Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices.

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates ("IM" and "TA") were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen-derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

Diploid and aneuploid sperm in tetraploid ginbuna, Carassius auratus langsdorfii.

Ginbuna (Carassius auratus langsdorfii (Teleostei: Cyprinidae)) occur in diploid, triploid, and tetraploid forms in wild populations. Diploid females reproduce bisexually, whereas polyploid (triploid and tetraploid) females reproduce gynogenetically with no contribution from sperm nuclei. However, tetraploid males produce diploid sperm. The mechanism responsible for the differences in egg and sperm ploidy has not been elucidated as tetraploid males are rare in wild populations. Here, we aimed to characterize the types of sperm and elucidate the mechanism of spermatogenesis in ginbuna. In the present study, we artificially produced tetraploid males by crossbreeding triploid ginbuna females with diploid goldfish (Carassius auratusauratus) males via accidental incorporation of sperm nuclei. We then examined spermatogenesis to reveal the process by which reduced diploid sperm are generated from tetraploid germ cells. DNA fingerprinting by random amplified polymorphic DNA (RAPD)-PCR indicated that the tetraploid progeny had a paternally derived genome. For the tetraploid male sperm, there were narrow (N-type) and broad (B-type) flow cytometrical histograms. The N-type were determined to be diploid with a low coefficient of variation (CV) by flow cytometry. The B-type were found to be aneuploid (hypodiploid to hexaploid) with a high CV. The head sizes of B-type sperm were variable, whereas those of the N-type sperm were uniform. Computer-assisted sperm analysis (CASA) revealed that both the haploid and diploid B-type sperm were weakly motile compared with the haploid sperm of goldfish and the diploid N-type sperm of tetraploid males. Bivalents and various multivalents were observed in the meiotic configurations of diploid spermatogenesis. In aneuploid spermatogenesis, most of the chromosomes were unpaired univalents and there were very few bivalents. Our findings provide empirical evidence for two different types of spermatogenesis in tetraploid C. a. langsdorfii males. Meiotic synapses might explain the observed differences in the ploidy status of the two sperm types.

Isolation characterization, virulence potential of Weissella ceti responsible for weissellosis outbreak in rainbow trout (Oncorhynchus mykiss) cultured in Mexico.

Weissella ceti, a Gram-positive nonmotile bacterium, is currently an emerging pathogen within rainbow trout (Oncorhynchus mykiss) farms in China, Brazil, the United States, and Japan. This study is the first to isolate, identify, and characterize W. ceti isolates from rainbow trout farmed in Mexico. In late 2015, a severe disease outbreak caused a 60% mortality rate among 20,000 fish. The diseased rainbow trout (100-300 g average) exhibited severe cachexia, body darkening, abdominal distension, exophthalmia, haemorrhages, and corneal opacity. Internally, diseased fish had pale gills; multifocal, disseminated whitish spots on the liver; haemorrhages in the swim bladder, ovary, and on the parietal surface of the muscle; and hearts with pseudo-membrane formation. Histologically, lesions were characterized by corneal oedema, degenerative and necrotic hepatitis, and meningitis. A brain (W-1) and kidney (W-2) isolate were identified as W. ceti through polyphasic taxonomy, which included phenotypic characterization and 16S rRNA sequencing. RAPD and ERIC-PCR analyses demonstrated genetic homogeneity among the Mexican isolates. Virulence tests in rainbow trout through intraperitoneal W. ceti injections at concentrations of 1 × 104 , 1 × 105 , and 1 × 106 CFU per fish resulted in cumulative mortality rates of 25%, 62.5%, and 87.5%, respectively, as well as the same clinical signs of hemorrhagic septicaemia as were recorded for the natural outbreak. The present report is the first to confirm the presence of W. ceti in Mexico, thus extending the known geographical distribution of this pathogen across the Americas.

