Salmonella in broilers

Salmonellae are gram-negative bacteria, which belong to the genus Salmonella which is part of the family of Enterobacteriaceae (bacteria living in the intestine). Salmonellae are non-encapsulated bacteria that can grow under either aerobic or anaerobic conditions. The optimum temperature for growth is 35 to 43ºC while the optimum pH is 6.6 to 8.2. However, Salmonellae can continue to grow at pH values between 4.5 and 9.5 and at temperatures between 5 and 54 ºC. A water activity above 0.94 is also required (Hanes, 2003).

Most of the salmonellae are motile bacteria that use flagella to move. Some serotypes like S. Gallinarum or S. Pullorum are non-motile. Among the different Salmonella serotypes, Salmonella Enteritidis and S. Typhimurium are presented separately from others because, on the one hand, these bacteria are often specifically cited in zoonosis control legislation, and, secondly, because there are differences in the epidemiology as compared to other salmonellae. Salmonella Enteritidis and S. Typhimurium are the predominant serotypes associated with human disease in most countries. Indeed, Salmonella spp. is one of the major causes of food poisoning in humans. According to the European Food Safety Authority (EFSA, 2009), a total of 154,099 cases of human salmonellosis were reported by the 25 EU Member States in 2004. The major sources of food borne salmonellosis are eggs and poultry meat (EFSA, 2005).

In the European Union, a report from EFSA published in 2007 indicates that an average of 23.7% of broiler farms in EU were Salmonella positive, with a wide variation (0 to 68%) between countries (see Figure 1). The most common serotypes were S. Enteritidis, S. Infantis, S. Mbandaka, S. Typhimurium and S. Hadar. As a consequence, the Commission Regulation (EC) No 646/2007 of 12 June 2007 states that The Community target shall be a reduction of the maximum percentage of flocks of broilers remaining positive for Salmonella Enteritidis and Salmonella Typhimurium to 1 % or less by 31 December 2011.

In the United States, non typhoidal Salmonella was reported to cause 1.3 million cases of food borne illness, 15,608 associated hospitalizations, and 553 deaths per year (Mead et al., 1999). A new Federal Regulation that is applicable since September 2009 requires egg producers with more than 3,000 laying hens to take steps to prevent the spread of Salmonella Enteritidis. Farms with more than 50,000 laying hens need to comply with the rules by July 2010, and the rest must comply by July 2012. The rules, which affect the purchase of chicks and young hens, sanitation in production facilities, testing for the bacteria, and storage of eggs at farms, are expected to affect 99% of the nation's egg production. The FDA estimates the measures will cost egg producers about $81 million annually.

In Thailand, different publications describe the extent of contamination at the farm level. For instance, Sasipreeyajan et al. (1996) investigated Salmonella contaminations in 35 different poultry farms. The bacteria were found in all broiler flocks (13) and all breeder farms (7) while 13 out of 15 layer farms were positive. A total of 1,488 samples were analyzed and salmonellae were found in samples of litter (42%), water in drinking troughs (36%), feed left over in the feed trays (28%), water in the main tanks (17%), cloacal swabs (13%) and stock feed (8%). Jerngklinchani et al. (1994) observed that 66% of 705 chicken meat samples collected from nine open markets, nine supermarkets and four poultry processing plants in Bangkok were positive for Salmonella. A similar survey was made by Boonmar et al. (1997) who found that 72% of chicken meat samples purchased from 10 retail markets in Bangkok were positive, while 10% of samples from one slaughterhouse for export were contaminated with the bacteria. Chicken faeces obtained from three farms located in the east region of Thailand were also analyzed and 7% were positive. In this report, the most predominant serotype was S. Enteritidis, which was isolated from 28% of the retail chicken meat, 5% of the chicken meat from slaughterhouse, and all the positive samples of chicken faeces.

