Vector-borne zoonotic blood parasites in wildlife from Ecuador: A report and systematic review.

BACKGROUND AND AIM: Ecuador is a hugely diverse country, but information on infectious diseases in local wild animals is scarce. The aim of this study was to screen the presence of blood parasites in free-ranging wild animals admitted to the Wildlife Hospital at Universidad San Francisco de Quito, from April 2012 to January 2019.
MATERIALS AND METHODS: We identified blood parasites by microscopic observation of blood smears from free-ranging wildlife species that attended the Wildlife Hospital of Universidad San Francisco de Quito (Ecuador) from April 2012 to January 2019.
RESULTS: The microscopic evaluations of animals as potential reservoirs for vector-borne zoonotic blood parasites revealed the presence of Anaplasma spp., Babesia spp., Ehrlichia spp., Hepatozoon spp., microfilaria, Mycoplasma spp., and Trypanosoma spp. in previously unreported wildlife species. In addition, we performed a systematic review to understand the current knowledge gaps in the context of these findings.
CONCLUSION: Our data contribute to the knowledge of blood parasites in wildlife from Ecuador. Furthermore, the potential transmission of these parasites to humans and domestic animals, current anthropogenic environmental changes in the region, and the lack of information on this suggest the importance of our results and warrant further investigations on infectious diseases in animals and humans and their relationship with environmental health as key domains of the One Health concept. Copyright: © Diaz, et al.

Introduction

From an ecological perspective, a parasite lives in association with another organism [1]. Parasites that live in the host’s blood are called blood parasites (hemoparasites), which include several species of the genera Anaplasma, Babesia, Ehrlichia, Haemoproteus, Hepatozoon, Leishmania, Leucocytozoon, Mycoplasma, Plasmodium, Schellackia, and Trypanosoma, among others [2-6]. Usually, hosts become infected through arthropod vectors such as fleas, flies, mosquitoes, or ticks [7]. Blood parasites are globally distributed and infect a wide variety of amphibians [8,9], reptiles [10], birds [11,12], and mammals, including humans [13].

Parasitism is considered by some scientists as a malevolent condition where parasites survive at some cost to host health status [14]. Some parasites have coevolved with their hosts, permitting prolonged interactions, and minimum parasite damage [15]. However, when these parasites are transmitted from their original hosts to humans (zoonotic transmission), the infection may produce damaging pathology, potentially leading to severe diseases and even death [16]. Thus, wildlife species are natural reservoirs of a wide range of zoonotic parasites and may serve as sentinel species for emerging diseases [8,17]. In this context, the One health paradigm gives the cornerstones of the relationship between wildlife and human health. This concept encompasses the provision of health for humans, animals, and environment through the interactions between all three pillars. To date, the One health triad application is being emphasized to control, prevent, and monitor infectious diseases[18]. Currently, increasing research efforts have been concentrated on selected blood parasites, mainly Leishmania spp., Plasmodium spp., and Trypanosoma spp. [7,18-21]. However, many vector-borne zoonotic hemoparasites from wildlife sources have been neglected; thus, information relating to their epidemiology and spillover related to human activity remains relatively uncharacterized [7,16-21].

In this study, we screened for blood parasites in blood smears from Ecuadorian free-ranging wildlife species admitted to the Wildlife Hospital at Universidad San Francisco de Quito, from April 2012 to January 2019. Furthermore, we complemented our findings with a systematic review of the literature investigating these parasites in wild animal species and their locations. The possible danger that the identified pathogens have in line with the One Health concept is also discussed, as some parasitic diseases are interconnected not only with animal health but also with human health through environmental relations [22].

Materials and Methods

Ethical approval

This study was conducted under the permit issued by Ecuador’s Ministry of Environment (019-2018-IC-FAU-DNB/MAE) and authorized by the Animal Ethics Committee of Universidad San Francisco de Quito USFQ (2018-011).

Study period and location

Laboratory results reported in this study were compiled in February 2019 from animals admitted from April 2012 to January 2019. A database search for the systematic review was performed in June 2020. The study was performed in the Wildlife Hospital of Universidad San Francisco de Quito (Quito, Ecuador).

