Volume 24, 2017
|Number of page(s)||5|
|Published online||12 May 2017|
First molecular investigation of Cryptosporidium spp. in young calves in Algeria
Première investigation moléculaire de Cryptosporidium spp. chez les veaux en Algérie
Laboratory of Exploration and Valorization of Steppic Ecosystems, Faculty SNV, University of Ziane Achour, 17000
2 Department of Infectious Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy
3 Biotechnology Laboratory of Bioactive Molecules and Cellular Physiopathology, Faculty of Biological Sciences, Department of Living Organisms, University of Batna 2, 05000 Batna, Algeria
* Corresponding author: firstname.lastname@example.org
Accepted: 13 April 2017
To date, no information is available on the prevalence and genetic identity of Cryptosporidium spp. in cattle in Algeria. In this study, 17 dairy farms in the province of Batna, located in the northeast of the country, were visited to collect 132 fecal samples from young calves (< 8 weeks old). Samples were examined microscopically using the modified Ziehl-Neelsen acid-fast staining method, and at least one sample per farm was submitted for molecular analysis. Amplification of a fragment of the small subunit ribosomal RNA gene was positive for 24 of the 61 samples (40%), and sequence analysis identified three species, namely Cryptosporidium bovis (n = 14), C. ryanae (n = 6), and C. parvum (n = 4). The C. parvum IIaA13G2R1 subtype, an uncommon zoonotic subtype, was identified in two isolates from a single farm by sequencing a fragment of the GP60 gene. This is the first report about genotyping and subtyping of Cryptosporidium in calves in Algeria.
Actuellement, aucune information n’est disponible sur la prévalence et l’identité génétique de Cryptosporidium spp. chez les bovins en Algérie. Dans ce travail, 17 fermes laitières de la province de Batna, située au nord-est du pays, ont été inspectées pour récolter 132 échantillons fécaux de jeunes veaux (< 8 semaines). Les échantillons ont été examinés microscopiquement en utilisant la méthode de Ziehl–Neelsen modifiée, et au moins un échantillon par ferme a été soumis à analyse moléculaire. L’amplification d’un fragment du gène de la petite sous-unité du RNA ribosomal a été positive pour 24 échantillons parmi 61 (40 %), et l’analyse des séquences a identifié trois espèces, Cryptosporidium bovis (n = 14), C. ryanae (n = 6) et C. parvum (n = 4). Le sous-type IIaA13G2R1 de C. parvum, un sous-type zoonotique peu répandu, a été identifié par séquençage d’un fragment du gène GP60 dans deux isolats d’une seule ferme. Ceci est le premier rapport sur le génotypage et le sous-typage de Cryptosporidium chez les veaux en Algérie.
Key words: Cryptosporidium / Cryptosporidiosis / Molecular characterization / Cattle / Algeria
© D. Benhouda et al., published by EDP Sciences, 2017
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Parasites of the genus Cryptosporidium cause diarrheal disease in many vertebrate hosts, including humans, and have a worldwide distribution [6, 7]. Currently, 31 Cryptosporidium species have been recognized based on biological and molecular characteristics, while many other genotypes are still of uncertain taxonomic status . In humans, infection is mostly caused by two species, Cryptosporidium hominis and C. parvum; the former is considered restricted to humans, whereas the latter also infects other mammals, in particular young ruminants. Other species commonly occur in cattle, including C. bovis, C. ryanae, and C. andersoni . The distribution of these species is age-dependent: C. parvum predominates in pre-weaned animals, whereas C. bovis and C. ryanae are more common in post-weaned animals and young stock, while C. andersoni is mostly found in adult cattle . Therefore, pre-weaned calves are considered important reservoirs of C. parvum oocysts infectious to humans, and outbreaks associated with exposure to calf feces are well documented .
In Africa, cryptosporidiosis is a particularly relevant health problem, and recent studies have shown that Cryptosporidium is second only to Rotavirus among etiological agents of moderate to severe diarrhea in very young children . Many studies have been conducted in African countries to estimate the burden of cryptosporidiosis in humans and animals, describe the circulating species and genotypes, and elucidate transmission routes [1, 8]. However, no information is currently available on human cryptosporidiosis in Algeria, and only a few studies have been conducted on animals, with a focus on horses and donkeys , and broiler chickens and turkeys .
