Post-mortem examination of a wild muntjac from Northern ...€¦ · Post-mortem of a wild muntjac in NI . Research and Development Series 12/12 A report commissioned by the Northern

Post-mortem examination of a wild muntjac from Northern Ireland.

Dick, J. T. A., McKillen, J., Chapman, N., Collins, L., Provan, J., Freeman, M., Hogg, K., & Reid, N. (2012). Post-mortem examination of a wild muntjac from Northern Ireland. (12 ed.) Northern Ireland Environment Agency.https://www.doeni.gov.uk/publications/post-mortem-examination-wild-muntjac-northern-ireland

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Post-mortem examination of a wild

muntjac from Northern Ireland

www.doeni.gov.uk/niea Post-mortem of a wild muntjac in NI

Research and Development Series 12/12 A report commissioned by the Northern Ireland Environment Agency

Post-mortem examination of a wild muntjac from Northern Ireland

Authors: Jaimie Dick, John McKillen, Norma Chapman, Lisa Collins, Jim Provan, Marianne Freeman, Kayleigh Hogg & Neil Reid

Contractor: Quercus NHRP contract number: CON 2/1 (241) Quercus Project: QU11-05

ISSN 1751 – 7796 (Online)

This report should be cited as:

Dick, J.T.A., McKillen, J., Chapman, N., Collins, L., Provan, P. Freeman, M. Hogg, K. & Reid, N. (2012) Post-mortem examination of a wild muntjac from Northern Ireland. Report prepared by the Natural Heritage Research Partnership (NHRP) between Quercus, Queen’s University Belfast and the Northern Ireland Environment Agency (NIEA). Northern Ireland Environment Agency Research and Development Series No. 12/12, Belfast

For further information on this report please contact: John Early,

Northern Ireland Environment Agency,

Biodiversity Unit, Klondyke Building,

Gasworks Business Park,

Lower Ormeau Rd,

Belfast.

BT7 2JA.

The opinions expressed in this report do not necessarily reflect the current opinion or policy of

the Northern Ireland Environment Agency.

1


EXECUTIVE SUMMARY

1. Deer are of major concern with regards to impacts on biodiversity, forestry and agriculture

as well as human health. The invasive Reeves’ muntjac deer Muntiacus reevesi, native to

Asia but established in Great Britain, has recently appeared in the wild in Ireland.

2. The first verified record in the wild in Northern Ireland was confirmed during June 2009 as

a result of a road traffic accident near Newtownards, Co. Down. The second record was a

culled animal shot in the grounds of Mount Stewart, Co. Down during June 2011.

3. The current report aimed to perform a detailed investigation of the most recently obtained

animal to establish a) its age, b) its genetic relationship with the first animal and c) the

threat it might pose in terms of carrying endo- or ecto-parasites or other micro-pathogens,

principally viruses and bacteria.

4. Analysis of its dentition suggested the culled animal was 56 weeks old (range 55-57

weeks).

5. Genetic analysis indicated that there was no possibility of a father-son relationship with the

buck killed in 2009. There was a 6.25% probability of both individuals being full-sibs and a

25% probability of a half-sib relationship.

6. The deer was free from all the major pathogens. Most significantly, a novel species of

Gammaherpesvirus was detected most closely matching type 2 ruminant rhadinovirus

(Gammaherpesvirinae) from mule deer. Further sequencing is required to provide a

definitive classification. There is no evidence suggesting this virus is pathogenic but its

detection is nonetheless of concern.

7. During late 2009 (after the first animal was recovered) and early 2010, there were a

number of sightings of muntjac within the vicinity of Mount Stewart. Our results indicate

that the animal shot in June 2011 could not have been this same animal, as the shot

animal must have been born in spring 2010. Moreover, genetic analysis indicates that the

two recovered individuals were highly unlikely to share the same mother and father,

suggesting they were the offspring of a minimum of three breeding adults (two fathers and

one mother or two mothers and one father). This brings the total number of known

individuals to 5 including the two offspring. The location of the breeding adults is unknown

and may be either in captivity with subsequent escapees, or deliberate releases, or be free

living in the wild. However, a further sighting during early 2012 may suggest a wild origin.

