STUDIES ON SOME BACTERIAL AND MYCOTOXIN CAUSES ASSOCIATED WITH ASCITES IN BROILERS AND THE SUBSEQUENT BIOCHEMICAL CHANGES

Animal Health Research Institute,

Damanhour Branch.

 

          Studies on some bacterial and mycotoxin causes associated with Ascites in broilers and the subsequent biochemical changes

(With 8 Tables)

 

By

A.A. Bkheet ; Seham F.G. and Hanna R.E.

(Received at 15/12/2011)

 

دراسات عن بعض المسببات البکتيرية والسموم الفطرية المصاحبة للاستسقاء فى دواجن التسمين والتغيرات البيوکميائية المصاحبة

 

أحمد ابوالمجد بخيت ، سهام فؤاد جورجى ، هناء رشاد الحوفى

 

يمثل الاستسقاء ظاهرة واسعة الانتشار کمسبب ملموس للنفوق فى بدارى التسمين ، لذلک تم اجراء هذه الدراسة للتعرف على المسببات البکتيرية او / والسموم الفطرية المصاحبة لهذه الظاهرة والتغيرات الکيميائية المصاحبة لها. حيث تم فحص عدد 60 مزرعة من بدارى التسمين فى محافظة البحيرة ذات الاعمار التى تتراوح من 1 – 6 أسابيع تعانى من ظاهرة الاستسقاء. حيث تم تجميع 2 – 3 من الطيور المصابة اضافة الى طائرين من الطيور السليمة ظاهريا کمجموعة ضابطة مع مراعاة کافة الاشتراطات اللازمة. تم تجميع عينات کاملة للفحوص البکتريولوجية والبيوکميائية اللازمة للدراسة. أوضحت الدراسة أن نسبة الحالات الايجابية بکتريولوجيا للحالات المصابة 52 (86.7 %). أظهرت الدراسة ايضا أن الميکروب القولونى يمثل 53 % من الحالات الايجابية تلاها المکورات العنقودية الذهبية بنسبة 26 % إضافة إلى بعض من عائلة الانتيروبکتيريسيى مثل البروتيس والکليبسيلا والستروباکتر بنسبة 23.1 ، 19.2 % ، 13.5 % على التوالى. وکمصاحب وحيد للظاهرة کان الميکروب القولونى والمکورات العنقودية الذهبية. والبروتيس والکليبسيلا والستروباکتر بنسبة 30.8 ، 11.5 ، 11.5، 7.5 ، 3.8 % على التوالى بنسبة إجمالية 65.3 % بينما الاصابات المشترکة کانت 34.6 % . کما أوضحت النتائج أن 57.1% من الميکروب القولونى کانت ممرضة وإيجابية لتفاعل النمو على صبغة الکونجو الاحمر. أظهرت النتائج التى أجريت على العلائق المستخدمة فى المزارع  فى المشتبه فيها للسموم الفطرية وجود سموم فطرية من نوعي الافلاتوکسين والاوکراتوکسين بمتوسط  12.38 ، 6.36 جزء فى البليون للنوعين على التوالى، کما أظهرت النتائج الکيميائية وجود تغيرات ملحوظة فى نسب الصوديوم والبوتاسيوم والکالسيوم والفوسفور والکلوريدات بين الحالات المصاية بالاصابات البکتيرية والسموم الفطرية عن تلک المصابة بالبکتيريا مقارنة بين حالات الاستسقاء الذى لم يتم عزل بکتيرى منها والحالات السليمة ظاهريا ، کما أظهرت النتائج أيضا نفس التغيرات المعنوية فى إنزيمات الکبد ووظائف الکلى وتغيرات معنوية فى البروتين بين المجموعات المختلفة.

