• View in gallery
    Figure 1.

    Geographical situation of sample collection sites. The map of Tunisia on the right shows the geographical situation of the study district Bizerte (square). The photo on the left gives details of the geographical locations of the different sample collection sites Joumine and Menzel Bourguiba regions (squares).

  • View in gallery
    Figure 2.

    Phylogenic analysis of the gp60 nucleotide sequences of C. parvum strains from humans and calves (tree obtained by the method of UPGMA with C. meleagridis as an outgroup and bootstrap values based on 1,000 replicates). Our samples: E459, E473, E21, and E424 were from humans and VB2, V7, V9, V10, V4, V17, V18, VB7, VB8, V11, V15, VB1, V14, VB22, and VB24 were from calves. Other nucleotide sequences were from GenBank.

  • 1.

    Alves M, Xiao L, Antunes F, Maltos O, 2006. Distribution of Cryptosporidium subtypes in human and domestic and wild ruminants in Portugal. Parasitol Res 99: 287292.

    • Search Google Scholar
    • Export Citation
  • 2.

    Chalmers RM, Katzer F, 2013. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol 29: 237251.

    • Search Google Scholar
    • Export Citation
  • 3.

    Xiao L, 2010. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol 124: 8089.

  • 4.

    El Sherbini GT, Mohammad KA, 2006. Zoonotic cryptosporidiosis in man and animal in farms, Giza Governorate, Egypt. J Egypt Soc Parasitol 36: 4958.

    • Search Google Scholar
    • Export Citation
  • 5.

    Essid R, Mousli M, Aoun K, Abdelmalak R, Mallouli F, Kanoun F, Derouin F, Bouratbine A, 2008. Identification of Cryptosporidium species infecting humans in Tunisia. Am Soc Trop Med Hyg 79: 702705.

    • Search Google Scholar
    • Export Citation
  • 6.

    Soltane R, Guyot K, Dei Cas E, Ayadi A, 2007. Prevalence of Cryptosporidium spp. (Eucoccidiorida: Cryptosporiidae) in seven species of farm animals in Tunisia. Parasite 11: 335338.

    • Search Google Scholar
    • Export Citation
  • 7.

    Gatei W, Hart CA, Gilman RH, Das P, Cama V, Xiao L, 2006. Development of a multilocus sequence typing tool for Cryptosporidium hominis. J Eukaryot Microbiol 53: 4348.

    • Search Google Scholar
    • Export Citation
  • 8.

    Wielinga PR, De Vries A, Vander Goot TH, Mank T, Mars MH, Kortbeek LM, Van der Giessen JW, 2008. Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands. Int J Parasitol 38: 809817.

    • Search Google Scholar
    • Export Citation
  • 9.

    Jaouad M, 2010. Constraints to improving forage feed resources and their impacts on the dynamics of the cattle breeding in Tunisia. Porqueddu C, Ríos S eds. The Contributions of Grasslands to the Conservation of Mediterranean Biodiversity. Zaragoza, Spain: CIHEAM/CIBIO/FAO/SEEP (Options Méditerranéennes: Série A. Séminaires Méditerranéens 92), 3943.

    • Search Google Scholar
    • Export Citation
  • 10.

    Allen AVH, Ridley DS, 1970. Further observations on the formol ether concentration technique for feacal parasites. J Clin Pathol 23: 545546.

    • Search Google Scholar
    • Export Citation
  • 11.

    Casmore DP, 1991. The epidemiology of human cryptosporidiosis and the water route of infection. Water Sci Technol 24: 157164.

  • 12.

    Henriksen SA, Pohlenz JF, 1982. Staining cryptosporidia by modified Ziehl-Neelsen technique. Acta Vet Scand 22: 594596.

  • 13.

    Coupé S, Safarti C, Hamane S, Dérouin F, 2005. Detection of Cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism. J Clin Microbiol 43: 10171023.

    • Search Google Scholar
    • Export Citation
  • 14.

    Sulaiman IM, Hira PR, Zhou L, Al Ali FM, Al Shelahi FA, Shweiki HM, Iqbal J, Khalid N, Xiao L, 2005. Unique endemicity of cryptosporidiosis in children in Kuwait. J Clin Microbiol 43: 28052809.

    • Search Google Scholar
    • Export Citation
  • 15.

