Abstract

In 2009, an outbreak of white nodular cutaneous lesions was detected in one of only two known breeding colonies of the critically endangered southern bentwing bat (Miniopterus schreibersii bassanii), at Naracoorte, South Australia. Necropsies were conducted on 10 euthanized bats in September 2009. In October 2009, 123 bats were examined under anesthesia, with skin biopsies collected from 18 affected bats. Prevalence of skin lesions was 45.2%. The prevalence among males was three times greater than among females. The majority of lesions examined histologically were granulomas, typically centered on a nematode. A single lesion had epidermal hyperplasia with intracytoplasmic inclusions consistent with a pox virus; pox virions were identified on electron microscopy. Nematodes dissected from frozen lesions were identified morphologically as Riouxgolvania beveridgei, previously described in the eastern bentwing bat (Miniopterus schreibersii oceanensis). The factors contributing to this apparent disease emergence and outbreak remain undetermined. Lesions consistent with white nose syndrome were not identified.

The southern bentwing bat, Miniopterus schreibersii bassanii (Miniopteridae), is a critically endangered species endemic to southeastern South Australia and southwestern Victoria, Australia. During spring the entire population aggregates for breeding in two caves at Naracoorte, South Australia, and Warrnambool, Victoria; this allows for intensive monitoring of the species, much of which is done remotely with the use of cameras installed in the caves. During winter the population disperses widely, occupying over 100 caves.

Over the last four decades, the total adult population of southern bentwing bat has declined from >150,000 to approximately 45,000. Threatening processes include a lack of appropriate management of wintering caves, drought and other impacts on wetland areas impacting upon food resources, and potential effects of agricultural chemicals on bats and the abundance of their invertebrate prey species (Department of Sustainability, Environment, Water, Population and Communities, 2013).

In September 2009, white nodules were observed on the wings of a large proportion of bats at the Naracoorte breeding cave (37°2′1″S, 140°47′42″E). Ten male bats with lesions were captured and anesthetized with the use of isoflurane in oxygen. Blood was collected via cardiac puncture from five animals; blood films were prepared and stained with Quick Dip™ (Fronine Quick Dip, Thermo Fisher Scientific, Norwood, South Australia), and blood was dried on 6-µm filter paper (Whatman plc, Maidstone, UK). Euthanasia was performed via intracardiac pentobarbital. Representative tissues were collected into 10% buffered formalin and processed routinely for histopathologic examination. Skin lesions and viscera were frozen at −80 C.

Nycteribiid flies were present on all bats. Hemoparasites morphologically consistent with Polychromophilus melanipherus, previously described in eastern bentwing bats (Miniopterus schreibersii oceanensis), were present in all five blood films (Dew, 1970).

At necropsy, all bats were in good body condition with good stores of abdominal fat. Skin lesions were present on the unfurred skin of all bats; subcutaneous tissues were not involved. Two macroscopic categories of skin lesion consistent with the identified syndrome of white nodules were defined.

  1. Raised white, often vesicular, 1–2-mm nodules on the dorsal aspect of the limbs, almost exclusively overlying the long bones; 2–6 lesions per bat with 90% prevalence. Lesions were most common over the radius/ulna, followed by the tibia/fibula/tarsus. Occasionally lesions had a small central crust (Fig. 1).

  2. Focal 1–2-mm areas of excoriation/ulceration on the skin on the dorsal aspect of the wings, most commonly in those areas with nodular lesions; 20% prevalence.

Figure 1.

Vesicular cutaneous lesion with central crusted pore on the hind limb of a southern bentwing bat (Miniopterus schreibersii bassani) associated with the nematode Riouxgolvania beveridgei. Naracoorte Caves, South Australia, 2009. Bar = 2.5 mm.

Figure 1.

Vesicular cutaneous lesion with central crusted pore on the hind limb of a southern bentwing bat (Miniopterus schreibersii bassani) associated with the nematode Riouxgolvania beveridgei. Naracoorte Caves, South Australia, 2009. Bar = 2.5 mm.

Additional focal areas of skin pallor of variable size (≤1 cm) were identified in the patagial skin of all bats; these lesions were considered consistent with fibrosis consequent to trauma, and were not investigated further. Internal lesions were limited to artifactual changes associated with cardiac puncture and euthanasia.

Only four lesions from three bats were examined histologically because of difficulties in processing the patagial skin to attain lesions in histologic section. Three of the lesions were focal areas of granulomatous to pyogranulomatous inflammation centered on degenerating nematode parasites (Fig. 2). The other lesion was a focal area of epidermal thickening with spongiosis, ballooning degeneration, and large eosinophilic intracytoplasmic inclusion bodies in large numbers of epidermal cells consistent with a poxvirus, with underlying pyogranulomatous inflammation (Fig. 3). Pox virions were identified in this lesion by electron microscopy with the use of the paraffin-embedded tissue. The histologic appearance of other tissues was unremarkable; trematode parasites were present in the small intestine of two bats with no associated inflammation.

Figure 2.

Granuloma with crusted pore centered on a nematode (Riouxgolvania beveridgei) in the skin of a southern bentwing bat (Miniopterus schreibersii bassani). Naracoorte Caves, South Australia, 2009.

Figure 2.

Granuloma with crusted pore centered on a nematode (Riouxgolvania beveridgei) in the skin of a southern bentwing bat (Miniopterus schreibersii bassani). Naracoorte Caves, South Australia, 2009.

Figure 3.

Epidermal hyperplasia with spongiosis and large eosinophilic intracytoplasmic inclusion bodies associated with a poxvirus in the skin of a southern bentwing bat (Miniopterus schreibersii bassani). Naracoorte Caves, South Australia, 2009.

