Herpesvirus-Associated Proliferative Skin Disease in Frogs and Toads: Proposed Pathogenesis.

A comparative study was carried out on common and agile frogs (Rana temporaria and R. dalmatina) naturally infected with ranid herpesvirus 3 (RaHV3) and common toads (Bufo bufo) naturally infected with bufonid herpesvirus 1 (BfHV1) to investigate common pathogenetic pathways and molecular mechanisms based on macroscopic, microscopic, and ultrastructural pathology as well as evaluation of gene expression. Careful examination of the tissue changes, supported by in situ hybridization, at different stages of development in 6 frogs and 14 toads revealed that the skin lesions are likely transient, and part of a tissue cycle necessary for viral replication in the infected hosts. Transcriptomic analysis, carried out on 2 naturally infected and 2 naïve common frogs (Rana temporaria) and 2 naturally infected and 2 naïve common toads (Bufo bufo), revealed altered expression of genes involved in signaling and cell remodeling in diseased animals. Finally, virus transcriptomics revealed that both RaHV3 and BfHV1 had relatively high expression of a putative immunomodulating gene predicted to encode a decoy receptor for tumor necrosis factor in the skin of the infected hosts. Thus, the comparable lesions in infected frogs and toads appear to reflect a concerted epidermal and viral cycle, with presumptive involvement of signaling and gene remodeling host and immunomodulatory viral genes.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

17Beta-estradiol affects the response of complement components and survival of rainbow trout (Oncorhynchus mykiss) challenged by bacterial infection.

Research on the endocrine role of estrogens has focused on the reproductive system, while other potential target systems have been less studied. Here, we investigated the possible immunomodulating role of 17ß-estradiol (E2) using rainbow trout (Oncorhynchus mykiss) as a model. The aims of the study were to examine a) whether estrogens can modulate immune gene transcription levels, and b) whether this has functional implications for the resistance of trout towards pathogens. Trout were reared from fertilization until 6 months of age under (1) control conditions, (2) short-term E2-treatment (6-month-old juveniles were fed a diet containing 20 mg E2/kg for 2 weeks), or c) long-term E2-treatment (twice a 2-h-bath-exposure of trout embryos to 400 µg 17ß-estradiol (E2)/L, followed by rearing on the E2-spiked diet from start-feeding until 6 months of age). Analysis of plasma estrogen levels indicated that the internal estrogen concentrations of E2-exposed fish were within the physiological range and analysis of hepatic vitellogenin mRNA levels indicated that the E2 administration was effective in activating the endogenous estrogen receptor pathway. However, expression levels of the hepatic complement components C3-1, C3-3, and Factor H were not affected by E2-treatment. In a next step, 6-month-old juveniles were challenged with pathogenic bacteria (Yersinia ruckeri). In control fish, this bacterial infection resulted in significant up-regulation of the mRNA levels of hepatic complement genes (C3-1, C3-3, Factor B, Factor H), while E2-treated fish showed no or significantly lower up-regulation of the complement gene transcription levels. Apparently, the E2-treated trout had a lower capacity to activate their immune system to defend against the bacterial infection. This interpretation is corroborated by the finding that survival of E2-treated fish under bacterial challenge was significantly lower than in the control group. In conclusion, the results from this study suggest that estrogens are able to modulate immune parameters of trout with functional consequences on their ability to cope with pathogens.

Improved polymerase chain reaction-based method to detect early-stage epitheliotropic T-cell lymphoma (mycosis fungoides) in formalin-fixed, paraffin-embedded skin biopsy specimens of the dog.

In the dog, early-stage epitheliotropic T-cell lymphoma (ETCL) can clinically and histologically mimic a large range of inflammatory dermatoses and often progresses rapidly to a more aggressive tumor stage. Early diagnosis of ETCL is essential to proceed with a specific oncologic therapy that is favorable for the prognosis. In the present study, an improved method for the detection of T-cell receptor gamma (TCRgamma) rearrangement was developed by designing a new set of consensus primers to amplify the different forms of rearranged canine TCRgamma gene sequences by polymerase chain reaction. The amplicons were analyzed by conventional polyacrylamide gel electrophoresis, which requires minimal specific equipment and may be performed in almost every pathology laboratory at low costs. The method proved to be highly specific and sensitive to detect early ETCL in formalin-fixed, paraffin-embedded biopsy specimens, providing an efficient tool for veterinary pathologists to distinguish early neoplastic from reactive cutaneous T-cell infiltrates (tumor-specific marker) or to discriminate T-cell lymphoma from B-cell lymphomas or nonlymphoid neoplasms (T-cell lineage marker). By direct sequencing analysis of amplified TCRgamma gene sequences, ETCL was found to rearrange exclusively the joining (J) 4 region, which suggests specific biology for primary cutaneous T-cell lymphomas. Also, a novel (seventh) functional J region in the TCRgamma gene, localized approximately 2.3 kb upstream of J5, was identified.

