Parasites of Free-Ranging and Captive American Primates: A Systematic Review

The diversity, spread, and evolution of parasites in non-human primates (NHPs) is a relevant issue for human public health as well as for NHPs conservation. Although previous reviews have recorded information on parasites in NHPs (Platyrrhines) in the Americas, the increasing number of recent studies has made these inventories far from complete. Here, we summarize information about parasites recently reported in Platyrrhines, attempting to build on earlier reviews and identify information gaps. A systematic literature search was conducted in PubMed, ISI Web of Science, and Latin American and Caribbean Health Sciences Literature (LILACS), and following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Ninety-three studies were included after the screening process. Records for 20 genera of NHPs, including 90 species were found. Most of the studies were conducted on captive individuals (54.1%), and morphological approaches were the most used for parasite identification. The most commonly collected biological samples were blood and stool, and Protozoa was the most frequent parasite group found. There is still scarce (if any) information on the parasites associated to several Platyrrhine species, especially for free-ranging populations. The use of molecular identification methods can provide important contributions to the field of NHPs parasitology in the near future. Finally, the identification of parasites in NHPs populations will continue to provide relevant information in the context of pervasive habitat loss and fragmentation that should influence both human public health and wildlife conservation strategies.


Introduction
Public health, animal welfare, and pathogen transfer to and from wild populations are among the current primary issues of concern in the framework of the One-Health concept. Such aspects are even more relevant in areas of the world such as South America, where biodiversity is declining at high rates and the rate of deforestation is growing. There is compelling evidence on how habitat loss and fragmentation may favor contact between humans and other animals, representing a potential threat for both [1]. In this scenario, non-human primates (NHPs) are of particular interest because of their close phylogenetic relationship with humans and their known role as reservoirs of zoonotic agents [2]. So far, six major groups of organisms have been found infecting NHPs: viruses, bacteria, fungi, protozoa, helminths, and arthropods [3]. For a series of multiple issues including behavioral ecology, public health, and NHPs conservation, it is important to

Materials and Methods
We carried out a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) to summarize information about parasites infecting American NHPs. The review protocol of this systematic review was not recorded into the International prospective register of systematics reviews (PROSPERO) (Supplementary File S2: Authors declaration and PRISMA checklist). We performed an independent search for each Platyrrhine genus, using the terms "parasite" and NHPs genus (e.g., parasite AND Cebus). The search was conducted in ISI Web of Knowledge and PubMed, including studies from June 2017 to 11 February 2021, thus collecting all the information published after the time frame used in the last available review regarding the subject [5]. Additionally, information from Latin American and Caribbean Health Sciences Literature (LILACS) was incorporated into the database, using the same search terms, until February 11th, 2021. In this way we collected information from a Latin American specific search engine, building on the review made by Solórzano-García and Pérez-Ponce de León [5].
We included studies performed in wild and captive Platyrrhines which reported parasite occurrence, while studies under laboratory conditions or the ones focused on fungi, bacteria, and viruses were not included. We used articles in English, Portuguese, or Spanish.
To organize the collected data, we considered specific taxonomy classifications: for Lagothrix, Saguinus, and Callicebus, we followed the classification proposed by Di Fiore et al. [15], Buckner et al. [16], and Byrne et al. [17], respectively. For all other NHPs genera, we followed the taxonomy of the "Handbook of the mammals of the world" [18]. Parasite taxonomy was included following the classification stated by the National Center for Biotechnology Information (NCBI).

Results
The literature review retrieved 720 searches: 444 from ISI Web of Knowledge, 214 from PubMed, and 62 from LILACS. Overall, we obtained 93 novel publications ( Figure 1) after eliminating duplicates, studies under laboratory conditions, studies already included in the review made by Solórzano-García & Pérez-Ponce de León [5]. Overall, the studies included in this review account for 20 Platyrrhine genera, including 90 species. The genus with most records was Alouatta (n = 51), while genera with the least records were Callimico (n = 1) and Cebuella (n = 1) ( Table 1). According to the parasite group, protozoa were overall the most reported along NHPs genera (Table 1). It was found that 54.1% studies were conducted on captive NHPs and 45.9% on free-ranging animals, while the source of biological sample and diagnostic method mostly used were blood and morphology, respectively (Table 2).   * Other: Serology, ELISA, indirect ELISA, indirect agglutination assays, Western blood IgG assays, immunochromatographic assays, sero K-SeT rapid diagnostic tests, indirect immunofluorescence assays, immunohistochemical assays, antigen-based rapid diagnostic tests, TESA-blot.

