Global epidemiology and species/genotype distribution of Cryptosporidium in camels: A systematic review and meta-analysis

This review analyzed reported data of Cryptosporidium prevalence in camels and the species/genotype distribution. Four databases (PubMed, Web of Science, Scopus, Google Scholar) were screened, and studies published by April 1, 2024, were included. Total estimates and 95% CIs were calculated using a random-effects model. The weighted prevalence of Cryptosporidium spp. in 7372 camels examined from 12 different countries was estimated at 13.8% with a 95% CI of 10.3–18.4%. The sensitivity analysis based on excluding the individual studies did not result in significant statistical changes in the final weighted prevalence. Subgroup prevalence of Cryptosporidium spp. in camels was analyzed by publication year, continent, WHO region, country, camel type, sample size, diagnostic method, age, and gender. A significant publication bias (P < 0.05) was reported in the present study. Limitations encountered in this study encompassed: insufficient study diversity, reliance on single study results, inadequate molecular and serological studies in comparison to microscopic studies, etc., all of which could impact the findings. The study identified eight Cryptosporidium spp. in camels: C. parvum, C. andersoni, C. bovis, C. muris, C. ratti, C. occultus, C. ubiquitum, and C. hominis. The first three species had pooled prevalence rates of 65.5%, 66%, and 19.2%, respectively. Each of the remaining five species was documented using a single dataset/study. Moreover, genotypes IIdA19G1, IIaA15G1R1, If-like-A15G2, IIdA15G1, IIaA15G2R1, IIaA17G2R1, and IIaA18G2R1 (C. parvum), genotype IV (C. ratti), genotype XIIa (C. ubiquitum), and genotype IkA19G1 (C. hominis) have been identified in camels globally. The findings suggest that camels can act as a source of infection for a variety of Cryptosporidium species/genotypes, and can therefore play a key role in disseminating this protozoan to humans and animals.


Introduction
With over 120 genotypes and 44 valid species, Cryptosporidium is a major public health concern due to its zoonotic nature (Ryan et al., 2021).In 2004, cryptosporidiosis was included in the World Health Organization's "Neglected Diseases Initiative," which covers diseases primarily affecting people in low-resource settings (Savioli et al., 2006).The association between immunocompromised individuals (AIDS/HIV) and cryptosporidiosis instances elevated Cryptosporidium to a prominent position as a common human infection.In an immunocompetent person, Cryptosporidium infection may not show any symptoms or may result in a transient diarrhea.However, Cryptosporidium can result in severe, persistent, and sometimes fatal diarrhea as well as acute malnourishment or wasting in immunocompromised people (Izadi et al., 2012;Utami et al., 2020).
In neonatal animals, cryptosporidiosis results in severe diarrhea.However, adult animals continue to be the primary source of infection and are typically asymptomatic carriers (Mosier and Oberst, 2000;Zhang et al., 2022).In recent years, there has been increased recognition of the role of camels as source of infection for Cryptosporidium spp., prompting a growing interest in understanding the prevalence, species/genotype distribution, and zoonotic potential of these parasites in camel populations.Given the close interaction between camels and humans in various parts of the world, the zoonotic potential of Cryptosporidium spp. in camels has significant implications for public health.Therefore, a comprehensive assessment of the global prevalence and species/genotype diversity of Cryptosporidium spp. in camels is essential for understanding the epidemiology of camel-associated cryptosporidiosis and for informing public health and veterinary interventions.This systematic review and meta-analysis aim to synthesize the available evidence on the prevalence and species/genotype distribution of Cryptosporidium spp. in camels, providing valuable insights into the global epidemiology of camel-associated cryptosporidiosis and its implications for zoonotic transmission.

Ethics approval
The present study was approved by the Ethics Committee of Ardabil University of Medical Sciences, Ardabil, Iran (approval no.IR.ARUMS.REC.1402.386).

Search strategy
In this study, the design, reporting, and interpretation of the data collected from published literature were conducted following the standard protocol of the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) checklist (Moher et al., 2015).Peer-reviewed published papers and abstracts on the prevalence of Cryptosporidium spp. in camels were identified through systematic searches in four international electronic databases (PubMed, Web of Science, Scopus, and Google Scholar).This search was conducted by two analysts, independently, without any time restrictions up to April 1, 2024.The search was conducted using Medical Subject Heading (MeSH) terms alone or in combination: ("Intestinal Parasites" OR "Parasitic Infections" OR "Cryptosporidium" OR "Cryptosporidium spp."OR "Cryptosporidiosis") AND ("Prevalence" OR "Epidemiology" OR "Frequency" OR "Occurrence") AND ("Subtype" OR "Genotype" OR "Genotyping") AND ("Ungulates" OR "Camelids" OR "Camels" OR "Animals").Additionally, the bibliographies of the original and review articles were thoroughly examined to identify other potential articles that were not retrieved during the database search.

