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Veterinary Pathology Online

http://vet.sagepub.com/content/early/2010/01/28/0300985809358036The online version of this article can be found at:

DOI: 10.1177/0300985809358036

published online 31 December 2009Vet PatholKennedy, F. Forster, B. Iulini, E. Bozzetta and C. Casalone

G. Di Guardo, U. Proietto, C.E. Di Francesco, F. Marsilio, A. Zaccaroni, D. Scaravelli, W. Mignone, F. Garibaldi, S.Coast of Italy

Cerebral Toxoplasmosis in Striped Dolphins (Stenella coeruleoalba) Stranded Along the Ligurian Sea

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Wildlife

Cerebral Toxoplasmosis in Striped Dolphins(Stenella coeruleoalba) Stranded Along theLigurian Sea Coast of Italy

G. Di Guardo1, U. Proietto1, C. E. Di Francesco1, F. Marsilio1,

A. Zaccaroni2, D. Scaravelli2, W. Mignone3, F. Garibaldi5,

S. Kennedy6, F. Forster6, B. Iulini4, E. Bozzetta4, and C. Casalone4

Abstract

This article reports the results of necropsy, parasitologic, microbiologic, histopathologic, immunohistochemical, indirect

immunofluorescence, biomolecular, and serologic investigations on 8 striped dolphins (Stenella coeruleoalba) found stranded

from August to December 2007 on the Ligurian Sea coast of Italy. Severe, nonsuppurative meningoencephalitis was found in4 animals, as characterized by prominent perivascular mononuclear cell cuffing and macrophage accumulations in neuropil.

These lesions were associated with mild lymphocytic–plasmacytic infiltration of choroid plexuses in 1 dolphin. Toxoplasma

gondii cysts and zoites, confirmed by immunohistochemical labeling, were scattered throughout the brain parenchyma of 2 of

the 4 dolphins. No viral inclusions were seen in the brain of any animal. Other findings included severe bronchointerstitial

pneumonia and pulmonary atelectasis, consolidation, and emphysema. Parasites were identified in a variety of organs, including

lung (Halocerchus lagenorhynchi). Microbiologic and serologic examinations for Brucella spp were negative on all 8 dolphins. The

4 animals with meningoencephalitis had serum antibodies against T gondii (titers ranging from 1:80 to 1:320) but not against

morbillivirus. In contrast, the other 4 dolphins were seropositive for morbillivirus (with titers ranging from 1:10 to 1:40) butseronegative for T gondii. No morbillivirus antigen or nucleic acid was detected in the tissues of any dolphin. It is concluded

that the severe lung and brain lesions were the cause of death and that T gondii was the likely etiologic agent of the cerebral

lesions. Morbillivirus infection was not considered to have contributed to death of these animals.

Keywords

striped dolphins, pathology, morbillivirus, Toxoplasma gondii, Brucella spp, Italy

Toxoplasma gondii, a protozoan agent infecting a range of mam-

malian species worldwide, has been implicated as a cause of

abortion and lethal systemic disease in several sea mammal spe-

cies.11Although the potential of this protozoan to affect cetacean

populations has not been thoroughly investigated thus far, a

number of reports clearly suggest that T gondii infection is of

potential concern to cetacean health and conservation.9,11,32

T gondii is commonly believed to be an opportunistic patho-

gen for aquatic mammals,24,26 as was the case in striped dolphins

(Stenella coeruleoalba) during the dolphin morbillivirus (DMV)

epidemic in the Mediterranean Sea from 1990 to 1992.7,21

Between late 2006 and early 2007 another morbillivirus

epidemic was reported in pilot whales (Globicephala melas)

in the Mediterranean Sea near Gibraltar12 and, in the following

months, in pilot whales and striped dolphins along the Spanish

coast.28 Apart from its less dramatic scale, this more recent

event shared many similarities with the Mediterranean striped

dolphin die-off in this area from 1990 to 1992.6,21 Nucleotide

sequence analysis indicated that the DMV strain causing the

recent mortality episodes in pilot whales and striped dolphins

is closely related to that involved in the 1990–1992 die-off.12,28

1Department of Comparative Biomedical Sciences, Faculty of Veterinary

Medicine, University of Teramo, Teramo, Italy2Department of Veterinary Public Health and Animal Pathology, Faculty of

Veterinary Medicine, University of Bologna, Ozzano Emilia, Bologna, Italy3 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta,

