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Phocine distemper virus (PDV) seroprevalence as predictor for future outbreaks in harbour seals
Veterinary Microbiology 183 (2016) 43–49
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Phocine distemper virus (PDV) seroprevalence as predictor for future outbreaks in harbour seals Eva Ludes-Wehrmeistera,** , Claudia Dupkea , Timm C. Harderb , Wolfgang Baumgärtnerc , Ludwig Haasd , Jonas Teilmanne , Rune Dietze, Lasse F. Jensenf , Ursula Sieberta,* a
Institute of Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine, Hannover, Foundation, Werftstrasse 6, 25761 Buesum, Germany O.I.E., FAO and National Reference Laboratory for Avian Influenza, Institute of Diagnostic Virology, Friedrich Loeffler Institut, Suedufer 10, D-17493 Greifswald-Insel Riems, Germany c Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Buenteweg 17, 30559 Hannover, Germany d Institute of Virology, University of Veterinary Medicine Hannover, Foundation, Buenteweg 17, 30559 Hannover, Germany e Department of Bioscience, Aarhus University, Frederiksborgvej 399, DK-4000 Roskilde, Denmark f Fisheries and Maritime Museum, Tarphagevej 2, 6710 Esbjerg V, Denmark b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 July 2015 Received in revised form 10 November 2015 Accepted 14 November 2015
Phocine distemper virus (PDV) infections caused the two most pronounced mass mortalities in marine mammals documented in the past century. During the two outbreaks, 23,000 and 30,000 harbour seals (Phoca vitulina), died in 1988/1989 and 2002 across populations in the Wadden Sea and adjacent waters, respectively. To follow the mechanism and development of disease spreading, the dynamics of Morbillivirus-specific antibodies in harbour seal populations in German and Danish waters were examined. 522 serum samples of free-ranging harbour seals of different ages were sampled between 1990 and 2014. By standard neutralisation assays, Morbillivirus-specific antibodies were detected, using either the PDV isolate 2558/Han 88 or the related canine distemper virus (CDV) strain Onderstepoort. A total of 159 (30.5%) of the harbour seals were seropositive. Annual seroprevalence rates showed an undulating course: Peaks were seen in the post-epidemic years 1990/1991 and 2002/2003. Following each PDV outbreak, seroprevalence decreased and six to eight years after the epidemics samples were tested seronegative, indicating that the populations are now again susceptible to new PDV outbreak. After the last outbreak in 2002, the populations grew steadily to an estimated maximum (since 1975) of about 39,100 individuals in the Wadden Sea in 2014 and about 23,540 harbour seals in the Kattegat area in 2013. A re-appearence of PDV would presumably result in another epizootic with high mortality rates as encountered in the previous outbreaks. The current high population density renders harbour seals vulnerable to rapid spread of infectious agents including PDV and the recently detected influenza A virus. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Harbour seal Common seal Phocine distemper virus Canine distemper virus Morbillivirus antibodies Epidemic
1. Introduction Following the ban on hunting of seals in the mid-70s, the harbour seal (Phoca vitulina) population in the Wadden Sea increased steadily and at present are considered to be the most common seal species in the Wadden Sea and Kattegat area (Reijnders, 1981; Reijnders et al., 2010; Teilmann et al., 2010). In 2014 the harbour seal population (Germany, Netherlands and Denmark) consisted of 26,576 individuals (maximum number
* Corresponding author. Fax: +49 511 856 8181. ** Corresponding author. E-mail addresses: [email protected] (E. Ludes-Wehrmeister), [email protected] (U. Siebert). http://dx.doi.org/10.1016/j.vetmic.2015.11.017 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.
counted during moult counts) resulting in an estimated total population size (based on the correction factor estimated by Ries et al. (1998)) of 39,100 individuals (Galatius et al., 2014). The estimated number of harbour seals in the Kattegat was 23,540 in 2013 (Galatius pers. comm.; correction factor derived from Härkönen et al., 1999). In 1987, prior to the first reported outbreak of the phocine distemper virus (PDV) infection, 8296 harbour seals were counted in the Wadden Sea (Reijnders et al., 2010) and in 1986, 4458 individuals were counted in the Kattegat area respectively (Olsen et al., 2010). During this first epizootic, more than 50% of the population died (Dietz et al., 1989; Härkönen et al., 2006). Thereafter, it recovered from a counted number of some 4400 animals in 1989 up to some 17,900 counted individuals in 2001 in the Wadden Sea (Reijnders et al., 2010) and from 2178 counted animals in 1989 up to 5941 individuals in 2000 in the
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Kattegat (Olsen et al., 2010). Another PDV outbreak caused a second mass mortality in 2002 (Härkönen et al., 2006) and decimated the population to 10,817 counted seals in the Wadden Sea and 4895 counted individuals in the Kattegat in 2003 (Reijnders et al., 2010; Olsen et al., 2010). During the second PDV outbreak a total of 30,000 individuals succumbed to the disease in European waters (Härkönen et al., 2006). Both epizootics started in spring on the small Danish island of Anholt in the Kattegat (Dietz et al., 1989; Härkönen et al., 2006) and spread throughout the seas of north-western Europe (Bergman et al., 1990; Härkönen et al., 2006). During both PDV outbreaks, only few grey seals (Halichoerus grypus) died of the disease (Bergman et al., 1990; Härkönen et al., 2006). Clinical signs of PDV infection in harbour seals are fever, coughing, dyspnoea, oculo-nasal discharge, conjunctivitis, ophthalmitis, keratitis, diarrhoea, abortion and increased buoyancy due to emphysema resulting in a reduced diving ability (Bergman et al., 1990; Kennedy, 1990, 1998). A few seals showed disturbances of the central nervous system (Bergman et al., 1990; Kennedy, 1990). Pneumonia with emphysema and oedema represented the main pathological findings (Bergman et al., 1990; Jauniaux et al., 2001; Kennedy, 1998). It has been suggested that the likely source of the 1988 epidemic was harp seals (Pagophilus groenlandicus), as it is known to host the virus and since mass migrations of this species took place into the southern Scandinavian waters in the winter of 1987– 1988 (Dietz et al., 1989). After the first outbreak grey seals (H. grypus) have been suggested to act as a reservoir for PDV or transferring the infection from the arctic seals so that outbreaks among harbour seals in European waters had been sparked by spill-over infections from these reservoirs (Härkönen et al., 2006; Kreutzer et al., 2008). For a comprised review of different PDV outbreaks all over the world we refer to Duignan et al. (2014). Apart from PDV, seals in the Caspian Sea, Lake Baikal and Antarctica were reported to have suffered from infection with the antigenically and genetically related but distinguishable Morbillivirus of terrestrial carnivores, canine distemper virus (CDV) (Mamaev et al., 1995; Kennedy 1998; Kreutzer et al., 2008;). Such infections obviously resulted from spill-over transmission of CDV from terrestrial carnivores (e.g. wolves (Canis lupus), dogs (Canis familiaris), foxes (Alopex lagopus)). As part of the health monitoring program established in 1990 following the first PDV outbreak, the aim of this study was to describe the Morbillivirus seroprevalence during the last 25 years in the population of harbour seals in the German and Danish waters. Data were used to predict the potential of a future outbreak of this disease.
