Role of preoperative retrograde enema on inflammatory and healing parameters in colonic anastomosis: experimental study in dogs / Papel do enema pré-operatório em parâmetro de inflamação e cicatrização em anastomose cólica: estudo experimental em cães

Objective: The purpose of this experimental study was to compare the inflammatory and wound healing response of dogs submitted to colonic anastomosis with and without preoperative retrograde enema. Methods: The study included two groups of 31 female dogs (Canis familiaris). G-I (control): no preoperative bowel preparation; G-II (study): preoperative retrograde enema using a 10% glycerin solution. All the animals were submitted to laparotomy and colotomy at 20 cm from the anal verge, followed by closure with a running extramucosal single-layer suture (Prolene® 000). The animals were then anesthetized and euthanized on the 7th (n = 10) or 21st (n = 20) postoperative day (POD) to remove the anastomosed colon segment for histological and immunohistochemical analysis evaluating the parameters: anastomotic edema, vasoproliferation, abdominal adhesions, type I and III collagen, nitric oxide and myeloperoxidase. The observed differences were analyzed with the Mann-Whitney test for nonparametric data and Student's t test for unpaired samples and parametric data. Results: One animal from G-I and one from G-II died on POD 7 and POD 10 due to anastomotic complications and sepsis, respectively The groups did not differ significantly with regard to inflammatory and healing parameters, although the levels of mature collagen were significantly lower in the animals submitted to preoperative bowel preparation. Conclusion: It has been shown that both procedures are safe to be used, however, the group with bowel preparation showed a lower amount of mature collagen in the immediate postoperative period and may be constituted a preventive factor for surgical complications for this type of surgical procedure, although no evidence in this study could be determined. (AU)

Objetivo: O objetivo deste estudo experimental foi comparar a resposta inflamatória e cicatrização de feridas em cães submetidos a anastomose cólica com e sem enema retrógrada pré-operatório. Métodos: O estudo incluiu dois grupos de 31 cães fêmeas (Canis familiaris). G-I (controle): sem preparo intestinal pré-operatório; G-II (estudo): Enema retrógrada pré-operatória com uma solução de glicerina a 10%. Todos os animais foram submetidos à laparotomia e colotomia a 20 cm da borda anal, seguido de fechamento em sutura extramucosa contínua (Prolene ® 000). Os animais foram anestesiados e, em seguida, submetidos à eutanásia no 7° (n = 10) ou 21 (n = 20) pós-operatório (DPO) para remover o segmento de cólon anastomosado para análise histológica e imunohistoquímica avaliando os parâmetros: edema da anastomose, vasoproliferação, aderências abdominais, colagénio tipo I e III, o óxido nítrico e a mieloperoxidase. As diferenças observadas foram analisadas com o teste de Mann-Whitney para os dados não paramétricos e teste t de Student para amostras não pareadas e dados paramétricos. Resultados: Um animal do GI e um do G-II morreu no dia 7 e 10° DPO devido a complições de anastomose e sepse, respectivamente. Os grupos não diferiram significativamente em relação aos parâmetros inflamatórios e de cura, embora os níveis de colágeno maduro foram significativamente menores nos animais submetidos ao preparo intestinal pré-operatório. Conclusão: Demonstrou-se que ambos os procedimentos são seguros para serem usados, no entanto, o grupo com a preparação do intestino mostrou uma menor quantidade de colágeno maduro no período pós-operatório imediato, podendo ser constituído um fator preventivo para compliçães cirúrgicas para este tipo de procedimento cirúrgico, embora nenhuma evidência neste estudo. (AU)

Antimicrobial drug resistance patterns among cattle- and human-associated Salmonella strains.

