Van Immerseel
email: filip.vanimmerseel@ugent.be

Pasmans

The use of organic acids to combat salmonella in poultry: a mechanistic explanation of the efficacy

Keywords: salmonella, organic acids, poultry, colonization

The use of organic acids to combat salmonella

Introduction

With the perception that antibiotics should no longer be used as animal growth promoters, there has been widespread interest in natural methods of inhibiting detrimental bacteria. Man has used fermentations as a method of food preservation for more than 6000 years [START_REF] Ohmomo | Silage and microbial performance, old story but new problems[END_REF], but now it appears that fermentation acids also have value as feed or drinking water additives.

Commercial preparations appear to enhance digestibility and diet palatability, thus improving feed conversion and growth of animals, including pigs and poultry. Some acid mixtures prevent mould growth on feed, and claims of increased egg production have been made. Pathogen control has also been reported, but the peer-reviewed scientific literature has few definitive studies. Until recently the use of short-chain fatty acids, medium-chain fatty acids and other organic acids was largely based on their antimicrobial activity outside the intestinal tract. This review: (1) describes the current use of organic acids to control Salmonella in poultry, (2) attempts to provide a rationale for assessing potential pathogen control, (3) summarizes in vivo experiments and field observations, and (4) discusses future applications of using acids, bacteria or dietary changes to control Salmonella.

Bacterial Metabolism Of Organic Acids

Bacteria can use organic acids as both carbon and energy sources. In Escherichia coli, the hydrophobic long chain fatty acids (LCFA, ≥C12) are transported across the cell membrane by carrier mechanisms, in which the fadL (outer membrane proteins) and the fadD proteins (inner membrane) are involved. FadL carries LCFA to the periplasmatic space and fadD is an acyl-CoA synthetase [START_REF] Dirusso | Bacterial long chain fatty acid transport: gateway to a fatty acid-responsive signalling system[END_REF]Van Den Berg, 2005). Once the acyl-CoA molecules are formed inside the cell, degradation occurs through the β-oxidation pathway, yielding multiple acetyl-CoA molecules (Clark and Cronan, 1996). Degradation of LCFA having odd number of carbon atoms also yields propionyl-CoA as an end product. Whether medium chain fatty acids (MCFA, C6 to C10) can be transported by carrier proteins or are able to diffuse freely across the cell membrane in undissociated form is less clear, but also the fadD protein and the β-oxidation (Clark and Cronan, 1996). The LCFA and MCFA can also be used for incorporation in the membrane as phospholipids. Short-chain fatty acids (SCFA, ≤ C4) presumably cross the outer membrane mainly through diffusion in the undissociated form (Clark and Cronan, 1996). Once inside the cell, they can be converted to their CoA thioester forms. Butyric acid is converted to butyryl-CoA by the acetoacetyl-CoA transferase system (AtoAD system), converted to acetoacetyl-CoA by the fadB/E system, and then further breakdown to acetyl-CoA is performed by the atoB gene product (Jenins andNunn, 1987a, 1987b;Clark and Cronan, 1996). Thus as an example, butyric acid is converted to 2 molecules of acetyl-CoA. Propionic acid, either taken up from the environment or generated as end-product of degradation of LCFA with odd number of carbon atoms is metabolized in Salmonella and E. Coli in the methylcitrate cycle. Propionyl-CoA reacts with oxaloacetate to form 2-methylcitrate, that is converted through a series of reactions, mediated by the Prp operon, to succinate and pyruvate (Horswill and Escalante-Semerena, 1999). These products can be used in the citric acid cycle.

Although it is thought that acetate can diffuse across the cell membrane, an acetate permease (ActP) was detected in E. coli [START_REF] Gimenez | The gene yjcG, cotranscribed with the gene acs, encodes an acetate permease in Escherichia coli[END_REF]. In E. Coli and Salmonella, acetate is converted to acetyl-CoA by either acetyl-CoA synthetase (encoded by acs gene) or the sequential action of acetate kinase and phophotransacetylase (encoded by ackA and pta, respectively) (Wolfe, 2005). Acetyl-CoA, generated by either the β-oxidation pathway, butyric acid breakdown or by acetate conversion, can be used for oxidation in the citric acid cycle and for replenishing intermediates of the citric acid cycle via the glyoxylate shunt.

Mechanism OF ANTIMICROBIAL ACTIVITY OF ORGANIC ACIDS

Fermentative bacteria produce organic acids when oxygen is not available as terminal electron acceptor, but they differ greatly in the types of acids that they produce. Because the oxidation of one molecule must be coupled to the reduction of another, anaerobic bacteria often produce several acids. The simplest fermentation is conversion of sugar to lactate, and many lactobacilli, streptococci, lactococci and enterococci have a scheme that is virtually homolactic when sugar is plentiful. However, when sugars are scarce, all of these bacteria are capable to switch to a fermentation that produces acetate, formate and ethanol, so ATP production can be enhanced. Butyric acid producing bacteria typically utilize the hydrogenases of butyrate (or other even longer chain fatty acids) production as a mechanism of reducing equivalent disposal. If the bacterium has a hydrogenase, interspecies hydrogen transfer to a methanogen decreases the need for dehydrogenase activity and acetate production typically is enhanced. Bacteria capable of utilising fatty acids are found in stagnant anaerobic environments, but these bacteria grow very slowly, and fermentative environments are typically acidic. Fermentation acids are inhibitory when the pH is low but some bacteria are much more resistant than others.

Traditionally, microbial growth inhibition by organic acids was explained by the ability of these acids to pass across the cell membrane, dissociate in the more alkaline interior and acidify the cell cytoplasm [START_REF] Kashket | Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance[END_REF]. Organic acids were compared to synthetic uncouplers that could remain membrane associated, and shuttle protons in a cyclic manner to dissipate the protonmotive force. The problem with this analogy was the fact that organic acid anions are charged and not lipid permeable. Indeed, the accumulation of benzoate anion is typically used to estimate intracellular pH. If benzoate acted like an uncoupler the pH gradient across the cell membrane would be dissipated by the tool being used to measure it! The uncoupling model of fermentation acid toxicity, also failed to address another practical question. Why are some bacteria so much more sensitive than others (Russell and Diez-Gonzalez, 1992)? For many years it was assumed that bacteria maintained a slightly alkaline intracellular pH, but this assumption was largely based on work with laboratory cultures of E. coli [START_REF] Padan | pH homeostasis in bacteria[END_REF]. It is now clear that many fermentative bacteria have the ability to let their intracellular pH decline when the extracellular pH becomes highly acidic. This decline in intracellular pH necessitates a metabolism that can tolerate a lower pH, but the strategy appears to be highly adaptive. When intracellular pH remains high, the pH gradient across the cell membrane can become very large. The protons can be pumped back out of the cell, but the pH gradient causes a logarithmic accumulation of the fermentation acid anions. By letting intracellular pH decrease, the bacterium has a much smaller pH gradient across the cell membrane and it is protected from anion accumulation.

Continuous culture studies with E. coli K-12 and O157: H7 indicated that the two strains diferred greatly in their sensitivity to acetate at pH 5.9, and the ability of O157: H7 to tolerate more acid than K-12 could be correlated with ability to decrease intracellular pH [START_REF] Diez-Gonzalez | The ability of Escherichia coli O157:H7 to decrease its intercellular pH and resist the toxicity of acetic acid[END_REF]. These experiments also revealed another important observation about Clostridium sporogenes, a silage and food contaminant, indicated that it accumulated lactate anion at acidic pH values in accordance with the pH gradient across the cell membrane, but lactate anion accumulation caused a secondary effect (Flythe and Russell, in review). When lactate anion increased, the cells lost intracellular glutamate, and its fermentation scheme of amino acid deamination is dependent on glutamate transaminase. The final result was a virtually complete inhibition of ammonia production. The antimicrobial activity of organic acids on other bacterial species has not been correlated with intracellular pH regulation, but bacteria that could be classified as neutrophiles seem to be more sensitive than those that are acid tolerant. For example, the minimal inhibitory concentration (MIC) of acetic acid is 250 times lower for Bacillus subtilis than lactobacilli (Hisiao and Siebert, 2002).

The anion model of organic acid toxicity explains why bacteria differ in their sensitivity to organic acids, but it does not provide information on the antibacterial effect of one acid versus another. The MIC for acetic, butyric, lactic and caprylic acid in E. coli are less than 4 g/l, but this same bacterium is approximately 10-times more resistant to malic, tartaric and citric acid (Hsiao and Siebert, 2002). This observation indicates that factors such as chain length, side chain composition, pKa values, hydrophobicity could affect the antimicrobial activity.

Effects Of Short-Chain Fatty Acids On Salmonella

[copy editor: please keep the heading below (and others) as bold font -EiC]

Antimicrobial activity against Salmonella. Less is known about the effect organic acids on Salmonella, but it should be noted that E. coli and Salmonella are both enteric bacteria and seem to have a similar physiology. Medium chain fatty acids (C6 to C12; caproic, caprylic, capric and lauric acid) appear to be much more effective against Salmonella than the short-chain fatty acids (formic, acetic, propionic and butyric acid), but it is important to differentiate bactericidal and bacteriostatic effects. As little as 25 mM C6 to C10 acids were bacteriostatic to a Salmonella Enteritidis, but the same strain tolerated 100mM of short-chain fatty acids (Van Immerseel et al., 2003;[START_REF] Hensel | Evolution of pathogenicity islands of Salmonella enterica[END_REF]. [START_REF] Sprong | Bactericidal activities of milk lipids[END_REF], reported that caprylic and capric acids were bactericidal, but C14:0, C18:1 and C18:2 acids were not. When Salmonella Enteritidis and Typhimurium were incubated with low concentrations ofmonocaprin (5 mM) that had been combined with an emulsifier, the bacteria did not survive (Thormar et al., 2006). In general, these data indicate that medium chain fatty acids have the greatest antibacterial activity against Salmonella, but large scale studies are lacking.

Effect of short-chain fatty acids on virulence of Salmonella. Salmonella is an opportunistic intracellular pathogen that has an elaborate set of virulence genes. These genes enable the bacterium to adapt to the environment and move between various micro-niches within a host. An early step in the pathogenesis of Salmonella is the penetration of intestinal epithelium [START_REF] Lostroh | The Salmonella pathogenicity island-1 type III secretion system[END_REF]. This activity is promoted by invasion genes that are located on a pathogenicity island (SPI-1), but several pathogenicity islands are required for full virulence [START_REF] Hensel | Evolution of pathogenicity islands of Salmonella enterica[END_REF]. SPI-1 has genes encoding regulatory proteins, structural components of a needle complex and additional effector proteins. Bacterial effector proteins facilitate the entry of Salmonella into the cytosol of epithelial cells, by inducing actin rearrangements that lead to uptake of the bacteria. SPI-1 is in turn activated by HilA, and this latter protein is environmentally regulated. When Salmonella Typhimurium was pre-incubated in growth media supplemented with various concentrations of butyrate and propionate, epithelial cell in evasion was suppressed. However, if the cells were preincubated in media supplemented with acetate invasion was still observed [START_REF] Durant | Short-chain fatty acids affect cell-association and invasion of Hep-2 cells by Salmonella Typhimurium[END_REF][START_REF] Lawhon | Intestinal short-chain fatty acids alter Salmonella Typhimurium invasion gene expression and virulence through BarA/SirA[END_REF]Van Immerseel et al., 2004). Similar results were obtained with Salmonella Enteritidis when primary caecal epithelial cells of the chicken were employed (Van Immerseel et al., 2004).