 

summary

 

Ascitis is world wide syndrome in growing broiler chickens responsible for significant economic losses. This study was conducted to investigate the bacterial, mycotoxin causes or in association with ascites and the biochemical changes. 60 farms of broiler chickens aged 1–6 week with complain of ascites in some members. Random sample of ascetic birds from each were examined and sample of apparently healthy as control. Ration samples were taken when mycotoxicosis were suspected. The study claimed that 86.7 % of cases were bacteriologically positive. E. coli was the predominant isolate from ascetic cases 53 % of the positive cases, followed by Staph. aureus 26 %. Members of Enterobactereacea Proteus, Klebsiella and Citrobacter Spp. were isolated in 23.1, 19.2, 13.5 %, respectively, as a sole isolate from ascetic cases. E. coli, Staph. aureus, Proteus, Klebsiella and Citrobacter were in incidence of 30.8,  11.5, 11.5, 7.5 and 3.8 %, respectively, with total incidence 65.3 %. Mixed infection represents 34.7 %. The gained E. coli were tested for pathogenicity and invasiveness as denoted 57.1 % of cases were positive for Congo red. The examined ration samples of some ascetic broiler flocks revealed aflatoxin and ochrotoxins with mean value of 12.38 and 6.36, respectively. The biochemical results revealed significant variations in electrolytes, minerals, liver enzymes and kidneys function between the bacterial & mycotoxin, bacterial, ascetic with no infection in comparison with apparently healthy broiler chicks.

 

Key words: Broilers, ascites, mycotoxin.

 

 

Introduction

 

Ascites is the condition in which the body cavity is filled with ascetic fluid leading to mortality and carcass condemnations (Riddell, 1997). The average incidence of ascitis in broiler flocks reached to 4.7 % (Nacamura et al., 1999; Soils Santos et al., 2005).

It has been suggested that right ventricular hypertrophy due to inadequate gas exchange and vasoconstriction of pulmonary arteries resulting in ascites formation (Buys and Barns, 1981). In addition, the increase of blood viscosity caused by high attitude, respiratory diseases and reduced oxygen transfer Cueve et al. (1974) and Huchzermeyer and De Ruyck, (1985). Moreover Baghbanzaden and Decuypere (2008); Singh et al. (2011) explained that ascites is a multi-faceted syndrome caused by interactions between physiological (O2 demand) environmental (altitude) and management (ventilation, nutrition and many diseased status) genetic factors. All seem responsible for the syndrome    

A considerable number of ascitis syndromes in broiler flocks caused by microorganisms, E. coli, Salmonella Spp. and many other organisms are considered pathogenic because of their lipopolysacharide layer which triggers pulmonary vasoconstriction leading to ascites (Chapman et al., 2005).  Moreover, some strains of E. coli which treated with some antibiotics lead to release of shigatoxin into blood stream of the infected individual affecting kidneys resulting in condition described (HUS) (Hemolytic Uremic Syndrome) which is principle cause of renal failure (Besser et al., 1999).

The over exposure of chicken to ammonia and dust resulted in damage to the epithelium of their respiratory tract which become deciliated and allowed inhaled pathogens especially E. coli (Oyetude     et al., 1978).

Mycotoxins are produced by certain filamentous fungi in rations as a result of fungal growth (Mujoni, 2003). The synergistic effects between mycotoxins exposure and some important diseases have been suggested. Mycotoxins concern has grown during the last few decades because of their implications to human and animal health as Welles productivity and economically (Wagacha and Muthomi, 2008). Recent studies reported that additionally combined administration of Aflatoxin and other toxins resulting in synergistic toxic effects in liver and kidneys (Dimitrokalls et al., 2008). Moreover, Raja et al. (2009); Eliana et al. (2010) added that, changes in clinical blood biochemistry including inhibition of liver enzymes and levels of plasma proteins mainly albumin and globulin.

The evidence of increased dietary sodium (Na+) can cause ascites in chickens. Sodium in drinking water is much harmful than that in feed as broilers consumed 1.5-2.5 times more water than feed. Moreover the effects of sodium from all nutrient sources is additive in chicken (Richard et al., 1992).   

