ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah Acta Herpetologica 6(1): 1-10, 2011 Widespread bacterial infection affecting Rana temporaria tadpoles in mountain areas Rocco Tiberti Department of Animal Biology, University of Pavia, Via Ferrata 1, I-27100 Pavia, Italy. E-mail: rocco. tiberti@unipv.it Submitted on: 2010, 29th January; Revised on:2010, 15th November; Accepted on:2010, 30th December. Abstract. Periodic mass die-offs of Rana temporaria tadpole populations have occurred in the ponds of prealpine mountain areas of Brescia (northern Italy) since the early 2000s. The author reports some observational data and analytical results from three sites: tadpoles from mortality events had erythema, especially on the legs, suggestive of septicemia. Bacterial culture of these tadpoles revealed Aeromonas hydrophila and Aeromonas sobria, two organisms often associated with Red leg dis- ease. Egg mass counts from 29 pastureland ponds did not revealed breeding activity declines over five years in the Monte Guglielmo area. Aeromonas hydrophila and Aer- omonas sobria usually behave as opportunistic bacteria that can become pathogenic after suppression of the immune system by endogenous or exogenous stressors. Thus, a plurality of environmental factors may contribute to mortality events; some of them are discussed, including loss of high altitude breeding ponds resulting in overcrowd- ing and poor water quality in remaining ponds and the presence of other pathogens. Keywords. mass die-offs, Red leg, Aeromonas sp., Rana temporaria, tadpoles. INTRODUCTION Since 1990, an increasing number of studies have reported declines in amphibian populations (Wake, 1991), and several contributing factors have been proposed (Gard- ner, 2001), including disease (Berger et al. 1998; Daszak et al., 2000; Kiesecker et al., 2001; Kiesecker et al., 2004). Therefore a lot of conservation efforts have been created by several organizations attempting to understand and monitor the decline. The need of taking full account of each episode of abnormal mortality has been emphasized. In the prealpine mountain areas of Brescia (Southern Italian Alps), mass die-offs among Rana temporaria tadpoles have been reported since the early 2000s. The purpose of this text is documenting the occurrence of these mortality events using retrospective data from Bagolino and Odeno and new data from Monte Guglielmo Massif. Therefore, 2 R. Tiberti from 2005 to 2009, in order to highlight any effect of mass die-offs on populations, Rana temporaria breeding activity has been monitored in the Monte Guglielmo area by count- ing egg masses in 29 ponds and collecting biological samples, including live and dead tad- ples, cloacal swambs on adult animals and water samples. MATERIAL AND METHODS Study area. All the samples have been collected in high altitude breeding ponds. Usually the ponds are artificially dug in high altitude pastures for watering cows; ponds persistence depends on human maintenance. The studied ponds are shallow (from a few centimeters to just over one meter) and subject to strong daily and seasonal variations of chemical and physical parameters; because of cattle presence the ponds are regularly subject to nutrient pollution in summer time. The studied areas have a typical mountain climate and the ponds freeze during the winter; in a few cases, they dry at the end of summer. Samples come from three distinct prealpine areas of Brescia (northern Italy): Bagolino, Odeno and Monte Guglielmo (Fig. 1). The sampling sites cover a wide longitudinal range in the prealpine area of Brescia. Bagolino (Lat.: 45°49’35’’; Long.: 10°27’38’’; Alt.: 769 m a.s.l.) is a village in the valley of the river Caffaro on the right side of Sabbia valley; Odeno (Lat.: 45°44’50’’; Long.: 10°20’12’’; Alt.: 917 m a.s.l.) is a hamlet of Pertica Alta in the Sabbia valley; Monte Guglielmo is a rather isolated mountain range that straddles the watershed