Osteomyelitis by Microsporum canis and Staphylococcus spp. in cat (Felis catus) - case report.

BACKGROUND: Staphylococcus spp and Microsporum canis are zoonotic microorganisms which can cause infections and systemic diseases. The bone infection is usually caused by invasion of pathogen through the hematologic route. Mixed osteomyelitis caused by bacteria and fungi is rare, and to date, there have been no reports of mixed osteomyelitis with Staphylococcus spp. and Microsporum canis. CASE PRESENTATION: This essay reports an atypical presentation of mixed osteomyelitis (Staphylococcus spp. and Microsporum canis) in a domestic cat. A 15-month-old female Persian cat was presented to a veterinary service; the main complaint was the appearance of a nodule in the mandibular ventral rostral region. A radiographic exam performed on the animal showed proliferative and osteolytic bone lesions. The patient was submitted to a biopsy for histopathological evaluation, along with bacterial and fungal cultures. Results showed mixed osteomyelitis by Staphylococcus spp. and Microsporum canis. Microbial Sensitivity Test was performed to choose a more suitable treatment. Two surgical procedures were executed to resect and curette the lesion, and treatments with anti-inflammatory, antibiotic, and antifungal drugs were established, showing a positive clinical evolution. After 8 months of treatment, the patient's owner moved to a different city, and the animal was seen by other veterinarians, who followed along with the same treatment. However, due to complications and a diminishing quality of life over 4 years of diagnosis, the patient was euthanized. CONCLUSION: Given the above, mixed osteomyelitis is difficult to treat and can cause losses of life quality resulting death, especially in infections where M. canis is the agent causing the disease. Bacterial osteomyelitis is more frequently reported. But the lack of investigation of microorganisms other than bacteria, such as fungal cases, may imply in underdiagnosed cases. Treatment of osteomyelitis can be difficult considering the difficulties in isolating the pathological agent, resistance to the drug used, prolonged treatment time, and cost.

The effect of priming and duration of oestradiol benzoate treatment before progesterone administration on embryo development and survival in anestrous recipient mares.

The effect of three different hormonal protocols to prepare anestrous recipient mares on embryo survival was evaluated. The first group consisted of only progesterone administration (NE) 4 days before embryo transfer, while the recipients from the other two groups received a single administration of 2.5 mg of oestradiol benzoate (SE) 2 days earlier or 8 mg of oestradiol split in increasing doses for 5 consecutive days (LE) ending 3 days before progesterone treatment. The likelihood of recovering an embryo 2 days after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the no oestrus, short and long oestrous groups respectively (p = .09). In conclusion, the presence and duration of oestradiol treatment before progesterone administration tended to influence the embryo survival in anestrous recipients 2 days after transfer. The surviving embryos recovered from the three different groups of recipients did not show any difference in size and morphology.

Artificial Long Estrus Protocols Administered Prior to Progesterone Increase Endometrial Uterocalin Expression in Anestrous Mares.

Uterocalin (P19) is a lipocalin protein that has binding activity for the provisioning of the embryo with lipids, including those essential for morphogenesis and pattern formation. Therefore, it is thought that P19 plays an important role in supporting the survival of the early embryo. Previous research has shown that the endometrium from acyclic mares expressed more P19 following a simulated long estrus: treatment of 8 mg of estradiol benzoate (EB) split over five consecutive days, followed by long-acting progesterone administration (LA-P4) 72 hours later, when compared to a single 2.5 mg EB treatment. The main objective of this study was to evaluate if a new long estrus protocol, with fewer EB administrations, also increases endometrial uterocalin expression. Fifteen mares were assigned to three groups: long estrus 5 administrations (LE5; a total of 8 mg of EB in increasing doses was given over five consecutive days, Day -7 to Day -3); long estrus 2 administrations (LE2; 3 and 5 mg EB were given on Day -7 and Day -4); and short estrus (SE; a single dose of 2.5 mg EB was administered on Day -2). All groups received LA P4 on day 0 (D0). Daily ultrasonography and blood collections were performed to assess uterine edema and estradiol and progesterone plasma concentrations, respectively. Endometrial biopsies were collected on Day 4 to evaluate P19 expression by RT-qPCR. There were no differences in P19 expression between groups LE2 and LE5. However, P19 expression was higher (P < .05) in the LE groups than in the SE group. Both LE protocols produced high uterine edema (score 2-3) for at least 6 days. There was no effect of group (LE2 or LE5) on overall estradiol and progesterone concentrations (P > .1). In conclusion, the two EB administrations protocol is more feasible for preparing acyclic recipient mares at field conditions. However, a positive effect of the LE protocols on embryo survival and viability in anestrous treated mares still needs to be confirmed.

