Identification of new semen trait-related candidate genes in Duroc boars through genome-wide association and weighted gene co-expression network analyses

Semen traits are crucial in commercial pig production since semen from boars is widely used in artificial insemination for both purebred and crossbred pig production. Revealing the genetic architecture of semen traits potentially promotes the efficiencies of improving semen traits through artificial selection. This study is aimed to identify candidate genes related to the semen traits in Duroc boars. First, we identified the genes that were significantly associated with three semen traits, including sperm motility (MO), sperm concentration (CON), and semen volume (VOL) in a Duroc boar population through a genome-wide association study (GWAS). Second, we performed a weighted gene co-expression network analysis (WGCNA). A total of 2, 3, and 20 single-nucleotide polymorphisms were found to be significantly associated with MO, CON, and VOL, respectively. Based on the haplotype block analysis, we identified one genetic region associated with MO, which explained 6.15% of the genetic trait variance. ENSSSCG00000018823 located within this region was considered as the candidate gene for regulating MO. Another genetic region explaining 1.95% of CON genetic variance was identified, and, in this region, B9D2, PAFAH1B3, TMEM145, and CIC were detected as the CON-related candidate genes. Two genetic regions that accounted for 2.23% and 2.48% of VOL genetic variance were identified, and, in these two regions, WWC2, CDKN2AIP, ING2, TRAPPC11, STOX2, and PELO were identified as VOL-related candidate genes. WGCNA analysis showed that, among these candidate genes, B9D2, TMEM145, WWC2, CDKN2AIP, TRAPPC11, and PELO were located within the most significant module eigengenes, confirming these candidate genes’ role in regulating semen traits in Duroc boars. The identification of these candidate genes can help to better understand the genetic architecture of semen traits in boars. Our findings can be applied for semen traits improvement in Duroc boars.     

Evaluation of tizanidine as a marker of canine CYP1A2 activity

The dog CYP1A2 enzyme is likely an important contributor to the metabolism of veterinary drugs. Dog CYP1A2 is expressed in liver, plus it is inducible and polymorphic, creating the potential for intersubject differences in pharmacokinetics. Hence, the ability to probe dog CYP1A2 activity and inhibition is relevant toward veterinary drug development and drug–drug interaction assessment. Previous studies have relied on human probes with questionable specificity for CYP1A2, so it was hypothesized that recombinant CYP1A2 could be used to find a specific CYP1A2 substrate. Intrinsic clearance experiments demonstrated that tizanidine was a substrate of CYP1A2. Profiling of tizanidine metabolites generated by CYP1A2 identified the imidazole metabolite that was detectable in dog plasma. The imidazole metabolite was subsequently used to evaluate tizanidine as a CYP1A2 probe. Co‐administration of the CYP1A inhibitor enrofloxacin with tizanidine significantly decreased (30%; n = 3) the formation of the imidazole metabolite vs. control experiments. As enrofloxacin is a weak inhibitor, further studies are required to confirm the sensitivity of tizanidine as an in vivo probe. However, tizanidine may be a more selective CYP1A2 probe than phenacetin when conducting in vitro studies due to the presence of other phenacetin‐metabolizing enzymes in dog liver microsomes.     

Transcriptome sequencing reveals two subtypes of cortisol-secreting adrenocortical tumours in dogs and identifies CYP26B1 as a potential new therapeutic target

Cushing's syndrome (CS) is a serious endocrine disorder that is relatively common in dogs, but rare in humans. In ~15%–20% of cases, CS is caused by a cortisol-secreting adrenocortical tumour (csACT). To identify differentially expressed genes that can improve prognostic predictions after surgery and represent novel treatment targets, we performed RNA sequencing on csACTs (n = 48) and normal adrenal cortices (NACs; n = 10) of dogs. A gene was declared differentially expressed when the adjusted p-value was <.05 and the log₂ fold change was >2 or < −2. Between NACs and csACTs, 98 genes were differentially expressed. Based on the principal component analysis (PCA) the csACTs were separated in two groups, of which Group 1 had significantly better survival after adrenalectomy (p = .002) than Group 2. Between csACT Group G1 and Group 2, 77 genes were differentially expressed. One of these, cytochrome P450 26B1 (CYP26B1), was significantly associated with survival in both our canine csACTs and in a publicly available data set of 33 human cortisol-secreting adrenocortical carcinomas. In the validation cohort, CYP26B1 was also expressed significantly higher (p = .012) in canine csACTs compared with NACs. In future studies it would be interesting to determine whether CYP26B1 inhibitors could inhibit csACT growth in both dogs and humans.     

