Prophylactic efficacy and safety evaluation of locally isolated strain Levilactobacillus brevis (MF179529), commercial probiotics and yeast: A comparison

doi:10.21203/rs.3.rs-4910533/v1

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Prophylactic efficacy and safety evaluation of locally isolated strain Levilactobacillus brevis (MF179529), commercial probiotics and yeast: A comparison

https://doi.org/10.21203/rs.3.rs-4910533/v1

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Aims:To anticipate the ban on antibiotic growth promoters in poultry researchers are trying to find safe alternatives from natural resources. The probiotics of Lactobacillus genus are among the promising candidates. The efficacy of probiotics is considered species, strain and source specific. In this study Levilactobacillus brevis, MF179529 comparison was performed with commercial probiotic and yeast.

Methods and results: This study provides a comparison of safety and prophylactic efficiency of (i) locally isolated potential probiotic strain, L. brevis, MF179529, (ii) commercial probiotics, Floramix plus , (iii) yeast, Saccharomyces cerevisiae and (iv) their two combinations on limiting avian pathogenic E. coli (APEC) induced colibacillosis in chicken. All probiotics inhibited infection induced anemia but results were more pronounced in groups I. Birds of group I displayed significantly (p < 0.05) higher general health scores, lower mortality and tissue bacterial load. APEC infection leads to reduction in relative weight of spleen. However, all probiotic treated groups displayed relatively normal spleen weight.

Conclusion:Animals receiving L. brevis MF179529 displayed lower blood cholesterol (p < 0.05), which might be considered a favorable characteristic of meat quality. No adverse effects were observed in other hematological and serological parameters.

Significance of study: L. brevis MF179529 and its combination with commercial probiotics is a better and safe alternative of antibiotic growth promoters in poultry.

Biological sciences/Immunology

Biological sciences/Zoology

Levilactobacillus brevis MF179529

avian pathogenic E. coli (APEC)

hematology

probiotics

serology

sheep red blood cells (SRBC)

Different broad spectrum antibiotics are commonly used as growth promoters and therapeutic agents which not only lead to emergence of antibiotic resistance in important poultry pathogens but also compromise meat quality and hamper the preparation and export of value-added products. FAO imposed ban on use of antibiotic growth promoters in the farm animals which in turn, resulted in risk of huge economical loss due to infectious diseases including fowl typhoid, enteritis, fowl cholera, colibacillosis and salmonellosis ¹. In current scenario, it necessitates exploring alternative growth promoters and probiotics appear as one of the best possible options ².

Probiotics are defined as the live microbes that when administered in adequate quantity, provide beneficial effect to host (FAO/WHO 2001). Most of probiotic bacterial strains belong to group of Lactobacilli genera. The lactobacilli are ubiquitous in nature; occur naturally in milk, fermented food, soil, water, manure and gastrointestinal tract (GIT)³. The probiotic lactobacilli use different mechanisms to inhibit the growth of pathogens viz., production of antimicrobial compounds, competitive exclusion by colonization in the gastrointestinal tract and masking of receptor site for binding of pathogen⁴. Numerous strains have been explored and reported from different sources ⁵ employing both in vitro and in vivo probiotic potential. A few products have been registered as safe growth promoters feed additive for poultry ⁶. Floramix plus is a commercial probiotic preparation recommended for use in poultry industry. It contains mixture of Bacillus licheniformis and Bacillus subtilis which are generally regarded as safe ². Similarly, Saccharomyces cerevisiae is also used in poultry to increase growth performance ⁷.

It is important to accentuate that the biological efficiency of probiotics is source and strain specific and cannot be generalized to all Lactobacillus species⁸. Researchers are characterizing more strains from diverse sources. L. brevis, MF179529 is one of our locally isolated strains. It displayed inhibitory activity against food borne and poultry pathogens including avian pathogenic E. coli (APEC, O1:K1), in in-vitro studies. The strain could grow at wide range of temperature (20–45 ̊ C); resist a range of pH (2–9) and NaCl salt concentration (1–8%) in in-vitro setting. Antilisterial efficiency and safety evaluation of L. brevis, MF179529 in mouse model has been reported⁹. On the basis of aforementioned characteristic, it was presumed to function as potential probiotic. Different studies emphasized the application of Lactobacillus probiotics but L. brevis has been least studied as probiotic in poultry. Moreover, data is available on autochthonous application of the probiotic strain¹⁰ but allocthonous application has not been properly addressed.

Colibacillosis is an important systemic infection that is caused by APEC and lead to acute fatal septicemia, pericarditis, peri-hepatitis and air sacculitis. APEC can infect birds at any age and lead to huge economic loss in terms of mortality (5–55%), weight loss and low quality of poultry products from colibacillosis and necrotic enteritis¹¹. Colibacillosis can be induced in chicks by intra-tracheal, intra-peritoneal or subcutaneous route. Previously, the pathogenicity of E. coli (O1:K1) has been confirmed through intra-peritoneal route¹². The current study is designed to compare safety and prophylactic efficacy of L. brevis MF179529 with Floramix plus and yeast using APEC induced colibacillosis infection model in poultry.

Chemicals and reagents

The chemicals used in this study were of technical grade. Culture media viz; MacConkey agar, de Man Rogosa Sharpe (MRS) agar, Blood agar (Oxide, UK), susceptibility disc, and PBS tablets (Sigma-Aldrich) were used.

