Lobomycosis: an emerging disease in humans and delphinidae.

Lobomycosis, a disease caused by the uncultivable dimorphic onygenale fungi Lacazia loboi, remains to date as an enigmatic illness, both due to the impossibility of its aetiological agent to be cultured and grown in vitro, as well as because of its unresponsiveness to specific antifungal treatments. It was first described in the 1930s by Brazilian dermatologist Jorge Lobo and is known to cause cutaneous and subcutaneous localised and widespread infections in humans and dolphins. Soil and vegetation are believed to be the chief habitat of the fungus, however, increasing reports in marine mammals has shifted the attention to the aquatic environment. Infection in humans has also been associated with proximity to water, raising the hypothesis that L. loboi may be a hydrophilic microorganism that penetrates the skin by trauma. Although its occurrence was once thought to be restricted to New World tropical countries, its recent description in African patients has wrecked this belief. Antifungals noted to be effective in the empirical management of other cutaneous/subcutaneous mycoses have proven unsuccessful and unfortunately, no satisfactory therapeutic approach for this cutaneous infection currently exists.

Skin diseases in Guiana dolphins (Sotalia guianensis) from the Paranaguá estuary, Brazil: a possible indicator of a compromised marine environment.

We report on the presence of lobomycosis-like disease (LLD) and nodular skin disease (NSD) in a community of Guiana dolphins (Sotalia guianensis) inhabiting the biologically and chemically contaminated Paranaguá estuary (Brazil) and on their absence in the community living in the cleaner Cananéia estuary. Prevalence rates of LLD and NSD were 3.9% and 12.6%, respectively, in 103 photo-identified (PI) dolphins from the Paranaguá estuary in the period 2006-2007. Adults and calves were affected. Lobomycosis-like lesions may be extensive and form large plaques. Skin nodules were sometimes ulcerated and associated with cutaneous traumas suggesting that traumatic injuries may play a role in the pathogenesis of this condition. In two adult dolphins, NSD evoked the beginning of LLD. In 1996-2007 none of the 200 PI Cananéia S. guianensis had LLD or NSD, a highly significant difference. Interestingly, these dolphins were reported to harbour relatively low concentrations of organochlorines. LLD and NSD are possibly indicators of environmental changes.

Serological evidence of morbillivirus infection in small cetaceans from the Southeast Pacific.

The presence of morbillivirus-specific serum antibodies was examined by an indirect enzyme linked immunosorbent assay (iELISA) and virus neutralization tests in serum samples from 30 dusky dolphins (Lagenorhynchus obscurus), 8 long-snouted common dolphins (Delphinus capensis), 2 inshore and 6 offshore bottlenose dolphins (Tursiops truncatus) and 20 Burmeister's porpoises (Phocoena spinipinnis) taken in fisheries off central Peru in 1993-1995. The sera from six dusky dolphins, one common dolphin and three offshore bottlenose dolphins were positive on a coat of dolphin morbillivirus (DMV) antigen in the iELISA. Several of these sera were also positive when tested against peste des petits ruminants and rinderpest virus antigen. Porpoise morbillivirus and/or DMV neutralizing antibodies were detected in the sera of two bottlenose and three dusky dolphins that reacted positively with DMV antigen in iELISA and also in the sera of one common, one dusky and one bottlenose dolphin that were negative in the iELISA. These results strongly suggest that viruses closely related, or identical, to the cetacean morbillivirus present in the North Atlantic and the Mediterranean Sea infect several species of Delphinidae of the Southeastern Pacific. No convincing morbillivirus-specific antibody positive reactions were detected in the sera from either the Burmeister's porpoises or the inshore bottlenose dolphins.

Molecular biology of bovine herpesvirus type 4.

Bovine herpesvirus type 4 (BHV-4) is a ubiquitous virus of cattle. Its genome is a 144 +/- 6 kb double-stranded DNA consisting of a unique central part (L-DNA) flanked at both ends by tandem repeats called polyrepetitive DNA (prDNA or H-DNA). The overall arrangement of genes has been obtained by the analysis of homologies between short BHV-4 DNA sequences and corresponding genes of Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). The gene expression is temporally regulated. Glycoprotein precursor p (gp10/gp17) is expressed as gamma 1 polypeptide. Glycoproteins gp1, gp8, gp11 and their precursors are gamma 2 proteins. The analysis of strain variations allows the definition of two types of strains, based on the DNA patterns: the Movar 33/63-like and the DN 599-like strains. Only the M40 strain, isolated in India, fails to fit this classification. The genomic variations have been compiled to build a dendrogram showing three levels of divergence between BHV-4 strains or isolates. The available molecular data indicate that the BHV-4 genome shares much similarity with the DNA of EBV and HVS, two representative members of the gammaherpesvirinae. BHV-4 may therefore be classified in the subfamily gammaherpesvirinae.

