Cryptosporidiosis and its potential risk factors in children and calves in Babol, north of Iran

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1 Tropical Biomedicine 28(1): (2011) Cryptosporidiosis and its potential risk factors in children and calves in Babol, north of Iran Ranjbar-Bahadori, Sh. 1 *, Sangsefidi, H. 2, Shemshadi, B. 1 and Kashefinejad, M. 3 1 Parasitology Department, Veterinary Faculty, Islamic Azad University, Garmsar branch, Garmsar, Semnan, Iran 2 Clinical Sciences Department, Veterinary Faculty, Islamic Azad University, Garmsar branch, Garmsar, Semnan, Iran 3 Statistic Department, Islamic Azad University, Garmsar branch, Garmsar, Semnan, Iran * Corresponnding author bahadori@iau-garmsar.ac.ir Received 31 August 2010; received in revised form 3 November 2010; accepted 4 December 2010 Abstract. This study was carried out during April-August 2009 to determine the prevalence of Cryptosporidium spp. and its potential risk factors in children and calves in Babol, north of Iran. A total of 150 faecal samples were taken directly from the rectums of calves which were no more than two months old. Information about age, breeding conditions, consistency of faeces (as diarrhoeic or normal) and contact with human were recorded. At the same time, 150 stool samples were taken from children aged one month to 6 years old in Amir Kola children hospital (Babol, north of Iran). All samples were stained with modified Ziehl-Neelsen s acid-fast and Auramine O techniques to detect for the presence of Cryptosporidium oocysts. Results revealed that the prevalence of Cryptosporidium in children and calves were 16% and 7.33% (Auramine O stain), and 10.67% and 4% (Modified Acid-fast stain), respectively. The prevalence of the infection according to age groups and consistency of faeces were found to be statistically significant with Auramine O stain. The prevalence of infection in urban and rural children was similar, but prevalence of Cryptosporidium was more in calves with native breeding. The young calves and children and type of animal breeding represent important risk factors for transmission of cryptosporidiosis. Moreover, there was no relationship between infection of Cryptosporidium in children and calves. Our finding revealed that clinical cryptosporidiosis cases exist in north of Iran and the most important infection route for Cryptosporidium spp. is anthroponotic transmission. INTRODUCTION Cryptosporidiosis is a zoonotic protozoan disease of worldwide distribution, affecting a wide range of vertebrate hosts. Zoonotic parasites are divided into 4 categories, such as direct-zoonotic, meta-zoonotic, cyclo-zoonotic, and sapro-zoonotic. Cryptosporidum is related to direct zoonotic and this parasite infects human directly from animals (Youn, 2009). Cryptosporidium parvum is the major species seen in humans and animals. Anthroponotic C. parvum type I (C. hominis) is found in humans, and C. parvum type II (C. bovis) is found in humans and animals (especially ruminants) (Sari et al., 2009). Its life cycle undergo two asexual generations in the epithelial cells of the intestine. Finally, gametogony phase ends with the formation of the oocyst, which is excreted with the faeces to the external environment (Del Coco et al., 2008). This phase is relevant for the detection and identification of the parasite (Fayer et al., 2000). As well as causing morbidity and mortality in animals, cryptosporidiosis in lambs and calves is considered to be an infection source for humans (Sari et al., 125