Spaying-induced coat changes: the role of gonadotropins, GnRH and GnRH treatment on the hair cycle of female dogs.

Although spaying can result in qualitative hair coat changes in dogs, the influence of spaying on the hair growth cycle has never been described. The study aims were to examine the effect of spaying and gonadotropin-releasing hormone (GnRH) treatment on canine hair coat, cycle stages of hair follicles, plasma gonadotropin concentrations and mRNA transcription of luteinizing hormone (LH) and GnRH receptors in hair follicles. Fifteen female dogs were examined before and 1 year after spaying and 24 spayed dogs before and after GnRH treatment. Spaying resulted in increased plasma gonadotropin concentrations and increased anagen : telogen ratio of hair follicles, but only 20% of the dogs developed coat changes. No differences were found in mRNA transcription of LH and GnRH receptors. GnRH treatment resulted in reduced plasma gonadotropin concentrations and improvement of coat changes in 79% of patients. This was associated with an increase in catagen hair follicles without changes in the anagen : telogen ratio. The present study demonstrated that spaying had an effect on the anagen : telogen ratio of hair follicles. Spaying-induced coat changes did not correlate with the anagen : telogen ratio. GnRH treatment reduced gonadotropin concentrations and reversed coat changes in some dogs, but had no effect on the hair growth cycle other than increasing the number of catagen hair follicles. A weak positive correlation between the plasma LH concentration and the anagen : telogen ratio was noted; however, our data did not suggest a direct receptor-mediated hormonal effect on the hair follicle. The present study did not identify the pathomechanism of spaying-induced coat changes.

Bufonid herpesvirus 1 (BfHV1) associated dermatitis and mortality in free ranging common toads (Bufo bufo) in Switzerland.

Here we report the discovery and partial characterization of a novel herpesvirus tentatively named Bufonid herpesvirus 1 (BfHV1) from severe dermatitis in free ranging common toads (Bufo bufo) in Switzerland. The disease has been observed in toads every year since 2014, in spring, during the mating season, at different and distant locations. The virus is found in the skin and occasionally in the brain of infected toads. The genome of the virus is at least 158 Kb long and contains at least 152 open reading frames with a minimal length of 270 nt. The genome of BfHV1 contains all the signature genes that are present in alloherpesviruses. Phylogenetic analysis based on the amino acid sequence of the DNA polymerase and terminase proteins positions the novel virus among the members of the genus Batrachovirus, family Alloherpesviridae. This is the first herpesvirus ever characterized in common toads.

A Genomic Approach to Unravel Host-Pathogen Interaction in Chelonians: The Example of Testudinid Herpesvirus 3.

We report the first de novo sequence assembly and analysis of the genome of Testudinid herpesvirus 3 (TeHV3), one of the most pathogenic chelonian herpesviruses. The genome of TeHV3 is at least 150,080 nucleotides long, is arranged in a type D configuration and comprises at least 102 open reading frames extensively co-linear with those of Human herpesvirus 1. Consistently, the phylogenetic analysis positions TeHV3 among the Alphaherpesvirinae, closely associated with Chelonid herpesvirus 5, a Scutavirus. To date, there has been limited genetic characterization of TeHVs and a resolution beyond the genotype was not feasible because of the lack of informative DNA sequences. To exemplify the potential benefits of the novel genomic information provided by this first whole genome analysis, we selected the glycoprotein B (gB) gene, for detailed comparison among different TeHV3 isolates. The rationale for selecting gB is that it encodes for a well-conserved protein among herpesviruses but is coupled with a relevant antigenicity and is consequently prone to accumulate single nucleotide polymorphisms. These features were considered critical for an ideal phylogenetic marker to investigate the potential existence of distinct TeHV3 genogroups and their associated pathology. Fifteen captive tortoises presumptively diagnosed to be infected with TeHVs or carrying compatible lesions on the basis of either the presence of intranuclear inclusions (presumptively infected) and/or diphtheronecrotic stomatitis-glossitis or pneumonia (compatible lesions) were selected for the study. Viral isolation, TeHV identification, phylogenetic analysis and pathological characterization of the associated lesions, were performed. Our results revealed 1) the existence of at least two distinct TeHV3 genogroups apparently associated with different pathologies in tortoises and 2) the first evidence for a putative homologous recombination event having occurred in a chelonian herpesvirus. This novel information is not only fundamental for the genetic characterization of this virus but is also critical to lay the groundwork for an improved understanding of host-pathogen interactions in chelonians and contribute to tortoise conservation.

