Introduction

Microsporidia are a diverse group of zoonotic pathogens parasitizing invertebrates (insects) and vertebrates (fish, birds and mammals) [1]. Enterocytozoon bieneusi and Encephalitozoon spp. (i.e., Enc. intestinalis, Enc. hellem, and Enc. cuniculi) are two well-known genera among microsporidian species [2], with E. bieneusi being responsible for over 90% of animal and human cases [3]. A distinctive stage in the microsporidian life cycle is the formation of infective spores, which potentially contaminate the environment including water supplies and foodstuff [4,5,6]. Clinical infection is frequently eminent in immunocompromised patients, manifesting as malabsorption with subsequent chronic diarrhea as well as wasting diathesis [7, 8]. Additionally, microsporidian infections in immunocompetent subjects are asymptomatic but important, since these individuals are carriers of infective spores as a significant epidemiological concern [7]. Previously, the global prevalence of microsporidia infections was estimated among HIV-positive patients, rendering a 11.8% (95% CI: 10.1–13.4%) pooled prevalence [9]. A considerably high total prevalence of microsporidia infection was, also, calculated among cat populations worldwide [29.7% (95% CI: 19.7–42.2%)] [10], rather than in dogs [23.1% (95% CI: 13.5–36.8%)] [11]. As mentioned previously, E. bieneusi is the most prevalent genus among other microsporidian species, which demands molecular approaches to be exactly identified and genotyped [12]. Molecular techniques based on the variations in the nucleotide sequence of the internal transcribed spacer (ITS) region of the rRNA gene are mostly preferred for the identification of E. bieneusi genotypes [12]. Until now, over 200 distinct genotypes of E. bieneusi have been identified in humans, animals or both [13]. Small ruminants (sheep and goat) contribute a major role in the production of various dairy products worldwide [14, 15]. Diarrhea is a common intestinal sequela of microsporidian infections in small ruminants, causing considerable mortality and production loss [5, 16]. As well, there are some reports of raw milk contamination by microsporidian agents in sheep and goats [5, 17]. However, little is known on the molecular prevalence and genotype distribution of microsporidia, particularly E. bieneusi genotypes, in small ruminants. Thereby, the present systematic review and meta-analysis was done to evaluate the molecular prevalence of microsporidian infections with emphasis on Enterocytozoon bieneusi genotypes among sheep and goats at a global scale.

Methods

Information sources and systematic search

The present systematic review and meta-analysis was performed based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement [18]. Four international databases (PubMed, Scopus, Web of Science and Google Scholar) were excavated to gather relevant records on the molecular prevalence of microsporidia infection in sheep and goats published between 1 January 2000 and 15 April 2021. The search process was accomplished using MeSH terms alone or in combination: (Microsporidium” OR “Microsporidia” OR “Microspora” OR “Enterocytozoon bieneusi” OR “Encephalitozoon spp.”) AND (“Prevalence” OR “Epidemiology”) AND ("Small Ruminant" OR "Sheep” OR “Goat”). In addition, the bibliographic list of initially found papers was manually searched to find other relevant citations.

Inclusion criteria, study selection and data extraction

The inclusion criteria for the present systematic review were as follows: (1) abstracts and/or full-texts published in English language; (2) cross-sectional original papers or short reports estimating the molecular prevalence of microsporidia infection in sheep and goats; (3) utilization of different molecular methods; (4) papers providing total sample size and positive samples; and (5) published online from 1 January 2000 until 15 April 2021. Two independent reviewers evaluated the articles based on determined inclusion criteria and possible contradictions in cases of study selection or extraction procedure were obviated by discussion and consensus. Also, those articles on microsporidia infection in humans or other animals, studies that used non-molecular diagnostics, experimental investigations in small ruminants, as well as review papers, cohort, case-reports, case series, and editorials were all excluded. In the following, a set of required information, including first author’s last name; year of publication; continent; country; small ruminant species (sheep or goats); number of examined animals; number of animals with a positive test result, age, gender, molecular methods, identified parasite species and gene targets were precisely extracted.

Study quality assessment

The Joanna Briggs Institute (JBI) checklist is a qualitative index for inclusion of articles [19], providing ten questions with four options including, Yes, No, Unclear, and Not applicable. Briefly, a study can be awarded a maximum of one star for each numbered item. Those papers with a total score of 4–6 and 7–10 points were assigned as moderate and high quality, respectively.