Microbial strains and their culture conditions

Commercial probiotics “Floramix Plus” (Hebei Junyu pharmaceutical Co., Ltd) and yeast were purchased (Batala medical store Gujranwala, Pakistan). L. brevis MF179529 was isolated in our laboratory from intestine of healthy cow (Bos torus indicus) and was assessed for in vitro probiotic properties and antagonistic ability. L. brevis MF179529 presented tolerance to acid, bile salt, NaCl, temperature (up to 45ºC), pH (2–9) and lysozyme (100mg/ml), displayed good adherence ability, self-aggregation, co-aggregation, antagonistic activity against foodborne and poultry pathogens including APEC (O1:K1)⁹. Identification was confirmed on the basis of 16s rRNA gene sequences. The sequences were submitted to NCBI and accession number was obtained. The unique name was generated by linking accession number with species name. The isolate was sub-cultured from glycerol stock by plating on de Man Rogosa Sharp (MRS) agar following incubation at 35^°C for 48 hours under anaerobic conditions. Fresh cultures were prepared by inoculation in 1:10 (V/V) in MRS broth. The cells were harvested by centrifugation at 3,380 xg for 15 minutes and stored at -20^°C for further use. Probiotic suspensions were prepared by suspending 1 g of probiotic freeze dry culture in one liter of drinking water.

Avian Pathogenic E. coli (APEC)

The E. coli strain used in the present study was isolated from diseased chicken showing typical pathological signs and lesions of colibacillosis. Detailed characteristics are mentioned in a study by Arshad et al.¹⁰. Glycerol stock of avian pathogenic E. coli (O1:K1) was stored at -40^°C. The APEC was revived by plating on MacConkey agar following incubation at 37^°C for 24 hours. Antibiogram study showed that isolate was resistant against vancomycin (30 µgml^− 1). Pure colonies were grown in LB broth until reached an optical density (OD₆₀₀) of 0.7. The cells were centrifuged for 15 minutes at 3,380 xg, washed twice and resuspended in PBS. The APEC count was determined by enumeration on MacConkey agar supplemented to vancomycin (30 µgml^− 1) using 10 fold serial dilutions. The media was made selective for the isolation of inoculated strain by supplementing MacConkey agar with vancomycin (30 µgml^− 1).

Preparation of sheep red blood cells (SRBC)

Blood samples from healthy sheep were collected in ethylene diamine tetra acetic acid (EDTA) coated vials. The blood samples were centrifuged and plasma along with buffy layer was discarded while RBCs were washed twice with normal saline and cell suspension (2% v/v) was prepared in normal saline.

Procurement, housing and management of chicks

One day old chicks (n = 126) of Cobb breed were purchased from a local hatchery. The experimental birds were kept in open pens with fixed feeders and drinking system under standard poultry practices. The feed and drinking water were provided ad libitum. Temperature was adjusted at 34 ± 01^°C during first week of the experiment and reduced gradually (02^°C per week) to 24 ± 01^°C at the end of experiment while humidity was maintained at 55–58% throughout the experiment.

Experimental design

Following study scheme of Festing ¹³ chicks were divided randomly into seven groups (I-VII) with 18 chicks per group and each group had three replicates with 6 chicks per replicate. Each group was placed into open pen housed in a separate room. A power analysis showed that the sample size of 18 has 80% power to effect size of 0.96 assuming a 5% significance level and a two sided test. The groups (I-V) were provided with different probiotics as L. brevis MF179529, commercial probiotics “Floramix Plus”, yeast, mixture of L. brevis MF179529 with commercial probiotics “Floramix Plus” and combination of L. brevis MF179529 with commercial probiotics “Floramix Plus” and yeast respectively. The probiotics were offered to birds throughout experiment in drinking water at a dose of 01g/10 liter. Groups VI and VII were kept as positive control (PC) and negative control (NC) respectively and kept on plan drinking water. The water suspension was replaced two times a day to keep constant supply of probiotics.

Exposure to SRBC and determination of primary and secondary antibody response

One ml of (2% v/v) SRBC was injected subcutaneously to chicken at the age of 15 days and booster dose was administered at the age of 25 days. Blood samples were collected through cutaneous ulnar vein at the age of 25 and 35 day for measuring primary and secondary antibody titer, respectively. Pre-challenge blood picture was recorded by using blood samples collected at the age of 35 days. The blood samples were collected in tubes with and without EDTA. Primary and secondary antibody titer was measured using haemagglutination test ¹⁴.

Challenge with APEC

At the age of 35 days, all groups except for group VII were infected with one ml of APEC suspension (10⁹ CFU/ml) through intra-peritoneal route. This dose was decided on the basis of prior studies. The group VII was treated similarly with normal saline. Birds were monitored on daily basis for mortality and general health conditions up to 6 days post infection (dpi).

Mortality and General health scoring

Mortality and general health conditions of each group were scored on daily basis. General health scoring was performed following Arshad et al.¹⁰ with slight modification, Briefly, the animals were scored 5 − 1; 5 = active, 4 = dropping wings, diarrhea, 3 = weak with ruffled feathers, 2 = lethargic and reluctant to walk and 1 = depressed with dropping neck and/or closed eyes. The animals at score 1 were culled by decapitation.

Blood sampling

Blood was collected at the age of 35 days (pre-infection) and 42 days (7 dpi in EDTA coated tubes (BD Franklin Lakes NJ USA) for hematological study and gel tubes (BD Franklin Lakes NJ USA) for serum separation. Blood samples were kept at 4^°C for 30 minutes. Serum was separated by centrifugation and stored at -40^°C until further analysis.

Necropsy and organ separation

At the age of 42 days animals were sacrificed under anesthesia with ketamine. Autopsies were performed and visceral organs were removed aseptically and weighed. Relative weight of internal organs such as gizzard, heart, kidneys, lungs, liver and spleen, was calculated by dividing tissue weight with body weight and expressed in percentage.