    Stibbs HH, Ongerth JE, 1986. Immunofluorescence detection of Cryptosporidium oocysts in fecal smears. J Clin Microbiol 24: 517521.

  • 16.

    Weber R, Bryan RT, Bishop HS, Wahlquist SP, Sullivan JJ, Juranek DD, 1991. Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. J Clin Microbiol 29: 13231327.

    • Search Google Scholar
    • Export Citation
  • 17.

    Trotz Williams LA, Martin DS, Gatei W, Cama V, Peregrine AS, Martin SW, Nydam DV, Jamieson F, Xiao L, 2006. Genotype and subtype analyses of Cryptosporidium isolates from dairy calves and humans in Ontario. Parasitol Res 99: 346352.

    • Search Google Scholar
    • Export Citation
  • 18.

    Xiao L, Zhou L, Santin M, Yang W, Fayer R, 2007. Distribution of Cryptosporidium parvum subtypes in calves in eastern United States. Parasitol Res 100: 701706.

    • Search Google Scholar
    • Export Citation
  • 19.

    Quilez J, Torres E, Chalmers RM, Hadfield SJ, Del Cacho E, Sanchez Acedo C, 2008. Cryptosporidium genotypes and subtypes in lambs and goat kids in Spain. Appl Environ Microbiol 74: 60266031.

    • Search Google Scholar
    • Export Citation
  • 20.

    Goh S, Reacher M, Casemore DP, Verlander NQ, Chalmers R, Knowles M, Williams J, Osborn K, Richards S, 2004. Sporadic cryptosporidiosis, North Cumbria, England, 1996–2000. Emerg Infect Dis 10: 10071015.

    • Search Google Scholar
    • Export Citation
  • 21.

    Hunter PR, Hughes S, Woodhouse S, Syed Q, Verlander NQ, Chalmers RM, Morgan K, Nichols G, Beeching N, Osborn K, 2004. Sporadic cryptosporidiosis case-control study with genotyping. Emerg Infect Dis 10: 12411249.

    • Search Google Scholar
    • Export Citation
  • 22.

    Robertson B, Sinclair MI, Forbes AB, Veith M, Kirk M, Cunliffe D, Willis J, Fairley CK, 2002. Case-control studies of sporadic cryptosporidiosis in Melbourne and Adelaide, Australia. Epidemiol Infect 128: 419431.

    • Search Google Scholar
    • Export Citation
  • 23.

    Roy SL, DeLong SM, Stenzel SA, Shiferaw B, Roberts JM, Khalakdina A, Marcus R, Segler SD, Shah D, Thomas S, Vugia DJ, Zansky SM, Dietz V, Beach MJ, 2004. Risk factors for sporadic cryptosporidiosis among immunocompetent persons in the United States from 1999 to 2001. J Clin Microbiol 42: 29442951.

    • Search Google Scholar
    • Export Citation
  • 24.

    Ashbolt RH, Coleman DJ, Misrachi A, Conti JM, Kirk MD, 2003. An outbreak of cryptosporidiosis associated with an animal nursery at a regional fair. Commun Dis Intell 27: 244249.

    • Search Google Scholar
    • Export Citation
  • 25.

    Harper CM, Cowell NA, Adams BC, Langley AJ, Wohlsen TD, 2002. Outbreak of Cryptosporidium linked to drinking unpasteurised milk. Commun Dis Intell 26: 449450.

    • Search Google Scholar
    • Export Citation
  • 26.

    Kiang KM, Scheftel JM, Leano FT, Taylor CM, Belle Isle PA, Cebelinski EA, Danila R, Smith KE, 2006. Recurrent outbreaks of cryptosporidiosis associated with calves among students at an educational farm programme, Minnesota, 2003. Epidemiol Infect 134: 878886.

    • Search Google Scholar
    • Export Citation
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Glycoprotein 60 Diversity in Cryptosporidium parvum Causing Human and Cattle Cryptosporidiosis in the Rural Region of Northern Tunisia

Ikram RahmouniLaboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, Tunis, Tunisia

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Rym EssidLaboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, Tunis, Tunisia

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Karim AounLaboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, Tunis, Tunisia

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Aïda BouratbineLaboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, Tunis, Tunisia

