Invasive species and their parasites: eastern cottontail rabbit Sylvilagus floridanus and Trichostrongylus affinis (Graybill, 1924) from Northwestern Italy.

The eastern cottontail Sylvilagus floridanus is a native American lagomorph. Within the genus Sylvilagus, the eastern cottontail is the species with the widest distribution. From 1950s, the species was introduced to several European countries. A rapid territorial expansion of the introduced eastern cottontails has been observed in many areas of Italy. The eastern cottontail has been demonstrated to play a main role as carrier of exotic parasites. To date, three nematode species, exotic in Italian ecosystems, have been reported from introduced S. floridanus. However, its parasite fauna biodiversity is richer in native populations of the American continent. The aim of this work was to further investigate the gastrointestinal parasites of S. floridanus, to evaluate the potential presence of other exotic species. During 2010, 101 hosts were examined, and three nematodes were collected from their digestive tract. Two parasite species (Obeliscoides cuniculi, Trichostrongylus calcaratus) were already reported in Italy; the isolation of Trichostrongylus affinis is instead the first report of this nematode in Italy and in Europe as a whole. This study wants to highlight the great risks related to the introduction of allochthonous species. The impact of the invasion by alien animal species may be particularly severe for public and animal health, due to the potential introduction of new pathogens. The good number of exotic parasites found in introduced eastern cottontails, together with the few sanitary surveys carried out, suggests that an epidemiological survey, with specimens from multiple localities on a wider geographic range, could lead to interesting findings on parasites of native and alien lagomorphs in Europe.

Causes of adult mortality in two populations of New Zealand sea lions (Phocarctos hookeri).

The New Zealand sea lion is an endangered species endemic to New Zealand. While causes of death are well described for pups of this species, mortality in adults is poorly characterised. This study investigated causes of death in 136 New Zealand sea lions in two different populations: a major breeding site on remote, uninhabited Enderby Island in the sub-Antarctic, and a slowly increasing recolonising population on the inhabited mainland. For animals with at least a partial diagnostic investigation (n = 112), the most frequently diagnosed causes of mortality were infectious disease (41/112; 37%), particularly tuberculosis due to M. pinnipedii (20/112; 18%), and conspecific trauma (27/112; 24%). Anthropogenic trauma was an important cause of death in mainland sea lions (9/33; 26%). Deliberate anthropogenic mortality has previously been identified as the greatest potential threat to population recovery for mainland sea lions, and as human and pinniped populations increase, managing interactions between these species will become increasingly important.

Humoral immune response to Klebsiella spp. In New Zealand sea lions (Phocarctos hookeri) and the passive transfer of immunity to pups.

During the 2001-02 and 2002-03 breeding seasons, epizootics of Klebsiella pneumoniae resulted in a dramatic increase of pup mortality in New Zealand sea lions (Phocarctos hookeri; NZSLs) on Enderby Island (Auckland Islands). To estimate the prevalence of infection in the NZSL population, a serologic test was developed using a Western blot and a polysaccharide antigen derived from a K. pneumoniae isolate from a NZSL pup. All archived serum samples collected between 1997 and 1998 and 2004 and 2005 at Sandy Bay Beach rookery, Enderby Island, were tested (314 pups and 302 adult females). Anti-Klebsiella antibodies were detected throughout this period, but overall, only 16% of NZSL pups between birth and 5 mo of age were seropositive compared with 95.7% of adults. There was no apparent change in antibody prevalence as a result of the two epizootics. A method to determine total immunoglobulin G (IgG) levels in sea lion serum also was developed to investigate passive immunoglobulin transfer to neonates and development of an acquired immune response. The IgG concentration was significantly lower in pups (median 2.1 mg/ml) than in adult females (median 80 mg/ml). Based on serologic results, it was not possible to determine whether K. pneumoniae was an endemic or a novel pathogen to the NZSL population because the test was not able to discriminate between Klebsiella species. However, this study suggested that the transfer of passive immunity to neonates was very low in the NZSL, especially for anti-Klebsiella antibodies.

