First description of a lesion in the upper digestive mucosa associated with a novel gammaherpesvirus in a striped dolphin (Stenella coeruleoalba) stranded in the Western Mediterranean Sea.

BACKGROUND: A wide variety of lesions have been associated with herpesvirus in cetaceans. However, descriptions of herpesvirus infections in the digestive system of cetaceans are scarce. CASE REPORT: A young female striped dolphin stranded in the Valencian Community (Spain) on the 6th August 2021. The animal showed external macroscopic lesions suggestive of an aggressive interaction with bottlenose dolphins (rake marks in the epidermis). Internally, the main findings included congestion of the central nervous system and multiple, well-defined, whitish, irregularly shaped, proliferative lesions on the oropharyngeal and laryngopharyngeal mucosa. Histopathology revealed lymphoplasmacytic and histiocytic meningoencephalitis, consistent with neuro brucellosis. The oropharyngeal and laryngopharyngeal plaques were comprised histologically of focally extensive epithelial hyperplasia. As part of the health surveillance program tissue samples were tested for cetacean morbillivirus using a real-time reverse transcription-PCR, for Brucella spp. using a real-time PCR, and for herpesvirus using a conventional nested PCR. All samples were negative for cetacean morbillivirus; molecular positivity for Brucella spp. was obtained in pharyngeal tonsils and cerebrospinal fluid; herpesvirus was detected in a proliferative lesion in the upper digestive mucosa. Phylogenetic analysis showed that the herpesvirus sequence was included in the Gammaherpesvirinae subfamily. This novel sequence showed the greatest identity with other Herpesvirus sequences detected in skin, pharyngeal and genital lesions in five different species. CONCLUSIONS: To the best of the authors' knowledge, this is the first report of a proliferative lesion in the upper digestive mucosa associated with gammaherpesvirus posititvity in a striped dolphin (Stenella coeruleoalba).

Systematic Determination of Herpesvirus in Free-Ranging Cetaceans Stranded in the Western Mediterranean: Tissue Tropism and Associated Lesions.

The monitoring of herpesvirus infection provides useful information when assessing marine mammals' health. This paper shows the prevalence of herpesvirus infection (80.85%) in 47 cetaceans stranded on the coast of the Valencian Community, Spain. Of the 966 tissues evaluated, 121 tested positive when employing nested-PCR (12.53%). The largest proportion of herpesvirus-positive tissue samples was in the reproductive system, nervous system, and tegument. Herpesvirus was more prevalent in females, juveniles, and calves. More than half the DNA PCR positive tissues contained herpesvirus RNA, indicating the presence of actively replicating virus. This RNA was most frequently found in neonates. Fourteen unique sequences were identified. Most amplified sequences belonged to the Gammaherpesvirinae subfamily, but a greater variation was found in Alphaherpesvirinae sequences. This is the first report of systematic herpesvirus DNA and RNA determination in free-ranging cetaceans. Nine (19.14%) were infected with cetacean morbillivirus and all of them (100%) were coinfected with herpesvirus. Lesions similar to those caused by herpesvirus in other species were observed, mainly in the skin, upper digestive tract, genitalia, and central nervous system. Other lesions were also attributable to concomitant etiologies or were nonspecific. It is necessary to investigate the possible role of herpesvirus infection in those cases.

Alpha- and gammaherpesviruses in stranded striped dolphins (Stenella coeruleoalba) from Spain: first molecular detection of gammaherpesvirus infection in central nervous system of odontocetes.

BACKGROUND: Herpesvirus infections in cetaceans have always been attributed to the Alphaherpesvirinae and Gammaherpesvirinae subfamilies. To date, gammaherpesviruses have not been reported in the central nervous system of odontocetes. CASE PRESENTATION: A mass stranding of 14 striped dolphins (Stenella coeruleoalba) occurred in Cantabria (Spain) on 18th May 2019. Tissue samples were collected and tested for herpesvirus using nested polymerase chain reaction (PCR), and for cetacean morbillivirus using reverse transcription-PCR. Cetacean morbillivirus was not detected in any of the animals, while gammaherpesvirus was detected in nine male and one female dolphins. Three of these males were coinfected by alphaherpesviruses. Alphaherpesvirus sequences were detected in the cerebrum, spinal cord and tracheobronchial lymph node, while gammaherpesvirus sequences were detected in the cerebrum, cerebellum, spinal cord, pharyngeal tonsils, mesenteric lymph node, tracheobronchial lymph node, lung, skin and penile mucosa. Macroscopic and histopathological post-mortem examinations did not unveil the potential cause of the mass stranding event or any evidence of severe infectious disease in the dolphins. The only observed lesions that may be associated with herpesvirus were three cases of balanitis and one penile papilloma. CONCLUSIONS: To the authors' knowledge, this is the first report of gammaherpesvirus infection in the central nervous system of odontocete cetaceans. This raises new questions for future studies about how gammaherpesviruses reach the central nervous system and how infection manifests clinically.