More recently, Boonprasert et al. (2009) evaluated the prevalence of Salmonella in two broiler breeder farms in Northern Thailand (Lamphun province). Samples were taken in a total of twenty nine flocks, at different ages of the birds. The overall Salmonella prevalence (at least 1 pair of boot swab faecal samples positive) of the breeding flocks was 82.8% and this increased with age, from 15.4% at the early stage (24-28 weeks of age), to 84.2% at middle (42-49 weeks of age) and 100.0% at late stage (57-69 week of age). Chaengprachak et al. (2009) made a similar study in broiler flocks of the 77 contract farms of an industrial poultry production company in Northern Thailand (Chiang Mai and Lamphun provinces). The Salmonella-positive flock prevalence was 91.6% in one-day-old chicks and 98.6% at 3 weeks before leaving for slaughter, respectively. The authors concluded that Salmonella infection was not due to the production farms themselves, but came from the one-day-old chicks, vertically infected from their breeder flocks or during transport from the hatchery. Tangkawattana et al. (2009) assessed the extent of contamination in chicken stalls in fresh markets of five upper northeastern provinces of Thailand. Out of more than 100 samples of meat and liver, 42% were contaminated.  The strains were evaluated for their resistance to antibiotics. Highest incidence of resistance was for tetracyclin (59.2%), followed by amoxicillin (36.7%), ciprofloxacin (32.7%), gentamicin (18.4%), norfloxacin (16.3%), chloramphenicol (12.2%) and sulfamethoxazole + trimethoprim (10.2%).

Indeed, of particular concern is the increasing number of human Salmonella infections that are resistant to antibiotics, and this is the main reason for phasing out antibiotics in animal farming. For instance, one strain of S. Typhimurium has emerged as resistant to five drugs: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (Helms et al., 2002). In Brazil, Duarte et al. (2009) observed that out of 19 Salmonella isolates found in broiler chicken carcasses and tested for antimicrobial resistance, 94.7% were resistant to at least one antimicrobial agent. Resistance to streptomycin (73.7%), nitrofurantoin (52.3%), tetracycline (31.6%), and nalidixic acid (21%) were the prevalent amongst Salmonella strains tested.

Reducing Salmonella contamination on poultry carcasses requires a complete approach that includes the entire integrated broiler operation, the breeding flocks, hatchery, broiler growers, feed mills and transporters. Therefore, in order to control Salmonella, one should consider the critical points of the farm to table continuum (see Figure 2).

According to the European Food Safety Agency, the primary production is the most important source of Salmonella spp. entering the food chain (EFSA, 2004a) and a strict control of Salmonella spp. in all parent flocks reduces the frequency of contamination at the broiler production stage (SCVPH, 2000). This was demonstrated in Denmark where a control programme based on top-down eradication, has permitted a reduction from more than 65% of Salmonella-infected broiler flocks in 1989 to less than 5% in 2000 (Wegener et al., 2003). Similar control programmes in Finland, Sweden and Norway have allowed reducing the prevalence of Salmonella spp. in broiler flocks since 1996 to less than 1% (EFSA, 2004a). According to Hald et al (2004), the reduction of prevalence in the broiler farms leads to a reduction of prevalence in meat and a reduction of human cases of salmonellosis.

The sources of salmonella infection in poultry farms are various (see Figure 3). Infection can take place via vertical transmission (poor hygiene and Salmonella contamination in the previous flock, contaminated chicks)  or horizontal transmission (feed, water, contact with rodents, insects, pets, birds or other domestic and wild animals, personnel or visitor, litter and faeces, dust, etc...). Some less obvious risk-factors like the size of the farm (farms with more than 3 houses have higher risks presumably because of increased human traffic between multiple sheds) or the climate (humid and warm) have been reported by Rose et al. (1999).

In order to efficiently control Salmonella spp. at the farm level, a set of biosecurity measures needs to be applied. More than often, people believe that a new technology or product will solve all their problems, while only a long term and complete strategy can help them to succeed. Presently, there is not enough data allowing prioritizing between the different intervention strategies. Therefore, a combination of different interventions will allow achieving significant reductions in the frequency of Salmonella-contaminated broilers sent to slaughter. The rest of this paper will review the possible actions that should be part of a comprehensive programme.

Vertical transmission can occur either by egg shell contamination or internal contamination of the yolk. Of course the first measure will be to ensure that non-contaminated day old chicks are delivered to the farm.

