Carp erythrodermatitis : host defense-pathogen interaction

<TT>The outcome of a bacterial infection depends on the interaction between pathogen and host. The ability of the microbe to survive in the host depends on its invasive potential (i.e. spreading and multiplication), and its ability to obtain essential nutrients and to resist the host's defense system. On the other hand, the host's resistance to a bacterial attack depends on its physiological state, the intensity of the bacterial attack and the efficacy of the defense system to neutralize toxins and eliminate the pathogen.</TT><TT>The objective of this thesis was to better understand the host- pathogen interaction in Carp Erythrodermatitis, an acute disease caused by the bacterial pathogen <u>Aeromonas salmonicida</u> . An overall picture of the disease process is outlined in the conclusions.</TT><TT>In chapter 3, carp sublethally infected with live virulent or avirulent <u>A. salmonicida</u> were tested for their ability to mount a humoral immune response to sheep erythrocytes. Fewer antibody forming cells in the pronephros and lower anti-SRBC titers in serum were found in infected fish, as compared to controls. Both avirulent and virulent bacteria elicited a suppression of the humoral immune system. In the case of virulent bacteria, this was not a delayed response to the antigen, since antibody titers ultimately tapered off. The same was not seen for the avirulent strain.</TT><TT>Virulent bacterial cells, as well as extracellular products (ECP) also had an effect on the cellular immune system of carp, particularly on leucocytes. Indeed, infected fish showed an increase in large peripheral white blood cells, but ultimately became leucopenic. Secondly, rejection of skin allografts was accelerated in infected fish, as compared with noninfected animals, suggestive of polyclonal leucocyte activation. A marked suppression of the inflammatory response to skin allografts was seen in infected animals. It was not possible in this first study to distinguish effects caused by the bacterium from those caused by ECP. An investigation of the separate effects of washed bacterial cells and of bacterial culture supernatant on carp leucocytes was therefore initiated.</TT> <TT>In chapter 4, bacteria-free supernatants from cultures of virulent or avirulent strains of <u>A.</u><u>salmonicida</u> . were obtained after different culture times. Early cultures contained mostly bacterial fragments and free LPS, while active exotoxins were also present in older cultures. The effects of these bacteria-free supernatants were tested on the mitogenic PHA response of carp pronephric leucocytes <u>in vitro</u> . Crude supernatants from virulent cultures modulated the response, whereas avirulent supernatants had no effect. The proliferative response of PHA was enhanced by early (20 hr) virulent supernatant, but severely depressed by supernatant from later (72-96 hr) cultures. Leucocyte proliferation also occurred in the absence of PHA, with early and heat- inactivated supernatants but at a much lower level, suggesting that only stimulated cells (blasts) responded to the mitogenic substance(s) present in the supernatants. Cytokines, such as IL2 were probably also involved in the proliferative response of these leucocytes.</TT><TT>We then checked if cell-associated material and/or purified LPS, known to be present in the bacterial culture supernatants, were responsible for the stimulation of carp leucocytes. Both were indeed found to be stimulatory. However, LPS-"depleted" supernatants still retained proliferative capacities, suggesting that inactivated bacterial proteins may also be mitogenic.</TT><TT>The modulating effects seen with ECP could explain the blastogenic response of carp leucocytes seen <u>in vivo</u> . Also, active exotoxins, found to be cytolytic to carp leucocytes, could explain the leucopenia seen during later stages of the disease.</TT><TT>In order to further investigate the role played by the non-specific defense system of carp in the disease process, a selection of several carp lines exhibiting a reproducible high (A4 and W49), moderate (WW) and low susceptibility (R3 and R8) to the bacterium was made (Chapter 5). Complement activation in sera from these five carp lines was studied because of its possible role in the neutralization of <u>A. salmonicida</u> ECP and in the killing of the bacteria ("alternative" route) . Washed cells, crude or LPS-"depleted" culture supernatants and purified <u>A. salmonicida</u> LPS were found to activate carp complement and deplete serum of active complement factors. A significant difference in complement activation was seen among the carp lines tested. In particular, an enhanced activation was seen in the more resistant carp line R3. The role of complement as a bactericidal and toxin-neutralizing agent is in agreement with our findings. Fish exhibiting low initial bactericidal potential would be from the start at risk of acute pathology.