Bat Species Identification and Alphacoronavirus, Beta- and Gammaherpesvirus Findings in Bat Colonies in Tuscany and Latium Regions During Lyssavirus Surveillance

Abstract

Chiroptera includes over 1400 bat species, with at least 35 of these present in Italy. Due to their role as Lyssavirus reservoirs, bats found dead, with and without signs suggestive of this infection, are routinely submitted to the laboratory network of the Istituti Zooprofilattici Sperimentali in the framework of the rabies national passive and active surveillance program. Carcasses and biological samples collected from January to December 2021 in Latium and Tuscany, regions of our jurisdiction, were further screened for the presence of Coronaviruses (CoVs) and Herpesviruses using pan-family virus PCR tests, and relative PCR products were Sanger sequenced. Genetic characterization through sequencing detected AlphaCoVs in Miniopterus schreibersii and Beta- and Gammaherpesviruses in Tadarida teniotis. Samples were also submitted to bat genetic species identification.

1. Introduction

Bats (order Chiroptera) represent over 1400 species worldwide, accounting for roughly 20% of all mammalian diversity [1]. In Italy, at least 35 species have been documented, spanning eleven genera and four families [2,3].

During the second half of the 20th century, bat populations in Europe declined significantly, probably due to different factors, such as agricultural intensification, the destruction of roosts, habitat loss and the massive use of toxic chemicals.

Beyond their ecological importance as pollinators and insect regulators, bats are increasingly recognized as reservoir hosts for a wide range of viruses. These include zoonotic agents of high concern, such as Lyssaviruses (LYSVs), Coronaviruses and Paramyxoviruses, many of which have been detected in European bat populations [4]. Bats are known to harbor viruses from at least 28 different families, including both RNA and DNA viruses, most of which are likely to be host-specific with limited zoonotic potential [5]. While many of these viruses appear to be host-adapted and pose minimal zoonotic risk, several—such as SARS-like Coronaviruses, Henipaviruses and Lyssaviruses—have demonstrated the capability to cross species barriers under the right ecological or genetic conditions [5,6].

In Europe, five different LYSVs, namely European bat 1 lyssavirus (EBLV-1), European bat 2 lyssavirus (EBLV-2), Bokeloh bat lyssavirus (BBLV), Lleida bat lyssavirus (LLEBV), Kotalahti bat lyssavirus (KBLV) [7] and the newly described Divača bat lyssavirus [8] are reported in specific bat host species [9]. In the same geographical area, some bat species were also found to be associated with other zoonotic pathogens, such as Coronavirus, Herpesvirus, Paramyxovirus and more [10]. A correct host species identification is of crucial importance to increase knowledge of the ecology and evolutionary pattern of bat viruses.

In the last two decades, the importance of the study of microorganisms detected in chiropteran species has grown, especially due to their potential role as vectors of zoonotic viral diseases, such as Betacoronaviruses, Henipaviruses and LYSV [11,12], as well as Paramyxovirus, in particular, in fruit bats [13]. The detection of microbes in some bat species that are close to those that cause human diseases may indicate only an evolutionary relationship and not that a pathogen currently circulating in bat populations will infect humans [14].

Chiroptera pathogens surveillance plays a crucial role in the management of public health safety, as the early detection of potentially newly emerging zoonotic pathogens is fundamental to prevent their diffusion, especially those with RNA genomes, in view of their higher mutation rate [15].

As bats play an important role as LYSV reservoirs and in their diffusion, even if the risk of LYSVs in both humans and bats is at present low [16], it is crucial to continue monitoring their presence, owing to the broad spectrum of affected bat species and the zoonotic threat of infection [7]. Respectively, since 1980 and 2008, passive and active surveillance in bats have been carried out to verify rabies-related LYSV circulation, also based on the analysis of dead bats, including those for which humans or domestic animals could have come in contact. A report on this activity relates LYSV serological evidence in at least six bat colonies present across the country, suggesting their circulation, even if no direct viral evidence was obtained, probably due to the low number of carcasses present or because the habitual LYSV host bat species are not present in great numbers in Italy [9]. In 2020, West Caucasian Bat Lyssavirus (WCBL) was detected in a cat living in Arezzo, Tuscany. For this, monitoring activities were immediately set up to verify the presence of further cases in the surrounding areas [17]. In parallel, passive surveillance was intensified in the neighboring regions, among which was Latium, in relation to the flight range of the potential bat reservoirs of this virus, which were indicated as being Miniopterus schreibersii [17,18,19].

