Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using groEL gene for Ehrlichia and Anaplasma species in rodents in Brazil.

New genotypes of Anaplasmataceae agents have been detected in wild carnivores, birds and deer in Brazil. The present work aimed to investigate the presence of Ehrlichia and Anaplasma species in rodents sampled in Brazil. Additionally, a newly designed quantitative 5' nuclease real-time multiplex PCR for Ehrlichia and Anaplasma spp. detection based on groEL gene amplification was designed, showing high specificity and sensitivity (10 groEL fragment copy/µL). Between 2000 and 2011, different rodent species [n=60] were trapped in 5 Brazilian biomes. Among 458 rodent spleen samples, 0.4% (2/458) and 2.4% (11/458) were positive for Ehrlichia and Anaplasma spp., respectively. Of 458 samples, 2.0% (9/458) and 1.1% (5/458) were positive for Anaplasma sp. and Ehrlichia sp., respectively, using conventional 16S rRNA PCR assays. Maximum Likelihood phylogenetic analyse based on a small region of 16S rRNA genes positioned the Anaplasma genotypes in rodents near Anaplasma phagocytophilum or Anaplasma marginale and Anaplasma odocoilei isolates. Ehrlichia genotypes were closely related to E. canis. There was a low occurrence of Anaplasma and Ehrlichia in wild and synanthropic rodents in Brazil, suggesting the circulation of new genotypes of these agents in rodents in the studied areas.

Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil.

OBJECTIVES: The objective of this study was to develop a quantitative 5' nuclease real-time polymerase chain reaction (PCR) assay to diagnose infections caused by Bartonella species. METHODS: Between January and April 2013 whole blood samples were collected by convenience from 151 cats (86 domiciled and 65 stray cats). The feline blood samples were subjected to a novel quantitative 5' nuclease real-time PCR (qPCR) for Bartonella species targeting the nictonamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) and conventional PCR assays targeting intergenic transcribed spacer, ribC, gltA, pap31 and rpoB, followed by sequencing and basic local alignment search tool analysis. RESULTS: The qPCR assay detected as few as 10 copies of plasmid per reaction. Forty-six (54.4% domiciled and 45.6% stray cats) of 151 sampled cats showed positive results in nuoG qPCR for Bartonella species. The absolute quantification of nuoG Bartonella DNA in sampled cats ranged from 1.1 × 10(4) to 1.3 × 10(4). Eighteen (39.1%) of 46 positive samples in the qPCR were also positive in conventional PCR assays. The sequencing confirmed that Bartonella henselae and Bartonella clarridgeiae circulate in cats in midwestern Brazil. CONCLUSIONS AND RELEVANCE: The present work provides details of a novel qPCR assay to diagnose infections caused by Bartonella species.

Molecular detection and analysis of spotted fever group Rickettsia in patients with fever and rash at a tertiary care centre in Tamil Nadu, India.

BACKGROUND: Detection of specific targets by PCR is used to confirm a diagnosis of spotted fever, but serological tests are still widely used. In this prospective study, nested PCR was performed on skin biopsy specimens to confirm the diagnosis of spotted fever. METHODS: In 58 clinically suspected cases of spotted fever, nested PCR, to detect gltA, 17 kDa lipoprotein antigen gene (17 kDa), ompA and ompB, from skin biopsy of the rash was performed. Sequencing was carried on amplicons representing the four targets to confirm specificity of amplification. This was followed by phylogenetic analysis using MEGA version 4.0 software. RESULTS: The gltA, 17 kDa, ompA, and ompB genes were detected from skin biopsy specimens in 38, 23, 27, and 22 individuals. Sequence analysis revealed that the gltA, 17 kDa, ompA, and ompB sequences belonged to spotted fever group (SFG) rickettsia. Of the six partial ompA gene sequences, only one was dissimilar to the previously reported 'Candidatus Rickettsia kellyi'. CONCLUSION: Further evidence indicates that SFG rickettsiae resembling 'Candidatus Rickettsia kellyi' cause fever and rash in southern India. More detailed phylogenetic analysis following isolation of rickettsia in culture is required for providing irrefutable proof for the occurrence of novel spotted fever rickettsiae in this region.

Study of infection by Rickettsiae of the spotted fever group in humans and ticks in an urban park located in the City of Londrina, State of Paraná, Brazil.

