CLINICOPATHOLOGICAL AND BACTERIOLOGICAL SALMONELLA TYPHIMURIUM EXPERIMENTALLY STUDIES ON THE EFFECT OF PROBIOTIC ON INFECTED CHICKEN

M. MOHAMED, SAMIA; I. AFIFI, SAMIA

doi:10.21608/avmj.2011.176939

CLINICOPATHOLOGICAL AND BACTERIOLOGICAL SALMONELLA TYPHIMURIUM EXPERIMENTALLY STUDIES ON THE EFFECT OF PROBIOTIC ON INFECTED CHICKEN

Assiut Veterinary Medical Journal

Article 25, Volume 57, Issue 131 - Serial Number 4, October 2011, Pages 1-17 PDF (1.08 M)

Document Type: Research article

DOI: 10.21608/avmj.2011.176939

Authors

SAMIA M. MOHAMED¹; SAMIA I. AFIFI²

¹Animal Health Research Institute, Beni Suef

²Animal Health Research Institute, Beni Suef.

Abstract

Sixty one day old chicks were used in this experiment. Chicks were divided into three equal groups. The first group used as control. The second and third group inoculated orally with one ml containing 10⁷cfu of Salmonella typhimurium for three successive days. The 1st and 2nd group fed ration without probiotic, while the third group fed ration contain probiotic (1 g per kg of ration). Mortalities were recorded and examined bacteriolgically and five birds from each group were killed at 3,7, and 14 day post infection for sampling and to evaluate cecal colonization, and organ invasion by S. typhimurium. RBCs count, Hb concentration and PCV were decreased in group II. Non significant decrease in WBCs count in groupI Ion the 7 and 14 days post infection. Significant increase in lymphocytes with significant decrease in neutrophils in group II. Values of total protein and albumin showed significant decrease in group II three days post infection. Activities of ALT and AST increase in group II after two weeks of experiment Values of GGT showed no significant changes in the different groups. Urea showed significant increase in group II at 7 and 14 day post infection. The changes in the blood parameters in group III were within the range of the control group. Pathological lesions were observed in the liver and kidney of group II. Pathomorphological changes were also observed in the intestine.

Keywords

Key words: Probiotics; chicken; S.typhimurium

Full Text

Animal Health Research Institute,

Beni Suef.

Clinicopathological and bacteriological Salmonellatyphimuriumexperimentally studies

on the effect of probiotic

on infected chicken

(With 6 Tables and 8 Figures)

Samia M.Mohamed and Samia I. Afifi

(Received at 3/9/2011)

دراسات باثولوجية اکلينيکية وبکتريولوجية على تاثيرالبروبيوتک على العدوى التجريبية للسالمونيللا فى الدواجن

سامية مصطفى محمد ، ساميه أبراهيم عفيفى

تم تقسيم عدد ستون کتکوت عمر يوم الى ثلاث مجموعات متساوية المجموعة الاولى تم استخدامها کمجموعة ضابطة تم عدوى المجموعة الثانية والثالثة بميکروب السالمونيللا 1ملى يحتوى على(⁷10) عترة عن طريق الفم لمدة ثلاث ايام غذيت المجموعة الثالثة على عليقه تحتوى على البروبيوتک اما المجموعتين الأولى والثانية غذيت على عليقة خالية من البروبيوتک وتم تسجيل نسبة الوفيات وتم اعادة العزل البکتريولوجى لميکروب السالمونيللا من الکتاکيت المصابه والنافقه وتعيين مدى قدرتها على الأنقسام فى الغشاء المبطن لللأمعاء وايضا قدرتها على غزو کل من خلايا الکبد والطحال تم ذبح 5 من الکتاکيت فى اليوم3و7و14بعد العدوى وتم اخذ عينات منها وقد وجد نقص معنوى فى عدد کرات الدم الحمراء والهيموجلوبين وحجم الکريات التکثفى کما وجد نقص غير معنوى فى کرات الدم البيضاء في المجموعة الثانية. وجد نقص فى البروتين والالبيومين وزيادة فى انزيمات الکبد واليوريا فى المجموعة الثانية0 أظهرت النتائج فى المجموعة الثالثة تحسنا فى صورة الدم ووظائف الکبد. اظهر الفحص الباثولوجى وجود تغيرات باثولوجية فى الکبد والکلى والامعاء.

Summary

Key words: Probiotics, chicken, S.typhimurium.

