Adaptive capacity of rearing hens : effects of early life conditions
Walstra, I. - \ 2011
Wageningen University. Promotor(en): Bas Kemp, co-promotor(en): Henry van den Brand; Jan ten Napel. - [S.l.] : S.n. - ISBN 9789461731265 - 147
hennen - opfoktechnieken - broeden - uitbroeden - embryogenese - experimentele infectie - warmtestress - immuniteitsreactie - immunologie - adaptatiefysiologie - hens - rearing techniques - incubation - hatching - embryogenesis - experimental infection - heat stress - immune response - immunology - adaptation physiology
The traditional strategy to deal with pathogens in the layer industry is based on monitoring and control methods, primarily aimed at minimizing the risk of infection with the pathogen. The aim of this thesis was to investigate whether the adaptive capacity of layers could be influenced by early life conditions as they may occur in layer practice, as an alternative strategy for improving layer health and disease resistance. The first study investigated whether suboptimal versus optimal incubation, hatch and early rearing conditions could influence the adaptive capacity during infectious challenges with Eimeria and Infectious Bronchitis (IB). The second study investigated effects of prenatal high temperature manipulation on postnatal temperature preference and adaptive response of layers to heat stress. The third study investigated effects of suboptimal and optimal incubation temperature on the adaptive response to Eimeria under normal circumstances or following exposure to a high (35oC) environmental temperature. The fourth study investigated effects of feed provision immediately after hatch (early feeding) and suppression of gram negative intestinal bacteria (by use of the antibiotic Colistin) for 21 d post hatch on microbial composition of the intestines, layer development and response to a mix challenge with lipopolysaccharide (LPS) and humane serum albumin (HuSA). Finally, effects of early feeding and Colistin treatment on organ weights and response to an infectious challenge with Eimeria were investigated. Results demonstrated that optimized incubation, hatch and rearing resulted in a better adaptive response to Eimeria and IB, as was shown by a higher feed intake and reduced weight loss. Optimal incubation as a single early life condition also had a positive influence on the adaptive response of layers toEimeria, as demonstrated by tendencies to higher feed intake and BW gain, less duodenal lesions and a lower oocyst production. Early feeding resulted in higher body and organ weights, a changed microbiota composition in the intestines, and a changed response to E. acervulina and LPS/HuSA. Colistin treatment resulted in a changed microbiota composition of the intestines and a changed response to E. acervulina and LPS/HuSA. These results confirmed the hypothesis that early life conditions can be used to influence the adaptive capacity to infectious challenges. In conclusion, improving the adaptive capacity with the use of particular early life conditions may be the first step towards an alternative method to maintain or improve layer health and disease resistance.
Transmission dynamics of Eimeria acervulina in broilers
Velkers, F.C. - \ 2011
Wageningen University. Promotor(en): Mart de Jong; J.A. Stegeman, co-promotor(en): A. Bouma. - [S.l.] : S.n. - ISBN 9789085859215 - 153
vleeskuikens - eimeria acervulina - coccidiose - ziekteoverdracht - experimentele infectie - oöcysten - polymerase-kettingreactie - ziektebestrijding - vaccins - vaccinatie - epidemiologie - broilers - eimeria acervulina - coccidiosis - disease transmission - experimental infection - oocysts - polymerase chain reaction - disease control - vaccines - vaccination - epidemiology
Control of the intestinal disease coccidiosis, caused by infections with Eimeria species, is a major challenge, especially for the broiler industry. Effective control strategies require a comprehensive understanding of processes that lead to infection and disease in a population. One of the key factors that determine infection dynamics in a flock is the rate of transmission between hosts. Therefore, transmission experiments were carried out to increase the understanding of the underlying mechanisms of Eimeria acervulina infections in broilers, to facilitate improvement of control strategies. An important outcome of the experiments was that the excreted oocyst dose, which may be related to severity of clinical signs, increased during successive generations of infection in the flock, but that the transmission rate was independent of the oocyst dose. This suggests that transmission is not determined by the number of oocysts excreted with faeces of infected birds but, most likely, by the probability of birds to come into contact with infectious faeces. Factors influencing the degree and dispersal of infectious faecal material in the environment, such as movements and (litter pecking) behaviour of chickens, environmental conditions and faeces characteristics, may have a large impact on infection dynamics and efficacy of control measures. Furthermore, it was demonstrated that a previous infection with a wild-type E. acervulina strain significantly reduced oocyst output and transmission after re-infection. After infection with a live vaccine