INTRODUCTION: Spotted fevers are emerging zoonoses caused by Rickettsia species in the spotted fever group (SFG). Rickettsia rickettsii is the main etiologic agent of Brazilian spotted fever (BSF)and it is transmitted by Amblyomma spp. ticks. METHODS: The study aimed to investigate SFG rickettsiae in the Arthur Thomas Municipal Park in Londrina, PR, by collecting free-living ticks and ticks from capybaras and blood samples from personnel working in these areas. Samples from A. dubitatum and A. cajennense were submitted for PCR in pools to analyze the Rickettsia spp. gltA (citrate synthase gene). RESULTS: All the pools analyzed were negative. Human sera were tested by indirect immunofluorescence assay with R. rickettsii and R. parkeri as antigens.Among the 34 sera analyzed, seven (20.6%) were reactive for R. rickettsii: four of these had endpoint titers equal to 64, 2 titers were 128 and 1 titer was 256. None of the samples were reactive for R. parkeri. An epidemiological questionnaire was applied to the park staff, but no statistically significant associations were identified. CONCLUSIONS: The serological studies suggest the presence of Rickettsiae related to SFG that could be infecting the human population studied; however, analysis of the ticks collected was unable to determine which species maybe involved in transmission to humans.

Study of infection by Rickettsiae of the spotted fever group in humans and ticks in an urban park located in the City of Londrina, State of Paraná, Brazil / Estudo da infecção por Rickettsias do grupo da febre maculosa em humanos e carrapatos de um parque urbano na Cidade de Londrina, Estado do Paraná

INTRODUCTION: Spotted fevers are emerging zoonoses caused by Rickettsia species in the spotted fever group (SFG). Rickettsia rickettsii is the main etiologic agent of Brazilian spotted fever (BSF) and it is transmitted by Amblyomma spp. ticks. METHODS: The study aimed to investigate SFG rickettsiae in the Arthur Thomas Municipal Park in Londrina, PR, by collecting free-living ticks and ticks from capybaras and blood samples from personnel working in these areas. Samples from A. dubitatum and A. cajennense were submitted for PCR in pools to analyze the Rickettsia spp. gltA (citrate synthase gene). RESULTS: All the pools analyzed were negative. Human sera were tested by indirect immunofluorescence assay with R. rickettsii and R. parkeri as antigens. Among the 34 sera analyzed, seven (20.6 percent) were reactive for R. rickettsii: four of these had endpoint titers equal to 64, 2 titers were 128 and 1 titer was 256. None of the samples were reactive for R. parkeri. An epidemiological questionnaire was applied to the park staff, but no statistically significant associations were identified. CONCLUSIONS: The serological studies suggest the presence of Rickettsiae related to SFG that could be infecting the human population studied; however, analysis of the ticks collected was unable to determine which species may be involved in transmission to humans.

INTRODUÇÃO: A febre maculosa é uma zoonose emergente causada por espécies de Rickettsia do grupo febre maculosa (GFM). Rickettsia rickettsii é o principal agente etiológico da febre maculosa brasileira (FMB) e é transmitida por Amblyomma spp. MÉTODOS: Com o objetivo de obter informações sobre GFM Rickettsiae no Parque Municipal Arthur Thomas em Londrina, PR, carrapatos de vida livre e de capivaras foram coletados, assim como amostras de sangue das pessoas que trabalham no parque. A. dubitatum e A. cajennense foram submetidos à PCR em pools para analises de Rickettsia spp. gltA (citrate synthase gene). RESULTADOS: Todos os pools de carrapatos analizados foram negativos. Soros de humanos foram testados pela imunofluorescência indireta com antigenos de R. rickettsii e R. parkeri. Entre os 34 soros analisados, 7 (20,6 por cento) foram positivos para R. rickettsii. Destes, quatro apresentaram títulos iguais a 64, dois iguais a 128 e um, igual a 256, mas nenhum soro reagiu com R. parkeri. Não houve nenhuma associação, estatisticamente significante, entre as variáveis analisadas no questionário epidemiológico fornecido às pessoas que participaram da pesquisa. CONCLUSÕES: Os estudos sorológicos sugerem a presença de alguma Rickettsiae relacionada ao GFM que poderiam estar infectando a população humana estudada. Entretanto, as análises dos carrapatos foram inconclusivas para determinar qual espécie poderia estar envolvida na transmissão para os humanos.

Serosurvey of antibodies against spotted fever group Rickettsia spp. in horse farms in Northern Paraná, Brazil.

Brazilian spotted fever (BSF) is an emerging disease most likely caused by Rickettsia rickettsii. The objective of the present study was to estimate the seroprevalence of BSF rickettsia infections in equines from six horse farms located in Londrina County, Paraná, Southern Brazil. Six owners of horse farms situated in Cambé, Santa Fé, Guaraci and Londrina municipalities participated in the study. All farms were located in areas where BSF has not been reported. A total of 273 horses were sampled and their sera were tested by indirect immunofluorescence assay (IFA) using R. rickettsii and R. parkeri antigens. Titers equal to and greater than 64 were considered positive. Of 273 sera tested, 15 (5.5%) reacted to R. rickettsii and 5 (1.8%) to R. parkeri. Five out of the six farms studied revealed seropositive animals and seropositivity rate ranged from 0 to 13%. The titers ranged from 64 to 512, and four samples had a titer of 512. Nine animals reacted to R. rickettsii with titers four-fold higher than those for R. parkeri. These results suggest that horses in Northern Paraná may have been exposed to rickettsiae identical or closely related to R. rickettsii.

