Bartonella spp. in different species of bats from Misiones (Argentina).

The aim of this study was to detect vector-borne pathogens (Anaplasmataceae family, Rickettsia genus, and Bartonella genus) in bats from Misiones (Argentina). Thirty-three specimens were captured over 8 days using mist nets. Twenty (60.6%) blood samples were positive (11/13 Artibeus lituratus, 4/10 Desmodus rotundus, 4/8 Carollia perspicillata, and 1/2 Myotis nigricans) by PCR for the gltA gene fragment of Bartonella. All samples were negative by PCR for the Anaplasmataceae family and Rickettsia genus. The phylogenetic analysis showed seven Bartonella genotypes. The three genotypes obtained from A. lituratus, 2 from C. perspicillata, and 1 from D. rotundus were related to Bartonella spp. from New World bats, while the sequence obtained from M. nigricans was related to Old World bats. We identified a considerable diversity of Bartonella genotypes in a small number of bats, thus further research is required to better understand the complex bat-pathogen interaction.

[Molecular detection of Saint Louis encephalitis virus in mosquitoes in Buenos Aires]. / Detección molecular de virus de encefalitis de Saint Louis en mosquitos de Buenos Aires, Argentina.

During March 2013 a population of eared doves (Zenaida auriculata) was established in the center of City of Buenos Aires. Considering the role of these birds as host competent for Saint Louis encephalitis virus (SLEV), a CDC light trap was put in place to perform entomologic surveillance. During this month 5 pools of mosquitoes (n = 48) were collected and taxonomically determined. Three of them were classified as Culex pipiens (n = 10) and the other two were Culex spp. (n = 38). In this case, the mosquitoes species could not be determined due to that individuals were damaged. One of the Culex spp. pool was found to be positive for Saint Louis encephalitis virus by molecular techniques. This was then sequenced and classified as genotype III.

[Molecular detection of Rickettsia massiliae and Anaplasma platys infecting Rhipicephalus sanguineus ticks and dogs, Bahía Blanca (Argentina)]. / Detección molecular de Rickettsia massiliae y Anaplasma platys en garrapatas Rhipicephalus sanguineus y caninos domésticos del municipio de Bahía Blanca (Argentina).

BACKGROUND: Rickettsioses, ehrlichioses and anaplasmoses are caused by Gram negative obligate intracellular bacteria and transmitted mainly by arthropods. AIM: To detect and perform the molecular characterization of these pathogens in ticks and domestic dogs in Bahia Blanca City (Buenos Aires, Argentina). METHODS: Fifty six blood samples from dogs and 82 ticks (75 Rhipicephalus sanguineus and 7 Amblyomma tigrinum) were studied. The samples were analyzed by PCR for Rickettsia (intergenic space 23S-5S rRNA), Ehrlichia/Anaplasma (16S rRNA), and Anaplasma platys (16S rRNA). RESULTS: 12% of R. sanguineus resulted positive for Rickettsia, identified by sequencing as Rickettsia massiliae; and 37.5% of the canine blood samples analyzed were positive for A. platys. Molecular characterization was also performed by amplification of the fragment of the citrate synthase gene (gltA) (Rickettsia genus) and the groESL gene (A. platys). Phylogenetic trees were constructed using the neighbor-joining method. These trees revealed that sequences obtained are similar to those from other geographical regions. CONCLUSION: The results indicate the presence of R. massiliae in R. sanguineus ticks for the second time in an urban area of South America and A. platys infection in dogs, being the southernmost region of Argentina where it has been notified.

A Novel Terrestrial Rabies Virus Lineage Occurring in South America: Origin, Diversification, and Evidence of Contact between Wild and Domestic Cycles.

The rabies virus (RABV) is characterized by a history dominated by host shifts within and among bats and carnivores. One of the main outcomes of long-term RABV maintenance in dogs was the establishment of variants in a wide variety of mesocarnivores. In this study, we present the most comprehensive phylogenetic and phylogeographic analysis, contributing to a better understanding of the origins, diversification, and the role of different host species in the evolution and diffusion of a dog-related variant endemic of South America. A total of 237 complete Nucleoprotein gene sequences were studied, corresponding to wild and domestic species, performing selection analyses, ancestral states reconstructions, and recombination analyses. This variant originated in Brazil and disseminated through Argentina and Paraguay, where a previously unknown lineage was found. A single host shift was identified in the phylogeny, from dog to the crab-eating fox (Cerdocyon thous) in the Northeast of Brazil. Although this process occurred in a background of purifying selection, there is evidence of adaptive evolution -or selection of sub-consensus sequences- in internal branches after the host shift. The interaction of domestic and wild cycles persisted after host switching, as revealed by spillover and putative recombination events.

