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Animal Health Research Institute-New Valley

Role of Cryptosporidial infection as a pathogen of neonatal calf diarrhea in Assiut Governorate

By

F. A. Osman and A. H. Sadiek* Dept. of Animal Medicine, Faculty of Vet. Medicine- Assiut University

Abstract The present study was conducted on 220 neonate diarrheic calves aged 1-60 days, during the period from November 2007 to April 2008, among of which 200 calves, were suffering profuse diarrhea. Complete clinical examination of diarrheic calves was carried out. Fecal and blood samples were collected for detection of Cryptosporidia by both direct standard fecal examination and by modified Ziel- Neelsen staining. Variable degrees of mucoid, blood stained profuse diarrhea, apathy, loss of suckling affinity, straining, dehydration and recumbency were the main signs observed. The prevalence of cryptosporidaial infection among diarrheic calves in this study was 21 %. The highest rate of infection was in calves aged 10 - 30 days (73.8 %), while the lowest rate of infection was in calves aged 1 - 10 days (7.14 %). Mixed infection with coccidial oocyst was noticed in 7 cases (16.8 %) and with Ascaris in 2 cases (4.8 %). The co-existent diarrhea and dehydration were associated with hemoconcentration which is represented by significant increase in PCV, blood urea nitrogen (BUN), hyponatremia, hypochloremia, hyperkalemia and hypoglycemia. Significant increase ( p<0.01) in TWBCs may suggest an associating bacterial infection. Some infected calves was complicated with bronchopneumonia, among of which 2 cases has been died. It could be concluded that Cryptosporidium is recognized as an important primary or secondary pathogen in neonates diarrheic calves, resulting in severe economic losses in neonates. Other causes such as bacteria and virus could not be excluded.

Keyword : Neonate calves, Cryptosporidium, Diarrhea Introduction

Neonatal calf diarrhea is considered one of the most common and economically devastating conditions encountered in raising calf industry. Diarrhea in neonate calves is facilitated by many hygienic, nutritional, environmental and managemental stress factors in addition to bacteria such as E. coli, rota and corona viruses (Mackenzie et al., 1994). Cryptosporidium is considered the most important protozoan causing

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neonate calf diarrhea and case fatalities either alone or in association with other bacteria, viruses and coccidia especially in the first 20 days of life (O'Donoghue, 1995 and Ambroise, 2000).

C. parvum is an intracellular protozoan causing enteritis and diarrhea in cattle, sheep, goat and man, the first report on bovine cryptosporidiosis was published in 1971. Now it has been identified as one of the primary etiological agents of neonatal calf diarrhea (Tzipori, 1985 a & b; Janoff and Reller 1987; and Naciri et al., 1999). The parasite can be transmitted by the direct fecal oral rout or through ingestion of contaminated, milk or milk replacer, water or grain (Kirkpatrick 1985). Herd level prevalence of C. Parvum has been reported to range from 13 to 100% (Wade et al, 2000 and Santin et al, 2004).

The most prominent signs of Cryptosporidiosis in calves are depression, anorexia, apathy, abdominal pain, fever and mainly diarrhea accompanied by the shedding of a large number of oocysts, dehydration and /or poor condition. Calves most often recovers spontaneously within 1-2 weeks even though there is a large variation between individuals in how they respond to and recover from infection (Tzipori,1985 a and Mackenzie et al., 1994). Concomitant infection with other enteric pathogens can aggravate the clinical signs and prolong the duration of disease. Morbidity and mortality can be very high in calves less than 2 weeks old (Mackenzie et al., 1994). The objectives of the present study were to investigate the prevalence and significance of C. Parvum in neonatal diarrheic calves and the extent of its concurrent clinical and laboratory consequences. Materials and methods 1- Animals and samples.

A total no. of 220 neonate Friesian calves aged 1-60 days was selected from different localities in Assiut Governorate during the period from Nov. 2007 – April, 2008 and used for this study, among of which 200 calves were suffering variable degrees of enteritis, diarrhea and dehydration. Twenty calves were proved to be clinically and laboratory healthy and kept as control group (tab. 1). Diarrheic calves were classified according to their ages into three groups group I: 1- 10 days, group II: 10 -30 days, and group III: 30 -60 days.

Sampling was collected from all calves during the above mentioned period to reduce any potential confusing effects of season on the prevalence of cryptosporidium as follow: a- Fecal samples were collected directly from calves and immediately placed in a screw caped bottles. The samples were labeled with the age of calves, duration and intensity of diarrhea and the associating clinical signs and sent to the laboratory.