Introduction

Salmonella, a genus of zoonotic bacteria, represents one of the primary causes of food poisoning throughout the world (Fantasia and Filetici, 1994) and is thus of considerable public health and economic importance (Uzzau et al., 2000), Salmonella is one of the major sources of toxi-infection in humans, most often because of consumption of poultry products. The main reason for this association is the presence in hen flocks of silent carriers. Many prophylactic means have been developed to reduce the prevalence of Salmonella carrier-state Zongo et al. (2010). Various prophylactic measures have been employed to controlSalmonella infection in poultry production, including the useof probiotics,to improve immune responses of poultry,(Jung et al., 2010),moreover they have potential antimicrobial effects against several pathogens including Listeria monocytogenes (Nomoto et al., 1985), Salmonella typhimurium (Simone et al., 1988) and Escherichia coli (Mack et al., 1999).

Probiotics are living naturally occurring microorganisms thatare employed as feed supplements. Consumption of probiotics benefits the host animal by improving intestinal microbial balance (Fuller, 1993) and by altering the immune system to reduce colonization by pathogens under certain conditions (Patterson and Burkholder, 2003).

Oral inculation of animal with lactobacilli led to elevated levels of total serum protein, globulin and increased white blood cell count (Pollmann et al., 1980) and increase production of anti-salmonella IgM antibodies (Dunham et al., 1993)

Aim of work:-

To determine the effect of an avian-specific probiotic on the cecal colonization, organ invasion of S. typhimurium, blood pictures, liver function, kidney function and pathological changes in experimentally infected chicken.

Materials and Methods

Chickens:

Sixty apparently healthy Balady chicks one-day-old were used in these experiment, it was proved that they were free from S. typhimurium through cultural and serological examination.

Bacterial strain

Salmonella typhimurium NCTC 12023 / ATCC 14028 obtained from Animal Health Research Institute - Dokky Gizza.

Probiotic:-

Bacillus subtilis 0.4 x10⁶ CFU / g of ration

Saccharomyees cerevisiae 3 x 10⁹ CFU / g of ration

Isolation and identification:-Isolation and identification of S. typhimurium was carried out according to Collee et al. (1996)

Experimental Design

Sixty (one-day-old) Balady chicks were divided into three groups each one contain 20 birds.

The first group (I) used as control fed ration without probiotic kept in a separate room (negative control).

- The second group (II) was fed ration without probiotic.

The third groups (III) was fed ration contain a commercial probiotic (1 g per kg of ration) (Alkhalfa et al., 2010).

At the 4^th day, the second and third group were inoculated orally with one ml containing 10⁷cfu of Salmonella typhimurium for three successive days. (according to Revolledo et al. (2009).

Mortalities were recorded and examined bactreiolgically and five birds from each group were killed at 3, 7, and 14 day post infection for sampling and to evaluate cecal colonization, and organ invasion.

To determine cecal colonization, the whole cecum was removed aseptically, weighed, homogenized and diluted 1:10 with a sterile solution of 0.1% in peptone water (Oxoid). One hundred microliters was spread on Salmonella-Shigella agar (Oxoid) and incubated at 37°C for 24 h. In cases in which growth was detected, a series of biochemical and serological confirmatory tests were performed according to Collee et al. (1996).

To determine organ invasion, after challenge, samples of liver and spleen were taken from birds of each experimental treatment. Samples were appropriately removed, and after recording liver and spleen weights, organs were homogenized separately. The homogenates of each organ were diluted 1:10 with a sterile solution of 0.1% peptone water (Oxoid) and 100 µL was spread on Salmonella-Shigella agar (Oxoid) as described above. Organ invasion was evaluated by enumeration of S. typhimurium in liver and spleen samples using the method as described above. Negative samples were placed in tetrathionate broth (1:10), incubated at 37°C for 24 h and streaked on Salmonella-Shigella agar (Oxoid).

Diagnostic kits.

Commercial diagnostic kits were purchased from Sinreact, Diamond, Egypt and Biodiagnostic for determination of hemoglobin (Hb), serum total protein, albumin, aspartateaminotranseferase (AST), alanine aminotransferase (ALT), Gammaglutamyl transeferase(GGT) and urea.

