# Zoonotic Cryptosporidium species and subtypes in lambs and goat kids in Algeria

Djamel Baroudi, Ahcene Hakem, Haileeyesus Adamu, Said Amer, Djamel Khelef, Karim Adjou, Hichem Dahmani, Xiaohua Chen, Dawn Roellig, Yaoyu Feng, et al.

► To cite this version:

Djamel Baroudi, Ahcene Hakem, Haileeyesus Adamu, Said Amer, Djamel Khelef, et al. Zoonotic Cryptosporidium species and subtypes in lambs and goat kids in Algeria. Parasites & Vectors, 2018, 11 (1), pp.582. 10.1186/s13071-018-3172-2. hal-04525279

HAL Id: hal-04525279

https://enva.hal.science/hal-04525279v1

Submitted on 12 Jul 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH

Open Access

<figure><img src="image_2.png" /></figure>

CrossMark

# Zoonotic *Cryptosporidium* species and subtypes in lambs and goat kids in Algeria

Djamel Baroudi¹,², Ahcene Hakem³, Haileeyesus Adamu⁴, Said Amer⁵, Djamel Khelef¹, Karim Adjou⁶, Hichem Dahmani⁷, Xiaohua Chen⁸, Dawn Roellig², Yaoyu Feng⁹ and Lihua Xiao⁹*

**Abstract**

**Background:** Little is known on the occurrence and identity of *Cryptosporidium* species in sheep and goats in Algeria. This study aimed at investigating the occurrence of *Cryptosporidium* species in lambs and goat kids younger than 4 weeks.

**Methods:** A total of 154 fecal samples (62 from lambs and 92 from kid goats) were collected from 13 sheep flocks in Médea, Algeria and 18 goat flocks across Algiers and Boumerdes. They were screened for *Cryptosporidium* spp. by nested-PCR analysis of a fragment of the small subunit (SSU) rRNA gene, followed by restriction fragment length polymorphism and sequence analyses to determine the *Cryptosporidium* species present. *Cryptosporidium parvum* and *C. ubiquitum* were further subtyped by sequence analysis of the 60 kDa glycoprotein gene.

**Results:** *Cryptosporidium* spp. were detected in 17 fecal samples (11.0%): 9 from lambs (14.5%) and 8 from goat kids (8.7%). The species identified included *C. parvum* in 3 lambs, *C. xiaoi* in 6 lambs and 6 goat kids, and *C. ubiquitum* in 2 goat kids. *Cryptosporidium* infections were detected mostly in animals during the first two weeks of life (7/8 for goat kids and 7/9 for lambs) and in association with diarrhea occurrence (7/17 or 41.2% goat kids and 7/10 or 70.0% lambs with diarrhea were positive for *Cryptosporidium* spp.). Subtyping of *C. parvum* and *C. ubiquitum* isolates identified the zoonotic IIaA13G2R1 and XIIa subtype families, respectively. Minor differences in the SSU rRNA gene sequences were observed between *C. xiaoi* from sheep and goats.

**Conclusions:** Results of this study indicate that three *Cryptosporidium* species occur in lambs and goat kids in Algeria, including zoonotic *C. parvum* and *C. ubiquitum*. They are associated with the occurrence of neonatal diarrhea.

**Keywords:** *Cryptosporidium parvum*, *Cryptosporidium ubiquitum*, *Cryptosporidium xiaoi*, Goat, Sheep, Algeria

**Background**

*Cryptosporidium* spp. are common enteric protozoa of humans and a wide range of animals [1]. They are involved in numerous outbreaks of diarrheal illness in humans and pre-weaned calves [2, 3]. However, studies of *Cryptosporidium* spp. in small ruminants are much smaller in numbers compared to those in cattle, especially from developing countries [4–6]. Data accumulated thus far indicate that cryptosporidiosis in small ruminants can lead to severe diarrhea, anorexia and weight loss in goat kids and lambs [5, 7–9]. Considerably

high infection rates have been reported in these animals in some areas [10–14].

Currently, over 30 *Cryptosporidium* species have been recognized based on morphological, biological and molecular characteristics (reviewed in [1]). Among them, *C. parvum*, *C. ubiquitum* and *C. xiaoi* are common species in small ruminants, although a small number of animals were reportedly infected with other *Cryptosporidium* species such as *C. andersoni* and *C. hominis* [4, 5, 14–18]. Geographical variations in the distribution of these *Cryptosporidium* spp. in small ruminants, however, have been described among the small number of studies conducted [19]. The common occurrence of zoonotic *C. parvum* and *C. ubiquitum* in goats and sheep has raised public health concerns over cryptosporidiosis. While C.

<figure><img src="image_3.png" /></figure>

BMC

© The Author(s). 2018 **Open Access** This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

<footnote>* Correspondence: lxiao1961@gmail.com
⁹Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Full list of author information is available at the end of the article</footnote>
*parvum* is well known for causing diarrhea in small ruminants, the pathogenicity of *C. ubiquitum* and *C. xiaoi* remains unclear [2].