[Diagnosis of a human rabies case in Jujuy, 2008]. / Diagnóstico de un caso de rabia humana en Jujuy, 2008.

On July 22, 2008, a previously healthy 8 years old boy from Jujuy, Argentina, died of encephalitis later confirmed as rabies. Diagnosis was made on the basis rabies-specific antibodies presence in a serum sample and it was confirmed by detection of the viral antigens in brain necropsy using the immunofluorescent test. Antigenic characterization identified dog as source of infection. Molecular analysis recognized the same genetic variant circulating in Jujuy since 2003. This report presents the patient's clinical course and the epidemiologic investigation that revealed a dog bite 60 days before the illness onset and the lack of rabies treatment.

Evaluation of a rapid immunochromatographic diagnostic test (RIDT) for diagnosis of rabies in samples from Argentina.

INTRODUCTION: Rabies is a globally widespread zoonosis of viral origin that causes fatal encephalitis in humans and animals. In countries where rabies is endemic and there is a lack of well-equipped diagnostic laboratories, a rapid immunochromatographic diagnostic test (RIDT) for detection of rabies could be an indispensable tool. In this study we evaluated the limit of detection, as well as specificity and sensitivity of RIDT, compared to the standard fluorescent antibody test (FAT). METHODOLOGY: A total of 174 samples were diagnosed by both RIDT and FAT. Fresh clinical samples, poorly conserved samples and brains in advanced state of decomposition generated under laboratory conditions were used to resemble field conditions. The sensitivity of RIDT was evaluated with CVS fixed strain of rabies virus (RABV), previously titrated in 21-day old albino mice and compared with the Reverse Transcription - Polimerase Chain Reaction (RT-PCR) technique in parallel. Additionally, the Mouse Inoculation Test (MIT) was used to perform the antigenic characterization of Rabies virus variants. RESULTS: The limit of detection of RIDT was 100 LD50 / 0.03 mL and its performance, as compared to that of FAT, showed a sensitivity of 97.96%, a specificity of 100% and a concordance by the Kappa test of 0.98 with 95% CI. CONCLUSIONS: RIDT provides results comparable to those of FAT and this test can be considered as an appropriate method under the field conditions, even in samples that are not suitable for FAT due to their state of decomposition.

Molecular characterization of Ehrlichia canis infecting dogs, Buenos Aires.

Canine monocytic ehrlichiosis (CME) is a worldwide potentially fatal tick-borne rickettsial disease of dogs caused by Ehrlichia canis and transmitted by Rhipicephalus sanguineus sensu lato. CME diagnosis includes indirect (serology) and direct (e.g. blood smears and PCR) methods. PCR is more sensitive and specific than direct microscopic examination and positive PCR results confirm infection, whereas positive serologic test results only confirm exposure. The aim of the present study was to perform a molecular characterization of E. canis from canine samples of the Metropolitan Area of Buenos Aires. We studied 223 blood samples of dogs submitted to our institute for CME diagnoses. The samples were initially screened for Anaplasmataceae family by PCR, resulting in 30 positive dogs (13.4%). Subsequently, positive DNAs were analyzed by nested PCR 16S rRNA specific for E. canis or Anaplasma platys, resulting in 15 (6.7%) and 16 (7.2%) positive dogs, respectively. For molecular characterization, samples positive for E. canis were subjected to amplification of a fragment of the dsb and p28 genes. The nucleotide sequences obtained for the dsb fragment resulted in 100% identity with others E. canis found in dogs from different regions of worldwide. The nucleotide sequences obtained for p28 gene resulted in 100% of identity with each other and closely with E. canis str. Jaboticabal (Brazil). Identity with others sequences of E. canis ranged from 76.9 to 79.7%. The occurrence of canine cases molecularly confirmed in Metropolitan Area of Buenos Aires highlights the need for more studies in order to understand epidemiological factors associated with CME, especially the disease transmission dynamic in South America given the existence of two lineages of R. sanguineus sensu lato with different vectorial capacity for transmission of E. canis.

