Aldosterone-progesterone relationship in sexually intact Chihuahua bitches.

BACKGROUND: Aldosterone represents an important target of heart failure therapy and may be a valuable indicator of the renin-angiotensin-aldosterone system activity. However, its assessment might be challenging because of the effect of individual factors. In a recent study, intact female dogs showed the highest value of urinary aldosterone-to-creatinine ratio (UAldo:C) compared to other sex categories. In humans and rodents, an influence of progesterone has been reported by several studies. To our knowledge, the relationship between aldosterone and progesterone has not yet been investigated in dogs. The aim of this prospective study was to investigate this relationship in sexually intact Chihuahua females, measuring both hormones twice in the same bitch, that is in anoestrus when progesterone concentrations are baseline and in dioestrus when they are high. RESULTS: The study population consisted of 14 sexually intact Chihuahua bitches. Serum progesterone (34.06 (21.17-44.90) vs. 0.19 [0.13-0.38] ng/ml; P < 0.001) and urinary aldosterone (9886.98 ± 5735.22 vs. 5005.72 ± 2127.73 pg/ml; P = 0.01) were significantly higher in dioestrus compared to anoestrous. Urinary aldosterone-to-creatinine ratio was higher in dioestrus compared to anoestrus (4.16 [3.17-6.80] vs. 3.39 ± 1.64 µg/g), but it did not reach the statistical significance (P = 0.056). Serum progesterone showed a moderate positive correlation with urinary aldosterone (ρ = 0.638, P < 0.001) and UAldo:C (ρ = 0.516, P = 0.005). CONCLUSIONS: The results of the present study suggest the existence of a progesterone-aldosterone relationship in canine species, indicating that sex and phase of reproductive cycle should be taken into account when interpreting aldosterone concentrations. Further studies are needed to confirm these results on a larger canine population and to identify the underlying mechanisms in this species.

Endocrine-Disrupting Chemicals and Their Effects in Pet Dogs and Cats: An Overview.

Over the past few decades, several pollutants classified as environmental endocrine-disrupting chemicals (EDCs) have become a matter of significant public health concern. Companion animals play a major role in human society, and pet ownership is substantially increasing worldwide. These intimate human-pet relationships imply sharing much of the same environment, thus including exposure to similar levels of EDCs in daily routine. Here, we review the current knowledge on the sources and routes of exposure to EDCs in domestic indoor and outdoor environments and discuss whether endocrine disruption is a health concern in pets. We summarize the phenomenon of endocrine disruption, providing examples of EDCs with a known impact on dog and cat health. Then, we propose an overview of the literature on the adverse effects of EDCs in domestic pets, with a special focus on the health of reproductive and thyroid systems. Finally, we explore the potential role of companion animals as unintentional sentinels of environmental exposure to EDCs and the implications for public health risk assessment in a "shared risk" scenario. Overall, this review supports the need for an integrated approach considering humans, animals, and the environment as a whole for a comprehensive assessment of the impact of EDCs on human and animal health.

Signs of Anxiety and Salivary Copeptin Levels in Dogs Diagnosed with Separation-Related Problems in a Short Separation Test.

The need for faster diagnosis and more accurate treatment decisions in separation-related problems (SRPs) in dogs is urgent, and a more precise behavioral phenotyping and the development of biomarkers may be of great value. Vasopressin could be a potential non-invasive biomarker of anxiety in dogs with SRPs, but reliable measurement of its concentration is challenging. Here, we compared the behavior and salivary concentrations of copeptin, an arginine vasopressin surrogate, in dogs with SRPs (Case group, n = 13) and with no problems (Control group, n = 15) as they were introduced to a novel environment and subjected to a short episode of separation and reunion with the owner. Dogs in the Case group had greater odds of showing locomotory or oral behaviors during the pre- and post-separation than Controls, while the odds were significantly lower during separation. They also had greater odds of being persistent in seeking attention and proximity from the stranger during reunion. Overall, dogs with SRPs were more likely to express an anxiety-like state during the entire test than Controls, with separation from the owner, and even its anticipation, possibly accounting for this group difference. Although salivary copeptin concentrations did not differ between the two groups, a different trend was detected in Cases and Controls that is worth exploring in further validation studies involving a larger sample.

