Novel probiotics: Their effects on growth performance, gut development, microbial community and activity of broiler chickens

 

The use of probiotics has become a field of science, medicine and business that is growing rapidly. In agricultural science, probiotic, prebiotics, feed enzymes and organic acids, have been seen as potential alternatives to in-feed antibiotics (IFA) (Choct, 2002).

 

The addition of either pure Lactobacillus cultures or mixtures of lactobacilli and other bacteria to broiler diets has produced variable results. Kalavathy et al. (2003) found an improvement in body weight gain (BWG) and feed conversion ratio (FCR) of broilers fed a mixture of different Lactobacillus strains from 1 to 42 days of age. A consistent improvement in BWG of chickens fed a culture of Lactobacillus has also been reported ( Awad et al., 2009). Feeding broiler chickens up to 6 weeks of age with a diet containing a single strain of Lactobacillus acidophilus or a mixture of lactobacilli significantly improved BWG and FCR ( Jin et al., 1998a). Cao et al. (2013) found that supplementation the broiler diets with a single strain of Lactobacillus (Enterococcus faecium) significantly improved the BW and BWG compared to the control. However, Ashayerizadeh et al. (2011) did not find any significant difference in the performance of chickens fed on diets containing a mixture of Lactobacillus cultures and other bacteria, compared with a non-supplemented diet. Variation in the effects of probiotics on growth performance of broiler chickens may be attributed to the differences in the strains of bacteria used as the dietary supplements.

 

In the present study, the effects of four strains of Lactobacillus spp. on pH, the concentrations of short chain fatty acids (SCFA) and lactic acid, and growth performance of broiler chickens were investigated; the populations of total anaerobic bacteria, lactic acid bacteria, Lactobacilli, Enterobacteria and Clostridium perfringens in gut environment were detected.

 

A total of 235 Lactobacillus isolates were tested using an antagonistic activity assay as described by Schillinger and Lucke, 1989 and Teo and Tan, 2005, and the four strains of Lactobacillus isolates were selected as probiotic candidates by largest inhibition zone appearance with indicator pathogenic strains of C. perfringens and Escherichia coli. These four strains of Lactobacillus were tentatively identified as Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus salivarius and one unidentified Lactobacillus sp.

 

All the strains were kept at −20°C in de Man, Rogosa, Sharpe (MRS) broth (Oxoid, CM0359) with 40% glycerol. The culture medium used for growth was MRS agar (Oxoid, CM0361). The overnight culture of each Lactobacillus isolate was used as a feed additive probiotic candidate after anaerobic incubation at 39°C for 24 h.

 

A total of 294 one-day-old male Cobb broiler chickens vaccinated against Marek?s disease, infectious bronchitis, and Newcastle disease were randomly assigned to 6 diets each with 7 replicates with 7 birds per replicate. Chickens were reared in multi-tiered brooder cages placed in a climate-controlled room up to 21 d, and then the birds were transferred to a metabolic cage room to 35 d. Feed and water were provided ad libitum. The room temperature was gradually decreased from 33°C on d 1 to 24°C on d 35. Eighteen hours of light was provided per day throughout the trial, excluding d 1 to 7 during which 23 h of light was provided. Each cage was equipped with a feeding and water trough placed outside and also an excreta collection tray. The commercial starter and finisher diets was formulated by Ridley AgriProducts (Tamworth, NSW, Australia) as shown in (Table 1) and fed as a one-phase mash feed to avoid inactivation of the probiotics. Four strains of Lactobacillus (No. 1286 tentatively identified as L. johnsonii, No. 709 tentatively identified as L. crispatus, No. 697 tentatively identified as L. salivarius and No. 461 unidentified Lactobacillus sp.) were selected as probiotic candidates and added to the feed to make up four different treatments. Two control treatments were also included, a negative control, with no additives and a positive control treatment with the antibiotic, zinc-bacitracin (ZnB, 50 mg/kg), added. The experimental diets with the probiotic candidates were mixed weekly. The individual strains were grown in MRS broth contained 5 g/L of yeast extract (powder, Oxoid, LP0021) and 20 g/L of glucose, for overnight (at 39°C) and harvested by centrifugation at 4,420× g for 15 min (Induction Drive Centrifugation, Beckman Model J2-21M, Beckman Instruments Inc., Palo Alto, California, USA), resuspended in phosphate buffered saline (PBS, pH 7.4) and mixed into a premix with the basal diet for 10 min using a miniature mixer. This pre-mixture of product with feed (1 kg) was then transferred into a larger mixer (total capacity 300 kg) where the final volume of the weekly feed batch was prepared. The mixer equipment was thoroughly cleaned between the mixing of different treatments by using a vacuum cleaner and a wash diet (basal feed).