between Trompia valley and Camonica valley. There are no further information on the exact location of sampling site from Bagolino and Odeno, while accurate data are available for Monte Guglielmo as shown in Fig. 1 and Table 2. Sampling and analytical methods. In order to closely observe any sign of disease, several frogs and tadpoles were captured using a landing net or with bare hands. Some of the caught animals underwent further treatments or were sampled: five adult frogs underwent cloacal swamb sam- pling for bacterial examination; fecal samples were collected gently rubbing the perianal area of adult frogs using sterilized swambs with culture medium; treated animals were immediatly released. Moreover several infected tadpoles (dead or still alive) were sampled; dead tadpoles were stored in test tube, while still alive tadpoles in containers allowing their survival until the refrigeration. Final- ly, five water samples from the infected sites were collected for bacteriological analysis. All samples were preserved at low temperature (4 °C) and examined within one day in the IZS (Istituto Zoo- profilattico Sperimentale) in Brescia. Each sample underwent bacteriological examination; parasito- logical and viral examinations were performed for the tadpoles sampled in Monte Guglielmo area. Details of the analytical methods are given in the following paragraphs. Parasitological examination. Microscopic examinations were performed to identify several parasites: 1-2 drops of sample were diluted in Lugol solution to observe flagellate and ciliate proto- zoans; nematodes and cestodes eggs were separated by flotation in a sodium nitrate satured solution; trematodes were separated by sedimentation and centrifugation and gross specimens were observed. Bacteriological examination. Since Rana temporaria is an ectothermic organism, the sample was obtained from internal tissues and organs and was directly cultured on Gassner Agar and Blood Agar plates and stored at variable temperature for 24-48 hours. Further, sub cultures were trans- ferred from Agar plates to differential and selective media and the morphology of the colony was observed; bacterial colonies were identified at the specific level thanks to specific biochemical test. Enterobacteria and non-enterobacteria identification was performed thank to identification systems as API-20E and API-20NE. Viral examination. Viral examination with Transmission Electron Microscopy (TEM) were performed directly on aqueous suspensions of internal organs and tissues to find mature virions. The samples were repeatedly frozen and thawed to facilitate the release of viral particles in the aque- http://en.wikipedia.org/wiki/Caffaro_(river) http://en.wikipedia.org/wiki/Valle_Sabbia 3Bacterial infection affecting Rana temporaria tadpoles ous suspension. The suspension was centrifuged twice at low speed (6000 and 10000 rpm, for 20 minutes) to remove coarse debris; a small amount of the second supernatant was added to micro- tubes containing formvar-coated copper grids for TEM and underwent ultracentrifugation (80000 rpm). The grid was drawn and coloured with 2% sodium phosphotungstate (NaPT) for 1.5 minutes and finally examined by TEM. Data analysis. Since 2005 to 2009, the author had been looking for the effects of mass die- offs on the abundance of Rana temporaria populations. Thus Rana temporaria breeding activity in the Monte Guglielmo massif was monitored by counting egg masses in 29 ponds. The presence of 4 metapopulations of Rana temporaria (A-D) was assumed to test for population declines; each metap-   Fig. 1. Study area; (1): position on the alpine range; (2): position in the provincial administration of Brescia; (3): studied ponds and groups of ponds (A-D) in Monte Guglielmo area. 4 R. Tiberti opulation was sustainded by a group of ponds less than one kilometer far away one from another (Fig. 1). Three different Linear Mixed-Effects model (LME) fitted by Restricted Maximum Likelihood had been used to explore the effects of time on the number of egg-masses layed in the ponds (Eggs), as a dependent variable. A time variable (Year) and a binary variable standing for Triturus carnifex pres- ence (Tricar) (which looked likely to be important in determining Rana temporaria breeding sites) had been added as fixed effects. The group of ponds (Group) had been added as random effect and each pond (Pond) as nested random effect. The models were compared each other using a Log-Likelihood Ratio Test and the accuracy of the last model was verified by testing the normality of the residuals. RESULTS Early signs of disease were observed in the advanced stage of larval development and rarely before the appearance of hind legs. Initial signs of disease included pettechia and ecchymosis within the caudoventral and lateroventral body and tail. In the later stages of the disease, larvae swam slowly (lethargy) even when pursued by for capture. In the ter- minal stage of the disease, moribund individuals hung listlessly at the surface or at the bottom of the pond. The disease was highly contagious, often involving the entire tadpole population in the infected pond and most of the ponds, even in large areas. Nevertheless adult Rana temporaria or other anuran (Bufo bufo) and urodele (Triturus carnifex) species, Table 1. Summary table of examination results. MG: samples from Monte Guglielmo area; O: samples from Pertica Alta – Odeno area; B: samples from Bagolino area; CFU: Colony-Forming Units; en dash: examination has not been performed. Sample Date Area Bacterial Examination Viral examination Parasitological examination 1 6 dying tadpoles 26 June 2006 MG Negative Negative Negative 2 6 died and 16 dying tadpoles 26 June 2006 MG Aeromonas Hydrophila infection Negative Negative 3 1 dying tadpole 03 July 2006 MG Negative Negatine Negative 4 2 dying tadpoles 03 July 2006 MG Aeromonas sobria infection Negative Negative 5 5 anal swambs 03 July 2006 MG Negative - - 6 Water 03 July 2006 MG Aeromonas sp. 52000 CFU - - 7 Water 03 July 2006 MG Negative - - 8 3 dying tadpoles 17 June 2003 O Negative - - 9 2 dead tadpoles 17 June 2003 O Negative - - 10 2 dead tadpoles 17 June 2003 O Aeromonas Hydrophila infection - - 11 10 dying tadpoles 31 May 2004 O Aeromonas sp. Infection - - 12 10 helthy tadpoles 31 May 2004 O Negative - - 13 Water 26 May 2003 O Aeromonas Hydrophila 160000 CFU - - 14 Water 26 May 2003 O Aeromonas Hydrophila 31000 CFU - - 15 Tadpoles 04 June 2003 B Aeromonas sp. Infection - - 16 Water 04 June 2003 B Aeromonas Hydrophila 13000 CFU - - 17 Water 04 June 2003 B Aeromonas sobria 16000 CFU - - 5Bacterial infection affecting Rana temporaria tadpoles cohabitating the same ponds, did not display clinical evidence of disease. Five anal tam- pons were collected as confirmatory test on apparently healthy adult frogs and no patho- logical bacteria were found. The examination of died or dying tadpoles, from all the study sites, always highlights a negative response for viral and parasitological examination, while the bacterial examina- tions often pointed out septicaemic infections caused by motile Aeromonas group (Aero- monas hydrophila and Aeromonas sobria) (Table 1). Similarly, the same pathogen was Table 2. Geographical data of ponds, Rana temporaria egg masses counting (Eggs) and Triturus carnifex presence (Tricar) in M. Guglielmo area, since 2005 to 2009. Lat: latitude; Long: Longitude; Alt: altitude; na: not available data; x: Triturus carnifex presence. POND Lat Long Alt (m) 2005 2006 2007 2008 2009 Eggs Tricar Eggs Tricar Eggs Tricar Eggs Tricar Eggs Tricar A1 45°47’26’’N 10°11’57’’E 1434 0 x 13 x 0 x 0 x 0 x A2 45°46’58’’N 10°11’14’’E 1389 3 40 7 6 0 x A3 45°47’16’’N 10°11’29’’E 1472 14 30 x 53 30 70 A4 45°47’14’’N 10°11’33’’E 1505 6 15 48 115 73 A5 45°47’33’’N 10°11’36’’E 1562 27 6 99 22 79 A6 45°47’33’’N 10°11’36’’E 1652 na na x 20 1 