Resistance of beef cattle of two genetic groups to ectoparasites and gastrointestinal nematodes in the state of São Paulo, Brazil

The resistance to infestations by ectoparasites and infections by gastrointestinal nematodes was studied in 45 animals (males and females) of two genetic groups: purebred Nelore (NI, n=28) and Three-Cross (1/2 Angus+1/4 Canchim+1/4 Nelore - TC, n=17). The animals were monitored for 24months, during which they were left to graze in tropical pastures without receiving treatment for parasites. Each month the animals were examined for infestations by external parasites, to count the numbers of cattle ticks Rhipicephalus microplus with diameter greater than 4.5mm present on the left side, horn flies (Haematobia irritans) present in the lumbar region and botfly larvae (Dermatobia hominis) present on the entire body. The H. irritans counts were performed with the aid of digital photographs. At the time of examination, fecal samples were collected to count the eggs per gram (EPG) and to perform coprocultures, and peripheral blood samples were drawn to determine the packed cell volume (PCV) and to count the eosinophils. For statistical analysis, the count data were transformed into log10 (n+1), where n is the number of parasites. For PCV, significant effects (P<0.05) were found for collection month (CO), genetic group (GG) and gender (SX), with means and respective standard errors of 41.5±0.65% for the NI animals, 39.3±0.83% for the TC, 41.5±0.72% for the females and 39.3±0.77% for the males. Regarding the eosinophil counts, only the effect of sex was significant (P<0.01), with means and respective standard errors of 926.0±46.2/μL, for males and 1088.0±43.8/μL of blood, for females. The NI animals presented lower mean counts for all the external parasites compared to the TC animals (P<0.01). For ticks, the transformed means followed by standard errors for the NI and TC animals were 0.06±0.01 and 0.34±0.02, while for horn flies these were 0.92±0.05 and 1.36±0.06 and for botfly larvae they were 0.05±0.03 and 0.45±0.05, respectively. The average EPG values were only influenced by CO (P<0.01). The coprocultures revealed the presence of the following endoparasites: Haemonchus spp., Cooperia spp., Oesophagostomum spp. and Trichostrongylus spp., the last in smaller proportion. There were no significant differences between the genetic groups for the endoparasite loads, except for Cooperia spp., which were present in greater number (P<0.05) in the NI group. The results obtained in this experiment confirm previous findings of greater susceptibility of the Nelore breed to Cooperia spp. and high resistance to ectoparasites. © 2013 Elsevier B.V.

Differential diagnosis of oocysts of Hammondia-like organisms of dogs and cats by PCR-RFLP analysis of 70-kilodalton heat shock protein (HSP70) gene

Organisms of the genera Toxoplasma, Hammondia and Neospora, the Hammondia-like organisms, are closely related coccidian with similarly sized oocysts. Therefore, a diagnosis based on microscopy of oocysts in feces is not a method of choice for species identification of these important parasites. In this paper, we present a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) method to differentially diagnose oocysts of Toxoplasma gondii from oocyst of Hammondia hammondi. Another PCR-RFLP was designed to differentiate oocysts of Hammondia heydorni from oocysts of Neospora spp. Both PCR-RFLP are based on nucleotide sequences of the Hsp70 coding gene. In conclusion, we presented two alternative molecular diagnostic assays that can be successfully applied for the differentiation of oocysts of Hammondia-like organisms shed by felids and canids.

Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces

In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA), was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5% by IMS-IFA and 15.7% by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage.

Biochemical analysis of female mice urine with reference to endocrine function: a key tool for estrus detection.

Species-specific chemical signals released through urine, sweat, saliva and feces are involved in communication between animals. Urinary biochemical constituents along with pheromones may contribute to variation across reproductive cycles and facilitate to estrus detection. Hence, the present study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lipids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogentreated female mice. In normal mice, the protein and lipid concentrations in urine were significantly higher in proestrus and estrus phases and the quantity of fatty acids was also comparatively higher in estrus. Furthermore, certain fatty acids, namely tridecanoic, palmitic and oleic acids, were present during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. However, the carbohydrate level was equally maintained throughout the four phases of estrous cycle. For successful communication, higher concentrations of protein and specific fatty acids in estrus are directly involved. The significant increase in estradiol at estrus and progesterone at metestrus seems to be of greater importance in the expression pattern of biochemical constituents and may play a notable role in estrous cycle regulation. Thus, we conclude that the variations observed in the concentration of the biochemical constituents depend on the phase of the reproductive cycle as well as hormonal status of animals. The appearance of protein and specific fatty acids during estrus phase raises the possibility to use these as a urinary indicators for estrus detection.

