Behavioural Diversity Study in Bottlenose Dolphin (Tursiops truncatus) Groups and Its Implications for Welfare Assessments.

In the recent past, animal welfare studies have tried to determine the best animal welfare measures and indicators. Expression of behavioural diversity is considered a potential positive welfare indicator, and to the authors' knowledge, it has not been validated nor studied in cetaceans. For the first time, a behavioural diversity study on bottlenose dolphins (Tursiops truncatus) groups was conducted at six European facilities. The study was carried out by the animal care staff, biologists and veterinarians and included 54 dolphins housed in several group compositions at the different participating facilities. The goal of our study was to analyse behavioural diversity in bottlenose dolphins at the group level to investigate how particular factors might impact the diversity of behaviours within the group and to discuss its implications for dolphin welfare assessments. Eight factors (i.e., "observer location", "number of individuals", "age class", "sex", "social grouping", "presence/absence of leading male", "presence/absence of visitors" and "enrichment provision") impacted the behavioural diversity of the observed groups, while no significant impact of the factors "time of day" and "activity before/after observation" could be found. Our study showed the feasibility of this kind of approach for cetaceans under professional care and the relevance to considering this parameter in dolphin welfare studies, despite certain limitations that warrant further research.

Physiological Parameters Monitored on Bottlenose Dolphin Neonates (Tursiops truncatus, Montagu 1821) over the First 30 Days of Life.

Bottlenose dolphins (Tursiops truncatus) have been bred under human care for more than 60 years. Calves up to 30 days of life have presented the highest mortality rate, although comparable data for free-ranging neonates within this age group is not available. Husbandry measures to increase survivability have therefore been constantly improved. This work shows the results of a structured veterinary program that established the procedures to collect relevant physiological parameters on 13 calves during their first 30 days of life. Standardized observation protocols facilitated statistical analysis of the respiratory pattern, nursing, morphometric measurements and bloodwork. These allow early detection of health issues. Healthy neonates had longer apnea duration, despite the shape and size of the facility. The nursing pattern showed that successful calves started nursing 3 to 18 h postpartum. Although with different individual patterns, a steady increase in suckling time occurs during the first 24 h of life. The healthy neonates grew 0.428 ± 0.102 kg per day and the complete blood count profile, glucose, iron, blood urea nitrogen (BUN), total protein, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (GGT), creatinine and electrolytes values considered normal for healthy calves are provided. Furthermore, cholesterol, triglycerides, α-amylase, lipase, magnesium and cortisol are reported for the first time for such young calves. A list of indications for prompt intervention is included.

The seal louse (Echinophthirius horridus) in the Dutch Wadden Sea: investigation of vector-borne pathogens.

BACKGROUND: Belonging to the anopluran family Echinophthiriidae, Echinophthirius horridus, the seal louse, has been reported to parasitise a broad range of representatives of phocid seals. So far, only a few studies have focused on the vector function of echinophthiriid lice, and knowledge about their role in pathogen transmission is still scarce. The current study aims to investigate the possible vector role of E. horridus parasitising seals in the Dutch Wadden Sea. METHODS: E. horridus seal lice were collected from 54 harbour seals (Phoca vitulina) and one grey seal (Halichoerus grypus) during their rehabilitation period at the Sealcentre Pieterburen, The Netherlands. DNA was extracted from pooled seal lice of individual seals for molecular detection of the seal heartworm Acanthocheilonema spirocauda, the rickettsial intracellular bacterium Anaplasma phagocytophilum, and the cell wall-less bacteria Mycoplasma spp. using PCR assays. RESULTS: Seal lice from 35% of the harbour seals (19/54) and from the grey seal proved positive for A. spirocauda. The seal heartworm was molecularly characterised and phylogenetically analysed (rDNA, cox1). A nested PCR was developed for the cox1 gene to detect A. spirocauda stages in seal lice. A. phagocytophilum and a Mycoplasma species previously identified from a patient with disseminated 'seal finger' mycoplasmosis were detected for the first time, to our knowledge, in seal lice. CONCLUSIONS: Our findings support the potential vector role of seal lice in the transmission of A. spirocauda and reveal new insights into the spectrum of pathogens occurring in seal lice. Studies on vector competence of E. horridus, especially for bacterial pathogens, are essentially needed in the future as these pathogens might have detrimental effects on the health of seal populations. Furthermore, studies on the vector role of different echinophthiriid species infecting a wide range of pinniped hosts should be conducted to extend the knowledge of vector-borne pathogens.

