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Heterophil phagocytic activity stimulated by Lactobacillus salivarius L61 and L55 supplementation in broilers with Salmonella infection.


Foodborne illness is usually caused by pathogenic bacteria contamination in food of animal origin such as Salmonella. Salmonellosis in poultry is mainly caused by Salmonella enterica serovar Enteritidis (SE) that can survive in the digestive tract of poultry for several months and contaminate both meat and eggs. Reduction of Salmonella infection in poultry is very important to minimize the loss of chicks and to prevent contamination of meat. Due to an overuse of antibiotic to treat diseases in animals, pathogens have developed a high resistance to antibiotics. Salmonella isolated from chicken has been reported resistant to many antibiotics such as tetracycline, erythromycin, and colistin (Cardoso et al., 2006). To avoid antibiotic residue in meat for consumption, animal rearing has turned to the use of alternatives to antibiotics, such as herbs and probiotics. The largest group of microorganisms used for probiotics is lactic acid-producing bacteria, such as Lactobacillus which is a normal flora found in the gastrointestinal tract of animals including humans (Gilliland et al., 1975). It has been recognized as a safe alternative. To qualify Lactobacillus strains as probiotics, in vivo studies are necessary to clarify their basic properties including survival in the gut environment, antagonistic property to other intestinal pathogens and the ability to adhere the intestinal epithelial cells (Nousiainen and Setala, 1998). However, some studies have reported that probiotic supplementation in chicken feed did not affect their growth (O'Dea et al., 2006). Limitation of probiotics used in animals may be due to uncertainty of their source and whether they specific to a particular animal species. Adhesion to animal epithelial cells of the gastrointestinal tract is supposed to determine host-specificity (Piatek et al., 2012). Immune response of broilers to SE infection has been reported in chicks aged 1 to 7 days after hatching; it is sensitive to infection and the broilers still may have a high mortality rate (Barrow et al., 1987). However, many studies reported a probiotic effect that reduces SE infection but there still is a lack information regarding some immune responses such as phagocytic activity in broiler chicks (Bai et al., 2013). Therefore, this study investigated the effect of a Lactobacillus strain isolated from chicken feces that had basic probiotic properties and anti- Salmonella activities in vitro (Sornplang and Leelavatcharamas, 2010), on treating of SE infection and also the effect on phagocytic activity of heterophils in broiler chicks.


Bactrial strains

SE was isolated from a broiler chicken farm in Khon Kaen province,

Thailand. Lactobacillus salivarius L61 and L. salivarius L55 were isolated from chickens feces, identified by nucleotide sequence analysis of 16S rRNA genes compared to GenBank with 99% to 100% similarity (accession number: CP0003233.1) and these strains can inhibit Salmonella growth in vitro, reported in our previous study (Sornplang and Leelavatcharamas, 2010). Both SE and Lactobacillus isolates were kept as freeze dried specimens at -70[degrees]C in a freezer in the Department of Veterinary Public Health, Faculty of Veterinary Medicine, Khon Kaen University, Thailand.

Experimental design

A total of 150 newborn Arbor Acres chicks of mixed sex were randomly divided into 5 groups (6 pens per group and 5 chicks/1.4 x 1.5 m/pen). Each group was raised in the open house system under natural conditions. Feed and water were provided ad libitum throughout the experimental period. The feeding trial lasted for 7 days. Nutrition provided by feed was as recommended by the National Research Council (1994) with no antibiotics and Salmonella free. Chicks were challenged with SE by oral gavage of 50% lethal dose ([10.sup.6] colony forming unit, CFU in 0.1 mL) (Pascual et al., 1999). The experimental groups were as follows: Group 1 provided feed and water only, group 2 provided water, feed and SE challenged (positive control group), group 3 provided water, feed, SE challenged and one hour later, L. salivarius L61 was supplemented by oral gavage at [10.sup.9] CFU (Chen et al., 2012), group 4 provided water, feed, SE challenged and one hour later, L. salivarius L55 was supplemented by oral gavage at [10.sup.9] CFU and group 5 provided water, feed, SE challenged and one hour later Lactobacillus L61 combination with L55 strains was supplemented by oral gavage at 107 CFU. Twenty four and 48 hours after Lactobacillus gavage, one chick was randomly selected from each of the 6 pens per treatment (6 chicks/treatment). Each chick was slaughtered by cervical dislocation and examined for the presence of SE. The experimental protocol was approved by Khon Kaen University Animal Ethics Committee.