Imperia, Italy4 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta,

Torino, Italy5Dip.Te.Ris., University of Genova, Genova, Italy6Agri-Food and Biosciences Institute for Northern Ireland, Stormont, Belfast,

Northern Ireland

Corresponding Author:

Giovanni Di Guardo, DVM, Dipl ECVP, Department of Comparative

Biomedical Sciences, Faculty of Veterinary Medicine, University of Teramo,

Piazza Aldo Moro, 45-64100, Teramo, Italy

Email: [email protected]

Veterinary Pathology

000(00) 1-9

ª The Author(s) 2010

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DOI: 10.1177/0300985809358036

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Veterinary Pathology OnlineFirst, published on January 29, 2010 as doi:10.1177/0300985809358036



As in the earlier event,4,5,21 unusually high striped dolphin

mortality was recorded along the coast of Italy during

this recent morbillivirus epidemic, including the Ligurian

coast of northwestern Italy from August to December 2007

(F. Garibaldi and W. Mignone, personal communication). The

aim of this study was to investigate possible causes of death of

these animals, with emphasis on four cases of T gondii infection.

Materials and Methods

The studywas carried out on 8 striped dolphins (Table 1) that had

been found dead along the Ligurian Sea coast of Italy (Imperia

province) from August to December 2007 (Fig. 1). The annual

mean number of cetaceans found stranded during the previous

22 years (1985–2006) in this region is 4, with S coeruleoalba

being the most commonly stranded species. The 2007 stranding

figures show an increase over previous years (Fig. 2).

At necropsy, tissue samples were collected from a range of

organs (lung, heart, stomach, intestine, liver, pancreas, kidney,

adrenal gland, testis, ovary, uterus, spleen, mesenteric, tracheo-

bronchial and pulmonary lymph nodes, skeletal muscle, and

skin), along with the entire brain from each animal. Tissues

were fixed in 10% neutral-buffered formalin or stored at 4�C

and –20�C until used. At necropsy, a blood sample was col-

lected from the heart chambers of each dolphin, and serum was

removed by immediate centrifugation at 1,000 to 1,500 revolu-

tions per minute for 15 minutes.

All tissues were embedded in paraffin wax, cut into 5-mm-

thick sections, and stained with hematoxylin and eosin for light

microscopic examination. Systematic tissue sampling was car-

ried out on the brain of each animal with sections prepared

from the telencephalon, diencephalon, mesencephalon, pons,

cerebellum, and medulla oblongata. Periodic acid–Schiff

(PAS) histochemical staining was performed on selected brain

sections, whereas Gram staining was carried out on brain slices

from 2 of the 8 dolphins under study.

Immunohistochemical and Indirect

Immunofluorescence Labeling

Immunohistochemical (IHC) labeling of T gondii was carried

out on brain and lung tissues from all dolphins. Antigen retrie-

val was performed by exposing poly-L-lysine-coated slides to a

Tris buffer solution (pH 7.6) containing 0.1% trypsin and 0.1%

calcium chloride. Endogenous peroxidases were inhibited by

transfering sections to a solution of hydrogen peroxide in

methanol, and nonspecific antigen binding sites were blocked

by applying normal horse serum. Sections were then incubated

with a commercially available goat polyclonal anti–T gondii

antiserum solution (1:500; VMRD Inc, Pullman, WA). A sec-

ondary biotinylated horse anti-goat immunoglobulin G (IgG)

solution (Sigma-Aldrich Inc, Saint Louis, MO) was applied

to sections, followed by signal amplification with avidin-

biotin-conjugated peroxidase. The reaction was visualized by

means of 3,30-diaminobenzidine (DAB) chromogen solution

(Sigma-Aldrich Inc), followed by counterstaining with Mayer

hematoxylin.

T gondii–infected ovine brain and canine lung were used as

positive control tissues. For negative controls, the IHC tech-

nique was applied to these tissues but with omission of the pri-

mary antiserum or to sections of normal or pathologically

unrelated ovine, canine, and dolphin tissues.

Indirect immunofluorescence (IIF) and IHC labeling for

morbillivirus antigen was carried out on brain, lung, spleen,

and lymph nodes from each animal. For IHC staining, antigen

retrieval was performed by exposing poly-L-lysine-coated

slides to a citrate buffer solution (pH 6). Endogenous per-

oxidases were inhibited by transferring sections to a solution

of hydrogen peroxide in methanol, and nonspecific antigen

binding sites were blocked by applying normal goat serum.

