Standardization of a SYBR Green Based Real-Time PCR System for Detection and Molecular Quantification of Babesia bovis and B. bigemina in Water Buffaloes (Bubalus bubalis)

Water buffalo (Bubalus bubalis) is a potential reservoir for Babesia bovis and B. bigemina in tropical regions, but the epidemiological evidence of their reservoir competence is limited, especially due to the lack of diagnostic tests capable of detecting and quantifying the low-level parasitemia present in the carrier animals. In this paper we present the standardization process of a SYBR Green based real-time PCR system (qPCR), consisting of two single qPCR assays, for the detection and quantification of B. bovis and/or B. bigemina. Both assays were optimized in similar protocols, including reagent concentrations and thermocycling parameters, so it is possible its use as a multiple qPCR in a single run. Both single assays showed a suitable analytical performance, especially by allowing detection of a greater number of carrier animals when compared with nested PCR assays (nPCR) against a reference panel of 60 DNA samples extracted from blood of both, infected- and non-infected buffaloes. Furthermore, a mathematical algorithm to convert the qPCR outcomes in percent of infected red blood cell was used, and was found that the estimated parasitemia in carrier buffaloes within the reference sample panels were close to those described in carrier cattle. This method could be a useful tool for epidemiological studies on the participation of the bubaline specie in the epidemic process of bovine babesiosis

Microsatellite analysis supports clonal propagation and reduced divergence of Trypanosoma vivax from asymptomatic to fatally infected livestock in South America compared to West Africa

Background: Mechanical transmission of the major livestock pathogen Trypanosoma vivax by other biting flies than\ud tsetse allows its spread from Africa to the New World. Genetic studies are restricted to a small number of isolates\ud and based on molecular markers that evolve too slowly to resolve the relationships between American and West\ud African populations and, thus, unable us to uncover the recent history of T. vivax in the New World.\ud Methods: T. vivax genetic diversity, population structure and the source of outbreaks was investigated through the\ud microsatellite multiloci (7 loci) genotype (MLGs) analysis in South America (47isolates from Brazil, Venezuela and\ud French Guiana) and West Africa (12 isolates from The Gambia, Burkina Faso, Ghana, Benin and Nigeria).\ud Relationships among MLGs were explored using phylogenetic, principal component and STRUCTURE analyses.\ud Results: Although closely phylogenetically related, for the first time, genetic differences were detected between\ud T. vivax isolates from South America (11 genotypes/47 isolates) and West Africa (12 genotypes/12 isolates) with no\ud MLGs in common. Diversity was far greater across West Africa than in South America, where genotypes from Brazil\ud (MLG1-6), Venezuela (MLG7-10) and French Guiana (MLG11) shared similar but not identical allele composition. No\ud MLG was exclusive to asymptomatic (endemic areas) or sick (outbreaks in non-endemic areas) animals, but only\ud MLGs1, 2 and 3 were responsible for severe haematological and neurological disorders.\ud Conclusions: Our results revealed closely related genotypes of T. vivax in Brazil and Venezuela, regardless of\ud endemicity and clinical conditions of the infected livestock. The MLGs analysis from T. vivax across SA and WA\ud support clonal propagation, and is consistent with the hypothesis that the SA populations examined here derived\ud from common ancestors recently introduced from West Africa. The molecular markers defined here are valuable to\ud assess the genetic diversity, to track the source and dispersion of outbreaks, and to explore the epidemiological\ud and pathological significance of T. vivax genotypes.This work was funded through projects within the PROAFRICA and PROSUL programs from the Brazilian agency CNPq. We are grateful to Professor Erney P. Camargo for the joint coordination of these projects and helpful commentaries on the manuscript. HAG was funded by a CDCH-UCV studentship from Venezuela; ACR is a postdoctoral fellow of PNPD-CAPES and CMFR is recipient of PhD scholarships from CNPq-PROTAX. The authors would like to acknowledge for clinical and epidemiological information, blood samples of T. vivax infected livestock and valuable help in the fieldwork several colleagues Garcia et al. Parasites & Vectors 2014, 7:210 Page 11 of 13\ud http://www.parasitesandvectors.com/content/7/1/210 from African countries, Venezuela and Brazil (Galiza GF, Da Silva A and Cadioli L\ud also for previous joint studies). We are grateful to The Wellcome Trust for making available sequences from the genome of T. vivax from Sanger Institute. We are deeply in debt to Wendy Gibson (Bristol University, UK) for helpful discussions and suggestions that much improved our manuscript

Variant antigen diversity in Trypanosoma vivax is not driven by recombination.

