MARINE AND FRESHWATER INVESTIGATION INTO THE FIRST DIAGNOSIS OF OSTREID HERPESVIRUS TYPE 1 IN PACIFIC OYSTERS Background
The Pacific oyster (Crassostrea gigas Thunberg 1793) is one of the three main aquaculture species in New Zealand (along with king salmon and green mussels). Pacific oysters were introduced into NZ in the last century and have been farmed commercially since the 1990s. For the year ending 31 March 2011 their export value was $18 million and domestic market value about $12 million (Anonymous, 2012). Oyster herpesvirosis is caused by a herpesvirus about 120 nm in diameter. It is believed that the bivalve herpesviruses are a completely separate family of herpesviruses with extremely limited genetic homology to the herpesviruses of mammals, birds and reptiles, or to the herpesviruses of amphibians and teleosts (Davison et al., 2005). As a result the family Malacoherpesviridae now includes the new genus Ostreavirus, containing the species Ostreid herpesvirus 1 (OsHV-1) (Davison et al., 2009). Strain differences within OsHV-1 may be significant and associated with virulence. Since the summer of 2008 increased mortality of young Pacific oysters on the French coast has been linked to a microvariant of the OsHV-1, named OsHV-1 μVar (Garcia et al., 2011; Martenot et al., 2012). OsHV-1 is not a notifiable or unwanted organism under the Biosecurity Act 1993, nor is it a listed disease with the World Organisation for Animal Health (OIE).
Epidemiology
The infectious dose of virus is unknown. The pathogenicity of the virus does vary with size of the host oyster (Burge, Griffin & Friedman, 2006). OsHV-1 DNA has been isolated from the water around infected Pacific oysters (Sauvage, Pépin, Lapègue, Boudry & Renault, 2009) and the disease can be experimentally transmitted in water (Schikorski et al., 2011). Indirect spread between infected and healthy individuals via water, by excretion of virus particles and from tissue remnants, appears to be the main method of local spread. Local spread of pathogens also appears to be very rapid in marine environments (McCallum, Harvell & Dobson, 2003). Movement of infected animals is likely to be the main mechanism of long-distance spread (e.g., from harbour to harbour). Whether vertical spread occurs is inconclusive but the virus has been detected in adult oyster tissues 20 SURVEILLANCE 40 (2) 2013
including gonad, and adult oysters could play the role of passive carriers (Barbosa-Solomieu et al., 2005). OsHV-1 is known to infect a number of different marine bivalve molluscs around the world (Hine & Thorne, 1997; Renault, Lipart & Arzul, 2001). In addition, the more intensive nature of commercial hatcheries, producing a number of bivalve species in recirculated water systems, could exacerbate this (Arzul, Renault, Lipart & Davison, 2001). It seems unclear, however, whether these different species are truly infected or acting as mechanical carriers, given that they are filter-feeders. In the northern hemisphere several European countries experienced annual mortality events correlated with high prevalence of OsHV-1 and as a consequence developed long-term studies to understand the risk factors associated with the die-offs. Peeler et al. (2012) performed a comprehensive study of the risk factors of Irish and French oyster mortality events, including agent factors of OsHV-1, host factors, management factors and environmental factors. They placed particular emphasis on the types of temperature stresses, both water and ambient, that correlated with the location and time of mortality events. Pacific oyster mortalities are likely to be influenced by many factors, with OsHV-1 as a necessary but not sufficient cause. Figure 1 lists some of the possible risk factors. Farming method (depth, racks, long lines) Timing (spring/autumn) Travel factors (duration, conditions) Biosecurity practices
OsHV-1 Other pathogenic bacteria
Agent
Management
Host Age/Size Genotype Plasticity/resilience Ploidy
Environment Increases in temperature (sudden) Location Tidal effects Water column Phytoplankton blooms Affected/unaffected
Figure 1: Possible risk factors for Pacific oyster mortality grouped around the epidemiological triangle of agent, host, and environment, with the addition of management factors.
First report in New Zealand
On 17 November 2010 the Investigation and Diagnostic Centre (IDC) Wallaceville was notified by the oyster aquaculture industry of mortality events occurring in Pacific oyster farms in Northland and the Coromandel. A similar event had been reported the previous autumn (March–May 2009). An investigation was initiated, primarily to determine whether the mortalities described were associated with an infectious agent and secondarily to rule out OIE-listed diseases that could result in oyster mortalities. IDC epidemiologists also began work to describe the magnitude and pattern of the outbreaks. This report summarises the initial investigation work.
