Application of some lactobacillus strains product for control of Salmonella typhimurium infection in diarrhoeic neonatal calves

A.M. NABIH^* and M.M. ARAFA^**

^* Mastitis and neonatal Diseases Department, Animal Reproduction Research Institute, Giza, Egypt.

^** Chemistry Department, Animal Heath Research Institute, Cairo, Egypt.

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                                            ABSTRACT

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استخدام بعض عترات اللاکتوباسلس في الوقاية من مرض الإسهال في العجول الرضيعة        المصابة بميکروب السالمونيللا تيفيميوريم

أشرف نبيه محمد ،  محمود محمد عرفة

تم عزلميکروب السالمونيللا المعوية النوع تيفيميوريم من عجول تعانى من اسهالات حادة ولها تاريخ مرضى للاصابة, کما تم عزل انواع اخرى منها سالمونيللا دوبليم وسالمونيللا مونيستر من عجول مصابة بالاسهال واخرى سليمة ظاهريا ومخالطة للعجول المصابة. کاناجمالىنسب العزل للعترات المختلفة من ميکروب السلمونيللا 34 من اصل 77 حالة مصابة بالاسهال (44.2%) و 12 ميکروب من اصل 97 حالة سليمة ظاهريا (12.9%). سجلت النتائج اعلى نسب عزل من اعداد الميکروبات المعزولة من نصيب السالمونيللا المعوية النوع تيفيميوريم , 21 ميکروب من اصل 34 (61.8 %) فى الحالات المصابة بالاسهال بينما کان العدد 9 من اصل 12 ميکروب فى الحالات السليمة ظاهريا والمخالطة للمصابة (75 %). کانت الاعداد المعزولة من السالمونيللا دوبليم والسالمونيللا مونيستر قليلة مقارنة بالتيفيموريم فتم عزل 11 من اصل 34 ميکروب (32.3 %) و 2 من اصل 12 (16.7 %) للسلمونيللا دوبليم , بينما کانت الاعداد 2 من اصل 34 ميکروب (5.9 %) و 1 من اصل 12 (8.3 %) للسالمونيللا مونيستر فى الحالات المصابة بالاسهال الحاد والسليمة ظاهريا والمخالطة للمصابة بالتتابع. تم تطبيق اختبار الاليزا على عدد 20 عينة بلازما دم  من عجول تعانى من إسهال حاد ومصابة بالسالمونيللا ومقارنتها بعينات من عجول تسمين تبدو سليمة صحيًا. اظهرت النتائج ارتفاع ملموس فى مستوى الاجسام المضادة لميکروب السلمونيللا فى العجول المصابة (1: 2400 -9600:1) عنها فى العجول السليمة ظاهريا والمخالطة للمصابة (400:1 - 1 : 7200) بالتتابع. تم تحليل نفس العينات بيوکيميائيا, وقد اظهرت التحاليل للعجول المصابة وجود إنخفاض ملحوظ فى نسب کلا من :- البروتين الکلى ، الألبيومين ، الجلوبيولين ، نسبة الألبيومين / الجلوبيولين ، کذلک نسبة الجلوکوز. بينما کان هناک إرتفاع معنوى فى نشاط إنزيمات الکبد وإنزيم الألکالين فوسفاتاز ونسبة الکرياتينين واليوريا وحامض البوليک. أيضا تغيرت نسب عناصر الأملاح المعدنية , حيث انخفضت نسبة کل من الکالسيوم ، الفوسفور ، الماغنسيوم ، الزنک ، النحاس ، الحديد ، الصوديوم والکلوريد. أما عنصر البوتاسيوم فقد زاد زيادة ملحوظة. تعزى جميع النتائج الى التأثير المباشر لحالات الاسهال الشديدة والتى تؤثر بدورها تأثير معنوى على مکونات الدم المختلفة. لوحظ أنه بعد إستخدام بکتيريا اللاکتوباسيلس کازياى فى العلاج , ظهر تحسن ملحوظ فى  بعض نسب ومعدلات مکونات بلازما الدم البيوکيميائية حيث تساوت تقريبا مع المعدلات الطبيعية.         کذلک انخفضت الاعداد البکتيرية انخفاضا ملحوظا للانواع المختلفة من السالمونيللا المعوية وکذلک الميکروب القولونى, مع زيادة ملحوظة فى اعداد بکتيريا اللاکتوباسلس. لذلک يوصى باستخدام مستحضرات بيولوجية طبيعية من بکتريا اللاکتوباسيليس کازياى کعلاج بديل للإسهال.

Key words: Diarrhoeic calves, Lipopolysaccarid (LPS), ALT, AST.

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INTRODUCTION

Salmonella infection occurs throughout the world and has a hazardous effect on human and animal health as well as great impact on farm economics. In calves the infection appears in the form of septicemia and diarrhoea and in pregnant cows many abortion cases were often manifested (Santos et al., 2001; Barrington et al., 2002). Salmonella enterica serovar typhimurium and dublin where the commonest serovars isolated from cattle. Other serovars as anatum, enteritidis, cerro, montevedio, saint paul, infantis rostock, newport and newington were also reported, but in lower incidences. Calves are highly susceptible to Salmonella infection especially when the pregnant dames were not vaccinated once or twice before parturition and if the neonatal did not receive colostrum (Visser et al., 1990; Konrad et al., 1994 and Santos et al., 2002).

Enzyme-linked immunosorbent assays (ELISAs) based on lipopolysaccharide (LPS) for different salmonella serovars has been evaluated by many researchers as a highly specific test for diagnosing Salmonella infection in bovine.

Combination Not only serum was tested in these ELISA but also milk samples and other body fluids were investigated (Hoorfar et al., 1995; Hoorfar and Wedderkoppe 1995; Smith et al., 1995; Seleim, 1999; Galland et al., 2000; Radke et al., 2002 and Veling et al., 2002).

Some authors declared, that serum biochemical profiles of diarrhoeic calves were affected as,total proteins,  albumin, globulin, A/G ratio and glucose were significantly decreased (Manaa et al., 1993 and Kaneko    et al., 1997). Liver and kidney profiles were on the contra`ry increased, whereas macro and microelements decreased significantly except for potassium which was significantly increased (Ragab et al., 1986; Aly et al., 1996 and Kaneko et al., 1977).

Recently many probiotic bacteria as lactic acid bacteria (LAB), Lactobacillus, Streptococcus, Bifidobacterium and some fungi as Aspergillus oryzae and yeast were used as health promoters for humans and animals. Certain preparations were used to boost immune status, increase resistance to infectious diseases, particularly of the intestine, decrease duration of diarrhea (Romond et al., 1998; sreekumar and hosono 1998; Tannock 2002; Ibnou-Zekri et al., 2003; Makras et al., 2006; Gratz et al., 2010). The mechanism of action of probiotics is not fully understood, either they migrate through the gut wall as viable cells and multiply to a limited extent or antigens released by the alive or dead organisms, that can be absorbed and stimulate the immune system directly. A third school of thought suggested that the Lactobacillus species acted indirectly through an effect on the other microbial components (as Coliform) of the gut flora. It was the product of this change which induced the immune response. Moreover, it appeared to be some relationship between the ability of Lactobacillus strain to translocate and the ability to be immunogenic (Fuller, 1989; Gibson, 1995; Roberfroid, 1998; Ibnou-Zekri et al., 2003).