Genetic relationship between Escherichia coli strains isolated from dairy mastitis and from the stable fly Stomoxys calcitrans / Caracterização genotípica de Escherichia coli isolados de leite com mastite e da mosca dos estábulos Stomoxys calcitrans

The stable fly Stomoxys calcitrans (Linnaeus, 1758) has been described as a potential spreader of infectious agents to cattle herds. Among the agents transmitted by this fly, Escherichia coli has attracted attention due to its potential to cause gastrointestinal disorders as well as environmental mastitis in dairy cows. Therefore, the aim of this study was to isolate and to assess the genetic diversity and the clonal relatedness among E. coli isolates from the milk of dairy mastitis and from stable flies anatomical sites by the Random Amplification of Polymorphic DNA (RAPD-PCR) technique. The molecular typing revealed a high degree of genetic polymorphism suggesting that these microorganisms have a non-clonal origin. Identical electrophoretic profiles were observed between E. coli isolates from different flies, different mammary quarters of the same cow and from cows on a single farm. These results reveal the circulation of the same bacterial lineages and suggest the role of the stable fly in bacterial dispersion. Considering the high pathogenic potential of this bacterial species, our findings alert to a more effective health surveillance.(AU)

A mosca dos estábulos Stomoxys calcitrans é descrita como um importante dispersor de agentes infecciosos aos bovinos. Dentre os agentes veiculados por esta mosca a bactéria Escherichia coli ganha relevância devido ao seu potencial em desenvolver alterações gastroentéricas, bem como mastite bovina ambiental. Desta forma, objetiva-se com este estudo isolar e acessar a diversidade genética e relação de clonalidade entre isolados de E. coli provenientes de casos de mastite e de moscas dos estábulos utilizando a técnica da Amplificação Randômica do DNA Polimórfico (RAPD). A tipagem molecular revelou elevado polimorfismo genético sugerindo que esses microrganismos têm origem não clonal. Perfis eletroforéticos idênticos entre si foram observados entre amostras isoladas de diferentes moscas, quartos mamários de uma mesma vaca, bem como de diferentes vacas dentro de uma mesma propriedade. Esses resultados revelam a circulação de uma mesma linhagem bacteriana e sugerem o papel da Stomoxys calcitrans na dispersão bacteriana. Considerando o elevado potencial patogênico dessa espécie bacteriana, nossos achados alertam para uma vigilância sanitária mais efetiva.(AU)

Molecular cloning and functional analysis of the goose FSHß gene.

The objective of this investigation was to clone goose FSHß-subunit cDNA and to construct a FSH fusion gene to identify the function of FSHß mRNA during stages of the breeding cycle. The FSHß gene was obtained by reverse transcription-PCR, and the full-length FSHß mRNA sequence was amplified by rapid-amplification of cDNA ends. FSHß mRNA expression was detected in reproductive tissues at different stages (pre-laying, laying period, and broody period). Additionally, the expression of 4 genes known to be involved in reproduction (FSHß, GnRH, GH, and BMP) were evaluated in COS-7 cells expressing the fusion gene (pVITRO2-FSHαß-CTP). The results show that the FSHß gene consists of a 16 base pair (bp) 5'-untranslated region (UTR), 396 bp open reading frame, and alternative 3'-UTRs at 518 bp and 780 bp, respectively. qPCR analyses revealed that FSHß mRNA is highly transcribed in reproductive tissues, including the pituitary, hypothalamus, ovaries, and oviduct. FSHß mRNA expression increased and subsequently decreased in the pituitary, ovaries, and oviduct during the reproductive stages. Stable FSH expression was confirmed using enzyme-linked immunosorbent assays after transfection with the pVITRO2-FSHαß-CTP plasmid. FSHß, GnRH, and BMP expression increased significantly 36 h and 48 h after transfection with the fusion gene in COS-7 cells. The results demonstrate that the FSHß subunit functions in the goose reproductive cycle and provides a theoretical basis for future breeding work.