SE detection and isolation

Cecal tonsil samples were dissolved in the tetrathionate enrichment broth (catalog no. 210420, Becton Dickinson, Sparks, Maryland, USA) and streaked on xylose lactose differential agar plate containing novobiocin 25 pg/mL and nalidixic acid 20 pg/mL. These plates were incubated at 37[degrees]C for 24 hours and then checked for SE (Higgins et al., 2007).

Heterophil isolation

Blood samples were randomly collected from ulnar vein for one chick from each of the 6 pens per treatment (6 chicks/treatment) at 24 and 48 hours after the treatment. Heterophils were isolated by the method of Lowry et al. (2005). In brief, 1 mL of blood sample was collected in a test tube containing ethylene diamine tetra-acetic acid, and red blood cells were sedimented with 3% Dextran 500 (Amersham Biosciences Co., Ltd., Uppsala, Sweden). The heterophil rich suspension was mixed with a discontinuous Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO, USA) and centrifugated at 250 x g for 30 min. The purity of the heterophil suspension consisted of 90% heterophils and 10% macrophages and > 95% viable as determined by microscopic examination of Wright-stained smears.

Heterophil phagocytosis

Heterophils were isolated from peripheral blood and adjusted to 5 x [10.sup.6] cells/mL, combination of live SE of 5 x [10.sup.7] cells/mL (ratio 1:10). The protocol was as described by Lowry et al. (2005). Briefly, the mixture was centrifuged and incubated at 39[degrees]C in 5% C[O.sub.2] for 1 h. Samples were submerged in an ice bath for 15 min to stop phagocytosis. Samples were pelleted by centrifugation, supernatants were decanted, and cell/bacteria pellets were resuspended in ice-cold clear RPMI 1640 (Carlsbad, CA, USA) and washed three times by repeating this process. Gentamicin solution was added to remove residual extracellular SE at the second wash. Sediment cells of the blood were smeared on a slide, stained with Wright's stain and examined microscopically. The results were expressed as percentage of heterophils with bacteria and phagocytic index (PI), where PI = (the percentage of heterophils containing bacteria) x (the average number of bacteria per ingesting heterophil). Phagocytosis and PI values were counted from 200 heterophil cells in different microscope fields.

Bacterial preparation

Stock freeze-dried culture of Lactobacillus was dissolved into 0.4 mL de Man, Rogosa, and Sharpe (MRS) broth. It was cultured in MRS agar, and then incubated at 37[degrees]C overnight or for 18 hours and bacterial growth (adjusted to 109 CFU/mL) was measured by the Optical Density ([OD.sub.600]). Stock culture of SE was inoculated into tryptic soy agar (Difco, Sparks, Maryland, USA) containing nalidixic acid, incubated at 37[degrees]C for 18 hours and dissolved in 0.1% peptone water to measure the OD (adjusted to [10.sup.6] CFU/mL).

Statistical analysis

Observation values of the mortality, SE recovery, phagocytosis and PI of chicks in each group was analyzed by analysis of variance. The means from these values among groups were further analyzed by Tukey HSD test at the 0.05 level of significance in a completely randomized design.


