Pacific Science, vol. 68, no. 1 July 16, 2013 (Early view) 

Environmental conditions associated with lesions in introduced free-ranging sheep in

Hawai‘i

By: Jenny G. Powers, Colleen G. Duncan, Terry R. Spraker, Bridget A. Schuler, Steven C. Hess*, Jonathan K.J. Faford and Hans Sin Abstract Wildlife species which have been translocated between temperate and tropical regions of the world provide unique opportunities to understand how disease processes may be affected by environmental conditions. European mouflon sheep (Ovis gmelini musimon) from the Mediterranean Islands were introduced to the Hawaiian Islands for sport hunting beginning in 1954 and were subsequently hybridized with feral domestic sheep (O. aries), which had been introduced in 1793. Three isolated mouflon populations have become established in the Hawaiian Islands but diseases in these populations have been little studied. The objective of this study was to evaluate and compare gross and histologic lesions in respiratory, renal, and hepatic systems of free-ranging sheep in two isolated volcanic environments on Hawai‘i Island. Tissue and fecal samples were collected in conjunction with population reductions during February 2011. We found gross or histologic evidence of lungworm infection in 44/49 sheep from Mauna Loa which were exposed to gaseous emissions from Kīlauea Volcano. In contrast, only 7/50 sheep from Mauna Kea had lesions consistent with lungworm, but Mauna Kea sheep had significantly more upper respiratory tract inflammation and hyperplasia consistent with chronic antigenic stimulation, possibly associated with exposure to fine airborne particulates during extended drought conditions. We hypothesize that gasses from Kīlauea Volcano contributed to severity of respiratory disease principally associated with chronic lungworm infections at Mauna Loa; however, there were numerous other potentially confounding environmental factors and interactions that merit further investigation. *Corresponding Author E-mail: shess@usgs.gov

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Introduction

Wildlife species which have been translocated between temperate and tropical regions of the

world provide unique opportunities to understand how disease processes may be affected by

environmental conditions (Woodford 1993, Cunningham 1996). Interactions between pathogens,

their hosts, and novel environments may mitigate or compound pathological responses;

producing unique situations which introduced species have not encountered during previous

evolutionary history, affecting viability, and contributing to invasive spread or ultimate

extirpation. Environmental conditions can change over time and rare events may occur which

affect the long-term health of introduced populations. Analyses of comparative pathological

responses between different environments may have major implications for other species,

including sympatric wildlife, livestock, and humans in the vicinity. The effects of natural

environmental conditions are often difficult to disentangle because they are not amenable to

controlled experimentation. Pathological responses may also be affected by heritage and

previous disease exposure of founding populations.

European mouflon sheep (Ovis gmelini musimon) from the Mediterranean Islands have

become abundant where they have been introduced to the Canary, Kerguelen, and Hawaiian

archipelagos (Chapuis et al. 1994, Hess et al. 2006, Nogales et al. 2006). Mouflon were first

introduced to the Hawaiian Island of Lāna‘i in 1954 for sport hunting (Tomich 1986). Feral

domestic sheep (O. aries), which had been introduced to Hawai‘i Island in 1793, were

intentionally hybridized with mouflon and released on Mauna Kea from 1962–1966 (Tomich

1986). Another Hawai‘i Island population of mouflon was founded at Kahuku on Mauna Loa

from only 11 individuals between 1968–1974 (Hess et al. 2006). Three isolated mouflon

populations have become established in the Hawaiian Islands to date and there has been little

study of disease within these populations, although lungworm (Muellerius capillaris) was

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previously documented in Mauna Loa mouflon (O’Gara 1994). Both free-ranging sheep

populations on Hawai‘i Island have become invasive, have caused severe degradation of native

vegetation, and as a consequence, have been lethally removed in large numbers to protect and

recover endangered flora and fauna (Stephens et al. 2008, Banko et al. 2013).

