Ectoparasites may serve as vectors for the white-nose syndrome fungus

Material collection

Using heat-sterilised forceps, we collected wing mites of the genus Spinturnix from 33 greater mouse-eared bats (Myotis myotis) at the end of the hibernation season (March – April 2014) at four sites in the Czech
Republic, i.e., Kate?inská Cave (Moravian Karst), the Šimon and Juda Mine (Jeseniky
Mountains) and the St. Kate?ina and Kristína Mines (Šumava Mountains). The mites were
determined morphologically using light microscopy 16], 17] in the pilot study as Spinturnix myoti. The sampled bat species did not host other Spinturnix taxa according to molecular genotyping 18].

In order to diagnose WNS and to quantify the fungus on bats, we swabbed the dorsal
side of the extended left wing and transilluminated the left wing membrane of each
bat using a UV lamp at 368 nm wavelength 19]. The wing was photographed and the number of fluorescent spots was counted from the
photographs. Nine bats were not photographed due to technical problems in the mine.
The total number of bats sampled for wing mites/swabbed/UV-examined at each site was:
Kate?inská Cave (4/4/4), Šimon and Juda Mine (19/19/10), St. Kate?ina Mine (6/6/6),
Kristína Mine (4/4/4).

UV-guided wing biopsy punches 19], stored in formalin, were embedded in paraffin, cut to 5 ?m tissue sections and stained
with periodic acid-Schiff. Fungal skin lesions diagnostic for WNS were identified.

Additional swabs collected to cultivate P. destructans were transferred onto Sabuoraud agar and incubated in dark at 10 °C. Selected isolates
were deposited into Culture Collection of Fungi, Prague, Czech Republic.

Ethical approval

Sampling was performed in compliance with Czech Law No. 114/1992 on Nature and Landscape
Protection, and was based on permits 01662/MK/2012S/00775/MK/2012, 866/JS/2012 and
00356/KK/2008/AOPK issued by the Agency for Nature Conservation and Landscape Protection
of the Czech Republic. The authors are authorised to handle free-living bats according
to Certificate of Competency No. CZ01297 (§17, Act No. 246/1992 Coll.).

DNA isolation and quantitative PCR

One to 17 wing mites were sampled from each bat. All mites found on a single bat were
pooled into a collection tube containing tissue lysis buffer (180 ?l, Qiagen DNeasy
Blood Tissue Kit, Qiagen, Halden, Germany) and transported to the laboratory in
a cooler. Directly after transfer, proteinase K (20 ?l) was added to the samples and
incubated at 56 °C for 2 h. Lysis buffer (200 ?l) was added and the samples further
incubated for 10 min. The manufacturer’s protocol was followed, and total isolated
DNA was eluted in 100 ?l of the elution buffer. DNA from swabs from dorsal side of
the left wing was isolated with Qiagen QIAamp DNA Mini Kit (Qiagen) according to the
manufacturer’s recommendation.

Fungal load on wing mites and bats was quantified with quantitative PCR (qPCR; 20]), using TaqMan® Universal Master Mix II with UNG (Uracil N-glycosylase; Life Technologies,
Foster City, CA, USA). In order to optimise the PCR reaction, bovine serum albumin
at final concentration of 0.05 mg/?l, and 0.025 U of Platinum® Taq DNA Polymerase
were supplemented. Forward and reverse primers were used at a final concentration
of 0.3 ?M. Species-specific and genus-specific fluorescently labelled custom probes
were used for the quantification of the PCR product, with final concentrations of
0.115 and 0.16 ?M, respectively. The reaction mix was prepared on ice with 2 ?l of
DNA and three replicates were mixed for each DNA sample. Dual probes used in the Shuey
et al.’s 20] protocol enable distinguishing true-positive samples with P. destructans from false-positives where high loads of related fungi occur 7].

A real-time qPCR reaction was performed on the LightCycler 480 PCR System (Life Technologies),
with initial inactivation at 50 °C for 2 min and a hot start at 96 °C for 10 min.
Nine cycles with a denaturation step at 95 °C for 15 s, and annealing at 62 °C for
1 min were followed by 43 identical cycles with quantification detection. The qPCR
was finalised with a dissociation at 95–60–95 °C for 15 s each and cooled to 40 °C
for 10 min. DNA isolated from a culture of the CCF3937 P. destructans strain 8] was used as a positive control and concentration reference during each run.

Data analysis

A DNA concentration calibration curve was calculated from a dilution series of the
CCF3937 P. destructans strain. The exact concentration of DNA in each dilution was determined using a Qubit
HS fluorometer (Invitrogen, Carlsbad, CA), using the manufacturer’s protocol. qPCR
effectivity was 1.96 and the sample concentration was calculated using custom scripts
in R 21]. Fungal load was estimated from equation log (q _PdDNA
)?=?3.194–0.287 Cp, R²
?=?0.9719, where q is DNA concentration and Cp is the cycle. Each result was adjusted according to the positive control in its run
and overall elution of the DNA. Fungal load on wing mites was calculated by dividing
the obtained fungal load from the whole sample with the number of mites collected
in that sample.