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background

In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro.

Methods

Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 μM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples ≥ 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors.

Results

Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction ≥ 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed.

Conclusion

Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.     

Candidate gene and genome-wide association studies of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep: a review

Paratuberculosis (Johne's disease), caused by Mycobacterium avium subspecies paratuberculosis, is responsible for significant economic losses in livestock industries worldwide. This organism is also of public health concern due to an unconfirmed link to Crohn's disease. Susceptibility to paratuberculosis has been suggested to have a genetic component. In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohn's disease. These genes include solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1), toll-like receptors, caspase associated recruitment domain 15 (CARD15, formerly NOD2), major histocompatibility complex (MHC) and cytokines (interleukin-10 and interferon-gamma) and their receptors. Genome wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. There are a number of limitations and difficulties in these approaches, some peculiar to paratuberculosis but others generally applicable to identification of genetic associations for complex traits. The technical approaches and available information for paratuberculosis have expanded rapidly, particularly relating to sheep and cattle. Here we review the current published evidence for a genetic association with paratuberculosis susceptibility, technological advances that have progressed the field and potential avenues for future research.     

Joint analysis of the influence of CYP11B1 and DGAT1 genetic variation on milk production, somatic cell score, conformation, reproduction, and productive lifespan in German Holstein cattle

Recent publications indicate genetic variation in milk production traits on proximal BTA14, which cannot be explained solely with genetic variation in the DGAT1 gene. To elucidate these QTL effects, animals from a German Holstein granddaughter design (18 families, 1,291 sons) were genotyped for CYP11B1 (V30A) and DGAT1 (K232A) polymorphisms. Frequencies of alleles of maternal descent were estimated for CYP11B1(V) (0.776) and DGAT1(K) (0.549). Allele substitution effects (alpha/2) were first calculated for both alleles in separate models and then in a joint model. From the joint analysis, CYP11B1(V) effects on fat content (+0.04%) and protein content (+0.01%) were positive. Effects on milk yield (-82 kg), fat yield (-0.5 kg), and protein yield (-1.9 kg) were negative. Compared with the individual analysis, DGAT1(K) effects on fat content (+0.28%), protein content (+0.06%), and milk yield (-258 kg) were reduced; fat yield (+10.8 kg) was enhanced; and protein yield (-3.8 kg) was reduced. In the joint analysis, allele substitution effects of CYP11B1(V) and DGAT1(K) together explained more of the variation in milk production traits than DGAT1(K) alone. Further significant effects were found for CYP11B1(V) and DGAT1(K) among 6 reproduction traits and 14 conformational traits. These observations indicate a possible negative influence of DGAT1(K) on maternal nonreturn rate, and thus, on length of productive life.     

CYP1B1 is polymorphic in cynomolgus and rhesus macaques

Cytochrome P450 (CYP) 1B1 is involved in the metabolic activation of various procarcinogens, and some CYP1B1 genetic variants alter CYP1B1-dependent procarcinogen metabolism. Cynomolgus and rhesus macaques are frequently used in toxicity tests due to their evolutionary closeness to humans. In this study, we attempted to identify CYP1B1 genetic variants in 13 cynomolgus and 4 rhesus macaques. A total of 17 genetic variants were identified, including 8 non-synonymous genetic variants, indicating that, similar to humans, CYP1B1 is polymorphic in macaques. These CYP1B1 genetic variants could be the basis for understanding potential inter-animal differences in macaque CYP1B1-dependent metabolism of promutagens.     

Reaction phenotyping of vinblastine metabolism in dogs

Vinblastine is a vinca alkaloid used either as a single agent or in combination therapy for the treatment of canine mast cell tumours and lymphomas. The objective of this study was to determine which isoform of cytochrome P450 enzyme is responsible for the majority of vinblastine metabolism in dogs. A panel of eight recombinant canine cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A12, CYP3A26, CYP2B11, CYP2C41, CYP2C21 and CYP2D15) were incubated in vitro with vinblastine. Findings were confirmed by the use of canine polyclonal antibodies of cytochrome P450 enzymes (CYP1A1, CYP3A12, CYP2B11 and CYP2C21) that were pre‐incubated with individual and pooled hepatic microsomes that were purified from canine liver. Substrate depletion was observed in the presence of recombinant CYP3A12, whereas depletion did not substantially occur when microsomes were pre‐incubated with polyclonal antibodies against CYP3A12. These findings confirmed that CYP3A12 is the major cytochrome P450 isoform responsible for the metabolism of vinblastine in dogs.     