Short communication: Proinflammatory gene expression relative to the collection technique of endometrial samples from cows with and without subclinical endometritis.

Uterine inflammation negatively affects reproductive performance and is an important cause of infertility and subfertility in dairy cows. Several studies have investigated the use of gene expression in endometrial samples collected by biopsy or cytology to evaluate the inflammatory response of the cow uterus. This study aimed to compare the expression of the CCL5, CXCL8, IL6, and IL1B genes in the bovine endometrium according to the site of sample collection [caruncular (C) or intercaruncular (IC)], the collection method (biopsy or cytology), and the category of inflammation based on endometrial cytology (zero, medium, or high) in subclinical endometritis. The reproductive tracts of dairy cows were collected from a slaughterhouse, and punch-biopsy samples of endometrial tissues were obtained from both regions (C and IC). Endometrial cells from these regions were collected with the cytobrush technique and then used for the analysis of mRNA expression by quantitative PCR. After counting polymorphonuclear cells (PMN) by endometrial cytology, 20 uteri with an ovary at stage I (d 1-4 of estrous cycle) were categorized into 3 groups. Uteri with 0% PMN (n = 10) were assigned to group zero, uteri with 5 to 15% PMN (n = 5) to group medium (12.2 ± 1.6% PMN), and uteri with >15% PMN (n = 5) to group high (53.8 ± 32.9% PMN). All data were analyzed with 2-way ANOVA with Bonferroni multiple comparison post test. The results from gene transcripts demonstrated that the region (C or IC) of the endometrial biopsy had no influence on any of the degrees of inflammatory reaction observed. However, gene expression was more elevated in the endometrium of cows with greater inflammation compared with those without inflammation (CCL5, CXCL8, IL6, IL1B) and those with medium inflammation (CCL5, IL6). Expression of the genes evaluated did not differ between the endometrium without inflammation and with medium inflammation. However, in the high inflammation group, all genes were comparatively more expressed in samples collected by cytology relative to those derived from biopsies for both anatomical regions. In conclusion, gene expression did not differ between the C and IC tissue. Samples collected from animals with greater inflammation had greater gene expression than those with zero or medium inflammation. In addition, cytology samples had greater gene expression than biopsy samples in the high inflammation group.

Biofilm Formation in Different Salmonella Serotypes Isolated from Poultry.

Little is known about Salmonella biofilm assembly, making the prevention of the disease a challenge in the poultry production chain. The objective of the present study was then to evaluate biofilm formation from different serotypes of Salmonella spp. in both polystyrene plates and eggshells. Salmonella Gallinarum and S. Minnesota were both classified as producers of biofilms of moderate intensity. Interestingly, S. Gallinarum produces biofilm even though being a serotype without flagellum and not having the lux gene in its genome, suggesting that there might be other important structures and genes associated with biofilm formation. Regarding Enteritidis, Typhimurium, Typhimurium variant, and Heidelberg serotypes, despite having high counts, BFI (Biofilm Formation Index) showed low biofilm production, probably due to the scarcity of extracellular matrix produced by such strains. A turkey eggshell model was then used for S. Enteritidis and S. Heidelberg biofilm formation. The results from the microbial count and scanning electron microscopy showed that Salmonella serotypes were also able to generate biofilm in eggshells, suggesting the presence of biofilms in poultry producing farms, a main concern for the poultry production industry.

Outbreak of cutaneous form of avian poxvirus disease in previously pox-vaccinated commercial turkeys / Surto de bouba aviária na forma cutânea em perus comerciais previamente vacinados

