ٍٍSTUDIES ON SOME BACTERIAL AND PARASITIC CAUSES OF LAMB DIARRHEA IN BOHAIRA PROVIENCE AND THE SUBSEQUENT BIOCHEMICAL CHANGES

Animal Health Research Institute,

Damanhour Branch.

ٍٍStudies on some bacterial and parasitic causes of lamb diarrhea in Bohaira provience and the subsequent

biochemical changes

(With 6 Tables)

By

A.A. Bkheet; R.S. Fadly and Hanaa R. ElHoffy

(Received at 25/8/2010)

دراسات على بعض المسببات البکتيرية والطفيلية المصاحبة للاسهالات في الحملان بمحافظة البحيرة والتغيرات البيوکيميائية المصاحبة

احمد ابو المجد بخيت ، رضا سمير فضلى ، هناء رشاد الحوفى

شملت هذه الدراسة 70 من الحملان تراوحت أعمارها من ثلاثة ايام حتى شهرين منها 60 حاله تعانى من الاسهال و (10) سليمة استخدمت کمجموعة ضابطة ومثلت هذه الحملان لانماط التربية المختلفة – مزرعة کبيرة وتجمعات صغيرة أضافة الى حالات مملوکة لأفراد بمحافظة البحيرة وکان الهدف من هذه الدراسة استبيان بعض المسببات البکتيرية والطفيلية المسببة للإسهال وکذلک دراسة التغيرات البيوکميائية المصاحبة. أظهرت الفحوص البکتيريولوجية أن الميکروب القولونى والکلبسيلا والانتيروباکتر والستروباکتر والبروتيس والسريتيا والمورجانيلا مورجانى وجدت بنسبة 43.3 و 28.3 و 16.6 و 15 و 13.3 و 13.3 و 8.3 % على التوالى ممثلة لمجموعة الانتيروباکترياسى وتم تصنيف عناصرها جميعا بيوکيمائيا. هذا اضافة للسودموناسى اروجينوزا والمکورات العنقودية الذهبية والمکورات السبحية الشرجية والمکورات المعوية البرازية بنسبة 8.3 و 13.3 و 3.3 و 6.7 % على التوالى. أظهرت الفحوص الطفيلية أن 83.3 % من الحملات مصابة بطفيليات معوية منها(42) 70 % ايجابية لاکياس الايميريا باستخدام طريقة التعويم و(11) 18.3 % ايجابية لحويصلات الکريبتوسبوريديا وأوضحت الدراسة أن (39) 65 % مصابة بالکوکسيديا منفرده و (8) 13.3 % مصابة بالکريبتوسبوريدا منفصلة و (3) 3 % مصابة بکلا الطفيليين وجميعها مصاحبة للإصابة البکتيرية وبعمل مزرعة لعينات البراز الايجابية لاکياس الايميريا فى 5و2% بوتاسيوم داى کرومات تم تصنيف سبعة أنواع من الحويصلات البوغية لايميريا الأغنام وکانت الايميريا کرندالز الاکثر انتشارا. تم إجراء اختبار حساسية لبعض البکتريا الهامة والأکثر عزلا لاختيار أنسب المضادات الحيوية. تمت دراسة التغيرات البيوکيميائية المصاحبة لحالات الإسهال ومقارنة التعبيرات بين السليمة ظاهريا والمصابة ببکتريا منفرده أو کربتوسبوريديا وکوکسيديا منفردتين او مشترکة وتم دراسة الأهمية الاقتصادية والصحية للاسهالات والسبل الکفيلة لمنعها.