During the year 2004, 178 human and 158 bovine clinical Salmonella isolates were collected across New York State to better understand the transmission dynamics and genetic determinants of antimicrobial resistance among human and bovine hosts. Serotyping, sequence typing, and pulsed-field gel electrophoresis typing results have been reported previously. Here we tested all isolates for phenotypic susceptibility to 15 antimicrobial drugs that are part of the National Antimicrobial Monitoring System bovine susceptibility panel. PCR was performed on a representative subset of unique isolates (n = 53) to screen for the presence of 21 known antimicrobial resistance genes (i.e., ampC, blaTEM-1, blaCMY-2, blaPSE-1, cat1, cat2, cmlA, flo, aadA1, aadA2, aacC2, strA, strB, aphA1-IAB, dhrfI, dhrfXII, sulI, sulII, tetA, tetB, and tetG); selected fluoroquinolone- and nalidixic acid-resistant (n = 3) and -sensitive (n = 6) isolates were also tested for known resistance-conferring mutations in gyrA and parC. Genes responsible for antimicrobial resistance were shared among isolates of human and bovine origin. However, bovine isolates were significantly more likely than human isolates to be multidrug resistant (P < 0.0001; Fisher's exact test). Our analyses showed perfect categorical agreement between phenotypic and genotypic resistance for beta-lactam and chloramphenicol. Our data confirm that resistance profiles of amoxicillin-clavulanic acid, chloramphenicol, kanamycin, and tetracycline were strongly associated with the presence of blaCMY or ampC, flo, aphA1-IAB, and tetA, respectively. Our findings provide evidence for the clinical value of genotypic resistance typing if incorporating multiple known genes that can confer a phenotypic resistance profile.

Pregnancy in Hystricomorpha: gestational age and embryonic-fetal development of agouti (Dasyprocta prymnolopha, Wagler 1831) estimated by ultrasonography.

Thirty-one pregnant agoutis, between Days 9 and 103 of gestation (Day 1 = day of detection of sperm in the vaginal smear), underwent B-mode ultrasonography; gestational sac diameter (GSD), crown-rump length (CRL), embryonic-fetal diameter (EFD), and placenta diameter (PD) were measured. There were positive correlations (P < 0.05) between GSD and CRL (r = 0.98), GSD and PD (r = 0.88), CRL and PD (r = 0.86), days of gestation (DG) and CRL (r = 0.85), and DG and PD (r = 0.73). The gestational sac was first observed on Day 14. The embryo was first seen on Day 18 in 9/31 of pregnant agoutis and on Day 22 in 20/31 of pregnant agoutis. Heartbeats were detected from the Day 25 and placentas were observed in 100% of the animals from Day 25. Early limb bud and ossification of the fetal skull were identified on Days 27 (15/31) and 45 (24/31), respectively. Fetal orientation (head and body) was evident from Day 40, the stomach, liver and lungs were identified on Day 50, the kidneys were reliably seen only on Day 55, and the aorta and vena cava were seen on Day 70. The fetal bowel and the urinary bladder were the last structures to be observed (Day 85). Ultrasonography was effective for early pregnancy diagnosis in agouti and for obtaining information on embryonic and fetal structures that could be used to predict gestational age and birth, thereby contributing to their reproductive management in captivity.

Listeria monocytogenes and hemolytic Listeria innocua in poultry.

Listeria monocytogenes is a ubiquitous, saprophytic, Gram-positive bacterium and occasional food-borne pathogen, often associated with ready-to-eat meat products. Because of the increased consumer interest in organic, all natural, and free range poultry products, it is important to understand L. monocytogenes in the context of such systems. Pasture-reared poultry were surveyed over the course of two 8-wk rearing periods. Cecal, soil, and grass samples were collected for Listeria isolation and characterization. Seven of 399 cecal samples (or 1.75%) were Listeria-positive. All positive cecal samples were obtained from broilers sampled at 2 wk of age. Grass and soil samples were collected from the pasture both before and after introduction of the poultry. Environmental samples collected after introduction of poultry were significantly more likely to contain Listeria (P < 0.001). The results of analytical profile index Listeria, sigB allelic typing, and hlyA PCR tests found that both L. monocytogenes and L. innocua, including hemolytic L. innocua, were recovered from the cecal and environmental (grass/soil) samples. The sigB allelic typing also revealed that (1) positive samples could be composed of 2 or more allelic types; (2) allelic types found in cecal samples could also be found in the environment; and (3) allelic types could persist through the 2 rearing periods. Our data indicate that both pasture-reared poultry and their environment can be contaminated with L. monocytogenes and hemolytic L. innocua.

