Antigenic characterization of morphologically distinct Anaplasma marginale isolates using a panel of monoclonal antibodies

The present study, describes the antigenic characterization of a Brazilian isolate of Anaplasma marginale with appendage (tail). A panel of monoclonal antibodies (McAbs) was produced and tested by the indirect fluorescent antibody test (IFAT), ELISA and Western blotting, and used to characterize two isolates of A. marginale (one with appendage and another without appendage). Among the clones produced, eight recognized antigenic proteins, with molecular weights varying from 18.4 to 66kDa. In Western blotting, the McAb reacted against a 45kDa antigen, which was shown, by the IFAT, to be located in the tail. Immunocytochemistry confirmed the tail specificity of the monoclonal reacting against the 45kDa antigen. The panel of McAb produced has a potential use in discriminating morphologically distinct A. marginale isolates. The present study, demonstrates the occurrence of antigenic diversity among Brazilian isolates of A. marginale.     

Efficient screening of the cystinuria-related C663T Slc3a1 nonsense mutation in Newfoundland dogs by denaturing high-performance liquid chromatography.

Cystinuria in Newfoundland dogs is a metabolic disease associated with a nonsense mutation in the exon 2 of the Slc3a1 gene. Similar to type I human cystinuria, heterozygote carriers are not affected by the disease and do not reveal differences in urinary concentration of dibasic amino acids when compared with normal dogs. However, through a recessive mode of inheritance, these dogs are able to transmit the disease to their offspring. Early detection of mutation carriers through cost-effective reliable methods is therefore essential for the implementation of breeding methods aimed at the eradication of the disease. Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid and efficient screening of nucleotide polymorphisms in polymerase chain reaction-amplified products. This technique was used for the identification of the C663T Slc3a1 mutation in Portuguese Newfoundland dogs. Polymerase chain reaction products amplified from a region containing the C663T locus were subjected to DHPLC analysis, and results were double checked by DNA sequencing. Results showed the presence of the mutation in 6 of the 22 dogs tested. Urine biochemical parameters correlated well with the number of mutated Slc3a1 copies, and homozygotes for the C663T mutation were the only dogs diagnosed with cystinuria. Sequence analysis confirmed the DHPLC results, demonstrating that the technique could be a reliable alternative to sequencing for the rapid and cost-effective identification of mutations in canine breeds.     

Cloning and expression of the M5 RNA segment encoding outer capsid VP5 of epizootic hemorrhagic disease virus Japan serotype 2, Ibaraki virus

The complete nucleotide sequence of a cDNA clone representing the M5 RNA segment of epizootic hemorrhagic disease virus Japan serotype 2 (EHDV-2), Ibaraki virus, was determined. The M5 segment is 1641 base pairs long with the single open reading frame which predicts a polypeptide of 527 amino acids. The comparison of the amino acid sequence of the VP5 with those of EHDV-1, bluetongue virus serotype 10, and African horse sickness virus serotype 4 revealed that the protein shared 67%, 57% and 42% homologies, respectively. In addition, the VP5 protein was expressed in insect cells by recombinant baculovirus, which could be recognized by the mouse anti-EHDV-2 sera at a position of the expected 59 kDa on immunoblot analysis.     

Classification of defects in cork planks using image analysis

Summary Image analysis techniques were used on cork surfaces of transverse sections of planks and multivariate analysis was applied to differentiate pores (lenticular channels) from important defects such as nail (horizontal and vertical) and insect galeries. The discriminant analysis showed that the variables area, perimeter, specific length and width, diameters in the radial and in the tangential direction, as well as the optical density could be combined in three discriminant functions that could make the classification. Defect specific classification functions gave good results for the automatic classification of pores, insect galeries and horizontal nail (error <10%), and acceptable for vertical nail (20% error).     

Accuracy of early fetal sex determination by ultrasonic assessment in goats

The objective of this study was to determine the accuracy of early fetal sex determination by ultrasonic assessment of the relative location of the genital tubercle (GT) in goats at different stages of pregnancy as well as by the identification of fetal external genitalia. Pregnant animals were divided into three experimental groups (EI: n=21, EII: n=28, EIII: n=33). In EI, fetuses (n=27) were transrectally monitored daily from days 40 to 60 of pregnancy with a linear transducer (6.0 and 8.0MHz). In EII, fetuses (n=40) were examined once between days 45 and 70 of pregnancy by transrectal ultrasonography. In EIII fetuses (n=52) between days 100 and 120 of pregnancy, were submitted to a single transabdominal ultrasonography using a convex transducer (5.0 and 7.5MHz). Regardless of fetal sex diagnosis, 15/15 (EI), 13/16 (EII) and 9/14 (EIII) of single pregnancies and 10/12 (EI), 20/24 (EII) and 21/38 (EIII) of twin pregnancies were correctly identified. The accuracy of sex identification among EI (92.6%), EII (82.5%) and EIII (57.7%) was not statistically different (P>0.05). Identification of the GT in male fetuses was possible from day 45 onward. Changes in the GT position were not observed between days 53 and 60 of pregnancy. Accuracy of fetal sexing under field conditions is high in goats when ultrasound imaging is properly timed during pregnancy and when it is performed with proper equipment by experienced operators.     

