ROLE OF CALVES AND LAMBS IN TRANSMITTING CRYPTOSPORIDIOSIS TO CHILDREN IN WEST DELTA REGION OF EGYPT

Dept. of Animal Hygiene and Zoonoses,

Faculty of Veterinary Medicine,

AlexandriaUniversity.

Role of Calves and Lambs in Transmitting Cryptosporidiosis to Children in West Delta Region of Egypt

(With 7 Tables)

By

H.A. Samaha; Y.N. Haggag; M.A. Nossair

and I. El-Radda

(Received at 23/11/2011)

دور العجول والحملان فى نقل مرض الکربتوسبورديوزس إلى الأطفال في منطقة غرب الدلتا بمصر

حامد سماحة ، ياسر حجاج ، محمد نصير ، إسلام الردة

تعتبر الأوليات المعوية المسببة للإسهال من أهم الأمراض المشترکة المنتقلة من الحيوانات المختلفة إلى الإنسان وعلى رأسها الکربتوسبوريديوم بارفم. يعتبر طفيل الکربتوسبوريديوم بارفم من أخطر هذه الأوليات المعوية وأکثرها انتشارا علي مستوي العالم يصيب الأطفال الصغار والحيوانات الصغيرة والطيور والزواحف ويتسبب في نزلات معوية قد تؤدى إلى الوفاة في مرحلة حديثي الولادة، أو من يعانون من تثبيط مناعي في الإنسان والحيوان کما يعتبر هذا الطفيلي من الأسباب الهامة للإسهال في العجول والحملان حديثة الولادة. وقد أجريت هذه الدراسة ببعض المناطق الريفية في محافظتي البحيرة والإسکندرية لتحديد مدى انتشار طفيل الکربتوسبوريديوم بارفم في کل من الأطفال والحيوانات الصغيرة ودراسة تأثير عدد من العوامل المختلفة فى انتشار المرض وعليه فقد تم تجميع عدد 688 عينة بواقع 240 عينة براز من أطفال أعمارهم أقل من 6 سنوات من المترددين على أقسام الأطفال في مستشفيات الإسکندرية والبحيرة بالإضافة إلى 248 عينة من العجول و200 عينة من الحملان تحت 3 شهور. وقد تم التعرف على حويصلات الکربتوسبوريديوم بارفم عن طريق صبغ مسحات الروث والبراز بصبغة الزيل نيلسن المعدلة وتم تأکيد نتائج العينات الايجابية باستخدام اختبارELISA على عينات البراز أو الروث مباشرة. وقد بلغ معدل الإصابة بطفيل الکربتوسبوريديوم بارفم في العجول والحملان والأطفال 9.3، 13.5 و 11.7 % على التوالى وکانت معدلات الإصابة المسجلة في العينات المجمعة من محافظة البحيرة أعلى منها في محافظة الإسکندرية وقد يرجع ذلک إلى الطبيعة الريفية لمحافظة البحيرة. هذا وقد تم دارسة تأثير العمر والحالة الصحية وفصول السنة على معدلات انتشار المرض ومن النتائج يتضح أن روث بعض حيوانات المزرعة قد يکون مصدرا للعدوى بتلک الطفيليات وأنها مازالت تلعب دورا محوريا في المحافظة على انتقال بعض الأوليات المعوية المسببة للإسهال إلى الأطفال في المناطق الريفية حيث الاتصال المباشر بين تلک الحيوانات والإنسان لذلک يجب إتباع بعض الإجراءات الصحية لمنع انتشار ونقل تلک الأمراض إلى الإنسان مثل الاهتمام بفحص الحيوانات على فترات وتقديم العلاج للحيوانات المصابة من أجل تقليل إفراز الأطوار المعدية في روث تلک الحيوانات مع ضرورة التخلص الصحي من روث الحيوانات بعد معاملتها کيميائيا باستخدام المطهرات مثل مستحضرات الجير الحي. هذا وقد تمت مناقشة هذه النتائج مناقشة علمية مستفيضة.