Trace elements, PCBs and organochlorine pesticides in New Zealand common dolphins (Delphinus sp.).

Trace elements, polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticide levels were determined in tissues collected from stranded and bycaught common dolphins (Delphinus sp.) from New Zealand waters between 1999 and 2005. The concentrations of mercury (Hg), selenium (Se), chromium (Cr), zinc (Zn), nickel (Ni), cadmium (Cd), cobalt (Co), manganese (Mn), iron (Fe), copper (Cu), tin (Sn), lead (Pb), arsenic (As) and silver (Ag) were determined in blubber, liver and kidney tissue. PCBs (45 congeners) and a range of OC pesticides including dieldrin, hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) and its metabolites DDE and DDD were determined in blubber samples. Cr and Ni were not detected in any of the samples and concentrations of Co, Sn and Pb were generally low. Concentrations of Hg ranged from 0.17 to 110 mg/kg wet weight. Organochlorine pesticides dieldrin, HCB, o,p'-DDT and p,p'-DDE were present at the highest concentrations. Sum DDT concentrations in the blubber ranged from 17 to 337 and 654 to 4430 microg/kg wet weight in females and males, respectively. Similarly, Sigma45CB concentrations ranged from 49 to 386 and 268 to 1634 microg/kg wet weight in females and males, respectively. The mean transmission of SigmaDDTs and ICES7CBs between a genetically determined mother-offspring pair was calculated at 46% and 42%, respectively. Concentrations of organochlorine pesticides determined in the present study are within similar range to those reported for Hector's dolphins (Cephalorhyncus hectori) from inshore New Zealand waters.

Neonatal mortality in New Zealand sea lions (Phocarctos hookeri) at Sandy Bay, Enderby Island, Auckland Islands from 1998 to 2005.

As part of a health survey of New Zealand sea lions (Phocarctos hookeri) on Enderby Island, Auckland Islands (50 degrees 30'S, 166 degrees 17'E), neonatal mortality was closely monitored at the Sandy Bay colony for seven consecutive years. Throughout the breeding seasons 1998-99 to 2004-05, more than 400 postmortem examinations were performed on pups found dead at this site. The primary causes of death were categorized as trauma (35%), bacterial infections (24%), hookworm infection (13%), starvation (13%), and stillbirth (4%). For most pups, more than one diagnosis was recorded. Every year, two distinct peaks of trauma were observed: the first associated with mature bulls fighting within the harem and the second with subadult males abducting pups. In 2001-02 and 2002-03, epidemics caused by Klebsiella pneumoniae increased mortality by three times the mean in nonepidemic years (10.2%). The increased mortality was attributed directly to acute suppurative infection due to the bacterium and also to an increase in traumatic deaths of debilitated pups. Parasitic infection with the hookworm Uncinaria spp. was a common finding in all pups older than three weeks of age and debilitation by the parasite may have contributed to increased susceptibility to other pathogens such as Klebsiella sp. or Salmonella sp. This study provides valuable quantitative data on the natural causes of neonatal mortality in New Zealand sea lions that can be used in demographic models for management of threatened species.

Outbreaks of sarcoptic mange in free-ranging koala populations in Victoria and South Australia: a case series.

OBJECTIVE: To describe outbreaks of sarcoptic mange caused by Sarcoptes scabiei in free-ranging koalas in Victoria (December 2008 to November 2015) and South Australia (October 2011 to September 2014). METHODS: Koalas affected by mange-like lesions were reported by wildlife carers, veterinary practitioners or State Government personnel to the Faculty of Veterinary and Agricultural Sciences at The University of Melbourne and the School of Animal and Veterinary Sciences at The University of Adelaide. Skin scrapings were taken from live and dead koalas and S. scabiei mites were identified. Tissues from necropsied koalas were examined histologically. RESULTS: Outbreaks of sarcoptic mange were found to occur in koalas from both Victoria (n = 29) and South Australia (n = 29) for the first time. The gross pathological and histopathological changes are described. CONCLUSION: We present the first reported cases of sarcoptic mange outbreaks in free-ranging koalas.

