Morphometric Characteristics of the Metacestode Echinococcus Vogeli Rausch & Bernstein, 1972 in Human Infections from the Northern Region of Brazil

Journal of Helminthology (2015) 89, 480–486 doi:10.1017/S0022149X14000376 q Cambridge University Press 2014

Morphometric characteristics of the metacestode Echinococcus vogeli Rausch & Bernstein, 1972 in human infections from the northern region of Brazil

F. Almeida1, F. Oliveira1, R. Neves2, N. Siqueira3, R. Rodrigues-Silva1*, D. Daipert-Garcia1 and J.R. Machado-Silva2 1Laboratory of Helminth Parasites of Vertebrates, Oswaldo Cruz Institute, Av. Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, Brazil: 2Laboratory of Helminthology Romero Lascasas Porto, Department of Microbiology, Immunology and Parasitology, Faculty of Medical Sciences, Biomedical Centre, State University of Rio de Janeiro, Rua Prof. Manoel de Abreu 444/5 Floor, Vila Isabel, 20511-070, Rio de Janeiro, Brazil: 3Acre State Hospital Foundation and Federal University of Acre, Rodovia BR 364, s/n km 02, Distrito Industrial, Rio Branco, 69914-220, Acre, Brazil

(Received 15 March 2013; Accepted 12 April 2014; First Published Online 22 May 2014)

Abstract

Polycystic echinococcosis, caused by the larval stage (metacestode) of the small-sized tapeworm, Echinococcus vogeli, is an emerging parasitic zoonosis of great public health concern in the humid tropical rainforests of South and Central America. Because morphological and morphometric characteristics of the metacestode are not well known, hydatid cysts from the liver and the mesentery were examined from patients following surgical procedures. Whole mounts of protoscoleces with rostellar hooks were examined under light and confocal laser scanning microscopy. Measurements were made of both large and small hooks, including the total area, total length, total width, blade area, blade length, blade width, handle area, handle length and handle width. The results conﬁrmed the 1:1 arrangement of hooks in the rostellar pad and indicated, for the ﬁrst time, that the morphometry of large and small rostellar hooks varies depending upon the site of infection. Light and confocal microscopy images displayed clusters of calcareous corpuscles in the protoscoleces. In conclusion, morphological features of large and small rostellar hooks of E. vogeli are adapted to a varied environment within the vertebrate host and such morphological changes in calcareous corpuscles occur at different stages in the maturation of metacestodes.

