Global prevalence of Neospora caninum in rodents: A systematic review and meta‐analysis

Abstract Background Neosporosis has been considered a cause of abortion in dairy and beef cattle worldwide. Rodents are reservoir hosts for several infectious diseases. It is necessary to determine the prevalence of Neospora caninum in rodents to improve the current understanding of the transmission dynamics of Neospora as well as its life cycle and risk of transmission to livestock. Therefore, the objective of the present study was to estimate the pooled global prevalence of N. caninum in different rodent species. Methods Published studies on the prevalence of N. caninum in different rodent species were searched in the MEDLINE/PubMed, ScienceDirect, Web of Science, Scopus and Google Scholar and the reference lists of the retrieved articles until July 30, 2022. The eligible studies were selected using inclusion and exclusion criteria. The extracted data were verified and analysed using the random‐effect meta‐analysis. Result For this meta‐analysis, a total of 4372 rodents from 26 eligible studies were included. The global prevalence of N. caninum in rodents was estimated at 5% (95% CI 2%–9%), with the highest prevalence in Asia (12%; 95% CI 6%–24%) and lowest prevalence in America (3%; 95% CI 1%–14%) and Europe (3%; 95% CI 1%–6%). N. caninum was more prevalent in females (4%; 95% CI 2%–9%) than in males (3%; 95% CI 1%–11%). The most common diagnostic test was polymerase chain reaction (PCR) (21 studies). The pooled prevalence of N. caninum in rodents based on the diagnostic method was as follows: immunohistochemistry: 11% (95% CI 6%–20%), NAT: 5% (95% CI 4%–7%), IFAT: 5% (95% CI 2%–13%) and PCR: 3% (95% CI 1%–9%). Conclusion The results of this study showed a relatively low but widespread prevalence of N. caninum infection in rodents.


INTRODUCTION
Neosporosis caused by Neospora caninum (Apicomplexa, Sarcocystidae) is an infectious disease of domestic and wild animals worldwide (Nazari et al., 2023).N. caninum is similar to another known Apicomplexan parasite called Toxoplasma gondii, which is of importance in medicine and veterinary medicine (Nazari et al., 2018).Neosporosis has been considered a cause of abortion in dairy and beef cattle worldwide.This disease can also cause abortion or neonatal mortality in sheep and goats, leading to high financial burdens and enormous economic losses (Benavides et al., 2022).
Domestic dogs (Canis familiaris), Australian dingoes (Canis lupus dingo), coyotes (Canis latrans) and grey wolves (Canis lupus) are definitive hosts of N. caninum and excrete oocysts in their feces.A wide range of domestic and wild animals as intermediate hosts become infected by ingesting food or water contaminated with sporulated oocysts (Marugan-Hernandez, 2017).However, host susceptibility to parasite is affected by many factors, such as the type of the host, mode of infection, physiological factors including age, sex, pregnancy and the parasite virulence (Fereig & Nishikawa, 2020).
The consumption of pray (rodents and other small mammals) that could contain N. caninum cysts is the main source of infection for definitive hosts.The rodents and other small animals may contribute to the spreading of N. caninum infection among domestic or wild dogs through the sylvatic cycle (Almería, 2013).The rodents (order Rodentia) are the largest order of living Mammalia, in the both number of species and the number of individuals.They have a short reproductive cycle and a high compatibility for living in various habitats.In addition, they act as reservoirs, carriers and hosts for several parasitic pathogens (Mohammadi et al., 2022).Various studies have suggested that rodents may play a role in the maintenance of the N. caninum life cycle and the dissemination of N. caninum in the environment (Benavides et al., 2022;Ferroglio et al., 2003Ferroglio et al., , 2005;;Hamidinejat et al., 2011).Although a high prevalence of N. caninum has been reported in a number of herbivores and carnivores (Anvari et al., 2020;Ying et al., 2022), limited data are available on the exposure of rodent to N. caninum.
Assessment the prevalence of N. caninum in rodent is necessary to improve current understanding of the transmission dynamics of Neospora and the life cycle and risk of transmission to livestock.Therefore, the objective of this study was to provide tangible evidence on the pooled global prevalence and risk factors of N. caninum in different rodent species using previously conducted studies.