This study showed that in the chicks group SE challenged without Lactobacillus treating the mortality was 100% by 7-day-old. Chick groups with SE challenged and treated by Lactobacillus exhibited significantly higher survival rate at 1, 2, and 7 days post-treatment compared to the SE challenge group and normal control group (Table 1). Chicks with SE challenged and supplemented with two Lactobacillus strains (L61+L55) showed 100% survival up to 7 days. Kramer et al. (2001) reported that chicks challenged with SE at the age of 1 day survived for 28 days; the mortality rate was higher than the chicks challenged with SE at the age of 7 days. This may be due to the immune system of 1-day-old chicks that are only partially developed as a similar study of Chen et al. (2012) who reported a cytokine stimulating neutrophils function was lower expression in neonate chicks than the older chicks. Heterophils of broiler chicks have been reported to move into the abdomen in significantly increased quantities after SE challenge (Swaggerty et al., 2005). In this study, survival rate of chicks depended on SE recovery in cecal tonsils. Chicks with SE challenge and treated by Lactobacillus groups showed significantly lower SE recovery in cecal tonsils compared to positive control group and group 1 (Table 2). This study showed one chick infected with SE in the non-infected control group (Table 2). This result can be explained by some broiler chicks being naturally contaminated with Salmonella from their environment, such as bedding materials (Gustafson and Kobland, 1984).

Day-of-hatch chicks inoculated with SE had no SE detected in their cecal tonsils when supplemented with probiotics for 24 h (Higgins et al., 2010) or for 3 d (Chen et al., 2012) before the SE inoculation. Our study showed a success of the probiotic treating SE infection in chicks that reduced SE in cecal tonsil when treated by probiotic within 1 h after the infection. Supplementation of Lactobacillus L61 and Lactobacillus L61 together with Lactobacillus L55 can eliminate SE infection at 48 and 24 h post-treatment, respectively. Phagocytosis is an innate immunity, first response to body inflammation or infection. In present study, chick heterophils were activated which challenged with SE. Although, some infected chicks may show a heterophil engulfed SE before treating with the Lactobacillus but all phagocytose heterophils engulfed only one SE per one heterophil (data not shown). Chick groups with SE challenged and treated by Lactobacillus supplementation can significantly improve (p < 0.05) phagocytosis and PI value at 24 and 48 h after treatment compared to positive control group and normal control group (Table 3). In this study, supplementation of Lactobacillus probiotic at 1 h post SE challenged can eradicate SE infection in broiler chicks by 24 h after treatment. This result was different from the study of Higgins et al. (2010) who reported Lactobacillus treatment after 24 h of Salmonella infection cannot reduce SE in chicks. The results of Lactobacillus supplementation within 1 h post SE infection of chicks in our study indicated that this was an advantageous time to eliminate the SE infection. Multi-strains or high dose ([greater than or equal to] [10.sup.6] CFU/mL) of probiotic supplementation showed a positive effect on eradication of intestinal infection as a similar report of Khodadad et al. (2013). In this study, chick heterophils were stimulated efficiently during 24 to 48 h post-treatment. Chick group with SE challenge and treated by combination of Lactobacillus L61 and Lactobacillus L55 exhibited a significantly higher PI value at 48 h than that the PI value at 24 h post-treatment (p < 0.05). In addition, supplementation with two strains of Lactobacillus in this study eradicated SE infection in chicks by 48 h after treatment. This result is due to the greater innate immune response to SE in probiotic-fed chickens. In conclusion, supplementation of Lactobacillus probiotic (Lactobacillus L61 or L55) can treat Salmonella infection in young chicks when supplemented within 1 h after infection. This result indicates that Lactobacillus probiotic may be used to prevent Salmonella infection in young chicks when supplemented at an optimal time of posthatch to 2-d-old chicks because their heterophils were more stimulated at this time.


We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.


The study was supported by Khon Kaen University, Thailand (grant research 2010 of new researcher development). We thank Dr. Yukifumi Nawa for helpful comments on the manuscript.


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Pairat Sornplang *, Vichai Leelavatcharamas * (1), and Chaiyaporn Soikum

Department of Veterinary Public Health, Khon Kaen University, Khon Kaen 40002, Thailand

* Corresponding Author: Pairat Sornplang. Tel: +66-43-364493, Fax: +66-43-364493, E-mail:

(1) Fermentation research center for value added agricultural products, Khon Kaen University, Khon Kaen 40002, Thailand.