Introduction disease worldwide, polycystic echinococcosis (PE) caused by E. vogeli is an emergent zoonosis of great public health Zoonotic larval cestode infections were recently significance in the humid and tropical rainforests of classified among the neglected tropical diseases (Budke South and Central America (D’Alessandro & Rausch, et al., 2009; WHO, 2012). While human echinococcosis 2008; Siqueira et al., 2010). Furthermore, the number of caused by E. granulosus is a serious life-threatening diagnosed cases of human PE may represent only the tip of the iceberg (D’Alessandro & Rausch, 2008). *Fax: þ 55-021-25621511 Echincoccus vogeli is considered to be the most E-mail: rsilva@ioc.fiocruz.br pathogenic species, responsible for a total of 179 reported Morphometrics of E. vogeli in human infections from Brazil 481 human cases in the past four decades (Knapp et al., 2009; Siqueira et al., 2010; Zegarra et al., 2010). Echinococcus vogeli is transmitted between canines, such as bush dogs (Speothos venaticus) and domestic dogs (Canis familiaris), that harbour the adult tapeworm, and rodents, such as the Agouti paca, that harbour E. vogeli during the larval cestode stage (D’Alessandro & Rausch, 2008). Wild canines become infected with E. vogeli through the consumption of infected pacas, which they hunt (Jenkins et al., 2005), while humans become infected accidentally after ingesting eggs present in canine faeces, which causes polycystic lesions that are found most frequently Fig. 1. Diagram of the rostellar hook of the protoscolex of in the liver and abdominal cavity (D’Alessandro & Echinococcus vogeli with measurements (mm) of the total Rausch, 2008). area (BA/HA), total width (TW), blade area (BA), blade width A number of studies have demonstrated that larval (BW), handle area (HA) and handle width (HW). hook morphology is a valid criterion, being faster and more cost effective than traditional techniques for the identification of E. granulosus (Gholami et al., 2011; Spring, Maryland, USA). For confocal laser scanning Mowlavi et al., 2012). Our previous study demonstrating microscopy (CLSM), protoscoleces were stained with the use of combining conventional and new morphologi- Langeron’s carmine. Whole mounts were examined cal tools, such as light microscopy, confocal laser scanning under a confocal microscope (LSM 510 – ZETA, Zeiss, microscopy, differential interference contrast and vari- Oberkochen, Germany) (Neves et al., 2004). All slides able-pressure scanning electron microscopy, helps were also examined under phase contrast by adding a prism to resolve matters in regard to protoscoleces from the (Zoom 3.8) to the laser scanning confocal microscope. E. granulosus metacestode (Almeida et al., 2009). However, Several linear measurements and areas of both large many of the phenotypic aspects of protoscoleces are and small hooks were analysed, including the total area, unknown. Parasitological diagnosis of human PE is based total length, total width, blade area, blade length, blade on morphometric features of rostellar hooks (D’Alessan- width, handle area, handle length and handle width dro & Rausch, 2008). The mean lengths of large and small (fig. 1). Images were captured with a digital camera hooks of E. vogeli are 41 and 33 mm respectively (Nikon Eclipse E200 camera; Nikon, Chiyoda, Japan) (D’Alessandro & Rausch, 2008). connected to a microscope (Olympus BX41 microscope; The aim of the present study was to extend our Olympus, Tokyo, Japan). The camera output was knowledge and understanding of E. vogeli with reference processed and analysed with Image Pro Plus 3 image to morphological and morphometric characteristics of the analysis software (Media Cybernetics). All measurements species, focusing on polymorphism of the rostellar hooks are in micrometres unless indicated otherwise. and the presence of calcareous corpuscles in the protoscolex. Data analysis Data analysis was carried out with GraphPad InStat Materials and methods (GraphPad Instat Software Inc. version 3.01, California, USA). Statistical analyses included analysis of variance Collection and examination of cysts (ANOVA) followed by a post-hoc Tukey’s test and Student’s The present investigation was carried out on hydatid t-test. Statistical significance was assessed at P # 0.05. cysts isolated from the livers and/or mesenteries of five female patients and three male patients, ranging in age Results from 4 to 49 years, from the cities of Feijo´ (0880905100S; 7082101300W), Sena Madureira (980305600S; 6883902500W), All microscopic techniques were necessary to examine Tarauaca´ (0880903900S; 7084505700W), Assis Brasil (1085602900S; the E. vogeli hooks and phenotypic aspects of proto- 6983400100W) and Pauini (0784205000S; 6685803300W) in the scoleces. Using light microscopy, it was possible to Amazonas and Acre states, Brazil. visualize some protoscoleces during different develop- Cysts were not pooled as each cyst was considered a mental stages and also visualize the high density single sample. The rostellar hooks of protoscoleces were and differential distribution of calcareous corpuscles aspirated under sterile conditions together with the (fig. 2A–D), which are mainly located close to the hydatid fluid from each cyst and then rinsed twice in a rostellar hooks (fig. 3A). In protoscoleces, no differences 0.85% NaCl solution. Each suspension of protoscoleces are shown in organization of the hooks (figs 2A and 3A). was passed through a sieve to remove larger debris. After There are two rows of hooks in the rostellar pad with a this step, the material was transferred and stored at room large hook intercalated by a small hook. Both large and temperature, in small tubes with 10% buffered formalin small rostellar hooks vary morphologically, although all (Almeida et al., 2007, 2009). hooks possess a handle, blade and guard (fig. 3B). Large For light microscopy, protoscoleces were mounted on and small hooks possess a central amorphous pulp glass slides coated with polyvinyl lactophenol and region. Large hooks frequently possess thin guards with crushed under a coverslip. Images were acquired using an irregular surface between the guard and the handle, Image Pro Plus software (Media Cybernetics Inc., Silver while small hooks are round and possess stout guards. 482 F. Almeida et al.

