Daniele Zucca

Retrospective Study of Etiologic Agents Associated with Nonsuppurative Meningoencephalitis in Stranded Cetaceans in the Canary Islands

ABSTRACT Nineteen natural cases of etiologically undetermined encephalitides in free-ranging cetaceans were studied retrospectively. Histological examination of the brains revealed variable degrees of nonsuppurative encephalitis or meningoencephalitis, characterized predominantly by perivascular lymphohistiocytic infiltrates. A PCR assay was used on brain and other available tissues to detect the presence of morbillivirus, herpesvirus, West Nile virus, Toxoplasma gondii, and Brucella spp. In addition, immunohistochemical (IHC) staining was performed on selected tissues to determine the presence of morbilliviral antigens. Six animals (5 striped dolphins and 1 common dolphin) showed IHC and/or molecular evidence of morbilliviral antigens and/or genomes, mainly in brain tissue. Conventional nested PCR detected herpesviral DNA in brain tissue samples from two striped dolphins. There was no evidence of West Nile virus, T. gondii, or Brucella spp. in any of the brain tissue samples examined. The information presented here increases the number of confirmed morbillivirus-positive cases within the Canarian archipelago from two previously reported cases to eight. Furthermore, a new nested-PCR method for the detection of morbillivirus is described here. Regarding herpesvirus, the phylogenetic analysis performed in the current study provides valuable information about a possible pathogenic branch of cetacean alphaherpesviruses that might be responsible for some fatal cases worldwide.

Fatal systemic morbillivirus infection in bottlenose dolphin, canary islands, Spain.

A systemic morbillivirus infection was diagnosed postmortem in a juvenile bottlenose dolphin stranded in the eastern North Atlantic Ocean in 2005. Sequence analysis of a conserved fragment of the morbillivirus phosphoprotein gene indicated that the virus is closely related to dolphin morbillivirus recently reported in striped dolphins in the Mediterranean Sea.

Cytokine expression in lungs of calves spontaneously infected with Mycoplasma bovis

Cytokine expression in the lung can play an important role during Mycoplasma bovis infection through leukocyte recruitment and activation, and the induction of a broad array of inflammatory mediators. To gain further insight into the pathogenesis of M. bovis-associated pneumonia, cytokine expression was examined, by immunohistochemical methods in formalin-fixed, paraffin wax-embedded tissues, in the lung of 20 calves spontaneously infected. Immunolabelling for tumor necrosis factor alpha (TNF)-α, interleukin-4 (IL-4), IL-10 and interferon-gamma (IFN-γ), was usually associated with pneumonia, particularly in macrophages and lymphocytes, and with the presence of M. bovis antigen. The expression was minimal in lungs from negative controls. The results demonstrated consistent upregulation of TNF-α, IL-4, IL-10 and IFN-γ expression during M. bovis-associated pneumonic lesions. These cytokines can participate in the immune and inflammatory responses during the pulmonary defense mechanisms against M. bovis infection.

Morbillivirus infection in Risso’s dolphin Grampus griseus: a phylogenetic and pathological study of cases from the Canary Islands

The earliest evidence of cetacean morbillivirus (CeMV) infection dates from 1982, when the dolphin morbillivirus strain (DMV) was identified in bottlenose dolphins Tursiops truncatus stranded in the mid-Atlantic region. Since then, CeMV has been detected globally in at least 26 species of mysticetes and odontocetes, causing widespread mortality and a wide range of pathological effects. In the Canary Islands, DMV and pilot whale morbillivirus have been detected in cetacean species, including short-finned pilot whales Globicephala macrorhynchus and bottlenose dolphins. Rissos dolphins Grampus griseus have been reported year-round in waters of the Canary Islands and are considered a resident species. No information is currently available on CeMV prevalence in this species in this ocean region. We searched for evidence of CeMV infection in 12 Rissos dolphins stranded in the Canary Islands from 2003 to 2015 by means of histopathology, PCR and immunohistochemistry. PCR revealed 2 CeMV-positive animals (16.6%). Phylogenetic analysis showed that the strains from the 2 positive specimens were phylogenetically quite distant, proving that more than 1 strain infects the Rissos dolphin population in this region. We also determined that the strain detected in one of the specimens mainly circulated in the northeastern Atlantic Ocean from 2007 to 2013.