Assessment of reproducibility of a VP7 Blocking ELISA diagnostic test for African horse sickness.

The laboratory diagnosis of African horse sickness (AHS) is important for: (a) demonstrating freedom from infection in a population, animals or products for trade (b) assessing the efficiency of eradication policies; (c) laboratory confirmation of clinical diagnosis; (d) estimating the prevalence of AHS infection; and (e) assessing postvaccination immune status of individual animals or populations. Although serological techniques play a secondary role in the confirmation of clinical cases, their use is very important for all the other purposes due to their high throughput, ease of use and good cost-benefit ratio. The main objective of this study was to support the validation of AHS VP7 Blocking ELISA up to the Stage 3 of the World Animal Health Organization (OIE) assay validation pathway. To achieve this, a collaborative ring trial, which included all OIE Reference Laboratories and other AHS-specialist diagnostic centres, was conducted in order to assess the diagnostic performance characteristics of the VP7 Blocking ELISA. In this trial, a panel of sera of different epidemiological origin and infection status was used. Through this comprehensive evaluation we can conclude that the VP7 Blocking ELISA satisfies the OIE requirements of reproducibility. The VP7 Blocking ELISA, in its commercial version is ready to enter Stage 4 of the validation pathway (Programme Implementation). Specifically, this will require testing the diagnostic performance of the assay using contemporary serum samples collected during control campaigns in endemic countries.

Phylogenomic analysis of the complete sequence of a gastroenteritis-associated cetacean adenovirus (bottlenose dolphin adenovirus 1) reveals a high degree of genetic divergence.

Adenoviruses are common pathogens in vertebrates, infecting a wide range of hosts, but only having rarely been detected and correlated with disease in cetaceans. This article describes the first complete genomic sequence of a cetacean adenovirus, bottlenose dolphin adenovirus 1 (BdAdV-1), detected in captive bottlenose dolphin population (Tursiops truncatus) suffering from self-limiting gastroenteritis. The complete genome sequence of BdAdV-1 was recovered from data generated by high-throughput sequencing and validated by Sanger sequencing. The genome is 34,080bp long and has 220 nucleotides long inverted terminal repeats. A total of 29 coding sequences were identified, 26 of which were functionally annotated. Among the unusual features of this genome is a remarkably long 4380bp E3 ORF1, that displays no sequence homology with the corresponding E3 regions of other adenoviruses. In addition, the fiber protein only has 26% identity with fiber proteins described in other adenoviruses. Three hypothetical proteins were predicted. The phylogenetic analysis indicates that the closest known relative to BdAdV-1 is an adenovirus detected in bottlenose dolphin (KR024710), with an amino acid sequence identity between 36 and 79% depending on the protein. Based on the phylogenic analysis, the BdAdV-1 appears to have co-evolved with its host. The results indicate that BdAdV-1 belongs to the Mastadenovirus genus of the Adenoviridae family, however, it is clearly different from other adenoviruses, especially in the 3'-end of the viral genome. The high degree of sequence divergence suggests that BdAdV-1 should be considered as a novel species in the Mastadenovirus genus. The study also demonstrates the usefulness of high-throughput sequencing to obtain full-length genomes of genetically divergent viruses.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA.

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

First molecular determination of herpesvirus from two mysticete species stranded in the Mediterranean Sea.

BACKGROUND: Herpesvirus can infect a wide range of animal species: mammals, birds, reptiles, fish, amphibians and bivalves. In marine mammals, several alpha- and gammaherpesvirus have been identified in some cetaceans and pinnipeds species. To date, however, this virus has not been detected in any member of the Balaenoptera genus. CASE PRESENTATION: Herpesvirus was determined by molecular methods in tissue samples from a male fin whale juvenile (Balaenoptera physalus) and a female common minke whale calf (Balaenoptera acutorostrata) stranded on the Mediterranean coast of the Region of Valencia (Spain). Samples of skin and penile mucosa from the fin whale and samples of skin, muscle and central nervous system tissue from the common minke whale tested positive for herpesvirus based on sequences of the DNA polymerase gene. Sequences from fin whale were identical and belonged to the Alphaherpesvirinae subfamily. Only members of the Gammaherpesvirinae subfamily were amplified from the common minke whale, and sequences from the muscle and central nervous system were identical. Sequences in GenBank most closely related to these novel sequences were viruses isolated from other cetacean species, consistent with previous observations that herpesviruses show similar phylogenetic branching as their hosts. CONCLUSIONS: To our knowledge, this is the first molecular determination of herpesvirus in the Balaenoptera genus. It shows that herpesvirus should be included in virological evaluation of these animals.