Since 2021, Lyssavirus active surveillance in Chiroptera is mandatory, in compliance with the Commission Implementing Regulation (EU) 2018/1882 and adopted with a national decree of the Italian President of the Republic 357/97 [20].

In view of the potential role of bats as reservoirs of SARS-CoV-2, during the COVID-19 pandemic, as indicated by the scientific literature and the sequences of the viruses available in the public databases [21], the presence of Coronavirus was also investigated in this study, as was the presence of another wide group of DNA viruses represented by Herpesviruses, which are extensively distributed in nature among several vertebrates [22] in which they can also cause important diseases.

2. Materials and Methods

Oral swabs of LYSV-susceptible bat species were collected during 2021 from a colony in Arezzo, Italy, within a national surveillance program set up subsequent to the detection of West Caucasian Bat Lyssavirus (WCBL) in 2020 in a cat living close by. During the same year, brain, lung and intestines were sampled from the carcasses of bats found dead in Latium and Tuscany; part of these samples were derived from a previous research project regarding a surveillance program for CoVs circulating in wildlife in Italy between 2020 and 2022 [17].

Nucleic acid extraction from biological tissues was performed using the automatic extractor QIAsymphony (DSP Virus/Pathogen Kit, Qiagen, GmbH, Hilden, Germany) according to the manufacturer’s instructions. Briefly, 0.1 g of each sample was added to 0.9 mL of ATL buffer (QIAGEN, GmbH, Hilden, Germany) and a 5 mm stainless steel bead and homogenized by Tissue Lyser II (QIAGEN, GmbH, Hilden, Germany), followed by centrifugation at 16,000× g. Oral swabs were suspended in 500 µL of a 1/1 phosphate saline buffer solution (PBS) and ATL buffer. A volume of 400 µL of supernatant from each sample was used for RNA/DNA extraction. The concentration and purity of the extracted nucleic acids were evaluated by spectrophotometric analysis based on the absorbance values (A), respectively, at the wavelengths of 260 and 280 nm as reported in the datasheet of the NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All individuals were genetically identified as described elsewhere [23]. To assess the absence of any inhibitor factor during the extraction protocol, an RT-PCR assay targeting the endogenous control of the beta actin gene was performed.

LYSV infection was first ruled out by testing brain tissues and oral swabs using real-time RT-PCR [17,24]. Lungs, intestines and oral swabs were tested using a panCoV RT-PCR assay, targeting the RNA-dependent RNA polymerase gene (RdRp) [25]. A subset of samples for which material was still available for analysis (n = 77/128 lungs and n = 64/111 oral swabs) was also tested using a pan-Herpes RT-PCR assay [26,27]. Positive amplicons were sequenced using an automated sequencer (3500 Genetic Analyzer, A. Biosystems, Foster City, CA, USA). The sequences were assembled and analyzed using the Geneious Prime 2021.0.3 (Biomatters, Auckland, New Zealand) and compared with those present for the viruses of this study in the National Center for Biotechnology Information (NCBI) GenBank database using the BLAST online tool (Rockville Oike, MD, USA).

Phylogenetic analysis was performed for our positive amplicons. Similar sequences were retrieved by aligning newly submitted sequences with the BLASTn program from NCBI against sequences obtained from the DBatVir [21] database and the NCBI (nt) database. In the case of Coronavirus amplicons, a taxonomic subgenera classification analysis was carried out using the R library MyCoV [28]. Sequence homology analysis was checked out with BLASTp and EMBOSS Needle (version 6.6.0, available on the European Bioinformatics Institute webpage [29]) programs.

Multiple sequence alignments were obtained using the MUSCLE program [30] and applying standard parameters. The jModelTest version 2.1.10 program took as input each multiple alignment to select the best model among all distributions, considering the smallest Bayesian Information Criterion (BIC) scores [31]. Phylogenetic relationships were inferred by using the Maximum Likelihood method with the PhyML version 3.3.20211231 program [32]. Tree images were plotted with the R-studio program [33] using the ape, ggtree, and tidytree libraries [34,35,36].