Abiotic factors which may affect the prevalence and severity of disease in free-ranging

sheep on Hawai‘i Island include insularity, climate, and volcanism. Introduced sheep

populations at Mauna Loa and Mauna Kea have been effectively isolated from each other and all

other populations, which may limit genetic diversity and heterozygosity (Allendorf 1986). The

tropical montane climate of Hawai‘i Island is aseasonal relative to the Mediterranean climate

where these sheep originated; however, there are strong local differences in precipitation in

Hawai‘i due to orographic effects. Another environmental factor of particular interest was

exposure to a continuing gaseous eruption from the Halema‘uma‘u vent of Kīlauea Volcano

which began in March of 2008 and increased sulfur dioxide (SO2) emissions by an order of

magnitude, causing the largest rise in mean atmospheric sulfate concentrations in the U.S. since

the early 1990s (Elias and Sutton 2012, Hand et al. 2012). Prevailing Northeasterly trade winds

concentrated SO2 emissions on the Southeastern flank of Mauna Loa, thus exposing mouflon on

Mauna Loa to consistently high SO2levels, whereas sheep on Mauna Kea had little SO2 exposure.

Biotic factors that may influence disease processes include differences in pathogen

exposure, genetics, and biological habitats between populations. Three liberations of mouflon

and domestic sheep may have contributed to differences in contagious diseases, parasites, genetic

founder effects, and limited initial genetic diversity in both populations. Due to the paucity of

information on the health of free-ranging animals in the region and the implications for health of

other wildlife species, livestock, and humans, we conducted post-mortem examinations and

collected tissue and fecal samples during population reductions of mouflon on Mauna Loa and

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hybrid sheep on Mauna Kea to study physiologic effects associated with biotic and abiotic

environmental conditions. The objective of this study was to evaluate and compare gross and

histologic lesions in respiratory, renal, and hepatic systems of free-ranging sheep in two isolated

environments on Hawai‘i Island.

Materials and Methods

Study Area

Sheep were collected from geographically isolated populations on Hawai‘i Island (Fig. 1).

European mouflon (n = 49; 12 male, 37 female) were collected on 3 February 2011 from the

Kahuku Unit of Hawai‘i Volcanoes National Park on Mauna Loa (Mauna Loa; 19° 18′ N, 155°

35′ W; 1,828–2,286 m elevation). Hybrid mouflon x domestic sheep (n = 50; 28 male, 22

female) were collected on 1–2 February 2011 from Palila Critical Habitat on Mauna Kea (Mauna

Kea; 19° 48′ N, 155° 36′ W; 1,828–2,740 m elevation). All animals were killed by gunshot

during population reductions in accordance with approved management protocols at Hawai‘i

Volcanoes National Park and the State of Hawai‘i Department of Land and Natural Resources

Division of Forestry and Wildlife. Both sheep populations reside primarily in subalpine

environments. Annual precipitation from 1983–2008 at 1,521 m elevation in the Mauna Loa

study area averaged 975.2 ± 341.3 (SD) mm (Hess et al. 2011). Average annual precipitation

from 1932–1977 at 2,268 m elevation on Mauna Kea was 490.4 ± 53.5 mm (Giambelluca et al.

2012). Extended drought conditions prevailed at both locations; rainfall in 2009, 2010, and 2011

was 67.8%, 46.2%, and 65.7% of the long-term annual mean rainfall at 21, 34, and 31 climate

stations, respectively, on Hawai‘i Island (National Weather Service, Honolulu, Hawai‘i). Mean

annual temperature on Mauna Kea was 11.1 ± 1.5º C (Juvik and Nullet 1993). Interpolated mean

minimum and maximum annual temperatures ranged from 7–18º C in the Mauna Loa study area

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(Schlappa et al. 2011). Seasonal frosts and occasional snow occurred in high elevation areas at

both locations.