Characterization of cathepsin B gene from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

The lysosomal cysteine protease cathepsin B of papain family is a key regulator and signaling molecule that involves in various biological processes, such as the regulation of apoptosis and activation of virus. In the present study, cathepsin B gene (Ec-CB) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CB cDNA was composed of 1918 bp and encoded a polypeptide of 330 amino acids with higher identities to cathepsin B of teleosts and mammalians. Ec-CB possessed typical cathepsin B structural features including an N-terminal signal peptide, the propeptide region and the cysteine protease domain which were conserved in other cathepsin B sequences. Phylogenetic analysis revealed that Ec-CB was most closely related to Lutjanus argentimaculatus. RT-PCR analysis showed that Ec-CB transcript was expressed in all the examined tissues which abundant in spleen, kidney and gill. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the mRNA expression of cathepsin B in E. coioides was up-regulated at 24 h post-infection. Subcellular localization analysis revealed that Ec-CB was distributed predominantly in the cytoplasm. When the fish cells (GS or FHM) were treated with the cathepsin B specific inhibitor CA-074Me, the occurrence of CPE induced by SGIV was delayed, and the viral gene transcription was significantly inhibited. Additionally, SGIV-induced typical apoptosis was also inhibited by CA-074Me in FHM cells. Taken together, our results demonstrated that the Ec-CB might play a functional role in SGIV infection.

Identification by culture, PCR, and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey.

This study used cultures, polymerase chain reaction (PCR), and immunoperoxidase to examine samples from 216 lungs from sheep and lambs with macroscopic pneumonia lesions for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and broth cultures with the help of a DNA extraction kit and replicated using genus-specific and species-specific primers for mycoplasma. The lung samples were examined by the immunoperoxidase method using hyperimmune Mycoplasma ovipneumoniae serum. The randomly amplified polymorphic DNA (RAPD) test was used for the molecular typing of M. ovipneumoniae isolates. Mycoplasma was isolated in the cultures of 80 (37.03 %) of a total of 216 lung samples. Genus-specific mycoplasma DNA was identified by PCR in 96 (44.44 %) samples in broth cultures and 36 (16.66 %) directly in the lung tissue. Of these 96 cases in which genus-specific identification was made, 57 (59.37 %) were positive for reaction with species-specific primers for M. ovipneumoniae and 31 (32.29 %) for Mycoplasma arginini. The DNA of neither of the latter two species could be identified in the remaining eight samples (8.33 %) where mycoplasma had been identified. As for the immunoperoxidase method, it identified M. ovipneumoniae in 61 of 216 lung samples (28 %). Positive staining was concentrated in the bronchial epithelium cell cytoplasm and cell surface. RAPD analysis resulted in 15 different profiles. Our results suggest that PCR methods could be successfully used in the diagnosis of mycoplasma infections as an alternative to culture method and identifying this agent at the species level.

Genetic diversity of the species Leporinus elongatus (Teleostei: Characiformes) in the Canoas Complex - Paranapanema River

Dams constructed along waterways interrupt the dispersion and migration of aquatic organisms, affecting mainly the abundance of migratory fish species. Translocation mechanisms have been constructed at dams aiming to minimize their impact on fish species migration behavior. There is little information available about the effect of the construction of dams on the genetic structure of the Neotropical migratory fish fauna. Therefore, RAPD molecular markers and microsatellites were utilized to evaluate the diversity and genetic structure of the migratory species Leporinus elongatus (piapara) in the Canoas Complex - Paranapanema River - Brazil. Ten groups were sampled in the fish ladders of the hydroelectric dam Canoas I and Canoas II during the reproductive period in three consecutive years. Both markers showed a high level of genetic diversity within these groups. The microsatellite markers demonstrated a loss of heterozygosity and a considerable level of inbreeding in the species. The genetic differentiation found among the groups with both markers utilized is within a range from low to moderate. The data obtained with the parameter of genetic diversity among the groups led to the conclusion that the groups of L. elongatus of the Canoas Complex are structured as a single population composed of sub-populations with low genetic diversity among them. The data on genetic diversity and population structure of L. elongatus are of great importance for the development of the species management and conservation programs in the Canoas Complex, which can also be utilized in aquaculture programs.