Here, we provide the first evidence of Cryptosporidium spp. infection in young calves (<2 months) from small, traditional farms in Algeria. We performed molecular investigations to define the parasite species.
The study was carried out in the Wilaya (province) of Batna, situated in the northeast of Algeria (Figure 1). This province covers about 12,192 km2 and has a population of approximately 1,120,000 inhabitants. During May 2016, a single visit was conducted at 17 dairy farms, and 132 samples were collected. These are traditional farms with a small number of adult cattle (from 6 to 40; age 2–9 years), mostly of the local breed known as Race Brune de l’Atlas. Calves are usually reared indoors with their mothers and fed with bottled milk.
Map of Algeria and of the Batna province showing the location of the dairy farms investigated in this study. The original maps were downloaded from http://www.d-maps.com/carte.php?num_car=176844&lang=en
A minimum of 5 g of feces was collected from each pre-weaned or post-weaned calf, either directly from the rectum, when possible, or from freshly deposited feces on the ground. Each sample was individually placed into a sterile plastic tube, mixed with an equal volume of 5% potassium dichromate, and transported to the laboratory of parasitology in a refrigerated box.
Fecal specimens were screened by microscopy for Cryptosporidium oocysts after staining with the modified Ziehl-Neelsen stain . A semi-quantitative score was used to distinguish between low (1–4 oocysts per field), moderate (5–10 oocysts per field), and high (>10 oocysts per field) levels of infection.
Fecal specimens were washed three times with distilled water by centrifugation to remove potassium dichromate prior to DNA isolation. Total DNA was extracted from ~200 mg of feces using a commercially available kit (QIAamp® DNA Stool Mini Kit, Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Purified DNA was stored at −20 °C prior to Polymerase Chain Reaction (PCR).
For species identification, a nested PCR assay was used to amplify a ~590 bp fragment of the small subunit rRNA (SSU rRNA) gene . For subtyping, a ~300 bp fragment of the glycoprotein 60 (GP60) gene, encompassing the microsatellite region at the 5′ of the gene, was amplified by nested PCR . Negative and positive controls (DNA from the C. parvum Moredun strain) were included in each experiment. PCR was performed using 25 μL of 2× GoTaq Green (Promega, Madison, WI, USA), 10 pmol of each primer, 2.5–5.0 μL of DNA, and nuclease-free water up to a final volume of 50 μL. Reactions were performed on a Perkin Elmer 9700 apparatus (Life Technologies, Carlsbad, CA, USA). Aliquots (5–10 μL) of the PCR products were loaded on 1.5% agarose gel stained with ethidium bromide. PCR products were purified using spin columns (Qiaquick PCR Purification kit, Qiagen, Milan, Italy) and sequenced directly on an ABI 3130 Genetic Analyzer. Bidirectional sequences were edited and assembled using the SeqMan 7.1 software package (DNASTAR, Madison, WI, USA). A BLAST search against the GenBank database was used to identify Cryptosporidium species and subtypes.
Based on microscopic analysis of fecal smears stained using the modified Ziehl-Neelsen acid-fast method, the majority of the samples (84%) at each of the 17 investigated farms were positive (Table 1), suggesting a herd prevalence of 100%. In the majority of the samples (58%), however, few oocysts were observed. Due to the possibility of false negative results of microscopy, and the higher sensitivity of PCR-based procedures, a panel of 66 samples, comprising at least one sample per farm, was selected for molecular analysis. Nested PCR amplification of a fragment of SSU rRNA resulted in the identification of 24 positive samples (36%). At the farm level, 14 of 17 had at least one sample positive by PCR (Table 1), suggesting a prevalence of 82% at the herd level and of 18% (24 of 132) at the animal level.
Cryptosporidium species identified in dairy cattle in Algeria.
Sequencing of the SSU rRNA amplicons identified Cryptosporidium bovis as the most common species (n = 14, present in nine farms), followed by C. ryanae (n = 6, present in six farms) and C. parvum (n = 4, present in three farms). In particular, the sequences obtained had 100% similarity to GenBank reference sequences for C. bovis (AY741305), C. ryanae (EU410344), and C. parvum (KY514062). No intra-species sequence variation in the SSU rRNA gene fragment was observed.