The objective of this study was to determine the different Salmonellaentericaserovars causing fatalities and diarrhoea in calves and to apply lipopolysaccaharide-ELISA for diagnosis. Moreover, the study included investigations on the serum biochemical profiles of diarrhoeic calves before and after competitive exclusion treatment with L.casei, as well as assessment of this biological treatment in curbing Salmonellaenterica-induced diarrhoea in beef calves.

MATERIALS and METHODS

Specimens:-

Internal organs from 5 recently dead calves  (5 Intestines, 5 gall bladders, 5 livers, 5 spleens, 5 lungs and 5 kidneys) aged from 1 week to 6 months, had a history of anorexia, pyrexia and sever diarrhoea sometimes tinged with blood, were examined bacteriologically. Another 174 faecal samples (77 from diarrhoeic calves and 97 from apparently healthy contact animals) and 174 blood samples (from the same animals) were examined bacteriologically for Salmonellaand serologically for Selmonellaantibodies respectively. Samples were collected during the period of one year from august 2010 to august 2011 form calves aged (1 week – 6 months) at governmental and private farms in Giza, Gharbia and Dakahlea governorates. All specimens were transferred to the laboratory in ice box with minimum delay.

Animals:-

In the second phase of investigation 20 diarrhoeic beef calves that were proven positive for S.typhimurieum were selected for treatment with L.caseias competitive exclusion treatment. Faecal and blood samples were collected from these calves on weekly bases and assessed for Salmonella, Coliform and lactobacillus content, while blood samples were assessed for humoral and cellular immune response as well as the serum biochemical analysis was carried out. Another 15 calves were apparently healthy, had no history of diarrhoea and proven negative forSalmonellawere selected as control.

Isolation and identification of salmonella enterica:-

Faecal samples were cultured into selenit-f. broth, and incubated at 37⁰C for 18hrs. Loopful from these broth cultures were then streaked onto MacConkey and S. S. agar plates, incubated at 37⁰C for 24 and 48hrs. Suspected colonies were identified morphologically, biochemically by the API 20E System (BioMereaus, France) and serologically according to the Kauffman-White Scheem by slide agglutination test using polyvalent and monovalent somatic (O) and flagellar (H) antisera (Wellcome Research Laboratories, UK.) according to (Edwards and Ewing 1972).

Isolation and identification of Lactobacillus casei for oral administration to calves:-

L. casei was isolated from the intestine of healthy calves on Togosa agar medium at 37⁰C and 10% CO₂. The isolation and identification was carried out according to Qin et al. (1995). The selected L. casei isolate was tested for bile and acid tolerance (growth at 1% bile salt rogosa agar and at pH5) then adjusted photometreally at (10¹⁰ CFU/ml PBS pH 7.4). One ml of adjusted L. casei was mixed into 250ml of 2% sterilized skim milk immediately before oral inoculation of the calves. The bacterial population was confirmed by enumeration of serial dilutions on rogosa agar plates in duplicate. The administration of the L. casei was carried out every other day for a period of 3 weeks.

Salmonella, Coliform as well as Lactobacilli counts were determined in the faeces before and after oral administration of the Lactobacilli. Salmonella count was determined by direct inoculation onto S.S. agar, Mac Conkey agar (for coliform) and Rogosa agar (for Lactobacillus). If the number of Salmonella was less than 500/g, enrichment in selenit-f. broth (Difco) could detect those samples which were  negative in direct plating (Zaho et al., 1998).

Lipopolysaccaharide (LPS) ELISA:-

ELISA to detect antibodies to LPS prepared from Salmonella enterica serovar typhimurium was carried out by extraction of LPS by phenol-chloroform-petroleum ether (extraction mixture) as described by Demarco de Hormacche et al. (1988). Each well of microtiter 96-well plates (Falcon) was coated with 5μg LPS/ml in carbonate bicarbonate buffer (pH 9.6). After overnight incubation at 37⁰C, the plates were incubated with blocking buffer, consisting of 3% bovine serum albumin (BSA-Sigma) in phosphate buffer saline pH 7.4 and 0.05% Tween 20 (PBS-T) to coat the unoccupied sites on the plates. After 1 hr at 37^°C. The plates were then washed with PBS-T and 100μl goat anti-bovine horseradish peroxidase conjugate (Dako) diluted 1:1000 in 0.3% BSA in PBS-T, was added to each well and incubated for 1 hr at 37^°C. The plates were washed with PBS-T then 100 μl 3,3’,5,5’-tetramcthylbenzidine (ICN), prepared according to the manufacturer’s instructions, were added to each well. After 10 min, 25 μl 5 M H₂SO₄ were added to each well and plates were read at 450 nm in ELISA reader. The cut off value was calculated as the average optical density (OD) value of the negative control values plus 2 standard deviations (SD). The antibody titer was calculated as the highest serm dilution that gives OD value above the cut off point (Ramos et al., 2000).

Estimation of cellular immunity (Macrophage activity):-

Calves leucocytes were harvested from the whole blood by density centrifugation on Histopaque 1077 (Sigma) according to, Lammler and Ding `(1994). Histopaque can separate the leucocytes in a buffy coat layer over the erythrocytes. After the separation of the leucocyte cell fraction over the histopaque surface, the RBCs among the harvested cells were lysed by adding 0.87% ammonium chloride solution pH 7.2 (1:5 v/v) with gentle shaking.

The leucocytes were then washed with Minimal Essential Medium (MEM, Sigma), and were finally adjusted to 10⁵ cells/ml MEM using a hemocytometer. Salmonella cultures were adjusted photometrically to 10⁹ bacteria /ml  in MEM medium, then equal volumes of leucocytes and S.typhimurium isolates were incubated at 37^°C for 1hr with gentle shaking. The leucocytes-S.typhemeurium mixtures were then spread on a microscope slide, fixed and stained with acridine orange and examined under the microscope. The phagocytosis index was measured according to (Shoshani et al., 2000)

Serum Biochemical Profile:-

Collected serum samples from 20 clinically diarrhoeic calves before and after administration of L. casei ( 10¹⁰ CFU/calf), as well as 15 control apparently normal calves were analysed biochemically for determination of total proteins (Hoffmann and Richterrich 1990), albumin and globulins (Doumas et al., 1971), ALT and AST aminotransferases (Reitman and Frankel 1957). Glucose (King and Woottin 1959), alkaline phosphatase (Kilchling and Fraiberg 1951), Magnesium (Neil and Nelly, 1956), sodium and potassium by using flame photometer (Oser, 1989), iron cupper and zinc were estimated spectrophotometrically by Fernandez and Kohn (1991) and chloride (Varley et al., 1980).