Absence of Sirtuin 1 impairs the testicular development in cattleyak by inactivating SF-1

Cattleyak, which are interspecific hybrids between cattle and yak, display much higher growth performances than yak. However, F1 male cattleyak are infertile due to defective testicular development. Sirtuin 1 (SIRT1) is a histone deacetylase that is essential for various biological processes, while the roles of testicular SIRT1 in yak and cattleyak are still poorly understood. Here, we found that SIRT1 was localized in various kinds of yak testicular cells except elongated spermatids while it was deficient in cattleyak testis. Further studies indicated that cattleyak testis exhibited decreased histone acetylation levels on H3 and H4. One of SIRT1 co-factors, steroidogenic factor-1 (SF-1), was lost in cattleyak testis at protein level. Expressions of several SF-1 target genes responsible for Sertoli cell development and steroidogenesis, including STAR, CYP11A1, CYP26B1, FDX1 and HSD3B, decreased significantly in cattleyak testis. In addition, SIRT1-mediated P53 acetylation was not responsible for the cell apoptosis in cattleyak testis. Taken together, our results suggested the deficiency of SIRT1 in yak testis caused inactivation of SF-1 and the impairment of testicular development. This research provides theoretical bases for understanding the mechanism of cattleyak sterility and gives new insights in revealing the roles of SIRT1 in regulating yak testicular development.     

Effect of 3,3',5-triiodothyronine and 3,5-diiodothyronine on progesterone production, cAMP synthesis, and mRNA expression of STAR, CYP11A1, and HSD3B genes in granulosa layer of chicken preovulatory follicles

In vitro studies were performed to assess whether stimulatory effects of triiodothyronine (T₃) on progesterone (P₄) production in a granulosa layer (GL) of chicken preovulatory follicles are associated with 3′,5′-cyclic adenosine monophosphate (cAMP) synthesis and mRNA expression of STAR protein, CYP11A1, and HSD3B. Effects of 3,5-diiodothyronine (3,5-T₂) on steroidogenic function in these follicles were also investigated. The GL of F3 to F1 follicles was incubated in medium supplemented with T₃ or 3,5-T₂, LH, or forskolin (F), and a combination of each iodothyronine with LH or F. Levels of P₄ and cAMP in culture media were determined by RIA. Expression of genes involved in P₄ synthesis (ie, STAR protein, CYP11A1, and HSD3B) in the GL of F3 to F1 follicles incubated in medium with T₃ or 3,5-T₂ and their combination with LH was performed by real-time PCR. Triiodothyronine increased basal and LH- and F-stimulated P₄ secretion by preovulatory follicles. The 3,5-T₂ elevated P₄ synthesis by F3, had no effect on F2 follicles, and diminished P₄ production by the GL of F1 follicles. It had no effect on LH-stimulated P₄ production; however, it augmented F-stimulated P₄ production by F2 and F1 follicles. Although T₃ did not affect basal and F-stimulated cAMP synthesis by the GL of preovulatory follicles, it increased LH-stimulated synthesis of this nucleotide. However, 3,5-T₂ elevated F-stimulated cAMP synthesis in F3 and F2 follicles; it did not change basal and LH-stimulated cAMP production. Triiodothyronine decreased basal STAR and CYP11A1 mRNAs in F3 follicles, increased them in F1 follicles, and elevated HSD3B mRNA levels in F1 follicles. Triiodothyronine augmented LH-stimulated STAR, CYP11A1, and HSD3B mRNA levels in F2 and CYP11A1 in F1 follicles. However, T₃ decreased LH-stimulated STAR and HSD3B mRNA levels in F1 follicles. The 3,5-T₂ did not affect basal STAR and CYP11A1 mRNA expression in all investigated follicles; however, it decreased LH-stimulated STAR expression in F2 and F1 ones. The effects of 3,5-T₂ caused elevated basal but diminished LH-stimulated HSD3B mRNA levels. In conclusion, data indicate that both iodothyronines are involved in P₄ production in the GL of chicken preovulatory follicles acting alone and additively with LH. Effects of iodothyronines depend on follicle maturation and are associated with modulation of cAMP synthesis and STAR, CYP11A1, and HSD3B mRNA expression. We suggest that iodothyronines participate in maturation and ovulation of chicken follicles.     