3. Results

In 2021, 128 bat carcasses were analyzed, of which 36 were from Tuscany and 92 were from Latium. Moreover, 111 oral swabs collected from the aforementioned Arezzo colony, where the presence of Miniopterus schreibersii was observed, were also examined [17]. Bats belonged mostly to Tadarida teniotis (n = 71, 29.7%), which were all collected from an urban colony that since 2008 had registered cyclical mass mortality [9], followed by Pipistrellus kuhlii (n = 23, 9.6%), Hypsugo savii (n = 18, 7.5%), Pipistrellus sp. (n = 10, 4.2%), Myotis daubentonii and Plecotus austriacus (both with n = 1, 0.4%) (

Table 1. Distribution of bat species per region (* 111 oral swab samples; ⁺ samples collected only from the Latium region) and PCR test performed.

Type of Surveillance	Bat Species	Total Number Per Species (%)	Region		Test Performed Investigated
			Latium	Tuscany	Pan-CoV [25]	Pan-Herpes [26,27]
Passive	Hypsugo savii	18 (7.5)	11	7	18	3 +
	Pipistrellus kuhlii	23 (9.6)	9	14	23	3 +
	Pipistrellus sp.	10 (4.2)	1	9	10	0
	Myotis daubentonii	1 (0.4)	0	1	1	0
	Plecotus austriacus	1 (0.4)	0	1	1	0
	Not identified	4 (1.7)	0	4	4	0
Colony investigation	Tadarida teniotis	71 (29.7)	71	0	71	71 +
Active	Miniopterus schreibersii	111 * (46.6)	0	111 *	111 *	64 *
Total		239 (100)	92	147	239	141

). A small percentage of bats (n = 4, 1.7%) were not identified, probably due to the storage conditions before submission to the laboratory, which could have caused nucleic acid degradation; probably for the same reason, it was not possible to arrive at species identification for some of the subjects.

All samples were negative for the presence of LYSVs, and three oral swabs were positive for CoV. Nine lung samples tested positive for Herpesvirus (n = 9/77, 11.68%), and due to the poor quality of some of the amplicon bands when visualized on electrophoresis gels, only two were confirmed by Sanger sequencing.

Three sequences were obtained for CoVs using Sanger sequencing: M. schreibersii Italy 1/2021, M. schreibersii Italy 2/2021 and M. schreibersii Italy 3/2021, while for Betaherpesviruses and Gammaherpesviruses, we obtained WJK71402.1 (OQ807168.1 nucleic) and WJK71403.1 (OQ807169.1 nucleic), respectively. Relative to CoV, two of three novel sequences are considered, as their length was sufficient for determining sub-genera taxonomic classification and an informative phylogenetic analysis. M. schreibersii Italy 1/2021 (OP776451.1 nucleotide and UZC49592.1 protein accession numbers) has a nucleotide length of 333 bp and a protein one of 110 amino acids, while M. schreibersii Italy 2/2021 (ON834690.1 nucleotide and UUG60948.1 protein accession numbers) and M. schreibersii Italy 3/2021 (OP627105.1 nucleotide and UYI58598.1 protein accession numbers) are long at 441 nucleotides and 147 amino acids, and they are identical. Comparing these novel isolates with the BLASTn program, OP776451.1 M. schreibersii Italy 1/2021 exhibits a 78.08% identity percentage with OP627105.1 M. schreibersii Italy 3/2021 and ON834690.1 M. schreibersii Italy 2/2021, aligning with a word size parameter of 15. Using BLASTp and EMBOSS Needle, UZC49592.1 M. schreibersii Italy 1/2021 aligned with UUG60948.1 M. schreibersii Italy 2/2021 and UYI58598.1 M. schreibersii Italy 3/2021, showing an 88.18% identity percentage and a similarity score of 102/110.

Table 2. Results of nucleotide and protein alignments of M. schreibersii Italy 2/2021 (ON834690.1 nucleotide and UUG60948.1 protein accession numbers) as query against M. schreibersii Italy 1/2021 (OP776451.1 nucleotide and UZC49592.1 protein accession numbers) and M. schreibersii Italy 3/20212021 (OP627105.1 nucleotide and UYI58598.1 protein accession numbers). New sequences obtained are marked in bold.

