• View in gallery

    Polymerase chain reaction–restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene of Cryptosporidium spp. A, Restriction patterns of C. parvum and C. hominis. Lanes 1a and 2a, undigested products; lanes 1b and 2b, products digested with Taq I; lanes 1c and 2c, products digested with Ase I; lane M, 100-basepair (bp) molecular mass markers. B, Restriction patterns of C. meleagridis. Lane 3a, undigested product; lanes 3b–d, products digested with Mse I, Ssp I, and Bst UI; Lane M, 100-bp molecular weight markers. *Partial digestion with Mse I.

  • View in gallery

    Alignment of DNA samples with deposited sequences of Cryptosporidium parvum, C. hominis, and C. meleagridis small subunit ribosomal RNA gene. Restriction sites of enzymes and their positions are shown in bold.

  • 1

    Tzipori S, Ward H, 2002. Cryptosporidiosis: biology, pathogenesis and disease. Microbes Infect 4 :1047–1058.

  • 2

    Huang DB, Chappell C, Okhuysen PC, 2004. Cryptosporidiosis in children. Semin Pediatr Infect Dis 15 :253–259.

  • 3

    Corso PS, Kramer MH, Blair KA, Addiss DG, Davis JP, Haddix JP, 2003. Cost of illness in the 1993 waterborne Cryptosporidium outbreak, Milwaukee, Wisconsin. Emerg Infect Dis 9 :426–431.

    • Search Google Scholar
    • Export Citation
  • 4

    Xiao L, Fayer R, Ryan U, Upton SJ, 2004. Cryptosporidium taxonomy: recent advances and implications for public health. Clin Microbiol Rev 17 :72–97.

    • Search Google Scholar
    • Export Citation
  • 5

    Belhadj S, Kallel K, Boussen N, Goubantini A, Bejaoui M, Ben Salem N, Zribi A, Ben Chaabane T, Chaker E, 1999. Role of cryptosporidia and microsporidia in diarrhea in immunocompromised patients. Tunis Med 77 :638–643.

    • Search Google Scholar
    • Export Citation
  • 6

    Bouratbine A, Aoun K, Barbouche R, Bejaoui M, Ben Ismaïl R, 1998. Fréquence d’isolement des oocystes de Cryptosporidium dans les selles diarrhéiques d’enfants tunisiens. Med Mal Infect 28 :308–310.

    • Search Google Scholar
    • Export Citation
  • 7

    Fathallah A, Saghrouni F, Madani B, Ben Rejeb N, Ben Said M, 2004. La cryptosporidiose dans la region de Sousse, Tunisie. Arch Pediatr 11 :207–211.

    • Search Google Scholar
    • Export Citation
  • 8

    Casemore DP, Amstrong M, Jackson B, 1984. Screening for Cryptosporidium in stools. Lancet I :734–735.

  • 9

    Coupe S, Sarfati C, Hamame S, Derouin F, 2005. Detection of Cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism. J Clin Microbiol 43 :1017–1023.

    • Search Google Scholar
    • Export Citation
  • 10

    Llorente MT, Clavel A, Goni MP, Varea M, Seral C, Becerril R, Suarez L, Gomez-Lus R, 2007. Genetic characterization of Cryptosporidium species from humans in Spain. Int J Parasitol 56 :201–205.

    • Search Google Scholar
    • Export Citation
  • 11

    Gatei W, Suputtamongkol Y, Waywa D, Ashford RW, Bailey JW, Greensill J, Beeching NJ, Hart CA, 2002. Zoonotic species of Cryptosporidium are as prevalent as the anthroponotic in HIV-infected patients in Thailand. Ann Trop Med Parasitol 96 :797–802.

    • Search Google Scholar
    • Export Citation
  • 12

    Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, Checkley W, Cabrera L, Gilman RH, Lal AA, 2001. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J Infect Dis 183 :492–497.

    • Search Google Scholar
    • Export Citation
  • 13

    Caccio SM, 2005. Molecular epidemiology of human cryptosporidiosis. Parasitologia 47 :185–192.

