Parasitology International xxx (2011) xxx–xxx

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Chinchilla laniger can be used as an experimental model for Taenia solium taeniasis

Pablo Maravilla a, Adriana Garza-Rodriguez a, Benjamin Gomez-Diaz a, Diego Emiliano Jimenez-Gonzalez a,Elizabeth Toral-Bastida a, Joel Martinez-Ocaña a, Brett West b, Nadia Molina b, Ramon Garcia-Cortes a,Simon Kawa-Karasik a, Mirza Romero-Valdovinos a, Guillermina Avila-Ramirez c, Ana Flisser a,⁎a Direccion de Investigacion, Hospital General “Dr. Manuel Gea Gonzalez”, 14080, Mexico DF., Mexicob International Program, Texas A & M University, College Station, TX, USAc Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, 04360, Mexico DF., Mexico

⁎ Corresponding author at: Departamento de Microbide Medicina, Universidad Nacional Autonoma de Mexic

E-mail address: flisser@servidor.unam.mx (A. Flisser

1383-5769/$ – see front matter © 2011 Elsevier Irelanddoi:10.1016/j.parint.2011.06.002

Please cite this article as: Maravilla P, et al,(2011), doi:10.1016/j.parint.2011.06.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 December 2010Received in revised form 2 June 2011Accepted 8 June 2011Available online xxxx

Keywords:ChinchillaCopro-antigen detectionExperimental modelInfection rateMethylprednisolone acetateOncopshere viabilityTaenia soliumTapeworms

Chinchilla laniger has been reported as an experimental definitive host for Taenia solium; however noinformation about its suitability and yield of gravid tapeworm proglottids containing viable and infective eggshas been published. In total 55 outbred female chinchillas were infected with 4 cysticerci each; hosts wereimmunodeppressed with 6 or 8 mg of methyl-prednisolone acetate every 14 days starting the day of infectionand their discomfort was followed. Kinetics of coproantigen ELISA or expelled proglottids was used to definethe infection status. Efficiency of tapeworm establishment was 21% and of parasite gravidity was 8%;chinchillas showed some degree of suffering along the infection. Viability of eggs obtained from gravidproglottids was tested comparing methods previously published, our results showed 62% viability withpropidium iodide, 54% with trypan blue, 34% with neutral red, 30% by oncosphere activation and 7% withbromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio (MTT) reduction; no statistical differences wereobtained between most techniques, except activation. Four piglets were infected with 50,000 eggs each,necropsy was performed 3 months later and, after counting the number of cysticerci recovered, thepercentage of infection was similar to data obtained with T. solium eggs recovered from humans. Our resultsdemonstrate that the experimental model of T. solium taeniasis in C. laniger is a good alternative for providingeggs and adult tapeworms to be used in different types of experiments; optimization of the model probablydepends on the use of inbred hosts and on the reduction of infected animals' suffering.

ologia y Parasitologia, Facultado, 04360, México DF., Mexico.).

Ltd. All rights reserved.

Chinchilla laniger can be used as an experime

© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Laboratory models to study taeniasis caused by Taenia solium areneeded because humans are the only natural hosts in which the adultcestode develops into a gravid tapeworm. This parasitic infection haslow incidence but causes relevant public health and animal husbandryproblems, i.e. human neurocysticercosis and swine cysticercosis.Furthermore: i) tapeworm carriers in the household are the mainrisk factor for acquiring neurocysticercosis; ii) taeniasis is generally asilent infection, which, in spite of the likelihood of self-diagnosis, isdifficult to identify; iii) availability of tapeworms for diagnosis isscanty; iv) studies focused on the host–parasite relationship and toevaluate vaccines are difficult to perform due to lack of tapewormmaterial [1–3].