Sections were subsequently incubated with a commercially

available mouse monoclonal antibody (MoAb) solution

(1:500) against canine distemper virus (CDV) nucleoprotein

antigen (VMRD Inc) that recognizes the same epitope from

different members of the Morbillivirus genus, including

DMV.28,34 A secondary biotinylated goat anti-mouse IgG solu-

tion (DAKO Corp, Carpinteria, CA) was applied to sections,

followed by signal amplification with avidin-biotin-

conjugated peroxidase. The reaction was visualized by means

of DAB chromogen solution (Sigma-Aldrich Inc), and slides

were counterstained with Mayer hematoxylin.22,30 For IIF

labeling of morbillivirus antigen, the same primary MoAb

(1:100 dilution) was applied to tissue sections, followed by

incubation for 3 hours with a goat anti-mouse IgG secondary

antibody conjugated with fluorescein isothiocyanate (FITC)

(KPL Inc, Gaithersburg, MD) and observation under a Zeiss

Axiophot fluorescence microscope.

For both IHC and IIF staining, morbillivirus-positive brain,

lung, spleen, and lymph node samples were used as control tis-

sues. These included CDV-infected dog tissues, DMV-infected

striped dolphin tissues, and phocine (phocid) distemper virus–

infected common seal tissues. For negative controls, the IHC

and IIF techniques were carried out on uninfected striped

dolphin tissues and on tissues from known DMV-infected

striped dolphins but with omission of the primary MoAb.

Microbiology

Detailed microbiologic investigations were carried out on brain

and lung tissues of all 8 dolphins. Representative tissue

Table 1. Details of the 8 Striped Dolphins

Dolphin No. Sex Age Class Date of Stranding

1 M Newborn 22 August 20072 F Adult 10 September 20073 F Adult 22 September 20074 F Subadult 25 September 20075 F Adult 4 November 20076 F Adult 18 November 20077 F Adult 1 December 20078 M Adult 6 December 2007

2 Veterinary Pathology 000(00)

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samples were cultured onto 5% blood agar and subsequently

incubated at 37�C for 48 hours under aerobic conditions (brain

and lung) or microaerobic (5% CO2) conditions (brain only).

Brain was also cultured on the following selective media

under both aerobic and microaerobic conditions: Gassner agar,

chocolate agar, Saboraud agar, Brucella agar, and Thayer-

Martin-modified agar. All inoculated media were incubated

at 37�C for 48 hours, except for Brucella-selective agar plates

(which were incubated at 37�C for 10 days under microaerobic

conditions) and Saboraud agar dishes (which were incubated

for 10 days at room temperature). All cultures were checked

daily.

Bacterial colonies were identified by means of commercial

API galleries (bioMerieux Inc, Durham, NC). Suspect fungal

colonies were subsequently transfered onto additional Saboraud

agar plates and identified by means of the rRNA 16s Microseq

500 sequencing kit (Applied Biosystems, Foster City, CA). Iden-

tification was based on amplification of a 500-base-pair frag-

ment from the 16s fungal ribosomal subunit2,15,23 and the D2

fragment from the large subunit (LSU) of fungal rRNA.16,17

Molecular Biology

A reverse transcription polymerase chain reaction (RT-PCR)

technique with morbillivirus-specific genomic sequences was

applied to tissue extracts from different regions of brain from

all 8 dolphins. In this technique, 2 nucleoprotein gene

fragments of 287 and 575 base pairs were selected as targets

based on the universal morbillivirus primers P1 (50-ACA-

GGATTGCTGAGGACCTAT-30) and P2 (50-GCACCG-

TACATGGTTATCTTG-30) (GenBank accession No.

EF451565),13,34 as well as the DMV-specific primers

DMV_REV (50-AGATGGGCGAGACTGCACC-30) and

DMV_FW (50-ATCAGGGCTCACTTTTGCATCCAGA-30)

(GenBank accession No. NC005283). Total RNA was

extracted from 25 mg of each tissue sample by means of the

RNeasy kit (Qiagen GmbH, Hilden, Germany) according to the

manufacturer’s instructions. The assay was subsequently per-

formed in 25 ml of reaction mixture containing 1X PCR Master

Mix (Invitrogen Corporation, Carlsbad, CA), 10 U of RNase

inhibitor, 50 U of MuLV RT (Applied Biosystems),

100 pmol/ml of each primer, and 2 ml of RNA. RT-PCR was

carried out in a single-step reaction of 60 minutes at 48�C, fol-

lowed by reverse transcriptase denaturation at 95�C for 2 min-

utes. Amplification was conducted by means of 35 cycles at

94�C for 45 seconds, at 48�C for 45 seconds, and at 72�C for

60 seconds followed by 7 minutes of final extension at 72�C.

