Development and validation of quantitative PCR for detection of Terrapene herpesvirus 1 utilizing free-ranging eastern box turtles (Terrapene carolina carolina)

Journal of Virological Methods 232 (2016) 57–61

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Development and validation of quantitative PCR for detection of Terrapene herpesvirus 1 utilizing free-ranging eastern box turtles (Terrapene carolina carolina) Lauren P. Kane a,∗ , David Bunick a , Mohamed Abd-Eldaim b , Elena Dzhaman a , Matthew C. Allender a a b

Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, United States Virology Department, College of Veterinary Medicine, Suez Canal University Faculty of Veterinary Medicine, Ismailia, Egypt

a b s t r a c t Article history: Received 8 October 2015 Received in revised form 8 February 2016 Accepted 8 February 2016 Available online 10 February 2016 Keywords: Herpesvirus Eastern box turtle Epidemiology qPCR Terrapene carolina

Diseases that affect the upper respiratory tract (URT) in chelonians have been well described as a significant contributor of morbidity and mortality. Specifically, herpesviruses are common pathogens in captive chelonians worldwide, but their importance on free-ranging populations is less well known. Historical methods for the diagnosis of herpesvirus infections include virus isolation and conventional PCR. Real-time PCR has become an essential tool for detection and quantitation of many pathogens, but has not yet been developed for herpesviruses in box turtles. Two quantitative real-time TaqMan PCR assays, TerHV58 and TerHV64, were developed targeting the DNA polymerase gene of Terrapene herpesvirus 1 (TerHV1). The assay detected a viral DNA segment cloned within a plasmid with 10-fold serial dilutions from 1.04 × 107 to 1.04 × 101 viral copies per reaction. Even though both primers had acceptable levels of efficiency and variation, TerHV58 was utilized to test clinical samples based on less variation and increased efficiency. This assay detected as few as 10 viral copies per reaction and should be utilized in free-ranging and captive box turtles to aid in the characterization of the epidemiology of this disease. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Viruses from the order Herpesvirales are large, icosahedral, enveloped, double stranded, DNA viruses (McGeoch and Gatherer, 2005; Wellehan et al., 2004; Sim et al., 2015). This order is divided into three families, Herpesviridae, Alloherpesviridae, and Malacoherpesviridae (Davison, 2010). The family Herpesviridae is further divided into three subfamilies, Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae encompassing approximately 90 different species (Davison, 2010; ICTV, 2014). Currently, chelonian herpesviruses, in the genus Scutavirus, have DNA sequences most homologous to viruses in the alphaherpesvirus family (Bicknese et al., 2010; Greenblatt et al., 2005; Ossiboff et al., 2015a,b; Herbst et al., 2004; Origgi et al., 2015). As has been seen in mammals and birds, several chelonian herpesviruses have been associated with clinical signs (Salinas et al., 2011; Stacy et al., 2008; Pettan-Brewer et al., 1996; Johnson et al.,

∗ Corresponding author at: College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, United States. E-mail address: [email protected] (L.P. Kane). http://dx.doi.org/10.1016/j.jviromet.2016.02.002 0166-0934/© 2016 Elsevier B.V. All rights reserved.

2005). In tortoises, this virus has a predilection for mucoepithelial cells, commonly associated with rhinitis, conjunctivitis, stomatitis and glossitis (Salinas et al., 2011; Pettan-Brewer et al., 1996; Johnson et al., 2005; Ossiboff et al., 2015a). Captive Greek tortoises inoculated with herpesvirus developed necrotizing stomatitis and rhinitis (Origgi et al., 2004). A captive desert tortoise diagnosed with a herpesvirus infection exhibited anorexia, lethargy, and caseous glossal plaques (Johnson et al., 2005). In aquatic turtles, herpesvirus infections have been linked to skin disease (Rebell et al., 1975; Cowan et al., 2015; Yonkers et al., 2015). Marine turtles affected with Gray Patch Disease, associated with Chelonid herpesvirus 1, develop circular gray skin lesions (Rebell et al., 1975). The most common clinical sign seen in marine turtles, with a herpesvirus infection due to Chelonid herpesvirus 5, is development of neoplastic fibropapillomas (Jacobson et al., 1991; Quackenbush et al., 2001; Chaloupka et al., 2009). Chelonid herpesvirus 6 has been associated with lung-eye-trachea disease in marine turtles (Curry et al., 2000; Coberely et al., 2002; Jacobson et al., 1986). However, some reported herpesvirus infections are fatal before any premonitory signs are noted (Harper et al., 1982; Jungwirth et al., 2014). Reports of box turtle herpesvirus are scarce. The presence of a unique virus has been demonstrated to occur in three different col-