Nucleotide			Protein
Accession Number	Identity	Querycov	Accession Number	Identity	Querycov
OP776451.1	78.08%	75%	UZC49592.1	88.2%	102/147
OP627105.1	100%	100%	UYI58598.1	100%	147/147

reports all the information about the comparison among the three novel CoVs, using as a query M. schreibersii Italy 2/2021.

The phylogenetic analysis was carried out using multiple sequence alignments of 129-amino acid and 397-nucleotide sequence lengths, obtained by including 29 sequences whose host organisms are bats. The nucleotide Alpha-CoVs tree (log likelihood −3518.55687) illustrated in Figure 1, which is mainly built from Miniopterus Alpha-CoVs, indicates a group belonging to the Minunacovirus subgenera and composed of HKU8 and HKU7—bat Coronavirus 1A and 1B taxonomy classifications—which separates from the HKU2, HKU6 and M.sch/A/Spain/2004 (HQ184049.1) isolates. Considering all the Minunacovirus subgenera, the uppermost part represents the Miniopterus bat Coronavirus 1 taxonomy, which consists of Bat Coronavirus 1A and 1B and other unclassified bat Coronaviruses, whereas the lowermost part includes the Miniopterus bat Coronavirus HKU8 classification. The other reference taxonomy classification, which is bat Coronavirus HKU7, stands between the two parts but is nearer to the HKU8 classification. The Minunacovirus subgenera tree topology is also confirmed by past studies, where the HKU7 and HKU8 classifications are close to each other [37,38] and more separated from the Miniopterus bat Coronavirus 1 (bat Coronavirus 1A and 1B classifications) [38]. The two samples considered, which are ON834690.1 M. schreibersii Italy 2/2021 and OP627105.1 M. schreibersii Italy 3/2021, fall next to the group of the Miniopterus bat Coronavirus HK8. Furthermore, they share the same clade with the French (KY423482.1 France 2014) and the Algerian (MN701038.1 Algeria 2017) isolates. M. schreibersii Italy 2/2021 and M. schreibersii Italy 3/2021 are identical to KY423482.1 France 2014, while they have a 98% identity percentage with MN701038.1 Algeria 2017. The branch of the Algerian isolate, which describes a short distance among M. schreibersii Italy 2/2021, M. schreibersii Italy 3/2021 and France 2014, is also caused by an insertion of an adenine in the seventh position of the pairwise alignment between it and one of the novel Italian isolates.

For Betaherpesviruses, the phylogenetic inference was achieved from a 466-nucleotide length multiple sequence alignment with 15 sequences, including different Betaherpesviruses and a Gammaherpesvirus, hosted by bats.

As reported in Figure 2, the phylogenetic tree (log likelihood −5280.60402) shows the submitted sequence with accession number OQ807168.1, mainly grouping with the other three European Betaherpesvirus sequences, sampled from Tadarida teniotis in 2008, having a percentage of identity of 100% with KR608296.1, 82.87% with KR608297.1 and 74.3% with KR608295.1. Even if only partial DNA polymerase B is considered, the isolates belonging to the same host taxonomic family are grouped in the same node, especially for Molossidae, Miniopteridae and Vespertilionidae, as already seen in a previous study [22].

As reported in

Table 3. Results of pairwise global alignments between the novel strain with accession number WJK71402.1 aligned with other European DNA polymerase sequences. * AN: accession number.

AN *	% Identity	Similarity Score	Starting Position	Amino Acids
AMY98779.1	48	97/150	INS 13	VRET
			INS 145	VI
AMY98857.1	54.0	100/150	INS 13	LRDS
			INS 137	VN
AMY98776.1	48.1	100/160	INS 13	LREE
			INS 22	DD
			INS 118	HAHFVDPEFR
			INS 131	FGERELSR
AMY98771.1	58.0	111/150	INS 13	VATD
			INS 139	ER
AMY98783.1	78.5	126/144	/	/
AMY98784.1	100	144/144	/	/
AMY98785.1	89.6	137/144	/	/
AMY98774.1	47.8	101/159	INS 5	FPENVDM
			DEL 10	GPG
			INS 22	AD
			DEL 121	PR
			DEL 126	LS
			INS 137	VEYLPG

, the main differences between our novel sequence WJK71402.1 (OQ807168.1) and other European DNA polymerases, with the exception of Tadarida teniotis hosts, constantly appear at the starting and ending positions. As expected, as shown in Figure 2, WJK71402.1 shows the highest values of percentage of identity and similarity scores with the other three European Betaherpesvirus sequences sampled from Tadarida teniotis in 2008, having a percentage of identity and a similarity score of 100% and 143/143 with AMY98784.1; 89.5% and 136/143 with AMY98785.1; and 78.3% and 125/143 with AMY98783.1

In

Table 4. Results of pairwise global alignment from the alignment of WJK71402.1 (nucleic accession number OQ807168.1) with other members of the taxonomic Betaherpesvirinae family. * AN: accession number.