Several mammal species have been evaluated as experimentalmodels of adult T. solium. Unsuccessful results were obtained withrhesus monkeys, pigs, dogs, cats, white rabbits, white rats, guinea pigs

and white mice under immunodeppressed conditions [3–6], in spiteof the fact that treatment with steroids facilitates the establishmentand development of adult T. solium and other related cestodes. Onlyrodents from Cricetidae, Heteromyidae and Chinchillidae are suscep-tible as experimental models. Golden hamsters (Mesocricetus auratus)and Mongolian gerbils (Meriones unguiculatus) are particularlyinteresting because mature T. solium adult worms can develop andthe host-parasite relation is different between them [4–7]. One gravidadult T. solium tapeworm was obtained in a chinchilla that was orallyinfected 12 weeks before with four T. solium cysticerci andmaintainedimmunodeppressed with 8 mg of methyl-prednisolone acetate (MPA;Depo-Medrol, Pharmacia Upjohn) every 14 days. [4]. The wholetapeworm was used to infect a healthy pig, 12 weeks later, atnecropsy, 14 cysticerci were found in skeletal muscle [4]. In thatstudy, 2 and 6 mg of MPA were also evaluated, chinchillas with 8 mgpresented longer tapeworms, while with 2 mg hosts became negativeby ELISA. Chinchillas without immunodeppression were not assayed;thus, the purpose of the present study was to reproduce theexperimental model of taeniasis due to T. solium in Chinchilla lanigerin order to evaluate its suitability, tapeworm recovery and eggviability.

ntal model for Taenia solium taeniasis, Parasitol Int

Table 2Data of four experiments performed in order to define several characteristics of adultTaenia solium obtained from infected Chinchilla laniger.

Experiment WPIa atnecropsy

Number oftapewormsper chinchilla

Length of parasites(cm)

Maximumdevelopmentof parasites

2 4 1 3 Immature11 4 68,76,84b,128b Gravid13c NDd ND Gravid

3 19 2 109b,117b Gravid22 2 71b,78b Gravid

4 1 1 ~0.5 Scolex1 4 1,1,1.5,1.5 Immature5 2 14,14 Immature8 4 20,33,76,90 Pregravide

15 4 125b,125b,130b,180b Gravid5 22 3 67b,71b,71b Gravid

22 1 4 Immature22 2 45,46 Pregravid22 2 41,70b Gravid22 1 23 Mature

a Weeks post infection.b Gravid tapeworm.c Necropsy was not performed but gravid proglottids were recovered in faeces.d Not determined.e Pregravid proglottids had lateral uterine branches protruding from a small central

branch and contained eggs with thin embryophores [3].

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2. Materials and methods

2.1. Animals

Before experiments were performed, the Internal Committee forUse and Care of Laboratory Animals of the Hospital General “Dr.Manuel Gea Gonzalez”, approved it on June 12, 2002. Outbred femaleC. laniger (55) three to four months old, were hosted individually inmetal battery-style caging with hardwood chip as bedding. Fiveindependent experiments along four years were performed. Based onprior observations that male sex hormone may reduce developmentof tapeworms [3,8], we decided to infect only female hosts; besides,stress is reduced when all animals in a unit are the same sex. Allchinchillas were maintained in an animal house with 12 h light and12 h darkness and fed with commercial pellets (Chinchilla Diet,Mazuri) and water ad libitum; gradually chinchilla food was changedto commercial monkey diet pellets (8714 Teklad 15% Monkey Diet,Harlan). Three weeks before infection, all hosts were treated forintestinal parasites with a single dose of 30 mg/kg body weight ofpraziquantel (Cesol, Merck) and a single dose of 20 mg/kg ofalbendazol (Zentel, SmithKline-Beecham). Chinchillas were assignedto the five experiments depending on its purpose and theiravailability, since these are not common laboratory animals.

2.2. Assessment of chinchilla suffering

In order to evaluate rodent suffering, along 8 weeks before and18 weeks after infection, at dusk the same researchers recordedweekly the behavior of each chinchilla during the last experiment(that included 60% of all animals used). Hiding, activity (movement),posture, vocalization, sneezing, and trembling were tabulated as low,moderate or high suffering according to the scale defined by Buckwell[9]. Afterwards, body weight was obtained using a scale for rodents,the number of breaths was visually determined and water consump-tion was calculated from the bottle level in each cage.

