In Memoriam

Abstract

Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) spleens immunized in vitro and exposed in culture to different concentrations of copper chloride. The sections were immunized with dinitrophenyl-Ficoll and cultured in Eagle's minimum essential medium with 2% fetal calf serum; half of the medium was withdrawn and replaced every other day. The passive hemolytic plaque assay was used to determine the number of antibody-producing cells 10 d after injection. In the sections cultured with the high copper concentration (100 μg/mL), all cells died; at copper concentrations of 0.1–10 μg/mL, leukocytes remained viable, but fewer antibody-producing cells were present than in organ sections cultured in medium without copper. This in vitro method reduces the number of animals needed and the length of time required to determine toxicity and immunosuppression, and it provides information on the effects of certain environmental pollutants on fish.     

Salmonid B lymphocytes demonstrate organ dependent functional heterogeneity

The passive hemolytic plaque assay was used to examine the functional heterogeneity of antibody producing cells in salmonid immune organs. In this study, the antibody response to Vibrio anguillarum antigens was induced by the injection of a somatic antigen extract. This antigen was also coated onto sheep red blood cells (SRBC) for plaque forming cell (PFC) determination. Previous studies have demonstrated that this response is antibody dependent and antigen specific (Kaattari and Irwin, 1985). The present study was focused upon the heterogeneity of antibody producing cells that arise in the spleen, anterior and posterior kidney of immunized coho salmon (Oncorhynchus kisutch). The functional heterogeneity of lymphocytes was assessed by histogram analysis of the antigen inhibition profiles of the plaque forming responses. These analyses have revealed that the anterior kidney lymphocytes possess a much more restricted profile of antibody specificities than do lymphocytes from the posterior kidney or spleen. These data suggest that B cell repetoires differ among the immune organs of salmonids.     

Toxicity of Haemophilus pleuropneumoniae to porcine lung macrophages

Viable Haemophilus pleuropneumoniae bacteria were toxic for porcine alveolar macrophages in vitro. This cytotoxic effect proved to be dose-related. A cell-free extract of H. pleuropneumoniae, heat-killed bacteria, and a Pasteurella multocida field strain were nontoxic. When macrophages were cultured with H. pleuropneumoniae bacteria in a ratio of 100 macrophages to six bacteria, ultrastructural signs of cellular degeneration were observed within 1 h. This degeneration was observed in macrophages with or without phagosomes containing H. pleuropneumoniae. A cytotoxic substance was filtered from a H. pleuropneumoniae culture in Eagle's minimal essential medium supplemented with Earle's salts (EMEM) and 10% foetal calf serum that was incubated for 10 h at 37 degrees C. This substance was destroyed by heating at 65 degrees C for 30 min. Macrophages were less susceptible to the toxic effect of H. pleuropneumoniae when serum of convalescent pigs was added.     

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.     

Co-culture of ovine ova with oviductal cells in medium 199

Three experiments were conducted to test the suitability of medium 199 supplemented with 10% fetal calf serum (M199FCS) as a medium for co-culture of one-cell sheep ova with sheep oviductal cells. In Exp. 1, ova were co-cultured for 5 d in 5 ml of M199FCS or in Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS). Co-culture did not increase the number of cleavages at the end of 5 d of culture, but M199FCS supported more cleavages than did F10FCS (P = .016). In Exp. 2, ova were cultured for 1 to 3 d in M199FCS alone or on oviductal, uterine or kidney cell monolayers from ewes 2 d postestrus and transferred to recipients from which they were recovered at 8 d postestrus. Co-culture with oviductal cells improved (P less than .001) the cleavage index of recovered embryos compared with culture in medium alone or co-culture with other cell types. In Exp. 3, monolayers of oviductal cells from ewes 2 d postestrus and from luteal-phase ewes were cultured as in Exp. 2. No difference was observed between the two sources of oviductal cells for their ability to support in vitro development of one-cell sheep eggs for 1 or 2 d. These studies suggest that M199FCS may be a good medium to use in an oviductal cell co-culture system for one-cell sheep ova. Results further suggest that specific secretions of oviductal cells may be important for early embryo development in vivo.     

