Efficacy and safety assessment of microbiological feed additive for chicken broilers in tolerance studies

Nowadays probiotics are widely used in animal nutrition, as evidenced by the variety of different types of bacterial feed additives on the EU market. Currently probiotics are used in feeding of poultry, pigs, sows, hens, turkeys, fattening cattle, dairy cows, lambs, sheep, goats, and rabbits (20). Probiotics are live microorganisms, mainly Gram-positive bacteria (Lactobacillus, Lactococcus, Enterococcus, Carnobacterium, Streptococcus, and Bacillus), gram-negative bacteria (Shewanella, Aeromonas, Vibrio, Enterobacter, and Pseudomonas), and fungi (Saccharomyces, Debaryomyces, and Phaffia). The mechanisms by which probiotics benefit the host’s health are known and have been confirmed by numerous studies (4). Probiotics can primarily be used to protect against bacterial pathogens, which is of great importance in large-scale rearing, where animals are exposed to stressful conditions and, for that reason, are more susceptible to infections (15). Colonisation of animals’ intestines by potentially pathogenic bacterial strains can be limited by competition with indigenous intestinal flora, mainly lactic acid bacteria (LAB) (8).

In order for a given probiotic strain to be marketed as feed additive, it must pass through the procedure for their authorization under Regulation (EC) No 1831/2003. Article 6 and Annex I of the Regulation provide categories and functional groups of feed additives. Probiotics are classified under the category “zootechnical additives”. In the case of registration of probiotic strains, the efficacy test of the additive must be performed on the target species. The positive effect of the probiotic supplement should be demonstrated by a decrease in the morbidity or mortality of the species tested and a more complete use of feed ingredients (20). Moreover, Commission Regulation (EC) No 429/2008 provides rules for the implementation of Regulation (EC) No 1831/2003 and imposes the obligation to provide a safety assessment (tolerance test) of the use of the probiotic additive by the target animals at the highest proposed levels in feed or water and at multiple levels.

The aim of a tolerance test is to provide a limited evaluation of the short-term toxicity of the additive to the target animals. It is also used to establish a margin of safety, if the additive is consumed at higher doses than recommended. Such tolerance tests must be conducted to provide evidence for the safety of the probiotic for the target animal. Test animals should be routinely monitored for visual evidence of clinical effects, performance characteristics, product quality, haematology and routine blood chemistry (7). Particular attention should also be paid to the acquisition of resistance to antibiotics and the transfer of resistance genes by probiotic strains used as feed additives (20).

The aim of the study was to provide an efficacy and safety assessment of the use of potentially probiotic lactic acid bacterium strains in the feed during broiler chickens rearing in the tolerance test.

A wide variety of bacterial species are used as probiotics in animal nutrition. Some of them are little known and their use may represent a risk for the target species. The European Food Safety Authority (EFSA) has proposed a system for a pre-market safety assessment of selected groups of microorganisms. Microorganisms which raise safety concerns could be granted QPS status (Qualified Presumption of Safety) and be released from a full safety assessment (toxins or virulence factors should be demonstrated to be absent or of no concern) (21). Lactobacillus plantarum and L. rhamnosus have QPS status, so a full package of toxicological studies is not necessary. No more is required than providing evidence of the safety of using bacteria from these species in the target animal nutrition, as stated in Commission Regulation (EC) No 429/2008.

According to published data the effect of probiotics on the performance of chickens can be various. No positive effects were reported of probiotic Lactobacillus johnsonii strain on weight gain or feed conversion irrespective of the method of its administration (in feed, drinking water, sprayed on litter, or administered directly to the beak) during five weeks of Cobb broiler rearing (18). In another study the probiotics Lactobacillus fermentum CCM 7158 and Enterococcus faecium M 74 were added to drinking water and were found to have a positive effect on weight gain compared to Ross 308 broiler chickens not administered probiotics. Probiotics did not affect body dimensions such as length of the back, body circumference, thigh length lower leg (11).

A beneficial effect of probiotics on broiler chicken body weight is not imparted in every instance and depends on the strain used or their particular combination. In this study the impact of the Lactobacillus rhamnosus KKP 825 strain with the Lactobacillus plantarum K KKP 529/p strain on body weight was not as clear as in another study, where Lactobacillus rhamnosus KKP 825 was used in combination with Lactobacillus paracasei KKP 824 and Lactobacillus rhamnosus KKP 826 (5).

The impact of probiotics on feed consumption can also vary depending on the strain used. The feeding of broiler chickens with probiotics resulted in a significant reduction in feed conversion ratio (FCR) and feed consumption per kg of bird weight gain compared to birds not fed probiotics (16). Meanwhile no effect of probiotic addition to feed (Lb. johnsonii, Lb. crispatus, and Lb. salivarius) was found on weight gain, feed intake, or FCR of Cobb broiler chickens during five weeks of rearing (17).

An important factor influencing the effectiveness of probiotics in poultry is also the manner and time of their administration. It has been shown that administration of probiotics in feed, compared to drinking water, contributed to a higher mean daily body weight gain (24).