Experimental infection of bulls with a genital isolate of bovine herpesvirus-4 and reactivation of latent virus with dexamethasone.

Five 13- to 18-month old Belgian Blue bulls were used in this experiment. Four bulls (Nos. 2, 3, 4 and 5) were inoculated intratesticularly with 10(5) plaque-forming units of bovine herpesvirus-4 (BHV-4) in each testicle (Day 0). The challenge BHV-4 strain was previously isolated from testicle cells of a bull exhibiting orchitis and azoospermia. The fifth bull (No. 1) was used as a control and received the same volume of uninfected cell culture supernatant. For 5 days, beginning on Day 51 post-infection, two bulls (Nos. 4 and 5) and the control bull (No. 1) received 0.1 mg kg-1 of dexamethasone. Unilateral castrations were then performed at regular intervals for viral examination. Treatment with dexamethasone reactivated latent BHV-4, but no clinical signs were observed in treated bulls until the end of the experiment (Day 93). Only Bull 3 showed conjunctivitis and temporary azoospermia. The virus was recovered from various samples showing that: (i) BHV-4 can be present in a latent state in the testicles and mononuclear blood cells; (ii) dexamethasone reactivates the virus; (iii) the virus is excreted by nasal and ocular routes. Each infected bull seroconverted and a booster antibody response appeared after dexamethasone treatment as shown by immunofluorescence. Neutralizing antibodies were detected in each bull by complement-dependent neutralization test with titres higher than those obtained by a classical neutralization test. No booster response of neutralizing antibodies was observed after dexamethasone treatment. The antigenically relevant envelope BHV-4 proteins were identified by Western blotting using sera samples from the animals. DNA restriction endonuclease profiles of viruses reisolated after primary infection and reactivation showed only small differences.

A review of virus infections of cataceans and the potential impact of morbilliviruses, poxviruses and papillomaviruses on host population dynamics.

Viruses belonging to 9 families have been detected in cetaceans. We critically review the clinical features, pathology and epidemiology of the diseases they cause. Cetacean morbillivirus (family Paramyxoviridae) induces a serious disease with a high mortality rate and persists in several populations. It may have long-term effects on the dynamics of cetacean populations either as enzootic infection or recurrent epizootics. The latter presumably have the more profound impact due to removal of sexually mature individuals. Members of the family Poxviridae infect several species of odontocetes, resulting in ring and tattoo skin lesions. Although poxviruses apparently do not induce a high mortality, circumstancial evidence suggests they may be lethal in young animals lacking protective immunity, and thus may negatively affect net recruitment. Papillomaviruses (family Papovaviridae) cause genital warts in at least 3 species of cetaceans. In 10% of male Burmeister's porpoises Phocoena spinipinnis from Peru, lesions were sufficiently severe to at least hamper, if not impede, copulation. Members of the families Herpesviridae, Orthomyxoviridae and Rhabdoviridae were demonstrated in cetaceans suffering serious illnesses, but with the exception of a 'porpoise herpesvirus' their causative role is still tentative. Herpes-like viruses and caliciviruses (Caliciviridae) give rise to cutaneous diseases in Monodontidae and Delphinidae. Antibodies to several serotypes of caliciviruses were found in odontocetes and mysticetes. An unrecognized Hepadnaviridae was detected by serology in a captive Pacific white-sided dolphin Lagenorhynchus obliquidens with chronic persistent hepatitis. Adenoviruses (Adenoviridae) were isolated from the intestinal tracts of mysticeti and a beluga Delphinapterus leucas but were not associated with any pathologies. We discuss the potential impact of Paramyxoviridae, Poxviridae and Papovaviridae on the dynamics of several odontocete populations.

Genital diseases in the peruvian dusky dolphin (Lagenorhynchus obscurus).

Ovarian cysts and one ovarian tumour, uterine tumours, vaginal calculi, abscesses of the broad ligament or undetermined testicular lesions were observed in 25 out of 502 female and male dusky dolphins (Lagenorhynchus obscurus) caught off Peru in 1985-1987 or 1993-1994. Tentative or definitive diagnoses included Graafian follicle cysts, luteinized cyst, ovarian parasitic granulomatous inflammation, dysgerminoma, leiomyoma, fibroleiomyoma and chronic fibrino-suppurative inflammation of the broad ligament. All lesions described represented first reports for L. obscurus, and the diagnosis of dysgerminoma was the first in a cetacean. It is also the first time that trematode eggs have been reported in the ovaries of cetaceans and that a vaginal calculus has been encountered in a sexually immature cetacean. The finding of struvite as a major component in two vaginal calculi suggested an infectious aetiology. Of 11 mature females with ovarian tumour or cysts or uterine tumours, only one (9.1%) was pregnant, i.esignificantly less than the expected pregnancy rate (53.3% in a random sample of Peruvian dusky dolphins). Several females with ovarian or uterine lesions and males with aberrant testes were large animals. It is possible that some of these lesions were associated with normal senescence of the reproductive system.