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The zoonotic potential of Cryptosporidium parvum was studied in an extensive cattle farming region of northern Tunisia. Seventy fecal samples from pre-weaning calves and 403 fecal samples from children were examined by microscopy after modified Ziehl–Neelsen (MZN) staining. Positive Cryptosporidium specimens were identified at a species level using an 18S rRNA nested polymerase chain reaction (PCR) followed by an Restriction Fragment Length Polymorphism (RFLP) analysis. C. parvum isolates were subgenotyped by sequence analysis of the glycoprotein 60 (gp60) gene. Among calf samples, 14 samples were positive by MZN method. C. parvum was identified in all cases. Twelve parvum isolates (85.7%) belonged to family subtype IIa. Subtype IIaA15G2R1 was more prevalent (50%). Two C. parvum isolates corresponded to the IIdA16G1 subtype. Seven human samples were positive by MZN method. C. parvum and C. meleagridis were identified in four and three cases, respectively. Intraspecific characterization of C. parvum identified two subtypes, the IIaA15G2R1 and the IIdA16G1, also found in calves.

Introduction

The protozoan Cryptosporidium is a major public and animal health concern.1 Immunocompromised people, young children, and pre-weaning animals are especially vulnerable. Until now, there is no affective treatment or vaccine commercially available to prevent the disease. Currently, 26 Cryptosporidium species have been named, and there is good evidence for 6 species as important causes of human cryptosporidiosis: C. hominis, C. parvum, C. meleagridis and occasionally, C. cuniculus, C. felis, and C. canis.2 Despite occasional reports in livestock, C. hominis seems to be anthroponotically transmitted.3 The other species are zoonotic and mainly transmitted from animals to human.4 In animals, C. parvum and C. meleagridis are the most clinically and economically important gastrointestinal species in pre-weaning ruminants and birds, respectively.2,3 In dairy cattle, C. parvum is mostly found in pre-weaning calves, whereas three other species, including C. andersoni, C. bovis, and C. ryanae, are found in older age groups.3 In Tunisia, Cryptosporidium spp. was identified as a prevalent parasite in human and farm animals.5,6 C. hominis and C. parvum were the dominant species in urban residents, whereas C. parvum and C. meleagridis were the causative species in children from rural areas.4 C. bovis was found in lambs, and C. meleagridis was found in one broiler chicken.6 Until now, no data are available about identification of subtypes of Cryptosporidium species.

Molecular characterization is essential in distinguishing human from non-human sources, understanding transmission, and strengthening the epidemiological evidence for causative links in outbreaks.2 To characterize the transmission dynamics and zoonotic potential of C. parvum, numerous studies have been conducted to subtype C. parvum in humans and farm animals, especially calves.3 One of the most popular subtyping tools is the DNA sequence analysis of 60 kDa glycoprotein (gp60). Sequence analysis of gp60 gene is widely used in Cryptosporidium subtyping because of its sequence heterogeneity and relevance to parasite biology. It is the single most polymorphic marker identified so far in the Cryptosporidium genome.7,8 The gp60 gene is similar to a microsatellite sequence, having tandem repeats of the serine-coding trinucleotide TCA, TCG, or TCT at the 5′ end of the gene, and it presents extensive sequence differences in the non-repeat regions, which categorize C. parvum to several subtype families.3 Among C. parvum, subtype families IIa and IId are found in both humans and ruminants to be responsible for zoonotic cryptosporidiosis. The IIc subtype family has, so far, been only found in humans.3 The present study aims to improve the understanding of the epidemiology of Cryptosporidium in Tunisia by studying the genetic diversity of C. parvum in both populations of pre-weaning calves and humans from the same area.

Materials and Methods

Fecal specimens and sample sites.

Cattle breeding in Tunisia is observed mainly in the northern areas of the country, where bovine farming is supported by favorable climatic conditions. Livestock statistics for 2006 show that 65% of the cattle are in the north and that more than 40% of the pure breed cattle population is found in the two northern districts of Beja and Bizerte.9 The study was performed in Bizerte District, which is located in northeast Tunisia (Figure 1). From April to October of 2007, 70 stool samples were collected immediately after defecation from calves less than 5 months in farms and private breeding units from the Joumine region (Figure 1). During the same period, 403 stool specimens were collected from children under 5 years of age; 258 stools (including 52 diarrheic specimens) were sampled from the pediatric rural communities (mean age = 33 months, SD = 17) living around farms animals of the Joumine region by a door to door survey, whereas 145 stools (including 51 diarrheic specimens) were collected in the healthcare unit of Menzel Bourguiba (mean age = 33 months, SD = 15), which is an important human settlement of Bizerte District (Figure 1).