Viral-antigen dependence and T-cell receptor expression in leucocytes from rhabdovirus immunized trout

This work describes the characterization of trout haematopoietic in vitro long-term cell cultures showing specific viral antigen-dependent cell (ADC) proliferation. The ADC cultures were developed from outbreed trout after surviving viral hemorrhagic septicaemia virus (VHSV) infections or after immunization with purified VHSV. For in vitro long-term proliferation of the ADC cultures, adherent (Ad) cells obtained from autologous trout were pulsed with VHSV recombinant glycoprotein G4 (G4-pulsed Ad cells) and added periodically to the cultures. ADC did not proliferate in cultures obtained from non-infected control trout treated in parallel with G4 or from VHSV survivor/VHSV immunized trout kidney donors treated with non-viral proteins. After months in culture, the ADC acquired an increasingly homogeneous morphology compatible with that of mature trout lymphocytes, secreted supernatant 'factors', and were stained with rabbit antibodies to the ectodomain of recombinant trout T-cell receptor (TcR) beta-chain. Together with all the above mentioned properties, the presence of TcR sequences in the ADC cultures confirmed by the expression of alpha- and beta-chain TcR by nested PCR amplification and sequencing of the amplified bands, suggests that these ADC cultures contain trout T-like cells engaged in a VHSV response. These trout ADC cultures offer a first opportunity to further analyze fish anti-viral immunological responses.     

Primary in vitro stimulation of antibody production by rainbow trout lymphocytes

Trinitrophenylated (TNP) forms of E. coli lipopolysaccharide (LPS) and keyhole limpet hemocyanin (KLH) were used to produce antigen specific plaque-forming cell (PFC) responses with rainbow trout (Salmo gairdneri) splenocytes from unprimed fish in vitro. The culture system that was developed is described and characterized with respect to the kinetics and dose responses for both the haptenated and unhaptenated forms of the carriers. The induction of the PFC response to TNP-LPS was inhibited with TNP-lysine. Exposure to graded levels of gamma-radiation demonstrated a low dose augmentation of the PFC response with both antigens. Antigen addition experiments reveal that both antigens appear to stimulate the same population of antibody-producing B lymphocytes.     

Influence of HMB (beta-hydroxy-beta-methylbutyrate) on antibody secreting cells (ASC) after in vitro and in vivo immunization with the anti-Yersinia ruckeri vaccine of rainbow trout (Oncorhynchus mykiss)

In practice, protection of fish against disease by immunization is of limited effectiveness. Therefore, research is concentrated on how to improve the potency and efficacy of vaccines and how to optimally activate the cell-mediated immunity and the specific antibody response. In the present study, the influence of HMB (beta-Hydroxy-beta-methylbutyrate) on the antibody secreting cells (ASC) after both in vitro and in vivo immunization of rainbow trout (Oncorhynchus mykiss) with the anti-yersiniosis vaccine was studied. For in vitro immunization, the spleens from 160 fish were sampled and placed each in 35 mm sterile wells with medium containing HMB at concentrations of 0, 0.1, 1, 5, 10, 25, 50 or 100 microg/mL of medium. The spleens from 80 fish were injected with the vaccine and incubated at 14 degrees C for 10 days. For the in vivo study, fish were fed pellets containing HMB at doses of 0, 10, 25 and 50 mg/kg bw per day. After 2 weeks of HMB supplementation, the fish were immunized by intraperitoneal injection of the vaccine. At 7, 14, 18, 21, 28 and 35 days after immunization, pronephros were taken from 10 fish in each group for testing. When analyzed by the ELISPOT assay, HMB increased the number of splenic ASC after in vitro immunization at concentrations between 10 and 100 microg/mL (P < 0.05). Dietary HMB also increased the number of total and specific ASC when the fish were vaccinated in vivo. In conclusion, the results of the present study showed that HMB increases the levels of specific ASC after both in vitro and in vivo immunization of rainbow trout with the anti-Yersinia ruckeri vaccine.     