28 A7 45°47’03’’N 10°11’11’’E 1371 na na 37 30 35 B1 45°45’43’’N 10°11’59’’E 1313 60 162 45 12 10 B2 45°45’35’’N 10°11’56’’E 1309 9 22 26 14 50 B3 45°45’28’’N 10°11’36’’E 1373 136 70 15 17 13 B4 45°45’22’’N 10°11’39’’E 1408 45 na 4 na 28 B5 45°44’43’’N 10°11’30’’E 1535 0 5 na 2 5 B6 45°44’53’’N 10°11’21’’E 1569 0 7 2 0 15 B7 45°45’14’’N 10°11’02’’E 1681 na 10 4 na 35 B8 45°44’55’’N 10°11’38’’E 1674 0 4 0 0 0 B9 45°44’60’’N 10°11’47’’E 1672 5 16 34 24 57 B10 45°44’48’’N 10°11’52’’E 1664 10 16 3 199 53 C1 45°46’46’’N 10°07’11’’E 1166 0 x 0 x 0 x 0 x 0 x C2 45°46’57’’N 10°07’16’’E 1175 0 x 0 x 0 x 0 0 x C3 45°46’40’’N 10°07’55’’E 1361 0 x 0 x 0 x 0 0 x D1 45°44’48’’N 10°09’52’’E 1564 17 30 0 0 1 D2 45°44’47’’N 10°09’52’’E 1563 6 4 1 0 1 D3 45°44’49’N 10°09’44’’E 1577 na 0 0 1 2 D4 45°45’14’N 10°09’44’’E 1734 18 7 na 0 0 D5 45°45’16’N 10°09’43’’E 1739 50 26 18 2 0 D6 45°45’20’N 10°09’46’’E 1760 na na na 0 0 D7 45°45’30’N 10°09’21’’E 1749 3 na na 6 7 D8 45°45’40’N 10°09’30’’E 1853 0 na na 0 0 D9 45°45’42’N 10°09’29’’E 1863 0 13 9 0 7 Tot. 409 496 425 481 569 6 R. Tiberti found in pond water samples by counting the number of bacterial colonies on a culture medium (Table 1). Testing the hypothesis that the number of egg masses per pond would have decreased from 2005 to 2009 (Table 2), no significant relationship between Eggs and Year was found (Table 3). In Mod1 we set Year and Tricar as fixed effects and Group as random effect. In Mod2, Pond has been added as nested random effect to test the effect of every pond on Eggs. Mod1 was compared with Mod2 with a Log-Likelihood Ratio Test (LRT = 18.06, P < 0.0001). Since the presence of Triturus carnifex influenced the variance structure of Mod2 (F = 22.16, P < 0.0001), Mod2 was weighted on the Tricar binary variable. The updated model (Mod3) was compared with Mod2 (LRT = 4.830825 , P < 0.05). The accu- racy of Mod3 was verified by testing the normality of the residuals (W = 0.99, P = 0.25). Based on the models, the population of Rana temporaria has not significantly declined since 2005; otherwise, the importance of Triturus carnifex as a factor in disturbing Rana temporaria breeding activity is detectable in all the three models. DISCUSSION The only pathogens isolated in the samples were Aeromonas hydrophila and Aero- monas sobria. Aeromonas sp. occurs widely in fresh and estuarine waters (Cahill, 1990; Hazen et al., 1978; Massa et al., 2001) and is considered a primary and secondary patho- gen of aquatic and terrestrial animals. These bacterial pathogens can infect anuran (Hub- bard, 1981; Carey, 1993; Bradford, 1991; Marquez et al., 1995; Razzetti and Bonini, 2001; Taylor et al., 1999; Nyman, 1986; Hird et al., 1981; Sherman and Morton, 1993; Colt et al., 1984; Hayes and Jennings, 1986; Glorioso et al., 1974; Rigney et al., 1978), urodeles (Kaplan and Glaczenski, 1965; Boyer et al, 1971), fishes, reptiles, birds, and mammals, including humans (Panigrahy et al., 1981; Cahill, 1990; Gorden et al., 1979; Davis et al., Table 3. Fixed effect results from mixed models testing the hypothesis that number of egg masses per pond will decrease since 2005 to 2009. The groups of pond and the ponds were included as a random effect and nested random effect in these models. NS: Not Significant. Model Source of variation Beta df F P Mod1 Intercept 149.95 121 13.4 < 0.001 YEAR -0.07 121 0.5 NS TRICAR -1.83 121 17.0 <0.001 Mod2 Intercept 116.35 96 11.5 <0.001 YEAR -0.06 96 0.6 NS TRICAR -1.26 96 7.1 <0.01 Mod3 Intercept 147.74 96 11.7 <0.001 YEAR -0.07 96 1.3 NS TRICAR -1.26 96 9.5 <0.01 7Bacterial infection affecting Rana temporaria tadpoles 1978). Aeromonas hydrophila is one of the pathogenic agents of Red leg disease. Gross signs observed during mortality events within the prealpine study sites included ettechia and ecchymosis within the caudoventral and lateroventral body and tail, lethargy and coma and were consistent with those reported for Red leg disease (Nace, 1968; Marquez et al., 1995; Nyman, 1986; Hird et al., 1981; Carey, 1993). However, Aeromonas sp. is not the only agent in this pathology, which is the result of a complex interaction between other gram negative bacteria including Pseudomonas spp., Proteus spp., Flavobacterium indolo- genes and F. meningosepticum (Hubbard, 1981; Taylor et al., 1993; Anver and Pond, 1984). Moreover, Aeromonas sp. presence by itself does not necessarily give evidence of disease; these bacteria can live in free waters, on the skin and in the digestive tract of amphibians without causing any disease (Hubbard, 1981; Carey, 1993; Hird et al., 1981). Consequently, even if Aeromonas sp. is also considered a primary pathogen, several stress- ors are often involved in outbreaks of disease and Aeromonas sp. is considered to behave as an opportunistic pathogen, infecting immunodepressed hosts. For example Carey (1993) reported that Aeromonas hydrophila infection occurred in Bufo boreas after the immune system of the amphibians was suppressed by environmental factors. Natural and anthropogenic stressors, including pre-existing diseases, may be involved in the occurrence of Red leg; as a matter of fact, Aeromonas sp. can behave as a secondary pathogen and its irregular isolation in the samples (Table 1) might suggest the presence of other primary pathogens. Moreover, the occurrence of the same above mentioned die- offs with gross signs of systemic hemorrhagic disease are considered to be indicators of viral infection (Gray et al., 2009; Converse and Green, 2005; Cunningham et al., 1996 ) and although Aeromonas sp. can cause Red leg disease, retrospective studies showed that ranaviruses are also often present and may be the primary pathogen. Nevertheless, viral examination seems to rule out ranaviruses presence at least in the samples from Monte Guglielmo, even if no confirmatory diagnosis (PCR and Immunohistochemistry meth- ods) (Hyatt et al., 2000; Reddacliff and Whittington, 1996) has been performed and the examined samples were few. Additionally, gross lesions, similar to the observed ones, were pointed out in diseases caused by Batrachochytrium dendrobatidis in adult amphibians (Green and Converse 2005). However chytridiomycosis is a fatal disease of post-meta- morphic frogs infecting keratinized skin of adults and can be ruled out as primary patho- gen of the observed mass die offs; since tadpoles have keratin only in mouthparts, chytrid infection on larval anurans commonly results in reduced developmental rate and foraging efficiency (Venesky et al., 2010) without causing mass die-offs. Lots of other factors may be implicated in determing the pathogenicity of Aeromonas sp. For example, during the last decades farmers reduced the number of cows in the moun- tain areas and consequently the demand for water; mountain ponds were often abandoned and underwent a drying process. This may result in a more extreme habitat for tadpoles. Still existing ponds are subject to wider depth fluctuations and the reduced basin volume is worsened by stronger seasonal and daily variations, resulting in increased total solids, eutrophication and tadpoles crowding; everyone of these factors can potentially suppress the tadpoles immune system and facilitate the disease. For instance, the abandonment of moun- tain ponds can affect the immune system of tadpoles in the wild and this process is poten- tially linked with diurnal oxygen supersaturation which can occur in eutrophic ponds: eval- uating the importance of dissolved gas, Colt et al. (1984) exposed Rana catesbeiana tadpoles to supersaturated water causing signs of Gas bubble disease followed by Red leg disease. 