2


INTRODUCTION

Deer, especially introduced species, are of major concern with regards to impacts on

biodiversity, forestry and agriculture (Putman & Moore 1998; Delahay, De Leeuw &

Claridge 2001). Deer also affect humans directly, causing fatalities and injury

through deer-vehicle collisions (Putman 1997). Consequently, expansion of

introduced and native deer populations will present considerable management

challenges in the future (Ward, 2005).

The rapidity of the spread of introduced Reeves’ muntjac Muntiacus reevesi (Ogilby

1839) is of great concern currently due to deliberate translocations and natural

dispersal from their initial inoculation site(s) in the south-east of Great Britain (Ward

2005).

The first verified record of a muntjac in the wild in Northern Ireland was confirmed on

the 3rd June 2009 as a result of a road traffic accident near Newtownards, Co. Down

(J554764). It was confirmed by a veterinary surgeon that the injuries presented were

consistent with a recent deer-vehicle collision, including broken limbs, blunt trauma

and scrape marks. The animal was a young buck. Identification was further verified

by two independent muntjac experts (Dick et al. 2011). This is the first record of a

new large mammal species in Northern Ireland since the introduction of Sika deer

Cervus nippon (Temminck 1838) in 1870.

This record followed the first reports of muntjac in the Republic of Ireland during

2007 when a free-living adult buck was shot near Avoca, Co. Wicklow, with a second

animal sighted alive shortly thereafter near Trooperstown (Dick et al. 2011). Muntjac

are held and bred in captivity throughout Ireland (Jaimie Dick pers. obs.) and

anecdotal sightings of the species in the wild in both Northern Ireland and Republic

of Ireland have increased in recent years. These include aggregations of sightings

near Mount Stewart on the Ards Peninsula, Co. Down (Freeman, Reid & Dick, 2010)

as recently as February 2012 (Nigel Lafferty pers. obs., Ballyhaft Road,

Newtownards).

3


Reeves’ muntjac was listed as one of the ‘most unwanted’ non-native species by

Invasive Species Ireland (www.invasivespeciesireland.com). The Government in the

Republic of Ireland have taken action to facilitate the eradication of any establishing

population(s) by placing the species on the Wildlife (Wild Mammals) (Open Seasons)

Amendment Order 2008. Dick, Provan & Reid (2009) reviewed candidate control

procedures for muntjac and outlined five ‘Options’ for response should the species

be confirmed in the wild. In Northern Ireland, muntjac has been added to Schedule 9

(Part I) of the Wildlife Order (NI) 1985 (as amended) which makes it an offence ‘if

any person releases or allows to escape into the wild any animal which is included in

Part I of Schedule 9’. In addition, to help facilitate control of muntjac, changes have

been made to Article 20 (Section 8a) in relation to the minimum calibre of gun

suitable for deer culling to account for the species-specific characteristics of muntjac.

In common with the Republic of Ireland, there is no closed season for muntjac in

Northern Ireland. Moreover, there is now a cross-border co-ordinated approach to

the species with the publication of an All-Ireland ‘Invasive Species Action Plan’

(ISAP) to further bolster recommendations for action if the species is confirmed in

the wild.

A second confirmed record of muntjac was made in Northern Ireland when a young

buck was shot at Mount Stewart, Co. Down on 11th June 2011 (Jaimie Dick, pers.

obs.). The current report aimed to perform a detailed investigation of this carcass to

establish:

1. The age of the culled animal

2. Whether this animal was related to the previous animal killed in a

road traffic accident

3. Whether the animal carried any endo- or ecto-parasites or other

micro-pathogens, principally viruses/bacteria e.g. bovine tuberculosis

(bTB)

4


METHODS

Ageing

The jaw of the animal was examined by Norma Chapman to describe dentition for

the purposes of ageing. The conformation of the teeth was recorded and compared

to a series of reference specimens of known age. The age was estimated in weeks

and is accurate to within 2 weeks.