Table 1 Overview of serum samples: location of catches (Germany: LS: Lower Saxony Wadden Sea, SH: Schleswig-Holstein Wadden Sea (Mittelplate, Lorenzensplate; Kolumbusloch,Westerhever), H: Helgoland, Denmark: R: Rømø, A: Anholt). Animals were grouped in sex and age classes: Age class I: pup (born in the year of catch; 1–7 months), II: yearling (seal born the year previous to the catch; 7–19 months); III older (than 19 months); *most of it published in Harder (1994). Year
Location Total Sex
Age class
Male Female Unknown I
II
III
Unknown
1990* SH
6
3
3
0
0
0
6
0
1991* SH LS
15 10
8 0
1 0
6 10
3
2
4
6 10
1992* SH LS
14 10
11 0
3 0
0 10
1 0
4 0
9 0 0 10
1993* SH LS
41 9
29 3
12 5
0 1
4 2
15 2
16 4
6 1
1996 1997 1998 2000 2001 2002 2003
SH SH SH SH SH SH SH
6 9 7 18 28 13 16
2 5 6 11 12 2 8
3 4 1 7 16 11 8
1 0 0 0 0 0 0
3 2 2 0 2 0 0
0 2 0 2 5 0 2
3 5 5 16 21 13 14
0 0 0 0 0 0 0
2004
SH R
27 16
18 14
9 2
0 0
4 0
1 1
22 15
0 0
2005
SH H A R
29 3 3 10
17 3 1 9
12 0 2 1
0 0 0 0
0 0 3 0
16 0 0 1
13 3 0 9
0 0 0 0
2006
SH H
16 7
12 7
4 0
0 0
0 0
3 2
13 5
0 0
2007
SH
31
23
8
0
0
13
18
0
2008
SH R A
27 8 6
15 6 3
12 2 3
0 0 0
0 0 0
8 3 3
19 5 3
0 0 0
2009 2010 2011 2012 2013
SH SH SH SH SH
10 22 15 31 27
2 5 11 14 14
8 17 4 17 13
0 0 0 0 0
0 0 0 0 0
2 0 6 4 5
8 22 9 27 22
0 0 0 0 0
2014
SH A
20 12
9 7
11 5
0 0
0 12
0 0
20 0
0 0
522
290
204
28
Sum
38 102 349 33
2. Material and methods 2.1. Sampling During the last 25 years, 522 serum samples were collected of free-ranging harbour seals of the German and Danish Wadden Sea, Helgoland and Anholt (Table 1 and Fig. 1) in connection with tagging or health programs and were examined for Morbillivirusspecific antibodies. The catches were performed as described by Hasselmeier et al. (2008) and Dietz et al. (2013). The animals experiment was approved under permit number Az V31272241.121-19 (70-6/07) at the Ministry of Energy, Agriculture, Environment and Rural Areas of Germany and from the Danish Nature Agency (SNS-3446-00054 and SN 2001-34461/SN-0005) and the Animal Welfare Division (Ministry of Justice, 2005/561976). After blood collection from the extradural intravertebral sinus or hind flipper the samples were transferred into 10 ml serum tubes. Based on the habitus, size and weight of the seals, animals were grouped into three age classes: (I) pups (0–7 months), (II)
yearlings (8–19 months) and (III) older (20 months). In 33 cases (1991–1993), no age of the animal was reported. Morbillivirus-specific antibodies were detected in standard neutralisation assays according to Appel and Robson (1973). Serum samples (obtained in 1990–1997 and 2002–2004) were tested by a neutralising peroxidase linked assay using PDV isolate 2558/Han 88 (Liess et al., 1989) modified by Harder et al. (1992). To investigate the presence of CDV neutralising antibodies in sera obtained in 1996, 1998, 2000, 2001 and since 2005, the CDV strain Onderstepoort (Frisk et al., 1999) was used. For providing an overview over almost all available data, both methods were used between 1990 and 2014 and were considered for data analysis. A small number of samples (n = 29) was tested for both PVD and CDV antibodies (1992; 1996–1998). If the results did not match, we used the value of PDV. Non-infected Vero cells, which were incubated with medium or 1:10 diluted serum, served as negative control. CDV-specific neutralising capacity of the sera was determined by
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Fig. 1. Map of north Germany and Denmark, black stars are locations of the harbour seal catches (522 samplings) conducted between 1990 and 2014.
inhibition of virus induced cytopathogenic effects and evaluated by the method of Reed and Muench (1938). The critical values of antibodies, which determine the animal as positive, differed between the labs (Institute of Virology, University of Veterinary Medicine Hannover, Foundation; Institute of Veterinary Pathology, Justus–Liebig-University Giessen; Landeslabor Schleswig-Holstein; Department of Pathology, University of Veterinary Medicine Hannover, Foundation) (1:10 (PDV and early CDV test) or >1:20 (CDV since 2000)).
2.3. Mathematical modelling The SIR model (Susceptible–Infected-Recovered-model) equations were based on Bodewes et al. (2013). For a detailed description of the mathematical modelling refer to the Supplementary material.
2.2. Statistical modelling The proportion of immune individuals over time was estimated using a generalised linear model. Response variable was the presence of either CDV- or PDV-antibodies. Time was the only predictor variable. We also tested for differences between females and males. The dataset was split into two parts; the first contained all samples taken before the outbreak in 2002 (before 04-052002), the second dataset consists of data after the outbreak. Samples for the modelling were only from Kolumbusloch, Lorenzensplate, Mittelplate, and Westerhever, all areas in the German Wadden Sea (Schleswig-Holstein). The prediction of this model was used to estimate coefficients of a simple disease dynamics model (SIR-type) explaining the transitions between susceptible (S), infected (I) and recovered (R) animals within the free-ranging harbour seal population (Grenfell et al., 1992). Measuring transition rates of such a dynamic process helps us to estimate the current immune status and the potential risk of infection during a new outbreak.
Fig. 2. Serum samples (n = 522) from free-ranging harbour seals of the German and Danish Wadden Sea, Helgoland and Anholt divided by percentage in positive and negative for each year. Red line indicates estimated percentage of immune animals in the population over time. The red band shows the standard error of the mean. Graphs were estimated by applying a simple logistic regression. In 1990 the proportion of immune animals was about 60% due to the outbreak in 1988/1989. Following the outbreak in 2002, the proportion of immune animals was at its maximum (>80%). After the outbreaks the proportion converges towards 0 with a rate of 0.4 (se: 0.1, p-value: <0.001) between 1991 and 2002 and 0.46 (se: 0.05, pvalue: <0.001) for the years 2003–2014. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3. Results 3.1. Overview of antibody development in the harbour seal population before, during and after the two PDV outbreaks (1990–2014) A total of 30.5% (n = 159) harbour seals in this study were seropositive for distemper virus (CDV and/or PDV). The Morbillivirus-neutralising antibody titres ranged from 1:10/>1:20 up to 1:1280 (PDV in 2002 in one animal). Fig. 2 shows the percentage of antibodies divided in positive and negative for each year, including the estimated percentage of immune animals in the population over time. A significant decrease over time in the percentage of immune or antibody positive individuals was observed both after the outbreak in 1988/1989 and after 2002 (Fig. 2). Such a trend was however, only found in (sub) adult animals (age class III) after the first outbreak and after the second outbreak also in yearlings (Table 2). No difference in the decline of seroprevalences over time was detected with respect to the sex of the animals (before the outbreak in 2002 p-value = 0.77, after the outbreak, p-value = 0.54). Three years after the last PDV outbreak in 2002, the number of tested seals with and without antibodies equalled. Since then, the number of seropositive individuals has steadily converged towards zero detectable antibodies. In 2014 the percentage of immunity in the population had declined to only 2% (0.9) (Fig. 2). A significant decrease of antibody positive individuals was seen over time after both PDV outbreaks, and the population is currently in a more or less naive state. 3.2. PDV antibody development of the three age classes When an age effect was not considered the coefficients of decline were estimated to be similar after both outbreaks (Fig. 2). In Fig. 3 all serum samples of seals were divided by age class I–III with (positive) and without (negative) antibodies against PDV/CDV and illustrated for each year. In the years (1991, 1996–1998, 2001, 2004, 2005, 2014) when seal pups (n = 38) were caught, seven had PDV antibodies (Fig. 3). These positive pups were all sampled between August and October (1991, 1993, 1996, 1997). Yearlings (n = 102), lacked Morbillivirus-specific antibodies with the exception of four animals in 1992, 2003 and 2006 (Fig. 3). The majority of caught seals were at least 20 month of age (Fig. 3, Table 1). This shows that some seals of age class III have experienced infection. 3.3. Antibodies against PDV and/or CDV In 1992 and 1996–1998 a small number of seals (n = 29) was tested for both PVD and CDV antibodies. In four of these animals the test for CDV was negative, whereas the test for PDV was positive. In these cases, the results of the PDV test were used. In all other sera (n = 25), for which both types of tests were performed,