Studies with regard to blood indices and biochemical variables indicated that, in the affected birds, there was a significant fall in serum total proteins, albumin, albumin globulin ratio (A:G), calcium and sodium. The elevation of serum alanine aminotransferase, asparate aminotransferase, alkaline phosphates and chlorine, phosphorus was reported in the ascitic birds. Also, there is haemoconcentration along with large fluctuations in serum enzyme and mineral levels in ascitic birds (Yersin et al., 1992; Julian, 1993; Dahiya et al., 2000; Daneshyar et al., 2009).

The present work was carried-out to study the ascites syndrome. Monitoring the bacterial causes or in association. The mycotoxin, in suspected cases, as cause or in association. Also, the biochemical alterations accompanied with ascitis in broiler flocks including liver and kidneys patterns and serum proteins.

 

Materials and Methods

 

I-Birds: A total number of 60 broiler flocks 1–6 weeks of age with complain of ascitis in many birds were used. Random samples from affected birds (2–3) were representing a case, also apparentally non ascetic birds were taken as control.

II-Sampling: Birds were sacrificed by slaughtering and blood serum was firstly departed for biochemical investigations.

After scarification, birds were desiccated and samples were taken from paranchymatus organs that include heart, liver, spleen, lung and kidneys then prepared for bacteriological investigations according to Cruckshank et al. (1975).

Bacteriological investigations:

The prepared samples were inoculated on nutrient broth and seleniet F broth (Oxoid, 1982) after incubation at 37 ^oC for 18 – 24 hours. Nutrient broth was inoculated on nutrient agar, blood agar and manitol salt agar. (Oxoid, 1982).

Selenite F. broth was inoculated on MacConkey agar, Eosin Methylen Blue (EMB), salmonella shegella agar (S. S. agar) (Oxoid, 1982) all were inoculated at 37 ^oC for 24 – 48 hrs.

- Suspected colonies were examined morphologically and cellular morphology after Gram stains. Gained isolates were identified according toCruickshank et al. (1975); Krig and Hoets (1984); Quinn et al. (2002).

- The identified E. coli strains were cultured on Congo red medium (Berkhoff and Vinal, 1988) to identify the pathogenic invasive E. coli.

Biochemical analysis was calorimetrically analyzed using biochemical kits responsible for these purposes:-

- Sodium and Potassium were determined according to the method of Glinderand King (1972). -Inorganic phosphorus was determined according to the method implied by Dally (1972).

- Chloride was determined according to the method of Feldkamp (1974)

- Total protein was determined according to the method of Peteres (1968), while the albumin level was determined according to the method of Dumas and Bigges (1972)while the globulin level was determined mathematically using the substraction of albumin from the total protein level.

- The level of Aspartate amino transferase activity (AST) was assayed calorimetrically using the method of Reitman and Frankel (1957).

- The determination of creatinine was carried-out using the method that described by Larsenc (1972).

- Determination of uric acid was carried-out according to the method of Fosti et al. (1980).

- Detection and calculation of Aflatoxin and Ochratoxin were carried florometrically, using Florometer according to the Florometer Protocol Manual (1997).

- Statistical analysis:

The statistical analysis was carried-out using Analysis of variance test (ANOVA) for determination of the effect of different bacterial and mycotic diseases of different biochemical and haematologiocal parameters under study according to SAS (1996).

 

Results

 

Table 1: Results of positive bacterial                                                                                                          

                         

Result	Number	%
positive	52	86.7
negative	8	13.3
Total	60	100

 

 

 

Table 2: Frequent isolates from ascitic broilers.

 

Isolate	Number	% (52*)	(% 60 **)
E. coli	28	53.8	46.7
Proteus Spp.	12	23.1	20
Proteus vulgaris	4	7.7	6.7
Proteus retegri	8	15.4	13.3
Klebsiella Spp.	10	19.2	16.6
Klebsiella ozaena	5	9.6	8.3
Klebsiella  oxytocia	5	9.6	8.3
Citrobacter Spp.	7	13.5	11.6
Citrobacter   Frundii	3	5.8	5
Citrobacte   Diversus	4	7.7	6.6
Staph aureus	14	26.9	23.3

 

Table 3: Bacterial aspects of ascitis in broilers (Number of positive samples, 52).