Meta-analysis

The comprehensive meta-analysis Bio stat v2.2 software was employed for meta-analysis procedure [10, 11, 20]. Calculation of the pooled prevalence of microsporidia infection among small ruminants and 95% confidence intervals (CIs) was done using random-effects model (REM), which enhances the distribution of true effect sizes among studies [21, 22]. Subgroup analysis was, also, performed in order to reveal the weighted prevalence based on continent, country, and type of ruminants (sheep and goats). Moreover, the probable association of microsporidia prevalence with age and gender was determined using REM-based odd ratio (OR) estimation. The heterogeneity between studies was computed via I2 index and the Cochrane’s Q statistics [10, 11, 23]. Funnel plot was used to show the probability of publication bias [24]. Forest plot diagram was utilized to represent the pooled prevalence (with 95% CI) of microsporidia infection in sheep and goats.

Results

Following comprehensive systematic search (Fig. 1), 1715 records were initially retrieved, among which many duplicate/non-eligible articles were removed and only 25 papers were finally eligible to undergo meta-analysis [16, 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48]. Of note, 9 out of 25 studies possessed more than one dataset (Table 1), so that 34 datasets (20 datasets for sheep and 14 for goat) were reviewed and required data were extracted. Table 1 shows the results of the quality assessment based on the JBI checklist, rendering acceptable quality for all articles.

Fig. 1
figure 1

PRISMA flow diagram describing included/excluded studies

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Table 1 All the studies investigating the global prevalence of microsporidia species in sheep and goats according to molecular methods
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All datasets represented molecular characterization of microsporidia infections in small ruminants from 8 countries located at 4 continents, including Asia (26 datasets, 9925 animals), Europe (four datasets, 169 animals), Africa (three datasets, 212 animals) and America (one dataset, 125 animals) (Tables 1 and 2). China possessed the most published literature with 17 studies and 24 datasets. Most studies focused on E. bieneusi and only one study reported Enc. intestinalis in goats [34] (Table 1). In addition, one study out of the total study focused only on the detection of Enc. cuniculi in goats [30]. A relatively moderate weighted prevalence of microsporidia infection was obtained for both sheep 17.4% (95% CI: 11.8–25%) and goats 16% (95% CI: 11.2–22.4%) (Additional file 1: Figs. S1 and 2). Similar pooled prevalence rates were estimated for E. bieneusi in both sheep 17.4% (95% CI: 11.8–25%) (Fig. 2) and goats 16.3% (95% CI: 11.3–22.8%) (Fig. 3). The molecular determination of E. bieneusi genotypes was frequently accomplished using ITS gene, and genotypes BEB6 (15 studies) and COS-1 (nine studies) in sheep, and CHG3 (six studies) and BEB6 (five studies) in goats were the most prevalent among all other genotypes (Table 1). America and Asia continents showed the highest total prevalence rates with 19.2% (95% CI: 13.2–27.1%) and 17.6% (95% CI: 13.1–23.3%), respectively, followed by Europe 10.2% (95% CI: 1.4–48.3%), and Africa 8.7% (95% CI: 2.9–23.6%) (Table 2). It is noteworthy that Table 2 demonstrates data on country-based prevalence of microsporidia infection.

Table 2 Subgroup analysis of continents, countries and animal type (sheep and goats), based on molecular methods
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Fig. 2
figure 2

The pooled molecular prevalence of E. bieneusi infection in sheep

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Fig. 3
figure 3

The pooled molecular prevalence of E. bieneusi infection in goats

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A positive association was observed between microsporidia infection with age (≤ 3 months) (OR = 2.044; 95% CI, 1.35–3.093%) and male gender (OR = 3.169; 95% CI, 2.215–4.535%) (Table 3). The included studies had a significant publication bias based represented in the funnel plot (Additional file 1: Fig. S3 for sheep and Additional file 1: Fig. S4 for goats).

Table 3 Gender and age associated with microsporidia infection among sheep and goats worldwide
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Discussion

The health of animals and human are tightly interconnected within the environmental context, what is called as the One Health approach [49]. Domestic animals such as sheep and goats are in close contact with humans in rural areas and may contribute to some zoonotic pathogens including microsporidia infections [46]. Hence, a global evaluation of the pooled prevalence of microsporidia infections in small ruminants seems necessary.

