Parasitismo natural de bufalinos em Botucatu, SP, Brasil: III. Dinâmica do parasitismo gastro-intestinal em vacas e suas crias

Gastro-intestinal parasitism of 24 buffalo cows before parturition, and post-parturition, their infection and that of their respective calves during the following 30 weeks were studied. Willis, Hoffmann and whenever possible, the modified Gordon & Whitlock techniques were used for fecal examinations. Toxocara vitulorum eggs were the earliest forms encountered in calves feces, as follows: during the 1st week after birth, 58.33% of the calves were positive, and in the 4th week, 100% of these animals were positive. Eggs of Strongyloides sp were in the 1st week after birth in two of the calves and in the 5th week, all for them were positive. The next parasites to appear were the Coccidia of which oocysts were detected in the feces of two calves in the 2nd week after birth, and 58.33% of the calves were positive for these in the 3rd week, and in the 6th week, all calves shed oocysts in their feces. On the other hand, eggs of Strongylids were the last forms to appear in calves feces. However, despite their sporadic appearance in the feces, eggs of these parasites were observed continuously from the 11th week onwards, and at this point, the percentage of positive samples began to increase to reach its peak. Relatively to adult animals, eggs of T. vitulorum were observed in the feces of 11 cows, one or twice at most; eggs of Strongyloides sp were seen only once in the feces of four buffalo cows and eggs of Strongylids in 21 out of 24 cows. Oocysts of Coccidia were observed in 16 cows. Mechanisms of infestation of calves with these parasites are discussed.

Development of a vaccine strategy against human and bovine schistosomiasis: background and update

Schistosomiasis is a chronic and debilitating parasitic disease that affects over 200 million people throughout the world and causes about 500,000 deaths annually. Two specific characteristics of schistosome infection are of primordial importance to the development of a vaccine: schistosomes do not multiply within the tissues of their definitive hosts (unlike protozoan parasites) and a partial non-sterilizing immunity can have a marked effect on the incidence of pathology and on disease transmission. Since viable eggs are the cause of disease pathology, a reduction in worm fecundity whether or not accompanied by a reduction in parasite burden is a sufficient goal for vaccine induced immunity. We originally showed that IgE antibodies played in experimental models a pivotal role for the development of protective immunity. These laboratory findings have been now confirmed in human populations. Following the molecular cloning and expression of a protein 28 kDa protein of Schistosoma mansoni and its identification as a glutathion S-transferase, immunization experiments have been undertaken in several animal species (rats, mice, baboons). Together with a significant reduction in parasite burden, vaccination with Sm28 GST was recently shown to reduce significantly parasite fecundity and egg viability leading to a decrease in liver pathology. Whereas IgE antibodies were shown to be correlated with protection against infection, IgA antibodies have been identified as one of the factors affecting egg laying and viability. In human populations, a close association was found between IgA antibody production to Sm28 GST and the decrease of egg output. The use of appropriate monoclonal antibody probes has allowed the demonstration that the inhibition of parasite fecundity following immunization was related to the inhibition of enzymatic activity of the molecule. Epitope mapping of Sm28 GST has indicated the prominent role of the N and C terminal domains. Immunization with the corresponding synthetic peptides was followed by a decrease of 70% of parasite fecundity and egg viability. As a preliminary step towards phase I human trials, vaccination experiments have been performed in cattle, a natural model for Schistosoma bovis. Vaccination of calves with the S. bovis GST has led to a reduction of ever 80% of egg output and tissue egg count. Significant levels of protection were also observed in goats after immunization with the recombinant S. bovis GST. Increasing evidence of the participation of IgA antibodies in protective immunity has prompted us toward the development of mucosal immunization. Preliminary results indicate that significant levels of protection can be achieved following oral immunization with live attenuated vectors or liposomes. These studies seem to represent a promising approach towards the future development of a vaccine strategy against one of major human parasitic diseases.

Ultrastructural Details of Cryptosporidium parvum Development in Calf Intestine

Cryptosporidium parvum and C. muris appear to be different species found in calves, with different oocysts size and distribution on the gastrointestinal tract. This work presents new images of C. parvum ultrastructure in calf intestine, mainly its development in nonmicrovillous cells and the presence of microtubular structures in the membrane enveloping the macrogamonts and immature oocysts.

Infection rate of Babesia spp. sporokinetes in engorged Boophilus microplus from an area of enzootic stability in the State of Minas Gerais, Brazil

The infection rates of Babesia sporokinetes in engorged Boophilus microplus were evaluated during a 2-year period in a dairy farm located in an area of enzootic stability. Every 14 days engorged females were collected from calves and from adult animals. Ticks were incubated at 27 ± 0.5ºC and 80-90% relative humidity and Babesia infection rates were determined by microscopic examination of Giemsa-stained hemolymph smears. After 52 collections, 2105 ticks were obtained, from which 982 were collected from calves and 1123 from cows. The total Babesia infection rate was 10%, however the incidence was higher (p < 0.05) in ticks collected from calves (17.5%) than in those collected from cows (3.6%). Females collected from cows showed the highest infection rates in January, March, and August, and absence of infection in April and May. Ticks feeding on calves were infected throughout the experimental period. The infection rates of engorged females collected from naturally infected calves that were artificially infested with Babesia-free-larvae of B. microplus gradually decreased until the calves were four months old. No differences were observed among infection rates of ticks collected from calves maintained under natural conditions.