strain, oocyst output following an infection with a wild-type strain was also significantly reduced. However, a significant reduction of transmission of the wild-type strain was not found in groups of broilers that had been infected with the vaccine strain. Nevertheless, it was demonstrated that the live vaccine was efficiently transmitted to initially unvaccinated birds. Furthermore, the level of reduction of oocyst output was equally high for directly vaccinated and the “contact-vaccinated” chickens, that became infected due to ingestion of oocysts excreted by vaccinated birds. These results indicate that transmission of the vaccine can induce protection against high oocyst output for the entire flock, even when not all birds receive the vaccine during the initial mass application. The results of these experiments indicate that influencing the rate of transmission of wild-type and vaccine strains can be important for reducing the adverse effects of flock infections with Eimeria. Furthermore, this thesis has increased insight into some of the underlying factors that determine transmission dynamics of E. acervulina in a broiler flock. Further investigation of these factors may reveal novel targets or facilitate improvement of current strategies for coccidiosis control.
Transferrin polymorphism of common carp: link with disease resistance
Jurecka, P.M. - \ 2008
Wageningen University. Promotor(en): Huub Savelkoul; A. Pilarczyk, co-promotor(en): Geert Wiegertjes; I. Irnazarow. - [S.l.] : S.n. - ISBN 9789085852438 - 178
karper - transferrine - polymorfisme - ziekteresistentie - genetisch bepaalde resistentie - trypanoplasma borreli - experimentele infectie - genexpressie - stikstofoxide - macrofagen - immunologie - carp - transferrin - polymorphism - disease resistance - genetic resistance - trypanoplasma borreli - experimental infection - gene expression - nitric oxide - macrophages - immunology
Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many
metabolic functions. Iron concentrations are tightly regulated, for example by ferritin,
because excessive iron leads to tissue damage. Iron cannot cross cellular membranes
directly and most cells acquire iron from the iron transporting protein transferrin (Tf),
via transferrin receptors. During nutritional immunity the body reacts with a metabolic
adjustment in order to render important nutrients unavailable to invading
microorganisms. However, pathogens also have evolved a range of mechanisms to
acquire iron from the host (chapter 1).
In the study described in this thesis, we used a natural host-parasite model of common
carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan
kinetoplastid, extracellular blood parasite of carp to get more insight in the competition
for iron between host and parasite. Transferrin of common carp is highly polymorphic
with several alleles identified according to differences in electrophoretic mobility. We
studied the implications of Tf polymorphisms for iron binding and modulation of
We performed a series of challenge experiments infecting five genetically different,
commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype
may influence the susceptibility to pathogens. We observed a significant association of
the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’
and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We
also showed that variation in resistance to T. borreli could be controlled by sex-related
genetic factors. Examination of parasite growth in vitro, in culture media supplemented
with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in
number of parasites in media supplemented with serum from DD-typed animals
In general, pathogens also have mechanisms to acquire iron from the host. We
developed a method for Tf depletion of carp serum using specific antibodies to carp Tf,
and compared T. borreli multiplication and survival in the presence or absence of Tf in
vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly
showed that Tf is essential for parasite growth and multiplication (chapter 3). We
isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol
precipitation and ion-exchange chromatography (chapter 5). We showed that parasite
growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in
culture media containing different sera typed differently for Tf genotypes (chapter 3).
We identified four complete coding sequences for common carp Tf alleles C, D, F and
G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs
of other species. We could show that carp Tf differs significantly in critical iron-binding
sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species
(chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe
indeed seems to affect the ability to bind iron, which may be compensated for by
higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene
expression of two Tf alleles D and G showed a comparably high gene expression level
in liver and small but consistent differences in gene expression for allele D over allele G
in other immunologically important organs (chapter 4). Our data suggest that the allelic
polymorphism is not related to differences in iron binding and/or binding to the host Tf
receptor but could be linked with other factors, such as competition for iron with
pathogens (chapter 4).