Molecular characterization of Rickettsia massiliae and Anaplasma platys infecting Rhipicephalus sanguineus ticks and domestic dogs, Buenos Aires (Argentina).

Rickettsioses, ehrlichioses and anaplasmoses are emerging diseases that are mainly transmitted by arthropods and that affect humans and animals. The aim of the present study was to use molecular techniques to detect and characterize those pathogens in dogs and ticks from Buenos Aires city. We studied 207 Rhipicephalus sanguineus ticks and 52 canine blood samples from poor neighborhoods of Buenos Aires city. The samples were molecularly screened for the genera Rickettsia, Ehrlichia, and Anaplasma by PCR and sequencing. DNA of Rickettsia massiliae (3.4%) and Anaplasma platys (13.5%) was detected in ticks and blood samples, respectively. For characterization, the positive samples were subjected to amplification of a fragment of the 190-kDa outer membrane protein gene (spotted fever group rickettsiae) and a fragment of the groESL gene (specific for A. platys). A phylogenetic tree was constructed using the neighbor-joining method, revealing that the sequences were closely related to those of strains from other geographic regions. The results indicate that human and animal pathogens are abundant in dogs and their ticks in Buenos Aires city and portray the potentially high risk of human exposure to infection with these agents, especially in poor neighborhoods, where there is close contact with animals in an environment of poor health conditions.

High diversity of rabies viruses associated with insectivorous bats in Argentina: presence of several independent enzootics.

BACKGROUND: Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory. METHODS: We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008. RESULTS: Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples. CONCLUSIONS: This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection.

Detección molecular de Rickettsia massiliae y Anaplasma platys en garrapatas Rhipicephalus sanguineus y caninos domésticos del municipio de Bahía Blanca (Argentina) / Molecular detection of Rickettsia massiliae and Anaplasma platys infecting Rhipicephalus sanguineus ticks and dogs, Bahía Blanca (Argentina)

Background: Rickettsioses, ehrlichioses and anaplasmoses are caused by Gram negative obligate intracellular bacteria and transmitted mainly by arthropods. Aim: To detect and perform the molecular characterization of these pathogens in ticks and domestic dogs in Bahia Blanca City (Buenos Aires, Argentina). Methods: Fifty six blood samples from dogs and 82 ticks (75 Rhipicephalus sanguineus and 7 Amblyomma tigrinum) were studied. The samples were analyzed by PCR for Rickettsia (intergenic space 23S-5S rRNA), Ehrlichia/Anaplasma (16S rRNA), and Anaplasma platys (16S rRNA). Results: 12% of R. sanguineus resulted positive for Rickettsia, identified by sequencing as Rickettsia massiliae; and 37.5% of the canine blood samples analyzed were positive for A. platys. Molecular characterization was also performed by amplification of the fragment of the citrate synthase gene (gltA) (Rickettsia genus) and the groESL gene (A. platys). Phylogenetic trees were constructed using the neighbor-joining method. These trees revealed that sequences obtained are similar to those from other geographical regions. Conclusion: The results indicate the presence of R. massiliae in R. sanguineus ticks for the second time in an urban area of South America and A. platys infection in dogs, being the southernmost region of Argentina where it has been notified.

Introducción: Las rickettsiosis, ehrlichiosis y anaplasmosis son causadas por bacterias gramnegativas, intracelulares obligadas y transmitidas principalmente por artrópodos. Objetivo: Detectar y caracterizar molecularmente estos patógenos en garrapatas y caninos domésticos del municipio de Bahía Blanca (provincia de Buenos Aires, Argentina). Material y Métodos: Se estudiaron 56 muestras sanguíneas de caninos, 75 garrapatas Rhipicephalus sanguineus y 7 Amblyomma tigrinum. Las muestras fueron analizadas por RPC para Rickettsia (espacio intergénico 23S-5S ARNr), Ehrlichia y Anaplasma (16S ARNr), y Anaplasma platys (16S ARNr). Resultados: Se detectó positividad a Rickettsia en 12% de R. sanguineus, identificándose por secuenciación a Rickettsia massiliae. Las muestras sanguíneas de los caninos resultaron en 37,5% positivas a A. platys. También se caracterizaron molecularmente por la amplificación del fragmento del gen citrato sintasa (gltA) (género Rickettsia) y del gen groESL (A. platys). Se construyeron árboles filogenéticos utilizando el método del vecino más cercano (neighbor-joining) revelando que las secuencias obtenidas son similares a las de otras regiones geográficas. Conclusión: Los resultados indican la presencia de R. massiliae en garrapatas R. sanguineus en una segunda zona urbana de Sudamérica y la infección por A. platys en caninos, siendo la región más austral de Argentina donde ha sido notificada.