As barragens construídas ao longo de sistemas hídricos interrompem a dispersão e a migração dos organismos aquáticos, afetando principalmente a abundância das espécies de peixes migradores. Mecanismos para transposição foram construídos em barragens visando minimizar esses impactos. Poucas são as informações disponíveis sobre o efeito da construção de barragens na estrutura genética populacional da fauna neotropical de peixes migradores. Nesse contexto, marcadores moleculares RAPD e microssatélites foram utilizados para avaliar a diversidade e a estrutura genética da espécie migradora Leporinus elongatus (piapara) no Complexo Canoas - rio Paranapanema - Brasil. Dez grupos foram amostrados nas escadas para transposição de peixes das UHEs Canoas I e Canoas II durante o período reprodutivo em três anos consecutivos. Ambos os marcadores evidenciaram uma alta diversidade genética para esses grupos. Os marcadores microssatélites mostraram uma perda de heterozigosidade e uma considerável taxa de endocruzamento para a espécie. A diferenciação genética encontrada entre os grupos, com ambos os marcadores utilizados, pode ser considerada de moderada a baixa. Os dados obtidos com os parâmetros de diversidade genética entre os grupos permitiram concluir que os grupos de L. elongatus do Complexo Canoas estão estruturados como uma única população composta por sub-populações com baixa diversidade genética entre elas. Os dados obtidos sobre a diversidade genética e estrutura populacional de L. elongatus são de grande importância para o desenvolvimento de programas de manejo e conservação da espécie no Complexo Canoas, podendo também ser utilizados em programas de aqüicultura.

Molecular cloning and differential expression of three GnRH genes during ovarian maturation of spotted halibut, Verasper variegatus.

In this study, the gonadotropin-releasing hormone (GnRH) genes in spotted halibut were cloned and sequenced by isolating their cDNAs. The species expressed three molecular forms of GnRH in the brain: chicken-type GnRH-II (cGnRH-II), seabream-type GnRH (sbGnRH), and salmon-type GnRH (sGnRH). Phylogenetic analysis divided the molecular forms of GnRHs into three branches: cGnRH-II branch, sGnRH branch, and fish-specific GnRH branch. The spatial expression showed that they had the highest expression levels in the brain. cGnRH-II was exclusively detected in the brain, while sbGnRH had a global expression pattern in all examined organs. sGnRH was detected in the brain, pituitary, and ovary. The temporal changes of brain GnRH mRNA expression levels were examined during ovarian maturation and postspawning, and the serum steroid hormones and gonadosomatic index (GSI) were recorded. Amounts of sbGnRH mRNA substantially elevated (P < 0.05) during ovarian maturation, which concomitant with considerable elevation of GSI and serum steroids levels. On the contrary, neither sGnRH nor cGnRH-II mRNA levels showed significant changes during ovarian maturation in this study. These results suggested that these three GnRH genes are the important regulators for the differential expression of GnRH in spotted halibut, and would help us better understand the reproductive endocrine mechanism of spotted halibut.

Bovine intramammary Escherichia coli challenge infections in late gestation demonstrate a dominant antiinflammatory immunological response.

Coliform mastitis that presents itself at parturition or in the early weeks of bovine lactation is often characterized by severe inflammation and impaired milk production and can lead to death of the animal. Chronic intramammary infections caused by persistent strains of Escherichia coli may result in high production losses. The aim of this study was to determine the inflammatory response to a teat-canal challenge of bovine mammary glands with a persistent strain of E. coli during late gestation (dry period) and into early lactation. Two weeks before parturition, animals were challenged in 2 quarters with 30 cfu of a persistent strain of E. coli; control quarters were vehicle-infused and not infused, respectively. Samples of dry cow secretions were taken from all quarters before challenge and at 6, 12, 18, 24, 48, 72, 96, and 120 h following challenge. Colostrum samples and milk samples were taken from all quarters at parturition and 6, 12, 18, 24, 48, 72, 96 and 120 h postpartum. Bacterial culture, combined with random amplified polymorphic DNA genetic strain-typing analysis, indicated recovery of the bacterial challenge strain until 48 to 96 h postchallenge, and again at parturition and up to 6 and 12h postpartum. One animal exhibited clinical mastitis and the bacterial challenge strain was evident to at least 12 d postpartum. During twice-daily milkings, production levels were lower in bacteria-challenged quarters compared with controls. Somatic cell counts decreased to normal levels at a slower rate in challenged quarters compared with control quarters. Cytokine analysis indicated a minimal proinflammatory cytokine response, including interleukin-1ß and tumor necrosis factor-α in challenged-quarter dry cow samples up to 120 h postchallenge. Interleukin-10 levels were significantly increased by 12h postchallenge in secretions from challenged and control quarters. These preliminary results in 2 cows indicate that proinflammatory signaling after intramammary bacterial infection may be actively suppressed during late gestation. We hypothesize that this immune-inhibitory response allows intramammary infections to become persistent in the dry period and cause clinical signs immediately after parturition.

