Use of equine embryo -derived mesenchymal stromal cells and their extracellular vesicles as a treatment for persistent breeding-induced endometritis in susceptible mares.

Persistent breeding induced endometritis (PBIE) is a significant cause of infertility in mares. The development of a safe, universal, readily available therapeutic to manage PBIE and facilitate an optimal uterine environment for embryo development may improve pregnancy rates in susceptible mares. Mesenchymal stromal cells (MSCs) are being used increasingly as a therapeutic mediator for inflammatory conditions such as endometritis, and early gestational tissue provides a unique source of multipotent stem cells for creating MSCs. Extracellular vesicles (EVs) are mediators of cell communication produced by many different cell types. This study utilized embryo-derived mesenchymal stromal cells (EDMSCs) and their EVs as a potential therapeutic modality for PBIE in two groups: a) PBIE-susceptible mares challenged with pooled dead sperm (n=5); and b) client-owned mares diagnosed as susceptible to PBIE (n=37 mares and 40 estrous cycles). Mares pre-treated with intrauterine EDMSCs or their EVs resulted in a significant reduction in the accumulation of intrauterine fluid post-breeding. Nine of 19 (47 %) mares treated with EDMSCs prior to natural breeding and 13 of 20 (65 %) mares treated with EDMSC derived EVs were pregnant after the first cycle and 12 of 18 (67 %) mares treated with EDMSCs, and 15 of 19 (79 %) mares treated with EVs conceived by the end of the breeding season. These preliminary clinical studies are the first reports of the use of EDMSCs or their EVs as a potential intrauterine therapy for the management of PBIE susceptible mares.

Observational cohort study of the effect of a single lubricant exposure during transvaginal ultrasound on cell-shedding from the vaginal epithelium.

The outer layers of the vaginal epithelium (VE) are important because they accumulate glycogen which, under optimal conditions, Lactobacillus spp. consume to grow and acidify the vaginal microenvironment with lactic acid. We hypothesized that exposure to lubricant, for example in the conduct of a transvaginal ultrasound (TVUS), may contribute to the shedding of mature epithelial cells, exposing immature cells. Cervicovaginal fluid (CVF) was sampled at four time points by menstrual cup (Softdisc™) from 50 women referred for TVUS, during which a controlled volume of lubricant was applied to the TVUS wand. Samples were collected (1) immediately before TVUS and (2) 6-12 hours, (3) within one week, and (4) two weeks after TVUS. Clinical vaginal lubricants are similar to commercial lubricants, and often have a high osmolality or pH, and contain bactericides such as methylparaben and propylparaben. The number and maturity of epithelial cells in each CVF sample were measured by quantitative and differential fluorimetry (maturity index, MI). Comparisons of cell-counts and maturity were made by paired Wilcoxon signed-rank tests. Among women with a high pre-TVUS MI (> 3), there was a decrease in median cell-count and mean MI in the sample collected 6-12 hours after TVUS (p<0.001, n = 26 and p < 0.001, n = 26, respectively). For these women, cell-count and MI remained lower in the sample collected within the subsequent week (p<0.001, n = 29 and p<0.01, n = 29, respectively), and MI remained lower in the sample collected within two weeks of TVUS (p<0.01, n = 25), compared to the pre-TVUS sample. Among participants with a low pre-TVUS MI (< 3), cell-count was higher in the sample collected within two weeks of TVUS compared to the pre-TVUS sample (p = 0.03, n = 15), but no significant changes in MI were observed. Results were similar when restricted to reproductive-age women. This preliminary data indicates hypertonic vaginal lubricants may increase vaginal epithelial cell shedding.

Factors influencing parentage ratio in canine dual-sired litters.