Population Pharmacokinetic Model of Iohexol in Dogs to Estimate Glomerular Filtration Rate and Optimize Sampling Time.

Monitoring iohexol plasma clearance is considered a useful, reliable, and sensitive tool to establish glomerular filtration rate (GFR) and early stages of kidney disease in both humans and veterinary medicine. The assessment of GFR based on iohexol plasma clearance needs repeated blood sampling over hours, which is not easily attainable in a clinical setting. The study aimed to build a population pharmacokinetic (Pop PK) model to estimate iohexol plasma clearance in a population of dogs and based on this model, to indicate the best sampling times that enable a precise clearance estimation using a low number of samples. A Pop PK model was developed based on 5 iohexol plasma samples taken from 5 to 180 minutes (min) after an intravenous iohexol nominal dose of 64.7 mg/kg from 49 client-owned dogs of different breeds, sexes, ages, body weights, and clinical conditions (healthy or presenting chronic kidney disease CKD). The design of the best sampling times could contain either 1 or 2 or 3 sampling times. These were discretized with a step of 30 min between 30 and 180 min. A two-compartment Pop PK model best fitted the data; creatinine and kidney status were the covariates included in the model to explain a part of clearance variability. When 1 sample was available, 90 or 120 min were the best sampling times to assess clearance for healthy dogs with a low creatinine value. Whereas for dogs with CKD and medium creatinine value, the best sampling time was 150 or 180 min, for CKD dogs with a high creatinine value, it was 180 min. If 2 or 3 samples were available, several sampling times were possible. The method to define the best sampling times could be used with other Pop PK models as long as it is representative of the patient population and once the model is built, the use of individualized sampling times for each patient allows to precisely estimate the GFR.

Factors affecting the urinary aldosterone-to-creatinine ratio in healthy dogs and dogs with naturally occurring myxomatous mitral valve disease.

BACKGROUND: Chronic renin-angiotensin-aldosterone system (RAAS) activation in course of heart diseases contributes to cardiac remodeling and heart failure. Myxomatous mitral valve disease (MMVD) is characterized by different stages of severity and trend of RAAS activity during the course of the disease is still uncertain. The urinary aldosterone-to-creatinine ratio (UAldo:C) has been proven to reflect RAAS activation in dogs and might be a useful marker in monitoring therapy and disease progression, but data about this parameter need to be expanded. The objective of this study was to evaluate the UAldo:C in healthy dogs and dogs with naturally occurring MMVD, and to investigate the relationships between this parameter and clinical, echocardiographic and laboratory variables. RESULTS: The study population consisted of 149 dogs: 49 healthy and 100 MMVD dogs (45 stage B1, 13 stage B2 and 42 stage C). Urinary aldosterone-to-creatinine ratio was not significantly different among healthy and MMVD dogs of any stages. Breed, sex and age showed a significant impact on UAldo:C. In particular, Chihuahua and Cavalier King Charles spaniel showed significantly higher UAldo:C than other breeds, as well as intact females than other genders. In stage C dogs, UAldo:C appeared to be increased by spironolactone and was positively associated with furosemide dose (P = 0.024). Aldosterone breakthrough (ABT) appeared to occur in 36% (8/22) of stage C dogs not receiving spironolactone. A significant positive association between UAldo:C and left atrium-to-aortic root ratio (LA/Ao) was found. CONCLUSIONS: Individual factors such as breed, sex and age appeared to influence UAldo:C, and therapy seemed to add further variability. In the light of these results, comparing the UAldo:C of a single patient with a population-based reference value might lead to wrong interpretations and an individual monitoring should be considered. The prevalence of ABT in the present study (36%) was in line with those previously reported. However, due to the high individual variability of UAldo:C found in the study, even this result should be re-evaluated in the setting of an individual longitudinal approach. The positive association between UAldo:C and LA/Ao supports the mutual relationship between RAAS and cardiac remodeling.

Regulation of the aryl hydrocarbon receptor activity in bovine cumulus-oocyte complexes during in vitro maturation: The role of EGFR and post-EGFR ERK1/2 signaling cascade.

