections. Also, a significant interaction was identified between gender and area of activity on the probability of T. gondii (p = 0.0324) infection. However, no significant interaction was observed between gender and area of activity on infections by either N. caninum or L. infantum. This study showed that cats from an area endemic for visceral leishmaniasis in Brazil are exposed to three different protozoans, two of which are causal agents of important zoonosis.

Molecular detection of hemotrophic mycoplasmas among domiciled and free-roaming cats in Campo Grande, state of Mato Grosso do Sul, Brazil.

Hemoplasmas are bacteria living in feline red blood cells. Feline hemoplasmosis is frequently associated with old male cats that have access to the streets. This study aimed to detect the presence of hemoplasma speciess in domiciled and free-roaming cats in Campo Grande, state of Mato Grosso do Sul (MS), Brazil, using molecular techniques. Between January 2013 and April 2013, EDTA-whole blood samples were collected from 151 domestic cats (65 free-roaming and 86 domiciled cats). Samples were subjected to PCR assays targeting hemoplasmas 16S rRNA, followed by sequencing, BLAST analysis and phylogenetic analysis. Results show an occurrence of 36.4% for hemoplasmas. Twenty-three cats (15.2%) were positive for 'Candidatus Mycoplasma haemominutum', 17 (11.2%) for M. haemofelis and 15 (9.9%) for 'Candidatus M. turicensis', from PCR. Coinfection by two or three hemoplasmas was found in 25 cats (16.6%). No statistically significant difference between genders or between lifestyles was observed for the presence of hemoplasmas among the cats. Results show different hemoplasma species are present in cat population (Campo Grande, MS, Brazil). It is suggested that a differential diagnosis for feline hemoplasmosis should be made when cats show nonspecific clinical signs of disease with systemic manifestation.

Phylogeography of msp4 genotypes of Anaplasma marginale in beef cattle from the Brazilian Pantanal / Filogeografia de genótipos msp4 de Anaplasma marginale em gado de corte no Pantanal brasileiro

Abstract The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1β gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.

Resumo O gene msp4 de A. marginale é unicodon, estável e pouco heterogêneo, sendo considerado como um marcador útil para caracterização filogeográfica desta bactéria. Este trabalho teve como objetivo analisar a filogeografia de A. marginale com base no gene msp4 em bovinos de corte do Pantanal Brasileiro, comparativamente a outra regiões do mundo. Alíquotas de sangue foram colhidas de 400 bovinos (200 vacas e 200 bezerros) em cinco propriedades de cria e recria extensiva. Como resultado, 56,75% (227/400) mostraram-se positivas para A. marginale pela qPCR para o gene msp1β e destas, 8,37% (19/227) amostras foram positivas na PCR convencional para o gene msp4. Na análise de distância Network, 14 sequências do Pantanal brasileiro foram agrupadas em um único grupo com as da Thailândia, Índia, Espanha, Colômbia, Paraná (Brasil), México, Portugal, Argentina, China, Venezuela, Austrália, Italia e Minas Gerais (Brasil). Dentre 68 sequências do Brasil e do mundo, constatou-se a presença de 15 genótipos, sendo o genótipo número um (#1) o mais distribuído. As sequências msp4 de A. marginale detectadas em bovinos de corte no Pantanal brasileiro apresentaram baixo polimorfismo com formação de dois genogrupos filogeneticamente relacionados àqueles encontrados em ruminantes de países das América do Sul e Central, Europa e Ásia.

Serological detection of Toxoplasma gondii, Leishmania infantum and Neospora caninum in cats from an area endemic for leishmaniasis in Brazil / Detecção sorológica de Toxoplasma gondii, Leishmania infantum e Neospora caninum em gatos de uma área endêmica para leishmaniose no Brasil

An investigation was made into the occurrence of antibodies to Toxoplasma gondii, Leishmania infantum and Neospora caninum in 151 domestic cats, based on the indirect fluorescent antibody test (IFAT). Serum samples were collected from 151 domestic cats (65 free-roaming and 86 domiciled cats; 55 males and 96 females) in Campo Grande, Mato Grosso do Sul, Brazil between January and April 2013. IgG antibodies to T. gondii, L. infantum and N. caninum were found, respectively, in 49 (32.5%), 34 (22.5%) and 10 (6.6%) sampled cats. A positive correlation was found between T. gondii and N. caninum, T. gondii and L. infantum, and N. caninum and L. infantum (p <0.05) infections. Also, a significant interaction was identified between gender and area of activity on the probability of T. gondii (p = 0.0324) infection. However, no significant interaction was observed between gender and area of activity on infections by either N. caninum or L. infantum. This study showed that cats from an area endemic for visceral leishmaniasis in Brazil are exposed to three different protozoans, two of which are causal agents of important zoonosis.