Protein AN *	Virus Name	% Identity	Similarity Score	Starting Position	Amino Acids	Corresponding Nucleic AN *
CCE57225.1:640-788	Murid betaherpesvirus 1	52.7	106/150	INS 7	EG	HE610455.1
				INS 138	PEA
AAK71288.1:10-157	Baboon cytomegalovirus	58.1	106/148	INS 9	G	AF387664.1
				INS 16	QV
				INS138	P
AAW57296.1:328-477	Murid betaherpesvirus 2	50.7	103/150	DEL 7	ND	AY728086.1
				INS 11	PEVSR
				INS 128	KVI
YP_008492977.1:583-733	Suid betaherpesvirus 2	53.0	100/151	INS 10	DVTGI	NC_022233.1
				INS 132	LS
YP_007969814.1:627-764	Elephantid betaherpesvirus 1	43.2	88/148	INS 13	LREE	NC_020474.2
				DEL 117	QNVLPRSDVL
AAP57912.1:454-594	Human betaherpesvirus 5	52.0	98/148	INS 7	PGGE	AY304055.1
				DEL 138	DVALKVI

, the Italian Betaherpesvirus strain protein sequence was also compared to several Betaherpesvirus sequences hosted by different animals, and all were classified within the Betherpesvirinae taxon family (Taxonomy ID: 10357).

The phylogenetic analysis of Gammaherpesvirus was performed with a multiple sequence alignment of 238 nucleic acid sequence lengths (from position 6 to 218 of the nucleic accession number OQ807169.1) using 23 sequences. Nevertheless, a limitation was a poor sequence length that affected the phylogenetic analysis.

Table 5. Results of pairwise global alignments between every Gammaherpesvirus bat sequence aligned with the complete novel strain with protein accession number WJK71403.1 (nucleic accession number OQ807169.1). The asterisk indicates that the deletion/insertion is at position 1 or 72 t of the sequence because the aligned sequence has the same length as WJK71403.1 but starts one amino acid after ours. * AN: accession number.

AN *	% Identity	Similarity Score	Starting Position	Amino Acids
AFM85234.1	66.7	57/72	/	/
AFM85236.1	69.4	63/72	/	/
AMA67369.1	68.1	61/72	/	/
ATA58242.1	59.0	59/78	DEL 51	KK
			INS 60	PHAPAA
ATU31554.1	57.5	52/73	*	*
BBB06458.1	70.8	63/72	/	/
ATU31556.1	64.4	61/73	*	*

reports identity percentages and similarity scores of the protein submitted sequence WJK71403.1 (OQ807169.1) against other bat-hosted virus sequences.

4. Discussion

In view of the results obtained, virus circulation in the limited number of bat species identified is sporadic and could be due to the low number of samples of these animals submitted to our laboratory, which is complicated by the fact that these carcasses can be predated before being found or could be in an advanced state of decomposition and, therefore, unsuitable for analysis. This could result in a probable underestimation of the number of bat viruses reported [39]. In Italy, the national passive surveillance network for rabies is based on the convenience sampling of dead, sick or injured animals. In particular, with reference to bats, prevalence studies are not feasible, as many of the species are protected.

Species identification was carried out using molecular methods due to the advanced state of decomposition in which some carcasses were found, and because some species have similar morphological features that do not allow an accurate species identification [1]. A correct bat species identification is essential to monitoring, especially in the case of an upsurge in their death rate, as well as the confirmation of the presence of bat species that are more susceptible to viral infections, such as LYSVs, as in the case of Miniopterus schreibersii and Myotis daubentonii reported in our study and others, such as Eptesicus serotinus, Myotis brandtii, Myotis dasycneme and Myotis nattereri, that were not detected [40]. This is another limiting factor, which underlines the importance of investigating bat samples for other viruses besides LYSVs in synergy with other laboratories of different regions that can host different bat species.

