Introduction

Giardia duodenalis (synonyms G. intestinalis and G. lamblia) is a flagellated protozoan in humans and a wide range of animals (Thompson and Ash 2016). Giardiasis is associated with pathophysiological alterations in the intestinal mucosa (Carmena et al. 2012), resulting in the occurrence of diarrhea, malabsorption, and weight loss of the infected hosts (Aloisio et al. 2006; Vivancos et al. 2018). The health and economic impacts are especially severe in children and young farm animals (Fletcher et al. 2012; Einarsson et al. 2016). Transmission of the pathogen is through the fecal-oral route, mostly via direct contact with infected hosts and ingestion of contaminated food and water (Squire and Ryan 2017).

Livestock, especially cattle, have often been implicated as a major source of environmental contamination and potential reservoirs for zoonotic infection (Feng and Xiao 2011). The role of sheep and goats, however, is less clear (Robertson et al. 2010; Conan et al. 2017). The prevalence of G. duodenalis infection in sheep varied from 1.5 to 42% among studies conducted in different countries (Geurden et al. 2010; Feng and Xiao 2011; Tzanidakis et al. 2014; Wang et al. 2016; Wegayehu et al. 2017). Some reports have indicated that the prevalence is higher during pre-weaned period (less of 3 months) and starts to decline as the age of the animals increases (Ye et al. 2015). In contrast, other studies reported higher infection rates in post-weaned lambs and adults (Yang et al. 2014; Wang et al. 2016). Giardia cyst shedding has been associated with reduced growth, carcass weight, and dressing efficiency in sheep (Jacobson et al. 2016).

To date, eight major genotypes known as assemblages (A–H) have been identified in G. duodenalis, with assemblages A and B being recognized as major human pathogens (Feng and Xiao 2011). Molecular biologic characterizations of isolates have thus far indicated a dominance of assemblage E, with some occurrences of assemblages A and B, in sheep in most countries (Gómez-Muñoz et al. 2009; Sahraoui et al. 2019).

Algeria has one of the largest populations of sheep flocks in Africa, mainly concentrated in the steppe (Baroudi et al. 2018). However, there are scant data on the occurrence and identity of G. duodenalis in sheep in this arid country (Sahraoui et al. 2019). The present study aimed at determining the infection rate and distribution of G. duodenalis genotypes in sheep in Djelfa, the steppe of Algeria.

Materials and methods

Study area and specimen collection

This study was carried out during the period of May 2014 to June 2016. A total of 346 fecal specimens (149 from males and 168 from females) were collected from 28 extensive sheep farms (Table 1) in the region of Djelfa in central steppe of Algeria (Fig. 1). Specimens were collected directly from the rectum from young animals up to 6 months of age (197 under 3 months and 149 over 3 months) using gloved fingers. Data on the animals including age, sex, and diarrhea status (presence or absence of diarrhea) were recorded for each specimen at the site of collection. The specimens were transported in an isotherm box to the laboratory. They were screened microscopically using zinc sulfate flotation method as described by Bartlett et al. (1978). A direct immunofluorescence assays (DFA) (MeriFluor® Cryptosporidium/Giardia, Meridian Bioscience Europe, Milano, Italy) was used in the confirmatory diagnosis of G. duodenalis infection. Positive specimens were stored in 2.5% potassium dichromate at 4 °C prior to molecular analysis.

Table 1 Occurrence of Giardia duodenalis in lambs in Djelfa by farm
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Fig. 1
figure 1

Geographic location of Djelfa province in Algeria showing the sheep farms sampled in the study

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DNA extraction

All specimens positive for G. duodenalis by microscopy were washed of potassium dichromate with distilled water by centrifugation at 1000×g for 10 min. Genomic DNA was extracted from approximately 0.2 mL of the sediment using the FastDNA® Spin Kit for Soil (MP Biomedicals, Santa Ana, CA) following manufacturer’s guidelines.

PCR analysis of Giardia duodenalis

Giardia duodenalis in 24 microscopy-positive specimens was characterized by PCR and sequence analyses targeting the triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes, using established procedures (Sulaiman et al. 2003; Read et al. 2004).