African trypanosomes (Trypanosoma) are vector-borne haemoparasites that survive in the vertebrate bloodstream through antigenic variation of their Variant Surface Glycoprotein (VSG). Recombination, or rather segmented gene conversion, is fundamental in Trypanosoma brucei for both VSG gene switching and for generating antigenic diversity during infections. Trypanosoma vivax is a related, livestock pathogen whose VSG lack structures that facilitate gene conversion in T. brucei and mechanisms underlying its antigenic diversity are poorly understood. Here we show that species-wide VSG repertoire is broadly conserved across diverse T. vivax clinical strains and has limited antigenic repertoire. We use variant antigen profiling, coalescent approaches and experimental infections to show that recombination plays little role in diversifying T. vivax VSG sequences. These results have immediate consequences for both the current mechanistic model of antigenic variation in African trypanosomes and species differences in virulence and transmission, requiring reconsideration of the wider epidemiology of animal African trypanosomiasis

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

A leishmaniose visceral é uma zoonose emergente, com elevado número de novos casos no Brasil, onde o cão doméstico é um importante reservatório do parasito no ciclo infeccioso da Leishmania chagasi. Um ensaio de imunoadsorção enzimática (ELISA) baseado em antígeno bruto de L. chagasi foi adaptado para a detecção de anticorpos IgM em soros de cães infectados. As diluições ótimas do antígeno e dos soros controles positivo e negativo foram determinadas através de titulação em bloco. A sensibilidade e especificidade do ELISA teste foram de 100%. Um total de 110 amostras de soros foram obtidas de cães oriundos de Belo Horizonte, Minas Gerais, Brasil, e avaliadas pelo ELISA e pela reação de imunofluorescência indireta (RIFI) para anticorpos IgM anti-L. chagasi. Aproximadamente 25% (n=27) dos cães testados foram sorologicamente positivos para L. chagasi pela RIFI, enquanto 89.09% foram soropositivos pelo ELISA. Os resultados obtidos pelo ELISA e pela RIFI foram significativamente diferentes (P<0.01). A associação do ELISA e da RIFI deve ser recomendada a fim de permitir a detecção mais eficiente da leishmaniose visceral canina pelos serviços veterinários.Visceral leishmaniasis is an emergent zoonosis with an increasing number of new cases in Brazil where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. An enzyme-linked immunosorbent assay (ELISA), based upon the use of a total soluble antigenic preparation of L. chagasi, was adapted for the detection of IgM antibodies in the serum of infected dogs. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 110 serum samples were taken from dogs in Belo Horizonte, Minas Gerais, Brazil, and examined for anti-L. chagasi IgM antibody by ELISA and indirect fluorescent antibody test (IFAT). About 25% (n=27) of all the dogs tested were found serologically positive for L. chagasi by IFAT, while 89.09% (n=98) were seropositive by ELISA. The results obtained by ELISA and IFAT were significantly different (P<0.01). The combined use of ELISA and IFAT is recommended in order to enable veterinary services to more efficiently detect canine visceral leishmaniasis.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Resposta imune-humoral de búfalos (Bubalus bubalis) contra Anaplasma marginale (Theiler, 1910)

O objetivo do presente estudo foi analisar a resposta imune humoral anti-Anaplasma marginale em búfalos (Bubalus bubalis) naturalmente infectados. Amostras de soro e de colostro/leite coletadas de búfalas adultas por um período de 335 dias após o parto e de soros dos seus bezerros do nascimento até 365 dias de vida, foram usadas para a realização do método ELISA indireto. Os dados foram analisados como a média de um grupo de animais, em diferentes faixas etárias, durantes os anos de 1999/2000 e individualmente (duas búfalas e dois bezerros), no ano de 2005. Os soros dos animais analisados em grupos apresentaram títulos de anticorpos abaixo do ponto de corte (D.O. = 0,265 e NE > 3) durante os primeiros 90 e 105 dias, para as búfalas e para os seus bezerros, respectivamente, mas em seguida, elevaram-se para níveis acima do ponto de corte até o final do trabalho, ou seja, um ano após, indicando uma imunidade ativa adquirida. Das duas búfalas examinadas individualmente, os anticorpos colostrais foram detectados em altos níveis, mas os sorológicos em baixos níveis durante os primeiros sete dias após o parto, sugerindo uma transferência de anticorpos do soro para a glândula mamária. da mesma forma, os dois bezerros tiveram títulos de anticorpos detectáveis já nas primeiras 24 horas após mamarem o colostro, indicando uma imunidade colostral passiva. em conclusão, os búfalos desenvolveram uma resposta imune humoral específica contra A. marginale e foram considerados portadores deste parasita.The aim of the present study was to analyze the humoral-immune response of water buffalo (Bubalus bubalis) naturally infected against Anaplasma marginale. For this work, colostrums/milk and blood samples were sequentially collected from buffalo cows prior and after partum for a period of 335 days and from buffalo calves from birth to 365 days after. The antibodies in the colostrums/milk and serum samples of these animals were determined using an ELISA indirect method and the data were analyzed as a mean of a group of animals with the matched ages during the period of 1999/2000 or individually during the year of 2005. The data from animals analyzed in group showed that the antibodies against A. marginale were in low concentration (below the cut off point: D.O. = 0.265 and ELISA levels, EL > 3), in the sera of buffalo, during the first 90 and 105 days, respectively for cows and calves. Then, the levels of antibodies in the serum samples of buffalo calves, slightly raised to above the cut off point and kept in higher levels up to approximately 365 days after birth, indicating active acquired immunity. Furthermore, when the animals were individually examined, the buffalo cows showed high antibody levels in the colostrums, but low levels in the blood stream during the first seven days post-partum, suggesting antibody transference from blood to mammary gland n addition to that, buffalo calves showed high antibody levels during the first 24 hours after suckling colostrum, indicating a colostral passive immunity. By conclusion, the buffalos were able to arm a humoral immune response against A. marginale and were considered reservoir of this parasite.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq