A SNP in the ovine cathepsin K (CTSK) gene is associated with yearling growth performance in a crossbred sheep population.

Cathepsin K (CTSK) is a lysosomal protease existent in the skeletal muscles which is involved in biochemical processes related to obesity. Several studies have reported the effects of CTSK gene on body weight and fat deposition in human, mice and pigs. However, information about its structure and functions in sheep is very limited. Thus, this study was performed to evaluate the association between CTSK gene variants and yearling growth performance in Afshari × Booroola-Merino crossbred sheep. A fragment of 500 bp in exon 6 and partial of intron 5 of CTSK gene was amplified with polymerase chain reaction (PCR). All animals were genotyped by single-stranded conformation polymorphism (SSCP) and further confirmed by sequencing. Association analysis using a fixed linear model indicated that g.106510225G > A SNP was significantly related to average daily weight gain (ADWG) per year, fat-tail weight to carcass weight ratio (FW/CW), muscle thickness (MT) and muscle cross-sectional area (MCSA) of animals (p < 0.05). Due to the low polymorphic information content (PIC <0.25) for targeted locus in studied population, more association studies are needed to confirm the CTSK gene effects on growth traits in sheep.

Diet-derived transmission of MicroRNAs from host plant into honey bee Midgut.

BACKGROUND: MicroRNA (miRNA) is a class of small noncoding RNAs, which targets on thousands of mRNA and thus plays important roles in many biological processes. It has been reported that miRNA has cross-species regulation functions between parasitoid-host, or plant-animal, etc. For example, several plant miRNAs enter into the honey bees and regulate gene expression. However, whether cross-species regulation function of miRNAs is a universal mechanism remains a debate question. RESULTS: We have evaluated transmission of miRNAs from sunflower and sedr plants into the midgut of honey bee using RNA-Seq analyses complemented with confirmation by RT-qPCR. The results showed that at least 11 plant miRNAs were found in the midgut of honey bee feeding by sunflower and sedr pollen. Among which, nine miRNAs, including miR-30d, miR-143, miR-148a, miR-21, let-7 g, miR-26a, miR-126, miR-27a, and miR-203, were shared between the sunflower- and sedr-fed honey bees, suggesting they might have essential roles in plant-insect interactions. Moreover, existence of these co-shared miRNAs presents a strong evidence to support the successful transmission of miRNAs into the midgut of the insect. In total, 121 honeybee mRNAs were predicted to be the target of these 11 plant-derived miRNAs. Interestingly, a sedr-derived miRNA, miR-206, targets on 53 honeybee genes. Kyoto Encyclopedia of Genes and Genome (KEGG) analyses showed that these target genes are significantly involved in hippo signaling pathway-fly, Wnt signaling pathway, and N-Glycan biosynthesis. CONCLUSIONS: In summary, these results provide evidence of cross-species regulation function of miRNA between honeybee and flowering host plants, extending our understanding of the molecular interactions between plants and animals.

Single-nucleotide polymorphism detecting of some candidate genes related to lipid metabolism in Booroola Merino-Afshari sheep by Bayesian model averaging.

This study was designed to identify single-nucleotide polymorphisms (SNPs) of some candidate genes related to lipid metabolism and their association with carcass fat in male crossbred lambs. Hence, 96 of almost 11-month-old Booroola Merino-Afshari crossbred lambs (first-generation backcross) were used by considering their phenotypic carcass traits. Then, DNA was extracted and DNA targets were amplified using designed specific primers by PCR procedure. Identification of potential SNPs was done by a direct sequencing method for LEP, FABP4, DGAT1, GH, and TRIB3 genes using the sequencing-RFLP procedure. Then, the most probable statistical models based on additive and genotypic effects of identified SNPs in each trait were obtained by the Bayesian model averaging (BMA) approach of R software (Ver. 3.3.1) to assess the association of SNPs with traits. Detected SNPs in this study included two SNPs in exon 3 of LEP, one SNP in exon 2 of TRIB3, one SNP in intron 2 of FABP4, one SNP in 5' UTR of DGAT1, and two SNPs in 3' UTR of GH genes. For carcass weight trait, one of the identified SNP genotypes in the LEP (c.587G > A) had a higher probability in the model. Carcass weight of lambs with GA genotype was 2.46 kg heavier than GG genotype. Also, two genes of TRIB3 and GH2 had the highest probability in the models of fat tail and waste weight, respectively. Based on the results, these polymorphisms can be used in the marker-assisted selection of breeding programs and designing DNA chips for genomic selection.

Comparative Computational Screening of Natural-based Partial Agonists for PPARγ Receptor.

INTRODUCTION: The nuclear transcription factor PPARγ, which can modulate cell growth via proliferation and apoptosis-related mechanisms, is a promising target in cancer therapy. This study aims to focus on PPARγ as the target and use virtual screening to find hits. METHODS: A set of 5,677 flavonoid compounds were filtered by subjecting them to descriptor-based drug-likeness and ADMET strategies to discover drug-like compounds. The candidates' modes of binding to PPARγ were then evaluated using docking and MD simulation. PharmMapper was used to identify the potential targets of selected hits. The pharmacological network was constructed based on the GO and KEGG pathway analysis. RESULTS: In primary screening, 3,057 compounds met various drug-likeness criteria and docked well as partial agonists in the PPARγ-LBD. Five compounds (euchrenone b1, kaempferol-7-Orhamnoside, vincetoxicoside B, morusin, and karanjin) were selected with the use of ADMET profiles for further MD simulation investigation. Based on the PharmMapper findings, 52 proteins were then submitted to GO and KEGG enrichment analysis. As expected by GO and KEGG pathway enrichment studies, core targets were enriched in the PI3K-Akt signaling pathway (p < 0.01), indicating that certain chemicals may be involved in cancer processes. CONCLUSION: Our results suggested that the selected compounds might have sufficient drug-likeness, pharmacokinetics, and in silico bioactivity by acting as PPARγ partial agonists. Although much work remains to illuminate extensive cancer therapeutic/ chemopreventive efficacy of flavonoids in vivo, in silico methodology of our cheminformatics research may be able to provide additional data regarding the efficacy and safety of potential candidates for therapeutic targets.

Prevalence of Chlamydophila psittaci infections in a human population in contact with domestic and companion birds.

Chlamydophila psittaci infections in humans are underestimated. We investigated the occurrence of C. psittaci in a Belgian population of 540 individuals. Data were from a population survey (n=2524) of apparently healthy community-dwelling subjects aged 35-55 years. Pharyngeal swabs and blood were taken. Individuals completed a questionnaire on professional and nonprofessional activities, smoking habits, medical history and contact frequency with different bird species. Swabs were analysed by a C. psittaci-specific and a Chlamydophila pneumoniae-specific PCR. Sera were tested by a recombinant C. psittaci major outer-membrane protein-based ELISA, a C. psittaci whole organism-based ELISA (Serion) and a micro-immunofluorescence test (Focus Diagnostics). Results confirmed our suspicion about the underestimation of psittacosis in Belgium. Psittaciformes and racing pigeons were the main infection source. Women with excessive alcohol intake defined as a mean intake of >2 units daily were more frequently infected than men. We analysed the effect of seropositivity and/or PCR positivity on inflammation (white blood cell count, high-sensitivity C-reactive protein, fibrinogen). In general, seropositivity showed a trend to slightly higher levels of inflammatory variables (all non-significant), whilst PCR positivity showed a trend to no effect or even lower inflammatory levels.

Chlamydophila psittaci infections in birds: a review with emphasis on zoonotic consequences.

The first part of the present review gives an overview on the history of infectious agents of the order Chlamydiales and the general infection biology of Chlamydophila (C.) psittaci, the causative agent of psittacosis. In the second part, the classification of C. psittaci strains, as well as issues of epidemiology of avian chlamydiosis., disease transmission routes, clinical disease, public health significance, present legislation and recommendations for prevention and control are reviewed.

Chlamydial infections in duck farms associated with human cases of psittacosis in France.

Five severe cases of psittacosis in individuals associated with duck farms were notified in France between January and March 2006. Diagnostic examination included serology and/or molecular detection by PCR from respiratory samples. As a consequence, we investigated all duck flocks (n=11) that were housed in the three farms where human infections occurred. While serology by complement fixation test was negative for all samples, cloacal and/or tracheal chlamydial excretion was detected by PCR in all three units. Notably, one duck flock was tested strongly positive in 2 of the 3 affected farms, and Chlamydophila (C.) psittaci strains were isolated from cloacal and/or tracheal swab samples from both farms. Human samples and duck isolates exhibited the same PCR-RFLP restriction pattern, which appeared to be an intermediate between genotypes A and B. Analysis of ompA gene sequences and comparison to those of the type strains showed that the isolates could not be strictly assigned to any of the generally accepted genotypes of C. psittaci. Further analysis by MLVA of the PCR-positive human samples revealed two distinct patterns, which were related to previously isolated C. psittaci duck strains.

Evaluation of the prophylactic use of ovotransferrin against chlamydiosis in SPF turkeys.

Chlamydophila (C.) psittaci infections are highly prevalent in turkeys and the economical and public health importance of these infections has been recognized since 1950. As there are no vaccines, antibiotic treatment (tetracylines, enrofloxacine) is often needed to allow marketing of poultry. In this study, we explored the use of ovotransferrin (ovoTF), a natural anti-microbial protein, in preventing an experimental C. psittaci infection in specific pathogen free (SPF) turkeys. Turkeys were treated with aerosolized ovoTF prior to the infection. Groups 1 and 2 received a single dose of 10 and 5 mg ovoTF per turkey, respectively. Group 3 received a daily dose of 5mg ovoTF per turkey during 12 days. Group 4 served as untreated, infected control group. Turkeys were aerosol infected using 10(6) TCID(50) of the virulent C. psittaci serovar/genotype D strain 92/1293. Birds were monitored (clinical signs, bacterial excretion) during 12 subsequent days before being necropsied. At necropsy, pathology and C. psittaci replication in various tissues was examined. A single dose of 10mg ovoTF and a repeated daily dose of 5mg ovoTF could not prevent the birds from becoming infected with C. psittaci, but they significantly reduced the outcome of the infection. A single dose of 5mg ovoTF had no influence on the outcome of the infection as compared to the non-treated infected controls. Our results demonstrate the anti-chlamydial effect of ovoTF in vivo and present a base for further research on practical applications of ovoTF on turkey farms.

Chlamydophila psittaci genotype E/B transmission from African grey parrots to humans.

Thirty-six birds from a parrot relief and breeding centre, as well as the manager, were examined for the presence of Chlamydophila psittaci. In the relief unit, 5 of 20 African grey parrots showed depression, ruffled feathers, loss of weight and mild dyspnoea. The birds received no antibiotic treatment. Birds of the breeding unit, 14 blue and gold macaws and 2 green-winged macaws, were healthy. They received doxycycline at the start of each breeding season. The manager complained of shortness of breath but took no medication. Using a nested PCR enzyme immunoassay (EIA), Cp. psittaci was detected in the faeces of all five sick birds, as well as in a nasal and pharyngeal swab from the manager. The veterinarian and her assistant became infected while sampling the parrots, as pharyngeal and nasal swabs from both were positive by nested PCR/EIA after visiting the parrot relief and breeding centre, but they showed no clinical signs of infection. Bacteria could be isolated from three of five nested PCR/EIA-positive birds, the manager and the veterinarian, but not from the veterinary assistant. Using an ompA genotype-specific real-time PCR, Cp. psittaci genotype E/B was identified as the transmitted strain. All breeding birds tested negative for Cp. psittaci. This is believed to be the first report on Cp. psittaci genotype E/B transmission from parrots to humans. In contradiction to genotype A strains, which are thought to be highly virulent to both birds and men, the currently described genotype E/B strain apparently caused no severe clinical symptoms in either parrots or humans.

Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System.

PURPOSE: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. METHODS: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. RESULTS: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. CONCLUSION: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

Evaluation of STAT5A Gene Expression in Aflatoxin B1 Treated Bovine Mammary Epithelial Cells.

PURPOSE: Aflatoxin B1 (AFB1) is a potent mycotoxin which has been produced by fungi such as Aspergillus flavus and Aspergillus parasiticus as secondary metabolites due to their growth on food stuffs and induces hepatocellular carcinoma in many animal species, including humans. In the present study, the effect of AFB1 on STAT5A gene expression was investigated in bovine mammary epithelial cells using real time RT-PCR. METHODS: Bovine mammary epithelial cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, cells were treated with AFB1 and incubated for 8 h. For real time PCR reaction, total RNA from the cultured and treated cells was extracted and used for complementary DNA synthesis. RESULTS: The expression of STAT5A gene was significantly down regulated by AFB1 in dose- dependent manner and led to the reduction of proliferation and differentiation of epithelial cells, which has direct effect in milk protein quantity and quality. CONCLUSION: According to the results, it seems that down regulation of STAT5A gene in AFB1-treated cells maybe due to DNA damage induced by AFB1 in bovine mammary epithelial cells.

Protection of budgerigars (Melopsittacus undulatus) against Chlamydophila psittaci challenge by DNA vaccination.

Plasmid DNA (pcDNA1::MOMP A) expressing the major outer membrane protein (MOMP) of Chlamydophila psittaci genotype A strain 89/1051 has been tested for its ability to induce protective immunity against Cp. psittaci challenge in budgerigars. Eight pairs of male and female budgerigars were housed in eight separate bird cages placed in two negative pressure isolators, four cages per group. All budgerigars were immunised twice intramuscularly with 100 microasmid DNA. Both groups received a primary DNA inoculation at day 0 followed by a booster inoculation 3 weeks later. Group 1 received pcDNA1::MOMP A, while group 2 received the placebo vaccine pcDNA1. Budgerigars were challenged by aerosol 2 weeks following the booster vaccination. The challenge consisted of 10(8) TCID(50) of the homologous Cp. psittaci genotype A strain. Cloacal and pharyngeal swabs of all budgerigars, taken prior to the experimental infection were negative in both PCR and culture. However, all budgerigars showed low pre-existing serum antibody titres. This indicates that animals were previously infected. Nevertheless, DNA immunisation could significantly reduce clinical signs, macroscopic lesions, pharyngeal and cloacal excretion as well as chlamydial replication, even in the presence of pre-existing serum antibodies, as compared to the placebo-vaccinated controls.

Chlamydophila psittaci transmission from pet birds to humans.

We studied zoonotic transmission of Chlamydophila psittaci in 39 breeding facilities for Psittaciformes (cockatoos, parrots, parakeets, lories) that frequently used antimicrobial drugs. Genotypes A or E/B were detected in 14.9% of humans at these facilities. Information on antimicrobial drug use in Psittaciformes and a C. psittaci vaccine are urgently required.

Specific-pathogen-free pigs as an animal model for studying Chlamydia trachomatis genital infection.

The purpose of the present study was to evaluate pigs as a large-animal model for female genital infection with two Chlamydia trachomatis human serovar E strains. Sixteen-week-old specific-pathogen-free female pigs (gilts) were intravaginally infected with the trachoma type E reference strain Bour or the urogenital serovar E strain 468. Several conclusions can be drawn from our findings on the pathogenicity of a primary C. trachomatis genital infection in gilts. First of all, we demonstrated that the serovar E strains Bour and 468 could ascend in the genital tract of gilts. The serovar E strains could replicate in the superficial columnar cervical epithelium and in the superficial epithelial layer of the uterus, which are known to be the specific target sites for a C. trachomatis genital infection in women. Second, inflammation and pathology occurred at the replication sites. Third, the organisms could trigger a humoral immune response, as demonstrated by the presence of immunoglobulin M (IgM), IgG, and IgA in both serum and genital secretion samples. Our findings imply that the pig model might be useful for studying the pathology, pathogenesis, and immune response to a C. trachomatis infection of the genital system.