Brain tissue extracts from a CDV-infected dog and a

DMV-infected striped dolphin were used as positive control

materials for the RT-PCR technique.

Serology

The sera from the 8 dolphins under study were evaluated by an

immunofluorescence antibody test (IFAT) for the detection of

anti–T gondii antibodies. A commercially available kit contain-

ing killed T gondii tachyzoites as substrate antigen and feline

T gondii–positive and T gondii–negative control sera (Fuller

Laboratories, Fullerton, CA) was used in this method. Briefly,

10 ml of serial twofold diluted sera, starting at 1:10, was added

to 12-well slides. After 30 minutes of incubation at 37�C, a

mixture of rabbit anti–bottlenose dolphin IgG (1:5000) was

added for another 30 minutes at 37�C. The reaction was visua-

lized by means of FITC fluorochrome-conjugated anti-rabbit

IgG, and slides were observed under a Zeiss Axiophot fluores-

cence microscope. As in a study that compared various serol-

ogic tests for T gondii,8 a cutoff titer of 1:40 was established,

and endpoint dilution titers of positive sera were determined.

A virus neutralization assay to detect anti-morbillivirus

antibodies was also performed, using the Onderstepoort strain

of CDV. Briefly, 50 ml of each serial twofold diluted serum,

starting at 1:5 dilution, and 50 ml of infectious culture medium

containing 100 TCID50 of the virus were mixed and incubated

in 96-well flat-bottomed plates for 1 hour at 37�C. One hundred

microliters (1� 105 cells/ml) of Vero cell suspension were then

added to each well. Cultures were finally incubated for 3 days

at 37�C with 5% CO2. Seropositivity was defined as a titer of 1

� 20.31,33

Finally, all sera were submitted to a rapid serum agglutina-

tion test for anti–Brucella spp antibodies based on Brucella

abortus and B melitensis antigens.19,29

Results

Table 2 summarizes the main pathologic findings in the 8 ceta-

ceans under study. All carcases were well preserved, and some

of the animals were in suboptimal nutritional status, as

indicated by reduced blubber thickness. Gross evidence of

meningeal hyperemia and brain edema was seen in 4 dolphins

(3 females and 1 male) (Fig. 3). Two of these animals had large

numbers of Phyllobothrium delphini andMonorygma grimaldii

larval cysts in the subcutaneous tissue of perineal region and

ventrolateral abdominal musculature, respectively (Table 3).

Six animals (75%) had severe bilateral bronchointerstitial

pneumonia, characterized by partial lung collapse, extensive

areas of consolidation, and prominent subpleural emphysema

Figure 1. Geographic area (Imperia province, Ligurian Sea) in whichthe 8 striped dolphins under study were found stranded.

Di Guardo et al 3



(particularly in the apical regions of both lungs). Adult

nematodes, morphologically consistent with Halocerchus

lagenorhynchi (formerly H delphini), were present in the

bronchial lumina of these dolphins (Fig. 4) and were associated

with moderate catarrhal exudate and frothy fluid, indicative of

pulmonary edema.

Severe multifocal, subacute to chronic, nonsuppurative

meningoencephalitis was present in 1 male and 3 female

adult dolphins (50%). These lesions mainly involved the

pons and mesencephalon and consisted of prominent peri-

vascular mononuclear cell cuffing and focal microglial

nodules scattered throughout the surrounding neuropil

(Fig. 5). Macrophage accumulations in proximity to blood

vessels and mild lymphoplasmacytic infiltration of choroid

plexuses were also seen in the adult male dolphin. A micro-

angiopathy in which the wall of affected vessels had an

‘‘onionlike’’ appearance was seen in all 4 of these animals.

Astrogliosis and mild to moderate neuronal degeneration

Figure 2. The number of cetaceans found stranded in the study area from 1985 to 2007, with striped dolphin mortality peaks in 1990 (blackbar) and 2007 (white bar) as a result of the dolphin morbillivirus epidemic.