SUMMARY

A total of 70 lambs aged from three days to 2 months were used in this study, 60 lambs showed symptoms of diarrhea while the remaining (10) lambs were healthy and used as control. The animals were belonged to a variable breeding model large farm, small holder and individually owned in Bohaira province. This work aimed to study some etiological agents, bacterial and parasitic, and studying the subsequent affects on some biochemical parameters. The bacteriological results revealed that. Ecoli, Klebsiella, Enterobacter Citrobacter, Proteus, Serratia spp and Morganella morganii as members of Enterobacteriaceae were detected in incidences of 43.3, 16.6, 15, 13.3 13.3, 8.3 % respectively. Pseudomonus aerogeneosa, Staph. aureus, Strept. ini and Enterococcus fecalis were detected in an incidences of 8.3, 13.3, 3.3 and 6.7 % respectively, the incidences were varied in variable breeding size. The parasitic investigations revealed 83.3 % positive samples 70% for Eimeria spp. by flotation technique and 18.3 % for Cryptosporidium spp. by modified Zeiehl Nelson technique, 65 % of cases were infested with Eimeria spp. Oocysts alone, 13.3 % of cases revealed Cryptosporidia parvum separately and 5 % were harboring mixed parasites all were coincide with bacterial infection. Seven spp. of Eimeria were identified by fecal culturing.           E. crandalis was the most common one. Sensitivity tests were carried for some of important and predominant isolates of bacteria. All members of the isolated Enterobacteriaceae strains were biochemicaly identified. The biochemical alterations accompanied with diarrhea including changes associated with bacterial, Coccidian, Cryptosposidia infections singly or mixed in relation to apparently healthy were done. The economic importance,  health hazards and reduce incidences or combating the problem were discussed.

Key words: Lambs, diarrhea, bacterial infection, parasitic infestation.

Introduction

Neonatal diarrhea is an important cause of death and considered to be one of the main hazards to lambs health (Mottelib et al., 1992; Sharif    et al., 2005).

Diarrhea is chemical entity which cause serious economic loss may lead to lamb mortality, weight loss or even lead to subsequent growth retardation (Bastarous et al., 2001). Diarrhea could be attributed to infection with single or multiple agents. Its severity depends partially on non infective contributing factors and on the nature of involved organisms.(Schoenian, 2007).Several bacterial species may be involved in diarrhea and losses of neonatal lambs. The most important being is E coli. and other members of Enterobacteriacae (Sharif et al., 2005; Wani et al., 2008)  Klebsiella species are incriminated in lamb diseases (Munoz et al., 1996). Different disease conditions were recorded in association with Klebsiella species from enteritis and pneumonia.(Mottelib et al., 1992; Abou EL Hassan, 1996)

Pseudomonas aeruginosa produces a wide variety of virulence which many play a role in the pathogencity of this organism. This virulence could be attributed to several extra cellular and cell associated substances(Goto, 1996; Marty et al., 1998).

Parasitic gastroenteritis is primarily a disease of lambs. The major of outbreaks of coccidiosis occur in lambs up to-4 months old. Although ewes have natural immunity, they act as a source of infection by contamination of bedding with coccidian oocysts (Foreyt, 1990; Qzdal      et al., 2009).Fifteen Eimerea spp. have the capacity of infesting sheep (Platzer et al., 2005).

Fourteen spp. infest sheep intestine, only one (E. gilruthi) in   abomasums.Clinical eimeriosis only occur in lowered resistance lambs or those subjected to heavy infections.

Cryptosporidiosis is one of the main causes of morbidity and mortality in young immune suppressed animal. Human being is considered as one of the major pathogens associated with diarrhea in ruminant (Hilali et al., 1998).

Hematological and biochemical alterations were recorded in newly born lambs suffering from diarrhea, moreover, significant changes in serum electrolytes, macro and microelements, some enzymes of blood serum protein were also recorded (Nassif et al. 2002; El-Sangery et al., 2004). The concentration of plasma protein reflects the health state because they are nutritive, carrier for the transport of components for most of the plasma constituents. (Jain, 1986).In addition they have an important function in body defenses (Kaneko, 1997)

Several outbreaks and sporadic cases of diarrhea occurred in neonatal lambs till weaning at BohairaProvince in variable breeding models, large farms, small holders causing economic losses. Therefore the goal of this study was aimed to throw some light on the variable bacterial and parasitic probable causes of diarrhea among lambs, studying the biochemical alterations and the changes in electrolytic pattern and their subsequent effects on lamb finding. The sensitivity tests for the predominant and important bacterial isolates were done.

Materials and method

I- Animals:A total of sixty (60) Egyptian sheep lambs of both sexes from three days up to two months old suffering from diarrhea were included in the present study and ten (10) lambs were apparently clinically healthy were served as a control. These lambs were belonged to private farm and small holders'. The diseased lambs under investigation were clinically suffered from diarrhea showing soft to fluid feces which may contain mucus and / or blood with as without foul odor.