Breeding a bitch with two different sires during a single estrous cycle has the potential to facilitate rapid genetic gain and improve reproductive performance within a canine breeding colony. There is limited data regarding the factors that contribute to the success of dual-sired litters in domestic dogs, and only anecdotal evidence suggesting that these litters rarely produce offspring from more than one sire. The objective of this prospective clinical study was to investigate multiple factors that likely affect the success of dual-sired litters on whelping rate, litter size and parentage ratio. These factors include: timing of artificial insemination (AI), order of sires, number of AI's per cycle, semen type, sperm quality and age of sire and bitch. Data collected over a 10 year period from twenty-nine estrous cycles (28 individual bitches of 10 different breeds) were evaluated after an initial AI with frozen semen from the 'genetically desired' sire and followed up with a second AI with either fresh (n = 9) or frozen (n = 16) semen or natural mating (n = 4) from a different, 'back up', sire. DNA parentage of each pup born was determined by using a primary panel of 288 SNPs. The whelping rate and litter size from previous single sire inseminations per estrous cycle, in the same bitches, (n = 16) over 25 estrous cycles using either fresh (n = 4) or frozen-thawed (n = 21) semen, were analyzed as controls. Of the 29 dual-sired breedings, 26 bitches whelped (89.7%), and 8 litters (30.8%) were of mixed parentage. In the litters of mixed parentage after a dual-sired breeding, a greater proportion of the offspring were from the second sire than the first sire (73.0% and 27.0% respectively; P < 0.05). Interestingly, in litters where all pups were of single sire parentage after a dual-sired breeding, 50.0% of the offspring were by the first sire and 50.0% were by the second sire. For litters of mixed or single paternity produced by dual-sired breeding there was no difference in average litter size. However, on a per estrous cycle basis for each bitch the whelping rate (89.7% v. 76.0%.) and litter size (5.5 ± 2.5 v. 4.0 ± 2.78) of all dual-sired breedings were greater (P < 0.05) compared to previous single-sired breedings (controls) respectively. This study demonstrates that offspring of mixed parentage derived from dual-sired breedings may be achieved. Furthermore, insemination with semen from two different sires may increase the whelping rate and litter size, which is an important consideration when using genetically valuable, or older individuals with potentially reduced fertility.

Asymptomatic Bacterial Vaginosis Is Associated With Depletion of Mature Superficial Cells Shed From the Vaginal Epithelium.

Previous studies have described bacterial vaginosis (BV) as associated with increased cell-shedding from the cervicovaginal epithelium. Cell-shedding in excess of cell-proliferation is thought to decrease epithelial barrier function and increase susceptibility to infection. This study evaluated the number of shed cells in mid-vaginal smears from women with a diagnosis of symptomatic BV (sBV, n = 17), asymptomatic BV (aBV, n = 71), or no BV (n = 104) by Amsel criteria. The sBV smears contained significantly more shed cells (median 158/100X field) than no BV smears (median 91/100X field), p = 7.2e-9. However, we observed that aBV smears contained significantly fewer shed cells (median 35/100X field) than no BV smears, p = 22.0e-16. The sizes of cell-aggregates (cells shed in sometimes multilayered sections with intact cell-cell attachments) followed the same pattern. Cell-aggregates in sBV smears were significantly larger (median ~220,000 µm2) than those in no BV smears (median ~50,000 µm2), p = 1.8e-6, but cell-aggregates in aBV smears were significantly smaller (median ~7,000 µm2) than those in no BV smears, p = 0.0028. We also compared the superficial cell index (SCI), a measure of cervicovaginal epithelial cell maturity, in no BV and aBV smears with relatively low numbers of shed cells (≤50/100X field). The SCI of no BV smears was significantly higher (median 0.86) than that of aBV smears (median 0.35), p = 4.3e-98, suggesting a depletion of mature cells with exposure and shedding of underlying immature cells in aBV with low number of shed cells. These results indicate that aBV may contribute disproportionately to the increased susceptibility to reproductive tract infections associated with BV. Our findings remained true when considering only those smears in which the microbiota comprised a diverse set of strict and facultative anaerobic bacteria [Community State Type IV (n = 162)], thus excluding those dominated by Lactobacillus spp. This is consistent with our developing hypothesis that high-shedding sBV and low-shedding aBV could be temporally separated phases of the same condition, rather than two separate forms of BV. These findings might inform future work on clinical management of symptomatic and asymptomatic bacterial vaginosis.

