Journal of Parasitology Strongyloides cebus (Nematoda: Strongyloididae) in Lagothrix cana (Primates: Atelidae) from the Brazilian Amazon: Aspects of clinical presentation, anatomopathology, treatment and parasitic biology --Manuscript Draft-Manuscript Number:

13-288R1

Full Title:

Strongyloides cebus (Nematoda: Strongyloididae) in Lagothrix cana (Primates: Atelidae) from the Brazilian Amazon: Aspects of clinical presentation, anatomopathology, treatment and parasitic biology

Short Title:

Strongyloidiasis in Lagothrix cana

Article Type:

Regular Article

Corresponding Author:

Vitor LT Mati, M.D., M.Sc., Ph.D student UFMG Belo Horizonte, Minas Gerais BRAZIL

Corresponding Author Secondary Information: Corresponding Author's Institution:

UFMG

Corresponding Author's Secondary Institution: First Author:

Vitor LT Mati, M.D., M.Sc., Ph.D student

First Author Secondary Information: Order of Authors:

Vitor LT Mati, M.D., M.Sc., Ph.D student Francisco C Ferreira Junior Hudson A Pinto Alan L Melo

Order of Authors Secondary Information: Abstract:

ABSTRACT: Seven cases of parasitism by Strongyloides cebus were identified in Lagothrix cana from Brazil. Aspects of the clinical presentation, treatment, pathology and parasitic biology of these infections are described. Moderate to severe disease was observed, requiring hospitalization of 3 primates, and diarrhea was the most common clinical sign described. One L. cana individual died, for which ulcerative enteritis was the major finding upon histopathological analysis. The use of ivermectin in these atelids was safe and effective against the parasite. Parallel attempts to experimentally infect gerbils with the parasite failed. Lagothrix cana is presented as a new host for S. cebus. The evidence that Strongyloides infections are common in nonhuman primates under free-living conditions, and even more prevalent in captive animals, likely represents a neglected problem.

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RH: MATI ET AL. – STRONGYLOIDIASIS IN LAGOTHRIX CANA STRONGYLOIDES CEBUS (NEMATODA: STRONGYLOIDIDAE) IN LAGOTHRIX CANA (PRIMATES: ATELIDAE) FROM THE BRAZILIAN AMAZON: ASPECTS OF CLINICAL PRESENTATION, ANATOMOPATHOLOGY, TREATMENT AND PARASITIC BIOLOGY Vitor Luís Tenório Mati, Francisco Carlos Ferreira Junior, Hudson Alves Pinto, and Alan Lane de Melo Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CP 486, CEP 30123-970, Belo Horizonte, MG, Brazil. Correspondence should be sent to: [email protected] ABSTRACT: Seven cases of parasitism by Strongyloides cebus were identified in Lagothrix cana from Brazil. Aspects of the clinical presentation, treatment, pathology and parasitic biology of these infections are described. Moderate to severe disease was observed, requiring hospitalization of 3 primates, and diarrhea was the most common clinical sign described. One L. cana individual died, for which ulcerative enteritis was the major finding upon histopathological analysis. The use of ivermectin in these atelids was safe and effective against the parasite. Parallel attempts to experimentally infect gerbils with the parasite failed. Lagothrix cana is presented as a new host for S. cebus. The evidence that Strongyloides infections are common in nonhuman primates under free-living conditions, and even more prevalent in captive animals, likely represents a neglected problem. The genus Strongyloides is comprised of nematode parasites of vertebrates (except for fishes) whose life cycles can include both homogonic and heterogonic routes of development (Little, 1966; Schad, 1989). Among the species of this genus isolated from primates, 1

Strongyloides stercoralis (Bavay, 1876), Strongyloides fuelleborni von Linstow, 1905, and Strongyloides kellyi (Viney et al., 1991) are found in Old World monkeys, apes and/or humans, while Strongyloides cebus Darling, 1911 is considered the only natural species found in New World monkeys (Little, 1966; Toft, 1982; Speare, 1989; Viney et al., 1991 not in LCS; Dorris et al., 2002). At least 20 species of Neotropical primates belonging to 11 genera and 3 families have been recorded as hosts of Strongyloides. These families (and genera) are Aotidae (Aotus), Atelidae (Alouatta, Ateles, Brachyteles, Lagothrix) and Cebidae (Callithrix, Cebuella, Cebus, Saguinus, Saimiri, Sapajus) (Darling, 1911; Little, 1966; Cosgrove et al., 1968; Kuntz and Myers, 1972; Stuart et al., 1993, 1998; Ximenes, 1997; Karesh et al., 1998; Phillips et al., 2004; Carrasco et al., 2008; Beltrán-Saavedra et al., 2009; Barrera et al., 2010). A diagnosis of S. cebus has usually been presumed, based on the observation of eggs in feces from Saimiri sciureus (Battles et al., 1988), Brachyteles arachnoides (Stuart et al., 1993), Alouatta palliata, Ateles geoffroyi and Cebus capucinus (Stuart et al., 1998; Rodríguez-Ortíz et al., 2004). Nematodes found in stool specimens from Ateles paniscus pentadactylus, Saguinus midas midas and Sapajus apella and identified as S. stercoralis by Leger (1921) may also be S. cebus according to Sandground (1925). On the other hand, specific diagnoses of S. cebus based on the analysis of the parasitic worms remain scarce and have only been carried out for the type host, C. capucinus, and for A. geoffroyi, Lagothrix lagotricha, S. sciureus and S. apella ssp. (Darling, 1911; Little, 1966; Campos, 1985). Despite S. cebus having been identified in some Neotropical primates, there is still a lack of knowledge about the geographic distribution and diversity of its hosts. Furthermore, the parasitic biology and pathology of natural strongyloidiasis in platyrrhines and appropriate 2

treatments for this disease are largely unknown. In fact, the little that is currently known about the histopathology of infection by these nematodes was provided by an analysis of intestinal fragments from infected C. capucinus in the original description of the parasite (Darling, 1911), and only a single study has evaluated the administration of ivermectin to a S. sciureus whose infection was considered to be caused by S. cebus (Battles et al., 1988). Thus, the efficacy and safety of this drug for the treatment of strongyloidiais in platyrrhines must be further investigated. In the present study, 7 cases of strongyloidiasis caused by S. cebus were identified in specimens of the gray wooly monkey, Lagothrix cana (É. Geoffroy, 1812), from the Brazilian Amazon. Clinical and pathological aspects of these infections were described, and characteristics related to the biology of the parasite and the management and treatment of these primates were addressed. In parallel, with the aim of better understanding the host specificity of the parasite and obtaining a rodent model of infection with this species, experimental infection of gerbils with S. cebus was attempted. MATERIALS AND METHODS Nonhuman primates Specimens of L. cana (n = 7; 4 males, 3 females) confiscated in the Brazilian Amazon (location undetermined) and initially maintained in the same holding area by the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) were sent to a conservation facility (Mantenedouro Silvestre Zoo ABALU) in the municipality of Inhaúma (19°29′27″S, 44°23′24″W), state of Minas Gerais (MG), southeastern Brazil. One male individual was juvenile and the remaining 6 primates were adults. All of the 7 nonhuman primates arrived at the same time and were placed in 2 wire mesh enclosures with a gravel floor, 3