Table 2 Estimated coefficients explaining the decrease of the proportion of immune individuals in the population over time using a generalised linear model. Only for older seals (age class III) and yearlings (age class II) after the latest outbreak estimates are significant. Age class
Before/after outbreak
I: Pup
Before After
0.05 0
0.2 0
0.8 1
II: Yearling
Before After
-1.16 -1.67
1.22 0.75
0.34 0.03
III: Older
Before After
-0.48 -0.52
0.11 0.06
<0.001 <0.001
Coefficient
Std. error
p-value
results concurred. Since 2010 only 9.4% of the tested animals had antibodies against CDV. Therefore, the results for PDV and CDV correspond in all but four animals. 3.4. Estimating the number of infected, susceptibles and recovered seals by an epidemiological model over the study time The SIR-model (Fig. 4) is an epidemiological model that generates the theoretical number of infected seals with PDV in the harbour seal population of the Wadden Sea over the time of the study. The population size was based on the total moult counted harbour seals in the Wadden Sea of Schleswig-Holstein (obtained from the Common Wadden Sea Secretariat). It was calculated only for the Wadden Sea of Schleswig-Holstein which represents the majority of the data and illustrates a constant increase of susceptible animals between 1990 and 2002, while the percentage of recovered animals declines due to a higher proportion of pups being born and the mortality of older seals. The estimated number of infected individuals between the two outbreaks and after 2004 is about zero. With the re-introduction of PDV in 2002, an estimated 87–99% (median 96%) of all animals became infected within the first year (Table 3), of which 38–52% (median 45%) died. The number of susceptible seals dropped considerably from 2002 to 2003 and declined further in the next year most likely due to virus transmission of PDV in the population. Simultaneously the number of recovered animals increased and included most of the population. As the infection extenuated soon after the outbreak, the number of susceptible seals decreased while the number of recovered individuals increased. In 2007, for the first time following the second PDV outbreak, the number of susceptible seals was slightly higher than the number of recovered animals. In 2014 the number of immune animals was down to 2% converging towards zero in the coming years. Thus, the number of susceptible or immune animals varied depending on the time passed since the last PDV outbreak. 4. Discussion Since 1990 serum samples of free ranging harbour seals from the Wadden Sea and the Kattegat area were collected and their antibody titres against Morbillivirus were tested. Peaks of Morbillivirus-seropositive harbour seals were detected after the two PDV outbreaks. Since then the antibody titres in the harbour seal population in the Wadden Sea decreased steadily. The absence of circulating Morbilliviruses generates PDV naïve offspring. Therefore, the PDV seroprevalences rate in the population will decrease to zero, which renders the harbour seal population fully susceptible to PDV. Based on our mathematical model, it is shown, that the harbour seal population off the coast of SchleswigHolstein will continue to grow. Assuming similar infection dynamics and mortality rates as reported in the two historic PDV outbreaks we calculate that 96% (of 9174 seals) become infected of which about 45% would die after the first year following the outbreak. In harbour seals sampled between 2002 and 2012 at the seal rehabilitation centre Pieterburen (the Netherlands), a similar dynamic of seroprevalences against PDV and CDV was described (Bodewes et al., 2013). These authors expected that during a new outbreak in the Wadden Sea about 82% of seals will be affected and more than 18,000 individuals will presumably die in 2013 (Bodewes et al., 2013). This matches the results of our model, where about 17,600 seals would die in 2015, if the whole Wadden Sea population is assessed using the current estimated population size of 39,100 individuals (Galatius et al., 2014). Virus-neutralising tests are appropriate tools to verify Morbillivirus antibody titres in marine mammals (Bodewes et al., 2013). Using different Morbillivirus species, titres against the homologous
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Fig. 3. Serum samples of seals divided in age class I–III with (positive) and without (negative) anitbodies against PDV/CDV. Between 1992 and 1998, 29 seals were tested for both PVD and CDV antibodies. If the results disagree, the PDV result was taken. In the years (1991, 1996–1998, 2001, 2004, 2005, 2014) when seal pups (n = 38) were caught, seven had Morbillivirus antibodies. These positive pups were all sampled between August and October; only four yearlings had antibodies against Morbillivirus.
virus show faster and higher responses (Duignan et al., 1994; Visser et al., 1990). Virus neutralisation tests against five different Morbilliviruses were performed in Atlantic walruses (Odobenus rosmarus) showing that all three animals tested were infected by PDV or a similar virus (Duignan et al., 1994). Bodewes et al. (2013) showed that the presence of antibodies against PDV and against CDV correlated, but in 18 out of 423 (4.3%) the result of both tests
differed. In the present study the test against PDV seems to be more sensitive but only a minor proportion of samples were tested for both virus types. Preferably, all samples would be tested in the same assay with the same antigen. However, serum of the used samples is no longer available, so a second analysis of either PDV or CDV is not possible. In order to investigate the complete timeline, we consider both results either from PDV or CDV.
Fig. 4. SIR model, population size based on the total moult counted harbour seals in the Wadden Sea of Schleswig-Holstein (numbers obtained from the Common Wadden Sea Secretariat).
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Table 3 95% central intervals and median of the normal posterior distribution of parameters estimated with the SIR model. Parameters refer to a time step of one year. Parameters are explained in detail in the supplementary material. Parameter
Interpretation
5%-Quantile
Median
95%-Quantile
a b g d r h
Reproduction rate Infection rate Recovery rate within one year Mortality rate due to infection Proportion that was infected during the first year (2003) Mortality rate (of non-infected)
0.222 0.00005 0.840 0.375 0.866 0.131
0.254 0.00016 0.955 0.452 0.964 0.153
0.288 0.00027 0.996 0.517 0.997 0.176
Besides the assay format used, the age of the sampled seals must be considered: Depending on the age of pups, Morbillivirus antibodies may not be the result of an active immune reaction, but rather be maternally-derived (Harder et al., 1993). Maternal antibodies, transferred by colostrum, will decline in a few weeks, whereas animals, which have been exposed to a Morbillivirus infection, will have longer lasting, constant levels of specific antibodies (Harder et al., 1993). Of the 38 pups tested, 34 were estimated to be between one and four months of age (tested August–October). Seven of these were PDV positive, in the years 1991, 1993, 1996, 1997, which could be explained by transference of maternal antibodies. Nevertheless, we did not exclude these samples because the existence of maternal antibodies also reflects, indirectly, the immune status of the current population, but not necessarily that they were exposed to the virus itself. A lack of maternal antibodies in the pup does not necessarily mean that the dam is likewise seronegative, since pups could have been separated from their mothers before antibody transmission appeared. In addition, antibody titres could be below the detection limit (Harder et al., 1993). Therefore we assumed no immunity of new-borns in the SIR model (census first of October) (according to Harder et al., 1993 and Harder, 1994). Infected seals surviving PDV infection are developing high levels of antibodies. For the statistical model it was assumed that animals with antibodies are immune throughout their life. But those antibodies may decrease during time if there is no booster, when PDV is not circulating in the environment. There is no clear reported correlation between the titre of neutralising antibodies and its protective efficacy. But, during the second epidemic almost no seals