The effects of organic acids on epithelial invasion can be explained by changes in SPI-1 expression. [START_REF] Durant | Short-chain volatile fatty modulate the expression of the hilA and invF genes of Salmonella Typhimurium[END_REF] measured hilA and invF (major activators of SPI-1) expression after exposure to acetate, butyrate and propionate in S. Typhimurium (2000). At pH 6, exposure of the bacteria to acetate increased the expression of these genes, but similar effects were not observed with propionate or butyrate. More recently, [START_REF] Lawhon | Intestinal short-chain fatty acids alter Salmonella Typhimurium invasion gene expression and virulence through BarA/SirA[END_REF] noted that butyrate and propionate, but not acetate led to a decrease in hilA, invF and sipC expression. Acetate, after its conversion to acetyl-phosphate, acts to phosphorylate BarA and subsequently SirA. BarA/SirA is a two component system thought to be involved in environmental sensing, and SirA enhanced transcription of hilA, finally resulting in increased invasion [START_REF] Jones | Salmonella invasion gene regulation: a story of environmental awareness[END_REF]. DNA microarrays of both Salmonella Typhimurium and Salmonella. Enteritidis indicated that low doses of butyric acid downregulated SPI-1, but it did not alter metabolic gene expression. HilD, a positive regulator of HilA, was also downregulated in both strains. S. Typhimurium and S. Enteritidis carrying plasmid-borne hilA::luxCDABE and hilD::luxCDABE transcriptional fusions confirmed the idea that butyrate down-regulated hilA and hilD [START_REF] Gantois | Butyrate specifically down-regulates Salmonella pathogenicity island I gene expression[END_REF]. The primary target of butyrate in the bacterial cell is still unknown but butyrate could interfere with HilA dependent regulation of SPI1 by altering the regulation of hilD transcription. These data indicate that shortchain fatty acids can regulate the invasive phenotype of Salmonella, and it should be mentioned that pre-incubation of Salmonella with short-chain fatty acids also increased acid resistance and survival in macrophages [START_REF] Kwon | Induction of acid resistance of Salmonella Typhimurium by exposure to short-chain fatty acids[END_REF].

Effects of short-chain fatty acids in Salmonella control in vivo. The use of acidic compounds to control Salmonella first appeared in the late 1960s, and mainly focused on decontamination of carcass meal (Khan and Katamy, 1969;[START_REF] Smyser | Evaluation of organic acids and other compounds as Salmonella antagonists in meat and bone meal[END_REF]Van Staden et al., 1980). [START_REF] Khan | Antagonistic effects of fatty acids against Salmonella in meat and bone meal[END_REF] evaluated the efficacy of 32 different acid preparations to decontaminate bone meal, and showed that low molecular weight volatile fatty acids were the most promising. Their results were a basis for the development of non-toxic, naturally occurring acidic compounds to control Salmonella. More than 35 years later, it is clear that their thoughts were prophetic. These acids have been added to feed, drinking water, and other matrices, in order to prevent Salmonella colonization of animal tissue and transmission through the food chain.

Poultry feed is a major source for Salmonella introduction to the farm (Williams, 1981).

When chickens are given artificially contaminated feed, the gut is colonized and Salmonella are shed into the environment [START_REF] Hinton | Salmonella infection in chicks following consumption of artificially contaminated feed[END_REF]. The original concept of incorporating acids into feed was based on the notion that the acids would decontaminate the feed itself and prevent Salmonella uptake by the chickens. When [START_REF] Iba | Studies on the use of a formic acid-propionic acid mixture (Bio-Add™) to control experimental Salmonella infection in broiler chickens[END_REF] inoculated feed with high doses of a S. Typhimurium strain, a commercial mixture of formic and propionic acid decreased the viability more than 1000-fold over seven days. When broiler chicks were given the acid treated feed that had been mixed with either Salmonella Enteritidis, Typhimurium or agona, the caecal Salmonella numbers were 7 log10 lower than control animals at day 5 (10 2 versus 10 9 cfu/g) [START_REF] Iba | Studies on the use of a formic acid-propionic acid mixture (Bio-Add™) to control experimental Salmonella infection in broiler chickens[END_REF]. Mixtures of formic and propionic acid were also effective when [START_REF] Hinton | Control of Salmonella infections in broiler chickens by the acid treatment of their feed[END_REF].

In a large scale study, (Humphrey and Lanning,1988) the number of Salmonella positive breeder feed samples decreased from 4.1 to 1.1% after the feed was supplemented with 0.5% formic acid. The antibacterial activities of organic acids were dependent on the temperature and moisture. Since the water content of poultry feed is generally low, the action of the acids is not always optimal, and it is not clear if in feed effects are the major reason of protection [START_REF] Hinton | Antibacterila activity of short-chain organic acids[END_REF]). In the 1980s, it became clear that the acid concentrations were also increased in the crop, and this antibacterial action could aid in controlling infection caused by horizontal transmission.

Indeed, when the acid treated feed is eaten by the chickens, it is both warmed and moistened and the activity of the short chain fatty acids should increase. It appears that supplemental acids are most apt to affect in the crop and gizzard rather than in the intestine. This point is illustrated in a study of Thompson and Hinton (1997), who fed laying hens a feed supplemented with a commercial mixture of formic and propionic acid. In these animals, pH values of the crop, gizzard, jejunum, caecum and colon were not altered relative to control animals, but formic and propionic acid concentrations in crop and gizzard were significantly increased. At the same time, the lactic acid concentration in the crop decreased significantly, suggesting that lactobacilli were either inhibited or killed (Thompson and Hinton, 1997). This is in accordance with results obtained by [START_REF] Hume | Effectiveness of dietary propionic acid in controlling Salmonella Typhimurium colonization in broiler chicks[END_REF], in which large increases in propionic acid concentration in the crop of 4-day old broilers were detected, when propionic acid was added to poultry feed, despite no observed changes in crop pH. Caecal short chain fatty acid patterns were not affected [START_REF] Hume | Effectiveness of dietary propionic acid in controlling Salmonella Typhimurium colonization in broiler chicks[END_REF].

Later in the 1980s, many studies examined the effects of supplemental acids on Salmonella colonization of chicken tissues. Action of formic and propionic acids were variable.

In a small-scale field trial, formic acid controlled shedding and caecal colonization by Salmonella serovars in naturally infected the animals. Indeed, 50% of all control animals had Salmonella positive cloacal swabs and caecal content samples, but Salmonella could not be detected in animals that consumed significant concentrations of formic acid [START_REF] Hinton | Control of Salmonella by acid disinfection of chicks' food[END_REF]. In 3 year study, the cumulative number of infections of newly hatched chicks with Salmonella decreased after breeder stocks were given formic acid treated feed [START_REF] Humphrey | The vertical transmission of Salmonella and formic acid treatment of chicken feed[END_REF].

Breeders that received acidified feed had fewer numbers of Salmonella in the breeder litter (4.3 These decreases were evident from the moment the breeders received acidified feed and illustrate the effects of vertical transmission [START_REF] Humphrey | The vertical transmission of Salmonella and formic acid treatment of chicken feed[END_REF]. The most striking proof of the efficacy of formic and propionic acid as feed additives to control Salmonella was given by [START_REF] Hinton | Control of Salmonella infections in broiler chickens by the acid treatment of their feed[END_REF]. In three independent experiments, no artificial infections or feed inoculations with Salmonella were performed. Formic acid supplemented feed, given from the day of hatch, decreased the number of positive faeces and caecal content samples dramatically.

The control groups had 25, 27 and 60% Salmonella positive faecal samples, but the treatment groups were 3, 0 and 0% [START_REF] Hinton | Control of Salmonella infections in broiler chickens by the acid treatment of their feed[END_REF]. When the formic acid treated feed was given at later age (16 or 32 days), no differences were detected between control and treated groups.

This illustrates that preventing initial colonization of Salmonella is most important. Once an infection is established, it is very difficult to counteract by using acid treated feed, at least in the same production round.

When a formic and propionic acid mixture (0.5 to 0.68% w/w) was added to broiler feed that was artificially contaminated with very low numbers of S. Kedougou (less than 50 cfu/g feed), only 1 of 30 groups (10 chickens each) became infected, compared with 22 out of 27 control groups [START_REF] Hinton | Control of Salmonella infections in broiler chickens by the acid treatment of their feed[END_REF]. These experiments were a basis for numerous publications on this topic. Izat et al. (1990a) found a significant effect when 0.4% propionic acid was added to broiler feed and the number of Salmonella bacteria on post-chill carcasses was assayed. No significant differences in Salmonella count in the small intestine could be detected, but the relevance of small intestinal sampling can be questioned because Salmonella mainly colonizes the caeca [START_REF] Desmidt | Pathogenesis of Salmonella Enteritidis phage type four after experimental infection of young chickens[END_REF]. Izat et al. (1990b) concluded that formic acid addition to the feed was not a reliable means of reducing the incidence or level of Salmonella in the caeca or processed carcasses. Some reductions in cecal Salmonella were detected after calcium formate addition, but no effects were observed in most treatment groups (Izat et al., 1990b). This can be attributed to the infection protocol. They infected the animals on day 2, 7, 14, 21 and 28 by adding Salmonella to the drinking water (approximately 10 5 per ml) rather than the feed. [START_REF] Mchan | Effect of feeding selected short-chain fatty acids on the in vivo attachment of Salmonella Typhimurium in chick ceca[END_REF] fed chickens a diet supplemented with 1% of a formic and propionic acid mixture and infected the animals at 2 days post-hatch with 10 6 cfu Salmonella Typhimurium.

They found significant reductions in caecal Salmonella colonization (>2.5 log units at 14 days of age, and > 3.5 log units at 21 days of age). [START_REF] Hume | Effectiveness of dietary propionic acid in controlling Salmonella Typhimurium colonization in broiler chicks[END_REF] concluded that propionic acid S. Typhimurium at day 4 of age, bu they were sampled 6 days post-infection. The low dosage and the short time between infection and sampling could explain these results. When chickens received feed containing mixtures of formic and propionic acid, mortality after oral challenge with S. Pullorum and Gallinarum declined (Berchieri and Barrow, 1995;[START_REF] Al-Tarazi | Effect of dietary formic and propionic acids on Salmonella Pullorum shedding and mortality in layer chicks after experimental infection[END_REF].

