Advances in Viral Aquatic Animal Disease Knowledge: The Molecular Methods' Contribution

Viruses are pervasive components of aquatic ecosystems, and most of them are harmless to humans and animals; however, several aquatic viruses can infect animals, leading to diseases, especially when fish are confined, such as in aquaculture facilities. Traditional methods used to detect and study viruses have been widely applied to aquatic animals' viruses, leading to the successful isolation, identification and understanding of several of them. However, they have limits, which can be overcome by molecular methods, such as polymerase chain reaction (PCR)-based assays, sequencing and in situ hybridisation. A standard PCR, followed by the sequencing of purified amplicons, is an effective method for both identifying well-known viruses and discovering new ones. In situ hybridisation, in which a labelled probe binds to a nucleic acid sequence in tissue, is able to correlate the presence of viruses to lesions. Novel molecular isothermal methods, such as loop-mediated isothermal amplification (LAMP), were also developed and applied to viral aquatic animal diseases, bringing molecular diagnosis into the field. This review considers the scientific literature dealing with the molecular methods employed hitherto to study the most relevant finfish and shellfish viral pathogens, stressing their advantages and disadvantages. Aquaculture is the fastest-growing food-producing sector, with a global production of 122.6 million tonnes in 2020. Nonetheless, aquatic animal production can be hampered by the occurrence of viral diseases. Furthermore, intensive farming conditions and an increasing number of reared fish species have boosted the number of aquatic animals' pathogens that researchers have to deal with, requiring the quick development of new detection and study methods for novel unknown pathogens. In this respect, the molecular tools have significantly contributed to investigating thoroughly the structural constituents of fish viruses and providing efficient detection methods. For instance, next-generation sequencing has been crucial in reassignment to the correct taxonomic family, the sturgeon nucleo-cytoplasmic large DNA viruses, a group of viruses historically known, but mistakenly considered as iridoviruses. Further methods such as in situ hybridisation allowed objectifying the role played by the pathogen in the determinism of disease, as the cyprinid herpesvirus 2, ostreid herpesvirus 1 and betanodaviruses. Often, a combination of molecular techniques is crucial to understanding the viral role, especially when the virus is detected in a new aquatic animal species. With this paper, the authors would critically revise the scientific literature, dealing with the molecular techniques employed hitherto to study the most relevant finfish and shellfish viral pathogens

Detection of Human and Fish Viruses in Marine Gastropods

Marine gastropods represent a major food source for higher trophic levels and an important source of animal protein for humans. Like bivalve molluscs, gastropods can accumulate several types of contaminants; however, the bioaccumulation of microorganisms, particularly viruses, has been poorly investigated in these animals. This study focused on gastropods (Tritia mutabilis, Bolinus brandaris and Rapana venosa) collected during the fishing season from 2017 to 2021 in the north-western Adriatic Sea, and on clams (Ruditapes philippinarum) harvested in the same geographical area, in order to evaluate the presence of human and fish viruses in their tissues. A virological investigation was carried out on the digestive gland using molecular methods. The presence of hepatitis A virus was detected in one sample, whereas noroviruses were not present in the investigated specimens. Regarding fish viruses, it was possible to detect the presence of nervous necrosis virus (NNV) in 26.5% of the analyzed gastropods; however, the histological examination did not show any pathological changes in the nervous tissue in both NNV-positive and -negative batches. As a whole, the investigated gastropods showed the ability to bioaccumulate viruses; however, lower contamination by human viruses compared to bivalve molluscs was pointed out, posing a minor concern to human health

Mutational Analysis of c-KIT and PDGFRA in Canine Gastrointestinal Stromal Tumors (GISTs)