Statistical Analysis:-

Statistical analysis of obtained serum values were carried out using the “t” test according to the method of (SSPS 14, 2006)

RESULTS

Only one serovar, Salmonellaenterica serovar typhimurium, 11 isolates were dected from the 30 collected organs of 5 dead calves. No haemolytic E.coliwas isolated from these 5 dead calves. Three different Salmonellaserovars were demonstrated from both clinical cases with diarrhoea as well as from the contact apparently healthy ones. Out of 77 diarrhoeic 34 were proven positive for Salmonellaisolation(44.2%) whereas the apparently healthy contact calves demonstrated lower rate of  isolation (12 out of 97, 12.4%).

Salmonellaentericaserovar typhimurium (S.typhimurium) was the most dominant serovar as revealed from the pattern of isolation. In clinical cases S.t.constituted 21 out of 34 isolates (61.8%) and 9 out of 12 (75%) in apparently healthy calves. Salmonellaenterica serovar dublin (S.dublin) and muenster (S.muenster) were revealed in lower isolation pattern, as 11 out of 34 (32.3%) and 2 out of 12 (16.7%) in case of S.dublin and 2 out of 34 (5.9%) and 1 out of 12 (8.3%) in case of S.muenster were detected in both diarrhoeic and apparently healthy calves respectively. All Salmonellaserovars were confirmed for its somatic (O) and flagellar (H) antigenic structure (Table 1). The cut off value for the LPS ELISA was calculated as 0.33 OD at 450 nm. Diarrhoeic calves recorded higher OD values (range 0.89 to 1.68) than apparently healthy (0.34 to 1.32). The high OD reading was expressed in high antibody titers in the diarrhoeic animals that ranged from 1:2400 to 1:9600, whereas the antibody titer ranged in the apparently healthy calves from 1:400 to 1:7200. LPS-ELISA testing generally revealed higher incidences of Salmonellainfection than the conventional culture method as 53 out of 77 (68.8%) and 37 out of 97 (38.1%) in diarrhoeic and apparently healthy calves respectively were tested positive for Salmonellaantibodies (Table-2).

Administration of L.casei (10¹⁰cfu) every other day for 3 weeks to 20 diarrhoeic calves elucidated significant improvement in the calves health conditions starting the first few days after administration. Salmonellacount was greatly reduced from 6.37 + 2.5 log₁₀ to zero. The Coliform count was also reduced but still within a limitted range of 5.28+2.1 to 6.21+2.3 (log₁₀). The remarkable increase in the Lactobacillascount was noticed immediately after administration from zero to 6.47+2.2 (log₁₀) (Table 3). Thehumoral as well as the cellular immune responses were also stimulated as the Salmonellaantibody levels were increased which was monitored by the elevated OD values from 1.68 to 1.94 OD at 450nm. The cellular immunity manifested in the macrophages activity was significantly enhanced by almost eight folds from 4.4+1.2 to 33.2 +5.1 cell/macrophange (Table – 3).

In diarrhoeic calves serum biochemical analysis showed significant decrease in total proteins, albumin, globulins, A/G ratio (hypoproteinemia), as well as glucose levels (Table 4 & 5). The enzyme activity of ALT, AST, alkaline phosphatase as well as the values of creatinine, urea and uric acid were significantly increased (Table 5). Minerals profiles were also changed, where serum calcium, phosphorus, magnesium, zinc, cupper, iron, sodium and chloride levels were significantly decreased. Potassium level was significantly increased (Table 6). After treatment with L.caseicertain improvements of most serum biochemical parameters were recorded, though in some cases some discrepancies were manifested compared to the control group (Table 4, 5, 6).

Table 1: Isolation and identification of different salmonella serovars from diarrhoeic and apparently healthy beef calves.

Table 2: Serum from diarrhoeic and apparently healthy, contact animals tested with LPS ELISA.

Table 3: Effect of lactobacillus casei administration on the intestinal microbial content, on humoral and cellular immunity of 20 salmonella enterica serovar typhimurium infected calves.

** Average number of cells inside the macrophage + standard deviation.

Table 4: Proteinogram in sera of 20 infected calves before and after treatment with probiotics (L.casei) in comparison with apparently healthy group.

Average value + standard error

a, b, c, values with different letters are significant (P<0.05). values with the same letters are not significant.

Table 5: Liver and Kidny Function indices in Serum of Calves Before and After Treatment

Average value + standard error

a, b, c, values with different letters are significant (P<0.05). values with the same letters are not significant.

Table 6: Serum Biochemical Analysis of Minerals Before and After Treatment.

Average value + standard error

a, b, c, values with different letters are significant (P<0.05). values with the same letters are not significant.

DISCUSSION

Salmonella infectious diarrhoea is an important cause of neonatal calf morbidity and mortality in different parts of the world including Egypt, which results in huge economic losses in the beef and dairy industries. Many risk factors were encountered with the occurrence of infection that was related to the calf, the pathogens involved and to the surrounding environment. The immune status of calves, specifically the level of passively acquired immunity through colostrum, is the major risk factor related to the calf and the occurrence of diarrhea (Abouzeed et al., 2000; Santos et al., 2001 and Barrington et al., 2002). Although numerous pathogens have been implicated in the occurrence of neonatal diarrhea as Salmonella, E. coli, Yersinia enterocolitica, Campylobacter and many others, only relatively limited numbers are commonly involved. Most should be viewed as secondary opportunists rather than primary pathogens, with the exception of Salmonella (Konrad et al., 1994; Barrington et al., 2002 and Santos et al., 2002). The isolation of Salmonella from the internal organs of 5 dead calves which had an episode of severe diarrhoea revealed the isolation of only one serovar (S.typhimureium), wheras 11 isolates were detected from the 30 collected organs and no haemolytic E. coli were detected in these dead calves. The further bacteriological examination of specimens collected from clinical cases with diarrhoea and the contact apparently healthy revealed isolation of 3 Salmonella serovars. Out of 77 diarrhoeic calves 34 were proven positive for Salmonella isolation (44.2%) whereas the apparently healthy contact calves demonstarted lower incidence of isolation, as 12 out of 97 (27.9%). S.typhimureium was the most dominant serovar as revealed in the pattern of isolation (Table 1). In clinical cases S.typhimureium constituted 21 out of 34 isolates (61.8%) and 9 out of 12 (75%) in apparently healthy calves. Salmonella enterica serovar dublin (S.dublin) and muenster (S.muenster) were also isolated in lower pattern, 11 out of 34 (32.3%) and 2 out of 12 (16.7%) in case of S.dublin and 2 out of 34 (5.9%) and 1 out of 12 (8.3%) in case of S.muenster were detected. Many researchers recorded similar isolation patterns and many other Salmonella serovars as enteritidis, infantis, Rostock, Saint paul, Newington, cerro, newport and muenster were incriminated in the induction of diarrhoea in calves with different incidences and clinical severalties of infection (Visser et al., 1990, Konrad et al., 1994; Abouzeed et al., 2000; Bishpham et al., 2001; Santos et al., 2002 and Ostad et al., 2009).