Initial investigation
An immediate field investigation was undertaken by a marine incursion investigator and aquatic scientist from the IDC. The clinical description was of acute mortality of Pacific oysters, particularly juvenile oysters (20–80 mm) and spat (free-swimming larvae less than 20 mm long) newly moved on to farms. Some animals displayed gaping (incomplete shell closure) on emersion and were slow to close when gently tapped while immersed. The most significant clinical findings, however, were large numbers of dead, gaping, empty shells or shells containing rapidly decomposing animals. From knowledge of animal movements onto properties an incubation period of less than seven days was evident. Samples were taken initially from five sites covering the far north, Bay of Islands, west coast and Auckland, amounting to about 250 oysters. Oysters selected for sampling were clinically unaffected owing to the difficulty of finding affected animals that were not already in a state of advanced decomposition. After laboratory testing at the IDC a presumptive identification of OsHV-1 was made by PCR on 27 November 2010 and this was confirmed on 6 December 2010 by DNA sequencing.
Testing limitations
Operating characteristics of the real-time PCR were unknown as the test was developed during the response. The analytical sensitivity was unknown and as a result caution needed to be exercised when interpreting any negative results. This means that although surveys for freedom from disease can be justified, freedom from the organism in apparently disease-free areas cannot be validated.
Histology was limited as a diagnostic tool because most of the samples obtained were healthy owing to the difficulty of finding affected oysters. As a result the samples obtained were not representative of the affected population. Possibly they represented latent infected, subclinical or recovered animals.
Data collection, analysis and results
Data on affected sites (numerator data) was collected opportunistically through industry assistance – initially from the minutes of industry meetings and, after the end of November, by voluntary weekly reporting. Estimates of the earliest time of onset and intra-farm prevalence by size-class were then made. The data is likely to be inaccurate and biased as it is based on visual inspection and recall, and limited by the numbers of farmers who participated. No baseline data was available to act as guidance as to the expected normal level of seasonal mortality. The data likely suffers from reporting biases, with most reports from the most severely affected or those with a greater frequency of observation. Caution is therefore necessary when making inferences from the data. This also limited analysis to being descriptive. Data on the overall population at risk (PAR) of being affected (denominator data) was extracted from the Aquaculture Readiness Data project (Brangenberg & Morrisey, 2010). Aggregate-level data (e.g., within growing area or geographic location) was good, as was knowledge of data sources and contacts available. The total number of oyster farm leases issued is 260 but there is no record of active leases, so the PAR was unknown at that level. No resources were available to perform active casing or surveillance. Owing to these limitations, the investigation was restricted to counting data largely collected after the event and it was not possible to determine the true prevalence, incidence, or incidence rates of disease within an aggregate level. Ideally the unit of interest would be at the lease or farm level, which would enable collection of intrinsic farm factors such as frequency of disease between and within farms and also management factors. Owing to the limitations of data collection described at both the numerator and denominator levels the reporting unit had to be confined to the harbour level. Also from the Aquaculture Readiness Data project, the defined dispersal SURVEILLANCE 40 (2) 2013
21
areas of pathogens was shown to typically encompass all the farms in areas like harbours and inlets, making them epidemiological units.
The earliest dates from the reports received for selected geographic locations and growing areas are shown in Figure 2, page 27. Most reports identified onset during the first three weeks of November.
It was apparent, however, that 18 locations (harbours, bays and inlets) were in the affected area, which included 260 farms. Of these, 15 locations were reported as affected. Seven locations were sampled (six affected, one unaffected), within which 14 different lease sites were sampled. Six of the seven locations sampled were positive for OsHV-1 and 11 of the 14 lease sites were positive (Table 1). No South Island farms were affected.