The Vaginal Microbiota and Behavioral Factors Associated With Genital Candida albicans Detection in Reproductive-Age Women.

BACKGROUND: Vulvovaginal candidiasis is commonly diagnosed and has been associated in prospective studies with the acquisition of HIV. Little data is available on how the composition of the vaginal microbiota, and other risk factors, are associated with the molecular detection of Candida albicans-a common cause of vulvovaginal candidiasis. METHODS: In a cross-sectional study, self-collected vaginal swabs were obtained from 394 nonpregnant, reproductive-age women. C. albicans was detected using polymerase chain reaction targeting C. albicans ITS1/2 region. Vaginal microbiota was characterized by 16S rRNA gene amplicon sequencing of the V3 to V4 hypervariable regions and clustered into community state types (CSTs). Multiple logistic regression identified factors associated with C. albicans detection. RESULTS: Twenty-one percent had C. albicans detected and 46% reported vaginal symptoms in the prior 60 days. There was a 2-fold increase in the odds of C. albicans if a woman was in a L. crispatus-dominated CST compared to CSTs with low-Lactobacillus levels (adjusted odds ratio, 2.05; 95% confidence interval, 0.97-4.37). History of self-treatment with antifungals, L. crispatus relative abundance, and receptive oral sex were also significantly associated with C. albicans detection. CONCLUSIONS: A L. crispatus-dominated vaginal microbiota is thought to protect women from both development of bacterial vaginosis and incidence of sexually transmitted infections; however, our data suggest that L. crispatus is associated with increased C. albicans detection. Receptive oral sex may also be a risk factor for vaginal C. albicans colonization.

Factors affecting the reproductive performance of bitches: A prospective cohort study involving 1203 inseminations with fresh and frozen semen.

The aim of this prospective cohort study was to utilize multivariable statistical methods to identify factors that significantly affected whelping rate, litter size and gestation length in a large population of bitches of many different breeds, presented for routine breeding management. In addition, we aimed to determine the incidence of dystocia and the proportion of bitches undergoing a caesarean section procedure. A total of 1146 individual bitches representing 84 different breeds contributed 1203 inseminations over the 9 year (2007-2015) study period. Bitches were inseminated with either frozen-thawed (n = 645), fresh (n = 543) or chilled (n = 15) semen from 1371 different males. The mean (SD) whelping rate was 74± 4% and the mean litter size was 5.8 ± 3.1 pups per litter for all bitches in the study. The whelping rate was significantly lower in bitches inseminated with frozen-thawed semen compared with bitches inseminated with fresh semen (71% vs 80% respectively; P < 0.001). Semen that was classified as having poor motility (<30% progressive) resulted in a significantly lower whelping rate (37%) than semen classified as good (30-65% progressive; whelping rate = 67%) or excellent (>65% progressive; whelping rate = 79%). There was a linear decline in whelping rate with advancing age. Greyhounds and Labradors demonstrated a significantly higher whelping rate (88% and 94% respectively) compared with all other breeds (71.3%, P < 0.001). Bitches inseminated with frozen-thawed semen had significantly smaller litter sizes than bitches inseminated with fresh semen (5.4 ± 3.1 vs 6.2 ± 3.0 pups per litter respectively; P = 0.02). Smaller breeds had significantly smaller litters (4.4 ± 2.1 pups) than medium (5.2 ± 2.9 pups), large (5.9 ± 2.9 pups) or giant (6.7 ± 3.8 pups) breeds. For each advancing year of age, litter size decreased by 0.13 pups per litter. The mean (SD) gestation length from LH0 was 65 ± 1.9 d. Greyhounds had a significantly longer pregnancy duration (68.0 ± 1.5 d) than other breeds. For each additional year of bitch age, gestation length increased by 0.11 days (P < 0.01), and for each additional pup per litter, gestation length was reduced by 0.08 days (P < 0.05). Of the 890 bitches for which whelping outcomes were recorded; 409 (46%) whelped normally without assistance, 249 (28%) had an elective C-section, 205 (23%) underwent an emergency C-section and 27 (3%) were medically managed or required veterinary assistance for dystocia. Brachycephalic breeds were 11.3 (95CI = 9.3-17.9; P < 0.001) times more likely to have a C-section compared to all other breeds. Bitches with litter sizes of one or two pups had a C-section rate of 83%, whereas bitches with litter sizes of three or more pups had a C-section rate of 43% (P < 0.001). This study provides important clinical information to optimise whelping rates, litter size and the prediction of whelping in certain breeds for clinicians working in canine reproduction.

