ORIGINAL PAPER

In vitro development of Eucoleus böhmi eggsin different environmental conditions

Stefania Perrucci & Angela Di Cesare & Gianluca Fichi

Received: 20 January 2014 /Accepted: 15 April 2014 /Published online: 29 April 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The development of Eucoleus böhmi eggs wasevaluated in vitro in order to acquire information on the lifecycle of this neglected respiratory nematode affecting canids.Fecal cultures were prepared using fecal samples from apositive dog and maintained at different conditions of temper-ature (20±1 and 5±1 °C) and relative humidity (RH) (85±5and 45±5 %). Egg development was microscopically exam-ined at days +7, +15, and +30. In addition, in order to assessthe vitality of eggs maintained at 5±1 °C for 30 days, theselatter cultures were moved, placed at 20±1 °C and 85±5 %RH, and observed for further 30 and 40 days. The resultsshowed that at 20±1 °C and 85±5 % RH, the totality of eggscompleted development in 30 days, while about 26 and 70 %of eggs were already fully developed after 7 and 15 days,respectively. No egg development occurred after 30 days at 5±1 °C, while 100 % of eggs placed at 5±1 °C for 30 days andthen moved at 20±1 °C and 85±5 % RH for further 40 dayswere found fully developed.

Keywords Eucoleus böhmi . In vitro development . Eggs .

Nasal eucoleosis . Dog

Introduction

Eucoleus böhmi (syn. Capillaria boehmi) (Gibbons 2010) isthe etiological agent of the nasal eucoleosis (capillariosis) ofcanids, a likely underestimated cause of upper respiratory tractsigns in dogs (Piperisova et al. 2010; Baan et al. 2011;Veronesi et al. 2013). Adults of E. böhmi are small, whitishnematodes measuring 12 to 64 mm in length and 0.08 to0.15 mm in diameter, which live threaded through the mucosaof nasal turbinates and frontal and paranasal sinuses of dogsand wild canids (Boch and Supperer 1980; Baan et al. 2011).This nematode is similar in morphology to Eucoleusaerophilus (syn. Capillaria aerophila), a closely related spe-cies that lives embedded underneath the epithelium of trachea,bronchi, and bronchioles of several carnivores (Traversa et al.2011), which is likely mistaken with E. böhmi andmisidentified in cases of nasal eucoleosis of dogs (Campbelland Little 1991; Conboy 2009). Eggs of E. böhmi may befound in nasal discharges or in the feces of infected animalsand are passed in a partial stage of embryonation, in which thedeveloping larva often appears square or rectangular(Campbell and Little 1991; Schoning et al. 1993). These eggs,45–60 μm (length) × 30–35 μm (width), are clear to golden incolor, barrel-shaped, plugged at each pole, often asymmetri-cal, and show a space between the embryo and the wall, whichtypically presents tiny pits on its surface (Schoning et al. 1993;Piperisova et al. 2010; Baan et al. 2011). Although in the lastfew years, symptomatic and asymptomatic cases of nasaleucoleosis have been frequently reported in dogs fromEurope and North America (Gajewska et al. 2004;Piperisova et al. 2010; Baan et al. 2011; Di Cesare et al.2012; Magi et al. 2012; Conboy et al. 2013; Veronesi et al.2013), the life cycle of E. böhmi is still completely unknown.It is believed (Campbell and Little 1991; Anderson 2000;Conboy 2009) that it can be either direct by the ingestion oflarvated eggs or possibly indirect by the ingestion of

S. Perrucci (*)Dipartimento di Scienze Veterinarie, Università di Pisa, Viale dellePiagge 2, 56124 Pisa, Italye-mail: perrucci@vet.unipi.it

S. Perruccie-mail: stefania.perrucci@unipi.it

A. Di CesareDipartimento di Scienze Biomediche Comparate, Università degliStudi di Teramo, Piazza Aldo Moro 45, 64100 Teramo, Italye-mail: adicesare@unite.it

G. FichiIstituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana,S.S. dell’Abetone e del Brennero 4, 56123 Pisa, Italye-mail: gianluca.fichi@izslt.it

Parasitol Res (2014) 113:2687–2691DOI 10.1007/s00436-014-3924-7

invertebrate facultative intermediate hosts, as in the case of thelife cycle of E. aerophilus (Traversa et al. 2011).