Specimen selection/collection/identification

The laboratory results of blood samples belonged to free-ranging wildlife species from Ecuador were compiled. All animals were admitted from April 2012 to January 2019 to the Wildlife Hospital of Universidad San Francisco de Quito and sample collection was performed exclusively for infectious disease diagnostics. Samples were analyzed by Lab-Vet (permit Ref. No.: AGR-AGROCALIDAD/CDL-2018-000052-OF), using published and standard laboratory methods based on blood smear visualization by light microscopy using a 40X magnifying lenses.

Literature review

PubMed (https://www.ncbi.nlm.nih.gov/pubmed), Scopus (https://www.scopus.com), and Web of Science (https://www.webofknowledge.com) databases were accessed and searched for published reports that match to our findings, search was performed on June 16, 2020. Search terms allowed us to retrieve original studies reporting Anaplasma, Babesia, Ehrlichia, Hepatozoon, microfilaria, Mycoplasma, and Trypanosoma in wild animal species. Search terms were used in all three databases to match the following combination of terms: (1) Anaplasma: Anaplasma AND ((Lagothrix AND lagotricha) OR (Saguinus AND fuscicollis) OR (Cebus AND albifrons)); (2) Babesia: Babesia AND ((Mazama AND rufina) OR (Lagothrix AND lagotricha) OR (Saguinus AND fuscicollis)); (3) Ehrilichia: Ehrilichia AND (Mazama AND rufina); (4) Hepatozoon: Hepatozoon AND (Melanosuchus niger OR Caiman crocodilus OR Boa constrictor); (5) Microfilaria: Microfilaria AND (M. niger OR Lagothrix lagotricha OR Nasua nasua); (6) Mycoplasma: ((Mycoplasma OR Haemobartonella) AND (N. nasua OR Panthera onca OR Puma concolor OR Leopardus tigrinus OR Leopardus pardalis OR Puma yagouaroundi OR Herpailurus yagouaroundi OR Potos flavus OR Lagothrix lagotricha OR Alouatta palliata OR Alouatta seniculus OR Cebus albifrons OR Sapajus apella OR Saimiri sciureus OR Saguinus fuscicollis OR Cebuella pygmaea OR C ebuella pygmaea OR Odocoileus virginianus OR Pudu mephistophiles OR Mazama rufina OR Choloepus didactylus OR Choloepus hoffmanni)); and (7) Trypanosoma: Trypanosoma AND (Choloepus AND hoffmanni). Searches were performed in July 2020 without publication year restrictions. Records in English, Portuguese, and Spanish were included in the study. The following were excluded from the study: (i) Studies that did not report the gender of blood parasites in animal species similar to this study, (ii) studies reporting vector-borne infections in animals other than the ones that we are reporting in this study (Table-1), (iii) studies conducted in laboratory animals or non-natural infections, (iv) duplicated titles or articles reporting results of other studies (ei. Reviews, not original research), and (v) studies that search for the parasites but do not find evidence of infection (negative reports) (Supplementary data can be available from the corresponding author). The country, publication year, blood parasite species, infected animal species, and diagnostic techniques (Supplementary data can be available from the corresponding author) were recorded for each manuscript.

Table-1

Hemoparasites found in blood smears of free-ranging wildlife animals attended at HDEV-USFQ.

Order	Family	Animal species	Anaplasma	Babesia	Ehrlichia	Hepatozoon	Microfilarias	Mycoplasma	Trypanosoma
Carnivora	Felidae	Puma yaguarondi						1
		Leopardus pardalis						15
		Leopardus tigrinus						2
		Puma concolor						6
		Panthera onca						3
	Procyonidae	Nasua nasua					1	2
		Potos flavus						2*
Primates	Atelidae	Lagothrix lagotrichia	1*A	1*			1*	5*A
		Alouatta palliata						1*
		Alouatta seniculus						1*
	Cebidae	Cebus albifrons	2*B					13*B
		Sapajus apella						1
		Saimiri sciureus						12
	Callitrichidae	Saguinus fuscicollis	1*	1*				10*
		Callithrix pygmaea						1*
Artiodactyla	Cervidae	Odocoileus virginianus						1*
		Pudu mephistophiles						1*
		Mazama rufina		1*C	2*C			1*C
Pilosa	Megalonychidae	Choloepus didactylus						1*
		Choloepus hoffmanni						1*
Crocodilia	Alligatoridae	Melanosuchus niger				5D	3*D
		Caiman crocodilus				3
Squamata	Boidae	Boa constrictor				2

First time reported

A Mycoplasma sp. and Anaplasma sp.found in the blood smear of the same animal.