Comparison of two commercial kits and two extraction methods for fecal glucocorticoid analysis in ocelots (Leopardus pardalis) submitted to ACTH challenge

The ocelot (Leopardus pardalis) is included in list of wild felid species protected by CITES and is part of conservation strategies that necessarily involve the use of assisted reproduction techniques, which requires practical and minimally invasive techniques of high reproducibility that permit the study of animal reproductive physiology. The objective of this study was to compare and validate two commercial assays: ImmuChem Double Antibody Corticosterone 125I RIA from ICN Biomedicals, Costa Mesa, CA, USA; and Coat-a-Count Cortisol 125I RIA from DPC, Los Angeles, CA, USA, for assessment of fecal glucocorticoid metabolites in ocelots submitted to ACTH (adrenocorticotropic hormone) challenge. Fecal samples were collected from five ocelots kept at the Brazilian Center of Neotropical Felines, Associação Mata Ciliar, São Paulo, Brazil, and one of the animals was chosen as a negative control. The experiment was conducted over a period of 9 days. On day 0, a total dose of 100 IU ACTH was administered intramuscularly. Immediately after collection the samples were stored at 20C in labeled plastic bags. The hormone metabolites were subsequently extracted and assayed using the two commercial kits. Previously it was performed a trial with the DPC kit to check the best extraction method for hormones metabolites. Data were analyzed with the SAS program for Windows V8 and reported as means ± SEM. The Schwarzenberger extraction method was slightly better when compared with the Wasser extraction method (103,334.56 ± 19,010.37ng/g of wet feces and 59,223.61 ± 12,725.36ng/g of wet feces respectively; P=0,0657). The ICN kit detected an increase in glucocorticoid metabolite concentrations in a more reliable manner. Metabolite concentrations (ng/g wet feces) on day 0 and day 1 were 66,956.28 ± 36,786.93 and 92,991.19 ± 28,555.63 for the DPC kit, and 205,483.32 ± 83,811.32 and 814,578.75 ± 292,150.47 for the ICN kit, respectively. The limit of detection for the ICN kit was 7.7 ng/mL for 100% B/Bo (25ng/mL for 88%B/Bo) and for the DPC kit it was 0.2ug/dL for 90.95% B/Bo (1ug/dL for 81.27% B/Bo). In conclusion it was confirmed that the Schwarzenberger extraction method and the ICN kit are superior for extracting and measuring fecal glucocorticoid metabolites in ocelot fecal samples.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Selenium supplementation of lactating dairy cows: Effect on selenium concentration in blood, milk, urine and feces

The objectives were to determine effects of graded levels of selenized yeast derived from a specific strain of Saccharomyces cerevisiae (CNCM I-3060) on animal performance and in selenium concentrations in the blood, milk, feces, and urine of dairy cows compared with sodium selenite; and to provide preliminary data on the proportion of selenium as selenomethionine in the milk and blood. Twenty Holstein cows were used in a 5 × 5 Latin square design study in which all cows received the same total mixed rations, which varied only in source or concentration of dietary selenium. There were 5 experimental treatments. Total dietary selenium of treatment 1, which received no added selenium, was 0.15 mg/kg of dry matter, whereas values for treatments 2, 3, and 4, derived from selenized yeast, were 0.27, 0.33, and 0.40 mg/kg of dry matter, respectively. Treatment 5 contained 0.25 mg of selenium obtained from sodium selenite/kg of dry matter. There were no significant treatment effects on animal performance, and blood chemistry and hematology showed few treatment effects. Regression analysis noted significant positive linear effects of increasing dietary selenium derived from selenized yeast on selenium concentrations in the milk, blood, urine, and feces. In addition, milk selenium results indicated improved bioavailability of selenium from selenized yeast, compared with sodium selenite. Preliminary analyses showed that compared with sodium selenite, the use of selenized yeast increased the concentration of selenomethionine in the milk and blood. There was no indication of adverse effects on cow health associated with the use of selenized yeast.

Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood, and feces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Chromic oxide as a digestibility marker for dogs - A comparison of methods of analysis

The study reported compared coefficients of apparent digestibility (CAD) obtained by total collection (TC) and by chromic oxide (Cr2O3) determined by visible absorption spectrometry (VIS) and flame atomic absorption spectrophotometry (FAAS). These quantification methods were also investigated to assess their precision. Nineteen adult dogs housed in individual digestibility cages were fed three commercial diets, each test including five or seven animals. The experiment was carried out in a completely randomized design with three among-subjects factors (foods) and three within-subject factors (methods), and the dogs were the experimental unit. Estimated CAD for all dietary components in the three diets by either chromium quantification methods did not differ and showed fair agreement with TC results (P > 0.05). Coefficients of chromic oxide recoveries in feces were 1.06 +/- 0.044 and 1.01 +/- 0.045 for VIS and FAAS, respectively, values significantly different (P < 0.05), indicating better recovery for FAAS. Significant differences (P<0.05) were found in the mean concentration of Cr2O3 for diets and feces (17 +/- 0.6 and 19 +/- 0.8g/kg for VIS and FAAS, respectively). Coefficient of variation for FAAS was 1.69% and 1.77% for VIS, which did not differ significantly (P > 0.05). Pearson's correlation coefficient (r=0.99; P < 0.01) indicated positive and significant association among the results for VIS and FASS, indicating reasonable precision and agreement of chromic oxide contents determined by these methods. Therefore, FAAS and VIS can be used successfully to determine Cr2O3 in dog foods and feces. (c) 2006 Elsevier B.V. All rights reserved.

Influence of husbandry systems on physiological stress reactions of captive brown brocket (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus)-noninvasive analysis of fecal cortisol metabolites

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Non invasive methods for genetic analysis applied to ecological and behavioral studies in Latino-America

Documenting the presence and abundance of the neotropical mammals is the first step for understanding their population ecology, behavior and genetic dynamics in designing conservation plans. The combination of field research with molecular genetics techniques are new tools that provide valuable biological information avoiding the disturbance in the ecosystems, trying to minimize the human impact in the process to gather biological information. The objective of this paper is to review the available non invasive sampling techniques that have been used in Neotropical mammal studies to apply to determine the presence and abundance, population structure, sex ratio, taxonomic diagnostic using mitochondrial markers, and assessing genetic variability using nuclear markers. There are a wide range of non invasive sampling techniques used to determine the species identification that inhabit an area such as searching for tracks, feces, and carcasses. Other useful equipment is the camera traps that can generate an image bank that can be valuable to assess species presence and abundance by morphology. With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in feces and amplify it to analyze the species diversity in an area, and the genetic variability at intraspecific level. This is particularly helpful in cases of sympatric and cryptic species in which morphology failed to diagnose the taxonomic status of several species of brocket deer of the genus Mazama.

A critical analysis of isolation of Paracoccidioides brasiliensis from soil

The present review is a critical analysis of positive and negative reports of the isolation of Paracoccidioides brasiliensis from soil. The strains isolated from soil or soil-contaminated material (dogfood, penguin feces) by Batista et al. in Recife, Brazil, Negroni in the Argentinian Chaco, Albornoz in rural Venezuela, Silva-Vergara et al, in Ibia, Brazil, Ferreira et al, in Uberlandia, Brazil, and Gezuele et al. at the Uruguayan base in the Antarctic region, presented mycological characteristics consistent with P. brasiliensis. In most of these studies, morphological characterization was complemented with an evaluation of virulence and antigenicity, and biochemical or molecular analysis. These isolations, therefore, can be considered true, supporting the concept of soil as an important element in the ecology of the pathogen. The large number of negative reports in attempts involving soil samples and the low repeatability of isolation of the fungus from the same area indicate that the specific conditions supporting growth of the pathogen in soil have not been fully clarified.