Given the complete lack of knowledge on E. böhmi lifecycle, the present study aimed to acquire novel information onthe development of E. böhmi eggs in different environmentalconditions.

Materials and methods

Collection, identification, and quantification of E. böhmi eggs

Fresh fecal samples (n. 16) were collected weekly from anasymptomatic privately owned dog ageing ~2 years fromJanuary and April 2012. All samples were quali-quantitatively analyzed for presence and number ofE. böhmi eggs per gram of feces (EPG) with a fecal flotationtest and a modified McMaster technique with a sensitivity of20 EPG (Permin and Hansen 1998; Taylor et al. 2007).Morphological and morphometric analyses of retrieved eggswere performed by using a Leica DM LB microscope and aLeica DFC 290 photocamera supported by the software LeicaLAS V 3.8.

Cultivation of E. böhmi eggs

Eggs were cultured the same day of collection as previouslydescribed (Henriksen and Korsholm 1983) for gastrointestinalstrongyle eggs. Briefly, each fecal culture was made by plac-ing 15 g of feces in a plastic drinking cup that had beenshortened so as to hold about 50 ml. The opening of the vesselwas covered with a double layer of gauze that was fixed withthe remaining part of the plastic cup. The vessel was placed ina reversed position inside an intact plastic drinking cup towhich about 30 ml of water was either added or not, depend-ing upon the relative humidity value at which each culture wasplaced. All cultures were then placed in an incubator (HighPerformance Incubator 2800, Galli, Milan, Italy) at differentconditions of temperature (20±1 and 5±1 °C) and relativehumidity (RH) (85±5 and 45±5 % measured with a

hygrometer) and microscopically examined to evaluate devel-opment progress at days +7, +15, and +30. In addition, inorder to assess the vitality of eggs maintained at 5±1 °C for30 days, these latter cultures were moved, placed at 20±1 °Cand 85±5 % RH, and observed for further 30 and 40 days.Through the study period, each experiment was conducted intriplicate. At microscopic evaluation, the percentage of devel-oped eggs in each culture was estimated on the basis of thenumber of eggs containing a mobile larva on the total eggnumber counted in 4 g of each culture at quantitative analysis.Morphological analyses of eggs were performed by using aLeica DM LB microscope and a Leica DFC 290 photocamerasupported by the software Leica LAS V 3.8.

Data analysis

The mean dimensions (±standard deviation) of E. böhmi eggsand the mean percentage (±standard deviation) of the EPGnumber found in all samples were calculated for each day ofexamination. The chi-square test (Glantz 2003) was used toevaluate the variability among the different percentages ofmature eggs found in cultures placed in the same environmen-tal conditions but made with fecal samples collected in differ-ent weeks and among the different percentages of mature eggsfound in samples placed at different environmental conditions.In addition, the confidence interval (95 % CI) of the percent-age of developed eggs was also calculated.

