Platynosomum illiciens (Trematoda: Dicrocoeliidae) in a hybrid marmoset ( Callithrix sp.) in the Municipality of Seropédica, RJ, Brazil – Case report

Platynosomum illiciens is a liver trematode encountered infecting mainly felids although it has also been reported in birds and in additional mammalian species, including non-human primates. The current study reports a natural P. illiciens infection primate of the genus Callithrix . The diagnosis was made using a combination of copro-parasitological techniques, morphological evaluation of adult specimens recovered from the liver during necropsy, and molecular analyses. Eggs were brown in color, oval, operculated, and contained a miracidium. Adult specimens recovered during necropsy were measured and showed dimensions compatible with P. illiciens. Molecular characterization of the trematode involved amplification by polymerase chain reaction (PCR), in combination with nucleotide sequencing, of an approximately 900 base pairs fragment corresponding to 18S-ITS1-5.8S ribosomal DNA. Sequenced amplicons showed 100% nucleotide identity with sequences deposited in the GenBank database as derived from specimens of P . illiciens recovered from cats in Malaysia and Brazil. It was concluded that the morphological and molecular analyses presented herein, confirmed the identification of the trematode recovered as P. illiciens . a definitive diagnosis of parasitism by P. illiciens in a captive specimen of the genus Callithrix.

Platynosomum illiciens (syn. Platynosomum fastosum, Platynosomum concinnum, Platynosomum amazonensis, Platynosomum marmoseti), is a biliary tract trematode encountered infecting mainly felids although it has been detected in other species of mammal, including non-human primates and also in birds (Rodrigues, 1963;Basu & Charles, 2014;Pinto et al., 2017). The life cycle begins with embryonated eggs being released in the feces of the definitive host with subsequent ingestion by the first intermediate host, namely terrestrial mollusks of the genera Subulina or Eulota. Miracidia are released and penetrate the connective tissue of the mollusk, wherein the process of cell division of the mother sporocyst to mature daughter sporocysts occurs, resulting in the formation of cercariae. The mature sporocysts containing the cercariae are released from the mollusk and are ingested by a second intermediate host, specifically terrestrial isopods, wherein the cercariae transform into infective metacercariae. Paratenic hosts (reptiles, amphibians) ingest the isopods infected with the metacercariae. Infection of the definitive host occurs via the ingestion of the second intermediate or paratenic hosts containing the metacercaria, which excyst and migrate through the duodenum to the bile ducts and gallbladder, wherein they develop into adults and reproduce, releasing embryonated eggs in the feces (Basu & Charles, 2014;Pinto et al., 2014).
Most infections of primates with P. illiciens are reported as post mortem findings, and as a result the clinical manifestations and process of pathogenesis are poorly understood. However, the diagnosis of platynosomiasis including ante mortem detection of asymptomatic infections can be achieved with the aid of centrifugation-flotation techniques including the use of saturated sucrose solution or via sedimentation with formalin-ether, with the latter being considered more effective (Basu & Charles, 2014). Confirmation occurs through the observation of brown, operculated eggs in the feces (Bielsa & Greiner, 1985;Basu & Charles, 2014). The number of eggs shed within the feces is influenced by the parasitic load, although it is important to consider that flukes of the genus Platynosomum are known to release eggs intermittently (Leal et al., 2011).
The current study reports a case of natural parasitism by P. illiciens in an adult female, hybrid marmoset of the genus Callithrix, held at the Center for Screening of Wild Animals (CETAS) in Seropédica, Rio de Janeiro, Brazil. The animal was admitted to the Veterinary Small Animal Hospital, located on the Seropédica Campus of The Federal Rural University of Rio de Janeiro (UFRRJ), with a history of head trauma. Parasitological examination of a stool sample was performed at the Experimental Chemotherapy Laboratory for Veterinary Parasitology (UFRRJ), as a component of the routine clinical protocols.