 

Table 1. Ingredient composition and calculated chemical composition of basal diets (as-fed basis).

 ¹Vitamin and mineral premix contained the following: vitamin A (as all-trans retinol), 12,000 IU; cholecalciferol, 3,500 IU; vitamin E (as D-a-tocopherol), 44.7 IU; vitamin B₁₂, 0.2 mg; biotin, 0.1 mg; niacin, 50 mg; vitamin K₃, 2 mg; pantothenic acid, 12 mg; folic acid, 2 mg; thiamine, 2 mg; riboflavin, 6 mg; pyridoxine hydrochloride, 5 mg; D-calcium pantothenate, 12 mg; Mn, 80 mg; Fe, 60 mg; Cu, 8 mg; I, 1 mg; Co, 0.3 mg; and Mo, 1 mg.

 

Representative feed samples of each feed batch were tested for bacterial concentrations on d 1, 3, and 7 of each week during the experimental period. Ten grams of sample feed were dissolved in 90 mL of peptone water (Oxoid, CM0009) and 10-fold dilutions were performed in Hungate tubes with 9 mL of peptone water. The numbers of lactic acid bacteria in the feed samples were determined on de MRS agar inoculated with 0.1 mL of diluted sample and after anaerobic incubation at 39°C for 48 h.

 

Representative samples from all experimental feeds were tested as above for bacterial concentrations before being added to the probiotic candidates to make up six different treatments.

 

Feed leftovers and birds were weighed on a weekly basis for calculation of average feed intake and body weight. Mortality was recorded when it occurred and FCR (feed intake/weight gain) was corrected for mortality. On d 21 and 35, two birds from each cage were randomly selected and killed by cervical dislocation. The abdominal cavity was opened and visceral organs were weighed. The weight and the length of the full small intestine and then the empty weight of each intestinal segment were recorded.

 

The contents of the gizzard were collected into plastic containers. An approximately 2 cm piece of the proximal ileum was flushed with ice-cold PBS at pH 7.4 and fixed in 10% formalin for morphological measurements. The contents of the ileum and caeca were collected, and then stored at −20°C until volatile fatty acids (VFA) analysis was performed.

 

About 1 g of fresh digesta samples from the ileum and caeca were transferred into 15 mL MacCartney bottles containing 10 mL of anaerobic broth. The suspension was homogenized for 2 min in CO2-flushed plastic bags using a bag mixer (Interscience, St. Norm, France) and serially diluted in 10-fold increments in anaerobic broth according to the technique of Miller and Wolin (1974). One millilitre of the homogenized suspension was then transferred into 9 mL of anaerobic broth and serially diluted from 10−1 to 10−5 (for the ileal samples) or 10−1 to 10−6 (for the caecal samples). From the last three diluted samples, 0.1 mL each was plated on the appropriate medium (10 mL) for enumeration of microbial populations.

 

Total anaerobic bacteria were determined using anaerobic roll tubes containing 3 mL of Wilkins-Chalgren anaerobe agar (Oxoid, CM0619) incubated at 39°C for 7 days. Lactic acid bacteria were enumerated on MRS agar (Oxoid, CM0361) incubated in anaerobic conditions at 39°C for 48 h. Coliforms and lactose-negative Enterobacteria were counted on MacConkey agar (Oxoid, CM 0007) incubated aerobically at 39°C for 24 h as red and colourless colonies, respectively. Lactobacilli were enumerated on Rogosa agar (Oxoid, CM 0627) after anaerobic incubation at 39°C for 48 h. Numbers of C. perfringens were counted on Tryptose-Sulfite-Cycloserine and Shahidi-Ferguson Perfringens agar base (TSC & SFP) (Oxoid, CM0587 OPSP) mixed with egg yolk emulsion (Oxoid, SR0047) and Perfringens (TSC) selective supplement (Oxoid, SR0088E) according to the pour-plate technique, where plates were overlaid with the same agar after spreading the inoculums and incubated anaerobically at 39°C for 24 h. All plates were incubated in the anaerobic cabinet (Model SJ-3, Kalter Pty. Ltd., Edwardstown, SA, Australia) and bacterial number counted using colony counter (Selby, Model SCC100, Biolab Australia, Sydney, NSW, Australia).