Cryptosporidium bovis was more common in calves aged 1–2 months (n = 11) than in those aged 15–20 days (n = 3); likewise, Cryptosporidium ryanae was found in five calves aged 1–2 months but only in a single 15-day-old calf. Finally, Cryptosporidium parvum was found only in calves younger than 1 month.
In a global African perspective, human-to-human transmission of Cryptosporidium is considered the main route of transmission, at least in Sub-Saharan countries, where C. hominis and anthroponotic (human host-restricted) subtypes of C. parvum account for the vast majority of cases observed in young children . Nevertheless, cryptosporidiosis is also common in a range of domestic and wild animal species, and evidence for zoonotic potential has been provided in many studies [1, 14].
In this context, molecular studies on human and animal cryptosporidiosis in North African countries are still scarce. In Egypt, calves are predominantly infected with C. parvum subtypes of the IId and IIa families , which are also found in humans in this country . In Tunisia, C. parvum IIaA15G2R1 and IIdA16G1 subtypes were identified in calves and children from a rural area in the north of the country . Another study identified C. hominis, C. parvum, and C. meleagridis in immunocompetent and immunocompromised individuals, mostly children, in Tunisia . Therefore, both zoonotic and anthroponotic species are involved in human cryptosporidiosis in these regions.
Here, we provide the first information on the prevalence and genetic identity of Cryptosporidium species in young calves (<2 months) reared in small, traditional farms in the north of Algeria. Our data show that calves aged 1–2 months are mostly infected with Cryptosporidium bovis followed by C. ryanae, whereas few animals were infected with C. parvum. This contrasts with the prevailing pattern of C. parvum dominance in young calves , but is consistent with data from less intensive management systems in different parts of the world [4, 23, 27], where C. bovis is the dominant species even in pre-weaned calves.
Four calves from three farms were positive for Cryptosporidium parvum, and subtyping of the GP60 gene in two isolates from a single farm identified subtype IIaA13G2R1. This uncommon subtype has been found in calves in Turkey , Canada , Belgium , and the Netherlands , and in people with HIV/AIDS in Malaysia  and in the United States .
In conclusion, the data presented suggest that cattle play a minor role in sustaining circulation of zoonotic Cryptosporidium species/genotypes. However, a better estimate of the prevalence and identity of the C. parvum genotypes in young calves, and a clarification of their role in clinical cryptosporidiosis are needed. Likewise, understanding the relative role of anthroponotic and zoonotic transmission in Algeria will require investigations into human cryptosporidiosis.
The authors have no competing interests.
This study was supported by the European Commission H2020 programme under Contract Number 643476 (www.compare-europe.eu) to SMC.
- Adamu H, Petros B, Zhang G, Kassa H, Amer S, Ye J, Feng Y, Xiao L. 2014. Distribution and clinical manifestations of Cryptosporidium species and subtypes in HIV/AIDS patients in Ethiopia. PLoS Neglected Tropical Diseases, 8, e2831. [CrossRef] [PubMed] [Google Scholar]
- Amer S, Honma H, Ikarashi M, Tada C, Fukuda Y, Suyama Y, Nakai Y. 2010. Cryptosporidium genotypes and subtypes in dairy calves in Egypt. Veterinary Parasitology, 169, 382–386. [CrossRef] [PubMed] [Google Scholar]
- Baroudi D, Khelef D, Goucem R, Adjou KT, Adamu H, Zhang H, Xiao L. 2013. Common occurrence of zoonotic pathogen Cryptosporidium meleagridis in broiler chickens and turkeys in Algeria. Veterinary Parasitology, 196, 334–340. [CrossRef] [PubMed] [Google Scholar]