SUMMARY

A total of 688 fecal and stool samples were collected from calves, lambs and children through out all seasons of the year including: 248 (62/ season) fecal samples of pre-weaned calves collected from diarrheic and apparently healthy animals from individually owned animals attending the veterinarian clinics in rural areas of Alexandria and Behera Provinces, 200 (50/ season) fecal samples of pre-weaned lambs were collected from diarrheic lambs and 240 (60/ season) stool samples were collected from children under 6 years old (the age of school entry) attending pediatric hospitals in Alexandria and Behera Provinces during the period extended from December 2008 to November 2009. All collected samples were subjected to microscopical examination by using Modified Ziehl-Neelsen (MZN) staining technique for detection of Cryptosporidium parvum oocysts. More over, all positive samples were confirmed by using RIDASCREEN Cryptosporidium ELISA kits supplied by GmbH, Darmstadt, Germany. It was found that the overall percentage of C. parvum infection in fecal samples of calves, lambs and children was 9.3, 13.5 and 11.7 %, respectively and the incidence of     C. parvum was higher in Behera Province than in Alexandria Province due to its rural nature. The effects of age, health condition and season of the year on the incidence of infection with C. parvum were studied and the public health significance of cryptosporidiosis was discussed.

Key words:Cryptosporidiosis, C.parvum, cales, lambs, children.

INTRODUCTION

Cryptosporidium species are of great public health concern as they may cause infection and severe illness in human specially children. Infections are self- limiting in people with normal immune systems but infection can be life threatening in people who have comprised immune system especially infection with Cryptosporidium parvum which always strongly associated with acute or chronic diarrhea in infants and HIV-infected adult patients worldwide as several recent waterborne outbreaks have shown, poses a significant threat to public health(Gabriela et al., 2005).C. parvum is considered one of the most important biological contaminants in drinking water. Dairy calves are thought to be the primary source of C. parvum contamination in watersheds. Understanding the spatial and temporal variation in the risk of               C. parvum infection in dairy cattle is essential for designing cost-effective watershed management strategies to protect drinking water sources (Szonyi et al., 2010).

The incidence of C. parvum in calves, lambs and children was investigated by many authors including: Noordeen et al. (2000); Wade   et al. (2000); El-Sibae et al. (2003); Zidan (2006); Castro-Hermida et al. (2007); Quilez et al. (2008); Coklin et al. (2009); Byomi et al. (2010); D­az-Lee et al. (2010).

Because of the public health significance of C. parvum, this work was applied to determine the incidence of C. parvum infection in calves, lambs and children in rural areas of Behera and Alexandria Provinces, compare using traditional methods in identifying C. parvum with using ELISA for detecting serological response, investigate seasonal patterns and age-associated trends of C. parvum in calves, lambs and children and throw a beam of light upon the public health significance of           C. parvum and the role of calves and lambs in continuation of its transmission.

MATERIALS and METHODS

1. Collection of samples:

A- Calves: 

A total of 248 fecal samples were collected from diarrheic (152) and apparently healthy calves (96) with age ranged from one day old to 3 months (pre- weaned calves) from individually owned animals attending the veterinarian clinics in rural areas of Alexandria and BeheraProvinces (62/Season).

B- Lambs:

A total of 200 fecal samples were collected from diarrheic lambs (71 males and 129 females) ranging in age from one day old to 3 months (pre- weaned lambs) from individually owned animals attending the veterinarian clinics in rural areas of Alexandria and Behera (50/ Season).

C. Children stool specimens:

Stool specimens were collected from 240 diarrheic children (120 males and 120 females) under 6 years old (the age of school entry) attending pediatric hospitals in Alexandria and BeheraProvinces (60/Season). Specimens were collected from cases in the same rural areas from which animal samples were collected.

Full history for each sample was taken including locality, age, health status and the characters of fecal samples then all samples are transferred directly as soon as possible to the laboratory of Animal Hygiene and Zoonoses Department, Faculty of Veterinary Medicine, Alexandria University and kept at 4 °C till examination.