Mixing different species or different batches of animals will increase the risk of contaminating a flock that was free of pathogens. Consequently, isolation practices such as mono-species segregation and all in-all out segregation by age will be helpful. Once again, relying on only one technique will increase risks of failure. Indeed, Lo Fo Wong et al. (2004) explained that the reason why limited and contradictory evidence exists to support the effectiveness of all in-all out is probably because the practice of personnel changing clothing and boots prior to entering or leaving the area is not always applied.

Another risk factor is the level of stress. Although this parameter is not easy to quantify and some stresses such as feed withdrawal or transport are inevitable, any management practice that will reduce stress can be helpful in reducing contamination levels. Feed withdrawal is used prior to slaughter to prevent the carcass contamination by gut content. However, Byrd et al. (1998) observed that feed withdrawal significantly increases Campylobacter and Salmonella contamination levels in the crops of broilers. Hinton et al. (2002) report that giving a glucose-based treatment to broilers helps to counter the undesirable effects of feed withdrawal.

An important target must be the reduction of exposure of the broilers to animals (wild or not), other birds, insects, and of course visitors. Different strategies such as using nets, fences, insecticides, etc... can be implemented.  For visitors, their access should be restricted to only essential personnel. They should use disinfectant foot dips that are frequently replaced. Inside the buildings, good management of litter can help reducing contamination. Carr et al. (1995) reported that litter moisture is normally between 25% and 35% and that limiting water activity to these levels creates a less favourable environment for the growth of Salmonella than more humid litters.

Vaccination of poultry against Salmonella uses live or inactivated vaccines. However, according to EFSA (2004b), vaccination of chickens should be considered as an additional measure to enhance the resistance of birds against Salmonella and reduce the shedding. Not all vaccines are effective against all serotypes and basic practices such as good farming and hygienic procedures (involving feed, birds, management, cleaning and disinfection, control of rodents etc.) combined with testing and removal of positive flocks remain necessary for a successful control of Salmonella in poultry.

Water can be a source of contamination for the broilers. Treating the water with disinfectants such as chlorine, ozone, or sodium chlorate will only be efficient if the water system is clean. Indeed, these chemicals will react with organic matter and be neutralized. Mohyla et al. (2007) report for instance that acidified sodium chlorite (600 g / 1000 litres of drinking water) reduced Salmonella in the crop. However, a double dosage was needed to reduce Salmonella in the lower digestive tract of the broilers. A shortcoming to the use of disinfectants is they reduce water intake by animals.  In order to reduce these negative consequences, a sporadic usage followed by a flushing is usually practiced.

Salmonella in broilers may also be reduced by acidifying the drinking water with organic acids, especially during feed withdrawal. For example Byrd et al. (2001) report that lactic acid or formic acid at 0.5% in drinking water before slaughter were effective to reduce Salmonella populations in broilers. Such dosages are high and it is advisable to gradually expose the birds to the acid in the water during the week before harvesting to prevent a drop in water intake. Combinations of organic acids such as Gustor Liquid, developed by Norel & Nature, allow reducing the dosage while maintaining the efficacy. Figure 4 shows the results obtained during a trial conducted on a broiler farm in Thailand. Using 2 kg / 1,000 liters water for the first 2 weeks, and then reducing the dosage to 1 kg / 1,000 liters (6 hours per day), has allowed to improve growth performance by 11% while mortality decreased by 24%.

It has been estimated that about 15% of the Salmonella contaminations in poultry products are caused by feed and that 90% of the Salmonella present in feed is caused by contaminated raw materials, especially those with high protein levels such as animal by-products or fishmeal, as well as heat treated products that are easily re-contaminated. Henken et al. (1992) calculated that farms receiving contaminated feed were 5.3 times more likely to produce Salmonella-positive flocks than farms supplied with microbiologically safe feed.

Besides raw materials, major risk factors in the feed mill are the bottom of the elevators, aspiration filters, top of cooler and cooler room, and top of bin for finished feed. Shut down periods usually increase the risk of proliferation of pathogens.

Different strategies have been suggested to control Salmonella in feed (Wray, 2001). Meal seems to reduce the risk when compared to pelleted feed (probably because of recontamination issues), but Salmonella prevalence is greater when finely ground as compared with coarsely ground.