Tissue bacterial load

APEC enumeration was performed in liver and spleen tissues only. Both tissues were removed aseptically and used in bacterial load determination assay. Weighed amount of liver and spleen was homogenized in sterilized PBS, serially diluted. A volume of 100 µl of last 2–3 dilutions was spread on selective MacConkey agar plates supplemented with vancomycin (30 µgml^− 1) for enumeration of APEC. Plates were incubated aerobically at 37°C for 24 h. Later on, resulting colonies were counted. The dilution, at which at least 100 colonies appeared, was used in calculations of viable bacterial cells enumeration. The viable bacterial counts were determined in duplicate for each sample. The viable cell count per gram of tissue was calculated as follows

CFU/ml = [(Number of colonies/0.1) x dilution factor] / weight of tissue (g)

The data was expressed in term of Log₁₀ CFU/g of tissue. The dilutions resulting in at least 100 CFU were used in calculation.

Hematological and serological analysis

The blood samples were analyzed for hematological as well as serological parameters using automatic hematology analyzer (Sysmax KX-21, USA) and automatic chemistry analyzer (Model HKTE0112, Guangzhou Hekang Biotechnology Co., Ltd China), respectively using commercial kits (Randox, Antrium, UK). Hematological parameters included Hb, RBC, HCT, MCV, MCH, MCHC, platelets, WBCs, ESR, neutrophil, lymphocytes, monocytes, and eosinophil count. Serological parameters included lipid profile (cholesterol, triglycerides, HDL, LDL and cholesterol/HDL ratio), liver function test (bilirubin, SGPT, SGOT, ALP, albumin) and renal profile (serum urea and creatinine).

Statistical analysis

Normality of quantitative data was confirmed by Shapiro-Wilk test. Parametric data were analyzed using one way ANOVA in SPSS software version 21.0. Mortality was analyzed using log rank test. The physical health score was analyzed using Kruskal Wallis H test. Pair wise comparison was performed using Mann-Whitney U test. Values were expressed as mean rank or arithmetic Mean ± SEM. Differences were considered significant at p < 0.05.

Mortality and General health scoring

Mortality of experimental birds was recorded on daily basis after APEC challenge and expressed in percentage. Highest mortality (44%) was observed in birds receiving commercial probiotic (group II), followed by group III receiving yeast and positive control group (22%) each. On the other hand, no mortality was noticed in groups I, IV and V which were receiving L. brevis alone or in combination with other probiotic strains (Table 1a). The mortality among groups was compared using log rank test, there was a significant difference in mortality of VI and VII (P = 0.036). The mortality of group II, III and VI was significantly higher than Group I. Likewise, mortality in Group II was significantly higher than IV, V and VII (log Rank values 0.002 for each group). Last but not the least group III displayed high mortality when compared with VI. Log rank test indicated no significant difference in mortality among groups II, III and VI (Table 1b).

Table 1

a Effects of different probiotics on the mortality pattern and percent survival of infected chicks days post challenge with APEC
Time post challenge (Days)	Groups (n = 18) (No. of mortalities in each group days post challenge)
Time post challenge (Days)	I	II	III	IV	V	VI	VII
1	0	0	0	0	0	0	0
2	0	2	0	0	0	0	0
3	0	0	0	0	0	0	0
4	0	3	2	0	0	0	0
5	0	1	2	0	0	3	0
6	0	2	0	0	0	1	0
% morality	0%	44.44%	22.22%	0%	0%	22.22%	0%
% Survival	100%	55.55%	77.77%	100%	100%	77.77%	100%
Group I = Lactobacillus brevis MF179529; II = commercial probiotics (B. licheniformis, B. subtilis) ; III = Yeast ; IV = Lactobacillus brevis MF179529 + commercial probiotics; V = Lactobacillus brevis MF179529 + commercial probiotics + Yeast; VI = positive control ; VII = negative control

Table 1

b Comparison of mortality among treatment groups as noticed using Log Rank (Mantel-Cox) test
GROUPS	II		III		IV		V		VI		VII
	χ²	Sig.	χ²	Sig.	χ²	Sig.	χ²	Sig.	χ²	Sig.	χ²	Sig.
I	10.074	0.002	4.375	0.036	0.00	1	0.00	1	4.381	0.036	0.00	1
II			2.061	0.151	10.074	0.002	10.074	0.002	2.417	0.12	10.074	0.002
III					4.375	0.036	4.375	0.036	0.015	0.903	4.375	0.036
IV							0.00	1	4.381	0.036	0.00	1
V									4.381	0.036	0.00	1
VI											4.381	0.036
Group I = Lactobacillus brevis MF179529; II = commercial probiotics (B. licheniformis, B. subtilis) ; III = Yeast ; IV = Lactobacillus brevis MF179529 + commercial probiotics; V = Lactobacillus brevis MF179529 + commercial probiotics + Yeast; VI = positive control ; VII = negative control

Physical health score was recorded on ordinal scale (5 − 1) and analyzed using Kruskal-Wallis H test. The results indicated positive influence of probiotic treatment on health status of birds with χ² > 27.77 and p < 0.000103 from 2 days post infection (dpi) onward. Pair wise comparison was performed using Mann-Whitney U test with Bonferroni corrections, which resulted in significance level of p < 0.007. Treatment with commercial probiotic could not protect animals from infection. Group II displayed significantly lower health score in comparison with group I (p < 0.000), IV (p < 0.007)), V (p < 0.000) and VII (p < 0.000) at 5 dpi. Group I, IV and V animals remained protected from infection. The health status of these groups was not significantly different from negative control group. Yeast treatment slightly improved the health score however it was not significantly different from either negative or positive control groups (Fig. 1).

Relative organ index

Infection with APEC led to significant (p < 0.05) shrivels in spleen. Lower values of spleen were noted in group IV and VI compared to group VII. However, organ index of gizzard, liver, heart, kidneys and lungs of all treated groups was similar to that of negative control group (Table 2).