older than 14 years were found so Härkönen et al. (2007) suggest, that survivors of the first PDV outbreak developed immunity lasting for at least 14 years. Very low titres may not protect seals during a new PDV outbreak. Therefore, the term “recovered” instead of “immune” was used, because no clear limittitre exists and the effects of a putatively protective T-cell mediated immunity were not considered here. In this study, seals considered seropositive had titres of >1:10 or 1:20 according to the different labs. It remains debatable whether or not individuals with a low titre (e.g. 1:40) are fully immune against PDV, implying that the proportion of susceptible seals may actually be higher than estimated. In addition to the peaks in seroprevalence following the PDV outbreaks, a third peak was identified in 1996 (Fig. 2). This could be an incidental finding due to the very low number of seals (n = 6) caught that year. There is, however, some evidence of a minor outbreak of PDV in the mid to late 1990s, as a few Morbillivirus infected harbour seals were found on the coast of Belgium and northern France in the summer of 1998 (Jauniaux et al., 2001). The present study summarises the course of seroprevalence of the German and parts of the Danish harbour seal populations. Apart from the recent influenza A (H10N7) outbreak (Bodewes et al., 2015), distemper viruses represent a recurring risk factor for elevated mortality in seals. Risk and timing of a new outbreak of PDV cannot be foreseen, but the risk increases over time and after
12 years (2002 until 2015) the humoral immunity against PDV has nearly disappeared (2%) in the harbour seal population, which means that the population has again become susceptible for PDV, carried e.g. by other seals species such as grey or harp seals. Hence, high mortalities among harbour seals in the Wadden Sea are expected, if PDV or CDV is introduced into the area. Acknowledgements The wild catches were supported by the Schleswig-Holstein’s Government-Owned Company for Coastal Protection, National Parks and Ocean Protection and Ministry of Energy Transition, Agriculture, Environment and Rural Areas Schleswig-Holstein. The authors are also grateful to all the helpers who participated in the seal catches in Germany and Denmark. Special thanks to all technicians (Miriam Hillmann, Kornelia Wolff-Schmidt and Danuta Waschke) who helped with the preparation and analyses of samples and Rogier Bodewes, Bianca Unger and Abbo van Neer for critically reading the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vetmic. 2015.11.017. References Appel, M., Robson, D.S., 1973. A microneutralization test for canine distemper virus. Am. J. Vet. Res. 34, 1459–1463. Bergman, A., Järplid, B., Svensson, B.-M., 1990. Pathological findings indicative of distemper in European seals. Vet. Microbiol. 23, 331–341. Bodewes, R., Morick, D., van de Bildt, M.W.G., Osinga, N., Garcia, A.R., Sanchez Contreras, G.J., Smits, S.L., Reperant, L.A.P., Kuiken, T., Osterhaus, A.D.M.E., 2013. Prevalence of phocine distemper virus specific antibodies: bracing for the next seal epizootic in north-western Europe. Emerg. Microbes. Infect. 2 (1), e3. doi: http://dx.doi.org/10.1038/emi.2013.2. Bodewes, R., Bestebroer, T.M., van der Vries, E., Verhagen, J.H., Herfst, S., Koopmans, M.P., Fouchier, R.A.M., Pfankuche, V.M., Wohlsein, P., Siebert, U., Baumgärtner, W., Osterhaus, A.D.M.E., 2015. Avian influenza A(H10N7) virus-associated mass deaths among harbor seals. Emerg. Infect. Dis. 21, 720–722. Dietz, R., Hansen, C.T., Have, P., Heide-Jørgensen, M.-P., 1989. Clue to seal epizootic? Nature 338, 627. Dietz, R., Teilmann, J., Andersen, S.M., Riget, F., Olsen, M.T., 2013. Movements and site fidelity of harbour seals (Phoca vitulina) in Kattegat, Denmark, with implications for the epidemiology of the phocine distemper virus. ICES J. Mar. Sci. 70, 186–195. Duignan, P.J., Saliki, J.T., St Aubin, D.J., House, J.A., Geraci, J.R., 1994. Neutralizing antibodies to phocine distemper virus in Atlantic walruses (Odobenus rosmarus rosmarus) from Arctic Canada. J. Wildl. Dis. 30, 90–94. Duignan, P.J., Van Bressem, M.F., Baker, J.D., Barbieri, M., Colegrove, K.M., De Guise, S., de Swart, R.L., Di Guardo, G., Dobson, A., Duprex, W.P., Early, G., Fauquier, D., Goldstein, T., Goodman, S.J., Grenfell, B., Groch, K.R., Gulland, F., Hall, A., Jensen, B.A., Lamy, K., Matassa, K., Mazzariol, S., Morris, S.E., Nielsen, O., Rotstein, D., Rowles, T.K., Saliki, J.T., Siebert, U., Waltzek, T., Wellehan, J.F., 2014. Phocine distemper virus: current knowledge and future directions. Viruses 6, 5093–5134. Frisk, A.L., König, M., Moritz, A., Baumgärtner, W., 1999. Detection of canine distemper virus nucleoprotein RNA by reverse transcription-PCR using serum, whole blood, and cerebrospinal fluid from dogs with distemper. J. Clin. Microbiol. 37 (11), 3634–3643.
E. Ludes-Wehrmeister et al. / Veterinary Microbiology 183 (2016) 43–49 Galatius, A., Brasseur, S., Czeck, R., Diederichs, B., Jensen, L.F., Körber, P., Siebert, U., Teilmann, J., Klöpper, S., 2014. Aerial surveys of harbour seals in the Wadden Sea in 2014. http://www.waddensea-secretariat.org/sites/default/files/downloads/ TMAP_downloads/Seals/harbour_seal_report_2014_b.pdf (accessed 22.06.15.). Grenfell, B.T., Lonergan, M.E., Harwood, J., 1992. Quantitative investigations of the epidemiology of phocine distemper virus (PDV) in European common seal populations. Sci. Total Environ. 115, 15–29. Harder, T.C., 1994. Nachweis morbilli- und herpesvirusspezifischer Antikörper in Seren und postmortal entnommenen Blutproben von Robben. In: Graumann, W., Drukker, J. (Eds.), Sonderdruck aus Histo- and Cytochemistry as a Tool in Environmental Toxicology. Gustav Fischer Verlag, Stuttgart, New York, pp. 88– 93. Harder, T.C., Stede, M., Willhaus, T., Schwarz, J., Heidemann, G., Liess, B., 1993. Morbillivirus antibodies of maternal origin in harbour seal pups (Phoca vitulina). Vet. Rec. 132, 632–633. Harder, T.C., Willhaus, T., Leibold, W., Liess, B., 1992. Investigations on course and outcome of phocine distemper virus infection in harbour seals (Phoca vitulina) exposed to polychlorinated biphenyls. J. Vet. Med. B 39, 19–31. Härkönen, T., Hårding, K.C., Lunneryd, S.G., 1999. Age- and sex-specific behaviour in harbour seals Phoca vitulina leads to biased estimates of vital population parameters. J. Appl. Ecol. 36, 825–841. Härkönen, T., Dietz, R., Reijnders, P., Teilmann, J., Harding, K., Hall, A., Brasseur, S., Siebert, U., Goodman, S.J., Jepson, P.D., Rasmussen, T.D., Thompson, P., 2006. A. review of the 1988 and 2002 phocine distemper virus epidemics in European harbour seals. Dis. Aquat. Org. 68, 115–130. Härkönen, T., Harding, K., Rasmussen, T.D., Teilmann, J., Dietz, R., 2007. Age- and sexspecific mortality patterns in an emerging wildlife epidemic: the phocine distemper in European harbour seals. PLoS One 9, 1–4. Hasselmeier, I., Fonfara, S., Driver, J., Siebert, U., 2008. Differential hematology profiles of free-ranging, rehabilitated, and captive harbor seals (Phoca vitulina) of the German North Sea. Aquat. Mammals 34, 149–156. Jauniaux, T., Boseret, G., Desmecht, M., Haelters, J., Manteca, C., Tavernier, J., van Gompel, J., Coignoul, F., 2001. Morbillivirus in common seals stranded on the coasts of Belgium and northern France during summer 1998. Vet. Rec. 148, 587– 591. Kennedy, S., 1990. A review of the 1988 European seal Morbillivirus epizootic. Vet. Rec. 127, 563–567.