2 2009). Infected calves can eliminate a large number of oocysts in their faeces and contaminate fresh food, drinking and recreational water causing infection affecting humans and other animals (Del Coco et al., 2008). The presence of Cryptosporidium spp. in faeces of calves and children has been reported in other countries in the world (Abu-Alrub et al., 2008; Bajer, 2008; Brook et al., 2008; Del Coco et al., 2008; Sari et al., 2009; Youn, 2009) and studies on risk factors have also been carried out, usually focusing on management factors at the herd level, derived from questionnaire gathered from farmers (Hamnes et al., 2006). The major clinical sign in cryptosporidiosis is gastroenteritis that is self-limiting. But in persons who are immunocompromised, it may cause chronic diarrhoea (Tzipori & Ward, 2002). Moreover, Cryptosporidium is considered as one of the most important enteric pathogen and is endemic in developing countries due to poor sanitation, crowding, and malnourishment. The rate of infection is predicted to be higher in children of developing countries (Abu-Alrub et al., 2008). The prevalence of this protozoan in children with diarrhoea in the neighboring countries was found to be 8.8% in Iraq, 16.6% to 27.9% in Egypt, 10.3% in Pakistan, and 7.3% in India (Abu-Alrub et al., 2008). The objective of this study was to evaluate the potential risk factors of cryptosporidiosis including age, type of animal breeding and human residence, the association of infection with consistency of faeces and the relationship between infection in calves and children in Babol, north of Iran. MATERIAL AND METHODS Study period and area description The study was performed between April and August 2009, in 8 dairy farms of 3 different localities of Babol, Mazandaran province, located 210 km from Tehran, and north of Iran. This area is located in the Caspian Sea zone which includes the coastal plains and northern aspect of Alborz range. It has suitable weather with high humidity and average temperature (15-25ºC). Therefore, this area is suitable for animal breeding. Sample collection and analysis A total of 150 faecal samples were taken directly from the rectums of calves which were no more than two months old using sterile latex gloves. Information about age, breeding conditions, consistency of faeces (as diarrhoeic or normal) and contact with human were collected and recorded. At the same time, a total of 150 stool samples were also taken from children aged one month to 6 years in Amir Kola children hospital (Babol, north of Iran). Each sample was collected in a dry, clean, and leak-proof plastic container labeled with child s name, gender, age, and living location and stored at 4ºC until they were processed. In the laboratory, all stool samples were processed by water-ether concentration (Bukhari & Smith, 1995) and smears of 20 µl of the sediment were prepared, dried and stained by modified Ziehl- Neelsen s acid-fast and Auramine O stains and were examined for the presence of Cryptosporidium oocysts. The Ziehl- Neelsen stained smears were examined by light microscopy under 1000X magnification. A sample was considered positive for cryptosporidiosis, if an oocyst was observed with the correct morphology including optical properties, internal structure, size, and shape. In the Auramine O method, samples were studied with dark field microscope and Cryptosporidium oocysts had fluorescent property. Statistical analysis Chi square (χ 2 ) was used to detect significant differences between the various groups at 5% level of significance. SPSS 10.0 for Windows was used to do the statistical analyses. 126

3 RESULTS The prevalence of Cryptosporidium in children and calves in Babol, north of Iran, during April-August 2009 were 16% and 7.33% (Auramine O stain), and 10.67% and 4% (Modified Acid-fast stain), respectively. The prevalence of the infection according to age groups is shown in Table 1. Results showed that 6% of positive samples (Auramine O stain) were from calves days old and 12% of samples with Cryptosporidium infection were in children in the age group 1-4 years old. Results showed that prevalence of Cryptosporidium infection in children and calves according to age groups was statistically significant only with Auramine O stain. Relationship between infection and consistency of faeces as diarrhoeic or normal is shown in Table 2. Results showed that it was significant with Auramine O stain. Furthermore, results showed that in both staining methods, there was no significant difference between the prevalence of cryptosporidiosis in urban and rural children (p>0.05). But the prevalence of cryptosporidiosis was more in calves with native breeding (4% and 2.67%) with Auramine O and Modified Acidfast stains, respectively (Table 3). Finally, the prevalence of cryptosporidiosis according to relationship between children and calves is shown in Table 4. Results revealed that it was not statistically significant (p>0.05) and there was no relationship between children and calves in infection to Cryptosporidium. DISCUSSION The results of this study confirmed that cryptosporidial infection was present in calves and children in Babol, north of Iran. Studies carried out in calves in Kars province, Turkey have reported a prevalence of 22.8%. The rate of infection was 30.3% in calves with diarrhoea and 10% in healthy calves (Sari et al., 2008). According to another study carried out in young calves in New Zealand, Brook et al. (2008) have reported a prevalence of 28% with PCR. For the first time in Argentina, infection to Cryptosporidium was reported in 17% of calves with <30 days old (Del Coco et al., 2008). Cryptosporidial infection rates in calves from other studies were as Table 1. The prevalence of Cryptosporidium infection in calves and children according to age group based on two staining methods: Auramine O and Modified Acid-fast Age Auramine O Modified Acid-fast Positive Negative Positive Negative N (%) N (%) N (%) N (%) Calves 1-30 days 02 (1.33) 072 (48) 02 (1.33) 072 (48) days 09 (6) 067 (44.67) 04 (2.67) 072 (48) Total 11 (7.33) 139 (92.67) 06 (4) 144 (96) Statistical analysis χ 2 = 4.609, sig = χ 2 = 0.640, sig = Children < one year 02 (1.33) 016 (10.67) 00 (0) 018 (12) 1-2 years 06 (4) 006 (4) 02 (1.33) 010 (6.67) 2-3 years 06 (4) 022 (14.67) 06 (4) 022 (14.67) 3-4 years 06 (4) 030 (20) 04 (2.67) 032 (21.33) 4-5 years 02 (1.33) 034 (22.67) 02 (1.33) 034 (22.67) 5-6 years 02 (1.33) 018 (12) 02 (1.33) 018 (12) Statistical analysis χ 2 = , sig = χ 2 = 7.010, sig =