Identificação sorológica e relação filogenética de Salmonella spp. de origem suína / Serological identification and phylogenetic relationship of Salmonella spp. pig origin

Salmonella spp. é um importante patógeno zoonótico que pode ser disseminado ao longo da cadeia produtiva de suínos. Objetivou-se avaliar a incidência de Salmonella spp. em fezes de suínos de terminação na granja, no pré-abate e amostras ambientais, identificar os sorovares e estabelecer a relação filogenética entre os isolados. Foram realizadas três coletas em lotes diferentes de suínos alojados na granja de terminação e nos mesmos animais após o transporte ao frigorífico totalizando 90 parcelas e 9 amostras ambientais. O transporte não influenciou na porcentagem de isolamento do microrganismo (p>0,05). Das 99 amostras, 50 (50,5 por cento) foram identificados como Salmonella spp., sendo identificado uma multiplicidade de sorovares: Agona (30 por cento), Typhimurium (26 por cento), Minnesota (24 por cento), Infantis (18 por cento) e Panama (2 por cento). Os dendrogramas demonstraram homologia entre isolados dos diferentes sorovares agrupados em clusters. A similaridade foi independente do local de isolamento indicando a presença de vários clones. As principais fontes de infecção determinadas foram a contaminação cruzada entre animais e ambiente e o consumo de ração contaminada. A diversidade de sorovares e a homologia entre eles indicam origem comum, demonstrando necessidade de monitoramento de bactérias zoonóticas e de implantação de medidas de controle mais eficazes para Salmonella spp. em suínos.

Salmonella spp. is an important zoonotic pathogen that can spread along the production chain of swines. The objective was to evaluate the incidence of Salmonella spp. in feces of swines in termination phase in the farm, in the pre-slaughter and environmental samples, identify the serotypes and establish a phylogenetic relationship among the isolates. Three collections were done in different batches of pigs housed in the termination pen and in the same animals after transport to the slaughterhouse totaling 90 plots and 9 environmental samples. The transport does not influenced the percentage of isolation of the microorganism (p>0.05). Of the total of 99 samples, 50 (50.5 percent) were identified as Salmonella spp., and was identified a variety of serovars: Agona (30 percent), Typhimurium (26 percent), Minnesota (24 percent), Infantis (18 percent) and Panama (2 percent). Dendrograms showed homology among isolates of different serovars grouped into clusters. The similarity was independent of the local of isolation, indicating the presence of several clones. The main sources of infection were cross-contamination between animals and environment and the consumption of contaminated feed. The diversity of strains and homology among the isolates indicates a common origin, demonstrating a need for monitoring of zoonotic bacterias and the deployment of more effective control measures for Salmonella spp. in swines.

Cloning, expression analysis and promoter structure of TBK1 (TANK-binding kinase 1) in Atlantic cod (Gadus morhua L.).