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lections (Sim et al., 2015; Yonkers et al., 2015). An eastern box turtle (Terrapene carolina carolina) which emerged early from brumation with lethargy, dehydration, and dyspnea died a week later and was diagnosed with herpesvirus on histopathology and PCR (Sim et al., 2015). A separate collection of turtles infected with ranavirus was opportunistically tested for herpesvirus via PCR and was found to have a 58% prevalence (Sim et al., 2015). A free ranging eastern box turtle with a papillomatous growth was diagnosed with a novel herpesvirus by nested PCR (Yonkers et al., 2015). Herpesvirus was diagnosed by PCR in a captive Australian Krefft’s river turtle (Emydura macquarii kreffti), which presented with severe proliferative and ulcerative skin and shell lesions (Cowan et al., 2015). A captive, juvenile, female northern map turtle (Graptemys geographica) was diagnosed with herpesviral pneumonia, which was confirmed by PCR, after exhibiting weakness and nasal discharge (Ossiboff et al., 2015a). Three novel herpesviruses were identified in free-ranging bog turtles (Glyptemys muhlenbergii), wood turtles (G. muhlenbergii), and spotted turtles (Clemmys guttata) by PCR (Ossiboff et al., 2015b). Additionally, two clinically healthy West African mud turtles (Pelusios castaneus) were diagnosed with a novel herpesvirus infection by PCR (Marschang et al., 2015). Nonetheless, the epidemiology in free-ranging eastern box turtle populations is still unknown. Several diagnostic methods have been used to diagnose herpesvirus infections in chelonians, including virus isolation and conventional PCR. Quantitative PCR (qPCR) has not been previously developed for the detection of Terrapene herpesvirus 1 (TerHV1) in chelonians and would provide increased sensitivity as compared to conventional PCR. This method would allow quantitation of viral copy numbers. Furthermore, it may allow detection of carriers with low levels of virus particles. Animals with subclinical infections may serve as important carriers or reservoirs for infectious disease; therefore, it is crucial for assay development to be capable of detecting pathogens in these animals. The quantification of viral copy numbers may also allow for therapeutic monitoring of infections. To date, no such assay has been reported for herpesvirus quantification in free-ranging or captive box turtles. The purpose of this study was to develop and evaluate diagnostic methods for detecting TerHV1 in eastern box turtles. The hypothesis tested in this study was that a Taqman qPCR assay would be sensitive for detecting Terrapene herpesvirus 1 (Order Herpesvirales, Family Herpesviridae, Subfamily Alphaherpesvirinae, Genus Scutavirus) in eastern box turtles. This quantitative method will be useful for early detection and disease monitoring in both captive and free-ranging chelonian populations.

2. Materials and methods 2.1. Conventional PCR, sequencing, and cloning An oral swab was collected from a suspect positive freeranging eastern box turtle in Tennessee determined by associated clinical signs, such as rhinitis, glossitis, or stomatitis. DNA was extracted according to the manufacturer’s instructions (QIAamp DNA blood mini kit, Qiagen, Valencia, California, USA). Nested PCR was performed as previously described for herpesvirus detection (VanDevanter et al., 1996) and later documented for detection of herpesviruses in chelonians (Marschang et al., 2006). Primary PCR contained two sense (DFA, 5 -GAYTTYGCNAGYYTNTAYCC3 ; and ILK, 5 -TCCTGGACAAGCAGCARNYSGCNMTNAA-3 ) and one anti-sense (KG1, 5 -GTCTTGCTCACCAGNTCNACNCCYTT-3 ) primers. Secondary PCR assays were performed with sense (TGV, 5 -TGTAACTCGGTGTAYGGNTTYACNGGNGT-3 ) and antisense (IYG, 5 -CACAGAGTCCGTRTCNCCRTADAT-3 ) primers targeting a herpesvirus-consensus region of the DNA polymerase gene.

