Aquatic Parasite Cultures and Their Applications.

In this era of unprecedented growth in aquaculture and trade, aquatic parasite cultures are essential to better understand emerging diseases and their implications for human and animal health. Yet culturing parasites presents multiple challenges, arising from their complex, often multihost life cycles, multiple developmental stages, variable generation times and reproductive modes. Furthermore, the essential environmental requirements of most parasites remain enigmatic. Despite these inherent difficulties, in vivo and in vitro cultures are being developed for a small but growing number of aquatic pathogens. Expanding this resource will facilitate diagnostic capabilities and treatment trials, thus supporting the growth of sustainable aquatic commodities and communities.

Monogenean Parasite Cultures: Current Techniques and Recent Advances.

Global expansion in fish production and trade of aquatic ornamental species requires advances in aquatic animal health management. Aquatic parasite cultures permit diverse research opportunities to understand parasite-host dynamics and are essential to validate the efficacy of treatments that could reduce infections in captive populations. Monogeneans are important pathogenic parasites of captured captive fishes and exhibit a single-host life cycle, which makes them amenable to in vivo culture. Continuous cultures of oviparous monogenean parasites provide a valuable resource of eggs, oncomiracidia (larvae) and adult parasites for use in varied ecological and applied scientific research. For example, the parasite-host dynamics of Entobdella soleae (van Beneden and Hesse, 1864) and its fish host, Solea solea (Linnaeus, 1758), is one of the most well-documented of all monogeneans following meticulous, dedicated study. Polystoma spp. cultures provide an intriguing model for examining evolution in monogeneans because they exhibit two alternative phenotypes depending on the age of infection of amphibians. Furthermore, assessments of the ecological, pathological and immunological effects of fish parasites in aquaculture have been achieved through cultures of Gyrodactylus von Nordmann, 1832 spp., Benedenia seriolae (Yamaguti, 1934), Neobenedenia Yamaguti, 1963 spp. and Zeuxapta seriolae (Meserve, 1938). This review critically examines methods to establish and maintain in vivo monogenean monocultures on finfish, elasmobranchs and amphibians. Four separate approaches to establish cultures are scrutinised including the collection of live infected hosts, cohabiting recipient hosts with infected stock, cohabiting hosts with parasite eggs or oncomiracidia (larvae) and direct transfer of live adult parasites onto new fish hosts. Specific parasite species' biology and behaviour permits predictive collection of parasite life stages to effectively maintain a continuous culture, while environmental parameters can be altered to manipulate parasite generation time. Parasite virulence and biosecurity are vital components of a well-managed culture to ensure appropriate animal welfare and uncontaminated surrounding environments. Contemporary approaches and techniques are reviewed to ensure optimised monogenean cultures, which ultimately can be used to further our understanding of aquatic parasitology and identify mechanisms to limit infestations in public aquaria, ornamental trade and intensive aquaculture.

In vitro equine embryo production using air-dried spermatozoa, with different activation protocols and culture systems.

The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P < 0.05). No differences in embryo development were seen between the two activation treatments nor between storage periods (P > 0.05). Blastocysts were obtained with cooled spermatozoa, and morulae were achieved using in vivo culture with 28-day storage spermatozoa and ionomycin-activated oocytes. When in vivo culture was performed, sperm DNA fragmentation was assessed using the sperm chromatin dispersion test and did not show statistical correlation with cleavage nor embryo recovery rates. In conclusion, equine embryos can be produced using air-dried spermatozoa stored for several weeks. Sperm extract activation increased cleavage rates but did not improve embryo development. In vivo culture allowed intrauterine stage embryos to be achieved.

Population doublings of murine CD4(+) memory T cells during continuous antigen stimulation in vivo.

We investigated the expansion rate of CD4(+) memory T cells using a newly developed in vivo system. Neonatal thymectomy abrogates the subsequent production of T cells and induces autoimmune gastritis (AIG) by the activation of CD4(+) T cells; this disease was transferred into athymic nude mice through the inoculation of splenic CD4(+) memory T cells. The transferred CD4(+) T cells increased logarithmically in number during the first 2months in the spleen of the recipients. The serial transfer of these splenocytes at two-month intervals revealed that the numbers of the AIG-transferable generations were inversely correlated with the age of the first AIG donors. The duration of the AIG-promoting capacity of CD4(+) T cells under continuous antigenic stimulation in vivo was approximately equivalent-one and a half years. These results indicate that there exists an intrinsic population doubling limit in memory CD4(+) T cells similar to that of self-renewing naïve ones.

The ART of studying early embryo development: progress and challenges in ruminant embryo culture.

The study of preimplantation mammalian embryo development is challenging due to difficulties in accessing in vivo-derived embryos in large numbers at the early stages and the inability to culture embryos in vitro much beyond the blastocyst stage. Nonetheless, embryos exhibit an amazing plasticity and tolerance when it comes to adapting to the environment in which they are cultured. They are capable of developing in media ranging in composition from simple balanced salt solutions to complex systems involving serum and somatic cells. At least a proportion of the blastocysts that develop in culture are developmentally competent as evidenced by the fact that live offspring have resulted following transfer. However, several studies using animal models have shown that such embryos are sensitive to environmental conditions that can affect future pre- and post-natal growth and developmental potential. This review summarises some key aspects of early embryo development and the approaches taken to study this important window in early life.

