Flow cytometric assay in peripheral blood of dogs--reference values for leukocytes from Brazilian beagles

Use of domestic reference values in the flow cytometry analysis is known to improve its accuracy by integrating local variations as gender, race and age. Up to date application of flow cytometry in veterinary medicine has been limited to describe the percentual values just for peripheral lymphocytes subsets of blood. We now report establishment of reference values for a wide range of proportional and absolute numbers of peripheral blood leukocytes, including T cells subsets, B cells, monocytes and eosinophils, applicable to the healthy population of Beagles in Brazil and other regions with similar demographic characteristics. Normal reference values were also established to estimate the gender-related differences. This information will provide clinical aid in the evaluation of immunologic status as well as standard values for experimental animals of dogs from Brazil and other similar regions.     

Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis

The vectorial competence of the tick Rhipicephalus sanguineus is discussed in relation to the epidemiology of canine visceral leishmaniasis, taking into account its strict association with dogs and the low indices of natural infection presented by its known vector, the phlebotomine sand fly Lutzomyia longipalpis. In order to evaluate natural infection by Leishmania chagasi and the infectivity of these parasites in the tick, 39 specimens (6 females, 11 males and 22 nymphs) of R. sanguineus were removed from 21 dogs showing diverse symptoms of zoonotic visceral leishmaniasis (ZVL). Six ticks (15.4%) gave positive results for the genus Leishmania using the PCR technique. To determine the infectivity of the parasites, 36 hamsters were inoculated orally and peritoneally with macerates of ticks removed from nine dogs symptomatic for visceral leishmaniasis. After 6 months the hamsters were sacrificed and necropsied. Serum was removed for IFAT, as well as spleen and liver fragments to make imprint smears and for PCR. Eight (88.9%) of these dogs presented ticks that were infective for 14 hamsters (41.2%), 12 (85.7%) of them infected peritoneally and two (14.3%) orally. PCR revealed 27 smears (40.9%) to be positive, 20 (62.5%) of them infected peritoneally and seven (20.6%) orally. IFAT showed 14 positive animals (41.2%). Based on these findings, we suggest that the vectorial capacity of R. sanguineus for L. chagasi should be evaluated further, opening new perspectives in the epidemiology of ZVL.     

Breed identity and leadership in a mixed flock of sheep

The aim of this study was to analyze the breed identity and leadership in a mixed flock of sheep. The flock consisted of 89 Suffolk adult ewes and 250 Columbia ewes and 45 Columbia rams. The animals were kept in the pasture during the day. Each hour the flock was allowed to feed for 20 minutes, and then was moved for 40 minutes to a small paddock near the grazing area. In the afternoon, all sheep were taken to the night pen. The animals were observed for 120 days, 4 times a day (480 leadership breed positions), with respect to the following conditions: (1) leave pen at front of flock, (2) return to pen at front of flock, (3) first to move to the grassland, (4) return to the paddock, (5) drink first (on arrival time at the night pen), and (6) preference for an area in the night barn. The Suffolk breed were first in leaving (94% of the times) and in returning to the night pen (65%) (P < 0.05), and 35% returned with the Columbia sheep. The Suffolk breed were first to move to the grazing area (90%) and 10% moved with the other breed (P < 0.05). When returning to the rest area, the Columbia sheep were first to reach 54% of the time, the 2 breeds were together 29% of the time, and the Suffolk sheep were alone 17% of the time (P < 0.05). When comparing who drank first, 50% of the time both breeds went together, 28% of the time the Columbia sheep were first, and 22% of the time the Suffolk sheep were first (P < 0.05). At night, most of the Suffolk sheep had a preference for 1 barn area (P < 0.05). The results of our study suggest that most animals of each breed performed activities together. Suffolk sheep were leaders when moving to the grazing area and when selecting the place to rest.     

Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis

We have performed a detailed investigation in 40 dogs naturally infected with Leishmania infantum (syn. chagasi), subdivided into three groups: asymptomatic (AD = 12), oligosymptomatic (OD = 12) and symptomatic (SD = 16), based on their clinical features. Twenty non-infected dogs (CD) were included as control group. Serological analysis, performed by IFAT and ELISA, demonstrated higher antibodies titers in SD in comparison to the AD. A positive correlation was found between parasite density in the spleen and skin smears as well as the bone marrow parasitism with clinical status of the infection. We observed that the progression of the disease from asymptomatic to symptomatic clinical form was accompanied by intense parasitism in the bone marrow. It is likely that this led to the impaired biochemical/hematological status observed. Finally, we believe that the follow-up of these parameters could be a relevant approach to be used as markers during therapeutic and vaccine evaluations.     

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.     

Activity of Ricinus communis (Euphorbiaceae) and ricinine against the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae) and the symbiotic fungus Leucoagaricus gongylophorus

The focus of this study was the identification of compounds from plant extracts for use in crop protection. This paper reports on the toxic activity of fractions of leaf extracts of Ricinus communis L (Euphorbiaceae) and isolated active compounds in the leaf-cutting ant Atta sexdens rubropilosa Forel and its symbiotic fungus Leucoagaricus gongylophorus (Singer) M?ller. The main compounds responsible for activity against the fungus and ant in leaf extracts of R communis were found to be fatty acids for the former and ricinine for the ants.     