Recently, researchers have attempted to transport the organic acids further down in the gastrointestinal tract by micro-encapsulation, which should prevent absorption of the acids in the upper tract and ensure release further down in the gastro-intestinal tract. Van Immerseel et al. (2004c) examined the effect of microbeads containing formic, acetic, propionic and butyric acid on colonization of S. Enteritidis in caeca, liver and spleen. Animals were infected (day 5 posthatch) with 5 x 10 3 cfu S. Enteritidis and samples were taken 3 days post-infection. Cecal colonization was significantly increased when acetic acid was added to the feed, but decreased when butyric acid was added. Internal organ colonization was increased if either formic or acetic acid were added to the feed, and this result is consistent with the idea that acids can enhance the virulence of Salmonella (see above). When powder and coated butyric acid additives (0.63 g/kg butyric acid) were compared using the same infection protocol, the coated form decreased colonization of the caeca, but the powdered form did not (Van Immerseel et al., 2005). The inability of the powered form to give a positive response, may have been due to the short time interval between infection and sampling. In an infection study using a seeder model in which 10 broilers were infected at day 5 post-hatch with 10 5 cfu S. Enteritidis and housed together with 40 non-inoculated broilers, 0.63 g/kg coated butyric acid in the feed significantly reduced shedding of S. Enteritidis in broilers until slaughter age. The effect of the acids on other members of the microbial community were not determined (Van Immerseel et al., 2005).

Drinking water is a source for infection, so it is important to keep drinking water free of Salmonella. Short-chain fatty acids have also been used as drinking water sanitizers. In a study where 'natural' infections were recorded, [START_REF] Al-Chalaby | Failure of drinking water sanitisation to reduce the incidence of natural Salmonella in broiler chickens[END_REF] evaluated a commercial product containing propionic acid. The acids eliminated Salmonella in drinking water, while more than 80% of the samples in control groups were positive. This result could not be explained by Salmonella carriage because litter, cloacal swab and caecal content samples were not reduced et al., 1985) Acetic, lactic or formic acid (0.5%) in drinking water reduced crop contamination after Salmonella Typhimurium challenge (10 8 cfu) when feed had been withdrawn to simulate pre-transport conditions to the slaughter house [START_REF] Byrd | Effect of lactic acid administration in the drinking water during preslaughter feed withdrawal on Salmonella and Campylobacter contamination of broilers[END_REF]. However, the number of positive caecal samples was not different between the treatment groups. Lactic and formic acid were even more effective than acetic acid. In a commercial farm study where broilers were provided 0.44% lactic acid in the drinking water during a 10h feed withdrawal period, crop contamination and incidence of Salmonella in pre-chill carcass rinses were decreased (Byrd et al., 2003). When moult was induced in layers 9 days of feed removal and the animals were inoculated with Salmonella on day 4 of the feed withdrawal, neither acetic nor lactic acid (0.5%) significantly reduced crop or caecal colonization (Byrd et al., 2004). These results seem to indicate that drinking water acidification is not as effective when chickens are moulted or highly stressed [START_REF] Holt | Molting and Salmonella enterica serovar Enteritidis infection : the problem and some solutions[END_REF].

Short-Chain Fatty Acids In The Gut: A Key To Control Pathogen Colonization?

Feed and drinking water sanitation, and the addition of acid to the crop appears to prevent pathogen colonization in the live animals, but the type of acid and its concentration can be very important. Salmonella colonization of the caeca and internal organs is not always affected by these treatments, especially if the infection pressure is high. Acids from feed or drinking water are not effective further down in the intestinal tract because Salmonella colonization is mainly in the caeca [START_REF] Desmidt | Pathogenesis of Salmonella Enteritidis phage type four after experimental infection of young chickens[END_REF]. Because the caecum is the main fermentation site, the concentrations of short-chain fatty acids are already higher there than in other intestinal segments [START_REF] Engberg | The influence of grinding and pelleting of feed on the microbial composition and activity in the digestive tract of broiler chickens[END_REF]. One-day-old broilers had no short chain fatty acids in their caeca, but concentrations were high by 10 days post-hatch (Van Der Wielen et al., 2000). Acetic acid is the predominant short-chain fatty acid in the caeca, with concentrations ranging between 70 and 90 µmol/g caecal content [START_REF] Engberg | The influence of grinding and pelleting of feed on the microbial composition and activity in the digestive tract of broiler chickens[END_REF], Van Der Wielen et al., 2002). In most studies, the caecal butyrate concentration ranges between 10 and 40 µmol/g in chicken caeca, and the propionate concentration is even less [START_REF] Engberg | The influence of grinding and pelleting of feed on the microbial composition and activity in the digestive tract of broiler chickens[END_REF], Van Der Wielen et al., 2002).

Because short-chain fatty acids can affect invasion and virulence gene expression of Salmonella [START_REF] Lawhon | Intestinal short-chain fatty acids alter Salmonella Typhimurium invasion gene expression and virulence through BarA/SirA[END_REF]Van Immerseel et al., 2004;Gantois et al., 2005), the natural quantities of It has already been shown in various animal species that Salmonella colonization of the gut is decreased when the bifidobacterial population is increased, either by administration of bifidobacteria as probiotic strains, or by addition of certain types of oligosaccharides that stimulation proliferation of these bacteria in the gut [START_REF] Asahara | Increased resistance of mice to Salmonella enterica serovar Typhimurium infection by symbiotic administration of[END_REF][START_REF] Buddington | Dietary oligofructose and inulin protect mice from enteric and systemic pathogens ant tumor inducers[END_REF][START_REF] Bovee-Oudenhoven | Dietary fructo-oligosaccharides and lactulose inhibit intestinal colonisation but stimulate translocation of Salmonella in rats[END_REF][START_REF] Silva | Effect of Bifidobacterium longum ingestion on experimental salmonellosis in mice[END_REF]Thitaram et al., 2005). When the caecal Bifidobacterium population in broilers was increased by isomalto-oligosaccharide addition to the feed, and the animals were infected with a high dose of Salmonella Typhimurium, large reductions in caecal colonization were observed (Thitaram et al., 2005).

Increases in lactic acid bacterial counts in the gut are correlated with increases in butyric acid concentrations [START_REF] Kleessen | Oligofructose and long-chain inulin: influence on the gut microbial ecology of rats associated with a human faecal flora[END_REF][START_REF] Humblot | Brussels sprouts, inulin and fermented milk alter the faecal microbiota of human microbiotaassociated rats as shown by PCR-temporal temperature gradient gel electrophoresis using universal, Lactobacillus and Bifidobacterium 16S rRNA gene primers[END_REF], and Salmonella colonization is decreased when butyric acid concentrations in the gut are increased (Van Immerseel et al., 2004, 2005). Bifidobacteria increase butyric acid concentrations, but these bacteria do not produce butyric acid themselves. Lactic acid bacteria, such as lactobacilli and bifidobacteria, stimulate proliferation of butyric acid producing bacteria. This mechanism is called cross-feeding. It has been shown that lactic acid, produced in vitro by Bifidobacterium adolescentis with starch as sole carbon source, is used by Anaerostipes caccae and Eubacterium hallii (in co-culture) for the production of large concentrations butyric acid [START_REF] Duncan | Contribution of acetate to butyrate formation by human faecal bacteria[END_REF]. Another approach would be a direct stimulation of butyric acid producing bacteria. In human gut samples, butyric acid producers are anaerobic bacteria belonging to the phylogenetic Clostridium clusters IV and XIVa [START_REF] Pryde | The microbiology of butyrate formation in the human colon[END_REF], and species related to Roseburia, Eubacterium, Faecalibacterium and Coprococcus can also produce butyrate [START_REF] Pryde | The microbiology of butyrate formation in the human colon[END_REF]. Many of the butyrate producing microbiota that are identified are net consumers of acetate [START_REF] Duncan | Contribution of acetate to butyrate formation by human faecal bacteria[END_REF]. It is not clear whether similar mechanisms exist in poultry gut microbiota. Random cloning and sequencing of 16S rDNA sequences isolated from chicken caeca revealed more than 85% of the clones belonging to eubacteria and clostridia [START_REF] Bjerrum | The intestinal of broiler chickens[END_REF]. Approximately 10% of the clones had high similarity with Faecalibacterium prausnitzii, a species that produces butyric acid in the human gut [START_REF] Bjerrum | The intestinal of broiler chickens[END_REF]. It is now evident that the addition of organic acids can have a beneficial effect on the quality of poultry by decreasing Salmonella and possibly other potentially pathogenic bacteria. The idea that probiotic and prebiotic applications, or simply rational design of feed composition could lead to similar effects producing favourable SCFA patterns is an interesting hypothesis, but research on this matter in poultry is still lacking. Recent studies have just started to generally describe the caecal microbiota of chickens (Lu et al., 2003a,b;[START_REF] Bjerrum | The intestinal of broiler chickens[END_REF]. , 84, 998-1003. Thompson, J.L. andHinton, M. (1997). Antibacterial activity of formic and propionic acids in the diet of hens on Salmonellas in the crop. British Poultry Science, 38, 59-65. Thormar, H., Hilmarsson, H. and Bersson, G. (2006). Stable concentrated emulsions of the 1monoglyceride of capric acid (monocaprin) with microbicidal activities against the food-borne bacteria Campylobacter jejuni, Salmonella spp., and Escherichia coli. Applied and Environmental Microbiology, 72, 522-526. Van Den berg, B. (2005). The FadL family : unusual transporters for unusual substrates. Current Opinions in Structural Biology, 15, 401-407. Van Der Wielen, P.W., Biesterveld, S., Notermans, S., Hofstra, H., Urlings, B.A. and Van Knapen, F. (2000). Role of volatile fatty acids in development of the cecal microflora in broiler chickens during growth. Applied and Environmental Microbiology, 71, 2206-2207. Van Immerseel, F., De Buck, J., Meulemans, G., Pasmans, F., Velge, P., Bottreau, E., Haesebrouck, F. and Ducatelle, R. (2003) Invasion of Salmonella Enteritidis in avian intestinal epithelial cells in vitro is influenced by short-chain fatty acids, International Journal of Food Microbiology, 85, 237 -248. Van Immerseel, F., De Buck, J., De Smet, I., Pasmans, F., Haesebrouck, F. and Ducatelle, R. (2004). Interactions of butyric acid and acetic acid-treated Salmonella with chicken primary cecal epithelial cells in vitro. Avian Diseases, 48, 384-391. Van Immerseel, F., De Buck, J., Boyen, F., Bohez, L., Pasmans, F., Volf, J., Sevcik, M., Rychlik, I., Haesebrouck, F. and Ducatelle, R. (2004b). Medium-chain fatty acids decrease colonization and invasion shortly after infection with Salmonella Enteritidis in chickens through hilA suppression. Applied and Environmental Microbiology, 70, 3582-3587. Van Immerseel, F., Fievez, V., De Buck, J., Pasmans, F., Martel, A., Haesebrouck, F. and Ducatelle, R. (2004c). Microencapsulated short-chain fatty acids in feed modify colonization and invasion early after infection with Salmonella Enteritidis in young chickens. Poultry Science, 83, 69-74. Van Immerseel, F., Boyen, F., Gantois, I., Timbermont, L., Bohez, L., Pasmans, F., Haesebrouck, F. and Ducatelle, R. (2005). Supplementation of coated butyric acid in the feed reduces colonization and shedding of Salmonella in poultry. Poultry Science, 84, 1851-1856. Van Staden, J.J., Van Der Made, H.N. and Jordaan, E. (1980). The control of bacterial contamination in carcass meal with propionic acid. Onderstepoort Journal of Veterinary Research, 47, 77-82. Williams, J.E. (1981). Salmonella in poultry feed : a worldwide review. World's Poultry Science Journal,37,[6][7][8][9][10][11][12][13][14][15][START_REF] Dirusso | Bacterial long chain fatty acid transport: gateway to a fatty acid-responsive signalling system[END_REF][17][18][START_REF] Louis | Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon[END_REF]

  . When the intracellular acetate concentration of K-12 increased there was a nearly equal molar increase in intracellular potassium. These results indicated that fermentation acid anion accumulation was at least in part an osmotic stress. Recent work with

  inoculated with low doses of S. kedougou, and the decrease was most obvious after several weeks of storage

  4%), hatchery waste (15.3 versus 1.2%) and insert paper samples (4.6 versus 1.4%).