 

Tissue samples were collected from the proximal ileum and flushed with buffered saline and fixed in 10% neutral buffered formalin for histomorphological analysis. Samples were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Sample sections were captured at 10× magnification using a Leica DM LB microscope (Leica Microscope GmbH, Wetzlar, Germany) and morphometric indices were determined as described by Iji et al. (2001). Each sample was measured in 15 vertically, well-oriented, intact villi, muscle depth and crypts photomicrographs of a stage micrometer recorded at 5× magnification.

Intestinal pH was measured immediately after death and excision of viscera at d 21 and 35. The pH of ileal and caecal contents was determined by the modified procedure of Corrier et al. (1990). After thawing at room temperature, the concentrations of SCFA and lactic acid of each digesta sample from the ileum and caeca were measured using gas chromatography (Varian CP-3800. Netherlands) according to the method described by Jensen et al. (1995).

 

Data were subjected to one-way analysis of variance (ANOVA) (StatGraphics Plus version 5.1 – Professional Edition, Manugistics Inc., Rockville, Maryland, USA) and the differences between mean values were identified by the least significant difference (LSD). Differences between treatments were deemed to be significant only if the P value was <0.05. All results were expressed as means. Bacterial counts were transformed to log10 values.

 

The Animal Ethics Committee of the University of New England approved this study (authority number AEC 06/093). Health and animal husbandry practices complied with the 'Australian code of the care of animals for scientific purposes’ issued by the National Health and Medical Research Council (NHMRC, 2004).

 

The experimental diets were prepared weekly. The concentration of LAB reached 8.57 lg cfu/mL (the highest) when the probiotic candidates were re-suspended in PBS solution (Table 2). Furthermore, the high concentrations of LAB (>5.04 lg cfu/g feed, the highest being 6.83 lg cfu/g feed) were observed from the probiotic treatments compared with the negative and positive control treatments.

 

Table 2. Lactic acid bacteria count (lg cfu/g) in feed samples from experimental diets during 1 to 42 d.

PBS = phosphate buffered saline; NC = negative control, with no additives added to the basal feed; PC = positive control, with the antibiotic, zinc-bacitracin (ZnB, 50 mg/kg) added; Iso461 = isolate treatments, with probiotic No. 461 unidentified Lactobacillus sp; Iso697 = isolate treatments, with probiotic No. 697 L. salivarius; Iso709 = isolate treatments, with probiotic No. 709 L. crispatus; Iso1286 = isolate treatments, with probiotic No. 1286 L. johnsonii added to the feed, respectively.

 

The concentration of LAB in feed decreased as each feeding week progressed (probiotic-containing diets were freshly made on a weekly basis, typically at the beginning of the week).

 

There were no significant (P > 0.05) effects on BWG, feed intake (FI) or FCR when the probiotic candidates were added into the feed during the 6-week experimental period ( Table 3). Although there were no major differences in mortalities in the different treatments (range 0 to 7.14%).

 

Table 3. Body weight gain (BWG), feed intake (FI), feed conversion rate (FCR) and mortality of broiler chickens during d 1 to 42.1

SE = standard error of means; NC = negative control, with no additives added to the basal feed; PC = positive control, with the antibiotic, zinc-bacitracin (ZnB, 50 mg/kg) added; Iso461 = isolate treatments, with probiotic No. 461 unidentified Lactobacillus sp; Iso697 = isolate treatments, with probiotic No. 697 L. salivarius; Iso709 = isolate treatments, with probiotic No. 709 L. crispatus; Iso1286 = isolate treatments, with probiotic No. 1286 L. johnsonii added to the feed, respectively.
¹Each value represents the mean of 7 replicates.

 

Probiotics increased (P < 0.01) the relative weight of the jejunum and ileum in 21-day-old chickens ( Table 4), as well as that of the ileum in 42-day-old birds compared with controls. The weights of liver, spleen, pancreas, bursa, gizzard and duodenum were not affected by the treatments.