4 Table 2. The prevalence of Cryptosporidium infection in calves and children according to consistency of faeces based on two staining methods: Auramine O and Modified Acid-fast Consistency of faeces Auramine O Modified Acid-fast Positive Negative Positive Negative N (%) N (%) N (%) N (%) Calves Diarrheic faeces 06 (4) 096 (64) 1 (0.67) 101 (67.33) Normal faeces 05 (3.33) 043 (28.67) 5 (3.33) 043 (28.67) Total 11 (7.33) 139 (92.67) 6 (4) 144 (96) Statistical analysis χ 2 = 7.569, sig = χ 2 = 0.988, sig = Children Diarrheic faeces 06 (4) 048 (32) 02 (1.33) 052 (34.67) Normal faeces 18 (12) 078 (52) 14 (9.33) 082 (54.67) Statistical analysis χ 2 = 4.293, sig = χ 2 = 1.500, sig = Table 3. The prevalence of Cryptosporidium infection in calves and children according to type of animal breeding and type of human residence based on two staining methods: Auramine O and Modified Acid-fast Type of animal breeding and human residence Auramine O Modified Acid-fast Positive Negative Positive Negative N (%) N (%) N (%) N (%) Calves Native breeding 06 (4) 030 (20) 4 (2.67) 032 (21.33) Industrial breeding 05 (3.33) 109 (72.67) 2 (1.33) 112 (74.67) Total 11 (7.33) 139 (92.67) 6 (4) 144 (96) Statistical analysis χ 2 = 6.072, sig = χ 2 = 6.230, sig = Children Urban 12 (8) 068 (45.33) 08 (5.33) 072 (48) Rural 12 (8) 058 (38.67) 08 (5.33) 062 (41.34) Statistical analysis χ 2 = 0.128, sig = χ 2 = 0.080, sig = Table 4. The prevalence of Cryptosporidium infection in calves and children according to relationship between them based on two staining methods: Auramine O and Modified Acid-fast Relationship between Auramine O Modified Acid-fast animal and Positive Negative Positive Negative human together N (%) N (%) N (%) N (%) Calves With human 09 (6) 125 (83.34) 5 (3.33) 129 (86) Without human 02 (1.33) 014 (9.33) 1 (0.67) 015 (10) Total 11 (7.33) 139 (92.67) 6 (4) 144 (96) Statistical analysis χ 2 = 0.704, sig = χ 2 = 0.236, sig = Children With animal 12 (8) 054 (36) 08 (5.33) 058 (38.67) Without animal 12 (8) 072 (48) 08 (5.33) 076 (50.67) Statistical analysis χ 2 = 0.417, sig = χ 2 = 0.262, sig =

5 follows: 20% in Canada (Olson et al., 1997) and 17.9% in France (Lefay et al., 2000). Most data on Cryptosporidium is in farm animals including cattle, sheep and goats. Sanz Ceballos et al. (2009) revealed the prevalence of Cryptosporidium infection in goats in southeast of Spain (19.1%). Other animals like camel are acknowledged as a potential source of contamination. For the first time, Razawi et al. (2009) isolated oocyst of Cryptosporidium from 37.9% camels (Camelus dromedarious) in central part of Iran. On the other hand; there are much data about cryptosporidial human infection in the world especially in children with or without diarrhoea. Al- Braiken (2008) showed that intestinal parasitic infection is a common health problem among children in Saudi Arabia and Cryptosporidium was reported from 8.3% inpatients and 2.3% outpatients. Haque et al. (2009) revealed that infection with Cryptosporidium species and Entamoeba histolytica were more prevalent in patients with acute diarrhoea than in healthy control subjects, for all ages and specifically for those 0-12 months of age in Dhaka, Bangladesh. In Philippines, the prevalence of Cryptosporidium and Giardia among 3,456 diarrheic patients was determined and of 133 (3.8%) positive samples, 67 (1.9%) were positive for Cryptosporidium (Natividad et al. 2008). Therefore, it appears that cryptosporidial infections in calves, children and other animals are distributed worldwide. Sari et al. (2009) showed that age is the major risk factor in the spread of cryptosporidiosis. Natividad et al. (2008) revealed that the prevalence of Cryptosporidium among pediatric patients (2.9%) was significantly higher compared to adult patients (0.2%). It appears that infection in cattle is highly age-dependent, with young calves showing the highest prevalence and intensity of shedding the organism (Quilez et al., 1996). Therefore, these young animals can shed the oocyst of Cryptosporidium which has a wide host range and is considered to be potentially zoonotic (Brook et al., 2008). In this present study, Auramine O stain is more specific for diagnosis of Cryptosporidium in prepared faecal samples and the prevalence rate of cryptosporidiosis was seen to be higher when this stain was used. The prevalence of infection according to age groups of calves or children was statistically significant with Auramine O stain. It was concluded that young calves and children play an important role in maintaining infection in nature and represent an important risk factor for transmission of infection to other humans and animals. This study also revealed the significant association between infection with Cryptosporidium and diarrhoea. However, the staining method used in the current study is important. On the other hand, there was correlation between age and consistency of the faeces, with younger animals and children tending to have loose faeces, perhaps due to the nature of the milk diet (Brook et al., 2008). Samie et al. (2009) reported that Cryptosporidium was one of the most common parasitic causes of diarrhoea among the hospital attendees in South Africa (25.5%). Of course, even when Cryptosporidium appears to be associated with loose faeces, it is possible that the parasite is not the only or primary enteropathogen. Type of animal breeding is another important risk factor in cryptosporidiosis. In the current study, results showed that the infection rate in calves with native breeding was higher compared to industrial breeding. So, our study revealed that the unhygienic condition of the calves is an important risk factor. From our observations, there was no remarkable difference between infection in rural and urban children and it was probably related to the similar hygienic conditions with clean and uncontaminated drinking water available in both locations. Abu-Alrub et al. (2008) revealed that the high prevalence rate of cryptosporidiosis in West Bank may be attributed to wastewater disposal. Notification rates showed large geographic variations, with rates in rural areas 2.8 times higher than in urban areas. At the territorial authority level, rates were also correlated with farm animal density and 129