B Both, Mycoplasma sp. and Anaplasma sp. was found in the blood smear of two animals.

C Mycoplasma sp., Babesia sp. and Ehrlichia sp found in blood smear of the same animal.

D Microfilaria and Hepatosoon was found in the blood smear of three animals.

Results

Hemoparasites in blood samples from free-ranging wildlife species

From April 2012 to January 2019, 290 free-ranging wildlife animals attended our veterinary facility, including 209 mammals, 52 reptiles, and 29 birds (Supplementary data can be available from the corresponding author). Blood parasites were detected in 97 (33.45%) animals, of which 87 were mammals and ten were reptiles. No positivity was identified in birds.

Anaplasma spp. was registered in four samples from three nonhuman primate species: Two White-fronted capuchin (C. albifrons) and one Brown woolly monkey (L. lagotricha) demonstrated coinfection with Mycoplasma spp. and also one Brown-mantled tamarin (S. fuscicollis) (Table-1). Babesia spp. was found in three mammal species: One Brown woolly monkey, one Brown-mantled tamarin, and one Little red brocket (M. rufina) that showed coinfection with Mycoplasma spp. and Ehrlichia spp., respectively. We also identified another Little red brocket infected with Ehrlichia spp. Mycoplasma spp. was the most frequent hemoparasite and was identified in 80 (91.95%) of the positive samples from 20 different mammal species (Table-1). Mycoplasma was followed by Hepatozoon spp. and was found in samples from ten reptiles (five Black caiman - M. niger, two snakes B. constrictor, and three Spectacled caimans - C. crocodilus). Three Black caiman showed coinfection with microfilariae. Microfilaria was also found in a South American coati (N. nasua) and a Brown woolly monkey. Finally, we identified one Hoffmann’s two-toed sloth (C. hoffmanni) positive for Trypanosoma spp. (Table-1).

Literature searches to investigate hemoparasites in animal species reported in this study

In total, 41 reports matched the hemoparasite findings in the 23 free-ranging wildlife species reported here (Supplementary data can be available from the corresponding author). Twelve studies reported more than one parasite or infected wild animal [23-34]. The search yielded studies from1979 to 2019, with Brazil having the most studies (n=26). The remaining studies were conducted in the USA (n=4), French Guiana (n=4), Colombia (n=2), Mexico (n=2), Costa Rica (n=2), and Peru (n=1).

Our literature review identified 16 Hepatozoon studies that matched our reptile findings. Reports of Hepatozoon fusifex, Hepatozoon terzii, and Hepatozoon cuestensis were identified for B. constrictor and Hepatozoon caimani for C. crocodilus and M. niger. The most common technique used to detect these parasites was the microscopic observation of blood smears. Electron microscopy and molecular detection (followed by sequencing) techniques were also used to identify Hepatozoon species [34-36]. For Mycoplasma, various species were previously reported in nine out of the21 searched mammal species (Table-2) [3,23,25-34,37-56]. Mycoplasma spp. detection and identification were primarily conducted using polymerase chain reaction (PCR) and sequencing. For Trypanosoma species, Trypanosoma leeuwenhoek and Trypanosoma rangeli were isolated and identified in C. hoffmanni using blood smear microscopic observations, histopathology, parasite culture, and PCR. Finally, microfilariae were previously observed in N. nasua and M. niger blood smears. Microfilaria in samples from N. nasua was identified as Dirofilaria repens, Dirofilaria immitis, Acanthocheilonema reconditum, Mansonella spp., and Brugia spp.

Table-2

Hemoparasites that have been previously reported in free-ranging wildlife animals.