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Kennedy, S., 1998. Morbillivirus infections in aquatic mammals. J. Comp. Pathol. 119, 201–225. Kreutzer, M., Kreutzer, R., Siebert, U., Müller, G., Reijnders, P., Brasseur, S., Härkönen, T., Dietz, R., Sonne, C., Born, E.W., Baumgärtner, W., 2008. In search of virus carriers of the 1988 and 2002 phocine distemper virus outbreaks in European harbour seals. Arch. Virol. 153, 187–192. Liess, B., Frey, H.R., Zaghawa, A., Stede, M., 1989. Morbillivirus infection of seals (Phoca vitulina) during the 1988 epidemic in the bay of Heligoland. J. Vet. Med. B 36, 601–608. Mamaev, L.V., Denikina, N.N., Belikov, S.I., Volchkov, V.E., Visser, I.K.G., Fleming, M., Kai, C., Harder, T.C., Liess, B., Osterhaus, A.D.M.E., Barett, T., 1995. Characterisation of Morbilliviruses isolated from Lake Baikal seals (Phoca sibirica). Vet. Microbiol. 44, 251–259. Olsen, M.T., Andersen, S.M., Teilmann, J., Dietz, R., Edrén, S.M.C., Linnet, A., Härkönen, T., 2010. Status of the harbour seal (Phoca vitulina) in southern Scandinavia. NAMMCO Sci. Publ. 8, 77–94. Reed, L.J., Muench, H., 1938. A simple method of estimating fity per cent endpoints. Am. J. Epidemiol./Am. J. Hyg. 27, 493–497. Reijnders, P.J.H., 1981. Management and conservation of the harbour seal (Phoca vitulina) population in the international Wadden Sea area. Biol. Conserv. 19, 213–221. Reijnders, P.J.H., Brasseur, S.M.J.M., Tougaard, S., Siebert, U., Borchardt, T., Stede, M., 2010. Population development and status of harbour seals (Phoca vitulina) in the Wadden Sea. NAMMCO Sci. Publ. 8, 95–106. Ries, E.H., Hiby, L.R., Reijnders, P.J.H., 1998. Maximum likelihood population size estimation of harbour seals in the Dutch Wadden Sea based on a markrecapture experiment. J. Appl. Ecol. 35, 332–339. Teilmann, J., Rigét, F., Harkonen, T., 2010. Optimizing survey design for Scandinavian harbour seals: population trend as an ecological quality element. ICES J. Mar. Sci. 67, 952–958. Visser, I.K.G., Kumarev, V.P., Örvell, C., de Vries, P., Broeders, H.W.J., van de Bildt, M. W.G., Groen, J., Teppema, J.S., Burger, M.C., UytdeHaag, F.G.C.M., Osterhaus, A.D. M.E., 1990. Comparison of two Morbilliviruses isolated from seals during outbreaks of distemper in North West Europe and Siberia. Arch. Virol. 111, 149–164.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Phocine distemper virus (PDV) seroprevalence as predictor for future outbreaks in harbour seals Eva Ludes-Wehrmeistera,** , Claudia Dupkea , Timm C. Harderb , Wolfgang Baumgärtnerc , Ludwig Haasd , Jonas Teilmanne , Rune Dietze, Lasse F. Jensenf , Ursula Sieberta,* a
Institute of Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine, Hannover, Foundation, Werftstrasse 6, 25761 Buesum, Germany O.I.E., FAO and National Reference Laboratory for Avian Influenza, Institute of Diagnostic Virology, Friedrich Loeffler Institut, Suedufer 10, D-17493 Greifswald-Insel Riems, Germany c Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Buenteweg 17, 30559 Hannover, Germany d Institute of Virology, University of Veterinary Medicine Hannover, Foundation, Buenteweg 17, 30559 Hannover, Germany e Department of Bioscience, Aarhus University, Frederiksborgvej 399, DK-4000 Roskilde, Denmark f Fisheries and Maritime Museum, Tarphagevej 2, 6710 Esbjerg V, Denmark b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 July 2015 Received in revised form 10 November 2015 Accepted 14 November 2015
Phocine distemper virus (PDV) infections caused the two most pronounced mass mortalities in marine mammals documented in the past century. During the two outbreaks, 23,000 and 30,000 harbour seals (Phoca vitulina), died in 1988/1989 and 2002 across populations in the Wadden Sea and adjacent waters, respectively. To follow the mechanism and development of disease spreading, the dynamics of Morbillivirus-specific antibodies in harbour seal populations in German and Danish waters were examined. 522 serum samples of free-ranging harbour seals of different ages were sampled between 1990 and 2014. By standard neutralisation assays, Morbillivirus-specific antibodies were detected, using either the PDV isolate 2558/Han 88 or the related canine distemper virus (CDV) strain Onderstepoort. A total of 159 (30.5%) of the harbour seals were seropositive. Annual seroprevalence rates showed an undulating course: Peaks were seen in the post-epidemic years 1990/1991 and 2002/2003. Following each PDV outbreak, seroprevalence decreased and six to eight years after the epidemics samples were tested seronegative, indicating that the populations are now again susceptible to new PDV outbreak. After the last outbreak in 2002, the populations grew steadily to an estimated maximum (since 1975) of about 39,100 individuals in the Wadden Sea in 2014 and about 23,540 harbour seals in the Kattegat area in 2013. A re-appearence of PDV would presumably result in another epizootic with high mortality rates as encountered in the previous outbreaks. The current high population density renders harbour seals vulnerable to rapid spread of infectious agents including PDV and the recently detected influenza A virus. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Harbour seal Common seal Phocine distemper virus Canine distemper virus Morbillivirus antibodies Epidemic
1. Introduction Following the ban on hunting of seals in the mid-70s, the harbour seal (Phoca vitulina) population in the Wadden Sea increased steadily and at present are considered to be the most common seal species in the Wadden Sea and Kattegat area (Reijnders, 1981; Reijnders et al., 2010; Teilmann et al., 2010). In 2014 the harbour seal population (Germany, Netherlands and Denmark) consisted of 26,576 individuals (maximum number
* Corresponding author. Fax: +49 511 856 8181. ** Corresponding author. E-mail addresses: [email protected] (E. Ludes-Wehrmeister), [email protected] (U. Siebert). http://dx.doi.org/10.1016/j.vetmic.2015.11.017 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.
counted during moult counts) resulting in an estimated total population size (based on the correction factor estimated by Ries et al. (1998)) of 39,100 individuals (Galatius et al., 2014). The estimated number of harbour seals in the Kattegat was 23,540 in 2013 (Galatius pers. comm.; correction factor derived from Härkönen et al., 1999). In 1987, prior to the first reported outbreak of the phocine distemper virus (PDV) infection, 8296 harbour seals were counted in the Wadden Sea (Reijnders et al., 2010) and in 1986, 4458 individuals were counted in the Kattegat area respectively (Olsen et al., 2010). During this first epizootic, more than 50% of the population died (Dietz et al., 1989; Härkönen et al., 2006). Thereafter, it recovered from a counted number of some 4400 animals in 1989 up to some 17,900 counted individuals in 2001 in the Wadden Sea (Reijnders et al., 2010) and from 2178 counted animals in 1989 up to 5941 individuals in 2000 in the
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Kattegat (Olsen et al., 2010). Another PDV outbreak caused a second mass mortality in 2002 (Härkönen et al., 2006) and decimated the population to 10,817 counted seals in the Wadden Sea and 4895 counted individuals in the Kattegat in 2003 (Reijnders et al., 2010; Olsen et al., 2010). During the second PDV outbreak a total of 30,000 individuals succumbed to the disease in European waters (Härkönen et al., 2006). Both epizootics started in spring on the small Danish island of Anholt in the Kattegat (Dietz et al., 1989; Härkönen et al., 2006) and spread throughout the seas of north-western Europe (Bergman et al., 1990; Härkönen et al., 2006). During both PDV outbreaks, only few grey seals (Halichoerus grypus) died of the disease (Bergman et al., 1990; Härkönen et al., 2006). Clinical signs of PDV infection in harbour seals are fever, coughing, dyspnoea, oculo-nasal discharge, conjunctivitis, ophthalmitis, keratitis, diarrhoea, abortion and increased buoyancy due to emphysema resulting in a reduced diving ability (Bergman et al., 1990; Kennedy, 1990, 1998). A few seals showed disturbances of the central nervous system (Bergman et al., 1990; Kennedy, 1990). Pneumonia with emphysema and oedema represented the main pathological findings (Bergman et al., 1990; Jauniaux et al., 2001; Kennedy, 1998). It has been suggested that the likely source of the 1988 epidemic was harp seals (Pagophilus groenlandicus), as it is known to host the virus and since mass migrations of this species took place into the southern Scandinavian waters in the winter of 1987– 1988 (Dietz et al., 1989). After the first outbreak grey seals (H. grypus) have been suggested to act as a reservoir for PDV or transferring the infection from the arctic seals so that outbreaks among harbour seals in European waters had been sparked by spill-over infections from these reservoirs (Härkönen et al., 2006; Kreutzer et al., 2008). For a comprised review of different PDV outbreaks all over the world we refer to Duignan et al. (2014). Apart from PDV, seals in the Caspian Sea, Lake Baikal and Antarctica were reported to have suffered from infection with the antigenically and genetically related but distinguishable Morbillivirus of terrestrial carnivores, canine distemper virus (CDV) (Mamaev et al., 1995; Kennedy 1998; Kreutzer et al., 2008;). Such infections obviously resulted from spill-over transmission of CDV from terrestrial carnivores (e.g. wolves (Canis lupus), dogs (Canis familiaris), foxes (Alopex lagopus)). As part of the health monitoring program established in 1990 following the first PDV outbreak, the aim of this study was to describe the Morbillivirus seroprevalence during the last 25 years in the population of harbour seals in the German and Danish waters. Data were used to predict the potential of a future outbreak of this disease.
