The Importance of Baseline Health Surveillance Efforts in Freshwater Fish Conservation Using the Threatened Iberian Leuciscids as an Example.

Freshwater fish species are experiencing the highest decline among vertebrates in this century. Although a great effort has been made to identify and tackle threats to the conservation of this taxa, several knowledge gaps still exist particularly for noncommercial endangered species, including considerations regarding fish health status. These species face deteriorating environmental conditions in their natural habitats that may lead to stress and increased risk for infectious disease outbreaks. Establishing health surveillance is crucial to identify and predict physiologic disruption in fish populations. Additionally, information retrieved may be used to direct targeted efforts to contribute to improving the conservation status of these species. We used threatened Iberian leuciscids as a case study to discuss the current knowledge regarding their health surveillance and to suggest recommendations for the establishment of practical health assessments that can benefit conservation plans for these species and be implemented in threatened or endangered freshwater fish species plans globally.

Variation of blubber thickness for three marine mammal species in the southern Baltic Sea.

Evaluating populational trends of health condition has become an important topic for marine mammal populations under the Marine Strategy Framework Directive (MSFD). In the Baltic Sea, under the recommendation of Helsinki Commission (HELCOM), efforts have been undertaken to use blubber thickness as an indicator of energy reserves in marine mammals. Current values lack geographical representation from the entire Baltic Sea area and a large dataset is only available for grey seals (Halichoerus grypus) from Sweden and Finland. Knowledge on variation of blubber thickness related to geography throughout the Baltic Sea is important for its usage as an indicator. Such evaluation can provide important information about the energy reserves, and hence, food availability. It is expected that methodological standardization under HELCOM should include relevant datasets with good geographical coverage that can also account for natural variability in the resident marine mammal populations. In this study, seasonal and temporal trends of blubber thickness were evaluated for three marine mammal species-harbor seal (Phoca vitulina), grey seal (Halichoerus grypus) and harbor porpoise (Phocoena phocoena)-resident in the southern Baltic Sea collected and investigated under stranding networks. Additionally, the effects of age, season and sex were analyzed. Seasonal variation of blubber thickness was evident for all species, with harbor seals presenting more pronounced effects in adults and grey seals and harbor porpoises presenting more pronounced effects in juveniles. For harbor seals and porpoises, fluctuations were present over the years included in the analysis. In the seal species, blubber thickness values were generally higher in males. In harbor seals and porpoises, blubber thickness values differed between the age classes: while adult harbor seals displayed thicker blubber layers than juveniles, the opposite was observed for harbor porpoises. Furthermore, while an important initial screening tool, blubber thickness assessment cannot be considered a valid methodology for overall health assessment in marine mammals and should be complemented with data on specific health parameters developed for each species.

Aeromonas spp. Prevalence, Virulence, and Antimicrobial Resistance in an Ex Situ Program for Threatened Freshwater Fish-A Pilot Study with Protective Measures.

Ex situ breeding programs are important conservation tools for endangered freshwater fish. However, developing husbandry techniques that decrease the likelihood of disease, antimicrobial resistance, and virulence determinants acquisition during this process is challenging. In this pilot study, we conducted a captivity experiment with Portuguese nase (Iberochondrostoma lusitanicum), a critically endangered leuciscid species, to investigate the influence of simple protective measures (i.e., material disinfection protocols and animal handling with gloves) on the dynamics of a potential pathogenic genus, Aeromonas, as well as its virulence profiles and antimicrobial resistance signatures. Our findings show that antimicrobial resistance in Aeromonas spp. collected from I. lusitanicum significantly increased during the extent of the assay (5 weeks), with all isolates collected at the end of the study classified as multidrug-resistant. Additionally, humans handling fishes without protective measures were colonized by Aeromonas spp. The use of protective measures suggested a decreasing trend in Aeromonas spp. prevalence in I. lusitanicum, while bacterial isolates displayed significantly lower virulence index values when virulence phenotypical expression was tested at 22 °C. Despite this study representing an initial trial, which needs support from further research, protective measures tested are considered a simple tool to be applied in ex situ breeding programs for aquatic animals worldwide. Furthermore, current results raise concern regarding antimicrobial resistance amplification and zoonotic transmission of Aeromonas spp. in aquatic ex situ programs.