 

Table 4. Relative organ weights (% body weight) of broiler chickens on d 21 and 35.

SE = standard error of means; NC = negative control, with no additives added to the basal feed; PC = positive control, with the antibiotic, zinc-bacitracin (ZnB, 50 mg/kg) added; Iso461 = isolate treatments, with probiotic No. 461 unidentified Lactobacillus sp; Iso697 = isolate treatments, with probiotic No. 697 L. salivarius; Iso709 = isolate treatments, with probiotic No. 709 L. crispatus; Iso1286 = isolate treatments, with probiotic No. 1286 L. johnsonii added to the feed, respectively.
Each value represents the mean of 7 replicates.
^a,b,c,d Means within a row not sharing a common superscript letter are significantly different (P < 0.05).

 

The probiotic treatments did not affect the intestinal pH (Table 5). As expected, the pH changed from acidic to alkaline from the proximal to the distal regions of the gastrointestinal tract (GIT), with a slight reversal of the trend in the caeca. Thus, at 3 weeks of age, digesta pH was 3.19, 7.45 and 6.67 in the gizzard, ileum and caeca, respectively. The corresponding values at 5 weeks of age were 3.06, 8.11 and 6.96. It was also observed that pH values in the ileum and caeca were generally higher in older birds (35 days of age) than younger birds (21 days of age).

 

Table 5. The pH and organic acids (µmol/g) in gizzard, ileum and caeca digesta on d 21 and 35 of birds fed on experimental diets.¹

SE = standard error of mean; NC = negative control, with no additives added to the basal feed; PC = positive control, with the antibiotic, zinc-bacitracin (ZnB, 50 mg/kg) added; Iso461 = isolate treatments, with probiotic No. 461 unidentified Lactobacillus sp; Iso697 = isolate treatments, with probiotic No. 697 L. salivarius; Iso709 = isolate treatments, with probiotic No. 709 L. crispatus; Iso1286 = isolate treatments, with probiotic No. 1286 L. johnsonii added to the feed, respectively; SCFA = short-chain fatty acid.
¹Each values represents the mean of 7 replicates.

 

The concentrations of VFA, formic acid and lactic acids did not differ in any part of the intestine.

 

The experimental diets did not affect the count of total anaerobic bacteria, LAB, Lactobacilli, Enterobacteria and C. perfringens in the digesta of the gizzard, ileum and caeca of birds at 21 days of age, except that the anaerobes and LAB tended to be higher in birds fed probiotics. At d 35, the number of Enterobacteria in the gizzard varied significantly (P < 0.05), with Iso697 and Iso1286 giving a lower count than the controls. The same was true in the caeca where all the isolates reduced enterobacterial counts, compared with the negative control ( Table 6).

 

Table 6. Effects of experimental diets on bacterial counts (lg cfu/g) in digesta of birds on d 21 and 35.

SE = standard error of means; NC = negative control; PC = positive control, with the antibiotic, zinc-bacitracin (ZnB, 50 mg/kg) added; Iso461 = isolate treatments, with probiotic No. 461 unidentified Lactobacillus sp; Iso697 = isolate treatments, with probiotic No. 697 L. salivarius; Iso709 = isolate treatments, with probiotic No. 709 L. crispatus; Iso1286 = isolate treatments, with probiotic No. 1286 L. johnsonii added to the feed.
Each value represents the mean of 7 replicates.
^a,b,c Means within a row not sharing same superscript letter are significantly different (P < 0.05).
¹Enterobacteria are coliform and lactose negative Enterobacteria.

 

The effects of different dietary treatments on villus height, crypt depth, muscle depth and villi:crypt ratio of the ileum on d 21 and 35 are shown in Table 7. The dietary treatments had no significant effect on villus height, crypt depth and muscle depth either on d 21 or 35. When the ratios of villus height to crypt depth were compared, a significantly higher (P < 0.05) ratio was obtained in the ileum of chickens fed diets containing probiotics on both d 21 and 35.

 

Table 7. Effects of experimental diets on the ileal morphometry.