Periparturient characteristics of mares and their foals on a New Zealand Thoroughbred stud farm.

AIMS: To describe selected periparturient variables in a sample of Thoroughbred mares and their foals on a commercial stud farm in New Zealand. METHODS: Data were collected for 969 foaling records, from 592 mares, by foaling attendants from one Thoroughbred stud farm in the Waikato region of New Zealand for the breeding seasons 2008-2013, inclusive, and collated retrospectively. The associations between mare- and foal-level variables and gestation length, time of birth (expressed as the number of hours after midday), fetal membrane retention time, time for the foal to stand and suckle after birth, and foal mortality were quantified using linear and logistic regression models. RESULTS: Mean gestation length for mares in this study was 350 (min 296, max 429) days, and was shorter for filly foals (348 (min 296, max 429) days) compared with colt foals (350 (min 310, max 404) days) (p=0.001). Overall, 517/968 (53%) foalings occurred between 19:00 and 01:00 and 212/968 (22%) foalings occurred during daylight. Overall, 815/855 (95%) expelled their fetal membranes within 4 hours of parturition. The mean interval from birth to standing and to suckling was shorter (p<0.001) for filly foals than colts by 13 (95% CI=9-17) and 17 (95% CI=8-26) minutes, respectively. Dystocia was reported for 81/968 (8.4%) foalings with relative fetal oversize being the most common cause (36/81 (44%) cases). Perinatal foal death was recorded for 26/966 (2.7%) foalings. The odds of foal mortality decreased with increasing length of gestation (p=0.027), and were greater for colt than filly foals (p=0.042) and when dystocia was reported (p<0.001). CONCLUSIONS: Gestation length was influenced by the sex of the foal, as was the time taken for the foal to stand and suckle after birth. One fifth of mares foaled during daylight hours. The majority of mares in this study population expelled their fetal membranes within 4 hours of foaling. CLINICAL RELEVANCE: Accurate measures for periparturient behaviour can be important in the management of mares and newborn foals. Clinically relevant differences appear to exist between mares and foals in New Zealand and those in the Northern Hemisphere for some key parameters, likely as a result of differences in management and environment. The results of this study suggest that mares should be considered to have retained their membranes if not passed within 4 hours of parturition.

Determination of the normal reference interval for anti-Müllerian hormone (AMH) in bitches and use of AMH as a potential predictor of litter size.

