Induction of Human Wharton's Jelly of Umbilical Cord Derived Mesenchymal Stem Cells to Be Chondrocytes and Transplantation in Guinea Pig Model with Spontaneous Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease commonly found in elderly people and obese patients. Currently, OA treatments are determined based on their condition severity and a medical professional's advice. The aim of this study was to differentiate human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) into chondrocytes for transplantation in OA-suffering guinea pigs. hWJ-MSCs were isolated using the explant culture method, and then, their proliferation, phenotypes, and differentiation ability were evaluated. Subsequently, hWJ-MSCs-derived chondrocytes were induced and characterized based on immunofluorescent staining, qPCR, and immunoblotting techniques. Then, early-OA-suffering guinea pigs were injected with hyaluronic acid (HA) containing either MSCs or 14-day-old hWJ-MSCs-derived chondrocytes. Results showed that hWJ-MSCs-derived chondrocytes expressed specific markers of chondrocytes including Aggrecan, type II collagen, and type X collagen proteins and ß-catenin, Sox9, Runx2, Col2a1, Col10a1, and ACAN gene expression markers. Administration of HA plus hWJ-MSCs-derived chondrocytes (HA-CHON) produced a better recovery rate of degenerative cartilages than HA plus MSCs or only HA. Histological assessments demonstrated no significant difference in Mankin's scores of recovered cartilages between HA-CHON-treated guinea pigs and normal articular cartilage guinea pigs. Transplantation of hWJ-MSCs-derived chondrocytes was more effective than undifferentiated hWJ-MSCs or hyaluronic acid for OA treatment in guinea pigs. This study provides a promising treatment to be used in early OA patients to promote recovery and prevent disease progression to severe osteoarthritis.

Cryostorage of silver barb (Barbodes gonionotus) semen in dry shipper: Efficacy, risk of bacterial cross-contamination and effects of Aeromonas hydrophila on post-thaw sperm quality.

This study was conducted to investigate the efficacy of dry shipper for the cryostorage of silver barb (Barbodes gonionotus) sperm, the subsequent risk of bacterial cross-contamination, and the effects of Aeromonas hydrophila on post-thaw sperm. Semen was diluted with calcium-free Hank's balanced salt solution containing 10% ME2SO, frozen at -8 °C/min and stored for 14 d in a dry shipper. A significant decline (P < 0.05) in the post-thaw sperm motility and viability of samples kept in the dry shipper for 14 d showed a reverse correlation (P < 0.05) with a slight increase in temperature within the dry shipper. The levels of contaminated bacteria in the compartments of the dry shipper were significantly (P < 0.05) lower than those detected in the liquid nitrogen tank. Bacteria from the atmosphere could recontaminate the chambers of the dry shipper and liquid nitrogen tank after 14 d. Bacillus was the most common bacteria isolated from the dry shipper, liquid nitrogen tank, circulating air, bench surface and outer surface of straws. There was no cross-contamination of A. hydrophila from contaminated straws to pathogen-free straws kept in either cryogenic tank. Post-thaw sperm motility and sperm viability significantly (P < 0.05) declined during cryostorage in the dry shipper and liquid nitrogen tank due to the introduction of A. hydrophila and the interaction effect of A. hydrophila and freezing. This study reports, for the first time, the efficacy of a dry shipper for the cryostorage of fish sperm for at least 14 d without a risk of bacterial cross-contamination.

Retail Baitfish in Michigan Harbor Serious Fish Viral Pathogens.