Morbillivirus infection in cetaceans of the western Atlantic.

We report serologic evidence of morbillivirus infection in eleven of fifteen species of odontocete cetaceans from the western Atlantic since 1986. Blood samples were obtained both from free-ranging and stranded animals. Virus neutralizing titers were higher against porpoise and dolphin morbilliviruses than against peste des petits ruminants virus, phocine distemper virus or canine distemper virus (CDV). Serum from five species, tested in a heterologous immunoprecipitation assay using radiolabelled CDV, precipitated the nucleocapsid (N) protein. Clinical morbillivirus infection may potentially impact already threatened species such as the harbour porpoise and precipitate mass strandings of socially cohesive odontocetes.

An insight into the epidemiology of dolphin morbillivirus worldwide.

Serum samples from 288 cetaceans representing 25 species and originating from 11 different countries were collected between 1995 and 1999 and examined for the presence of dolphin morbillivirus (DMV)-specific antibodies by an indirect ELISA (iELISA) (N = 267) or a plaque reduction assay (N = 21). A total of 35 odontocetes were seropositive: three harbour porpoises (Phocoena phocoena) and a common dolphin (Delphinus delphis) from the Northeastern (NE) Atlantic, a bottlenose dolphin (Tursiops truncatus) from Kent (England), three striped dolphins (Stenella coeruleoalba), two Risso's dolphins (Grampus griseus) and a bottlenose dolphin from the Mediterranean Sea, one common dolphin from the Southwest (SW) Indian Ocean, three Fraser's dolphins (Lagenodelphis hosei) from the SW Atlantic, 18 long-finned pilot whales (Globicephala melas) and a bottlenose dolphin from the SW Pacific as well as a captive bottlenose dolphin (Tursiops aduncus) originally from Taiwan. The presence of morbillivirus antibodies in 17 of these animals was further examined in other iELISAs and virus neutralization tests. Our results indicate that DMV infects cetaceans worldwide. This is the first report of DMV-seropositive animals from the SW Indian, SW Atlantic and West Pacific Oceans. Prevalence of DMV-seropositives was 85.7% in 21 pilot whales from the SW Pacific and both sexually mature and immature individuals were infected. This indicates that DMV is endemic in these animals. The same situation may occur among Fraser's dolphins from the SW Atlantic. The prevalence of DMV-seropositives was 5.26% and 5.36% in 19 common dolphins and 56 harbour porpoise from the NE Atlantic, respectively, and 18.75% in 16 striped dolphins from the Mediterranean. Prevalence varied significantly with sexual maturity in harbour porpoises and striped dolphins; all DMV-seropositives being mature animals. The prevalence of seropositive harbour porpoise and striped dolphins appeared to have decreased since previous studies. These data suggest that DMV is not endemic within these populations, that they are losing their humoral immunity against the virus and that they may be vulnerable to new epidemics.

Comparative antibody response in harbour and grey seals naturally infected by a morbillivirus.

The antibody response of free-ranging harbour and grey seals, naturally infected by a morbillivirus, was assessed using a virus neutralizing test and a radio-immunoprecipitation assay. The prevalence of antibody was similar between species, however, grey seals had significantly higher virus neutralizing titers. Serum from clinically healthy grey seals precipitated the nucleocapsid (N) protein along with the hemagglutinin (H) and fusion (F) glycoproteins. By contrast, significantly fewer harbour seal sera precipitated the envelope glycoproteins and responses were weaker than those of grey seals. One harbour seal with acute morbillivirus pneumonia, and two with encephalitis precipitated only the N protein. Serum from four harbour seals with encephalitis weakly recognized the envelope glycoproteins. Thus, the antibody response of grey seals appears more competent than that of harbour seals with respect to morbillivirus antigens. We speculate that this difference between the species may be an important determinant of morbillivirus susceptibility.