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b- Two blood samples were collected from jugular vein, in clean sterile centrifuge tubes. The first blood sample was collected with anticoagulant (sodium salt of EDTA) for estimation of TRBCs, WBCs and PCV %. The second blood sample was collected without anticoagulant and stored for biochemical study. 2- Clinical examination. Clinical examination of diarrheic calves was carried out according to Rosenberger, (1979) with special reference to description of diarrhea, body temperature, suckling affinity, posture and vitality of calves as well as signs and degree of dehydration. 3- Parasitological examination. Samples were stored at 4◦c in the laboratory and processed immediately at the same day of collection. The standard concentration floatation technique was initially applied on all samples. For each sample, 1g of feces was processed using sugar solution (SG 1.33) as the flotation medium to recover C. parvum like oocysts and other parasites. Calves were considered positive, if more than 10 oocysts /HPF with the correct morphology (i.e. optical properties, internal structure, size, shape) were found. The modified acid- fast Ziehl –Neelsen stain is most reliably and traditionally used to detect the presence of Cryptospordial oocysts (Webster et al., 1998) as follow: 1- Prepare a thin smear by spreading a small amount of feces on slide

with a wooden dowel. 2-Fix with gentle heat until the slide is uncomfortable to the touch and

cool the slide before proceeding. 3- Stain with carbol- fuchsin solution for 3 minutes. 4- Wash in running water until no more stain appears in the wash water . 5- Rinse with decolorizer until no more stain appears in (decolorizer can

easily made by adding 3 ml HCl to 97 ml 95% ethanol). 6- Rinse gently in running tape water. 7- Counter stain with the Methylene blue (0.03%) stain for 30 sec. 8- Wash gently under running water, stand the slides on end and air dry. 9- Oil immersion lenses are corrected for use with cover slide. Permanent

reference slide can be made by dipping the dry stained slide in xyline. 4- Hematological and biochemical analysis.

The total RBCs, total WBCs, Hb concentration and PCV were estimated according to Coles (1986). Blood glucose, blood urea and creatinine levels in blood sera were estimated using test kits supplied by bio–Meraux. Serum sodium and potassium levels were estimated photometerically by a flam photometer (Corning Model 400 England ESSEX). Blood serum chloride level was estimated using chloride analyzer (corning Model 925, England Essex).

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Results. Diagnosis of C. Parvum using acid –fast modified Ziel –Neelsen

staining technique revealed that the investigated Cryptospordium oocysts appeared as acid –fast (red – pink) on a blue back ground . The oocysts are sporulated, spherical to ovoid in shape with smooth wall, containing 4 sporozoites and residuum measuring 4 -5 um (fig. 1, 2). Results of microscopic examination of 200 stained fecal smears (Tab. 3) revealed that 42 (21%) were harbored C . Parvum oocysts, while 158 (79%) gave negative results. Positive cases of Cryptosporidium oocysts were associated also with 7 cases of coccidian oocysts (16.66%) and Ascaris vitulorum eggs (4.17 %) in 2 cases.

Greenish yellow mucoid diarrhea, often bloody stained with, straining, lack of appetite, rough coat and dehydration were the major signs observed. Lastly most calves lay down in a profound depression and harried respiration (tab. 2). Five calves (16.12 %) declared signs of bronchopneumonia and eventually 2 calves (6.5 %) were found dead 5-8 days after appearance of respiratory symptoms in G II (tab.2). Non significant changes were observed in RBCs, Hb meanwhile significant increase in WBCs and PCV, blood urea and serum K associated with significant decrease in serum Na and Cl were reported.

Discussion The greenish yellow profuse watery diarrhea, colicy pains, reduced suckling affinity and the occurring dehydration of diarrheic calves reflect the destructive lesions occurred in the intestine due to its infection with Cryptosporidia. Similar observations were reported by El –Khodery, (1996), Sadiek and Schlerka (1996), El –Sherif et al (2000) and EL –Dessouky, 2005). In the present study, the prevalence of cryptosporidiosis was 21 % out of 200 diarrheic calves resembling the previous reports of EL –Khodery, (1996) and EL-Sherif et al (2000). The obtained results is considered lower than that reported by Abdel- Salam, et al (1993). Such variation could be explained by different variables, such as age, feeding, seasons, individual immunity, housing and managemental factors. The oocysts recovered are spherical in shape within a clear holes, 4-5 um in diameter, stained pink and the back ground appeared blue in color.