Figure 1.
Figure 1.

Geographical situation of sample collection sites. The map of Tunisia on the right shows the geographical situation of the study district Bizerte (square). The photo on the left gives details of the geographical locations of the different sample collection sites Joumine and Menzel Bourguiba regions (squares).

Citation: The American Society of Tropical Medicine and Hygiene 90, 2; 10.4269/ajtmh.13-0522

Oocyst detection and species identification.

Fresh stool specimens were examined for Cryptosporidium spp. oocysts. Microscopic examination was carried out on smears of fecal concentrates (simplified version of Ritchie's formalin-ether sedimentation method)10 after staining with the modified Ziehl–Neelsen technique (MZN).11,12 DNA was extracted from all positive specimens using the QIAmp DNA Stool Mini-Kit (Qiagen Inc., Hilden, Germany) according to the manufacturer's recommendations. Cryptosporidium species were identified using a two-step 18S rRNA nested polymerase chain reaction (PCR) followed by an RFLP analysis as described by Coupé and others5,13 with some modifications.

Intraspecific characterization of C. parvum isolates.

Subgenotyping of C. parvum isolates was performed using nested PCR to amplify a fragment of the gp60 gene as described elsewhere.1,7 Briefly, two-step nested PCR was used. AL3531 (5′-ATAGTCTCCGCTGTATTC-3′) and AL3535 (5′-GGAAGGAACGATGTATCT-3′) primers were used for the first-round PCR and AL3532 (5′-TCCGCTGTATTCTCAGCC-3′) and AL3534 (5′-GCAGAACCAGCATC-3′) primers were used for the second-round PCR to amplify a 840-bp fragment. Extracted DNA (1 μL) was mixed with a solution containing 200 nmol each primer, 200 μM 2′-deoxynucleoside 5′-triphosphate, 1.5 mM MgCl2, and 2.5 U HotStar Taq polymerase (Qiagen GmbH, Hilden, Allemagne), with a final volume of 50 μL. Cycling conditions were an initial denaturation at 94°C for 10 minutes followed by 35 cycles of a three-step program (94°C for 45 seconds, 50°C for 45 seconds, and 72°C for 1 minute). The amplified DNA fragments were purified using the Wizard Genomic DNA Purification Kit (Promega, Charbonnières, France) and sequenced in both directions with a Big Dye Terminator Cycle Sequencing Kit on an ABI Prism 377 DNA Sequencer (Applied Biosystems, Foster City, CA). For each isolate, the C. parvum family group was assigned by sequence comparison with those isolates available in the GenBank database and published in peer-reviewed international scientific journals (www.ncbi.nlm.nih.gov/blast). Subtypes were named on the basis of the number of TCA (A), TCG (G), and ACATCA (R) as described by Sulaiman and others.14 and Gatei and others.7 Nucleotide sequences were aligned with reference genotypes from GenBank using ClustalW and analyzed using Mega5 software.

The phylogenetic tree was created using the unweighted pair group method with arithmetic mean (UPGMA) based on evolutionary distances calculated by the Kimura two parameters. The reliability of branches was assessed by bootstrap analysis using 1,000 replicates. The topology of the C. parvum tree was constructed using C. meleagridis as outgroup.

Results

Among calf samples, 15 (21.4%) samples were positive by the MZN method. PCR confirmed positivity, and RFLP analysis yielded typical restriction patterns for C. parvum in all cases. Intraspecific characterization of C. parvum isolates identified only two subtype families: IIa and IId (Figure 2); 13 of 15 C. parvum isolates (86.7%) belonged to family subtype IIa. The IIaA15G2R1 was the more prevalent (46.2%) subtype within this family (Table 1). The two latter C. parvum isolates corresponded to the IIdA16G1 subtype (Table 1).

Figure 2.
Figure 2.

Phylogenic analysis of the gp60 nucleotide sequences of C. parvum strains from humans and calves (tree obtained by the method of UPGMA with C. meleagridis as an outgroup and bootstrap values based on 1,000 replicates). Our samples: E459, E473, E21, and E424 were from humans and VB2, V7, V9, V10, V4, V17, V18, VB7, VB8, V11, V15, VB1, V14, VB22, and VB24 were from calves. Other nucleotide sequences were from GenBank.