All Salmonella serovars were confirmed for its somatic (O) as well as flagellar (H) antigenic structure (Table 1). The serological examination of blood samples for detection of Salmonella antibodies by LPS-ELISA, revealed a cut off value of 0.33 OD at 450nm. Diarrhoeic calves recorded higher OD values (range 0.89 to 1.68 OD) than apparently healthy (range 0.34 to 1.32 OD). These results were in agreement with other authors who employd different ELISAs in monitoring Salmonella antibodies in the serum. In some cases the OD values which usually reflect the antibody titers in the samples did not match the severity of the clinical status of the animal as some severely diarrhoeic lethargic animals has low OD values and vice versa. (Hoorfar  et al., 1995 Hoorfar and Wedderkoppe 1995; Smith et al., 1995; Galland et al., 2000; Radke et al., 2002 Veling et al., 2002 and Fayol-Messaoudi et al., 2007).

The high OD reading was expressed in high antibody titer in the diarrhoeic animals that ranged from 1:2400 to 1:9600, whereas the antibody titer ranged in the apparently healthy calves from 1:400 to 1:7200. LPS-ELISA revealed higher incidences of Salmonella infection than the conventional culture method as 53 out of 77 (68.8%) and 37 out of 97 (38.1%) in diarrhoeic and apparently healthy calves respectively were detected with LPS-ELISA positive (Table 2.). These discrepancies between the culture and serological methods were explained due to the intermittent sheding of the micro-oganism as well as the eliciting of antibodies not instant with the onset of infection (Hoorfar and Wedderkoppe 1995; Smith et al., 1995; Radke et al., 2002; Chart et al., 2002; Veling et al., 2002; Ibnou-Zekri et al., 2003 and Galland et al., 2000).

Administration of L. caseiwas based on its resistance to culture on 1% bile salts media as well as its acid tolerance (pH 5). Diarrhoeic calves administered L.casei (10¹⁰ cfu) every other day for 3 weeks elucidated significant improvement in the health conditions starting the first few days after administration. Salmonella counts were greatly reduced from 6.37+2.5 log₁₀ to zero. The Coliform counts were also reduced but stayed in the range of 5.28+2.1 to 6.21+ 2.3 (log₁₀). The remarkable increase in the lactobacillus counts was noticed immediately after administration from zero to 6.47+2.2 (log₁₀) (Table 3). The change in the bacterial counts after the administration of L. casei could be attributed to the competitive exclusion of the Salmonella on the enterocytes receptors, production of lactic acid and many other metabolites which shift the pH in the intestine to acidic. This acidity was considered crucial in colonization of      L. casei and hindering the growth of Salmonella and other enteropathogenic bacteria. Some researchers used the organic acids and other probiotic substances as lactulose and lactitol (synthetic disaccharides) to produce prophylactic effect against enteropathogenic bacteria (Fuller, 1989; Roberfroid 1998; Zaho et al., 1998; Tannock, 2002 and Ibnou-Zekri et al., 2003).

The humoral as well as the cellular immune responses were also stimulated after the         L casei administration as the Salmonella antibody levels were increased as monitored by the elevated OD values from 0.68 to 1.94 OD. The cellular immunity manifested in the macrophages activity was also significantly enhanced by almost eight folds from 4.4+1.2 to 33.2 + 5.1 cell macrophage. All these signs of health and immune status improvement were due to the direct or indirect action of the probiotic L.casei, that stimulated B-lymphocytes to produce different immunoglobulins isotypes. Other cytokines, interleukines and interferon were also produced due to direct stimulation of certain cell receptors triggered by the L. casei (Roberfroid 1998; Zaho et al., 1998; Tannock, 2002; Ibnou-zekri et al., 2003 and Pengcheng et al., 2011).

On the other hand when investigating the serum biochemical analysis of diarrhoeic calves, significant decrease in total proteins, albumin, globulins, A/G ratio (hyprproteinemia) as well as glucose was manifested due to the action of Salmonella enterotoxines (Table4 & 5). These toxines activated the adenyl cyclase enzyme, which lead to producation of cyclic adenosine monophosphate (cAMP). This cAMP instantly increased the intestinal fluid secretion from the systemic circulation resulting in varying degrees of dehydration, electrolyte imbalance and acidosis. These results were supported with many other authors (Blood et al., 1983; Manna et al., 1993 and Kaneko et al., 1997). Also the enterotoxines induced intestinal secretion may be blocked by cycloheximide which is an inhibitor of protein synthesis Serebro et al. (1969). The significant decrease in glucose level (Table 5) was due to decrease in glycogenesis and increase an aerobic glycolysis which was induced by the effect of diarrhoea as (Tennant et al., 1968). It was also manifested in diarrhoeic calves, that the enzyme activity of ALT, AST, alkaline phosphatase as well as the values of creatinine, urea and uric acid were significantly increased (Table 5), that could be explained due to the direct damaging effect of Salmonella toxines on hepatic and renal cells. These results were also confirmed by Manaa et al. (1993); Aly et al. (1996).

The serum minerals profiles were also altered in diarrhoeic calves, where serum calcium, phosphorus, magnesium, zinc, cupper, iron sodium and chloride levels were significantly decreased, whereas potassium level was significantly increased (Table 6). The decrease in calcium and magnesium levels could be due to secondary nutritional and metabolic disturbances, that was caused by excessive faecal losses, malabsorption that results from vairous types of bowel diseases including Salmonella infection, or due to intrinsic biochemical effect in the mucosal cells that interfere with digestion and absorption (Kaneko et al., 1997). Hypomagnesimia may also be exacerbated by the server diarrhoea (Groutides and Michell 1990). The significant decrease in serum sodium, chloride, iron, zinc and cupper (Table 6) was due to the increased intestinal secretion of water and electrolytes (Kaneko  et al., 1997). Moreover, diarrhoea is a common cause of metabolic acidosis due to direct loss of bicarbonate via faeces (Lewis and Phillips, 1972; Ragab et al., 1986) and therefor, during diarrhoea the increase in hydrogen ions were buffered by intracellular and extracellular buffers. In exchange for the intracellular movement of the hydrogen and potassium ions to the extracellular compartment predisposing to hyperkalemia (Robinson and Hauxtable 1988; Aly et al., 1996). This hyperkalemia continued due to the increased movements of cellular potassium into the extra cellular fluid and decreased renal excretion (Fisher, 1965).

The oral administration of L. casei could also amelurate the damaging effect of Salmonella toxines on the microvilli, entrocytes as well as the liver and kidney cells. Significant improvement was noticed on the serum biochemical parameters (Table 4,5,& 6) though in some cases the values did not reach those of the control apparently healthy calves. L. casei could optimize the permeability of the mucosal epithelium as well as colonizing on the intestinal mucosa (Zaho et al., 1998; Tannock, 2002; Ibnou-Zekri et al., 2003 and Musa et al., 2009). Though Salmonella infection in calves could produce heavy economic losses in the animal wealth, yet we could conclude, that by regular monitoring of animals with bacteriological examination of faecal samples as well as serological (by LPS-ELISA) and biochemical analysis of serum, we could control Salmonella infection in calves. Also oral administration of L. casei could improve the health and the immune status of diarrhoeic calves and could be considered as value-added to the farmers and breeders economics if they widely use it.