Table 1 Overview of serum samples: location of catches (Germany: LS: Lower Saxony Wadden Sea, SH: Schleswig-Holstein Wadden Sea (Mittelplate, Lorenzensplate; Kolumbusloch,Westerhever), H: Helgoland, Denmark: R: Rømø, A: Anholt). Animals were grouped in sex and age classes: Age class I: pup (born in the year of catch; 1–7 months), II: yearling (seal born the year previous to the catch; 7–19 months); III older (than 19 months); *most of it published in Harder (1994). Year
Location Total Sex
Age class
Male Female Unknown I
II
III
Unknown
1990* SH
6
3
3
0
0
0
6
0
1991* SH LS
15 10
8 0
1 0
6 10
3
2
4
6 10
1992* SH LS
14 10
11 0
3 0
0 10
1 0
4 0
9 0 0 10
1993* SH LS
41 9
29 3
12 5
0 1
4 2
15 2
16 4
6 1
1996 1997 1998 2000 2001 2002 2003
SH SH SH SH SH SH SH
6 9 7 18 28 13 16
2 5 6 11 12 2 8
3 4 1 7 16 11 8
1 0 0 0 0 0 0
3 2 2 0 2 0 0
0 2 0 2 5 0 2
3 5 5 16 21 13 14
0 0 0 0 0 0 0
2004
SH R
27 16
18 14
9 2
0 0
4 0
1 1
22 15
0 0
2005
SH H A R
29 3 3 10
17 3 1 9
12 0 2 1
0 0 0 0
0 0 3 0
16 0 0 1
13 3 0 9
0 0 0 0
2006
SH H
16 7
12 7
4 0
0 0
0 0
3 2
13 5
0 0
2007
SH
31
23
8
0
0
13
18
0
2008
SH R A
27 8 6
15 6 3
12 2 3
0 0 0
0 0 0
8 3 3
19 5 3
0 0 0
2009 2010 2011 2012 2013
SH SH SH SH SH
10 22 15 31 27
2 5 11 14 14
8 17 4 17 13
0 0 0 0 0
0 0 0 0 0
2 0 6 4 5
8 22 9 27 22
0 0 0 0 0
2014
SH A
20 12
9 7
11 5
0 0
0 12
0 0
20 0
0 0
522
290
204
28
Sum
38 102 349 33
2. Material and methods 2.1. Sampling During the last 25 years, 522 serum samples were collected of free-ranging harbour seals of the German and Danish Wadden Sea, Helgoland and Anholt (Table 1 and Fig. 1) in connection with tagging or health programs and were examined for Morbillivirusspecific antibodies. The catches were performed as described by Hasselmeier et al. (2008) and Dietz et al. (2013). The animals experiment was approved under permit number Az V31272241.121-19 (70-6/07) at the Ministry of Energy, Agriculture, Environment and Rural Areas of Germany and from the Danish Nature Agency (SNS-3446-00054 and SN 2001-34461/SN-0005) and the Animal Welfare Division (Ministry of Justice, 2005/561976). After blood collection from the extradural intravertebral sinus or hind flipper the samples were transferred into 10 ml serum tubes. Based on the habitus, size and weight of the seals, animals were grouped into three age classes: (I) pups (0–7 months), (II)
yearlings (8–19 months) and (III) older (20 months). In 33 cases (1991–1993), no age of the animal was reported. Morbillivirus-specific antibodies were detected in standard neutralisation assays according to Appel and Robson (1973). Serum samples (obtained in 1990–1997 and 2002–2004) were tested by a neutralising peroxidase linked assay using PDV isolate 2558/Han 88 (Liess et al., 1989) modified by Harder et al. (1992). To investigate the presence of CDV neutralising antibodies in sera obtained in 1996, 1998, 2000, 2001 and since 2005, the CDV strain Onderstepoort (Frisk et al., 1999) was used. For providing an overview over almost all available data, both methods were used between 1990 and 2014 and were considered for data analysis. A small number of samples (n = 29) was tested for both PVD and CDV antibodies (1992; 1996–1998). If the results did not match, we used the value of PDV. Non-infected Vero cells, which were incubated with medium or 1:10 diluted serum, served as negative control. CDV-specific neutralising capacity of the sera was determined by
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Fig. 1. Map of north Germany and Denmark, black stars are locations of the harbour seal catches (522 samplings) conducted between 1990 and 2014.
inhibition of virus induced cytopathogenic effects and evaluated by the method of Reed and Muench (1938). The critical values of antibodies, which determine the animal as positive, differed between the labs (Institute of Virology, University of Veterinary Medicine Hannover, Foundation; Institute of Veterinary Pathology, Justus–Liebig-University Giessen; Landeslabor Schleswig-Holstein; Department of Pathology, University of Veterinary Medicine Hannover, Foundation) (1:10 (PDV and early CDV test) or >1:20 (CDV since 2000)).
2.3. Mathematical modelling The SIR model (Susceptible–Infected-Recovered-model) equations were based on Bodewes et al. (2013). For a detailed description of the mathematical modelling refer to the Supplementary material.
2.2. Statistical modelling The proportion of immune individuals over time was estimated using a generalised linear model. Response variable was the presence of either CDV- or PDV-antibodies. Time was the only predictor variable. We also tested for differences between females and males. The dataset was split into two parts; the first contained all samples taken before the outbreak in 2002 (before 04-052002), the second dataset consists of data after the outbreak. Samples for the modelling were only from Kolumbusloch, Lorenzensplate, Mittelplate, and Westerhever, all areas in the German Wadden Sea (Schleswig-Holstein). The prediction of this model was used to estimate coefficients of a simple disease dynamics model (SIR-type) explaining the transitions between susceptible (S), infected (I) and recovered (R) animals within the free-ranging harbour seal population (Grenfell et al., 1992). Measuring transition rates of such a dynamic process helps us to estimate the current immune status and the potential risk of infection during a new outbreak.
Fig. 2. Serum samples (n = 522) from free-ranging harbour seals of the German and Danish Wadden Sea, Helgoland and Anholt divided by percentage in positive and negative for each year. Red line indicates estimated percentage of immune animals in the population over time. The red band shows the standard error of the mean. Graphs were estimated by applying a simple logistic regression. In 1990 the proportion of immune animals was about 60% due to the outbreak in 1988/1989. Following the outbreak in 2002, the proportion of immune animals was at its maximum (>80%). After the outbreaks the proportion converges towards 0 with a rate of 0.4 (se: 0.1, p-value: <0.001) between 1991 and 2002 and 0.46 (se: 0.05, pvalue: <0.001) for the years 2003–2014. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3. Results 3.1. Overview of antibody development in the harbour seal population before, during and after the two PDV outbreaks (1990–2014) A total of 30.5% (n = 159) harbour seals in this study were seropositive for distemper virus (CDV and/or PDV). The Morbillivirus-neutralising antibody titres ranged from 1:10/>1:20 up to 1:1280 (PDV in 2002 in one animal). Fig. 2 shows the percentage of antibodies divided in positive and negative for each year, including the estimated percentage of immune animals in the population over time. A significant decrease over time in the percentage of immune or antibody positive individuals was observed both after the outbreak in 1988/1989 and after 2002 (Fig. 2). Such a trend was however, only found in (sub) adult animals (age class III) after the first outbreak and after the second outbreak also in yearlings (Table 2). No difference in the decline of seroprevalences over time was detected with respect to the sex of the animals (before the outbreak in 2002 p-value = 0.77, after the outbreak, p-value = 0.54). Three years after the last PDV outbreak in 2002, the number of tested seals with and without antibodies equalled. Since then, the number of seropositive individuals has steadily converged towards zero detectable antibodies. In 2014 the percentage of immunity in the population had declined to only 2% (0.9) (Fig. 2). A significant decrease of antibody positive individuals was seen over time after both PDV outbreaks, and the population is currently in a more or less naive state. 3.2. PDV antibody development of the three age classes When an age effect was not considered the coefficients of decline were estimated to be similar after both outbreaks (Fig. 2). In Fig. 3 all serum samples of seals were divided by age class I–III with (positive) and without (negative) antibodies against PDV/CDV and illustrated for each year. In the years (1991, 1996–1998, 2001, 2004, 2005, 2014) when seal pups (n = 38) were caught, seven had PDV antibodies (Fig. 3). These positive pups were all sampled between August and October (1991, 1993, 1996, 1997). Yearlings (n = 102), lacked Morbillivirus-specific antibodies with the exception of four animals in 1992, 2003 and 2006 (Fig. 3). The majority of caught seals were at least 20 month of age (Fig. 3, Table 1). This shows that some seals of age class III have experienced infection. 3.3. Antibodies against PDV and/or CDV In 1992 and 1996–1998 a small number of seals (n = 29) was tested for both PVD and CDV antibodies. In four of these animals the test for CDV was negative, whereas the test for PDV was positive. In these cases, the results of the PDV test were used. In all other sera (n = 25), for which both types of tests were performed,
Table 2 Estimated coefficients explaining the decrease of the proportion of immune individuals in the population over time using a generalised linear model. Only for older seals (age class III) and yearlings (age class II) after the latest outbreak estimates are significant. Age class
Before/after outbreak
I: Pup
Before After
0.05 0
0.2 0
0.8 1
II: Yearling
Before After
-1.16 -1.67
1.22 0.75
0.34 0.03
III: Older
Before After
-0.48 -0.52
0.11 0.06
<0.001 <0.001
Coefficient
Std. error
p-value
results concurred. Since 2010 only 9.4% of the tested animals had antibodies against CDV. Therefore, the results for PDV and CDV correspond in all but four animals. 3.4. Estimating the number of infected, susceptibles and recovered seals by an epidemiological model over the study time The SIR-model (Fig. 4) is an epidemiological model that generates the theoretical number of infected seals with PDV in the harbour seal population of the Wadden Sea over the time of the study. The population size was based on the total moult counted harbour seals in the Wadden Sea of Schleswig-Holstein (obtained from the Common Wadden Sea Secretariat). It was calculated only for the Wadden Sea of Schleswig-Holstein which represents the majority of the data and illustrates a constant increase of susceptible animals between 1990 and 2002, while the percentage of recovered animals declines due to a higher proportion of pups being born and the mortality of older seals. The estimated number of infected individuals between the two outbreaks and after 2004 is about zero. With the re-introduction of PDV in 2002, an estimated 87–99% (median 96%) of all animals became infected within the first year (Table 3), of which 38–52% (median 45%) died. The number of susceptible seals dropped considerably from 2002 to 2003 and declined further in the next year most likely due to virus transmission of PDV in the population. Simultaneously the number of recovered animals increased and included most of the population. As the infection extenuated soon after the outbreak, the number of susceptible seals decreased while the number of recovered individuals increased. In 2007, for the first time following the second PDV outbreak, the number of susceptible seals was slightly higher than the number of recovered animals. In 2014 the number of immune animals was down to 2% converging towards zero in the coming years. Thus, the number of susceptible or immune animals varied depending on the time passed since the last PDV outbreak. 