6 most transmission of Cryptosporidium in New Zealand appears to be zoonotic from farm animals to humans. But our findings are not in agreement with those reported by Snel et al. (2009) and showed that there was no relationship between cryptosporidiosis in calves and children. Nascimento et al. (2009) reported that the most important infection route for Cryptosporidium spp. is person-to-person transmission, which is well illustrated in daycare centers. Immaturity, deficiencies of the immune system and inadequate hygiene habits are factors that also contribute towards this type of infection. Findings by Areeshi et al. (2008) showed that 11 cases of cryptosporidiosis were caused by Cryptosporidium hominis and only one by C. parvum and most of the cases were probably the result of anthroponotic transmission. As a conclusion, in this study area, clinical cryptosporidiosis cases exist and this area is contaminated with Cryptosporidium spp. Therefore molecular studies need to be carried out to specify its species and to identify the genotypes to determine the source of infection. REFERENCES Abu-Alrub, S.M., Abusada, G.M., Farraj, M.A. & Essawi, T.A. (2008). Prevalence of Cryptosporidium spp. in children with diarrhoea in the West Bank, Palestine. The Journal of Infection in Developing Countries 2(1): Al-Braiken, F.A. (2008). Is intestinal parasitic infection still a public health concern among Saudi children? Saudi Medicine Journal 29(11): Areeshi, M., Dove, W., Papaventsis, D., Gatei, W., Combe, P., Grosjean, P., Leatherbarrow, H. & Hart, C.A. (2008). Cryptosporidium species causing acute diarrhoea in children in Antananarivo, Madagascar. Annals of Tropical Medicine and Parasitology 102(4): Bajer, B. (2008). Cryptosporidium and Giardia spp. infections in human, animals and the environment in Poland. Parasitology Research 104: Brook, E., Hart, C.A., French, N. & Christley, R. (2008). Prevalence and risk factors for Cryptosporidium spp. infection in young calves. Veterinary Parasitology 152: Bukhari, Z. & Smith, H.V. (1995). Effect of three concentration techniques on viability of Cryptosporidium parvum oocysts recovered from bovine feces. Journal of Clinical Microbiology 33(10): Del Coco, V.F., Cordoba, M.A. & Basualdo, J.A. (2008). Cryptosporidium infection in calves from a rural area of Buenos Aires, Argentina. Veterinary Parasitology 158: Fayer, R., Morgan, U. & Upton, S.J. (2000). Epidemiology of Cryptosporidium: transmission, detection and identification. International Journal of Parasitology 301(12-13): Hamnes, I.S., Gjerde, B. & Robertson, L. (2006). Prevalence of Giardia and Cryptosporidium in dairy calves in three areas of Norway. Veterinary Parasitology 140: Haque, R., Mondal, D., Karim, A., Molla, I.H., Rahim, A., Faruque, A.S., Ahmad, N., Kirkpatrick, B.D., Houpt, E., Snider, C. & Petri, W.A.Jr. (2009). Prospective case-control study of the association between common enteric protozoal parasites and diarrhea in Bangladesh. Clinical Infectious Diseases 48(9): Lefay, D., Naciri, M., Poirier, P. & Chermette, R. (2000). Prevalence of Cryptosporidium infection in calves in France. Veterinary Parasitology 89(1-2): 1-9. Nascimento, W.R., Cavalcanti, I.M., Irmão, J.I. & Rocha, F.J. (2009). Presence of Cryptosporidium spp in children with acute diarrhea in a public daycare center in Recife, State of Pernambuco. Revista da Sociedade Brasileira de Medicina Tropical 42(2): (in Portuguese with English abstract). 130

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