 

Sole cause			Mixed infection
Isolate	No	%	Isolate	No	%
E. coli	16	30.8	E. Coli + Staph aureus	4	7.70
Staph. aureus	6	11.5	E. coli + Proteus	4	7.70
Proteus Spp.	6	11.5	E. coli + Klebsiella	4	7.70
Klebsiella Spp.	4	7.7	Klebsiella + Staph	2	3.84
Citrobacter Spp.	2	3.8	Citrobacter + Proteus	2	3.84
			Staph. aureus + Citrobacter	2	3.84
Total	34	65.3	Total	18	34.6

 

 

Table 4: Results of in vitro differentiation between pathogenic invasive E. coli

 

Examined E coli	Congo Red Reaction
	Positive		Negative
Total	N	%	N	%
28	16	57.1	12	42.9

 

 

Table 5: Aflatoxin and Ochratoxin in rations of some ascetic broiler flocks rations.

 

 

 

Table 6: Sodium, Potassium, Calcium, Phosphorus and Chlorine Levels among different groups.

 

 

Means within the same column of different litters are significantly different at (P < 0.01).

 

 

 

 

 

Table 7: Uric acid, Creatinin, S GPT and S. GOT Levels among different groups.

 

Means within the same column of different litters are significantly different at (P < 0.01).

 

Table 8: Albumin, Globulin, Total protein Levels and Albumin/ Globulin ratio among different groups.

 

Group	N	Albumin mg/dl	Globulin  mg/dl	Total protein mg/dl	Albumin/ Globulin ratio
		Mean±SEM	Mean±SEM	Mean±SEM	Mean±SEM
Control negative	10	A 1.46±0.07	A 1.48±0.08	A 2.94±0.07	C 1.03±0.09
Control positive	8	B 1.43±0.06	B 1.41±0.08	B 2.83±0.07	B 1.10±0.10
Bacterial isolates groups	33	B 1.40±0.07	C 1.30±0.10	C 2.70±0.08	A 1.41±0.11
Bacterial isolates + Mycotoxins	19	C 1.34±0.05	D 1.10±0.07	D 2.44±0.09	B 1.15±0.15

 

Means within the same column of different litters are significantly different at (P < 0.01).

 

Discusion

 

Ascitis syndrome in broiler flocks have been increasing at an alarming rate and this condition has become one of leading causes of mortality and whole carcass contaminations through the world (Julian, 2002).

The present study was conducted to study the ascitis syndrome. A total of 60 broiler flocks of 1–6 week of age in Behaira province have complain of ascitis in some birds, 3–4 birds of each flock represented a sample and supplemented for the present study. Ration samples from some farms suspected to have mycotoxicosis were obtained. Results as illustrated in Table 1, revealed that 86.7 % of cases were bacteriologically positive and the remnant 13.3 % was negative for bacterial isolation these results agreed with the suggestion of right ventricular hypertrophy due to inadequate gas exchange and the vasoconstriction of pulmonary arteries as declared by Huchzermeyer and De-Ryck (1985). The higher incidence of bacterial association agreed with Chapman et al. (2005) who claimed that considerable number of ascitis syndrome caused by microorganisms. Also, the opinion of Oyetude et al. (1978) who claimed that the over exposure of chicken to ammonia, hypoxia and the damage of respiratory tract and deciliation allowed inhaled pathogens.

Table 2 revealed that variable detected bacterial isolates E. coli could be isolated in total incidence of 53.8 % from positive cases and 46.7 % for all examined.  E. coli were detected as sole cause in 30.8 %. These results meet with many literatures as discussed by Oyetunde et al. (1978) who claimed that E. coli represent the main pathogen inhaled to infect respiratory tissue after deceliation of respiratory tract.