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Application of some lactobacillus strains product for control of Salmonella typhimurium infection in diarrhoeic neonatal calves

A.M. NABIH^* and M.M. ARAFA^**

^* Mastitis and neonatal Diseases Department, Animal Reproduction Research Institute, Giza, Egypt.

^** Chemistry Department, Animal Heath Research Institute, Cairo, Egypt.

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                                            ABSTRACT

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استخدام بعض عترات اللاکتوباسلس في الوقاية من مرض الإسهال في العجول الرضيعة        المصابة بميکروب السالمونيللا تيفيميوريم

أشرف نبيه محمد ،  محمود محمد عرفة

تم عزلميکروب السالمونيللا المعوية النوع تيفيميوريم من عجول تعانى من اسهالات حادة ولها تاريخ مرضى للاصابة, کما تم عزل انواع اخرى منها سالمونيللا دوبليم وسالمونيللا مونيستر من عجول مصابة بالاسهال واخرى سليمة ظاهريا ومخالطة للعجول المصابة. کاناجمالىنسب العزل للعترات المختلفة من ميکروب السلمونيللا 34 من اصل 77 حالة مصابة بالاسهال (44.2%) و 12 ميکروب من اصل 97 حالة سليمة ظاهريا (12.9%). سجلت النتائج اعلى نسب عزل من اعداد الميکروبات المعزولة من نصيب السالمونيللا المعوية النوع تيفيميوريم , 21 ميکروب من اصل 34 (61.8 %) فى الحالات المصابة بالاسهال بينما کان العدد 9 من اصل 12 ميکروب فى الحالات السليمة ظاهريا والمخالطة للمصابة (75 %). کانت الاعداد المعزولة من السالمونيللا دوبليم والسالمونيللا مونيستر قليلة مقارنة بالتيفيموريم فتم عزل 11 من اصل 34 ميکروب (32.3 %) و 2 من اصل 12 (16.7 %) للسلمونيللا دوبليم , بينما کانت الاعداد 2 من اصل 34 ميکروب (5.9 %) و 1 من اصل 12 (8.3 %) للسالمونيللا مونيستر فى الحالات المصابة بالاسهال الحاد والسليمة ظاهريا والمخالطة للمصابة بالتتابع. تم تطبيق اختبار الاليزا على عدد 20 عينة بلازما دم  من عجول تعانى من إسهال حاد ومصابة بالسالمونيللا ومقارنتها بعينات من عجول تسمين تبدو سليمة صحيًا. اظهرت النتائج ارتفاع ملموس فى مستوى الاجسام المضادة لميکروب السلمونيللا فى العجول المصابة (1: 2400 -9600:1) عنها فى العجول السليمة ظاهريا والمخالطة للمصابة (400:1 - 1 : 7200) بالتتابع. تم تحليل نفس العينات بيوکيميائيا, وقد اظهرت التحاليل للعجول المصابة وجود إنخفاض ملحوظ فى نسب کلا من :- البروتين الکلى ، الألبيومين ، الجلوبيولين ، نسبة الألبيومين / الجلوبيولين ، کذلک نسبة الجلوکوز. بينما کان هناک إرتفاع معنوى فى نشاط إنزيمات الکبد وإنزيم الألکالين فوسفاتاز ونسبة الکرياتينين واليوريا وحامض البوليک. أيضا تغيرت نسب عناصر الأملاح المعدنية , حيث انخفضت نسبة کل من الکالسيوم ، الفوسفور ، الماغنسيوم ، الزنک ، النحاس ، الحديد ، الصوديوم والکلوريد. أما عنصر البوتاسيوم فقد زاد زيادة ملحوظة. تعزى جميع النتائج الى التأثير المباشر لحالات الاسهال الشديدة والتى تؤثر بدورها تأثير معنوى على مکونات الدم المختلفة. لوحظ أنه بعد إستخدام بکتيريا اللاکتوباسيلس کازياى فى العلاج , ظهر تحسن ملحوظ فى  بعض نسب ومعدلات مکونات بلازما الدم البيوکيميائية حيث تساوت تقريبا مع المعدلات الطبيعية.         کذلک انخفضت الاعداد البکتيرية انخفاضا ملحوظا للانواع المختلفة من السالمونيللا المعوية وکذلک الميکروب القولونى, مع زيادة ملحوظة فى اعداد بکتيريا اللاکتوباسلس. لذلک يوصى باستخدام مستحضرات بيولوجية طبيعية من بکتريا اللاکتوباسيليس کازياى کعلاج بديل للإسهال.

Key words: Diarrhoeic calves, Lipopolysaccarid (LPS), ALT, AST.

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INTRODUCTION

Salmonella infection occurs throughout the world and has a hazardous effect on human and animal health as well as great impact on farm economics. In calves the infection appears in the form of septicemia and diarrhoea and in pregnant cows many abortion cases were often manifested (Santos et al., 2001; Barrington et al., 2002). Salmonella enterica serovar typhimurium and dublin where the commonest serovars isolated from cattle. Other serovars as anatum, enteritidis, cerro, montevedio, saint paul, infantis rostock, newport and newington were also reported, but in lower incidences. Calves are highly susceptible to Salmonella infection especially when the pregnant dames were not vaccinated once or twice before parturition and if the neonatal did not receive colostrum (Visser et al., 1990; Konrad et al., 1994 and Santos et al., 2002).

Enzyme-linked immunosorbent assays (ELISAs) based on lipopolysaccharide (LPS) for different salmonella serovars has been evaluated by many researchers as a highly specific test for diagnosing Salmonella infection in bovine.

Combination Not only serum was tested in these ELISA but also milk samples and other body fluids were investigated (Hoorfar et al., 1995; Hoorfar and Wedderkoppe 1995; Smith et al., 1995; Seleim, 1999; Galland et al., 2000; Radke et al., 2002 and Veling et al., 2002).

Some authors declared, that serum biochemical profiles of diarrhoeic calves were affected as,total proteins,  albumin, globulin, A/G ratio and glucose were significantly decreased (Manaa et al., 1993 and Kaneko    et al., 1997). Liver and kidney profiles were on the contra`ry increased, whereas macro and microelements decreased significantly except for potassium which was significantly increased (Ragab et al., 1986; Aly et al., 1996 and Kaneko et al., 1977).

Recently many probiotic bacteria as lactic acid bacteria (LAB), Lactobacillus, Streptococcus, Bifidobacterium and some fungi as Aspergillus oryzae and yeast were used as health promoters for humans and animals. Certain preparations were used to boost immune status, increase resistance to infectious diseases, particularly of the intestine, decrease duration of diarrhea (Romond et al., 1998; sreekumar and hosono 1998; Tannock 2002; Ibnou-Zekri et al., 2003; Makras et al., 2006; Gratz et al., 2010). The mechanism of action of probiotics is not fully understood, either they migrate through the gut wall as viable cells and multiply to a limited extent or antigens released by the alive or dead organisms, that can be absorbed and stimulate the immune system directly. A third school of thought suggested that the Lactobacillus species acted indirectly through an effect on the other microbial components (as Coliform) of the gut flora. It was the product of this change which induced the immune response. Moreover, it appeared to be some relationship between the ability of Lactobacillus strain to translocate and the ability to be immunogenic (Fuller, 1989; Gibson, 1995; Roberfroid, 1998; Ibnou-Zekri et al., 2003).