SE = standard error of means. NC = negative control, with no additives added to the basal feed; PC = positive control, with the antibiotic, zinc-bacitracin (ZnB, 50 mg/kg) added; Iso461 = isolate treatments, with probiotic No. 461 unidentified Lactobacillus sp; Iso697 = isolate treatments, with probiotic No. 697 L. salivarius; Iso709 = isolate treatments, with probiotic No. 709 L. crispatus; Iso1286 = isolate treatments, with probiotic No. 1286 L. johnsonii added to the feed, respectively.
Each value represents the mean of 7 replicates.
^a,b,c Means within a row not sharing a common superscript letter are significantly different (P < 0.05).

 

All the birds were in very good health during the experimental period of 6 weeks, and dietary supplementation with probiotics resulted in numerically higher BWG compared to the negative control group. There was no significant effect on growth performance of broiler chickens when the probiotic candidates were administered via feed. These results were in line with those of Huang et al. (2004) who supplemented either Lactobacillus casei or L. acidophilus with or without cobalt in the diets of broiler chickens. There have also been several studies in which no positive results were found when broilers were fed with probiotic supplements. For example, Watkins and Kratzer, 1984, Maiolino et al., 1992 and Panda et al., 2000 did not find any significant difference in the BWG of chickens given feed containing host-specific probiotics (KTM, 74/1 and 59), L. acidophilus and Streptococcus faecium compared with those given a non-supplemented diet.

 

On the other hand, there are numerous studies that report positive effects of various probiotics on bird performance. For example, BWG of broiler was improved by a culture of L. acidophilus ( Jin et al., 2000), and by a single strain of Lactobacillus (E. faecium) ( Cao et al., 2013) or a mixture of Lactobacillus ( Jin et al., 1998b and Kalavathy et al., 2003). The magnitude of improvement depends on the type of probiotics added and the conditions under which they are used. It was reported by Mohan et al. (1996) that the BWG could range from 5 to 9% higher and FI 2% lower when chickens were fed with probiotic supplements.

 

Variation in the effects of probiotics on growth performance of broiler chickens may be attributed to differences in the strains of bacteria used as the dietary supplements. Several health benefits, resulting from improved digestion, have been claimed for both Lactobacillus spp. and Bifidobacterium spp. At the nutritional level, they increase the digestibility of fermented milk products in humans ( Deeth and Tamine, 1981) and increase the bioavailability of calcium, iron, copper, phosphorus, zinc and manganese in rats (McDonough et al., 1983). Furthermore, Yeo and Kim (1997) reported that feeding a diet containing a probiotic (L. casei) significantly increased average intake of broiler chickens during the first 3 weeks but not during 4 to 6 weeks of age. Chickens gained more weight as a result of mixing L. salivarius with another two Lactobacillus spp. in their diets ( Lan et al., 2003). Yeo et al. (2008) reported that L. johnsonii improved growth performance significantly, acting as an antimicrobial addition in feed for broiler chickens.

 

In the current study, strains of L. johnsonii, L. crispatus, L. salivarius and one unidentified L. sp. tended to improve BWG, FI and FCR in broiler chickens. It is viewed that the effects of probiotics on the growth performance, feed conversion or production of farm animals are, even in specific situations, not consistent enough to consider their use due to economic considerations ( Veldman, 1992). The current study was based on a laboratory scale experiment under clean conditions, which may have masked any growth promoting effect of the probiotics. Another possibility is the concentration of the probiotics in the diet. In the current study, the concentration of the probiotic candidates in the experimental feed was around 106 cfu/g of feed, which were few folds lower than is usually recommended as the inclusion rate (108 cfu/g of products) of commercial probiotic feed additives. This was due to the limited fermentation capacity for amplification of the probiotic candidates in the current study. It is possible that higher concentrations of the probiotic candidates in the feed may exert a more profound positive response on growth performance, especially if the infection pressure from pathogenic bacteria, such as C. perfringens, is high. However, this needs to be investigated in future studies.

 

In the current study, the relative weights of the major digestive and immune organs were not affected by probiotic treatments compared with the controls. However, probiotic supplementation significantly increased the relative weight of the jejunum and ileum on d 21 and that of the ileum on d 42. Such findings have been reported in the literature. For example, Pedroso et al. (2003) added Lactobacillus reuteri and L. johnsonii into drinking water and reported a significant increase in intestinal weight in 21-day-old broilers. The mechanism by which this occurs is not known as the effect of probiotics on organ weights in animals is equivocal. Thus, Jin et al. (1998a) and Guan et al. (2003) found that supplementation of broiler diets with lactobacilli did not affect the weight of the intestine.