2. Materials:

• Modified Ziehl-Neelsen stain was prepared as mentioned by Henrikson and Pohlenz (1981)

• RIDASCREENCryptosporidium ELISA kits supplied by GmbH, Darmstadt, Germany.

3. Methods:

• Preparation of collected samples: 5 g of each fecal and stool sample was placed in 2.5 % potassium dichromate solution and stored at       2–8°C until it is used in the ELISA test. If the material is not going to be used within 3 days, it is recommended to store it at –20°C. Multiple freezing and thawing of the sample must be avoided.

• Microscopic examination using MZN staining techniquewas performed according toHenrikson and Pohlenz (1981).

• ELISA was done using RIDASCREENCryptosporidium kits according to the manual supplied by GmbH, Darmstadt, Germany

RESULTS

Table 1: Incidence of C. parvum infection in examined fecal and stool samples of investigated speciesby MZN staining technique and confirmed by ELISA:

Table 2: Incidence of C. parvum infection in calves in relation to their health status:

Table 3: Incidence of C. parvum infection in total examined samples in relation to locality:          

Table 4: Incidence of C. parvum infection in examined samples in relation to seasons of the year:

Table 5: Incidence of C. parvum infection in examined fecal samples of calves and lambs in relation to age groups:

Table 6: Incidence of C. parvum infection in stool samples of children in relation to age groups:

Table 7: Incidence of C. parvum infection in examined stool samples of children in relation to history of animal contact:

DISCUSSION

Cryptosporidiosis remains among the most prevalent zoonotic pathogens in spite of the great strides made in its control and extirpation. It causes gastroenteritis and diarrhea in several species specially calves, lambs and human. The infection may be transmitted from man to man or from animals to man. Moreover, cryptosporidiosis is also of well known veterinary importance as it may cause economic losses in calves and lambs.

In the current study, the role of calves and lambs as a source of children infection with C. parvum was investigated.

The total incidence of C. parvum infection in examined fecal and stool samples collected from calves, lambs, dogs and children by MZN staining and confirmed by ELISA assay was presented inTable 1; firstly the obtained results revealed that the total incidence of C. parvum in calves fecal samples using MZN staining teachnique was 9.3 %. This result was lower to that obtained byQuilez et al. (2008) (57.8%), Byomi et al. (2010) (10.24 %). On the other hand, this result was higher that obtained Wade et al. (2000) (0.9 %), Castro-Hermida et al. (2007) (8.4 %), Coklin et al. (2009) (6.2 %). Moreover, Ralston et al. (2003) found nearly similar results. Secondly, the obtained results revealed that the total incidence of C. parvum in lambs was 13.5 %. This result was lower than that obtained by Ozmen et al. (2006)(66.7 %), Paoletti et al. (2009)(17.5 %) and Yang et al. (2009) (24.5 %), while it was higher than that obtained by Zidan (2006) (12.4 %), Castro-Hermida et al. (2007) (5.3 %) and Wang et al. (2010) (4.8 %). Finally, the obtained results in Table 1 revealed that the total incidence of C. parvum in children was 11.7 %. This incidence was higher than that obtained by Handousa et al. (1991) (10.9 %) andEl-Sibae et al. (2003) (8 %) on the other hand this incidence was lower than that recorded by Soliman (1992)(13.5 %), Dupont et al. (1995) (20 %) and Rozej et al. (2010)(47.5 %). Moreover, this result was nearly equal with that obtained byZidan (2006) (11.95 %) andFreites et al. (2009) (11.8 %).

The result of comparison between the performance of the enzyme linked immunoassay (ELISA) with MZN staining technique in diagnosis of C. parvum infection in different animal species and children was demonstrated also in Table 1. All positive cases by MZN staining technique were retested by commercial ELISA kits that detected all positive cases of children (100 %) then calves (95.7 %) and lambs (92.6 %) with overall sensitivity of 96.2 %. These results agreed with Marques et al. (2005) and Zidan (2006) who concluded that the commercial ELISA and MZN technique proved to be valuable, sensitive and specific diagnostic tools for C. parvum.

