Detection and Characterization of Hepatitis E Virus in Domestic Pigs of Different Ages in Portugal

This study represents the primary hepatitis E virus (HEV) surveillance in domestic pigs in Portugal, five pig farms were investigated in 5 different Portuguese regions, ten faecal samples were collected at four different stages of the production. All faecal samples were tested for hepatitis E virus by real-time RT-PCR. At least one sample from each farms of all age groups tested positive for HEV. The prevalence in the pig herds varied from 10% to 30% and the mean prevalence was 32% in weaners, 20% in growers, 32% in fatteners and 4% in adult dry sows. Phylogenetic analysis of the detected HEV sequences indicated that the circulating virus strains belong under the genotype 3.     

Supplementation of citric acid in plant protein‐based diets for juvenile tambaqui,Colossoma macropomum

We evaluated citric acid supplementation (0, 10, 20, and 30 g/kg) in plant protein‐based diets for juvenile tambaqui, Colossoma macropomum. In Experiment 1, fish (n = 160; 27.56 ± 2.73 g) were distributed in 310 L tanks (n = 16) for 80 days. Zootechnical performance, hematological parameters, and mineral composition (muscle and bones) did not differ significantly (p > .05). The inclusion of citric acid‐reduced pH of diets (p = .001) and stomach (p = .035). In Experiment 2, fish (n = 60; 48.00 ± 1.98 g) were distributed in fecal collection aquaria (200 L) for 21 days. The following were observed: a linear effect for the crude protein (p = .0004) and crude fat (p = .0037) digestibility coefficients; a quadratic effect for crude energy (p = .0037) and dry matter (p = .0004); an optimal supplementation level of 18.5 g/kg. Calcium (p = .0060) and magnesium (p = .0222) showed a linear effect, while phosphorus (p = .0001) had a quadratic effect with the 20 g/kg optimum supplementation level. To conclude, the supplementation of this organic acid in diets does not negatively affect fish nutritional status, health or welfare, and increases nutrient and mineral availability.     

Inferred longevity of Amazonian rainforest trees based on a long-term demographic study

We used data from a long-term (14–18 years) demographic study to infer the maximum longevity for populations of 93 relatively abundant tree species in central Amazonia. We also assessed the influence of several life-history features (wood density, growth form, mortality rate, recruitment rate, stem diameter, growth increment, population density) on tree longevity. Data on 3159 individual trees were collected in 24 permanent, 1 ha plots in undisturbed forest arrayed across a large (ca. 1000 km²) study area. For each species, three estimates of longevity were generated (by dividing the stem diameter of the largest tree by the median, upper quartile, and upper decile of observed diameter-growth rates), and the mean of these three values was used as a longevity estimate. Longevity values ranged from 48 years in the pioneer Pourouma bicolor (Cecropiaceae) to 981 years for the canopy tree Pouteria manaosensis (Sapotaceae), with an overall mean of 336±196 years. These growth-based estimates of maximum tree age were concordant with those derived from analyses of mean mortality rates. Tree longevity was positively correlated with wood density, maximum stem diameter, and population density, and negatively correlated with annual mortality, recruitment, and growth rates. On average, pioneer species had much lower longevity than did non-pioneers, whereas among old-growth trees, emergent species had greater longevity than did canopy species. Our results are consistent with radiocarbon-based studies that suggest that Amazonian trees can occasionally exceed 1000 years of age.     

A comparative study of methods for diagnosis of obesity in an urban mixed-race population in Minas Gerais, Brazil

BACKGROUND: Obesity is defined as an excess of total body fat and may be assessed by different methods. The objective of the present study was to establish the discriminatory power of anthropometric data in determining obesity. METHODS: The subjects comprised 685 individuals, aged 20-79 years, sampled from a population-based survey. The following indicators were used: body mass index (BMI), waist circumference (WC) and total body fat percentage estimated with both Siri's equation (%BF Siri) and foot-to-foot bioelectrical impedance analysis (%BF BIA). Sensitivity and specificity of different cut-off points for each method were determined using %BF BIA as reference. RESULTS: Of 685 participants, 57.6% were aged >/= 40 years, 69.9% were women and 72.6% self-referred themselves as non-white. To classify obesity based on sex and age among women aged 0.05). Classifying according to skin colour did not change cut-off points in any indicator. CONCLUSION: BMI and WC better discriminate obesity among women and men aged >/= 40 years from a mixed-race population.     