Reactions were performed in 25 ␮L total volume using Premix taq (Takara Taq version) containing hot start Taq (TaKaRa Bio Inc.). Five ␮l of template DNA from the first step was used in the second reaction. Feline herpesvirus 1 and canine herpesvirus 1 were used as positive controls, while DNase/RNase-free water was used as a negative control. PCR products were resolved by electrophoresis in 1.4% agarose gels. Products were sequenced in both directions using the Molecular Biology Resource Facility at the University of Tennessee and compared to known sequences in GenBank using TBLASTX. Phylogenetic analysis of herpesvirus sequences obtained from clinical samples and the literature (Sim et al., 2015; KJ004665) were used to design a consensus 480 base pair gene segment (Integrated DNA Technologies, Coralville, Iowa) that was then cloned in Escherichia coli (TOPO TA Cloning® kit, Invitrogen, Carlsbad, CA). The cloning products were verified through sequencing in both directions (Keck Biotechnology Center at the University of Illinois). Plasmids were linearized with BamH1 (Clontech Laboratories, Mountain View, CA), purified (QIAfilter plasmid Maxi kit, Qiagen, Valencia, CA), and quantified using a spectrophotometer (Nanodrop spectrophotometer, Thermo Scientific, Wilmington, DE). Ten-fold serial dilutions of linearized plasmids were made from 1.04 × 109 ng/␮l to 1.04 × 10◦ ng/␮l. Viral genome (DNA) copy number was calculated using the following formula:



#copies/␮L =

  6.022 × 1023 copies/mol  

ng DNA of plasmid ± clone/␮L



9 (base per length) 1 × 10 ng/g

650g/mol of base pair

The final copy number for ten-fold serial dilutions ranged from 1.04 × 107 to 1.04 × 101 viral copies per reaction. 2.2. Real time qPCR assay Two TaqMan-MGB (Taqman® primers, FAM dye labeled, Applied Biosystems, Carslbad, CA) qPCR assays, TerHV58 and TerHV64, for TaqMan-MGB (Taqman® primers, FAM dye labeled, Applied Biosystems, Carslbad, CA) qPCR assays were designed using a commercial software program (Primer Express® , Applied Biosystems, Carlsbad, CA) based on the sequence of the TerHV1 DNA polymerase gene segment in our clone. The TaqMan with assay TerHV58 was performed using forward (5 -TCTATTGGGCGAGCTGTTGACreverse (5 -GAAAAGCCATACGCCTACGC-3 ), and 3 ), probe (5 -ACTGGCTGGCCTTG-3 ), targeting a 58 base pair segment of the herpesvirus DNA polymerase gene. The TaqMan with assay TerHV64 was perusing forward (5 -GGCCACCCGAGATTATATCCA-3 ) formed  reverse (5 -TTCTGGCCGACTTCCCG-3 ), and probe (5 CCCATTGGAGCGAACGGGAAAAG-3 ), targeting a 64 base pair segment of the herpesvirus DNA polymerase gene. Real-time qPCR assays were performed using a real-time PCR thermocycler (7500 ABI real-time PCR System, Applied Biosystems, Carlsbad, CA) and data was analyzed using associated software (Sequence Detection Software v2.05, Applied Biosystems, Carlsbad, CA). Each reaction contained 12.5 ␮l of 20x TaqMan Platinum PCR Supermix-UDG with ROX (TaqMan Platinum PCR Supermix-UDG with ROX, Invitrogen, Carlsbad, CA), 1.25 ␮l TaqMan primer-probe, 2.5 ␮l plasmid dilution, and water to a final volume of 25 ␮l. Cycling parameters were as follows: 1 cycle at 50 ◦ C for 2 min followed by 95 ◦ C for 10 min, then 40 cycles at 95 ◦ C for 15 seconds and 60 ◦ C for 60 seconds, and a final cycle of 72 ◦ C for 10 min. 2.3. Standard curve and sensitivity To determine the sensitivity, assays were performed in four technical repeats on dilutions of turtle-derived positive control plasmid of TerHV1 DNA (1.04 × 107 –1.04 × 101 copies/rxn) within a single run. Standard curves were generated using the cycle thresh-