Table 2

Relative organ weights of broilers challenged with APEC (O1:K1) (Mean ± SE)
Groups	Gizzard	Liver	Spleen	Heart	Kidney	Lungs
I	1.28 ± 0.08	2.58 ± 0.09	0.15 ± 0.01^bc	0.63 ± 0.04	0.43 ± 0.09	0.74 ± 0.04
II	1.01 ± 0.13	2.98 ± 0.55	0.17 ± 0.02^bc	0.66 ± 0.11	0.36 ± 0.08	0.61 ± 0.10
III	1.44 ± 0.15	2.7 ± 0.39	0.15 ± 0.01^bc	0.79 ± 0.05	0.37 ± 0.06	0.62 ± 0.14
IV	1.67 ± 0.24	2.54 ± 0.36	0.13 ± 0.02^b	0.72 ± 0.17	0.42 ± 0.11	0.73 ± 0.12
V	1.85 ± 0.21	2.87 ± 0.35	0.19 ± 0.00^c	0.77 ± 0.14	0.41 ± 0.07	0.66 ± 0.03
VI	1.52 ± 0.02	3.08 ± 0.04	0.05 ± 0.01^a	0.97 ± 0.05	0.45 ± 0.02	0.48 ± 0.01
VII	1.51 ± 0.21	2.38 ± 0.66	0.17 ± 0.03^c	0.74 ± 0.10	0.48 ± 0.02	0.64 ± 0.07
Data was analyzed using one way ANOVA followed by Tukey’s test and expressed as Mean ± SE. Values having different letters are significantly different at p < 0.05 (in a column). In table treatments are as follows;
Group I = Lactobacillus brevis MF179529; II = commercial probiotics (B. licheniformis, B. subtilis); III = Yeast; IV = Lactobacillus brevis MF179529 + commercial probiotics; V = Lactobacillus brevis MF179529 + commercial probiotics + Yeast; VI = positive control; VII = negative control.

Bacterial load

A significant difference (p < 0.05) was observed in the bacterial load among treatment groups at 7th dpi. In liver, the lowest bacterial count was recorded in group IV (1.95 Log₁₀ CFU/g), followed by group II (2.85 Log₁₀ CFU/g) and group V (3.1 Log₁₀ CFU/g). However, in liver tissue of yeast treated groups, the bacterial count was similar to that of positive control group. Similarly, APEC could not be recovered from spleen of group IV, whereas, lowest APEC count was observed in group I (1.60 Log₁₀ CFU/g) followed by group II (2.72 Log₁₀ CFU/g). Group V and group III (3.86 and 4.22 Log₁₀ CFU/g), respectively (Fig. 2).

Hematology

Hematological parameters of different groups were compared before and after infection. At day 28, group I was found to have higher (p < 0.05) Hb level and RBC count as compared to negative control group. However, after infection the Hb level of all groups dropped significantly (p < 0.05) but in group I its level (11.47 ± 0.38 g/dl) was in recommended normal range. Similar trends were observed in the values of RBCs before and after infection in all groups. Among the probiotic groups, no significant difference (p > 0.05) was found in the values of neutrophil before infection. But after infection, a significant rise (p < 0.05) was found in neutrophil count of group VI as compared to other treatment groups. However, values of hematocrit (HCT), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), erythrocyte sedimentation rate (ESR), mean cell volume (MCV), white blood cells count (WBC), monocytes, eosinophil, and lymphocytes remained unaltered. Data of hematological parameters is presented in Table 3.