Heat treatment of feed, combined with increased moisture, is a common practice in feed mills. In theory, Salmonella is inactivated by a temperature of 77°C for one second, but in practice it is not always easy to guarantee a homogeneous heat treatment and therefore stricter conditions are applied. Cox et al. (1986) reported a good control during pelleting of poultry feeds at temperatures exceeding 83°C. A supplier of equipment thermal conditioners recommends a minimum retention time of 90 seconds, with a temperature of 80 to 88°C (Rousseaux, personal communication, 2009).

Appropriate heat treatment of feed is efficient, but recontamination, in the feed mill, during transport or on the farm, is likely to happen. Moreover, Salmonella is able to survive prolonged periods in dry environments. Therefore it is advisable to use also a chemical treatment. Products such as Salmonat, developed by NOREL&NATURE, are based on buffered organic acids, to minimize corrosion of feed equipment and negative effects to animal growth or health. They will reduce pathogen contamination and ensure protection of the feed even after pelleting. Last but not least, we should not forget that excessive heat treatment of feed will usually result in reduced animal performance (reduced growth and higher FCR).

Use of specific formulation strategies or additives will modify the environment of the gastrointestinal tract and may help control pathogens such as Salmonella. For instance, using 10% egg yolk powder in feed of broilers reduced S. Typhimurium in faces to undetectable levels at 2 weeks of age (Kassaify and Mine, 2004).

Several types of feed additives can help controlling Salmonella and other Enterobacteriaceae in the gut of broilers. They can be categorized as antibiotics, organic acids and their salts, plant extracts, prebiotics and probiotics. Antibiotics, especially when used at low doses, are raising concerns that broiler food products may be a source of resistant salmonella for humans. The appearance of cases of multi-resistance in food borne Salmonella isolates suggests the need for more prudent use of antibiotics by farmers, veterinarians, and physicians (Carraminana et al., 2004). Indeed, the trend is that fewer antibiotics can be used in broiler feed as it becomes a strong demand from the consumer in many countries.

Organic acids are typical products of microbial metabolism and have a long history as food preservatives due to their antimicrobial properties. In solution, organic acids exist in pH-dependant equilibrium between uncharged, acid molecules and their respective charged anions (for example acetic acid / acetate). The key basic principle of their mode of action is that when non-dissociated (non-ionized, more lipophilic) they can penetrate the bacteria cell wall and disrupt the normal physiology of certain types of bacteria. Since the proportion of dissociated acid increases as pH increases, once inside the cell, they will be exposed to the near neutral intracellular pH of the bacteria and dissociate, liberating an anion (A-) and a proton (H+) in the cytoplasm (Russell and Diez-Gonzalez 1998). The internal pH will decrease and because pH sensitive bacteria do not tolerate a big difference between the internal and the external pH, a specific mechanism (H+ -ATPase pump) will act to bring the pH inside the bacteria to a normal level. This phenomenon consumes energy and eventually can stop the growth of the bacteria or even kill it. The anionic (A-) part of the acid is trapped inside the bacteria because it will diffuse freely through the cell wall only in its non-dissociated form. The accumulation of (A-) becomes toxic to the bacteria (Russell 1992) by complex mechanisms resulting in to inhibition of metabolic reactions (Krebs et al. 1983), reduction the synthesis of macromolecules (Cherrington et al. 1991), or disruption membranes (Freese et al. 1973). On the contrary, the non-pH sensitive bacteria (such as lactic acid bacteria) will tolerate a larger differential between the internal and the external pH, if the internal pH becomes low enough, organic acids will re-appear in a non-dissociated form and exit the bacteria by the same route it went in. Another explanation for this may be that Gram-positive bacteria have a high concentration of intracellular potassium, which provides a counter cation for the acid anions (Russell and Diez-Gonzalez 1998). An important parameter to take into account is the constant of dissociation of the acid (pKa), which is the pH value for which the concentrations of dissociated and undissociated species are equal. This means for example that formic acid (pKa = 3.75) will be 50% dissociated and 50% undissociated at a pH value of 3.75. So anti-bacterial activity of organic acids will also be influenced by the pH of the intestinal tract. The higher the pH value, the more they will tend to dissociate.