TBK1, also termed NAK or T2K, is a ubiquitous member of the IκB kinase (IKK) family that is required for innate and adaptive immune responses. We have identified and characterized the full-length TBK1 cDNA in Atlantic cod. The cod TBK1 gene consists of 2190 bp open reading frame encoding a polypeptide of 729 amino acids. According to a BLAST search, the cloned TBK1 gene has a high degree of sequence similarity (80.7-92%) to the various members of the TBK1 family, indicating that it is conserved during evolution. RT-PCR showed that the largest quantity of TBK1 transcripts was found in spleen, followed by the liver, gill, head kidney, gut, pyloric caeca, while the expression of TBK1 mRNA in muscle and skin was low. Both PMA, poly I:C and ß-glucan promoted expression of TBK1 transcripts in vivo. Furthermore, we determined an 875 bp sequence upstream of the transcriptional start site (TSS) and found a number of sequence motifs that matched known transcription factor-binding sites. Activities of the presumptive regulatory regions of this gene were assessed by transfecting different 5'-deletion constructs in CHSE-214 cells. After the expression experiments, the results showed that the basal promoters and positive transcriptional regulator activities of cod TBK1 gene were dependent by sequences located from -875 to -425 bp and from -245 to +28 bp upstream of TSS. This study provides further insights into the transcriptional regulation of cod TBK1.

Resistência aos antimicrobianos e análise da diversidade genética de Staphylococcus aureus por PCR-RAPD / Resistance to antimicrobials and analysis of Staphylococcus aureus genetic diversity using RAPD-PCR

O objetivo deste trabalho foi analisar a sensibilidade antimicrobiana in vitro de cepas de Staphylococcus aureusisoladas de tetos de vacas e mãos de retireiros, além de verificar o polimorfismo entre elas pela técnica de PCR-RAPD. Os testes foram realizados pela técnica de difusão em discos e, após a extração do material genético foram desenvolvidas as técnicas de PCR e RAPD, usando para isso 40 iniciadores diferentes. A análise do polimorfismo foi realizada empregando-se o programa de taxonomia numérica NTSYS. As sensibilidades dos antimicrobianos nas cepas obtidas de tetos de vacas foram 4% para a penicilina, 88% para a tetraciclina, 92% para a gentamicina, 96% para a vancomicina e 100% ao cloranfenicol. Para as cepas provenientes das mãos de retireiros, os resultados de sensibilidade foram zero para a penicilina, 70% para a tetraciclina e 90% para a vancomicina e 100% para os antimicrobianos gentamicina e cloranfenicol. A realização do E-teste indicou uma concentração inibitória mínima (CIM) maior que 256 mg/mL para as cepas resistentes ao antimicrobiano vancomicina. Os estudos permitiram detectar a resistência dos S. aureus mediante o uso dos antimicrobianos testados e determinar a diversidade genética entre as cepas de estafilococos devido à presença de muitas bandas polimórficas encontradas em todos os iniciadores.

The aim of this study was to analyze in vitro antimicrobial susceptibility of Staphylococcus aureus strains isolated from teats of cow udders and milking workers' hands as well as to verify polymorphism among them by using RAPD-PCR technique. Tests were conducted by disk diffusion technique and after the collection of the genetic material PCR and RAPD techniques were performed with the use of 40 different initiators. The analysis of polymorphism was conducted by using the NTSYS program of numerical taxonomy. The susceptibility of antimicrobials in the strains collected from teats of cow udders was 4% to penicillin, 88% to tetracycline, 92% to gentamicine, 96% to vancomycin and 100% to chloranfenicol. As for the strains collected from milking workers' hands, susceptibility results were 0% to penicillin, 70% to tetracycline and 90% to vancomycin and 100% to gentamicine and chloranfenicol antimicrobials. E-test showed minimum inhibitory concentration (MIC) greater than 256 ?g/mL to strains resistant to the antimicrobial vancomycin. The studies made it possible to detect S. aureus resistance upon the use of the tested antimicrobials and to determine the genetic diversity found among strains of staphylococcus due to the presence of many polymorphic bands found in all initiators.

Molecular cloning and characterization of bloodthirsty from Atlantic cod (Gadus morhua).