<<Figure 1 near here>>

Field Methods

Field necropsies were performed by veterinary pathologists. Relative body condition and

approximate age were assessed by postmortem external examination. Three levels of body

condition were assigned by palpation of the proximal dorsal rib cage; poor (ribs prominent),

good (ribs palpable), or fat (ribs not easily palpable). Age was based on tooth eruption and wear

of incisor teeth; presence of deciduous incisors indicated lambs, unworn permanent incisors

indicated sub-adults, worn permanent incisors indicated adults (Montana Veterinary Research

Laboratory 1936, Hess et al. 2011). Reproductive status was determined by evidence of a fetus,

recent uterine involution, or lactation during necropsy. All major organ systems were visually

assessed for gross abnormalities. Samples of upper and lower respiratory tract (i.e., nasal

mucosa, trachea, bronchial bifurcation, and lung lobes), kidney, and liver were collected, and

fixed in 10% neutral buffered formalin. Fresh feces were placed on ice in the field and then

frozen at -20°C at the end of each day.

Laboratory Assays

Formalin fixed samples were trimmed, embedded in paraffin, sectioned at five micrometers and

stained with hematoxylin and eosin at Colorado State University Veterinary Diagnostic

Laboratory for histologic examination. Fecal samples were thawed at 4°C prior to preparation,

and analyzed using standard McMaster fecal float techniques in a saturated magnesium sulfate

solution and scanned at 40x to detect lungworm and other internal parasite larvae and ova which

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were identified to species level when possible (Urquhart et al. 1996). Preservation by freezing

precluded quantitative assessment of lungworm larvae using the Baermann technique (Zajac and

Conboy 2006), thus prevalence, but not the number of larvae, was compared. Chi-square tests

were used to compare categorical variables including age, body condition, sex, frequency of

lesions, and lungworm presence between populations (SPSS v. 19, IBM, Chicago IL).

Results

There were no differences in age (χ2 = 3.40; P = 0.18) or body condition score (χ2 = 2.21; P =

0.14) between sheep from Mauna Loa and Mauna Kea. Lambs and sub-adults comprised 43% of

all animals and both populations were in good or fat body condition. A lower proportion of

males (23%) were collected from Mauna Loa than Mauna Kea (56%; χ2 = 11.19; P < 0.01).

Pregnancy in females (77% overall) did not differ between populations (χ2 = 0.34; P = 0.56).

Lungworm granulomas, characterized by multifocal to coalescing pulmonary nodules,

were more prevalent in sheep from Mauna Loa (44/49) than from Mauna Kea (7/50; χ2 = 56.92;

P < 0.01). Granulomas were firm, raised, subpleural, focal, pale tan or greenish nodules ranging

from 0.5–5 cm in diameter, and were most commonly located in the caudodorsal lung.

Histologically, lesions consisted of large aggregates of eosinophils with a peripheral rim of

macrophages and substantial fibrosis and mineralization. Inflammation was often focused

around collections of nematode eggs and larvae. Smooth muscle hyperplasia was observed

concurrently with parasitism in severely affected areas. Larvae and eggs were also present in

less severely affected areas of lungs without significant inflammation, but adult nematodes were

rare in these areas. The severity and chronicity of lesions varied within and between individuals,

but was generally less severe in Mauna Kea sheep than the Mauna Loa population, where up to

50% of the pulmonary parenchyma was affected in some animals.

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No gross evidence of disease was seen in the upper respiratory tract, however, the most

common histological lesion of nasal turbinates was increased submucosal, mononuclear

inflammation with variable presence of eosinophils; areas of mucosal thickening and goblet cell

hyperplasia were less commonly observed. These abnormalities were more common in Mauna

Kea (38%) than Mauna Loa sheep (10%; χ2 = 10.39; P < 0.01). Similar mid-tracheal lesions

occurred in only 13% of all animals and did not differ between populations (χ2 = 1.43; P = 0.23).

Lesions were also seen at the tracheal bifurcation in 26% of animals, but frequency did not differ

between populations (χ2 = 1.49; P = 0.22).