Among organic acids, butyric acid possesses interesting characteristics that make it "not just an acid". Butyric acid is a natural product of the bacterial fermentation of the carbohydrates in the intestine of monogastrics, or in the rumen of ruminants. With acetic and propionic acid, butyric acid belongs to the group of VFAs (Volatile Fatty Acids). It is usually applied in feed as a salt of sodium (sodium butyrate) which makes its handling easier since it is solid, stable and much less odorous. In the large intestine, sodium butyrate is rapidly absorbed to provide energy to the epithelial cells and promote sodium and water assimilation. Over many years, different researchers have shown positive effects of sodium butyrate on the intestinal epithelium, such as increase in the villi length and crypt depth, which result in a better absorption of nutrients. More recent research demonstrates an anti-inflammatory effect of sodium butyrate on gastric and intestinal mucosal cells. The immune system seems less challenged, which results in a better overall use of the nutrients absorbed. This explains the positive influence of sodium butyrate on the body weight gain and feed conversion of poultry, swine or calves.

For several years already Norel & Nature has been investigating the benefits of using butyric acid in different species (swine, poultry, ruminants but also aquaculture) as well as the production and handling aspects. Indeed, the free form of this acid is difficult to handle due to its highly corrosive properties. Since 2005, when the Spanish company presented Gustor B-coated (micro-encapsulated sodium butyrate) the R&D department has continued their investigations with the aim of producing a more concentrated, though still protected, product to add to their range of slow-release, natural growth promoters. This has led to the development of Gustor BP-70.

In Gustor BP-70, a specific fat coating is used to protect the active ingredients. This allows the sodium butyrate to reach the distal sections of the GI tract, not only acting as a natural growth promoter but also reducing the levels of pathogenic bacteria, especially Salmonella. This was demonstrated during a recent trial conducted at the University of Leon in Spain. Two groups of 100 broilers each were given either a standard diet (control) or the same feed containing 1.3 kg of Gustor BP-70 per ton feed. The test lasted for 42 days. At day 1, all birds were confirmed being negative for Salmonella Enteritidis and at day 5, twenty percent of birds from each group were challenged by oral inoculation with 10⁵ UFC of Salmonella. Cloacal samples were taken on days 6, 9, 13, 27, and 41 to monitor infection levels. At day 42 all birds were slaughtered and crop, caecum, liver and spleen were tested. It was observed that the number of birds shedding Salmonella (positive birds) increased steadily during the experiment in the control group. At the end of the trial, all broilers from the control group were positive for Salmonella. On the contrary, birds fed with Gustor BP-70 showed a consistent and significant (p<0.001) reduction of Salmonella Enteritidis infection after the second week (Figure 5). At the end of the trial, 90 % of the birds from the control group had caeca and crops colonized with Salmonella at slaughter, while 90% of the broilers receiving the feed containing Gustor BP-70 were negative at the caecum level, and 80% of them were negative at the crop level. At the internal organs level, it was observed that 20% of the birds from the control group had their liver and spleen colonized with Salmonella. No colonization at all was found in liver from birds treated with the butyrate-protected additive, and only 10% of the broilers were positive at the spleen level. These results demonstrate that the specific production process used in the design of Gustor BP-70 allows a unique coating which ensures a slow release of the sodium butyrate during digestion, and an effect all along the gastrointestinal tract. Therefore, Gustor BP-70 has a positive effect on bird health by preventing Salmonella colonization at intestinal and systemic phases. (Fernández-Rubio et al., 2009)

According to literature, it is known that essential oils from certain plants possess antimicrobial activity against different pathogens, including Salmonella. For instance Koscova et al. (2006) report the beneficial effect of essential oils from oregano (Origanum vulgare) and thyme (Thymus vulgaris). Phadungkit (2005) demonstrated that extract from the fruit of Ardisia elliptica, a tropical plant that can reach heights of up to 5 metres was helpful in controlling Salmonella in broilers.