The fecal sample was evaluated using a centrifugal-flotation method employing sucrose solution (Sloss et al., 1999) and also by formalin-ether sedimentation (Ritchie, 1948). Microscopic examination revealed the presence of brown, oval, operculated eggs with an internal miracidium, indicating they were trematode eggs ( Figure 1A). Thereafter, eggs (n=30) were measured using an Olympus SZX16 binocular microscope, with photomicrographs generated via a coupled UC30 camera and analyzed using the cellSens Dimension software. The dimensions of the largest and smallest diameters were 41.24μm (32.62-46.76) and 28.13μm (22.05-37.67), respectively, with an average wall thickness of 1.79μm.
As reported previously (Basu & Charles, 2014), the formalin-ether sedimentation technique (Leal et al., 2015) showed greater sensitivity for the recovery of P. illiciens eggs from feces than did the centrifugal flotation technique (Sloss et al., 1999). Ritchie (1948) proposed that the enhancement in recovery reflected the fact that heavy eggs, such as those of P. illiciens, are more likely to sediment than to float. Nevertheless, it is pertinent to note that the findings of the present study corroborate the observation of Shell et al. (2015), that the centrifugal flotation technique is an effective method for the detection of P. illiciens eggs in feces.
Owing to the severe clinical condition resulting from the head trauma and as a consequence of unresponsiveness to treatment, the animal died and was subjected to parasitological necropsy (protocol number; ICMBio/n°57694-1). A total of 13 specimens of adult trematodes were encountered in the liver, together with two in the jejunum and two in the gallbladder. Specimens for morphological and morphometric analyses were fixed in acetic formaldehyde at room-temperature, stained with hydrochloric carmine, dehydrated in a series of ascending ethanol solutions (alcohol 70, 90 GL and absolute) and mounted in balsam added to beech-wood creosote according to the procedure of Amato et al. (1991). Microscopic examinations employed an Olympus BX51 binocular microscope, with a coupled UC30 camera and the cellSens Dimension software.
Fragments of liver, jejunum, gallbladder, and lung were placed in petri dishes containing saline solution and observed under a Nikon YS2-T binocular microscope. Thereafter, they were held overnight in Hoffman's goblets in an attempt to recover eggs or larvae. However, no parasitic forms were found in any of the organs examined. In contrast, examination of biliary and fecal contents, collected post mortem, showed the presence of numerous eggs.
Adults of P. illiciens are characterized by the distribution of the supracecal vitellines in the median region of the body, lobed and opposite testicles, round to lobed ovary (located below the testicles) and, by the acetabulum being slightly larger than the oral sucker (Lenis et al., 2009). Those features were confirmed by the observations recorded in the present report ( Figure 1B). The morphological data, obtained for the adult specimens of P. illiciens examined herein, were compared (Table 1) with the values provided for proposed species of Platynosomum, recorded by Brazilian researchers, naturally infecting felids and birds (Travassos, 1944), or from non-human primates (Kingston & Cosgrove, 1967;Pinto et al., 2017). In addition, we included morphological data registered in mice (Mus musculus) artificially infected with P. illiciens . The results of those comparisons provided support for the proposition of Pinto et al. (2017) that the parasites employed in the description of the species P. amazonensis and P. marmoseti (Kingston & Cosgrove, 1967) were most likely not fully developed. As such, rather than representing new species, they simply reflected the phenomenon of extensive morphological variation that has been widely documented for specimens of P. illiciens infecting different hosts (Travassos, 1944;Rodrigues, 1963;Pinto et al., 2016). Thus, we agree with the recommendation of Pinto et al. (2017) that P. amazonensis and P. marmoseti should be considered synonyms of P. illiciens.