located close to secondary vegetation. Objects for environmental enrichment were available continuously, and the mean stocking density was 1 animal/30 m3. The animals were fed daily with fruits and vegetables, and water was provided ad libitum. Clinical examinations In the conservation unit, the animals were monitored daily, and a detailed veterinary evaluation was performed monthly as well as when any clinical complication occurred in a primate. When more severe cases were observed, the animals were hospitalized in a veterinary hospital located at Belo Horizonte, MG (19°55'57"S, 46°56'32"W). Necropsy and anatomopathology One hospitalized primate that died (P1) was necropsied, and the gross lesions observed during necropsy were recorded. Fragments of different organs and the entire intestines were fixed in 10% neutral buffered formalin, and the tissues were subsequently paraffin embedded, cut into 4-μm sections and stained via the hematoxylin-eosin histological technique. Images of the microscopic slides containing the sections were captured using a Leica DM500 microscope (Leica Microsystems, Heerbrugg, St. Gallen, Switzerland) with a video camera attached to a PC, and the Leica Application Suite (EZ LAZ), version 2.0, was employed for analysis. Swabs were collected aseptically from the lungs and liver and conserved in a transport medium until being processed in the laboratory shortly thereafter. These samples were streaked on plates with 5% sheep blood agar and on McConkey agar (Difco, Detroit, Michigan) and incubated at 37 C for 24 hr. Coproparasitological monitoring Fresh fecal samples from each gray wooly monkey were collected serially at intervals ranging from 9 to 40 days throughout the period of the investigation. The first fecal sample was 4

examined 13 days after arrival of the primates at the conservation unit during hospitalization of the animal P1. To perform qualitative analyses, the spontaneous sedimentation (Lutz, 1919) and Baermann (modified by Moraes, 1948) methods were used. In the first method, approximately 2 g of feces was processed, and fecal cultures were set up with the remainder of the samples for use in the latter technique. In preparing these cultures, feces were mixed with vermiculite and subsequently incubated in a chamber heated to 27 C for a period of between 72 and 96 hr to allow hatching and larval development of the nematode. All of the material obtained via the modified Baermann method was examined. In the spontaneous sedimentation assays, 3 slides were evaluated per sample. Considering that the amount of stool used in these assays and the volume analyzed on each slide were approximately constant, a semi-quantitative estimation of the number of nematode forms was conducted, and positive results were categorized by convention as + (< 10 eggs and/or L1 per slide), ++ (10 to 100 eggs and/or L1 per slide) and +++ (> 100 eggs and/or L1 per slide) by the same author. In parallel, coproparasitological monitoring by spontaneous sedimentation was also performed for other primates (Alouatta caraya and Alouatta seniculus) whose cages in the conservation facility were relatively near those of L. cana. Nematode identification and experimental infection of gerbils Parasitic females were obtained from the small intestine specimens that were fixed during the necropsy of the monkey that died. Additionally, larvae and free-living forms obtained from positive coprocultures of material from all the living monkeys were fixed in 10% formalin at 70 C. The parasitic forms and the larvae and free-living adults of the nematode were diaphanized in lactophenol and analyzed with aid of a light microscope. 5

Taxonomic identification of the specimens was based on morphological criteria, and the main taxonomic features considered were the following: morphometric data of parasitic females and free-living forms; shape of ovaries of parasitic female; characteristics of vulva and tail of parasitic and free-living specimens; width of third-stage filariform larvae (L3i), according different authors (Darling, 1911; Little, 1966; Campos, 1985). As biological characteristics are also useful in the study of nematode species, attempts were made to experimentally infect gerbils (Meriones unguiculatus) (n=10). A total of 500 infective L3i obtained from positive fecal cultures of L. cana and counted as previously described (Melo et al., 2012) were inoculated via the subcutaneous route in each rodent. From the fifth to the 21st day of experimental infection, samples of feces were collected from all of the animals daily and subjected to serial qualitative analyses using the Lutz method. After this period, the gerbils were euthanized by cervical dislocation according to the protocol of the local animal experimentation ethics committee (CETEA/UFMG), and necropsies of all of the rodents were performed. The intestines were collected, opened longitudinally with scissors and analyzed via stereomicroscope. Thereafter, intestines and lungs were sliced finely and placed in a Baermann apparatus for 3 hr in saline at 37 C. Drug treatment All of the infected nonhuman primates were treated with 2 doses of ivermectin (1% water solution, 200 µg/Kg, subcutaneously) with an interval of 21 days. When the results of the fecal analysis remained positive, additional doses were administered. Subsequent to treatments, parasitological examinations were continued until the fecal tests were deemed to be completely negative after at least 2 consecutive negative fecal results. RESULTS 6

Clinical aspects During the first weeks of captivity in the conservation unit, all of the monkeys showed clinical changes that were primarily characterized by episodes of semiliquid to liquid diarrhea, occasional bristling of the fur, different degrees of appetite loss, reduction of the panniculus adiposus, dehydration, prostration and dyspnea. Their stools exhibited abundant mucus and a foul odor and were grossly off-color (yellowish to blackened). Although the primates had received veterinary assistance during their captivity, 3 of the animals (42.9%) showed abrupt clinical deterioration that was primarily correlated with dehydration, and supportive care and observation were required. The first hospitalization occurred 12 days after arrival of the animals to the conservation facility. A summary of the clinical data from the 7 individuals of L. cana studied, including information about the applied therapy and the results of any laboratory tests performed during hospitalization, is provided in Table I. Necropsy and identification of parasitic females The first primate (P1) to be hospitalized, the young male, died prior to parasitological diagnosis. In general, the postmortem examination of this monkey revealed gross lesions and mucus hypersecretion, together with edema, hyperemia and petechial hemorrhages, which were especially marked in the duodenum and proximal jejunum (Fig. 1). Furthermore, areas with multiple superficial erosions and, slightly less frequently, ulcers, which were rounded or irregular and characterized by breaks in the mucosa, were observed in the small intestine. The lungs displayed rare, small bleeding points, and the peritoneal cavity contained free fluid, but no blood. Microscopic analysis (Figs. 2–5) confirmed the presence of severe enteritis with inflammation, epithelial disruption and destruction of enterocytes as well as an increase in the 7

number of goblet cells in some areas of the mucosa. Lesions confirming the existence of ulcerative duodenitis were observed, as multifocal erosions and some ulceration of the mucosa observed under macroscopic examination were also observed via microscopy. These injuries were associated, at least in part, with the occurrence of villus detachment and destruction, often with only some basal cells surviving, and this destruction sometimes reached the submucosa. The pathological involvement of the villi varied from slight damage and atrophy to dramatic changes in the villous architecture due to the necrosis of most enterocytes. Intense hyperemia, areas of hemorrhage due to the destruction of villi and vessel damage and a moderate inflammatory infiltrate containing lymphocytes, plasma cells, eosinophils and predominantly neutrophils were observed. The inflammatory infiltrate exceeded the muscularis mucosae, reaching the submucosal layer (another histopathological characteristic of ulcerative enteritis) and less frequently extending into the muscular and serosa layers. We further observed fibrosis disorganizing the submucosal layer and a diffuse exudate that primarily consisted of eosinophilic fibrillar material and some eosinophils covering the mucosal surface. In addition, multiple cross-sectional structures presenting a morphology compatible with nematodes were observed in histological sections, most often in the basal portion of the mucosa and surrounded by leukocytes. A small number of eggs were also observed. The lung histopathology showed minor alterations. Multifocal hyperemia, edema and a histiocytic inflammatory infiltrate as well as some areas of atelectasis with adjacent emphysema were observed in the lungs. Microbiological cultures from both the lungs and liver were negative under the conditions employed in the present study. Specimens of minuscule nematodes from the small intestine of this monkey were visualized under a stereomicroscope. Only parasitic females were observed, and their 8

