Transovarial Transmission of Anaplasma marginale in Rhipicephalus (Boophilus) microplus Ticks Results in a Bottleneck for Strain Diversity.

Anaplasma marginale is an obligate intraerythrocytic bacterium of bovines, responsible for large economic losses worldwide. It is mainly transmitted by Rhipicephalus (Boophilus) microplus ticks and, despite mounting evidence suggesting transovarial transmission, the occurrence of this phenomenon remains controversial. We evaluated the vector competence of R. microplus larvae vertically infected with A. marginale to transmit the bacterium to a naïve bovine. A subgroup of engorged female ticks collected from an A. marginale-positive animal was dissected and the presence of the pathogen in its tissues was confirmed. A second subgroup of ticks was placed under controlled conditions for oviposition. After confirming the presence of A. marginale in the hatched larvae, an experimental infestation assay was conducted. Larvae were placed on an A. marginale-free splenectomized calf. The bacterium was detected in the experimentally infested bovine 22 days post-infestation. We analyzed the A. marginale diversity throughout the transmission cycle using the molecular marker MSP1a. Different genotypes were detected in the mammalian and arthropod hosts showing a reduction of strain diversity along the transmission process. Our results demonstrate the vertical transmission of A. marginale from R. microplus females to its larvae, their vector competence to transmit the pathogen, and a bottleneck in A. marginale strain diversity.

[Enteroparasitosis in a child population of the Hurlingham District, Buenos Aires]. / Enteroparasitosis en una población infantil del Municipio de Hurlingham, Buenos Aires.

INTRODUCTION: Intestinal parasites have the greatest impact on child populations as they can, in the long term, lead to stunted growth with advances in cognitive development. The transmission of these parasites is favored by environments of social vulnerability. METHODS: In a cross-sectional study carried out in the District of Hurlingham, in the suburbs of Buenos Aires, the prevalence of intestinal parasitoses and its associated risk factors in a child population attending Primary Health Care Centers was estimated. Coproparasitological survey, Graham test and a socio-environmental and labor survey were performed. RESULTS: A total of 130 children, 50.8% women and 49.2% men (0-12 years) were analyzed; 89.1% of those over 4 years of age were in school. In 28.5% of households, the mother or father had a formal job and 63.8% received the Universal Child Allowance. Conditions of social vulnerability were observed in the analyzed population: poor access to network water and sewage and environmental sanitation. The parasitological diagnosis revealed that 75 children [57.7% (95% CI: 48.7-66.3)] were parasitized. Protists were more frequent than helminths and the most prevalent species were Blastocystis spp, Enterobius vermicularis, Giardia lamblia and Cryptosporidium sp. The parasites in children were associated with schooling (p <0.01) and age (p <0.05). DISCUSSION: This work constitutes the first survey on intestinal parasitoses in the District of Hurlingham and the socioeconomic and environmental context where it is developed.

Introducción: Los parásitos intestinales tienen mayor impacto en las poblaciones infantiles ya que pueden, a largo plazo, conducir a un retraso en el crecimiento con alteración del desarrollo cognitivo. Sumado a esto, los entornos de vulnerabilidad social favorecen la transmisión y permanencia de estos parásitos en el ambiente. Métodos: En un estudio transversal realizado en el Municipio de Hurlingham (conurbano bonaerense) se estimó la prevalencia de enteroparasitosis y sus factores de riesgo asociados en una población infantil concurrente a Centros de Atención Primaria para la Salud locales mediante un análisis coproparasitológico, un test de Graham y una encuesta socioambiental y laboral. Resultados: Un total de 66 niñas (50.8%) y 64 niños (49.2%) entre 0 a 12 años fueron analizados. El 89.1% de los mayores de 4 años estaban escolarizados. En el 28.5% de los hogares la madre o el padre tenía trabajo formal y el 63.8% percibía la asignación universal por hijo (AUH). Se observaron condiciones de vulnerabilidad social en la población analizada, tales como: falta de acceso al agua de red y cloacas y un saneamiento ambiental deficiente. El diagnóstico parasitológico reveló un total de 75 casos [57.7% (IC 95%: 48.7-66.3)] de parasitosis. Los protistas fueron los parásitos más frecuentes y las especies más prevalentes: Blastocystis spp, Enterobius vermicularis, Giardia lamblia y Cryptosporidium sp. La presencia de parásitos estuvo asociada a la escolarización (p <0.01) y a la edad del niño (p <0.05). Discusión: Este fue el primer relevamiento sobre las enteroparasitosis en el municipio de Hurlingham y el contexto socioeconómico y ambiental donde se desarrollan.