Primary in vitro stimulation of antibody production by lizard splenocytes

Single-cell suspensions of adult lizard (Chalcides ocellatus) spleen have been induced, in vitro, to produce a primary immune response. Using rat red cells (RRBC) as antigen and the culture conditions normally used in most vertebrate species but new for reptilia, it has been found that, in vitro at 37 degrees C, lizard spleen cells produce an antibody-forming response optimal at day 10. The response depends on the number of cultured cells and the dose of antigen, and parallels that obtained in vivo. Leibovitz (L-15) medium supplemented with 10% normal adult lizard serum was a satisfactory culture medium. 2-mercaptoethanol (2-ME), an ingredient used in mammalian cell culture, enhanced antibody production in lizard cells.     

Evaluation of different media and a BGM cell culture assay for isolation of Borrelia burgdorferi sensu lato from ticks and dogs

A co-culture assay for isolation of Borrelia burgdorferi sensu lato (sl.) from naturally infected ticks and dogs suspected of Lyme borreliosis (LB) was evaluated using buffalo-green-monkey (BGM) cells as the mammalian component. Four different media were tested for their ability to provide sufficient growth conditions for spirochetes and BGM cells. A total of 176 Ixodes ricinus ticks and 268 specimens from 98 dogs were used to compare cell-free culture with the BGM co-culture. A 1:1 mixture of Barbour-Stoenner-Kelly medium (BSK) and Eagle's minimum essential medium (EMEM) supported the growth of the two test strains, B. burgdorferi sensu stricto B31 and B. valaisiana VS116 to the same extent as BSK medium and the growth as well as the viability of BGM cells in this medium were the same as in EMEM. Using the 1:1 mixture of BSK and EMEM, borrelial growth measured in co-culture with BGM cells did not differ significantly from corresponding values obtained in cell-free cultures. In cell-free culture the isolation rate of B. burgdorferi sl. from ticks was significantly higher in BSK/EMEM 1:1 than in BSK medium (P < 0.01). Co-culture with BGM cells had no significant influence on the isolation rate of borreliae from ticks. However, a significant amount of isolates were obtained by one of the procedures only. Analysing canine specimens accordingly, spirochetes were grown from the blood of one dog after four weeks in BGM cell co-culture. The isolate was classified as B. afzelii by PCR-coupled restriction fragment length polymorphism analysis.     

鸡胚胎干细胞及其应用研究进展

鸡胚胎干细胞是一种多能性干细胞,从X期胚盘分离胚盘细胞或早期鸡胚的生殖嵴分离原始生殖细胞,经体外长期抑制分化培养可得到鸡胚胎干细胞。为维持细胞在培养过程中的未分化状态,需要采用饲养层细胞培养,同时设计合理的培养液配方并添加多种抑制分化或促进增殖的细胞因子。通过碱性磷酸酶活性检测、胚胎表面特异性抗原检测、分化试验及嵌合体试验等方法,可对鸡胚胎干细胞进行准确鉴定。文章主要就鸡胚胎干细胞的分离、培养与鉴定方法的研究进展及其应用前景进行简要综述,为进一步发展更高效的鸡胚胎干细胞培养体系并应用于生产实践提供一定的借鉴。     

Cellular immunity in salmonids infected with the microsporidial parasite Loma salmonae or exposed to non-viable spores

Following a per os challenge of naive rainbow trout with live spores of Loma salmonae, head kidney mononuclear cells (MNC) in culture were able to proliferate in response to crude soluble parasite extract or intact dead spores. A significant response was seen by week 2 post-exposure and a maximum response developed by week 6 or 8, respectively. During this initial challenge, spore filled cysts developed on the gills of challenged fish, and the cysts ruptured by week 12 as is typical for microsporidial gill disease of salmonids (MGDS). Two weeks following this, fish were re-challenged with live spores, and in these fish an enhanced in vitro proliferative response of MNC was immediately apparent, and spore filled cysts did not develop. In contrast, when naive trout were given dead spores by intraperitoneal injection, the most pronounced proliferative responses of MNC developed earlier (week 2 PE) and the response was greater when cells were incubated in vitro with dead spores rather than with crude soluble extract. When these fish were re-challenged per os with live spores, a heightened proliferation in MNC was observed 4 weeks after this exposure and the fish likewise resisted development of xenomas. In fish infected orally or injected intraperitoneally with spores, a marked increase in the response to the mitogen concanavalin A was seen for 22 weeks post-exposure when compared to controls not receiving any spores.     