Anti-Müllerian hormone (AMH) is a reliable endocrine marker of ovarian reserve in many species with extensive literature in both humans and cattle. However, there are no known hormonal predictors of ovarian reserve and potential reproductive performance in the bitch. A prospective cohort study was performed involving 155 intact bitches of various ages (range 1.2-7.6 years) and breeds that were presented for routine breeding management over a one-year period. All bitches were artificially inseminated with frozen or fresh semen using the transcervical insemination (TCI) technique. AMH concentrations were measured using a commercially available canine AMH ELISA (Ansh Labs® , Texas, USA), which we validated prior to performing the study. The reference interval (RI) for AMH for all bitches in the study, regardless of body weight, was 2.9-21.1 ng/ml. There was a significant effect of bitch size and age on AMH concentrations. The RI for giant breeds was significantly (p < .01) lower (1.75-15.6 ng/ml) than small-sized (5.6-24.2 ng/ml), medium-sized (4.3-23.7 ng/ml) and large-sized (4.3-21.0 ng/ml) bitches. The mean AMH concentration in bitches less than 4 years of age was 12.4 ng/ml, whereas the mean AMH concentration in bitches older than 4 years of age was 10.5 ng/ml (p < .05). For each additional year of age above 1 year of age, AMH concentrations fell by 0.5 ng/ml. There was no effect of AMH concentration on the whelping rate. Smaller breeds had smaller litters (and higher AMH concentrations), but within each breed size category, bitches with higher AMH concentrations had significantly larger litter sizes (p < .01). For each 1 ng/ml increase in AMH, litter size increased by 0.3 pups/litter. In conclusion, we determined a normal reference interval for AMH in bitches based on body weight using a canine-specific assay. In agreement with findings in humans and other species, we found that there is a decline in AMH concentrations with advancing age in bitches. Finally, the significant positive correlation between AMH concentrations and litter size indicates that AMH may be a useful management tool for the selection of bitches in breeding programmes.

Revealing the nature of excitons in liquid exfoliated monolayer tungsten disulphide.

Transition metal dichalcogenides (TMD) hold promise for applications in novel optoelectronic devices. There is therefore a need for materials that can be obtained in large quantities and with well understood optical properties. In this report, we present thorough photoluminescence (PL) investigations of monolayer tungsten disulphide obtained via liquid phase exfoliation. As shown by microscopy studies, the exfoliated nanosheets have dimensions of tens of nanometers and thickness of 2.5 monolayers on average. The monolayer content is about 20%. Our studies show that at low temperature the PL is dominated by excitons localized on nanosheet edges. As a consequence, the PL is strongly sensitive to the environment and exhibits an enhanced splitting in magnetic field. As the temperature is increased, the excitons are thermally excited out of the defect states and the dominant transition is that of the negatively charged exciton. Furthermore, upon excitation with a circularly polarized light, the PL retains a degree of polarization reaching 50% and inherited from the valley polarized photoexcited excitons. The studies of PL dynamics reveal that the PL lifetime is on the order of 10 ps, which is probably limited by non-radiative processes. Our results underline the potential of liquid exfoliated TMD monolayers in large scale optoelectronic devices.

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand.

CASE HISTORY AND CLINICAL FINDINGS: On 9 January 2014 (Day 0) a mare from a stud farm in the Waikato region presented with urinary incontinence without pyrexia. Over the following 33 days 15 mares were clinically affected with neurological signs. All but one mare had a foal at foot. The most commonly observed clinical signs were hind limb paresis and ataxia. In some cases recumbency occurred very early in the course of disease and seven mares were subject to euthanasia for humane reasons. LABORATORY FINDINGS: Equid herpesvirus (EHV) type 1 was detected using PCR in various tissues collected post mortem from two mares with neurological signs. DNA sequencing data from the DNA polymerase gene of the virus showed a nucleotide transition at position 2254, a mutation encoding amino acid D752 that is highly associated with the neuropathogenic genotype of EHV-1. In total 12/15 mares were confirmed positive for EHV-1 on PCR. Results from a virus neutralisation test and ELISA on paired serum samples, and PCR on whole blood and nasal swabs, indicated that of four paddocks in a high-risk area where a cluster of cases had occurred, 20/21 (95%) horses were likely to have been exposed or were confirmed infected with EHV-1. Subsequent to the outbreak two mares aborted, one at 9 months and one at 10 months of gestation. The cause of abortion was confirmed as EHV-1 with the same genotype as that involved in the outbreak. DIAGNOSIS: Equine herpesvirus myeloencephalopathy. CLINICAL RELEVANCE: The outbreak described shows the considerable impact that can occur in outbreaks of equine herpesvirus myeloencephalopathy in New Zealand. Early biosecurity controls not only reduced the effect on the farm but mitigated the potential for the virus to spread to other horse enterprises.