Gene expression profiling of porcine mammary epithelial cells after challenge with Escherichia coli and Staphylococcus aureus in vitro

Postpartum Dysgalactia Syndrome (PDS) represents a considerable health problem of postpartum sows, primarily indicated by mastitis and lactation failure. The poorly understood etiology of this multifactorial disease necessitates the use of the porcine mammary epithelial cell (PMEC) model to identify how and to what extent molecular pathogen defense mechanisms prevent bacterial infections at the first cellular barrier of the gland. PMEC were isolated from three lactating sows and challenged with heat-inactivated potential mastitis-causing pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for 3 h and 24 h, in vitro. We focused on differential gene expression patterns of PMEC after pathogen challenge in comparison with the untreated control by performing microarray analysis. Our results show that a core innate immune response of PMEC is partly shared by E. coli and S. aureus. But E. coli infection induces much faster and stronger inflammatory response than S. aureus infection. An immediate and strong up-regulation of genes encoding cytokines (IL1A and IL8), chemokines (CCL2, CXCL1, CXCL2, CXCL3, and CXCL6) and cell adhesion molecules (VCAM1, ICAM1, and ITGB3) was explicitly obvious post-challenge with E. coli inducing a rapid recruitment and activation of cells of host defense mediated by IL1B and TNF signaling. In contrast, S. aureus infection rather induces the expression of genes encoding monooxygenases (CYP1A1, CYP3A4, and CYP1B1) initiating processes of detoxification and pathogen elimination. The results indicate that the course of PDS depends on the host recognition of different structural and pathogenic profiles first, which critically determines the extent and effectiveness of cellular immune defense after infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0178-z) contains supplementary material, which is available to authorized users.     

Expression of retinoic acid‐metabolizing enzymes,ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1 in canine testis during post‐natal development

Mammalian spermatogenesis involves highly regulated temporal and spatial dynamics, carefully controlled by several signalling processes. Retinoic acid (RA) signalling could have a critical role in spermatogenesis by promoting spermatogonia differentiation, adhesion of germ cells to Sertoli cells, and release of mature spermatids. An optimal testicular RA concentration is maintained by retinaldehyde dehydrogenases (ALDHs), which oxidize RA precursors to produce RA, whereas the CYP26 class of enzymes catabolizes (oxidize) RA into inactive metabolites. The objective was to elucidate gene expression of these RA‐metabolizing enzymes (ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1) and their protein presence in testes of young, peripubertal and adult dogs. Genes encoding RA‐synthesizing isozymes ALDH1A1, ALDH1A2 and ALDH1A3 and RA‐catabolizing isomers CYP26A1, CYP26B1 and CYP26C1 were expressed in testis at varying levels during testicular development from birth to adulthood in dogs. Based on detailed analyses of mRNA expression patterns, ALDH1A2 was regarded as a primary RA‐synthesizing enzyme and CYP26B1 as a critical RA‐hydrolysing enzyme; presumably, these genes have vital roles in maintaining RA homeostasis, which is imperative to spermatogenesis and other testicular functions in post‐natal canine testis.     

Genetic variations of BRCA1 and BRCA2 genes in dogs with mammary tumours

Mammary tumours are the most common tumour type in female dogs. The formation of the mammary tumours is multifactorial but the high incidence of tumour disease in certain canine breeds suggests a strong genetic component. BRCA1 and BRCA2 are the most important genes significantly associated with mammary tumours. The aim of this study was to determine the association between the variations of these two genes and canine mammary tumours. 5′-untranslated region, intron 8 and exon 9 of BRCA1 and exons 12, 24, 27 of BRCA2 were sequenced in order to detect the genetic variations. In addition to six previously identified polymorphisms, six novel single nucleotide polymorphisms (SNPs) were detected. Five of the coding SNPs were synonymous and three of them were non-synonymous. The comparison of the sequences from 25 mammary tumour bearing and 10 tumour free dogs suggested that the two SNPs in intron 8 and exon 9 of BRCA1 and two SNPs in exon 24 and exon 27 of BRCA2, which are firstly identified in this study, might be associated with mammary tumour development in dogs. Especially one SNP in exon 9 of BRCA1 and one SNP in exon 24 of BRCA2 were found to be significantly associated with canine mammary tumours.     

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.     