Transferrin itself may also exert effects that are not directly linked with maintaining
iron levels and Tf cleavage products have been shown to stimulate macrophages to
produce large amounts of nitric oxide (NO). To study the induction of NO in carp head
kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G)
to purity and showed that the level of activation of macrophages by Tf was different for
the D and G allele (chapter 5). Differences in NO levels induced could be related to
different cleavage forms of the two alleles D and G, as shown by Western blot,
confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products
induced significantly higher nitric oxide (NO) production than cleavage products of Gtype
Tf. (chapter 5).
Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The
TfR-Tf complex then is internalised and transported to lysosomes, where Tf is
proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like
cysteine proteinase from T. borreli and production of a recombinant and biologically
active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is
able to digest host transferrin. Likely, Tf cleavage fragments are released from the
trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages
Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in
immunity of common carp and the influence of allelic polymorphism on competition for
iron between host and pathogen (chapter 7). Further investigations should shed more
light on the selective advantage of particular alleles to provide a basis for incorporating
Tf as a genetic marker in marker-assisted selection programmes for increased resistance
to diseases. This could contribute to improved survival of carp kept under semiintensive
farming systems in ponds.
Foot and mouth disease virus in different host species; the effect of vaccination on transmission
Orsel, K. - \ 2007
Utrecht University. Promotor(en): Mart de Jong; J.A. Stegeman, co-promotor(en): A. Bouma. - Utrecht : Universiteit Utrecht - ISBN 9789039345443 - 140
rundveehouderij - varkenshouderij - schapenhouderij - mond- en klauwzeer - infectieziekten - virusziekten - vaccinatie - experimentele infectie - cattle husbandry - pig farming - sheep farming - foot and mouth disease - infectious diseases - viral diseases - vaccination - experimental infection
Foot and mouth disease (FMD) is a contagious disease, affecting important livestock species like cattle, sheep and pigs. Therefore, FMD is listed as a notifiable disease to the Office International des Epizooties. The outbreaks of FMD in Europe in 2001 triggered the discussion about the use of vaccination as an additional control measure. The aim of vaccination during an epidemic of FMD is to stop virus transmission among animals or between herds. The main purpose of the work presented in this thesis was to quantify the efficacy of vaccination against FMDV in cattle, sheep and pigs, and to compare virus excretion and virus spread before clinical signs become apparent in vaccinated and non-vaccinated populations of the three host species. Transmission was quantified with reproduction ratio R, i.e. the expected number of secondary cases produced by a typical infected individual during its entire period of infectiousness in a completely susceptible population. With a reproduction ratio significantly below 1, no major outbreaks are likely to occur. Also, the size of an outbreak on a herd to be expected at the moment of first detection of clinical signs was estimated. Differences were observed between vaccine efficacy in the various species. In vaccinated calves R was significantly below 1. In vaccinated dairy cows no transmission was observed. In sheep, R was estimated below 1, although not significantly. Limited vaccine effectiveness in pigs was found; R was estimated above 1. When the vaccination capacity is limited, these findings indicate that a differentiated vaccination programme for various species might be applied. At the moment of clinical detection, most infected individuals can be expected in a pig herd. Combined with the limited vaccine efficacy priority might best be given to culling of pig herds
Residual feed intake in young chickens : effects on energy partitioning and immunity
Eerden, E. van - \ 2007
Wageningen University. Promotor(en): Bas Kemp; Mart de Jong, co-promotor(en): Henry van den Brand. - [S.l.] : S.n. - ISBN 9789085045939 - 168
jonge kippen - voeropname - efficiëntie - middelentoewijzing - energiegebruik - experimentele infectie - immuniteitsreactie - immuniteit - pullets - feed intake - efficiency - resource allocation - energy consumption - experimental infection - immune response - immunity