Indigenous small cyprinid fish species play an important role in Great Lakes ecosystems and also comprise the backbone of a multimillion-dollar baitfish industry. Due to their widespread use in sport fisheries of the Laurentian Great Lakes, there are increasing concerns that baitfish may introduce or disseminate fish pathogens. In this study, we evaluated whether baitfish purchased from 78 randomly selected retail bait dealers in Michigan harbored fish viruses. Between September 2015 and June 2016, 5,400 baitfish divided into 90 lots of 60 fish were purchased. Fish were tested for the presence of viral hemorrhagic septicemia virus (VHSV), spring viremia of carp virus (SVCV), golden shiner reovirus (GSRV), fathead minnow nidovirus (FHMNV), fathead minnow picornavirus (FHMPV), and white sucker bunyavirus (WSBV). Using the epithelioma papulosum cyprini cell line and molecular confirmation, we demonstrated the presence of viruses in 18 of the 90 fish lots (20.0%) analyzed. The most prevalent virus was FHMNV, being detected in 6 of 30 lots of Fathead Minnow Pimephales promelas and 3 of 42 lots of Emerald Shiners Notropis atherinoides. We also confirmed GSRV in two fish species: the Golden Shiner Notemigonus crysoleucas (5 of 11 lots) and Fathead Minnow (3 of 30 lots). Two VHSV (genotype IVb) isolates were recovered from a single lot of Emerald Shiners. No SVCV, FHMPV, or WSBV was detected in any of the fish examined. Some of the infected fish exhibited clinical signs and histopathological alterations. This study demonstrates that live baitfish are a potential vector for the spread of viral pathogens and underscores the importance of fish health certifications for the Great Lakes baitfish industry.

Laboratory investigation into the role of largemouth bass virus (Ranavirus, Iridoviridae) in smallmouth bass mortality events in Pennsylvania rivers.

BACKGROUND: Mortality episodes have affected young-of-year smallmouth bass (Micropterus dolomieu) in several river systems in Pennsylvania since 2005. A series of laboratory experiments were performed to determine the potential role of largemouth bass virus (Ranavirus, Iridoviridae) in causing these events. RESULTS: Juvenile smallmouth bass experimentally infected with the largemouth bass virus exhibited internal and external clinical signs and mortality consistent with those observed during die-offs. Microscopically, infected fish developed multifocal necrosis in the mesenteric fat, liver, spleen and kidneys. Fish challenged by immersion also developed severe ulcerative dermatitis and necrotizing myositis and rarely panuveitis and keratitis. Largemouth bass virus-challenged smallmouth bass experienced greater mortality at 28 °C than at 23 or 11 °C. Co-infection with Flavobacterium columnare at 28 °C resulted in significant increase in mortality of smallmouth bass previously infected with largemouth bass virus. Aeromonas salmonicida seems to be very pathogenic to fish at water temperatures < 23 °C. While co-infection of smallmouth bass by both A. salmonicida and largemouth bass virus can be devastating to juvenile smallmouth bass, the optimal temperatures of each pathogen are 7-10 °C apart, making their synergistic effects highly unlikely under field conditions. CONCLUSIONS: The sum of our data generated in this study suggests that largemouth bass virus can be the causative agent of young-of-year smallmouth bass mortality episodes observed at relatively high water temperature.

In vitro inoculation of Aeromonas hydrophila and Pseudomonas fluorescens in cryopreserved silver barb (Barbodes gonionotus) milt: Effect on fertilization capacity and transmission potential to embryos.

Purposive use of cryopreserved sperm contaminated with pathogenic agents has increased the risk of spreading of fish diseases. The objective of this study was to investigate the effects of A. hydrophila subsp. hydrophila and P. fluorescens inoculations into cryostored milt on fertilization capacity and transmission potential to embryos of silver barb (Barbodes gonionotus) with or without 0.25% penicillin-streptomycin (PS) administration. The experiment comprised six treatments: addition of milt into T1) extender only, T2) extender with 0.25% PS, T3) extender with A. hydrophila subsp. hydrophila (BG19), T4) extender with A. hydrophila subsp. hydrophila (BG19) and 0.25% PS, T5) extender with P. fluorescens (BG20) and T6) extender with P. fluorescens (BG20) and 0.25% PS. Milt were loaded into 0.25-mL straws and cryostored in the controlled-rate programmable freezer. After a cryostorage for 28 d, post-thawed sperm were evaluated for the fertilization capacity and risk of pathogen transmission to embryos. Inoculation of A. hydrophila subsp. hydrophila and P. fluorescens into extended milt (T3 and T5) caused a reduction (P < 0.05) in fertilization capacity of cryopreserved sperm. Cryopreserved sperm inoculated with the two pathogenic bacteria and 0.25% PS (T4 and T6) did not fertilize the eggs. The two pathogenic bacteria could be transmitted into embryos after artificial insemination of eggs with bacterial-inoculated cryopreserved sperm, suggesting that the risks of disease transmission via cryopreserved fish sperm would exist. This is the first study reporting pathogenic bacterial transmission on in vitro fish embryos through artificial insemination of cryopreserved sperm.

