Dept. of Bacteriology.

Animal Health Research Institute, Mansoura Lab.

STUDIES ON SOME BACTERIAL CAUSES

AND BLOOD SERUM BIOCHEMICAL CHANGES

OF RESPIRATORY AFFECTIONS IN LAMBS

(With 6 Tables)

By

M.M. Abd El–Latif and S.A. El-Dessouky*

*Dept. of Biochemistry, Animal Health Research Institute Mansoura Lab .

(Received at 17/12/2005)

دراسات عن بعض المسببات البکتيرية والتغيرات البيوکيميائية لمصل الدم

فىالإصابات التنفسية للحملان

محمود محمد محمود عبد اللطيف , صبري عبده الدسوقي

أجريت هذه الدراسة علي عدد 36 من الحملان في إحدى المزارع الخاصة بمحافظة الدقهلية منها 7 من الحملان السليمة ظاهريا , 11 من الحملان المريضة إکلينيکيا و18 من الحملان المذبوحة. وقد تم أخذ عينات للفحص البکتريولوجي وکذلک عينات دم وسيرم للفحوصات الدموية والبيوکيميائية وبالفحص البکتريولوجي للعينات تبين أن 35 عينة إيجابية للعزل البکتيري بنسبة 6 (85.17%) ,11(100%) و18(100%) من السليم ظاهريا, والمريض إکلينيکيا والحملان المذبوحة علي التوالي. وتم عزل 66 معزولة بکتيرية صنفت بيوکيميائيا إلي باستريلا مالتوسيدا 19(28.79%) , باستريلا هيموليتيکا 5 (7.58 %) , الميکروب القولوني 15 (22.73%) , کليبسيلا نيمونيى8 ( 12.12%) , سودوموناس إرجينوزا      7(10.61%) والميکروب الذهبي العنقودي 12 (18.18 %). وبإجراء اختبار ضراوة معزولات الباستريلا ملتوسيدا کانت کل المعزولات من النوع الممرض. وبإجراء الفحوصات الدموية والبيوکيميائية لعينات الدم المأخوذة من الحيـوانات السليمة ظاهريا والمريضة إکلينيکيا تبين أن التغيرات في صورة ومصل الدم في الحملان المصابة باضطرابات تنفسية عند مقارنتها بالسليم ظاهريا هـي: 1- وجود نقص معنوي عالي في العد الکلي لکرات الدم الحمراء وترکيز الهيموجلوبين , حجم الخلايا المضغوطة وخلايا الليمفوسيت وفي الجانب الآخر وجدت زيادة معنوية عالية في العد الکلي لکرات الدم البيضاء ومعنوية للخلايا المتعادلة والحمضية وخلايا المونوسيت. 2- وجود زيادة معنوية عالية في مستوى کل من أنزيمات الترانس أمينيز بالکبد (S.GGT, S.ALT, S.AST) وکذلک بولينا الدم وزيادة معنوية في مستوى الکرياتينين. 3- وجود نقص معنوي عالٍ في مستوي الالبيومين ونقص معنوي في مستوي کل من البروتين الکلي, والصوديوم , الکلورايد والکالسيوم. وعلي النقيض وجدت زيادة معنوية في مستوى سکر الدم والبوتاسيوم. مما سبق يتضح أن الإصابات التنفسية في الحملان ذات الأصل البکتيري تسبب تغيرات معنوية في صورة الدم وفي وظائف الکبد والکلى وکذلک تؤثر علي مستوى کل من عناصر الصوديوم , البوتاسيوم, الکلورايد والکالسيوم.