Prevalence of morbilliviruses among pinniped and cetacean species.

Since 1987, morbillivirus infections have caused serious disease outbreaks with high mortality among aquatic mammals. Baikal seals (Phoca sibirica) in Siberia were involved in an outbreak caused by a virus closely related to canine distemper virus (CDV) in 1987. Harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) in north-western Europe were struck by a newly recognised morbillivirus of seals (PDV1). A serological survey has indicated that these morbillivirus infections frequently occur among several pinniped species. Besides pinnipeds, the presence of morbillivirus infections among cetaceans, including whales, dolphins and porpoises, has been demonstrated since the outbreak of PDV among seals in north-western Europe. Morbillivirus was isolated from several stranded harbour porpoises (Phocoena phocoena). This virus proved to be very similar to a virus which was isolated during a disease outbreak with high mortality among striped dolphins (Stenella coeruleoalba) in the Mediterranean area. The viruses isolated from these cetacean species were quite different from the viruses isolated from the seals. They proved more related to the ruminant morbilliviruses, peste des petits ruminants virus and rinderpest virus. The potential transmission of the dolphin morbillivirus to the endangered population of the Mediterranean monk seal (Monachus monachus) has been considered. Studies are presently being conducted into the possibility of inducing protection against morbillivirus infection in this species by vaccination with an immune stimulating complex (ISCOM) preparation based on CDV.

Evidence of poxvirus in dusky dolphin (Lagenorhynchus obscurus) and Burmeister's porpoise (Phocoena spinipinnis) from coastal Peru.

In 1990, 74 dusky dolphins (Lagenorhynchus obscurus) and 10 Burmeister's porpoises (Phocoena spinipinnis) were examined for the presence of hyperpigmented marks and pinhole lesions on the skin (tattoo lesions) at the fishing terminal of Pucusana, central Peru. Prevalences of tattoo lesions were 8.1% and 30% in the dolphins and porpoises, respectively. Intracytoplasmic poxviruses were demonstrated by transmission electron microscopy in ultrathin sections of three of eight samples of infected epidermis from both species. The reason for the negative results in others is unclear but may be related to stages of infection with low virus density or even incorrect classification of some lesions as genuine viral tattoos. An irregular arrangement of tubules on the outer viral membrane, similar to those in orthopoxviruses, was visible in negative contrast preparations for P. spinipinnis. This is the first record of poxvirus in porpoises (Phocoenidae) and also the first report for dusky dolphins, and generally for cetaceans of the southern hemisphere.

Serological evidence of Brucella species infection in odontocetes from the south Pacific and the Mediterranean.

Sera from 58 odontocetes taken in fisheries off Peru in 1993 to 1995 and from 24 cetaceans stranded along the Spanish coast of the Mediterranean in 1997 to 1999 were tested for the presence of Brucella species antibodies in competitive and indirect ELISAS (cELISA and iELISA). Among the animals from Peru, 21 of 27 (77.8 per cent) Lagenorhynchus obscurus, three of six Delphinus capensis, one of two inshore and two of three offshore Tursiops truncatus and five of 20 (25 per cent) Phocoena spinipinnis were positive in the cELISA. Brucella species antibodies were also observed in two of 16 (12.5 per cent) Stenella coeruleoalba and in one of two Ttruncatus from the Mediterranean. These data provide the first evidence for the presence of cetacean brucellae in the south Pacific Ocean and the Mediterranean Sea.

The biology of bovine herpesvirus-4 infection of cattle.

The biology of bovine herpesvirus-4 (BHV-4) infection of cattle is reviewed. The infection is distributed worldwide. Most of isolated viruses are non-pathogenic in cattle; some of them are able to produce a genital disease. Twenty-nine structural polypeptides were described; ten of them are glycosylated. Two major glycoproteins were characterized by monoclonal antibodies. Restriction maps of BHV-4 DNA are available for the enzymes EcoRI, BamHi and HindIII. The strain variations studied by restriction analysis are very weak. The virus is able to persist in a latent state after primary infection. The identified sites of latency are nervous ganglia and mononuclear blood cells. The immune response of cattle after BHV-4 infection is characterized by low or undetectable levels of neutralizing antibodies. Four envelope proteins are recognized by convalescent sera and are the main antigenic components. Skin test remains negative in immunized cattle. Bovine herpesvirus-4 is not strictly species-specific: infection was proved in American bison (Bison bison), African buffalo (Syncerus caffer), sheep and probably cat, because feline herpesvirus-2 is in fact a BHV-4 strain. Finally BHV-4 shares antigenic and genomic relationships with alcelaphine herpesvirus-1, the causal agent of the African form of malignant catarrhal fever.