Eventually animal age plays a great role in calf susceptibility to Cryptosporidium infection in the present study. It has been observed that 31 calves, aged 10-30 days have the highest rate of infection (73.8%) followed by calves aged 30-60 days (19.04%) while those aged 1-10 days have the lowest rate of infection (7.14%) as in tab. 3. The present data agreed with ỎHanley et al (1999) and EL –Sherif et al (2000) who stated that Cryptosporidium was an important pathogen when calves were less than one month old and confirms previous reports from various

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researchers that Cryptosporidium is a very common enteric protozoan in calves where about 73% were found in calves less than two months of age and decreased to about 0- 1% after weaning (Santin et al., 2004). Signs of bronchopneumonia and deaths among some calves of G II may reflect association of bacteria and virsus with Cryptosporidial infection. Similar findings were found by Sadiek and Schlerka, (1996). Haematological and biochemical parameters (tab. 4).

The non significant changes in the mean values of TRBCs and Hb values in diseased calves agreed with those previously reported by Awadalla (1996) and EL- Dessouky (2005). The expected reduction of TRBCs and Hb values due to anorexia and diarrhea could be masked by the expected increase in their values due to longstanding diarrhea and dehydration and the subsequent hemoconcentration (Sadiek and Schlerka, 1996). The significant increase in TWBCs could be declared according to Malina et al (1994) stating that the infection with cryptospordia or any protoza like parasites lead to increase in the TWBCs or it may reflect the associating bacterial infection. Mean values of PCV, showed highly significant (p<0.01) increase, reflecting excessive loss of body fluid in diarrheic calves, in addition to inadequate intake of milk and fluids during diarrhea (Fadl-Alla 1989).

The high significant (p<0.01) decrease in the glucose agreed with those obtained by EL-Dessouky (2005) that could be related to lack of glucose absorption from damaged intestine where the cryptospordia causes damage of villi which impair digestion and absorption (Anderson,1981). The significant increase (p<0.05) in blood urea with insignificant increase in serum creatinine may be attributed to the deficit in renal blood perfusion and reduced urine formation in calves (Walt, 1965 and Sadiek and Schlerka, 1996).The significant decrease in sodium and chloride levels (p<0.05) could be attributed to the loss of large amounts of sodium and chloride ions with intestinal secretion and diarrhea (Fadl-Alla (1989) and EL-Dessouky (2005)..

The highly significant (p<0.01) increase in potassium (K) level could be attributed to the subsequent acidosis associating long standing enteritis in which false positive increase in K values in attempt to compensate the occurring metabolic acidosis which known as K/H exchange (Radostits et al., 2000). Hyperkalemia is a common problem associated with the acid-base and electrolyte disturbances that occur in neonate diarrheic calves (EL. Dessouky, 2005 and Alan et al., 2008). Lastly it could be concluded that Cryptosporidium is considered a problem of neonate calves resulting in diarrhea, dehydration and deaths. Its destructive effect on the intestinal villi resulted in hyponatremia, hypochloremia and hypoglycemia, hemoconcentration and azotemia.

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Cryptosporidiosis in calves in Sohag Governorate, Assiut. Vet. Med. J. 29 (57): 151 – 163.

Alan D. W, Moise N. S. and Rebhun W. C. (2008): Hyperkalemic Atrial Standstill in Neonatal Calf Diarrhea. Journal of Veterinary Internal Medicine. Volume 6 Issue 5, Pages 294 – 297 (online published 2008 )

Ambroise-Thomas, P. (2000): Emerging parasite zoonoses: the role of host-parasite relationship. Int. J. Parasitol. ,; 30: 1361-

Anderson, B. C. (1981): Patterns of shedding of cryptosporidial oocysts in Idaho calves. JAVMA; 178; 982 -984.

Awadalla, S. A. M. (1996): Studies on enteritis in newly –born buffalo calves. Ph. D. Thesis. Fac. of Vet. Med. Zagazig University.

Coles, E. H. (1986): Veterinary Clinical Pathology 4th Ed. W. B. Saunders Company, Philadelphia, London and Toronto.

El –Dessouky S. A. and Nabila. M. El – Masry. (2005): Effect of Crypt. Parvum infection on the haematological and blood biochemical changes of buffalo calves with special reference to the prevalence of infection among buffaloes. Assiut. Vet. Med. J; 51 (105). 108 -123.