SUMMARY

This study was carried out on 36 lambs in private farm at Dakahlia governorate, 7 of them apparently healthy, 11 clinically diseased suffering from respiratory affections and 18 slaughtered lambs. Bacteriological examination of the samples revealed that 35 samples were positive for bacterial isolates, distributed as 6 (85.71%), 11 (100.00%) and 18 (100.00%) of apparently healthy, clinically diseased and slaughtered lambs respectively. 66 bacterial isolates identified biochemically into P. multocida 19 (28.79%), P. heamolytica 5 (7.58%), E. coli 15 (22.73%), Klebsiella pneumonia 8 (12.12%), Pseudomonas aeruginosa 7 (10.61 %) and staph. aureus 12 (18.18%). Pathogenicity test for P. multocida isolates indicated that all isolates were pathogenic. Blood samples were collected from the clinically  healthy and diseased groups of lambs. Two blood samples were obtained from each animal, one as a whole blood and the other in the form of blood for obtaining clear non-haemolysed serum. Haematological studies revealed that presence of high significant decrease in the total erythrocytic count, haemoglobin concentration, packed cell volume and blood lymphocytes (per mm³ blood) in diseased animals when compared with clinically healthy ones. Also high significant increase in total leucocytic count, significant increase in neutrophil, oesinophil and monocyte cells were recorded in diseased cases.The studied biochemical parameters revealed high significant elevation in the values of AST, ALT, GGT and blood urea in diseased animals. Also creatinine showed marked elevation. Presence of high significant decrease in the level of albumin, while  total protein, sodium chlorides and calcium levels were significantly decreased. On contrary, presence of significant increase in the glucose and potassium levels. From previously mentioned data, it was cleared that respiratory affections in lambs especially those of bacterial origin cause significant changes in blood picture, liver and kidney functions and the level of both sodium, potassium, chloride and calcium.

Key words: Bacterial causes, Blood serum, Respiratory affection, Lambs.

INTRODUCTION

Respiratory affections constitute a common problem in sheep, particulary lambs causing serious economic losses and mortalities. Wilson et al., (1985) and Radostits et al., (2002). The causes of respiratory affections in lambs are bacteria, viruses, fungi, parasitesand and other bacteria are the main cause of respiratory affections of sheep Soroor (1999). While poor hygienic measures and climatic disorders were the most predisposing factors to such infection Sharma and Woldeh, (1995). Respiratory troubles to all domestic animals were attributed to mixed infection with different bacteria isolates. (Yehia, 2000 and Wafaa, 2003). Pasteurella spp., klebsiella spp., E. coli,  Pseudomonas spp. and staphylococcus aureus are the most bacterial causes of. Lambs pneumonia (Martin 1996 and Bedier 2003). Pasteurella spp. were the main causative agents of the respiratory affection Sadiek  et al., (1993). The inflammatory lung diseases were generally accompanied by noticable drop of erthrocytic counts and marked elevation of total leucocytic counts, further more in advanced cases hepatic and renal dysfunctions were noticed (Soroor, 1999 and Rodostitis et al., 2002).

The aim of the present work was directed to study :–

1- Isolation and identification of bacterial causes of respiratory  affections in lambs.

2-  Pathogenicity of the isolates to laboratory animals.

3- Some haematological and biochemical changes associated with respiratory affections to aid in early diagnosis of such affections.

MATERIALS and METHODS

Animals:

The present study was carried out on 36 lambs aged from 12- 18 months located at a private farm in Dakahlia Governorate. The animals were fed on barseem and dry ration. Eighteen of these lambs were suffering from respiratory disorders were slaughtered. The rest were divided into two groups, 7 clinically healthy and 11 diseased lambs showing signs of respiratory troubles including rapid breathing, moist rales, congested mucous membranes, mucopurulent nasal discharge, severe dysponea and pyrexia in (Rectal temperature 40 – 41°C). Parasitological examination was done for the diseased lambs to exclude those infested with internal parasites.

Samples:

Bacteriological samples:

Nasal swabs were collected from all animal groups. The tracheal swabs and lung tissues were only collected from the slaughtered lambs. The samples were taken under aseptic conditions and sent without delay to the laboratory for bacteriological examinations as follow:

Nasal and tracheal swabs were inoculated into nutrient broth and incubated at 37^oC for 24 hr. and then subcultured into 5% blood sheep agar, MacConkeys agar and nutrient agar and incubated at 37^oC for 24- 48 hr. The surface of the lung tissues were sterilized with a hot spatula then, the tissue was incisized with sterile scalpel and with sterile platinum loop samples were taken and inoculated in the previously mentioned media. The produced colonies were identified by their morphology and biochemicals activities according to Koneman et al., (1997) and Quinn (2002).