Fig. 2. Echinococcus vogeli metacestode to show stages of (A) the invaginated protoscolex with the rostellar pad and calcareous corpuscles irregularly localized; (B) evaginated protoscolex with suckers ( ), rostellum (arrowhead), neck region (*) and a high density of calcareous corpuscles (arrow); (C) evaginated protoscolex with scolex (*), neck region (arrowhead), developing proglottid ( ) and cluster of calcareous corpuscles (arrow); (D) evaginated protoscolex showing the scolex with suckers (*), neck (arrowhead) with a lower density of calcareous corpuscles (arrow) than in (C) and the developing proglottid ( ). All scale bars ¼ 21 mm.

In most cases, the surface between the guard and the When comparing hook measurements according to handle is irregular. The blade does not show any relevant the infected organ, hooks from the liver are larger for all features. parameters analysed, with the exception of the blade CLSM images confirm the 1:1 arrangement of hooks in length. When comparing small hooks with regard to the the rostellar pad (fig. 3C, D). There are protoscoleces in infected organ, all parameters analysed possess signifi- different stages of development with a high density of cant differences (P , 0.05), except for the blade length calcareous corpuscles (fig. 3E, F). In evaginated proto- and the handle width. Large hooks possess only two scoleces, we note the presence of both dorsal and ventral significantly different characteristics, namely the blade suckers, along with clusters of calcareous corpuscles in area and total area (P , 0.05). this region (fig. 3E). Morphometric approaches allowed the discrimination of differences between large and small hooks. All Discussion measurements are similar to those reported previously in the literature (table 1), although there are no previous The taxonomy of the Echinococcus genus has been data for mesentery samples. For the next step of this resolved based on morphological characteristics viewed study, a comparison of morphometric characteristics of under light microscopy together with scanning and hooks from the liver and mesentery was undertaken transmission electron microscopy (Rausch et al., 1978, (table 2). A phenotypic polymorphism was found in the 1984; Kumaratilake et al., 1986; Antoniou & Tselentis, 1993). linear and area biometrical parameters based on the In addition, confocal microscopy provides new morpho- analysis of 250 large and small hooks. logical features of E. granulosus (Almeida et al., 2009). Morphometrics of E. vogeli in human infections from Brazil 483

Fig. 3. Light (A, B) and confocal laser scanning micrographs (C–F) of a whole mounts of the Echinococcus vogeli metacestodes, to show (A) large hooks in the upper row and small hooks in lower row (arrow) and calcareous corpuscles close to the rostellar hooks (arrowhead), scale bar ¼ 15 mm; (B) polymorphism of large (arrow) and small (arrowhead) hooks, scale bar ¼ 15 mm; (C) and (D) 1:1 arrangement of hooks in the rostellar pad (arrows), scale bars ¼ 12 mm; (E) evaginated (*) and invaginated ( ) protoscoleces, calcareous corpuscles (arrow), and dorsal and ventral suckers (arrowhead), scale bar ¼ 20 mm; (F) dense arrangement of calcareous corpuscles (arrow), scale bar ¼ 12 mm. 484 F. Almeida et al.

Table 1. Rostellar hook morphometrics (mm) of Echinococcus vogeli metacestodes in the liver and mesentery (*) undertaken in the present study, compared with values reported by previous authors.

D’Alessandro Meneghelli Somocurcio Abdul-Hadi Knapp Present study et al., 1979 et al., 1992 et al., 2004 et al., 2007 et al., 2009 (n ¼ 197) (n ¼ 197) (n ¼ 300) (n ¼ 100) (n ¼ 500) (n ¼ 1000)

Large hook length 41.4 ^ 3.8 41.4 ^ 3.8 40–45 41 39.9 ^ 0.8 40.6 ^ 1.4 39.9 ^ 0.8* Small hook length – – 28–35 33 31.8 ^ 0.4 32.7 ^ 0.7 31.8 ^ 0.4* n, Number of hooks measured.