</TT><TT>In chapter 6, we investigated the role of carp serum transferrin in the resistance of carp to CE. The analysis of the Tf polymorphism in 4 carp lines resulted in several distinctive variants. The DD genotype seemed to correlate with a lower susceptibility to the bacterium, whereas the PC genotype seemed to correspond with a higher disease susceptibility. Other results on the other hand, could not be interpreted at this point. In particular, the serum Tf genotype GD was found in both highly susceptible and more resistant carp lines. More work, using carp showing these specific genotypes seemed warranted.</TT><TT>Results on the iron-binding capacity of the fish serum showed a higher binding potential for the DD genotype, found only in the more resistant carp. However, the lowest iron-binding capacity was seen for the GG genotype, which was also found in relatively resistant carp. Finally, the relative concentration of Tf in the serum of the various carp lines did not seem to correlate with their susceptibility to the disease. With these preliminary data, several Tf genotypes were identified in our carp lines, some of which may correlate with lower and higher susceptibilities to the bacterium. However, Tf did not seem to be the most important factor in resistance of carp to <u>A. salmonicida</u> .</TT><TT>Without an efficient mechanism to neutralize the proteolytic enzymes present in the blood circulation during bacterial infection, not only the immune system, but also important physiological systems such as the coagulation, fibrinolytic, and kinin pathways are put at risk. Also, irreversible proteolytic degradation of connective and muscle tissues begins to occur, as seen during gross clinical observation of infected carp. In this disease, proteases in the blood of the fish had several origins: bacterial ECP, complement activation products, and other inflammatory products released by activated granulocytes.</TT><TT>In chapter 7, carp were found to possess at least two serum antiproteases, i.e. α ₁ -antitrypsin (α ₁ -AT) and α ₂ -macroglobulin (α ₂ -M). Using the zymography technique, a third pro tease/antiprotease complex could be identified in the serum of the more resistant carp line R3, after infection with <u>A. salmonicida</u> . This complex was not found in the sera of the other more susceptible carp lines. More work is needed to identify this antiprotease and to investigate its significance in disease resistance.</TT><TT>Carp from the 4 selected carp lines were given a lethal challenge with <u>A. salmonicida</u> and monitored at regular time intervals for their serum α ₁ -AT and α ₂ -M levels. Serum (α ₁ -AT) levels remained at baseline level in all carp lines, throughout the infection. The absence of (α ₁ -AT) involvement in ECP neutralization was conspicuous, since the antiprotease has a broad antiproteolytic activity. Oxygen metabolites at the site of inflammation may have inactivated the antiprotease. In contrast, serum α ₂ -M levels dropped drastically in all carp, shortly after challenge. The recovery of antiprotease levels was slow in all animals. Serum α ₂ -M of the carp line A4 remained at a critically low level for at least 5 days post-challenge. Maximum α ₂ -M levels reached after 13 days were highest for the more resistant R3 carp, and lowest for the susceptible W49 carp. The recovery time of serum antiprotease levels could play an important role in the resistance of carp to <u>A. salmonicida</u> , by prolonging the survival time of the fish and by allowing the specific immune system to respond.</TT><TT></TT><strong><TT>CONCLUSIONS</TT></strong><TT>Two main factors account for the acute pathology, in a disease like Carp Erythrodermatitis (CE): 1) the virulence of the bacterium and 2) the ineffectiveness of the defense mechanisms of the fish. Differences seen in the extent of the pathological symptoms and in the outcome of the infection relate to one or both of these factors.</TT><TT><u>1. Virulence of the bacterium</u></TT><TT>- The virulence of <u>Aeromonas salmonicida</u> is based upon its ability to invade the host, to resist the defense mechanisms and to obtain essential nutrients.</TT><TT>- The additional membrane layer (A-layer) of virulent <u>A. salmonicida</u> strains plays a major role in the resistance against the bactericidal activity of carp serum (i.e. hindrance of antibody and complement binding to target cells), as well as against the attack by phagocytes.