Hemoparasite	Animal
Hepatozoon spp. [3,34,37-44]	Caiman crocodilus, Boa constrictor
Hepatozoon caimani [45,46]	Caiman crocodilus
Hepatozoon fusifex [47]	Boa constrictor
Hepatozoon terzii [48]	Boa constrictor
Hepatozoon cuestensi [49]	Boa constrictor
Mycoplasma spp. [26, 50]	Puma concolor, Odocoileus virginianus
Mycoplasma haemocanis [28]	Nasua nasua
Mycoplasma haemofelis [23,25, 32-33,51]	Nasua nasua, Pantera onca, Puma concolor, Leopardus tigrinus
Mycoplasma haemocanis/Mycoplasma haemofelis [28,32]	Leopardus pardalis
Candidatus Mycoplasma turicensis [23,32,51]	Nasua nasua, Pantera onca, Leopardus pardalis
Candidatus Mycoplasma haemominutum [23,25,26,29-32]	Pantera onca, Puma concolor, Leopardus tigrinus, Leopardus pardalis
Candidatus Mycoplasma haemomacaque [27]	Sapajus apella
Candidatus Mycoplasma kahaneii [27,30]	Saimiri sciureus
Trypanosoma leeuwenhoek [52]	Choloepus hoffmanni
Trypanosoma rangeli [53,54]	Choloepus hoffmanni
Dirofilaria repens [55]	Nasua nasua
Dirofilaria immitis[55]	Nasua nasua
Acanthocheilonema reconditum [55]	Nasua nasua
Mansonella spp. [55]	Nasua nasua
Brugia spp. [55]	Nasua nasua
Non identified mirofilaria species [56]	Melanosuchus niger

Our literature search did not identify Hepatozoon spp. in M. niger or Mycoplasma spp. in P. flavus, L. lagotricha, A. palliata, A. seniculus, C. albifrons, P. mephistophiles, S. fuscicollis, C. pygmaea, M. rufina, C. didactylus, or C. hoffmanni. In addition, no publications reported the presence of Babesia spp. in M. rufina, L. lagotricha, or S. fuscicollis or Ehrlichia in M. rufina.

Discussion

The detection of vector-borne blood parasitized wildlife species may provide valuable information on the transmission cycles of pathogens in nature. Knowing the potential parasite reservoirs could inform authorities to implement preventative measures to reduce the risk to wildlife, domestic animal, and human health [57]. From a public health perspective, this approach should be of particular relevance to environments with high animal diversity, where habitat fragmentation has affected the abundance and distribution of vectors and reservoirs involved in the transmission cycle of zoonotic parasites [58].

This study reported vector-borne zoonotic blood parasites in several wild mammal and reptile species from Ecuador. The findings were complemented by data from a systematic review where we searched studies of hemoparasites that infect the wild species identified in our work. For the first time, we report the occurrence of Anaplasma, Babesia, Ehrlichia, Hepatozoon, and Mycoplasma in various wild animal species. These data suggest knowledge gaps of infectious diseases circulating in wildlife from different ecosystems in our region.

All hemoparasites identified here could infect numerous animal species, including humans, and may be biologically or mechanically transmitted by arthropods. Anaplasmosis and ehrlichioses are two tick-borne infectious diseases and are well known in veterinary medicine circles and were recently shown to cause mild, moderate, and severe disease in humans [59]. Both bacteria are obligate intracellular pathogens vectored by ticks, which serve as vectors for vertebrate hosts [60,61]. Babesiosis is an emerging zoonotic disease caused by tick-borne apicomplexan protozoa of the genus Babesia [62]. This pathogen infects a wide range of animals, with wildlife species being the principal reservoir hosts for zoonotic Babesia species [63]. Hepatozoon spp. is intraerythrocytic tick-borne protozoa; infections occur when an infected invertebrate host is ingested by a vertebrate host. Infection with Hepatozoon spp. can affect birds mammals and reptiles and can produce mild to severe disease including symptoms such as anorexia, weight loss, fever, and polyuria, or remain asymptomatic [64-66]. Humans infected with Hepatozoon have been reported [67], suggesting a possible but rare infection incidence. Filarial nematodes are vector-borne worms that release microfilariae into the host blood; these pathogens are considered to be a threat for animal and human health [68]. Most have adapted to the transmission through hematophagous arthropods [69] and can be found in surgical tissue biopsy specimens or removed intact from superficial sites from infected animals [70]. Globally, Mycoplasma spp. represent emerging pathogens in animals and humans, with wildlife animals being important bacterial reservoirs [71,72]. These organisms are epicellular erythrocytic pathogens, transmitted by blood-feeding arthropod vectors [73,74] and causing asymptomatic to mild and severe disease in a wide variety of mammalian species [75]. Finally, trypanosomes, the most common hemoparasites in the world, are transmitted by hematophagous invertebrate vectors [76]. They infect humans and domestic and wild animal species, often causing chronic and fatal diseases [77].