morphology was consistent with the classical description of S. cebus (Figs. 6, 7) (see Little (1966) and Campos (1985) for taxonomic details). Eggs with a morphology suggestive of Strongyloides were observed via coproscopy. Coproparasitological evaluation and efficacy of drug treatment Following the postmortem diagnosis of the first monkey, analysis of feces from the other 6 nonhuman primates (P2 to P7) confirmed that they were also infected with the parasite. Morphological analysis of free-living stages obtained from stool cultures of these primates allowed specific identification of S. cebus (Figs. 8-10). A total of 33 stool samples were collected, and the data obtained from the semiquantitative analysis of the effects of treatment with ivermectin are reported (Table II). There was a progressive reduction of the number of eggs in the feces of L. cana individuals after each dose of the anthelmintic compound. The proportions of negative results after the first, second and third doses of ivermectin were 16.6 (1/6), 83.3 (5/6) and 100% (6/6), respectively. Only 1 animal (P6) that had shown a negative result previously was positive for S. cebus a second time, but after the third dose of the drug, the tests were again negative. The results obtained using the Baermann-Moraes method corresponded qualitatively to those observed via spontaneous sedimentation. The monkeys tolerated the treatment with ivermectin well and remained clinically stable, without showing signs of side effects or toxicity. Clinical improvement of all animals and a better consistency of the feces were observed after their parasitological tests had become negative. Eggs or larvae of S. cebus were never observed in feces from specimens of Alouatta spp. in the present study. 9

Free-living forms of S. cebus and attempts to infect gerbils Embryonated parasite eggs were observed in newly evacuated feces. Less than 4 hr later, first-stage rhabditiform larvae (L1) were identified, which invariably developed into secondstage rhabditiform larvae (L2) and then into either L3i stage larvae (direct, or homogonic development) or a free-living generation with third (L3)-and fourth (L4)-stage rhabditiform larvae and male and female adults (indirect, or heterogonic development). Two molts occurred during the direct route of development (from L1 to L3i) and 4 in the indirect cycle (from L1 to adult worms). The development of S. cebus was asynchronous, as in the analyzed cultures, all stages of larval development were present. Under microscopy of fresh material obtained from cultures, constriction in the region of the vulva was easily observable in free-living females and was evident in older nematodes when eggs were not found in the uteri. Interestingly, indirect development was found to be predominant in the examined specimens. Among all of the stages evaluated (from both the first and second generations), the L3i recovered after 96 hr of culture only represented approximately 20% of the total population. In these cultures, male and female free-living adults occurred at a ratio of 1 to 4, and second-generation L1 and L2 larvae were also observed. Over time, some of these rhabditiform larvae developed into free-living adults, which were found in all of the analyzed cultures. Finally, the specimens of M. unguiculatus were not susceptible to infection with S. cebus L3i larvae according to the methodology employed in the present study. No eggs or larvae of the parasite were found in their feces, and during necropsy, neither larvae nor juvenile and adult parasitic females were recovered from the rodents. DISCUSSION 10

The data obtained in the present study complement existing information on the hostparasite relationship and elucidate clinical and pathological aspects of infection of platyrrhines with Strongyloides. However, efforts should be made towards specific diagnosing of this nematode, which has not been achieved frequently. This report represents the first record of S. cebus in L. cana, and excluding presumed cases, the second report of the occurrence of this species in Brazil, which is the country with the world's largest variety of primate species (118 species; Paglia et al., 2012). The only previous specific record of S. cebus in Brazil came from Goiânia, state of Goiás, Brazilian Central-West region (Campos, 1985). The identification of the nematode in monkeys that had recently been brought from the Brazilian Amazon in the present study confirmed that the distribution S. cebus can cover a wide area extending from southeastern Brazil to Central America, especially when presumed identifications are also considered (Darling, 1911; Stuart et al., 1993, 1998; Rodríguez-Ortíz et al., 2004; Barrera et al., 2010). The absence of records of Strongyloides in New World monkeys further south (e.g., in Argentina, Paraguay and Uruguay) and north (e.g., in Mexico) may be due to the lack of studies addressing this topic. In addition to the description (Darling, 1911) and redescription (Little, 1966) of S. cebus, specific identification based on its parasitic forms was performed only by Campos (1985) and in the present report. Nevertheless, the morphological characteristics of specimens of Strongyloides recovered from A. geoffroyi (Kreis, 1932) are compatible with S. cebus. In general, the previously established morphological features recommended for the identification of Strongyloides spp. from primates and also used in the present study were adequate and easy to determine, and due to this, there was no need for further study of these characteristics. In the 11

parasitic females of S. cebus, ovaries are spiraled around intestine and lips of vulva are prominent; these forms generally display a greater number of eggs in the uteri, and the tail tapers more gradually and is more sharply pointed than S. fuelleborni (Little, 1966; Campos, 1985). With respect to the morphology of free-living females of Strongyloides obtained from monkeys, the constriction of the body near the vulva has historically been an object of controversy, including discussion regarding the validity of Strongyloides simiae (currently a junior synonym of S. fuelleborni; Little, 1966). The extent of post-vulvar constriction in S. fuelleborni has been shown to be variable and sometimes nonexistent, depending on the temperature of culture (Premvati, 1958a), and similar variations in the shape of heterogonic females have been observed, even when the changes in the culture temperature were not evaluated (Little, 1966). This constriction found in free-living females of S. cebus is not part of the species description. However, it usually occurs (Little, 1966; Campos, 1985), which corroborates our findings with respect to the heterogonic females obtained from the L. cana stool cultures, as this feature was clearly evident in all of the examined worm specimens. Thus, this marked constriction observed in heterogonic females of S. cebus, when present, may also be a useful feature in the identification of species, but there are other characters of nematode forms recovered from cultures that are necessary for the identification at specific level. In fact, the specific diagnosis of Strongyloides from the L. cana individuals that did not die was also carried out via analysis of L3i larvae and adult free-living forms obtained from stool cultures. Only Barrera et al. (2010) previously identified S. cebus based on these adult forms obtained from Saguinus oedipus after culturing. The S. cebus L3i exhibit a greater diameter than other Strongyloides spp. from primates, and the tail of free-living adults is shorter

12

and in males the postanal papillae closer than in S. fuelleborni (Darling, 1911; Little, 1966; Campos, 1985). Although Little (1966) suggested that the form passed in the feces is an important criterion in the taxonomic study of Strongyloides, it does not seem prudent to affirm that an infection is truly caused by S. cebus based only on the finding of eggs in the feces of platyrrhines, as these monkeys are also naturally or experimentally susceptible to other species of Strongyloides (Yamashita, 1963; Hershkovitz, 1977; Toft, 1982; Pissinatti, 2001; Melo et al., 2012). Furthermore, hatching of S. cebus larvae occurs shortly after defecation, as demonstrated here, and the existence of unknown species of Strongyloides in Neotropical primates should not be completely disregarded, although S. cebus is still accepted as the single species from platyrrhines that presents a spiral ovary (Little, 1966). Nevertheless, the presumed diagnosis of strongyloidiasis has been quite commonly applied in both New World and Old World monkeys, and errors in taxonomic identification have occurred that are sometimes difficult to resolve. For example, there was a report of parasitism of Macaca mulatta by Strongyloides papillosus whose diagnosis was later questioned (File and Kessler, 1989). Furthermore, Anderson et al., (2012) recently performed a molecular study of Strongyloides collected in a free-raging baboon colony and verified differences at multiple sites compared to published sequences of S. fuelleborni, which was the most probable presumed diagnosis. To explain this finding, it was suggested that recent acquisition of the parasite from a local host or an error in the published sequence of the parasite, perhaps due to another species misidentification, had occurred. Thus, studies aimed at obtaining additional molecular sequences for S. cebus and other Strongyloides from primates are still necessary.