Estrogen modulates hepatic gene expression and survival of rainbow trout infected with pathogenic bacteria Yersinia ruckeri.

In the aquatic environment, fish are exposed to various stimuli at once and have developed different response mechanisms to deal with these multiple stimuli. The current study assessed the combined impacts of estrogens and bacterial infection on the physiological status of fish. Juvenile rainbow trout were exposed to two different concentrations of 17ß-estradiol (E2) (2 or 20 mg/kg feed) and then infected with three concentrations of Yersinia ruckeri, a bacterial pathogen causing massive losses in wild and farmed salmonid populations. Organism-level endpoints to assess the impact of the single and combined treatments included hepatic vitellogenin transcript expression to evaluate the E2 exposure efficiency and survival rate of pathogen-challenged fish. The two E2 doses increased vitellogenin levels within the physiological range. Infection with Y. ruckeri caused mortality of trout, and this effect was significantly enhanced by a simultaneous exposure to high E2 dose. The hormone reduced survival at intermediate and high (10(4) and 10(6) colony forming units, cfu) bacterial concentrations, but not for a low one (10(2) cfu). Analysis of hepatic gene expression profiles by a salmonid 2 k cDNA microarray chip revealed complex regulations of pathways involved in immune responses, stress responses, and detoxicification pathways. E2 markedly reduced the expression of several genes implicated in xenobiotic metabolism. The results suggest that the interaction between pathogen and E2 interfered with the fish's capability of clearing toxic compounds. The findings of the current study add to our understanding of multiple exposure responses in fish.

Immunohistochemical localization and quantitative assessment of GnRH-, FSH-, and LH-receptor mRNA Expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying.

It has been proposed that gonadotropins and/or gonadotropin releasing hormone (GnRH) could be involved in the pathophysiology of the side effects after spaying in bitches, such as urinary incontinence and an increased production of a woolly undercoat. In order to provide tools to investigate the role of these hormones in dogs we developed immunohistochemical techniques and real-time RT-PCR to study whether GnRH-, LH-, and FSH-receptors exist in canine skin and urinary bladder. Tissue samples from the skin of the flank region and the ventral midline of the urinary bladder from euthanised dogs were examined. We were able to quantify mRNA expression of GnRH-, FSH-, and LH-receptors in canine skin and bladder biopsies with a high primer efficacy. Immunohistochemical studies showed that GnRH-, FSH-, and LH-receptors are expressed in vessel walls, the epidermis, the hair follicle and in sebaceous and sweat glands in canine skin and in transitional epithelium, and smooth muscle tissue in the urinary bladder. Our data provide the fundamentals to examine the distribution of FSH-, LH-, and GnRH-receptors in canine skin and urinary bladder and to assess gene activity at the transcriptional level by real-time RT-PCR.

Association studies using random and "candidate" microsatellite loci in two infectious goat diseases.

We established a set of 30 microsatellites of Bovidae origin for use in a biodiversity study in Swiss and Creole goats. Additional microsatellites located within or next to "candidate" genes of interest, such as cytokine genes (IL4, INF-gamma) and MHC class II genes (DRB, DYA) were tested in the caprine species in order to detect possible associations with two infectious caprine diseases. Microsatellite analysis was undertaken using automated sequencers (ABI373 & 3100). In the first study, a total of 82 unrelated Creole goats, 37 resistant and 45 susceptible to Heartwater disease (Cowdriosis) were analysed. In this study, the two microsatellite loci DRBP1 (MHCII) and BOBT24 (IL4) were positively associated with disease susceptibility, demonstrating a corrected P-value of 0.002 and 0.005, respectively. In a second investigation, we tested 36 goats, naturally infected with the nematode parasite Trichostrongylus colubriformis. These animals were divided into a "low" and "high" excreting group on the basis of two independently recorded fecal egg counts. For this nematode resistance study, we detected a significant association of one of the alleles of the microsatellite locus SPS113 with "low" excretion (resistance). The MHC class II locus DYA (P19), was weakly associated with susceptibility in both diseases (Pc = 0.05). In future experiments, we will extend the sample size in order to verify the described associations.