Lungworm seroprevalence in free-ranging harbour seals and molecular characterisation of marine mammal MSP.

Harbour seals (Phoca vitulina) are frequently infected with the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus. The infection is often accompanied by secondary bacterial infections and can cause severe bronchopneumonia and even death in affected animals. Hitherto, the detection of lungworm infections was based on post mortem investigations from animals collected within stranding networks and a valid detection method for live free-ranging harbour seals was not available. Recently, an ELISA was developed for detecting lungworm antibodies in harbour seal serum, using major sperm protein (MSP) of the bovine lungworm, Dictyocaulus viviparus as recombinant diagnostic antigen. To determine lungworm seroprevalence in free-ranging harbour seals, serum was taken from four different seal age groups (n = 313) resulting in an overall prevalence of 17.9% (18.9% of males, 16.7% of females). 0.7% of harbour seals up to six weeks of age were seropositive, as were 89% of seals between six weeks and six months, 53.6% between six and 18 months and 24.2% of seals over 18 months of age. In the 18 months and over age group, seropositive animals showed statistically significant reductions in body weight (P = 0.003) and length (P < 0.001). Sera from lungworm infected harbour seals in rehabilitation (n = 6) revealed that duration of antibody persistence may be similar to that of lungworm infected cattle, but further studies are needed to confirm this. Phylogenetic analyses of MSP sequences of different marine and terrestrial mammal parasitic nematodes revealed that lungworm MSP of the genus Dictyocaulus (superfamily Trichostrongyloidea) is more closely related to metastrongylid marine mammal lungworms than to trichostrongylid nematodes of terrestrial hosts.

Vertebral column deformities in white-beaked dolphins from the eastern North Atlantic.

Five white-beaked dolphins Lagenorhynchus albirostris with outwardly vertebral kyphosis, kyphoscoliosis or lordosis were identified during a photo-identification survey of over 400 individuals (2002-2013) in Faxaflói and Skjálfandi Bays, Iceland. In addition, 3 stranding reports from Denmark, The Netherlands and the UK were analysed, providing both external observation and post mortem details of axial deviations of the vertebral column in this species. Two of the free-ranging cases and 2 of the stranded specimens appeared to have an acquired disease, either as a direct result of trauma, or indirectly from trauma/wound and subsequent infection and bony proliferation, although we were unable to specifically identify the causes. Our data represent a starting point to understand vertebral column deformations and their implications in white-beaked dolphins from the eastern North Atlantic. We recommend for future necropsy cases to conduct macro- and microscopic evaluation of muscle from both sides of the deformed region, in order to assess chronic or acute conditions related to the vertebral deformations and cause of death.

Harbour seal (Phoca vitulina) PMN and monocytes release extracellular traps to capture the apicomplexan parasite Toxoplasma gondii.

Extracellular traps (ETs) are composed of nuclear DNA as backbone adorned with histones, cytoplasmic antimicrobial peptides/proteins which are released from a range of vertebrate and invertebrate host immune cells in response to several invading pathogens. Until now this ancient novel innate defence mechanism has not been demonstrated in any marine mammal. Interactions of harbour seal (Phoca vitulina)-PMN and -monocytes with viable tachyzoites of Toxoplasma gondii were investigated in this respect in vitro. For the demonstration and quantification of harbour seal PMN- and monocyte-derived ETs, extracellular DNA was stained with Sytox Orange. Fluorescence assays as well as scanning electron microscopy (SEM) analyses demonstrated PMN- and monocyte-promoted ET formation rapidly being induced upon contact with T. gondii-tachyzoites. The co-localisation of extracellular DNA decorated with histones (H3), neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite entrapping structures confirmed the classical characteristics of PMN- and monocyte-promoted ETs. Exposure of harbour seal PMN and monocytes to viable tachyzoites resulted in a significant induction of ETs when compared to negative controls. Harbour seal-ETs were efficiently abolished by DNase I treatment and were reduced after PMN and monocytes pre-incubation with the NADPH oxidase inhibitor diphenilane iodondium. Tachyzoites of T. gondii were firmly entrapped and immobilised within harbour seal-ET structures. To our best knowledge, we here report for the first time on T. gondii-induced ET formation in harbour seal-PMN and -monocytes. Our results strongly indicate that PMN- and monocyte-triggered ETs represent a relevant and ancient conserved effector mechanism of the pinniped innate immune system as reaction against the pathogenic protozoon T. gondii and probably against other foreign pathogens occurring in the ocean environment.