The tripartite motif (TRIM) proteins are involved in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, apoptosis and antiviral activity. In this study, we report the identification and characterization of an Atlantic cod tripartite motif-containing protein named bloodthirsty from a poly I:C subtractive cDNA library. The Atlantic cod bloodthirsty (Acbloodthirsty) has a predicted open reading frame of 541 amino acids, encoding a putative 64-kDa protein. The N-terminal region contains the three motifs typical of TRIM proteins, a RING finger, one B-box, and a coiled-coil domain, which together form the TRIM motif found in this large family of proteins, whereas the C-terminal region contains a PRYSPRY domain. The intracellular localization of Acbloodthirsty in CHSE-214 cells showed mostly diffuse staining with some discrete compartments in the cytoplasm. The induction of bloodthirsty transcripts by poly I:C was seen in spleen using quantitative reverse transcriptase PCR (RT-qPCR). The expression pattern indicates that Acbloodthirsty is involved in antiviral immune response in Atlantic cod.

Echinococcus granulosus genotypes in livestock of Iran indicating high frequency of G1 genotype in camels.

In this study, 112 Echinococcus granulosus isolates from different livestock of Iran were genotyped by PCR amplification of ribosomal DNA-internal transcribed spacer 1 (rDNA-ITS1) region followed by restriction fragment length polymorphism (RFLP) with the enzyme RsaI. The possibility of intra-genotype variation was also investigated using randomly amplified polymorphic DNA (RAPD) analysis. Isolates from sheep, goats, cattle and the majority of camels (12 of 18; 66.7%) were identified as the G1 genotype and a few camel isolates (6 of 18; 33.3%) belonged to the G6 genotype. Overall G1 and G6 genotypes were identified in 94.6% (106 of 112) and 5.3% (6 of 112) of all isolates, respectively. RAPD analysis based on 15 separate primers showed 7-14 bands of 200-3000bp for strain G1. Considering each individual primer, no differences observed among isolates from different hosts and between livers and lungs. This study confirmed the existence of G1 and G6 genotypes in Iran. Moreover, G1 is much more prevalent even in camels, indicating the importance of sheep-dog cycle in public health. Studying intra-genotypic variation of E. granulosus warrants more research using other primers and methods.

Interleukin-17D in Atlantic salmon (Salmo salar): molecular characterization, 3D modelling and promoter analysis.

IL-17 is a proinflammatory cytokine that plays an important role in the clearance of extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. Much work on IL-17 has been done in humans and higher vertebrates while little work has been conducted in lower vertebrates including fish. In this study, we have cloned and characterized the full-length cDNA and genomic sequence of IL-17D from Atlantic salmon. The Atlantic salmon IL-17D (AsIL-17D) cDNA possessed an open reading frame of 621 bp encoding a putative protein of 206 aa with a predicted molecular weight of 23 kDa. The AsIL-17D gene has two exons and one intron showing the same (genome) organisation compared to zebrafish IL-17D. The encoded protein showed 97.6-48.8% identities to other IL-17D homologues, eight conserved cysteine residues were found within this group. Conserved residues believed to be important in receptor binding were also confirmed in salmon IL-17D by homology modelling. Phylogenetic analysis also confirmed the close relationship with other IL-17D homologues. Functional characterization of the 5' flanking region indicated that the region between -1552 and -150 contained sufficient elements for promoter activity. Tissue expression studies by real-time PCR showed a predominant expression of IL-17D transcript in gonads, skin, intestine, thymus of Atlantic salmon. The involvement of IL-17D during proinflammatory responses was demonstrated by investigating the time-dependent expression profile of IL-17D in head kidney and spleen following intraperitoneal injection of live Aeromonas salmonicida, LPS, and beta-glucan. This study provides further evidence for the existence of distinct homologue of IL-17D isoform in fish showing early expression induced by immunostimulants and bacterial infection that supports the fact that IL-17D is regulated by inflammatory processes in fish.

Cloning and expression analysis of Atlantic halibut (Hippoglossus hippoglossus) CD3 genes.