Keywords: chicken, residual feed intake, resource allocation, immune response, Salmonella Enteritidis, energy partitioning.The continuous selection in farm animals for efficient production and high production levels may have led to animals that are "programmed" to put a lot of resources in production processes, at the expense of resources for maintenance processes, among which the immune system. When efficiently and non-efficiently producing animals in a population are discriminated, it is hypothesized that non-efficient animals are better able to reallocate resources from production processes to maintenance processes than efficient animals. Non-efficient animals may, thus, be better off than efficient animals when maintenance processes are under pressure.Residual feed intake is used as a trait to discriminate efficient and non-efficient animals. It is defined as the difference between observed feed intake and expected feed intake; in this thesis, expected feed intake is based on metabolic body weight and growth. Animals that eat more than expected have a high residual feed intake and are considered non-efficient, whereas animals that eat less than expected have a low residual feed intake and are considered efficient.The research described in this thesis was carried out with pullets. Pullets are young, growing female chickens that do not produce eggs yet. Pullets were rated from high to low residual feed intake as a phenotypic trait; animals with the highest and lowest values for residual feed intake were selected for further research. Immune responses to non-replicating antigens and to live Salmonella Enteritidis bacteria were investigated.The results showed that non-efficient pullets had a higher feed intake than efficient pullets, but body weight and growth were equal in efficient and non-efficient pullets. Energy partitioning trials showed that non-efficient pullets spent more energy on maintenance processes than efficient pullets. However, an infection with Salmonella Enteritidis did not lead to repartitioning of energy from production processes to maintenance processes. It was concluded that a Salmonella Enteritidis infection is not energetically costly. Efficient pullets had a lower immune status than non-efficient pullets in situations where the animals were not infected with Salmonella Enteritidis, whereas during a Salmonella Enteritidis infection the efficient pullets had higher immune responses than non-efficient pullets. It is suggested that efficient and non-efficient pullets, as measured by residual feed intake, may have different "immune coping styles".
Gene expression profiling of chicken intestinal host responses
Hemert, S. van - \ 2007
Wageningen University. Promotor(en): Martien Groenen; Mari Smits, co-promotor(en): Annemarie Rebel. - [S.l.] : S.n. - ISBN 9789085045816 - 159
kippen - genexpressie - darmen - darmziekten - ziekteresistentie - experimentele infectie - immuniteitsreactie - immunologie - genetica - fowls - gene expression - intestines - intestinal diseases - disease resistance - experimental infection - immune response - immunology - genetics
Chicken lines differ in genetic disease susceptibility. The scope of the research described in this thesis was to identify genes involved in genetic disease resistance in the chicken intestine. Therefore gene expression in the jejunum was investigated using a microarray approach. An intestine specific cDNA microarray was generated from a normalized and subtracted library. Gene expression in young chickens was studied using two different disease models, malabsorption syndrome and Salmonella enteritidis . For each model two different chicken lines were studied, which differed in susceptibility to the specific diseases. Gene expression differences between the chicken lines were found under control and under infected conditions. In the studies described here the main focus was on genes that could be involved in disease susceptibility. Large differences between the chicken lines with different genetic backgrounds were found in their gene expression responses to the infections. After malabsorption syndrome the more susceptible chicken line regulated immune related genes, genes involved in food absorption and genes with unknown functions. The chicken line most susceptible for salmonella upregulated genes involved in inflammation, or with unknown functions, whereas the more resistant chicken line regulated genes involved in acute phase response, the fibrinogen system, actin polymerisation, and also genes with unknown functions. Most gene expression responses to both infection models were found 1 day post infection. Gene expression differences between the two chicken lines lead to the hypothesis that immunological differences could be the basis of differences in susceptibility for Salmonella . Therefore the two chicken lines were studied for the phagocytic properties of intestinal mononuclear cells and these properties were different for the two chicken lines. Also, a decrease in the number of CD4 + T-cells and macrophages in response to the Salmonella infection was found only in one chicken line. In both chicken lines the number of CD8 + T-cells increased, but faster in the susceptible chicken line. So genetic background influences intestinal gene expression responses and immunological responses.