The Asian fish tapeworm Schyzocotyle acheilognathi is widespread in baitfish retail stores in Michigan, USA.

BACKGROUND: The Asian fish tapeworm Schyzocotyle acheilognathi (Yamaguti, 1934) is an important fish pathogen because of its wide range of intermediate and definitive hosts and its pathological consequences. This study was designed to determine if baitfish are a likely vector contributing to the expansion of the invasive Asian fish tapeworm. RESULTS: We collected live baitfish for examination from 78 retail stores in Michigan between September 2015 and June 2016. A total of 5400 baitfish (90 lots, 60 fish/lot) were examined, including 42 emerald shiners [Notropis atherinoides (Rafinesque, 1818)] lots, 30 fathead minnow [Pimephales promelas (Rafinesque, 1820)] lots, 11 golden shiners [Notemigonus crysoleucas (Mitchill, 1814)] lots, 3 sand shiners [Notropis stramineus (Cope, 1865)] lots, 1 lot each of spottail shiners [Notropis hudsonius (Clinton, 1824)], Northern redbelly dace [Phoxinus eos (Cope, 1861)], and blacknose dace [Rhinichthys atratulus (Hermann, 1804)] and 1 lot of mixed two species: weed shiners [Notropis texanus (Girard, 1856)] and sand shiners. CONCLUSIONS: Based on its scolex and strobilar morphology combined with gene sequence analysis, S. acheilognathi was only found in emerald shiners, golden shiners and sand shiners. The mean within lot prevalence and abundance of infection was highest in emerald shiners (20.3 ± 14.0 and 1.15 ± 1.34), followed by golden shiners (8.3 ± 10.7 and 0.89 ± 1.27) and sand shiners (1.3 ± 2.6 and 0.02 ± 0.05). However, the mean intensity of S. acheilognathi in emerald shiners was lower (4.3 ± 2.6) than that of golden shiners (6.6 ± 6.7). S. acheilognathi-infected fish exhibited enlargement of the abdomen, distension of the intestinal wall, and intestinal occlusion and hemorrhage. This finding suggests that live baitfish are a likely vector by which the invasive Asian tapeworm's range is expanding.

Susceptibility of Representative Great Lakes Fish Species to the North Carolina Strain of Spring Viremia of Carp Virus (SVCV).