The present systematic review and meta-analysis showed that microsporidia infection, with particular emphasis on E. bieneusi, is more prevalent in sheep (17.4%) than in goats (16.3%). Most microsporidia species are able to infect the gastrointestinal tract, while some species occupy the urinary tract, hence being found in urine samples. In this meta-analysis, only one study examined the molecular prevalence of Enc. cuniculi in urine samples, which was negative for all samples [30].

Although most studies used the nested PCR technique, some studies used the PCR and real-time SYBR green techniques. The most important advantage of nested PCR compared to the other two methods is that it could detect low amounts of microsporidia due to its high specificity [50, 51]. Moreover, nested PCR with the ITS gene is able to identify different E. bieneusi genotypes [51], whereas PCR with SSU rRNA gene fails to identify genotypes [52]. Genotyping of E. bieneusi using ITS gene sequence has been the most preferred and the gold standard method in recent decades, offering adequate information on pathogenicity and source of the organism [53]. Reportedly, BEB6, COS-1, and CHG3 of E. bieneusi have been the most prevalent genotypes among ruminants, in particular sheep and goats [39, 42, 53]. Of note, other less common zoonotic genotypes (Peru 6 and I), were also found in the present review, mostly isolated from humans and small ruminants [53]. This indicates to the possible environmental transmission of infective spores between humans and small ruminants. However, many samples from these animals and humans should be genotyped to endorse the zoonotic transmission of the genotypes.

China possessed the largest dataset (24 datasets) with a pooled prevalence rate of 17.9%, while only 7 other countries had reported microsporidia infection in sheep and goats. Still little is known regarding microsporidian infections in small ruminants in many countries worldwide, particularly in those nations having traditional animal husbandry system. As shown in Table 2, some key countries have few studies which implicates the need for further studies and more attention to sheep and goats microsporidiosis in these countries. It is noteworthy that information derived from the Europe (three studies), Africa (two studies), and America (one study) must be interpreted cautiously, because of paucity of studies (Table 2). There are several risk factors involved in the distribution of the microsporidian agents, including climatic variation, type of animal husbandry, parasite control measures, Human Development Index (HDI), etc. [11, 54]. Traditional animal husbandry systems facilitate the access of small ruminants to other domestic, wild and stray animals or close contact with environmental sources (e.g., consumption of spores contaminated water and food) [1, 7, 20, 51]. As such, different animals, water resources, and vegetables play a crucial role in maintaining the microsporidia cycle. Therefore, sheep and goats may be considered as a major reservoir of microsporidia, which subsequently may be responsible for the outbreaks of human microsporidiosis.

In the present meta-analysis, we found a higher microsporidia prevalence in ≤ 3 months and male animals, being statistically significant. Younger animals have immature and/or deficient immune status, hence they may be more susceptible to the microsporidia infection [26, 27], as substantiated by the higher prevalence in this review.

This systematic review and meta-analysis has some limitations and the results presented here should be interpreted with respect to these limitations, comprising lack of prevalence information in many countries; low sample size in some studies; and lack of risk factor (i.e., age and gender) and clinical symptoms (i.e., gastrointestinal disorders) assessment in most studies. Moreover, although this is a global meta-analysis on the molecular prevalence of microsporidia in sheep and goats, only eligible published studies were included, and it is possible that useful data were missed from the ‘grey’ literature. Also, online registration in PROSPERO failed, because data were already extracted. Considering these limitations, it is noteworthy to say that our results may be not precisely reflect the true prevalence, and the presented numbers are apparent prevalence rates. Nevertheless, it is believed what we had reported here is close to true microsporidia prevalence in sheep and goats within a global context.

Conclusion

This study showed a relatively high prevalence of microsporidia infection in sheep and goats worldwide, which could be directed towards better control and prevention of microsporidia infection in sheep and goats. Further, the findings of the present study should be taken into account by the health care authorities, physicians, veterinarians of the countries. The high-risk groups including immunocompromised patients must receive accurate and valid information about the risk of contact with the infected these ruminants. We suggest performing further studies to clarify the global prevalence of microsporidiosis based on molecular methods, which would be a guide to the establishment of appropriate public health interventions.