 

On the other hand, probiotics appear to influence the microstructure of the gut more consistently. The current study showed that probiotics significantly affected villus height to crypt depth ratio in the ileum compared with control diets. This indicates that the absorptive function in the ileum of these chickens was higher compared with control treatments. Iji et al. (2001) found that, at d 21, the ileal villi were significantly longer in chickens fed a less viscous diet although they were not different during the first 7 days of the experiment. The intestine can change its surface area by growing in length, and/or by increasing or decreasing the height of its villi when probiotics are supplied in the diet. Shortening and fusion of villi will result in loss of surface area for digestion and absorption of food (van Dijk et al., 2002), whereas the converse is true with longer villi and shallower crypts (Chiou et al., 1996).

 

The GIT has the ability to adapt or to react morphologically to changing conditions such as altered diet (Huisman et al., 1990 and van der klis and Van der voorst, 1993). Of course, it is well-known that dietary probiotics lead to marked changes in the gut microflora, often favouring the host. The influence of probiotics on the gut microflora will be discussed in the following section.

 

The current study demonstrates that Enterobacteria make up only a minor proportion of the ileal and caecal microflora in broilers on the sampling days (d 21 and 35). Probiotic supplementation reduced the population of Enterobacteria in the ileum and caeca compared to the control groups. This is in agreement with the findings of Mulder et al. (1997) who reported that inoculation with a probiotic strain of L. reuteri significantly reduced the number of Enterobacteria in broiler chickens. A similar finding was presented by Ln et al. (2003) with a mixture of L. acidophilus/gallinarum, Lactobacillus agilis, L. salivarius, and Lactobacillus spp.

 

Probiotics, such as L. crispatus, L. salivarius and L. johnsonii, have antimicrobial activities against Enterobacteria ( Garriga et al., 1998, Pascual et al., 1999, Veldman, 1992 and Van der Wielen et al., 2002). Cao et al. (2013) reported that broiler chickens fed diets supplemented with Lactobacilli spp. were more resistant to the pathogenic effects of E. coli. The antimicrobial effects of probiotics come from the VFA and other organic acids such as lactate and succiniate produced ( Thompson et al., 1998 and Kubena et al., 2001) and through the production of bacteriocins and phage-displayed peptides ( Ingham et al., 2003, Joerger, 2003 and Sakai et al., 2006). The probiotic candidates used in the current study tended to increase the number of lactic acid bacteria and lactobacilli in the ileum and caeca on d 21. Furthermore, all probiotic candidates, except Iso461, tended to increase the concentration of acetic and lactic acids in the ileum compared with the control treatments. Other potential antimicrobial agents such as bacteriocins and decencies were not measured in the current study.

 

Although the population of lactobacilli was larger in the ileal and caecal contents of the treatment groups fed probiotic supplements, the current study does not demonstrate an improvement in growth performance of birds. The impact of lactobacilli on animal health and performance is controversial. Whilst many Lactobacillus spp. act via a number of mechanisms, including competitive exclusion, to reduce the number of pathogens in the GIT, leading to improvement in bird performance ( Jin et al., 1998ab; Schneits and Hakkinen, 1998), other species seem to be neutral in their effects on birds performance (Gunal et al., 2006). The metabolic activity of common lactobacilli results in the production of end-products such as lactate, succinate, H2, CO2 and CH4 and SCFA, acetate, propionate and butyrate, as well as bacterial biomass. It was shown by De Vries and Stouthammer (1968) that most of the VFA formed by intestinal bacteria are absorbed and metabolized by the birds, thus contributing to host energy requirements (Fooks and Gibson, 2002). However, it is possible that the competition for nutrients by a large number of lactobacilli in the GIT of birds may offset some or all of the beneficial effects of probiotics on nutrient digestibility and absorption. This hypothesis will require investigation in the future.

 

Four Lactobacillus probiotic candidates had no adverse on the general health status of broiler chickens and altered the gut microflora of birds resulting in a reduction in the number of entrobacteria in the ileum and an increase in the weight of the jejunum and the ileum. However, there were no other significant effects of these probiotics on the growth performance and gut development of birds, due probably to the hygienic experimental conditions of the current study.

 

 

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