  • 14

    Learmonth JJ, Ionas G, Ebbett KA, Kwan ES, 2004. Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand. Appl Environ Microbiol 70 :3973–3978.

    • Search Google Scholar
    • Export Citation
  • 15

    Alves M, Xiao L, Antunes F, Matos O, 2006. Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal. Parasitol Res 99 :287–292.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Identification of Cryptosporidium Species Infecting Humans in Tunisia

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  • 1 Laboratoire de Recherche 05SP03, Laboratoire de Parasitologie et Laboratoire d’Immunologie-Vaccinologie-Genetique Moléculaire, Institut Pasteur de Tunis, Tunis, Tunisia; Service des Maladies Infectieuses, la Rabta, Tunis, Tunisia; Laboratoire d’Immuno-Hématologie Pédiatrique, Centre de Greffe de Moelle Osseuse, Tunis, Tunisia; Laboratoire de Parasitologie-Mycologie, Hopital Saint Louis, Assistance Publique Hôpitaux de Paris et Faculté Denis Diderot, Paris, France

Prevalence and species distribution of Cryptosporidium spp. were determined among 633 immunocompetent children less than five years of age and 75 patients hospitalized for immunodeficiency who lived in northern Tunisia. Microscopy was used for initial screening to detect positive samples and a nested polymerase chain reaction and restriction fragment length polymorphism analysis was used to determine the species. Cryptosporidium spp. was identified in 2.7% of cases (19 stool samples), and there was a significant difference between samples collected from immunocompromised patients and those collected from healthy children (10.7% versus 1.7%). Prevalence was also significantly higher in diarrheal specimens than in formed specimens (6.3% versus 1.6%). Cryptosporidium hominis and C. parvum were responsible for most Cryptosporidium spp. infections (78.9%). Cryptosporidium hominis was more prevalent in children from urban areas than in those from rural areas, and C. parvum was found with similar prevalence rates in the two populations. Cryptosporidium meleagridis was identified in four children on farms.

INTRODUCTION

Cryptosporidium is a genus of enteric protozoons commonly associated with persistent diarrhea in immunocompromised persons that involves mostly persons with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome and primary immunodeficiency. It is also known to be a common cause of a self-limited diarrheal disease in all age groups of immunologically healthy people.1 In developing countries, the disease probably exerts most of its impact on pediatric health.2 In developed countries, cryptosporidiosis is frequently associated with water-borne outbreaks.3 In recent years, the development of genetic tools as part of the taxonomic characterization of Cryptosporidium spp. has promoted our understanding of the transmission mechanism and epidemiology of human cryptosporidiosis. Thus, numerous studies have identified five species of Cryptosporidium (C. parvum, C. hominis, C. meleagridis, C. felis, and C. canis) with different reservoir hosts as being responsible for most human Cryptosporidium spp. infections.4

Modified acid-fast staining has been used in Tunisia to identify Cryptosporidium spp. as a prevalent parasite in HIV-infected patients and immunocompromised and apparently immunocompetent children.57 However, microscopic examination of oocysts in clinical specimens does not enable identification of species and the assessment of the public health significance of Cryptosporidium spp. in the environment. The aim of this study was to determine the prevalence of Cryptosporidium species in different human populations. This information will contribute to a better understanding of transmission routes in Tunisia. A better understanding of the epidemiology will be helpful in devising appropriate control measures.

MATERIALS AND METHODS

Samples.

From 2006 through 2007, 708 stools samples were collected from 633 apparently immunocompetent children less than five years of age (477 living in farming areas in northeastern Tunisia and 156 originally from Tunis City) and from 75 patients hospitalized for immunodeficiency (39 HIV-infected patients and 36 children with primary immunodeficiency). Informed consent was obtained from parents and adult patients before stool collection.

Fresh stool specimens (549 formed and 159 diarrheal) were examined for Cryptosporidium spp. oocysts (Table 1). Microscopic examination was carried out on smears of fecal concentrates (formalinethyl acetate concentration) after staining with the modified Ziehl-Neelsen technique.8 When Cryptosporidium oocysts were identified microscopically, an aliquot of the stool specimen was diluted [v/v] in distilled water, placed into tubes, and stored frozen for molecular analysis.