The aryl hydrocarbon receptor (AhR) has been extensively characterized as an environmental sensor with major roles in xenobiotic-induced toxicity. Evidence is accumulating that these functions serve as adaptive mechanisms overlapping its physiological roles. We previously described a critical role of constitutive AhR activation for the correct progress of mammalian oocyte maturation but the signaling pathway through which AhR controls maturation remains unclear. The aim of this study was to investigate whether the AhR interacts with the epidermal growth factor receptor (EGFR) and p42/44 extracellular regulated kinases (ERK1/2), both key factors in the signaling network that finely regulates the oocyte maturation. As experimental model we used bovine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM). Blocking ERK1/2 signaling in COCs during IVM with the specific EGFR inhibitor AG1478 or the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 downregulated the expression of the AhR-target gene Cyp1a1. Inhibition of AhR activity was associated with a reduction in the oocytes' ability to progress in meiosis resumption. In contrast, exposure to the AhR antagonist resveratrol reduced both CYP1A1 expression and the oocytes' maturation competence, without affecting ERK1/2 signaling. These findings strongly indicate the EGFR/ERKs signaling network as an upstream regulator of the AhR activation in COCs, offering a new understanding of the finely tuned physiological mechanism leading to oocyte maturation. This information may provide fresh opportunities for improving oocyte in vitro maturation, and therefore boosting the efficiency of assisted reproduction techniques in mammals.

Diagnostic potential of simplified methods for measuring glomerular filtration rate to detect chronic kidney disease in dogs.

BACKGROUND: Glomerular filtration rate (GFR) is the most sensitive indicator of initial renal function decline during chronic kidney disease (CKD), but conventional protocols for measuring GFR are labor-intensive and stressful for the dog. OBJECTIVES: To assess the diagnostic potential for detecting CKD with simplified GFR protocols based on iohexol plasma clearance. ANIMALS: Seventeen CKD-positive and 23 CKD-negative dogs of different breeds and sex. METHODS: Prospective nonrandomized study. Plasma iohexol was measured 5, 15, 60, 90, and 180 minutes after injection. Glomerular filtration rate was calculated using 5 samples (GFR5 ) or simplified protocols based on 1, 2, or 3 samples. The GFR5 and simplified GFR were compared by Bland-Altmann and concordance correlation coefficient (CCC) analysis, and diagnostic accuracy for CKD by receiver operating characteristic curves. A gray zone for each protocol was bounded by the fourth quartile of the CKD-positive population (lower cutoff) and the first quartile of the CKD-negative population (upper cutoff). RESULTS: All simplified protocols gave reliable GFR measurements, comparable to reference GFR5 (CCC >0.92). Simplified protocols which included the 180-minutes sampling granted the best GFR measure (CCC: 0.98), with strong diagnostic potential for CKD (area under the receiver operating characteristic curve ± SE: 0.98 ± 0.01). A double cutoff including a zone of CKD uncertainty guaranteed reliable diagnosis outside the gray area and identified borderline dogs inside it. CONCLUSIONS: The simplified GFR protocols offer an accurate, hands-on tool for CKD diagnosis in dogs. The gray zone might help decision-making in the management of early kidney dysfunction.

Anti-Müllerian hormone receptor type 2 is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion.

Preantral and small antral follicles may secret anti-Müllerian hormone (AMH) to control gonadotrophin secretion from ruminant gonadotrophs. The present study investigated whether the main receptor for AMH, AMH receptor type 2 (AMHR2), is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion. Expression of AMHR2 mRNA was detected in anterior pituitaries (APs) of postpubertal heifers using reverse transcription-polymerase chain reaction. An anti-AMHR2 chicken antibody was developed against the extracellular region near the N-terminus of bovine AMHR2. Western blotting using this antibody detected the expression of AMHR2 protein in APs. Immunofluorescence microscopy using the same antibody visualised colocalisation of AMHR2 with gonadotrophin-releasing hormone (GnRH) receptor on the plasma membrane of gonadotrophs. AP cells were cultured for 3.5 days and then treated with increasing concentrations (0, 1, 10, 100, or 1000pgmL-1) of AMH. AMH (10-1000pgmL-1) stimulated (P<0.05) basal FSH secretion. In addition, AMH (100-1000pgmL-1) weakly stimulated (P<0.05) basal LH secretion. AMH (100-1000pgmL-1) inhibited GnRH-induced FSH secretion, but not GnRH-induced LH secretion, in AP cells. In conclusion, AMHR2 is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion.