The most common hemoparasite observed in this study was Mycoplasma, infecting a variety of wildlife species from Artiodactyla, Carnivora, Crocodilia, Pilosa, Primates, and Squamata orders (Table-1). Multiple Mycoplasma species have been reported in Nasua spp., including coinfection with Trypanosoma cruzi in Nasua narica from Costa Rica [78]. We also observed this microorganism in Odocoileus virginuanus. About Mycoplasma, the two species that commonly infect cervids are M. ovis and M. haemocervae, and we were able to identified a report that describes a genetically closely related species to M ovis infecting O. virginuanus [79]. In addition, Mycoplasma infection in free-ranging felids was higher than in wild felids from zoos [26]. Several studies described Mycoplasma circulation among domestic cats and free-ranging felids [26,80,81]. Here, we reported for the first time the presence of Mycoplasma in 27 wild felids from Ecuador. These felids belonged to five different species, which are all free-ranging animals. Felids are important top predators; thus, the identification of hemoparasites infections may suggest these animals are bioaccumulating pathogens from prey species [82]. This may be why we did not find Mycoplasma in M. niger. Likewise, Mycoplasma reports were not identified for A. palliata and A. seniculus. However, this was expected as previous studies have reported the presence of Candidatus Mycoplasma kahane in Alouatta caraya and Sapajus flavius [24,74]. Our systematic review found that Mycoplasma was not reported in species such as Choloepus spp., Lagothrix lagotricha, Mazama rufina, P. flavus. and P. mephistophiles among others, agreed with our laboratory findings. This information identified knowledge gaps in the prevalence and dynamics of Mycoplasma species in wildlife animals.

Despite Trypanosoma spp. being one of the most common hemoparasite genera in wildlife [83-86] and its repeated identification in multiple sloth species [3,54,87-89], we identified it only in C. hoffmanni. For microfilaria species, they were previously reported in M. niger and N. nasua. In the latter, reports of D. repens, D. immitis, A. reconditum, Mansonella spp., and Brugia spp. suggested its importance as a reservoir, while the filaria infecting M. niger remains known [55]. Here, we reported for the first time the presence of microfilaria in L. lagotricha. However, further studies are required to identify filaria diversity infecting other wildlife species, including M. niger and L. lagotricha.

Hepatozoon mainly infects reptiles and, to a lesser extent, mammals [3,90]. We observed this genus in the reptiles, C. crocodilus, B. constrictor, and M. niger. Interestingly, only H. caimani was identified in Caiman spp. This result is notoriously contrasting with the reports of a high diversity of Hepatozoon species (H. fusifex, H. terzii, and H. cuestensis) that could infect B. constrictor [43,91-93]. We also reported the presence of Ehrlichia in M. rufina, even though we were unable to identify previous reports of this infection. However, Ehrlichia studies exist for other deer species [94]. Likewise, we did not find reports of Anaplasma in S. fuscicollis, C. albifrons, and L. lagotricha nor reports of Babesia in S. fuscicollis, L. lagotricha, and M. rufina, although these two hemoparasites have been reported in other nonhuman primates and ruminants [95-97]. Therefore, these data contribute to the distribution knowledge of these hemoparasites.

The analytical methodology used in our study was based on blood smear visualization using light microscopy, a method traditionally used for blood parasite diagnostics in veterinary medicine [98]. Although microscopic diagnostic sensitivity and specificity for this method are generally lower than PCR [71], it generates fewer false positives, which is often a major concern in large-scale PCR studies [99,100]. However, the microscopic identification of some blood parasites, for example, Babesia spp. [101], Hepatozoon spp. [102], or Microfilaria spp. [68], remains the “gold standard” diagnostic tool. Therefore, optical microscopy could be a simple and low-cost method for determining hemoparasite prevalence in wildlife [103-105]. We readily acknowledge that the results from this research may be underestimated due to our favoring only microscopic techniques. Therefore, further studies using combined morphological and molecular analyses are required to provide more precise identifications of blood parasites in wildlife [95,103-104].