Submitted Apr. 24, 2015; Revised Jun. 1, 2015; Accepted Jun. 24, 2015
Table 1. Number of surviving chicks after Salmonella
Enteritidis (SE) challenge and fed Lactobacillus in
posthatch chicks

Treatments                    Numbers of
                               chick (%)

                                 24 h

1. Feed and water only       29/30 (96.67)
  (basal diet, B)
2. B+SE challenged           18/30 (60.00)
  (positive control group)
3. B+SE+L. salivarius L61    29/30 (96.67)
4. B+SE+L. salivarius L55    29/30 (96.67)
5. B+SE+L. salivarius         30/30 (100)
  L61+L. salivarius L55

Treatments                    Numbers of
                               chick (%)

                                48 h *

1. Feed and water only       22/23 (95.65)
  (basal diet, B)
2. B+SE challenged           7/12 (58.33)
  (positive control group)
3. B+SE+L. salivarius L61     23/23 (100)
4. B+SE+L. salivarius L55    22/23 (95.65)
5. B+SE+L. salivarius         24/24 (100)
  L61+L. salivarius L55

Treatments                    Numbers of
                               chick (%)

                                7 d **

1. Feed and water only       14/16 (87.50)
  (basal diet, B)
2. B+SE challenged            0/1 (0.00)
  (positive control group)
3. B+SE+L. salivarius L61    16/17 (94.12)
4. B+SE+L. salivarius L55    15/16 (93.75)
5. B+SE+L. salivarius         18/18 (100)
  L61+L. salivarius L55

* Total chicks per group were counted at 24 h
survival and excluded from randomly slaughtered
for each group of 6 chicks.

** Total chicks per group were counted at 48 h
survival and excluded from randomly slaughtered
for each group of 6 chicks.

Table 2. The Salmonella Enteritidis (SE) recovery from cecal
tonsils of experimental chicks (6 chicks/group) at 24 h and
48 h after treatment

Treatments                                Number of chicks
                                          with SE found per
                                          chicks examined (%)

                                             24 h          48 h

1. Feed and water only (basal diet, B)      0/6 (0)     1/6 (16.67)
2. B+SE challenged (positive control       6/6 (100)     6/6 (100)
3. B+SE+Lactobacillus salivarius L61      1/6 (16.67)     0/6 (0)
4. B+SE+L. salivarius L55                 2/6 (33.33)   1/6 (16.67)
5. B+SE+L. salivarius L61+L.                0/6 (0)       0/6 (0)
  salivarius L55

Table 3. Effects of Lactobacillus treatment on chicks' heterophil
phagocytosis at 24 h and 48 h after Salmonella Enteritidis (SE)

Treatments                    Percentage of     Average SE:
                              heterophils+SE    heterophil

                              24 h     48 h     24 h    48 h

1. Feed and water only        60       62        1.3    1.27
  (basal diet, B)
2. B+SE chalenged (positive   50       51       1.12    1.12
  control group)
3. B+SE + Lactobacillus       74       75       2.30    2.29
  salivarius L61
4. B+SE + L.salivarius L55    72       73       2.19    2.19
5. B+SE + L.salivarius        91       96       4.78    5.20
  L61+ L55

Treatments                    Phagocytic

                                24 h        48 h

1. Feed and water only         78.00        78.74
  (basal diet, B)
2. B+SE chalenged (positive      56         57.12
  control group)
3. B+SE + Lactobacillus       170.20 *    171.75 *
  salivarius L61
4. B+SE + L.salivarius L55    157.68 *    159.87 *
5. B+SE + L.salivarius        434.98 *   499.20 */**
  L61+ L55

* Indicates significant (p<0.05) differences were observed
between normal control, or positive control and treated in each

** Indicates significant (p<0.05) differences were observed
between 24 and 48 h treated in each row.
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Author:Sornplang, Pairat; Leelavatcharamas, Vichai; Soikum, Chaiyaporn
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9THAI
Date:Nov 1, 2015
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