Pathogenicity and virulence of isolated Pasteurella multocida:

This was done according to Wessman (1964) where 51 Swiss Webster white  mice of (18 – 22 gram weight) were used. The mice was injected intra peritoneally by 0.1 ml of bacterial suspension (1.5 x 10⁸ organism per ml.). All dead mice showed postmortem changes.   Reisolation of inoculated strains from heart blood of dead mice was carried out.The prepared blood films were stained with leshiman's stain for showing the characteristic features of P. multocida organisms.

Blood Samples:

Two blood samples were collected from each lamb via jugular vein puncture. The first samples were whole blood collected in vacutainer tubes containing EDTA as anticoagulant and were used for haematological studies according to Jain (2000). The second samples were collected in centrifuge tubes and allowed to clot at 37°C and then  nonhaemlysed blood serum was separated used for measuring total protein according to (Doumas et al., 1981), albumin (Frank, 1950), glucose (Trinder, 1969), blood urea (Coulmobe 1963), creatinine (Husdan and Rapoport 1968), serum AST, ALT (Reitman and Frankel 1957) and Serum GGT (Szasz, 1969). Using commercial diagnostic kits supplied by BioMerieux France, while total globulin calculated mathematically by subtracting albumin from total protein. Blood serum  Sodium, potassium and chloride were determined using flame photometer (Oser, 1979). Serum Calcium was determined according to (Gindler and King 1972).

Statistical analysis :-

All Data were subjected to statistical analysis according to Snedcor and Cochran, (1982).

RESULTS

All bacteriological, haematological and serum biochemical results were illustrated in Tables (1 – 6).

Table 3: Pathogenicity of isolated Pasteurella multocida in mice.

Table 4: Mean values of haematological parameters in clinically healthy and respiratory affected lambs.

* : significant at (p < 0.05).

** : highly significant at (p < 0.01).

N.S : non significant . 

Table 5: Mean values of liver and kidney functions in clinically healthy and respiratory affected lambs.

* : significant at (p < 0.05).

** : highly significant at (p < 0.01).

Table 6: Mean values of some biochemical parameters in clinically healthy and respiratory affected lambs.

* : significant at ( p < 0.05 ).

** : highly significant at ( p < 0.01 ).

NS : Non significant.

DISCUSSION

Commensal bacteria present in the respiratory system may cause respiratory disease when animals are subjected to stress factors (Palotay and Newhall 1985). The prominent clinical signs which were observed in this study were similar to those recorded by (Attia and Eassa 1997 and Hatem et al., 2003). Results recorded in Table (1) revealed that the higher incidence of bacteria was obtained from clinically 11 diseased lambs (100%) and (100%) from 18 slaughtered lambs as compared to healthy 6 ones (85. 71%). While Ibrahim and Selim (2003) isolated bacteria from clinically healthy, diseased and dead lambs with an incidence of 20%, 66.67 and 78.26% respectively.

The results of identification of the obtained isolates using biochemical tests revealed that isolation of P. multocida, P. haemolytica, E. coli, klebsiella pneumoniae, Pseudomonas aeruginosa and staph. aureus as shown in (Table, 2). These findings agreed with those reported by Yehia (2000) and Bedier (2003).

In our investigation, the bacteriological incidence revealed that isolation of P. multocida. and pseudomonas aeruginosa were 2 (5.71%) for each one and. 1 (2.86%) for each E.coli and Klebsiella pneumoniae from clinically healthy lambs as shown in (Table, 2). On theother hand Elyas (1993) who isolated Staph. aureus (26%), E. coli (16%) and         P. multocida (3%) from clinically normal lambs. Bacteriological examination (Table 2) of clinically diseased lambs revealed that the isolation of P. multocida 6 (17.14%), P. haemolytica 2 (5.71%), Klebiella pneumoniae 3 (8.57%), Pseudomonas aeruginosa ( 2.86 %) and 5 (14.29%) for each E. coli and staph. aureus. These resrlts were nearly similar to those reported by Hatem et al., (2003) who isolated     P. multocida, p. haemolytica, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and staph. aureus from diseased sheep with respiratory affections. The data present in (Table 2) showed that the positive bacteriological examination of nasal, tracheal. and lung samples, from slaughtered lambs were 15 (42.86), 7 (20.0%) and 16 (45.71%) respectively. Where the types of isolates were P. multocida,   P. haemolytica, E. coli, klebsiella pneumoniae, pseudomonas aeruginosa and staph. aureus. Similar findings were recorded by Elyas (1993) and Hatem et al., (2003).