   The health status of calves under investigation affected the incidence of C. parvum. This illustrated in Table 2 where the incidence of C. parvum in diarrheic calves (12.5 %) was higher than apparently healthy calves (4.2 %). This result agreed withNaciri et al. (1999) who recorded the presence of C. parvum oocysts in the faeces of diarrheic suckling and dairy calves in France at the percentages of 34.7 and 2.4 %, respectively, while C. parvum were detected in faeces of non diarrheic suckling and dairy calves at a percentages of 13.8 and 3.9 %, respectively, Uga et al. (2000) who reported that calves infected with   C. parvum had a significant higher rate of diarrhea (33 %) than non infected calves (8 %) suggesting that C. parvum infection is the likely cause, Zidan (2006) recorded an incidence of 14 % in diarrheic calves and 4.8 % in apparently healthycalvesandD­az-Lee et al. (2010)found that a high number of infected animals suggesting the parasite C. parvum as a major parasitic disease agent of neonatal calves with diarrhea in dairy farms of the Metropolitan Region of Chile.

   The data presented in Table 3 firstly, highlighted the incidence of C. parvum in calves in the two investigated provinces. The incidence was higher in BeheraProvince (10.5 %) than in AlexandriaProvince (8.1 %) that may reflect the rural nature of BeheraProvince as compared with AlexandriaProvince where hygienic conditions might be suspected. This result agreed with Zidan (2006) who recorded an incidence of 10.94 % in BeheraProvince that confirmed the endemic nature C. parvum infection of calves of Behera Province that may represent a zoonotic threat to human being in this province which need effective control measures to eliminate that zoonotic threat. Data presented in Table 3 secondly, clarifiedthat the incidence of C. parvum in lambs was higher in Behera Province (16 %) than in Alexandria Province (11 %). The higher incidence in Behera than in Alexandria similar to that of calves' incidence that may note that calves and lambs may act as reservoirs of   C. parvum infection. This result was higher than that obtained by Zidan (2006) who recorded an incidence of 11.8 % in Behera Province that also confirmed the endemic nature C. parvum infection of lambs of BeheraProvince.

   The incidence of C. parvum in the examined stool samples of children collected from different localities was present also in Table (3). The incidence of C. parvum in examined stool samples from children of Behera Province (14.2 %) was higher than in Alexandria Province (9.2 %). This result was in agreement with results of examination of calves and lambs from the same localities that reflected the close association between the incidence of infection in animals and children that highlighted the zoonotic significance of C. parvum. These results confirmed by Hira et al. (1989)who statedthat contaminated water supplies and contact of animals such as sheep and goats were of the known risk factors facilitating transmission. Moreover, Keusch et al. (1992) indicated that persons at greatest risk were immunocompromised adults and children, especially those with AIDS, children in day care and travelers to endemic regions. The obtained result of C. parvum incidence in children of Alexandria Province under investigation was lower than that recorded by Soliman (1992) who detected a prevalence of 13.51% among school children in a rural area in Alexandria Province while the obtained result of C. parvum incidence in children of Behera Province under investigation was nearly similar to that obtained by Zidan (2006) (13.84 %).