Heifers express G-protein coupled receptor 61 in anterior pituitary gonadotrophs in stage-dependent manner.

Gonadotropin-releasing hormone receptors (GnRHRs) colocalize with insulin and glucocorticoid receptors in lipid rafts of the gonadotroph plasma membrane, where they facilitate downstream signaling. We recently found that orphan G-protein-coupled receptor (GPR)61 is expressed in the anterior pituitary (AP) of heifers, leading us to speculate that GPR61 colocalizes with GnRHR in the plasma membrane of gonadotroph and is expressed at specific times of the reproductive cycle. To test this hypothesis, we examined the coexpression of GnRHR, GPR61, and either luteinizing hormone (LH) ß subunit or follicle-stimulating hormone (FSH) ß subunit in AP tissue and cultured AP cells by immunofluorescence microscopy. GPR61 was detected in gonadotrophs, with a majority of them being colocalized with GnRHR and the remainder present at other parts of the cell surface or in the cytoplasm. We obtained a strong positive overlap coefficient (0.71±0.01) between GPR61 and GnRHR on the cell-surface of cultured GnRHR-positive AP cells. Real-time PCR and western blot analyses found that expression was lower (P<0.05) in AP tissues during early luteal phase as compared to pre-ovulation or mid- or late luteal phases. Additionally, the 5ꞌ-flanking region of the GPR61 gene contained several sites with response elements similar to those of estrogen or progesterone. These data suggested that GPR61 colocalizes with GnRHR in the plasma membrane of gonadotrophs, and its expression changes stage-dependently in the bovine anterior pituitary gland.

Maternal exposure to di(2-ethylhexyl)phthalate (DEHP) promotes the transgenerational inheritance of adult-onset reproductive dysfunctions through the female germline in mice.

Endocrine disruptors (EDs) are compounds known to promote transgenerational inheritance of adult-onset disease in subsequent generations after maternal exposure during fetal gonadal development. This study was designed to establish whether gestational and lactational exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) at environmental doses promotes transgenerational effects on reproductive health in female offspring, as adults, over three generations in the mouse. Gestating F0 mouse dams were exposed to 0, 0.05, 5mg/kg/day DEHP in the diet from gestational day 0.5 until the end of lactation. The incidence of adult-onset disease in reproductive function was recorded in F1, F2 and F3 female offspring. In adult F1 females, DEHP exposure induced reproductive adverse effects with: i) altered ovarian follicular dynamics with reduced primordial follicular reserve and a larger growing pre-antral follicle population, suggesting accelerated follicular recruitment; ii) reduced oocyte quality and embryonic developmental competence; iii) dysregulation of the expression profile of a panel of selected ovarian and pre-implantation embryonic genes. F2 and F3 female offspring displayed the same altered reproductive morphological phenotype and gene expression profiles as F1, thus showing transgenerational transmission of reproductive adverse effects along the female lineage. These findings indicate that in mice exposure to DEHP at doses relevant to human exposure during gonadal sex determination significantly perturbs the reproductive indices of female adult offspring and subsequent generations. Evidence of transgenerational transmission has important implications for the reproductive health and fertility of animals and humans, significantly increasing the potential biohazards of this toxicant.

Q fever seroprevalence and risk factors in sheep and goats in northwest Italy.