Samples.

a - Blood samples:

Blood samples were collected from the wing vein after three days, one week and two weeks. Blood samples were divided into 2 parts; the first part was collected on EDTA for erythrocytes (RBCs) and leucocytes (WBCs) count, Hb concentration, packed cell volume (PCV) and differential leucocytic count according to (Feildman et al., 2000). The second part was collected into plain centrifuge tube for serum separation and determination of total protein according to (Henry et al., 1974), serum albumin according to (Doumas, 1971), aspartate amino transeferase and alanin aminotransferase activites according to (Reitman and Frankel, 1957), serum gamma glutayltransferase (GGT) urea according to Patton and Crouch (1977).

b -Tissues samples:

Tissues specimens were taken from liver, kidney, spleen and intestine. The specimens were fixed in 10%formaline solution, trimened then processed sectioned at 5 Mu and stained with haematoxylin and eosin according to Bancroft et al. (1994).

Statistical analysis:

Collected data from the different groups of chickens were statistically analyzed for the mean and standard error according to Selvin (1996).

Results

Mortality rate of each group, colonization of the ceca at d 3,7 and 14 after (inoculation with 10⁷cfu S. typhimurium) and invasion of liver and spleen at d 3,7 and 14 after (inoculation with 10⁷cfu S. typhimurium) were shown in Tables 1,2,3 and 4.

Hemogram:

Results of hemogram in experimental group of chickens were shown in (Table 5). Results revealed that values of RBCs count, Hb concentration and PCV were significantly decreased in group II in comparison to group I and III .Non significant decrease in WBCs count in group II on the 7^th and 14^th days post infection. Significant increase in lymphocytes with significant decrease in neutrophils in group II was observed 3 days post infection. Parameters of hemogram in group III were non significantly changed from that of the control group.

Serum biochemical:

Results of serum biochemical parameters in chickens of different experimental groups are shown in Table 6.

Values of total protein and albumin showed significant decrease in group II after three days of experiment in comparison with control group. Activities of ALT showed significant increase in group II after two weeks of experiment while AST activities was increased insignificantly in group II at three days, one week and two week of the experiment. Values of GGT showed no significant changes in the different groups. Determination of urea showed significant increase on the 7^th and 14^th days post infection in group II in comparison to group I and III.

Changes in the parameters of serum biochemistry in group III were not significantly differ from that of the control group.

Histopathological finding:

Comparative study of the microscopical picture of organs from normal control non infected chicken with those of salmonella- infected ones revealed pathological changes mainly in the liver, kidney, spleen and intestine.

In cases examined in the early stages of infection, ie.,3 and 7 days post- infection, sheets of hepatocytes were widely separated from each other's and the sinusoidal spaces showed the presence of increased number of macrophages (Fig.1). The hepatic and portal veins were congested and the liver parenchyma was oedematous. Circumscribed, single or multiple foci of aggregated mononuclear cells were distributed in the hepatic parenchyma (Fig. 2) .In later stages, ie., 14 days post-infection, pathological changes consisted of a relatively widened area of necrosis with accumulation of debris and fragmented cells and infiltration of polymorphnuclear leucocytes (Fig. 3 and 4). Fibrinous exudates may be found in the liver capsule mixed with mononuclear inflammatory cells.

At all stages, the kidneys showed acute lymphocytic interstitial nephritis with infiltration of mononuclear cells in the intertubular connective tissue of the renal parenchyma (Fig. 5 and 6). This inflammatory change was accompanied by degenerative and necrotic changes in the epithelial lining of the renal tubules and presence of bacterial colonies in the glomeruli. The spleen showed accumulation of vesicular macrophages in a circumscribed manner in the parenchyma ((Fig. 8 and 9). The intestinal villi were relatively short, and epithelial cells showed necrotic changes and were desquamated and accumulated in the lumen intermixed with fibrinous exudates (Fig. 10 and 11). The lamina propria was infiltrated with few inflammatory cells.

Table 1: Mortality rate of each group

Group	N. of birds	N .of died birds 2^nd day 3^rd day total	Mortality rate
I – Negative control	20	0 0 0	0%
II - (S. typhimurium without a probiotic)	20	1 2 3	15%
III - (S. typhimurium + a probiotic)	20	0 3 3	15%

Table 2: Colonization of the ceca at d 3, 7 and 14 after (inoculation with 10⁷cfu S. typhimurium)

Group	3days post infection	7 days post infection	14 days post infection
I – Negative control	0	0	0
II - (S. typhimurium without a probiotic)	2.5 x 10⁴	2.2 x 10⁴	1.4 x 10⁴
III -(S. typhimurium + a probiotic)	1.7 x 10⁴	1.3 x 10⁴	0.5 x 10⁴

Group	3days post infection	7days post infection	14days post infection
I – Negative control	0	0	0
II -(S. typhimurium without a probiotic)	1.2 x 10⁴	0.7 x 10⁴	0
III -(S. typhimurium + a probiotic)	0.6 x 10⁴	0.2 x 10⁴	0