Meningoencephalitis associated with an unidentified apicomplexan protozoan in a Pacific harbor seal.

A Pacific harbor seal (Phoca vitulina richardsii) was found on the central California coast with neurologic signs and labored breathing, which were unresponsive to treatment. Necropsy revealed a nonsuppurative necrotizing meningoencephalitis, a multilocular thymic cyst, and nonsuppurative cystitis and renal pyelitis. Microscopic examination revealed protozoans in the brain, thymic cyst, and bladder mucosa. Ultrastructurally, the protozoal tachyzoites were different from those of Neospora caninum, Toxoplasma gondii, and Sarcocystis neurona; the rhoptries were small and had electron-dense contents, and the organism divided by endodyogeny. Specific antibodies were not detected in serum using agglutination (N. caninum, T. gondii) and immunoblot assays (S. neurona). Immunohistochemistry for these organisms was negative. Polymerase chain reaction on brain tissue using specific primers did not amplify T. gondii deoxyribonucleic acid. The meningoencephalitis in this seal thus appears to have been caused by a novel protozoan.

Meningoencephalitis due to a Sarcocystis neurona-like protozoan in Pacific harbor seals (Phoca vitulina richardsi).

Seven Pacific harbor seals with meningoencephalitis associated with Sarcocystis neurona-like protozoa are described. Six of the 7 seals were free-ranging and were found stranded over an 80-km stretch of central California coastline; the other was captive. All had marked to severe nonsuppurative meningoencephalitis, most severe in the cerebellar cortex. Immunohistochemistry for S. neurona antigens was positive on brain tissue in all cases, revealing numerous merozoites as well as developing and mature schizonts, including rosette forms. Electron microscopy performed on 3 animals revealed merozoites and schizonts consistent with Sarcocystis sp., with the absence of rhoptries in merozoites, lack of a parasitophorous vacuole around schizonts, and division by endopolygeny. Serology using western blotting revealed the presence of anti-S. neurona immunoglobulins in the sera of 4 of 5 seals tested. Four animals also had a concurrent mild to moderate nonsuppurative myocarditis; in 1 seal, rare sarcocysts of undetermined species were present within cardiomyocytes.

Patterns of cell proliferation and growth rate in limb bones of the domestic fowl (Gallus domesticus).

The growth rates of each extremity of the tibiotarsus (TibT) and tarsometatarsus (TMT) bones and of a phalangeal bone were measured in Rhode Island red chicks. Histological sections of the growth plates at each of these sites were made for measurements of the thickness of the flattened dividing cell layer and counts of the number of cells between its boundaries. The diameter of the mature chondrocytes in the metaphysis was also measured. The extent of the dividing cell population within the flattened cell layer and the proportion of cells in division were measured from autoradiographs prepared after labelling dividing cells in vitro or in vivo (three birds in each group) with tritiated thymidine. From the results the rate of cell production in the plane of growth at each growth plate was estimated. It varied from about six cells per day at each end of the phalanx to just over 50 cells per day at the proximal end of the TibT and TMT. The duration of the cycle time was estimated to be 13 to 21 hours at the proximal TibT and TMT and distal TibT but considerably longer at the distal TMT and at both ends of the phalanx. Variation in growth rate between extremities of the bones examined was associated largely with variation in the dividing cell populations and the thickness of the flattened cell layer, but it appears that there were also significant differences in cell cycle times between sites. The in vitro and in vivo labelling techniques gave comparable results.

Comparative cell kinetics of avian growth plates.