Figure 3.

Epidermal hyperplasia with spongiosis and large eosinophilic intracytoplasmic inclusion bodies associated with a poxvirus in the skin of a southern bentwing bat (Miniopterus schreibersii bassani). Naracoorte Caves, South Australia, 2009.

A month later, 123 bats (45.5% male, 54.5% female) were captured in a harp trap and mist net at the entrance to the Naracoorte maternity cave, and examined for active and healing lesions representing categories 1 and 2 above, respectively. The prevalence of active lesions was 33.0%. The combined prevalence of active and healing lesions was 45.2% in the sample, 77% in males, and 23% in females. Lesions were present in 8 of 57 females determined to be pregnant by abdominal palpation, and in five of eight nonpregnant females.

Eighteen bats with prominent vesicular lesions were anesthetized as described above. Lesions were individually identified, photographed, and biopsied for histopathologic examination (n = 38) or dissection to harvest parasites (n = 3 in formalin, n = 4 frozen at −20 C). Blood was collected from the cephalic vein. Hemoparasites consistent with those identified previously were present in 11 of 18 blood films.

Histologic sections of lesions were obtained from 33 of the 38 biopsies. All lesions were inflammatory, predominantly eosinophilic and granulomatous dermatitis. Eighteen lesions were centered on a nematode. Some nematodes were well preserved, others were degenerated. Two lesions with parasites had an open pore through the epidermis, consistent with the gross appearance of some lesions (Figs. 1, 2). The remaining 15 lesions had no parasite in section, though were considered consistent with a parasitic granuloma—possibly tangential sections, or at a stage of the disease following elimination of the parasite. Some lesions without parasites in section had lymphocytic or neutrophilic infiltrates. No lesions consistent with a poxvirus infection were identified.

Frozen lesions were dissected under low-power light microscopy. Extracted nematodes were fixed in 70% ethanol, cleared in glycerol, and identified morphologically as Riouxgolvania beveridgei (Bain and Chabaud, 1979). Specimens have been deposited in the Muséum National d'Histoire Naturelle, Paris, France (Bocal 779, 132 YU). During the three breeding seasons following this outbreak very few bats with lesions were observed, though captures were not undertaken to quantify prevalence.

Nematodes of five described genera in the Order Muspiceida have been described in many parts of the world in a diverse range of mammalian hosts including bats, rodents, marsupials, and reindeer (Anderson and Bain, 1982). Four Riouxgolvania spp. have been described worldwide, all skin parasites of bats (Bain and Chaboud, 1968, 1979; Hasegawa et al., 2012).

The life histories of the Muspiceida are not well understood. Modes of transmission that have variably been proposed include mucocutaneous penetration, ingestion via lactation or grooming, and cannibalism. Muspicea borreli is reported to have a direct life cycle in Mus domesticus, likely involving penetration of the skin or mucous membranes of the host (Spratt et al., 2002). The Riouxgolvania spp. are protandrous hermaphrodites; the eggs hatch in the uterus, and infective larvae develop within the body of the female (Bain and Chaboud, 1979). The vulva of the gravid Riouxgolvania spp. appears to be nonfunctional (Hasegawa et al., 2012). Rausch and Rausch (1983) suggested that infective larvae of the bat-associated muspiceid Maseria vespertilionis exit the host through a cutaneous fistula; the open pore identified histologically in two lesions in this investigation suggest a similar strategy for R. beveridgei. Four spear-shaped, cuticular ornamentations on the infective third-stage larva of Riouxgolvania kapapkumi may facilitate skin penetration of the host (Hasegawa et al., 2012). The close contact between cave-dwelling bats, which typically congregate in large numbers, would be conducive to this mode of transmission (Rausch and Rausch, 1983; Hasegawa, 2012). Transmission via hematophagous parasites may be an alternative mechanism of transmission for infective larva of the Muspiceida (Beaver and Burgdorfer, 1987). Nycteribiid flies, found on a large proportion of the bats examined in this study, are known to transmit hemoparasites and could contribute to the transmission of R. beveridgei (Dew, 1970).

It is unclear if this nematode parasite was historically endemic in the population and had suddenly increased in prevalence, or if it has been recently introduced to a naïve population. With limited understanding of the life cycle of the nematode parasite in this population, it is difficult to speculate on what factors might have allowed such a dramatic, yet short-lived, increase in prevalence. No significant adverse impact to affected individuals or to the population in association with this parasite was identified.

Anecdotally, white nodular lesions have been observed very occasionally in M. s. bassanii in both the Naracoorte and Warrnambool populations, though the etiology apparently has not been investigated previously. Riouxgolvania beveridgei was described in M. s. oceanensis by Bain and Chabaud (1979). The ranges of the two subspecies overlap in south central Victoria, so cross-species transmission could have occurred.

Lesions consistent with white nose syndrome were not identified in this investigation. White nose syndrome has caused significant population declines of multiple species of cave-dwelling bat in North America (Blehert et al., 2009). It has not been reported in bats in Australia. Demonstrating the absence of white nose syndrome was an important component of this investigation of white skin lesions in a cave-dwelling bat.

ACKNOWLEDGMENTS

We acknowledge staff and volunteers at the Naracoorte Caves who assisted in monitoring and capture of bats for this investigation. Ian Beveridge and Odile Bain identified the parasite. Electron microscopy was performed by Alex Hyatt and Sandra Crameri at the Australian Animal Health Laboratory. Funding for the investigation was provided by Biosecurity SA: Animal Health.

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