Enteroparasitosis en una población infantil del Municipio de Hurlingham, Buenos Aires / Enteroparasitosis in a child population of the Hurlingham District, Buenos Aires

Resumen Introducción: Los parásitos intestinales tienen mayor impacto en las poblaciones infantiles ya que pueden, a largo plazo, conducir a un retraso en el crecimiento con alteración del desarrollo cognitivo. Sumado a esto, los entornos de vulnerabilidad social favorecen la transmisión y permanencia de estos parásitos en el ambiente. Métodos: En un estudio transversal realizado en el Municipio de Hurlingham (conurbano bonae rense) se estimó la prevalencia de enteroparasitosis y sus factores de riesgo asociados en una población infantil concurrente a Centros de Atención Primaria para la Salud locales mediante un análisis coproparasitológico, un test de Graham y una encuesta socioambiental y laboral. Resultados: Un total de 66 niñas (50.8%) y 64 niños (49.2%) entre 0 a 12 años fueron analizados. El 89.1% de los mayores de 4 años estaban escolarizados. En el 28.5% de los hogares la madre o el padre tenía trabajo formal y el 63.8% percibía la asignación universal por hijo (AUH). Se observaron condiciones de vulnerabilidad social en la población analizada, tales como: falta de acceso al agua de red y cloacas y un saneamiento ambiental deficiente. El diagnóstico parasitológico reveló un total de 75 casos [57.7% (IC 95%: 48.7-66.3)] de parasitosis. Los protistas fueron los parásitos más frecuentes y las especies más prevalentes: Blastocystis spp, Enterobius vermicularis, Giardia lamblia y Cryptosporidium sp. La presencia de parásitos estuvo asociada a la escolarización (p<0.01) y a la edad del niño (p<0.05). Dis cusión: Este fue el primer relevamiento sobre las enteroparasitosis en el municipio de Hurlingham y el contexto socioeconómico y ambiental donde se desarrollan.

Abstract Introduction: Intestinal parasites have the greatest impact on child populations as they can, in the long term, lead to stunted growth with advances in cognitive development. The transmission of these parasites is favored by environments of social vulnerability. Methods: In a cross-sectional study carried out in the District of Hurlingham, in the suburbs of Buenos Aires, the prevalence of intestinal parasitoses and its associated risk factors in a child population attending Primary Health Care Centers was estimated. Coproparasitological survey, Graham test and a socio-environmental and labor survey were performed. Results: A total of 130 children, 50.8% women and 49.2% men (0-12 years) were analyzed; 89.1% of those over 4 years of age were in school. In 28.5% of households, the mother or father had a formal job and 63.8% received the Universal Child Allowance. Conditions of social vulnerability were observed in the analyzed population: poor access to network water and sewage and environmental sanitation. The parasitological diagnosis revealed that 75 children [57.7% (95% CI: 48.7-66.3)] were parasitized. Protists were more frequent than helminths and the most prevalent species were Blastocystis spp, Enterobius vermicularis, Giardia lamblia and Cryptosporidium sp. The parasites in children were associated with schooling (p<0.01) and age (p<0.05). Discussion: This work constitutes the first survey on intestinal parasitoses in the District of Hurlingham and the socioeconomic and environmental context where it is developed.

Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis.

Bovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10-12 % parasitemia for B. bigemina and of 1 × 10-6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.

Closing the Gaps to Understand the Tick Transmission of Anaplasma marginale among Giant Anteaters (Myrmecophaga tridactyla) in Argentina.