Sequence and phylogenetic analyses

All positive secondary PCR products were purified using Montage PCR Cleanup Filters (Millipore, Bedford, MA) and directly sequenced in both directions using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on an ABI 3130 Genetic Analyzer (Applied Biosystems). The nucleotide sequences obtained were read and assembled using ChromasPro (http://technelysium.com.au/ChromasPro.html) and aligned with reference sequences of each target using ClustalX 2.1 (http://www.clustal.org/). The maximum likelihood (ML) implemented in MEGA7 (http://www.megasoftware.net/) with the general time reversible model was used in phylogenetic analysis. Bootstrap analysis with 1000 replicates was used to estimate support of the cluster formation. Unique nucleotide sequences generated in this study were deposited in GenBank under accession numbers MT321644 to MT321652.

Statistical analysis

The chi-square test was used in the comparison of infection rates between age groups, sex category, and the presence or absence of diarrhea. The analysis was conducted using XLSTAT 2014 (Microsoft®, WA, USA). Differences were considered significant at p ≤ 0.05.

Results

Prevalence of G. duodenalis by microscopy

Microscopic examination of the fecal specimens by the zinc sulfate flotation method revealed that 24/346 (7.0%) contained Giardia cysts, all of which were confirmed by direct immunofluorescence assay. Giardia cysts were detected on 9 of the 28 (32.1%) farms examined, with infection rates by farm varying from 0 to 42.9% (Table 1). Giardia infection rate was 6.7% (10/149) in specimens from males and 7.7% (13/168) in specimens from females (χ2 = 0.12; p = 0.72). In addition, the infection rate was 9.1% (18/197) in animals under 3 months of age, compared with 4.0% (6/149) in animals of over 3 months of age (χ2 = 3.14; p = 0.063). The G. duodenalis infection rate was significantly higher in lambs with diarrhea (7/44, 15.9%) than those without diarrhea (17/302, 5.6%) (χ2 = 6.28; p = 0.02).

Distribution of G. duodenalis assemblages

PCR analysis of 24 microscopy-positive specimens yielded the expected PCR products from 15 of the specimens. The failure in PCR amplification in nine microscopy-positive specimens could be due to low parasite burdens and long storage of the specimens prior to PCR analysis. Sequence analysis of the PCR products indicated the presence of both assemblages A and E. In tpi PCR, assemblage A was detected in 8 specimens and assemblage E in 7 specimens, whereas in gdh PCR, they were detected in 5 and 9 specimens, respectively. One specimen failed to yield the expected gdh PCR product. Among the PCR-positive specimens, two (42165 and 42166) were identified as having assemblage A at the tpi gene locus but assemblage E at the gdh locus (Table 2). Assemblage A was seen in younger animals (≤ 6 weeks) with diarrhea. In contrast, assemblage E was mostly seen in older asymptomatic animals (Table 2).

Table 2 Distribution of Giardia duodenalis assemblages and subtypes in lambs based on nucleotide sequence analysis of the triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes
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Phylogenetic relationship of G. duodenalis assemblages

At the tpi gene locus, the assemblage A sequences detected belonged to A1-like (n = 1) and A4 (n = 7). The sequence of subtype A1-like had an A to T substitution compared with JF792423 from sheep in Spain, while the subtype A4 had sequences identical to KR075934 from sheep in China. Furthermore, the assemblage E sequences detected showed complete identity to MK442913 from sheep in China.

At the gdh locus, the assemblage A sequences detected belonged to A1 (n = 5). The sequences of subtype A1 had 100% identity to reference sequence KR075940 from sheep in China. The assemblage E specimens generated three types of sequences. The first type included three sequences and had one difference from MK442909 from sheep in China. The second type included two sequences and had one nucleotide difference from MH621339 from goats in China. The third type of sequences included four sequences and had one nucleotide difference from MK561344 from sheep in Greece.

In phylogenetic analysis of the tpi sequences, the assemblage A sequences clustered with reference sequences of A4 from sheep (KR075934) and prairie dogs (KP780958 and KP780971) and humans (GQ329677) and one sequence clustered with A1 reference sequences from sheep (JF792423) and humans (GU564275 and HQ836660). The assemblage E sequences obtained clustered with reference sequences from cattle (KJ124917), sheep (MK442913 and KT922262), and pigs (MH644773) (Fig. 2).

Fig. 2
figure 2

Phylogenetic relationship among Giardia duodenalis genotypes from sheep in Algeria inferred based on sequences of the triosephosphate isomerase (tpi) gene using the maximum likelihood method implemented in MEGA7. Cluster formation support on the ML tree was calculated based on 1000 bootstrap replicates. Representative sequences obtained in this study are marked with diamonds. The sequence label includes the host animal followed by GenBank accession number and genotype identity

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In phylogenetic analysis of the gdh sequences, the five assemblage A sequences clustered with numerous A1 sequences from humans and animals in the GenBank database. In contrast, the assemblage E sequences were placed in two clusters containing reference sequences from sheep in different geographical areas (Fig. 3).