The seasonal distribution of C. parvum in calves was recorded in Table 4 where the highest incidence of C. parvum was observed in spring season (12.9 %), followed by summer (9.9 %) then winter and autumn (8.1 %) and (6,5 %), respectively. This distribution was agreed with results obtained by Wade et al. (2000); Byomi (2010)who foundinsignificant association between infection with C. parvum in calves and season of the year. On the other hand, these results disagreed withMann et al. (1986); Sahal et al. (2005) who recorded that the occurrence of the disease was more common in winter (56.4 %) than during other seasons (autumn 0 %, summer 15.4 % and spring 28.2 %). The seasonal distribution of C. parvum in lambs was also recorded in Table 4 where the highest incidence of C. parvum was observed in autumn season (16 %), followed by winter (14 %) then similar incidence in both spring and summer (12 %). The effect of season on C. parvum infection in children was presented in Table 4 that revealed a higher incidence in summer season (21.9 %), followed by spring (13.3 %) then winter and autumn (6.8 %) and (5.0 %), respectively. This seasonal distribution was agreed with results obtained byTzipori (1983); Mata et al. (1984); Marc et al. (1990) who reported that the higher incidence of infection was during warmer months (May to August)andZidan (2006) who recorded an incidence of 18.46 %, 14 %, 10.71 % and 6.25 % during summer, spring, winter and autumn, respectively. These results suggested that the incidence rates were slightly higher during spring and summer seasons than during autumn and winter seasons. On the other hand, these results disagreed withBaxby and Hart (1986)who found that the peak incidence of Cryptosporidiun infection was in the late winter and early spring andSiwila et al. (2011) who reported that Cryptosporidium infections were significantly more common in the wet compared to the dry season.

The effect of age of calves and lambs on the incidence of              C. parvum infection was illustrated in Table 5.Concerning calves, the highest incidence was observed in the age group (1- <2 months)        (11.5 %) followed by the age group (<1 month) (9 %) and finally the age group (2- <3 months) (7.7%). This result disagreed withZidan (2006) who found that the highest incidence was in the age group (up to 1 month) (15.2 %) followed by the age group (1- 2 months) (5 %)andInpankaew et al. (2010) who found that calves less than 2 months old were more frequently infected by C. parvum than others. This result proved that the age of calves was an important factor affecting the incidence of C. parvum infection in calves. McCluskey et al. (1995) indicated that the period of prevalence of cryptosporidiosis was 100% in calves from 1-30 days of age. This proved that infection occurred early in the neonatal period and the environment was heavily contaminated with oocysts. Initial exposure to infective oocysts appears to occur in the maternity pen or shortly after placement in outdoor cages. However the role of the apparently healthy carriers in the epidemiology of the disease has more recently described. Excretion of oocysts has been found in apparently healthy adult cows (Villacorta et al., 1991; Scott et al., 1994). Concerning lambs, the highest incidence was observed in the age group (<1 months) (22.6 %) followed by the age group (1- <2 month) (7.8 %) and finally the age group (2- <3 months) (5.8 %). This result agreed withOlson et al. (1997) in Canada, Majewaska et al. (2000) in West-Central region of Poland, Noordeen et al. (2000) in SirLanka and Zidan (2006) who found that the highest incidence was in the age group (< 1 month) (19.63 %) followed by the age group (< 2 months) (8.75 %). This study indicated that there was a clear association between infection with        C. parvum and the age of lambs.

The effect of the age group distribution on the infection rates with C. parvum infection in was illustrated in Table 6. The highest incidence was observed in the age group (<2 years) (18.2 %) followed by the age group (2- <4 years) (9.6 %) and finally the age group (4- <6 years) (5.6 %). This result agreed withZidan (2006) who found that the highest incidence was in the age group (<2 years old) (20.2 %) followed by the age group (3- 4 years) (7.5 %)then the age group (< 6 years old) (5.5 %).These results indicated that the age of children at risk is an important factor in C. parvum infection. These results were in agreement with results obtained by Das et al. (1993) who observed that the highest detection rate of Cryptosporidium oocysts was in the first two years of life in both diarrheic and control children and Henry et al. (1995) who concluded that the highest risk of persistent diarrhea was in children of 2-3 years.

Incidence of C. parvum infection in examined stool samples of children in relation to history of animal contactwas illustrated in Table 7. It was clear that animal contact affected incidence of C. parvum in children where incidence was higher in children with history of animal contact (14.9 %) than others with no history of animal contact (6.5 %). This finding was in agreement withSoliman (1992); Younis (1995); Zidan (2006) who recorded an incidence of 16.7 % in contact group and 4.2 % in non contact group. Theses results confirmed the association between the influence of contact with animals and infection with          C. parvum as most cases inhabited rural communities in Behera and AlexandriaProvinces.

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