Sampling
From September to November 2018, 567 samples [189 rectal swabs (R), 189 conjunctival swabs (C) and 189 oral swabs (O)] were collected from 189 individuals of 4 different species of troglophile bats (Permit of Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA) Prot. N. 14589/T-A31) (Table n. 1).
Myotis myotis lives in forest environments, open pastures and meadows. Its diet is mostly based on insects caught on the ground.
Myotis capaccinii feeds on insects, particularly Trichoptera, Neuroptera, Diptera and occasionally on fish fry caught just above water surfaces or just below them.
Miniopterus schreibersii feeds on insects caught in flight. With a highly specialized trophic regime, it preys mainly on Lepidoptera. Non-flying preys are also reported in its diet.
Rhinolophus hipposideros feeds on small insects (Diptera, Lepidoptera and Neuropoptera), which they capture in flight or on the ground (e.g. spiders). They generally hunt individually, very close to the ground.
The sampling was carried out in six different caves of the southern Italy territory (Table n. 2).
-
Grotta dei Pipistrelli (SR). Although located in a protected area (Natural Reserve "Pantalica"), this cave is not far from the town of Sortino, where semi-wild breeding is practiced. The cave is known for its large colonies of bats and it is the only systematically monitored Sicilian cave. The maximum number of bats recorded was around 10.000 in 2013. Minioptera and large Myotis are the predominant genera, with a lower prevalence of Rhinolophids. Below the entrance to the cave flow two streams of water (Anapo and Calcinara) from which the bats drink after emerging from the cave. The waste water from the municipality of Palazzolo Acreide (SR) flows into the Anapo.
-
Grotta Palombara (Melilli, SR). Fossil karst cavity that develops for about 800 meters. Palombara cave is located near the biggest petrochemical plant of Italy, known as “Augusta-Priolo-Melilli”. The cave hosts a colony of bats belonging to the species Myotis myotis, Miniopterus schreibersii, Rhinolophus euryale, and Rhinolophus ferrumequinum. The maximum number of Chiroptera recorded was about 1000 specimens. Human activity, especially the presence of numerous illegal landfills, has significantly degraded the environment around the cave.
-
Grotta Chiusazza (Floridia, SR). The total development is about 250 m. The area in which the cave opens is characterized by the presence of intensive monocultures and, arable land and small ponds, fed by the runoff from farmland, where the bats go to feed and drink. The cave is populated by a large colony of Minioptera and dozens of Rhinolophids.
-
Grotta del Burrò (Randazzo, CT). Volcanic cave created as a result of the eruptive activity of Etna. It is a large lava flow tunnel, over 200 m long. The surrounding area has a dense shrub-herbaceous vegetation cover and no arable land. In the territory, semi-wild cattle breeding is commonly practiced. The cave is inhabited by a large mixed colony of bats (about 600–700 Minioptera along with several dozen large Myotis and Rhinolophids).
-
Grotta dei Pipistrelli (Cassano allo Jonio, CS). Large cavity consisting of a succession of caverns whose bottom is occupied by debris and guano. It hosts a breeding colony of bats with a prevalence of Myotis myotis and Miniopterus schreibersii. The territory in which the cavity opens is hilly in the western part and slopes down towards the Piana di Sibari in the East, characterized by the presence of agricultural activity.
-
Grave Grubbo (Verzino, KR). Opens at 285 m a.s.l. and extends for 1926 m. The cave is active and water flows into one of its branches from an inlet point during rainy periods. The cave hosts a large, mixed breeding colony of bats (Minioptera and Rhinolophids). The surrounding area is characterized by extensive crops and olive groves, as well as cattle and sheep breeding.
The samples were collected between September and November 2018.The bats were caught, in some cases, simply with the hands in order to minimize the risk of disturbance to the colony. In some cavities a hand-held net with a telescopic handle was used to capture static and non-flying bats. Only in 2 cavities (Grotta dei Pipistrelli of Sortino and Grotta dei Pipistrelli of Cassano allo Ionio) it was possible to use the harp-trap to catch specimens that flew off the roosts. Rectal, conjunctival and oral swabs for bacteriological survey were obtained from each bat using individually packed sterile microbiological swabs pmoistened with sterile saline solution 09%, inserting the tip and gently rotating it against the mucosa. The swabs were subsequently inserted into tubes containing Amies transport medium (Copan Italia, Brescia, Italy) and kept in a cooler with frozen gel packs for purposes of transport for a maximum of 8 h before culture-plate inoculation, or further storage in a refrigerator at 4°C for a maximum of another 24 h, if no earlier processing was possible due to logistical reasons. All bats were released immediately after sampling.
Bacterial Isolation and Identification
The samples were transported in conditions of refrigeration to the laboratory and examined for potentially pathogens detection. All samples (n. 567) were examined for Gram - bacteria; conjunctival and oral swabs (n. 378) were also submitted to bacteriological examination for Gram positive bacteria. Rectal swabs (n. 189), after an enrichment in buffered peptone water, were streaked into MacConkey Agar plates (Biolife Italiana, Milano, Italy). Conjunctival and oral swabs were cultured in nutritive broth and, after, streaked into MacConkey Agar plates and into Staphylococci 110 Medium plates (Biolife Italiana, Milano, Italy). Colonies demonstrating distinctive macroscopic appearance were considered separate organisms and isolated on new plates. Isolates were subcultured in Blood Agar plates for identification by mass spectrometry MALDI-TOF (matrix assisted laser desorption/ionisation - time of fligt mass spectrometry). The isolated colonies were seeded in a 48-well metal plate with disposable loops, using as a reference strain Escherichia coli ATCC 8739. The spectra are analysed by VITEK MS system (bioMérieux SA, Marcy l'Etoile, France), using the software Axima (Shimadzu Kyoto, Japan)-SARAMIS database (Spectral ARchive And Microbial Identification System) (AnagnosTec, Berlin, Germany). Eighty-eight strains, unidentified by MALDI-TOF mass spectrometry, after being grown on Blood Agar Base (Biolife Italiana, Milano, Italy) and diluted in physiological solution were typed at the Laboratory of Specialized Bacteriology of the Zooprophylactic Institute of Sicily, using the traditional macro test tube method. The bacteria of the genus Bacillus spp. (POS BAT 19/Rev 0) have been characterized by carbohydrates oxidation and fermentation, motility, urease, gelatinase, nitrate reduction and Voges Proskauer (VP) tests; Staphylococcus and Streptococcus spp. (POS BAT 05 /Rev 0 and POS BAT 30/Rev 0) were characterized by catalase, hemolysis, coagulase, oxidase, VP tests and carbohydrate fermentation. The enterobacteria and gram-negative glucose nonfermenting bacteria (POS BAT 09 /Rev 0) were identified by OF, mobility, catalase, oxidase, urease and triptophanase tests and utilization/ fermentation/ oxidation of carbohydrates. The serological typing of Salmonella spp. strains (POS BAT 04/Rev.4) was performed following the Kauffmann-White-Le Minor method in agreement with the National Salmonellosis Center of Padua, Italy (Grimont and Weill, 2007).
Statistical analysis
We evaluated the difference in the number of strains belonging to pathogenic species isolated from different sampling sites and different bat species using Fisher’s Exact Test, fixing the significance limit at P = 0.05.