13

The data obtained in the present study confirm that heterogonic development appears to play a central role in the biology of S. cebus, possibly leading to a significant increase in the length of residence and the number of L3i in the environment. In fact, the occurrence of at least 2 generations in the free-living cycle of this species, with both involving the development of adults, as observed in the analyzed cultures, has been reported (Little, 1966), though it is possible that this phenomenon does not occur invariably (Campos, 1985). However, Beach (1936), studying the heterogonic cycle of a species of Strongyloides isolated from the feces of A. geoffroyi and C. capucinus, which he considered to be S. simiae, reported the occurrence of third-generation adults that gave rise to fourth-generation eggs and larvae. Thus, practically indefinite propagation of the free-living phase was suggested to occur when environmental conditions are favorable. In the L. cana stool cultures (96 hr) examined in the present study, only approximately 20% of the nematodes were L3i larvae, corroborating previous observations (Darling, 1911). In contrast, after 15 days of culturing fecal material from S. oedipus infected with S. cebus, this value was 55%, and many free-living forms were still observed (Barrera et al., 2010). On the other hand, it has been observed that S. fuelleborni only presents 1 free-living generation (Premvati, 1958b; Little, 1966; Rego, 1972), though an exception to this pattern was reported by Beg (1968). The clinical manifestations observed in nonhuman primates infected with Strongyloides range from mild to severe and can include death, which might indicate a greater propensity of these hosts to develop severe forms of strongyloidiasis or, more likely, a high parasite load. There is 1 previous report available of the death of an individual of L. lagotricha following marked gastrointestinal symptoms, and the recovered parasitic females were identified as S. papillosus in the broadest sense (Pillers and Southwell, 1929). In an experimental study 14

performed in A. geoffroyi, 1 individual died 52 days after infection with 10,000 L3i larvae from an autochthonous strain of Strongyloides (Faust, 1931). As observed in L. cana infected with S. cebus, severe diarrhea associated with dehydration appears to be related to increased morbidity and, ultimately, the death of monkeys with strongyloidiasis. Previous studies examining the natural infection of platyrrhines with S. cebus have not focused on clinical aspects, and there is a lack of information about the severity of such infections in these primates. However, in nonhuman catarrhines, it has been suggested that the presence of Strongyloides other than S. stercoralis has little measurable effect on their health. In fact, in an isolated population of M. mulatta from an unnatural free-ranging environment, animals harboring S. fuelleborni were found to be in excellent physical condition and showed high reproductive and low mortality rates (File and Kessler, 1989). Furthermore, in a large colony of baboons exhibiting high levels of infection with S. fuelleborni, only a few animals required hospitalization (Anderson et al., 2012). On the other hand, there is no doubt about the high potential severity of S. stercoralis infection in nonhuman primates due to the possibility of autoinfection and dissemination of the parasite (McClure et al., 1973; De Paoli and Johnsen, 1978; Penner, 1981; Harper et al., 1982). This innate inability of other species of Strongyloides to carry out autoinfection, which is the genesis of complicated disease associated with S. stercoralis, may have contributed to an extrapolated conception that considers infections by these parasites that display a lower potential for complications as benign. It is possible that severe cases of strongyloidiasis caused by S. cebus or S. fuelleborni are indeed not the most common, but they certainly occur and may be more frequent in housed monkeys. The intestinal pathology of infections with S. fuelleborni and S. cebus in primates remains unclear. During an extensive survey of parasitic lesions in M. mulatta, M. fascicularis 15

and Papio spp., specimens of Strongyloides were observed in the epithelium of the small intestines of monkeys, and an inflammatory response in the lamina propria was reported, though not described in detail (Abbott and Majeed, 1984). The only available description of the intestinal histopathology during infection with S. cebus is also brief, reporting many “lymphoid and plasma cells” and “eosinophilic leucocytes” in the intestinal mucosa of C. capucinus (Darling, 1911). Furthermore, slides analyzed by Darling showed imbedded ova and crosssections of adult nematodes in the lumen of the organ and under the epithelium of the mucosa, whereas in the deeper layers of the mucosa and in the submucosa, no invasion was observed. In general, these histopathological findings were corroborated in the present study, and additional details were observed in tissues from the necropsied L. cana individual. In this individual, the lesions were more severe and profound and were usually related to the presence of adult worms. Moreover, submucosal involvement was frequent and was characteristic of ulcerative enteritis. In addition to some clinical aspects, the intestinal pathology observed in the L. cana individual that died from S. cebus infection exhibited similar aspects to that described in nonhuman catarrhines (De Paoli and Johnsen, 1978; Penner, 1981; Harper et al., 1982) and humans (Andrade and Caymmi-Gomes, 1964; Caymmi-Gomes, 1980) infected with S. stercoralis. Considering only primates as hosts, only the gross intestinal lesions caused by this species of parasite have been studied previously, and their characteristics are consistent with the injuries induced by S. cebus. According to the predominant characteristics of intestinal strongyloidiasis lesions, they can be classified as catarrhal enteritis (showing preserved villi, a mild inflammatory infiltrate with goblet cell hyperplasia and worms limited to the mucosal crypts), edematous enteritis (displaying occasional atrophic villi, moderate inflammation and edema in the lamina propria) and ulcerative enteritis (the epithelium exhibits erosions and 16

ulcerations, an intense inflammatory infiltrate and many parasites) (Caymmi-Gomes, 1980; Genta and Caymmi-Gomes, 1989). The last condition corresponds best to what was observed in the fatal case described here. However, as expected, unlike the anatomopathology of complicated strongyloidiasis caused by S. stercoralis (Andrade and Caymmi-Gomes, 1964; De Paoli and Johnsen, 1978; Caymmi-Gomes, 1980), the observed lung injuries were minor and may have resulted from migrating larvae of S. cebus that were acquired later, which could be an indication of the occurrence of continuous reinfection of L. cana in captivity. The cell types observed via microscopic analysis indicate both cellular and humoral responses to the parasite, which is similar to what has been observed in experimental and human strongyloidiasis, as macrophages, lymphocytes, eosinophils, mast cells and goblet cells have been identified as possible effectors of Strongyloides spp. expulsion (Onah and Nawa, 2000). In relation to the humoral response, plasma cells were also observed during infection with S. cebus. These cells are B lymphocytes that differentiate after binding to a specific antigen and signaling from a helper T lymphocyte (Th2 type), and they produce specific immunoglobulins (Ig) against the parasite, particularly IgA (Coutinho et al., 1996). Although eosinophils were observed in intestinal lesions in the necropsied monkey, there was no peripheral eosinophilia identified in this animal, and the most significant finding of blood analysis performed shortly before its demise was severe anemia, which may have caused or aggravated probable hypoxemia and led to death. In this case, the intestinal wall might have become thinner due to the severe inflammatory reaction, which can lead to successive ulceration and villus destruction, resulting in hemorrhages, potentially explaining the anemia. In a previous study, the blood of 2 free-ranging Ateles chamek individuals from Bolivia that were naturally infected with Strongyloides sp. was analyzed. A high eosinophil count was observed in 1 of 17

these specimens, in which the presence of lice eggs was also reported, while the other monkey did not exhibit a high eosinophil count (Karesh et al., 1998). Laboratory findings and pathological conditions may vary depending on the type of host involved, the duration of the parasitism process and the occurrence of co-infections, and thus, additional studies addressing these aspects of strongyloidiasis in nonhuman primates are required. The effectiveness of ivermectin for treating S. fuelleborni has been demonstrated in rhesus monkeys (Dufour et al., 2006; Wang et al., 2008). The best-understood effect of ivermectin is the paralysis of parasites via potentiating the release and binding of GABA (gamma-aminobutyric acid) at some nerve synapses, but suppression of the reproductive capacity of target organisms has also been described (Campbell and Benz, 1984). The safety and action spectrum of ivermectin in platyrrhines have received little attention, and the single relevant study (Battles et al., 1998) addressing its effectiveness for the treatment of strongyloidiasis in these primates was conducted in a colony of S. sciureus whose members had a history of periodic diarrhea. In this study, after a treatment regimen consisting of 3 doses of intramuscular ivermectin at approximately 10-day intervals, 2 monkeys continued to shed eggs in their feces, although later, all of the stool specimens were negative. In the present study, parasitological cure of L. cana infected with S. cebus was also obtained after the administration of 3 doses, although the route and treatment intervals employed were different, indicating that successive treatments are necessary to control this parasite, possibly depending on the conditions of captivity. Ivermectin was found to be safe and effective in eliminating S. cebus from these atelids. The literature on helminth infections in monkeys exhibits many frustrating shortcomings, and most of the relevant host-parasite records have come from zoo or captive animals 18