The Characteristics of Limbal Epithelial Cells Auto-Cultivated In Vivo on Amniotic Membrane in Rabbits

PURPOSE: To investigate the characteristics of the limbal epithelial cells auto-cultivated in vivo on amniotic membrane (LIVAMs) designed for the treatment of limbal stem cell deficiency. METHODS: We removed the epithelium of AM with a No.15 blade after it was blotted with 20% ethanol and made a 360 degrees stromal flap along the epithelial defect. We then mounted over-sized AM (1 mm larger in diameter than the defect) over the defect with the border of AM inserted under the flap, and performed interrupted suture with 10-0 nylon. A therapeutic contact lens was fitted over the AM and a temporary tarsorrhaphy was performed. To examine whether the limbal epithelial cells grew well onto AM, we observed the cornea after fluorescein dye staining using a slit lamp. To explore the characteristics of LIVAMs, we performed hematoxylin-eosin (H&E) staining, immunochemical staining with AK-2, AE-5, AM-3 monoclonal antibodies, and transmission electron microscopy. RESULTS: Three of four rabbits had successful epithelial growth on the amniotic membrane. The epithelial growth on the amniotic membrane was stained using immunohistochemical staining (AK-2, AE-5). Electron microscopy showed a structure similar to that of a normal corneal epithelium. CONCLUSIONS: The technique of auto-cultivation of limbal epithelial cells in vivo on amniotic membrane can be an efficient and convenient method and preserves the characteristics of limbal epithelial cells for the treatment of limbal stem cell deficiency.

Study on Basic Culture Method of Tumor Chondrocytes / 대한정형외과학회잡지

In vivo and in vitro culture of rat tumor chondrocytes is a good culture model for physiologic, pathologic and biochemical studies of tumor chondrocytes. Human tumor chondrocyte culture is another method, but it is difficult to maintain a cell line and in vivo culture. So called, Swarm Rat Chondrosarcoma, which wss developed spontaneously in a Spra-gue-Dawley rat and maintained by Dr. R. Swarm, can be cultured in vivo and in vitro at the same time. It is easy to maintain Swarm Rat Chondrosarcoma and is said that there are nearly no notable changes in cellular characteristics during consecutive cultures. In this study, in order to establish the basic culture method of rat tumor chondrocytes, the inoculated tumor mass were studied with swarm rat chondrosarcoma cell line which had been preserved at Orthopedic Research Laboratory of the Msssachusetts General Hospital. In vivo culture, injection of digested 1×10(6) cells, 1×10(7) cells and direct inoculation of tumor of mass were done at each 10 Sprague-Dawley rats group and examination was done at postinoculation 2, 3, 4, 5, 6, 7 and 8 weeks. In vitro culture, 1×10(6)/ml, 2×10(6)/ml and 4×10(6)/ml concentration cell suspensions were plated at 96-well-plate and observed at 2, 3, 4 and S days with inverted microscope. The results of this study are as follows. 1. In vivo culture, the best result was observed at direct inoculation of tumor fragments among 1×10(6) cells, 1×10(7) cells and direct tumor fragments inoculation. 2. The ideal time of obtaining tumor mass growing in rsts is between 4 to 6 weeks after inoculation. 3. In vitro culture, the proper cell density in 96-well-plate was 1 x 10(6)/ml among 1×10(6)/ml, 2×10(6)/ml snd 4 × 10(6)/ml.

Autonomous yolk protein synthesis in ovaries ofDrosophila cultured in vivo.

Immature ovaries ofDrosophila mercatorum were injected into young larvae and into adult males ofD. mercatorum, D. melanogaster, D. hydei, D. virilis, andZaprionius vittiger. These homo- and heteroplastic transplantations allow normal vitellogenesis to occur in the donor ovary. By SDS gel electrophoresis, we identified the major species-specific yolk proteins of mature eggs (stage 14) which were exclusively of donor-specific origin. Other experiments withD. hydei andZ. vittiger showed that, when females were used as hosts, the host-specific yolk proteins became incorporated into the donor eggs. When two immature ovaries, one ofD. mercatorum and one ofD. hydei, were co-cultured in males, again only the donor-specific yolk proteins were found in the mature eggs implying that these yolk proteins were not released into the host hemolymph.A parthenogenetic strain ofD. mercatorum was used to demonstrate the ability of transplanted immature ovaries to produce viable eggs which can give rise to fertile adults.The role of the species-specific yolk proteins is discussed with respect to the dual origin of these proteins during normal vitellogenesis, i.e., an autonomous synthesis within the ovary itself in addition to the well-known production by the fat body. Further experiments with pupae as hosts indicate that even in the absence of juvenile hormone and in the presence of high doses of ecdysone, vitellogenesis can proceed within the donor ovary.Based on these experiments, a new hyopthesis on the hormonal control of vitellogenesis inDrosophila is presented. We propose that yolk proteins derived from the fat body are controlled by juvenile hormone, whereas the independent and autonomous vitellogenesis within the ovary itself is controlled by endogenously synthesized ecdysone.