Mild liver inflammation occurred in 67% of Mauna Loa sheep and 54% of sheep from

Mauna Kea, but frequency of lesions did not differ between populations (χ2 = 1.85; P = 0.17).

Five animals had mineralized liver nodules during gross examination (n = 3 Mauna Loa, n = 2

Mauna Kea). The most common microscopic liver lesion was mild periportal mononuclear

hepatitis that did not disrupt limiting plates. Moderate biliary hyperplasia in conjunction with

mild periportal fibrosis occurred in 24% of Mauna Kea sheep but was not observed in any

Mauna Loa sheep (χ2 = 13.38; P < 0.01). The kidneys of all animals were grossly unremarkable.

Mild renal abnormalities occurred in 44% of all animals with no difference between populations

(χ2 = 1.70; P = 0.19) and included mononuclear interstitial nephritis, lymphoid aggregates in the

renal pelvis, and occasional mineralization in collecting tubules.

Trematode, nematode, cestode, and coccidia ova or larvae occurred more often in feces of

Mauna Loa sheep (67%) than Mauna Kea sheep (24%; χ2 = 18.76; P < 0.001). Lungworm larvae

were more prevalent in feces of sheep from Mauna Loa (24%) than Mauna Kea (2%; χ2 = 10.97;

P < 0.001). Lungworm larvae could be definitively identified as M. capillaris in only five cases

based on an accessory spine and characteristic S-shaped kink at the end of the tail (Pybus and

Shave 1984). The possibility of morphologically identical Parelaphostrongylus spp. was ruled

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out because the Hawai‘i sheep had no contact with the definitive host of Parelaphostrongylus,

white-tailed deer (Odocoileus virginianus), and these larvae are not shed in the feces of aberrant

hosts (Bowman et al. 1999). Protostrongylus spp. was not definitively identified or excluded

from diagnosis. In addition to lungworm larvae, Mauna Loa sheep also had fecal ova consistent

with the genera Nematodirus, Strongylus, and Eimeria. Mauna Kea sheep had ova consistent

with the genera Nematodirus, Trichuris, Moniezia, and Eimeria (Bowman et al. 1999, Urquhart

et al. 1996).

Discussion

We found major differences between Mauna Loa mouflon and Mauna Kea hybrid sheep in

pathological lesions of respiratory systems. The most common respiratory lesion observed was

eosinophillic inflammation associated with lungworm infestation. Gross and/or histologic

evidence of pulmonary nematodiasis infection occurred in nearly all Mauna Loa mouflon we

examined. In contrast, only 20% of Mauna Kea hybrid sheep had lesions consistent with

lungworm. Mauna Kea hybrid sheep had significantly more upper respiratory tract inflammation

and hyperplasia consistent with chronic antigenic stimulation than Mauna Loa mouflon. We

observed no evidence, however, of erosions in nasal turbinates, trachea, or bronchi in either

population. There were numerous potential factors which may have affected the prevalence and

severity of pulmonary disease between the two populations.

Potential contributing factors include major differences in volcanic substrates,

precipitation between the two otherwise aseasonal environments, and the combined effects of

these abiotic factors on available forage species within plant communities. Differences in

abundance of lungworms, rather than susceptibility of sheep, may have also affected differences

in respiratory health between populations. While the two sheep populations reside ≤ 60 km of

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each other, biological habitats differ strongly between the two sites. Biotic interactions which

may mitigate or compound these pathological responses include differences in vegetation,

rainfall, invertebrates, soil microflora and microfauna, or genetic heritage of the founding sheep

populations. A more humid and warmer environment with a wider variety of plant species may

have been favorable for intermediate snail or slug hosts of lungworms, contributing to the higher

prevalence of infestation in the Mauna Loa mouflon, although it is not known if founders were

infected prior to arrival. The Mauna Loa mouflon were descended from a small number of

individuals, probably with limited initial genetic variation (Allendorf 1986, Hess et al. 2006). In

bighorn sheep (O. canadensis) populations that have undergone genetic bottlenecks, decreased

heterozygosity in disease resistance genes is associated with higher lungworm parasite loads