Q fever is a zoonosis caused by the bacterium Coxiella burnetii; domestic ruminants, mainly goats and sheep, are the main source of Q fever outbreaks in humans. From both a public and an animal health perspective, providing reliable prevalence data is extremely relevant for the decision processes by policymakers and food producer organizations. Information on Q fever seroprevalence in small ruminants in Italy is currently incomplete and largely based on reports of reproductive disorders in livestock farms. To estimate animal and flock seroprevalence of C. burnetii in small ruminants (sheep, goats and mixed flocks), a cross-sectional study with a two-stage design was carried out in northwest Italy. Between January and December 2012, sera from 5738 animals (2553 sheep and 3185 goats) belonging to 411 flocks (206 goats, 111 sheep, and 94 mixed flocks) were examined for specific anti-C. burnetii IgG antibodies by a commercial ELISA kit. A questionnaire investigating possible associations between farm management and C. burnetii seropositivity was administered. At the flock level, the overall true seroprevalence adjusted for test sensitivity and specificity was 31.2% (95% confidence interval [CI]: 24.8-37.7). Sheep-farm and goat-farm true seroprevalence was 38.7% (95% CI 25.5-51.9) and 19.5% (95% CI 11.5-27.6), respectively. Interestingly, the true seroprevalence (48.5%; 95% CI 34.7-62.3) was higher in the mixed flocks (sheep and goats). At the animal level, the overall true seroprevalence was 15.9% (95% CI 15.4-16.4). No difference was found between the two species, but the true seroprevalence was significantly higher (χ(2)=7.49; p<0.007) among the goats in mixed flocks (25.7%; 95% CI 24.4-27.1) than the sheep (16.3%; 95% CI 15.1-17.4), suggesting a potential difference in susceptibility between the two species or the result of factors affecting their immune response or related to the livestock management system as the period of exposure to C. burnetii. A multivariable logistic model that controlled for farm-level clustering identified five main risk factors associated with farm seropositivity (p≤0.05): flock size of more than 12 animals (odds ratio [OR] 4.2; 95% CI 2.6-6.7), contact with other flocks (OR 2.1; 95% CI 1.2-3.6), mixed flock type (OR 2.4; 95% CI 1.4-4.2), farms located in the western area (OR 2.4; 95% CI 1.4-4.2), and infertility during the previous year (OR 2.6; 95% CI 1.2-5.2). The results of this study yielded baseline information that may be useful to set up future epidemiologic, flock management, and public health policies for the prevention and control of Q fever in Italy.

Maternal exposure to a mixture of di(2-ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) causes reproductive dysfunction in adult male mouse offspring.

We investigated the effects of maternal exposure to the plasticizer di(2-ethylhexyl) phthalate (DEHP) and the organic industrial compounds polychlorinated biphenyls (PCBs), singly and combined, on the reproductive function of male mouse offspring. Mice dams were exposed throughout pregnancy and lactation to 1µg PCBs (101+118)/kg/day, 50µg DEHP/kg/day, or the DEHP/PCB mixture in the diet. The mixture induced permanent alterations in adult F1 males' reproductive health in a way, differently from the single compounds. Depending on the endpoint, we observed: (1) synergy in altering the gross and histological morphology of the testis; (2) antagonism on the expression levels of genes involved in pituitary-gonadal cross-talk; (3) non-interactions on sperm parameters and testosterone production. This study illustrates the complex action of a DEHP/PCB mixture, leading to a unique panel of effects on the male reproductive system, indicating the need for research on the reproductive hazards of combined endocrine disruptors.

Follicular fluid leptin concentrations and expression of leptin and leptin receptor in the equine ovary and in vitro-matured oocyte with reference to pubertal development and breeds.

There is no published information about follicular-fluid leptin concentrations or the presence of leptin and leptin receptor in the equine ovary or oocyte. Three groups of mares - adult draft mares, draft fillies and adult Standardbred mares - were included in the study. Leptin and leptin receptor were detected in all immature oocytes by immunofluorescence with higher intensity in oocytes from draft mares compared with draft fillies and Standardbred mares. After in vitro maturation a higher proportion of oocytes reached metaphase II in draft mares than in draft fillies and Standardbred mares, and in all groups both leptin and leptin receptor became localised in the oocyte cortex but with higher immunopositivity in draft mares compared with draft fillies and Standardbred mares. These intensities were confirmed by the expression profiles of leptin and leptin receptor mRNA. Moreover, leptin was detected in ovarian blood vessels in all three types of animal and within the corpora lutea in adult mares. Serum and follicular-fluid concentrations of leptin were similar in draft and Standardbred mares but higher in draft mares than in draft fillies. This study supports the hypothesis that expression of leptin and leptin receptor mRNA and the rate of maturation can be related either to adiposity or to puberty.

Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring.