Citation: The American Society of Tropical Medicine and Hygiene 90, 2; 10.4269/ajtmh.13-0522

Table 1

Cryptosporidium species and C. parvum subtypes identified in human population according to epidemiological data

Study area and code Age group (years) Sex Stool consistency Cryptosporidium species C. parvum subtype family C. parvum subtypes
Joumine region
 E21 3–4 Female F C. parvum IId A16G1
 E424 3–4 Male D C. parvum IId A16G1
 E183 3–4 Male F C. meleagridis    
 E204 4–5 Female F C. meleagridis    
Menzel Bourguiba region
 E459 2–3 Male D C. parvum IIa A15G2R1
 E473 2–3 Male D C. parvum IIa A15G2R1
 E250 2–3 Male F C. meleagridis    

D = diarrheic; F = formed.

Among human samples, seven (1.7%) samples were positive by the MZN method. PCR confirmed positivity, and RFLP analysis yielded typical restriction patterns for C. parvum in four cases and C. meleagridis in three cases. Intraspecific characterization of C. parvum isolates identified only two subtypes: IIaA15G2R1 in two cases and IIdA16G1 in two other cases (Table 1). Cryptosporidium species and C. parvum subtypes according to epidemiological data are reported in Table 2.

Table 2

C. parvum subtypes identified in calves from Bizerte District

Subtype family and subtype Number of isolates in humans Number of isolates in calves
IIa
 A15G2R1 2 6
 A16G2R1 0 3
 A13G2R1 0 2
 A20G3R1 0 1
IId
 A16G1 2 2

Phylogenic analysis of all C. parvum strains was shown in Figure 2.

Discussion

Conventional procedures for oocyst concentration and detection in stool specimens, namely formalin-ether sedimentation and acid-fast staining, were used for Cryptosporidium screening. However, the high threshold necessary for oocyst detection by these coprodiagnosis methods, particularly in formed or semiformed stool specimens, could limit oocyst detection in asymptomatic individuals and underestimate the number of positive samples.15,16

Prevalence rate of Cryptosporidium spp. infection among children less than 5 years old was particularly low (1.7%). This rate may be partly because of the sample processing methodology but also could have been related to an absence of close contact between the human population sampled and animals. C. meleagridis was found almost as frequently as C. parvum. This finding suggests that zoonotic transmission from poultry is one of the most important causes of human cryptosporidiosis in rural communities of Tunisia, where most families have their own poultry breeding close to their houses. However, even in this farming region, families do not necessarily have their own cattle, which suggest a low direct contact with these animals. Nevertheless, C. parvum was the only specie identified in the positive calf stool specimens collected in the same region, which provides evidence of possible association of infected calves and human infection with C. parvum. The role of cattle in the zoonotic transmission of C. parvum in northern Tunisia was further supported by gp60 subgenotyping data.

As reported by other studies, family subtype IIa was the dominant family in calves.3 Also, the IIaA15G2R1 seemed to be the most common subtype on the dairy farms.17,18 Interestingly, two of four (50%) C. parvum isolates from children belonged to family subtype IIa and were similar to the IIaA15G2R1 subtype (the most prevalent subtype identified in calves during the same period), which suggests that family subtype IIa and particularly, IIaA15G2R1 subtype can spread easily within cattle populations and be transmitted to humans as well. In fact, this variant is frequently observed in C. parvum populations worldwide and has been widely reported in zoonotic infections.1 As a risk factor for human cryptosporidiosis, contact with cattle was implicated in the neighboring countries, such as Egypt and Spain,4,19 as well as the United States, United Kingdom, Ireland, and Australia.2023 However, in our study, taking into account the age of children and their kind of life (human settlement), contact with livestock is possible; however, drinking raw (unpasteurized) milk may also represent a probable risk factor for Cryptosporidium transmission in this area.2426 More investigations should be performed with large and more representative samples.

As reported in European countries, the IId subtype family was occasionally found in calves in addition to IIa subtypes.3 In this study, the IIdA16G1 was identified in two calves as well as two children living in the rural environment around farm animals. Interestingly, IId subtypes of C. parvum have never been found in humans in the United States and Canada, where they are absent in calves.3 Thus, the less common bovine C. parvum subtype family IId may potentially also be responsible for some zoonotic infections in northern Tunisia. However, the relatively high proportion of cases (50%) in humans highlights the need for additional epidemiological investigations. In fact, in regions where both subtypes IIa and IId are found (i.e., Spain), family subtype IIa infects preferentially calves, whereas family subtype IId has a tropism for lambs and kids.19 Although an earlier study conducted in Tunisia suggested that C. parvum was absent in sheep,22 studies involving more animal samples from lambs are needed for a better understanding of the sources of Cryptosporidium human infections in this northern area.