2.3. Immunodeppression, infection and follow up

Immunodeppression was induced at the day of infection, andevery 14 days thereafter, initially with 8 mgMPA and afterwards with6 mg. The change in dose was becausewe observed a general decreasein the health status due to MPA. T. solium cysticerci were excised fromskeletal muscle of naturally infected pigs. Prior to infection, somecysticerci were assayed for viability by in vitro evagination afterincubation in culture medium with 25% pig bile in 0.01 M phosphate-buffered 0.15 M saline, pH 7.2 (PBS) for 3 h[3,4]. Infection wasperformed per os with four translucid, fluid filled viable cysticerci.Starting five weeks post-infection (WPI) feces from each animal were

Table 1Details of all five experiments performed in order to define several recovery parameters of

Experiment Steroid dose(mg)

Number ofchinchillasuseda

Number ofinfectedchinchillasb

(%)

Total numberof cysticerciused forinfection

1 0 3 2 (66) 122 8 5 3 (60) 203 6 6 2 (33) 244 6 11 5 (45) 445 6 30 5 (17) 120

a Each animal received per os 4 cysticerci.b In all experiments evagination rate of cysticerci used was 100%, except in experiment 4c Total tapeworms recovered at necropsy/total cysticerci inoculated ×100.d Gravid tapeworms recovered at necropsy/total cysticerci inoculated ×100.e Gravid tapeworms recovered at necropsy/total tapeworms recovered at necropsy.

Please cite this article as: Maravilla P, et al, Chinchilla laniger can be use(2011), doi:10.1016/j.parint.2011.06.002

recovered three times per week, hydrated during one day, and sievedto look for proglottids. After screening for parasites in feces, thehardwood chips of all cages were sterilized and picked up by acommercial service of biohazard collection and sanitary processing.

Individual or small chains of proglottidswerewashed exhaustivelywith water, kept in PBS with an antibiotic-antimycotic solution(Gibco) and stored at 4 °C. Some fresh gravid proglottids were stainedwith Mayer's Carmin in order to count the number of eggs. Alldisposable materials used to recover feces, proglottids and eggs, weresubmerged in 10% sodium hypochlorite for approximately one hour toeliminate infective T. solium eggs. Except for the last experiment,ELISA for detection of coproantigens (CpAg ELISA) was used to followthe infection as previously described [4,10]. Necropsies wereperformed along the study in order to observe the development ofthe tapeworms and to avoid the suffering of chinchillas. Inexperiments 2, 3 and 4, rodents with evident damage due to MPA,were euthanized, and in experiment 5, based on waste parametersdefined by Buckwell [9], when animals presented moderate or highsuffering they were euthanized according to the recommendations ofthe American Veterinary Medical Association [11]. Tapeworms thatwere recovered along the experiments were placed in distilled water,stored overnight at 4 °C for relaxation, measured over a rule in a Petridish, fixed in 70% ethanol, stainedwithMayer's Carmin, andmounted.

adult Taenia solium obtained from infected Chinchilla laniger.

Tapeworms recoveredat necropsy

% infectionc Overall % ofgravidtaeniasd

% yield ofgravidtaeniase

Total Gravid

ND 0 ND 0 ND5 2 25 10 404 4 17 17 10015 4 34 9 279 4 7.5 3 44

, where it was 70%.

d as an experimental model for Taenia solium taeniasis, Parasitol Int

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2.4. In vitro tests for oncosphere viability

Gravid proglottids stored at 4 °C for less than 7 days were thinly cutin small pieces with fine sharp point scissors over a sieve of 200 μmmesh size and washed with 20 ml fresh water, egg solution wascentrifuged at 1000 g for 5 min and the pellet was resuspended in 3 mlPBS. The number of eggs/ml was estimated on a Neubauer countingchamber; eggs were hatched according to Wang et al. [12]. Briefly,500 μl of egg solution (with approximately 5000 eggs) were poured inper well a 24 well culture cell plate, 800 μl PBS plus 100 μl 0.5% sodiumhypochlorite solution were added and gently mixed with a Pasteurpipette. Disruption of embryophoric blocks was followed under aninverted microscope; when most oncospheres were released from theembryophore they were transferred to a conic plastic tube, PBS wasadded and the suspension centrifuged (1,000 g, 5 min). The pelletcontaining oncospheres was washed and resuspended in 3 ml PBS,500 μl were placed in threewells of one plate and the remaining volumewas placed in twowells of two different plates. Five distinct procedureswere tested for the in vitro oncosphere viability assay; the three wellswith oncospheres were added with 1:10 solution of 0.4% trypan bluesolution (TB; Sigma); 0.3% neutral red solution (NR; Sigma) or 0.001%propidium iodide solution (PI; Sigma). One of the other plates received1 mlof 0.025%bromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio(MTT; Amresco) in glucose–saline solution. Samples were gentlymixedwith a Pasteur pipette, 5 min later oncosphere viabilitywas determinedby counting stained and unstained oncospheres under light microscopyfor TBandNR,while IPwasobservedunderUV light.MTTwas incubated