4. Discussion Since 1990 serum samples of free ranging harbour seals from the Wadden Sea and the Kattegat area were collected and their antibody titres against Morbillivirus were tested. Peaks of Morbillivirus-seropositive harbour seals were detected after the two PDV outbreaks. Since then the antibody titres in the harbour seal population in the Wadden Sea decreased steadily. The absence of circulating Morbilliviruses generates PDV naïve offspring. Therefore, the PDV seroprevalences rate in the population will decrease to zero, which renders the harbour seal population fully susceptible to PDV. Based on our mathematical model, it is shown, that the harbour seal population off the coast of SchleswigHolstein will continue to grow. Assuming similar infection dynamics and mortality rates as reported in the two historic PDV outbreaks we calculate that 96% (of 9174 seals) become infected of which about 45% would die after the first year following the outbreak. In harbour seals sampled between 2002 and 2012 at the seal rehabilitation centre Pieterburen (the Netherlands), a similar dynamic of seroprevalences against PDV and CDV was described (Bodewes et al., 2013). These authors expected that during a new outbreak in the Wadden Sea about 82% of seals will be affected and more than 18,000 individuals will presumably die in 2013 (Bodewes et al., 2013). This matches the results of our model, where about 17,600 seals would die in 2015, if the whole Wadden Sea population is assessed using the current estimated population size of 39,100 individuals (Galatius et al., 2014). Virus-neutralising tests are appropriate tools to verify Morbillivirus antibody titres in marine mammals (Bodewes et al., 2013). Using different Morbillivirus species, titres against the homologous
E. Ludes-Wehrmeister et al. / Veterinary Microbiology 183 (2016) 43–49
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Fig. 3. Serum samples of seals divided in age class I–III with (positive) and without (negative) anitbodies against PDV/CDV. Between 1992 and 1998, 29 seals were tested for both PVD and CDV antibodies. If the results disagree, the PDV result was taken. In the years (1991, 1996–1998, 2001, 2004, 2005, 2014) when seal pups (n = 38) were caught, seven had Morbillivirus antibodies. These positive pups were all sampled between August and October; only four yearlings had antibodies against Morbillivirus.
virus show faster and higher responses (Duignan et al., 1994; Visser et al., 1990). Virus neutralisation tests against five different Morbilliviruses were performed in Atlantic walruses (Odobenus rosmarus) showing that all three animals tested were infected by PDV or a similar virus (Duignan et al., 1994). Bodewes et al. (2013) showed that the presence of antibodies against PDV and against CDV correlated, but in 18 out of 423 (4.3%) the result of both tests
differed. In the present study the test against PDV seems to be more sensitive but only a minor proportion of samples were tested for both virus types. Preferably, all samples would be tested in the same assay with the same antigen. However, serum of the used samples is no longer available, so a second analysis of either PDV or CDV is not possible. In order to investigate the complete timeline, we consider both results either from PDV or CDV.
Fig. 4. SIR model, population size based on the total moult counted harbour seals in the Wadden Sea of Schleswig-Holstein (numbers obtained from the Common Wadden Sea Secretariat).
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Table 3 95% central intervals and median of the normal posterior distribution of parameters estimated with the SIR model. Parameters refer to a time step of one year. Parameters are explained in detail in the supplementary material. Parameter
Interpretation
5%-Quantile
Median
95%-Quantile
a b g d r h
Reproduction rate Infection rate Recovery rate within one year Mortality rate due to infection Proportion that was infected during the first year (2003) Mortality rate (of non-infected)
0.222 0.00005 0.840 0.375 0.866 0.131
0.254 0.00016 0.955 0.452 0.964 0.153
0.288 0.00027 0.996 0.517 0.997 0.176
Besides the assay format used, the age of the sampled seals must be considered: Depending on the age of pups, Morbillivirus antibodies may not be the result of an active immune reaction, but rather be maternally-derived (Harder et al., 1993). Maternal antibodies, transferred by colostrum, will decline in a few weeks, whereas animals, which have been exposed to a Morbillivirus infection, will have longer lasting, constant levels of specific antibodies (Harder et al., 1993). Of the 38 pups tested, 34 were estimated to be between one and four months of age (tested August–October). Seven of these were PDV positive, in the years 1991, 1993, 1996, 1997, which could be explained by transference of maternal antibodies. Nevertheless, we did not exclude these samples because the existence of maternal antibodies also reflects, indirectly, the immune status of the current population, but not necessarily that they were exposed to the virus itself. A lack of maternal antibodies in the pup does not necessarily mean that the dam is likewise seronegative, since pups could have been separated from their mothers before antibody transmission appeared. In addition, antibody titres could be below the detection limit (Harder et al., 1993). Therefore we assumed no immunity of new-borns in the SIR model (census first of October) (according to Harder et al., 1993 and Harder, 1994). Infected seals surviving PDV infection are developing high levels of antibodies. For the statistical model it was assumed that animals with antibodies are immune throughout their life. But those antibodies may decrease during time if there is no booster, when PDV is not circulating in the environment. There is no clear reported correlation between the titre of neutralising antibodies and its protective efficacy. But, during the second epidemic almost no seals older than 14 years were found so Härkönen et al. (2007) suggest, that survivors of the first PDV outbreak developed immunity lasting for at least 14 years. Very low titres may not protect seals during a new PDV outbreak. Therefore, the term “recovered” instead of “immune” was used, because no clear limittitre exists and the effects of a putatively protective T-cell mediated immunity were not considered here. In this study, seals considered seropositive had titres of >1:10 or 1:20 according to the different labs. It remains debatable whether or not individuals with a low titre (e.g. 1:40) are fully immune against PDV, implying that the proportion of susceptible seals may actually be higher than estimated. In addition to the peaks in seroprevalence following the PDV outbreaks, a third peak was identified in 1996 (Fig. 2). This could be an incidental finding due to the very low number of seals (n = 6) caught that year. There is, however, some evidence of a minor outbreak of PDV in the mid to late 1990s, as a few Morbillivirus infected harbour seals were found on the coast of Belgium and northern France in the summer of 1998 (Jauniaux et al., 2001). The present study summarises the course of seroprevalence of the German and parts of the Danish harbour seal populations. Apart from the recent influenza A (H10N7) outbreak (Bodewes et al., 2015), distemper viruses represent a recurring risk factor for elevated mortality in seals. Risk and timing of a new outbreak of PDV cannot be foreseen, but the risk increases over time and after
12 years (2002 until 2015) the humoral immunity against PDV has nearly disappeared (2%) in the harbour seal population, which means that the population has again become susceptible for PDV, carried e.g. by other seals species such as grey or harp seals. Hence, high mortalities among harbour seals in the Wadden Sea are expected, if PDV or CDV is introduced into the area. Acknowledgements The wild catches were supported by the Schleswig-Holstein’s Government-Owned Company for Coastal Protection, National Parks and Ocean Protection and Ministry of Energy Transition, Agriculture, Environment and Rural Areas Schleswig-Holstein. The authors are also grateful to all the helpers who participated in the seal catches in Germany and Denmark. Special thanks to all technicians (Miriam Hillmann, Kornelia Wolff-Schmidt and Danuta Waschke) who helped with the preparation and analyses of samples and Rogier Bodewes, Bianca Unger and Abbo van Neer for critically reading the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vetmic. 2015.11.017. References Appel, M., Robson, D.S., 1973. A microneutralization test for canine distemper virus. Am. J. Vet. Res. 34, 1459–1463. Bergman, A., Järplid, B., Svensson, B.-M., 1990. Pathological findings indicative of distemper in European seals. Vet. Microbiol. 23, 331–341. Bodewes, R., Morick, D., van de Bildt, M.W.G., Osinga, N., Garcia, A.R., Sanchez Contreras, G.J., Smits, S.L., Reperant, L.A.P., Kuiken, T., Osterhaus, A.D.M.E., 2013. Prevalence of phocine distemper virus specific antibodies: bracing for the next seal epizootic in north-western Europe. Emerg. Microbes. Infect. 2 (1), e3. doi: http://dx.doi.org/10.1038/emi.2013.2. Bodewes, R., Bestebroer, T.M., van der Vries, E., Verhagen, J.H., Herfst, S., Koopmans, M.P., Fouchier, R.A.M., Pfankuche, V.M., Wohlsein, P., Siebert, U., Baumgärtner, W., Osterhaus, A.D.M.E., 2015. Avian influenza A(H10N7) virus-associated mass deaths among harbor seals. Emerg. Infect. Dis. 21, 720–722. Dietz, R., Hansen, C.T., Have, P., Heide-Jørgensen, M.-P., 1989. Clue to seal epizootic? Nature 338, 627. Dietz, R., Teilmann, J., Andersen, S.M., Riget, F., Olsen, M.T., 2013. Movements and site fidelity of harbour seals (Phoca vitulina) in Kattegat, Denmark, with implications for the epidemiology of the phocine distemper virus. ICES J. Mar. Sci. 70, 186–195. Duignan, P.J., Saliki, J.T., St Aubin, D.J., House, J.A., Geraci, J.R., 1994. Neutralizing antibodies to phocine distemper virus in Atlantic walruses (Odobenus rosmarus rosmarus) from Arctic Canada. J. Wildl. Dis. 30, 90–94. Duignan, P.J., Van Bressem, M.F., Baker, J.D., Barbieri, M., Colegrove, K.M., De Guise, S., de Swart, R.L., Di Guardo, G., Dobson, A., Duprex, W.P., Early, G., Fauquier, D., Goldstein, T., Goodman, S.J., Grenfell, B., Groch, K.R., Gulland, F., Hall, A., Jensen, B.A., Lamy, K., Matassa, K., Mazzariol, S., Morris, S.E., Nielsen, O., Rotstein, D., Rowles, T.K., Saliki, J.T., Siebert, U., Waltzek, T., Wellehan, J.F., 2014. Phocine distemper virus: current knowledge and future directions. Viruses 6, 5093–5134. Frisk, A.L., König, M., Moritz, A., Baumgärtner, W., 1999. Detection of canine distemper virus nucleoprotein RNA by reverse transcription-PCR using serum, whole blood, and cerebrospinal fluid from dogs with distemper. J. Clin. Microbiol. 37 (11), 3634–3643.