Our results agreed also with Wooley et al. (1994) who claimed that E. coli strains produce heat labile enterotoxins and lipopolysacharide which trigger pulmonary vasoconstriction leading to ascitis (Chapman et al., 2005).

Table 2, 3 revealed that Staphylococcus aureus is the second following pathogen could be isolated in ascetic flocks in incidence of 26.9 % from positive bacteriological cases and as single sole isolates in 11.5 %.

Growth lesions of septicemia that caused by Staph. aureus infection are vascular and congestion of many organs including liver and lung and spleen (Bickford and Resenwald, 1975). These septicemic lung congestion leads to hypoxia and imbalance of oxygen exchange revealing to ascetic syndrome.

Table 2, 3 revealed that proteus Spp. were isolated from 33.1 % of positive bacterial and represented 11.5 % as sole isolate. Proteus septicemia occurs in broilers suspected of having immunologic deficiency (Randal et al., 1987). Also has been associated with respiratory diseases in chicken (Lin et al., 1993) More over (Ye et al., 1995) recorded, 50 % mortalities in experimentally inoculated chickens with, proteus mirabilis, reisolated from lung, trachea, kidneys of chicken expressing respiratory signs, paralysis and high mortalities.

Table 2, 3 declared that Klebsiella Spp. were detected in 19.2% as associated isolate it was detected in incidence of 7.7 % as sole isolate in positive bacterial Ascites cases Klebsiella is an environmental contaminant that occasionally cause mortalities in chicken. The organism has been associated with respiratory, ocular, septicemic and reproductive disease of poultry Plesser et al. (1975). Chicken inoculated with three Klebsiella pneumonia has the highest mortality as declared by(Desouky et al., 1982).

Citrobacter Spp. was detected in 13.5% of positive bacterial ascetic cases and represents a sole isolate in (2) 3.8 % of positive cases. Citrobacter is a genus in the enterobacteriacae family, commonly colonized mucous membrane of respiratory and digestive tract (Saif, 2009).Citrobacter is one of many environmental bacteria that are occasionally isolated from weak chick and yalk sac infection (Lin et al., 1996)it has been isolated from liver of turkey pullet with respiratory disease (Fales et al., 1978).

Citrobacter species are known to cause a wide variety of nesocomal infection of the respiratory tract, urinary tract and the blood, hepatic, bileary and pancreatic disease that caused by Citrobacter frundii  (Morco et al., 1985).

All the previously discussed bacterial pathogens may be the main cause of Ascites initiators as many of E. coli strains on the action on cilleary system of respiratory tract, pneumonia which antagonism oxygen exchange and subsequent hypoxia.

Also, the pneumonia generated due to these pathogens coalesced with bad environmental and anemia generation could be the main precursor of Ascites.

Table 4 tabulated the in vitro differentiation of invasive E. coli by culturing on Congo red medium. Out of 28 isolates of E. coli 16 (57.1) were Congo red positive.

The growth status on Congo red is indicator for virulence, pathogenic invasive E. coli as recorded by Berkhoff and Vinal (1986). Our results lower than those reported by Moawad et al. (2008), who claimed that 83.7 % of E. coli strains causing diarrhea in calves.  Congo red, a simple acid dye has been used by Berkhoff and Vinal (1986)to distinguish between virulent and a virulent E. coli. They observed that direct correlation between the ability of certain E. coli to bind Congo red and their ability to induce septicemia infection in poultry (Gjesssing and Berkoff, 1989) However Spears et al. (1992) observed that the virulence of avian E. coli could not be absolutely predicted by the Congo red binding test.

Table 5 declared the individual mycotoxin affections of 19 examined suspected ration samples florometrically for Aflatoxin and Ochratoxin. Aflatoxin ranged from 0 up to 70 ppb with mean value of 12.38 and Ochratoxin from 0 up to 21 ppb with mean 6.36 these values even within permissible limit or slightly elevated but the summation and cumulative effects rendered it to be harmful (Dimitrokalls et al., 2008;Wagacha and Muthomi, 2008).     