Because E. vogeli is related to all other Echinococcus larger number of discriminatory criteria than large hooks taxa (Knapp et al., 2011), this investigation demonstrated and that blade length and handle width were not useful several phenotypic characteristics shared with E. granu- parameters, as had been reported previously for losus. We showed that E. vogeli has two rows of hooks E. granulosus (Almeida et al., 2007). in the rostellar pad, arranged 1:1, with a large hook To the best of our knowledge, this is the first study intercalated by a small hook (Rausch, 2006; Almeida et al., describing measurements of mesenteric metacestodes. 2009). In addition, large and small hooks presented as a Surprisingly, all measurements of mesenteric metacestodes central, amorphous pulp region. Large hooks frequently were less than those of hepatic metacestodes. A possible had thin guards, in contrast to the slender guards explanation for this is that E. vogeli metacestodes display observed in E. granulosus (Almeida et al., 2009). Small phenotypic plasticity, in which morphological diversi- hooks were rounded and had stout guards, and the fication may be organ-dependent or organ-induced. In surface between the guard and the handle was irregular, this case, the liver and mesentery appear to represent confirming previous studies (Almeida et al., 2009). heterogenic niches. Previously, we demonstrated that Differences in hook morphology are related to their E. granulosus hooks recovered from lungs were larger function in the digestive systems of predatory carnivores than those collected from the liver (Almeida et al., 2007). It (Antoniou & Tselentis, 1993). is important to note that physiological and biochemical The present study confirmed that morphometry is conditions are determinants of infection sites (Almeida a powerful tool for the analysis of rostellar hooks of et al., 2007). Type I PE is the most common presentation of cestodes belonging to the genus Echinococcus (Ponce- polycystic echinococcosis and includes the liver as a site of Gordo & Cuesta-Bandera, 1997; Almeida et al., 2009; infection (D’Alessandro & Rausch, 2008), which suggests Mowlavi et al., 2012). Results from liver metacestodes that the liver provides better physiological conditions for show that the total lengths of small and large hooks agree metacestode development than the mesentery. with previous observations (D’Alessandro et al., 1979; Tissue from cestode species, such as Taenia solium Meneghelli et al., 1992; Somocurcio et al., 2004; Abdul- (Xifeng & Baodong, 1998; Vargas-Parada et al., 1999; Yang, Hadi et al., 2007; Knapp et al., 2009). From a phenotypic 2004), E. granulosus metacestodes (Smith & Richards, point of view, parameters relating to large and small 1993; D’Alessandro, 1997; Vargas-Parada & Laclette, 1999; hooks, such as total area, total width, blade area, blade Martıńez et al., 2005), T. taeniaeformis and Dipylidium length, blade width, handle area, handle length and caninum proglottids (Khalifa et al., 2011), contains mineral handle width, can assist in differentiating E. granulosus concretions termed calcareous corpuscles. An established metacestodes from various sites of infection (Almeida body of literature states that calcareous corpuscles are of et al., 2007). This investigation aimed to verify whether cellular origin (Pawlowski et al., 1988; Xifeng & Baodong, these parameters were reliable tools for distinguishing 1998; Vargas-Parada et al., 1999), irregularly spherical between hepatic and mesenteric metacestodes. Most or ovoid in shape (Smith & Richards, 1993) and are of importantly, we observed that small hooks provided a different sizes (Vargas-Parada & Laclette, 1999).

Table 2. Comparative morphometrics (mm) with mean values ^ standard deviation of large and small hooks of Echinococcus vogeli metacestodes, relative to location within the liver and mesentery.

Large hook Small hook

Mesentery Liver P Mesentery Liver P

Total area 211.85 ^ 11.27 231.16 ^ 14.12 0.04* 140.20 ^ 6.54 155.36 ^ 4.71 0.00* Total length 39.86 ^ 0.80 40.64 ^ 1.43 0.31 31.84 ^ 0.43 32.68 ^ 0.69 0.05* Total width 12.42 ^ 0.48 13.04 ^ 0.63 0.11 10.36 ^ 0.32 11.18 ^ 0.62 0.02* Blade area 152.34 ^ 8.20 165.53 ^ 7.81 0.03* 97.80 ^ 3.71 107.31 ^ 3.90 0.00* Blade length 27.45 ^ 0.30 27.57 ^ 0.39 0.59 20.26 ^ 0.60 20.23 ^ 0.23 0.92 Blade width 12.47 ^ 0.54 13.06 ^ 0.54 0.12 10.29 ^ 0.37 11.13 ^ 0.71 0.05* Handle area 57.92 ^ 4.87 62.86 ^ 7.42 0.25 40.75 ^ 4.02 45.99 ^ 1.84 0.03* Handle length 13.19 ^ 0.45 13.79 ^ 1.15 0.31 11.59 ^ 0.33 12.41 ^ 0.65 0.04* Handle width 7.07 ^ 0.47 7.29 ^ 0.46 0.47 5.40 ^ 0.44 5.80 ^ 0.26 0.12