El –Khodery, S. A. (1996): Effect of Cryptospordiosis on some blood parameters in calves. M, V Sc. Thesis. Fac. Vet. Med. Kafr EL Sheih, Tanta University.

El –Sherif, A. M; Abdel –Gawad, M. A; Lotfy, H. S. and Shokier, K. A. M. (2000): Impact of gasterintestinal nematodes and Same enteric protozoal affections on the health of buffalo calves. Assiut. Vet. Med. J.; 43 (86): 260 -270 .

Fadl –Alla, M. G. E. (1989): Clinical, hematological and biochemical studies on calves suffering from diarrhea. M. V. Sc. Thesis, Faculty. Vet. Med. Cairo University

Janoff, E.N and L.B. Reller. (1987): Cryptosporidium species, a Protean Protozoan. J. Clin. Microbiol. 25: 967-975.

Kirkpatrick , C. E and Farrel, J. P. (1985): Cryptospordiosis continuing Education. Article. , 1 (3) 154- 161

Mackenzie. W. R. N. J. Hoxic, M. E. Proctor. M. S. Gradus. K. A. Blair, D. E Peterson. J. J Kamieszak. D. G Addis. K. R. Fox. J. B. Rose. and J. p. Davis. (1994): A massive outbreak of Cryptosporidium infection transmitted through the public water supply. New England. J. Med 331: 161 -167

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Malina, J. M; Rodriguez –ponce, E; Ferrer, O; Cutierrez, A. C. and Herndez, S, (1994): Biopathological data of goat kids with cryptosporidiosis. Vet. Rec. , 135 (67) :67 68,

Naciri, M, L., Mancassola M, P; Poirier P. R., Chermette R. (1999): Role of Cryptosporidium parvum as apathogen in neonatal diarrhea complex in suckling and dairy calves in France. Vet. Parasitol. 85: 245 -257.

O, Handley, R. M; Cockwill, C. Mcallister, T. A; Jelinski, M Morck, D. W. and Olson, M. E. (1999): Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. J. Am. Vet. Med. Assoc. Feb; 1; 214 (3): 391 -396.

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Sadiek, A. H. and Schlerka, G. (1996): Studies on Rehydration Therapy in diarrheic milk fed calves, Tierartztliche Umscahu, 51(9): 544-552

Santín, M. , J. Trout, L. Xiao, L. Zhou, E. Greiner, and R. Fayer. (2004). Prevalence and agerelated variation of Cryptosporidium species and genotypes in dairy calves. Vet. Parasit. 122:103-117 .

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Tables and figures

Tab. 1: Sources and No. of Diarrheic calves selected for the study. % of positive case

No. of negative case

No. of positive case

No . of examined animals

Sources and No.

37.5% 10 6 16 Arab- El madabk

19.23% 21 5 26 Shodb 18.75% 26 6 32 El - zawia 21.87% 25 7 32 Dronka 22.22% 28 8 36 Rifa 22.53% 110 32 142 Total number

Assiut city

25% 6 2 8 Abo –korse 14.28% 6 1 7 El-Akadema 20% 8 2 10 El-Baliza 20% 20 5 25 Total

Abu-Tig city

13.33% 13 2 15 Sahel - selem 16.66% 15 3 18 El -Khanyem 21% 158 42 200 Total

Tab. 2: The clinical signs observed in diarrheic calves under study.

G1 (1-10 days) G2 (20-30 days) G3 (30-60 days) Age groups Signs N % N % N % Diarrhea 3 100 31 100 8 100 Anorexia 3 100 25 80.6 3 37.5 Apathy 2 75 28 90.3 3 37.5 Straining 2 75 15 48.4 4 50 Mild fever 1 33 11 35.5 2 25 Dehydration 3 100 22 71 4 50 Recumbency 2 75 18 58 2 25 Bronchopneum. - - 5 16.12 - Deathes - - 2 6.5 - -

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Tab. 3:Incidence of Cryptosporidial infection among calves in relation to age and sex as well as the co-existence coocidia and ascaris.

Sex Age groups (days) Variables ♂ ♀ G 1:1-10 G 2: 10-30 G 3: 30-60 N % F % N % N % N % C.parvum 17 40.4 25 60.6 3 7.14 31 73.8 8 19.04 C. parvum + Coccidia

3 7.1 4 9.5 - - 2 4.8 5 9.6

C. parvum + Ascaris

- - 2 4.7 - - - - 2 4.8

Tab. 4: Mean values of some hematological and biochemical parameters in healthy (control) and infected neonatal calves.