Fig. 1. Kinetics of coproantigen detection by ELISA (CpAg ELISA, shown as points with a cimmunosuppressed chinchillas found since week 6 (A). Relationship between CpAg ELISAstandard deviations.

Please cite this article as: Maravilla P, et al, Chinchilla laniger can be use(2011), doi:10.1016/j.parint.2011.06.002

for 12 h at 37 °C in 5% CO2. All samples were again gently mixed with apipette and observed under light microscopy, except PI that wasanalyzed in UV conditions. Oncospheres used for activation wereincubated in artificial intestinal fluid (1% pancreatin, 0.2% anhydroussodiumbicarbonate, 10% pig bile, 10% bovine fetal serum inRPMI-1640)for 1 h at 37 °C, under gentle continuous shakingwith a Pasteur pipette;the number of oncospheres that were changing shape and extrudinghooks were recorded. All analysis were performedwith 4–5 repetitions,except MTT that was assayed twice (using the original egg solution).

2.5. In vivo test for oncosphere viability

In order to confirm that oncospheres were viable, two month old,four female hybrid pigs were infected per os with 50,000 eggs eachfrom the same batch of eggs that was used for the in vitro viabilitytests. Eggs were concentrated by centrifugation and placed in twogelatin capsules that were introduced in each pig's pharynx; to assureswallowing a trickle of water was followed. Pigs were maintained in apen with cement floor and a food collector, and were fed withcommercial diet and water ad libitum. Prior to infection, bloodsamples were taken for detection of anti-cysticercus antibodies byWestern-blot as described by Tsang et al. [13] with an enrichedglycoprotein fraction used as antigen, and commercial anti-pig IgG(Zymed). At 17 and 27 WPI pigs were killed, sera were collected andall skeletal muscle was sliced in search of cysticerci. The parasiteswere categorized as being viable when a clear-fluid-filled vesicle

ontinuous line) since week 5 and number of released proglottids in feces (bars) fromabsorbance value and number of released proglottids (B). Vertical lines correspond to

d as an experimental model for Taenia solium taeniasis, Parasitol Int

Fig. 2. Negative image of one Taenia solium gravid proglottid recovered from sievedchinchilla's feces; it contains around two hundred eggs that look like white dots.

4 P. Maravilla et al. / Parasitology International xxx (2011) xxx–xxx

containing a detectable scolex was present, while necrotic or calcifiedlesions without a recognizable scolex were designated nonviable.

2.6. Statistical analysis

Descriptive statistics are expressed as mean and standarddeviation (SD). Statistical analysis was performed using one wayANOVA and Dunnett's test was used for multiple comparisons. Thevalues were considered to be significantly different when PN0.05.Data analysis was performedwith SSPS Version 15.0 forWindows andPrimer of Biostatistics software [14].

Table 3Parameters used for in vitro assessment of oncosphere viability.

Technique Numbers of oncospheresanalyzed in each assay.

Numbers of oncospheresalive in each assay

Activation 19 320 212 217 919 10

Trypan blue stain 97 4588 5081 4351 32

Neutral red stain 62 1870 27

Propidium iodide stain 77 5096 58

MTT reduction 119 1389 373 354 4

a Standard deviation.b No statistical differences were found between activation and MTT reduction, the remain

Dunnett's post hoc test.