E. Ludes-Wehrmeister et al. / Veterinary Microbiology 183 (2016) 43–49 Galatius, A., Brasseur, S., Czeck, R., Diederichs, B., Jensen, L.F., Körber, P., Siebert, U., Teilmann, J., Klöpper, S., 2014. Aerial surveys of harbour seals in the Wadden Sea in 2014. http://www.waddensea-secretariat.org/sites/default/files/downloads/ TMAP_downloads/Seals/harbour_seal_report_2014_b.pdf (accessed 22.06.15.). Grenfell, B.T., Lonergan, M.E., Harwood, J., 1992. Quantitative investigations of the epidemiology of phocine distemper virus (PDV) in European common seal populations. Sci. Total Environ. 115, 15–29. Harder, T.C., 1994. Nachweis morbilli- und herpesvirusspezifischer Antikörper in Seren und postmortal entnommenen Blutproben von Robben. In: Graumann, W., Drukker, J. (Eds.), Sonderdruck aus Histo- and Cytochemistry as a Tool in Environmental Toxicology. Gustav Fischer Verlag, Stuttgart, New York, pp. 88– 93. Harder, T.C., Stede, M., Willhaus, T., Schwarz, J., Heidemann, G., Liess, B., 1993. Morbillivirus antibodies of maternal origin in harbour seal pups (Phoca vitulina). Vet. Rec. 132, 632–633. Harder, T.C., Willhaus, T., Leibold, W., Liess, B., 1992. Investigations on course and outcome of phocine distemper virus infection in harbour seals (Phoca vitulina) exposed to polychlorinated biphenyls. J. Vet. Med. B 39, 19–31. Härkönen, T., Hårding, K.C., Lunneryd, S.G., 1999. Age- and sex-specific behaviour in harbour seals Phoca vitulina leads to biased estimates of vital population parameters. J. Appl. Ecol. 36, 825–841. Härkönen, T., Dietz, R., Reijnders, P., Teilmann, J., Harding, K., Hall, A., Brasseur, S., Siebert, U., Goodman, S.J., Jepson, P.D., Rasmussen, T.D., Thompson, P., 2006. A. review of the 1988 and 2002 phocine distemper virus epidemics in European harbour seals. Dis. Aquat. Org. 68, 115–130. Härkönen, T., Harding, K., Rasmussen, T.D., Teilmann, J., Dietz, R., 2007. Age- and sexspecific mortality patterns in an emerging wildlife epidemic: the phocine distemper in European harbour seals. PLoS One 9, 1–4. Hasselmeier, I., Fonfara, S., Driver, J., Siebert, U., 2008. Differential hematology profiles of free-ranging, rehabilitated, and captive harbor seals (Phoca vitulina) of the German North Sea. Aquat. Mammals 34, 149–156. Jauniaux, T., Boseret, G., Desmecht, M., Haelters, J., Manteca, C., Tavernier, J., van Gompel, J., Coignoul, F., 2001. Morbillivirus in common seals stranded on the coasts of Belgium and northern France during summer 1998. Vet. Rec. 148, 587– 591. Kennedy, S., 1990. A review of the 1988 European seal Morbillivirus epizootic. Vet. Rec. 127, 563–567.
49
Kennedy, S., 1998. Morbillivirus infections in aquatic mammals. J. Comp. Pathol. 119, 201–225. Kreutzer, M., Kreutzer, R., Siebert, U., Müller, G., Reijnders, P., Brasseur, S., Härkönen, T., Dietz, R., Sonne, C., Born, E.W., Baumgärtner, W., 2008. In search of virus carriers of the 1988 and 2002 phocine distemper virus outbreaks in European harbour seals. Arch. Virol. 153, 187–192. Liess, B., Frey, H.R., Zaghawa, A., Stede, M., 1989. Morbillivirus infection of seals (Phoca vitulina) during the 1988 epidemic in the bay of Heligoland. J. Vet. Med. B 36, 601–608. Mamaev, L.V., Denikina, N.N., Belikov, S.I., Volchkov, V.E., Visser, I.K.G., Fleming, M., Kai, C., Harder, T.C., Liess, B., Osterhaus, A.D.M.E., Barett, T., 1995. Characterisation of Morbilliviruses isolated from Lake Baikal seals (Phoca sibirica). Vet. Microbiol. 44, 251–259. Olsen, M.T., Andersen, S.M., Teilmann, J., Dietz, R., Edrén, S.M.C., Linnet, A., Härkönen, T., 2010. Status of the harbour seal (Phoca vitulina) in southern Scandinavia. NAMMCO Sci. Publ. 8, 77–94. Reed, L.J., Muench, H., 1938. A simple method of estimating fity per cent endpoints. Am. J. Epidemiol./Am. J. Hyg. 27, 493–497. Reijnders, P.J.H., 1981. Management and conservation of the harbour seal (Phoca vitulina) population in the international Wadden Sea area. Biol. Conserv. 19, 213–221. Reijnders, P.J.H., Brasseur, S.M.J.M., Tougaard, S., Siebert, U., Borchardt, T., Stede, M., 2010. Population development and status of harbour seals (Phoca vitulina) in the Wadden Sea. NAMMCO Sci. Publ. 8, 95–106. Ries, E.H., Hiby, L.R., Reijnders, P.J.H., 1998. Maximum likelihood population size estimation of harbour seals in the Dutch Wadden Sea based on a markrecapture experiment. J. Appl. Ecol. 35, 332–339. Teilmann, J., Rigét, F., Harkonen, T., 2010. Optimizing survey design for Scandinavian harbour seals: population trend as an ecological quality element. ICES J. Mar. Sci. 67, 952–958. Visser, I.K.G., Kumarev, V.P., Örvell, C., de Vries, P., Broeders, H.W.J., van de Bildt, M. W.G., Groen, J., Teppema, J.S., Burger, M.C., UytdeHaag, F.G.C.M., Osterhaus, A.D. M.E., 1990. Comparison of two Morbilliviruses isolated from seals during outbreaks of distemper in North West Europe and Siberia. Arch. Virol. 111, 149–164.