Eligibility criteria, study selection, and data extraction
The eligibility evaluation process proceeded as follows: 1) initial screening using title and abstract, 2) eliminating duplicate records, 3) acquiring full text of relevant papers, and final eligibility verification.Subsequently, three analysts extracted essential information for the meta-analysis stage, which was then validated by two other analysts.Any discrepancies or disagreements were resolved through consensus and discussion with the project's principal investigator.The inclusion criteria for this study were: (1) the study population was restricted to camels, (2) all cross-sectional and epidemiological studies without language or geographical restrictions, (3) studies investigating Cryptosporidium spp. in camel feces using molecular, microscopic, and/or serological detection methods, (4) studies published until April 1, 2024, and (5) reporting total sample size and prevalence rates for Cryptosporidium spp.Articles that did not mention the prevalence of Cryptosporidium spp. in camels, studies on Cryptosporidium spp. in non-camel species, research on tissue and blood samples, experimental infections in camels, case studies, reviews, letters, and articles with unclear information were excluded from this study.Variables extracted from each record included: the first author's last name, study implementation time, publication year, WHO regions, countries where the study was done, camel types, age groups, genders, diagnostic methods, total sample sizes, infected samples, and Cryptosporidium prevalence rates.Using molecular data, we also assessed the global distribution of different Cryptosporidium species and genotypes isolated from camels.

Quality assessment
The quality of the articles was evaluated using the "Joanna Briggs Institute (JBI) critical appraisal checklist" (Institute, J.B, 2017).Papers scoring 4-6 and > 6 points were deemed moderately and highly qualified, respectively.Articles with ≤3 points were excluded from the systematic review.
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Data synthesis and meta-analysis
In this study, all statistical analyses were performed using the Comprehensive Meta-Analysis (CMA) v3 software.P-values <0.05 were deemed statistically significant.The random-effects model was utilized to evaluate the prevalence of Cryptosporidium spp. in camels by estimating pooled prevalence and 95% CIs (Asghari et al., 2023).Sub-group analysis was conducted to assess the weighted prevalence of infection in camels according to camel types, WHO regions, countries, publication years, continents, sample size, diagnostic methods, genders, and age groups.A forest plot diagram was created to display the pooled prevalence with 95% CIs.The funnel plot was used to assess publication bias in the analysis.Heterogeneity among studies was evaluated using the I 2 index, with values below 25%, 25-50%, and over 50% considered as low, moderate, and high heterogeneity, respectively (Mahdavi et al., 2021).Furthermore, sensitivity analysis was conducted to evaluate variations in the final weighted prevalence of Cryptosporidium infection after excluding individual studies.

Study selection
The four searched global databases yielded a total of 6127 initial records.After eliminating duplicates and conducting a final review of the remaining 4682 records, 53 articles were ultimately included.Additionally, a quality evaluation based on JBI criteria led to the exclusion of six more studies.Finally, 43 highly qualified papers with 43 datasets met the criteria for inclusion in the present study (Fig. 1).

Table 1
The main details of 43 articles about the occurrence of Cryptosporidium spp. in camels.These articles were screened as highly relevant and data extracted from them were used in this study and listed in the table.

Qualitative and quantitative characteristics of included papers
The key features of the articles included are outlined in Table 1.These studies span from 1996 to 2023 and analyzed 7372 camel fecal samples globally.A total of 25 studies/datasets were related to one-humped camels (Camelus dromedarius), five datasets were related to two-humped camels (Camelus bactrianus), and 13 datasets were related to unknown camels (Camel spp.).Geographically, the research was distributed as follows: 10 studies in Egypt, six in Iran, six in Iraq, six in Algeria, five in China, three in Saudi Arabia, two in Ethiopia, one in Azerbaijan, one in Kuwait, one in Nigeria, one in Tunisia, and one in the USA.The sample size ranged from 4 to 1097 camels examined.Out of 43 studies/datasets on Cryptosporidium infection in camels, 12 papers (12 datasets) detailed the species/ genotype distribution of this parasite.Among diagnostic methods, microscopy was predominantly used in most studies (27 datasets), with molecular and serological techniques accounting for 12 and four datasets, respectively.A total of 11 datasets mentioned the age group of the studied camels, while nine datasets specified the gender.The JBI checklist showed that 22 papers (22 datasets) had high quality (>6 points), while the other 21 articles (21 datasets) had moderate quality (4-6 points) (Supplementary Table 1).

Global prevalence of Cryptosporidium spp. infection in camels
The overall frequency of Cryptosporidium spp.infection in camels was estimated at 13.8% with a 95% CI of 10.3-18.4% (Fig. 2).Heterogeneity analysis indicated significant level of heterogeneity in this meta-analysis (Q = 854.1,I 2 = 95.1%,P = 0.000).

Sensitivity analysis
After performing the sensitivity analysis, removing specific camel-related datasets did not report any significant changes in the final frequency (Supplementary Fig. 10).

Global distribution of Cryptosporidium genotypes in camels
From three datasets, C. parvum genotypes IIdA19G1, IIaA15G1R1, If-like-A15G2, IIdA15G1, IIaA15G2R1, IIaA17G2R1, and IIaA18G2R1 have been documented in Egypt, China, and Algeria.In Egypt, genotype IV of C. ratti has been identified.In China, genotypes XIIa and IkA19G1 have been found in C. ubiquitum and C. hominis, respectively (Table 3).