- Budu-Amoako E, Greenwood SJ, Dixon BR, Barkema HW, McClure JT. 2012. Giardia and Cryptosporidium on dairy farms and the role these farms may play in contaminating water sources in Prince Edward Island, Canada. Journal of Veterinary and Internal Medicine, 26, 668–673. [CrossRef] [Google Scholar]
- Casemore D, Armstrong M, Sands R. 1985. Laboratory diagnosis of cryptosporidiosis. Journal of Clinical Pathology, 38, 1337–1341. [CrossRef] [PubMed] [Google Scholar]
- Checkley W, White AC Jr, Jaganath D, Arrowood MJ, Chalmers RM, Chen XM, Fayer R, Griffiths JK, Guerrant RL, Hedstrom L, Huston CD, Kotloff KL, Kang G, Mead JR, Miller M, Petri WA, Priest JW, Roos DS, Striepen B, Thompson RC, Ward D, Van Voorhis WA, Xiao L, Zhu G, Houpt ER. 2015. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infectious Diseases, 15, 85–94. [CrossRef] [Google Scholar]
- Cho YI, Han JI, Wang C, Cooper V, Schwartz K, Engelken T, Yoon KJ. 2013. Case-control study of microbiological etiology associated with calf diarrhea. Veterinary Microbiology, 166, 375–385. [CrossRef] [PubMed] [Google Scholar]
- Desai NT, Sarkar R, Kang G. 2012. Cryptosporidiosis: An under-recognized public health problem. Tropical Parasitology, 2, 91–98. [CrossRef] [PubMed] [Google Scholar]
- Duranti A, Cacciò SM, Pozio E, Di Egidio A, De Curtis M, Battisti A, Scaramozzino P. 2009. Risk factors associated with Cryptosporidium parvum infection in cattle. Zoonoses Public Health, 56, 176–182. [CrossRef] [PubMed] [Google Scholar]
- Essid R, Mousli M, Aoun K, Abdelmalek R, Mellouli F, Kanoun F, Derouin F, Bouratbine A. 2005. Identification of Cryptosporidium species infecting humans in Tunisia. American Journal of Tropical Medicine and Hygiene, 79, 702–705. [Google Scholar]
- Fayer R, Santín M, Dargatz D. 2010. Species of Cryptosporidium detected in weaned cattle on cow-calf operations in the United States. Veterinary Parasitology, 170, 187–192. [CrossRef] [PubMed] [Google Scholar]
- Geurden T, Berkvens D, Martens C, Casaert S, Vercruysse J, Claerebout E. 2007. Molecular epidemiology with subtype analysis of Cryptosporidium in calves in Belgium. Parasitology, 134, 1981–1987. [CrossRef] [PubMed] [Google Scholar]
- Grinberg A, Pomroy WE, Squires RA, Scuffham A, Pita A, Kwan E. 2011. Retrospective cohort study of an outbreak of cryptosporidiosis caused by a rare Cryptosporidium parvum subgenotype. Epidemiology and Infection, 139, 1542–1550. [CrossRef] [PubMed] [Google Scholar]
- Hunter PR, Hadfield SJ, Wilkinson D, Lake IR, Harrison FC, Chalmers RM. 2007. Subtypes of Cryptosporidium parvum in humans and disease risk. Emerging Infectious Diseases, 13, 82–88. [CrossRef] [PubMed] [Google Scholar]
- Ibrahim MA, Abdel-Ghany AE, Abdel-Latef GK, Abdel-Aziz SA, Aboelhadid SM. 2016. Epidemiology and public health significance of Cryptosporidium isolated from cattle, buffaloes, and humans in Egypt. Parasitology Research, 115, 2439–2448. [CrossRef] [PubMed] [Google Scholar]
- Iqbal A, Lim YA, Surin J, Sim BL. 2012. High diversity of Cryptosporidium subgenotypes identified in Malaysian HIV/AIDS individuals targeting gp60 gene. PLoS One, 7, e31139. [CrossRef] [PubMed] [Google Scholar]
- Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acacio S, Biswas K, O’Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, Levine MM. 2013. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet, 382, 209–222. [CrossRef] [PubMed] [Google Scholar]
- Laatamna AE, Wagnerová P, Sak B, Květoňová D, Xiao L, Rost M, McEvoy J, Saadi AR, Aissi M, Kváč M. 2015. Microsporidia and Cryptosporidium in horses and donkeys in Algeria: detection of a novel Cryptosporidium hominis subtype family (Ik) in a horse. Veterinary Parasitology, 208, 135–142. [CrossRef] [PubMed] [Google Scholar]