The present study examined the effects in mice of exposure to di(2-ethyl-hexyl) phthalate (DEHP) throughout pregnancy and lactation on the development and function of the pituitary-gonadal axis in male and female offspring once they have attained adulthood. Groups of two to three dams were exposed with the diet from gestational d 0.5 until the end of lactation, at 0, 0.05, 5, and 500 mg DEHP/kg · d. The experiment was repeated three times (total: seven to 10 dams per treatment). The 500-mg dose caused complete pregnancy failure, whereas exposure to doses of 0.05 and 5 mg did not affect pregnancy and litter size. In total, about 30 male and 30 female offspring per group were analyzed. Offspring of the DEHP-treated groups, compared with controls, at sexual maturity showed: 1) lower body weight (decrease 20-25%, P < 0.001); 2) altered gonad weight (testes were ∼13% lighter and ovaries ∼40% heavier; P < 0.001); 3) poor germ cell quality (semen was ∼50% less concentrated and 20% less viable, and ∼10% fewer oocytes reached MII stage, P < 0.001); 4) significant lower expression of steroidogenesis and gonadotropin-receptor genes in the gonads; and 5) up-regulated gonadotropin subunit gene expression in the pituitary. In conclusion, our findings suggest that, in maternally exposed male and female mice, DEHP acts on multiple pathways involved in maintaining steroid homeostasis. Specifically, in utero and lactational DEHP exposure may alter estrogen synthesis in both sexes. This, in turn, induces dysregulation of pituitary-gonadal feedback and alters the reproductive performance of exposed animals.

Effects of polychlorinated biphenyls in CD-1 mice: reproductive toxicity and intergenerational transmission.

Several studies indicate that in utero and perinatal exposure to polychlorinated biphenyls (PCBs) induces adverse reproductive effects, but it remains unclear whether such effects may be transmitted to subsequent generations. We therefore investigated the association between maternal exposure to PCBs and reproductive health in male and female offspring over three generations. Mouse dams were fed 0, 1, 10, and 100 µg/kg/day of a PCB mixture (101 + 118) during pregnancy and lactation. PCB levels were measured in the tissues of both dams and offspring. PCB concentrations at all doses investigated were greater in the offspring than in the dams (p ≤ 0.0001) confirming that the progeny were exposed as a result of maternal exposure. In F1 offspring, exposure to PCBs resulted in reductions in (1) testis weight (p ≤ 0.05) and seminiferous tubule diameter (p ≤ 0.05), (2) sperm viability (p ≤ 0.0001) and developmental capacity (p ≤ 0.05), (3) ovary weight (p ≤ 0.05), (4) oocyte developmental capacity (p ≤ 0.05), and (5) increased follicular atresia (p ≤ 0.0001). In females, adverse effects were observed only in the F1 animals. In contrast, male offspring exhibited reduced sperm viability and altered seminiferous tubule distribution up to the third generation, showing intergenerational transmission. In summary, our data indicate that exposure to PCBs at the time of gonadal sex determination perturbed, significantly, the reproductive physiology of male and female offspring in adulthood. Furthermore, male reproductive deficiencies may be observed in at least two further generations. These findings have significant implications for reproductive health and fertility of animals and humans.

Relationship between S. aureus gene pattern and dairy herd mastitis prevalence.

The importance to evaluate Staphylococcus aureus virulence analysing the combination of virulence genes is largely recognised, and the recent availability of simplified microarray tools allows performing these analyses also in the dairy field. The combined availability of herd-specific S. aureus mastitis prevalence data, isolates from these herds, and microarray technology offered the opportunity to investigate the relationship between S. aureus genetic pattern and their prevalence among dairy herds. Eleven commercial dairy herds following a S. aureus control programme were enrolled in the study, and 33 S. aureus isolates were collected from these herds. Diagnostic DNA microarrays based on the array-tube platform were used for genotyping of staphylococcal DNA. The genetic analysis of the 157-genes microarray showed as only 19 genes were present in all the isolates considered, and among them the genes coding for the leukocidin subunits (LukF, LukS and LukY), haemolysins (hla, hld and an unnamed haemolysin) and enterotoxin X. Several genes considered in the arrays were absent in all the isolates, including the ones encoding the resistance to most of the antimicrobials, except for tetracycline. In our isolates, some agr alleles were never identified (B-III, C-III, D-III, C-IV and D-IV). The comparison of epidemiological data with the genetic pattern suggests that agr type II is associated to the most diffusive isolates, being recovered from the largest number of herds and with the highest frequency. Microarray technique showed to be a useful method to assess the characteristics of virulence of S. aureus isolated in dairy herds and to investigate the relationship with the prevalence of the microorganism. These results support previous evidence that specific gene patterns could be associated to S. aureus mastitis.

The role of teat skin contamination in the epidemiology of Staphylococcus aureus intramammary infections.