inlA premature stop codons are common among Listeria monocytogenes isolates from foods and yield virulence-attenuated strains that confer protection against fully virulent strains.

Previous studies showed that a considerable proportion of Listeria monocytogenes isolates obtained from foods carry a premature stop codon (PMSC) mutation in inlA that leads to production of a truncated and secreted InlA. To further elucidate the role these mutations play in virulence of L. monocytogenes, we created isogenic mutants, including (i) natural isolates where an inlA PMSC was reverted to a wild-type inlA allele (without a PMSC) and (ii) natural isolates where a PMSC mutation was introduced into a wild-type inlA allele; isogenic mutant sets were constructed to represent two distinct inlA PMSC mutations. Phenotypical and transcriptional analysis data showed that inlA PMSC mutations do not have a polar effect on the downstream inlB. Isogenic and natural strains carrying an inlA PMSC showed significantly reduced invasion efficiencies in Caco-2 and HepG2 cell lines as well as reduced virulence in oral guinea pig infections. Guinea pigs were also orally infected with a natural strain carrying the most common inlA PMSC mutation (vaccinated group), followed by challenge with a fully virulent L. monocytogenes strain 15 days postvaccination to probe potentially immunizing effects of exposure to L. monocytogenes with inlA PMSC mutations. Vaccinated guinea pigs showed reduced bacterial loads in internal organs and improved weight gain postchallenge, indicating reduced severity of infections in guinea pigs exposed to natural strains with inlA PMSC mutations. Our data support that (i) inlA PMSC mutations are causally associated with attenuated virulence in mammalian hosts and (ii) naturally occurring virulence-attenuated L. monocytogenes strains commonly found in food confer protective immunity.

Multilocus sequence typing supports the hypothesis that cow- and human-associated Salmonella isolates represent distinct and overlapping populations.

A collection of 179 human and 156 bovine clinical Salmonella isolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA, manB, and mdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specific Salmonella enterica serotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related to Salmonella serotype Typhimurium. Salmonella serotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number of Salmonella isolates carried two copies of manB (33 isolates) or showed small deletion events in fimA (nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification of Salmonella surface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission of Salmonella.

Ecology and transmission of Listeria monocytogenes infecting ruminants and in the farm environment.

A case-control study involving 24 case farms with at least one recent case of listeriosis and 28 matched control farms with no listeriosis cases was conducted to probe the transmission and ecology of Listeria monocytogenes on farms. A total of 528 fecal, 516 feed, and 1,012 environmental soil and water samples were cultured for L. monocytogenes. While the overall prevalence of L. monocytogenes in cattle case farms (24.4%) was similar to that in control farms (20.2%), small-ruminant (goat and sheep) farms showed a significantly (P < 0.0001) higher prevalence in case farms (32.9%) than in control farms (5.9%). EcoRI ribotyping of clinical (n = 17) and farm (n = 414) isolates differentiated 51 ribotypes. L. monocytogenes ribotypes isolated from clinical cases and fecal samples were more frequent in environmental than in feed samples, indicating that infected animals may contribute to L. monocytogenes dispersal into the farm environment. Ribotype DUP-1038B was significantly (P < 0.05) associated with fecal samples compared with farm environment and animal feedstuff samples. Ribotype DUP-1045A was significantly (P < 0.05) associated with soil compared to feces and with control farms compared to case farms. Our data indicate that (i) the epidemiology and transmission of L. monocytogenes differ between small-ruminant and cattle farms; (ii) cattle contribute to amplification and dispersal of L. monocytogenes into the farm environment, (iii) the bovine farm ecosystem maintains a high prevalence of L. monocytogenes, including subtypes linked to human listeriosis cases and outbreaks, and (iv) L. monocytogenes subtypes may differ in their abilities to infect animals and to survive in farm environments.

Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes.

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.

Lipid peroxidation induced by Clinostomum detruncatum in muscle of the freshwater fish Rhamdia quelen.

The effect of Clinostomum detruncatum metacercaria infection on the activities of the antioxidant enzymes superoxide dismutase and catalase in muscle of the freshwater fish Rhamdia quelen was analyzed. Tert-butyl hydroperoxide-initiated chemiluminescence, a measure of lipid peroxidation, was also investigated. Enzyme activities were similar in infected and uninfected fishes. However, the chemiluminescence was almost 2-fold higher in muscle of infected fishes than in muscle of uninfected ones. These results indicate that parasite infection induces oxidative stress and a higher level of membrane damage in the fish muscle due to an imbalance between pro-oxidants and non-enzymatic antioxidants. Our results suggest that fish response to parasite infection could involve, as in other vertebrates, reactive oxygen intermediates.

Expression and regulation of mitochondrial uncoupling protein 1 from brown adipose tissue in Leishmania major promastigotes.

Rat uncoupling protein 1 (UCP1) was successfully translated in transfected Leishmania major promastigotes. Immune electron microscopy revealed that the protein was exclusively in the mitochondria. UCP1 expression was about 350,000 copies per promastigote, accounting for 4.7% of the total mitochondrial protein. In intact parasites, expression of UCP1 induced a slight increase in respiratory rate and a modest decrease in mitochondrial membrane potential (delta psi(m)). In contrast, in digitonin-permeabilized parasites, a significantly lower value both in delta psi(m) (57 +/- 10 vs 153 +/- 12 mV) and respiratory control ratio (0.99 vs 1.54) were observed for UCP1 versus control parasites, although when UCP1 activity was inhibited by bovine serum albumin (BSA) and GDP, control values were restored. Therefore, a fully functional UCP1 was present and only partially inhibited in vivo by endogenous purine nucleotides. However, neither ATP levels, growth rate nor mitochondrial protein import differed significantly between both types of parasites. Expression of the pore-like mutant UCP1 delta 9 was deleterious to the organism. Consequently, Leishmania was capable of expressing and importing into mitochondria proteins from higher eukaryotes lacking an N-terminal targeting pre-sequence as UCP1. As described previously, parasite metabolism had only a limited tolerance to mitochondrial disfunction. Transfection of Leishmania with foreign proteins which play an important regulatory role in metabolism is a useful tool to study both parasite metabolism in general, and alternative pathways involved in maintaining internal homeostasis.

Nocturnal urinary growth hormone excretion as a criterion for growth hormone deficiency.

Nocturnal urinary growth hormone (U-hGH) levels measured by a sensitive immunoenzymometric assay were compared with hGH levels in serum before and after a clonidine test in healthy children and in children with short stature to determine whether U-hGH measurement is useful for the screening of hGH deficiency. The study was carried out on 19 healthy children (10 prepubertal and 9 pubertal subjects) and on 20 children with short stature, 10 with growth hormone deficiency (hGHD) and 10 with constitutional growth retardation. The diagnosis of hGHD was based on a blunted response to two provocative hGH tests in the appropriate clinical setting. Overnight urinary hGH secretion (mean of 3 collections) was measured by an immunoenzymometric assay. The best discrimination was obtained when the results were expressed as ng/h. Only one individual in the prepubertal group (U-hGH, 0.05 ng/h) and one patient in the growth retardation group (U-hGH, 0.08 ng/h) had a urinary hGH value below the highest value (0.17 ng/h) observed in the growth hormone deficiency group. The coefficient of correlation between urinary hGH in ng/h and post-clonidine peak was 0.50 (P = 0.0015), between urinary hGH in ng/l and post-clonidine peak was 0.48 (P = 0.0025), between urinary hGH in ng/l per hour and post-clonidine peak was 0.47 (P = 0.0027). The highest specificity (0.93), sensitivity (0.90), false negative rate (0.96) and false positive rate (0.82) were obtained when U-hGH was expressed as ng/h per night. Measurement of urinary nocturnal hGH excretion is a useful, simple, noninvasive method for the diagnosis of hGH deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Nocturnal urinary growth hormone excretion as a criterion for growth hormone deficiency