(Yamashita, 1963; Dunn, 1970; Kuntz and Myers, 1972; Hershkovitz, 1977). These characteristics of the literature have not changed sufficiently, and with respect to cases of nonhuman primate strongyloidiasis, the situation can be considered critical. The few existing studies on the occurrence of Strongyloides in wild platyrrhines show highly variable values. For example, in Brazil, the prevalence of S. cebus (presumed identification) in B. arachnoides was reported to be 47 and 89% in 2 zones of the states of São Paulo and Minas Gerais, respectively, whereas no infected primates were identified in 2 other localities in these states (Stuart et al., 1993). In Costa Rica, the rates of infection by Strongyloides in wild Howler monkeys (Alouatta spp.) from 4 different areas were found to be 9, 12, 13 and 73% (Stuart et al., 1998), and in the same country, depending on the geographical area studied, parasite eggs were found in 40.7 and 50% of stool specimens from Saimiri oerstedii (Chinchilla et al., 2010). In Cebus, Ateles and Alouatta in Costa Rica, the mean prevalence of S. cebus infections (presumed identification) was reported to be 0, 7 and 15%, respectively (Stuart et al., 1998). During a survey performed in wild monkeys in Peru, Strongyloides were observed in 10, 18, 20, 31 and 36% of Sapajus apella, Cebus albifrons, Aotus vociferans, A. seniculus and Ateles belzebuth, respectively, while neither eggs nor larvae of the parasite were detected in stool samples from Callicebus brunneus, Saguinus fuscicollis and S. sciureus (Phillips et al., 2004). Another study conducted in Peru in groups of wild A. belzebuth showed that strongyloidiasis may be present in up to 61.7% of these animals (Carrasco et al., 2008). Natural infection with Strongyloides in nonhuman catarrhines has been somewhat better studied. In this group, parasitological surveys of free-ranging monkeys living in natural or semi-natural environments have similarly shown varying prevalences of Strongyloides infection, often of approximately 50% or more, in different host genera such as Papio, Cercopithecus, Chlorocebus and Macaca (File and Kessler, 1989; 19

Munene et al., 1998; Hope et al., 2004; Legesse and Erko, 2004; Pourrut et al., 2011; Ryan et al., 2012). However, among these animals, the number of infected individuals in nature can be small or null depending on the locality and studied host because some hosts, such as some arboreal monkeys, may live in habitats that hinder infection by this parasite (Muriuki et al., 1998; Kooriyama et al., 2012). Higher percentages of infection have been observed in captive nonhuman primates than in wild ones, especially before the development of more effective anthelmintics. For instance, there have been reports indicating that almost 100% of laboratory rhesus monkeys from Calcutta, India, are infected with Strongyloides (Chandler, 1925), and in a colony from New Orleans, USA, 87.5% of the rhesus monkeys and 66.7% of the spider monkeys produced stools that contained eggs of these parasites (Faust, 1931). The present study was not conducted on L. cana under natural conditions and it therefore cannot be determine whether all of animals had already been infected prior to their captivity in the conservation unit. While some of the primates were likely infected with S. cebus before captivity, the 100% prevalence observed prior to specific treatment and the presumptively higher intensity of infection associated with a greater disease severity described above may be correlated with the physical conditions of captivity, together with the stress that results from transport and captivity. These circumstances may trigger an immunosuppression process, which in experimental studies with other Strongyloides spp., has been shown to increase susceptibility to infection and the release of parasite eggs or L1 larvae in the host’s stool (Bailenger et al., 1976; Olson and Schiller, 1978; Martins et al., 2009). Munene et al. (1998) noted that housed baboons displayed a lower percentage of infection with S. fuelleborni than free-ranging baboons. These authors remarked that this finding could not be easily explained, although the regular deworming of the captive 20

primates, which reduces the prevalence of nematodes, may have produced the observed result. The parasite was absent in baboons housed in cages hanging above the floor, while infection was moderate in primates housed in cement cages and more common in animals housed in cages with a gravel floor. This last housing condition was similar to that of the L. cana individuals infected with S. cebus described here. This higher prevalence in animals associated with ground contact is consistent with the biology of the parasite. Factors such as host characteristics and the parasite species or strain involved can influence the prevalence of Strongyloides in nonhuman primates. Moreover, environmental factors can favor the development of free-living stages and increase the density of L3i larvae within a particular small area, which may result in higher infection ratios in captive monkeys. The maintenance of the life cycle is related to the number and proximity of infected and uninfected primates. For instance, the lower prevalence of S. fuelleborni found in pet monkeys than in bushmeat samples (wild monkeys, mostly Cercopithecus spp.) is likely due to the isolation of the former animals beginning in infancy, which substantially reduces the possibility of natural infection (Pourrut et al., 2011). Furthermore, housed rhesus monkeys from larger social groups show up to twice the prevalence of helminths found in groups with fewer animals (Phillippi and Clarke, 1992), and a negative correlation between home range size and helminth species richness among primates has been proposed based on ecological data (Nunn et al., 2003). In the present study, animal P6 showed a negative parasitological test postchemotherapy, but 30 days after the second dose of ivermectin, a small number of eggs was again observed in its feces. Although this finding might be explained by an interruption of the fertility of the parasite induced by the administration of the drug, the occurrence of a new 21

infection cannot be ruled out. Considering the known absence of an autoinfection process during infection with S. cebus and that the negative results found in this monkey occurred when the coproscopy results for all of the other animals were still positive, the possibility of a reinfection process, as discussed above based on the lung lesions observed in the animal that died, should be taken into account. Moreover, the cycle of S. cebus is direct and relatively brief; for example, prepatent periods of 7-9 (Campos, 1985), 14 (Little, 1966) and 18 (Faust, 1931) days postinfection have been observed in experimental infections using monkeys. These issues highlight the fact that some aspects of the biology of S. cebus require further study, and the development of a rodent model for this parasite would be desirable. However, specimens of M. unguiculatus were found to not be susceptible to the parasite in the present study, although this species is permissive to rodent parasites such as S. venezuelensis, S. robustus and S. callosciureus (Tsuji et al., 1993; Sato et al., 2008) and S. stercoralis from dogs (Nolan et al., 1993). This result is interesting because based on molecular analysis, S. cebus and S. venezuelensis were grouped in the same phylogenetic clade, while S. stercoralis was in a different clade (Dorris et al., 2002). The viability of the experimental infection of dogs, rats and mice with S. cebus has been previously described in detail. However, patency of infection has never been observed in any of these animals, which may be useful in studying species of Strongyloides from monkeys and could indicate a high specificity of these parasites for primate hosts (Campos, 1985). On the other hand, specimens of Callithrix penicillata were shown to be susceptible to experimental infection with S. venezuelensis; some of these primates developed a lasting infection and presented eggs in their stools for more than 6 mo after infection with this rodent parasite, which is naturally found in areas inhabited by marmosets (Melo et al., 2012).

