The roles of haemocytes and the lymphoid organ in the clearance of injected Vibrio bacteria in Penaeus monodon shrimp.

In order to study the reaction of Penaeus monodon haemocytes, live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled. Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that large numbers of haemocytes encapsulated the bacteria at the site of injection. A rapid decrease of live circulating bacteria was detected in the haemolymph. Bacterial clearance in the haemolymph was induced by humoral factors, as observed by agglutinated bacteria, and followed by uptake in different places in the body. Bacteria mainly accumulated in the lymphoid organ (LO), where they, or their degradation products, could be detected for at least 7 days after injection. The LO consists of folded tubules with a central haemal lumen and a wall, layered with cells. The haemolymph, including the antigens, seemed to migrate from the central tubular lumen through the wall, where the bacteria are arrested and their degradation is started. Electron microscopy of the LO revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It is proposed that haemocytes settle in the tubule walls before they phagocytose. Immunostaining suggests that many of the haemocytes degranulate in the LO, producing a layer of fibrous material in the outer tubule wall. These findings might contribute to the reduced haemocyte concentration in the haemolymph of diseased animals or following injection of foreign material. It is proposed that the LO is a filter for virtually all foreign material encountered in the haemolymph. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessel in most decapod crustaceans that do not possess a LO. The experimental shrimp appeared to contain many LO spheroids, where bacterial antigens were finally observed as well. It is proposed that the spheroids have a degradation function for both bacterial and viral material, and that their presence is primarily related to the history of the infectious burden of the shrimp.

Immune modulation by fish kinetoplastid parasites: a role for nitric oxide.

Trypanoplasma borreli and Trypanosoma carassii are kinetoplastid parasites infecting cyprinid fish. We investigated the role of nitric oxide (NO) in immune modulation during T. borreli and T. carassii infection of carp. Phagocytic cells from different organs produced NO and serum nitrate levels increased, demonstrating that T. borreli activates NO production in vivo. In contrast, T. carassii did not induce NO production in vivo and inhibited LPS-induced NO production in vitro. Production of NO was detrimental to the host as T. borreli-infected carp treated with the inducible NO synthase inhibitor aminoguanidine had a higher survival than infected control carp. This detrimental effect can be explained (in part) by the toxicity of NO to cells in vitro as NO inhibited the proliferative response of blood and spleen leukocytes. Head-kidney phagocytes were resistant to the immunosuppressive effects of NO in vitro. The NO-inducing activity of T. borreli may be an adaptation developed to ensure survival and immune evasion in the fish host. Apparently, T. carassii has adopted another strategy by deactivating specific functions of phagocytes. Both strategies may ensure long-term survival of the parasite.

The gill is a major organ for antibody secreting cell production following direct immersion of sea bass (Dicentrarchus labrax, L.) in a Photobacterium damselae ssp. piscicida bacterin: an ontogenetic study.

Extremely high numbers of antibody secreting cells (ASC) were observed in the gills of sea bass fry immunised at three different age/sizes (initial weight of 0.1, 2 and 5 g) by direct immersion in a Photobacterium damselae spp. piscicida bacterin. The relatively low ASC production in the head kidney and spleen suggests that the systemic compartment was only slightly stimulated upon immersion vaccination. There was no response of corresponding magnitude in the gut as the one observed in the gills. A clear age effect was observed in the ASC response of the different groups, especially visible in the gills. Significantly higher numbers of specific ASC were observed in the gills of the two oldest groups (initial weight of 2 and 5 g) compared with the youngest fish (initial weight of 0.1 g), but the oldest groups were not significantly different from each other. Additionally, a more rapid response was observed with the ageing of the fish, with peak responses in all the organs at day 18, 16 and 8 post-immunisation in the smallest to largest fish, respectively. There was no evidence that direct immersion exposure to P. damselae ssp. piscicida at the earliest stages used in the present study (0.1 g) was tolerogenic. In the context of present knowledge, this study strongly supports the importance of the route of immunisation to locally stimulate ASC and the importance that the gills might have in specific responses.

Molecular and functional characterization of a fish inducible-type nitric oxide synthase.