  22%) in the feed was ineffective in reducing the number of Salmonella in the crop and caeca of broilers. There work was based on 8 trials in which the broilers were inoculated with 10 4 cfu

  chain fatty acids could play an important role in Salmonella colonization. If short-chain fatty acid production in the caeca could be altered by changes in feed composition, producers would have a very cost-effective and efficient way of controlling Salmonella.

  .N.,Chung, C.H., Day, D.F., Hinton, A.Jr., Bailey, J.S., Siragusa, G.R. (2005).Isomaltooligosaccharides increases cecal Bifidobacterium population in young broiler chickens. Poultry Science

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp

Acknowledgements

Research on controlling Salmonella in poultry using short-chain fatty acids performed by the authors of this manuscript has been funded by the Federal Service Public Health, Safety of the Food Chain and Environment, Belgium, under contract S6134/1, the European Commission, under contract 505523 (SUPASALVAC), and a Marie Curie Training Fellowship under contract QLK2-CT-2001-60081. Dr. F. Van Immerseel is funded by a post-doctoral research grant of the Ghent University (BOF).


Průková Dana

Vernerová Zdeňka

Pilčík Tomáš

Stepanets Volodymir

Indrová Marie

Geryk Josef

Plachý Jiří

Hejnar Jiří

Svoboda Jan

Jan Svoboda

Differences in pathogenicity among strains of the same or different of avian leukosis virus subgroups

Keywords: Avian leukosis virus (ALV), wasting disease, course of wasting disease, immunopathology of tissues from infected animals

An efficient induction of the wasting disease in chickens by avian leukosis virus (ALV), particularly ALV subgroup C, requires >10 2 IU of virus inoculated in mid embryogenesis.

The most conspicuous symptoms of the disease were induced by ALV subgroup C; however, significant differences in the occurrence of wasting disease were found among individual members of this subgroup. Almost comparable pathogenicity was exhibited by ALV subgroup D, whereas viruses of subgroups B and A proved to be moderately and almost nonpathogenic, respectively. Using antibodies to cellular antigens, tissue alterations were shown clearly in ALV-C-infected chickens. An essential feature was depletion of lymphocytes in the thymus, bursa and spleen. While the number of dendritic cells (DC) in the bursa was increased, their representation in the thymus and spleen was reduced. In the spleen, however, the reduction of DC concerned only an ellipsoid compartment, which in itself was also markedly reduced. An increased number of macrophages in the thymus and spleen corresponded with the observed general activation of the monocyte-macrophage system. In the spleen CD4 + T-cells were reduced while CD8 + T-cells were increased. In agreement with this finding was a failure of chickens to respond to Brucella antigen and inability of their splenocytes to respond to ConA, both of which pointed to the damage of immune reactivity.

Avian leukosis viruses (ALV) are the most common naturally occurring avian retroviruses associated with neoplasia and adverse effects on immunity in domestic chickens. They represent a distinct group of simple alpharetroviruses that are classified into ten subgroups (A to J) according to the properties of viral envelope glycoprotein underlying their immunological cross-reactivity, viral cross-interference and host range [START_REF] Hunter | Viral entry and receptors[END_REF]. Some of them carry a viral oncogene responsible for rapid malignant conversion of target cells (acutely transforming viruses). Slowly transforming viruses, which do not possess an oncogene, are able to induce a broad spectrum of tumours after a long latency by a mechanism of insertional mutagenesis based on the provirus integration into the genome near a cellular protooncogene (Payne, 1992).

In the acute phase of infection, particularly in ALV-congenitally infected chickens, a non-neoplastic syndrome, the wasting disease, with the latency of 2-3 weeks after hatching with an incidence approaching nearly 100%, is induced. The main clinical sign of the wasting syndrome is progressive weight loss, which is accompanied by metabolic and hormonal disorders, depletion of lymphatic organs and immunosuppression, and in some cases also by osteopetrosis, anaemia and occasionally diarrhoea [START_REF] Meyers | Phytohemagglutinin-induced leukocyte blastogenesis in normal and avian leukosis virus-infected chickens[END_REF]Smith and Eldic, 1978;[START_REF] Price | Inhibition of concanavalin A response during osteopetrosis virus infection[END_REF]Rupp et al., 1982;[START_REF] Fadly | Immunocompetence of chickens during early and tumorigenic states of Rous-associated virus-1 infection[END_REF]Carter and Smith, 1983;Carter et al., 1983;[START_REF] Carter | Specificity of avian leukosis virus-induced hyperlipidemia[END_REF]Landmann et al., 2002;reviewed in Svoboda et al., 2003). [START_REF] Carter | Specificity of avian leukosis virus-induced hyperlipidemia[END_REF] pointed out a specific pathogenic effect of subgroup C viruses, characterized by stunting, hypothyroidism, hyperinsulinemia, alteration of lipid metabolism and lymphoblastoid infiltration of liver, thyroid and pancreas. However, a Under natural conditions ALVs spread in the flock by three modes: (1) horizontal transmission, when the virus is passed from bird to bird by direct contact or indirectly, (2) vertical or congenital transmission, when the virus is passed from hen to offspring by the egg albumen, and (3) genetic transmission, which refers to endogenous viral genome transmission from parents to offspring. The mode of transmission has a strong impact upon the pattern of infection: depending on the presence of viraemia, antibodies, or both, infected birds can be classified into four serological classes: (1) viraemia, no antibody, (2) no viraemia, with antibody, (3)viraemia with antibody, (4) no viraemia, no antibody (reviewed in Payne, 1992a). Recently, a novel infection profile was identified in ALV-J-infected broiler breeder flocks, which includes hens shedding viral antigens into the cloaca intermittently [START_REF] Witter | Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny[END_REF]. The most prevalent in the field are exogenous viruses of subgroup A, to a lesser extent viruses of subgroup B, and the occurence of subgroup C and D viruses was reported only rarely. However, epidemic outbreaks of newly generated viral recombinants occurred at the present time, causing significant economical losses (Payne, 1992b). Based on the knowledge of ALV biology and host response to the infection, eradication programs were developed for viruses of subgroups A and B and are successfuly applied to commercial and experimental breeding flocks. For original eradication programs criteria proposed by Payne (1992a) were employed. They include selection of hens negative for viral antigens in the egg albumen and vaginal schwabs, hatching of chicks in small groups, and testing for the presence of ALV in blood followed by breeding of ALV-free chicks in isolation. Additional approaches for selection of resistant chicken lines such as use of resistance to ALV, a recessive genetic trait, and PCR techniques are being introduced. As we have shown previously, a heterologous avian species, ducks are sensitive to wasting disease induction by [START_REF] Stepanets | Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching[END_REF]. However, the scarcity of diagnostic tools such as monoclonal antibodies to duck cellular antigens and the sensitivity of ducks to viruses of subgroup C only [START_REF] Duff | Characteristic of two new avian tumor virus subgroups[END_REF]Vogt, 1969, Nehyba et al. 1990) limit the exploitation of this model. On the other hand, such limitations did not hold for chickens, where a series of diagnostic antibodies as well as chicken strains sensitive to ALVs of all subgroups are available.

Using the chicken model we aimed at clarification of additional features, especially pathomorphological and immunological characteristics of the wasting disease induced by different strains of ALV-C. In order to achieve a proper comparison, we included additional ALV subgroups (A, B, D) as a control.

Material and Methods

Experimental design and animals. In order to prevent cross-contamination with ALV, newly hatched control and infected animals were kept separately. Because of this requirement, the experiments were spread over a period of two years and, during this period 12 experimental groups were set (Table 1). It should be noted that in each experimental group one control group was included. Data from individual experiments were pooled to obtain representative values. Chickens were inoculated intraembryonally via the chorioallantoic vein with 0.1 ml of virus suspension and/or culture medium or post-hatching with the same volume into the jugular vein.)

We defined wasting disease induced by ALV as the reduced ability of infected chickens to grow. The first symptoms of the wasting could be noted by gross physical examination as soon as one week after hatching; however, it turned into a fully developed disease a week later. As a quantitative parameter of the disease we selected the body mass of Virus strains. RAV-1, virus progeny of molecularly cloned RAV-1/OR [START_REF] Felder | Functional and biological properties of an avian variant long terminal repeat containing multiple A to G conversion in the U3 sequence[END_REF] was kindly provided by Dr. G. Calothy, Institute Curie-Biologie, Orsay, France. Viruses RAV-2, RAV-7, RAV-49 and RAV-50 were kindly supplied by Dr. V. Nair, Institute of Animal Health, Compton, United Kingdom. The origin of td daPR-C representing the progeny of a molecular clone of a transformation-defective mutant of the Prague strain Rous sarcoma virus of subgroup C was described previously [START_REF] Stepanets | Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching[END_REF]. td B77-C, a leukosis virus of subgroup C specificity, was kindly provided by Dr. K. Toyoshima, University of Osaka, Japan. (For virus strains see Table 2). Viruses were grown on chicken embryo fibroblast cell line DF-1 cell line [START_REF] Himly | The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses[END_REF] [START_REF] Stepanets | Amyloid A amyloidosis in non-infected and avian leukosis virus-C persistently infected inbred ducks[END_REF]. As measured by the 16Q test (see below), standard production of 10 6-7 infectious units (IU) per ml was obtained with all virus strains.