Disease frequency within individual farms
TABLE 1: Reported, sampled and laboratory-tested status of harbours and growing areas during the oyster mortality event, from the first known occurrences to 18 January 2011. GROWING AREA (GA)
201
GEOGRAPHIC LOCATIONS
REPORTED HARBOUR STATUS FROM 1/11/10 TO 18/1/11
NUMBER OF FARMS SAMPLED
NUMBER OF AT-RISK FARMS
NUMBER OF OsHV-1 CONFIRMED FARMS
0
34
0
Parengarenga Harbour
Affected
202
Whangaroa Harbour
Affected
1
21
1
204
Kerikeri Inlet
Affected
0
12
0
204A
Kerikeri Inlet
Affected
0
4
0
205
Orongo Bay
Affected
1
23
1
206
Waikare Harbour
Affected
3
29
3
208 (North)
Kaipara Harbour
Affected
0
36
0
209 (South)
Kaipara Harbour
Affected
1
1
1 suspicious
215
Houhora Harbour
Unaffected
2
16
0
218
Rangaunu
Affected
1
7
1
301
Mahurangi Harbour
Affected
5
43
4
412
Hauraki Gulf
Affected
0
3
0
413, 414
Hauraki Gulf
Affected
0
3
0
207
Whangarei Harbour
Affected
0
1
0
Hokianga Harbour
Affected (unconfirmed)
0
2 (spatcatching
0
611, 612
Coromandel
Affected
0
12
0
6101, 6102
Coromandel
Unaffected
0
6
609
Whangapoua Harbour
Affected
0
2
0
602
Whitianga Harbour
Unaffected
0
1
0
608
Kawhia Harbour
Unaffected
0
1
0
701
Ohiwa Harbour
Affected
0
2
0
502
Kauri Bay
Affected
0
1
0
22 SURVEILLANCE 40 (2) 2013
During the voluntary weekly reporting periods in December 2010, 78 reports were received from 15 farmers (Table 2). Mortality was significant on some farms and throughout all age-groups of oysters. From the average cumulative mortality figures there appears to be a gradient of effect by age (which equates to size) of oyster, with greater mortality in younger animals, but the normal mortality range by age is unknown. TABLE 2: Disease mortality presence and cumulative mortality by age, December 2010. MORTALITY*
AVERAGE CUMULATIVE MORTALITY
RANGE
Spat
92%
50%
15–100%
Small
78%
34%
0–80%
Large
89%
14%
5–60%
*Percentage of reports with mortality present in that size class
Environmental risk factors
Environmental risk factors (temperature, phytoplankton, salinity changes) were examined. Data to fully investigate these factors (matched to sites and times of interest and measuring the appropriate parameters) was very limited or in some cases not available. From the data available it appears sea temperatures at times preceding outbreaks of disease were higher in 2010 than in preceding years at some of the affected sites. This is probably not unexpected and due to the La Niña event, and supports other evidence that temperature is the environmental risk factor most often linked with oyster mortalities (Soletchnik et al., 2007). No other conclusions about environmental risk factors could be drawn from the data. It is possible that the rate of temperature rise rather than the actual temperature reached is a risk factor. Figure 3 shows the trend in temperature over time at the Waiheke weather station.
in one area are likely to be present in many or most other areas, though interestingly no disease has been reported in the South Island to date. Perhaps OsHV-1 is a necessary but insufficient cause of disease and other risk factors are required.
Conclusions
Evidence at the time that suggested OsHV-1 was directly associated with the mortality events described included: • the finding of OsHV-1 in oysters from all affected sites tested; • the massive increase in prevalence of OsHV-1 (detected by real- time PCR) by day 5 after transfer of susceptible animals into an affected growing area, with mortalities suddenly increasing on day 6; • evidence from overseas outbreaks that implicates OsHV-1 as a necessary cause of spat mortality and hatchery larval mortality; and • laboratory work at IDC that has revealed a close genetic association to the µ-var strain of OsHV-1, which has been linked to Pacific oyster mortalities in Europe since 2008.
Figure 2: Timeline of earliest observed oyster mortality
Evidence that OsHV-1 was likely to be widespread throughout the industry included: Figure 3: Temperature readings for the Waiheke weather station, October–November 2010. Note the higher mean temperature from 26 October to 5 November in 2010, indicated by the solid red trendline when compared with the black trendline for preceding years.