</TT><TT>- Our virulent atypical strain of <u>A. salmonicida</u> (V234/81) has a low invasive capacity (i.e. slow growth rate, strict nutritional and metabolic requirements for maintaining virulence). It secretes proteolytic enzymes with collagenase and gelatinase activity, which break down host skin, connective tissue and muscle. These enzymes are released in order to facilitate penetration. However, the bacteria remain at the skin surface and are usually not recovered from blood or internal organs of infected carp. A local release of proteases leads to the hemorrhagic and necrotic surface lesions. Variation in the type and amount of extracellular products (ECP) released by <u>A. salmonicida</u><u>in vivo</u> and the survival time of the fish leads to differences in the extent of the disease symptoms.</TT><TT>- The soluble bacterial factors with proteolytic and hemolytic activities are also means for the pathogen to overcome nutritional deprivation, by digestion of collagen and muscle tissue (providing essential amino acids) and by liberation of hemoglobin from lysed red blood cells (providing essential iron).</TT><TT></TT><TT><u>2. Defense mechanisms of the carp</u></TT><TT>- While bacterial cells do not seem to penetrate into the blood or internal organs of the fish, free LPS and exotoxins are probably responsible for the disturbance in the carp's inflammatory and immune system.</TT><TT>- Polyclonal leucocyte activation is induced by the bacterial LPS and other ECP, but this response is counteracted by the activity of the cytolytic enzymes, leading first to the presence of blasts in the peripheral blood of infected fish and later leucopenia. In both cases, regulation of B and T cell responses is affected.</TT><TT>- <u>A. salmonicida</u> ECP (i.e. LPS and probably also proteases) strongly activate carp complement and granulocytes. The release of inflammatory products, at a distance from the proliferating bacteria, exhausts the neutralizing activity of carp serum and seems to contribute to toxicity. Uncontrolled proteases result in disturbances in the coagulation system and in changes in vascular permeability, leading to edema and internal hemorrhages.</TT><TT>- The continuous exposure to bacterial and endogenous inflammatory proteases also cause a rapid decrease of the total serum proteins. As some serum antiproteases (i.e. α ₂ -M), immunoglobulins and complement factors become depleted, carp serum not only loses its neutralizing but also its bactericidal potential.</TT><TT>- In the final stage of the disease, the severe skin ulceration, combined with a general weakening of the defense system, rapidly leads to an invasion by opportunistic pathogens. The fish die of acute respiratory distress and shock due to septicemia.</TT><TT>- In addition to environmental effects (i.e. water temperature and quality, handling stress) and physiological state of the fish (i.e. nutritional and reproductive status), which are known to influence disease resistance, the extent of the disease symptoms and the survival of carp infected with A. salmonicida are also influenced by differences in genetics.</TT><TT>- Genetic differences in the susceptibility to <u>A. salmonicida</u> observed among various carp lines are probably due to the ECP- neutralizing or bacterial killing potential of the carp serum or mucus.</TT><TT>- Carp serum antiproteases clearly play a significant role in the disease outcome. The relatively fast recovery of normal serum a2-M levels and the possible presence of an additional antiprotease type(s) in resistant carp lines suggest that both quantitative as well as qualitative differences may be important in disease resistance.</TT><TT>- Serum transferrin genotype and iron binding capacity may also contribute to disease resistance, as atypical <u>A. salmonicida</u> show a lower capacity to compete for iron as compared with the typical bacterial strains. However, preliminary results suggest that transferrin may not be a major factor in resistance to CE. Transferrin genotypes were identified, which seemed to correlate with lower or higher resistance to CE. More work using carp with these specific transferrin genotypes and crossbreeding of these carp lines is needed to elucidate the exact role of transferrin in this disease.</TT><TT>- In the future, it will be important to develop carp lines with an efficient first or second line of defense against <u>A. salmonicida</u> , allowing fish more time for specific immune responses and immunological memory. If memory can be obtained, effective vaccination should be one of the promising developments.</TT>