New data on the cell kinetics of the cartilage growth plates in the chicken, budgerigar and rhea derived from studies with tritiated thymidine labelling are given. Quantitative histological measurements on growth plates from leg bones (tibiotarsus, tarsometatarsus and a phalangeal bone) in a further five species of birds are presented. Counts of flat cells and measurements of the average diameter of hypertrophic cells were made for each growth plate. These data are compared with values for the overall growth rates of the bones. The variation in sizes of hypertrophic cells was small and it is concluded that the largest factor in determining the growth rate of an avian bone is the size of the zone of flat cells.

Pathology of morbillivirus infection in striped dolphins (Stenella coeruleoalba) from Valencia and Murcia, Spain.

During the summer and fall of 1990 hundreds of striped dolphins (Stenella coeruleoalba) died in the Spanish Mediterranean as a result of morbillivirus infection. A pathological investigation was carried out on dolphins from Valencia and Murcia which were among the first to die in the epizootic. The dolphins were in poor body condition and pneumonia was the main necropsy finding. Microscopic lung lesions characterized by necrosis of bronchial and bronchiolar epithelium and infiltration of alveoli with macrophages, lymphocytes, neutrophils and multinucleated syncytia were seen in most dolphins. Cytoplasmic and nuclear eosinophilic viral inclusions were present in bronchial and bronchiolar epithelium and in syncytia. Focal granulomatous inflammation associated with nematodes was also present. Brain lesions included diffuse degeneration and necrosis of neurons, microgliosis, perivascular cuffing, formation of syncytia and focal demyelination. Cytoplasmic and nuclear eosinophilic inclusions were present in neurons and glial cells. There was severe lymphoid necrosis and depletion of spleen and lymph nodes and syncytia also occurred in lymph nodes. Biliary and transitional epithelium contained nuclear and cytoplasmic eosinophilic inclusions. Immunoperoxidase staining using monoclonal antibodies to phocine distemper virus confirmed the presence of morbillivirus antigens in lung and brain. The distribution and severity of lesions in striped dolphins are similar to those of distemper in seals, harbor porpoises and terrestrial mammals. The formation of syncytia in the lung and brain may be a useful pathological indicator of morbillivirus infection and may be used in the investigation of pinniped and cetacean strandings in North America.

Phocine distemper in harbor seals (Phoca vitulina) from Long Island, New York.

The first occurrence of phocine distemper (PD) disease in harbor seals (Phoca vitulina) from the United States is reported. Two seals stranded on Long Island, New York (USA) in February 1992 with clinical signs of respiratory distress, fever, and depression. Pneumonia and diffuse pulmonary congestion were the most significant post mortem findings. On histologic examination one seal had a diffuse broncho-interstitial pneumonia with formation of syncytia. The principal lesion in the second animal was nonsuppurative meningoencephalitis. Using immunoperoxidase staining, PD viral antigen was found in the cytoplasm of bronchiolar epithelium and cerebral cortex neurons. With a differential virus neutralization test, there were higher titers against phocine distemper virus (PDV) than against canine distemper virus. Thus, PDV is the most likely agent responsible for the observed lesions.

Neutralizing antibodies to phocine distemper virus in Atlantic walruses (Odobenus rosmarus rosmarus) from Arctic Canada.

The first evidence of phocine distemper virus (PDV) infection in Atlantic walruses (Odobenus rosmarus rosmarus) from Nottingham Island, Northwest Territories, Canada, is reported. Blood samples were collected from three male walruses killed by Inuit hunters in the fall of 1990. Differential virus neutralization test for each animal yielded higher titers against PDV than against other members of the Morbillivirus genus including canine distemper, peste des petits ruminants, rinderpest and measles viruses. Thus, PDV infection may be enzootic in walruses of the eastern Canadian Arctic.

A comparison of Salmonella serotypes isolated from New Zealand sea lions and feral pigs on the Auckland Islands by pulsed-field gel electrophoresis.