(Luikart et al. 2008). Domestic sheep ancestry, a lengthier period of local adaptation, and

enhanced vigor among Mauna Kea hybrid sheep may have also conferred resistance to common

parasites or other diseases. Muellerius spp. infections typically do not produce clinical signs in

domestic sheep (Pugh 2002) but may be more pathogenic in non-adapted hosts such as bighorn

(Pybus and Shave 1984, Demartini and Davies 1977) and possibly mouflon (Panayotova-

Pencheva and Alexandrov 2010). Lungworm prevalence in wild mouflon of Europe is reported

to be high and M. capillaris is considered the most important pneumonematode of mouflon in the

Czech Republic (Lamka et al. 1996). In two studies in Bulgaria, M. capillaris was found in nine

of 11 mouflon in combination with other protostrongylid species, all of which had mild to severe

lung lesions (Panayotova-Pencheva 2006, Panayotova-Pencheva and Alexandrov 2010). Four

protostrongylid genera including M. capillaris were found in ten mouflon in a study conducted in

Spain (Meana et al. 1996). While lungworm infestations in mouflon are apparently common, the

severity of lung lesions observed in Mauna Loa mouflon was surprising. In other wild sheep

such as bighorn, severe lungworm associated pathology rarely occurs in the absence of

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underlying respiratory tract damage. It occurs most often in conjunction with bacterial and/or

viral infections or other stressing events characteristic of bighorn sheep pneumonia complex

(Forrester 1971, Spraker et al. 1984, Monello et al. 2001). Mauna Loa mouflon were regularly

exposed to high concentrations of volcanic gases after the 2008 eruption of Kīlauea which may

have contributed to compromised lung health, lower resistance to lungworm infections, and

increased propensity for severe inflammatory reactions to pulmonary nematodiasis.

The higher prevalence of lesions associated with chronic antigenic stimulation in Mauna

Kea hybrid sheep despite their lack of exposure to volcanic gasses may have been associated

with exposure to fine airborne particulates as a result of reduced plant ground cover during

extended drought conditions. Exposed surface substrates of Mauna Kea primarily consist of

volcanic cinder and ash composed of vitrandept (from Latin vitrium “glass” and depositum

“deposit”) particles which form fine wind-blown particulates and occasional dust storms,

whereas surface substrates of Mauna Loa are primarily rocky basaltic lava flows resistant to

wind erosion (Hess and Banko 2011, Trusdell et al. 2005). Effects of Muellerius spp. infections

typically appear as gross lesions and nodules on bronchioles, bronchioli, alveoli, and alveolar

septa, (Pybus and Shave 1984, Demartini and Davies 1977, Panayotova-Pencheva and

Alexandrov 2010) but not as inflammation of nasal turbinates, tracheae, or hyperplasia of upper

respiratory tracts.

The hepatic periportal fibrosis and biliary hyperplasia we observed in Mauna Kea hybrid

sheep may have also been related to extended drought conditions. Although ten naturalized plant

species on Hawai‘i Island may contain pyrrolizidine alkaloids (Wagner et al. 2005), none were as

abundant as the invasive pasture weed Senecio madagascarensis, which was widespread

throughout Mauna Kea, but not yet established at Mauna Loa (Benitez et al. 2008). These

alkaloids inhibit mitosis of hepatocytes and can cause liver damage (McGavin and Zachary

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2007), particularly if consumed in large quantities in the absence of other available forage during

extended drought conditions. We did not directly examine other potential physiologic

differences in exposure to the minor components of volcanic products such as hydrogen sulfide,

chloride, and fluoride; however, there was no gross indication of dental fluorosis or other toxicity

related effects.