Analysis of PPP1R11 expression in granulosa cells during developmental follicles of yak and its effects on cell function

The aims of this study were to analyse the protein phosphatase 1 regulatory subunit 11 (PPP1R11) expression and cellular localization in yak follicles and investigate its effects on cell proliferation, apoptosis and oestrogen secretion in granulosa cells (GCs). Ten healthy and non-pregnant female yaks (4-year-old) were used as experimental animals. The mRNA relative expression level of PPP1R11 in GCs from small (<3.0 mm), medium (3.0–5.9 mm) and large (6.0–9.0 mm) follicles was detected by RT-qPCR, and the cellular localization of PPP1R11 protein was detected by immunohistochemistry staining (IHC). After isolation, culture and identification of yak GCs in vitro, si-PPP1R11 and si-NC (negative control) were transfected into GCs. RT-qPCR and immunofluorescence staining were used to evaluate the interference efficiency, and ELISA was performed to detect oestrogen concentration. Then, EdU staining and TUNEL staining were conducted to analyse cell proliferation and apoptosis. In addition, the oestrogen synthesis, proliferation- and apoptosis-related genes were detected by RT-qPCR after knockdown PPP1R11. The results showed that PPP1R11 is mainly located in ovarian GCs, and the expression levels of PPP1R11 in GCs from large follicles were significantly higher than that from medium and small follicles. Transfection of si-PPP1R11 into GCs could significantly inhibit the expression of PPP1R11. Interestingly, the oestrogen secretion ability and the expression level of oestrogen pathway-related genes (STAR, CYP11A1, CYP19A1 and HSD17B1) were also significantly downregulated. Moreover, the proportion of positive cells was decreased, and cellular proliferation-related genes (PCNA, CCNB1 and CDC25A) were significantly downregulated after knockdown PPP1R11. However, the proportion of apoptotic cells was increased, and apoptosis-related genes (BAX, CASP3 and P53) were significantly upregulated. Taken together, this study was the first revealed the expression and cellular localization of PPP1R11 in yak follicles. Interference PPP1R11 could reduce oestrogen secretion, inhibit proliferation and promote apoptosis in GCs, which provided a basis for further studies on the regulatory mechanism of PPP1R11 in follicle development.     

Variation in the SLC16A1 and the ACOX1 Genes Is Associated with Gallop Racing Performance in Arabian Horses

Arabian horses are not only one of the most ancient breeds in the world, but they are also one of the most appreciated racehorse breeds today. The breed generates attention for their phenomenal endurance ability and their capability for gallop racing. Consequently, genetic testing to select the best individuals is attracting ever increasing interests from the Arabian industry. As such, the aim of this study was to further investigate associations between performance and variation at candidate genes suspected of having a key role in Arabian gallop racing performance. Generalized linear models were fit to test associations between eight candidate gene variants and a variety of gallop racing performance traits in a sample of Arabian racehorses (n = 287). Two genes, solute carrier family 16 member 1 (SLC16A1) and acyl-CoA oxidase 1 (ACOX1), were significantly associated with multiple gallop racing performance traits, whereas another gene, actinin alpha 3 (ACTN3) was associated with best race distance. Previously established associations between these three genes and equine metabolism strongly suggest further investigation of these genes, and their relationship with Arabian horse performance is warranted.     

Serogroup distribution,diversity of exotoxin gene profiles,and phylogenetic grouping of CTX-M-1- producing uropathogenic Escherichia coli

The emergence of CTX-M-1 producing Uropathogenic Escherichia coli (UPEC) has become a serious challenge. In addition to antimicrobial resistance, a number of virulence factors have been shown. Therefore, this study was designed to determine the prevalence of O- serogroups, phylogenetic groups, exotoxin genes, and antimicrobial resistance properties of CTX-M-1- producing UPEC. A total of 248 UPEC isolates were collected. The antibiotic resistance was performed, and PCR was used to detect the bla_CTX-M1, exotoxins, serogroups and phylogroups of UPEC. Of 248 isolates, 95 (38.3%) harbored bla_CTX-M-1. Of them, serogroups O1 and O25 were predominant, accounting for 20% and 13.7%, respectively. The hlyA was the dominant exotoxin gene (32.6%), followed by sat (28.4%), vat (22.1%), cnf (13.7%), picU (8.4%), and cdt (2.1%). The hlyA gene was significantly associated with pyelonephritis (P = 0.003). Moreover, almost half of the isolates (45.4%) belonged to phylogenetic group B2. Most of exotoxin genes were present in significantly higher proportions in group B2 isolates except cdt gene (P < 0.05). All of the isolates were susceptible to imipenem, nitrofurantoin, and fosfomycin. The CTX-M-1-producing UPEC strains causing nosocomial infections are more likely to harbor certain exotoxin genes, raising the possibility that this increase in virulence genes may result in an increased risk of complicated UTI.     

Subclinical,chronic intramammary infection lowers steroid concentrations and gene expression in bovine preovulatory follicles

Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.