Prevalence of canine herpesvirus 1 and associated risk factors among bitches in Iranian breeding kennels and farms.

BACKGROUND: Canine herpesvirus 1 (CHV-1) is an important cause of death in newborn puppies and of fertility problems in adult dogs. Identification of risk factors may help to reduce infection rates and alleviate concerns for dog owners and breeders. This study was designed to screen for CHV-1 infection in bitches of breeding kennels and farms in Iran and relate this to possible risk factors. METHODS: A total of 63 vaginal samples were collected from dogs in 5 breeding kennels (n = 47) and from 7 farms (n = 16). Real-time polymerase chain reaction was used to detect the CHV-1 specific glycoprotein B (gB) gene. Prevalence rates were evaluated in relation to various risk factors, including region, housing, vaccination, deworming, pregnancy, reproductive problems, number of dogs living together and hygiene conditions. RESULTS: In total, 21 (33.3%) of 63 vaginal samples were positive for CHV-1 DNA. The prevalence rate in farms (7/16; 43.7%) was higher than in kennels (14/47; 29.7%). No association was found between CHV-1 prevalence and potential risk factors. CONCLUSIONS: CHV-1 is highly prevalent in dogs in Iranian farms and kennels. Since the CHV1 vaccine is unlicensed in Iran, effective management strategies are essential to reduce the consequences of this pathogen.

Enhancing the Butyrylcholinesterase Activity in HEK-293 Cell Line by Dual-Promoter Vector Decorated on Lipofectamine.

PURPOSE: Human butyrylcholinesterase (BChE) serves as a bio scavenger to counteract organophosphate poisoning. It is also a potential drug candidate in several therapeutic fields. Therefore, in the present study, we constructed a new dual-promoter plasmid consisting of Cytomegalovirus (CMV) and human elongation factor 1α (EF-1α) promoters and transfected that into HEK-293 cells using Lipofectamine to enhance the BChE secretion. METHODS: The new dual-promoter construction (pBudCE dual BChE) including two copies of the BChE gene was designed and transfected into cells by liposomal structures. The cloned plasmids were evaluated by enzyme digestion and gel electrophoresis analysis. Experimental groups were categorized into the cells transfected by pBudCE dual BChE (treatment), pCMV (positive control) vectors, and nontransfected cells (negative control). BChE gene expression was evaluated by qRT-PCR and the enzyme activity was assessed using modified Ellman's method. The freeze-thaw process was carried out for analyzing the stability of the pBudCE dual BChE vector. RESULTS: Validation examination of the cloned plasmids confirmed the successful cloning process. The gene expression level and Ellman's method value in pBudCE dual BChE was higher than the other groups. CMV promoter has also increased the enzyme activity, although the difference was not significant compared with the control group. Interestingly, freeze-thaw cycles followed by several passages did not affect the enzyme activity. CONCLUSION: The designed construction with CMV and EF-1α promoters could increase BChE gene expression and the activity of the BChE enzyme in HEK-293 cell line. Large-scale production of BChE enzyme can be achieved by using dual-promoter plasmid construction compared to a single-promoter vector to be used in clinical trials.

First molecular study of Canine herpesvirus-1 in reproductive specimens of adult dogs in southeast of Iran.

Canine herpesvirus-1 (CHV-1) is recognized to be enzootic in the dog population with a widespread distribution. This pathogen leads to a lethal generalized illness in newborn puppies and is associated with reproductive disorders. CHV-1 should be considered as an important pathogen of neonatal death and infertility; so, it appears to pose a threat for breeding kennels. Although serologic data point to the circulation of CHV-1 among dogs of Iran are available, no definitive diagnosis has been conducted based on the molecular assay. So, this research was done to detect the prevalence of CHV-1 in dogs of Kerman. In this study, the presence of CHV-1 in vaginal specimens and biopsies of the uterus of dogs referring to the Veterinary Hospital of Shahid Bahonar University of Kerman was determined. Fifteen uterine samples and seven vaginal samples were included in group of the pregnant dogs. Moreover, thirteen uterine samples and twenty vaginal samples were related to the dogs displaying suspicious clinical signs such as reproductive disorders. Samples were collected and evaluated using real-time PCR. Viral DNA was detected in 21 samples from a total of 140 (15 %) collected samples which were related to 14 uterine samples (20 %) and 7 (10 %) vaginal specimens. The association of this virus with age, breed, housing, pregnancy and reproductive disorders was not significant. Five positive reproductive samples were belonged to the dogs with a history of reproductive disorders including pyometra, metritis, stillbirths, vaginitis and vaginal prolapse. This study is the first molecular detection of CHV-1 in reproductive samples of dogs in Iran. Considering the significant prevalence of this virus, it is necessary to carry out management measures in controlling and preventing this disease. Tracing CHV-1 requires further research on this virus in dogs of this region.

Bayesian estimation of sensitivity and specificity of a PCR method to detect Coxiella burnetii in milk and vaginal secretions in sheep and goat samples.