Spring viremia of carp virus (SVCV) is a notifiable pathogen of the World Organization of Animal Health. Since SVCV was isolated in Lake Ontario in 2007, concern has grown about its spread in the Great Lakes basin and its potential negative impacts on fish species of importance in stock enhancement programs basinwide. The susceptibility of representative fish species from the families Cyprinidae (Fathead Minnow Pimephales promelas, Golden Shiner Notemigonus crysoleucas, Spotfin Shiner Cyprinella spiloptera, and Creek Chub Semotilus atromaculatus), Centrarchidae (Largemouth Bass Micropterus salmoides), Percidae (Walleye Sander vitreus), Salmonidae (Rainbow Trout Oncorhynchus mykiss), and Esocidae (Muskellunge Esox masquinongy) to SVCV was evaluated by experimental infection under laboratory conditions. Morbidity and mortality were recorded, and virus re-isolation, seminested reverse transcription PCR, and histopathological assessments were performed. Using intraperitoneal (i.p.) injection, Fathead Minnows and Golden Shiners were highly susceptible to SVCV (40-70% mortality). All dead or moribund and apparently healthy surviving Fathead Minnows and Golden Shiners were SVCV positive. The SVCV was also detected in challenged but healthy Spotfin Shiners (30%) and Creek Chub (5%). However, noncyprinid species exhibited no morbidity or mortality and were free of SVCV following an observation period of 30 d. In a follow-up experimental challenge, Fathead Minnows and Golden Shiners were SVCV challenged at 103 and 105 PFU/mL by means of waterborne immersion. After immersion, Fathead Minnows and Golden Shiners exhibited characteristic SVCV disease signs, but mortality was less (30% and 10% mortality, respectively) than that in fish with i.p. injections. The SVCV was detected in all mortalities and a subset of healthy Fathead Minnows and Golden Shiners. Necrotic changes were observed in the kidneys, liver, spleen, ovaries, and heart, and other histopathological lesions also occurred. These findings suggest that two of the four cyprinids tested are susceptible to SVCV-induced disease and that all four can act as potential carriers of SVCV in the Laurentian Great Lakes. Received January 11, 2017; accepted July 17, 2017.

Influence of Aeromonas hydrophila and Pseudomonas fluorescens on motility, viability and morphometry of cryostored silver barb (Barbodes gonionotus) sperm.

This objective of the study was to evaluate the effects of A. hydrophila subsp. hydrophila and P. fluorescens on sperm motility, sperm viability and sperm morphometry of cryopreserved silver barb (Barbodes gonionotus) semen and survival of tested bacteria after cryostorage. Semen was diluted in a calcium-free Hank's balanced salt solution (Ca-F HBSS) supplemented with or without 0.25% penicillin-streptomycin (PS) after which A. hydrophila subsp. hydrophila or P. fluorescens was immediately added into extended semen prior to freezing. Extended semen and cryostored semen kept for 20 min, 24 h, 7 d, 14 d and 28 d were assessed for sperm motility, sperm viability, sperm morphometry, survival of challenged bacteria and the relationship between bacteria and sperm. Bacterial-exposed semen with or without 0.25% PS supplementation showed a significant reduction (P < 0.05) in sperm motility and viability during a cryostorage of 28 d, compared to semen without bacterial supplementation (control groups). Addition of A. hydrophila subsp. hydrophila and P. fluorescens resulted in a significant (P < 0.05) alteration of sperm morphometry of cryopreserved semen, especially flagellum width. The two pathogens were detected at a level of 10(5) CFU ml(-1) in cryostored semen with or without antibiotic supplementation. There were significant correlations among bacterial number, percentage of sperm motility and viability and flagellum width. In conclusion, the presence of A. hydrophila subsp. hydrophila and P. fluorescens had a deleterious effect on cryopreserved silver barb sperm based on a reduction in sperm motility and viability and alteration of sperm morphometry, especially flagellum width.

Effect of antibiotic supplementation on the quality of cryopreserved fish sperm of silver barb (Barbodes gonionotus): Sperm motility and viability, bacterial quality and fertilization.