The pathogenicity of P. multocida isolates to white mice     (Table 3) revealed that all isolates were highly pathogenic to mice producing acute septicemia and death within 48 hours post inoculation. This finding agreed with the result obtained by Aliaa (2002).

Dealing with the changes in the haematological parameters in diseased lambs (Table 4), showed microcytic hypochromic anemia represented by high significant reduction (p> 0.01) in erythrocytic counts (RBCs), hemoglobin concentration (HB), and packed cell volume (PCV%). These findings could be attributed to the failure of bone marrow cells and hepatocytes in utilization and hemoglobin synthesis resulting in inhibition of erythropoiesis during bacterial infection Kaneko et al, (1997). These results agreed with those recorded by       El- Bealawy and Marouf (2002) and Radostits et al., (2002).

            Table (4) showed high significant increase (p>0.01) in total leucocytic count and significant increase (p>0.05) in neutrophils, oesinophils and monocytes. On the contrary marked reduction (p>0.01) in lymphocytes was observed. These findings were supported by those obtained by Sadiek et al., (1993) who referred such haematological changes to bacterial infection and inflammatory lesions that acts as severe stimuli for production of mature and immature neutrophils.   

            The obtained data concerning liver functions tests, (Table 5) showed a marked elevation (p>0.01) of all measured liver enzymes including Serum AST, Serum AlT and Serum GGT. Haziroglue and Kul (2004) declared that such elevation in liver enzymes referred to the degeneractive and necrotic changes of the liver following the bacterial infection and its circulating toxins. These results come in accordance with those reported by Attia and Eassa (1997), Kodary and Abdalla, (2001).

            The respiratory affected lambs showed high significant and significant increase (p>0.01 & p>0.05) in serum urea and cretinine levels respectively (Table, 5). These findings fitted closely with those of Abdalla and Emam, (2005). Radostitis et al., (2002) referred the increases of blood. urea and certinine values to the increase in protein catabolism, febrile conditions disease, causing impaired cardiac function and decreased renal blood flow as well as renal damage from bacterial effects.

            Significant drop were observed in both blood serum, total protein and albumin (Table 6). Omran et al., (2005) said that the hypoproteinaemia and hypoalbuminaemia could be due to the state of anorexia and inability of the liver to synthesise protein. The result of protein pattern in the current work agreed with those recorded by El-Seidy et al., (2003) and Hatem et al., (2003).

            The results of the respiratory affected lambs revealed significant increase (p>0.05) in glucose levels. These findings coincide with those obtained by Abdalla and Emam (2005). Coles (1986) attributed the hyperglycemia to anorexia on one hane and to liver glycogen unstability in deficient oxygen supply in diseased lambs on the other side. 

Concerning serum electrolytes, (Table 6) showed significant hyponatraemia, hypochloremia (p>0.05) and hyperkalamia (p>0.01) in diseased lambs. The recorded changes might be attributed to the hyperpyrexia in the acute course of the disease and metastatic infection of liver and kidneys resulting in hepatic  and renal dysfunction (Soroor 1999) and Novert (2004). Similar results were recorded by Omran et al., (2005). Radostits et al., (2002) attributed the hypocalcemia to the anorexic state and intestinal malabsorption.

Finally, it could be concluded thatthe recovered pathogenic and potentially pathogenic isolates have an important role in the respiratory affection, So adequate hygienic measures and proper management may reduce the degree of animals exposure to disease producing agents. Respiratory affections specially those of bacterial origin were accompanied with some reversible adverse effects on animal health represented by hepatic, renal dysfunction and disturbance of haematological patterns, so we can put the previously concluded points in our consideration in diagnosis and treatment respiratory affections.

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           Table 1: Incidence of bacteriologically positive cases in examined lambs.

          Table 2: Type, incidence and frequency of bacteria recovered from examined Lambs.

         *The percentage was calculated according to the positive number of samples (35)

         ** The percentage was calculated according to the total number of isolates (66)