This study describes an outbreak of avian poxvirus disease in previously pox-vaccinated turkeys in Brazil. The turkeys had suggestive gross lesions of cutaneous avian poxvirus in the skin of the head and cervical area without changes in the flock mortality rates. In the slaughterhouse, 30 carcasses were removed from the slaughter line to collect tissue from cutaneous lesions for histological analyses and characterization of the virus. The virus was identified by conventional polymerase chain reaction (PCR) and subsequent gene sequencing. Acanthosis, hyperkeratosis, and hydropic degeneration were seen on skin histopathology. Eosinophilic intracytoplasmic inclusion bodies (Bollinger) on keratinocytes were observed in 46.6% of the samples. Avian poxvirus DNA was detected on PCR in 83.3% of the total samples. PCR associated with histopathology had 93.3% of positivity for avian poxvirus. In the phylogenetic study, samples show 100% matching suggesting that the outbreak occurred by a single viral strain and was different from those strains affecting other wild birds such as canaries and sparrows. A single mutation (Adenine for Guanine) was detected in our study's strain and in the strains of turkey, chickens, and vaccine strains published in GenBank. Also, when the sequence strain of the present study and sequences from GenBank of canarypox and sparrowpox strains were aligned, a Thymine was found replacing the Adenine or Guanine. The in ovo vaccination method as single-use in turkeys of this study apparently did not provide adequate protection against avianpox disease, but additional vaccination administered by wing-web when turkeys were 45-60 days old in the new flocks controlled the disease. In the subsequent year, new cases of this disease were not found. It was not possible to confirm the source of the virus strain, but infection with a field strain derived from chickens is one possibility, considering the poultry farm population in the area and biosecurity aspects. For wide characterization of avipoxvirus and differentiation among strains, the complete sequence of the viral genome is required.(AU)

Este estudo descreve um surto de bouba aviária em perus previamente vacinados contra poxvirus aviário no Brasil. Os perus apresentaram lesões macroscópicas, sugestivas de bouba aviaria cutânea, na pele da cabeça e região cervical sem alteração nas taxas de mortalidade do lote. No abatedouro, 30 carcaças foram retiradas da linha de abate para coleta de dois fragmentos de pele com lesões para análise histológica e caracterização do vírus. A identificação do vírus foi realizada por PCR convencional e posterior sequenciamento. No exame histopatológico das lesões de pele, houve acantose, hiperqueratose e degeneração hidrópica. Corpúsculos de inclusão intracitoplasmáticos eosinofílicos (Bollinger) foram encontrados em 46,6% das amostras. A técnica de PCR detectou o DNA do vírus da bouba aviária em 83,3% do total de amostras. PCR associado com a histopatologia resultou em 93,3% de positividade para o vírus da bouba aviária. No estudo filogenético, as sequências resultaram em 100% de identidade, sugerindo que o surto ocorreu por uma única estirpe de vírus diferenciada das outras estirpes que acometem canários e pardais. Uma única mutação (Adenina para Guanina) foi detectada nas estirpes deste estudo e nas sequências de perus, galinhas e estirpes vacinais publicadas no GenBank. Além disso, quando a sequência da estirpe do presente estudo e as sequências das estirpes de canarypox e sparrowpox foram comparadas, a Timina foi encontrada em substituição a Adenina ou Guanina. A vacinação in ovo em dose única utilizada nos perus deste estudo aparentemente não forneceu proteção adequada contra a doença causada pelo poxvirus aviário. Entretanto, a revacinação na membrana da asa em perus com 45-60 dias de idade dos novos lotes controlou a doença. No ano subsequente, novos casos desta doença não foram registrados. Não foi possível confirmar a origem da estirpe viral, mas estirpes de campo oriundas de galinhas seria uma possibilidade, considerando a população na área e os aspectos de biosseguridade. Para caracterização ampla do avipoxvirus e diferenciação entre as estirpes, a sequência completa do genoma viral é requerida.(AU)

Ação do extrato etanólico da casca do pequi (Caryocar brasiliense) na cardiotoxicidade crônica induzida por doxorrubicina em ratos / Caryocar brasiliense shell ethanol extract in chronic cardiotoxicity induced by doxorubicin in rats