Halophilic Carotenoids and Breast Cancer: From Salt Marshes to Biomedicine

Breast cancer is the leading cause of death among women worldwide. Over the years, oxidative stress has been linked to the onset and progression of cancer. In addition to the classical histological classification, breast carcinomas are classified into phenotypes according to hormone receptors (estrogen receptor—RE—/progesterone receptor—PR) and growth factor receptor (human epidermal growth factor receptor—HER2) expression. Luminal tumors (ER/PR-positive/HER2-negative) are present in older patients with a better outcome. However, patients with HER2-positive or triple-negative breast cancer (TNBC) (ER/PR/HER2-negative) subtypes still represent highly aggressive behavior, metastasis, poor prognosis, and drug resistance. Therefore, new alternative therapies have become an urgent clinical need. In recent years, anticancer agents based on natural products have been receiving huge interest. In particular, carotenoids are natural compounds present in fruits and vegetables, but algae, bacteria, and archaea also produce them. The antioxidant properties of carotenoids have been studied during the last years due to their potential in preventing and treating multiple diseases, including cancer. Although the effect of carotenoids on breast cancer during in vitro and in vivo studies is promising, clinical trials are still inconclusive. The haloarchaeal carotenoid bacterioruberin holds great promise to the future of biomedicine due to its particular structure, and antioxidant activity. However, much work remains to be performed to draw firm conclusions. This review summarizes the current knowledge on pre-clinical and clinical analysis on the use of carotenoids as chemopreventive and chemotherapeutic agents in breast cancer, highlighting the most recent results regarding the use of bacterioruberin from haloarchaea.     

Fuzzy-logic-based feeder system for intensive tilapia production (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In high-fish-density aquaculture systems, tilapia producers are compelled to provide 100% of food required to obtain profitable growth rates. It is well known that fish have a low food conversion rate and feeding represents the most important expenditure, approximately 40% of total production cost. Therefore, precise quantities of food should be provided to avoid water pollution and economic losses due to food waste when water conditions are inadequate for fish feeding. A way to control food provisions in this work was determined by the conditions of temperature, dissolved oxygen, fish age, and body weight, since these variables have a direct effect on fish metabolism and growth. Thus, a change in metabolism is reflected in a modification of energy requirements and, as a consequence, in variations of food consumption. In this work, a new feeder with fuzzy-logic control algorithms is proposed for fish feeding; this technique allows farmer knowledge to be taken into account in a series of if–then-type rules. To define these rules the temperature and dissolve oxygen were considered in order to provide precise food quantities. The results show minimal differences in growth (P > 0.05) between treatments, important food saving of 29.12% (equivalent to 105.3 kg), and lower water pollution (reduced water dissolved solids and ammonium components) compared with timed feeders. This system provides an important contribution to sustainability of intensive aquaculture systems, increasing productivity and profitability, and optimizing water use.     

Growth performance, survival and maturation of Litopenaeus vannamei (Boone) in an inland CRS with no water reposition

Closed recirculation systems (CRS) present an alternative for providing organisms to the aquaculture. A CRS with zero water exchange was used in the present study; the CRS consisted of a culture and maturation facility, biofilter system and reservoirs tanks. During two consecutive trials, the CRS efficiency was evaluated by assessing the growth, survival and maturation of juveniles into adults Litopenaeus vannamei (Boone). Throughout the study, water quality parameters (temperature, pH, salinity, NH₃, NO₂ and NO₃) and ion concentration were monitored. Most parameters showed fluctuations without significant differences. However, a decrease in pH was observed during the maturation phase, and an increase in phosphorus was detected, in both the trials, compared with that in initial seawater. Growth and survival for juvenile and pre-adult shrimps presented similar variations without significant differences. Female gonadal maturation and spawning rate were not significantly different between trials. Unfortunately, shrimp eggs underwent lysis 6 h after spawning. These results show that the growth, survival and maturation obtained under CRS conditions are reproducible, and suggest the possibility of using these systems for biosecure shrimp culture, protected against eventual diseases outbreaks. The results of this study also suggest the importance of future studies addressing ion concentration changes in a CRS with zero water exchange.     

Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean

Biological nitrogen fixation plays a key role in agriculture sustainability, and assessment of rhizobial diversity contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. Although in recent decades the use of molecular techniques has contributed greatly to enhancing knowledge of rhizobial diversity, concerns remain over simple issues such as the effects of sampling on estimates of diversity. In this study, rhizobia were isolated from nodules of plants grown under field conditions, in pots containing soil, or in Leonard jars receiving a 10⁻² or a 10⁻⁴ serially-diluted soil inoculum, using one exotic (soybean, Glycine max) and one indigenous (common bean, Phaseolus vulgaris) legume species. The experiments were performed using an oxisol with a high population (10⁵ cells g⁻¹ soil) of both soybean rhizobia, composed of naturalized strains introduced in inoculants and of indigenous common-bean rhizobia. BOX-PCR was used to evaluate strain diversity, while RFLP-PCR of the ITS (internally transcribed spacer) region with five restriction enzymes aimed at discriminating rhizobial species. In both analyses the genetic diversity of common-bean rhizobia was greater than that of soybean. For the common bean, diversity was greatly enhanced at the 10⁻⁴ dilution, while for the soybean dilution decreased diversity. Qualitative differences were also observed, as the DNA profiles differed for each treatment in both host plants. Differences obtained can be attributed to dissimilarity in the history of the introduction of both the host plant and the rhizobia (exotic vs. indigenous), to host-plant specificity, rhizobial competitiveness, and population structure, including ease with which some types are released from microcolonies in soil. Therefore, sampling method should be considered both in the interpretation and comparison of the results obtained in different studies, and in the setting of the goals of any study, e.g. selection of competitive strains, or collection of a larger spectrum of rhizobia. Furthermore, effects of sampling should be investigated for each symbiosis.