II - Sampling:  

1- Fecal swabs: Fecal swaps were taken aseptically from diarrheic lambs and apparently healthy for bacterial isolation and identifications according to Knoneman et al. (1994); Quinn et al. (1994).

2- Fecal samples: Weretaken directly from the rectum in a clean plastic bags from diarrheic and control (apparently health) lambs and examined microscopically

3- Blood samples: Were collected from Jugular vein without anticoagulant for serum separation, from diarrhoeal lambs (n = 60), as well as clinically healthy (n=10) which were used as a control group. The collected serum samples were stored at -20°C until biochemical analysis.

Bacteriological media:

1- Liquid media, Nutrient broth, Muller Hinton broth, Seleniet -F broth, Brain heart inf. broth (Oxoid, 1982)

2- Semisolid media (soft agar): Semisolid nutrient agar (Cruickshank et al., 1975)

3- Solid media: Nutrient agar, Blood agar base, MacConky agar Eosin Ethylene Blue (EMB) agar Manitol salt agar, Salmonella Shegella agar, Muller Hinton agar (Oxoid, 1982)

4- Media used for bacteriological identifications Peptone water, M.R. and V.P broth Simons citrate agar, Christensen's urea agar base, Triple sugar Iron agar, Lysine iron Agar (Oxoid, 1982)

Methods     

Bacteriological examinations, isolations and identifications were carried according toCruickshank (1975); Krig and Halts (1984); Quinn    et al. (2002).

Parasitological investigations:

1- Concentration floatation technique: Usingsaturated salt solution to detect Eimeria Spp. Oocysts and using Sheathers solution to demonstrate the presence of Cryptosporidium spp Oocysts according to Soulsby, (1982)

2- Fecal culture for positive Eimeria spp. Oocysts using (2.5%) pot. dichromate solution.

3- Thin fecal  smear stained with modified Ziel Nilson  technique to detect Cryptosporidium parvuma according to Henricksen and pohlenz (1981)

4- Identification of Oocysts (morphology and morphometry according to Levine (1985)

Biochemical analysis: Was calorimetrically analyzed using test kits.

 - For measuring glucose level according (Siet et al., 1981)

 - Total protein were carried according to (Peteres, 1968)

 - Calcium was determined according to Glinder and king (1972)

 - Inorganic phosphorus was estimated accorsing to Daly (1972)

 - Sodium and Potassium were determined according Oser (1979)

 - Anti- microbial sensitivity, was carried according to test diffusions technique as described by Koneman et al. (1995) on Muller Hinktion agar. A total of 12 anti-bacterial agents discs (Oxoid)were used in this investigation including Florophencol, Marbofloxacine, Enrofloxacine, Sulpha Trimethprime, Gentamycine, Streptomycine. The results were interpreted according to Quinn et al. (1994). Statistical analysis was carried according toSnaedecor and Cochran (1982)

Results

Table 1: Incidences of isolated bacteria from diarrhoeal and apparently healthy lambs at variable breeding models

Table 2: Prevalence of bacterial infections among variable breeding models

Table 3: Result of anti biogram of the important predominant isolated bacteria.

Table 4: Incidence of parasitic infestation in fecal samples of diarrhoeal lambs (n=60)

Table 5: Prevalence and characters of different Eimeria species isolated from diarrhoeal infested lambs (n=50)

Table 6: Effect of bacterial infection or concurrent with Eimerea or Cryptosporidium on serum glucose, protein serum and mineral levels of diarrheic lams.

Values are means and their standard errors.

Means within a row without a common letter differ significantly 41(P < 0.05)

Discussion

Diarrhea is of common occurrence in animals of all ages in the flock. In lambs it could resulted in significant mortalities, weight loss or even lead to subsequent growth retardation (Bastarous et al., 2001; Sharif, et al., 2005). Table 1 shows that the, Enterobacteriaceae constitute the main causes of diarrhea in lambs as 81.4 % among diarrheal lambs. The incidence varied from 78.9 % in individual breeder and reached to 85.2 % in small holder's model. These results approached to these reported by EL.Ged, et al. (1994); Bastarous, et al. (2001); Andres et al. (2009); Ismail and Yousef (2009).