The CD3 complex is in higher vertebrates shown to be important for the activation of T-cells. The T-cell system in fish is believed to be similar to that in higher vertebrates, and the CD3 chains could therefore be an important marker for identification of T-cells in fish. Here, we report the cDNA and corresponding gene sequence of Atlantic halibut (Hippoglossus hippoglossus) CD3gammadelta, CD3varepsilon, and CD3zeta chains, and the tissue-specific expression pattern of CD3 and T- cell receptor (TCR) genes. Important structural characteristics defining the CD3 genes seemed to be conserved in the halibut CD3 chains, such as a signal peptide, an extracellular region, a transmembrane helix having a negatively charged residue, and an ITAM bearing cytoplasmic tail. The extracellular domain of halibut CD3gammadelta and CD3varepsilon included two cysteines presumably involved in Ig-fold stabilisation and the CxxCxE motif important for dimerization. A spliced variant of CD3varepsilon was identified, lacking the Ig-fold, but with the CxxCxE motif intact. The real time RT-PCR analysis revealed a highly similar expression pattern of the CD3 genes and the TCRalpha and TCRbeta genes, indicating that the functional relationship between the TCR and the CD3 genes are preserved in teleosts.

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes.

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

Molecular cloning of the canine fragile histidine triad (FHIT) gene and Fhit protein expression in canine peripheral blood mononuclear cells.

A fragile histidine triad (FHIT) gene has been studied as a tumor-associated gene in humans. The aberrant FHIT gene and its protein expression have been reported in many types of human cancers. The present study explored the canine FHIT gene structure and its protein expression in the peripheral blood mononuclear cells of healthy dogs by RT-PCR, RACE and immunoblot analysis. The obtained canine FHIT gene contained nine small exons and was located on canine chromosome 20. Furthermore, we identified an alternative splicing form of the FHIT transcript. The deduced amino acid sequence was well conserved between species, and anti-human Fhit antibody could be used to detect the canine Fhit protein. These findings will be useful for future research.

Caracterização fenotípica e molecular de amostras de Burkholderia mallei isoladas na Região Nordeste do Brasil / Phenotypic and molecular characterization of Burkholderia mallei isolated in northeastern Brazil

Objetivou-se com este trabalho realizar o estudo bioquímico e molecular de amostras de Burkholderia mallei isoladas de eqüídeos com diagnóstico clínico e sorológico para o mormo e provenientes da Região Metropolitana do Recife-PE e Zona da Mata dos Estados de Alagoas e Pernambuco. Foram realizadas as técnicas microbiológicas para o isolamento e identificação fenotípica de B. mallei e as técnicas moleculares de ribotipagem-PCR e RAPD-PCR. Das oito amostras estudadas, quatro apresentaram pequenas variações fenotípicas. Nas técnicas moleculares, as amostras formaram quatro grupos de diferentes perfis ribotípicos, demonstrando também quatro perfis genotípicos. Houve associação nos resultados da Ribotipagem-PCR e RAPD-PCR. As variações nos perfis ribotípicos e genotípicos foram associadas às diferentes regiões estudadas. De acordo com os resultados obtidos, conclui-se que as pequenas variações bioquímicas não estão associadas aos diferentes perfis moleculares e que essas diferenças demonstram uma heterogeneidade que está associada à procedência das amostras, indicando que a infecção nos animais ocorre por clones diferentes das amostras analisadas.

The objective of this paper was to study the molecular performance and phenotypic characterization of Burkholderia mallei isolated from horses with clinical and serological diagnosis of glanders, originating from the Metropolitan District of Recife and Zona da Mata of Pernambuco and Alagoas. The isolation and biochemical identification of B. mallei was carried out by microbiological and molecular techniques of PCR-fingerprinting and RAPD-PCR. From the eight samples studied, four showed little phenotype variations. In the molecular tests, the samples formed 4 groups of different ribotype profiles and 4 genotype profiles. There was some association of PCR-fingerprinting with RAPD-PCR results. It was concluded that the slight biochemical variations were not associated with different molecular profiles. They also indicated that these differences show heterogeneity associated with the origin of the sample, indicating that the infection was caused by clones of different strains and that the polymorphism of DNA observed could make it difficult to choose one standard strain for an immune prophylactic treatment of glanders.