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old (Ct ) values of the positive control plasmid dilutions. Intra-assay variation was determined for both assays by calculating the mean Ct values, standard deviations (SD), and coefficient of variations (CV) separately for each control plasmid DNA dilution (107 –101 ). Efficiency curves of the dilutions were performed in uninfected cell culture lysates (spiked with plasmid dilutions) and turtle oral swab extracts from negative samples, with high (67.56 ng/␮l) and low (8.61 ng/␮l) total DNA concentrations, as measured by spectrophotometry. 2.4. Box turtle samples DNA extracts from twenty-seven eastern box turtles from Tennessee were assayed using conventional PCR in 2007. These samples were assayed by qPCR and repeated for conventional PCR seven years later. The level of agreement was compared between the results of the conventional PCR and qPCR. The quantity of TerHV1 target DNA in oral swabs was determined using a standard curve method. The copy number of the target DNA was determined from the standard curve generated with ten-fold dilutions of the positive control plasmid for each assay. 2.5. Statistical analysis Copy numbers were tabulated and evaluated for normality using the Shapiro–Wilk test. Mean, median, standard deviation, 95% confidence interval, and 10–90% percentiles were determined for positive cases (copy number) for each assay. The prevalence of TerHV1 was determined (categorical variable assigned; 1 = positive, 0 = negative) and 95% binomial confidence intervals were determined. Level of agreement (kappa) was determined between both the real-time PCR assays and the conventional PCR previously reported based on prevalence. All statistical analysis was performed using statistical software (SPSS Statistics 22, IBM, Chicago, IL). 3. Results 3.1. PCR, standard curve, reproducibility Serial ten-fold dilutions of positive control plasmids assayed with our TaqMan assay generated standard curves based on Ct values (Fig. 1). The linear range for TerHV58 was between 1.04 × 107 to 1.04 × 101 with an R2 of 1 (slope = −3.389) (Fig. 1). Low and high DNA dilution efficiency curves carried out on spiked-uninfected cell lysates and turtle oral swab extracts resulted in an equal performance between the low (slope = −3.327; R2 = 0.998) and the high DNA samples (slope = −3.354; R2 = 1). The amplification plot of TaqMan qPCR TerHV58 are presented in Fig. 2. The intra- and inter-assay reproducibility was evaluated for the serial dilutions of the control plasmids. The intra-assay CVs for TaqMan TerHV58 was between 0.04–0.27% and the inter-assay CVs were 0.28–1.70%, respectively (Table 1). The intra-assay CVs for TaqMan TerHV64 was between 0.15–0.33% and the inter-assay CVs were 0.34–1.95%, respectively. The results indicate high reproducibility between assays at all dilutions. The dynamic range for qPCR assays at which point the Ct value of the technical repeats was from 1.04 × 107 to 1.04 × 101 . The TaqMan primer sets, TerHV58 and TerHV64, were successful in detecting either a 58 or 64 base pair length gene segment, respectively, of a conserved portion of the DNA polymerase gene of TerHV1. Both primers had acceptable level of efficiency and variation but TerHV58 was more efficient, based on the standard curve slope, and had less variation, R2 value, as compared to TerHV64, and was the primer utilized in testing clinical samples.

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3.2. Box turtle samples Twenty-seven oral swabs were collected from eastern box turtles that presented to the University of Tennessee from March through October 2007. Previous conventional PCR assay determined an 11% (n = 3) prevalence rate for these samples (data not shown). The same samples were re-tested by conventional PCR for this study and a 3.4% (n = 1) prevalence rate was determined. Quantitative PCR utilizing primer TerHV58 determined an 11% (n = 3) prevalence of TerHV1, in the same three individuals as conventional PCR in 2007. Observed kappa statistic was 1.0 when comparing 2007 results with qPCR, demonstrating perfect agreement, however agreement (kappa = 0.36) was low when comparing 2015 conventional PCR results with qPCR. The DNA viral copy numbers of the three positive samples determined by qPCR was 890.17, 25.80, 112.83 with a mean purity (A260/A280) of 1.74, as measured by spectrophotometry.