Signiﬁcant differences (in bold) given as P , 0.05. Morphometrics of E. vogeli in human infections from Brazil 485

In the present investigation, it appears that calcareous Financial support corpuscles from the protoscolex of E. vogeli follow a common, basic pattern, similar to that of E. multilocularis The authors gratefully acknowledge the financial (Ohnishi & Kutsumi, 1991) and E. granulosus (Smith & support of the Fundaçaõ Carlos Chagas Filho de Amparo Richards, 1993). The present results are consistent a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and with earlier studies on the description of the cellular the Universidade do Estado do Rio de Janeiro (UERJ). organization of developing stages of E. granulosus (Martıńez We would also like to thank the Conselho Nacional de et al., 2005). The latter indicated that cell buds generated Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) for a during the formation of protoscoleces are concentrated scholarship (to J.R.M.S.). mainly in suckers and the rostellar pad (Martıńez et al., 2005). Despite the observation that both invaginated and Conflict of interest evaginated protoscoleces contain calcareous corpuscles, None. there are variations in their density and location, as shown by light and confocal microscopy. In the invaginated stage, protoscoleces containing the rostellar Ethical standards pad and calcareous corpuscles were irregularly localized, The authors assert that all procedures contributing whereas in the evaginated stage, calcareous corpuscles to this work comply with the ethical standards of the were concentrated in the rostellum, suckers, neck Instituto de Pesquisa Evandro Chagas, Fundaçaõ region and developing proglottids. Furthermore, during Oswaldo Cruz, Rio de Janeiro, RJ, Brazil and with morphogenesis, younger and evaginated protoscoleces the Helsinki Declaration of 1975, as revised in 2008, and contained a high density of calcareous corpuscles, but a have been approved by the institutional committee reduced density of clusters was observed in protoscoleces (no. 002.1.000.009-04). with some evidence of strobilization. Despite a number of exhaustive studies, the physiology References of calcareous corpuscles in various cestode taxa remains poorly understood (Vargas-Parada & Laclette, 1999). Abdul-Hadi, S., Chacoń, N.J., Bruces, A.C., Gutierrez, Calcareous corpuscles are the principal sources of J.E., Safar, J.A., Egui, M.A., Falco, A. & Cantele, H.E. calcium carbonate, magnesium and phosphate (Smith & (2007) Autochthonous polycystic hepatic echinococco- Richards, 1993), with a possible role in excretory activity sis due to Echinococcus vogeli at the Venezuela amazon: (Vargas-Parada & Laclette, 1999) or in the secretion of case report. Revista da Sociedade Venezuelana de glycoproteins (Yang, 2004). As their sites, such as suckers, Microbiologia 27, 120–126. rostellar pads, hooks and proglottids, present intense Almeida, F.B., Rodrigues-Silva, R., Neves, R.H., biological activity, it may be hypothesized that calcareous Romani, E.L. & Machado-Silva, J.R. (2007) Intra- corpuscles are involved in parasite differentiation and specific variation of Echinococcus granulosus in live- growth (Rodrigues et al., 1997). Although mechanisms by stock from Peru. Veterinary Parasitology 143, 50–58. which calcareous corpuscles contribute to metacestode Almeida, F.B., Rodrigues-Silva, R., Neves, R.H., Lessa, differentiation are not fully understood, these structures M.M.G., Sanchez, E.R.L. & Machado-Silva, J.R. may act as a source of ions (Rodrigues et al., 1997) or (2009) Morphological and morphometric studies on thioredoxin peroxidase, which suggests that they protect protoscoleces rostellar hooks of Echinococcus granulosus against oxidative damage, and may have other biological from Peru visualized by several microscopic techni- roles, such as energy transformation and differentiation ques. Neotropical Helminthology 3, 65–71. (Rodrigues et al., 1997; Li et al., 2004). Antoniou, M. & Tselentis, Y. (1993) Studies on To date, morphological characteristics of calcareous Echinococcus granulosus using the scanning electron corpuscles from E. vogeli metacestodes have not been microscope. II. The hooks. Parasitology Research 79, sufficiently studied. 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