Reproductive performance of Thoroughbred mares in the Waikato region of New Zealand: 2. Multivariable analyses and sources of variation at the mare, stallion and stud farm level.

AIM: The objective of this study was to utilise multivariable statistical methods appropriate for clustered data to identify mare-related explanatory variables that significantly affected the reproductive performance of Thoroughbred mares in the Waikato region of New Zealand. In addition, we aimed to determine the relative contribution of the mare, stallion and stud farm to reproductive performance. METHODS: A prospective cohort study was performed involving five stud farms in the Waikato region of New Zealand during three consecutive breeding seasons (2006-2008). A total of 1,482 individual mares contributed 2007 mare years and 3,402 oestrous cycles over the three breeding seasons. Reproductive performance was measured using three parameters; (a) first-cycle pregnancy rate (FCPR), (b) end-of-season pregnancy rate (SPR), and (c) the start-of-mating to conception interval. RESULTS: When controlled for the effects of serving stallion, stud farm and year of study the only significant mare-related variables included in the final models of FCPR, SPR and conception interval were the age of the mare and her reproductive status (classified as dry or foaling). Advancing mare age significantly reduced reproductive performance regardless of reproductive status and foaling mares had significantly poorer reproductive outcomes compared with dry mares when controlled for age. For each additional increase in year of age, the FCPR was reduced by a factor of 0.94 (95% CI=0.92-0.96) and the SPR was reduced by a factor of 0.91 (95% CI=0.88-0.93). Mares older than 14 years of age took longer to conceive after the start-of-mating compared with younger mares. The daily hazard of conception for mares 14 years and older was 0.64 (95% CI=0.47-0.83) times less than mares younger than 9 years of age. Determining the relative contribution of the mare, stallion and stud farm to the FCPR indicated that 95.9% of the variation was at the mare level, 4.1% was at the stallion level and 0% was at the stud farm level. For the SPR the variance components indicated that 92.5% of the variation was at the mare level, 6.7% was at the stallion level and 0.8% was at the stud farm level. CONCLUSIONS: The reproductive performance of Thoroughbred mares in the Waikato region of New Zealand is influenced by two main mare-related factors; the age of the mare and her reproductive status (dry or foaling). The majority of variation in reproductive performance was associated with mare-level factors and the contribution of the stallion and stud farm was relatively minor.

Reproductive performance of Thoroughbred mares in the Waikato region of New Zealand: 1. Descriptive analyses.

AIM: To describe the reproductive performance of a population of Thoroughbred mares on stud farms in the Waikato region of New Zealand. METHODS: A prospective cohort study was performed involving five stud farms in the Waikato region of New Zealand during three consecutive breeding seasons (2006-2008). A total of 1,482 individual mares contributed 2007 mare years and 3,402 oestrous cycles over the three breeding seasons. Mares were served by 87 individual stallions. RESULTS: The mean first-cycle pregnancy rate (FCPR) was 53.6%, the end-of-season pregnancy rate (SPR) was 85.3% and the foaling rate was 80.2%. There were significant differences amongst farms for FCPR (range 47.3-68.2%; p<0.01), SPR (range 81.4-92.6%; p<0.001) and foaling rate (range 77.8-90.1%; p<0.001). There were significant differences amongst stallions in their FCPR (range 38-74%; p<0.001) and SPR (range 72-97%; p<0.001).The proportion of pregnancies lost between day 14-16 and day 42 of gestation was 5.5% and the proportion of pregnancies lost between day 42 and term was 3.0%. Mares older than 14 years of age and barren mares had the highest pregnancy loss rates. Of all pregnancies diagnosed at 14-16 days, 12.9% were multiple pregnancies. The length of the breeding season was relatively short with 87.3% of services occurring in the 91-day period between 1 September and 30 November. CONCLUSIONS: The reproductive performance of Thoroughbred mares in the Waikato region of New Zealand was similar to that reported from other major Thoroughbred breeding countries. Although the per-cycle pregnancy rates in this study were lower than those reported in some studies, the foaling rate was similar to previous reports and this was achieved during a relatively short breeding season.