Literature review and data sources
Relevant studies on the prevalence of N. caninum infection in rodent were searched through the MEDLINE/PubMed, ScienceDirect, Web of Science, Scopus and Google Scholar electronic databases.This metaanalysis study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (see PRISMA check-list in Additional file 1: Table S1).Two investigators (P.A. and M.A.) searched the relevant studies up to July 30, 2022.The search was limited to studies published in English language.

Search strategy and study selection
A combination of 'Neospora' , 'caninum' , 'neosporosis' , 'prevalence' , 'epidemiology' and 'rodent' was used as search terms.All search results were imported into the Endnote X9 software library, and all duplicates were removed using the same software.

Quality assessment
The quality of the included studies was assessed using The Joanna Briggs Institute (JBI) critical appraisal checklist for crosssectional/prevalence studies.This checklist evaluates six aspects of a study: sampling process, data analysis process and statistical methods, study settings, measurement tools and response rate.Each item is rated on a scale of 0 (No) to 1 (Yes).The studies that obtained a minimum score of 6 out of 9 were considered high quality and were included in the analysis.Quality assessment was independently done by two reviewers (Y.H. and A.B.).Discussion and consensus were used to resolve disagreements.In this study, all studies were included because their minimum score was 6.

Statistical analysis
Data were imported to the STATA software version 14 (version 14; Stata Corp).Random-effects meta-analysis model using logit transformation was used to estimate the pooled prevalence rates with 95% confidence intervals.Forest plots were used to visualize the heterogeneity among the included studies.The heterogeneity was determined using the τ 2 and Cochrane Q tests.Subgroup analysis was also performed to investigate the overall pooled prevalence of neosporosis in rodents based on the gender, diagnostic method, continent, published year, sample size and tissue type for polymerase chain reaction (PCR).A p value less than 0.05 was considered statistically significant.

Study characteristics
We identified a total of 3922 records following the initial search of databases; after removing duplicates and/or non-eligible papers, 26 articles were eligible to be included in this systematic review and meta-analysis (Figure 1).

DISCUSSION
Rodents can play an important epidemiological role in the spread of neosporosis because they are preyed by canids, which in turn shed resistant oocysts into the environment through their feces and spread the infection.In addition, they are found in almost every terrestrial habitat and can easily move between habitats and possibly spread the infection within different communities.Considering the veterinary and economic importance of neosporosis, we conducted the first systematic review and meta-analysis to evaluate the prevalence of N. caninum in rodents worldwide.Abbreviations: CI, confidence interval; PCR, polymerase chain reaction.a The prevalence of N. caninum using other diagnostic methods, such as PAS staining, histopathology, bioassay and parasitology was zero.
b The number of studies examining N. caninum in blood, spleen, kidney and lymph nodes was <2.
Based on the results of the present meta-analysis, 5% (95% CI 2%-9%) of rodents are infected with N. caninum across the world.This prevalence rate is lower than N. caninum seroprevalence in cattle (23%; 95% CI 19%-27%) (Ansari-Lari, 2021), domestic cats and wild felids (15%; 95% CI 10%-21%) (Nazari et al., 2023) and sheep (13%; 95% CI 10%-15%) (Morales et al., 2022).A possible explanation for the low prevalence found is that the most common diagnostic method used in the studies included in the present study was molecular tests.The molecular technique is rarely used in larger living mammals due to the ethical limitations of parasite isolation.The PCR Neospora suffers from lack of standardization and variable performance according to the laboratory.(Akya et al., 2020).Therefore, the overall estimate may not be a true estimate of the infection rate in rodents and should be interpreted with caution due to this unavoidable limitation.
The findings of this systematic review and meta-analysis indicated that Asia (12%) and Oceania (7%) had the highest prevalence estimates compared to America and Europe (3% both  et al., 2023;Yamage et al., 1996).Overall, in present systematic review and meta-analysis, NC-5 was used for detection of N. caninum DNA in 13 molecular studies.Beside, N. caninum DNA detection was investigated in different tissues, including the blood, liver, spleen, kidney, lymph node, brain, heart or skeletal muscle, whereas the parasite DNA was identified more frequently in the skeletal muscle sample (6%) compared to the brain, liver and heart tissue samples from the same animals (2%, 5%, 2%, respectively).On the contrary, some researchers believe that protozoan cysts and tachyzoites are most likely to be found in the brain and spinal cord, and intracellular tachyzoites can also be detected in myocytes and myocardial Purkinje fibres (Donahoe et al., 2015;Nishimura et al., 2013).However, it should be noted that in the present systematic review, the detection of the parasite DNA in the skeletal muscle was investigated in only two studies; therefore, the results should be interpreted with caution.
A key strength of this systematic review and meta-analysis is that it determined the pooled prevalence estimates of N. caninum among rodents across the world for the first time.Nevertheless, this metaanalysis had several limitations, including a small number of studies and also uneven distribution of eligible studies across different continents, lack of information about risk factors, and variations in diagnostic methods with different sensitivity and specificity indexes.Overall, the estimated prevalence rates should be interpreted considering these limitations.