Coxiella burnetii is a gram-negative and polymorphic rod bacterium that causes Q fever, a common zoonotic disease distributed worldwide. Widespread occurrences of the disease outbreaks indicate the importance of coordinated animal and human health efforts to control these outbreaks. Different tests are available to determine the C. burnetii infection status of a flock, but false negative responses may occur, as infectious animals can shed bacteria in milk intermittently, especially during an asymptomatic infection. In this study, a Bayesian latent class model was implemented to estimate the sensitivity (Se) and specificity (Sp) of a PCR method for the detection of C. burnetii in milk samples (PCRM) and vaginal swabs (PCRV) from Iranian sheep and goats. A nested PCR assay was conducted to detect infected animals among 170 milk samples and 170 vaginal swabs from goat flocks and 170 milk samples and 170 vaginal swabs from sheep flocks. We implemented a Bayesian latent class model to estimate the Se and Sp of a PCR method for the detection of C. burnetii in milk samples and vaginal swabs from sheep and goats. Estimations were based on the cross-classified results of PCRM and PCRV from the sheep and goat subpopulations. Positivity was 17.6 and 33.5%, respectively, for PCRM and PCRV samples among sheep. In goats, the apparent prevalence was 32.9 and 56.4% in PCRM and PCRV samples testing positive, respectively. This indicated the lower sensitivity of PCRM. The Se of PCRV was significantly higher than Se of PCRM, which corresponded to a higher rate of vaginal-positive, milk-negative PCR samples. In contrast, Sp of PCRV was lower than Sp of PCRM, representing the higher false-positive rate of vaginal swabs. The PCRV outperformed PCRM in terms of identifying latently infected sheep and goats; however, neither method could identify all latently infected sheep and goats, thus the combination is recommended to maximize our ability to identify infected animals. The true prevalence of C. burnetii infection was higher in Iranian goats than sheep.

Histomorphometric evaluation of mice testicular tissue following short- and long-term effects of lipopolysaccharide-induced endotoxemia.

OBJECTIVES: Lipopolysaccharide (LPS)-induced endotoxemia is known to cause male infertility. This study was designed to explore the effects of bacterial LPS on histomorphometric changes of mice testicular tissues. MATERIALS AND METHODS: In experiment 1, a pilot dose responsive study was performed with mice that were divided into five groups, receiving 36000, 18000, 9000, and 6750 µg/kg body weight (B.W) of LPS or only saline (control). White blood cells (WBC) were observed for 3 days after LPS inoculation. In experiment 2, two groups of mice were treated with 6750 µg/kg B.W of LPS or only saline (control). Five cases from each experimental group were sacrificed at 3, 30, and 60 days after LPS inoculation. Left testes were fixed in Bouin's solution, and stained for morphometrical assays. RESULTS: Time-course changes of WBC obtained from different doses of LPS-treated mice showed that inoculation of 6750 µg/kg B.W produced a reversible endotoxemia that lasts for 72 hr and so it was used in the second experiment. In experiment 2, during the first 3 days, no significant changes were observed in the evaluated parameters instead of seminiferous tubules diameter. Spermatogenesis, Johnsen's score, meiotic index, and epithelial height were significantly affected at 30th day. However, complete recovery was only observed for the spermatogenesis at day 60. Interestingly, deleterious effects of LPS on spermatogonia were only seen at 60th day (P<0.05). CONCLUSION: Endotoxemia induced by LPS has long-term detrimental effects on spermatogonia and later stage germ cells, which are reversible at the next spermatogenic cycle.

The effects of long-term leptin administration on morphometrical changes of mice testicular tissue.

OBJECTIVES: Leptin is a novel and interesting hormone for anyone trying to lose weight, but its effects on male gonad structure in longitudinal study is unknown. The present study was designed to explore morphometrical changes of mouse testicular tissue after long-term administration of leptin. MATERIALS AND METHODS: Thirty healthy mature male mice were randomly assigned to either control (n=15) or treatment (n=15) groups. Leptin was intraperitoneally injected to the treatment group (0.1 µg/100 µl of physiological saline) once a day for 30 consecutive days, and control animals received normal saline with the same volume and route. Five mice from each experimental group were sacrificed at 15, 30 and 60 days after the beginning of treatments. Left testes were removed, weighted and then fixed in 10% buffered formalin, and stained with hematoxylin and eosine for morphometrical assays. RESULTS: Except for sertoli cell nucleus diameter, which was affected from 30(th) day, evaluation of other morphometrical parameters such as Johnsen's score, meiotic index, spermatogenesis, epithelial height, seminiferous tubules diameter and spermatogonial nucleus diameter revealed significant decrease from 15(th) day after leptin administration compare to those of the control group (P<0.05). Thus, meiotic index and spermatogonial cell nucleus diameter were two parameters that were further disturbed on 30(th) day compare to the day 15 (3.09±0.03 vs. 3.23±0.03, P=0.006 and 5.50±0.09 vs. 6.08±0.14, P=0.007, respectively). CONCLUSION: Our results showed that long-term administration of leptin could disturb testicular tissue structure and delay spermatogenesis process.