Novel adenovirus detected in captive bottlenose dolphins (Tursiops truncatus) suffering from self-limiting gastroenteritis.

BACKGROUND: Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated with fatal hepatitis in sea lions. However, only in rare cases have adenoviruses been detected in cetaceans, where no clear correlation was found between presence of the virus and disease status. CASE PRESENTATION: A novel adenovirus was identified in four captive bottlenose dolphins with self-limiting gastroenteritis. Viral detection and identification were achieved by: PCR-amplification from fecal samples; sequencing of partial adenovirus polymerase (pol) and hexon genes; producing the virus in HeLa cells, with PCR and immunofluorescence detection, and with sequencing of the amplified pol and hexon gene fragments. A causative role of this adenovirus for gastroenteritis was suggested by: 1) we failed to identify other potential etiological agents; 2) the exclusive detection of this novel adenovirus and of seropositivity for canine adenoviruses 1 and 2 in the four sick dolphins, but not in 10 healthy individuals of the same captive population; and 3) the virus disappeared from feces after clinical signs receded. The partial sequences of the amplified fragments of the pol and hexon genes were closest to those of adenoviruses identified in sea lions with fatal adenoviral hepatitis, and to a Genbank-deposited sequence obtained from a harbour porpoise. CONCLUSION: These data suggest that adenovirus can cause self-limiting gastroenteritis in dolphins. This adenoviral infection can be detected by serology and by PCR detection in fecal material. Lack of signs of hepatitis in sick dolphins may reflect restricted tissue tropism or virulence of this adenovirus compared to those of the adenovirus identified in sea lions. Gene sequence-based phylogenetic analysis supports a common origin of adenoviruses that affect sea mammals. Our findings suggest the need for vigilance against adenoviruses in captive and wild dolphin populations.

First molecular detection and characterization of herpesvirus and poxvirus in a Pacific walrus (Odobenus rosmarus divergens).

BACKGROUND: Herpesvirus and poxvirus can infect a wide range of species: herpesvirus genetic material has been detected and amplified in five species of the superfamily Pinnipedia; poxvirus genetic material, in eight species of Pinnipedia. To date, however, genetic material of these viruses has not been detected in walrus (Odobenus rosmarus), another marine mammal of the Pinnipedia clade, even though anti-herpesvirus antibodies have been detected in these animals. CASE PRESENTATION: In February 2013, a 9-year-old healthy captive female Pacific walrus died unexpectedly at L'Oceanografic (Valencia, Spain). Herpesvirus was detected in pharyngeal tonsil tissue by PCR. Phylogenetic analysis revealed that the virus belongs to the subfamily Gammaherpesvirinae. Poxvirus was also detected by PCR in skin, pre-scapular and tracheobronchial lymph nodes and tonsils. Gross lesions were not detected in any tissue, but histopathological analyses of pharyngeal tonsils and lymph nodes revealed remarkable lymphoid depletion and lymphocytolysis. Similar histopathological lesions have been previously described in bovine calves infected with an alphaherpesvirus, and in northern elephant seals infected with a gammaherpesvirus that is closely related to the herpesvirus found in this case. Intracytoplasmic eosinophilic inclusion bodies, consistent with poxviral infection, were also observed in the epithelium of the tonsilar mucosa. CONCLUSION: To our knowledge, this is the first molecular identification of herpesvirus and poxvirus in a walrus. Neither virus was likely to have contributed directly to the death of our animal.

Two cases of pseudohermaphroditism in loggerhead sea turtles Caretta caretta.

Two juvenile (curved carapace lengths: 28 and 30 cm) loggerhead sea turtles Caretta caretta with precocious male external characteristics were admitted to the ARCA del Mar rescue area at the Oceanogràfic Aquarium in Valencia, Spain, in 2009 and 2010. Routine internal laparoscopic examination and subsequent histopathology confirmed the presence of apparently healthy internal female gonads in both animals. Extensive tissue biopsy and hormone induction assays were consistent with female sex. To the best of our knowledge, this is the first report of pseudohermaphroditism in loggerhead sea turtles based on sexual external characteristics and internal laparoscopic examination. Our findings suggest that the practice of using external phenotypical characteristics as the basis for gender identification in sea turtles should be reevaluated. Future research should focus on detecting more animals with sexual defects and their possible effects on the sea turtle population.