Two decades of monitoring in marine debris ingestion in loggerhead sea turtle, Caretta caretta, from the western Mediterranean

Anthropogenic marine debris is one of the major worldwide threats to marine ecosystems. The EU Marine Strategy Framework Directive (MSFD) has established a protocol for data collection on marine debris from the gut contents of the loggerhead sea turtle (Caretta caretta), and for determining assessment values of plastics for Good Environmental Status (GES). GES values are calculated as percent turtles having more than average plastic weight per turtle. In the present study, we quantify marine debris ingestion in 155 loggerhead sea turtles collected in the period 1995-2016 in waters of western Mediterranean (North-east Spain). The study aims (1) to update and standardize debris ingestion data available from this area, (2) to analyse this issue over two decades using Zero-altered (hurdle) models and (3) to provide new data to compare the only GES value available (off Italian waters). The composition of marine debris (occurrence and amounts of different categories) was similar to that found in other studies for the western Mediterranean and their amounts seem not to be an important threat to turtle survival in the region. Model results suggest that, in the study area, (a) period of stranding or capture, (b) turtle size and (c) latitude are significant predictors of anthropogenic debris ingestion (occurrence and amount) in turtles. The GES value for late juvenile turtles (CCL>40 cm) has decreased in the last ten years in the study area, and this is very similar to that obtained in Italian waters. We also provide a GES value for early juvenile turtles (CCL≤40 cm) for the first time. Recommendations arising from this study include ensuring use of (1) the standardized protocol proposed by the MSFD for assessing marine debris ingestion by loggerhead sea turtles and (2) the ecology of the turtles (neritic vs oceanic), rather than their size, to obtain GES values.

Prostatic Lesions in Odontocete Cetaceans

The prostate is the only accessory male genital gland described in cetaceans. Although few studies describe the gross and histologic anatomy of the prostate in cetaceans, there is no information on pathological findings involving this organ. The prostate glands of 45 cetaceans, including 8 different odontocete species (n = 44) and 1 mysticete, were evaluated. The main pathologic diagnoses were verminous prostatitis, septic prostatitis, viral prostatitis, benign prostatic hyperplasia, and prostatitis of unknown etiology. Verminous prostatitis (n = 12) was caused by nematodes of the genus Crassicauda, and different presentations were observed. Septic prostatitis, identified in 2 cases, both involved nematode infestation and Clostridium spp coinfection. One case of viral prostatitis was identified and was associated with morbillivirus infection. In prostatitis of unknown cause (n = 7), varying degrees of prostatic lesions, mostly chronic inflammation, were identified. Impacts at individual levels (eg, localized disease, loss of reproductive capacity) and population levels (eg, decreased reproductive success) are plausible. Our results indicate a high occurrence of prostatic lesions in free-ranging odontocetes. For this reason, the prostate should be routinely inspected and sampled during necropsy of odontocete cetaceans.

Pathologic findings and causes of death of stranded cetaceans in the Canary Islands (2006-2012)

This study describes the pathologic findings and most probable causes of death (CD) of 224 cetaceans stranded along the coastline of the Canary Islands (Spain) over a 7-year period, 2006–2012. Most probable CD, grouped as pathologic categories (PCs), was identified in 208/224 (92.8%) examined animals. Within natural PCs, those associated with good nutritional status represented 70/208 (33.6%), whereas, those associated with significant loss of nutritional status represented 49/208 (23.5%). Fatal intra- and interspecific traumatic interactions were 37/208 (17.8%). Vessel collisions included 24/208 (11.5%). Neonatal/perinatal pathology involved 13/208 (6.2%). Fatal interaction with fishing activities comprised 10/208 (4.8%). Within anthropogenic PCs, foreign body-associated pathology represented 5/208 (2.4%). A CD could not be determined in 16/208 (7.7%) cases. Natural PCs were dominated by infectious and parasitic disease processes. Herein, our results suggest that between 2006 and 2012, in the Canary Islands, direct human activity appeared responsible for 19% of cetaceans deaths, while natural pathologies accounted for 81%. These results, integrating novel findings and published reports, aid in delineating baseline knowledge on cetacean pathology and may be of value to rehabilitators, caregivers, diagnosticians and future conservation policies.

Retrospective study of foreign body-associated pathology in stranded cetaceans, Canary Islands (2000–2015)

Marine pollution, overrepresented by plastic, is a growing concern worldwide. However, there is little knowledge on occurrence and detrimental impacts of marine debris in cetaceans. To partially fill in this gap of knowledge, we aimed to investigate the occurrence and pathologies associated with foreign bodies (FBs) in a large cohort of cetaceans (n = 465) stranded in the Canary Islands. The Canary Islands shelter the greatest cetacean biodiversity in Europe, with up to 30 different species, of which nine are regularly present year around. We found at least one ingested FB in 36 out of 465 (7.74%) studied cetaceans, involving 15 different species, including eight out of the nine (80%) cetacean species present year-round in the Canary Islands. Rissos dolphin was the species most affected, followed by sperm whale, beaked whale and mysticetes. Plastic FB were the most common item found (80.56%). FB was directly associated with death in 13/36 (36.11%) animals. Poor body condition and deep diving behavior were found to be risk factors for FB ingestion, whereas the adult age was a protective factor. To the authors knowledge this is the first study that use statistical analysis to investigate risk and protective factors for FB ingestion. This study also provides insights of the potential impact caused by ingested FBs on the animals health and mortality. This knowledge is critical to better understand and assess the impact of FB in cetaceans setting the scientific basis for prospective impact monitoring and future conservation policies.