Using an oligonucleotide primer based on a partial goldfish inducible nitric oxide synthase (iNOS) sequence, a complete carp iNOS cDNA was isolated from an activated carp phagocyte cDNA library. Nucleotide and predicted amino acid sequence analysis indicate that carp iNOS encodes a 1,127-amino acid protein with 57% sequence identity to human iNOS. Like mammalian NOSs, carp iNOS protein contains putative binding sites for heme, tetrahydrobiopterin, calmodulin, flavine mononucleotide, flavine adenine dinucleotide, and NADPH. Phylogenetic analysis, using neighbor joining, showed that the carp iNOS protein clustered together with the other vertebrate iNOS proteins. Inducibility of carp iNOS was confirmed by reverse transcription-polymerase chain reaction after stimulation of carp phagocytes with lipopolysaccharide or the protozoan blood flagellate Trypanoplasma borreli. These stimulators produced high amounts of nitric oxide that were toxic for T. borreli in vitro. The nuclear transcription factor NF-kappaB was shown to play a role in the induction of iNOS transcription.

Ontogeny of the thymus in a teleost fish, Cyprinus carpio L.: developing thymocytes in the epithelial microenvironment.

A monoclonal antibody, WCL9, specific for membrane molecules of a thymocyte subpopulation was used to detect these cells in situ during the ontogeny of thymus. Cryo-sections revealed WCL9+ cells in the rudiment of the thymus (day 4 post fertilization); thereafter, the positive cells were observed exclusively in the cortex from the first appearance of thymic regionalization (week 4 post fertilization) until adult age. Whole-mount immunostaining of the thymus with WCL9 revealed the three-dimensional structure of the cortex by specific staining. The presence and distribution of apoptotic cells during thymus development was studied by in situ end-labelling of fragmented DNA. From week 4 post fertilization onwards, apoptotic cells were more frequently detected in the cortex than medulla, suggesting a continuous selection of thymocytes in the cortex. Ultrastructural studies confirmed the presence of numerous cortical apoptotic cells inside macrophages. Electron microscopy provided evidence for the existence of epithelial heterogeneity in the thymus. During the ontogeny, the differentiation of epithelial cells was followed from the first weeks until the juvenile age. Cell types were classified on the basis of their localization and cytological characteristics as: i) limiting epithelial cells located in subcapsular, perivascular and peritrabecular zones; ii) reticular epithelial cells situated in medullary and cortical zones; iii) nurse-like cells at the border between the cortex and medulla, iiii) Hassall's body-like structures localized in the medulla. This study could suggest the occurrence of a wide range of lympho-epithelial interactions throughout thymocytes differentiation.

Immune responses after injection vaccination of fish.

During the last 20 years considerable progress has been made in describing and understanding the immune system of fish. Fish are the phylogenetically oldest vertebrate group with an immune system showing clear similarities to the defence systems of mammals and birds. Both innate immunity (non-specific responses) and acquired immunity (specific responses) are important for the defence against invading pathogens. Antigen injection will evoke humoral and cellular responses, which show the expected characteristics of specificity and memory. Variability in the results can be caused by external factors such as antigen dose, temperature and handling stress. Moreover, the genetic background of the fish may also play a role. The use of standardised inbred fish lines is recommended for the optimal development and evaluation of vaccines and vaccination procedures.

Immunogenetics of disease resistance in fish: a comparative approach.

The study of the genetic regulation of infectious disease resistance depends on the availability of inbred lines or selection lines of the species under investigation. The small numbers of such lines of fish has limited the strategy in teleosts to studies of associations between disease and immune/health traits. Attempts to correlate genetic differences in immune responsiveness with survival after experimental challenge with pathogenic bacteria have failed to define immune parameters that can substantially aid selection for genetic resistance to infectious diseases. Advantages and disadvantages of selection strategies as illustrated by mouse and chicken models are discussed. In this study we summarize the present situation in fish as well as our attempts to develop gynogenetic lines of carp for immunogenetic research.

Characterization of isogenic carp (Cyprinus carpio L.) lines with a genetically determined high or low antibody production.