Antigen recognition by rainbow trout (Oncorhynchus mykiss) of whole cell proteins expressed by Lactococcus garvieae when obtained directly from fish and under iron limited culture conditions

Infection by Lactococcus garvieae has become a widely recognised problem associated with intensively cultured fish. Long-term control of fish infections may be possible by vaccination providing a suitable and efficacious epitope is expressed during production of cells used for vaccine preparation. The identification of novel vaccine candidates must, therefore, consider how the host species recognises and responds to bacterial cell components. L. garvieae was cultured in iron deficient, limited and haem iron enriched media and the whole cell proteins expressed under these conditions were compared with those expressed in bacteria extracted with Percoll gradients directly from spleen tissue of infected rainbow trout (Oncorhynchus mykiss). SDS-PAGE of the cell proteins showed the existence of several different electropherotypes according to the iron status of the culture media. Only minor differences in cell protein profile were detected in bacteria obtained directly from fish spleens, but when the electropherograms were analysed by Western blots using L. garvieae hyperimmune fish sera, several proteins could be identified that were expressed only when L. garvieae was growing in vivo. Siderophore could be detected in culture supernatant of iron deficient, limited and haem iron enriched media but not in media with higher nutrient concentrations. The siderophore could not be identified as a type of catechol or hydroxymate. Rainbow trout recognise proteins in the range of approximately 50-80 kDa for bacterial cells obtained without subculture from infected fish and culture conditions can influence protein profiles for this pathogen.     

The in vitro effect of bovine lactoferrin on the activity of organ leukocytes in rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis)

Lactoferrin (LF) is a glycoprotein found in milk, neutrophil granules, secretions and selected organs of mammals. Lactoferrin exhibits antibacterial, antiviral, fungicidal, immunoregulatory and other functions. Although fish are devoid of this protein and its cell receptors, LF effect on the immune mechanisms of fish has been demonstrated. The objective of this study was to investigate the effect of bovine lactoferrin, applied in vitro, on the activity of head kidney and spleen leukocytes in three freshwater fish species: rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis). The obtained results validate LF beneficial effect on the respiratory burst of phagocytes in rainbow trout and wels catfish despite the fact that the potential killing activity against Aeromonas hydrophila was not stimulated in any of the studied species. Bovine lactoferrin enhanced the proliferation of T-lymphocytes in rainbow trout and European eel, as well as of B-lymphocytes in rainbow trout.     

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.     

A passage and storage system for isolated bovine endometrial epithelial and stromal cells

To establish a storage system for isolated endometrial cells, we investigated the basal, oxytocin (OT)- and tumor necrosis factor (TNF) alpha-stimulated production of prostaglandin (PG) F(2alpha) in bovine-passaged and frozen-thawed endometrial cells. Stromal and epithelial cells obtained from cows in the early stage of the estrous cycle (Days 2-5) were frozen at -80 C or further cultured and/or passaged until passage 4 in DMEM/Ham's F-12 supplemented with 10% calf serum. A fresh-unfrozen primary culture and one-time passaged fresh-unfrozen cells were used as the control. When both unfrozen and frozen cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and the cells were stimulated with OT (100 ng/ml) or TNFalpha (1 ng/ml) for 4 h. The passage and freezing of the endometrial cells did not affect their morphology. In primary culture of frozen and unfrozen endometrial cells, OT strongly stimulated PGF(2alpha) production in epithelial cells, and TNFalpha strongly stimulated PGF(2alpha) production in stromal cells (P<0.05). The basal output of PGF(2alpha) in frozen stromal cells was similar to that in unfrozen stromal cells. However, the basal output of PGF(2alpha) in frozen epithelial cells was significantly lower than that unfrozen cells (P<0.05). On the other hand, in passaged cells, the basal level of PGF(2alpha) production was retained until passage 1 in epithelial cells, whereas it was retained until passage 4 in stromal cells. Although epithelial cells responded to OT in PGF(2alpha) production until passage 2 (P<0.05), the stromal cells showed a significant response to TNFalpha until passage 4 (P<0.05). These results suggest that stored cells could be used for studying the physiology of bovine endometrium in vitro until passage 1 in endometrial epithelial cells, and until passage 4 in stromal cells.     