Results

All fecal samples collected from the examined dog resultedpositive for the presence of trichuroid eggs at qualitativeanalysis. Eggs measured (mean ± standard deviation of 74eggs) 56.65±2.25 μm by 30.62±1.14 μm, while plugs mea-sured about 4.49±1.46 μm in height and 7.47±1.16 μm inwidth. Each egg contained a developing larva that wassurrounded by a space separating it from the shell (Fig. 1a).Tiny pits were clearly visible on the surface of the egg shell(Fig. 1b). For their size and morphological features, they were

Fig. 1 Eucoleus böhmi eggsisolated from fresh dog fecalsamples. a E. böhmi eggcontaining a typical developingembryo (×1,000 magnification;scale bar 7 μm). b Outer smallpits clearly visible on the surfaceof E. böhmi egg shell (×1,000magnification; scale bar 7 μm)

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identified as typical E. böhmi eggs (Schoning et al. 1993;Piperisova et al. 2010; Baan et al. 2011). At the quantitativeanalysis, an average number of 828±595 EPG was counted inthe examined samples. Each culture contained about 12,420±8,925 eggs and about 3,300±2,400 eggs/culture at each envi-ronmental condition were examined for development.

All eggs were found still completely undeveloped after 7, 15,and 30 days of cultivation at 5±1 °C, both at 85±5 and at 45±5 % RH. Similarly, undeveloped eggs were found in +7-day

cultures placed at 20±1 °C and 45±5 % RH, while 100 % ofeggs were completely developed after 30 days at 20±1 °C and85±5 % RH (Table 1; Fig. 2a, b). No eggs or a few number, inaverage less than 1 % of the EPG number found in the respec-tive sample, of morphologically altered eggs (Fig. 2c) werefound in samples cultured at 20±1 °C and 45±5 % RH andexamined after 30 days (Table 1). At 20±1 °C and 85±5%RH,the rate of complete development after 7 days (25.83 %, 95 %CI 14.51–37.16) was low (Fig. 2d), while most eggs (69.77 %,95 % CI 59.13–80.40) contained a mobile larva at day +15(Table 1). Indeed, the rate of development resulted highlydifferent (P<0.01) between +7- and +15-day cultures. On thecontrary, no significant differences emerged among repetitionsof cultures kept at 20±1 °C and 85±5 % RH for 7 or for15 days. A high mean percentage (53.81 %, 95 % CI 42.48–65.15) of fully developed eggswas counted in samples placed at5 °C and 85±5% or 45±5%RH for 30 days and then shifted at20±1 °C and 85±5%RH for further 30 days, while in the samecultures, 100 % of eggs were fully developed after 40 days.

Discussion

E. böhmi is a neglected and poorly known nematode respon-sible for nasal eucoleosis in domestic and wild canids.

Fig. 2 Eucoleus böhmi eggs aftercultivation in different conditionsof temperature and relativehumidity (RH). a Fully developedE. böhmi egg after 30 days ofcultivation at 20±1 °C and 85±5 % RH (×1,000 magnification;scale bar 7 μm). b Outer smallpits clearly visible on the surfaceof the shell of a fully developedE. böhmi egg after 30 days at 20±1 °C and 85±5 % RH (×1,000magnification; scale bar 7 μm). cMorphologically altered egg ofE. böhmi found after 30 days incultures placed at 20±1 °C and 45±5 % RH (×1,000 magnification;scale bar 7 μm). dDeveloped (onthe left) and undeveloped (on theright) E. böhmi egg found after7 days of cultivation at 20±1 °Cand 85±5 % RH (×400magnification; scale bar 15 μm)

Table 1 Mean percentages (%) and 95 % confidence intervals (95 % CI)of mature Eucoleus böhmi eggs found at +7, +15, and +30 days incultures placed at different conditions of temperature (20±1 and 5±1°C) and relative humidity (RH) (85±5 and 45±5 %)