Anaplasma marginale, a well-known cattle pathogen of tropical and subtropical world regions, has been previously molecularly characterized in a giant anteater (Myrmecophaga tridactyla) from Corrientes, Argentina. Ticks or other hematophagous arthropod involved in the wild transmission cycle remained unknown. The aim of the present study was to analyze the simultaneous occurrence of A. marginale in blood samples and ticks from giant anteaters from Corrientes in order to investigate if ticks could be relevant in the transmission among these mammals. Blood samples from 50 giant anteaters collected in different years and 26 ticks Amblyomma dubitatum and A. sculptum were studied through the molecular amplification of two unequivocal species-specific genes from A. marginale: msp5 and msp1ß. Twenty five giant anteaters and tick organs (salivary glands, gut and oviduct) from 11 ticks tested positive to the A. marginale DNA amplification. The further molecular characterization through MSP1a tandem repeats analysis revealed the presence of genotypes circulating among giant anteaters that had been previously identified in cattle blood samples from the same geographical region. These results confirm the presence of A. marginale in giant anteaters in Corrientes and suggests that A. dubitatum and A. sculptum ticks could be involved in the transmission among giant anteaters. Future studies will determine the role of these tick species in the wild transmission cycle in the study area and the eventual connection with the domestic cycle.

Detección de bacterias de los géneros Ehrlichia Anaplasma y Rickettsia en garrapatas rhipicephalus Sanguineus sl en Pergamino Argentina / Detection of Bacteria of the Genera Ehrlichia, Anaplasma and Rickettsia in Rhipicephalus sanguineus s.l Ticks in the City of Pergamino, Argentina

Las rickettsiosis, ehrlichiosis y anaplasmosis son causadas por bacterias gramnegativas intracelulares obligadas y transmitidas por artrópodos. El objetivo de este trabajo fue determinar especies de garrapatas presentes en caninos de la ciudad de Pergamino y detectar presencia de patógenos de los géneros Ehrlichia, Anaplasma y Rickettsia. MÉTODOS: Se estudiaron 21 garrapatas Rhipicephalus sanguineus s.l obtenidas de caninos de diferentes ambientes de la ciudad de Pergamino. Las muestras fueron analizadas mediante PCR, amplificando un fragmento del gen gltA para Rickettsia spp. y del 16SrRNA para los géneros Ehrlichia/Anaplasma. RESULTADOS: Se detectó positividad a Rickettsia spp. en el 4,76%, identificándose por secuenciación a la especie Rickettsia massiliae. Para la familia Anaplasmataceae se detectó positividad para Ehrlichia canis (23,8%) y Anaplasma platys (19,04%). CONCLUSIONES: Se trata del primer reporte de patógenos de interés zoonótico y/o veterinario pertenecientes a los géneros Rickettsia, Ehrlichia y Anaplasma en garrapatas Rh. sanguineus s.l para el norte de la provincia de Buenos Aires

Immunisation of cattle against Babesia bovis combining a multi-epitope modified vaccinia Ankara virus and a recombinant protein induce strong Th1 cell responses but fails to trigger neutralising antibodies required for protection.

Protection against the intraerythrocytic protozoan parasite Babesia bovis depends on both strong innate and adaptive immune response, this latter involving the presentation of parasite antigens to CD4+ T-lymphocytes by professional antigen-presenting cells. Secretion of Th1 cytokines by CD4+ T cell is also very important for isotype switching to IgG2, the best opsonising antibody isotype in cattle, to target extracellular parasites and parasite antigens displayed at the erythrocyte surface. In the field of vaccinology, heterologous prime-boost schemes combining protein-adjuvant formulations with a modified vaccinia Ankara vector expressing the same antigen have demonstrated the induction of both humoral and cellular immune responses. It has been previously demonstrated that MVA-infected dendritic cells can present antigens in the context of MHC II and activate CD4+ T cell. These results support the use of the MVA viral vector for a pathogen like Babesia bovis, which only resides within erythrocytes. In this study, 13-15-months-old Holstein-Friesian steers were immunised with a subunit vaccine as a prime and a modified vaccinia Ankara vector as a boost, both expressing a chimeric multi-antigen (rMABbo - rMVA). This antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: merozoite surface antigen - 2c (MSA - 2c), rhoptry associated protein 1 (RAP - 1) and heat shock protein 20 (HSP20). Responses were compared with the Babesia bovis live attenuated vaccine used in Argentina (R1A). Eleven weeks after the first immunisation, all bovines were challenged by the inoculation of a virulent B. bovis strain. All groups were monitored daily for hyperthermia and reduction of packed cell volume. Both the rMABbo - rMVA and R1A vaccinated animals developed high titters of total IgG antibodies and an antigen-specific Th1 cellular response before and after challenge. However, all rMABbo - rMVA steers showed clinical signs of disease upon challenge. Only the R1A live vaccine group developed an immune response associated with in vitro neutralising antibodies at a level that significantly inhibited the parasite invasion. The lack of protection observed with this recombinant formulation indicates the need to perform further basic and clinical studies in the bovine model in order to achieve the desired effectiveness. This is the first report in which a novel vaccine candidate against Babesia bovis was constructed based on a recombinant and rationally designed viral vector and evaluated in the biological model of the disease.

Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis.

The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known-positive and 300 known-negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen's kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68-80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present.

Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens.

Molecular identification of Anaplasma marginale in two autochthonous South American wild species revealed an identical new genotype and its phylogenetic relationship with those of bovines.

BACKGROUND: Anaplasma marginale is a well-known cattle pathogen of tropical and subtropical world regions. Even though, this obligate intracellular bacterium has been reported in other host species different than bovine, it has never been documented in Myrmecophaga tridactyla (giant anteater) or Hippocamelus antisense (taruca), which are two native endangered species. METHODS: Samples from two sick wild animals: a Myrmecophaga tridactyla (blood) and a Hippocamelus antisense (blood and serum) were studied for the presence of A. marginale DNA through msp5 gene fragment amplification. Further characterization was done through MSP1a tandem repeats analysis and MLST scheme and the genetic relationship among previously characterized A. marginale sequences were studied by applying, eBURST algorithm and AMOVA analysis. RESULTS: Anaplasma marginale DNA was identified in the Myrmecophaga tridactyla and Hippocamelus antisense samples. Through molecular markers, we identified an identical genotype in both animals that was not previously reported in bovine host. The analysis through eBURST and AMOVA revealed no differentiation between the taruca/anteater isolate and the bovine group. CONCLUSIONS: In the present publication we report the identification of A. marginale DNA in a novel ruminant (Hippocamelus antisense) and non-ruminant (Myrmecophaga tridactyla) host species. Genotyping analysis of isolates demonstrated the close relatedness of the new isolate with the circulation population of A. marginale in livestock. Further analysis is needed to understand whether these two hosts contribute to the anaplasmosis epidemiology.

Social and environmental health determinants and their relationship with parasitic diseases in asymptomatic children from a shantytown in Buenos Aires, Argentina.

Health inequities are a common problem for all countries and are the result of not only adverse social conditions but also poor public policies. Today chronic diseases represent the most relevant threats and are a current challenge. Parasitic infections, a leading cause of child morbidity affecting low-income populations, can be transmitted because of an unhealthy environment. Notwithstanding, scarce data have been published on the epidemiological profile of intestinal parasitoses in asymptomatic children living in shantytowns. Vulnerable populations settled in slums are growing in Argentina, particularly in Buenos Aires city. Consequently, this work intended to screen healthy carriers of enteric parasites and determine the epidemiologic profile in asymptomatic children residing in one of those communities, to explore risk factors associated with the transmission of parasites, and to initiate a basic health education campaign to promote healthy behavior in the community. Fecal samples (n = 138) were analyzed by conventional parasitological methods and a survey gathered data on symptoms, family composition, and environmental and hygiene-related variables. High prevalence of feco-orally-transmitted parasitoses (83·3%) and polyparasitism were remarkable findings. The main environmental health determinants were those related to excreta disposal and water provision. Health promotion actions were performed through the diffusion of a set of posters with iconic images and brief messages for health education. Results suggest the need for an environmental sanitation policy to complement health promotion actions. It is essential to spread the results of investigations that address inequities and social determinants of health in order to integrate data with local political processes and alert on acceptable actions for developing appropriate interventions.