Background

Cryptosporidium spp. are common enteric protozoa of humans and a wide range of animals [4,5,6]. 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, 7,8,9]. Considerably high infection rates have been reported in these animals in some areas [10,11,12,13,14].

Currently, over 30 Cryptosporidium species have been recognized based on morphological, biological and molecular characteristics (reviewed in [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 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).

Table 1 Cryptosporidium species in goat kids and lambs in Algiers, Boumerdes and Médea provinces, Algeria
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Table 2 Occurrence of Cryptosporidium spp. in goat kids by age and diarrhea status
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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.

Fig. 2
figure 2

Differentiation of Cryptosporidium ubiquitum (Lanes 1, 4), C. xiaoi (Lanes 2, 3, 5–8), and C. parvum (Lanes 9, 10) from lambs and goat kids by RFLP analysis of the SSU rDNA PCR products using restriction enzymes SspI (upper panel) and MboII (lower panel). Lane 11: positive control (C. baileyi); Lane M: 100 bp molecular markers. The extra MboII band in Lane 10 is due to the presence of a non-specific band in the PCR product

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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 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).

Table 3 Occurrence of Cryptosporidium spp. in lambs by age and diarrhea status
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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, 29,30,31,32,33,34,35]. Few comparable data are available from develo** 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 [11,12,13, 17, 36,37,38,39,40,41]. 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, 17, 37], Tunisia [52] and Ethiopia [53], where C. parvum 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. ubiquitum 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 infections occurred mostly in animals younger than three weeks. This agrees with observations in previous studies [10, 17, 18, 34, 37,38,39,40, 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 diarrhea 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 occurrence 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 cryptosporidiosis in small ruminants in Algeria.