Haemocytic defence in black tiger shrimp (Penaeus monodon)
Braak, K. van de - \ 2002
Wageningen University. Promotor(en): E.A. Huisman; W.B. van Muiswinkel; W.P.W. van der Knaap; J.H.W.M. Rombout. - S.l. : S.n. - ISBN 9789058086518 - 159
penaeus monodon - crustacea - garnalen - immuunsysteem - immuniteitsreactie - immuniteit - verdedigingsmechanismen - via de cel overgebrachte immuniteit - rode bloedcellen - hemolymfe - monoclonale antilichamen - experimentele infectie - infectieziekten - garnalenteelt - penaeus monodon - crustacea - shrimps - immune system - immune response - immunity - defence mechanisms - cell mediated immunity - haemocytes - haemolymph - monoclonal antibodies - experimental infection - infectious diseases - shrimp culture
Tropical shrimp culture is one of the fastest growing aquaculture sectors in the world. Since this production sector is highly affected by infectious pathogens, disease control is nowadays a priority. Effective prevention methods can be developed more efficiently when quantitative assays for the evaluation and monitoring of the health status of shrimp are available. The defence mechanisms of crustaceans are poorly understood, but knowledge about these is a prerequisite for the development of such health parameters. Therefore, the aim of this thesis was to obtain a better understanding of the defence system of the major cultured shrimp species in the world, Penaeus monodon . The present study emphasised the cellular components of the circulatory system, which play a central role in the haemolymph defence, i.e. the haemocytes.
To study the usefulness of haemolymph for shrimp health assessment, several cellular and humoral characteristics of P. monodon were determined after haemolymph sampling from the ventral part of the haemocoel (chapter 2). Among other things, five different haemocyte types were distinguished by light microscopy, while electron microscopy revealed granular cells, semigranular cells and hyaline cells. It was concluded that haemolymph characterisation might be a useful tool for health estimation of P. monodon , but that standardisation of the techniques is a prerequisite.
The use of monoclonal antibodies (mAbs) was proposed as a potential approach for the characterisation of haemocytes. Therefore, a set of mAbs specific for P. monodon haemocytes was produced by immunising mice with haemocyte membrane lysates (chapter 3). Four mAbs (WSH 6, WSH 7, WSH 8 and WSH 16) were selected and extensively characterised. For all mAbs, differences in amount and intensity of the labelling were found between immediately fixed haemocytes and non-fixed cells that were kept in Alsever's solution (AS, an anticoagulant which reduces haemocyte activation) and kept in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of the majority of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mAbs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS or in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different sizes and electron densities. Immuno-reactive extracellular fibrous material could be observed when cells were kept in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and lowest for WSH 16. Double labelling revealed that all mAbs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis was put forward that immuno-reactive molecules recognised by these mAbs, were related to haemocyte activation factors and that the mAbs could be used in studying haemocyte differentiation, behaviour and function in P. monodon shrimp. Later on, WSH 8 indeed proved suitable for this in immuno-histochemical studies.
A better characterisation of the immuno-reactive molecules would support the interpretation of the results. In order to investigate whether the mAbs reacted with well-conserved molecules and with haemocytes in animals with molecules that were better characterised than those of P. monodon , a comparative study was carried out (chapter 4). The mAbs also reacted on haemocyte monolayers of the freshwater shrimp Macrobrachium rosenbergii and the two freshwater crayfish Procambarus clarkii and Pacifastacus leniusculus . Immuno-labelling on haemolymph monolayers of the terrestrial isopod crustacean Porcellio scaber (woodlouse) and on coelomic fluid of the annelid Lumbricus terrestris (earthworm) showed partial reactivity. Immuno-reactivity was not observed on haemolymph monolayers of the insect Spodoptera exigua (Florida moth) and the mollusc Lymnaea stagnalis (pond snail), or on blood cell monolayers of the freshwater fish Cyprinus carpio (carp) and of human. On histological sections of M. rosenbergii and P. clarkii , mAb labelling was observed on the haemolymph plasma and on a proportion of the haemocytes. This comparative study showed reactivity of the mAbs in a wide range of crustaceans and related animals and suggests that well conserved molecules were recognised, which may indicate functional importance. Later on, molecules of P. leniusculus that reacted with WSH 6 were better characterised and it was indicated that this molecule could be clotting protein or filamin, which both could be involved in coagulation processes. Unfortunately, the immuno-reactive molecules of P. monodon with WSH 8 could not be characterised further.