Discussion
The most recent list (2018-2020) of the World's 25 Most Endangered NHPs Species includes six Platyrrhines: Ateles geoffroyi, Cebus aequatorialis, Saguinus bicolor, Plecturocebus olallae, Alouatta guariba, and Callithrix aurita [112]. After the systematic review process, there were retrieved publications with parasitological data for A. geoffroyi (n = 4), S. bicolor (n = 4), A. guariba (n = 11), and C. aurita (n = 1), while there were no articles mentioning C. aequatorialis and P. olallae. Additionally, there were no records for Plecturocebus caquetensis, P. olallae, Leontopithecus caissara, and Callicebus barbarabrownae, listed in the IUCN Red List as Critically Endangered [6], neither for Cebus malitiosus, Saimiri vanzolinii, Callicebus coimbrai, Alouatta ululata, or Cebus cesarae, listed as Endangered [6]. The amount of information is probably biased by the availability of different species in captivity, a condition that strongly facilitate parasitological investigations. It can be speculated that the lack of information for endangered species could be related to their scarcity in captive conditions. Although other kinds of studies (e.g., behavioral, genetic) may have been carried out for those species during the time range considered in this study, it must be highlighted that parasitological studies are also very important, representing a useful insight for monitoring the health status of NHPs in contexts of human-NHPs interfaces, as human-induced forest loss increase the exposition of NHPs to human and domesticated animal pathogens [8]. Additionally, even if non-lethal parasite infections are common in wild NHPs, parasite infections could cause sickness behaviors that may be adaptative in the short-term but have longer-term fitness consequences [113]. Note that for some Critically Endangered and Endangered NHPs species which had no parasitological studies until 2017, data have been recorded between 2017 and 2021, as is the case of Cebus kaapori, Sapajus flavius, and Ateles marginatus. Moreover, even if there are reports for specific NHPs species, the observation is limited to a specific area implying that not all the geographic range of the species has been covered.
Overall, just over half of the studies were conducted on captive NHPs (54.1%), however, for the genera Alouatta, Cacajao, Callithrix, and Leontocebus there were more records on free-ranging NHPs. Studies in both free-ranging and captive NHPs are important, for instance, in the design of conservation strategies, reintroduction programs, and NHPs acquisition for research laboratories or zoos. Determining the composition of parasite communities in captive NHPs allows the identification of parasites of concern regarding the introduction of novel parasites to potentially susceptible wildlife populations during reintroduction programs, and also lead to a better understand parasite ecology, for instance, it has been observed that vector-borne parasites are more likely found in free-ranging NHPs, while parasites transmitted through either close and non-close contact, including the fecal-oral transmission, are more likely detected in captive NHPs [114].
Regarding the diagnostic method, morphological approaches were found to be the most used, followed by molecular procedures.
The most common biological samples were blood and stool, and ectoparasites corresponded to the least reported. Sampling NHPs, specially free-ranging, is logistically challenging as invasive sampling techniques such as the collection of blood, requires field anesthesia; therefore, optimization of non-invasive surveillance on NHPs is critical for understanding disease ecology of pathogens and identifying zoonotic diseases likely to emerge [115]. In this context, non-invasive methods such as stool collection are among the safest alternatives to study multiple aspects of the biology of NHPs [2]. However, even the collection of stool samples requires considerable efforts for their assignment to a specific individual, as well as to avoid multiple sampling for the same individual and later calculate the prevalence of parasites.
Parasitological surveys of NHPs contribute to the understanding of the epidemiology, zoonotic emergence risk and transmission dynamics [41]. In this context, parasitological studies using adequate tools to evaluate the zoonotic potential are necessary. In the present review, as most studies are based on parasite morphology, some parasite species and/or genetic variants could not be determined, thus not allowing to assess their zoonotic potential. In future studies, the use of molecular tools will become essential, not only to identify and determine the presence/absence of parasites, but also to identify species/variants of the parasites circulating in each NHPs species and in each sampling site in order to better understand their distribution in NHPs and to evaluate transmission dynamics. Although there are challenges related to the molecular processing of the samples (e.g., disruption of the Ascaris and Trichuris eggshells prior to DNA extraction), efforts should be made to develop efficient protocols especially in stool samples. These are considered reliable for the non-invasive detection of pathogens, opening up new possibilities in the molecular epidemiology and evolutionary analysis of infectious diseases [2].
As habitat loss and forest fragmentation are currently a concerning global trend, and NHPs are in closer contact with humans, consequent ecological changes need to be monitored. Forest fragmentation is one of the main factors threatening NHPs [8], affecting all but not only the six Neotropical species included into the World's 25 Most Endangered NHPs List [112]. However, only two studies included in the present review accounted for forest fragmentation as a variable during the analyses [25,45], even if some were performed in fragmented areas [67,81,85,91,105]. We strongly encourage the inclusion of this crucial factor as a variable for future studies as a way of better understanding parasite ecology, taking into account that some studies had reported a higher parasite prevalence in NHPs living in fragmented habitats [62,118], while other authors had found a lower presence of parasites [69], in comparison to prevalence found in continuous forests. Additionally, parasite taxa composition may vary according to NHPs living condition [118,119].
Not only more efforts aimed to broaden the knowledge of parasites infecting NHPs are required but we also suggest the standardization of the result presentation/display. For instance, it is necessary to include the coordinates of the sampling sites and show information separately for each NHPs species and study site when sampling is simultaneously carried out in different sites, involving more than one NHPs species. Therefore, the availability of the necessary information to perform meta-analysis, spatial analyses, and calculate parasite prevalence is facilitated, allowing to draw conclusions usable to a better understanding of infection patterns.

Conclusions
In the present review, parasitological records for 20 genera of NHPs mainly conducted on captive animals were retrieved. Morphological approaches were found to be the most used, and Protozoa was the most frequent parasite group reported. Parasitological studies on American NHPs still need to be performed, especially for some genera and species with several information gaps, as well as Critically Endangered and Endangered primates, in both free-ranging and captive conditions. Parasitological studies using adequate tools to evaluate potential zoonoses are necessary in order to better understand the distribution of parasites in NHPs and to evaluate transmission dynamics, also taking considering factors as habitat loss and forest fragmentation.  Acknowledgments: We thank the authors who have shared useful information or details for the development of the database generated in this review. We also thank the anonymous reviewers of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.