Prebiotics can be defined as non-digestible feed ingredients that stimulate the growth or activity of beneficial bacteria and limit the development of pathogens in the digestive tract of the animal. For instance, fructooligosaccharides, mannose-oligosaccharides, and isomaltooligosaccharides are potential prebiotics as they promote the development of Bifidobacterium and Lactobacillus species and reduce the prevalence of Salmonella in broilers (Fernandez et al., 2002, Fukata et al., 1999). Ishihara et al. (2000) observed beneficial effects of partially hydrolyzed guar gum, a source of galactomannans, in layers.

Probiotics can be defined as a preparation or product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonisation) in a compartment of the host, and by that exert beneficial health effects in this host (Schrezenmeir and de Vriese, 2001).

The concept of probiotics goes back to Elie Mechnikoff who suggested in 1907 that replacing or diminishing the number of 'putrefactive' bacteria in the gut with lactic acid bacteria could improve health of the intestinal tract. In animal nutrition, the first preparations appeared on the market about 40 years ago and usually had the image of miracle products. Nowadays, most of the strains available are clearly identified and have demonstrated their efficacy through scientific trials, especially if they are registered in European Union. One example is Ecobiol, a strain of Bacillus amyloliquefaciens developed and owned by Norel & Nature. One of the first characteristics that a probiotic must demonstrate to be considered as a viable alternative to AGP is a good stability. Being a sporulated bacteria, Ecobiol is very stable during storage but also resistant to heat treatment. We have for instance observed that when a temperature of 90 degrees Celsius was applied for one minute, Ecobiol recovery rate was around 90%. Trials in complete feed have shown that pelleting did not affect the stability of Ecobiol. This probiotic also shows a good tolerance to several antibiotics commonly used in animal husbandry, such as Colistine, Amoxicilline, Oxytetracycline, Sulfamide, Tiamuline, Neomicine, Zinc Bacitracine, or Avilamycine.

One of the unique features of Bacillus amyloliquefaciens is that it is able to produce a natural antibiotic protein called barnase. Barnase is a ribonuclease (commonly abbreviated as Rnase). This is a specific type of enzyme (nuclease) that catalyzes the degradation of RNA into smaller components. When Ecobiol produces Barnase in its environment, replication of pathogenic bacteria is prevented. This was demonstrated in vitro and in vivo on bacteria such as E. coli, Salmonella, or Clostridium perfringens. At the same time, beneficial bacteria such as Lactobacilli grew faster. In a trial conducted at the Leon University in 2008, Ecobiol was able to prevent the spreading of Salmonella in a flock of broiler after oral contamination of 20% of the birds (Figure 6). It also prevented successfully colonization at crop (p<0.001) and liver (p<0.05) levels, with fewer positive Salmonella samples in caecum (p=0.14) and no differences in the spleen.

 

Salmonella are one of the major sources of food-borne infections in humans, and poultry meat and eggs are among the major Salmonella carriers in the food chain. Reducing Salmonella contamination in poultry products requires a complete approach that includes the entire integrated broiler operation, the breeding flocks, hatchery, broiler growers, feed mills and transporters. Therefore, in order to obtain a substantial reduction of pathogen loads, the application of a combination of intervention strategies is required. A variety of promising intervention practices are available, but, to be useful on the farm they must demonstrate their efficacy, being practical and economically acceptable. They also need to be safe and not interfere with animal growth and development. Last but not least, they must be acceptable to the consumer.

 

Figure 1: Prevalence (%) of Salmonella-positive flocks in different countries of the European Union. Data between brackets indicate the number of farms tested. Source: EFSA, 2007.

 

Figure 2: The farm to table continuum (adapted from Codex Committee on Food Hygiene, 2007).

 

Figure 3: Infection routes of Salmonella in broiler production (Adapted from Oostenbach, 2004).

Figure 4: Performances of broilers when using Gustor Liquid in the drinking water. Results obtained on a broiler farm in Thailand (Norel & Nature SA, Madrid, Spain)

 

Figure 5: Faecal shedding of Salmonella Enteriditis-infected broilers fed with the partially protected Gustor XXI BP70 butyrate-based additive (Norel & Nature SA, Madrid, Spain)

Figure 6: Faecal shedding of S. Enteritidis-infected broilers after 6, 9, 13, 20, 27, 34 and 41 days post-hatching in control and ECOBIOL supplemented group.

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