The circulating haemocytes of crustaceans are generally divided into hyaline, semigranular or granular cells, however, this classification is still ambiguous. Not much is known about haemocyte production in penaeid shrimp, but for a better haemocyte classification it is useful to establish how these cells are produced and mature. In order to clarify this, the localisation and (ultra)structure of the haematopoietic tissue and its relation with the circulating haemocytes were studied in chapter 5. The haematopoietic tissue is located in many lobules dispersed in different areas in the cephalothorax, mainly at the dorsal side of the stomach and at the base of the maxillipeds. In order to study the haemocyte production and maturation, shrimp were either injected with LPS, while mitosis was inhibited by vinblastine, or were repeatedly sampled for haemolymph. The presumed precursor cells in the haematopoietic tissue were located towards the exterior of the lobules and maturing young haemocytes towards the inner part, where they can be released into the haemal lacunae. It was proposed that the presumed young haemocytes were generally known as the hyaline cells. Moreover, a new model was proposed where the hyaline cells gave rise to two haemocytic developmental series, i.e., the large- and small-granular cell line. In addition, indications were found that the granular cells of at least the large-granular cell line mature and accumulate in the connective tissue and are easily released into the haemolymph. Light and electron microscopical observations supported the regulation of the haemocyte populations in the circulation by (stored) haemocytes from the connective tissue.
In order to investigate the clearance reaction of P. monodon haemocytes live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled (chapter 6). Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that many haemocytes encapsulated the bacteria at the site of injection. Furthermore, 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, where they, or their degradation products, could be detected for at least seven days after injection. The lymphoid organ 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. The lymphoid organ of penaeids is also poorly studied. Electron microscopy of the lymphoid organ revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It was proposed that haemocytes settle in the tubule walls before they phagocytose. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessels in most decapod crustaceans that do not possess a lymphoid organ.
Immuno-staining suggested that many of the haemocytes degranulate in the lymphoid organ, 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 lymphoid organ is a filter for virtually all foreign material encountered in the haemolymph. Haemocyte degranulation in the lymphoid organ tubule walls could contribute to the filtering capacity of this organ.
The experimental shrimp appeared to contain many lymphoid organ spheroids, where bacterial antigens were finally also observed. 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.
White spot syndrome virus (WSSV) is the pathogen that is a major cause of mortality in shrimp culture in the past decade. In contrast to the extensive study of the morphology and genome structure of the viral pathogen, the defence reaction of the host during WSSV infection is hardly studied. Therefore, the haemocyte response upon experimental WSSV infection was examined in P. monodon shrimp (chapter 7). A strong decline in free circulating haemocytes was detected during severe WSSV infection. The combination of in situ hybridisation with a specific DNA probe to WSSV and immuno-histochemistry with a specific antibody against haemocyte granules was carried out on tissue sections. Haemocytic reactions have never been reported in chronic or acute viral infections in shrimp, but the present results showed that many haemocytes leave the circulation and migrate to tissues where many virus-infected cells are present. However, a subsequent response to the virus-infected cells was not detected. During virus infection, the number of cells in the haematopoietic tissue was also reduced. Moreover, it was suggested that many haemocytes degranulated in the lymphoid organ, producing a similar but more obvious layer of fibrous material in the outer tubule wall than after bacterial injection.
The obtained results are summarised and discussed in chapter 8. Furthermore, the results described in chapters 6 and 7 were used to refine the proposed model of chapter 5. The haemocytes of the small-granular cell line are suggested to mature and carry out their function in the lymphoid organ. The results of the present research emphasise the rapid activation of the haemocytes after stimulation of the animal and illustrate several relevant functions of those cells. The present knowledge provides reliable grounds for further discussions about production, maturation and activation of the haemocytes in penaeid shrimp and possibly also in related animals like other shrimp species, crayfish, lobsters and crabs. Knowledge of the functioning of the defence system is of extreme importance since stimulation of this system is considered as a potential intervention strategy in shrimp culture to overcome the infectious diseases.