Relation between Physical Properties of the Zona Pellucida and Viability of Bovine Embryos after Slow-freezing and Vitrification

In vitro-produced bovine morulae/blastocyst embryos (n = 119) were slow-frozen and vitrified and the physical alterations of the zona pellucida (ZP) was observed by scanning electron microscopy (SEM) to find an explanation for the loss of developmental capacity of the embryos after freezing/thawing. A control group was provided, in which embryos (n = 38) were neither frozen nor vitrified. Embryos were in vitro-cultured in a standard CO2 Heraeus incubator and their viability was assessed 24 and 48 h after the start of culture, evaluating their morphological aspect. After 24 h of culture, embryo survival rate for slow-freezing/thawed (n = 23), vitrified/thawed (n = 20) and control embryos (n = 20) was 39, 27 and 90%, and 35, 14 and 65% after 48 h of culture, respectively. For evaluation of physical changes occurring in ZP, 20 embryos were slow-frozen, 18 were vitrified and 18 were used as control. All embryos were fixed, dried and examined under an SEM. Embryo's diameter, as well as the number of pores and their diameter was measured in squares of 6.4 microm width. We observed that, on average, the diameter of the embryos (92.26 +/- 10.15 microm) did not differ significantly among all embryos. As far as the diameter of the pores in the outer surface of the ZP is concerned, the results revealed a significant difference (P < 0.05) between control (0.48 +/- 0.0025 microm), slow-frozen (0.34 +/- 0.0007 microm) and vitrified (0.27 +/- 0.0006 microm) embryos. For the number of pores, statistical differences (p < 0.05) were observed between control and vitrified embryos (45.4 +/- 7.3 vs 38.2 +/- 8.2). It is possible that ZP functions as a barrier which is positive when dealing with pathogens, but is harmful when nutrients were supplied from the outside, especially at 48 h of culture. Results indicate that the steps of cryopreservation cause alterations in ZP, with irreversible damage on the further developmental competence of bovine embryos.     

Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil

A typing procedure based on polymorphism of the coagulase gene (coa) was used to discriminate Staphylococcus aureus isolated from Minas Gerais dairy cows with mastitis. Amplification of the gene from the 64 S. aureus isolates produced 27 different polymerase chain reaction (PCR) products; 60 isolates showed only 1 amplicon, and 4 showed 2 amplicons. The isolates were grouped into 49 types by analyzing the restriction fragment length polymorphism (RFLP) of the coa gene; the 10 most common types accounted for 39% of the isolates. The results demonstrate that many variants of the coa gene are present in the studied region, although only a few predominate.     

Histomorphometry and quantification of nucleolar organizer regions in bovine thyroid containing methylthiouracil residues

In order to study the morphology and morphometry and to characterize and quantify the nucleolar organizer regions (NORs) of bovine thyroids containing methylthiouracil (MTU) residues, five animals were orally treated with a suspension of MTU (5 g/animal/day) for 20 days (group A). This treatment protocol was interrupted 5 days before the the animals were slaughtered. Six animals receiving placebos composed group B. A third group (group C) was composed of normal thyroids obtained from a slaughterhouse. All glands were previously assessed for detection of antithyroid residues by chromatography, and only those glands from MTU-treated animals were positive. Follicles of glands from group A showed wide variation in size and shape. There was a predominance of small follicles covered by multiple layers of columnar cells, sometimes forming papillary projections into the lumen, characterizing severe interfollicular and intrafollicular adenomatosis. Many follicles had vacuolated cells with nuclei showing karyolysis or pyknosis and reduced amounts of a low-density and very excavated colloid. They also showed higher follicular epithelia and larger proportions of their structural components when compared with glands of groups B and C. In the thyroids from group A, the argyrophilic nucleolar organizer region-associated proteins (AgNORs) were greater in number, with small ones scattered all over the nucleus. Although the size of AgNORs in thyroids from groups B and C was variable, these AgNORs were fewer and larger than were those in glands from group A. In conclusion, the MTU induces proliferation and regressive changes in follicular cells, and the AgNOR technique is efficient to distinguish different degrees of thyroid hyperplasia.     

Effects of low-intensity pulsed ultrasound on wound healing in corneas of dogs following keratoplasty

The effects of low-intensity pulsed ultrasound on wound healing were evaluated at the graft-cornea transition in dogs following lamellar keratoplasty using tunica vaginalis preserved in 98% glycerin. Twenty-one dogs were subdivided into three groups of seven animals. The first group (W/US) received daily treatment of low-intensity pulsed ultrasound (20 mW/cm2) for 15 min for the first 10 days post surgery. The second group (N/US) was submitted to the same procedure but with the ultrasound apparatus turned off. The third group, the control (CO), underwent the surgical procedure only. The animals were clinically evaluated during the initial (1-15 days), intermediate (16-30 days) and late (31-120 days) postoperative period. The corneas were evaluated by light microscopy at 1, 3, 7, 15, 30, 60 and 120 days after surgery. Clinically, there were no differences which would promote an advantage to any of the treatments. Light microscopy, however, revealed more extensive vascularization and more advanced wound healing in the W/US group, as well as a tendency towards early graft incorporation. Based on the present results, low-intensity pulsed ultrasound shows advantages, especially in situations where trophic support is a mandatory condition, facilitating better graft incorporation and rapid recovery of stromal organization.     

Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis

Although the nephropathy of visceral leishmaniasis (VL) is known both in humans and dogs, histopathologic alterations have not been thoroughly studied. We examined renal alterations in 55 dogs with naturally acquired VL compared with five noninfected dogs from an endemic area in northeastern Brazil. Glomerulonephritis was found in 55 dogs, interstitial alterations in 53 dogs, and tubular changes in 43 dogs with VL. The glomerular alterations found were minor glomerular abnormalities (n = 8, 14.5%), focal segmental glomerulosclerosis (n = 10, 18.2%), mesangial proliferative glomerulonephritis (n = 17, 32.7%), membranoproliferative glomerulonephritis, (n = 18, 30.9%), crescentic glomerulonephritis (n = 1, 1.8%), and chronic glomerulonephritis (n = 1, 1.8%). Morphometric and ultrastructural studies complemented the analysis. The five control animals exhibited no glomerular alterations. The glomerular lesions were related to functional alterations. Considering that the alterations of canine and human nephropathy in VL are very similar, the data obtained in this study constitute an important contribution to the understanding of canine and human VL nephropathy.     

Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS)

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.     

Virulence factors of avian pathogenic Escherichia coli isolated from broilers from the south of Brazil

Sixty-three Escherichia coli strains isolated from broilers with respiratory problems were examined for virulence factors, hemolysin synthesis ability, motility, hemagglutination capacity, operon pap presence, colicin production, and serum resistance. The capacity to hemagglutinate guinea pig erythrocytes was found in 53 (84.1%) of the samples, but only 30 (47.6%) agglutinated chicken erythrocytes. D-mannose-sensitive hemagglutination against guinea pig erythrocytes was found in 19 (30.2%) samples and against chicken erythrocytes, in 15 (23.8%) samples, whereas the D-mannose-resistant hemagglutination with guinea pig erythrocytes was found in 34 (54%) samples, and 13 of these (20.6%) showed this characteristic against chicken erythrocytes. Operon pap, P fimbria codifier, was detected in 26 samples in a total of 34 D-mannose-resistant samples. Colicin production was observed in 55 (87.3%) of the strains, and 41.8% presented V colicin production. Of the samples analyzed, 56 (88.9%) presented serum resistance, six (9.5%) were intermediate, and only one (1.6%) was sensitive to the action of the complement. The diversity of virulence profiles detected in the samples in this study explains in part the multifactorial characteristics of avian colibacillosis.     

Use of intraluminal nitinol stents in the treatment of tracheal collapse in a dog

Tracheal collapse is a common problem that is typically observed in middle-aged and older small-breed dogs. It is a structural, obstructive airway disease with a dynamic component that can affect the intra- and extrathoracic portions of the trachea and mainstem bronchi. Many methods of treatment have been suggested, including medical management and provision of extraluminal and intraluminal support. All techniques used to treat intrathoracic and mainstem bronchial collapse have been associated with major complications or limitations. This report describes the implantation of intraluminal nitinol stents to successfully treat intrathoracic as well as extrathoracic tracheal collapse in a dog. The stents are composed of material that has characteristics similar to those of the trachea; nitinol stents may provide a method of supporting intrathoracic tracheal and mainstem bronchial collapse in dogs.     

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.     

Variability of resistance in goats infected with Haemonchus contortus in Brazil

The variability between and within breeds with respect to nematode egg counts (EPG), packed cell volume (PCV) and hemoglobin (Hb) was studied in 36 yearling female goats of the Caninde (15), Bhuj (6) and Anglo-Nubian (15) breeds, exposed to Haemonchus contortus. Nematode-free goats were turned to a contaminated paddock in late February. From then on, fecal egg per gram counts (EPG), packed cell volume (PCV) and hemoglobin (Hb) were determined at 2-week intervals up to Week 18. The EPG, transformed as [log(EPG + 75)], varied (P < 0.01) between goats within breeds and between weeks of exposure, but not between goat breeds (P > 0.05). PCV and Hb were affected by goat breeds (P < 0.05), by goats within breeds (P < 0.01) and by weeks of exposure (P < 0.01). Anglo-Nubians had higher (P < 0.01) PCV and Hb than Caninde; Bhuj had intermediate values. There were two EPG rises; one between Weeks 6 and 10 and the other between Weeks 14 and 16. The within breed variability was marked during the EPG rise on Week 6, when individual egg counts ranged from 130 to 2500. The EPG rises coincided with drops in Hb. PCV presented a similar trend, though not as marked. Haemonchus was responsible for more than 95% of nematode eggs counted. Considering the goat as experimental unit, the correlation coefficients (r) were: -0.45, P = 0.0064, between log(EPG + 75) and PCV; and -0.53, P = 0.0009, between log(EPG + 75) and Hb. The negative correlation between egg counts and blood values suggested breed differences in PCV and Hb were related to resistance to H. contortus infection and/or to its effects.