Industry movement patterns
Movement data was collected before this outbreak through the Aquaculture Readiness Data project (Brangenberg & Morrissey, 2010). The industry appears to be very connected by movements that would lead to widespread dissemination of risk organisms. Organisms
1. The high prevalence of areas affected during this event as well as the autumn mortality.
2. The high proportion of the affected farms sampled that tested positive for the virus in the spring event, together with the distribution of sampling. 3. The fact that the compressed time period of first reported onset of mortality in affected farms (or SURVEILLANCE 40 (2) 2013
23
areas) is not consistent with a new introduction and propagating epidemic, but rather with an environmental trigger factor, or (less likely) a multiplepoint-source epidemic. 4. Normal movement patterns and lack of biosecurity practices in the industry. 5. Mortalities in C. gigas larvae were reported to have occurred in February 1991 in a New Zealand hatchery (Hine et al., 1992). This paper reported the detection of a herpes-like virus. 6. Evidence from overseas outbreaks suggested that OsHV-1 can be present in the absence of disease, and that enabling factors (including environmental factors) are required to initiate an outbreak.
REFERENCES Anon. (2012) Situation and outlook for primary industries, 50. http://tinyurl. com/lqcl88w. Accessed 20 June 2013 Arzul I, Renault T, Lipart C, Davison AJ (2001) Evidence for interspecies transmission of oyster herpesvirus in marine bivalves. Journal of General Virology 82(4): 865–870. Barbosa-Solomieu V, Dégremont L, Vazquez-Juarez R, Ascencio-Valle F, Boudry P, Renault T (2005) Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas). Virus Research 107(1): 47–56. Brangenberg N, Morrisey D (2010) Aquiculture readiness data project. Surveillance 37(4): 41–43. Burge CA, Griffin FJ, Friedman CS (2006) Mortality and herpesvirus infections of the Pacific oyster (Crassostrea gigas) in Tomales Bay, California, USA. Disease of Aquatic Organisms 72: 31–43. Davison A J., Eberle R., Ehlers B., Hayward G S., McGeoch D J., Minson A C., et al. (2009). The order herpesvirales. Archives of Virology; 2009.154: 1, 171-177, Davison AJ, Trus BL, Cheng N, Steven AC, Watson MS, Cunningham C, et al. (2005) A novel class of herpesvirus with bivalve hosts. Journal of General Virology 86(1): 41–53. Hine P, Thorne T (1997) Replication of herpes-like viruses in haemocytes of adult flat oysters Ostrea angasi: an ultrastructural study. Diseases of Aquatic Organisms 29: 189–196. Keeling S E. New Zealand juvenile oyster mortality associated with ostreid herpesvirus - a longitudinal study. MPI internal document. McCallum H, Harvell D, Dobson A (2003) Rates of spread of marine pathogens. Ecology Letters 6(12): 1062–1067. Peeler E J, Allan Reese R, Cheslett DL, Geoghegan F, Power A, Thrush MA (2012) Investigation of mortality in Pacific oysters associated with ostreid herpesvirus- 1μVar in the Republic of Ireland in 2009. Preventive Veterinary Medicine, 105(1): 136–143 24 SURVEILLANCE 40 (2) 2013
Renault T, Lipart C, Arzul I (2001) A herpes-like virus infects a non-ostreid bivalve species: Virus replication in Ruditapes philippinarum larvae. Diseases of Aquatic Organisms 45(1) 1–7. Sauvage C, Pépin JF, Lapègue S, Boudry P, Renault T (2009) Ostreid herpesvirus 1 infection in families of the Pacific oyster, Crassostrea gigas, during a summer mortality outbreak: Differences in viral DNA detection and quantification using real-time PCR. Virus Research 142(1): 181–187. Schikorski D, Faury N, Pepin JF, Saulnier D, Tourbiez D, Renault T (2011) Experimental ostreid herpesvirus 1 infection of the Pacific oyster Crassostrea gigas: Kinetics of virus DNA detection by q-PCR in seawater and in oyster samples. Virus Research 155(1): 28–34.
ACKNOWLEDGEMENTS
The authors would like to thank members of the New Zealand oyster industry association who contributed to this article by providing data. Special thanks go to Colin Johnston, and Ministry for Primary Industries staff at the Animal Health Laboratory, Wallaceville and the animals response team at Pastoral House, Wellington. Paul Bingham
[email protected] Naya Brangenberg
[email protected] Rissa Williams
[email protected] Mary Van Andel
[email protected] Investigation and Diagnostic Centres and Response Ministry for Primary Industries 66 Ward Street Upper Hutt New Zealand