A doxorrubicina (DOX) é um quimioterápico utilizado no tratamento de neoplasias malignas, porém possui a cardiotoxicidade como efeito colateral. O objetivo deste trabalho foi verificar quanto à ação do extrato etanólico da casca do pequi (Caryocar brasiliense) (EECP) por meio de avaliação morfológica (macroscópica, microscópica e ultramicroscópica), bem como avaliar a expressão de metaloproteinases (MMP2 e MMP9) e seus inibidores teciduais (TIMP1 e TIMP2) no miocárdio de ratos submetidos à cardiotoxicidade crônica pela DOX, tratados ou não com o EECP. O experimento teve duração de três meses e foram utilizados 30 ratos da raça Wistar, distribuídos em seis grupos de cinco animais. G1 e G2 receberam como pré-tratamento 300mg/kg e 600mg/kg de EECP, respectivamente, por gavagem, durante sete dias e mantiveram o tratamento durante os 21 dias de aplicação da DOX. Em G1, G2, G3, G4 e GC, a cardiotoxicidade foi induzida com aplicações semanais de 2mg/kg de DOX, via intraperitoneal, totalizando quatro aplicações (8mg/kg) e, nos ratos do grupo Sham (GS), foi aplicado 1ml de solução fisiológica. Os animais do G3 receberam diariamente 300mg/kg e os do G4 600mg/kg de EECP, por gavagem, durante os 21 dias de aplicação da DOX. Os do GC e GS receberam 1 ml de água, diariamente, também por gavagem. Após o término das aplicações, os animais foram mantidos por dois meses, totalizando três meses de experimento. A avaliação macroscópica foi realizada após 90 dias, momento em que foram colhidas amostras para análise em microscopia eletrônica, histopatologia e imunoistoquímica. Ao exame necroscópico foi observada ascite nos animais que receberam DOX. Houve baixo índice de mortalidade (3,33%), representado pela morte de um rato que desenvolveu pneumonia por falsa via. Não foi observada alteração no peso e nas medidas do coração dos ratos. Nas doses de 300 e 600mg/kg, o EECP atenuou a degeneração vacuolar miocítica. Na dose de 600mg/kg, o EECP reduziu a quantidade de células de Anitschkow e a fragmentação das miofibrilas. Não houve resultado significativo quanto à imunomarcação das MMP e, quanto a seus inibidores (TIMP), houve maior imunomarcação de TIMP2 no GC, grupo que recebeu apenas DOX. Concluiu-se que o extrato etanólico da casca do pequi (EECP) é eficiente em minimizar os efeitos da cardiotoxicidade crônica induzida pela DOX no miocárdio de ratos, considerando que nas doses de 300 e 600mg/kg o EECP atenua a degeneração vacuolar miocítica e, na dose de 600mg/kg, o EECP reduz a quantidade de células de Anitschkow e a fragmentação das miofibrilas.(AU)

Doxorubicin (DOX) is a chemotherapic drug used in the treatment of malignancies, but has the cardiotoxicity as collateral effect. The objective of this study was to evaluate the action of pequi shell etanolic extract (Caryocar brasiliense) (PSEE) through morphological evaluation (macroscopic, microscopic and ultramicroscopic), and to evaluate the expression of metalloproteinases (MMP2 and MMP9) and its tissue inhibitors (TIMP1 and TIMP2) in the myocardium of rats with chronic cardiotoxicity by DOX and treated or not with PSEE. The experiment lasted three months and 30 Wistar rats were divided into six groups of five animals. G1 and G2 received 300mg/kg and 600mg/kg of PSEE, respectively, as pretreatment, by gavage for seven days and continued treatment for 21 days of application of DOX. In G1, G2, G3, G4 and GC, cardiotoxicity was induced with weekly applications of 2mg/kg DOX, intraperitoneally, totaling four applications (8 mg/kg), and in the Sham group (GS) 1ml of saline solution was applied. G3 animals received daily 300mg/kg of PSEE, and G4, 600mg/kg, by gavage, for 21 days of application of DOX. The GC and GS received 1ml of water daily by gavage also. After the completion of the application, the animals were kept for two months, with three months of experiment. Macroscopic evaluation was performed after 90 days, at which time samples were taken for analysis in electron microscopy, histopathology and immunohistochemistry. At necropsy, ascites was observed in animals that received DOX. There was a low mortality rate (3.33%), being one mouse that developed false road pneumonia. There was no change in weights and measures of the rat hearts. At doses of 300 and 600mg/kg, the PSEE attenuates myocyte vacuolar degeneration. At a dose of 600mg/kg, PSEE reduces amount Anitschkow cells. There was no significant result on the immunostaining of MMP, but considering their inhibitors (TIMP) there was a greater immunostaining of TIMP2 in GC, the group that received only DOX. It was concluded that PSEE is effective in minimizing effects of chronic cardiotoxicity induced by DOX in the myocardium of rats, whereas at doses of 300 and 600mg/kg, PSEE attenuates vacuolar degeneration in myocytes and at the dose of 600mg/kg the PSEE reduces the amount of Anitschkow cells and myofibrils fragmentation.(AU)

Selected sperm traits are simultaneously altered after scrotal heat stress and play specific roles in in vitro fertilization and embryonic development.