Antibody production to dinitrophenyl-keyhole limpet haemocyanin (DNP-KLH) served as the immune parameter to divergently select carp (Cyprinus carpio L.) to produce high- and low-responder F1 hybrid lines. Antibody production to trinitrophenyl-lipopolysaccharide (TNP-LPS) and to DNP-KLH were similar in magnitude. By contrast, some high-responder lines were low responders to DNP-human serum albumin, and vice versa. Low-responder carp were relatively susceptible to infection with the parasite Trypanoplasma borreli. This suggested that at least one gene with a major influence on resistance differed between the two homozygous parents (69, 85) used to generate the high- and low-responder homozygous families, respectively. The isogenic lines showed no within-line variation in DNA fingerprints, but differed with respect to their MhcCyca-DAB genes.

Investigations into the ubiquitous nature of high or low immune responsiveness after divergent selection for antibody production in common carp (Cyprinus carpio L.).

This paper reports on the selection of individual carp with a high or low antibody response, in combination with reproduction by gynogenesis, in order to develop well-characterised inbred carp lines consisting of practically unlimited numbers of carp with the same genotype. Two homozygous progenies, previously characterised as having a high or low immune response to dinitrophenyl keyhole limpet haemocyanin (DNP-KLH), were immunised with either a T-dependent (DNP-human serum albumin (DNP-HSA)) or T-independent (trinitrophenyl lipopolysaccharide (TNP-LPS)) hapten-carrier complex. In comparison with the antibody response after DNP-KLH immunisation, the response to DNP-HSA was observed to be highly variable and did not differ between the divergently selected progenies. This suggests that the divergent selection for antibody production to DNP-KLH has been carrier-specific. Immunisation with T-independent TNP-LPS induced a very rapid response which differed between the high and low responders, and likely measured changes in the DNP-specific precursor pool of B cells caused by the selection. A number of selected individuals with a high immune response to DNP-KLH were infected with Trypanoplasma borreli, a haemoflagellate parasite of carp, to examine a possible relationship between the increase in immune responsiveness and disease resistance, but no change could be detected. However, individual homozygous carp were able to escape inbreeding depression and survive the infection. Such carp would be likely candidates for gynogenetic reproduction to obtain viable inbred carp lines.

Genetic variation in susceptibility to Trypanoplasma borreli infection in common carp (Cyprinus carpio L.).

Gynogenetic reproduction of homozygous females, or crossbreeding two homozygous animals, results in fish lines without genetic variation. Hybrid crosses are expected to express a more stable development than homozygous lines, the latter may have an important value for gaining insight into genetic components of host resistance to parasite infection. We examined the antibody response of carp (Cyprinus carpio L.) to infection with Trypanoplasma borreli. Outbred carp responded with a production of specific antibodies, but highly susceptible isogenic hybrid carp did not. This suggests an apparent relationship between susceptibility and the lack of specific antibody production. This relation was partially confirmed by the passive transfer of immunity with immune plasma. In addition, two isogenic homozygous carp lines were highly susceptible to the trypanoplasm (100% mortality), in contrast with outbred carp, the majority of which survived infection. None of the carp in either homozygous carp line produced an antibody response to parasite-unrelated antigen (DNP-KLH). This suggests that the low antibody response was not entirely due to a poor state of health, but that these carp have a genetically predetermined low antibody response.

Divergent selection for antibody production in common carp (Cyprinus carpio L.) using gynogenesis.

A base population (n = 101) of carp, consisting of a single hybrid cross, was immunized with the hapten-carrier complex DNP-KLH, to perform a divergent selection for antibody response. Measurement of the DNP-specific antibody response at 12 and 21 days postimmunization, allowed the classification of a low number of individual carp as early/high (10%) or late/low (13%) responders. Three individuals defined as early/high and three defined as late/low responding, were gynogenetically reproduced to obtain corresponding homozygous progenies within one generation only. Upon immunization with DNP-KLH, the antibody response was found to be significantly higher in the early/high responder homozygous offspring. Although the homozygosity of the offspring apparently caused a (s)lower antibody response (compared with the base population), the differences between the high and low responder offspring to indicate a genetic influence on the antibody response. The realized heritability (h2) for antibody production was estimated at 0.37 +/- 0.36. The present study provides the basis for a divergent selection of homozygous inbred carp lines with a genetically controlled difference in antibody response. These inbred lines will allow us to investigate relationship(s) between immune responsiveness and resistance to infectious diseases in fish.

Fish as biomarkers in immunotoxicology.