The interaction of chronic exposure to volcanic gasses with likely greater intermediate

host abundance for lungworms could have contributed to the extensive lungworm associated

pathology we observed. The severity of pulmonary disease we observed also has implications

for sympatric wildlife, livestock, and humans in the vicinity. Areas downwind of the volcanic

plume have experienced vegetation damage, negative effects on lung function and reproduction

in domestic livestock, and adverse effects on human respiratory health; however, effects on free-

ranging animals have not been well studied. Anecdotal information gathered from local ranchers

in the vicinity of Kīlauea suggested low livestock birth weights and a number of reproductive

and general health abnormalities became widespread after the 2008 eruption. Lamb/ewe ratios

of Mauna Loa mouflon in 2008 and 2009 were substantially lower than that of 2007 (S. Hess,

unpubl. data) and population abundance at higher elevations was noticeably reduced relative to

the number of mouflon removed (J. Faford, personal observation). Studies of human health near

Kīlauea indicated that the odds of cardiovascular disease were significantly greater in individuals

exposed to high concentrations of volcanic gasses relative to unexposed individuals (Longo

2009). Similarly, exposed humans had a significantly higher risk of acute bronchitis than

unexposed cohorts (Longo and Yang 2008). Retrospective evaluations of hospital visits

associated with volcanic gasses revealed a strong association with individuals for asthma or

chronic obstructive pulmonary disease (Michaud et al. 2004) suggesting preexisting lung disease

may be a risk factor for respiratory symptoms following exposure to volcanic gasses. Humans

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experimentally exposed to SO2 showed mucosal erythaema in the distal part of tracheae and

proximal main bronchi (Sandström et al. 1989, Petruzzi et al. 1994). Laboratory mice exposed to

SO2 exhibited lesions in the nasomaxillary turbinates consisting of edema, necrosis, and

desquamation of the respiratory and olfactory epithelium (Petruzzi et al. 1994). These findings

are inconsistent with our observations of less frequent turbinate lesions in Mauna Loa mouflon

regularly exposed to high SO2 concentrations than in unexposed Mauna Kea hybrid sheep,

suggesting SO2 exposure was not sufficient in concentration or duration to cause acute upper

respiratory contact lesions.

Based on our finding and the effects of SO2 studies in laboratory animals and humans

(Sandström et al. 1989, Petruzzi et al. 1994), we hypothesize that airway irritation and possible

lung compromise compounded lungworm infections in these sheep. Further study of long-term

effects of exposure to volcanic emissions while controlling for numerous other potentially

confounding environmental factors and their interactions is warranted in other sympatric wildlife

and humans. Nonetheless, pathology observed in the respiratory, hepatic, and renal systems did

not affect body condition or contribute to conception failure or early pregnancy loss in this study.

Pregnancy rates were similar and unlikely to limit either population, and all animals were either

in “good” or “fat” body condition. Despite major differences in respiratory health between

populations, there was no indication that that either population was limited in reproduction,

survival, or ability to increase in numbers or geographic range under novel conditions of tropical

montane environments in which these sheep have no evolutionary history.

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Acknowledgments

We thank E. Wheeler and J. Spaak for laboratory support, D. Pacheco and C. Cabral for field

expertise, D. Okita for helicopter services, and two anonymous reviewers for many helpful

comments. Special thanks to B. Nagamine and L. Ballweber for assistance with parasitology and

to L. Petrie for advice on this project. Use of trade, product, or firm names in this publication is

for descriptive purposes and does not imply endorsement by the U.S. Government. This research

was funded by the National Park Service Biological Resource Management Division and the

Invasive Species Programs of U.S. Geological Survey.

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Figure 1. Study locations of lesions in free-ranging sheep (Ovis spp.) on Hawai‘i Island.

Outlined regions are Palila Critical Habitat on Mauna Kea, the Kahuku Unit, Mauna Loa, and

Kīlauea sections of Hawai‘i Volcanoes National Park. Elevation contours are at 500 m intervals.

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