Global gene expression analysis in skin biopsies of European red deer experimentally infected with bluetongue virus serotypes 1 and 8.

Bluetongue virus (BTV) is a double-stranded RNA virus transmitted by blood-feeding biting midges of the genus Culicoides to wild and domestic ruminants, causing high morbidity and variable mortality. The aim of this study was to characterize differential gene expression in skin biopsies of red deer (Cervus elaphus) hinds experimentally infected with BTV serotypes 1 and 8. Skin biopsies were collected from BTV-1 and BTV-8 experimentally infected and control hinds at 14 and 98 days post-infection (dpi). Global gene expression profile in response to BTV infection was characterized at 14 dpi using a bovine microarray together with real-time RT-PCR analysis of differentially expressed genes at 14 and 98 dpi. Eighteen genes were upregulated and three were downregulated in response to virus infection, with no significant differences between BTV-1 and BTV-8 infected hinds. Seven unique genes, six upregulated (ISG15, PSMB8, PSMB9, BOLA, C1qA, C4) and one downregulated (FOS) were over-represented after conditional test for biological process gene ontology, which affected five molecular pathways (RIG-1, proteasome, MHC-1, complement, TLR) implicated in host immune response. BTV infection had a minor and transient effect on gene expression in hinds, as shown by the very few genes that were differentially expressed in response to infection at 14 dpi, most of which had similar expression levels between infected and uninfected animals at 98 dpi. These results suggested that red deer could control BTV infection with little effect on host molecular pathways.

Molecular diagnosis of lobomycosis-like disease in a bottlenose dolphin in captivity.

We report the diagnosis and molecular characterization of lobomycosis-like lesions in a captive bottlenose dolphin. The clinical picture and the absence of growth in conventional media resembled the features associated with Lacazia loboi. However sequencing of ribosomal DNA and further phylogenetic analyses showed a novel sequence more related to Paracoccidioides brasilensis than to L. loboi. Moreover, the morphology of the yeast cells differed from those L. loboi causing infections humans. These facts suggest that the dolphin lobomycosis-like lesions might have been be caused by different a different fungus clustered inside the order Onygenales. A successful treatment protocol based on topic and systemic terbinafine is also detailed.

Role of hepatic macrophages during the viral haemorrhagic fever induced by African Swine Fever Virus.

To ascertain the role played by the various liver monocyte-macrophage populations in the course of a viral hemorrhagic fever, fifteen pigs were inoculated intramuscularly with the highly virulent isolate of African Swine Fever Virus (ASFV) España-70 and slaughtered at 1-7 days post-inoculation (dpi). Samples of liver were fixed in different solutions and routinely processed for morphological, immunohistochemical and ultrastructural studies. Viral antigen (vp73) was detected from 3 dpi onward, mainly in circulating monocytes of sinusoid and Kupffer's cells (KC), as well as in portal macrophages and hepatocytes from 5 dpi. Anti-SWC3 immunolabelled cells were increased from 1 dpi, peaking between 3 and 5 dpi, thereafter declining until the end of the experiment. The significant increase in the number of sinusoidal circulating monocytes and KC expressing IL-1alpha, TNFalpha and IL-6 from 1 dpi, confirmed the secretory activation of these cells. The results show that in the course of an ASFV-induced hemorrhagic syndrome, hepatic macrophage populations undergo major quantitative and biosynthetic changes prior to virus detection, suggesting the existence of a mechanism by which the virus concentrates infectable cells, which subsequently spread the virus around the body.

Antigenic properties and diagnostic potential of African swine fever virus protein pp62 expressed in insect cells.

African swine fever (ASF) is an infectious and economically important disease of domestic pigs. The absence of vaccine renders the diagnostic test the only tool that can be used for the control of new outbreaks of the disease. At present, the enzyme-linked immunosorbent assay (ELISA) test is the most useful method for large-scale ASF serological studies, although false positives have been detected, mainly on poorly preserved sera. In order to improve the current diagnostic test available for ASF, we have studied the antigenic properties of the ASF virus polyprotein pp62 and its suitability for use in a novel ELISA. Two well-known antigenic proteins of ASF virus, p32 and p54, were also included in this study. These proteins were expressed in the baculovirus expression system and used as antigens in ASF serological tests. Our results indicate that the use of these recombinant proteins as antigens in the ELISAs improves the sensitivity and specificity obtained with the conventional diagnosis test used to detect antibodies against ASF virus. Furthermore, the use of polyprotein pp62 in an ELISA for testing poorly preserved sera allows performance of the diagnosis of ASF without the need to confirm the results by the immunoblot test. These features make pp62 one of the most interesting viral proteins to be used for serological ASF diagnosis.