The objective of this study was to determine the different Salmonellaentericaserovars causing fatalities and diarrhoea in calves and to apply lipopolysaccaharide-ELISA for diagnosis. Moreover, the study included investigations on the serum biochemical profiles of diarrhoeic calves before and after competitive exclusion treatment with L.casei, as well as assessment of this biological treatment in curbing Salmonellaenterica-induced diarrhoea in beef calves.

MATERIALS and METHODS

Specimens:-

Internal organs from 5 recently dead calves  (5 Intestines, 5 gall bladders, 5 livers, 5 spleens, 5 lungs and 5 kidneys) aged from 1 week to 6 months, had a history of anorexia, pyrexia and sever diarrhoea sometimes tinged with blood, were examined bacteriologically. Another 174 faecal samples (77 from diarrhoeic calves and 97 from apparently healthy contact animals) and 174 blood samples (from the same animals) were examined bacteriologically for Salmonellaand serologically for Selmonellaantibodies respectively. Samples were collected during the period of one year from august 2010 to august 2011 form calves aged (1 week – 6 months) at governmental and private farms in Giza, Gharbia and Dakahlea governorates. All specimens were transferred to the laboratory in ice box with minimum delay.

Animals:-

In the second phase of investigation 20 diarrhoeic beef calves that were proven positive for S.typhimurieum were selected for treatment with L.caseias competitive exclusion treatment. Faecal and blood samples were collected from these calves on weekly bases and assessed for Salmonella, Coliform and lactobacillus content, while blood samples were assessed for humoral and cellular immune response as well as the serum biochemical analysis was carried out. Another 15 calves were apparently healthy, had no history of diarrhoea and proven negative forSalmonellawere selected as control.

Isolation and identification of salmonella enterica:-

Faecal samples were cultured into selenit-f. broth, and incubated at 37⁰C for 18hrs. Loopful from these broth cultures were then streaked onto MacConkey and S. S. agar plates, incubated at 37⁰C for 24 and 48hrs. Suspected colonies were identified morphologically, biochemically by the API 20E System (BioMereaus, France) and serologically according to the Kauffman-White Scheem by slide agglutination test using polyvalent and monovalent somatic (O) and flagellar (H) antisera (Wellcome Research Laboratories, UK.) according to (Edwards and Ewing 1972).

Isolation and identification of Lactobacillus casei for oral administration to calves:-

L. casei was isolated from the intestine of healthy calves on Togosa agar medium at 37⁰C and 10% CO₂. The isolation and identification was carried out according to Qin et al. (1995). The selected L. casei isolate was tested for bile and acid tolerance (growth at 1% bile salt rogosa agar and at pH5) then adjusted photometreally at (10¹⁰ CFU/ml PBS pH 7.4). One ml of adjusted L. casei was mixed into 250ml of 2% sterilized skim milk immediately before oral inoculation of the calves. The bacterial population was confirmed by enumeration of serial dilutions on rogosa agar plates in duplicate. The administration of the L. casei was carried out every other day for a period of 3 weeks.

Salmonella, Coliform as well as Lactobacilli counts were determined in the faeces before and after oral administration of the Lactobacilli. Salmonella count was determined by direct inoculation onto S.S. agar, Mac Conkey agar (for coliform) and Rogosa agar (for Lactobacillus). If the number of Salmonella was less than 500/g, enrichment in selenit-f. broth (Difco) could detect those samples which were  negative in direct plating (Zaho et al., 1998).

Lipopolysaccaharide (LPS) ELISA:-

ELISA to detect antibodies to LPS prepared from Salmonella enterica serovar typhimurium was carried out by extraction of LPS by phenol-chloroform-petroleum ether (extraction mixture) as described by Demarco de Hormacche et al. (1988). Each well of microtiter 96-well plates (Falcon) was coated with 5μg LPS/ml in carbonate bicarbonate buffer (pH 9.6). After overnight incubation at 37⁰C, the plates were incubated with blocking buffer, consisting of 3% bovine serum albumin (BSA-Sigma) in phosphate buffer saline pH 7.4 and 0.05% Tween 20 (PBS-T) to coat the unoccupied sites on the plates. After 1 hr at 37^°C. The plates were then washed with PBS-T and 100μl goat anti-bovine horseradish peroxidase conjugate (Dako) diluted 1:1000 in 0.3% BSA in PBS-T, was added to each well and incubated for 1 hr at 37^°C. The plates were washed with PBS-T then 100 μl 3,3’,5,5’-tetramcthylbenzidine (ICN), prepared according to the manufacturer’s instructions, were added to each well. After 10 min, 25 μl 5 M H₂SO₄ were added to each well and plates were read at 450 nm in ELISA reader. The cut off value was calculated as the average optical density (OD) value of the negative control values plus 2 standard deviations (SD). The antibody titer was calculated as the highest serm dilution that gives OD value above the cut off point (Ramos et al., 2000).

Estimation of cellular immunity (Macrophage activity):-

Calves leucocytes were harvested from the whole blood by density centrifugation on Histopaque 1077 (Sigma) according to, Lammler and Ding `(1994). Histopaque can separate the leucocytes in a buffy coat layer over the erythrocytes. After the separation of the leucocyte cell fraction over the histopaque surface, the RBCs among the harvested cells were lysed by adding 0.87% ammonium chloride solution pH 7.2 (1:5 v/v) with gentle shaking.

The leucocytes were then washed with Minimal Essential Medium (MEM, Sigma), and were finally adjusted to 10⁵ cells/ml MEM using a hemocytometer. Salmonella cultures were adjusted photometrically to 10⁹ bacteria /ml  in MEM medium, then equal volumes of leucocytes and S.typhimurium isolates were incubated at 37^°C for 1hr with gentle shaking. The leucocytes-S.typhemeurium mixtures were then spread on a microscope slide, fixed and stained with acridine orange and examined under the microscope. The phagocytosis index was measured according to (Shoshani et al., 2000)

Serum Biochemical Profile:-

Collected serum samples from 20 clinically diarrhoeic calves before and after administration of L. casei ( 10¹⁰ CFU/calf), as well as 15 control apparently normal calves were analysed biochemically for determination of total proteins (Hoffmann and Richterrich 1990), albumin and globulins (Doumas et al., 1971), ALT and AST aminotransferases (Reitman and Frankel 1957). Glucose (King and Woottin 1959), alkaline phosphatase (Kilchling and Fraiberg 1951), Magnesium (Neil and Nelly, 1956), sodium and potassium by using flame photometer (Oser, 1989), iron cupper and zinc were estimated spectrophotometrically by Fernandez and Kohn (1991) and chloride (Varley et al., 1980).