Morbillivirus infections in aquatic mammals.

Infections with morbilliviruses have caused heavy losses among different populations of aquatic mammals during the last 5 years. Two different morbilliviruses were isolated from disease outbreaks among seals in Europe and Siberia: phocid distemper virus-1 (PDV-1) and phocid distemper virus-2 (PDV-2) respectively. PDV-1 was characterized as a newly identified morbillivirus, most related to canine distemper virus (CDV), whereas PDV-2 most probably is a strain of CDV. Morbilliviruses were also isolated from porpoises--porpoise morbillivirus (PMV)--and dolphins--dolphin morbillivirus (DMV)--which had stranded on the coasts of Europe. PMV and DMV proved to be closely related to, but distinct from 2 ruminant morbilliviruses, rinderpest virus (RPV) and peste-des-petits-ruminants virus (PPRV). Serological surveys carried out among pinniped and cetacean species in the seas of Europe and North America indicated that infections with these newly discovered morbilliviruses or closely related viruses commonly occur among aquatic mammal species.

Bovine herpesvirus 4 genome: cloning, mapping and strain variation analysis.

The restriction map of the bovine herpesvirus 4 (BHV-4) genome (V. Test strain) was established for the restriction enzymes EcoRI, BamHI and HindIII by analysis of clones from a lambda library (Sau3AI partial digestion) and from a plasmid library (EcoRI fragments). One genome unit was defined as the length of the unique central part, flanked at both ends by one of the terminal tandem repeats called polyrepetitive DNA (prDNA) and was estimated to be 113 +/- 2 kbp. A restriction map of the prDNA of the V. Test strain showed internal 200 bp tandem repeats of different sequences. This region in the prDNA was highly polymorphic between BHV-4 strains, even in a viral DNA preparation from a plaque-purified strain. The right junction between the repeated and the unique sequence of the genome occurred at an almost constant site, but the left junction contained a modified prDNA and was variable between BHV-4 strains. The unique central part of the genome was very similar in the four strains under consideration, with a few variations due to the presence or absence of a restriction site and four length variations were observed, located at positions 0.006 to 0.034 (left end), 0.211 to 0.225, 0.864 to 0.881 and 0.962 to 0.984 (right end). The total length variation of 1 genome unit does not exceed 1 kbp.

Dolphin and porpoise morbilliviruses are genetically distinct from phocine distemper virus.

The morbilliviruses recently isolated from two cetacean species in the North and Mediterranean Seas have been shown to differ from phocine distemper virus isolated from European seals using monoclonal antibodies. We have identified a "universal" morbillivirus primer set, based on highly conserved regions of the morbillivirus phosphoprotein (P) gene and used this to amplify a region surrounding the RNA editing site from all known members of the group. Sequence analysis of this region of the gene shows that the dolphin and porpoise viruses are related but quite different from all other members of the group, forming a distinct lineage more closely related to the ruminant morbilliviruses than to the carnivore viruses.

Antigenic and genomic identity between simian herpesvirus aotus type 2 and bovine herpesvirus type 4.

Herpesvirus aotus type 2 (HVA-2) was isolated from a culture of kidney cells from a healthy owl monkey (Aotus trivirgatus). Bovine herpesvirus type 4 (BHV-4) is frequently isolated from diseased and even healthy cattle and occasionally from sheep, wild ruminants and cats. The two viruses are related antigenically, as was revealed by an indirect fluorescent antibody test using polyclonal antisera from experimentally infected rabbits or monoclonal antibodies raised against six BHV-4 proteins, three of which were glycosylated. The genome structures of the two viruses consist of a unique central sequence flanked at both ends by G + C-rich tandem repeats. Restriction maps (produced using EcoRI, BamHI and HindIII) of these two viruses were nearly identical but the unique sequence of the HVA-2 genome possessed two additional BamHI sites. Four genomic regions of variable size were detected, two located in the unique part, one in the repetitive part and one in the left junction between the unique and the repeated part of the genome; these slight variations were similar to those observed between various BHV-4 isolates. These results suggest that HVA-2 and BHV-4 belong to the same virus species; HVA-2 could be either a BHV-4 contaminant of owl monkey kidney cell cultures or an isolate from an owl monkey accidentally infected with BHV-4.