Table 3

Effects of different probiotic treatments on hematological parameters before and after challenge with APEC
	(age)	Treatments groups
Parameters	Days	I	II	III	IV	V	VI	VII
Hb(g/dl)	35th	13.20 ± 0.40^ab	14.67 ± 0.81^b	12.87 ± 0.43^ab	13.03 ± 0.23^ab	14.27 ± 0.32^ab	NA	11.29 ± 0.24^a
Hb(g/dl)	42nd	10.93 ± 0.27^abc	11.47 ± 0.38^c	10.83 ± 0.23^abc	9.90 ± 0.06^a	11.13 ± 0.03^bc	10.33 ± 0.23^ab	11.34 ± 0.35^b
RBC(10⁶/µl)	35th	2.82 ± 0.07^ab	3.37 ± 0.20^b	2.69 ± 0.05^ab	2.87 ± 0.02^ab	2.73 ± 0.29^ab	NA	2.54 ± 0.16^a
RBC(10⁶/µl)	42nd	3.04 ± 0.07^a	3.71 ± 0.13^b	2.93 ± 0.12^a	2.98 ± 0.06^a	3.14 ± 0.23^ab	2.71 ± 0.03^a	2.78 ± 0.11^a
HCT (%)	35th	44.50 ± 1.01	43.83 ± 3.73	42.54 ± 3.23	40.37 ± 0.49	42.70 ± 3.27	NA	44.73 ± 1.09
HCT (%)	42nd	36.93 ± 1.84	34.53 ± 1.33	36.50 ± 1.33	34.30 ± 0.45	41.63 ± 3.53	36.30 ± 0.72	37.90 ± 0.64
MCV(fl)	35th	130.63 ± 1.96	136.30 ± 3.45	137.17 ± 3.93	132.00 ± 1.04	131.80 ± 1.77	NA	121.67 ± 2.01
MCV(fl)	42nd	149.90 ± 4.06	146.53 ± 3.33	150.17 ± 3.99	146.53 ± 2.79	151.70 ± 6.10	146.83 ± 1.48	124.60 ± 2.57
MCH (pg)	35th	47.80 ± 1.61	47.80 ± 0.76	48.91 ± 0.35	47.07 ± 0.52	49.17 ± 0.86	NA	42.57 ± 0.38
MCH (pg)	42nd	37.00 ± 1.11^b	36.57 ± 0.73^ab	36.93 ± 0.73^b	29.93 ± 2.91^a	36.00 ± 0.00^ab	34.63 ± 1.18^ab	32.57 ± 2.11
MCHC (mg/d)l	35th	31.87 ± 0.18	32.70 ± 0.25	32.63 ± 0.63	32.20 ± 0.21	32.47 ± 0.73	NA	31.67 ± 0.27
MCHC (mg/d)l	42nd	29.67 ± 0.23	29.70 ± 0.36	29.67 ± 0.41	28.53 ± 0.29	29.03 ± 0.03	28.47 ± 0.55	27.70 ± 0.64
Platelets (cmm)	35th	17.33 ± 0.88^ab	18.67 ± 0.33^b	16.00 ± 0.58^a	17.33 ± 0.33^ab	17.00 ± 0.58^ab	NA	15.33 ± 0.10^a
Platelets (cmm)	42nd	21.07 ± 1.03^ab	23.05 ± 0.48^b	18.68 ± 1.02	20.81 ± 0.67^b	21.32 ± 0.56^b	19.33 ± 0.48^a	20.97 ± 0.55^b
WBC (10³/ ml)	35th	160.30 ± 4.08^ab	146.50 ± 3.65^ab	164.98 ± 3.86^b	142.74 ± 0.90^a	161.58 ± 8.60^ab	NA	137.62 ± 4.92^a
WBC (10³/ ml)	42nd	297.60 ± 8.68	279.97 ± 22.05	291.83 ± 9.55	268.33 ± 17.75	285.97 ± 5.51	308.37 ± 11.00	246.50 ± 3.65
ESR (mm/hr)	35th	6.67 ± 0.67	4.67 ± 1.33	3.34 ± 0.67	5.33 ± 0.67	6.01 ± 1.15	NA	5.13 ± 1.33
ESR (mm/hr)	42nd	4.67 ± 1.76	6.00 ± 1.15	8.67 ± 0.67	4.67 ± 0.67	6.67 ± 0.67	6.00 ± 1.15	5.33 ± 0.33
Neutrophil (10³/µl)	35th	6.33 ± 0.88	9.33 ± 0.88	10.00 ± 1.53	8.00 ± 0.58	8.33 ± 0.88	NA	6.00 ± 0.58
Neutrophil (10³/µl)	42nd	7.67 ± 0.88^a	10.00 ± 1.00^ab	11.00 ± 2.00^bc	6.67 ± 0.67^a	8.33 ± 0.88^a	11.33 ± 2.73^c	6.23 ± 1.53^a
Lymphocyte(10³/µl)	35th	89.33 ± 0.67	85.00 ± 8.14	88.67 ± 1.67	82.66 ± 1.76	75.67 ± 2.96	NA	75.00 ± 6.2
Lymphocyte(10³/µl)	42nd	76.00 ± 2.51	73.33 ± 3.18	67.33 ± 4.33	77.33 ± 1.45	72.00 ± 2.65	67.67 ± 5.24	71.67 ± 1.20
Monocyte (10³/µl)	35th	13.33 ± 2.33^ab	20.00 ± 2.52^ab	15.67 ± 1.21^ab	14.33 ± 0.88^ab	24.67 ± 2.41^b	NA	10.67 ± 3.93
Monocyte (10³/µl)	42nd	15.67 ± 0.88	14.67 ± 1.45	20.00 ± 2.00	14.00 ± 0.58	18.00 ± 2.52	19.67 ± 2.337	11.67 ± 0.33
Eosinophil (10³/µl)	35th	2.00 ± 0.00^b	2.00 ± 0.00^b	1.00 ± 0.01^a	1.33 ± 0.33^ab	2.01 ± 0.01^b	NA	1.33 ± 0.33
Eosinophil (10³/µl)	42nd	1.67 ± 0.33	2.00 ± 1.00	1.67 ± 0.33	1.33 ± 0.33	1.67 ± 0.33	1.33 ± 0.33	1.00 ± 0.00
Data was analyzed using one way ANOVA followed by Tukey’s test and expressed as Mean ± SE. Values having different letters are significantly different at p < 0.05 (in a row). In table treatments are as follows; Group I = Lactobacillus brevis MF179529; II = commercial probiotics (B. licheniformis, B. subtilis); III = Yeast; IV = Lactobacillus brevis MF179529 + commercial probiotics; V = Lactobacillus brevis MF179529 + commercial probiotics + Yeast; VI = positive control; VII = negative control.

Serum biochemistry

Probiotic treatment led to significant reduction in serum cholesterol level (p < 0.05) that was noticed after feeding probiotics for 4 weeks. APEC infection resulted in a rise of 20 mg/dl cholesterol, but in negative control group change was much pronounced. At 7 dpi the serum cholesterol of treated animals was similar to that of negative control group while positive control group presented highest level (193.33 ± 3.18 mg/dl) as compared to all experimental groups. However, no significant difference was observed among different probiotic treatments.

Similar picture was noticed for triglycerides, where slight reduction was noticed among treatment groups at 4 week age compared to negative control. APEC infection led to evaluation in triglycerides as compared to positive control group but no significant difference was observed among treatment groups after infection. Similarly, value of cholesterol/HDL ratio in positive control group VI (5.23 ± 0.15) was higher. The probiotic treatments resisted this change and value remained similar to group VII (Table 4).