Fossil psychodoid flies and their relation to parasitic diseases

Psychodid sand flies are blood-sucking fly vectors of several parasitic diseases. The oldest definitive record of this group is from the Lower Cretaceous amber of Lebanon (circa -135 to -125 My), but the high diversity within this group supports the idea that the psychodoids originated much earlier in history. The palaeontology demonstrates that the Lower Cretaceous representatives of the different subfamilies of Psychodidae had similar morphology and were blood-feeders, which supports Hennig's hypothesis on the ground plan structure of this family. Historical relationship between sand flies and diseases is unclear up to the present time, but this relationship could be as old as the origin of psychodoids because of the blood-feeding life mode.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

A Neospora caninum 17 kDa protein fraction (p17) has been described as an immunodominant antigen (IDA) under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17) was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86).

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment lenght polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10% IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71% at 48 h vs 14.5%), even after two weeks (47% vs 1.9%). Also, the percentage of extracellular stages augmented (25.3% vs 1.1% at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

Molecular characterization of G and P-types bovine rotavirus strains from Goiás, Brazil: high frequency of mixed P-type infections

In this study, 331 samples from calves less than one month old from a dairy herd in the district of Piracanjuba, state of Goiás, Brazil were tested for rotavirus. Thirty-three samples (9.9%) tested positive for rotavirus. Out of those, 31 were submitted to G and P characterization by reverse transcription followed by semi-nested polymerase chain reaction. Two samples were characterized as G6P[1], three as G10P[11] and five as G6P[11]. The majority of the samples (51.6%) displayed multiple P genotypes (P-genotype mixtures), including typical human genotypes P[4] and P[6M], suggesting the occurrence of co-infections and genetic reassortment. Also, the detection of human genotypes in bovine samples may be considered evidence of the zoonotic potential of rotaviruses. To our knowledge, this is the first report of such a high frequency of P genotype mixtures in bovine rotavirus samples. It also increases data on G and P rotavirus genotypes circulating in dairy herds in Brazil and can help in the development of more efficient immunization approaches, thereby controlling infection and reducing economical losses.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

Clostridium difficile ribotypes in humans and animals in Brazil

Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies aboutC. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficileribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen.

Termite assemblages in five semideciduous Atlantic Forest fragments in the northern coastland limit of the biome

Termites are abundant organisms in tropical ecosystems and strongly influence the litter decomposition and soil formation. Despite their importance, few studies about their assemblage structures have been made in Brazilian Atlantic Forest fragments, especially in the area located north of the São Francisco River. This study aims to analyze the assemblage composition of five Atlantic Forest fragments located in the northern biome limit along the Brazilian coast. A standardized sampling protocol of termites was applied in each fragment. Thirty-three termite species belonging to twenty genera and three families were found in the forest fragments. The wood-feeder group was dominant both concerning to species richness and number of encounters in all areas. In sites northern to 7°S, there is an evident simplification of the termite assemblage composition regarding species richness and number of encounters by feeding group. This fact is apparently due to a higher sandy level in soils and to semideciduous character of the vegetation in the northern fragments. Thus, even on the north of São Francisco River, termite biodiversity is heterogeneously spread with highest density of species in the portion between 07°S and São Francisco River mouth (10°29'S).

Behaviour of pre-parturient sows housed in intensive outdoor or indoor systems

This work compared the behaviour of pre-parturient sows housed in intensive confined and outdoor systems, during the morning and afternoon prior to delivery. Eight sows were kept individually in farrowing crates or in paddocks with access to fresh pasture from 8 to 10 days before expected parturition. All outdoor sows built a nest within 24 hours before farrowing. On the morning and afternoon before farrowing they spent 6.5% of the time collecting grass and 7.5% of the time organizing the nest. Outdoor sows were observed more often than confined sows rooting (4.60% vs. 0.25%), feeding (10.2% vs. 3.0%), standing (51% vs. 13%) and walking (8% vs. 0%). Indoor sows redirected the nesting behaviour to other behaviours like attempts to dig the ground, nosing, biting and rooting parts of the crate, feeder or drinker, during 4.7% of the time. They also spent more time than outdoor sows inactive (85% vs. 60%), lying (72% vs. 41%), drinking (2.1% vs. 0.5%) and vacuum chewing (3.7% vs. 0.1%). The pre-parturient behaviour of sows was considerably affected by the husbandry system. The outdoor system appears to be more appropriate for the sows' welfare than the conventional confinement.

Body condition score to predict the postpartum fertility of crossbred beef cows

The relationship between changes in body condition score (BCS) during the postpartum and fertility in beef cows suckling calves under extensive conditions were investigated. Cows were subjected to four BCS evaluations over the postpartum period, starting around one month after calving. In the second evaluation cows were treated with medroxy-progesterone acetate impregnated pessaries and received an injection of estradiol benzoate. At the third evaluation, pessaries were removed and calves were separated from the cows for 96 hours, during which time estrous was observed twice a day, and animals artificially inseminated 12 hours after detection. When calves returned to their dams, bulls were introduced until a 60-day mating period was reached. The distribution of BCS differed among calving groups and evaluations. Results indicated that only cows comprising a BCS 3 (1 to 5 scale) around the first month postpartum can be used in an artificial insemination program with possibilities of becoming pregnant. There was no statistical difference between the calving groups in pregnancy rate. The evolution of the BCS of the cows during postpartum can be used to adjust the start of the breeding season to coincide with the time of the year where herd pregnancy rates will be highest.