Discussion
Evaluating Cryptosporidium infection in animals, particularly camels, is significant for several reasons.Firstly, Cryptosporidium is a parasite that can cause gastrointestinal illness in animals, leading to symptoms such as diarrhea, dehydration, and weight loss.By identifying and monitoring Cryptosporidium infection in animals, veterinarians and researchers can better understand the prevalence and impact of the parasite in different populations.Additionally, cryptosporidiosis is a zoonotic disease, meaning it can be transmitted from animals to humans.Camels are commonly used for milk production and as working animals in certain regions, so evaluating Cryptosporidium infection in camels is important for assessing the potential risk of transmission to humans.This information can help inform public health measures to prevent and control the spread of the parasite (Saleh and Mahran, 2007;Sazmand et al., 2012;Wang et al., 2021).
There has been no comprehensive review focusing on the prevalence, species/genotypes distribution, and zoonotic importance of Cryptosporidium in camels.Non-animal meta-analyses have indicated a global prevalence of Cryptosporidium in humans (Dong et al., 2020) and water reservoirs (Daraei et al., 2021) at 7.6% (95% CI: 6.9-8.5%) and 36% (95% CI: 31.4-40.7%),respectively.The reported prevalence of this parasitic infection is 8% (95% CI: 5-11%) in dogs (Taghipour et al., 2020a), 6% (95% CI: 4-8%) in cats (Taghipour et al., 2021), 17% (95% CI: 13-20%) in rodents (Taghipour et al., 2020b), 16.3% (95% CI: 15-17.6%) in pigs (Chen et al., 2023), and 7.6% (95% CI: 4.8-10.8%) in equines (Li et al., 2022).Our findings revealed that the global prevalence of cryptosporidiosis in camels [13.8% (95% CI: 10.3-18.4%)] is relatively high compared to animals like dogs, cats, and horses, but lower compared to rodents and pigs.It also indicated that camels could serve as a proper source of infection for Cryptosporidium infection, highlighting the importance of considering public health and zoonotic infection transmission.Discrepancies in reported prevalences across studies may be attributed to variations in study numbers, sample quality and sizes, animal species, geographical locations, animal husbandry practices, and diagnostic method sensitivity.The sensitivity analysis results showed that excluding any of the studies (datasets) on cryptosporidiosis in camels did not lead to significant statistical changes in the final weighted prevalence.This indicates that no outlier data is present in the studies analyzed in this review that would significantly affect the overall prevalence of Cryptosporidium spp. in camels.
A high rate of heterogeneity was identified as publication bias in this study, potentially impacting the outcomes (Thornton and Lee, 2000).This could be due to variations in geographical region, publication year, number of studies, and sample size as shown in Table 2. Other factors not addressed in this review, such as animal health status, sampling methods, sample preservation, and animal-rearing practices, could also contribute to publication bias.Therefore, the findings of this study should be interpreted carefully.Despite the valuable epidemiological data collected in our current study, future research could further illuminate the occurrence and distribution of species and epidemiological trends of Cryptosporidium infection in camels worldwide.

Conclusion
This review and meta-analysis study on Cryptosporidium spp. in camels revealed a moderate pooled prevalence (13.8%) of this protozoan infection.The findings indicate that camels can serve as a source of infection for range of Cryptosporidium species and genotypes, highlighting the need for preventive measures and medical and veterinary attention in areas with camels.Limitations encountered in this study encompassed: insufficient study diversity, reliance on single study results, inadequate molecular and serological studies in comparison to microscopic studies, etc., all of which could impact the findings.Therefore, it is advised to interpret the results of this study with caution.Extensive and detailed research is required to understand the epidemiology of Cryptosporidium and the distribution of its species/genotypes in camels.

Declaration of competing interest
The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Fig. 1 .
Fig. 1.Flowchart depicting the process of included studies in the present review.

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.Mahdavi et al.

Fig. 3 .
Fig. 3.The funnel plot shows the publication bias in the present study.

Table 1
(continued ) a UC: Unclear.bMicroscopicdetectionmethod and Ziehl-Neelsen staining.cPolymeraseChainReaction.dEnzyme-LinkedImmunosorbentAssay.e Multilocus Sequence Typing.fImmunochromatographicassay.gRestrictionFragmentLength Polymorphism.hIndirectFluorescentAntibody.F.Mahdavi et al.Fig.2.The overall prevalence of Cryptosporidium spp. in camels, based on data from the included studies, using a random-effects model and 95% confidence intervals.*Blue colors indicate the event rate/prevalence reported in each study, while the red color represents the final weighted prevalence.(Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)F.Mahdavi et al.

Table 2
Subgroup analysis of Cryptosporidium spp. in examined camels according to publication year, continent, WHO region, country, camel type, sample size, diagnostic method, age, and gender.

Table 3
Summary of the reported data on Cryptosporidium species and genotypes in camels.