Algeria is adopting intensive farming of small ruminants to cope with the high demand for meat and milk. A recent estimate from the Ministère De L'Agriculture et du Développement Rural indicates that the country has approximately 2,800,000 sheep and 490,000 goats (http://www.minagri.dz/contacts.html). Although *Cryptosporidium* spp. from cattle, horses, camels and chickens have been recently characterized using molecular biological tools [20–23], little information is available on the identity of *Cryptosporidium* spp. in goats and sheep. In the present study we therefore generated some preliminary data on the occurrence of *Cryptosporidium* species in goat kids and lambs in Algeria.

# Methods

## Collection of samples

This study was conducted between January 2012 and January 2014 on 13 sheep flocks in Ksar el Boukhari of Médea Province (No. 1), and 18 goat flocks from seven localities in the provinces of Algiers (Nos 2, 3, 4, 5) and Boumerdes (Nos 6, 7, 8) (Fig. 1). A total of 92 and 62 fecal samples were collected directly from the rectum of goat kids and lambs, respectively. Only animals aged 4 weeks or younger were sampled. Fecal consistency and demographic data on the animals were recorded at the site of sample collection. The samples were transported to the laboratory in ice boxes and preserved in 2.5% potassium dichromate at 4 °C until molecular analysis.

## DNA extraction and PCR analysis

Potassium dichromate was washed off fecal samples with distilled water by centrifugation. Genomic DNA was extracted from 0.2 ml of fecal slurry without further pathogen concentration using the FastDNA SPIN Kit for Soil (BIO 101, MP Biomedicals, Carlsbad, CA, USA). DNA preparations were screened for *Cryptosporidium* spp. by using a small subunit (SSU) rRNA-based nested PCR, with DNA of *C. baileyi* as the positive control and reagent-grade water as the negative control. The detection limit of the approach was ~10 oocysts per gram of feces. *Cryptosporidium* species in positive PCR products were determined by restriction fragment length polymorphism (RFLP) analysis using restriction enzymes SspI and MboII as described [24] and by DNA sequencing. *Cryptosporidium parvum* and *C. ubiquitum* were subtyped by nested-PCR-sequence analysis of the *gp60* gene as previously described [25, 26].

## DNA sequence analysis

To confirm the identification of *C. ubiquitum* and *C. xiaoi*, the secondary PCR products of the *SSU* rRNA gene from the two *Cryptosporidium* species were sequenced in both directions on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The *SSU* rRNA gene products of *C. parvum* were not sequenced because it has a well-known SspI and MboII RFLP pattern. In addition, all PCR products of the *gp60* gene were sequenced to identify *C. parvum* and *C. ubiquitum* subtypes. The generated sequences were assembled using the

<figure><img src="image_4.png" /></figure>
ChromasPro v.1.5 software (http://www.technelysium.com.au/ChromasPro.html) and aligned with each other and reference sequences downloaded from GenBank using ClustalX (http://www.clustal.org/). Representative sequences generated in the study were submitted to GenBank under accession numbers LC414387-LC414393 for the *SSU* rRNA gene and LC414394 and JX412917 for the *gp60* gene of *C. parvum* and *C. ubiquitum*, respectively.

## Statistical analysis

*Cryptosporidium* infection rates between diarrheic and non-diarrheic animals were compared statistically using Fisher's exact test implemented in the Statistical Package for the Social Sciences (SPSS version 22.0). Differences were considered significant at *P* ≤ 0.05.

## Results

### Occurrence of *Cryptosporidium* spp. in goat kids and lambs

*Cryptosporidium* spp. were detected in 8/92 (8.7%) fecal samples from goat kids and in 9/62 (14.5%) fecal samples from lambs, with an overall infection rate of 11.0%. They were present on 3/18 goat farms and 4/9 sheep farms (Table 1). Most *Cryptosporidium*-positive samples were from animals up to 3 weeks of age with diarrhea for both goats and sheep (Table 2).

### *Cryptosporidium* species and subtypes

The RFLP analysis of the *SSU* rDNA PCR products identified two *Cryptosporidium* species in goat kids, including *C. xiaoi* in 6 of 8 *Cryptosporidium*-positive samples and *C. ubiquitum* in 2 of 8 *Cryptosporidium*-positive samples (Fig. 2). *Cryptosporidium xiaoi* was detected in Rouiba (Algiers) and Zemouri (Boumerdes), while *C. ubiquitum* was seen in Khemis-elkhechna (Boumerdes). In lambs, *C. parvum* was present in 3 of 9 *Cryptosporidium*-positive and *C. xiaoi* was identified in the 6 of 9 *Cryptosporidium*-positive samples from Ksar-elBoukhri (Médea). DNA sequencing of *SSU* rDNA PCR products

confirmed the detection of *C. ubiquitum* and *C. xiaoi* in these samples.

Data from 12 samples of *C. xiaoi* (six each from sheep and goats) generated two sequence types. The first type was represented by six sequences from sheep and was identical to one *C. xiaoi* sequence (DQ871346) first obtained from a yak in China [24]. The second sequence type was represented by six sequences from goats and was identical to one *C. xiaoi* sequence (EF362478) first obtained from a sheep in the USA [27]. There were two nucleotide differences in the partial *SSU* rRNA gene between the two sequence types (substitution of TT in the first type by CA in the second sequence type at position 420 and 421 of the reference sequence EF362478). The two sequences of the *SSU* rRNA gene fragment of *C. ubiquitum* were identical to each other and to AF442484 initially detected in lemurs in the USA [28].