Extraction of DNA.

DNA was extracted from samples using the QIAamp DNA Stool MiniKit (Qiagen Inc., Hilden, Germany) according to the manufacturer’s recommendations using approximately 200 μL of diluted stools for the first buffer step. After quantification, extracted DNA was frozen at −20°C until analyzed.

Analysis by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP).

A Cryptosporidium genus-specific nested PCR was used to amplify a 214-basepair fragment of the Cryptosporidium 18S ribosomal RNA gene encompassing the polymorphic region between nucleotides 179 and 271 as described.9 The first PCR amplification was performed with forward primer SCL1 (5′-CTGGTTGATC-CTGCCAGTAG-3′) and reverse primer CBP-DIAGR (5′-TAAGGTGCTGAAGGAGTAAGG-3′) complementary to nucleotides 4–23 and 1016–1036, respectively (GenBank accession no. AF093489). The second-round PCR was carried out using forward primer SCL2 (5′-CAGTTATAGTT-TACTTGATAATC-3′) and reverse primer SCR2 (5′-CAATACCCTACCGTCTAAAG-3′) complementary to nucleotides 106–128 and 299–318 respectively. The PCR amplification was performed in a 25-μL volume containing 5 μL of DNA template, 10× PCR buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 μM of each primer, and 1.25 units of Ampli-Taq Gold Polymerase (Roche, Indianapolis, IN). Cycling conditions were an initial denaturation at 94°C for 5 minutes, followed by 30 cycles of a three-step program (94°C for 30 seconds, 60°C for 34 seconds, and 72°C for 90 seconds). The reactions were then held at 72°C for 10 minutes (PXE 0.2 Thermal cycler; Thermo Fisher Scientific Inc., Waltham, MA).

The second-round PCR conditions were as described above using slight modifications. Briefly, 5 μL of the initial PCR product was used as DNA template and was amplified with 0.4 μM of each primer. Amplification included 30 cycles at 94°C for 30 seconds, 52°C for 45 seconds, and 72°C for 60 seconds. Amplified products were separated by electrophoresis on a 2% agarose gel and visualized after staining with ethidium bromide.

A DNA sample testing positive (kindly provided by Dr. Francis Derouin, Laboratoire de Parasitologie-Mycologie, Université Paris VII, Paris, France) was used as positive control. Oocyst-positive samples from which no specific DNA was amplified were retested at a 1:10 dilution and checked for inhibition by spiking these samples with 1 μL of the positive control.

Restriction assays.

Amplified product was initially digested with the restriction endonuclease Taq I as reported.9 The reaction contained 5 μL PCR product, 3 μL of the recommended 10× buffer, 2 units of enzyme, and distilled water to give a volume of 30 μL. Reactions were incubated for 2 hours at 65°C. Fragments were separated by electrophoresis on a 3% agarose gel in 1× Tris-borate-EDTA buffer, and the fragments were visualized after staining with ethidium bromide. The products that were digested by Taq I were digested with Ase I to differentiate C. hominis from C. parvum. In cases of negative results, the restriction enzymes Mse I, Bst UI, and Ssp I were used to identify other species.9 All restriction assays were conducted according to the manufacturer’s recommendations (New England Biolabs, Hitchin, United Kingdom).

Sequencing.

Three second-round PCR products identified as C. hominis, C. parvum, and C. meleagridis by the restriction profile were purified using the Wizad Purification Kit (Promega, Charbonnières, France). The PCR products were then sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). Sequences were compared with those available in the GenBank database with the BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST).

Statistical analysis.

Chi-square and Fisher exact tests were used to compare percentages (Epi-Info Software, version 6.04; Centers for Disease Control and Prevention, Atlanta, GA).

RESULTS

Detection of Cryptosporidium spp.