The Salmonella serotypes S. Cerro and S. Newport were isolated from New Zealand sea lions (Phocarctos hookeri) and feral pigs on the Auckland Islands in the New Zealand subantarctic region. The isolates were typed by pulsed-field gel electrophoresis using Xba1 as the restriction enzyme. The isolates were indistinguishable, which suggests that Salmonella infection cycles between sea lions and pigs in this environment. Apart from a previous isolation from a single New Zealand fur seal (Arctocephalus forsteri), S. Newport has not been recorded in any animals from New Zealand, but it is associated with gastroenteritis in humans. Contamination of the marine environment by human waste is a possible source of infection for marine mammals and warrants further investigation.

Phocine distemper in a harp seal (Phoca groenlandica) from the Gulf of St. Lawrence, Canada.

The first case of phocine distemper in a seal from Canadian waters and the first case of clinical phocine distemper in a harp seal, Phoca groenlandica, is reported. A two-month-old female harp seal stranded on Prince Edward Island in May 1991. Significant clinical findings were lethargy and severe conjunctivitis. Pulmonary congestion was the main necropsy finding, and histological lesions included diffuse demyelinating nonsuppurative encephalitis and mild multifocal interstitial pneumonia. Acidophilic intracytoplasmic and intranuclear inclusions were present in cerebral neurons and astrocytes. Immunoperoxidase staining confirmed phocine distemper virus (PDV) antigen in the cytoplasm and nuclei of neurons, bronchiolar gland epithelium and transitional epithelium of the bladder. Infectivity titers of canine distemper virus (CDV) (Onderstpoort strain) and a morbillivirus isolated from a grey seal were significantly reduced by serum from the harp seal.

Epizootiology of morbillivirus infection in North American harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus).

A longitudinal study of morbillivirus infection among harbor (Phoca vitulina) and gray (Halichoerus grypus) seals on the Atlantic coast of North America was carried out between 1980 and 1994. Serology also was carried out on harbor seals from the Pacific northwest coast collected in 1992 and 1993. The prevalence of morbillivirus neutralizing antibodies was significantly (P < 0.0001) higher in gray (73%, n = 296) than in harbor seals (37%, n = 387) from the Atlantic. Titers were significantly (P < 0.0001) higher against phocine distemper (PDV) compared to any other morbillivirus. Antibodies were not detected in serum from Pacific harbor seals. During the winter of 1991 to 1992 an epizootic occurred among harbor seals on the northeast coast of the United States. The event was characterized by an increase in strandings and by a significant (P = 0.001) increase in PDV antibody prevalence to 83% (n = 36) in seals stranded that winter. Morbillivirus lesions and antigen were observed in six animals found stranded from southern Maine to Long Island, New York (USA), between November 1991 and April 1992. In addition, morbillivirus encephalitis was detected in tissues from a harbor seal that stranded in 1988. Enzootic infection appeared to be present in both seal species, although with a different prevalence of disease. We propose that enzootic infection among gray seals is facilitated by population size, high annual recruitment and innate resistance to clinical disease. Infection may be maintained in the smaller harbor seal population through casual contact with gray seals.

Epizootiology of morbillivirus infection in harp, hooded, and ringed seals from the Canadian Arctic and western Atlantic.

Using a virus neutralization technique, we found phocine distemper virus (PDV) antibody in 130 (83% of 157) harp seals (Phoca groenlandica) from the western North Atlantic sampled between 1988 and 1993 inclusive. In contrast, only 44 (24% of 185) hooded seals (Cystophora cristata) had antibodies against PDV even though they were sympatric with harp seals and were sampled over a similar period, from 1989 to 1994 inclusive. Antibodies occurred in 106 (41%) of 259 ringed seals (Phoca hispida); this prevalence was higher than expected given the solitary behavior and territoriality characteristic of this species. Seropositive ringed seals were found at each of seven locations across Arctic Canada from Baffin Bay to Amundsen Gulf at which samples were collected between 1992 and 1994. However, the prevalence of infection was highest where ringed seals are sympatric with harp seals in the eastern Canadian Arctic.