Determination of the replicating virus in plasma

The presence of infectious virus in chicken plasma was determined by the complementation test with 16Q cells (quail cells harboring the provirus of high-titer Bryan strain Rous sarcoma virus (BH-RSV) lacking the env gene) [START_REF] Murphy | A new-replication-defective variant of the Bryan high-titre strain of Rous sarcoma virus[END_REF], essentially as described previously [START_REF] Stepanets | Amyloid A amyloidosis in non-infected and avian leukosis virus-C persistently infected inbred ducks[END_REF].

Briefly, chicken embryo fibroblasts (CEF) were incubated with 100 µl of chicken plasma, subcultured twice and co-cultivated with 16Q cells for one week. Transforming pseudotypes of BH-RSV were detected by the focus assay.

Immunization of chickens with heat-inactivated Brucella abortus. Chickens were immunized with Brucella abortus essentially as described by [START_REF] Stepanets | Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching[END_REF]. Briefly, preimmune sera were collected two days before the immunization started at the age of 26 days. Chickens were inoculated with heat-inactivated Brucella abortus, strain Weybridge S99 twice a week for a period of one month. A single immunization dose represented 6 x 10 7 bacteria per 10 g of the body weight and was administered in 0.2 ml of phosphate-buffered saline (PBS) intravenously. Three days after the last immunization, chicken sera were collected and antibody titers determined using the agglutination method as described previously [START_REF] Stepanets | Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching[END_REF]. The titers were expressed as reciprocals of the terminal serum dilution producing the positive reaction. Isolation of splenocytes and mitogenesis assay. Spleens were harvested from 14-day-old chickens at autopsy. After cutting with scissors, single-cell suspensions were made by straining the pieces through a 70-µm nylon mesh (Falcon, Becton Dickinson Labware, NJ, USA). Splenic lymphocytes were resuspended in RPMI 1640 (Sigma, St. Louis, USA) supplemented with 1% fetal bovine serum (FCS) (Gibco BRL Life Technologies, Germany), purified by centrifugation for 20 min at 150 g over a Ficoll-Paque (Amersham Pharmacia Biotech, Buckinghamshire, UK) gradient and the collected cells were washed twice in RPMI 1640 supplemented with 1% FCS. 3 H-TdR incorporation assay was performed as described earlier [START_REF] Indrová | IFNα therapy of renal cell carcinoma: defect of lymphocyte sensitivity to mitogenic and activating cytokine signals in patients non-responding to therapy[END_REF][START_REF] Sobota | Use of cryopreserved lymphocytes for assessment of the immunological effects of interferon therapy in renal cell carcinoma patients[END_REF]. Briefly, for priming of proliferative spleen cell responses, freshly isolated lymphocytes were cultivated in a concentration of 0.5 x 10 6 /ml in complete RPMI 1640 medium supplemented with 10% FCS, 0.5 ml of 200 mM Lglutamine (Sigma, St. Louis, USA), antibiotics and 10 -5 M of mercaptoethanol (Sigma, St.

Louis, USA) alone or supplemented with 1 µg/ml of ConA (Pharmingen, San Diego, USA).

Cells were resuspended in a total volume of 0.2 ml of culture medium and incubated minimally in quadruplicate in 96-well round bottom microtiter plates (Nunc, Roskilde, Denmark) for 48 hrs, at 37° C in a humidified atmosphere with 5% CO 2 . Eighteen hours before harvesting, the cells were labelled with 0.04 MBq 3 H-TdR per well and the uptake of 3 H-TdR was measured by LS 6000 Liquid Scintillation Counter (Beckman, Fullerton, CA, USA) and recorded as counts per minutes (cpm). Proliferative responses were determined as the stimulation index (SI) = average cpm value in samples incubated with mitogenic agent/average cpm value in samples incubated in medium alone.

Histology, histochemistry and immunohistochemistry. Animals were weighed and euthanasied at 14 th day after hatching. Their lymphatic organs were quickly removed, weighed and trimmed into two parts. One was fixed in buffered 4% paraformaldehyde, pH Paraformaldehyde-fixed samples were embedded in paraffin, stained with hematoxylin and eosin and Gomori stain. For immunohistochemistry 4 % paraformaldehyde-fixed 6 µm cryostat sections were used. The antibodies used, their origin, working dilutions and cell types with which they react are listed in Table 3. The reaction product was visualized by incubation with En Vision detection kit (Dako, Cytomation, Denmark). Only the antibody against CD3 was visualized with LSAB+ detection kit (Dako, Cytomation, Denmark). Acid phosphatase activity was demonstrated by the simultaneous azo-coupling method with naphthol AS BI phosphate (Sigma) as a substrate and hexazotized pararosanilin as diazonium salt according to Lojda et al. (1976).

Western blot analysis. Two-week-old control and infected chickens (RAV-1 and td daPR-C) were sacrificed and thymus, bursa and spleen were collected, immediately frozen and stored at -80 o C. Protein extracts were prepared using Triton lysis buffer (25 mM HEPES, 1% Triton, 2 mM EDTA, 0.1 M NaCl, 25 mM NaF, 1 mM Na 3 VO 4 ) supplemented with leupeptin, pepstatin and antipain (Sigma-Aldrich, St. Louis, MI, USA) (1 µg/ml of each) and PMSF (Sigma-Aldrich, St. Louis, MI, USA) (3 mg/ml). Total protein concentration was assessed and normalized by the standard Bradford assay. The amount of 30 µg of total protein was separated on 15% SDS PAGE, transferred onto the PVDF membrane (Amersham Biosciences, Piskataway, NJ, USA), blocked with 5% non-fat milk in PBS-T buffer (PBS supplemented with 0.1% TWEEN, Sigma-Aldrich, St. Louis, MI, USA) and incubated for 60 min with anti-p27 gag and anti-alpha-tubulin TU-01. After washing the membranes were incubated with anti-mouse IgG -HRP (horse raddish peroxidase)-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) for 60 min at room temperature, washed, and immunoreactive proteins were detected with the chemiluminescence system ECLplus (Amersham Biosciences, Piskataway, NJ, USA) according to the manufacturer`s instructions.

Statistical data analysis.

To test the null hypothesis -i.e. no difference exists in the abovementioned parameters between chickens infected with leukosis viruses and chickens that belong to the control group -we used the Dunnett test as an extension to ANOVA (GLM), implemented in: http://www.R-project.org.Version 2.1.0 (R, 2005).

Results

Role of the age and the administered virus dose on ALV-C-induced wasting disease in chickens.

ALVs have a large potential to induce a broad spectrum of neoplastic and nonneoplastic disorders, particulary when congenitaly transmitted or intraembryonally inoculated, since this mode of infection provides optimal conditions for induction of persistent viraemia after hatching. Actively replicating virus reintegrates into the genome of target cells, which is a prerequisite for its accidental integration into the vicinity of a cellular protooncogene, leading finally to oncogene activation and tumour conversion of the cell. ALV-induced tumours could therefore be detected after a long latent period. In the meantime, however, the infected organism is continually supplied by a large quantity of viral antigens, which may disturb either directly or indirectly by so far unknown mechanisms different physiological functions. The outcome of this disturbance is acute non-neoplastic diseases such as wasting disease detected soon after hatching. To evaluate the potential of the highly pathogenic td daPR-C in wasting disease induction, we inoculated chickens at early stages of their development with 10 5 IU of virus (Table 4).

We observed a significant decrease (p<0.01) of the body mass in all intraembryonally infected chickens at two weeks after hatching. In addition, the mass of thymus and bursa was 12 also decreased, while the mass of the spleen was increased (p<0.01). The significance of the observed parameters was not influenced by the sex of experimental animals. Moderate changes, yet still significant (p<0.05), were obtained when newly hatched chickens were inoculated. However, chickens inoculated later at 7th or 14th day of age and sacrificed on day 21 or 28, respectively, exhibited only a significant increase of the spleen mass (p<0.05) .

Since different time periods of virus inoculation during embryogenesis (10th to 14th day) did not play a role in the induction of wasting disease, we selected mid embryogenesis (12th day) as an optimal time and used it in all further experiments.

To reveal possible virus-dose dependent induction of wasting disease we infected experimental animals with decimally diluted td daPR-C. As given in Table 5, the most pronounced changes were observed in the group inoculated with the highest dose of the virus (10 5 IU); however, virus diluted up to 10 2 IU still exerted significant changes in all monitored parameters. This data agreed with the occurrence of viraemia detected actually in all animals infected with a highly diluted virus (10 2 IU). In the group of birds inoculated with 10 1 IU, 85% of animals were viremic, displaying significantly increased spleen mass; however, no wasting and only moderate impairment of the thymus were found. Interestingly, also in the group receiving 10 0 IU some viremic animals were present (18%), but no wasting was detected. Viraemia at two weeks of age was not tested; however, the absence of any wasting or alteration of lymphoid organs at this time (Table 5) would also indicate the absence of efficiently replicating virus in these animals. Since it is known that the course of infection is variable (Payne, 1992a[START_REF] Witter | Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny[END_REF], we cannot exclude the possibility that chickens inoculated with highly diluted virus and tested negatively on the 2nd day after hatching might shed virus later in life.

Wasting disease induced by intraembryonic inoculation of different ALV strains.

To estimate the potential of individual virus strains in wasting disease induction, we decided to 6, RAV-1 of subgroup A proved to be quite non-pathogenic. Pathogenicity of RAV-2 of subgroup B was clearly demonstrated since significant changes could be observed in the thymus and bursa, which, however, were not accomapnied by wasting and spleen alteration. On the contrary, viruses of subgroup C (RAV-7, td daPR-C and td B77-C) and RAV-50 of subgroup D turned out to be highly pathogenic, producing significant changes of both the body and lymphatic organ mass. The only exception was RAV-49 (subgroup C), producing more moderate, yet still significant decrease of the body mass. As it has been already well documented (Carter and Smith, 1983, 1984, Carter et al., 1983), RAV-7 appeared to be most virulent among the studied viruses, since both body and organ mass were strikingly decreased in infected chickens. This conclusion is also supported by the highest post-hatching mortality found in RAV-7-infected chickens. It should be noted that in comparison to control animals no markedly decreased hatchability was found in infected chickens. Only in some pathogenic viruses (RAV-50, RAV-49 and td daPR-C), an increased embryonic mortality could be registered (Table 6). . All viruses replicated well in vivo (Table 6). Only moderate differences in the level of viraemia were observed between individual viral strains. Surprisingly, RAV-1 and RAV-2, both of which did not induce wasting disease , replicated even better (10 6 -10 7 IU/ml) than viruses of subgroups C and D (10 5 IU/ml), which exhibit a higher pathogenicity. This finding was in accord with the higher and lower amount of the viral Gag protein detected by the Western blotting in lymphatic tissues of chickens infected with RAV-1 and td daPR-C, respectively (not shown).