E.coli, Klebsiella, Enterobacter, Citrobacter, Proteus spp were the predominant members from both diarrhoeal and apparently healthy as represented 4.366, 29.3, 16.66, 15, 13.3% respectively in diarrhoeal and 20, 10, 30, 30, 20% in apparently health respectively. These results agreed with those reported by Bastarous et al. (2001) and Wani et al. (2008). E.coli was isolated in pure culture from 26 cases (43.14 %), these results approached to those reported by Fogan and Desmatchelier (1999) who recorded 36% from abattoirs yards. Higher incidences were reported by Bastarous et al. (2001) 50.59% and Ismail & Yousef (2009) with incidence 56.14%. The highest incidence 100 % was recovered by Blanco et al. (1996) among   (5–21) days old diarrhoeal lambs. Generally in E.coli infection, diarrhea occurs through the effect of enterotoxins which stimulate granulates cyclase activity of elial epithelium, heat stable toxin, (S.T) or adenyle cyclase activity of intestinal and capillary epithelium, heat labile toxin, (LT) resulting in hyper secretion of electrolytes and increased water diffusion into intestinal lumen resulting in acidosis and dehydration Wani et al. (2008).   

Table 1 revealed the bacteriological examination of fecal samples (83) isolates of Enterobacteriaceae from diarrhoeal (26) 43.3 % of E.coli, (17) 28.3 % of Klebsiella belonged to 8 Kleb. Oxytocea, Kleb. ozaenae (8) for each and single Kleb. pneumoniae, Enterobacter spp (10) 16.6 % belonged to 4 for each Ent. agglemerans and Ent. aerogenes and only 2 Ent. coloaca. Citrobacter 9(15 %) belonged to Cit. diversus (6), Cit. amalonaticuc (2) and only (1) Cit.freundii, Proteus spp. were recovered (8) 13.3 % 6 Prot. vulgaris and (2) Prot mirabilis, Serratia were detected (8) 13.3 % including Serratia marcescens (6) and Serratia funtecola (2) finally Morganella morganii were detested (5) 8.3 % These results agreed with those reported by Ashraf (1996)

Klebsiella species are incriminated with lamb diseases specially enteritis among neonatal mortalities (Mottelib et al., 1992; Abo EL Hassan (1996). The severity and significance depends on the system of management and the degree of intensification, colostrums intake, birth weight, age susceptibility, housing factor and the climatic conditions (Meltzer and Shpight, 1996).

Pseudomonas aeruginosa were isolated in (5) 8.5% from diarrhoeal lambs. These results are nearly to that recorded by Amal et al. (2002). Pseudomonas aeruginosa is an opportunistic pathogen that rarely cause disease in healthy animals. That pathogen cause abroad spectrum of infection as urinary, respiratory and intestinal tract infection and other sites although it contributes to high morbidity and mortality rates (Hotack and Majt, 1997).

Tables 1 and 2 show that Staphylococcus auereus were detected in infection rate of 13.3 % among diarrheal lambs while it was negative in apparently healthy lambs. Staphylococcus auereus is an important pathogen responsible for mortalities in newly born lambs as septicemic pathogen (Nevin et al., 2001)

Tables 1 and 2 revealed Streptococcus ini and Enterococcus fecales in incidences of 3.3, 6.6 % in diarrheal lambs, these organisms presented as commensally pathogens become invadors when disturbed immunity.

The pattern of antibiotic sensitivity of the most prevalent and harmful intestinal pathogens were done in vitro and the obtained data as in Table 3 revealed that Florofenecol, Cephteofure, Marbofloxacine were the most sensitive drug with incidence of 80, 70, 70% among 10 isolates of gained E.coli isolates followed by Enrofloxaciu and Lincospectin with 60%, these results approached to those recorded by Khaled (2004).