Please cite this article as: Maravilla P, et al, Chinchilla laniger can be use(2011), doi:10.1016/j.parint.2011.06.002

3. Results

3.1. Infection and follow up

Recovery of T. solium adults in different experimental infections ischronologically summarized in Table 1. Experiment 1 consisted ofthree non-deppressed chinchillas, only two of them were parasitizedaccording to CpAg ELISA, but after 2 WPI they became negative, thustheywere not euthanized. In experiment 2, five rodents received 8 mgof MPA and three of them exhibited five tapeworms during necropsy.In experiments 3 to 5, considered as replicate assays, 6 mg steroidwere administrated. In all infections cysticerci used showed 100% invitro evagination, except in experimental group 4, where it was 70%.Interestingly in experiment 4 the highest percentage of tapewormrecovery (34%) but the lowest number of gravid tapeworms (27%)were found. In contrast, in experiment 3, only 33% chinchillas wereparasitized, four Taenia adults were recovered at necropsy, all gravid.Finally, experiment 5 showed the lowest percentage of infectedchinchillas (17%); however 44% recovered tapeworms were gravidsimilar to experimental group 2.

Table 2 shows the length and development of tapewormsrecovered at necropsy. After 10 WPI gravid parasites were found inmost infected chinchillas, although some parasites at 22 WPI weresmaller and showed less development, probably because sections oftheir strobila were released before necropsy. As seen in Fig. 1Aproglottids were released in feces since 6 WPI until the end, asmeasured in experiments 2, 3 and 4. Absorbance values exhibited awave-like profile; similarly to all expelled proglottids during sievingof feces. Gravid proglottids in feces were seen since 10WPI. No strongcorrelation (R2=0.054) was identified between the absorbance valueand the number of released proglottids (Fig. 1B). Gravid proglottidscontained small numbers of eggs, ranging from around ten to almostone thousand eggs (Fig. 2).

3.2. In vitro tests for oncosphere viability

The viability of oncospheres recovered from gravid proglottidswasassessed in experiment 4 by in vitro tests; results are summarized inTable 3. Low numbers of oncospheres were assayed because gravidproglottids released by chinchillas have few eggs and several werelost during treatment (hatching and washing) as has been observedby others [15]. All techniques showed unequivocal interpretations, asseen in the examples given in Fig. 3. Viability values between tests

Average number ofoncospheres analyzed (SDa)

Mean of viableoncospheres (SD)

Percentage of viableoncospheresb

17.4 (3.21) 5.2 (3.96) 30

79.2 (19.94) 42.5 (7.59) 54

66.0 (5.66) 22.5 (6.36) 34

86.5 (13.43) 54.0 (5.65) 62

83.7 (27.51) 5.7 (4.86) 7

ing techniques were statistically different between them (Pb0.05) by one-way ANOVA,

d as an experimental model for Taenia solium taeniasis, Parasitol Int

Fig. 3. Photographic images showing in vitro tests for oncosphere viability measurement. Gravid proglottid with somemature eggs (A); activated oncospheres are seenwith differentshapes (B); trypan blue staining (C); neutral red staining (D); MTT reduction (E) and propidium iodide staining (F). From C to F viable oncospheres are in the left side. Bar 20 μm.

5P. Maravilla et al. / Parasitology International xxx (2011) xxx–xxx

were statistically different, except activation and MTT reduction thatwere similar but showed low viability values. TB and PI dyes showedthe highest percentages of viability. Some techniques were problem-atic: intensity of oncosphere mobility was very variable in theactivation assay; neutral red crystals precipitated and hid someoncospheres, while with MTT there was spontaneous activation,although no change in color was evident.

3.3. In vivo assessment for oncosphere viability

Eggs obtained from experiments 2, 3 and 4were used to infect fourpiglets, each with 50,000 as described in Table 4. Eggs fromexperiments 2 and 3 produced a low rate of infection in two pigletsand only nonviable cysticerci were found (0.01 and 0.1% respectively),while in the other two piglets, infected with eggs from experiment 4,0.3% and 0.4% viable cysticerci were found respectively. Western blotfor diagnosis of cysticercosis performed with serum samples obtainedat 12 WPI showed some diagnostic bands in all four pigs (Fig. 4).

Table 4Data of the cysticerci recovered from pigs after experimental infection with eggsobtained from chinchillas harboring T. solium adult worms.