Table 4

Comparison of APEC induced changes in serological parameters in birds kept on different probiotics supplements and anti SRBC titer of primary and secondary antibody.
Blood serum parameters	Treatment groups
Blood serum parameters	Day	I	II	III	IV	V	VI	VII
Cholesterol (mg/dl)	35th	139.67 ± 3.18 ^a	142.00 ± 5.51^a	149.67 ± 4.10 ^a	149.67 ± 3.71 ^a	148.67 ± 4.67 ^a	NA	159.00 ± 4.00^b
Cholesterol (mg/dl)	42nd	154.67 ± 1.33 ^a	154.67 ± 3.93^a	161.67 ± 2.91 ^a	167.33 ± 7.97 ^a	162.33 ± 3.84 ^a	193.33 ± 3.18^b	165.67 ± 11.39 ^a
Triglycerides (mg/dl)	35th	72.33 ± 1.76^ab	70.00 ± 1.00 ^a	78.67 ± 1.20^abc	83.67 ± 3.93^bc	76.00 ± 4.04^ab	NA	77.00 ± 5.86^a
Triglycerides (mg/dl)	42nd	79.33 ± 4.06	79.67 ± 2.03	86.67 ± 1.33	95.00 ± 4.04	88.00 ± 1.00	95.33 ± 7.22	71.00 ± 5.00
HDL (mg/dl)	35th	37.33 ± 0.33	35.33 ± 0.88	35.33 ± 1.20	38.00 ± 1.15	38.33 ± 1.45	NA	36.00 ± 0.58
HDL (mg/dl)	42nd	36.33 ± 0.33	36.67 ± 0.88	35.67 ± 0.88	36.67 ± 0.33	38.67 ± 0.67	37.00 ± 0.58	37.33 ± 0.33
LDL (mg/dl)	35th	83.67 ± 1.86	78.67 ± 4.48	97.33 ± 3.18	95.00 ± 7.09	88.67 ± 2.91	NA	80.67 ± 7.86
LDL (mg/dl)	42nd	124.67 ± 9.49	97.33 ± 4.91	114.33 ± 18.66	113.67 ± 12.99	104.67 ± 2.19	123.67 ± 6.69	91.00 ± 12.22
Cholesterol/HDL	35th	3.74 ± 0.11 ^a	4.03 ± 0.24 ^a	4.25 ± 0.19^ab	3.95 ± 0.21 ^a	3.88 ± 0.06 ^a	NA	4.42 ± 0.13^b
Cholesterol/HDL	42nd	4.26 ± 0.07 ^a	4.23 ± 0.20 ^a	4.53 ± 0.04 ^a	4.57 ± 0.26^ab	4.20 ± 0.15 ^a	5.23 ± 0.15^b	3.32 ± 0.33 ^a
Bilirubin (mg/dl)	35th	0.73 ± 0.03	0.73 ± 0.03	0.67 ± 0.03	0.63 ± 0.89	0.80 ± 0.06	NA	0.71 ± 0.06
Bilirubin (mg/dl)	42nd	0.77 ± 0.03	0.73 ± 0.03	0.83 ± 0.7	0.73 ± 0.03	0.73 ± 0.03	0.73 ± 0.03	0.74 ± 0.06
SGPT(IU/L)	35th	23.00 ± 1.15	24.33 ± 0.33	24.33 ± 2.03	25.67 ± 1.86	25.33 ± 2.73	NA	25.00 ± 3.00
SGPT(IU/L)	42nd	20.33 ± 0.67	22.67 ± 3.18	23.00 ± 0.58	22.67 ± 2.19	22.67 ± 3.18	30.33 ± 2.03	26.67 ± 0.88
SGOT(IU/L)	35th	104.00 ± 37.81	162.33 ± 1.86	148.00 ± 14.00	167.00 ± 34.26	144.67 ± 26.39	NA	137.33 ± 1.20
SGOT(IU/L)	42nd	166.00 ± 10.97	164.67 ± 17.02	197.33 ± 25.15	211.67 ± 31.83	190.00 ± 17.95	236.33 ± 62.32	187.67 ± 3.53
ALP(IU/L)	35th	280.00 ± 20.01	345.67 ± 31.79	351.00 ± 48.44	403.33 ± 11.46	344.33 ± 25.31	NA	281.00 ± 14.01
ALP(IU/L)	42nd	412.00 ± 26.50	865.33 ± 63.56	837.33 ± 176.95	760.00 ± 198.91	771.33 ± 14.19	837.33 ± 227.50	463.67 ± 12.14
Albumen (g/dl)	35th	3.27 ± 0.27	3.40 ± 0.15	3.07 ± 0.27	3.43 ± 0.09	3.17 ± 0.29	NA	3.30 ± 0.12
Albumen (g/dl)	42nd	1.27 ± 0.12	0.87 ± 0.15	1.03 ± 0.13	1.07 ± 0.03	1.03 ± 0.03	0.80 ± 0.15	1.17 ± 0.17
Urea serum(mg/dl)	35th	22.67 ± 0.88^a	28.67 ± 0.67^ab	25.33 ± 3.33^ab	32.67 ± 2.03^b	26.00 ± 2.52^ab	NA	29.00 ± 5.57^ab
Urea serum(mg/dl)	42nd	20.67 ± 0.67^a	21.00 ± 0.00^a	21.0 ± 0.58^a	22.00 ± 0.58^a	23.0 ± 0.58^a	28.00 ± 1.15^b	23.33 ± 1.45^a
Creatinine (mg/dl)	35th	0.73 ± 0.03	0.77 ± 0.03	0.77 ± 0.07	0.83 ± 0.03	0.67 ± 0.03	NA	0.80 ± 0.06
Creatinine (mg/dl)	42nd	0.50 ± 0.00^a	0.57 ± 0.03^a	0.60 ± 0.00^a	0.53 ± 0.03^a	0.60 ± 0.00^a	0.73 ± 0.03^b	0.80 ± 0.06^b
IgM (log₂)	25th	2.33 ± 0.33^b	2.00 ± 0.58^b	0.67 ± 0.33^a	1.00 ± 0.58^a	1.00 ± 0.00^a	NA	2.00 ± 0.00^b
IgG (log₂)	35th	3.67 ± 0.67^b	3.00 ± 0.58^b	0.33 ± 0.33^a	2.00 ± 0.58^b	0.33 ± 0.33^a	NA	4.00 ± 1.15^b
Data was analyzed using one way ANOVA followed by Tukey’s test and expressed as Mean ± SE. Values having different letters are significantly different at p < 0.05 (in a row). In table treatments are as follows;
Group I = Lactobacillus brevis MF179529; II = commercial probiotics (B. licheniformis, B. subtilis); III = Yeast; IV = Lactobacillus brevis MF179529 + commercial probiotics; V = Lactobacillus brevis MF179529 + commercial probiotics + Yeast; VI = positive control; VII = negative control.
N/A- not applied/determined. Group VI was not infected at day 35 and values of VI and VII were same.

