The global molecular epidemiology of microsporidia infection in sheep and goats with focus on Enterocytozoon bieneusi: a systematic review and meta-analysis

Background Microsporidia is a zoonotic pathogen with health consequences in immunocompromised patients. Small ruminants are a potential reservoir of microsporidia for humans in their vicinity. Hence, we aimed to evaluate the molecular prevalence of microsporidian infections with emphasis on Enterocytozoon bieneusi genotypes among sheep and goats at a global scale through systematic review and meta-analysis approach. Methods The standard protocol of preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were followed. Eligible prevalence studies on small ruminant microsporidiosis, published from 1 January 2000 until 15 April 2021 were gathered using systematic literature search in PubMed, Scopus, Web of Science and Google Scholar databases. Inclusion and exclusion criteria were applied. The point estimates and 95% confidence intervals were calculated using a random-effects model. The variance between studies (heterogeneity) was quantified by I2 index. Results In total, 25 articles (including 34 datasets) were included for final meta-analysis. The pooled molecular prevalence of microsporidia in sheep and goats was estimated to be 17.4% (95% CI: 11.8–25%) and 16% (95% CI: 11.2–22.4%), respectively. Likewise, the overall prevalence of E. bieneusi was estimated to be 17.4% (95% CI: 11.8–25%) for sheep and 16.3% (95% CI: 11.3–22.8%) for goats. According to internal transcribed spacer (ITS) gene analysis, E. bieneusi with genotypes BEB6 (15 studies) and COS-1 (nine studies) in sheep, and CHG3 (six studies) and BEB6 (five studies) in goats were the highest reported genotypes. Conclusion The present results highlight the role of sheep and goats as reservoir hosts for human-infecting microsporidia. Therefore, this global estimate could be beneficial on preventive and control measures. Supplementary Information The online version contains supplementary material available at 10.1186/s41182-021-00355-7.


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

Open Access
Tropical Medicine and Health *Correspondence: alitaghipor71@yahoo.com 1 Zoonoses Research Center, Jahrom University of Medical Sciences, Jahrom, Iran Full list of author information is available at the end of the article Page 2 of 12 Taghipour et al. Trop Med Health (2021) 49:66 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.

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 I 2 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.

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.
Additional file 1. Figure S1. The pooled molecular prevalence of microsporidia infection in sheep. Figure S2. The pooled molecular prevalence of microsporidia infection in goats. Figure S3. Publication bias using funnel plot. Publication bias in sheep datasets. Funnel plot displaying prevalence data for all included publications (n = 20). Each circle represents reported prevalence from one individual study. Please note wide value distribution outside the funneled area indicating significant publication bias. Figure S4. Publication bias using funnel plot. Publication bias in goat's datasets. Funnel plot displaying prevalence data for all included publications (n = 14). Each circle represents reported prevalence from one individual study. Please note wide value distribution outside the funneled area indicating significant publication bias.