O presente estudo teve como objetivo investigar a ocorrência de anticorpos anti-Toxoplasma gondii, Leishmania infantum e Neospora caninum, em 151 gatos, por meio da Reação de Imunofluorescência Indireta (RIFI). Entre os meses de janeiro e abril de 2013, amostras de soro foram coletadas de 151 gatos domésticos (65 gatos errantes e 86 gatos domiciliados; 55 machos e 96 fêmeas), de Campo Grande, Mato Grosso do Sul, Brasil. Anticorpos IgG anti-T. gondii, anti-L. infantum e anti-N. caninum foram encontrados em 49 (32,5%), 34 (22,5%) e 10 (6,6%) gatos amostrados, respectivamente. Verificou-se uma associação estatisticamente significativa entre as infecções por T. gondii e N. caninum, T. gondii e L. infantum e N. caninum e Leishmania infantum (p <0,05). Além disso, foi observada uma interação significativa entre sexo, área de atividade na probabilidade de infecção por T. gondii (p = 0,0324). No entanto, não foi observada interação significativa entre sexo e área de atividade nas infecções por N. caninum e L. infantum. Este estudo mostrou que os gatos de uma área endêmica brasileira para leishmaniose visceral são expostos a três diferentes protozoários, sendo dois deles importantes agentes zoonóticos.

Molecular detection of hemotrophic mycoplasmas among domiciled and free-roaming cats in Campo Grande, state of Mato Grosso do Sul, Brazil / Detecção molecular de micoplasmas hemotróficos em gatos domiciliados e errantes em Campo Grande, estado do Mato Grosso do Sul, Brasil

Hemoplasmas are bacteria living in feline red blood cells. Feline hemoplasmosis is frequently associated with old male cats that have access to the streets. This study aimed to detect the presence of hemoplasma speciess in domiciled and free-roaming cats in Campo Grande, state of Mato Grosso do Sul (MS), Brazil, using molecular techniques. Between January 2013 and April 2013, EDTA-whole blood samples were collected from 151 domestic cats (65 free-roaming and 86 domiciled cats). Samples were subjected to PCR assays targeting hemoplasmas 16S rRNA, followed by sequencing, BLAST analysis and phylogenetic analysis. Results show an occurrence of 36.4% for hemoplasmas. Twenty-three cats (15.2%) were positive for ‘Candidatus Mycoplasma haemominutum’, 17 (11.2%) for M. haemofelis and 15 (9.9%) for ‘Candidatus M. turicensis’, from PCR. Coinfection by two or three hemoplasmas was found in 25 cats (16.6%). No statistically significant difference between genders or between lifestyles was observed for the presence of hemoplasmas among the cats. Results show different hemoplasma species are present in cat population (Campo Grande, MS, Brazil). It is suggested that a differential diagnosis for feline hemoplasmosis should be made when cats show nonspecific clinical signs of disease with systemic manifestation.

Hemoplasmas são bactérias encontradas aderidas aos eritrócitos de felinos. A hemoplasmose felina está frequentemente associada a gatos velhos machos, sem raça definida e com acesso à rua. O presente estudo objetivou realizar a detecção molecular de espécies de hemoplasmas em gatos domiciliados e errantes em Campo Grande, estado do Mato Grosso do Sul (MS), Brasil. Entre janeiro/2013 e abril/2013, amostras de sangue foram colhidas de 151 gatos domésticos (65 errantes e 86 domiciliados) e avaliadas por PCR frente à presença de sequências do gene do 16S rRNA de hemoplasmas, seguidas de sequenciamento, análise pelo BLAST e análise filogenética. Os resultados deste estudo mostraram uma ocorrência de 36,4%. Vinte e três (15,2%) gatos mostraram-se positivos na PCR para ‘Candidatus Mycoplasma haemominutum’, 17 (11,2%) para Mycoplasma haemofelis, e 15 (9,9%) para ‘Candidatus Mycoplasma turicensis’. A co-infecção por dois ou três hemoplasmas ocorreu em 25 gatos (16,6%). Não foi observada diferença estatística significativa entre sexo e estilo de vida dos gatos amostrados e a presença de hemoplasmas. O estudo mostrou que diferentes espécies de hemoplasmas circulam na população de gatos (domiciliados e errantes) na cidade de Campo Grande, MS, Brasil. Sugere-se o diagnóstico diferencial para hemoplasmose felina em gatos que apresentam sinais clínicos inespecíficos de doença com manifestação sistêmica.