No significant variation was observed (p > 0.05) in indicators of liver function [bilirubin, serum glutamine pyruvic transaminase (SGPT), serum glutamine oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), and albumin] of all probiotic treated groups before or after challenge with APEC. Similarly, no difference in serum urea and creatinine levels was observed following 4 week feeding with different probiotics. However, APEC challenge led to elevation in serum urea concentration of Group VI but no such change could be noticed in probiotic fed groups. The lower values of creatinine were observed in all probiotic treated groups compared with negative control group (Table 4) and (Fig. 3).

Anti-SRBC primary and secondary antibody titer against SRBC

Serum samples of groups III, IV and V showed lower levels of IgM compared to group VII. Regarding IgG, low antibody titer was observed in groups III and V compared to groups IV, VI and VII. On the other hand, no reduction in antibody titer could be recorded in group I and II (Table 4).

Recent ban on use of antibiotic growth promoters in farm animals accelerated search for potent prophylactics from natural resources. Nature has bestowed commensal strains with antagonistic ability that directly or indirectly provides protection from pathogens. Following detailed characterization and study, beneficial microbes, can be employed as growth promoters without risk of emergence and spread of antibiotic resistance in pathogens. Escherichia coli is an important pathogenic bacterium commonly found in the broiler surroundings and result in billion-dollar losses annually from colibacillosis and necrotic enteritis¹¹. Physical signs of colibacillosis include ruffled feathers weakness, lethargy, reduced appetite, poor growth, diarrhea, depression, droopy head and mortality which can be correlated with severity of infection¹⁵. Current study provides data on antagonistic and prophylactic application of recently reported strain, L. brevis MF179529 and compares its potential with commercial probiotics (Floramix plus) and Yeast against APEC induced colibacillosis infection in poultry.

. Commercial probiotics “Floramix plus” and yeast supplementation could not protect birds against APEC infection that was evident by 22–44% mortality in these groups. Consistent with these observations, physical health scores of birds receiving commercial probiotic were comparable to positive control group. However, the birds which were kept on L. brevis MF17952 alone or in combination with commercial probiotic and yeast remained active and did not show severe signs of infection. The differences in general health scores among treatment groups might be linked with antagonistic potential of L. brevis MF17952 and strain specificity ⁷. Lactobacilli are known to counter pathogen directly by competitive exclusion or indirectly by immuno-modulation and production of lactic acid and other antimicrobial metabolites¹⁵. Higher levels of IL-4, IL-6 and IL-10 cytokines have been reported in birds receiving L. rhamnosus ¹⁶. In current study, APEC was administered through intra-peritoneal route and protective effect of L. brevis MF179529, observed in this study, is probably due to the production of antimicrobial compounds or other metabolites that regulate systemic immune response ¹⁷.

Bacterial load is quantitative measure of efficacy of treatment. It is hypothesized that if probiotics have some therapeutic efficiency, it will help in reducing bacterial load in tissues. A significant difference in bacterial load among different groups was noticed. In parallel with findings of clinical signs and mortality, animals of group I and II displayed significantly lower bacterial load as compared to other groups. Pathogen could not be recovered from spleen of group II birds and bacterial counts were very low in group I (1.60 Log₁₀ CFU/g). Group II was unable to control the dissemination of pathogens. The differences in efficiency might be explained on the basis of strain specificity and its mechanism of action against each pathogen ¹⁸. Collectively, the decrease in infection (group I), points towards antagonistic efficiency of L. brevis MF179529 in vivo.

Relative organ indices are used as indicators of hostile effects of pathogens in experimental organisms¹⁹. In this study, lower value of spleen index was noticed in PC group. The spleenomegaly and spleen shrivel have been associated with severe infections²⁰. Shrinkage of spleen was reported in patients having pneumococcal infection ²¹. In contrast to our findings, Steinberg et al. ²² reported spleenomegaly in birds infected with salmonellosis. This variation in results may be linked to pathogenic strain used in this study and its mode of administration. No significant difference (p > 0.05) was observed in relative weights of gizzard, liver, heart, kidneys and lungs in experimental groups (Table 2). The findings of organ index are in line with Samtiya et al. ²³(2019) who reported that organ index remains unaltered in bacterial infection.

The level of hematological parameters was recorded before and after infection. Among treatment groups, group I displayed significantly higher Hb and RBC values after 4 weeks of feeding. The Hb of all groups dropped by 2–3 units following the APEC infection. Despite that the L. brevis treated groups displayed values within physiological norms. These findings further strengthen the view that L. brevis positively influences general health of organisms. The increase in Hb, RBC and platelets in group I could be due to better absorption of nutrients from intestine and assertive effects on erythropoiesis²⁴. Our results are in agreement with Hidayat et al.²⁵, who reported increase in Hb level, RBC and hematocrit value following probiotic supplementation. Another commercial probiotic (Protexin Boost) has been documented to stimulate erythropoiesis that leads to increase the hematological parameters ²⁶. In contrast, Dimcho et al ²⁷ and Alkalaf et al ²⁸ reported that probiotic supplementation does not affect hematological parameters. Differences in outcomes might be related to strain specificity. Positive control group VI displayed low level of Hb and RBC that reflects damage and disruption of RBC membrane ²⁹. Pathogens are known to damage RBCs to meet their iron requirement ³⁰.