Sequence analysis of the *gp60* gene indicated that the three *C. parvum*-positive samples from lambs had the IIaA13G2R1 subtype, whereas the two *C. ubiquitum*-positive samples from goat kids had the XIIa subtype.

### Occurrence of *Cryptosporidium* spp. by age

In goat kids, *Cryptosporidium* was detected in 2/22 (9.1%), 5/25 (20.0%), 1/27 (3.9%) and 0/18 (0%) of the animals sampled in the first, second, third and fourth week of age, respectively. In contrast, *Cryptosporidium* was detected in 3/15 (20.0%), 4/18 (22.2%), 1/13 (7.7%) and 1/16 (6.3%) lambs sampled in the first, second, third and fourth week of age, respectively. In goat kids, *C. xiaoi* was mostly seen during the first three weeks of age, with two cases (2/22; 9.1%) in the first week, three cases (3/25; 12.0%) in the second week, and one case in the third week (1/27; 3.7%). The two *C. ubiquitum* cases identified in goat kids were seen in the second week of age (2/25; 8.0%) (Table 2). In lambs, one *C. parvum* infection was detected in the first week of age (1/15; 6.7%) and the other two infections in the second week (2/18; 11.1%), while *C. xiaoi* was detected in four animals

<table><caption>**Table 1** *Cryptosporidium* species in goat kids and lambs in Algiers, Boumerdes and Médea provinces, Algeria</caption><thead><tr><th>Location</th><th>No. of farms</th><th>Host</th><th>No. of samples</th><th>No. positive for <i>Cryptosporidium</i> (no. of positive farms)</th><th><i>Cryptosporidium</i> spp. (no. of samples)</th><th>Subtype (no. of samples)</th></tr></thead><tbody><tr><td>Ksar-el Boukhari (Médea)</td><td>13</td><td>Sheep</td><td>62</td><td>9 (4)</td><td><i>C. xiaoi</i> (6), <i>C. parvum</i> (3)</td><td>IIaA13G2R1(3)</td></tr><tr><td>Rouiba (Algiers)</td><td>3</td><td>Goats</td><td>16</td><td>3 (1)</td><td><i>C. xiaoi</i> (3)</td><td>–</td></tr><tr><td>Bordj-El-Kiffan (Algiers)</td><td>2</td><td>Goats</td><td>11</td><td>0</td><td>0</td><td>–</td></tr><tr><td>Heraoua (Algiers)</td><td>2</td><td>Goats</td><td>9</td><td>0</td><td>0</td><td>–</td></tr><tr><td>Khemis-elkhechna (Boumerdes)</td><td>2</td><td>Goats</td><td>11</td><td>2 (1)</td><td><i>C. ubiquitum</i> (2)</td><td>XIIa (2)</td></tr><tr><td>Hamadi (Boumerdes)</td><td>3</td><td>Goats</td><td>17</td><td>0</td><td>0</td><td>–</td></tr><tr><td>Zemouri (Boumerdes)</td><td>4</td><td>Goats</td><td>15</td><td>3 (1)</td><td><i>C. xiaoi</i> (3)</td><td>–</td></tr><tr><td>Oued-smar (Algiers)</td><td>2</td><td>Goats</td><td>13</td><td>0</td><td>0</td><td>–</td></tr><tr><td>Total</td><td>31</td><td></td><td>154</td><td>17 (7)</td><td>3 species</td><td>2 subtypes</td></tr></tbody></table>
<table><caption>**Table 2** Occurrence of *Cryptosporidium* spp. in goat kids by age and diarrhea status</caption><thead><tr><th>Distribution</th><th>No. of samples</th><th>No. positive for <i>Cryptosporidium</i> (%)</th><th><i>Cryptosporidium</i> spp. (no. of samples)</th></tr></thead><tbody><tr><td colspan="4">By age (days)</td></tr><tr><td>1–7</td><td>22</td><td>2 (9.1)</td><td><i>C. xiaoi</i> (2)</td></tr><tr><td>8–14</td><td>25</td><td>5 (20.0)</td><td><i>C. xiaoi</i> (3), <i>C. ubiquitum</i> (2)</td></tr><tr><td>15–21</td><td>27</td><td>1 (3.7)</td><td><i>C. xiaoi</i> (1)</td></tr><tr><td>22–28</td><td>18</td><td>0</td><td></td></tr><tr><td colspan="4">By age (days) and diarrhea status</td></tr><tr><td>1–7</td><td></td><td></td><td></td></tr><tr><td>Diarrheic</td><td>8</td><td>2 (25.0)</td><td><i>C. xiaoi</i> (2)</td></tr><tr><td>Non-diarrheic</td><td>14</td><td>0 (0)</td><td></td></tr><tr><td>8–14</td><td></td><td></td><td></td></tr><tr><td>Diarrheic</td><td>9</td><td>5 (55.6)</td><td><i>C. xiaoi</i> (3), <i>C. ubiquitum</i> (2)</td></tr><tr><td>Non-diarrheic</td><td>16</td><td>0 (0)</td><td></td></tr><tr><td>15–21</td><td></td><td></td><td></td></tr><tr><td>Diarrheic</td><td>4</td><td>1 (25.0)</td><td><i>C. xiaoi</i> (1)</td></tr><tr><td>Non-diarrheic</td><td>23</td><td>0 (0)</td><td></td></tr><tr><td>22–28</td><td></td><td></td><td></td></tr><tr><td>Diarrheic</td><td>2</td><td>0 (0)</td><td></td></tr><tr><td>Non-diarrheic</td><td>16</td><td>0 (0)</td><td></td></tr><tr><td>Total</td><td>92</td><td>8 (8.7)</td><td><i>C. xiaoi</i> (6), <i>C. ubiquitum</i> (2)</td></tr></tbody></table>