8 R. Tiberti Finally, several limitations should be pointed out in the current study, since the obtained results show that it is not possible to state unequivocally whether Aeromonas sp. acts as a primary pathogen or whether there are pre-existing environmental or infective stressors in the studied populations. Several unexplored factors could be involved in the observed mass die-offs, weakening the immune system of tadpoles; even some short-term acute stressors can induce immunosuppression lasting for days (Ellsaesser and Clem, 1986; Pickering et al., 1982; Carey, 1993), making even more difficult to determine conclusively which environmental factor (or which combination of environmental factors) may have caused a sufficient degree of stress to induce disease in the prealpine populations. However, in Monte Guglielmo area, the number of egg masses has not significantly declined since 2005 and the consequences of the disease were not found in a reduced breeding activity of Rana temporaria, suggesting that this species is able to withstand high larval mortality occurred in the last years. Regardless, evidence of widespread mass die-offs in prealpine areas has been reported since the early 2000s and previous declines should not be ruled out. The measured abundance could be the result of a decline lasting for years and reduc- ing the population to the current minimum. Finally, the extent of the phenomenon and the amplitude of the affected area should arouse the interest for other conservation issues, such as the health and the consevation status of other amphibians living in the same areas (including Bombina variegata and Triturus carnifex, listed in Annex II of the Habitat Direc- tive) and the preservation of important habitats such as high altitude ponds. ACKNOWLEDGEMENTS I am particularly grateful to Mr. Giuseppe Tomasini and Dr. Franco Panunzi for their kind help in data collection. I want to thank Prof. Giuseppe Bogliani for his support, Dr. Cristian Pas- quaretta for the precious help in data analysis and Tito Tiberti for his kind proofreading of the text. I thank the Comunità Montana of Valle Trompia and the provincial administration of Brescia. Sam- ple examination would not have been possible without the contribution of the IZS (Istituto Zoopro- filattico Sperimentale) of Brescia. REFERENCES Anver, M.R., Pond, C.L. (1984): Biology and diseases of amphibians. In: Laboratory ani- mal medicine, p. 427-447. Fox, J. G., Cohen, B. J., Loew, F. M., Eds, Academic Press Inc, Orlando, Florida. Berger, L., Speare, R., Daszak, P., Green, D.E., Cunningham, A.A., Goggin, C.L., Slocombe, R., Ragan, M.A., Hyatt, A.D., McDonald, K.R., Hines, H.B., Lips, K.R., Marantelli, G., Parkes, H. (1998): Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. PNAS 95: 9031-9036. Boyer, C.L., Blackler, K., Delanney, L.E. (1971): Aeromonas hydrophila infection in the Mexican axolotl, Sirenodon mexicanum. Lab. Anim. Sci. 21: 372-375. 9Bacterial infection affecting Rana temporaria tadpoles Bradford, D.F. (1991): Mass mortality and extinction in a high elevation population of Rana muscosa. J. Herpetol. 25: 174-177. Cahill, M.M. (1990): Virulence factors in motile Aeromonas species. J. Appl. Bacteriol. 69: 1-16. Carey, C. (1993): Hypothesis concerning the causes of the disappearence of boreal toads from the mountains of Colorado. Conserv. Biol. 7: 355-362. Carey, C., Heyer, R.W., Wilkinson, J., Alford, R.A., Arntzen, J.W., Halliday, T., Hunger- ford, L., Lips, K.R., Middleton, E.M., Orchard, S.A., Rand, A.S. (2001): Amphibian declines and environmental change: use of remote sensing data to identify environ- mental correlates. Conserv. Biol. 15: 903-913. Colt, J., Orwicz, K., Brooks, D. (1984): Effects of gas-supersatured water on Rana catesbei- ana tadpoles. Acquaculture 38: 127-136. Converse, K.A., Green, D.E. (2005): Diseases of tadpoles. In: Wildlife diseases: landscape epidemiology, spatial distribution and utilization of remote sensing technology, p. 72-88. Majumdar, S.K., Huffman, J.E., Brenner, F.J., Panah, A.I., Eds, Pennsylvania Academy of Science, Easton. Cunningham, A.A., Langton, T.E., Bennett, P.M., Lewin, J.F., Drury, S.E., Gough, R.E., Macgregor, S.K. (1996): Pathological