ACKNOWLEDGMENTS

We are grateful to Dr. R. Hamza and all the staff of the Regional Directory of Public Health of the Bizerte governorate for their contribution to the achievement of this work. We also thank Dr. R. Ben Omrane, Dr. M. Sayari, and H. Abassi for their help in calf stools collection and Dr. S. Benabderrazak for her help in phylogenic analysis.

  • 1.

    Alves M, Xiao L, Antunes F, Maltos O, 2006. Distribution of Cryptosporidium subtypes in human and domestic and wild ruminants in Portugal. Parasitol Res 99: 287292.

    • Search Google Scholar
    • Export Citation
  • 2.

    Chalmers RM, Katzer F, 2013. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol 29: 237251.

    • Search Google Scholar
    • Export Citation
  • 3.

    Xiao L, 2010. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol 124: 8089.

  • 4.

    El Sherbini GT, Mohammad KA, 2006. Zoonotic cryptosporidiosis in man and animal in farms, Giza Governorate, Egypt. J Egypt Soc Parasitol 36: 4958.

    • Search Google Scholar
    • Export Citation
  • 5.

    Essid R, Mousli M, Aoun K, Abdelmalak R, Mallouli F, Kanoun F, Derouin F, Bouratbine A, 2008. Identification of Cryptosporidium species infecting humans in Tunisia. Am Soc Trop Med Hyg 79: 702705.

    • Search Google Scholar
    • Export Citation
  • 6.

    Soltane R, Guyot K, Dei Cas E, Ayadi A, 2007. Prevalence of Cryptosporidium spp. (Eucoccidiorida: Cryptosporiidae) in seven species of farm animals in Tunisia. Parasite 11: 335338.

    • Search Google Scholar
    • Export Citation
  • 7.

    Gatei W, Hart CA, Gilman RH, Das P, Cama V, Xiao L, 2006. Development of a multilocus sequence typing tool for Cryptosporidium hominis. J Eukaryot Microbiol 53: 4348.

    • Search Google Scholar
    • Export Citation
  • 8.

    Wielinga PR, De Vries A, Vander Goot TH, Mank T, Mars MH, Kortbeek LM, Van der Giessen JW, 2008. Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands. Int J Parasitol 38: 809817.

    • Search Google Scholar
    • Export Citation
  • 9.

    Jaouad M, 2010. Constraints to improving forage feed resources and their impacts on the dynamics of the cattle breeding in Tunisia. Porqueddu C, Ríos S eds. The Contributions of Grasslands to the Conservation of Mediterranean Biodiversity. Zaragoza, Spain: CIHEAM/CIBIO/FAO/SEEP (Options Méditerranéennes: Série A. Séminaires Méditerranéens 92), 3943.

    • Search Google Scholar
    • Export Citation
  • 10.

    Allen AVH, Ridley DS, 1970. Further observations on the formol ether concentration technique for feacal parasites. J Clin Pathol 23: 545546.

    • Search Google Scholar
    • Export Citation
  • 11.

    Casmore DP, 1991. The epidemiology of human cryptosporidiosis and the water route of infection. Water Sci Technol 24: 157164.

  • 12.

    Henriksen SA, Pohlenz JF, 1982. Staining cryptosporidia by modified Ziehl-Neelsen technique. Acta Vet Scand 22: 594596.

  • 13.

    Coupé S, Safarti C, Hamane S, Dérouin F, 2005. Detection of Cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism. J Clin Microbiol 43: 10171023.

    • Search Google Scholar
    • Export Citation
  • 14.

    Sulaiman IM, Hira PR, Zhou L, Al Ali FM, Al Shelahi FA, Shweiki HM, Iqbal J, Khalid N, Xiao L, 2005. Unique endemicity of cryptosporidiosis in children in Kuwait. J Clin Microbiol 43: 28052809.

    • Search Google Scholar
    • Export Citation
  • 15.