This paper presents an overview on the state of the art in the development and application of biomarkers for immunotoxicology in fish. There are several reasons for developing this field: many fish diseases are related to environmental quality, various environmental pollutants have immunotoxic potential and many fish diseases have an immunological component. As in immunotoxicology in general, this aspect, in fish, has received ample attention in the recent past. Much benefit has been obtained from progress in related fields of science, such as fish immunology and rodent immunotoxicology. Meanwhile there is a broad spectrum of potential biomarkers for immunotoxicology in fish, from which macrophage parameters seem to be most widely used. The application of others, such as lymphoid cell parameters is still limited, probably due to practical problems such as lack of experience with conduct, validation and interpretation. Specific problems include the paucity of background data in the case of epidemiological field studies and the important role of other (non-chemical) stress factors in the immune response, and hence the lack of specificity of potential biomarkers.

Estimation of the genetic variation in complement activity of common carp (Cyprinus carpio L.).

The complement status of hybrid, laboratory raised carp was determined by an in vitro approach of the alternate complement activity (ACH50) and total haemolytic activity (CH50), and by measurement of serum C3 levels. The lysis of target sheep red blood cells (RBC) in the haemolytic assay for CH50 activity depended, amongst others, on the haemolysin concentration in the assay. Rocket electrophoresis showed a mean serum C3 concentration of 0.95 mg ml-1. The variation for both ACH50 and CH50 haemolytic activity was approximately 30%. The degree of genetic determination of the parameters was investigated by estimation of their repeatabilities, which were relatively high for CH50 (0.71) and ACH50 activity (0.72), but lower for C3 levels (0.54). Correlations between ACH50 values and C3 levels were significant, but moderate (0.54-0.58).

Effects of inoculum route and dose on the immune response of common carp, Cyprinus carpio to the blood parasite, Trypanoplasma borreli.

Carp were inoculated intramuscularly (im) or intraperitoneally (ip) with different doses of live Trypanoplasma borreli. Prepatent period was shortened by using the im route and by increasing the dose. Haematocrit was reduced following im inoculation with 9.5 x 10(5) parasites. Most fish recovered between 8 and 13 weeks post-inoculation (wpi). Neither parasitaemia nor anaemia was detected in any recovered fish following homologous challenge. An enzyme-linked immunosorbent assay was developed to quantitate antibody response elicited by the parasite. Antibody levels rose rapidly during the first 4 wpi. The response elicited by increased dose was significantly higher only at 4 wpi. Route of exposure did not affect antibody response. Peak responses coincided with decline and eventual absence of parasitaemia.

Skin grafting in gynogenetic common carp (Cyprinus carpio, L.). The development of histocompatible clones.

The fate of skin allografts exchanged among heterozygous and homozygous gynogenetic common carp siblings, and among newly developed inbred strains and F1 hybrids, is described. Heterozygous gynogenetic offspring were produced by fertilizing eggs with UV-irradiated sperm and by treating the resulting zygote with a cold shock (0 degree C, 45 min). The temperature shock causes retention of the second polar body, which allows the eggs to develop into normal diploid fry. Homozygous gynogenetic offspring were similarly produced by using a heat shock (40 degrees C, 2 min), which suppresses the first mitotic division. Skin allografts exchanged among heterozygous gynogenetic carp exhibited prolonged survival, with some allografts (21.8%) surviving for over 28 days. Furthermore, a strong histocompatibility locus was seen to segregate in this group. In contrast, skin allografts exchanged among homozygous gynogenetic siblings were all rejected within 14 days (MST 9.4 days). New homozygous inbred strains, designated JJ and MM, were produced by gynogenetic reproduction of homozygous female carps, while F1 hybrids were produced by crossing of these homozygous females with homozygous male siblings. All grafts exchanged among members of the same strain were permanently accepted. Likewise grafts from homozygous strain members were accepted by fish from the related F1-hybrids, while the reverse grafts were rejected. These results provide evidence for the idea that in the carp histocompatibility genes exist at least one major locus and multiple minor loci, which are codominantly expressed.

A major histocompatibility locus in fish: serological identification and segregation of transplantation antigens in the common carp (Cyprinus carpio L.).