4. Discussion Herpesviruses have been well described in various reptilian species, especially chelonians, and may pose a threat to affected populations (Johnson et al., 2005; Stacy et al., 2008; Sim et al., 2015; Ossiboff et al., 2015b). Overall, there is limited published information regarding herpesviruses in box turtle species, particularly in free-ranging populations (Sim et al., 2015; Yonkers et al., 2015; Ossiboff et al., 2015a,b; Cowan et al., 2015; Marschang et al., 2015). Epidemiologic surveys that characterize the species range and disease extent rely on a sensitive diagnostic assay. A quantitative PCR assay was developed that allows detection of a DNA polymerase gene segment of TerHV1 with as few as 10 viral copies per reaction. The detection and quantification of viral copies may allow for earlier therapeutic intervention to reduce morbidity and mortality rates in box turtles as has been observed with other species (Staton et al., 2010). Taqman qPCR assays utilize a third hybridization or probe in the selection step, which allows for increased specificity of qPCR when testing clinical samples. The assay had highly reproducible intra- and inter-assay variability coefficient of variation of less than 5%. Assay TerHV58 was more sensitive than assay TerHV64, as seen with a lower coefficient of variation at all dilutions. TerHV58 was designed to be specific for a segment of DNA polymerase gene which previous studies have targeted (Murakami et al., 2001; Marschang et al., 2006). One of the biggest limitations of this assay was lack of specificity determination. While this assay was developed specifically for the detection of a unique sequence of TerHV1, future studies should evaluate the specificity of this assay and its application in diagnosing presence of viral DNA in turtle samples that may possess additional herpesviruses. DNA from twenty-seven turtle oral swabs collected in 2007 was screened for TerHV1 using conventional at the time of collection and again in 2015, as well as with the developed qPCR. Conventional PCR detection decreased in the 8 subsequent years since collection. Differences between prevalence detection by conventional PCR may be attributed to the DNA samples being stored at −20 ◦ C with multiple freeze-thaw cycles (Shao et al., 2012). In contrast, use of qPCR revealed equal prevalence detection as the original conventional PCR screening. Despite the high agreement, as stated by kappa, between the conventional PCR eight years prior and the qPCR assay, qPCR allowed quantification of the viral copies where conventional PCR could not. The storage method may also have degraded DNA of weak positive samples overtime and prevalence may have been higher if qPCR was available at the time of collection. Dual testing (conventional and qPCR methods) on the

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Fig. 1. Standard curve for a Taqman probe-based primer TerHV58 obtained with 10-fold serial dilutions from 1.04 × 107 to 1.04 × 101 viral copies per reaction. The graph is plotted against a logarithmic concentration of the serial dilutions.

Fig. 2. Amplification plot for the standard curve for TaqMan probe-based primer TerHV58 obtained with 10-fold serial dilutions from 1.04 × 107 to 1.04 × 101 viral copies per reaction.

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Table 1 Intra- and inter-assay variability for a Taqman probe-based primer TerHV58 obtained with 10-fold serial dilutions from 1.04 × 107 to 1.04 × 101 viral copies per reaction for detection of Terrapene herpesvirus 1 DNA polymerase gene. Intra-assay