    Stibbs HH, Ongerth JE, 1986. Immunofluorescence detection of Cryptosporidium oocysts in fecal smears. J Clin Microbiol 24: 517521.

  • 16.

    Weber R, Bryan RT, Bishop HS, Wahlquist SP, Sullivan JJ, Juranek DD, 1991. Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. J Clin Microbiol 29: 13231327.

    • Search Google Scholar
    • Export Citation
  • 17.

    Trotz Williams LA, Martin DS, Gatei W, Cama V, Peregrine AS, Martin SW, Nydam DV, Jamieson F, Xiao L, 2006. Genotype and subtype analyses of Cryptosporidium isolates from dairy calves and humans in Ontario. Parasitol Res 99: 346352.

    • Search Google Scholar
    • Export Citation
  • 18.

    Xiao L, Zhou L, Santin M, Yang W, Fayer R, 2007. Distribution of Cryptosporidium parvum subtypes in calves in eastern United States. Parasitol Res 100: 701706.

    • Search Google Scholar
    • Export Citation
  • 19.

    Quilez J, Torres E, Chalmers RM, Hadfield SJ, Del Cacho E, Sanchez Acedo C, 2008. Cryptosporidium genotypes and subtypes in lambs and goat kids in Spain. Appl Environ Microbiol 74: 60266031.

    • Search Google Scholar
    • Export Citation
  • 20.

    Goh S, Reacher M, Casemore DP, Verlander NQ, Chalmers R, Knowles M, Williams J, Osborn K, Richards S, 2004. Sporadic cryptosporidiosis, North Cumbria, England, 1996–2000. Emerg Infect Dis 10: 10071015.

    • Search Google Scholar
    • Export Citation
  • 21.

    Hunter PR, Hughes S, Woodhouse S, Syed Q, Verlander NQ, Chalmers RM, Morgan K, Nichols G, Beeching N, Osborn K, 2004. Sporadic cryptosporidiosis case-control study with genotyping. Emerg Infect Dis 10: 12411249.

    • Search Google Scholar
    • Export Citation
  • 22.

    Robertson B, Sinclair MI, Forbes AB, Veith M, Kirk M, Cunliffe D, Willis J, Fairley CK, 2002. Case-control studies of sporadic cryptosporidiosis in Melbourne and Adelaide, Australia. Epidemiol Infect 128: 419431.

    • Search Google Scholar
    • Export Citation
  • 23.

    Roy SL, DeLong SM, Stenzel SA, Shiferaw B, Roberts JM, Khalakdina A, Marcus R, Segler SD, Shah D, Thomas S, Vugia DJ, Zansky SM, Dietz V, Beach MJ, 2004. Risk factors for sporadic cryptosporidiosis among immunocompetent persons in the United States from 1999 to 2001. J Clin Microbiol 42: 29442951.

    • Search Google Scholar
    • Export Citation
  • 24.

    Ashbolt RH, Coleman DJ, Misrachi A, Conti JM, Kirk MD, 2003. An outbreak of cryptosporidiosis associated with an animal nursery at a regional fair. Commun Dis Intell 27: 244249.

    • Search Google Scholar
    • Export Citation
  • 25.

    Harper CM, Cowell NA, Adams BC, Langley AJ, Wohlsen TD, 2002. Outbreak of Cryptosporidium linked to drinking unpasteurised milk. Commun Dis Intell 26: 449450.

    • Search Google Scholar
    • Export Citation
  • 26.

    Kiang KM, Scheftel JM, Leano FT, Taylor CM, Belle Isle PA, Cebelinski EA, Danila R, Smith KE, 2006. Recurrent outbreaks of cryptosporidiosis associated with calves among students at an educational farm programme, Minnesota, 2003. Epidemiol Infect 134: 878886.

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Author Notes

* Address correspondence to Aïda Bouratbine, Laboratoire de Parasitologie-Mycologie, LR 11-IPT-06, Institut Pasteur de Tunis, 13 place Pasteur, 1002 Tunis, Tunisia. E-mail: aida.bouratbine@pasteur.rns.tn
† These authors contributed equally.

Authors' addresses: Ikram Rahmouni, Rym Essid, Karim Aoun, and Aïda Bouratbine, Laboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, Tunis, Tunisia, E-mails: ikra_rah@hotmail.fr, essidrym@hotmail.com, karim.aoun@pasteur.rns.tn, and aida.bouratbine@pasteur.rns.tn.

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