Fig. 3
figure 3

Phylogenetic relationships among Giardia duodenalis genotypes from sheep in Algeria inferred based on sequences of the glutamate dehydrogenase (gdh) gene using the maximum likelihood method implemented in MEGA7. Cluster formation support on the ML tree was calculated based on 1000 bootstrap replicates. Representative sequences obtained in this study are marked with diamonds. The sequence label includes the host animal followed by GenBank accession number and genotype identity

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Discussion

Results of the study have demonstrated the occurrence of G. duodenalis in sheep in Algeria. In the present study, the overall infection rate of G. duodenalis in lambs was 6.9%. Similar results have been reported in sheep in the USA (Santin et al. 2007), China (Zhang et al. 2012; Ye et al. 2015; Jin et al. 2017; Wu et al. 2018; Qi et al. 2019), Spain, Ethiopia, and Ghana (Diaz et al. 1996; Wegayehu et al. 2017; Squire et al. 2017). Younger animals and animals with diarrhea were shown in this study to have higher G. duodenalis infection rates. Previously, higher infection rates of G. duodenalis were reported in pre-weaned lambs in China, in Algeria, in Brazil, in Canada, and in Greece (Olson et al. 1997; Paz e Silva et al. 2014; Tzanidakis et al. 2014; Ye et al. 2015; Chen et al. 2019; Sahraoui et al. 2019). This could be due to the lack of acquired immunity in young animals. Similarly, previous studies had shown higher occurrence of G. duodenalis infections in lambs with diarrhea compared than in healthy animals (Olson et al. 1995; Skirnisson and Hansson 2006; Geurden et al. 2010). This is expected, as giardiasis is a known cause of outbreaks of diarrhea in pre-weaned lambs (Olson et al. 1997; Carmena et al. 2012).

Molecular analysis of G. duodenalis isolates in the present study indicated the occurrence of assemblages A and E, with almost an equal distribution. These results are in contradiction to the occurrence of assemblages E and D reported recently by Sahraoui et al. (2019) in lambs in Algeria. In most studies elsewhere, assemblage E has been reported as the dominant G. duodenalis genotype in sheep (Geurden et al. 2008; Ryan et al. 2005; Yang et al. 2009; Robertson et al. 2010; Jafari et al. 2014; Minetti et al. 2014; Paz e Silva et al. 2014; Tzanidakis et al. 2014; Wegayehu et al. 2017; Qi et al. 2019). However, a common occurrence of both assemblages A and E has been reported in sheep in China (Zhang et al. 2012; Liu et al. 2014; Ye et al. 2015; Wang et al. 2016) and some European countries (Geurden et al. 2008; Gómez-Muñoz et al. 2012). Of note, assemblage E is mainly found in cloven-hoofed domestic mammals (cattle, water buffaloes, sheep, goats, and pigs) (Feng and Xiao 2011). Recently, we have identified the occurrence of both assemblages A and E in cattle in Algeria, with assemblage A as the dominant genotype (Baroudi et al. 2017), indicating that the transmission of G. duodenalis in ruminants Algeria could be different from other areas. In this study, assemblage A was only found in pre-weaned lambs with diarrhea, while assemblage E was mostly found in post-weaned lambs without diarrhea. Thus far, as assemblage E is the dominant G. duodenalis genotype in lambs, there have been no reports of age and diarrhea-associated differences in the distribution of assemblages A and E in lambs.

The assemblages A and E of G. duodenalis identified in the study have zoonotic potential. The assemblage A subtype A4 at the tpi locus and A1 at the gdh gene locus was reported in humans previously (Feng and Xiao 2011). Although assemblage E is mostly a genotype in hoofed animals, it was detected in human specimens from Egypt (Foronda et al. 2008), Brazil (Fantinatti et al. 2016), and Australia (Zahedi et al. 2017). Therefore, assemblage E can potentially infect humans and threat public health.

In conclusion, G. duodenalis appears to be a common pathogen in lambs in Djelfa, Algeria. As both assemblages A and E have been identified as common G. duodenalis genotypes in lambs, this raises questions about the public health risk of acquiring G. duodenalis infection from sheep. More epidemiological studies are needed to confirm this hypothesis and to improve our understanding of the epizootiology and epidemiology of giardiasis in small ruminants in Algeria.