Table 2. Nutritional Status, Gastric Content, and Main Lesions in the 8 Striped Dolphinsa

Dolphin No. Nutritional Status Gastric Content MENG BP VB Extrapulmonary Parasitic Lesions

1 Good Partially digested milk – þ þ –2 Poor None – þ þ Parasitic nodules in subcutis and abdominal

musculature; parasitic hepatitis3 Good Partially digested fish – þ þ –4 Good Partially digested fish – þ – –5 Good Partially digested fish þ – – Parasitic hepatitis and pancreatitis6 Poor None þ – þ Parasitic nodules in subcutis and abdominal

musculature; parasitic hepatitis and pancreatitis7 Poor None þ þ þ Parasitic hepatitis and pancreatitis8 Good Partially digested fish þ þ þ Parasitic hepatitis and pancreatitis

a MENG, meningoencephalitis; BP, bronchointerstitial pneumonia; VB, verminous bronchopneumonia; lesions present (þ), lesions absent (–).




and necrosis characterized by vacuolation, chromatolysis,

and shrinkage were also observed. Single, round, basophilic

PAS-positive structures morphologically consistent with

T gondii cysts (Fig. 6) were scattered throughout the brain

parenchyma of 2 of these dolphins (Table 3). Viral inclusions

were not seen in the brain of any animal, and no bacteria were

apparent in Gram-stained brain tissue of 2 animals.

Microscopic lung lesions were characterized by seromu-

cous exudation and macrophage infiltration of bronchial,

bronchiolar, and alveolar lumina and by the presence of occa-

sional double- to triple-nucleated alveolar syncytia. Lesions

of severe chronic interstitial pneumonia, as characterized by

lymphoid cell infiltration, extensive alveolar atelectasis,

and marked septal fibrosis, were present in these animals

(Figs. 7, 8). Parasitic granulomas were found scattered

throughout both lungs.

Mild to moderate, multifocal, subacute to chronic lesions of

nonsuppurative portal hepatitis were seen in 4 female and

1 male adult dolphins (62%). Four of these animals (50%) also

had severe pancreatitis (Table 2), characterized by an infiltrate

of lymphocytes, plasmacytes, and eosinophils and by the pres-

ence of extensive hemorrhagic and necrotic foci. Many

metazoan parasites and ova, morphologically consistent with

Campula spp, were observed in the hepatic and pancreatic

(Fig. 9) parenchyma and ductal lumina of all 5 of these dol-

phins (Table 3).

No other significant microscopic findings were observed in

any animal.

Immunohistochemistry and Indirect

Immunofluorescence

Immunohistochemical labeling of T gondii cysts and zoites was

seen scattered throughout the brain tissue of 2 dolphins that had

meningoencephalitis (Fig. 10). T gondii antigen was not

identified in any tissue from any of the other 6 dolphins. Immu-

nohistochemical and IIF labeling for morbilliviral antigen was

negative on all dolphins.

Microbiology

Several bacteria were isolated from the lung tissue of 3 dolphins

and the brain tissue of 2 dolphins (Table 3). No Brucella spp or

fungi were recovered from any animal.

Molecular Biology

Morbillivirus nucleic acid was not detected in the tissues of any

dolphin.

Serology

The sera from the 4 dolphins that had severe lesions of

meningoencephalitis were positive for anti–T gondii antibo-

dies, at titers ranging from 1:80 to 1:320, but did not contain

anti-CDV neutralizing antibodies. Conversely, anti-CDV

neutralizing antibodies at titers ranging from 1:10 to 1:40 were

detected in the sera of the 4 dolphins that did not have menin-

goencephalitis. No anti–T gondii antibodies were detected in

sera of these animals. Antibodies to B abortus or B melitensis

were not detected in any animal.

Discussion

All the dolphins investigated had parasitic infection and asso-

ciated lesions in either lung or in liver, pancreas, subcutis, and

abdominal skeletal musculature. Four (50%) had severe

nonsuppurative meningoencephalitis and 6 (75%) had severe

chronic bilateral pneumonia. These lesions almost certainly

contributed to death, and their frequencies were similar to those

previously reported in stranded cetaceans.5 In this respect, the

Figure 3. Brain; striped dolphin, adult female (No. 5). Marked congestion of meningeal vessels in animal that had nonsuppurativemeningoencephalitis. Figure 4. Lung; striped dolphin (No. 3). Adult nematodes morphologically consistent with Halocerchus lagenorhyinchi

(arrows) within bronchial lumina of an adult female that had bilateral bronchopneumonia.