Inter-assay

Viral copy

CT mean

CT SD

CV

CT mean

CT SD

CV

10,400,000 1,040,000 104,000 10,400 1040 104 10 NTC

15.93 19.32 22.89 26.32 29.79 32.88 36.13 38.81

0.03 0.03 0.01 0.03 0.08 0.08 0.06

0.19% 0.16% 0.04% 0.11% 0.27% 0.24% 0.17%

15.81 19.15 22.8 26.12 29.39 32.67 35.81

0.1 0.2 0.1 0.2 0.5 0.3 0.1

0.63% 1.04% 0.44% 0.77% 1.70% 0.92% 0.28%

same samples suggests that indeed the prevalence of infection was low in this population of box turtles. The current study demonstrates that a TaqMan assay is reliable and sensitive for the detection of a DNA polymerase gene segment of TerHV1. The TaqMan assay has low variability and is a recommended assay for free-ranging eastern box turtle herpesvirus surveys. This assay is capable of detecting a low number of viral copies, which may allow for early or subclinical identification. This assay should be used as a conservation tool in eastern box turtles and can help characterize the epidemiology of herpesvirus. References Bicknese, E.J., Childress, A.L., Wellehan, J.F.X., 2010. A novel herpesvirus of the proposed genus chelonivirus from an asymptomatic bowsprit tortoise (Chersina angulata). J. Zoo Wildl. 41, 353–358. Chaloupka, M., Balazs, G.H., Work, T.M., 2009. Rise and fall over 26 years of a marine epizootic in Hawaiian green sea turtles. J. Wildl. Dis. 45, 1138–1142. Coberely, S.S., Condit, R.C., Herbst, L.H., Klein, P.A., 2002. Identification and expression of immunogenic proteins of a disease-associated marine turtle herpesvirus. J. Virol. 76, 10553–10558. Cowan, M.L., Raidal, S.R., Peters, A., 2015. Herpesvirus in a captive Australian Krefft’s river turtle (Emydura macquarii krefftii). Aust. Vet. J 93, 46–49. Curry, S.S., Brown, D.R., Gaskin, J.M., Jacobson, E.R., Ehrhart, L.M., Blahak, S., Herbst, L.H., Klein, P.A., 2000. Persistent infectivity of a disease-associated herpesvirus in green turtles after exposure to seawater. J. Wildl. Dis. 36, 792–797. Davison, A.J., 2010. Herpesvirus systematics. Vet. Microbiol. 143, 52–69. Greenblatt, R.J., Quackenbush, S.L., Casey, R.N., Rovnak, J., Balazs, G.H., Work, T.M., Casey, J.W., Sutton, C.A., 2005. Genomic variation of the fibropapilloma-associated marine turtle herpesvirus across seven geographic areas and three host species. J. Virol. 79, 1125–1132. Harper, P.A., Hammond, D.A., Heuschele, W.P., 1982. A herpesvirus-like agent associated with a pharyngeal abscess in a desert tortoise. J. Wildl. Dis. 18, 491–494. Herbst, L., Ene, A., Desalle, R., Lenz, J., 2004. Tumor outbreaks in marine turtles are not due to recent herpesvirus mutations. Curr. Biol. 14, R697–699. ICTV, International Committee on Taxonomy of Viruses. (2014). Herpesviridae: Virus Taxonomy 2014 Release. Jacobson, E.R., Gaskin, J.M., Roelke, M., Greiner, E.C., Allen, J., 1986. Conjunctivitis, tracheitis, and pneumonia associated with herpesvirus infection in green sea turtles. J. Am. Vet. Med. Assoc. 189, 1020–1023. Jacobson, E., Buergelt, C., Williams, B., Harris, R.K., 1991. Herpesvirus in cutaneous fibropapillomas of the green turtle Chelonia mydas. Dis. Aquat. Organ. 12, 1–6. Johnson, A.J., Pessier, A.P., Wellehan, J.F.X., Brown, R., Jacobson, E.R., 2005. Identification of a novel herpesvirus from a California desert tortoise (Gopherus agassizii). Vet. Microbiol 111, 107–116. Jungwirth, N., Bodewes, R., Osterhaus, A.D., Baumgartner, W., Wohlsein, P., 2014. First report of a new alphaherpesvirus in a freshwater turtle (Pseudemys concinna concinna) kept in Germany. Vet. Microbiol. 170, 403–407. Marschang, R.E., Gleiser, C.B., Papp, T., Pfitzner, A.J., Bohm, R., Roth, B.N., 2006. Comparison of 11 herpesvirus isolates from tortoises using partial sequences from three conserved genes. Vet. Microbiol 117, 258–266. Marschang, R.E., Heckers, K.O., Heynol, V., Weider, K., Behncke, H., 2015. Herpesvirus detection in clinically healthy West African mud turtles (Pelusios castaneus). Tierarztl. Prax. Ausg. K. Kleintiere. Heimtiere 43, 166–169.

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