Molecular epidemiology of seal parvovirus, 1988-2014.

A novel parvovirus was discovered recently in the brain of a harbor seal (Phoca vitulina) with chronic meningo-encephalitis. Phylogenetic analysis of this virus indicated that it belongs to the genus Erythroparvovirus, to which also human parvovirus B19 belongs. In the present study, the prevalence, genetic diversity and clinical relevance of seal parvovirus (SePV) infections was evaluated in both harbor and grey seals (Halichoerus grypus) that lived in Northwestern European coastal waters from 1988 to 2014. To this end, serum and tissue samples collected from seals were tested for the presence of seal parvovirus DNA by real-time PCR and the sequences of the partial NS gene and the complete VP2 gene of positive samples were determined. Seal parvovirus DNA was detected in nine (8%) of the spleen tissues tested and in one (0.5%) of the serum samples tested, including samples collected from seals that died in 1988. Sequence analysis of the partial NS and complete VP2 genes of nine SePV revealed multiple sites with nucleotide substitutions but only one amino acid change in the VP2 gene. Estimated nucleotide substitution rates per year were 2.00 × 10(-4) for the partial NS gene and 1.15 × 10(-4) for the complete VP2 gene. Most samples containing SePV DNA were co-infected with phocine herpesvirus 1 or PDV, so no conclusions could be drawn about the clinical impact of SePV infection alone. The present study is one of the few in which the mutation rates of parvoviruses were evaluated over a period of more than 20 years, especially in a wildlife population, providing additional insights into the genetic diversity of parvoviruses.

Novel B19-like parvovirus in the brain of a harbor seal.

Using random PCR in combination with next-generation sequencing, a novel parvovirus was detected in the brain of a young harbor seal (Phoca vitulina) with chronic non-suppurative meningo-encephalitis that was rehabilitated at the Seal Rehabilitation and Research Centre (SRRC) in the Netherlands. In addition, two novel viruses belonging to the family Anelloviridae were detected in the lungs of this animal. Phylogenetic analysis of the coding sequence of the novel parvovirus, tentatively called Seal parvovirus, indicated that this virus belonged to the genus Erythrovirus, to which human parvovirus B19 also belongs. Although no other seals with similar signs were rehabilitated in SRRC in recent years, a prevalence study of tissues of seals from the same area collected in the period 2008-2012 indicated that the Seal parvovirus has circulated in the harbor seal population at least since 2008. The presence of the Seal parvovirus in the brain was confirmed by real-time PCR and in vitro replication. Using in situ hybridization, we showed for the first time that a parvovirus of the genus Erythrovirus was present in the Virchow-Robin space and in cerebral parenchyma adjacent to the meninges. These findings showed that a parvovirus of the genus Erythrovirus can be involved in central nervous system infection and inflammation, as has also been suspected but not proven for human parvovirus B19 infection.

Prevalence of phocine distemper virus specific antibodies: bracing for the next seal epizootic in north-western Europe.

In 1988 and 2002, two major phocine distemper virus (PDV) outbreaks occurred in harbour seals (Phoca vitulina) in north-western European coastal waters, causing the death of tens of thousands seals. Here we investigated whether PDV is still circulating among seals of the Dutch coastal waters and whether seals have protective serum-antibodies against PDV. Therefore seal serum samples, collected from 2002 to 2012, were tested for the presence of PDV-neutralizing antibodies. Antibodies were detected in most seals in 2002 and 2003 while after 2003 antibodies were detected only in seals less than two month-old and adult seals that probably had survived the 2002 PDV-epizootic. We estimated the current proportion of seals with antibodies against PDV at 11%. These findings suggest that at present the vast majority of seals are not immune to PDV infection. PDV re-introduction in this area may cause a major epizootic with infection of >80% and mass-mortality of >50% of the population.