Knowledge of the epidemiological pattern and the potential sources of infections is important to control Staphylococcus aureus in dairy herds. This paper reports the results of a study applying both pulse field gel electrophoresis (PGFE) and the assessment of a selected number of virulence genes to investigate the role of teat skin on Staph. aureus transmission among cows and on the contamination of milk. Overall 61 isolates were considered, 23 from teat skin, 33 from milk samples and 5 from curd samples. Teat swabs were taken in five herds, but in only three of them could Staph. aureus be isolated. Curd was sampled in three herds, but Staph. aureus could be isolated in only two herds. The distribution of isolates among herds confirmed the presence of herd-specific Staph. aureus strain in most of the herds. The same pattern was observed in teat skin samples, in quarter milk samples, and in the curd samples. Our findings are consistent with other studies showing the role of teat skin as a potential reservoir. Moreover, Staph. aureus was isolated from teat skin of confirmed Staph. aureus-negative cows that were segregated from infected ones. Our findings also suggest that some strains have higher chances to survive on teat skin and therefore to increase the risk for contamination of milk and milk products due to the persistence of intramammary infections.

Mink growth hormone structural-functional relationships: effects of renaturing and storage conditions.

Fourier-transform infrared spectroscopy, in vitro bioassay and enzyme-linked immunoassay were used to study the structural-functional relationships of recombinant mink growth hormone (mGH), refolded and stored under different conditions. Porcine GH (pGH) was synthesized and used as an example. These two hormones, when refolded and stored the same way, had the same secondary structures, biological and immunological efficacy, and biological potency. Only the immunological potency differed, mGH being significantly less potent than pGH. Renaturation pH and storing frozen or at 4 degrees C in 5% glycerol did not affect either the secondary structure or the activity. However, freeze-drying raised the content of buried alpha-helices and lowered that of solvated alpha-helices and of unordered structures. These conformational changes were associated with a reduction of immunological and biological potency of mGH and of immunological potency of pGH. These findings provide original information on the secondary structure of mGH, and show that conformational changes induced by lyophilization adversely affect its activity.

Production of recombinant mink growth hormone in E. coli.

Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular protein after 3 h of induction with 1 mM isopropyl beta-D-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4-2.1 mg/ml in the presence of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed. Some 25-30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink in vivo and availability in fur industry.

Relationship between some Staphylococcus aureus pathogenic factors and growth rates and somatic cell counts.

Staphylococcus aureus isolates produce several pathogenic factors. The combination of these products influences the pathogenic role of different isolates, but their specific effects are well known in the pathogenesis of udder infections. This study focused on the association of polymorphism of the coagulase gene, protein A gene, collagen-binding protein gene, and of fibrinogen-binding protein gene on somatic cell count (SCC) and on Staph. aureus growth rate. Fifty Staph. aureus isolates from 13 dairy cow herds, located in seven different provinces, were considered. The results showed a low frequency of cna gene, similar to the one observed in human isolates. Meanwhile, the high frequency of efb gene indirectly confirmed the role of this factor in bacterial pathogenesis, being associated with adhesion to epithelia. The association of these two single genes with SCC and growth rate showed to be not significant. The polymorphism of spa gene was confirmed to be significantly associated with inflammatory response and growth rate, albeit with a pattern different from the one suggested for human isolates. Sorting of isolates based on the clusters obtained by combining polymorphisms of spa and coa genes and the presence of cna and efb genes, showed that a single cluster (cluster V) was prevalent in the different herds and provinces, while the other six clusters identified were widely spread among the remaining 60% of the isolates. Results showed that clusters VI and VII had significantly higher growth rates at 3, 4, and 6 h in comparison with the other clusters. Meanwhile, quarters infected with these strains showed significantly lower SCC levels. The frequency of isolates from cluster V, suggested that they should possess pathogenic factors increasing their invasiveness, even if in the presence of a stronger inflammatory response. These results indirectly confirm previous findings on the different interactions between isolates and the udder immune system. They also suggest that isolates with higher growth rates and inducing a lower inflammatory response have better chances to spread among the herd. The relatively simple genomic method proposed in this study could be applied by an increasing number of diagnostic laboratories and could be useful in studying the epidemiology of Staph. aureus intra-mammary infections in dairy herds when collecting data from the field.