Diarrheic calves N= 200

Control healthy calves N= 20

Items Parameters

9.40 ± 0.22* 8.57 ± 0.17 TWBCs (T/L) 8.11 ± 0.18 7.95 ± 0.19 TRBCs (G/L) 11.17 ± 0.28 10.59 ± 0.23 Hb (g/dl) 38.83 ± 0.69 ** 35.68 ± 0.42 PCV (%) 61.43 ± 2.15** 72.21 ± 1.29 Glucose (mg/dl) 27.49 ± 0.99** 23.36 ± 0.75 Blood urea (mg/dl) 0.95 ± 0.02 0.89 ± 0.01 Creatinine (mg/dl) 125.47 ± 1.78* 133.14 ± 2.09 Sodium(mmol/l) 5.77 ± o.23** 4.39 ± 0.25 Potassium (mmol /l) 94.24 ± 0.64* 96.35 ± 0.38 Chloride (mmol /l)

* Significant at P < 0.05 ** Highly significant at P < 0.01

Fig. 1: Stained fecal smear with ziel-Neelson (to the left) and a non stained fecal smear (to the right).

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Fig. 2: Prevalence of Cryptosporidia, Coccidia and Ascaris

infection among neonate diarrheic calves in Assiut

01020304050607080

Males Females G I GII G III

Groups

% o

f inf

ectio

n

Crypt. Crypt. & Coccid. Crypt & Ascaris

الملخص العربى

Animal Health Research Institute دور الكريبتوسبوريديا آأحد مسببات االسهال فى العجول حديثة الوالدة بمحافظة أسيوط

بواسطة

*فتحي عثمان وعلي صديق آلية الطب البيطري، جامعة أسيوط-قسم طب الحيوان-*

دة بمحافظـة أسـيوط تتـراوح من العجول حديثة الوال 220أجريت هذه الدراسة على عدد 200، منهم 2008 الى ابريل 2007 من نوفمبر يوما فى الفترة 60-1أعمارها ما بين عمر

أخـذت . عجال سليما أخذت للمقارنـة 20ومضاعفاته العجول المصابة باإلسهال الحاد و منك بفحـص بالطرق التقليدية وكـذل في البراز للكربتوسبوريديا للفحص الطفيلي عينات البراز

. مصبوغة بالزيل نيلسون وأخذت عينات دم للفحـص الـدموي والبيوكيميـائي براز مسحة تلخصت األعراض اإلكلينيكية في وجود درجات متفاوتة من اإلسهال الحاد والمـدمم أحيانـا

وعدم القدرة والمصحوب بفقد تام للرغبة في الرضاعة والحزق أثناء التبرز واإلنهاك والجفاف وجـود حويـصالت الطفيلـي أظهـرت نتـائج الفحـص . على الوقوف والرقـاد أحيانـا

فى إصابةوكانت أعلى نسبة , حاالت اإلصابة باإلسهال إجماليمن % 21الكربتوسبوريديا في أيـام كمـا %) 7.14( 10-0وأقلها في العمر مـن %) 73.71( يوما 30- 10العمر من وحالتـان ) %16.6( حاالت منها 7 حويصالت الكوكسيديا في مختلطة مع إصاباتلوحظت

5كما لوحظ ظهور أعراض التهاب شـعبي فـي ) %4.8(من االصابة بطفيل االسكارس أوضح الفحص الخلوي للدم ارتفاعا غير معنويا في عدد كريات .حاالت ونفوق حالتين منهما

المصمت مما يعكس وجود الجفاف بين الدم الحمراء والهيموجلوبين ومعنويا في حجم الخاليا ، كما لوحظ انخفاضا معنويا في رتفاعات معنوية في البولينا أيضا العجول المصابة تم تأكيده با

والجفـاف اإلسـهال كأحد مضاعفات كل من الصوديوم والكلوريد والجلوكوز في مصل الدم كريات البيضاء والـذي قـد لوحظ أيضا ارتفاعا معنويا في العدد الكلي لل . والحموضة الدموية

البكترية المصاحبة من ناحية ويعكس حالة الجفاف وتركيز الدم مـن ناحيـة اإلصاباتيعكس اب .أخــرى ة لاللته باب الهام د االس ر أح بوريديا تعتب ل الكربتوس والخالصة ان االصابة بطفي

ى خ ؤدي ال ي ت ية والت ة والفيروس باب البكتري هال باالضافة لألس وي واالس صادية المع سارة اقت .فادحة في العجول الصغيرة