Serosurvey of antibodies against spotted fever group Rickettsia spp. in horse farms in Northern Paraná, Brazil / Soroprevalência de anticorpos contra Rickettsia spp. do grupo febre maculosa em equinos de haras no Norte do Paraná, Brasil

Brazilian spotted fever (BSF) is an emerging disease most likely caused by Rickettsia rickettsii. The objective of the present study was to estimate the seroprevalence of BSF rickettsia infections in equines from six horse farms located in Londrina County, Paraná, Southern Brazil. Six owners of horse farms situated in Cambé, Santa Fé, Guaraci and Londrina municipalities participated in the study. All farms were located in areas where BSF has not been reported. A total of 273 horses were sampled and their sera were tested by indirect Immunofluorescence assay (IFA) using R. rickettsii and R. parkeri antigens. Titers equal to and greater than 64 were considered positive. Of 273 sera tested, 15 (5.5 percent) reacted to R. rickettsii and 5 (1.8 percent) to R. parkeri. Five out of the six farms studied revealed seropositive animals and seropositivity rate ranged from 0 to 13 percent. The titers ranged from 64 to 512, and four samples had a titer of 512. Nine animals reacted to R. rickettsii with titers four-fold higher than those for R. parkeri. These results suggest that horses in Northern Paraná may have been exposed to rickettsiae identical or closely related to R. rickettsii.

Febre Maculosa Brasileira (FMB) é uma doença emergente, sendo Rickettsia rickettsii o seu principal agente etiológico. O objetivo deste estudo foi determinar a soroprevalência de rickettsia do grupo da febre maculosa em equinos de seis haras localizados nos municípios de Cambé, Santa Fé, Guaraci e Londrina. As propriedades eram localizadas na região Norte do Paraná onde casos de FMB ainda não foram diagnosticados. Foram colhidas amostras de sangue de 273 equinos, e os soros foram testados pela RIFI, usando R. rickettsii e R. parkeri como antígenos, considerando-se como positivos títulos >64. Entre 273 soros, 15 (5,5 por cento) reagiram contra R. rickettsii e 5 (1,8 por cento) para R. parkeri. Cinco de seis haras estudados tinham animais reativos, e a taxa de sororreatividade variou de 0 a 13 por cento. Os títulos variaram de 64 para 512, e três amostras apresentaram título de 512. Nove animais reagiram para R. rickettsii com títulos quatro vezes maiores que para R. parkeri. Esses resultados sugerem que equinos no Norte do Estado do Paraná, Brasil, podem ter sido expostos a uma rickettsia idêntica ou muito próxima a R. rickettsii.

Demonstration of OspC type diversity in invasive human lyme disease isolates and identification of previously uncharacterized epitopes that define the specificity of the OspC murine antibody response.

Outer surface protein C (OspC) of the Lyme disease spirochetes is an important virulence factor that has potential utility for vaccine development. Of the 21 OspC types that have been identified, it has been postulated that types A, B, I, and K are specifically associated with invasive infections. Through an analysis of isolates collected from patients in Maryland we found that OspC types C, D, and N are also associated with invasive infections. This observation suggests that there is greater diversity in the group of OspC types associated with invasive infection than has been previously suggested. Detailed knowledge of the antigenic structure of OspC is essential for vaccine development. To determine if the antibody response to OspC is type specific, recombinant proteins of several different OspC types were immunoblotted and screened with sera from mice infected with isolates having known OspC types. These analyses revealed a high degree of specificity in the antibody response and suggested that the immunodominant epitopes of OspC reside in the variable domains of the protein. To localize these epitopes, OspC fragments were generated and screened with serum collected from infected mice. These analyses led to identification of previously uncharacterized epitopes that define the type specificity of the OspC antibody response. These analyses provide important insight into the antigenic structure of OspC and also provide a basis for understanding the variable nature of the antibody response to this important virulence factor of the Lyme disease spirochetes.

Detection of Anaplasma phagocytophilum DNA in Ixodes ticks (Acari: Ixodidae) from Madeira Island and Setubal District, mainland Portugal.

A total of 278 Ixodes ticks, collected from Madeira Island and Setubal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setubal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setubal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.