GFAAS Determination of Zinc in Fish Feed and Feces Using Slurry Sampling

This paper presents a simple, fast, and sensitive method to determine zinc in samples of feces and fish feed by electrothermal atomic absorption spectrometry through the direct introduction of slurries of the samples into the spectrometer's graphite tube. The procedure is based on the injection of 10 mu L of an acidified aqueous solution containing 0.50% w/v of feces or feed and 0.50% v/v HNO(3) into graphite tube. The limits of detection and quantification calculated for 20 readings of the blank of the standard slurries (0.50% w/v of feces or feed devoid of zinc) were 0.04 and 0.13 mu g L(-1) for the standard feces slurries and 0.05 and 0.17 mu g L(-1) for the standard feed slurries. The proposed method was applied in studies of digestibility of zinc in different fish feeds, and their results proved compatible with that obtained from samples mineralized by acid digestion using microwave oven.

Clinical, serological, and parasitological analysis of snakes naturally infected with Cryptosporidium serpentis

Infection by Cryptosporidium serpentis is one of the most important diseases in reptiles and is characterized by chronic clinical or subclinical infection and the presence of hypertrophic gastritis, food regurgitation, progressive weight loss, mortality, and intermittent or continuous shedding of oocysts in the feces. The objectives of this study were to standardize an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against C. serpentis and to evaluate the clinical, parasitological, and humoral immune response in snakes naturally infected with C. serpentis. Twenty-one snakes naturally infected with C. serpentis and housed at the Butantan Institute, São Paulo, Brazil, underwent clinical and parasitological analyses for C. serpentis infection through daily records of clinical signs and a monthly survey of fecal shedding of oocysts using the Kinyoun's acid-fast staining. The serological evaluation was performed monthly by indirect ELISA using crude total antigen from oocysts of C. serpentis to detect anti-C. serpentis antibodies. Clinical symptoms consisted of food regurgitation, inappetence, and progressive weight loss. The parasitological analysis revealed intermittent fecal shedding of a variable number of oocysts in all snakes, with positivity in 85.32% (157/184) of the samples. The indirect ELISA was positive in 68.25% (86/126) of the samples. A humoral immune response was observed in most animals; however, fluctuating antibodies levels, leading to alternating positive and negative results, were observed in most snakes. © 2013 Elsevier B.V. All rights reserved.

Molecular investigation of zoonotic genotypes of Giardia intestinalis isolates in humans, dogs and cats, sheep, goats and cattle in Araçatuba (Sãoo Paulo State, Brazil) by the analysis of beta-giardin gene fragments

In the period from July 2009 to October 2010, fecal samples from 61 animals and 154 humans from the municipality of Aracatuba (São Paulo State, Brazil) were studied. Fecal samples from animals were collected in the Municipal Animal Shelter and the Veterinary Hospital of the Universidade Estadual Paulista. Human fecal specimens were collected in playschools in the outskirts of the city by the private network of clinical analysis laboratories of the municipal. Diagnosis was done by optical microscopy using the Faust and Hoffmann, Pons and Janer techniques. The genotypes of Giardia intestinalis were characterized by PCR-RFLP and confirmed by sequencing the ß-giardin gene. Human specimens were positive in 25.3% (39/154) of the cases with 26.8% (36/134) of the specimens from children and 15% (3/20) from adults being positive. The frequency of G. intestinalis among the animals was 23.0% (14/61). A total of 32 isolates of G. intestinalis obtained from human feces and six from dogs and cats were characteristic of the A genotype (AI and AII/AIII). The results of this study in respect to frequency of giardiasis are similar to reported in most studies in Brazil. The prevalence observed in animal populations conforms to worldwide infection rates. G. intestinalis genotypes considered zoonotic were detected in both pets and humans from the city of Aractuba, suggesting a possible zoonotic transmission of the parasite in the northwestern region of São Paulo State. The absence of these genotypes in farm animals may imply that they are not involved in the chain of transmission to humans in this region.