Higher neutrophil count is an indicator of persistence of bacterial infection ³¹. Higher neutrophil count observed in group VI and yeast treated groups only can be linked to persistence of infection in positive control group and impotency of yeast in controlling infection. No significant difference (p > 0.05) in other hematological indicators viz., HCT, MCV, MCHC, ESR and lymphocytes corroborate with findings by Abudabos et al. ³².

Interestingly, 4 week feeding of different probiotics resulted in significant reduction in serum cholesterol level of all treatment groups compared to negative control. However, the values of all groups were in normal physiological ranges. It might be due to hypo-cholesteromic influence of probiotics ³³. Hypocholesteromic effects can be due to bile salt hydrolase (BSH) enzyme of LB. It is assumed that probiotics microbes contain BSH activity which converts bile into less soluble bile salts whereas cholesterol is used in de novo synthesis of bile leading to overall depletion in serum cholesterol level. In contrast to our findings, Olumide et al. ³⁴ found no significant change in cholesterol level among experimental birds. This variation in results of probiotics on lipid profile might be explained on the basis of strain specificity, experimental conditions or dosage of probiotics ³⁵. On the other hand, APEC infection led to elevation in cholesterol which was noticed in all treatment groups; however probiotic treatment protected animals from infection induced hyper-cholesteremia.

In serum biochemistry liver and renal function tests are considered gold standards of toxicity or stress depending on nature of pathogen. The toxicity and infection may lead to higher values of hepatic markers in certain conditions ³⁶. The data on liver profile (bilirubin, SGPT, SGOT, ALP and albumin) was evaluated to monitor safe nature of probiotic treatment (at day 28) and any adverse effects of the APEC (at 7 dpi) in birds fed with probiotics. In current study, no significant difference (p > 0.05) was observed in the values of liver enzymes among experimental groups at both times. The pre-challenge data points towards the safe nature of all probiotic’s supplements including L. brevis. Following challenge with APEC, levels of SGPT and SGOT were observed higher (p ˂ 0.05) in group VI only compared with their counterparts. This indicated the protective nature of probiotics against liver dysfunction ³⁷. The levels of SGPT, SGOT and ALP in group I were comparable with negative control group validating its hepatoprotective property. Our findings are in agreement with Owosibo et al.³⁸ ,who reported safe nature of commercial probiotic strains.

The renal function can be assessed by serum urea and creatinine³⁹. Both indicators remained unaltered following 4-week probiotic feeding. APEC exposure resulted elevation in urea and depletion in creatinine level. The elevation in urea was noticed only in group VI while depletion in serum creatinine was recorded in all groups. Although low level of creatinine does not create any malfunctioning yet it might reflect some shift in nitrogen metabolism. These findings are in line with Firouzi et al. ⁴⁰ who reported that probiotics have no effect on blood creatinine.

To assess the influence of probiotics on immune response, primary and secondary anti SRBC antibody titer was recorded. Lower antibodies were observed in group III, IV and V while the titers in I, II and VII displayed similar antibody titer. In contrast, various authors have reported that probiotics boost protective immune responses of host⁴¹. Although, the study provides evidence of protection against infection, but its influence on immunomodulation could not be recorded. It might be due to low sample size used in this study. Further studies on large sample size and use of more sensitive techniques are suggested to check immunoregulatory potential of L. brevis.

The current study provides evidence that responses of probiotics are strain specific. Based on findings of mortality, clinical signs, bacterial load and blood picture, group I and group IV are found better than other probiotics used in study. Our study further supports the allocthonous use of probiotics can also be useful. The study provides rational that probiotic strains for farm animals can be isolated from different sources.

SRBC, sheep red blood cells; IgG, immunoglobulin G; IgM, immunoglobulin M; FAO, food and agriculture organization; APEC, avian pathogenic E.coli; MRS, de Man Rogosa Sharpe; dpi, days post infection;

Acknowledgement

Authors are thankful to the Department of Zoology, University of the Punjab, Lahore, Pakistan for providing research facilities to prepare this valuable document.

Ethical statement

Animal experiment was approved by Institutional Animal Care and Use Committee (Bioethic-10-3245), University of the Punjab, Lahore, Pakistan. All methods were followed according to approved experiment (Bioethic-10-3245) and ethical guideline of University of the Punjab, Lahore, Pakistan. It is also confirmed that this experiment performed according to arrive guidelines.

Animals were kept in standard conditions to minimize the distress in animals during experimentation. For euthanasia, ketamine was administered following Pakistan Poultry Association Guidelines and arrive guidelines.

Conflict of interest

It is declared that there is no conflict of interest among authors and others.

Consent for publication

All authors give permission to the publisher to publish this research work.

Availability of data and materials

All data is presented in this manuscript. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no known competing financial interest.

Author’s contribution

[AR]and[SA]: equally contributed in experimental work and drafting, [MA]: Manuscript revision, [NA]: Research design and statistical analysis. [AAS] added valuable suggestions during manuscript drafting. [MI] rewrite and verified the analyzed data of manuscript. Validation, funding contributed and final approval by [SS], [MKG].

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No competing interests reported.

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Prophylactic efficacy and safety evaluation of locally isolated strain Levilactobacillus brevis (MF179529), commercial probiotics and yeast: A comparison

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Abstract

Figures

Introduction

Material and methods

Chemicals and reagents

Microbial strains and their culture conditions

Preparation of sheep red blood cells (SRBC)

Procurement, housing and management of chicks

Experimental design

Exposure to SRBC and determination of primary and secondary antibody response

Challenge with APEC

Mortality and General health scoring

Blood sampling

Necropsy and organ separation

Tissue bacterial load

Hematological and serological analysis

Statistical analysis

Results

Mortality and General health scoring

Relative organ index

Bacterial load

Hematology

Serum biochemistry

Anti-SRBC primary and secondary antibody titer against SRBC

Discussion

Abbreviations

Declarations

Ethical statement

References

Additional Declarations

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