Disease prevention is a key aspect in developing cryopreservation procedures for fish sperm and in improving the reproductive biotechnology for commercially important aquatic species. The purpose of this study was to evaluate the effects of an antibiotic supplementation (0.25% penicillin-streptomycin, PS and 0.25% penicillin-gentamicin, PG) on sperm motility and viability, bacterial profile and fertilization capacity of cryopreserved silver barb (Barbodes gonionotus) semen. The experimental protocol involved three treatments: addition of PS alone; addition of PG alone; and no addition of antibiotics (Control). Semen samples were frozen and cryostored for 12-mo. Administration of 0.25% PS significantly (P<0.05) improved sperm motility and viability and reduced (P<0.05) total heterotrophic bacteria, Gram negative bacteria and pseudomonads bacteria in cryopreserved semen. Post-thawed semen treated with 0.25% PS did not contain contaminating bacteria including Bacillus subtilis subsp. inaquosorum, Bacillus safensis, Aeromonas punctata subsp. caviae, Serratia plymuthica, Pseudomonas azotoformans and Pseudomonas sp. Post-thawed semen supplemented with 0.25% PG showed degenerative changes in motility and viability of sperm. Eggs fertilized with 0.25% PS or antibiotic-free cryopreserved semen had similar fertilization rates, lower (P<0.05) than those of fresh semen. Incorporation of 0.25% PS was suitable for cryopreservation of silver barb semen based on the presence of good quality of post-thawed sperm and elimination of bacterial contaminants: B. subtilis subsp. inaquosorum, B. safensis, A. punctata subsp. caviae, Ser. plymuthica, P. azotoformans and Pseudomonas sp. This is the first study to investigate the antibiotic effect on the number of bacteria and their profile in cryopreserved semen of fish.

Semen collection methods affect the bacterial composition of post-thawed semen of silver barb (Barbodes gonionotus).

Biosafety issue associated with the risk of pathogenic contamination of cryopreserved semen is a common concern because of associated declines in sperm quality, storage period and disease transmission. This study was conducted to evaluate the effects of methods of semen collection on sperm quality and bacterial composition of post-thawed semen of silver barb (Barbodes gonionotus). Semen collection methods consisted of four treatments: (1) hand-stripping of abdomen without rinsing of urogenital area with water, (2) hand-stripping of abdomen after rinsing of urogenital area with water, (3) catheterization without rinsing of urogenital area with water and (4) catheterization after rinsing of urogenital area with water. Semen diluted with calcium-free Hank's balanced salt solution containing 10% dimethylsulfoxide (DMSO) was frozen at a freezing rate of -8°Cmin(-1) before plunging in liquid nitrogen. Post-thawed semen collected by catheterization after rinsing urogenital area had the lowest bacterial number, about 2-log reduction of total heterotrophic, Gram negative and pseudomonad bacteria, compared with the other three collection treatments. However, percentages of motile and viable sperm were not significantly (P>0.05) different among treatments. This method eliminated Flavobacterium aquatile, Bacillus megaterium, Kocuria varians, Staphylococcus haemolyticus and Aeromonas media in cryopreserved semen. This is the first report demonstrating the effects of semen collection methods on bacteriological quality of frozen-thawed fish semen.

Potential Bacillus probiotics enhance bacterial numbers, water quality and growth during early development of white shrimp (Litopenaeus vannamei).

Epidemics of epizootics and occurrence of multiresistant antibiotics of pathogenic bacteria in aquaculture have put forward a development of effective probiotics for the sustainable culture. This study examined the effectiveness of forms of mixed Bacillus probiotics (probiotic A and probiotic B) and mode of probiotic administration on growth, bacterial numbers and water quality during rearing of white shrimp (Litopenaeus vannamei) in two separated experiments: (1) larval stages and (2) postlarval (PL) stages. Forms of Bacillus probiotics and modes of probiotic administration did not affect growth and survival of larval to PL shrimp. The compositions of Bacillus species in probiotic A and probiotic B did not affect growth and survival of larvae. However, postlarvae treated with probiotic B exhibited higher (P<0.05) growth than probiotic A and controls, indicating Bacillus probiotic composition affects the growth of PL shrimp. Total heterotrophic bacteria and Bacillus numbers in larval and PL shrimp or culture water of the treated groups were higher (P<0.05) than in controls. Levels of pH, ammonia and nitrite of the treated shrimp were significantly decreased, compared to the controls. Microencapsulated Bacillus probiotic was effective for rearing of PL L. vannamei. This investigation showed that administration of mixed Bacillus probiotics significantly improved growth and survival of PL shrimp, increased beneficial bacteria in shrimp and culture water and enhanced water quality for the levels of pH, ammonia and nitrite of culture water.