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the canine gastrointestinal tract and are diagnosed by the immunohistochemical expression of the receptor tyrosine kinase (RTK) KIT. Activating mutations of the proto-oncogenes c-KIT and PDGFRA drive GIST oncogenesis and are used to predict the response to RTK-inhibitors in human oncology. Currently, the frequency and significance of these mutations in canine GIST have not been adequately explored. Therefore, we investigated the mutational status of c-KIT (exons 9, 11 and 13) and PDGFRA (exons 12 and 18) genes by PCR followed by fragment analysis for c-KIT deletions and PCR followed by screening with DHPLC and direct sequencing confirmation for single nucleotide variations in 17 formalin-fixed paraffin-embedded canine GISTs confirmed by KIT immunopositivity. c-KIT mutations were detected in 47% of cases, with a mutation detection rate significantly higher (p = 0.0004, Fisher's exact test) and always involving exon 11. A PDGFRA gene mutation (exon 18) was identified in one case. Even if follow-up data were not available for all cases, four cases with documented abdominal metastases displayed c-KIT mutations. These data confirm that c-KIT exon 11 mutations occur frequently in canine GISTs, and identify the presence of a PDGFRA mutation similar to human GISTs. This study also suggests a potential association of c-KIT mutation with more aggressive biological behavior

Feeding common sole (Solea solea) juveniles with increasing dietary lipid levels affects growth, feed utilization and gut health

Knowledge about the nutritional requirements of common sole (Solea solea) is limited and no information regarding the optimal dietary lipid level is available yet. Thus, this study was undertaken to assess the growth response and feed utilization of common sole juveniles fed diets with increasing lipid levels. Four isonitrogenous (59% protein) pelletized diets with different dietary lipid levels (8%, 12%, 16% and 20%; L8, L12, L16 and L20, respectively) were fed to triplicate fish groups of 80 individuals to apparent satiation over 150 days. A one-way ANOVA, Tukey's post hoc test and linear regression were used to analyse the data (P 64 0.05). At the end of the trial, final body weight was significantly higher in fish fed L8 (40.7 \ub1 1.7 g), followed by those fed L12 (35.1 \ub1 1.2 g), L16 (27.9 \ub1 2.5 g) and L20 (22.1 \ub1 0.3 g). The specific growth rate was higher in fish fed L8 and L12, compared to the other treatments, and it was lowest in L20. Voluntary feed intake decreased with an increase in the dietary lipid level. The feed conversion rate, the protein efficiency ratio and the gross protein efficiency were lower in fish fed L20, while no significant differences were observed among L8, L12 and L16. Gross lipid efficiency was significantly higher in fish fed low lipid diets. Histological observations showed that 19 of 36 observed subjects had lipid droplets in the cytoplasm of enterocytes at the apex of the mucosal folds and, in some cases, also along the entire fold (intestinal steatosis). The number of fish with intestinal steatosis in groups L20 and L16 was significantly higher than the number in group L8. Ultrastructure showed large electrondense lipid droplets within the cytoplasm of enterocytes and warping of the cytoplasmic membrane (steatosis); in some cases, lipid droplets were also present within the Golgi apparatus. In conclusion, the results of this trial suggest that the diet for S. solea juveniles should include no more than 12% lipids. Higher lipid inclusions not only led to a substantial decline in performance but also affected gut health. This should be taken into consideration in formulating specific practical diets for common sole

Swim Bladder Disorders in Koi Carp (Cyprinus carpio)

Swim bladder disorders and consequent buoyancy problems are encountered in ornamental fish, including koi carp. Nevertheless, beyond clinical and pharmacologicalmanagement, they are largely underdiagnosed. In this study, nine koi carp showing abdominal swelling and abnormal swimming behavior were investigated. Clinical approach, varying from case to case, included ultrasonographic and X-ray investigations, bacteriological analysis of the collected fluid, antimicrobial susceptibility pattern, and possibly histological analysis. Diagnostic imaging, corroborating gross examination, documented swim bladder deformation/dislocation and serous fluid within the swim bladder chambers of most animals. Bacteria belonging to the Aeromonas hydrophila/caviae group and Shewanella xiamenensis were identified. S. xiamenensis strains showed a sensibility to all tested molecules except for one strain, which was resistant to tetracycline and cyprofloxacin. Antibiotic treatment succeeded in the full recovery of three cases in which S. xiamemensis infection was detected. Chronic aerocystitis was histologically documented where tissue was available. The swim bladder histopathological findings highlighted a chronic process that had compromised the quality of life of the animals. A multidisciplinary clinical\u2013pathological and microbiological approach is highly suggested to recognize swim bladder conditions as early as possible, aiming to drive medical intervention and raising the chances of fish survival

Porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus alone or associated are frequent intralesional detected viruses in porcine respiratory disease complex cases in Northern Italy

Methods: This study aimed to examine the pathological impact of Porcine Circovirus type 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) through histological and immunohistochemical analysis of 79 cases of Porcine Respiratory Disease Complex (PRDC) collected from 22 farms in Northern Italy. Lung tissue and several lymphoid organ samples were deployed to associate PCV2-positive stain with Circovirus-associated Diseases (PCVD). Results: The most common lung lesion observed was interstitial pneumonia, alone or combined with bronchopneumonia. By immunohistochemistry, 44 lungs (55.7%) tested positive for PCV2, 34 (43.0%) for PRRSV, 16 (20.3%) for both viruses and in 17 cases (21.5%) neither virus was detected. Twenty-eight out of 44 (63.6%) PCV2-positive cases had lymphoid depletion or granulomatous inflammation in at least one of the lymphoid tissues examined; thus, they were classified as PCV2 Systemic Diseases (PCV2-SD). In the remaining 16 out of 44 cases (36.4%), PCV2-positive lung lesions were associated with hyperplastic or normal lymphoid tissues, which showed PCV2-positive centrofollicular dendritic cells in at least one of the lymphoid tissues examined. Therefore, these cases were classified as PRDC/PCV2-positive. In the PCV2-positive animals, 42.9% of the PCV2-SD cases (12/28) showed immunohistochemistry (IHC) positivity for PRRSV in the lung tissue, while 25.0% of PRDC/PCV2-positive cases (4/16) showed double positivity for PCV2 and PRRSV. Discussion: In light of the caseload presented in this study, characterized by the high proportion of PCV2-SD cases alongside the overall respiratory symptomatology, it is imperative to emphasize the crucial role of a comprehensive sampling protocol. This is critical to avoid underestimating the harm caused by PCV2 in farms, particularly with respect to the systemic form of the disease. PCV2 and PRRSV remain the primary infections associated with PRDC in Italy that can significantly impact farm health and co-infections in the field can worsen the pathology, thus the selection of appropriate preventive measures is critical

Degenerative Myelopathy in Hovawart Dogs: Molecular Characterization, Pathological Features and Accumulation of Mutant Superoxide Dismutase 1 Protein

Degenerative myelopathy (DM) is an adult-onset, progressive neurological disease affecting several breeds of dog. Homozygosity or compound heterozygosity for the canine superoxide dismutase 1 (SOD1) gene mutations, possibly modulated by the modifier SP110 locus, are associated with a high risk for DM. Although the pathophysiological mechanisms are largely unknown, a role for mutant SOD1 in causing neuronal degeneration has been postulated. Three Hovawart dogs, 9e12 years of age, developed slowly progressive incoordination and weakness of the pelvic limbs leading to non-ambulatory flaccid paraparesis and muscle atrophy. Neuropathological lesions comprised axonal degeneration and loss of ascending and descending spinal pathways, which were most severe in the mid- to caudal thoracic segments. Accumulation of mutant SOD1 protein in neurons and reactive astrocytes was demonstrated by immunolabelling with the 16G9 antibody against the mutant SOD1 protein (p.E40K amino acid substitution). All three dogs were homozygous for the c.118A allele, but none had the SP110 ‘risk’ haplotype, suggesting a weak association of SP110 with the onset of DM in this breed. Our data suggest that the Hovawart breed is predisposed to the SOD1:c.118G>A mutation, which is associated with the development of DM. Prevention of DM could be achieved with the help of strategies based on epidemiological and genetic testing