Sympatric threatened Iberian leuciscids exhibit differences in Aeromonas diversity and skin lesions' prevalence.

Assessments regarding health aspects of Iberian leuciscids are limited. There is currently an information gap regarding effects of infectious diseases on these populations and their role as a possible conservation threat. Moreover, differences in susceptibility to particular agents, such as Aeromonas spp., by different species/populations is not clear. To understand potential differences in Aeromonas diversity and load, as well as in the prevalence and proportion of skin lesions, in fishes exposed to similar environmental conditions, an observational study was implemented. Using a set of 12 individuals belonging to two sympatric Iberian leuciscid species (Squalius pyrenaicus and Iberochondrostoma lusitanicum), the skin lesion score in each individual was analyzed. Furthermore, a bacterial collection of Aeromonas spp. isolated from each individual was created and isolates' load was quantified by plate counting, identified at species level using a multiplex-PCR assay and virulence profiles established using classical phenotypic methods. The similarity relationships of the isolates were evaluated using a RAPD analysis. The skin lesion score was significantly higher in S. pyrenaicus, while the Aeromonas spp. load did not differ between species. When analyzing Aeromonas species diversity between fishes, different patterns were observed. A predominance of A. hydrophila was detected in S. pyrenaicus individuals, while I. lusitanicum individuals displayed a more diverse structure. Similarly, the virulence index of isolates from S. pyrenaicus was higher, mostly due to the isolated Aeromonas species. Genomic typing clustered the isolates mainly by fish species and skin lesion score. Specific Aeromonas clusters were associated with higher virulence indexes. Current results suggest potential differences in susceptibility to Aeromonas spp. at the fish species/individual level, and constitute important knowledge for proper wildlife management through the signalization of at-risk fish populations and hierarchization of conservation measures.

Climatic Alterations Influence Bacterial Growth, Biofilm Production and Antimicrobial Resistance Profiles in Aeromonas spp.

Climate change is expected to create environmental disruptions that will impact a wide array of biota. Projections for freshwater ecosystems include severe alterations with gradients across geographical areas. Life traits in bacteria are modulated by environmental parameters, but there is still uncertainty regarding bacterial responses to changes caused by climatic alterations. In this study, we used a river water microcosm model to evaluate how Aeromonas spp., an important pathogenic and zoonotic genus ubiquitary in aquatic ecosystems, responds to environmental variations of temperature and pH as expected by future projections. Namely, we evaluated bacterial growth, biofilm production and antimicrobial resistance profiles of Aeromonas species in pure and mixed cultures. Biofilm production was significantly influenced by temperature and culture, while temperature and pH affected bacterial growth. Reversion of antimicrobial susceptibility status occurred in the majority of strains and tested antimicrobial compounds, with several combinations of temperature and pH contributing to this effect. Current results highlight the consequences that bacterial genus such as Aeromonas will experience with climatic alterations, specifically how their proliferation and virulence and phenotypic resistance expression will be modulated. Such information is fundamental to predict and prevent future outbreaks and deleterious effects that these bacterial species might have in human and animal populations.

Molecular Epidemiology, Virulence Traits and Antimicrobial Resistance Signatures of Aeromonas spp. in the Critically Endangered Iberochondrostoma lusitanicum Follow Geographical and Seasonal Patterns.

Despite the fact that freshwater fish populations are experiencing severe declines worldwide, our knowledge on the interaction between endangered populations and pathogenic agents remains scarce. In this study, we investigated the prevalence and structure of Aeromonas communities isolated from the critically endangered Iberochondrostoma lusitanicum, a model species for threatened Iberian leuciscids, as well as health parameters in this species. Additionally, we evaluated the virulence profiles, antimicrobial resistance signatures and genomic relationships of the Aeromonas isolates. Lesion prevalence, extension and body condition were deeply affected by location and seasonality, with poorer performances in the dry season. Aeromonas composition shifted among seasons and was also different across river streams. The pathogenic potential of the isolates significantly increased during the dry season. Additionally, isolates displaying clinically relevant antimicrobial resistance phenotypes (carbapenem and fluroquinolone resistance) were detected. As it inhabits intermittent rivers, often reduced to disconnected pools during the summer, the dry season is a critical period for I. lusitanicum, with lower general health status and a higher potential of infection by Aeromonas spp. Habitat quality seems a determining factor on the sustainable development of this fish species. Also, these individuals act as reservoirs of important antimicrobial resistant bacteria with potential implications for public health.