Statistical Analysis:-

Statistical analysis of obtained serum values were carried out using the “t” test according to the method of (SSPS 14, 2006)

RESULTS

Only one serovar, Salmonellaenterica serovar typhimurium, 11 isolates were dected from the 30 collected organs of 5 dead calves. No haemolytic E.coliwas isolated from these 5 dead calves. Three different Salmonellaserovars were demonstrated from both clinical cases with diarrhoea as well as from the contact apparently healthy ones. Out of 77 diarrhoeic 34 were proven positive for Salmonellaisolation(44.2%) whereas the apparently healthy contact calves demonstrated lower rate of  isolation (12 out of 97, 12.4%).

Salmonellaentericaserovar typhimurium (S.typhimurium) was the most dominant serovar as revealed from the pattern of isolation. In clinical cases S.t.constituted 21 out of 34 isolates (61.8%) and 9 out of 12 (75%) in apparently healthy calves. Salmonellaenterica serovar dublin (S.dublin) and muenster (S.muenster) were revealed in lower isolation pattern, as 11 out of 34 (32.3%) and 2 out of 12 (16.7%) in case of S.dublin and 2 out of 34 (5.9%) and 1 out of 12 (8.3%) in case of S.muenster were detected in both diarrhoeic and apparently healthy calves respectively. All Salmonellaserovars were confirmed for its somatic (O) and flagellar (H) antigenic structure (Table 1). The cut off value for the LPS ELISA was calculated as 0.33 OD at 450 nm. Diarrhoeic calves recorded higher OD values (range 0.89 to 1.68) than apparently healthy (0.34 to 1.32). The high OD reading was expressed in high antibody titers in the diarrhoeic animals that ranged from 1:2400 to 1:9600, whereas the antibody titer ranged in the apparently healthy calves from 1:400 to 1:7200. LPS-ELISA testing generally revealed higher incidences of Salmonellainfection than the conventional culture method as 53 out of 77 (68.8%) and 37 out of 97 (38.1%) in diarrhoeic and apparently healthy calves respectively were tested positive for Salmonellaantibodies (Table-2).

Administration of L.casei (10¹⁰cfu) every other day for 3 weeks to 20 diarrhoeic calves elucidated significant improvement in the calves health conditions starting the first few days after administration. Salmonellacount was greatly reduced from 6.37 + 2.5 log₁₀ to zero. The Coliform count was also reduced but still within a limitted range of 5.28+2.1 to 6.21+2.3 (log₁₀). The remarkable increase in the Lactobacillascount was noticed immediately after administration from zero to 6.47+2.2 (log₁₀) (Table 3). Thehumoral as well as the cellular immune responses were also stimulated as the Salmonellaantibody levels were increased which was monitored by the elevated OD values from 1.68 to 1.94 OD at 450nm. The cellular immunity manifested in the macrophages activity was significantly enhanced by almost eight folds from 4.4+1.2 to 33.2 +5.1 cell/macrophange (Table – 3).

In diarrhoeic calves serum biochemical analysis showed significant decrease in total proteins, albumin, globulins, A/G ratio (hypoproteinemia), as well as glucose levels (Table 4 & 5). The enzyme activity of ALT, AST, alkaline phosphatase as well as the values of creatinine, urea and uric acid were significantly increased (Table 5). Minerals profiles were also changed, where serum calcium, phosphorus, magnesium, zinc, cupper, iron, sodium and chloride levels were significantly decreased. Potassium level was significantly increased (Table 6). After treatment with L.caseicertain improvements of most serum biochemical parameters were recorded, though in some cases some discrepancies were manifested compared to the control group (Table 4, 5, 6).

Table 1: Isolation and identification of different salmonella serovars from diarrhoeic and apparently healthy beef calves.

Table 2: Serum from diarrhoeic and apparently healthy, contact animals tested with LPS ELISA.

Table 3: Effect of lactobacillus casei administration on the intestinal microbial content, on humoral and cellular immunity of 20 salmonella enterica serovar typhimurium infected calves.

** Average number of cells inside the macrophage + standard deviation.

Table 4: Proteinogram in sera of 20 infected calves before and after treatment with probiotics (L.casei) in comparison with apparently healthy group.

Average value + standard error

a, b, c, values with different letters are significant (P<0.05). values with the same letters are not significant.

Table 5: Liver and Kidny Function indices in Serum of Calves Before and After Treatment

Average value + standard error

a, b, c, values with different letters are significant (P<0.05). values with the same letters are not significant.

Table 6: Serum Biochemical Analysis of Minerals Before and After Treatment.

Average value + standard error

a, b, c, values with different letters are significant (P<0.05). values with the same letters are not significant.

DISCUSSION

Salmonella infectious diarrhoea is an important cause of neonatal calf morbidity and mortality in different parts of the world including Egypt, which results in huge economic losses in the beef and dairy industries. Many risk factors were encountered with the occurrence of infection that was related to the calf, the pathogens involved and to the surrounding environment. The immune status of calves, specifically the level of passively acquired immunity through colostrum, is the major risk factor related to the calf and the occurrence of diarrhea (Abouzeed et al., 2000; Santos et al., 2001 and Barrington et al., 2002). Although numerous pathogens have been implicated in the occurrence of neonatal diarrhea as Salmonella, E. coli, Yersinia enterocolitica, Campylobacter and many others, only relatively limited numbers are commonly involved. Most should be viewed as secondary opportunists rather than primary pathogens, with the exception of Salmonella (Konrad et al., 1994; Barrington et al., 2002 and Santos et al., 2002). The isolation of Salmonella from the internal organs of 5 dead calves which had an episode of severe diarrhoea revealed the isolation of only one serovar (S.typhimureium), wheras 11 isolates were detected from the 30 collected organs and no haemolytic E. coli were detected in these dead calves. The further bacteriological examination of specimens collected from clinical cases with diarrhoea and the contact apparently healthy revealed isolation of 3 Salmonella serovars. Out of 77 diarrhoeic calves 34 were proven positive for Salmonella isolation (44.2%) whereas the apparently healthy contact calves demonstarted lower incidence of isolation, as 12 out of 97 (27.9%). S.typhimureium was the most dominant serovar as revealed in the pattern of isolation (Table 1). In clinical cases S.typhimureium constituted 21 out of 34 isolates (61.8%) and 9 out of 12 (75%) in apparently healthy calves. Salmonella enterica serovar dublin (S.dublin) and muenster (S.muenster) were also isolated in lower pattern, 11 out of 34 (32.3%) and 2 out of 12 (16.7%) in case of S.dublin and 2 out of 34 (5.9%) and 1 out of 12 (8.3%) in case of S.muenster were detected. Many researchers recorded similar isolation patterns and many other Salmonella serovars as enteritidis, infantis, Rostock, Saint paul, Newington, cerro, newport and muenster were incriminated in the induction of diarrhoea in calves with different incidences and clinical severalties of infection (Visser et al., 1990, Konrad et al., 1994; Abouzeed et al., 2000; Bishpham et al., 2001; Santos et al., 2002 and Ostad et al., 2009).