22

Presumed identifications are certainly important because the specific identification of a parasite is not always necessary to determine management measures, such as the treatment to be administered to infected hosts. However, in the absence of a specific diagnosis, valuable information about species diversity and host-parasite relationships between Strongyloides and nonhuman primates is often lost. Dunn (1970) has argued that host-parasite data, including information on host specificity and species distributions, may reflect ecological/environmental (e.g., host habits, habitat and dietary preferences) and physiological/genetic factors. We would like to make it clear that we are not suggesting the deliberate collection and necropsy of wild monkeys to allow specific identification of their parasites, but in the case of Strongyloides spp., there is a possibility of studying and identifying free-living forms, as shown in the present study. Taking into account that knowledge regarding the infection of platyrrhines with S. cebus is still incipient, diagnosis of Strongyloides from these hosts to the specific level, which is, in fact, feasible, should be encouraged. In conclusion, we described clinical and pathological aspects of strongyloidiasis caused by S. cebus in gray wooly monkeys. Severe and even fatal disease can occur in captive animals if not treated, but the use of ivermectin in these atelids was found to be safe and effective against the parasite. Lagothrix cana is a new host for S. cebus, and it was confirmed that there is a reasonable degree of specificity between the parasite and its primate hosts because S. cebus infection did not develop in the tested gerbil model. We also discussed evidence that Strongyloides infections are very common in nonhuman primates under natural conditions and are possibly even more prevalent in captive animals, which is a problem that may be underestimated and neglected, including in conservation units, when adequate control is not achieved. 23

ACKNOWLEDGMENTS To Alice de Sá Lopes, Wander Ulisses Mesquita and Bady Elias Curi Neto for technical support, and to CNPq (Conselho Nacional de Pesquisa e Tecnologia) for partial financial support. VLTM and HAP are supported by doctoral degree fellowships from the CNPq, and FCFJ by a doctoral degree fellowship from the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). ALM is supported by CNPq fellowship. LITERATURE CITED Abbott, D. P., and S. K. Majeed. 1984. A survey of parasitic lesions in wild-caught, laboratorymaintained primates (rhesus, cynomolgus, and baboon). Veterinary Pathology 21: 198–207. Anderson, J., R. Upadhayay, D. Sudimack, S. Nair, M. Leland, J. T. Williams, and T. J. Anderson. 2012. Trichuris sp. and Strongyloides sp. infections in a free-ranging baboon colony. Journal of Parasitology 98: 205–208. Andrade, Z. A., and M. Caymmi-Gomes. 1964. Pathology of fatal strongyloidiasis. Revista do Instituto de Medicina Tropical de São Paulo 6: 28–34. Bailenger, J., F. Carcenac, G. Faraggi, and A. Cabannes. 1976. Rôle de I’ hypocorticostéronémie dans le mécanisme de la ‘‘self-cure’’ des rats, parasités par Strongyloides ratti. Annales de Parasitologie Humaine et Comparee 51: 653–665. Barrera, D., V. Pereira-Bengoa, F. Nassar-Montoya, A. Savage, L. Soto, H. Giraldo, F. García, and O. C. Ramírez. 2010. Parásitos em uma población natural de tití cabeza blanca (Saguinus oedipus), Hacienda el Ceibal, Colombia. In Primatología em Colombia: Avances al principio del milenio, V. Pereira-Bengoa, P. R. Stevenson, M. L. Bueno, and F. Nassar-Montoya (eds.). Fundación Universitaria San Martín, Bogotá, Colombia, p. 161–169.

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Battles, A. H., E. C. Greiner, and B. R. Collins. 1988. Efficacy of ivermectin against natural infection of Strongyloides spp. in squirrel monkeys (Saimiri sciureus). Laboratory Animal Science 38: 474–476. Beg, M. K. 1968. Studies on the life-cycle of Strongyloides fulleborni von Linstow, 1905. Annals of Tropical Medicine and Parasitology 62: 502–505. Beltrán-Saavedra, L. F., P. M., Beldomenico, and J. L. Gonzales. 2009. Estudio coproparasitológico de mamíferos silvestres en cautiverio con destino a relocación en Santa Cruz, Bolivia. Veterinaria y Zootecnia 3: 51–60. Campbell, W. C., and G. W. Benz. 1984. Ivermectin: a review of efficacy and safety. Journal of Veterinary Pharmacology and Therapeutics 7: 1–16. Campos, D. M. 1985. Strongyloides cebus Darling, 1911, confirmação de espécie. Revista de Patologia Tropical 14: 173–219. Carrasco, F., M. Tantaleán, K. N. Gibson, and M. Williams. 2008. Prevalencia de helmintos intestinales de una población de monos maquisapas silvestres Ateles belzebuth chamek en el Parque Nacional de Manu, Perú. Neotropical Helminthology 2: 19–26. Caymmi-Gomes, M. 1980. Mecanismos patológicos relacionados à auto-endo-infecção na estrongiloidose humana fatal. Revista de Patologia Tropical 9: 165–262. Chandler, A. C. 1925. The species of Strongyloides (Nematoda). Parasitology 17: 426–433. Chinchilla, M., I. Valerio, O. M. Guerrero, G. Gutiérrez, and R. Sánchez. 2010. Parasitismo intestinal en monos tití o ardilla Saimiri oerstedii (Primates: Cebidae) de Costa Rica. Revista Ibero-Latinoamericana de Parasitologia 69: 106–111. Cosgrove, G. E., B. Nelson, and N. Gengozian. 1968. Helminth parasites of the tamarin, Saguinus fuscicollis. Laboratory Animal Care 18: 654–656. 25

Coutinho, H. B., T. I. Robalinho, V. B. Coutinho, J. R. Almeida, J. T. Filho, G. King, D. Jenkins, Y. Mahida, H. F. Sewell, and D. Wakelin. 1996. Immunocytochemistry of mucosal changes in patients infected with the intestinal nematode Strongyloides stercoralis. Journal of Clinical Pathology 49: 717–720. Darling, S. T. 1911. Strongyloides infections in man and animals in the isthmian canal zone. Journal of Experimental Medicine 14: 1–24. De Paoli, A., and D. O. Johnsen. 1978. Fatal strongyloidiasis in gibbons (Hylobates lar). Veterinary Pathology 15: 31–39. Dorris, M., M. E. Viney, and M. L. Blaxter. 2002. Molecular phylogenetic analysis of the genus Strongyloides and related nematodes. International Journal for Parasitology 32: 1507–1517. Dufour, J. P., F. B. Cogswell, K. M. Phillippi-Falkenstein, and R. P. Bohm. 2006. Comparison of efficacy of moxidectin and ivermectin in the treatment of Strongyloides fulleborni infection in rhesus macaques. Journal of Medical Primatology 35: 172–176. Dunn, F. L. 1970. Natural infection in primates: helminths and problems in primate phylogeny, ecology, and behavior. Laboratory Animal Care 20: 383–388. Faust, E. C. 1931. Infection experiments in monkeys with human, macaque and Ateles strains of Strongyloides. Experimental Biology and Medicine 28: 919–920. File, S., and M. J. Kessler. 1989. Parasites of free-ranging Cayo Santiago macaques after 46 years of isolation. American Journal of Primatology 18: 231–236. Genta, R. M., and M. Caymmi-Gomes. 1989. Pathology. In Strongyliodiasis, a major roundworm infection of man, D. I. Grove (ed.). Taylor and Francis, London, U.K., p. 105–132. Harper III, J. S., J. M. Rice, W. T. London, D. L. Sly, and C. Middleton. 1982. Disseminated strongyloidiasis in Erythrocebus patas. American Journal of Primatology 3: 89–98. 26