Among 708 samples examined by microscopy, 19 (2.7%) were positive for Cryptosporidium spp. oocysts. Oocysts were found in 10 (6.3%) diarrheal stools and 9 (1.6%) formed stools (P < 0.01) (Table 1). Positive samples were collected from 11 immunocompetent children (7 living in rural communities and 4 in urban communities) and from 8 immunocompromised patients (6 HIV-infected patients and 2 children with cellular immunodeficiency caused by defective expression of major histocompatibility complex class II molecules). Prevalence was significantly higher in immunocompromised patients than in immunocompetent children (10.7% versus 1.7%; P < 0.01) (Table 1). In this latter pediatric population, there was no significant difference between urban and rural prevalence rates (2.5% versus 1.4%) (Table 2).

Species identification.

Nested PCR analysis of microscopy-positive samples confirmed the presence of Cryptosporidium species. The RFLP analysis yielded typical restriction patterns for C. parvum in 8 cases, C. hominis in 7 cases, and C. meleagridis in 4 cases (Figure 1). Sequences of 3 samples identified as C. parvum, C. hominis, and C. meleagridis have been stored in GenBank under the accession nos. EU675852, EU675853, and EU675854, respectively. These samples showed 100% similarity with C. parvum sequence (GenBank accession no. AF308600) and 99% similarity with C. hominis sequence (GenBank accession no. DQ388386) and C. meleagridis sequence (GenBank accession no. AF112574), respectively. Mutations were not detected around restriction enzyme sites (Figure 2).

Cryptosporidium parvum and C. hominis were responsible for 15 cases of Cryptosporidium spp. infections (78.9%). Their distribution was not different on the basis of stool consistency (five cases with C. parvum and four cases with C. hominis in formed stools versus three cases from each species in diarrheic specimens). Their distribution was not different by immune status (five and three cases of C. parvum and C. hominis, respectively, in immunocompetent children versus three and four cases, respectively, in immunocompromised persons). Cryptosporidium hominis was more prevalent in children from urban areas than in children from rural areas (1.9% versus 0%; P < 0.05), and C. parvum was found with similar prevalence rates in the two populations (0.6% versus 0.8%) (Table 2).

Cryptosporidium meleagridis was identified only in children from rural areas; three immunocompetent non-diarrheic children and a four-year-old diarrheic immunocompromised child. In rural areas, no significant difference between the prevalence rate of C. meleagridis and that of C. parvum was found (0.6% versus 0.8%) (Table 2).

DISCUSSION

As previously reported, this study has showed that Cryptosporidium spp. are frequently identified in diarrheic stools of immunocompetent children and immunocompromised patients in Tunisia.5,6 However, this study is the first in Tunisia and as far as we know in north Africa to attempt identification of Cryptosporidium species infecting humans.

Despite the relative small number (19) of isolates characterized, we identified three species: C. hominis, C. parvum, and C. meleagridis. Similar to another report, C. hominis and C. parvum were responsible for most infections (15 cases, 78.9%).4 These parasites were found with a similar frequency, 8 cases of C. parvum (53.3%) and 7 cases of C. hominis (46.7%). In agreement with previous reports, distribution of these species was similar in immunocompetent and immunocompromised persons and also in formed and diarrheic stools.10 Cryptosporidium meleagridis was more prevalent in our study (4 cases, 21.1%) than it had been previously reported.11 Immunosuppression is apparently not a prerequisite for infections with this species because it was found, similar to other reports,12 in immunocompetent children.

In immunocompetent children, although the prevalence was as high in urban communities (2.5%) as in rural communities (1.4%), variation in prevalence rates of species was noted within the 2 populations. Cryptodporidium hominis was absent in rural pediatric communities, which suggests that anthroponotic transmission is less common in this population. The presence of C. parvum and C. meleagridis is sugestive of zoonotic transmission and direct contact with farm animals.13,14 In urban pediatric communities, the high prevalence of C. hominis indicates that humans are a major source of infection and that person-to-person transmission probably plays a major role in the spread of Cryptosporidium. The concomitant presence of C. parvum could be the result of environmental transmission (contaminated water or food) or the result of an anthroponotic transmission.4 Caution should be used in interpreting the significance of finding parasites traditionally associated with animals in humans. There is good evidence that subpopulations of C. parvum, e.g., GP60 type IIc, have a strictly anthroponotic transmission cycle.15 Thus, further analyses, such as sequence analysis of the GP60 gene,15 are needed to confirm the origin of these strains.