T° and RH +7 days +15 days +30 days

20±1 °C and85±5 % RH

25.83 %*; 95 %CI 14.51–37.16

69.77 %*; 95 %CI 59.13–80.40

100 %*

20±1 °C and45±5 % RH

0 0 –a

5±1 °C and85±5 % RH

0 0 0

5±1 °C and45±5 % RH

0 0 0

aNo eggs or a few numbers of morphologically altered eggs

*P<0.01

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Although often responsible for subclinical infections(Campbell and Little 1991; Schoning et al. 1993; Di Cesareet al. 2012), in the last years, E. böhmi has been frequentlyreported as a cause of respiratory symptoms with varyingdegrees of severity in dogs (Piperisova et al. 2010; Baanet al. 2011; Conboy et al. 2013; Veronesi et al. 2013).Aberrant intracranial migration of E. böhmi has also beenrecently reported in a dog showing generalized convulsiveseizures (Clark et al. 2013). Diagnosis of E. böhmi infectionis usually achieved by detection of eggs in the feces or of adultworms and/or eggs during cytologic evaluation of nasalflushes, rhinoscopy, nasal biopsy, and on postmortem grossand histopathological evaluation of the nasal cavities or si-nuses (King et al. 1990; Campbell and Little 1991; Schoninget al. 1993; Piperisova et al. 2010; Baan et al. 2011; Clark et al.2013; Veronesi et al. 2013). The present results, even thoughnot comparative, showed that copromicroscopic assays maybe indeed useful to diagnose nasal eucoleosis, given therelative number of eggs detectable at the quali-quantitativetechniques applied.

Knowledge of E. böhmi life cycle, including its environ-mental development, is fundamental to develop effective con-trol measures useful to prevent the infection and the frequentrecurrence after an anthelmintic treatment (Baan et al. 2011;Veronesi et al. 2013). The present study provides novel infor-mation on the biology of this little known trichuroid nema-tode. The results may indicate that environmental tempera-tures mimicking spring (20±1 °C) and winter (5±1 °C) inareas of temperate zones where E. böhmi is reported (DeLiberato et al. 2009; Piperisova et al. 2010; Baan et al. 2011;Di Cesare et al. 2012; Magi et al. 2012; Conboy et al. 2013;Veronesi et al. 2013) may favor or inhibit the environmentaldevelopment of this parasite, respectively. Additionally, theherein data show that E. böhmi eggs are able to survive for atleast 30 days in winter-like conditions and to continue theirdevelopment in more favorable temperature conditions inwhich they mature in about 30 to 40 days. Considering thatE. böhmi life cycle is hypothesized (Campbell and Little 1991;Conboy 2009; Anderson 2000) to be direct, via the ingestionof larvated eggs, the evidence that some eggsmay develop in avery short time (i.e., 7 days) may explain the high frequencyof recurrence episodes observed in infected dogs after thetreatment (King et al. 1990; Baan et al. 2011; Conboy et al.2013; Veronesi et al. 2013). Moreover, RH appeared an im-portant factor limiting the survival of E. böhmi eggs in theenvironment at spring temperature values, because in thisstudy no eggs or morphologically altered eggs were foundafter 30 days at 20±1 °C and 45±5 % RH. From the compar-ison of these results with those reported for the closely relatedspecies E. aerophilus, some differences emerged. In fact, arecent study (Traversa et al. 2011) demonstrated that at 20±1 °C and 80–5 % RH, most E. aerophilus eggs reach theinfective stage after 60 days, while the rate of complete

development after 35 and 45 days was about 30 % and about50–70 %, respectively. In addition, no eggs of E. aerophiluswere found completely mature after 15 days. Hence, thepresent results suggest that the environmental developmentof E. böhmi could be shorter of about 4 weeks if compared tothat of E. aerophilus. In particular, differently fromE. aerophilus (Traversa et al. 2011; Taylor et al. 2007),E. böhmi eggs are eliminated in a partial stage ofembryonation, i.e., contain a developing larva. Therefore, thisdifference could represent an important factor responsible forthe shorter duration of E. böhmi environmental cycle.

In conclusion, data from this study on E. böhmi egg devel-opment in different environmental conditions provide usefulinformation on the environmental cycle of this little knowntrichuroid nematode and represent the first step towards theknowledge of E. böhmi life cycle, which still needs to beaddressed and elucidated.

Conflict of interest The authors declare that they have no conflict ofinterest.

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