Nocturnal urinary growth hormone (U-hGH) levels measured by a sensitive immunoenzymometric assay were compared with hGH levels in serum before and after a clonidine test in healthy children and in children with short stature to determine whether U-hGH measurement is useful for the screening of hGH deficiency. The study was carried out on 19 healthy children (10 prepubertal and 9 pubertal subjects) and on 20 children with short stature, 10 with growth hormone deficiency (hGHD) and 10 with constitutional growth retardation. The diagnosis of hGHD was based on a blunted response to two provocative hGH tests in the appropriate clinical setting. Overnight urinary hGH secretion (mean of 3 collections) was measured by an immunoenzymometric assay. The best discrimination was obtained when the results were expressed as ng/h. Only one individual in the prepubertal group (U-hGH, 0.05 ng/h) and one patient in the growth retardation group (U-hGH, 0.08 ng/h) had a urinary hGH value below the highest value (0.17 ng/h) observed in the growth hormone deficiency group. The coefficient of correlation between urinary hGH in ng/h and post-clonidine peak was 0.50 (P = 0.0015), between urinary hGH in ng/l and post-clonidine peak was 0.48 (P = 0.0025), between urinary hGH in ng/l per hour and post-clonidine peak was 0.47 (P = 0.0027). The highest specificity (0.93), sensitivity (0.90), false negative rate (0.96) and false positive rate (0.82) were obtained when U-hGH was expressed as ng/h per night. Measurement of urinary nocturnal hGH excretion is a useful, simple, noninvasive method for the diagnosis of hGH deficiency. However, the day-to-day variability and wide normal range limit its usefulness in mild forms of hGH insufficiency

Utilizaçäo do esfregaço de coágulo sanguíneo para o diagnóstico de Babesia e Anaplasma em bovinos / Utilization of blood clot smear for diagnosis of Babesia and Anaplasma in cattle

O desempenho da técnica de esfregaço de fragmento de coágulo de sangue, com as coloraçöes de Giemsa diluído e puro, é avaliado em relaçäo à apresentaçäo dos elementos figurados e na determinaçäo de parasitemia por Babesia bovis, B. bigemina e Anaplasma marginale. Utilizou-se um bovino, seis meses de idade e inoculado com os agentes da tristeza parasitária bovina como doador das amostras de coágulo. A técnica mostrou-se adequada ao diagnóstico dos parasitos, com restriçöes à observaçäo de eritrócitos e leucócitos

Diagnóstico de hemoparasitose através da técnica de distensäo de gota de coágulo sanguíneo / Diagnosis of hemoparasitosis trough the spread-smear technique using a drop of blood clot

O desempenho da técnica de distensäo de Gota do Coágulo de sangue, entre as coloraçöes de Giemsa diluído e puro, é avaliado em relaçäo à possibilidade de observaçäo dos elementos figurados e da presença de Babesia bovis, Babesia bigemina e Anaplasma marginale. Utilizou-se um bovino, com 6 meses de idade inoculado com os agentes da Tristeza Parasitária, como doador das amostras de sangue. A técnica permitiu a observaçäo dos eritrócitos, leucócitos e dos hemoparasitas