The biochemical changes in ascetic cases in comparison with control showed several significant changes. Table 6 revealed a significant increase in serum sodium (Na), Potassium (K) and chloride (Cl) in all ascetic groups (Bacterial,Bacterial and mycotoxin associated and no-bacterial, non mycotoxic associated) in comparison with control non ascetic broiler. These results agreed with those reported by Richard et al. (1992); Wideman (2001); Julian (2002); Rezvan and Adel (2008).

The mono-valant minerals (Na⁺, K⁺ and Cl) determine the dietary electrolyte balance (DEB). Sometimes referred to as strong ions due to their greater effect on acid-base balance of body fluids than divalent ions such as Ca, Mag, PO4 or SO4 (Hooge, 2003).The increase in serum Na⁺ values were directly proportional to the serum K⁺. This results indicates that osmo-regulation of the body fluid is mainly achieved with the help of these two cations (Mustaq et al., 2005).

Water and dietary salt greatly increase plasma sodium concentration, therefore have a greater blood volume expansion, the avian blood capillaries are small and can dilate only every little to accommodate the extra blood volume produced Koike et al. (1979). In chickens a rise in plasma osmolality and/or Na⁺ concentration stimulate plasma vasotocin (AVT) which functions as an antidiuritic hormone and might further reduce Na⁺ excretion (Richard et al., 1992).

Our results showed that, the increase in Na⁺ level causes increasing body temperature probably by altering the hypothalamic thermoregulatory set prints, this would increase the body oxygen requirement and increase blood flows through the lung, increased interstitial fluid in the lung Na⁺ induced fluid retention could compress blood capillaries and increase resistance to blood flow. Sodium affects red blood cell rigidity and size and could increase resistance to flow in the small blood capillaries.

The significant increase in sodium, potassium and chloride may be due to kidney lesions that kidneys unable to excrete Na⁺ , K , Cl. These results agreed with those recorded by Siller (1981); Damron et al. (1986).

The serum calcium and phosphorus as illustrated in Table 6 showed significant decrease in ascetic broilers with bacterial and bacterial and Mycotoxins. These results agreed with those of Bailey et al. (1989) who claimed that in Ochratoxin affected birds. Moreover, Fernandez et al. (1994) reported that aflatoxins lower the serum calcium and phosphorus concentration in broilers. Lik et al. (2011) suggested that accumulation of intracellular calcium and inhibition of Ca⁺ ATPase might be important factors for the reduce deformability of the erythrocytes of ascetic broilers.

The total protein and albumin were significantly reduced in ascetic broilers as in Table 8, similar observations were reported by Yersin et al. (1992); Enkvetchkul et al. (1993). Moreover, Biswas et al. (1995); Tankson et al. (2002), Metwally et al. (2003), Daneshyar et al. (2009); Hashem and Mahmoud (2009) claimed that, the reduction in serum protein may be adaptive physiological response to the empending loss of extracellular fluids via ascities. Meanwhile, Diaz-Cruz et al. (1996); Daneshyar et al. (2009) attribute the decrease in serum protein to the glucogeneses because this pathway was substrates rather than carbohydrates such as amino acids to produce glucose. Other explanation by Singh et al. (2011) who declared that, the decreased plasma protein could be attributed to the loss of high protein lymph from the liver or stop eating prosess. 

Liver enzymes as tabulated in Table 7 revealed significant elevation. Glutamic-pyruvic transaminase (GPT) and Glutamic oxalacitic transaminase (GOT) as well as serum uric acid and creatinine which reflect tissue damage or chronic passive congestion. These results were in agreement with that reported by Enkvetcha et al. (1993); Julian (1993); Balog et al. (1994); El-Sayed (1995); Metwally et al. (2003); Hashem and Mahmoud (2009). The uric acid is the primary catabolic product of protein, non-protein nitrogen and purines in birds. The avian kidney excrete uric acid could be attributed to renal damage leading to decrease in the elimination rate of these waste products.

 

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