Concerning sensitivity of Klebsiella spp. as in Table 3, Florofenecol, Cephteofure, Marbofloxacine and Gentamycine were the more sensitive with 80, 60, 60 and 60 % followed by Lincospectin and cephotaxim with 40% lowest sensitivity were detected for Sulpha- methexazol, Amoxicillin and Ampicilline as 20% of isolates revealed sensitive. Moreover, Oxytetracycline was resistant for all Klebsiella spp isolate as E.coli. These results agreed with and Khaled (2004).

In the present investigation the antibiogram of Pseudomonas aeruginosa isolates were studied. The selection for treatment of it is often problematic due to its antibiotic resistance Kovac, et al.  (1998).

Table 3 denotes that Revampicine, Sulpha methexazol, Tetracycline, Amoxicillin and Gentamycine were resistant for the isolated Pseudomonas. These results agreed with Ghoniem et al. (2002) Pseudomonas aeruginosa were sensitive for Cephteofur Marbocyl, florophenecal and Lincospectin in 40 %, these results agreed with Bert       et al. (1998). However lower incidence could be attributed to low number of examined isolates.

The sensitivity of Staphylococcus aureus as in Table 3 revealed that Cephteofur Marbofloxacine, Cephotaxim, Lincospectin and Gentamycine are sensitive with rate 80, 60, 60, 60 % respectively, these results agreed with Guta et al. (2002) while disagreed with Hatem (2003).

Amoxicillin, Tetracycline, Sulpha methanol and Revampicine were lower as only (1) 20 % were sensitive. These results agreed with the finding of Hatem et al. (2003) and Ashraf (1996).

Those protozoan parasites Eimeria and Cryptosporidia are important enteric diseases of lambs resulting in diarrhea, inefficient weight gain and occasionally death (Foreyt 1990).

The parasitological examination of fecal samples revealed that    50 (83.3 %) out of 60 examined lambs were infested with Eimeria and Cryptosporidium (Table 4). These results are in accordance with Foreyt (1990); Causapé et al. (2002); Ozdal et al. (2009). Eimeria oocysts were identified in 42(70%) by floatation technique. This result agreed with Yakhchali and Golomi, (2008) (69.6%) in Iran and relatively similar to Ozdal et al. (2009) 60.6% in Turkey, but our results was lower than  those reported by Khalifa et al. (1986) 84% in Egypt. Boshra, (1994) 91.33% in Egypt and Bastarous, et al. (2001) 100 % in Egypt, However a Lower percentage than our results was recorded by Aly (1990) 24.56 % in upper Egypt, Mottelib, et al. (1992) 20% at AL-Quassim, Abou EL Hadid and Lotfy (2007) 29% in Egypt and Yakhchali and Zarei (2008) 16.7 % in Iran., these different  results, may be attribute to location, season, age, sex and health condition of examined lambs Seven species of Eimerea were detected in the present study the predominant was E.crandalis 59.5 % (Table 5). This result agreed with that reported by Reginsson and Richter (1997); Bastouerous et al. (2001) in Egypt. Lambs could harbored mixed infection with at least three species of Eimera Dittmar et al. (2010). Parasitological examination of fecal samples for the presence of Cryptosporidium parvum by Modified Zeihl Niealsen Technique (acid fast stain) revealed spherical red-pink stained oocysts with granular appearance and single layer wall 4.5-5 micron in diameter against green background this results agreed with description of Abu-Eisha (1994), Selim, (1995) and, Abu-Elwafa, et al. (2007) Cryptosporedium spp. were detected in 11(18.3 %) of 60 lambs. Table (4), the same result recorded by Abu- Elwafa et al. (2007) in Egypt.

The pervious studies that have been conducted on prevalence of Cryptosporidium spp. in lambs, based on microscopy, have reported prevalence ranging from 10.10% to 68.3% for Cryptosporidium spp.    (Abu Eisha (1994); Selim (1995); Abd. ELwahed (1999); Nassif, et al. (2002) in Egypt, Majewska, et al. (2000) in Poland, Ulutas and Voyvada (2004) Sari, et al. (2008); Ozdal et al. (2009); Turkey, Misic et al. (2006); Serbia, Soltan et al. (2007) Tunisia) Our result Fall within the range reported in pervious studies that mentioned above. These differences between results might attribute to geographical Location, overcrowded or bad hygienic condition, health conditions of examined Lambs and environmental conditions, breading size.