Genetics

Genetic analysis was performed on both the original buck killed in a road-traffic

accident during 2009 and the second buck shot at Mount Stewart during 2011.

Microsatellite markers developed for M. crinifrons were used (Wu et al. 2008).

Post-mortem examination

A standard post-mortem examination was conducted of the animal culled in June

2011 by the Veterinary Science Division, Agri-food and Biosciences Institute (AFBI).

Routine bacteriology and mycobacterial analysis was performed. Parasitological

examination was conducted for a range of common infections. Virology PCR was

conducted on a range of tissues including for a large range of standard bovine

viruses and using degenerate ‘broad spectrum’ assays for other/novel viruses.

Sequencing was used for confirmation of PCR positives.

5


RESULTS

Ageing

Muntjac have lost their first premolar during their recent evolution, thus the first tooth

in the row is called second premolar, in both upper and lower deciduous and

permanent dentitions.

1. The right mandible was in 2 pieces and had a loose molar.

Conformation of the teeth were as follows: I1 I2 I3 c pm2 pm3 broken, pm4 (3 cusps confirms it is deciduous), M1 M2 and separate from bone M3 (partly erupted). The tip of permanent replacement of pm2 was just

appearing from the inner (lingual).

2. In the portion of right maxilla there were as follows: pieces of pm2 pm3

pm4 M1 M2 not fully erupted and M3 erupting. Above pm4 there was its

permanent replacement developing. One upper canine of which 14mm

protruded below gum.

Given the conformation of the teeth, this specimen was judged to be 56 weeks of

age, falling within the variation typically seen from reference specimens that are 55

and 57 weeks of age.

Genetics

Genetic analysis indicated that there was no possibility of a father-son relationship

between the original buck killed in a road-traffic accident during 2009 and the second

buck shot at Mount Stewart during 2011. There was a 6.25% probability of both

individuals being full-sibs and a 25% probability of a half-sib relationship.

6


Post-mortem examination

The deer was free from gross signs of disease and all major pathogens. No gross

lesions present in oeseophagus, trachea, lungs, heart, rumen, reticulum, omasum,

abomasum, small and large bowel, liver, kidney, spleen, mesenteric lymph node,

pancreas or thyroid gland. Alimentary tract contents normal.

Routine bacteriology found low levels of Streptoccocus spp. and Bacillus

licheniforms but this is what is expected in a healthy animal. Results indicate that

there was no E. coli which is highly unusual for an ungulate species.

Mycobacterial analysis was negative in mesenteric lymph node and heart. Faeces

negative for Johne’s disease.

The parasite count was zero for worms, Fasciola eggs, paramphistome eggs,

Coccdia and worm eggs.

Virological PCR and sequencing was carried out on kidney, heart, spleen, lung,

bronchial lymph node thyroid, caecum, faeces, oesophogus, colon, small intestine

regions 1-6, pancreas, liver, abomasun, medistinal lymph node, mesenteric lymph

node, trachea, rumen contents, blood clot and blood.

The carcass was negative for bovine herpesvirus 1 (infectious broncial rhinitis virus),

bovine parainfluenza virus 3, bovine viral diarrhoea virus, bovine respiratory syncytial

virus, bluetongue virus, kobuvirus, calicivirus (norovirus and sapovirus), anellovirus,

adenovirus, coronavirus, reovirus, orthoreovirus, rotavirus and hepatitis E virus.

However, colon, mediastinal lymph node, lung and blood were weakly positive for

herpesvirus. The band amplified from the colon was sequenced and identified as

matching rhadinovirus of mule deer (gammaherpesvirus). A number of tissues were

also positive for picornavirus and further sequencing is needed determine the correct

classification.

7


Other PCR or RT-PCRs tests were carried out for astrovirus, bocavirus, circovirus,

paramyxovirus, bovine hokovirus and bovine parvovirus type 2. Assays were

negative, but results were inconclusive due to lack of fully and properly validated

assays/controls.

DISCUSSION

The chronology of events surrounding muntjac in the Ards peninsula, Co. Down,

Northern Ireland is thus:

1. From May 2009, anecdotal muntjac sightings began to increase in frequency near

Mount Stewart, with a photograph nearby at Spring Lane (25th May 2009).