CONCLUSION
The results of this study showed a relatively low but widespread preva-

F
Forest plot of the global prevalence of Neospora caninum in rodents.
lence of N. caninum infection in rodents.The findings of the present study provide a better picture of the epidemiology of Neospora among rodents and may be used to improve the existing knowledge of the role of these small mammals as intermediate hosts in the life cycle of Neospora.AUTHOR CONTRIBUTIONS Arezoo Bozorgomid and Yazdan Hamzavi designed the study.Mobin Ahmadi, Parvaneh Adimi, Sahab Falahi and Yazdan Hamzavi collected the data.Yahya Salimi conducted the statistical analyses.Arezoo Bozorgomid and Yazdan Hamzavi drafted the first version of the paper.All co-authors made a substantial contribution to interpreting data, critically revised the article and approved the final version, including the authorship list.
Important characteristics of the included studies (n = 26).
others methods.The DNA of N. caninum was either detected by PCR amplification of ITS1 (1 study), NC5 (13 studies), SAG3 (1 study) or both ITS1 and NC5 (2 studies) loci, in different tissues.All studies were considered eligible for the final meta-analysis.The references for the included studies and their detailed characteristics are listed in Table1.TA B L E 1Abbreviations: IHC, immunohistochemistry; PCR, polymerase chain reaction.a It is not a rodent.
Pooled prevalence of Neospora caninum in rodents in different subgroups.
TA B L E 2 (Correa-Castro et al., 2021;Mamaghaniid to the small number of studies from Oceania (two studies) and lack of any reports from Africa.A high prevalence of N. caninum in intermediate hosts is assumed to be associated with the presence of dogs.N. caninum only occurs where canids are present.Zanet et al.   andprimers and the organ of choice for detection of infection can influence the outcome of PCR(Rostamian et al., 2021).Thus, PCR results for the detection of cyst-forming coccidia in tissue samples should be interpreted with caution because of false negative results.Many PCR-based methods have been developed to target different genes or gene regions specific to N. caninum such as SAG3, 18S rDNA, 28S rDNA, ITS1 and Nc-5.The most popular gene target is the Nc-5 gene because it is highly specific and not found in the genome of Toxoplasmatinae subfamily, such as T. gondii, Hammondia spp., Sarcocystis spp.andBesnoitia besnoiti(Correa-Castro et al., 2021;Mamaghani (3%).PCR was the most common test for the diagnosis of N. caninum (21 studies).With respect to the applicability of PCR for the diagnosis of cyst-forming parasites in tissue specimens, it is worth noting that this approach has intrinsic difficulties for the diagnosis of this type of infection; for example, small amount of the parasite in the tissue, the target DNA