Di Guardo et al 5



Figure 5. Brain; striped dolphin, adult female (No. 5). Prominent perivascular cuffing with mononuclear cells and microglial cell nodules inneuropil. HE. Bar ¼ 100 mm. Figure 6. Brain; striped dolphin, adult female (No. 5). A single basophilic structure morphologically consistentwith a mature Toxoplasma gondii cyst in thalamus (arrow). HE. Bar ¼ 55 mm. Figure 7. Lung; striped dolphin, adult female (No. 2). Evidence ofchronic lymphoid interstitial pneumonia. HE. Bar ¼ 200 mm. Figure 8. Lung; striped dolphin, adult female (No. 2). Extensive consolidation withmarked alveolar atelectasis and prominent septal fibrosis. HE. Bar ¼ 100 mm. Figure 9. Pancreas; striped dolphin, adult female (No. 6). Anadult parasite morphologically consistent with Campula spp in perilobular interstitium with associated hemorrhage, necrosis, and inflammatorycell infiltration. HE. Bar ¼ 100 mm. Figure 10. Brain; striped dolphin, adult female (No. 5). Immunohistochemical labeling of Toxoplasma gondiicyst (large arrow) and zoites (small arrows) in thalamus. Mayer hematoxylin counterstain. Bar ¼ 50 mm.

Table 3. Microbiologic and Parasitologic Findings in the 8 Striped Dolphinsa

MorbillivirusToxoplasma

gondii Brucella spp

DolphinNo. IHC IIF RT-PCR SN IFAT Culture RSA

Other PathogenicBacteria Lung Parasites Extrapulmonary Parasites

1 Neg Neg Neg 1:10 Neg Neg Neg Neg Halocerchus

lagenorhynchi

Neg

2 Neg Neg Neg 1:40 Neg Neg Neg Staphylococcus

hominis (lung)H lagenorhynchi Phyllobothrium delphini

(subcutis), Monorygma grimaldii

(abdominal musculature),Campula spp (liver)

3 Neg Neg Neg 1:20 Neg Neg Neg Neg H lagenorhynchi Neg4 Neg Neg Neg 1:20 Neg Neg Neg Neg H lagenorhynchi Neg5 Neg Neg Neg Neg 1:320 Neg Neg Aeromonas salmoni-

cida (lung)Neg Campula spp (liver, pancreas),

Toxoplasma gondii cysts and zoites(brain)

6 Neg Neg Neg Neg 1:80 Neg Neg Enterococcus

casseliflavus (brain)Neg P. delphini (subcutis), M. grimaldii

(abdominal musculature), Campulaspp (liver, pancreas)

7 Neg Neg Neg Neg 1:320 Neg Neg Streptococcus

parasanguinis

(brain), S gordoni

(brain)

H lagenorhynchi Campula spp (liver, pancreas),T gondii cysts and zoites (brain)

8 Neg Neg Neg Neg 1:320 Neg Neg E faecalis (lung) H lagenorhynchi Campula spp (liver, pancreas)

a IHC ¼ immunohistochemistry; IIF ¼ indirect immunofluorescence, RT-PCR ¼ reverse transcriptase-polymerase chain reaction; SN ¼ serum neutralization;IFAT ¼ immunofluorescence antibody test; RSA ¼ rapid serum agglutination.




suboptimal nutritional status observed in some of the present

dolphins is consistent with a subacute to chronic disease

process.