In vitro evaluation of porcine lymphocyte response to phytohaemagglutinin using a modified “whole blood” technique

The purpose of these investigations was to develop a modified whole blood technique for measuring quantitatively the responsiveness of pig peripheral blood lymphocytes to phytohaemagglutinin (PHA) in vitro. Washed blood cells from a fixed volume of blood were suspended in culture medium supplemented with foetal calf serum and stimulated with a pure mitogenic PHA preparation. The stimulation was measured by the incorporation of ³H-thymidine.Data are presented to show the effect of different variables on the culture system. The transformation response was measured at different levels of PHA and the reproducibility of the different responses from repeated investigations was used to evaluate the usefulness of the test.The best reproducibilities of the stimulation response were found at the high PHA concentrations.Also the calculated PHA concentration giving maximum stimulation response had a relatively high reproducibility and indicates that this value is a convenient and reliable alternative measure of the functional capacity of the lymphocytes.     

Yersinia ruckeri septicaemia in experimentally infected carp (Cyprinus carpio L.) fingerlings.

The presence of Yersinia ruckeri, the causal agent of enteric redmouth disease (ERM) in salmonids and a few other freshwater fish, has so far been reported from a variety of sources including the intestine of healthy carp. Since there are no data on the pathogenicity of this bacterium for carp, 15 fingerlings were experimentally infected by intraperitoneal injection of about 5 x 10(5) cells. Thirteen injected fish were moribund or died within 4 days with septicaemic lesions. Two survivors were sampled on Day 28 after infection. Yersinia ruckeri was reisolated from the internal organs of all experimental fish. By histopathological examination moribund fish had generalised bacteriaemia with inflammation, degeneration and necrotic foci in kidney, liver and spleen, corresponding to findings described previously in ERM of rainbow trout. Survivors of challenge on Day 28 had a chronic disease characterised by prominent peritonitis and enteritis, exhaustion of the erythroid, granuloid and lymphoid components in haematopoietic kidney tissue as well as focal degeneration and necrosis in organs. These data indicate a high sensitivity of carp to intraperitoneal infection with a relatively low dose of Y. ruckeri.     

In vitro effects of beta-hydroxy-beta-methylbutyrate (HMB) on cell-mediated immunity in fish

beta-Hydroxy-beta-methyl butyrate(HMB) has been shown to counteract many of the negative effects of intensive animal production methods and results in increased growth and protection against diseases. In the present study, the effect of HMB on the immunocompetence cell activity in rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio) was examined. Pronephric phagocytes and lymphocytes were isolated from the fish and grown in culture medium (RPMI-1640) containing either 0, 0.1, 1, 5, 10, 25, 50 or 100 microg HMB/ml of medium. The effects of HMB on the respiratory burst activity (RBA) stimulated by phorbol myristate acetate (PMA), the potential killing activity (PKA) and lymphocyte proliferation stimulated by either concanavalin A (Con-A) or lipopolysaccharide (LPS) were examined. The addition of HMB to the culture medium increased the RBA by up to 84% (p<0.01) over that of cells grown without HMB. Similarly, the PKA of the phagocytes was also increased with HMB addition to the medium by up to 140% (p<0.01) over that of cells grown without HMB. Lymphocyte proliferation stimulated by both ConA and LPS was also increased approximately two-fold (p<0.01) when HMB was added to the culture medium at concentrations between 10 and 100 microg HMB/ml in both rainbow trout and carp. The greatest effects of HMB on RBA and PKA activities were observed at a concentration >50 microg HMB/ml while lymphocyte proliferation was maximally stimulated at 25 microg HMB/ml. In conclusion, the current study shows that HMB could potentially improve immunocompetence cell activity in fish through increased cell proliferation and functionality.