Detección molecular de virus de encefalitis de Saint Louis en mosquitos de Buenos Aires, Argentina

Durante el mes de marzo de 2013 una población de palomas torcazas (Zenaida auriculata) se instaló en una zona céntrica de la ciudad de Buenos Aires. Conociendo el rol que poseen estas aves como hospedadores competentes del virus de la encefalitis de Saint Louis (SLEV), fue colocada en el lugar una trampa de luz tipo CDC, a fin de realizar una vigilancia entomológica. Durante ese mes,fueron capturados 5 grupos de mosquitos (n = 48), 3 correspondieron a la especie Culex pipiens (n = 10) y 2 a Culex spp.(n = 38), no pudiéndose determinar en estos últimos con precisión la especie por encontrarse dañados. En un grupo de mosquitos Culex spp. se detectó el SLEV por técnicas moleculares. Posteriormente fue secuenciado y clasificado como perteneciente al genotipo III.

During March 2013 a population of eared doves (Zenaida auriculata) was established in the center of City of Buenos Aires. Considering the role of these birds as host competent for Saint Louis encephalitis virus (SLEV), a CDC light trap was put in place to perform entomologic surveillance. During this month 5 pools of mosquitoes (n = 48) were collected and taxonomically determined. Three of them were classified as Culex pipiens (n = 10) and the other two were Culex spp. (n = 38). In this case, the mosquitoes species could not be determined due to that individuals were damaged. One of the Culex spp. pool was found to be positive for Saint Louis encephalitis virus by molecular techniques. This was then sequenced and classified as genotype III.

Molecular detection of Neorickettsia risticii in Brazilian free-tailed bats (Tadarida brasiliensis) from Buenos Aires, Argentina

Neorickettsia risticii is the causative agent of Potomac Horse Fever, a severe febrile disease affecting horses, transmitted by trematodes species with a complex life cycle. A total of 30 insectivorous bats (Brazilian free-tailed bat Tadarida brasiliensis) were analyzed by PCR for presence of genus Anaplasma, Ehrlichia, Neorickettsia and Rickettsia. Three samples showed positive reactions for genus Anaplasma, Ehrlichia and Neorickettsia, and the sequences were 99.67% identical to Neorickettsia risticii. The role of bats in the life cycle of N. risticii has yet to be elucidated; however bats may be reservoirs for this bacterium. To our knowledge, this is the first evidence of N. risticii in Argentina.

Diagnóstico de un caso de rabia humana en Jujuy, 2008 / Diagnosis of a human rabies case in Jujuy, 2008

El 22 de Julio de 2008, un niño de 8 años de edad, residente en la provincia de Jujuy, Argentina, falleció por una encefalitis producida por el virus de la rabia. El diagnóstico se realizó mediante la detección de anticuerpos en suero y se confirmó por inmunofluorescencia en el cerebro. La tipificación antigénica correspondió a la variante 1 trasmitida por perros. El análisis molecular estableció que el virus detectado es de la misma variante genética que circula en Jujuy desde 2003. Este trabajo resume la evolución clínica del paciente y la posterior investigación epidemiológica que reveló el antecedente de mordedura por un perro 60 días antes de la iniciación de la enfermedad y la ausencia de un tratamiento antirrábico post-exposición.

On July 22, 2008, a previously healthy 8 years old boy from Jujuy, Argentina, died of encephalitis later confirmed as rabies. Diagnosis was made on the basis rabies-specific antibodies presence in a serum sample and it was confirmed by detection of the viral antigens in brain necropsy using the inmunofluorescent test. Antigenic characterization identified dog as source of infection. Molecular analysis recognized the same genetic variant circulating in Jujuy since 2003. This report presents the patient's clinical course and the epidemiologic investigation that revealed a dog bite 60 days before the illness onset and the lack of rabies treatment.