Antibiotic Resistance and Virulence Profiles of Gram-Negative Bacteria Isolated from Loggerhead Sea Turtles (Caretta caretta) of the Island of Maio, Cape Verde.

Previous studies revealed high levels of antimicrobial resistance (AMR) in loggerhead sea turtles (Caretta caretta), describing this species as prime reservoir of antimicrobial-resistant bacteria. This study aimed to characterise, for the first time, the AMR and virulence profiles of Gram-negative bacteria isolated from 33 nesting loggerhead turtles of the island of Maio, Cape Verde. Cloacal, oral, and egg content swab samples (n = 99) were collected and analysed using conventional bacteriological techniques. Shewanella putrefaciens, Morganella morganii, and Vibrio alginolyticus were isolated from the samples under study. The isolates obtained from this loggerhead subpopulation (North-East Atlantic) revealed lower levels of AMR, compared with the results of studies performed in other subpopulations (e.g., Mediterranean). However, the detection of resistance to carbapenems and multiple antimicrobial resistance indices higher than 0.20, raises concern about the potential association of these animals to points of high antimicrobial exposure. Furthermore, virulence phenotypic characterisation revealed that the isolates presented complex virulence profiles, including the ability to produce biofilms. Finally, due to their pathogenic potential, and considering the evidence of illegal consumption of turtle-related products on the island of Maio, the identified bacteria may represent a significant threat to public health.

Predators in northern Germany are reservoirs for parasites of One Health concern.

Urbanisation and invasion of wildlife into urban areas as well as human leisure activities create diverse wildlife-domestic animal-human interfaces, increasing the risk of (zoonotic) parasite spillover from sylvatic to domestic and synanthropic cycles. This study investigated the endo- and ectoparasite fauna, emphasising on parasites of One Health Concern, of the most common predators in northern Germany between November 2013 and January 2016. Eighty red foxes (Vulpes vulpes), 18 stone martens (Martes foina) and nine raccoon dogs (Nyctereutes procyonoides) were available for the study. Overall, 79 (73.8%) of the examined predators (n=107) harboured at least one endoparasite. The most frequently detected endoparasites in red foxes were Toxocara canis (43.8% positive individuals), Capillaria spp. (36.3%), Alaria alata (25.0%), Echinococcus multilocularis (26.3%) and Uncinaria stenocephala (25.0%). Furthermore, Toxascaris leonina, Trichuris vulpis, Taenia ssp., Mesocestoides spp. and coccidian oocysts were observed. The endoparasite species richness in raccoon dogs was comparable to red foxes, while in stone martens, only Capillaria spp. were found. Muscle digestion for detection of Trichinella spp. and antigen testing for Giardia spp. did not show positive results. Ectoparasite analyses revealed infestations with ticks species of the genus Ixodes as well as Dermacentor reticulatus. Scabies mites were not present in digested skin samples, while Demodex spp. mites were observed by faecal flotation in one red fox. Furthermore, fleas (Archaeopsylla erinacei and Chaetopsylla globiceps) were observed in the fur of red foxes, while lice were not present in any predator species. However, infestation frequency with ectoparasites was with 19.2% generally low in available predator skins (n=99). Overall, the present study showed that predators in northern Germany serve as reservoirs for parasites of One Health concern, with four of the five most frequent endoparasites being zoonotic, highlighting the need of parasite surveillance in wildlife predators in order to implement measures avoiding spillovers to domestic animals and humans.

Assessing seal carcasses potentially subjected to grey seal predation.