Hershkovitz, P. 1977. New World monkey parasites. In Living New World monkeys (Platyrrhini) with an introduction to primates, P. Hershkovitz (ed.). University of Chicago Press, Chicago, Illinois, p. 368–396. Hope, K., M. L. Goldsmith, and T. Graczyk. 2004. Parasitic health of olive baboons in Bwindi Impenetrable National Park, Uganda. Veterinary Parasitology 122: 165–170. Karesh, W. B., R. B. Wallace, R. L. Painter, D. Rumiz, W. E. Braselton, E. S. Dierenfeld, and H. Puche. 1998. Immobilization and health assessment of free-ranging black spider monkeys (Ateles paniscus chamek). American Journal of Primatology 44: 107–123. Kooriyama, T., H. Hasegawa, M. Shimozuru, T. Tsubota, T. Nishida, and T. Iwaki. 2012. Parasitology of five primates in Mahale Mountains National Park, Tanzania. Primates 53: 365– 375. Kreis, H. A. 1932. Studies on the genus Strongyloides (Nematodes) (1932) American Journal of Epidemiology 16: 450–491. Kuntz, R. E., and B. J. Myers. 1972. Parasites of South American primates. International Zoo Yearbook 12: 61–68. Leger, M. 1921. Anguillulose intestinale des singes a la Guyane Francais. Compte Rendu de la Societe de Biologie 84: 555–558. Legesse, M., and B. Erko. 2004. Zoonotic intestinal parasites in Papio anubis (baboon) and Cercopithecus aethiops (vervet) from four localities in Ethiopia. Acta Tropica 90: 231–236. Little, M. D. 1966. Comparative morphology of six species of Strongyloides (Nematoda) and redefinition of the genus. Journal of Parasitology 52: 69–84. Lutz, A. 1919. O Schistosomum mansoni e a schistosomatose, segundo observações, feitas no Brazil. Memórias do Instituto Oswaldo Cruz 11: 121–155. 27

Martins, W. A., J. R. Nicoli, L. M. Farias, M. A. Carvalho, D. C. Cara, and A. L. Melo. 2009. Strongyloides venezuelensis: Efeito de antimicrobiano e imunossupressor no curso da infecção em camundongos da linhagem AKR/J. Revista de Ciências Médicas e Biológicas 8: 315–324. McClure, H. M., L. M. Strozier, M. E. Keeling, and G. R. Healy. 1973. Strongyloidosis in two infant orangutans. Journal of the American Veterinary Medical Association 15: 629–632. Melo, A. L., V. L. Mati, and W. A. Martins. 2012. Callithrix penicillata as a nonhuman primate model for strongyloidiasis. Primates 53: 303–309. Moraes, R. G. 1948. Contribuição para o estudo do Strongyloides stercoralis e da estrongiloidíase no Brasil. Revista do Serviço Especial de Saúde Publica 1: 507–624. Munene, E., M. Otsyula, D. A. Mbaabu, W. T. Mutahi, S. M. Muriuki, and G. M. Muchemi. 1998. Helminth and protozoan gastrointestinal tract parasites in captive and wild-trapped African non-human primates. Veterinary Parasitology 78: 195–201. Muriuki, S. M., R. K. Murugu, E. Munene, G. M. Karere, and D. C. Chai. 1998. Some gastrointestinal parasites of zoonotic (public health) importance commonly observed in old world nonhuman primates in Kenya. Acta Tropica 71: 73–82. Nolan, T. J., Z. Megyeri, V. M. Bhopale, and G. A. Schad. 1993. Strongyloides stercoralis: the first rodent model for uncomplicated and hyperinfective strongyloidiasis, the Mongolian gerbil (Meriones unguiculatus). Journal of Infectious Disease 168: 1479–1484. Nunn, C. L., S. Altizer, K. E. Jones, and W. Sechrest. 2003. Comparative tests of parasite species richness in primates. American Naturalist 162: 597–614. Olson, C. E., and E. L. Schiller. 1978. Strongyloides ratti infection in rats. I. Immunopathology. American Journal of Tropical Medicine and Hygiene 27: 521–526.

28

Onah, D. N, and Y. Nawa. 2000. Mucosal immunity against parasitic gastrointestinal nematodes. Korean Journal of Parasitology 38: 209–236. Paglia, A. P., G. A. Fonseca, A. B. Rylands, G. Herrmann, L. M. Aguiar, A. G. Chiarello, Y. L. Leite, L. P. Costa, S. Siciliano, M. C. Kierulff et al. 2012. Annotated checklist of Brazilian mammals, 2nd ed. Conservation International, Arlington, Virginia, 76 p. Penner, L. R. 1981. Concerning threadworm (Strongyloides stercoralis) in great apes – lowland gorillas (Gorilla gorilla) and chimpanzees (Pan troglodytes). Journal of Zoo Animal Medicine 12: 128–131. Phillippi, K. M., and M. R. Clarke. 1992. Survey of parasites of rhesus monkeys housed in small social groups. American Journal of Primatology 27: 293–302. Phillips, K. A., M. E. Haas, B. W. Grafton, and M. Yrivarren. 2004. Survey of the gastrointestinal parasites of the primate community at Tambopata National Reserve, Peru. Journal of Zoology 264: 149–151. Pillers, A. W., and T. Southwell. 1929. Strongyloidosis of the woolly monkey (Lagothrix humboldti). Annals of Tropical Medicine and Parasitology 23: 129–129. Pissinatti, A. 2001. Medicine, selected disorders. In Biology, medicine and surgery of South American wild animals, M. E. Fowler, and Z. S. Cubas (eds.). State University Press, Ames, Iowa, p. 272–274. Pourrut, X., J. L. Diffo, R. M. Somo, C. F. Bilong-Bilong, E. Delaporte, M. LeBreton, and J. P. Gonzalez. 2011. Prevalence of gastrointestinal parasites in primate bushmeat and pets in Cameroon. Veterinary Parasitology 175: 187–191.

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Premvati. 1958a. Studies on Strongyloides of primates. IV. Effect of temperature on the morphology of the free-living stages of Strongyloides fülleborni. Canadian Journal of Zoology 36: 623–628. _____. 1958b. Studies on Strongyloides of primates. III. Observation on the free-living generations of S. fülleborni. Canadian Journal of Zoology 36: 447–452. Rego, A. A. 1972. Observações sobre a biologia do Strongyloides fulleborni von Linstow, 1905 e tentativas de infecção experimental de roedores. Atas da Sociedade de Biologia do Rio de Janeiro 15: 99–103. Rodríguez-Ortíz, B., L. García-Prieto, and G. P. León. 2004. Checklist of the helminth parasites of vertebrates in Costa Rica. Revista de Biologia Tropical 52: 313–354. Ryan, S. J., J. S. Brashares, C. Walsh, K. Milbers, C. Kilroy, and C. A. Chapman. 2012. A survey of gastrointestinal parasites of olive baboons (Papio anubis) in human settlement areas of Mole National Park, Ghana. Journal of Parasitology 98: 885–888. Sandground, J. H. 1925. Speciation and specificity in the nematode genus Strongyloides. Journal of Parasitology 12: 59–80. Sato, H., S. Tanaka, Y. Une, H. Torii, M. Yokoyama, K. Suzuki, A. Amimoto, and H. Hasegawa. 2008. The stomal morphology of parasitic females of Strongyloides spp. by scanning electron microscopy. Parasitology Research 102: 541–546. Schad, G. A. 1989. Morphology and life history of Strongyloides stercoralis. In Strongyloidiasis, a major roundworm infection of man, D. I. Grove (ed.). Taylor and Francis, London, U.K., p. 85–104. Speare, R. 1989. Identification of species of Strongyloides. In Strongyloidiasis, a major roundworm infection of man, D. I. Grove (ed.). Taylor and Francis, London, U.K., p. 11–83. 30