Morphological picture of wasting disease in ALV-infected chickens. In all but control and RAV-1-infected animals examined at two weeks after hatching (see Table 6 As severe alterations of thyroid, pancreas and liver by ALVs have already been recognized and described in detail, including disturbances in blood chemistry (Carter and Smith, 1983, 1984, Carter et al., 1983), we concentrated on lymphoid tissue alterations induced by ALVs of different subgroup specificity and above all by viruses of subgroup C.

The gross morphological changes of lymphatic organs corresponded well with tissue alterations revealed by light microscopy (Table 7). The thymic cortex of ALV-C-infected chickens was totally disintegrated with pronounced lymphatic depletion, accentuated cortical epithelial cells and activated monocyte-macrophage system, as demonstrated on the sample from td daPR-C-infected chicken (Fig. 1a,b). In animals infected with RAV-7 and td B77-C the lobular architecture of thymuses vanished. The majority of cells in the cortex was stained positively for the T-cell-specific antigen CD3. Depletion of lymphocytes was not accompanied by marked differences in representation of CD4 + and CD8 + T lymphocytes. Morphological changes in the spleen were observed only in ALV-C and ALV-Dinfected chickens and were present predominantly in the white pulp. The red pulp was only slightly deteriorated. B-cell areas (the peri-ellipsoid lymphocyte sheaths -PELS and the germinal centers) were reduced or in the case of RAV-49 and td B77-C-infected chickens nearly absent (Fig. 1c,d). T-cell areas, which together with B cells participate in the formation of periarteriolar lymphocyte sheaths (PALS), were also slightly reduced (Fig. 1e,f). The majority of T cells had the CD8 + and TCR2 + phenotype. In the red pulp CD8 + T cells predominated, too. Splenic ellipsoid-associated reticulum cells (EARC; they form a sheath of spindle-shaped cells around penicilliform capillaries) identified by CVI 68.2 antibody were markedly reduced or even disintegrated in chickens infected with RAV-7 and td daPR-C (Fig. 1g,h). Spleens of all infected chickens contained increased numbers of macrophages arranged mostly around the ellipsoid (Fig. 1i,j). Slight fibrous changes of the reticular tissue might have contributed to the observed increased splenic mass in ALV-C and D-infected chickens.

Lymphatic tissues were stained positively for viral p27 gag antigen in all infected chickens; however, a weaker signal was observed in the thymus and bursa in ALV-C and Dinfected chickens. At the cellular level p27 gag was regularly revealed in macrophages of all tissues examined, thymic epithelium, follicular dendritic cells, splenic ellipsoid cells and in some B lymphocytes. Morphological alteration of lymphoid organs of diseased chickens, indicated a heavily impaired immune system. To evaluate the level of immunosuppression we performed a blastogenic assay with ConA-stimulated spleen lymphocytes of two-week-old chickens.

Infection with td daPR-C clearly inhibited blastogenic response of splenocytes stimulated with 10 µg of the lectin. On the contrary, only moderate inhibition was recorded in splenocytes from RAV-1-infected chickens (Table 8). Similarly, a difference between both viruses was scored when responsiveness to the bacterial antigen was tested in one-month-old chickens. After repeated administration of heat-inactivated Brucella abortus, about 70% of td daPR-C-infected chickens did not produce any antibodies and only a minority of them was able to mount a potent humoral response to the antigen (Table 9). In contrast, RAV-1-infected chickens responded to the antigen as efficiently as control animals, with the exception of a small fraction of birds (13%) producing a lower titre (40-80) of antibodies.

Discussion. Retroviruses belong to infectious agents that can efficiently establish persistent infection, the outcome of which is frequently wasting disease and immunosuppression leading to the general devastation and finally to the death of the host. Pathogenesis of the wasting disease appears to be associated with both, hormonal imbalance manifested by a reduced thyroxine level [START_REF] Carter | Specificity of avian leukosis virus-induced hyperlipidemia[END_REF] and apparently a direct toxic effect of the virus.

Cytotoxicity has been well documented in ALVs of subgroup B (ALV-B) and D (ALV-D)

employing for cellular entry a receptor (tvb) related to the tumor necrosis factor receptor [START_REF] Brojatsch | TVB receptors for cytopathic and noncytopathic subgroups of avian leukosis viruses are functional death receptors[END_REF]. Also ALVs of subgroup C (ALV-C), whose the cellular receptor has been identified recently [START_REF] Elleder | The receptor for the subgroup C avian sarcoma and leukosis viruses, Tvc, is related to mammalian butyrophilins, members of the immunoglobulin superfamily[END_REF], are well known as potent inducers of the wasting disease (Carter et al., 1983;Carter and Smith, 1983;[START_REF] Carter | Specificity of avian leukosis virus-induced hyperlipidemia[END_REF] and anemia, the severity of which, however, appears to be strain-dependent [START_REF] Smith | Induction of anemia by avian leukosis viruses of five subgroups[END_REF]; [START_REF] Karakoz | In vivo effect of three transformation-defective mutants of subgroup C avian sarcoma viruses[END_REF][START_REF] Stepanets | Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching[END_REF]. The function of the Tvc protein, a new member of the butyrophilin family, is not known. However, the documented cythopathogenicity of ALV-C for DF-1 cells [START_REF] Himly | The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses[END_REF] indicates, that some death-promoting activity of the tvc receptor might be stimulated upon binding of the retrovirus. Obviously, the signalling pathway would be different from those one activated by ALV-B and -D viruses. It should be noted that in adition to the genetic outfit of the virus and tissue tropism determined by a functional receptor, other tissue-specific cellular factors supporting efficient retroviral expression (Svoboda, 1998, Sorin and[START_REF] Sorin | Dynamics of virus-host interplay in HIV-1 replication[END_REF] decide upon the pathogenicity of the particular viral strain.

In the present study we have attempted to define the potential of several ALV-C virus strains to induce wasting disease within the first two weeks after hatching and to compare it to other ALVs of subgroups A, B, and D.

For efficient wasting disease induction, the embryonic stage of the host plays a critical role [START_REF] Smith | Pathogenesis of virus-induced osteopetrosis in the chicken[END_REF]. As shown in Table 5 the lowest virus dose administered intraembryonally into chicken embryos and exerting the pathological activity approaches the value of 1 -10 IU. This observation is in accordance with data obtained in another experimental system, juvenile pigtail macaques infected with highly pathogenic simian immunodeficiency virus variant SIVsmmPBj14, where 1 TCID 50 (tissue culture infecting dose) produced acutely lethal entheropathic syndrome (O'Neill et al., 1999).

Clearly, the threshold amount of inoculated virus is a necessary, but not sufficient, prerequisite for induction of the acute wasting disease. We can specially imagine that the virus in terminal dilutions produces a low degree of viraemia over the observation period, which is not sufficient for triggering wasting disease. Similarly, chicken endogenous virus RAV-0 equipped with a deficient enhancer and not efficiently replicating shows no signs of pathogenicity [START_REF] Robinson | Subgroup-E Avian-leukosis-virus-associated disease in chickens[END_REF]Cullen etal., 1983). In our case, a high virus dose was used for inoculation and all tested ALVs replicated well in vivo (Table 6). Hence, they should readily produce wasting disease. Surprisingly, the opposite is true since RAV-1 and RAV-2, reaching the highest titres in vivo, exhibit no or moderate pathogenicity, respectively, and viruses of subgroups C and D, which replicate less efficiently, are highly pathogenic. Apparently, viral gene products participate in the acute pathogenicicty of ALVs and, of them, the Env protein appears to be the main candidate. An abundant knowledge has been accumulated so far on the role of the Env protein in retrovirus pathogenicity (reviewed in [START_REF] Svoboda | Heterogenous pathogenicity of retroviruses: lessons from birds, primates, and rodents[END_REF].

When comparing the acute pathogenicity of ALV viruses of A, B, C and D subgroup specificity we have encountered notable differences among individual viral strains. In agreement with published data [START_REF] Carter | Specificity of avian leukosis virus-induced hyperlipidemia[END_REF][START_REF] Stepanets | Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching[END_REF], the most distinctive changes were produced by viruses of subgroup C, among them only RAV-49 appeared less eficient in wasting disease induction (Table 6). A moderate pathogenicity not accompanied by wasting was scored in RAV-2-infected chickens, in which involution only of the thymus and bursa were found. The moderate pathogenicity of RAV-2 was surprising, as other viral strains employing the same receptor associated with cytotoxicity, MAV-2(O) of subgroup B [START_REF] Hirota | Immunopathology of chickens infected in ovo and at hatching with the avian osteopetrosis virus MAV.2-0[END_REF][START_REF] Smith | Pathogenesis of virus-induced osteopetrosis in the chicken[END_REF] and RAV-50 of subgroup D (this study), were proven to be highly pathogenic in vivo. The data on RAV-2 and RAV-49 document that within both subgroups, B and C, differences in pathogenicity exist between individual viral strains and indicate that both, wasting and the lymphoid tissue alteration may be induced independently. This conclusion is also supported by recently published data on ALV-J pathogenicity for in ovo infected chickens, in which significant growth suppression was not accompanied by alteration of immune responsiveness [START_REF] Landman | Effect of an in ovo infection with a Dutch avian leukosis virus subgroup J isolate on the growth and immunological performance of SPF broiler chickens[END_REF].

Endogenous avian retroviral sequences (EAV) that participated in ALV-J formation [START_REF] Bai | HPRS-103 (exogenous avian leukosis virus, subgroup J) has an env gene related to those of endogenous elements EAV-0 and E51 an E element found previously only in sarcoma viruses[END_REF] represent a potential source of genetic material for the occurrence of recombinants The most conspicuous histopathological changes were found in chickens infected with subgroup C and D, and less distinctively with subgroup B viruses. It should be stressed that accidental infection of chickens with immunosuppressive agents such as CIAV and IBDV seems to be unlikely since no antiviral antibodies could be detected in our breeding flock of BL chickens. The pathological lesions found in ALV-infected chickens differed clearly from those induced by CIAV or IBDV infection [START_REF] Von Bülow | Chicken infectious anemia[END_REF]Schacht, 1997, Lukert and[START_REF] Lukert | Infectious Bursal Disease[END_REF]. We also never observed clinical symptoms of arthritis induced by reovirus infection, which can be accompanied by other disease conditions, e.g. growth retardation, bursal and thymic atrophy (Rosenberger and Olson, 1997). Alterations revealed in the thymus tissue of these animals, accompanied in some cases even with collapsed lobular architecture and slight fibrosis, resembled precocious puberty [START_REF] Seemayer | Precocious thymic involution manifest by epithelial injury in the acquired immune deficiency syndrome[END_REF] or thymic epithelial injury found in both children and adult patients suffering acquired immunodeficiency syndrome [START_REF] Ye | The thymus during HIV disease: Role in pathogenesis and in immune recovery[END_REF]Hazra and Mackal, 2005). Detection of p27 gag protein indicated replication of the virus in thymic epithelial cells. Expressed retroviral proteins may injure functions of this cellular compartment, which plays an important role in promotion and maturation of thymocytes through the action of secreted humoral substances or through the direct contact with thymocytes [START_REF] Lobach | Ontogeny of the human thymus during fetal development[END_REF]. An observed increase of TCR2 + T cells in the thymus might represent a consequence of such functional thymic disturbance, the detailed mechanism of which remains, however, unclear.