Table 1

Prevalence rate of Cryptosporidium spp. among immunocompetent children and immunocompromised patients according to stool consistency in Tunisia*

Formed stoolsDiarrheic stools
PatientsNo.MZN+, no. (%)No.MZN+, no. (%)
* MZN = Modified Ziehl-Neelsen technique.
Immunocompetent children5117 (1.36)1224 (3.2)
Immunocompromised children90 (0)272 (7.4)
Immunocompromised adults292 (6.8)104 (40)
549915910
Total(1.6%)(6.2%)
Table 2

Prevalence rate of Cryptosporidium species among children from urban and rural communities in Tunisia

CommunityC. hominis , no. cases (prevalence rate)C. parvum , no. cases (prevalence rate)C. meleagridis, no. cases (prevalence rate)Cryptosporidium spp., no. cases (prevalence rate)
Urban (n = 156)3 (1.9%)1 (0.6%)0 (0%)4 (2.5%)
Rural (n = 477)0 (0%)4 (0.8%)3 (0.6%)7 (1.4%)
Total (n = 633)3 (0.45%)5 (0.8%)3 (0.45%)11 (1.7%)
Figure 1.
Figure 1.

Polymerase chain reaction–restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene of Cryptosporidium spp. A, Restriction patterns of C. parvum and C. hominis. Lanes 1a and 2a, undigested products; lanes 1b and 2b, products digested with Taq I; lanes 1c and 2c, products digested with Ase I; lane M, 100-basepair (bp) molecular mass markers. B, Restriction patterns of C. meleagridis. Lane 3a, undigested product; lanes 3b–d, products digested with Mse I, Ssp I, and Bst UI; Lane M, 100-bp molecular weight markers. *Partial digestion with Mse I.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 5; 10.4269/ajtmh.2008.79.702

Figure 2.
Figure 2.

Alignment of DNA samples with deposited sequences of Cryptosporidium parvum, C. hominis, and C. meleagridis small subunit ribosomal RNA gene. Restriction sites of enzymes and their positions are shown in bold.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 5; 10.4269/ajtmh.2008.79.702

*

Address correspondence to Aïda Bouratbine, Laboratoire de Parasitologie, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002 Tunis Belvedère, Tunisia. E-mail: aida.bouratbine@pasteur.rns.tn

Authors’ addresses: Rym Essid, Karim Aoun, and Aïda Bouratbine, LR Parasitoses Emergentes, Laboratoire de Parasitologie, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002 Tunis Belvedère, Tunisia. Mohamed Mousli, Laboratoire d’ Immunologie-Vaccinologie-Genetique Moléculaire, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002 Tunis Belvedère, Tunisia. Rim Abdelmalek and Fakher Kanoun, Service des Maladies Infectieuses, la Rabta, 1007 Jebbari Tunis, Tunisia. Fethi Mellouli, Service d’Immuno-Hématologie Pédiatrique, Centre de Greffe de Moelle Oseuse, 2 Rue Djebel Lakhdhar, Tunis, Tunisia. Francis Derouin, Laboratoire de Parasitologie-Mycologie, Hopital Saint Louis, Assistance Publique Hôpitaux de Paris et Faculté Denis Diderot, 1 Avenue Claude Vellefaux 75 010, Paris, France.

Acknowledgments: We thank Dr. Ezzedine Brahem, Dr. Ridha Hamza, doctors and health agents from the Regional Directory of Public Health of Bizerte, doctors and nurses from PMI Cité Hllal, Tunis, and PMI Ibn Khaldoun, Tunis, for facilitated data and stool collection from healthy children. We also thank Professor Taoufik Ben Chaabane (Department of Infectious Diseases, La Rabta Hospital, Tunis, Tunisia), Ikram Rahmouni, and Nejla Chabchoub (Department of Parasitology, Pasteur Institute of Tunis, Tuynis, Tunisia) for cooperation and help.