The 4 dolphins that had lesions of meningoencephalitis had

high serum titers of anti–T gondii antibodies, and histologic

and immunohistochemical evidence of T gondii cysts and

zoites was found in the brains of these animals. In a previous

study in bottlenose dolphins, anti–T gondii antibody titers

(detected via the modified agglutination test) were confirmed

by IFAT and 1:40 identified as the prevalent positive titer.8Our

serologic data were obtained by means of IFAT, which has

been validated as a confirmatory assay for T gondii infection

in sea otters (Enhydra lutris nereis),26 a species in which T gon-

dii meningoencephalitis is a common finding.3 Toxoplasma

infection, which appears to be common in some cetacean

populations, including bottlenose dolphins from the coasts of

Florida, South Carolina, and California,9,11 has recently been

linked to contamination of coastal waters with oocysts.9,27 The

epidemiology of T gondii infection of offshore species such as

Risso’s dolphins (Grampus griseus) and striped dolphins is

more difficult to explain but has been speculatively linked to

the release of ship ballast waters contaminated by onboard

rodents, cats, or contaminated soil.32 Although T gondii is

commonly regarded as an opportunistic pathogen in aquatic

mammals,24,26 as clearly documented in striped dolphins dur-

ing the 1990–1992 Mediterranean Sea DMV epidemic,7,21 the

present findings suggest that T gondii had a primary etiologic

role in the meningoencephalitis seen in the seropositive animals

under study. A similar role has been suggested for captive and

feral pinniped and cetacean species.9,10,25 This hypothesis is sup-

ported by the negative results of all laboratory investigations for

morbillivirus in these 4 animals, as well as by the negative

results of the serologic and microbiologic investigations in all

8 dolphins for Brucella infection, which has been associated

with meningoencephalitis in cetaceans, including striped dol-

phins.14,19 The severe brain lesions in the present dolphins dif-

fered from those associated with Brucella infection in that

meningitis was less prominent. In contrast, the severity of ence-

phalitis was greater than that reported in Brucella-infected ceta-

ceans.14,19Relatively high concentrations of immunotoxic heavy

metals were measured in tissues from the liver (mercury, lead)

and the kidney (cadmium) from the 8 dolphins under study.35

However, it is not known if these metals had any etiologic role

in the lesions found in these animals.

Enterococcus casseliflavus was recovered from the brain tis-

sue of 1 dolphin that had meningoencephalitis, and Strepto-

coccus parasanguinis and S gordoni were recovered from

another. Although no information is available on the pathogeni-

city of these bacteria in sea mammals, E casseliflavus has been

recently implicated as a cause of meningitis in human beings.20

No microscopic alterations compatible with a microbial etiology

were observed, and Gram staining did not reveal the presence of

any bacteria in the cerebral tissue from these animals; as such,

perimortem bacterial colonization seems likely.

The 8 striped dolphins under investigation were found

stranded in a localized geographic area and over a limited

period during which there was a considerable increase

in reported strandings in that region (F. Garibaldi and

W. Mignone, personal communication). These observations

strongly support the hypothesis of a common source exposure

as the cause of the increased striped dolphin mortality during

2007 in the Ligurian Sea. A similar pattern occurred in 2007

along the Spanish coastline in the western Mediterranean,

where dozens of striped dolphins and several pilot whales died

as a result of a DMV epidemic.12,28 Morbillivirus infection was

also recently reported in the brain tissue of a young striped dol-

phin found stranded in June 2007 on the northwestern Sardi-

nian coast of Italy.1 Again in 2007, a mass die-off of

common seals (Phoca vitulina) and harbour porpoises (Pho-

coena phocoena) occurred along the regions of the Danish and

Swedish coasts in which two major morbilliviral epidemics

occurred in 1988 and 2002, respectively. The cause of that

recent die-off is unknown.18

Anti-morbillivirus antibodies were detected in the sera of

4 dolphins investigated in this study. Because the serologic test

did not permit discrimination between immunoglobulin M and

IgG antibodies, the timing of morbillivirus infection could not

be determined. However, neither morbilliviral antigen nor

nucleic acid was detected in the tissues of these dolphins, and

the tissue lesions were not consistent with those of morbilli-

virus infection.6,21

In conclusion, (1) the high antibody titers against T gondii in

the 4 dolphins that had lesions of meningoencephalitis, (2) the

presence of T gondii cysts and zoites in the brain of 2 of these

animals, and (3) the absence of concurrent morbillivirus and

Brucella spp infections support the hypothesis that this proto-

zoan played a primary etiologic role in the development of the

severe meningoencephalitis. The hypothesis that these lesions

almost certainly caused the death and subsequent stranding of

these animals is also supported by the lack of anti-Toxoplasma

antibodies in the 4 dolphins that had no cerebral lesions.

Declaration of Conflict of Interest

The authors declared that they had no conflicts of interest with respect

to their authorship or the publication of this article.

Financial Disclosure/Funding

This work was carried out with a research grant (Progetto di Ricerca

Cause di Mortalit�a e Studi Patogenetici in Cetacei Spiaggiati sulle

Coste Italiane) from Ministero dell’Ambiente e della Tutela del

Territorio e del Mare, Italy.

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