In this study, experiments are described that were designed to investigate whether fish have an immune regulatory systems similar to the major histocompatibility complex (MHC) in higher vertebrate species. From combinations of gynogenetic carp showing either slow or fast rejection of skin transplants, the latter were chosen for alloantiserum production by hyperimmunization with peripheral blood leucocytes. The resulting alloantisera were analyzed for hemagglutinating reactivity with gynogenetic siblings and proved to be operationally monospecific in absorption experiments. The serologically determined carp erythrocyte specificities were shown to correspond to two codominantly expressed allelic products of a single locus and were designated K1 and K2, respectively. Flow cytometer analysis revealed that the same products are also present on leucocytes from peripheral blood, thymus, spleen, and pronephros. K1- and K2-homozygous second-generation gynogenetic siblings were used to study the histocompatibility nature of the K locus products. Skin transplants between K-allogeneic gynogenetic siblings were rejected significantly faster than within K-syngeneic combinations. Taken together, these data suggest that the K locus incorporates MHC class I-like characteristics.

The use of artificial gynogenesis in studies on the immune system of carp (Cyprinus carpio L.).

Gynogenetic carp were obtained by using gamma-irradiated sperm for fertilization followed by a cold shock. The gynogenetic fish and normal siblings of the same parents were analysed for 3 genetic markers: scale pattern, transferrin (Tf) and allograft rejection. Gynogenetic offspring showed only the maternal scale pattern (scattered). In the majority of the gynogenetic fish a heterozygous Tf pattern identical to the mother was observed. Animals homozygous for Tf were rare in this group. A typical paternal Tf band was only seen in normal siblings. An exchange of skin grafts between normal animals was always followed by rejection (median survival time, 12 days at 23 OC). However, the rejection was delayed in the gynogenetic group (MST, 19,6 days). There were even signs of acceptance in some animals. This approach can be used for the identification of 2nd and 3rd generation gynogenetic fish, which are homozygous for certain MHC haplotypes.

Blood changes in carp (Cyprinus carpio) induced by ulcerative Aeromonas salmonicida infections.

Only recently Aeromonas salmonicida has been recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. Our attempts to formulate a vaccine based on bacterial surface antigens were unsuccessful in conferring reliable protection against lethal challenge. This lead us to study pathological changes in the humoral defense system during ulcerative A. salmonicida infection in carp. High numbers of opportunist pathogens such as A. hydrophila and Pseudomonas sp. were frequently recovered from the internal organs of moribund fish, in addition to A. salmonicida. These findings together with leucopenia in moribund fish suggest that pathogenesis is characterized by a state of immune suppression. In addition, fish which had sustained a sublethal infection were not protected against a subsequent lethal challenge. However, fish previously injected with a concentrated and inactivated culture supernatant showed protection. Differential blood cell counts did not differ between experimental and control groups during sublethal infection in contrast to serum proteins. Furthermore infected non-immune carp showed a progressive decrease of immunoglobulin and total serum protein levels before the day of peak mortality whereas protected carp maintained the immunoglobulin concentration despite a decrease in protein. Our observations suggest the involvement of multiple pathogenic events, affecting different parts of the humoral defense system during ulcerative A. salmonicida infection. The immunosuppressive effects can be minimized by prior vaccination with culture supernatant.

Does Aeromonas salmonicida affect the immune system of carp, Cyprinus carpio L.?

Aeromonas salmonicida is a significant bacterial pathogen of cyprinid and salmonid fishes causing the systemic disease furunculosis. Several observations led us to believe that A. salmonicida was able to evade or suppress the immune system of the fish: injection of whole bacteria or surface antigens was unsuccessful at protecting fish against lethal challenges; memory did not develop in survivors of sublethal infections; diseased fish often carried other opportunistic bacterial pathogens in addition to A. salmonicida, and serum protein and particularly immunoglobulin significantly decreased during A. salmonicida infections. We tested the ability of fish sublethally infected with virulent and avirulent A. salmonicida to mount a humoral immune response to sheep erythrocytes and found fewer plaque forming cells in the pronephros and lower serum anti-SRBC antibodies in infected fish as compared to controls. We also monitored the cellular immune response of diseased fish by skin allograft rejection and found an enhancement of the response that increased as the disease progressed. However, the extend of inflammation was reduced in infected fish as compared to non-infected animals. At this moment these preliminary observations are difficult to explain. Our future research will focus more specifically on cell populations that may be affected by A. salmonicida.