Experiment Necropsy(WPIa)

Number of cysticercirecovered from each pig

Percentinfectionb

Viable Nonviable

2 17 0 5 0.013 17 0 49 0.14 17 65 133 0.44 27 160 0 0.3

a WPI, weeks post-infection.b Calculated from the total number of cysticerci recovered from each pig after

infection with 50,000 eggs.

Fig. 4. Western blot for the diagnosis of human cysticercosis used in four pigs at12 weeks post infection; lane 1, positive control; lane 2, negative control; lanes 3–6,serum samples from pigs experimentally infected with eggs obtained from tapewormsrecovered from chinchillas. Arrows point to diagnostic bands.

Please cite this article as: Maravilla P, et al, Chinchilla laniger can be used as an experimental model for Taenia solium taeniasis, Parasitol Int(2011), doi:10.1016/j.parint.2011.06.002

6 P. Maravilla et al. / Parasitology International xxx (2011) xxx–xxx

3.4. Discomfort of chinchillas

Assessment of pain in infected chinchillas was evaluated inexperiment 5; results are summarized in Table 5. Body weight andbreathing where the clinical features that exhibited a statisticallysignificant detriment indicating moderate suffering, the remainingmorphological and behavioral features recorded (hiding, activity,posture, vocalization, sneezing and trembling) showed no evidentdifferences.

4. Discussion

The use of animal models for the study of taeniasis due to T. soliumhas been critical for understanding the host-parasite relationshipduring infection. Three rodent species have been successfully used:Syrian golden hamsters, Mongol gerbils and chinchillas. The first twoprovided significant results on the host's immune response and on thebiology of this cestode parasite in immunodeppressed and in non-deppressed hosts [3,7].The third one, chinchillas, is the only hostwhere tapeworms become gravid. It has been suggested that steroidsact as growth hormones for cestodes instead of being immunodep-pressants to the hosts (Taeniasis/Cysticercosis and EchinococcosisInternational Symposiumwith Focus on Asia and the Pacific and ThirdCongress of the Federation of Asian Parasitologists focused on CestodeZoonoses, www.pasean-zoonet.asahikawa-med.ac.jp). In fact, femalesex steroids facilitate infection, while male sex steroid hormonesreduce parasite loads [8]. Nevertheless, glucocorticoids, as MPA, havespecific receptors, target cells and biological effects and are welldocumented as potent anti-inflammatories and immunodeppressors[16]. No glucocorticoid receptor has been reported for helminths, thusthe effect found in rodents harboring T. solium is due to MPA'sconsequences on the immune system of the hosts. Since only onepaper related to chinchillas was published 12 years ago showing thata gravid tapeworm developed [4], here we report five experimentsperformed along four years in which outbred chinchillas and differentpigs with cysticercosis were used. Previous studies in other rodenthosts have shown that in immunodeppressed hamsters, the overallrecovery of non-gravid tapeworms is between 67 and 80%, and inimmunodeppressed gerbils it is 18–75% [3,4,7]. In the present studyan average of 21% recovery, similar to 27% reported by Maravilla et al.[4] was obtained. Although less efficient than the other two models,chinchillas, being outbred, are capable of allowing the full develop-ment of the tapeworm, since 8% of gravid parasites were obtained,

Table 5Scale to assess pain in rodents and lagomorphs under experimentation (Buckwell, 1999 [9]

Description Suffering

Low Moderate High

Body weight Without loss 20% loss N25%

Breathing Normal or fast Occasionally abnormal Dysp

Water consumption 25% less than normalduring 72 hr

40% less than normal during72 hr

60% l7 dayby 72

Hiding Partial hair-erection Standing on the edge, totalhair-erection, white nose,ears and palms

Totaldehyand c

Activity Depressed, with normalresponse under stimulation

Depressed, with low responseunder stimulation. Weakpartner's interaction

Indiffunde

Posture Occasionally shy Frequently shy Imm

Vocalization Occasional Frequent Contihand

Sneezing Low and occasional Frequent FrequTrembling Occasional Frequent with transitory

prostration (b1 h)Persiself m

Please cite this article as: Maravilla P, et al, Chinchilla laniger can be use(2011), doi:10.1016/j.parint.2011.06.002

indicating that this is the only experimental model (with theexception of one gibbon [17]) in which gravid T. solium tapewormsdevelop. Therefore, the data obtained show that chinchillas are astable and reproducible model. An important addition to this studywas the evaluation of discomfort in the immunodeppressed hosts. Ourdata indicate some degree of suffering; nonetheless a more stringentand less aggressive way to measure discomfort should be evaluated,because, added to the immunodeppressive treatment, frequentanimal handling in these evaluations was by itself violent.