Impairment of bursal tissue in animals infected with subgroup C viruses resembled the changes induced by intraembryonic infection with MAV-2(0), subgroup B virus [START_REF] Smith | Pathogenesis of virus-induced osteopetrosis in the chicken[END_REF][START_REF] Hirota | Immunopathology of chickens infected in ovo and at hatching with the avian osteopetrosis virus MAV.2-0[END_REF]. In accord with altered bursal morphology was a finding of severe impairment of humoral immunity in td daPR-C-infected chickens that failed in mounting an antibody response to Brucella abortus antigen. Surprisingly, a small fraction of animals were, however, able to respond to the antigen in the same manner as controls (Table 9). Since outbred chickens were used, probably host factors would influence their resistance to immunosuppresive activity of the virus. MHC-dependent variations in immune response to viral antigens and the rate of progression of Rous sarcoma virus-induced tumours in congenic chicken lines have been well established [START_REF] Plachý | src-specific immunity in inbred chickens bearing v-src DNA-and RSV-induced tumors[END_REF][START_REF] Hoffman | v-src oncogene-specific carboxy-terminal peptide is immunoprotective against Rous sarcoma growth in chickens with MHC class I allele B-F12[END_REF]. Two weeks after hatching RAV-1-infected chickens have apparently preserved intact humoral immunity.

The most remarkable changes in the spleen were encountered again in chickens infected with subgroup C and D viruses. Namely ALV-C viruses produced an impairment of PELS (considered for a functional equivalent of the mammalian marginal zone, [START_REF] Jeurissen | Histological and functional differentiation of non-lymphoid cells in the chicken spleen[END_REF] and PALS regions, which is characterized by a marked reduction of B-and T cells with increased CD8 + and depleted CD4 + lymphocytes. These changes resembled both damage of the marginal zone and shifts in CD8 + and CD4 + T-lymphocyte representation in the spleen of HIV-infected patients [START_REF] Wilkins | Splenic marginal zone atrophy and progressive CD8+ T-lymphocytosis in HIV infection: a study of adult postmortem spleens from Cote d´Ivoire[END_REF]. The observed disruption of a sheath of ellipsoidal cells is apparently in accord with the presence of live virus in the spleen as similar results were found by [START_REF] Jeurissen | In situ detection by monoclonal antibody D-35.1 of cells infected with Marek's disease virus that interact with splenic ellipsoid-associated reticulum cells[END_REF] in chickens infected with Marek's disease virus.

However, intact ellipsoids in RAV-1-and RAV-2-infected chickens would indicate some selectivity of this cellular compartment in responding to different viral antigens.

The severe alteration of lymphoid tissues agreed well with the inefficiency of splenic lymphocytes isolated from td daPR-C-infected chickens in responding properly to ConA (Table 6). Subgroup C viruses thus appear as immunosupressive as MAV-2(O) [START_REF] Smith | Characterization of the immunosuppression accompanying virus-induced avian osteopetrosis[END_REF][START_REF] Price | Inhibition of concanavalin A response during osteopetrosis virus infection[END_REF]. Contrary to that, splenic lymphocytes of RAV-1- (Rupp et al., 1982;[START_REF] Fadly | Immunocompetence of chickens during early and tumorigenic states of Rous-associated virus-1 infection[END_REF].

The affinity of ALVs for lymphoid tissues resulting in severe morphological alteration of the thymus, bursa and spleen followed by subversion of immune regulatory functions appears to be a unifying aspect of ALV acute pathogenicity. However, in tissue sections from infected animals irrespective of the viral strain, only some B lymphocytes stained positively for the viral Gag protein, indicating active viral replication. Instead, other lymphoid cellular compartments such as macrophages, thymic epithelium, follicular dendritic cells and splenic ellipsoid cells were largely infected. In the pathogenesis of ALV-induced immune damage the direct toxic effect of viruses, namely those of subgroup B and D, has been invoked. However, the direct toxicity may not be a principal cause of lymphoid tissue depletion; more likely lymphocytes are attacked in an indirect way, as it has been shown in another retroviral infection [START_REF] Alimonti | Mechanisms of CD4 + T lymphocyte cell death in human immunodeficiency virus infection and AIDS[END_REF]. The striking differences in acute pathogenicity of individual strains of ALVs offer a unique experimental system for analysis of viral genome regions playing a significant role in establishing wasting disease. Despite a large body of our knowledge about adverse effects of retroviral infections, the mechanism responsible for wasting disease in infected hosts has not yet been fully clarified. In this respect, comparative studies of the wasting disease and immunosuppression employing different experimental models appear valuable, because they could bring new insights into the pathogenesis of acute retrovirus-induced diseases. Chicken embryos and young chickens inoculated with 10 5 IU of td daPR-C on10th, 12th, and14th day of incubation or 1st, 7th and 14th day post hatching. Determination of body and organ mass two weeks after hatching (intraembryonic infection) or on14th, 21st and 28th day of age (post-hatching infection).

Table 2 2 and 3 Chicken embryos inoculated in mid embryogenesis with different doses of td daPR-C (10 5 to 10 -2 IU). Determination of body and organ mass two weeks after hatching.

F o r P e e r R e v i e w

O n l y

  pathogenicity among individual ALV strains provides ground for depicting gene sequences playing an important role in ALV acute pathogenicity.

  of pathomorphological changes in lymphoid tissues induced by ALVs of different subgroup specificity, has not been performed.

  after hatching, when the body mass data of infected and control chickens were significantly different.Brown Leghorn (BL) chickens of the phenotype C/E from the breeding colony of the Institute of Molecular Genetics were used in all experiments. They are free of avian leukosis viruses (ALV), chicken anaemia virus (CIAV), infectious bursal disease virus (IBDV) and other common poultry pathogens. The BL stock meets the SPF status except for reovirus infection since only reovirus-specific antibodies can be detected; however, no clinical symptoms of tenosynovitis or infectious stunting syndrome associated with avian reovirus infection has ever been found in the flock. No vaccination has been applied in our breeding colony of chickens. The animals are regularly examined by the state veterinarian.Experimental chickens were reared on wire floored isolation units and fed commercial feed mixture "400 AGK K1" (complete feed mixture for breeding young chickens of the first period; metabolizable energy, 12,031 MJ/kg: ZZN, Slaný, Czech Republic) and tap water ad libitum. All experiments were carried out in accordance with the Home Office of the Czech Republic Guidelines.

  and the other was frozen in liquid nitrogen vapor and stored at -80 o C before use.

  stocks (10 5 -10 6 IU/0.1ml) for infection of chicken embryos. As shown in Table

  was characterized by involution of the thymus and bursa. In some cases, mainly in subgroup C-infected chickens, the thymic lobes were represented only by a narrow strip of lymphoid tissue hardly discernible from the surrounding connective tissue. Strikingly increased spleens were regularly found in all chickens inoculated with both, subgroup C and D viruses. No other pathological changes were identified even in chickens that died before killed for examination. Only some RAV-1-infected animals developed peritonitis accompanied by ascites at the time of sacrifice.

  Only a slight increase (up to 50% in comparison with 30% in controls) of thymic TCR2 + Tlymphocytes was noted in RAV-7, RAV-49 and td daPR-C-infected chickens. A slight reduction of follicular dendritic cells and paucity of Hassal's corpuscles was revealed in the thymic medulla of all ALV C-infected chickens.In bursa of Fabricius (BF) of ALV-C-infected chickens, a markedly decreased number of lymphoid follicles having a reduced amount of cortical lymphocytes was partially fibrous changes. The most impaired architecture of lymphatic follicles was produced by RAV-7 and td B77-C. The connective tissue around follicular residues contained a slightly increased amount of T lymphocytes and the monocytomacrophage system appeared to be activated. Follicular dendritic cells were moderately increased in follicular centers of chickens infected with td daPR-C and RAV-49.

  Impairment of the immune system in chickens with ALV-induced wasting disease.

  properties different from those of non-recombinant viral strains. Whether and how the differences in pathogenicity are related to certain mutations in the viral genome, especially in the env gene is being investigated.

  to stimulation by ConA, which is in accord with published data

  Also, providing the B. abortus antigen and determination of agglutination titers in chicken sera by Dr. Plačkov, P. (Bioveta Ivanovice na Hané, Czech Republic) is greatly acknowledged. The authors wish to thank Mikušová, L., Takáčová, Š. and Kolářová, M., for excellent technical assistance. This work was suported by Grant No. 523/04/0489 (Grant Agency of the Czech Republic) and in part by project no. AV0Z50520514 awarded by the Academy of Sciences of the Czech Republic.

Figure legends Fig. 1

 legends1 Figure legends

  for immunohistochemical analysis were collected from at least two independent experiments. b Number of animals examined by immunohistochemistry. c No alteration. d Decreased number of cells or size of the cellular compartment: -, slightly, --, intermediately, ---, considerably, ----, strongly. e CK AE1/AE3 antibody. f Increased number of cells or size of the cellular compartment: +, slightly, ++, intermediately, +++, considerably, ++++, strongly. g Kul-1 antibody. h MAb6 antibody. i CVI-74.3 antibody. j B-cells detected by HIS-C1 antibody. k PELS, peri-ellipsoid lymphocyte sheaths, B cells detected by HIS-C1 antibody. l PALS, periarteriolar lymphocyte sheaths, T cells detected by CD3 antibody. m CD8 + T cells detected by 11-39 antibody. n CD4 + T cells detected by 2-6 antibody. o EARC, ellipsoid-associated reticulum cells detected by CVI 68.2 antibody.

  except for subgroup C viruses, which duck embryo fibroblasts (DEF, isolated from inbred Khaki Campbell ducks of the D/ABDE phenotype from the breeding colony of the Institute of Molecular Genetics,

	were propagated on
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	8

Table 1 .

 1 Design of twelve experiments perfomed

	Experiment	Design	Data referred-to
	1

Table 7

 7

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 2 .

 2 List of viruses used Indicated virus doses (in 0.1 ml) were used for inoculation of animals in all experiments. Only td daPR-C was serially diluted up to 10 -2 IU (see Table5) when testing the relation of virus pathogenicity to the injected virus dose.

