Morbillivirus were isolated from Mediterranean striped dolphins (Stenella coeruleoalba) dying along the coasts of Italy and Greece in 1991. They were antigenically identical to the morbilliviruses isolated from striped dolphins in Spain in 1990

A Aguilar

A. D. M. E. Osterhaus

ADME Osterhaus

BWJ Mahy

C Örvell

C. Örvell

CR Pringle

E. Androukaki

F Black

I. K. G. Visser

IKG Visser

J Kovamees

JM Bompart

JR Heimlich-Boran

K. Siakavara

L. Stanzani

M Domingo

M. -F. Van Bressem

MF Bressem Van

P Vries De

R Ellis

R Lindmark

R. L. De Swart

S Kennedy

Bressem, M.F. van

Visser, I.K.G. (Ilona)

Swart, R.L. (Rik) de

Örvell, C.

Stanzani, L.

Androukaki, E. (Eugenia)

Siakavara, K.

Osterhaus, A.D.M.E. (Albert)

Erasmus University Digital Repository

Arch Virol (1993) 129:235-242 _Archives Virrology © Springer-Verlag 1993 Printed in Austria Dolphin morbillivirus infection in different parts of the Mediterranean Sea Brief Report M.-F. Van Bressem 1, I. K. G. Visser 2, R. L. De Swart 2, C. Orveli 3, L. Stanzani 4, E. Androukaki 5, K. Siakavara 6, and A. D. M. E. Osterhaus 1' 7 1 Division of Virology, Institute ofInfectious Diseases and Immmunology, Veterinary Faculty, State University of Utrecht, Utrecht, 2 Seal Rehabilitation and Research Center, Pieterburen, The Netherlands 3 Department ofVirology, Central Microbiology Laboratory of Stockholm City Council, Stockholm, Sweden 4 Fondazione Cetacea, Riccione, Italy 5 Hellenic Society forthe Study and Protection of the Monk Seal, Athens 6 Institute of Marine Biology ofCrete, Heraklion, Greece 7 Laboratory of Immunobiology, National Institute of Public Health and Envrionmental Protection, Bilthoven, The Netherlands Accepted August 28, 1992 Summary. Morbillivirus were isolated from Mediterranean striped dolphins (Stenella coeruleoalba) dying along the coasts of Italy and Greece in 1991. They were antigenically identical to the morbilliviruses i olated from striped olphins in Spain in 1990. Within the genus Morbillivirus of the family Paramyxoviridae, five different viruses have been recognised, which infect members of different mammalian orders under natural conditions: measles virus (MV) of humans, canine dis- temper virus (CDV) of dogs and other carnivores, rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) of artiodactyls [4, 7]. The fifth member of the genus, phocid distemper virus-1 (PDV-1) [21], caused a devastating epizootic among the harbour seals (Phoca vitulina) of North Western Europe in 1988 [18, 19], PDV-1 proved to be antigenically and genetically closely related to CDV [13, 15, 24]. Since this die-off, morbillivirus infections have been described in other pinniped species and cetaceans [11]. In ceteceans, morbilliviruses were isolated from harbour porpoises (Phocoena 236 M.-F. Van Bressem et al. phocoena) stranded on the Irish 1-12] and Dutch coasts 1-20]. In the summer of 1990, a morbillivirus associated ie-off occurred among the striped dolphins (Stenelta coeruteoalba) of the western part of the Mediterranean Sea [1, 5, 8, 22]. From June to August 1991, a high mortality was observed among striped dolphins along the southern coast of Italy [6]. From September 1991 to January 1992, a similar die-off was noticed among striped olphins in the Greek waters. On the basis of epizootiological data, the mortality was suspected to be the result of an extension of the morbillivirus-associated disease outbreak which previously occurred in the western part of the Mediterranean Sea. We now document the presence of morbillivirus antigen a d morbillivirus- specific serum antibodies in samples from several striped dolphins stranded during this recent die-off. We also report he results of the preliminary antigenic characterization f the morbilliviruses isolated from the lungs of three of these animals. From May to December 1991 organ material was collected from 23 striped