Early fetal sexing of Saanen goats by use of transrectal ultrasonography to identify the genital tubercle and external genitalia

OBJECTIVE: To define the optimum period for sexing of Saanen goat fetuses by use of transrectal ultrasonography. ANIMALS: 82 Saanen goats pregnant with 124 fetuses. PROCEDURES: Fetal sexing was performed on the basis of the final location of the genital tubercle or identification of external genitalia. In experiment 1, fetuses (n = 78) were monitored every 48 hours from days 40 to 60 of gestation, whereas for experiment 2, 46 fetuses were examined only once between days 47 and 77 of gestation. RESULTS: For experiment 1, accuracy of fetal sexing was 20 of 20 (100%) for a single fetus, 39 of 42 (92.8%) for twin fetuses, and 10 of 16 (62.5%) for triplet fetuses. Diagnostic accuracy was significantly lower for triplet fetuses than that for single or twin fetuses. Final location of the genital tubercle was detected between 45 and 55 days of gestation (mean +/- SEM, 48.9 +/- 1.8 days). For experiment 2, accuracy of fetal sexing for a single fetus (24/24 [100%]) was significantly higher than the accuracy for twin fetuses (16/22 [72.7%]). Considering all fetuses that were born, accuracy of diagnosis was 69 of 78 (88.4%) for experiment 1 and 40 of 46 (86.9%) for experiment 2. Accuracy did not differ significantly between experiments. CONCLUSIONS AND CLINICAL RELEVANCE: Real-time ultrasonography after day 55 of gestation is a suitable method for determination of sex of Saanen goat fetuses by observation of the genital tubercle or identification of external genitalia.     

Study of stayability in Nellore cows using a threshold model

The objectives of the current study were to assess the feasibility of using stayability traits to improve fertility of Nellore cows and to examine the genetic relationship among the stayabilities at different ages. Stayability was defined as whether a cow calved every year up to the age of 5 (Stay5), 6 (Stay6), or 7 (Stay7) yr of age or more, given that she was provided the opportunity to breed. Data were analyzed based on a maximum a posteriori probit threshold model to predict breeding values on the liability scale, whereas the Gibbs sampler was used to estimate variance components. The EBV were obtained using all animals included in the pedigree or bulls with at least 10 daughters with stayability observations, and average genetic trends were obtained in the liability and transformed to the probability scale. Additional analyses were performed to study the genetic relationship among stayability traits, which were compared by contrasting results in terms of EBV and the average genetic superiority as a function of the selected proportion of sires. Heritability estimates and SD were 0.25 +/- 0.02, 0.22 +/- 0.03, and 0.28 +/- 0.03 for Stay5, Stay6, and Stay7, respectively. Average genetic trends, by year, were 0.51, 0.34, and 0.38% for Stay5, Stay6, and Stay7, respectively. Estimates of EBV SD, in the probability scale, for all animals included in the pedigree and for bulls with at least 10 daughters with stayability observations were 7.98 and 12.95, 6.93 and 11.38, and 8.24 and 14.30% for Stay5, Stay6, and Stay7, respectively. A reduction in the average genetic superiorities in Stay7 would be expected if the selection were based on Stay5 or Stay6. Nonetheless, the reduction in EPD, depending on selection intensity, is on average 0.74 and 1.55%, respectively. Regressions of the sires' EBV for Stay5 and Stay6 on the sires' EBV for Stay7 confirmed these results. The heritability and genetic trend estimates for all stayability traits indicate that it is possible to improve fertility with selection based on a threshold analysis of stayability. The SD of EBV for stayability traits show that there is adequate genetic variability among animals to justify inclusion of stayability as a selection criterion. The potential linear relationship among stayability traits indicates that selection for improved female traits would be more effective by having predictions on the Stay5 trait.     

A histological study of the seminal vesicle of the armoured catfish Corydoras aeneus

Most species of Corydoras exhibited a reproductive behaviour called 'T-position', and exhibited an accessory gland in the male genital tract, called the seminal vesicle. It appeared that both the structure and the composition of the fluid varied considerably between the species investigated. Consequently, different opinions were proposed regarding the possible role of seminal vesicle on this particular reproductive behaviour. Male adults of Corydoras aeneus were collected, anaesthetized, and samples of seminal vesicle were fixed in Bouin's solution. The sections were stained with haematoxylin-eosin and periodic acid Schiff. The seminal vesicle showed a system of anastomosed secretory tubules, forming a vesicular collective network, which gave rise to the vesicular ducts. The latter fused with the testicular efferent ducts and formed the spermatic ducts. Considering this fusion, when the sperm cells reached the spermatic ducts, the fluid produced at the seminal vesicle covered them. Histochemical studies evidenced the presence of neutral and acid glycosaminoglycans in the seminal fluid. Considering the reproductive behaviour of C. aeneus, it is believed that the protection associated with the immobilization of the sperm cells assures the sperm integrity during the passage through female's intestine until fertilization.     

Ultrastructural changes in Tritrichomonas foetus after treatments with AlPcS4 and photodynamic therapy

The Tritrichomonas foetus is an amitochondrial parasitic protist which causes bovine trichomoniasis, a major sexually transmitted disease in cattle. No effective drugs for this disease have been approved to this date. Photodynamic therapy (PDT) is an experimental treatment that shows great potential for treating bacteria, fungi, yeasts, and viruses. However, the cytotoxic effect of PDT on protozoan has been poorly studied. In this study, PDT with aluminum phthalocyanine tetrasulfonated (AlPcS4) photosensitizer was efficient in killing T. foetus. The mode of cell death in T. foetus after PDT was investigated by transmission electron microscopy. Morphological changes, such as membrane projections, nucleus fragmentation with peripheral masses of heterochromatin, endoplasmic reticulum proliferation, intense cytoplasmic vacuolization, fragmented axostyle-pelta complex, and internalized flagella could be observed. This is the first report to demonstrate cell death in T. foetus after PDT, and thus will open up new lines of investigation to develop new treatments for bovine trichomoniasis.