Bacterial species and bacterial counts in the intestinal tract of poultry are influenced by a number of factors. Due to the complexity of the interactions affecting simultaneously the intestinal ecosystem, it is difficult to evaluate the outcome of individual actions.
In general, the use of low doses of antibiotics decreases the number of the most susceptible bacterial communities and enhances the growth of the resistant bacteria. Nutritional composition of feed and thus local availability of raw materials as well as seasonal changes will impact the intestinal flora.
By Gino Lorenzoni, technical manager Biomin, Austria
Through the years the assessment of microbial populations has relied on in vitro culture techniques using selective or non-selective media. One of the main disadvantages of this method is that it can only assess culturable microorganisms. Recently, other culture-independent methods that rely on molecular techniques have been developed to study bacterial populations. Through these modern approaches we have learnt that there are many species of bacteria that we are not able to grow in vitro, including many unknown genera of bacteria.
Actually, it has been estimated that less than 25% of the intestinal bacteria have been cultured. Consequently, when analysing intestinal bacterial populations we must consider that we are observing a modest portion of the real ecosystem and conclusions drawn from diverse studies must be taken carefully. It has been reported that the microbial communities present in the intestinal tract of poultry are modified by a number of factors including stocking density, diet, feeding practices, housing conditions, age of birds and pathogens. Even though bacterial ecosystems among flocks tend to be similar, quantitative and qualitative differences exist among individuals even if raised in the same pen with a common source of feed and water. It is also very well established that bacterial communities change radically between the different anatomical segments of the digestive tract.
Crop, stomach and small intestine
Lactobacilli establish in the crop after a few days of hatching. Depending on the time that the feed remains in the crop, lactobacilli may have some influence in fermentation. The passage from the crop to the small intestine involves drastic changes in the luminal environment. By means of pH variation and enzymatic action the pro-ventricle plays a significant role as a chemical barrier against pathogens. Actually, E. coli, and Campylobacter have been found in higher numbers in the crop than in the gizzard. In broilers fed a corn-soy based diet deprived of antibiotics and additives, nearly 70% of the rRNA sequences found in the ileum corresponded to Lactobacillus.
A population of Lactobacillus is present in birds of two days of age and it remains without drastic changes until market age. The main species of Lactobacillus present in chickens are L. acidophilus, L. salivarius, and L. fermenti. Host specificity has been described for Lactobacillus, in fact, the small intestine of germ free chickens is not effectively colonised by human stains of L. acidophilus. The majority of non-Lactobacillus sequences detected in the small intestine belong to the family Clostridiaceae (11%), and to the genera Streptococcus (6,5%) and Enterococcus (6.5%). Using conventional microbiologic techniques Streptococci, lactobacilli, and E. coli accounted for 60-90% of the bacteria in the duodenum and upper and lower ileum. Surprisingly 9-39% of the bacterial isolates obtained from the small intestine corresponded to strict anaerobes. Within the anaerobes Eubacterium species were the most commonly isolated.
Differences in ages
The use of antibiotics as therapeutic or preventive doses impacts chickens of different ages differently. For example, three day old birds treated with bacitracin and virginiamycin at 4.4 and 11 ppm, respectively, increased L. salivarius while 22 ppm of virginiamycin almost completely inhibited the presence of these bacteria in the ileum of chickens.
Similarly, treatment with antibiotics such as growth promoters also increased the numbers of Enterococcus sp. in the same experiment. On the other hand, virginiamycin at 11 and 22 ppm inhibited L. salivarius in two week old chickens. Lactobacillus population decreases in the ileum of two and three week old broilers fed with a diet supplemented with salinomycin 40 ppm and avilamycin 10 ppm. It is important to note that not all Lactobacilli have the same properties; actually important differences like bacteriocin production are often single-strain characteristics and thus extra benefit can be achieved by using probiotic strains that are known to possess a particular feature.
In birds raised without antibiotics the number of C. perfringens seems to increase with age; salinomycin 40 ppm and avilamycin 10 ppm decrease the number of C. perfringens and this effect seems more pronounced when the diet is supplemented with soy oil than with lard and tallow. It has been noted that tylosin phosphate (100 ppm) increases the number of Lactobacillus gasseri in detriment of C. perfringens, which was detected in higher numbers in control birds. Feed withdrawal also impacts bacterial population in the small intestine. It has been determined that the longer the feed withdrawal is the more severe the decrease in bacterial uniformity, assessed by a reduction in the number of bacterial species detected.
Different bacterial populations
As mentioned above the microflora is influenced by several factors and thus the results of different studies also vary among the literature. Using molecular techniques, Lue and co-workers reported that at three days of age there was no significant difference between the bacteria present in the ileum and ceca with a large proportion of the bacteria corresponding to Lactobacillus. In chickens cecal counts of Lactobacilli average 1 x 109. Using traditional microbiological techniques, along with Lactobacilli large amounts of Enterobacteriaceae and enterococci were found.
In the ceca of juvenile birds, the bacterial population is different from that found in the small intestine. Actually, as early as three days of age the number of Enterobacteriaceae and enterococci start to decline probably due to the increase in volatile fatty acids (acetate, butyrate, and propionate) in the ceca. Starting at 12 days of age the total count of facultative anaerobic plus absolute anaerobic bacteria is 10 to 15 times greater than that of aerobic bacteria, accounting for as many as 1.6 x 1011/g of dry tissue. The majority of the anaerobic bacteria corresponded to Gram-positive bacteria. By two weeks of age the concentration of volatile fatty acids and Enterobacteriaceae and enterococci stabilise in the ceca.In the ceca 65% of the isolated rRNA sequences corresponded to Clostridiaceae.
Other abundant bacterial sequences corresponded to Fusobacterium (14%), Lactobacillus (8%) and Bacteroides (5%). Zhu reported that the ceca of mature birds fed a standard commercial diet was mainly populated by Clostridium leptum (20%), Clostridium coccoides (27%), Sporomusa sp. (21%), and gamma proteobacteria groups (20%). It was also determined that Atopobium, Bacteroides, and bifidobacteria accounted for 3.6, 2 and 1% of the bacterial population, respectively.
Impact of housing conditions
Dietary protein source and level of inclusion affect the numbers of C. perfringens in ileum and ceca. In general terms, the increment of crude protein levels in poultry diets is correlated with an increase in C. perfringens in fishmeal-base diets (which are higher in glycine and methionine than soy based diets). This correlation however, is not always found in birds consuming a soy-based diet. It has been established that housing conditions also impact bacterial population in poultry. Actually, the effect of floor pens vs. battery brooders has more influence modifying the microflora than the antibiotics avilamycin, bacitracin methylene disalicylate and enramycin.
Bacteria which are normally pathogenic for poultry, like Clostridium in young birds and Salmonella, Campylobacter, and E. coli in older birds, can be regularly isolated from healthy individuals. The fact that these bacteria do not produce intestinal disturbances in most of the cases could be attributed to a healthy balance of the intestinal microflora. In fact, in poultry, enteric disorders are routinely reported after the normal microflora is disturbed by antibiotic treatment.