dolphins, two common dolphins (Delphinus delphis), one bottlenose dolphin (Tursiops truncatus) and one Cuvier's beaked whale (Ziphius cavirostris) stranded on the coasts of southern Italy and Greece as indicated in Table 1. Morbillivirus antigen was detected by a previously described antigen capture ELISA [22] in lung suspensions of four out of the eleven striped dolphins stranded in Italy, of four out of the eight striped dolphins beached in Greece, and not in organ materials of the bottlenose dolphin and the Cuvier's beaked whale. Morbilliviruses were isolated from the lungs of one of the Greek (D 6) and two of the Italian (D 2, D 4) animals positive in the antigen detection assay. Virus isolation was performed in peripheral blood mononuclear cell(PBMC) cultures of a bottlenose dolphin (TT. PBMC). TT. PBMC were inoculated with 10% suspension prepared from the lungs of four striped dolphins stranded in Italy and two dolphins beached in Greece during the die-off of 1991 (Table 1). They were co-cultivated with Veto cells after 7 days. TT. PBMC and Vero cell monolayers were checked at regular intervals for the development of cytopathic changes. Syncytia were detected in the TT. PBMC cultures inoculated with the lung suspensions of these dolphins within seven days. In the Veto cells co- cultivated with these cultures, a cytopathic effect was subsequently observed within the following ten days. Morbillivirus antigen was detected in cell-free supernatants of all of these cultures, using the antigen capture ELISA. Nu- cleocapsids typical for the members of the family Paramyxoviridae were ob- served in infected cetl-lysates by negative contrast electron microscopy (not shown). In order to assess whether an epizootiological link had existed between the outbreaks in the eastern and western parts of the Mediterranean Sea, the reac- tivities of CDV and PDV-t-specific MoAbs with the morbillivirus i olates were tested in an indirect ELISA [-16, 17]. For preparation of ELISA antigens the isolates from Italy (DMV/Lucca/0691, DMV/Trapani/0891) and Greece (DMV/ Zakinthos/0991) were passaged at least four times in Veto cell monolayer cul- Table 1. Data gathered on materials collected from cetaceans stranded in Italy and Greece Organ Serum Species Sex SL Site of stranding Date Ag Virus ELISA VN material hr. (cm) (month/ detec- isola- Ab Ab nr. year) tion tion titer titer I-D 3 1935 S. coeruleoalba F Lucca, Italy 05/91 - - 20 160 I-D 2 NA S. coeruleoalba F Lucca, Italy 06/91 + + NT NT NA 1951 S. coeruleoalba Reggio Calabria, 08/91 NT NT - 160 Italy I-D 4 2086 S. coeruleoalba M Trapani, Italy 08/91 + + 160 160 NA 2087 S. coeruleoalba F Italy 08/91 NT NT - - I-D 5 NA S. coeruleoalba M 94 Brindisi, Italy 08/91 - - NT NT I-D 6 NA S. coeruleoalba M 201 Lecce, Italy 08/91 - NT NT NT I-D 28 NA S. coeruleoalba M 193 Brindisi, Italy 08/91 + NT NT NT I-D29 NA S. coeruleoalba M 198 Brindisi, Italy 08/91 - NT NT NT I-D 30 NA S. coeruleoalba M 214 Monopoli, Italy 08/91 - NT NT NT I-D 032 NA S. coeruleoalba M 199 Molfetta, Italy 08/91 - NT NT NT I-D 03 NA S. coeruleoalba F Lecce, Italy 08/91 + NT NT NT I-D 04 NA S. coeruleoalba F Lecce, Italy 08/91 - NT NT NT NA 2117 S. coeruleoalba F Ravenna, Italy 04/90 NT NT - - I-D 31 NA T. truncatus M 237 Foggia, Italy 08/91 - NT NT NT NA 2116 D. delphis Cagliari, Italy 07/90 NT NT 80 640 G-D 1 NA S. coeruleoalba F 88 Zakinthos, Greece 08/91 - - NT NT G-D 6 2124 S. coeruleoalba M 164 Zakinthos, Greece 09/91 + + 80 640 G-D 7 2125 S. coeruleoalba M 204 Zakinthos, Greece 09/91 NT NT 80 40 G-D 9 2198 S. coeruleoalba 11/91 - NT 160 320 G-D 10 2199 S. coeruleoalba M Aegina, Greece 11/91 + NT 320 2560 G-D 11 NA S. eoeruleoalba 200 Crete, Greece 12/91 - NT NT NT G-D 12 NA S. coeruleoalba F 140 Crete, Greece 12/91 + NT NT NT G-D 13 2227 S. coeruleoalba M 180 Crete, Greece 12/91 + NT 80 160 G-D 14 NA S. eoeruleoalba 122 Crete, Greece 12/91 - NT NT NT G-BW 1 2126 Z. cavirostris F ~ 500 Alonyssos, Greece 09/91 - NT - < 80* G-D 8 NA D. delphis Crete, Greece 09/91 - NT NT NT O ~,.,o O O t-.) k.o NA Not available, NT not tested, SL standard length, + positive result in the assay, - negative result in the assay, * sample toxic to the cells at lower dilutions, F female, M male 238 M.-F. Van Bressem et al. tures. No  di f ferences were observed between the react iv i ty pat terns  o f  these viruses with the panel  o f  MoAbs  used. The  react iv ity pat terns  o f  the I ta l ian and Greek  isolates were also identical  to the prev ious ly  descr ibed pat terns  o f  the Spanish isolates DMV-16  A and DMV-MUC [20] (Table 2). Morbi l l iv i rus neutra l iz ing ant ibodies  present  in the serum samples were detected in a mic ro -neut ra l i za t ion  assay [23] using about  100 TCIDs0 o f  DMV 16 A and serial two fold di lut ions o f  the serum samples (start ing at 1:40).  Neut ra l i za t ion  titers were expressed as the reciprocals  o f  the d i lut ion still giving complete  reduct ion  o f  cy topath ic  changes. Morbi l l iv i rus  neutra l iz ing serum ant ibodies  were detected in three out  o f  four  str iped do lph ins  which died in I ta ly dur ing the 1991 outbreak ,  in five out  o f  five str iped do lph ins  s t randed in Greece and  in a common do lph in  beached in I ta ly in July 1990. Virus neu- t ra l izat ion titers ranged f rom 40 to 2560 (Table  1). Wi th  the except ion  o f  one serum sample (1951), all samples posit ive in the VN test were also posit ive in an EL ISA  detect ing morbi l l iv i rus specific ant ibodies  [23] on  a coat  o f  DMV 16 A antigen. Morbit l iv i rus-speci f ic  ant ibodies  were not  detected on the serum f rom a Cuvier 's  beaked whale and a str iped do lph in  s t randed in I ta ly in Apr i l  1990 (Table  1). Table 2. Reaction of monoclonal antibodies raised against CDV with the Spanish (DMV 16 A and DMV MUC) and the Italian (DMV/Lucca/0691, DMV/Trapani/0891) and Greek (DMV/Zakinthos/0991) isolates of DMV Designation Specificity CDV DMV Spain DMV Italy DMV Greece of clone (n = 2) (n = 2) (n = 1) 3.662 NP 1 + - - - 3.958 NP 3 + + + NT 3.991 NP 4 + - - - 4.100 NP 5 + - - - 3.568 P 2 + + + NT 3.695 P 3 + - - - 4.051 P 5 + - - - 4.088 P6 + + + + 1.347 H 1 + - - - 2.267 H 2 + - - - 3.734 H 3 + - - - 3.775 H 4 + - - - 4.074 H 5 + - - - 4.275 H 6 + - - - 4.941 H 7 + - - - 3.633 F 1 + + + + 3.697 F 2 + - - - 5.148 F3 + - - - + Reactivity in the assay, - no reactivity in the assay; NT not tested Morbillivirus infection in Mediterranean striped dolphins 239 The detection of morbillivirus antigen in the lungs of several affected striped dolphins and the subsequent isolation of a morbillivirus from capture ELISA positive lung suspensions indicated the role of a morbillivirus infection during the die-off of 1991. Although the DMV isolates originated from striped olphins stranded in different regions, they exhibited the same reaction patterns i  the indirect ELISA with a set of MoAbs raised against CDV and PDV-1 (Tables 2 and 3). These patterns were also identical to those of the two morbilliviruses isolated uring the outbreak among striped dolphins in the western part of the Mediterranean Sea [20]. This indicates the existence of an epizootiological link between this outbreak and a similar morbillivirus-related die-off which occurred in the same species, in the western part of the Mediterranean Sea in 1990. The detection of morbillivirus-specific antibodies in serum samples from several striped dolphins further confirmed the circulation of a morbillivirus in these populations. The presence of VN serum antibodies in absence of ELISA antibodies in one of the sera, may be explained by the fact that the morbillivirus- specific IgM antibodies produced in the acute stage of the infection, were probably not recognized by protein-A used in the