Interestingly most parasites found in one host had similardevelopment and size, probably those recovered at 22 WPI (exper-iment 5) that were small and non-gravid could have released most ofits strobila and, although it was not possible to determine, it couldreflect the simultaneous release of hundreds of proglottids found insome chinchillas of experiments 2, 3 and 4. The presence of severalgravid worms in chinchillas also suggests that if more than onecysticercus is ingested, more than one tapeworm can develop up togravity, contradicting the knowledge that T. solium is a solitarytapeworm [1]. Unexpectedly gravid proglottids containedmuch lowernumbers of eggs than those found in those obtained from T. soliumexpelled from humans (around 60,000 [2]) probably becausechinchillas are less suitable hosts for this tapeworm. Also noassociation was found in the number of proglottids released and theabsorbance values obtained by the CpAg ELISA. This may be due to thefact that ELISA becomes positive even with small tapeworms, whichmakes it useful for diagnosis of taeniasis in prepatent humaninfections [10,18].

To our knowledge this is the only study where 5 viability methods,previously published individually as useful for defining the percent-age of viable oncospheres (except PI), were assayed with the samebatch of eggs, and an aliquot was also used to infect pigs and confirmthat they were infective. The best method, defined by the percent ofviability, the low variance and the easiness to perform, was stainingdead cells with propidium iodide, reported for Cryptosporidium[19],sperm and other cells. This evaluation was especially importantbecause there are studies that report adequate viability of tapewormeggs using activation [12,14,20–22], neutral red [22] or MTT [23,24].In the present study very low values were obtained with thesetechniques, indicating that the percent of viability reported by thedifferent authors is not comparable. Trypan blue performed almost aswell as Propidium Iodide, therefore either one can be used forassessing oncosphere viability; however, although these stains mayprovide a good approximation, it is important to have in mind that

) and results obtained in infected chinchillas during experiment 5.

Mean score

Before infection At 18 WPI

loss 622.6±79.3 517.6±25.9 (17% loss)P=0.002

noea or abnormal Normal (102.5±10.6) Occasionally abnormal(90.4±11.2) P=0.032

ess than normal durings or cessation of eatinghr

Normal Normal

standing on edge anddration, white nose, earsool palms

Partial hair-erection Evident areas ofalopecia in legs,face and abdomen

erence; non-responsiver stimulation and handling

Depressed, with low responseunder stimulation

Adynamic with lowresponse understimulation

ovable and paralyzed Occasionally shy Adynamic andfrequently shy

nuous, even withoutling

Occasional Occasional

ent and persistent Low and occasional Frequentstent prostration (N1 h),utilation and seizures

Occasional Frequent

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these techniques have been described to establish the viability insingle cells and not in multicellular organisms as are T. soliumoncospheres. Experimental cysticercosis in outbred hybrid Europeanpigs showed some diagnostic bands in western blot similar to naturalinfections [25] and had an efficiency of up to 0.4% comparable toexperimental infections using T. solium eggs recovered from humans[26]; indicating that eggs released by chinchillas are as infective asthose expelled by human carriers.

In summary, the main achievements of the present study are:i) reproducible infections at different times in outbred experimentalhosts; ii) follow up of infections by copro-antigen ELISA and byproglottid release counts; iii) comparison of different techniques forevaluation of oncosphere viability, iv) evaluation of rodent suffering;and v) reproducible pig infection. Our results demonstrate that theexperimental model of T. solium taeniasis in C. laniger is an alternativefor providing eggs and adult tapeworms to be used in different typesof experiments. Nevertheless optimization of the model probablydepends on the use of inbred hosts and on reduction of infectedanimals' suffering.

Acknowledgements

To Jorge Campos, Luis Fernandez and Pedro Palomares for theirsupport in animal care and handling.

References

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