Kinetics of energy source utilization in Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) embryonic development

The present work evaluates the kinetics of utilization of the main potential energy sources throughout the embryonic developmental stages of Boophilus microplus. The embryonic development of this arthropod is completed in 21 days. Cellularization of the blastoderm occurs on the 6th day and is rapidly followed by germ band extension and segmentation, whose first signs are visible on the 7th day. Cellularization is typically a maternal-driven process, carried out by molecular determinants deposited in the oocyte during oogenesis. On the other hand, segmentation is of zygotic nature, being the consequence of the synthesis of various components by the growing embryo. The enhancement in total B. microplus RNA was observed after cellularization, corroborating the replacement of maternal-driven processes by embryonic zygotic expression. An abrupt increase in oxygen consumption was observed from cellularization until the 8th day of development. The reduction in dry weight at the same period and the susceptibility of oxygen consumption to KCN suggest that the respiration process is activated during early embryonic development. A marked decrease in total lipid content occurred between the 5th and 7th days of development, suggesting this is the main energy source for cellularization. A major reduction in carbohydrate content occurred later, between the 7th and 9th days, and it could be assigned to the morphological segmentation of the embryo. Although the total amount of proteins remains unchanged from oviposition to hatching, a 15% reduction in vitellin (VT) content was observed before cellularization, up to the 4th day after egglaying. This observation was correlated to the synthesis of new proteins needed to support early embryo development. Additional 20% of VT was consumed thereafter, mainly at the end of embryogenesis, and in this case VT is probably used as energy source to the older embryo. Altogether, these data indicate different energy sources for maternal and zygotic driven processes.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Apparent tick paralysis by Rhipicephalus sanguineus (Acari: Ixodidae) in dogs

Certain tick species including Ixodes holocyclus can inoculate neurotoxins that induce a rapid, ascending flaccid paralysis in animals. Rhipicephalus sanguineus, the most widespread tick of dogs, is recognized as a vector of several pathogens causing diseases in dogs and humans. A single report suggests its role as cause of paralysis in dogs. This study presents the clinical history of 14 young dogs heavily infested by R. sanguineus (intensity of infestation, 63-328) in an endemic area of southern Italy. During May to June of 2011, dogs were presented at the clinical examination with neurological signs of different degrees (e.g., hind limb ataxia, generalized lethargy, and difficulty in movements). All animals were treated with acaricides and by manual tick removal but ten of them died within a day, displaying neurological signs. The other 4 dogs recovered within 3 days with acaricidal and supportive treatment. Twelve dogs were positive by blood smear examination for Hepatozoon canis with a high parasitemia, two also for Babesia vogeli and two were negative for hemoparasites. Low-grade thrombocytopenia, hypoalbuminemia, and pancytopenia were the haematological alterations most frequently recorded. Other causes of neurological disease in dogs were excluded and the diagnosis of tick paralysis by R. sanguineus was confirmed (ex juvantibus) by early and complete recovery of 4 dogs following acaricidal treatment and tick removal.     

First report and molecular characterization of Trypanosoma vivax in cattle from state of Pernambuco, Brazil

This paper describes an outbreak of Trypanosoma vivax for the first time in the state of Pernambuco, Brazil, affecting dairy cattle in the municipality of Itambé in the northern coastal zone of the state. Clinical signs compatible with infection by blood protozoa and epidemic miscarriages were observed. The diagnosis of T. vivax was confirmed through biometric microscopy and molecular analysis with PCR and DNA sequencing. The T. vivax isolate detected in the present study proved to be genetically very close to other Brazilian isolates of the protozoan despite being geographically distant.     

Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis

Control of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE and AdjuPrime. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE or AdjuPrime resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE in efficacy field trials against canine VL.