Blood examination is performed as a screening procedure to assess general health. Clinical signs of illness in birds are frequently subtle, so clinical chemistry is necessary to evaluate cellular changes (23). However, it is difficult to compare the results of blood or biochemical parameters of birds with other authors’ results because of the influence upon them of diet, age, rearing behaviour, environmental conditions, bird species, and sex (2, 12). On the other hand, according to Mazurkiewicz (15), the reference values of blood biochemical parameters for broiler chickens are the same for both sexes.

A key tool in the diagnosis of certain diseases is the determination of enzyme activity in the metabolic profile. Increasing enzyme activity in blood may indicate damage to cellular structures and is proportional to its degree (13). Interpretation of the assay results, however, is difficult due to the wide range of enzyme activities (9).

AST activity is currently considered to be a very sensitive but nonspecific biomarker of hepatocellular disease and is used with the muscle-specific enzyme creatine kinase (CK) to differentiate between liver and muscle damage (10). In the presented study CK was slightly above the reference value in all experimental groups, but AST was at a very high level (384–563.5 U L^–1), much higher than the maximum reference value (15). AST and CK activities increase under pressure (e.g. during slaughtering), and these stressful circumstances being impossible to completely eliminate under experimental conditions may explain these raised levels. AST was also higher than in other described studies (2, 3). AST levels did not differ significantly depending on age and sex of chickens (2).

Total protein is important information used to establish supportive care. A decline in total protein may indicate protein-losing nephropathy, enteropathy or liver failure. In the case of total protein insufficiency, increasing dehydration should be investigated (10). Total protein in all experimental groups in this study was consistent with the reference values reported in the literature (6, 15).

Uric acid is a product of the catabolic breakdown of proteins and its concentration in serum depends on age, diet, and reproduction (23). According to Harr (10), increasing concentration of uric acid is due to renal disease and decreasing concentration is caused by liver failure. The concentration of uric acid in all experimental groups in this study was in accordance with the references values (6, 15), so no damage to said organs was observed. Recorded by Albokhadaim et al., the concentration of uric acid in the blood of four-week- old chickens was independent of sex and was higher than in this study but the chickens were reared at much higher temperature (32°C) (2). Single administration of probiotic to one-day-old chicks resulted in increasing concentration of blood-carbohydrate, glucose, protein, and haemoglobin levels after 35 days of rearing (22).

In this study no significant escalatory effect of LAB administration on bile acids in chickens’ blood was observed. Bile acids are used to assess liver function. A concentration of bile acids higher than 75 μmol L¹ suggests hepatic insufficiency, while a concentration higher than 100 μmol L^–1 is diagnostic for decreased liver function, which could be caused by exposure to environmental hazards such as feed contamination (10).

In literature amassed data on haematological values of broiler chickens’ blood are limited. Haemoglobin, PCV and eosinophils of male chickens were higher than appropriate parameters of females but the percentage of lymphocytes in females was higher than in male chickens (23). Values for PCV, WBC, heterophils, lymphocytes, and basophils of the males differed from those of the females. Moreover, biochemical parameters, such as ALT, AST, and total protein were also different depending on sex (1).

Even less information is available about the impact of probiotics on haematological parameters of broiler chickens’ blood. Male broilers were fed probiotic (Enterococcus faecium) and no significant differences were observed in the number of erythrocytes or lymphocytes between chickens receiving and not receiving the probiotic (14). In this study no apparent effect of LAB on the physiological condition of broilers as might be manifested by changes in haematological parameters was observed and the marked parameters were within the standard range of values (6, 15).

Many studies have so far confirmed the ability of Lactobacillus spp. to reduce the growth of undesirable microorganisms in the gastrointestinal tract of broiler chickens. The probiotic strain (L. johnsonii) was used in drinking water, feed and sprayed on litter or administered directly to the beak, during 21 days of Cobb broiler rearing. The bacterium had a significant lessening effect on Clostridium perfringens and Enterobacteriaceae in the small intestine (17).

In another study the probiotics (L. crispatus, L. salivarius, and L. johnsonii) have been shown to have an effect on increasing the total number of anaerobic and lactic bacteria and decreasing the number of Enterobacteriaceae in the small intestine of Cobb broiler chickens (18). The literature provides a further account of L. johnsonii: chickens were infected with Salmonella sofia while receiving probiotic L. johnsonii directly to the beak. Probiotic bacteria were shown to significantly reduce the number of Clostridium perfringens and Salmonella sofia in the gastrointestinal tract of birds compared to the control group not receiving probiotics (19).

In conclusion, the positive effect of the addition to diets of a bacterial preparation consisting of the two potentially probiotic lactic acid bacteria strains: Lactobacillus plantarum K KKP 592/p and Lactobacillus rhamnosus KKP 825 on performance parameters is evidenced in the first feeding period (starter feed). However, feeding chickens with bacteria does not have a significant effect on the final body weight, total feed intake, or feed conversion.

It can also be assumed that feeding broiler chickens with LAB addition influences mortality by reducing the number of deaths caused by various diseases. Moreover, feeding chickens with a bacterial preparation has a positive impact on the intestinal microbiota, due to inhibition of the growth of Salmonella and Clostridium in the intestine.

The safety of the proposed bacterial feed additive was attested to by the failure of a dose of bacterial additive ten times higher than the maximum recommended to have any greater influence on examined health indicators, such as biochemical and haematological parameters of blood, than the maximum recommended dose.