2. On 3rd June 2009, a buck muntjac was killed in a road-traffic accident on the Killaughy

Road approximately 3.5km from the photographed animal the preceding month.

3. However, anecdotal sightings continued in the area throughout late 2009 and early 2010

(Freeman et al. 2010).

4. On 11th June 2011, a 55-57 week old buck was shot at Mount Stewart (the subject of

this report). This animal could not have been responsible for earlier sightings during late

2009 or early 2010 (subsequent to the first animal being killed in a road-traffic accident)

as it must have been born during spring 2010.

5. Genetic analysis indicates that the two individuals were highly unlikely to share the

same mother and father, suggesting parentage from a minimum of three breeding adults

(two fathers and one mother or two mothers and one father). This brings the total

number of known individuals to 5 including the two offspring. The location of the

breeding adults at present is unknown and they may be either held in captivity or may

represent an establishing feral population.

6. A further muntjac sighting was made during February 2012 near Mount Stewart on the

Ballyhaft Road (Nigel Lafferty pers. obs.) supporting the latter hypothesis that there may

be multiple males and females in the wild raising the possibility of an establishing feral

population.

8


The post-mortem carried out here suggests that the animal recovered poses little

threat in terms of parasites or pathogens (i.e. no risk from bovine TB, Johne’s

disease or salmonella). Notably, there were low levels of bacteria in the animal

digestive tract (specifically, no E. coli) and no parasite fauna which may be

characteristic of an animal that had been treated recently with antibiotics and

anthelmintics. However, there may be natural reasons for this lack bacterial and

parasite communities so it is not certain that this can be taken as an indication that

the animal had been in captivity prior to being shot.

However, the animal was positive for picornavirus and herpesvirus, the latter of

which closely matched a sequence of type 2 ruminant rhadinovirus

(Gammaherpesvirinae) from mule deer. Other Rhadinovirus members include

bovine herpesvirus-4, implicated in post-partum metritis (Donofrio et al., 2009) and a

number of primate viruses associated with cancer in immunosuppressed individuals

such as Kaposi sarcoma herpesvirus i.e. human herpesvirus 8 (Cesarman, 2011).

The sequence matched with lower homology to a wide range of Gammaherpesvirus

from the Rhadinovirus and Macavirus genera, including ovine herpesvirus 2, genus

Macavirus, which causes sheep-associated malignant catarrhal fever (Russell et al.,

2009), and caprine herpesvirus 2, genus Macavirus, which is known to cause

malignant catarrhal fever in some species of deer. However, there are a number of

ruminant Macaviruses that are known to cause no clinical disease (Li et al. 2005).

The discovery of a novel species of Gammaherpesvirus is notable but its

significance as a pathogen of deer is unknown at this time. While there is no

evidence to suggest that this virus is pathogenic, and while this taxonomic group

contains non-pathogenic viruses of ruminants, the relatedness of the virus to known

pathogens of ruminants is of some concern as there is always a possibility, however

small, of a benign virus evolving to become more pathogenic or to expand its host

range. In addition it is well known for apparently harmless viruses proving pathogenic

when introduced to immunologically naive or susceptible populations. So while the

risks associated with this virus are probably small the fact that DNA from a

completely novel virus has been identified in such a small sample would suggest that

there is significant value in future virological work involving invasive species,

9


particularly muntjac deer. Further sequencing of the viral genome through standard

molecular methods would allow definitive classification of the virus and a

metagenomic analysis of other samples using next generation sequencing would be

a powerful method of further virus discovery.

RECOMMENDATIONS

We make 3 recommendations for action:

1. Integrated and co-ordinated species surveillance and monitoring to document

the establishment, spread and impact of muntjac on the Ards Peninsula.

Specifically, proactive detection involving baited camera trap stations in Mount

Stewart estate and surrounding area coordinated by the ‘Mount Stewart

Eradication Plan’ members (principally National Trust, Forest Service, Local

Shooting Syndicate, Invasive Species Ireland, QUB and NIEA) in order to

detect further individuals in the wild and initiate culling.