		Subgroup	Virus dose used in the
	Virus strain	specificity	experiments a	Origin of virus strains
	RAV-1	A	10 6	Felder et al., 1994
	RAV-2	B	10 6	Nair,V., Institute for
				Animal Health,
				Compton, United
				Kingdom
	RAV-7	C	10 6	Nair,V., Institute for
				Animal Health,
				Compton, United
				Kingdom
	RAV-49	C	10 6	Nair,V., Institute for
				Animal Health,
				Compton, United
				Kingdom
	td daPR-C	C	10 5	Geryk et al., 1979,
				Kashuba et al., 1993
	td B77-C	C	10 6	Toyoshima et al.,
				1970
	RAV-50	D	10 6	Nair,V., Institute for
				Animal Health,
				Compton, United
				Kingdom
	a

Table 3 .

 3 List of primary antibodies useda MAb against Ch, monoclonal antibody directed against chicken antigen; b MAb against H, monoclonal antibody directed against human antigen; c Rb against H, polyclonal rabbit antibody directed against human antigen.

	Antibody Type	Source	Diluti-	Recognized
				on	molecule and/or
					cells
	11-39	MAb against Ch a , IgG 1 ,	Dr.O. Vainio, University of	1:200	CD8 + T-cell
		ascites	Turku, Turku, Finland		subpopulation
			(Luthala et al., 1995)
	2-6	MAb against Ch, IgG 1 ,	Dr.O. Vainio, University of	1:200	CD4 + T-cell
		ascites	Turku, Turku, Finland		subpopulation
			(Luthala et al., 1993)
	MAb6	MAb against Ch, IgG 1 ,	Dr. J. Čihák, University of	1:300	TCR2 + T-cell
		ascites	Munich, Munich, Germany		subpopulation
			(Cihak, J. et al., 1988)
	Kul 01	MAb against Ch,	Dr. J. Mast, Veterinary and	1:200	monocytes,
		ascites	Agrochemical Research		macrophages,
			Centre, Brussels, Belgium.		interdigitating cells
			(Mast et al., 1998)
	HY 21.1	MAb against Avian	Dr. E. Humphries, West	1:1000	epitope on the
		leukemia/sarcoma	Virginia University, WV,		retroviral p27 gag
		viruses, IgG 2a , culture	U.S.A
		supernatant	(unpublished)
	CK	MAb against H b , IgG 1 ,	DakoCytomation, Denmark	1:50	thymic epithelial
	AE1/AE3	ascites	A/S, Denmark		cells
	HIS-C1	MAb against Ch, IgG 1	Cedi-Diagnostics B.V.,	1:10	Bu-1/B cells
			Lelystad, The Netherlands
	CVI-68.2 MAb against Ch, IgG 1	Cedi-Diagnostics B.V.,	1:10	ellipsoid-associated
			Lelystad, The Netherlands		reticulum cells
	CD 3	Rb against H c	DakoCytomation, Denmark	1 : 200	T cells
			A/S, Denmark
	CVI-74.3 MAb against Ch, IgG 1	Cedi-Diagnostics B.V.,	1:10	follicular dendritic
			Lelystad, The Netherlands		cells
	TU-01	MAb against pig alpha-	EXBIO Praha, Prague, Czech	1:2000	epitope on the N-
		tubulin, IgG1	Republic		terminal structural
					domain of alpha-
					tubulin

Table 4 .

 4 Body mass and relative mass of lymphoid organs in chickens infected in different periods of embryogenesis and post-hatching with td daPR-C aa Chickens were inoculated intravenously in different periods of embryogenesis into a chorioallantoic vein or into jugular vein after hatching with medium (non-infected) or td daPR-C. Intraembryonically infected chickens were sacrificed two weeks after hatching, newly hatched chickens two weeks after administration of the virus (ie. on 14th, 21st and 28th day of age). At the time of sacrifice the mass of the body and lymphoid organs was determined. b Mean ± SD; Relative organ mass: (organ mass in mg/body mass in g) x 100. c Mean ± SD. . of infected chickens. f The estimates for control and infected chickens marked ** are significantly different (Dunnett test; p<0.01), those marked * are slightly significantly different (Dunnett test; p<0.05). g ns, not significant.

	Hatchability: 10-day-old embryos

d no

Table 5 .

 5 Body mass and relative mass of lymphoid organs in chickens infected in mid embryogenesis with serial 10-fold dilutions of td daPR-C aRelative organ mass c Chickens were inoculated intravenously in mid embryogenesis with medium (non-infected) or decimally diluted td daPR-C. Two weeks after hatching the animals were sacrificed and the mass of the body and lymphatic organs was determined. b Infectious virus present in the sample of blood plasma collected on the second day after hatching was detected by 16Q test (see Material and Methods). c Mean ± SD; relative organ mass see legend to Table4. d Hatchability: non-infected animals 94%, 10 5 IU td daPR-C 78%, in all other groups infected with diluted virus it ranged from 85 to 100% without any relation to the inoculated virus dose. e Mean ± SD. f The estimates for control and infected chickens marked **

			No. of	Viraemia b
		Virus dose	chickens	(viraemic/	Body mass
	Experiment	(IU)	tested d	tested)	(g) e	S f	Thymus	S	Bursa	S	Spleen	S
	I.	Non-infected	11	na g	84.7 ± 8.4	na	348 ± 68	na	245 ± 51	na	79 ± 11	na
		10 5	7	nt h	57.5 ± 15.8	**	149 ± 80	**	137 ± 45	**	190 ± 38	**
		10 4	13	nt	61.4 ± 10.1	**	101 ± 38	**	134 ± 22	**	187 ± 22	**
		10 3	15	nt	74.4 ± 7.4	**	142 ± 37	**	161 ± 41	**	219 ± 61	**
		10 2	11	nt	72.2±11.9	**	152 ± 33	**	151 ± 19	**	164 ± 29	**
	II.	Non-infected	12	nt	106.9 ± 14.2	na	352 ± 69	na	264 ± 64	na	113 ± 15	na
		10 2	11	11/11	76.4 ± 8.6	**	184 ± 80	**	192 ± 45	**	193 ± 27	**
		10 1	13	11/13	91.9 ± 11.7	ns h	284 ± 86	*	232 ± 58	ns	190 ± 61	**
		10 0	11	2/11	89.0 ± 11.1	ns	363 ± 105	ns	275 ± 68	ns	125 ± 67	ns
		10 -1	12	0/12	117.3 ± 25.3	ns	396 ± 95	ns	288 ± 38	ns	120 ± 24	ns
		10 -2	13	0/13	117.7 ± 11.1	ns	390 ± 61	ns	266 ± 85	ns	109 ± 21	ns
	are significantly different (Dunnett test; p<0.01), those marked * are slightly significantly different (Dunnett test; p<0.05). g na, not applicable. h
	not tested

a

Table 6 .

 6 Body mass and relative mass of lymphoid organs in two-week-old chickens infected in mid embryogenesis with avian leukosis viruses of different subgroup specificity aRelative organ mass b Chickens were inoculated intravenously in mid embryogenesis with medium (non-infected) or avian leukosis viruses of subgroups A to D. Two weeks after hatching the animals were sacrificed and the mass of the body and lymphatic organs was determined. b See legend to Table2. c Animals died within two weeks after hatching. d The titer of infectious virus present in the blood plasma collected two weeks after hatching was determined by 16Q test (see Material and Methods). e Mean ± SD. f S, see legend to Table4. g na, not applicable. h ns, not significant.

			Hatchability/Post-
		No. of	hatching death c								Viraemia d
		chickens	(%)	Body mass (g) e							(IU/ml)
	Virus strain	tested			S f	Thymus	S	Bursa	S	Spleen	S	[median(min /max) (n)]
	Non-infected	177	89/0	100.5 ± 20.8	na g 334 ± 86	na 257 ± 70 na 114 ± 40 na	na
	RAV-1	71	86/0	101.9 ± 17.5	ns h 328 ± 73	ns 247 ± 61 ns 124 ± 32 ns	1.0 x 10 7 (10 5 -10 7 ) (4)
	RAV-2	32	87/3	93.7 ± 26.1	ns	241 ± 80	** 199 ± 42 ** 119 ± 37 ns 5.5 x 10 6 (10 6 -10 7 ) (4)
	RAV-7	41	87/11	64.4 ± 13.7	** 132 ± 48	**	99 ± 44	**	166 ±51 ** 1.0 x 10 5 (10 5 -10 6 ) (6)
	RAV-49	29	75/0	91.6 ± 14.6	*	171 ± 56	** 133 ± 41 ** 186 ± 51 ** 5.5 x 10 5 (10 5 -10 6 ) (4)
	td daPR-C	114	81/2	73.4 ± 16.5	** 156 ± 59	** 121 ± 36 ** 192 ± 54 ** 5.5 x 10 5 (10 5 -10 6 ) (4)
	td B77-C	67	89/0	70.5 ± 14.1	** 155 ± 67	** 119 ± 47 ** 156 ± 52 ** 1.0 x 10 5 (10 4 -10 6 ) (6)
	RAV-50	25	70/4	81.2 ± 24.0	** 168 ± 48	** 157 ± 40 ** 164 ± 53 ** 5.5 x 10 5 (10 5 -10 6 ) (4)

a

Table 7 :

 7 Summary of morphological findings in lymphatic organs of ALV-infected chickens a

Table 8 .

 8 Proliferative spleen cell responses of control and virus-infected chickens a3 H -thymidine incorporation x 10 3[mean ± SD (n)] b Chickens were inoculated intravenously in mid embryogenesis with a culture medium (noninfected) and td daPR-C or RAV-1. Two weeks after hatching the animals were sacrificed and spleens were used to prepare of cell suspensions. b Incorporation of 3 H-TdR in non-stimulated and Con-A-stimulated cells was determined and the stimulation index (SI) was calculated. c S, see legend to Table4.

	Spleen cell	Hatchability
	donor	(%)	Non-stimulated Con A-stimulated	SI	S c
	Non-infected	90	0,39 ± 0,22 (8)	3,73 ± 2,21 (8)	9,6	**
	td daPR-C	75	0,27 ± 0,09 (8)	0,36 ± 0,15 (9)	1,3	ns
	RAV-1	87	0,60 ± 0,17 (5)	2,26 ± 0,89 (5)	3,8	*

a

Table 9 .

 9 Antibody response of Brown Leghorn chickens intraembryonally infected with avian leukosis viruses to Brucella abortus antigen aa Chickens were inoculated intravenously in mid embryogenesis with a culture medium (noninfected), td daPR-C and RAV-1. Bacterial antigen was administered into jugular vein twice a week in a period from 4 to 8 weeks of age. b The titer of antibodies represents the reciprocals of the terminal dilution of sera producing the positive reaction. (Note that titer of antibodies corresponding to 20 signifies almost a lack of immune response.)

	No. of animals producing antibody

150x122mm (300 x 300 DPI)

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Acknowledgements

The authors are very thankful to Dr. V. Nair, (Viral Oncogenesis Group, Institute for Animal Health, Compton, United Kingdom), to Dr. G. Calothy (Institute Curie-Biologie, Orsay, France) and to Dr. K. Toyoshima (University of Osaka, Japan) for providing avian leukosis