during the first two weeks (2/15; 13.3% and 2/18; 11.1, respectively) and two animals during the third and fourth weeks (1/13; 7.7% and 1/16; 6.3%, respectively) (Table 2).

### Occurrence of *Cryptosporidium* spp. by diarrhea status

Altogether, 23 of the 92 fecal samples (25.0%) were collected from goat kids with diarrhea, including 8/92 (8.7%) in the first week, 9/92 (9.8%) in the second week, 4/92 (4.3%) in the third week, and 2/92 (2.2%) in the fourth week of age (Tables 2, 3). All *Cryptosporidium* infections in goat kids were detected in animals with diarrhea. At the first week of age, *Cryptosporidium* infection was detected in 2/8 (25.0%) diarrheic kids, with *C. xiaoi* as the only *Cryptosporidium* species involved. At the second week of age, *Cryptosporidium* was observed in 5/9 (55.6%) diarrheic animals, including *C. xiaoi* (3/5) and *C. ubiquitum* (2/5). At the third week of age, *Cryptosporidium* was present in 1/4 (25.0%) diarrheic ones, with the species being diagnosed as *C. xiaoi*. The occurrence of cryptosporidiosis in diarrheic goat kids (34.8%) was statistically higher compared to that in non-diarrheic (0.0%) ones (P = 0.0000001).

Among the 62 fecal samples collected from lambs, 13 (21.0%) were from diarrheic animals, including 4 in the first week, 6 in the second week, and 3 in the third week of age. *Cryptosporidium* was detected in 3/4 (75.0%), 4/6 (66.7%) and 1/3 (33.3%) of the diarrheic lambs in the first, second and third weeks, respectively (Tables 2, 3). Among them, *C. parvum* was detected in 1/3 and 2/4 of the *Cryptosporidium*-positive samples in the first and second week, respectively. *C. xiaoi* was found in diarrheic animals up to 3 weeks of age and in non-diarrheic ones after that (Table 2). Thus, the overall infection rate in lambs was 14.5%, with the infection rate in diarrheic ones reaching 61.5% (8/13), compared with 2.0% (1/49) in non-diarrheic ones (P = 0.0000003).

### Discussion

In the present study, the occurrence and genotype and subtype identity of *Cryptosporidium* spp. in goat kids and lambs in Algeria were examined. The overall infection rate of *Cryptosporidium* spp. was 11.0% (8.7% in goat kids and 14.5% in lambs). Previous studies reported *Cryptosporidium* infection rates of 5.1–82.0% in sheep and 7.1–93.0% in goats in industrialized nations [4, 10, 14, 16, 29–35]. Few comparable data are available from
<figure><img src="image_5.png" /></figure>

developing countries, but infection rates of 2.5–67.5 and 2.9–72.5% have been reported in sheep and goats, respectively, in Zambia, Egypt, China, Bangladesh, Iran, Argentina and México [11–13, 17, 36–41]. Variations in infection rates among studies could be attributed to the differences in animal age, diagnostic methods, sample sizes, animal management and climates.

In this study, three *Cryptosporidium* species were identified in small ruminants, including *C. parvum*, *C. xiaoi* and *C. ubiquitum*. In both goats and sheep *C. xiaoi* appeared to be the dominant species (6/8 in goats and 6/9 in sheep), with *C. ubiquitum* being detected only in two of the eight *Cryptosporidium*-positive goats and *C. parvum* in three of the nine *Cryptosporidium*-positive sheep. In concordance with this, *C. xiaoi* was detected as a dominant species in small ruminants in other African countries including Egypt [37] and Tanzania [42] as well as Asian countries such as Bangladesh [40] and China [11, 17, 41, 43]. Similarly, *C. xiaoi* was the major *Cryptosporidium* species in small ruminants in some developed countries such as France [34], Greece [18], Norway [44], Poland [14] and Australia [35, 45]. In the presence study, two types of *SSU* rDNA sequences were obtained from *C. xiaoi*, with sheep and goats having different types. Both sequence types, however, have been observed in both sheep and goats in previous studies based on BLAST analysis of GenBank sequences. As sheep and goat samples from the present study were collected from

<table><caption>**Table 3** Occurrence of *Cryptosporidium* spp. in lambs by age and diarrhea status</caption><thead><tr><th>Distribution</th><th>No. of samples</th><th>No. positive for <i>Cryptosporidium</i> (%)</th><th><i>Cryptosporidium</i> spp. (no. of samples)</th></tr></thead><tbody><tr><td>By age (days)</td><td></td><td></td><td></td></tr><tr><td>1–7</td><td>15</td><td>3 (20.0)</td><td><i>C. xiaoi</i> (2), <i>C. parvum</i> (1)</td></tr><tr><td>8–14</td><td>18</td><td>4 (22.2)</td><td><i>C. xiaoi</i> (2), <i>C. parvum</i> (2)</td></tr><tr><td>15–21</td><td>13</td><td>1 (7.7)</td><td><i>C. xiaoi</i> (1)</td></tr><tr><td>22–28</td><td>16</td><td>1 (6.3)</td><td><i>C. xiaoi</i> (1)</td></tr><tr><td>By age (days) and diarrhea status</td><td></td><td></td><td></td></tr><tr><td>1–7</td><td></td><td></td><td></td></tr><tr><td>Diarrheic</td><td>4</td><td>3 (75.0)</td><td><i>C. xiaoi</i> (2), <i>C. parvum</i> (1)</td></tr><tr><td>Non-diarrheic</td><td>11</td><td>0 (0)</td><td></td></tr><tr><td>8–14</td><td></td><td></td><td></td></tr><tr><td>Diarrheic</td><td>6</td><td>4 (66.7)</td><td><i>C. xiaoi</i> (2), <i>C. parvum</i> (2)</td></tr><tr><td>Non-diarrheic</td><td>12</td><td>0 (0)</td><td></td></tr><tr><td>15–21</td><td></td><td></td><td></td></tr><tr><td>Diarrheic</td><td>3</td><td>1 (33.3)</td><td><i>C. xiaoi</i> (1)</td></tr><tr><td>Non-diarrheic</td><td>10</td><td>0 (0)</td><td></td></tr><tr><td>22–28</td><td></td><td></td><td></td></tr><tr><td>Diarrheic</td><td>0</td><td>0 (0)</td><td></td></tr><tr><td>Non-diarrheic</td><td>16</td><td>1 (6.25)</td><td><i>C. xiaoi</i> (1)</td></tr><tr><td>Total</td><td>62</td><td>9 (14.5)</td><td><i>C. xiaoi</i> (6), <i>C. parvum</i> (3)</td></tr></tbody></table>
different areas, it is unclear whether the two types repre-
sent different types of *C. xiaoi* circulating in different
areas.

*Cryptosporidium parvum* was seen in three sheep
among the small number of animals examined. These
findings are in agreement with previous common find-
ings of the pathogen in sheep in European countries and
Australia [4, 10, 18, 31, 36, 46]. Among developing
countries, a small number of *C. parvum* infections have
been reported in goats and sheep from Asia, including
China [17, 47, 48], India [49], Jordan [50] and Turkey
[51]. Our results, however, are in contrast to those from
studies conducted in most African countries including
Egypt [37], Tunisia [52] and Ethiopia [53], where *C. par-
vum* has thus far not been reported in small ruminants.
*Cryptosporidium parvum* was also absent in sheep and
goats in other studies in China [38, 41].

The *C. parvum* identified in the study belonged to the IIaA13G2R1 subtype. Although IIaA13G2R1 subtype is not a common *C. parvum* subtype and has not reported previously in sheep, it was detected in some calves in Belgium and Algeria [54, 55], ponies in the USA [56], calves and goat kids in Turkey [51], and humans in Malaysia [57], indicating that it is likely a zoonotic pathogen in a broad range of areas. Similarly, the *C. ubi-quitum* in goats in the present study was subtyped as XIIa, a well-known subtype family in goats elsewhere, including Greece [18], China [17, 38] and Australia [45]. It is also commonly reported in sheep in many countries [26, 35, 41]. It is responsible for zoonotic *C. ubiquitum* infection in humans in industrialized countries, especially the UK [26].

In this study, *C. parvum*, *C. xiaoi* and *C. ubiquitum* in-
fections occurred mostly in animals younger than three
weeks. This agrees with observations in previous studies
[10, 17, 18, 34, 37–40, 47]. Currently, controversy exists
on the clinical significance of *C. ubiquitum* and *C. xiaoi*
[2]. In the present study, most *C. xiaoi* cases (11/12), the
two *C. ubiquitum* and the three *C. parvum* cases all had
diarrhea. *Cryptosporidium* infections in lambs and goat
kids have been associated with the occurrence of diar-
rhea in some studies [10, 16, 32, 34, 39, 40].
Case-control studies are needed to confirm the role of
*C. ubiquitum* and *C. xiaoi* in the induction of diarrhea
in infected animals.

Conclusions

Results of this study showed a relatively common occur-
rence of *C. xiaoi* in lambs and goat kids in association
with the occurrence of diarrhea. The additional presence
of zoonotic *C. parvum* and *C. ubiquitum* indicates that
cryptosporidiosis in small ruminants may have further
public health implications. More extensive molecular
epidemiological studies are needed to substantiate these

observations and to improve our understanding of the
epidemiology and public health significance of crypto-
sporidiosis in small ruminants in Algeria.

Acknowledgements

We thank Laathamna Abdelkarim of the University Ziane Achor of Djelfa for statistical analysis. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Funding

This work was supported in part by the National Natural Science Foundation of China (31425025).

Availability of data and materials

The data supporting the conclusions of this article are included within the
article. Representative sequences generated in the study were submitted to
GenBank under the accession numbers JX412917 and LC414387-LC414394.