Stuart, M. D., V. Pendergast, S. Rumfelt, S. Pierberg, L. Greenspan, K. Glander, and M. Clarke. 1998. Parasites of wild howlers (Alouatta spp.). International Journal of Primatology 19: 493– 512. _____, K. B. Strier, and S. M. Pierberg. 1993. Coprological survey of parasites of wild Muriquis, Brachyteles arachnoids, and Brown Howling monkeys, Alouatta fusca. Journal of the Helminthological Society of Washington 60: 111–115. Toft II, J. D. 1982. The pathoparasitology of the alimentary tract and pancreas of nonhuman primates: A review. Veterinary Pathology 19: 44–92. Tsuji, N., Y. Nakamura, and N. Taira. 1993. Long-lasting parasitism of Strongyloides venezuelensis in Mongolian gerbils (Meriones unguiculatus). Journal of Parasitology 79: 305– 307. Viney, M. E., R. W. Ashford, and G. Barnish. 1991. A taxonomic study of Strongyloides Grassi, 1879 (Nematoda) with special reference to Strongyloides fuelleborni von Linstow, 1905 in man in Papua New Guinea and the description of a new subspecies. Systematic Parasitology 18: 95-109. Wang, T., G. Y. Yang, H. J. Yan, S. Wang, Y. Bian, A. C. Chen, and F. J. Bi. 2008. Comparison of efficacy of selamectin, ivermectin and mebendazole for the control of gastrointestinal nematodes in rhesus macaques, China. Veterinary Parasitology 153: 121–125. Ximenes, M. F. 1997. Parasitismo por helmintos e protozoários no sagui comum (Callithrix jacchus). In A Primatologia no Brasil, 6, M. B. Sousa, and A. A. Menezes (eds.). Editora da UFRN and Sociedade Brasileira de Primatologia, Natal, Brazil, p. 249–256. Yamashita, J. 1963. Ecological relationships between parasites and primates. I. Helminth parasites and pimates. Primates 4: 1–96. 31

Figure 1. Gross lesions from the small intestine of an individual of Lagothrix cana infected with Strongyloides cebus showing enteritis with edema, hyperemia and petechial hemorrhages, and multiple superficial erosions. Bar = 1 cm. Figures 2–5. Histopathology of the small intestine of an individual of Lagothrix cana infected with Strongyloides cebus. (2) Enteritis associated with changes in the villous architecture due to villus detachment and partial destruction of the mucosa. Note the presence of multiple crosssections of parasites (arrows) and tissue debris in the lumen. HE stain, bar = 500 µm. (3) Erosions and an eosinophilic fibrillar exudate (necrosis) covering the mucosal surface (arrows). Hemorrhage, a diffuse inflammatory infiltrate in the mucosa and fibrosis in the submucosal layer are also observed. HE stain, bar = 200 µm. (4) Detail of the boundary between the mucosa (M) and the submucosa (SM). The destruction of enterocytes, inflammatory cells reaching the submucosal layer, areas of hemorrhage (arrows) and intense hyperemia (arrowheads) can be observed. HE stain, bar = 50 µm. (5) A parasite in the basal mucosa surrounded by a mixed inflammatory infiltrate, including plasma cells (arrows). HE stain, bar = 20 µm. Figures 6–10. Morphological characteristics of Strongyloides cebus from Lagothrix cana. (6) Vulvar region of parasitic female, lateral view. Lips of vulva are prominent (arrow); uteri contain a row of eggs and present width slightly larger than half the width of the body of the parasite. (7) Tail of parasitic female, lateral view. (8) Vulvar region of free-living female, lateral view. Note the constriction of the body near the vulva (arrow) that is not prominent. (9) Tail of free-living female, lateral view. (10) Tail of free-living male, lateral view. Bars = 30µm.

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Table I Click here to download Table: Table I.doc

TABLE I. Clinical data on and the requirement for hospital care among captive Lagothrix cana naturally infected with Strongyloides cebus. Primate

Gender

Need for hospital care

Clinical findings

Summary from hospitalization

P1

♂

Yes

Severe catarrhal diarrhea and dehydration, reduction of the panniculus adiposus and bristling of the fur, intense pallor of the skin and mucous membranes, anorexia, lethargy and acute respiratory distress.

P2

♂

No

P3

♀

Yes

Intermittent diarrhea with changes in stool consistency and a reduction of the panniculus adiposus. Moderate and continuous catarrhal diarrhea, prostration, loss of appetite, reduction of the panniculus adiposus and subsequent substantial dehydration

Supportive care and volume replacement. Death of the animal one day after admission, before the results of laboratory tests were released [RBC: 1.1x10 6/mm3, hemoglobin: 2.2 g/dl, hematocrit: 6%, WBC: 19,000/mm3 (neutrophils: 71%, lymphocytes: 22%, monocytes: 5%, eosinophils: 2%), platelets: 43,000/mm3, normal liver function tests (enzymes, bilirubins and proteins), urea: 79.6 g/dl, creatinine: 1.6 g/dl, hemoparasite screening: negative, coproscopy: Strongyloides eggs]. Animal's body was sent for necropsy. NA*

P4

♀

No

P5

♂

Yes

P6

♂

No

P7

♀

No

* NA = Not applicable.

Intermittent diarrhea with changes in stool consistency, prostration and a reduction of the panniculus adiposus. Intermittent diarrhea with alterations in stool consistency and a reduction of the panniculus adiposus. The primate progressed to a poorer general condition, including a loss of appetite, diarrhea and dehydration. Intermittent diarrhea with changes in stool consistency and reduction of the panniculus adiposus. Moderate watery diarrhea, prostration and reduction of the panniculus adiposus.

Veterinary management included supportive care, a bland diet, fluid therapy and treatment with B complex vitamins, metronidazole, trimethoprim-sulfamethoxazole and, ultimately, after parasitological diagnosis, ivermectin. After 7 days of hospitalization, clinical improvement was observed, and the primate was discharged, but a few eggs and larvae were still present in feces (see table 2). NA

This primate was also treated with fluid therapy, B complex vitamins, metronidazole and trimethoprim-sulfamethoxazole prior to specific treatment with ivermectin. After 10 days of hospitalization and partial clinical improvement, the primate was discharged, although eggs and larvae were not completely eliminated from the feces (see table 2). NA

NA

Table II Click here to download Table: Table II.doc

TABLE II. Results of a semi-quantitative fecal analysis and the response of captive Lagothrix cana infected with Strongyloides cebus to treatment with ivermectin. Primate

Days post-first ivermectin dose* -17

0

+12#

(1st dose)

+21

+51

(2nd dose)

(3rd dose)

+91

+142

P1||

NA†

NA

NA

NA

NA

NA

NA

P2

++

NA

NA

+

-

-

-

P3

+++

NA

+

++

-

-

-

P4

+

NA

NA

+

-

-

-

P5

+++

NA

+

++

-

-

-

P6

++

NA

NA

-

+

-

-

P7

+++

NA

NA

+

-

-

-

* Each dose of ivermectin (200 µg/Kg) was administered subcutaneously. † NA = Not available; +, ++ and +++ indicate low, moderate and high numbers of eggs in feces, respectively, and - indicates negative result. || The feces obtained during necropsy contained a high number of parasite eggs. # Analyses performed during hospitalization.

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