Pseudogymnoascus destructans load on the mites was correlated with the fungal load on the left wing and with the
number of UV fluorescent spots diagnostic for WNS. The number of UV fluorescent spots
corresponds with the amount of damage caused by the fungal infection 19].

Results and discussion

All 33 screened bats and all 33 wing mite samples were positive for P. destructans DNA. The fungal load sampled from bat wings was higher than that from ectoparasites
(mean?±?SD: P. destructans load on bat wing (ng)?=?7.95?±?15.26, P. destructans load on all mites sampled (pg)?=?133.27?±?170.37, P. destructans per mite (pg)?=?49.38?±?75.03). The fungal load on bat wing was significantly correlated
both with fungal load on pooled wing mites (Pearson’s r?=?0.69, p??0.001) and with fungal load per wing mite (Pearson’s r?=?0.74, p??0.001; Fig. 1a). Furthermore, fungal load per wing mite was positively correlated with the number
of fluorescent lesions on bat wings (Pearson’s r?=?0.46, p?=?0.02; Fig. 1b).

Fig. 1. Pseudogymnoascus destructans load on ectoparasites. a Fungal load on wing mites in relation to P. destructans load on bat, and b number of UV-fluorescent spots representing WNS lesions, on the left wing. Both axes
are log
₁₀
. Solid line shows linear regression function, dashed lines delimit its 95 % confidence
intervals

The sampled bats suffered from WNS as evidenced with the UV-fluorescent spots corresponding
to WNS skin lesions (Fig. 2) that were found on all bats in this study. The pathogenic fungus was viable on them
as proved by culture experiments (P. destructans isolates CCF4987-CCF4992).

Fig. 2. White-nose syndrome lesions on bat skin. a Orange-yellow spots displayed with ultraviolet light transillumination representing
WNS skin lesions in M. myotis bat wing. b Histopathologic cross section from a UV-guided wing punch biopsy stained for fungi
with periodic acid-Schiff shows three cupping erosions surrounded by necrotic tissue
and neutrophilic infiltration (arrows) and fungal invasion in a hair follicle (arrowhead), confirming WNS

Our data provide direct evidence for the presence of spores and/or hyphae from the
fungus causing WNS on bat ectoparasites. Furthermore, we observed a positive relationship
between amount of fungal infection on bats and fungal load present on wing mites.

Up until now, WNS fungus transmission has been assumed to occur between bats either
by physical contact or by contact with the environment 22]. Given that spinturnicid mites switch hosts horizontally by crawling from one bat
to another 23], our data demonstrate the potential for wing mites to play a role in the transmission
of P. destructans spores between bats.

Ectoparasites in general play an important role as vectors of many diseases in vertebrates,
including such emerging diseases as plague, malaria, leishmaniasis, trypanosomiasis,
haemorrhagic fevers, babesiosis, borreliosis, tularemia, tick-borne encephalitis and
many others 24], 25]. Unlike WNS, however, these are all caused by haemoparasitic agents, meaning that
they are transmitted inside the bodies of the vectors. To our knowledge, there is
no known disease transported by ectoparasite vectors on the outside of its body. Having
said this, there are numerous examples of mechanical transport of pathogens by arthropods,
such as the transport of rotaviruses, protozoan parasites or salmonellosis by non-biting
flies and cockroaches 26]–28]. We hypothesise that mechanical transport of P. destructans propagules between bats on the bodies of spinturnicid mites is enabled by their specialisation
of living on bat wing membranes, i.e., the body region most typically affected by
fungal growth 29]. This is supported by our finding of a positive relationship between fungal load
on wing mites and fungal load and infection intensity on bat wings. An analogous situation
may also hold true for other bat ectoparasites infesting bats during the time of fungal
growth, such as Macronyssidae, fleas and nycteribiid flies; however, this remains
to be investigated.

Positive findings of P. destructans in bat hibernacula over the summer period 30], and tests of its nutritional abilities, suggest that it is able to grow and sporulate
on a variety of organic substrates, including dead fish, insects and mushroom tissues
31]. It is quite likely, therefore, that P. destructans can grow on bat ectoparasites, at least in periods when bats roost in cold environments
and/or enter body torpor.

The transmission mode of wing mites, and their inability to survive off the host’s
body, requires physical contact between bats 32]. Consequently, bat species that hibernate in clusters may be at higher risk of becoming
infected by WNS than solitary hibernating bats. WNS prevalence is higher in bats forming
clusters than in those hibernating solitarily, but solitary hibernators are also susceptible
to the disease 11]. In this case, it is possible that P. destructans propagule transmission between bats takes place prior to hibernation, i.e., during
the swarming period when bats are mating 33].

Last but not least, in addition to transport of fungal spores and/or fragments of
mycelium, mites may facilitate entry of the fungal hyphae through the epidermis of
bats via injuries caused by their bites. These injuries could prove very important
for the pathogenesis of P. destructans skin infections as no signs of fungus keratinolytic activity were observed in the
stratum corneum of bats under ultramicroscopy 34]. Our confirmation of the potential for wing mites to serve as vectors for P. destructans suggests a previously unknown transmission mode for WNS and stresses the importance
of further research focused on testing this hypothesis.