Authors' contributions

DB, AH, DK, YF and LX conceived the study. DB, AH, HA, SA, KA, HD, XC and DR conducted the experiments. DB, AH, SA and LX analyzed the data; DB, SA, AH and LX prepared the report. All authors read and approved the final manuscript.

Ethics approval

The procedures used in this investigation comply fully with ethics regulations in Algeria. The study protocol was approved by ethics committee of Ecole Nationale Supérieure Vétérinaire, Algiers, Algeria (PROJET CNEPRU/ CODE: F02620130033).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author details

¹École Nationale Supérieure Vétérinaire, Rue Issaad Abbes, El Alia, Alger, Algérie. ²Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329, USA. ³Laboratoire exploration et valorisation des écosystèmes steppique, Université Ziane Achor, 17000 Djelfa, Algérie. ⁴Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia. ⁵Department of Zoology, Faculty of Science, Kafr El Sheikh University, Kafr El Sheikh 33516, Egypt. ⁶UMR-BIPAR, ANSES-Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, Paris, France. ⁷Université Saad Dahleb Blida, Blida, Algérie. ⁸Beijing Tropical Medicine Research Institute, Beijing Friendship Hospital, Beijing 100050, China. ⁹Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.

Received: 9 August 2018 Accepted: 25 October 2018

Published online: 06 November 2018

References

1. Ryan U, Fayer R, Xiao L. *Cryptosporidium* species in humans and animals: current understanding and research needs. *Parasitology*. 2014;141:1667–85.

2. Xiao L. Molecular epidemiology of cryptosporidiosis: an update. *Exp Parasitol*. 2010;124:80–9.

3. Santin M. Clinical and subclinical infections with *Cryptosporidium* in animals. *N Z Vet J*. 2013;6:1–10.

4. Quilez J, Torres E, Chalmers R, Hadfield S, Del Cacho E, Sánchez-Acedo C. *Cryptosporidium* genotypes and subtypes in lambs and goat kids in Spain. *Appl Environ Microbiol*. 2008;74:6026–31.
5. Robertson L. Giardia and *Cryptosporidium* infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination. *Epidemiol Infect*. 2009;137:913-21.

6. Kotkova M, Nemejk C, Sak B, Hanzal V, Kvetonova D, Hlaskova L, et al. *Cryptosporidium ubiquitum*, *C. muris* and *Cryptosporidium* deer genotype in wild cervids and caprines in the Czech Republic. *Folia Parasitol (Praha)*. 2016;63:3.

7. Olson M, Ralston B, O'Handley R, Guselle N, Appelbee A. What is the clinical and zoonotic significance of cryptosporidiosis in domestic animals and wildlife. In: Thompson RC, Armson A, Ryan UM, editors. *Cryptosporidium: From Molecules to Disease*. Amsterdam: Elsevier B.V; 2003.

8. Noordeen F, Rajapakse R, Horadagoda N, Abdul-Careem M. *Cryptosporidium*, an important enteric pathogen in goats - a review. *Small Rumin Res*. 2012; 106:77-82.

9. Paraud C, Chartier C. Cryptosporidiosis in small ruminants. Small Rumin Res. 2012;103:93-7.

10. Díaz P, Quilez J, Prieto A, Navarro E, Pérez-Creo A, Fernández G, et al. *Cryptosporidium* species and subtype analysis in diarrhoeic preweaned lambs and goat kids from north-western Spain. *Parasitol Res*. 2015;114: 4099-105.

11. Peng XQ, Tian GR, Ren GJ, Yu ZQ, Lok JB, Zhang LX, et al. Infection rate of *Giardia duodenalis*, *Cryptosporidium* spp. and *Enterocytozoon bieneusi* in cashmere, dairy and meat goats in China. *Infect Genet Evol*. 2016;41:26-31.

12. Romero-Salas D, Alvarado-Esquivel C, Cruz-Romero A, Aguilar-Dominguez M, Ibarra-Priego N, Merino-Charrez JO, et al. Prevalence of *Cryptosporidium* in small ruminants from Veracruz, Mexico. BMC Vet Res. 2016;12:14.

13. Shafieyan H, Alborzi A, Hamidinejat H, Tabandeh M, Hajikolaei M. Prevalence of *Cryptosporidium* spp. in ruminants of Lorestan province, Iran. J Parasit Dis. 2016;40:1165-9.

14. Kaupke A, Michalski M, Rzeżutka A. Diversity of *Cryptosporidium* species occurring in sheep and goat breeds reared in Poland. *Parasitol Res*. 2017; 116:871-9.

15. Giles M, Chalmers R, Pritchard G, Elwin K, Mueller-Doblies D, Clifton-Hadley F. *Cryptosporidium hominis* in a goat and a sheep in the UK. *Vet Rec*. 2009; 164:24-5.

16. Díaz P, Quilez J, Robinson G, Chalmers R, Díez-Baños P, Morrondo P. Identification of *Cryptosporidium xiaoi* in diarrhoeic goat kids (*Capra hircus*) in Spain. *Vet Parasitol*. 2010;172:132-4.

17. Mi R, Wang X, Huang Y, Zhou P, Liu Y, Chen Y, et al. Prevalence and molecular characterization of *Cryptosporidium* in goats across four provincial level areas in China. PLoS One. 2014:e111164.