Table 3: Invasion of liver at d 3, 7 and 14 after (inoculation with 10⁷cfu S. typhimurium)

Group	3days post infection	7days post infection	14days post infection
I – Negative control	0	0	0
II -(S. typhimurium without a probiotic)	1.3 x 10⁴	0.9 x 10⁴	0.5 x 10⁴
III - (S. typhimurium + a probiotic)	0.8 x 10⁴	0.5 x 10⁴	0

Table 4: Invasion of spleen at d 3, 7 and 14 after (inoculation with 10⁷cfu S. typhimurium)

Table 5: Mean values ± S.E. of hemogram in different experimental group of chickens.

Mono. %	Neut. %	Lymph. %	WBCs x10³/ul	PCV %	HB g/dl	RBCs x10⁶/ul	Group	Time
5.00±0.40	28.60± 1.00	66.40 ± 1.30	25.80 ± 2.22	27.75 ± 1.30	9.90 ± 0,30	2.63 ± 0.09	I	3days post infection
6.00 ± 0.40	24.00 ± 1.30*	70.00 ± 1.10*	26.75± +3.09	23.75 ± 0.30*	7.83 ± 0.50*	1.69± 0.09*	II
5.30±0.60	26.00 ± 0.70	68.70 ± 0.70	24.50 ± 3.27	24.33 ± 2.80	9.2 0± 0.50	2.14 ± 0.14	III
5.30±0.40	27.70 ± 0.70	67.00 ± 0.90	26.23 ± 2.00	26.2 ± 1.70	9.74 ± 0.20	2.23 ± 0.20	I	7days post infection
6.30±0.33	32.00 ± 1.20	64.67 ± 0.60	22.80 ± 2.22	19.00 ± 1.70*	7.34 ± 0.50*	1.83± 0.20*	II
4.40±0.20	30.35 ± 1.80	65.25 ± 2.00	30.00 ± 6.25	25.75 ± 2.10	10.09 ± 0.60	1.99 ± 0.60	III
5.05±0.28	27.45 ± 0.85	67.50 ± 0.95	26.00 ± 1.00	28.25 ± 0.80	9.74 ± 0.20	2.23 ± 0.20	I	14days post infection
6.00±0.94	23.20 ± 2.47*	70.80 ± 2.87	21.00 ± 3.05	21.67 ± 0.60*	8.68 ± o.60	1.72 ± 0.04	II
5.60±1.20	26.60 ± 0.97	67.80 ± 1.62	23.00 ± 1.52	27.00 ± 0.57	11.46 ± 0.70	1.89 ± 0.08	III

Group I: Normal control. Group II: Salmonella infected group.

Group: Probiotic and salmonella treated group.

*Significant different from control, p 0.05

Table 6: Mean values ± S.E. of some serum biochemical parameters in different experimental group of chickens.

Urea mg/dl	GGT U/ml	ALT U/ml	AST U/ml	Glob. g/dl	Alb. g/dl	T. P g/dl	Group	Time
6.81 ± 0.63	16.67 ± 0.22	33.14 ± 4.86	58.71 ± 0.71	1.60 ± 0.17	2.44 ± 0.28	4.04 ± 0.38	I	3days post infection
7.97 ± 0.47	16.10 ± 0.51	45.91 ± 4.81	57.08 ± 1.51	0.90± 0.09*	1.37± 0.07*	2.27 ± 0.07*	II
6.75 ± 0.39	16.03 ± 0.38	33.84 ± 1.08	58.36 ± 0.95	1.84 ± 029	2.04 ± 0.12	3.88 ± 0.29	III
7.34 ± 0.50	16.82 ± 0.17	33.98 ± 4.80	57.79 ± 0.64	1.69 ± 0.21	2.29 ± 0.26	3.9 9± 0.20	I	7days post infection
9.78± 1.87*	17.59 ± 1.23	39.14 ± 5.55	59.58 ± 0.70	2.02± 0.33	1.03 ± 0.04	3.05 ± 0.15	II
7.28 ± 1.12	15.66± 0.831	29.53 ± 6.11	57.81 ± 1.59	1.37 ± 0.32	2.38 ± 0.04	3.75 ± 0.37	III
7.80 ± 0.77	16.84 ± 0.19	34.79 ± 4.11	58.71 ± 0.71	1.82 ± 0.23	2.12 ± 0.22	3.94 ± 0.24	I	14days post infection
10.38± 1.95*	17.64 ± 0.78	52.96 ± 5.75*	62.29 ± 1.49	1.05 ± 0.14	1.75 ± 0.03	2.80 ± 0.04	II
7.69 ± 0.52	17.24 ± 0.20	37.03 ± 7.28	57.96 ± 1.44	1.67± 0.265	2.29 ± 0.13	3.96 ± 0.42	III

Group I: Normal control. Group II: Salmonella infected group. Group: Probiotic and salmonella treated group.