and microbiological findings from incidents of unusual mortality of the common frog (Rana temporaria). Philos. Trans. R. Soc. Lond. B Biol. Sci. 351: 1539-1557. Daszak, P., Cunningham, A.A., Hyatt, A.D (2000): Emerging infectious diseases of wild- life: threats to biodiversity and human health. Science 287: 443-449. Davis, W.A., Kane, J.G., Garagusi V.F. (1978): Human Aeromonas infections: a review of the literature and a case of endocarditis. Medicine 57: 267-277. Ellsaesser, C.F., Clem, L.W. (1986). Haematological and immunological changes in channel catfish stressed by handling and transport. J. Fish Biol. 28: 511–521. Gardner, T. (2001): Declining amphibian populations: a global phenomenon in coserva- tion biology. Anim. Biodiv. Conserv. 24: 25-44. Glorioso, J.C., Amborski, R.L., Amborski, G.F., Culley, D.D. (1974): Microbiological stud- ies on septicemic bullfrogs (Rana catesbeiana). Am. J. Vet. Res. 35: 1241-1245. Gorden, R.W., Hazen, T.C., Esch, G.W., Fliermans, C.B. (1979): Isolation of Aeromonas hydrophila from the american alligator, Alligator mississipiensis. J. Wildlife Dis. 15: 239-243. Gray, M.J., Miller, D.L., Hoverman, J.T. (2009): Ecology and pathology of amphibian rana- viruses. Dis. Aquat. Organ. 87: 243-266. Green, D.E., Converse K.A. (2005): Diseases of frogs and toads. In: Wildlife diseases: land- scape epidemiology, spatial distribution and utilization of remote sensing technol- ogy, p. 89-117. Majumdar, S.K., Huffman, J.E., Brenner, F.J., Panah, A.I., Eds, Penn- sylvania Academy of Science, Easton. Guarino, F.M., Di Già, I., Sindaco, R. (2008): Age structure in a declining population of Rana temporaria from northern Italy. Acta Zool. Hung. 54: 99-112. Hayes, M.P., Jennings M.R. (1986): Decline of ranid species in Western North America: are Bullfrogs (Rana catesbeiana) responsible? J. Herpetol. 20: 490-509. Hazen, T.C., Fliermans, C.B., Hirsch, R.P., Esch, G.W. (1978): Prevalence and distribution of Aeromonas hydrophila in the USA. Appl. Environ. Microbiol. 36: 731-738. 10 R. Tiberti Hird, D.W., Diesch, S.L., McKinnel, R.G., Gorham, E., Martin, F.B., Kurtz, S.W., Dubrovol- ny, C. (1981): Aeromonas hydrophila in wild-caught frogs and tadpoles (Rana pipi- ens) in Minnesota. Lab. Anim. Sci. 31: 166-169. Hubbard, G.B. (1981): Aeromonas hydrophila infection in Xenopus laevis. Lab. Anim. Sci. 31: 297-300. Hyatt, A.D., Gould, A.R., Zupanovic, Z., Cunningham, A.A., Hengstberger, S., Whitting- ton, R.J., Coupar, B.E.H. (2000): Characterisation of piscine and amphibian iridovi- ruses. Arch. Virol. 145: 301-331. Kannan, S., Nair, G.B. (2000): Aeromonas: an emerging pathogen associated with evolv- ing clinical spectrum and potential determinants of pathogenicity. Indian J. Med. Microbiol. 18: 92-97. Kaplan, H.M., Glaczenski, S.S. (1965): Salamanders as laboratory animals: Necturus. Lab. Anim. Care 15: 151-155. Kiesecker, J.M., Blaustein, A.R., Belden, L.K. (2001): Complex causes of amphibian popu- lation declines. Nature 410: 681-684. Kiesecker, J.M., Belden, L.K., Shea, K., Rubbo, M.J. (2004): Amphibian decline and emerg- ing disease. Am. Sci. 92: 138-147. Marquez, R., Olmo, J.L., Bosh, J. (1995): Recurrent mass mortality of larval midwife toads Alytes obstetricans in a lake in the Pyrenean Mountains. Herpetol. J. 5: 287-289. Massa, S., Alitiera, C., d’Angela, A. (2001): The occurrence of Aeromonas sp. in natural mineral water and well water. Int. J. Food Microbiol. 63: 169-173. Nace J.W. (1968): The amphibian facility of the university of Michigan. BioScience 18: 767- 775. Nyman, S. (1986): Mass mortality in larval Rana selvatica attributable to the bacterium, Aeromonas hydrophila. J. Herpetol. 20: 196-201. Panigrahy, B., Mathewson, J.J., Hall, C.F. (1981): Unusual disease conditions in pet of avi- ary birds. J. Am. Vet. Med. Assoc. 