Relative to the viral characterization, it would be interesting to correlate the migratory behavior of this species to the phylogenetic data obtained, as the strains detected are related to those identified in geographically close regions [41]. In fact, in the phylogenetic reconstructions, significant differences reveal that M. schreibersii Italy 2/2021 and M. schreibersii Italy 3/2021, which are identical to each other, are located in a group composed of two strains sampled in France and Algeria. Moreover, they were classified with the R library MyCoV [28] as a Minunacovirus taxonomic subgenus of the Alphacoronavirus genus, which are more related to the Miniopterus bat Coronavirus HKU8. On the other hand, for M. schreibersii Italy 1/2021, even if the amplicon obtained was too short to be analyzed phylogenetically, the software used still classified it as Minunacovirus taxonomic subgenus by MyCoV [28]. In addition, even if it presents differences with M. schreibersii Italy 2/2021 and M. schreibersii Italy 3/2021, the three CoV-positive swab samples were collected at the same moment within the same colony. From the data obtained, it can be noted that different Minunacovirus taxonomic subgenus viruses can circulate within the same colony, as previously reported, which could be due to a high genetic variability of these viruses [42].

Betaherpesvirus phylogenetic relationships demonstrate a strict association between novel strain and virus species hosted by European Tadarida teniotis bats, especially with the Tadarida teniotis Betaherpesvirus 2. In consideration of the importance of the DNA pol B region, when our novel strain was aligned with other Betaherpevirinae taxon family strains hosted by other species (Table 4), the majority of similar and matching amino acids were located in the central part of its protein sequence, with the 5′ and 3′ ends being the most dissimilar parts. In fact, the DNA-directed DNA polymerase family B multifunctional domain (InterPro IPR006134) in our novel strain starts from 18 and goes to the 119 aa position. The inference of phylogenetic relationships of novel Gammaherpesvirus is very limited, as the sequence length is too short for a reliable reconstruction. Comparing its short sequence with other deposited sequences, as shown in Table 5, does not show huge differences, with the exception of a vespertilionid Gammaherpesvirus 3 (ATA58242.1).

Although the risk of disease transmission is due to a close interrelationship between humans and animals, it is crucial to follow correct procedures to prevent such events. In relation to the recent COVID-19 pandemic, monitoring the presence of other pathogens, such as CoVs, of which bats are known to be reservoirs, is fundamental due to their ability for genetic reassortment, and this possibly could be responsible for interspecies transmission [6]. The zoonotic potential of viruses discovered in bats has not yet been demonstrated, with the exception of Lloviu virus (LLOV), a filovirus closely related to Ebola virus. Despite this, bats are often in contact with humans and domestic animals, and they host a number of pathogens that have ecological opportunities to emerge in other species, including humans [43].

Due to the reduction in natural habitats and the increase in urbanization, many species have become anthropophilic, with a greater probability of contact and interaction with humans [44]. These flying mammals are able to host many pathogens without showing clinical signs of disease, probably because they are capable of developing antiviral immune responses that control virus replication, thus limiting self-damaging inflammatory responses [15]. Monitoring the presence of viruses and potentially susceptible species is useful in the early detection of virus introduction and circulation for the timely adoption of suitable sanitary measures of containment and the prevention of spread, as proven by the detection in Italy of an LYSV [9] reported only once in bats in the Caucasian region, many years earlier [18].

5. Conclusions

This study provides useful data about the situation of virus circulation and species identification in bats present in the Latium and Tuscany regions in Italy. This is a crucial point to investigate the presence of different viruses that also have a zoonotic potential, which can be hosted by different bat species. These findings can enhance our knowledge of viruses infecting wildlife and the risks related to the possible spillover from wildlife to people.

As our country is part of the agreement on the Act for the Conservation of Population of European Bats (UNEP/EUROBATS), which aims to protect all 51 European bat species through specific legislation, education and conservation measures and international co-operation, species identification using genetic sequencing is useful for monitoring species present in a specific area, especially whenever the morphological identification could be complicated. Furthermore, the submission of dead bats for laboratory analysis is of crucial importance to detect and identify pathogenic agents, as well as the bat species involved, and to be proactive in the prevention of the transmission of pathogens within and between different host species to prevent the spillover of microorganisms.