There is no totally effective therapy for Cryptosporidiosis Other than a healthy immune system good management, sanitary conditions and quarantine of sick lambs (Foryet, 1990; Misic, 2006) Veterinarians and breeders should be aware of the disease in order to avoid greet losses among lambs and to prevent its transmassion to human (Selim 1995; Abd. ELwahed 1999). 

Concerning the biochemical alterations in diarrhoeal lambs which accompanied with Bacterial and / or parasitic affections. Serum glucose levels showed significant decrease in variable affections compared with those apparently healthy (Table 6), these findings agreed with those reported by El-Sengary, et al. (2004); El-Dessouky and Nabila, (2005); Osman and Sadick (2008); Ismail et al. (2009); Shahira and Sahar (2009). who postulated factors causing hypoglycemia as anorexia, decrease intestinal glucose absorption, a low level of glucose reserves is young age and alterations in tissue metabolism caused by decreased blood flow and oxygenation associated with the hypovolemic shock which present in the hypoglycemic diarrheic animals.

Concerning the results of total serum protein the results as illustrated in Table 6 revealed significant hypoproteinemia, hypoalbuminemia and hypo globulinemia. These results agreed with the findings of Jain (1986); Ahmed (2002); Nassif, et al. (2002); Shahira and Sahar (2009).

The decrease in blood serum protein in the present study may be attributed to inability of the gut of infected animal to absorb and assimilate the heamobiotic principals regarding blood serum total protein, albumin and globulin. Jain (1986) declared that the loss of protein during inflammation or ulcerations of gastro intestinal tract lead to impaired absorption as well as increase protein loss due to injured mucosal cell.     

Serum electrolytes showed significant decrease in serum sodium among the variable causative agent of diarrhea in lambs where, the highest reduction in those affected by bacteria, Eimerea and Cryptosporidium. The reduction of values was less in single than double causes of diarrhea in comparison with apparently healthy one. That significant decrease in sodium level (P<0.05) hyponatremia could be attributed to losses of body fluids and the impairment of absorption through the damaged intestinal mucosa (Ismail et al., 2009; Shahira and Sahar, 2009). Nearly similar results were also obtained by Assad and Nizar (2004).

Concerning blood serum potassium level in the present study as illustrated in Table 6 revealed a significant increase (hyper Kalemia) compared with apparently healthy lambs. These results were supported by the finding of El Dessouky and Nahila (2005); Osman and Sediek (2008); Shahira and Sahar (2009). The results could be attributed to the subsequent acidosis associating with long standing enteritis in which false positive increase in K. values in attempts to compensate the occurring metabolic acidosis which known as K/H exchange (Radostits et al., 2000).

Hyper Kalmia is a common problem associate with the acid base and electrolyte disturbances that occurs in diarrheic lambs (Alan et al., 2008)   

In the  present results, Table 6 showed significant decrease in serum calcium level when compared with those control apparently healthy lambs, these results agreed with Shigehero et al. (2003); Shahira and Sahar (2009); Ismael and Fatma (2009). The decrease in blood serum calcium level could be attributed to losses of body fluid in hyper secretary diarrhea and impaired absorption through the damaged intestinal mucosa as reported by the previous authors.

Duncan et al. (1994) attributed the decrease in calcium level to the hypo albuminemia where decrease albumin concentration lower the total calcium level while both ionized and complex calcium levels remain normal.

In conclusion it is obvious that diarrhea is clinical sign of major importance. Enterobacteriacae specially E.coli, infection, must be considered in diarrheic lambs, other bacterial causes as Pseudomonas and Staph.aureus infection singly or combined with parasite specially Crytosporidia and coccidiosis cause biochemical alterations firstly attention to the mangemental factors and nutrition of the dams to insure adequate colostrums intake will help in the minimization of the infection. Moreover the proper planning and adequate knowledge of the back ground of the diseases and application of appropriate preventive programs at right time leading to elimination of diarrhea, the hygiene and good management will lead to combating, prevention of parasitic infestations. The adequate knowledge of biochemical alterations and proper interference will compate the lethal effects of diarrhoeal acidosis and dehydration.

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