The reproductive performance of Thoroughbred mares treated with intravaginal progesterone at the start of the breeding season.

The objective of this study was to investigate the effects of intravaginal progesterone on the reproductive performance of transitional Thoroughbred mares on commercial stud farms. Two hundred twenty-seven (227) non-lactating transitional Thoroughbred mares aged between 4 and 18 y (mean 9.4 ± 3.2 y) located on three stud farms in the Waikato region of New Zealand were used in the study performed during four consecutive breeding seasons (2007-10). Mares were age-matched in pairs and either treated with an intravaginal progesterone releasing device (Cue-Mare, 1.72 g progesterone, 10% w/w) for up to 10 d (Treated; n = 126) or left untreated (Control; n = 101). In both groups, 1,667 iu of hCG was given IV when an ovarian follicle ≥35 mm was detected (in conjunction with estrous behavior) and each mare was bred by natural service. Treated mares were served earlier in the breeding season (mean ± SD interval to first service was 13.9 ± 3.0 vs 26.7 ± 13.2 d for Treated and Control groups, respectively; P < 0.001). In the Treated and Control groups, 95.2 and 42.6% of mares were served within the first 21 d of the season (P < 0.001). Treated mares conceived earlier in the breeding season (mean number of days to conception 37.5 ± 14.2 vs 50.8 ± 21.3 d, P = 0.01). There was no difference between groups in the first service pregnancy rates (53.9 and 50.5% for Treated and Control mares, P = 0.89). Treatment with an intravaginal device increased the number of mares conceiving by the end of the breeding season (91.3 vs 82.3% for Treated and Control groups, P = 0.04). Therefore, this treatment protocol appeared to offer a convenient, economical and reliable method for managing transitional mares on commercial Thoroughbred stud farms.

Kinetics of Equid herpesvirus type 2 infections in a group of Thoroughbred foals.

The significance of infection with Equid herpesvirus 2 (EHV-2) remains unresolved, mainly due to its widespread distribution, and frequent isolation of the virus not only from diseased animals, but also from clinically normal horses. It has been suggested that EHV-2 exerts its effects on the host indirectly, through predisposition to secondary infections. The aim of this study was to determine kinetics of EHV-2 infection among foals and to investigate the role that EHV-2 may play in development of Rhodococcus equi pneumonia on one farm. Serial blood samples were collected from 43 foals over a period of 10 months. Viral load of EHV-2 in blood was determined using quantitative realtime PCR assay. All but 2 foals were positive for EHV-2 on at least one occasion. The majority (88%) of foals became infected with EHV-2 within the first 2 months of life. Once infected, most foals (86%) remained positive for EHV-2 on all subsequent samplings. The load of EHV-2 varied between individual foals and between different sampling times for each foal. There was a significant difference in EHV-2 load between samples collected from healthy foals and foals suspected of R. equi pneumonia only for 2-month-old foals, but not for foals of other ages. The results of this study extend our knowledge of EHV-2 epidemiology among foals.

Calcium, parathyroid hormone, oxytocin and pH profiles in the whelping bitch.