2. Proactive detection and reaction throughout Northern Ireland at sites of other

possible innocula i.e. locations of escapees or releases (see Freeman, Reid &

Dick, 2010). Data on anecdotal sightings are held by Queen’s University Belfast

and the National Biodiversity Data Centre, Waterford.

3. Any future carcasses should be subjected to similar analyses as presented

here in order to estimate population sizes and further assess disease profiles.

Specifically, further sequencing of the novel Gammaherpesvirus genome

through standard molecular methods to facilitate definitive classification of the

virus and a metagenomic analysis of future muntjac samples using next

generation sequencing would be a powerful method of further virus discovery.

10


ACKNOWLEDGEMENTS

Thanks to Jason Barle and Jim McCarthy, Disease Surveillance and Investigation

Branch, Agri-Food and Biosciences Institute for carrying out the post-mortem

examination (2011-20287). Conrad Watson (Disease Surveillance and Investigation

Branch) and Eric Walton (Bacteriology) carried out routine bacteriology and

mycobacterial analyses respectively, Hillary Edgar (Disease Surveillance and

Investigation Branch) preformed parasitiological tests and Paula Lagan and Michael

McMenamy (Virology) carried out virology PCR and sequencing. John Early acted as

NIEA Client Officer.

11


REFERENCES

Cesarman E. Gammaherpesvirus and lymphoproliferative disorders in immunocompromised patients. Cancer Lett. 2011 Jun 28;305(2):163-74. Review.

Delahay, RJ; De Leeuw, ANS; Claridge, M, et al. (2001) First report of Mycobacterium bovis in a muntjac. Veterinary Record, 149: 95-96.

Dick, J.T.A, Provan, J. & Reid, N. (2009) Muntjac Knowledge Transfer: Ecology of introduced muntjac deer and appraisal of control procedures. Report prepared by the Natural Heritage Research Partnership, Quercus for the Northern Ireland Environment Agency, Northern Ireland, UK.

Dick, J.T.A., Freeman, M., Provan, P. & Reid, N. (2011) First record of a free-living Reeves' muntjac deer (Muntiacus reevesi (Ogilby 1839)) in Northern Ireland. Irish Naturalists' Journal, 31(2); 151.

Donofrio G, Franceschi V, Capocefalo A, Cavirani S, Sheldon IM. Isolation and characterization of bovine herpesvirus 4 (BoHV-4) from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome. Reprod Biol Endocrinol. 2009 Aug 19;7:83.

Freeman, M., Reid, N. & Dick, J.T.A. (2010) Evaluation of the veracity of anecdotal muntjac records in Northern Ireland. Report prepared by the Natural Heritage Research Partnership, Quercus, Queen’s University Belfast for the Northern Ireland Environment Agency. Northern Ireland Environment Agency Research and Development Series No. 10/XX.

Li H, Keller J, Knowles DP, Taus NS, Oaks JL, Crawford TB. Transmission of caprine herpesvirus 2 in domestic goats. Vet Microbiol. 2005 Apr 25;107(1-2):23-9.

Putman, R.J. (1997). Deer and road traffic accidents: options for management. Journal of Environmental Management, 51, 43-57.

Putman, R.J. and Moore, N.P. (1998) Impact of deer in lowland Britain on agriculture, forestry and conservation habitats. Mammal Review, 28(4): 141-163.

Russell GC, Stewart JP, Haig DM. Malignant catarrhal fever: a review. Vet J. 2009 Mar;179(3):324-35.

Ward, A.I. (2005) Expanding ranges of wild and feral deer in Great Britain. Mammal Review, 35: 165-173.

Wu HL, Meng K, Zhu-GP (2008) Isolation and characterization of microsatellite markers in black muntjac (Muntiacus crinifrons). Molecular Ecology Resources 8, 584-586.

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Post-mortem examination of a wild muntjac from Northern ...€¦ · Post-mortem of a wild muntjac in NI . Research and Development Series 12/12 A report commissioned by the Northern