Improvements in the estimation of male fertility indicators require advances in laboratory tests for sperm assessment. The aims of the present work were (1) to apply a multivariate analysis to examine sperm set of alterations and interactions and (2) to evaluate the importance of sperm parameters on the outcome of standard IVF and embryonic development. Bulls (n = 3) were subjected to scrotal insulation, and ejaculates were collected before (preinsulation = Day 0) and through 56 days (Days 7, 14, 21, 28, 35, 42, 49, and 56) of the experimental period. Sperm head morphometry and chromatin variables were assessed by a computational image analysis, and IVF was performed. Scrotal heat stress induced alterations in all evaluated sperm head features, as well as cleavage and blastocyst rates. A principal component analysis revealed three main components (factors) that represented almost 89% of the cumulative variance. In addition, an association of factor scores with cleavage (factor 1) and blastocyst (factor 3) rates was observed. In conclusion, several sperm traits were simultaneously altered as a result of a thermal insult. These sperm traits likely play specific roles in IVF and embryonic development.

Método imunomagnético associado ao meio MesenCult® na obtenção de células mononucleares da medula óssea de coelhos negativas para o anticorpo monoclonal CD45 / Immunomagnetic method associated with MesenCult® medium to obtain bone marrow mononuclear cells from rabbits negative for CD45 monoclonal antibody

O objetivo detse artigo é de descrever um protocolo de isolamento das células mononucleares da medula óssea de coelhos, seguido de purificação celular por depleção negativa com o anticorpo monoclonal CD45 e posterior expansão em meio de cultura MesenCult®. Dez coelhos machos adultos, da raça Nova Zelândia, com idade média de 1,0±0,2 anos e peso médio 3,5±0,24kg, foram utilizados para padronização da metodologia. O isolamento das células mononuclares da medula óssea foi realizado pelo gradiente de densidade Ficoll-paque® e a purificação e obtenção das células- pela depleção negativa com o anticorpo monoclonal CD45 em base imunomagnética. A população celular obtida foi expandida posteriormente em meio de cultura MesenCult®. No isolamento pelo gradiente de icoll-Paque® foi obtido um rendimento médio de 7,31x106 células/mL. Após purificação e obtenção das possíveis células-tronco mesenquimais pela base imunomagnética, houve um decréscimo do rendimento para 2,28x106 células/mL, mas o processo de expansão foi incrementado pelo cultivo celular. Os resultados indicaram que as células obtidas da fração mononuclear da medula óssea, cultivadas in vitro foram capazes de gerar células aderentes 24 horas após o cultivo, com predominância de células fibroblastóides sugestivas de células-tronco mesenquimais. Concluiu-se que a obtenção de células-tronco mesenquimais pode ser alcançada após purificação das células mononucleares da medula óssea de coelhos pelo método imunomagético, o meio de cultura MesenCult® proporciona um ambiente adequado para a rápida expansão in vitro e o número de passagens exerce influência negativa sobre as características morfológicas das células.

The objective of this study was to describe guidelines for the isolation of bone marrow mononuclear cells from rabbits, followed by cell purification by negative depletion with CD45 monoclonal antibody, and further expansion in MesenCult® medium. Ten adult male New Zealand White rabbits, age average of 1.0±0.2 years and weighting 3.5±0.24kg, were used to obtain a standardized method. The mononuclear cells of the bone marrow were isolated with Ficoll-paque® density gradient centrifugation, and the cell purification and acquisition was completed by negative depletion with CD45 monoclonal antibody in immunomagnetic base. The cell population obtained was expanded in MesenCult® medium. Through isolation with Ficoll-paque® density gradient was possible to obtain an average yield of 7.31x106 cells/mL. After purification and acquisiton of potential mesenchymal stem cells by the immunomagnetic base, there was a yield decrease to 2.28x106 cells/mL; however the expansion process was increased in cell culture. The results indicated that cells obtained from the mononuclear fraction of bone marrow and cultivated in vitro were capable to generate adherent cells 24 hours after culture, with predominance of fibroblastoid cells suggestive of mesenchymal stem cells. It can be concluded that mesenchymal stem cells can be achieved with purified rabbit bone marrow mononuclear cells through the immunomagnetic method, as the MesenCult® medium provides a suitable environment for a quick in vitro expansion, and the number of passages exerts negative influence on the morphological characteristics.

Rapid purification of a new P-I class metalloproteinase from Bothrops moojeni venom with antiplatelet activity.