Effects of supplement therapy on sperm parameters, protamine content and acrosomal integrity of varicocelectomized subjects.

PURPOSE: Surgical repair of varicocele has long been a procedure to correct spermatogenesis. However, the outcome has been reported to be inadequate. We combined varicocelectomy with supplement therapy to evaluate the concurrent effect of these procedures. METHODS: A prospective randomized controlled study was undertaken to investigate the effects of zinc sulfate, folic acid and zinc sulfate/folic acid on sperm quality, protamine content and acrosomal integrity following surgical repair of varicocele. Male subjects with palpable varicocele were included in the study and randomized into four groups. Subjects received Zinc sulfate, Folic acid, Zinc sulfate/Folic acid or placebo for 6 months. A semen sample was obtained before surgery and 3 and 6 months after surgical repair. Semen samples were evaluated for sperm parameters as well as chromatin content and acrosomal integrity. RESULTS: Most of the evaluated parameters showed a mild improvement after varicocelectomy in the placebo group. Interestingly, co-administration of Zinc sulfate and folic acid improved most factors significantly. Folic acid administration but not zinc sulfate could increase sperm number. Hence, Zinc sulfate was better than folic acid when change in morphology was assessed, and none of them was significantly effective in sperm motility. In Zinc sulfate and Folic acid groups, protamine content and halo formation rate significantly improved. CONCLUSIONS: We may conclude that co-administration of zinc and folic acid significantly improved sperm parameters and increased varicocelectomy outcomes. So, medical treatment with compatible drugs after surgery might be advantageous for obtaining acceptable results.

Zinc therapy improves adverse effects of long term administration of copper on epididymal sperm quality of rats.

BACKGROUND: Industrial copper ingest is a common form of poisoning in animals. Zinc has an important role in the physiology of spermatozoa, in sperm production and viability. OBJECTIVE: This study was set to investigate whether the adverse effects of long term copper consumption on quality of rat spermatozoa could be prevented by zinc therapy. MATERIALS AND METHODS: Forty eight mature (6-8 weeks old) male rats were randomly allocated to either control (Cont, n=12) or three treatment groups each containing twelve animals. Animals in the first treatment group was gavaged with copper sulfate, the second treatment group was injected with zinc sulfate, and the third treatment group was given combined treatment of copper and zinc. Control animals received normal saline using the same volume and similar methods. Six rats from each group were sacrificed on day 28 and 56 after treatments for sperm quality evaluations. RESULTS: In spite of testicular weight reduction 56 days after copper consumption in comparison to the control group (p=0.002), there was not a significant difference between the control and combined treatment of copper and zinc group (31.40±0.55 vs. 28.63±0.55, p=0.151). Administration of copper caused a significant decrease in the sperm count, viability and motility after 56 days compared to the control group. However, a complete recovery in sperm count was seen in combined treatment of copper and zinc group after 56 days compared to the control group (p=0.999) and a partial improvement was seen about the percentage of viability and motility (p<0.001). CONCLUSION: Adverse effects of long term consumption of copper on sperm quality could be prevented by zinc therapy in rats.

Effects of transplanted mesenchymal stem cells isolated from Wharton's jelly of caprine umbilical cord on cutaneous wound healing; histopathological evaluation.

The aim of this study was to investigate the effects of transplanted Wharton's jelly mesenchymal stem cells (WJMSCs) of caprine umbilical cord on cutaneous wound healing process in goat. After collection of caprine pregnant uterus of mixed breed goats from abattoir, the Wharton's jelly (WJ) of umbilical cord was harvested. The tissues were minced in ventilated flasks and explant culture method was used for separating mesenchymal stem cells (MSCs). The isolated cells were immunostained for Actin protein, histochemically assayed for the presence of alkaline phosphatase activity, and analyzed for detection of matrix receptors (CD44) and hematopoetic lineage markers (CD34), using flow cytometery. After The isolated cells, 3×10(6) MSCs were stained with BrdU and prepared for transplantation to each wound. Four 3-cm linear full thickness skin incisions were made on both sides of thoracic vertebrate of four Raeini goats (two wounds on each side). The left wounds were implanted with MSCs in 0.6 ml of Phosphate buffer saline (PBS), and the right wounds considered as control group that received 0.6 ml of PBS. The samples were taken from the wounds 7 and 12 days after the wounding, and healing process was compared histologically between the two groups. Anti-BrdU staining showed that the transplanted cells were still alive in the wound bed during the study. The histopathological study revealed that re-epithelialization was complete at days 7 in treated wounds with WJMSCs, whereas in control wound the wounds still showed incomplete epithelialization 12 days after wounding. Also, microscopic evaluation showed less inflammation, thinner granulation tissue formation with minimum scar in the treated wounds in comparison with control wounds. In conclusion, this study demonstrates the beneficial effect of caprine WJMSCs in cutaneous wound healing in goat.