In order to conduct an objective evaluation of potential ecological effects of grey seal predation on marine mammals, it is essential to establish a broad knowledge base helping in the thorough identification of such cases during post-mortem examination. The aim of this work is to report and discuss outcomes resulting from a retrospective evaluation of harbour (Phoca vitulina) and grey seal (Halichoerus grypus) stranding and necropsy data (n = 3274). In addition, the results are compared to a recent case of definite grey seal predation from Germany as well as reports from other countries. Carcasses potentially subjected to grey seal predation show severe lacerations with a circular pattern leaving a smooth, linear and cut-like wound margin. Large parts of skin and underlying tissue are detached from the body and loss of blubber is common. Occurrence frequencies of encountered lesions are presented and a list of parameters to be used for the assessment of similar cases as well as a complementary decision tree are suggested. With the proposed parameters, categories and tools, a baseline can be built in order to facilitate the standardised recognition of predation cases during post-mortem examinations of seals between groups working with populations across several geographic ranges.

Assessing harbour porpoise carcasses potentially subjected to grey seal predation.

As a follow-up on the data presented for seals, we herein report and discuss outcomes resulting from a retrospective evaluation of harbour porpoise stranding and necropsy data from Schleswig-Holstein, Germany (n = 4463) to enable an objective evaluation of potential ecological effects of grey seal predation on porpoises. Results are compared to a recent case of definite grey seal predation as well as to reports from other countries. Porpoise carcasses potentially subject to grey seal predation show severe lacerations, with large parts of skin and underlying tissue being detached from the body. Loss of blubber tissue is common. Based on the occurrence frequencies of encountered lesions, a list of parameters as well as a complementary decision tree are suggested to be used for future assessments. The results shown add to an increasingly standardised assessment protocol of suspected grey seal predation cases making respective results comparable between different areas and countries. The usage of a standardised protocol may increase the awareness of grey seal predation and the reporting of such cases. By this, differences in the predation and feeding patterns as well as the potential ecological relevance of this behaviour may be elucidated.

Pathological Findings in White-Beaked Dolphins (Lagenorhynchus albirostris) and Atlantic White-Sided Dolphins (Lagenorhynchus acutus) From the South-Eastern North Sea.

In the North Sea, white-beaked dolphins (Lagenorhynchus albirostris) occur regularly and are the second most common cetacean in the area, while their close relative, the Atlantic white-sided dolphin (Lagenorhynchus acutus), prefers the deeper waters of the northern North Sea and adjacent Atlantic Ocean. Though strandings of both species have occurred regularly in the past three decades, they have decreased in the southern North Sea during the last years. Studies describing necropsy findings in stranded Lagenorhynchus spp. are, to date, still scarce, while information gained through post-mortem examinations may reveal valuable information about underlying causes of this decline, including age structure and the reproduction status. Therefore, we retrospectively assessed and compared the necropsy results from fresh Lagenorhynchus spp. stranded along the southeastern North Sea between 1990 and 2019. A full necropsy was performed on 24 white-beaked dolphins and three Atlantic white-sided dolphins from the German and Dutch coast. Samples of selected organs were taken for histopathological, bacteriological, mycological, parasitological and virological examinations. The most common post-mortem findings were emaciation, gastritis and pneumonia. Gastritis and ulceration of the stomach was often associated with an anisakid nematode infection. Pneumonia was most likely caused by bacterial infections. Encephalitis was observed in three animals and morbillivirus antigen was detected immunohistochemically in one case. Although the animal also showed pneumonic lesions, virus antigen was only found in the brain. Parasitic infections mainly affected the gastro-intestinal tract. Lungworm infections were only detected in two cases and no associations with pathological alterations were observed. Stenurus spp. were identified in two of three cases of parasitic infections of the ears. Twelve of the 26 white-beaked dolphins stranded in Germany were found between 1993 and 1994, but there was no evidence of epizootic disease events or mass strandings during the monitored period.

CRYPTOSPORIDIUM SPECIES AND GIARDIA SPECIES PREVALENCE IN MARINE MAMMAL SPECIES PRESENT IN THE GERMAN NORTH AND BALTIC SEAS.