All Salmonella serovars were confirmed for its somatic (O) as well as flagellar (H) antigenic structure (Table 1). The serological examination of blood samples for detection of Salmonella antibodies by LPS-ELISA, revealed a cut off value of 0.33 OD at 450nm. Diarrhoeic calves recorded higher OD values (range 0.89 to 1.68 OD) than apparently healthy (range 0.34 to 1.32 OD). These results were in agreement with other authors who employd different ELISAs in monitoring Salmonella antibodies in the serum. In some cases the OD values which usually reflect the antibody titers in the samples did not match the severity of the clinical status of the animal as some severely diarrhoeic lethargic animals has low OD values and vice versa. (Hoorfar  et al., 1995 Hoorfar and Wedderkoppe 1995; Smith et al., 1995; Galland et al., 2000; Radke et al., 2002 Veling et al., 2002 and Fayol-Messaoudi et al., 2007).

The high OD reading was expressed in high antibody titer in the diarrhoeic animals that ranged from 1:2400 to 1:9600, whereas the antibody titer ranged in the apparently healthy calves from 1:400 to 1:7200. LPS-ELISA revealed higher incidences of Salmonella infection than the conventional culture method as 53 out of 77 (68.8%) and 37 out of 97 (38.1%) in diarrhoeic and apparently healthy calves respectively were detected with LPS-ELISA positive (Table 2.). These discrepancies between the culture and serological methods were explained due to the intermittent sheding of the micro-oganism as well as the eliciting of antibodies not instant with the onset of infection (Hoorfar and Wedderkoppe 1995; Smith et al., 1995; Radke et al., 2002; Chart et al., 2002; Veling et al., 2002; Ibnou-Zekri et al., 2003 and Galland et al., 2000).

Administration of L. caseiwas based on its resistance to culture on 1% bile salts media as well as its acid tolerance (pH 5). Diarrhoeic calves administered L.casei (10¹⁰ cfu) every other day for 3 weeks elucidated significant improvement in the health conditions starting the first few days after administration. Salmonella counts were greatly reduced from 6.37+2.5 log₁₀ to zero. The Coliform counts were also reduced but stayed in the range of 5.28+2.1 to 6.21+ 2.3 (log₁₀). The remarkable increase in the lactobacillus counts was noticed immediately after administration from zero to 6.47+2.2 (log₁₀) (Table 3). The change in the bacterial counts after the administration of L. casei could be attributed to the competitive exclusion of the Salmonella on the enterocytes receptors, production of lactic acid and many other metabolites which shift the pH in the intestine to acidic. This acidity was considered crucial in colonization of      L. casei and hindering the growth of Salmonella and other enteropathogenic bacteria. Some researchers used the organic acids and other probiotic substances as lactulose and lactitol (synthetic disaccharides) to produce prophylactic effect against enteropathogenic bacteria (Fuller, 1989; Roberfroid 1998; Zaho et al., 1998; Tannock, 2002 and Ibnou-Zekri et al., 2003).

The humoral as well as the cellular immune responses were also stimulated after the         L casei administration as the Salmonella antibody levels were increased as monitored by the elevated OD values from 0.68 to 1.94 OD. The cellular immunity manifested in the macrophages activity was also significantly enhanced by almost eight folds from 4.4+1.2 to 33.2 + 5.1 cell macrophage. All these signs of health and immune status improvement were due to the direct or indirect action of the probiotic L.casei, that stimulated B-lymphocytes to produce different immunoglobulins isotypes. Other cytokines, interleukines and interferon were also produced due to direct stimulation of certain cell receptors triggered by the L. casei (Roberfroid 1998; Zaho et al., 1998; Tannock, 2002; Ibnou-zekri et al., 2003 and Pengcheng et al., 2011).

On the other hand when investigating the serum biochemical analysis of diarrhoeic calves, significant decrease in total proteins, albumin, globulins, A/G ratio (hyprproteinemia) as well as glucose was manifested due to the action of Salmonella enterotoxines (Table4 & 5). These toxines activated the adenyl cyclase enzyme, which lead to producation of cyclic adenosine monophosphate (cAMP). This cAMP instantly increased the intestinal fluid secretion from the systemic circulation resulting in varying degrees of dehydration, electrolyte imbalance and acidosis. These results were supported with many other authors (Blood et al., 1983; Manna et al., 1993 and Kaneko et al., 1997). Also the enterotoxines induced intestinal secretion may be blocked by cycloheximide which is an inhibitor of protein synthesis Serebro et al. (1969). The significant decrease in glucose level (Table 5) was due to decrease in glycogenesis and increase an aerobic glycolysis which was induced by the effect of diarrhoea as (Tennant et al., 1968). It was also manifested in diarrhoeic calves, that the enzyme activity of ALT, AST, alkaline phosphatase as well as the values of creatinine, urea and uric acid were significantly increased (Table 5), that could be explained due to the direct damaging effect of Salmonella toxines on hepatic and renal cells. These results were also confirmed by Manaa et al. (1993); Aly et al. (1996).

The serum minerals profiles were also altered in diarrhoeic calves, where serum calcium, phosphorus, magnesium, zinc, cupper, iron sodium and chloride levels were significantly decreased, whereas potassium level was significantly increased (Table 6). The decrease in calcium and magnesium levels could be due to secondary nutritional and metabolic disturbances, that was caused by excessive faecal losses, malabsorption that results from vairous types of bowel diseases including Salmonella infection, or due to intrinsic biochemical effect in the mucosal cells that interfere with digestion and absorption (Kaneko et al., 1997). Hypomagnesimia may also be exacerbated by the server diarrhoea (Groutides and Michell 1990). The significant decrease in serum sodium, chloride, iron, zinc and cupper (Table 6) was due to the increased intestinal secretion of water and electrolytes (Kaneko  et al., 1997). Moreover, diarrhoea is a common cause of metabolic acidosis due to direct loss of bicarbonate via faeces (Lewis and Phillips, 1972; Ragab et al., 1986) and therefor, during diarrhoea the increase in hydrogen ions were buffered by intracellular and extracellular buffers. In exchange for the intracellular movement of the hydrogen and potassium ions to the extracellular compartment predisposing to hyperkalemia (Robinson and Hauxtable 1988; Aly et al., 1996). This hyperkalemia continued due to the increased movements of cellular potassium into the extra cellular fluid and decreased renal excretion (Fisher, 1965).

The oral administration of L. casei could also amelurate the damaging effect of Salmonella toxines on the microvilli, entrocytes as well as the liver and kidney cells. Significant improvement was noticed on the serum biochemical parameters (Table 4,5,& 6) though in some cases the values did not reach those of the control apparently healthy calves. L. casei could optimize the permeability of the mucosal epithelium as well as colonizing on the intestinal mucosa (Zaho et al., 1998; Tannock, 2002; Ibnou-Zekri et al., 2003 and Musa et al., 2009). Though Salmonella infection in calves could produce heavy economic losses in the animal wealth, yet we could conclude, that by regular monitoring of animals with bacteriological examination of faecal samples as well as serological (by LPS-ELISA) and biochemical analysis of serum, we could control Salmonella infection in calves. Also oral administration of L. casei could improve the health and the immune status of diarrhoeic calves and could be considered as value-added to the farmers and breeders economics if they widely use it.

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