18. Tzanidakis N, Sotiraki S, Claerebout E, Ehsan A, Voutzourakis N, Kostopoulou D, et al. Occurrence and molecular characterization of *Giardia duodenalis* and *Cryptosporidium* spp. in sheep and goats reared under dairy husbandry systems in Greece. *Parasite*. 2014;21:45.

19. Xiao L, Feng Y. Molecular epidemiologic tools for waterborne pathogens *Cryptosporidium* spp. and *Giardia duodenalis*. Food Waterborne Parasitol. 2017;8:9-14-32.

20. Baroudi D, Khelef D, Goucem R, Adjou K, Adamu H, Zhang H, Xiao L. Common occurrence of zoonotic pathogen *Cryptosporidium meleagris* in broiler chickens and turkeys in Algeria. *Vet Parasitol*. 2013;196:334-40.

21. Laatamna AE, Wagnerová P, Sak B, Květoňová D, Xiao L, Rost M, et al. Microsporidia and *Cryptosporidium* in horses and donkeys in Algeria: detection of a novel *Cryptosporidium hominis* subtype family (Ik) in a horse. *Vet Parasitol*. 2015;208:135-42.

22. Baroudi D, Khelef D, Hakem A, Abdelaziz A, Chen X, Lysen C, et al. Molecular characterization of zoonotic pathogens *Cryptosporidium* spp., *Giardia duodenalis* and *Enterocytozoon bieneusi* in calves in Algeria. *Vet Parasitol* Reg Stud Rep. 2017;8:66-9.

23. Baroudi D, Zhang H, Amer S, Khelef D, Roellig DM, Wang Y, et al. Divergent *Cryptosporidium parvum* subtype and *Enterocytozoon bieneusi* genotypes in dromedary camels in Algeria. *Parasitol Res*. 2018;117:905-10.

24. Feng Y, Ortega Y, He G, Das P, Xu M, Zhang X, et al. Wide geographic distribution of *Cryptosporidium bovis* and the deer like genotype in bovines. *Vet Parasitol*. 2007;144:1-9.

25. Alves M, Xiao L, Sulaiman I, Lal A, Matos O, Antunes F. Subgenotype analysis of *Cryptosporidium* isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol. 2003;41:2744-7.

26. Li N, Xiao L, Alderisio K, Elwin K, Cebelinski E, Chalmers R, et al. Subtyping *Cryptosporidium ubiquitum*, a zoonotic pathogen emerging in humans. Emerg Infect Dis. 2014;20:217-4.

27. Fayer R, Santin M. *Cryptosporidium xiaoi* n. sp. (Apicomplexa: *Cryptosporidiiidae*) in sheep (*Ovis aries*). *Vet Parasitol*. 2009;146:192-200.

28. da Silva AJ, Caccio S, Williams C, Won KY, Nace EK, Whittier C, et al. Molecular and morphologic characterization of a *Cryptosporidium* genotype identified in lemuris. *Vet Parasitol*. 2003;111:297-307.

29. Causapé AC, Quilez J, Sánchez-Acedo C, del Cacho E, López-Bernad F. Prevalence and analysis of potential risk factors for *Cryptosporidium parvum* infection in lambs in Zaragoza (northeastern Spain). *Vet Parasitol*. 2002;104: 287-98.

30. Santín M, Trou J, Fayer R. Prevalence and molecular characterization of *Cryptosporidium* and *Giardia* species and genotypes in sheep in Maryland. *Vet Parasitol*. 2007;146:17-24.

31. Mueller-Doblies D, Giles M, Elwin K, Smith RP, Clifton-Hadley FA, Chalmers R. Distribution of *Cryptosporidium* species in sheep in the UK. *Vet Parasitol*. 2008;154:214-9.

32. Imre K, Luca C, Costache M, Sala C, Morar A, Morariu S, et al. Zoonotic *Cryptosporidium parvum* in Romanian newborn lambs (*Ovis aries*). *Vet Parasitol*. 2013;191:119-22.

33. Cacciò SM, Sannella AR, Mariano V, Valentini S, Berti F, Tosini F. Pozio E. A rare *Cryptosporidium parvum* genotype associated with infection of lambs and zoonotic transmission in Italy. *Vet Parasitol*. 2013;191:128-31.

34. Rieux A, Paraud C, Pors I, Chartier C. Molecular characterization of *Cryptosporidium* spp. in pre-weaned kids in a dairy goat farm in western France. *Vet Parasitol*. 2013;192:268-72.

35. Yang R, Jacobson C, Gardner G, Carmichael I, Campbell AJ, Ng-Hublin J, Ryan U. Longitudinal prevalence, oocyst shedding and molecular characterization of *Cryptosporidium* species in sheep across four states in Australia. *Vet Parasitol*. 2014;200:50-8.

36. Goma FY, Geurden T, Siwila J, Phiri I, Gabriel S, Claerebout E, Vercruysse J. The prevalence and molecular characterization of *Cryptosporidium* spp. in small ruminants in Zambia. Small Rumin Res. 2007;72:77-80.

37. Mahfouz M, Mira N, Amer S. Prevalence and genotyping of *Cryptosporidium* spp. in farm animals in Egypt. *J Vet Med Sci*. 2014;76:1569-75.

