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.

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.

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.

Sizing the lung in dogs: the inspiratory capacity defines the tidal volume. / Avaliação do tamanho do pulmão em cães: a capacidade inspiratória define o volume corrente.

OBJECTIVE: To evaluate a novel physiological approach for setting the tidal volume in mechanical ventilation according to inspiratory capacity, and to determine if it results in an appropriate mechanical and gas exchange measurements in healthy and critically ill dogs. METHODS: Twenty healthy animals were included in the study to assess the tidal volume expressed as a percentage of inspiratory capacity. For inspiratory capacity measurement, the mechanical ventilator was set as follows: pressure control mode with 35cmH2O of inspired pressure and zero end-expiratory pressure for 5 seconds. Subsequently, the animals were randomized into four groups and ventilated with a tidal volume corresponding to the different percentages of inspiratory capacity. Subsequently, ten critically ill dogs were studied. RESULTS: Healthy dogs ventilated with a tidal volume of 17% of the inspiratory capacity showed normal respiratory mechanics and presented expected PaCO2 values more frequently than the other groups. The respiratory system and transpulmonary driving pressure were significantly higher among the critically ill dogs but below 15 cmH2O in all cases. CONCLUSIONS: The tidal volume based on the inspiratory capacity of each animal has proven to be a useful and simple tool when setting ventilator parameters. A similar approach should also be evaluated in other species, including human beings, if we consider the potential limitations of tidal volume titration based on the calculated ideal body weight.

OBJETIVO: Avaliar uma nova abordagem fisiológica para a determinação do volume corrente em ventilação mecânica, de acordo com a capacidade inspiratória, e determinar se isso resulta em medidas mecânicas e de troca gasosa adequadas em cães saudáveis e em estado crítico. MÉTODOS: Incluíram-se, neste estudo, 24 animais para avaliar o volume corrente expresso como porcentagem da capacidade inspiratória. Para mensuração da capacidade inspiratória, o ventilador mecânico foi regulado como segue: modo controle de pressão, com 35cmH2O de pressão de inspiração e pressão expiratória final de zero, por 5 segundos. Subsequentemente, estudaram-se dez cães em condições clínicas críticas. RESULTADOS: Cães saudáveis ventilados com volume corrente que correspondia a 17% da capacidade inspiratória demonstraram mecânica respiratória normal e apresentaram os valores previstos de PaCO2 mais frequentemente do que os animais nos demais grupos. A pressão no sistema respiratório e a pressão transpulmonar foram significantemente mais elevadas nos cães em condição crítica, porém em todos os casos, estiveram abaixo de 15cmH2O. CONCLUSÕES: O volume corrente calculado com base na capacidade inspiratória de cada animal comprovou ser uma ferramenta útil e simples para o estabelecimento dos parâmetros do ventilador. Convém também realizar abordagem semelhante em outras espécies, inclusive no ser humano, quando se consideram as potenciais limitações da titulação do volume corrente, com base no peso corpóreo ideal calculado.

Avaliação do tamanho do pulmão em cães: a capacidade inspiratória define o volume corrente / Sizing the lung in dogs: the inspiratory capacity defines the tidal volume

RESUMO Objetivo: Avaliar uma nova abordagem fisiológica para a determinação do volume corrente em ventilação mecânica, de acordo com a capacidade inspiratória, e determinar se isso resulta em medidas mecânicas e de troca gasosa adequadas em cães saudáveis e em estado crítico. Métodos: Incluíram-se, neste estudo, 24 animais para avaliar o volume corrente expresso como porcentagem da capacidade inspiratória. Para mensuração da capacidade inspiratória, o ventilador mecânico foi regulado como segue: modo controle de pressão, com 35cmH2O de pressão de inspiração e pressão expiratória final de zero, por 5 segundos. Subsequentemente, estudaram-se dez cães em condições clínicas críticas. Resultados: Cães saudáveis ventilados com volume corrente que correspondia a 17% da capacidade inspiratória demonstraram mecânica respiratória normal e apresentaram os valores previstos de PaCO2 mais frequentemente do que os animais nos demais grupos. A pressão no sistema respiratório e a pressão transpulmonar foram significantemente mais elevadas nos cães em condição crítica, porém em todos os casos, estiveram abaixo de 15cmH2O. Conclusões: O volume corrente calculado com base na capacidade inspiratória de cada animal comprovou ser uma ferramenta útil e simples para o estabelecimento dos parâmetros do ventilador. Convém também realizar abordagem semelhante em outras espécies, inclusive no ser humano, quando se consideram as potenciais limitações da titulação do volume corrente, com base no peso corpóreo ideal calculado.

ABSTRACT Objective: To evaluate a novel physiological approach for setting the tidal volume in mechanical ventilation according to inspiratory capacity, and to determine if it results in an appropriate mechanical and gas exchange measurements in healthy and critically ill dogs. Methods: Twenty healthy animals were included in the study to assess the tidal volume expressed as a percentage of inspiratory capacity. For inspiratory capacity measurement, the mechanical ventilator was set as follows: pressure control mode with 35cmH2O of inspired pressure and zero end-expiratory pressure for 5 seconds. Subsequently, the animals were randomized into four groups and ventilated with a tidal volume corresponding to the different percentages of inspiratory capacity. Subsequently, ten critically ill dogs were studied. Results: Healthy dogs ventilated with a tidal volume of 17% of the inspiratory capacity showed normal respiratory mechanics and presented expected PaCO2 values more frequently than the other groups. The respiratory system and transpulmonary driving pressure were significantly higher among the critically ill dogs but below 15 cmH2O in all cases. Conclusions: The tidal volume based on the inspiratory capacity of each animal has proven to be a useful and simple tool when setting ventilator parameters. A similar approach should also be evaluated in other species, including human beings, if we consider the potential limitations of tidal volume titration based on the calculated ideal body weight.

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.