- Rahmouni I, Essid R, Aoun K, Bouratbine A. 2014. Glycoprotein 60 diversity in Cryptosporidium parvum causing human and cattle cryptosporidiosis in the rural region of Northern Tunisia. American Journal of Tropical Medicine and Hygiene, 90, 346–350. [CrossRef] [Google Scholar]
- Ryan U, Xiao L, Read C, Zhou L, Lal AA, Pavlasek I. 2003. Identification of novel Cryptosporidium genotypes from the Czech Republic. Applied Environmental Microbiology, 69, 4302–4307. [CrossRef] [PubMed] [Google Scholar]
- Ryan U, Fayer R, Xiao L. 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology, 141, 1667–1685. [CrossRef] [PubMed] [Google Scholar]
- Robertson LJ, Bijorkman C, Axen C, Fayer R. 2014. Cryptosporidiosis in farmed animals, in Cryptosporidium: parasite and disease. Cacciò SM, Widmer G, Editors. Springer-Verlag: Wien. p. 149–235. [CrossRef] [Google Scholar]
- Silverlås C, Näslund K, Björkman C, Mattsson JG. 2010. Molecular characterisation of Cryptosporidium isolates from Swedish dairy cattle in relation to age, diarrhoea and region. Veterinary Parasitology, 169, 289–295. [CrossRef] [PubMed] [Google Scholar]
- Sow SO, Muhsen K, Nasrin D, Blackwelder WC, Wu Y, Farag TH, Panchalingam S, Sur D, Zaidi AK, Faruque AS, Saha D, Adegbola R, Alonso PL, Breiman RF, Bassat Q, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ahmed S, Qureshi S, Quadri F, Hossain A, Das SK, Antonio M, Hossain MJ, Mandomando I, Nhampossa T, Acácio S, Omore R, Oundo JO, Ochieng JB, Mintz ED, O’Reilly CE, Berkeley LY, Livio S, Tennant SM, Sommerfelt H, Nataro JP, Ziv-Baran T, Robins-Browne RM, Mishcherkin V, Zhang J, Liu J, Houpt ER, Kotloff KL, Levine MM. 2016. The burden of Cryptosporidium diarrheal disease among children < 24 months of age in moderate/high mortality regions of Sub-Saharan Africa and South Asia, utilizing data from the Global Enteric Multicenter Study (GEMS). PLoS Neglected Tropical Diseases, 10, e0004729. [CrossRef] [PubMed] [Google Scholar]
- Taylan-Ozkan A, Yasa-Duru S, Usluca S, Lysen C, Ye J, Roellig DM, Feng Y, Xiao L. 2016. Cryptosporidium species and Cryptosporidium parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey. Experimental Parasitology, 170, 16–20. [CrossRef] [PubMed] [Google Scholar]
- 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. Parasitology Research, 99, 346–352. [CrossRef] [PubMed] [Google Scholar]
- Wang R, Wang H, Sun Y, Zhang L, Jian F, Qi M, Ning C, Xiao L. 2011. Characteristics of Cryptosporidium transmission in preweaned dairy cattle in Henan, China. Journal of Clinical Microbiology, 49, 1077–1082. [CrossRef] [PubMed] [Google Scholar]
- Wielinga PR, de Vries A, van der Goot TH, Mank T, Mars MH, Kortbeek LM, van der Giessen JW. 2008. Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands. International Journal for Parasitology, 38, 809–817. [CrossRef] [PubMed] [Google Scholar]
Cite this article as: Benhouda D, Hakem A, Sannella AR, Benhouda A & Cacciò SM: First molecular investigation of Cryptosporidium spp. in young calves in Algeria. Parasite, 2017, 24, 15.
Map of Algeria and of the Batna province showing the location of the dairy farms investigated in this study. The original maps were downloaded from http://www.d-maps.com/carte.php?num_car=176844&lang=en
|In the text|
Current usage metrics show cumulative count of Article Views (full-text article views including HTML views, PDF and ePub downloads, according to the available data) and Abstracts Views on Vision4Press platform.
Data correspond to usage on the plateform after 2015. The current usage metrics is available 48-96 hours after online publication and is updated daily on week days.
Initial download of the metrics may take a while.