The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMP α -II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMP α -II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMP α -II cleaves A α -chain of fibrinogen followed by B ß -chain, and does not show any effect on the γ -chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30-50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMP α -II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMP α -II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMP α -II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders.

Ovarian activity and oocyte quality associated with the biochemical profile of serum and follicular fluid from Girolando dairy cows postpartum.

This study was designed to evaluate the influence of heat stress (HS) on the metabolic profile of serum and follicular fluid (FF), ovarian follicle development, and oocyte quality of Girolando dairy cows. Oocytes, blood, and FF (follicles ≥9mm) samples were obtained at 30, 45, 60, 75, and 90 days postpartum in the summer and winter seasons. During transvaginal follicular aspiration, rectal temperature (RT), body condition score (BCS), number of ovarian follicles, and quality of oocytes were recorded. The ambient air temperature (AT) and relative humidity (RH) were also recorded to calculate the temperature humidity index (THI). Glucose, total cholesterol (TC), triglycerides (TG), urea, sodium (Na), potassium (K), and calcium (Ca) concentrations were determined using serum and FF samples. The RT, THI, and BCS loss were greater (P<0.01) in the summer; however, glucose, Na, and K serum concentrations decreased in the same season (P<0.05). Degenerated oocytes were positively associated (P<0.05) with THI (r=0.14) and AT (r=0.13), and negatively associated with glucose (r=-0.12) and K (r=-0.11) serum concentrations. HS induces metabolic changes, which compromise the number of ovarian follicles and the follicular environment, thus resulting in morphologically damaged oocytes.

Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom.

A fibrinogenolytic metalloproteinase from Bothrops moojeni venom, named moojenin, was purified by a combination of ion-exchange chromatography on DEAE-Sephacel and gel filtration on Sephacryl S-300. SDS-PAGE analysis indicated that moojenin consists of a single polypeptide chain and has a molecular mass about 45 kDa. Sequencing of moojenin by Edman degradation revealed the amino acid sequence LGPDIVSPPVCGNELLEVGEECDCGTPENCQNE, which showed strong identity with many other snake venom metalloproteinases (SVMPs). The enzyme cleaves the Aα-chain of fibrinogen first, followed by the Bß-chain, and shows no effects on the γ-chain. Moojenin showed a coagulant activity on bovine plasma about 3.1 fold lower than crude venom. The fibrinogenolytic and coagulant activities of the moojenin were abolished by preincubation with EDTA, 1,10-phenanthroline and ß-mercaptoethanol. Moojenin showed maximum activity at temperatures ranging from 30 to 40 °C and its optimal pH was 4.0. Its activity was completely lost at temperatures above 50 °C. Moojenin induced necrosis in liver and muscle, evidenced by morphological alterations, but did not cause histological alterations in mouse lungs, kidney or heart. Moojenin rendered the blood uncoagulatable when it was intraperitoneally administered into mice. This metalloproteinase may be of medical interest because of its anticoagulant activity.

Structural changes of fat tissue after nonaspirative ultrasonic hydrolipoclasy.

BACKGROUND: Hydrolipoclasy is an alternative technique less invasive than liposuction. Hydrolipoclasy uses normal saline or hypotonic solution and ultrasound waves that act directly on local adiposity. In the literature there are few reports of morphostructural changes on adipose tissue. MATERIALS AND METHODS: This study was aimed to evaluate the amount of fat cells injured immediately after treatment and after three days and also cell migration to the area treated using 8 pigs as experimental models, as well as cellular changes by transmission electron microscopy (TEM). RESULTS: The Wilcoxon test was conducted, and a difference was found between the treated side and the corresponding control side on the number of viable cells. The treated side showed a smaller number of viable cells compared to the control side both immediately after treatment and 3 days later. Also occurring 3 days after treatment was the migration of lymphoid cells and fibroblasts, which shows the local inflammatory process and conjunctive neoformation. Soon after treatment there was fluid accumulation within adipocytes. CONCLUSIONS: The results shown in this paper demonstrate Ultrasonic Hydrolipoclasy as a viable alternative for the treatment of localized fat deposits without the side effects of traditional surgical procedures. Better results are expected when hypotonic solution is used, since it penetrates into the cell.

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa.

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

Low-intensity ultrasound application in distal radius metaphyseal bone defects of dogs.