Financial support: This study was supported by the Institut de Medecine et d’Epidémiologie Appliqué e–Fondation Internationale Leon Mba, France, and the Ministry of Higher Education, Research and Technology, Tunisia in the setting of the Research Laboratory Parasitoses Emergentes (LR 05SP03).

REFERENCES

  • 1

    Tzipori S, Ward H, 2002. Cryptosporidiosis: biology, pathogenesis and disease. Microbes Infect 4 :1047–1058.

  • 2

    Huang DB, Chappell C, Okhuysen PC, 2004. Cryptosporidiosis in children. Semin Pediatr Infect Dis 15 :253–259.

  • 3

    Corso PS, Kramer MH, Blair KA, Addiss DG, Davis JP, Haddix JP, 2003. Cost of illness in the 1993 waterborne Cryptosporidium outbreak, Milwaukee, Wisconsin. Emerg Infect Dis 9 :426–431.

    • Search Google Scholar
    • Export Citation
  • 4

    Xiao L, Fayer R, Ryan U, Upton SJ, 2004. Cryptosporidium taxonomy: recent advances and implications for public health. Clin Microbiol Rev 17 :72–97.

    • Search Google Scholar
    • Export Citation
  • 5

    Belhadj S, Kallel K, Boussen N, Goubantini A, Bejaoui M, Ben Salem N, Zribi A, Ben Chaabane T, Chaker E, 1999. Role of cryptosporidia and microsporidia in diarrhea in immunocompromised patients. Tunis Med 77 :638–643.

    • Search Google Scholar
    • Export Citation
  • 6

    Bouratbine A, Aoun K, Barbouche R, Bejaoui M, Ben Ismaïl R, 1998. Fréquence d’isolement des oocystes de Cryptosporidium dans les selles diarrhéiques d’enfants tunisiens. Med Mal Infect 28 :308–310.

    • Search Google Scholar
    • Export Citation
  • 7

    Fathallah A, Saghrouni F, Madani B, Ben Rejeb N, Ben Said M, 2004. La cryptosporidiose dans la region de Sousse, Tunisie. Arch Pediatr 11 :207–211.

    • Search Google Scholar
    • Export Citation
  • 8

    Casemore DP, Amstrong M, Jackson B, 1984. Screening for Cryptosporidium in stools. Lancet I :734–735.

  • 9

    Coupe S, Sarfati C, Hamame S, Derouin F, 2005. Detection of Cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism. J Clin Microbiol 43 :1017–1023.

    • Search Google Scholar
    • Export Citation
  • 10

    Llorente MT, Clavel A, Goni MP, Varea M, Seral C, Becerril R, Suarez L, Gomez-Lus R, 2007. Genetic characterization of Cryptosporidium species from humans in Spain. Int J Parasitol 56 :201–205.

    • Search Google Scholar
    • Export Citation
  • 11

    Gatei W, Suputtamongkol Y, Waywa D, Ashford RW, Bailey JW, Greensill J, Beeching NJ, Hart CA, 2002. Zoonotic species of Cryptosporidium are as prevalent as the anthroponotic in HIV-infected patients in Thailand. Ann Trop Med Parasitol 96 :797–802.

    • Search Google Scholar
    • Export Citation
  • 12

    Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, Checkley W, Cabrera L, Gilman RH, Lal AA, 2001. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J Infect Dis 183 :492–497.

    • Search Google Scholar
    • Export Citation
  • 13

    Caccio SM, 2005. Molecular epidemiology of human cryptosporidiosis. Parasitologia 47 :185–192.

  • 14

    Learmonth JJ, Ionas G, Ebbett KA, Kwan ES, 2004. Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand. Appl Environ Microbiol 70 :3973–3978.

    • Search Google Scholar
    • Export Citation
  • 15

    Alves M, Xiao L, Antunes F, Matos O, 2006. Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal. Parasitol Res 99 :287–292.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Aïda Bouratbine, Laboratoire de Parasitologie, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002 Tunis Belvedère, Tunisia, Tel: 216-71-890-827, Fax: 216-71791-833, E-mail: aida.bouratbine@pasteur.rns.tn.
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