Marine mammals are known to harbor Cryptosporidium spp. and Giardia spp., especially species or genotypes commonly associated with humans. In animals of the North and Baltic Seas, this information is missing. In this study, fecal samples ( n = 97) from harbor porpoises ( Phocoena phocoena), harbor seals ( Phoca vitulina), and grey seals ( Phocoena phocoena) present in German waters were examined via microscopic examination (by using a modified Ziehl-Neelsen technique [MZN]) to estimate the prevalence of these parasites. Additionally, intestinal tissue samples ( n = 1,049) from specimens of 10 marine mammal species were submitted for histopathologic examination (HP). A low prevalence was detected (2.1 and 1.3%, for MZN and HP, respectively), associated with Cryptosporidium spp. Giardia spp. was not observed. Current results suggest a low prevalence of these parasites in marine mammals present in Germany. However, methods with higher sensitivity, such as molecular methods, are crucial to confirm the prevalence, establish origin routes, and determine epidemiologic factors associated with the low prevalence observed.

IDIOPATHIC IRON OVERLOAD IN A HARBOR SEAL ( PHOCA VITULINA).

Iron overload has been described in various wild species. The majority of cases involve captive animals, often associated with increased dietary iron uptake. Here a case of idiopathic iron overload in a female adult harbor seal under human care is presented. The animal displayed a progressive anorexia, apathy, and increased serum iron levels. Radiographs showed radiopaque foreign bodies in the stomach. The seal died during an elective laparotomy. Twenty-five coins and two metal rings were removed from the stomach. Histopathologic examination revealed iron storage without cellular damage in liver, spleen, kidney, and pulmonary and mesenteric lymph nodes. Atomic absorption spectrophotometry analysis for iron revealed values thirty times above the reference ranges in spleen and liver; however, the coins only contain minor levels (parts per million) of iron. The etiology of the iron overload in this animal remains unclear. A multifactorial process cannot be excluded.

Dolphin Morbillivirus in a Fin Whale (Balaenoptera physalus) in Denmark, 2016.

We studied the etiology of encephalitis in a fin whale (Balaenoptera physalus) that stranded in 2016 on the coast of Denmark. Dolphin morbillivirus (DMV) was detected in the brain and other organs. Phylogenetics showed close relation to DMV isolated from a striped dolphin (Stenella coeruleoalba) from Spain in 2012.

Pathological findings in the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides), with special emphasis on infectious and zoonotic agents in Northern Germany.

Anthropogenic landscape changes contributed to the reduction of availability of habitats to wild animals. Hence, the presence of wild terrestrial carnivores in urban and peri-urban sites has increased considerably over the years implying an increased risk of interspecies spillover of infectious diseases and the transmission of zoonoses. The present study provides a detailed characterisation of the health status of the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides) in their natural rural and peri-urban habitats in Schleswig-Holstein, Germany between November 2013 and January 2016 with focus on zoonoses and infectious diseases that are potentially threatening to other wildlife or domestic animal species. 79 red foxes, 17 stone martens and 10 raccoon dogs were collected from traps or hunts. In order to detect morphological changes and potential infectious diseases, necropsy and pathohistological work-up was performed. Additionally, in selected animals immunohistochemistry (influenza A virus, parvovirus, feline leukemia virus, Borna disease virus, tick-borne encephalitis, canine adenovirus, Neospora caninum, Toxoplasma gondii and Listeria monocytogenes), next-generation sequencing, polymerase chain reaction (fox circovirus) and serum-neutralisation analysis (canine distemper virus) were performed. Furthermore, all animals were screened for fox rabies virus (immunofluorescence), canine distemper virus (immunohistochemistry) and Aujeszky's disease (virus cultivation). The most important findings included encephalitis (n = 16) and pneumonia (n = 20). None of the investigations revealed a specific cause for the observed morphological alterations except for one animal with an elevated serum titer of 1:160 for canine distemper. Animals displayed macroscopically and/or histopathologically detectable infections with parasites, including Taenia sp., Toxocara sp. and Alaria alata. In summary, wildlife predators carry zoonotic parasitic disease and suffer from inflammatory diseases of yet unknown etiology, possibly bearing infectious potential for other animal species and humans. This study highlights the value of monitoring terrestrial wildlife following the "One Health" notion, to estimate the incidence and the possible spread of zoonotic pathogens and to avoid animal to animal spillover as well as transmission to humans.