We assessed the repair of transverse, 3-mm wide bone gaps created at the distal radius in 28 dogs that were randomly divided into two 14-animal groups; one was the control group and the other received a daily, 20-min application of low-intensity pulsed ultrasound for 100 days. Sequential radiographs, histomorphometrics, bone fluorescent histology and bone vascularity assessments found that all animals from both groups obtained a stage of hypertrophic-type nonunion with fibrocartilage tissue formation throughout the region of osteotomy. However, treated animals exhibited areas of endochondral ossification within the fibrocartilage region. There was no difference in type of vascularity or the newly formed bone process obtained by tetracycline labeling. Application of low-intensity ultrasound was not capable of significantly changing the reparative process and it may not be sufficiently powerful to overcome a combination of local deleterious effects on bone healing, created by gapping, excessive motion and periosteal resection.

Age-related association of rDNA and telomeres with the nuclear matrix in mouse hepatocytes.

Transcribed sequences have been suggested to be associated with the nuclear matrix, differing from non-transcribing sequences, which have been reported to be contained in DNA loops. However, although a dozen of genes have their expression level affected by aging, data on chromatin-nuclear matrix interactions under this physiological condition are still scarce. In the present study, liver imprints from young, adult and old mice were subjected to FISH (fluorescence in situ hybridization) for 45S rDNA and telomeric sequences, with or without a lysis treatment to produce extended chromatin fibres. There was an increased amount of 45S rDNA sequences located in DNA loops as the animals grow older, while telomeric sequences were always observed in DNA loops irrespective of the animal age. We assume that active rRNA genes associate with the nuclear matrix, while DNA loops contain silent sequences. Transcription of each 45S rDNA repeat unit is suggested to be dependent on its interaction with the nuclear matrix.

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom.

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.

Different isolates from Leishmania braziliensis complex induce distinct histopathological features in a murine model of infection.

The aim of this study was to evaluate the histopathological features in tissues of mice infected by human isolates (I, II, and III) or the reference M2903 strain of Leishmania braziliensis complex. BALB/c and C57Bl/6 mice were infected in the hind footpad with 10(6) stationary-phase promastigotes of L. braziliensis complex. The evolution of lesions was observed for 10 weeks and the animals were then euthanized and liver, spleen and popliteal lymph nodes were collected. Tissues were stained with hematoxylin and eosin and analyzed by immunohistochemistry assay. Increased thickness of infected footpads was observed in all animals, lesions were nodular and non-ulcerated. Mice infected with isolate I presented inflammatory infiltrates consisting predominantly of mononuclear cells in all tissues examined, and also a great number of megakaryocytes, compared with other isolates. Infection with isolate II led to an infected footpad enlargement not seen in other isolates. In addition, mononuclear infiltrates in the liver and hemosiderin in spleen were noted. Conversely, mice infected with either isolate III or M2903 strain only showed an increased number of megakaryocytes in spleen. All tissues examined had detectable amastigote forms of Leishmania by immunohistochemistry in all groups. Taking together, our results showed an unforeseen behavior of different isolates of L. braziliensis complex that led to diverse pathological findings.

Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice.

Strongyloides stercoralis is an intestinal nematode capable of chronic, persistent infection and hyperinfection of the host; this can lead to dissemination, mainly in immunosuppressive states, in which the infection can become severe and result in the death of the host. In this study, we investigated the immune response against Strongyloides venezuelensis infection in major histocompatibility complex (MHC) class I or class II deficient mice. We found that MHC II(-/-) animals were more susceptible to S. venezuelensis infection as a result of the presence of an elevated number of eggs in the faeces and a delay in the elimination of adult worms compared with wild-type (WT) and MHC I(-/-) mice. Histopathological analysis revealed that MHC II(-/-) mice had a mild inflammatory infiltration in the small intestine with a reduction in tissue eosinophilia. These mice also presented a significantly lower frequency of eosinophils and mononuclear cells in the blood, together with reduced T helper type 2 (Th2) cytokines in small intestine homogenates and sera compared with WT and MHC I(-/-) animals. Additionally, levels of parasite-specific immunoglobulin M (IgM), IgA, IgE, total IgG and IgG1 were also significantly reduced in the sera of MHC II(-/-) infected mice, while a non-significant increase in the level of IgG2a was found in comparison to WT or MHC I(-/-) infected mice. Together, these data demonstrate that expression of MHC class II but not class I molecules is required to induce a predominantly Th2 response and to achieve efficient control of S. venezuelensis infection in mice.