*Significant different from control, p 0.05.

Discussion

S. typhimuriumis a gram-negative enteropathogenic bacterium, which have been most frequently associated with human infections (Porwollik et al., 2004). Poultry are among the most common reservoir of salmonellae that can be transmitted through the food chain to humans. In young chicks, S. typhimuriuminfection can lead to increased incidence of illness, while older birds are less susceptible to the effects of this pathogen, often experiencing intestinal colonization and even systemic dissemination without significant morbidity or mortality, (Gast 2003). Various prophylactic measures have been employed to controlSalmonella infection in poultry production, including the useof probiotics,(Jung et al., 2010). Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host (Borchers et al., 2009).

The present study showed that the oral infection of three days old chicks with S. typhimurium produce 15% mortalaties in chicks fed on ration either supplemented with a probiotic or without a probiotic (Table 1), the results disagreed with (Jung et al., 2010), these may contribute to the difference in age of chicks, they used 20 day old chicks while we used one day old chicks with ill developed immune system. Table (2) clearing that there was a difference between cecal colonization of S. typhimurium in chicks fed on ration supplemented with a probiotic and chicks fed on ration without a probiotic, thus supplementation of ration with a probiotic decrease cecal colonization of S. typhimurium in chicks, the results agreed with results recorded by Simone et al. (1988) and Jung et al. (2010), also lower number of S. typhimurium colonized the ceca in both infected groups at 14 day post infection and this due to the immune response of birds.

Regarding to Tables 3 and 4, invasion of liver and spleen by S. typhimurium decreased in the group feed on ration supplemented with a probiotic, similar results were recorded by Revolledo et al. (2009) and Jung et al. (2010), S. typhimurium began to disappear from liver and spleen 14 day post infection but more rapidly in the group feed on ration supplemented with a probiotic, these results agreed with the results recorded by Simone et al. (1988) and Revolledo et al. (2009), but the sever lesions in the liver and spleen may be contribute to the endotoxins released after death of the bacterial celles as explained by Saif et al. (2003). From the results of the experiment we noticed that S. typhimurium was cleared early from organs than ceca, the results agreed with results recorded by Jones et al. (2007) and Taseen et al. (2009), they explained that S. typhimurium was recovered at lower levels from the ceca over a period of 14 days postinfection, while the recovery from the liver and spleen were lower at 3 days post infection.

In this study, we evaluate the effect of probiotic on chickens after challenge by S.typhimurium. The results of this study showed that values of RBCs count, Hb concentration and PCV were significantly decrease in group II and non significant decrease in WBCs count. These results agree with Maková et al. (2008).

The decreased values of total protein and albumin with increase in ALT, AST and urea could be due to the pathological effect of salmonella endotoxin on liver Saif et al. (2003) and kidney. The histopathological result coincide these result.

With regard to the influence of probiotic on haematological and serum biochemical parameters investigated in the experiment, no significant changes on RBCs count, haemoglobin concentration and PCV in group III all times of experiments as illustrated in Table 5 and 6. This is in agreement with Dimcho et al. (2005) and Alkhalf et al. (2010). In contrast, the finding disagree with Cetin et al. (2005) who observed that the probiotic supplementation caused statistically significant increase in the RBCs count, Hb concentration and hematocrit values of turkeys. The difference may be attributed to type and number of species of bacteria present in probiotics.

It is clear that the us\he pathogen for adhesion sites or nutritional sources Guillot (2003) immuonomodulation of the host Isolauri et al. (2001) and inhibition of the production of bacterial toxins Brandao (1998).

In conclusion supplemention of poultry ration with a probiotic decrease cecal colonization and organs invasion by S. typhimurium and the probiotic strains could increase the protective effects against S. typhimurium infection.

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Fig. 4: Section of the liver showing necrotic hepatocytes and disintegrated leucocytes (H&EX400)

Fig. 5: Section of the kidney shows interstitial nephritis with infiltration of mononuclear cells in interstitial areas (H&EX400)

Fig. 1 b: - Hepatic cords widely separated; the sinusoidal spaces are filled with phagocytic Kupffers cells (H&E X 400).