ELISA [14]. Furthermore, Table 3. Reaction of monoclonal ntibodies raised against PDV-I with the Spanish (DMV 16 A and DMV MUC) and the Italian (DMV/Lucca/0691, DMV/Trapani/0891) and Greek (DMV/Zakinthos/0991) isolates of DMV Designation Specificity PDV-1  DMV Spain DMV Italy DMV Greece of clone (n = 2) (n = 2) (n = 1) 1.064 C 5 NP 1 + 1.071 E 2 NP 1 + 1.069 G 2 NP 2 + 1.062 G 5 H 1 + 1.063 C 3 H 1 + 1.063 E 9 H 1 + 1.069 D 9 H 1 + 1.071 E5 H 1 + t.067 E 5 H 2 + 1.068 F 2 H 3 + 1.070 B 5 H 4 + 1.072 C 4 H 4 + 1.085 C 4 H 5 + 1.t22D 11 H6 + i m m b i m m h m m m m m m i 1.062 E 2 F 1 + - - - 1.068 B 2 F 1 + - - - 1.067 D 2 F 2 + + + + 1.073 G 10 F 3 + + + + 1.092 C 3 F 3 + + + + + Reactivity in the assay, - no reactivity in the assay 240 M.-F. Van Bressem et al. the relatively low levels of antibodies demonstrated in the ELISA vs. VN antibodies may have been caused by a limited reactivity of protein A for Ig of cetacean origin. The presence of morbillivirus neutralizing and ELISA-reactive antibodies in a common dolphin indicated that this animal had also suffered from a morbillivirus infection. Unfortunately, from this animal no materials were available for antigen detection or virus isolation. The limited number of samples from other cetacean species makes it difficult to conclude whether they are also susceptible to DMV infection. Our success in isolating DMV in lym- phocytes from a bottlenose dolphin suggests that other cetacean species could be infected by DMV. However, although the striped dolphin can be seen swim- ming together with other cetaceans like the common dolphin [9] and the short- finned pilot whale (Globicephala macrorhynchus) [10], the die-off was so far only reported to affect the endangered Mediterranean striped dolphin [3]. Besides a possible higher susceptibility o DMV infection and DMV associated disease development, the tendency of the striped dolphin to form groups of dozens of animals [2] may have favoured the spread of the infection in this species. The high mortality rate during the die-off indicates that DMV had entered a non-immune population [4]. Speculations that the recent morbillivirus related mortality in striped ol- phins was the result of transmission of PDV-1 from seals to dolphins can now be contradicted on the basis of the antigenic differences between these two viruses. Also the natural tendency of the morbillivirus to be confined to one mammalian order, and the biological differences shown between PDV-1 and DMV [20] indicate that there has been no etiological link between the PDV- 1 and DMV related outbreaks. The identification f at least two new members of the genus Morbillivirus causing devastating outbreaks among aquatic mammals raises questions about the origin of these viruses, the reason for their sudden appearance and their antigenic or genetic relationships with the other members of the genus. These questions are subject of our present investigations in this field. Acknowledgements We kindly acknowledge J. S. Teppema for the lectron microscopy, R. Basso, A. Bortolotto, L. Casini, A. Di Natale, G. Gnone, M. G. Hartmann, the Italian Centre for Cetacean Studies, the Italian Merchant Ministry, V. Kouroutos, L. Lino, C. Meotti, E. Tounta, L. Vedder and V. Zavras, for their help in collecting the samples, Dr. P J. H. Van Bree for the identification of the Cuvier's beaked whale, A. Vecchione forher appreciated assistance during the processing of the samples, and C. Kruyssen for help in preparing the manuscript. References 1. Aguilar A (1990) Dolphin die-off in the Western Mediterranean. 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Dolphin morbillivirus infection in different parts of the Mediterranean Sea

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Dolphin morbillivirus infection in different parts of the Mediterranean Sea

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