Sequence analysis of duplicate 18S-ITS1-5.8S rDNA amplicons revealed 100% (841/841 bases) nucleotide identity with the sequences MT015696 and MH156567 deposited in the GenBank as derived from specimens of P. illiciens recovered from cat liver in Malaysia and in Brazil, respectively. In addition, the novel sequence was identical to the sequences KU987672 and KU987674 deposited as originating in specimens of P. fastosum recovered from cats in Vietnam. Pinto et al. (2018) included the same P. fastosum sequences from Vietnam in a comparative analysis of ITS-1 sequences derived from adult specimens of P. illiciens recovered from primates and mice, together with sequences generated from metacercariae in geckos. All sequences, with the exception of a single nucleotide polymorphism (SNP) observed in one of the sequences obtained from an experimentally infected mouse, were identical. Those results provided definitive evidence that P. illiciens can naturally infect felines and non-human primates as well as rodents experimentally, corroborating the findings of earlier morphological studies (Pinto et al., , 2017. Importantly, the sequencing data demonstrated that Platynosomum infections in cats in the Americas and Asia are caused by the same parasite, substantiating the proposal of Rodrigues (1963), for the recognition of P. fastosum and P. illiciens as synonyms. As such, the marmoset trematode examined herein was considered to represent P. illiciens and the sequence was deposited in the GenBank with accession number MK166042.
Although extensive morphological variability was reported for specimens of P. illiciens observed in hosts with natural (Travassos, 1944) or experimental infections , factors such as parasite age, the precise location in the biliary system and the fixation technique used may influence the dimensions of the internal structures used for specific morphological diagnosis. For those reasons, it is of value to consider that such morphological variations may explain the various reports of different species of Platynosomum in animals (Kingston & Cosgrove, 1967;Sitko, 1998;Pinto et al., 2016), when, in fact, based on molecular analyses they were most likely the same species (Pinto et al., 2018).
The importance of members of the genus Platynosomum as parasites of captive, non-human primates of the genus Callithrix has been illustrated in numerous studies conducted in Brazil (Silva et al., 2012;Mati et al., 2015;Pinto et al., 2017Pinto et al., , 2018Pereira et al., 2021). However, with the exception of a single study (Pinto et al., 2018), molecular characterization was not used to corroborate the morphological identification of the parasites. As shown in the current study, sequence-based identification of ribosomal DNA can provide increased precision and
Pinto et al.
Present study confidence in the diagnosis of Platynosomum infections. Therefore, its use should be encouraged as a means to definitively determine the species that affect members of the genus Callithrix and other non-human primates in order to provide epidemiological data relevant to health surveillance. The possibility of applying existing molecular methods to monitor fecal samples, using DNA extracted from eggs, should also be investigated as a potential method for detecting asymptomatic infections and for determining the necessity for anti-parasitic treatments, including praziquantel, which in themselves may be detrimental to the health of marmosets (Mati et al., 2021).
Knowledge concerning physiopathology and the clinical manifestations of natural P. illiciens infections in nonhuman primates is inadequate. This deficiency results from the fact that most cases occurred as postmortem findings, based primarily upon the detection of adult trematodes in the liver and gall bladder, as in the present study. However, detailed histopathological data generated in two studies conducted by Brazilian primatologists (Sousa et al., 2008;Pereira et al., 2021) provided insight into the most common histopathological alterations and hepatobiliary disorders in different species of captive, non-human primates naturally parasitized by P. illiciens. Sousa et al. (2008) examined six common marmosets (Callithrix jacchus) suffering from severe non-diarrheic weight loss, and reported ascending cholangitis, cholestasis, and portal fibrosis that in some animals culminated in secondary biliary cirrhosis. In the more recent study, portal fibrosis, biliary epithelial hyperplasia, and inflammatory reactions with a predominance of lymphocytes and plasmocytes were considered the principal lesions and disorders encountered in 11 animals, classified by the authors as naturally infected with P. illiciens (Pereira et al., 2021).
In conclusion, contrary to reports of more than one species of Platynosomum as the etiological agent of platynosomiasis in non-human primates in Brazil, the morphological and molecular data presented herein provided a definitive diagnosis of parasitism by P. illiciens in a captive specimen of the genus Callithrix.