178: 394-395. Pickering, A.D., Pottinger, T.G., Christie, P. (1982): Recovery of the brown trout, Salmo trutta L., from acute handling stress: a time-course study. J. Fish Biol. 20: 229-244. Razzetti, E., Bonini, L (2001): Infezioni e parassitosi negli anfibi: il possibile impatto delle ricerche erpetologiche. Atti Soc. Sci. Nat. Museo civ. Stor. Nat. Milano 142: 97-102. Reddacliff, L.A., Whittington, R.J. (1996): Pathology of epizootic haematopoietic necrosis virus (EHNV) infection in rainbow trout (Oncorhynchus mykiss Walbaum) and red- fin perch (Perca fluviatilis L.). J. Comp. Pathol. 115: 103-115. Rigney, M.M., Zilinsky, J.W., Rouf, M.A. (1978): Pathogenicity of Aeromonas hydrophila in Red Leg Disease in Frogs. Curr. Microbiol. 1: 175-179. Sherman, C.K., Morton, M.L. (1993): Population declines of Yosemite toads in the eastern Sierra Nevada of California. J. Herpetol. 27: 186-198. Taylor, S.K., Williams, E.S., Mills, K.W. (1999): Effects of malathion on disease susceptibil- ity in woodhouse’s toads. J. Wildlife Dis. 35: 536-541. Venesky, M.D., Parris, M.J., Storfer, A. (2010): Impacts of Batrachochytrium dendrobatidis infection on tadpole foraging performance. Ecohealth 6: 565-75. Wake, D. (1991): Declining Amphibian populations. Science 253: 860. http://www.ncbi.nlm.nih.gov/pubmed/20135192 http://www.ncbi.nlm.nih.gov/pubmed/20135192 bbib67 OLE_LINK1 OLE_LINK2 bbib28 OLE_LINK5 OLE_LINK6 OLE_LINK7 OLE_LINK8 _GoBack OLE_LINK1 OLE_LINK2 OLE_LINK3 OLE_LINK4 OLE_LINK1 OLE_LINK2 OLE_LINK19 OLE_LINK20 OLE_LINK21 OLE_LINK29 OLE_LINK3 OLE_LINK4 OLE_LINK5 OLE_LINK31 OLE_LINK14 OLE_LINK15 OLE_LINK12 OLE_LINK13 OLE_LINK16 OLE_LINK17 OLE_LINK22 OLE_LINK23 OLE_LINK24 OLE_LINK8 OLE_LINK9 OLE_LINK10 OLE_LINK11 OLE_LINK18 OLE_LINK27 OLE_LINK28 OLE_LINK25 OLE_LINK26 OLE_LINK6 OLE_LINK7 OLE_LINK34 OLE_LINK37 OLE_LINK38 Acta Herpetologica Vol. 6, n. 1 - June 2011 Firenze University Press Widespread bacterial infection affecting Rana temporaria tadpoles in mountain areas Rocco Tiberti Extreme feeding behaviours in the Italian wall lizard, Podarcis siculus Massimo Capula1, Gaetano Aloise2 Lissotriton vulgaris paedomorphs in south-western Romania: a consequence of a human modified habitat? Severus D. Covaciu-Marcov*, Istvan Sas, Alfred Ş. Cicort-Lucaciu, Horia V. Bogdan Body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (Ommatotriton ophryticus) Eyup Başkale1, Ferah Sayım2 , Uğur Kaya2 Genetic characterization of over hundred years old Caretta caretta specimens from Italian and Maltese museums Luisa Garofalo1, John J. Borg2, Rossella Carlini3, Luca Mizzan4, Nicola Novarini4, Giovanni Scillitani5, Andrea Novelletto1 The phylogenetic position of Lygodactylus angularis and the utility of using the 16S rDNA gene for delimiting species in Lygodactylus (Squamata, Gekkonidae) Riccardo Castiglia*, Flavia Annesi Localization of glucagon and insulin cells and its variation with respect to physiological events in Eutropis carinata Vidya. R. Chandavar1, Prakash. R. Naik2* The Balearic herpetofauna: a species update and a review on the evidence Samuel Pinya1, Miguel A. Carretero2 Effects of mosquitofish (Gambusia affinis) cues on wood frog (Lithobates sylvaticus) tadpole activity Katherine F. Buttermore, Paige N. Litkenhaus, Danielle C. Torpey, Geoffrey R. Smith*, Jessica E. Rettig Food composition of Uludağ frog, Rana macrocnemis Boulenger, 1885 in Uludağ (Bursa, Turkey) Kerim Çiçek Preliminary results on tail energetics in the Moorish gecko, Tarentola mauritanica Tommaso Cencetti1,2, Piera Poli3, Marcello Mele3, Marco A.L. Zuffi1 Climate change and peripheral populations: predictions for a relict Mediterranean viper José C. Brito1, Soumia Fahd 2, Fernando Martínez-Freiría1, Pedro Tarroso1, Said Larbes3, Juan M. Pleguezuelos4, Xavier Santos5 Assessing the status of amphibian breeding sites in Italy: a national survey Societas Herpetologica Italica* Osservatorio Erpetologico Italiano ACTA HERPETOLOGICA Journal of the Societas Herpetologica Italica ACTA HERPETOLOGICA Rivista della Societas Herpetologica Italica