An interesting finding from this study was that most wildlife species (22 out of 23), where zoonotic blood parasites were detected, were distributed in the Amazon Basin: Alouatta. seniculus [105], B. constrictor [106], C. crocodilus [107], C. pygmaea [108], C. albifrons [109], Cebus macrocephalus [110], C. didactylus [111], C. hoffmanni [112], L. lagotricha [113], Leopardus pardalis [114], L. tigrinus [115], M. rufina [116], M. niger [117], N. nasua [118], O. virginianus [119], P. onca [120], P. flavus [121], P. mephistophiles [122], P. concolor [123], P. yagouaroundi [124], S. fuscicollis [125], and S. sciureus [126]. Only A. palliata is not distributed in the Amazon basin, it lives in the Choco Tropical Rain Forest, and in the deciduous and piemontan forest of the Western Cordillera of the Ecuadorian Andes [127].

The Amazon Basin comprises many of the most diverse global ecosystems across seven countries, namely, Bolivia, Brazil, Colombia, Ecuador, Guyana, Peru, and Venezuela [128]. These complex ecosystems contain natural foci of vector-borne human parasitic diseases. However, disease dynamics have been greatly affected due to deforestation and climate change [129,130]. Vector-borne diseases are particularly sensitive to climate warming, as arthropod vector development rates, geographical distribution, and transmission dynamics are altered [131,132]. Along with increased global temperatures, anthropogenic factors, such as increased urbanization, agriculture, and livestock, are invading wildlife habitats, with increased interactions between humans, domestic, and wild animals[133,134]. These changes in wild ecosystems create new opportunities for vector establishment or increases in existing populations [7,20,101,135]. Specifically, Ecuador has suffered the highest deforestation rate in South America [136], with evidence showing that 46% of South Ecuador’s original forest cover had been converted by 2008 into pastures and other anthropogenic land covers [137]. Furthermore, projected models for future climate change scenarios in Ecuador have demonstrated potential shifts in mosquito distribution, that could lead to an increase of vector-borne infectious diseases [138].

Our systematic review revealed a dearth of publications on selected hemoparasites in the animal species we identified from the Amazon Basin. Specifically, no publications emanated from Bolivia, Ecuador, and Venezuela, whereas most of the research came from Brazil. Therefore, an important vector-borne zoonotic blood parasitology research gap exists in these regions. For these reasons, research efforts are required in these regions where current environmental changes could lead to new epidemiological patterns in vector-borne disease, generating negative impacts on wildlife, domestic animals, and human health. Indeed, pathogen transmission from a vertebrate animal to a human (zoonotic spillover) represents a global but poorly understood public health issue [134]. Thus, a better understanding of the evolution and ecology of zoonotic blood parasites is crucial to determine the foci of possible emerging infectious diseases and to prevent the outbreak of diseases [139,133]. Therefore, as other authors have indicated [38,129], epidemiological surveillance is required to address vector-borne zoonotic blood parasites in the Amazon Basin and other highly diverse ecosystems. In addition, as interfaces between wildlife, domestic animals, and human populations interact and become more complex, more interdisciplinary collaborations will be required to prevent disease transmission at these junctures [140,141]. The only way authorities and policymakers will make informed decisions and prioritize action issues is to foster projects framed under the one health initiative, contributing to public health improvement, wildlife conservation, and livestock sustainability through this holistic strategy [142].

Conclusion

We described vector-borne zoonotic blood parasite diversity in wildlife species from Ecuador and highlighted the lack of information in the region. Indeed, zoonotic spillover is a poorly understood public health issue; thus, our work emphasizes how research can determine if infected wildlife are important reservoirs of hemoparasites distinct to the common Leishmania spp., Plasmodium spp., and Trypanosoma spp. In addition, the fact that all the blood parasites identified in this work are transmitted by arthropod vectors suggests that their ecology and distribution may be affected by habitat fragmentation and climate change. Our data add relevant information to the literature on infectious diseases in wildlife and spotlights the need to understand diseases and its consequences in animal and human health within ecosystem conservation and dynamics, which are the pillars of the one health framework.

Authors’ Contributions

ED: Conceptualization, methodology, writing – original draft, review, and editing. AH, CV, GD: Methodology, data compilation. VAB: Conceptualization, methodology, writing – review and editing, and supervision. All authors have read and approved the final manuscript.