Despite the high prevalence of primary uterine inertia in whelping bitches, the underlying pathogenesis remains unclear. The objectives were to i) determine serum concentrations of total calcium, ionized calcium (iCa), parathyroid hormone (PTH), and blood pH in normally whelping bitches throughout the peri-parturient period; and ii) investigate relationships among iCa, PTH, and acid-base status, and the role that they and oxytocin may have in the underlying pathogenesis of canine uterine inertia. Bitches were randomly selected from a population of German Shepherd Dog bitches with a history of uncomplicated parturition (Group 1; n=10), and from a population of Labrador bitches with a clinical history of an increased incidence of uterine inertia and stillbirths (Group 2; n=20). Jugular blood samples were collected daily from -4 d to the onset of whelping (t=0 h), and then every 4h until the last pup was born. Overall, bitches from Group 2 had higher mean+/-SEM serum concentrations of PTH (4.72+/-2.45 pmol/L, P<0.001), lower iCa (1.31+/-0.08 pmol/L, P<0.05), and higher venous pH (7.41+/-0.03, P<0.005) than bitches from Group 1 (2.9+/-1.44 pmol/L, 1.38+/-0.06 mmol/L, and 7.33+/-0.02, respectively) during the periparturient period. However, there was no significant difference between Groups 1 and 2 for serum oxytocin concentrations during the periparturient period (45.5+/-40 and 65.5+/-82 pg/mL). We inferred that low iCa resulting from a rising pH and decreasing PTH during the periparturient period may have contributed to decreased uterine contractility and increased risk of stillbirths. Therefore, manipulating the cationic/anionic difference in diets of pregnant bitches, similar to the bovine model for hypocalcamia, may reduce the incidence of stillbirths in the bitch.

Polymer nanoparticles for immunotherapy from encapsulated tumor-associated antigens and whole tumor cells.

Encapsulation of tumor-associated antigens (TAA) in polymer nanoparticles is a promising approach to increasing the efficiency of antigen (Ag) delivery for antitumor vaccines. We optimized a polymer preparation method to deliver both defined tumor-associated proteins and the complex mixtures of tumor Ags present in tumors. Tumor Ags were encapsulated in a biodegradable, 50:50 poly(D,L-lactide co-glycolide) copolymer (PLGA) by emulsification and solvent extraction. Two particular Ags were studied, gp100 (a melanoma-associated antigen) and ovalbumin (OVA), as well as mixtures of proteins and lysates of tumor cells. The efficiency of encapsulation was measured by protein assays of dissolved nanoparticles. Ag stability after release from nanoparticles was verified by SDS-acrylamide gel electrophoresis and Western blot analysis. Molecular weight and protein loading interact to define the encapsulation efficiency and release rate of nanoparticles formulated from 50:50 PLGA. A midrange molecular weight polymer had more desirable release properties at 100 mg/mL than at 50 mg/mL protein loading, indicating the need for optimization of nanoparticle formulation for preparations with different particle loadings. Mixtures of proteins derived from cell lysates were reliably encapsulated into nanoparticles, which released the spectrum of proteins contained in lysates. Antigenic proteins were co-encapsulated with cell lysate and released from nanoparticles; these Ags retained their antigenicity and functioned better than soluble Ags when tested in in vitro assays of T cell cytokine formation and in vivo tumor vaccination challenge.

Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus.

Nanoparticles larger than the reported mesh-pore size range (10-200 nm) in mucus have been thought to be much too large to undergo rapid diffusional transport through mucus barriers. However, large nanoparticles are preferred for higher drug encapsulation efficiency and the ability to provide sustained delivery of a wider array of drugs. We used high-speed multiple-particle tracking to quantify transport rates of individual polymeric particles of various sizes and surface chemistries in samples of fresh human cervicovaginal mucus. Both the mucin concentration and viscoelastic properties of these cervicovaginal samples are similar to those in many other human mucus secretions. Unexpectedly, we found that large nanoparticles, 500 and 200 nm in diameter, if coated with polyethylene glycol, diffused through mucus with an effective diffusion coefficient (D(eff)) only 4- and 6-fold lower than that for the same particles in water (at time scale tau = 1 s). In contrast, for smaller but otherwise identical 100-nm coated particles, D(eff) was 200-fold lower in mucus than in water. For uncoated particles 100-500 nm in diameter, D(eff) was 2,400- to 40,000-fold lower in mucus than in water. Much larger fractions of the 100-nm particles were immobilized or otherwise hindered by mucus than the large 200- to 500-nm particles. Thus, in contrast to the prevailing belief, these results demonstrate that large nanoparticles, if properly coated, can rapidly penetrate physiological human mucus, and they offer the prospect that large nanoparticles can be used for mucosal drug delivery.