Next-generation sequencing characterization of the gut bacterial community of gilthead sea bream (Sparus aurata, L.) fed low fishmeal based diets with increasing soybean meal levels

The present study was carried out to evaluate growth, gut histology and gut bacterial community of gilthead sea bream (Sparus aurata) fed with increasing dietary soybean meal (SBM) levels in a low fishmeal (FM) based diet, in comparison with a control diet. Five isoproteic and isolipidic experimental diets were formulated to contain increasing levels of SBM (0, 100, 200, and 300 g kg 121 named S0, S10, S20 and S30, respectively) with 150 g kg 121 of FM, and one control diet (C) without SBM and containing 350 g kg 121 of FM. Sixty sea bream (initial body weight 75.9 \ub1 1.9 g, n = 900) per tank were reared in a recirculation system at 23.0 \ub1 1.0 \ub0C and fed to satiation. The trial was run in triplicate and lasted 100 days. At the end of the trial fish fed the S30 diet showed a higher (P 64 0.05) specific growth rate (SGR) compared to S0 (SGR, 1.17 \ub1 0.03, 1.20 \ub1 0.01, 1.22 \ub1 0.01, 1.25 \ub1 0.01 and 1.21 \ub1 0.04 for S0, S10, S20, S30 and C, respectively), and a higher feed intake (FI) compared to S0, S10 and S20. Sea bream fed the C diet had a higher (P 64 0.05) FI compared to S0 (FI, 1.40 \ub1 0.01, 1.45 \ub1 0.01, 1.44 \ub1 0.03, 1.51 \ub1 0.03 and 1.46 \ub1 0.02 for S0, S10, S20, S30 and C, respectively). No significant differences in feed conversion rate, protein efficiency ratio, gross protein efficiency and gross lipid efficiency among the treatments were detected. No specific histopathological changes indicative of soy-induced enteritis were observed in the intestine of any fish examined. Gut bacterial community of the distal intestine content was analyzed by Next-Generation Sequencing. At the phylum level, the gut bacterial community was dominated by Firmicutes (relative abundance 71%), while the most represented family was Lactobacillaceae (26%). Even if no significant differences (P 64 0.05) in the gut bacterial community \u3b1 and \u3b2-diversity according to the different diets were detected, Cyanobacteria and Lactobacillaceae progressively increased from diet C to diet S30. In conclusion results of growth, nutrient utilization, gut histology and gut bacterial community indicate that SBM can be successfully incorporated up to a level of 300 g kg 121 with the inclusion of 150 g kg 121 of FM, without any deleterious effects on growth, protein utilization and gut health during the on-growing of sea bream

Histochemical Analysis of Herniated Disc Tissue Surgically Removed from 27 Dogs

Hernias of intervertebral discs are a common canine disease that is usually treated surgically. Recently, a histological scoring system for surgically removed canine intervertebral herniated discs has been developed by scoring the lesions of both the anulus fibrosus (AF) and the nucleus pulposus (NP). Since the proportion of AF and NP in the surgical samples may vary, depending on the surgical approach, the aim of this study is to grade separately the lesions in AF and NP and to modify the previously described scoring system adding three parameters: inflammation, mineralization and neovascularization. Possible association of the modified grading system with clinical parameters were statistically assessed. Herniated disc material was collected from 27 dogs. AF was present in 10/27 cases and was classified as grade 2 in 4 cases, grade 3 in 5 cases and grade 4 in 1 case. The NP was present in all 27 cases and was classified as grade 2 in 1 case, grade 3 in 5 cases, grade 4 in 9 cases and grade 5 in 12 cases. A statistically significant association was evidenced between short pre-operative period and higher grade of both the NP and of the AF (P<0.01). Separating the grades of the AF and NP can be useful for a fair assessment of degeneration, and circumventing the limitation of the qualitative and quantitative variability of samples