38. Wang R, Li G, Cui B, Huang J, Cui Z, Zhang S, et al. Prevalence molecular characterization and zoonotic potential of *Cryptosporidium* spp. in goats in Henan and Chongqing, China. *Exp Parasitol*. 2014;142:11-6.

39. Zucatto A, Aquino M, Inácio S, Figueiredo R, Pierucci J, Perri S, et al. Molecular characterization of *Cryptosporidium* spp. in lambs in the South Central region of the State of São Paulo. Arq Bras Med Vet Zootec. 2015;67: 441-6.

40. Siddiki A, Mina S, Farzana Z, Ayesa B, Das R, Hossain M. Molecular characterization of *Cryptosporidium xiaoi* in goat kids in Bangladesh by nested PCR amplification of 18S rRNA gene. Asian Pac J Trop Biomed. 2015; 5:202-7.

41. Li P, Cai J, Cai M, Wu W, Li C, Lei M, et al. Distribution of *Cryptosporidium* species in Tibetan sheep and yaks in Qinghai, China. *Vet Parasitol*. 2016;215: 58-62.

42. Parsons MB, Travis D, Lonsdorf EV, Lipende I, Roellig DM, Collins A, et al. Epidemiology and molecular characterization of *Cryptosporidium* spp. in humans, wild primates, and domesticated animals in the Greater Gombe Ecosystem, Tanzania. PLoS Negl Trop Dis. 2015;9:e0003529.

43. Wang Y, Feng Y, Cui B, Jian F, Ning C, Wang R, Zhang L, Xiao L. Cervine genotype is the major *Cryptosporidium* genotype in sheep in China. *Parasitol Res*. 2010;106:341-7.

44. Robertson L, Björkman C, Axén C, Fayer R. Cryptosporidiosis in farmed animals. In: Cacciò SM, Widmer G, editors. *Cryptosporidium*: Parasite and Disease. Vienna: Springer; 2014. p. 149-236.

45. Al-Habsi K, Yang R, Williams A, Miller D, Ryan U, Jacobson C. Zoonotic *Cryptosporidium* and *Giardia* shedding by captured rangeland goats. *Vet Parasitol* Reg Stud Rep. 2017;7:32-5.

46. Drumo R, Widmer G, Morrison LJ, Tait A, Grelloni V, D'Avino N, et al. Evidence of host-associated populations of *Cryptosporidium parvum* in Italy. *Appl Environ Microbiol*. 2012;78:3523-9.

47. Ye J, Xiao L, Wang Y, Wang L, Amer S, Roellig DM, et al. Periparturient transmission of *Cryptosporidium xiaoi* from ewes to lambs. *Vet Parasitol*. 2013;197:627-33.

48. Mi R, Wang X, Huang Y, Mu G, Zhang Y, Jia H, et al. Sheep as a potential source of cryptosporidiosis in China. *Appl Environ Microbiol*. 2018;84:17.

49. Maurya PS, Rakesh RL, Pradeep B, Kumar S, Kundu K, Garg R, et al. Prevalence and risk factors associated with *Cryptosporidium* spp. infection in young domestic livestock in India. *Trop Anim Health Prod*. 2013;45:941-6.
50. Hijjawi N, Mukbel R, Yang R, Ryan U. Genetic characterization of *Cryptosporidium* in animal and human isolates from Jordan. Vet Parasitol. 2016;228:116-20.

51. Taylan-Ozkan A, Yasa-Duru S, Usluca S, Lysen C, Ye J, Roellig DM, et al. *Cryptosporidium* species and *Cryptosporidium parvum* subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey. Exp Parasitol. 2016;170:16-20.

52. Soltane R, Guyot K, Dei-Cas E, Ayadi A. Prevalence of *Cryptosporidium* spp. (Eucoccidiorida: Cryptosporiidae) in seven species of farm animals in Tunisia. Parasite. 2007;14:335-8.

53. Wegayehu T, Karim MR, Li J, Adamu H, Erko B, Zhang L, Tilahun G. Prevalence and genetic characterization of *Cryptosporidium* species and *Giardia duodenalis* in lambs in Oromia Special Zone, Central Ethiopia. BMC Vet Res. 2017;13:22.

54. Geurden T, Berkvens D, Martens C, Casaert S, Vercruysse J, Claerebout E. Molecular epidemiology with subtype analysis of *Cryptosporidium* in calves in Belgium. Parasitology. 2007;134:1981-7.

55. 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.

56. Wagnerová P, Sak B, McEvoy J, Rost M, Sherwood D, Holcomb K, Kváč M. *Cryptosporidium parvum* and *Enterocytozoon bieneusi* in American Mustangs and Chincoteague ponies. Exp Parasitol. 2016;162:24-7.

57. Iqbal A, Lim YA, Surin J, Sim BL. High diversity of *Cryptosporidium* subgenotypes identified in Malaysian HIV/AIDS individuals targeting gp60 gene. PLoS One. 2012;7:e31139.

Ready to submit your research? Choose BMC and benefit from:

* fast, convenient online submission

* thorough peer review by experienced researchers in your field

* rapid publication on acceptance

* support for research data, including large and complex data types

* gold Open Access which fosters wider collaboration and increased citations

* maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions