OCCURRENCE OF GERM CELL NEOPLASIA IN MALE STRIPED BASS (MORONE SAXATILIS) UNDER HUMAN CARE IN A PUBLIC AQUARIUM: SURGICAL TREATMENT AND OUTCOME.

The striped bass (Morone saxatilis) has been a fish species of special concern in Canada since its marked decline in the early 21st century in the St. Lawrence River. Individuals kept in public aquaria contribute to public education and could support conservation efforts through research. Over a 3-yr period, 12 male striped bass housed in a multispecies exhibit developed coelomic distension. The testes were enlarged (12/12), cystic (2/12), and heterogeneous (3/12) on coelomic ultrasound. Upon coeliotomy, enlarged (12/12), partially (4/12) or totally white discolored (6/12) testes were noted. These were associated with coelomic hemorrhage (8/12), effusion (3/12) or adhesions to surrounding organs (9/12). Orchiectomies were performed in all fish. Among these, seven fish survived 2 mon postsurgery, and four fish were still alive 900 d postsurgery. Germ cell neoplasia was diagnosed on histopathological examination in 9 of 12 individuals, but no abnormalities were found in the three other cases. Preventive orchiectomies were performed on the remaining six male striped bass in this exhibit. Germ cell neoplasms were present in two of these six fish. No anesthetic or surgical complications were noted; all six cases were alive 2 mon postsurgery and four of the fish survived 900 d postsurgery. Survival times were not significantly different between fish that underwent preventive or curative orchiectomy (P = 0.19). Although risk factors associated with the development of these gonadal tumors remain unknown, a genetic or environmental origin is suspected. Orchiectomy should be considered in suspected cases of testicular tumors.

Host Dependent-Transposon for a Plasmid Found in Aeromonas salmonicida subsp. salmonicida That Bears a catB3 Gene for Chloramphenicol Resistance.

Plasmids that carry antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen with severe consequences in salmonid farming. Here, we describe a 67 kb plasmid found in the A. salmonicida subsp. salmonicida Strain SHY15-2939 from Quebec, Canada. This new plasmid, named pAsa-2939 and identified by high throughput sequencing, displays features never found before in this bacterial species. It contains a transposon related to the Tn21 family, but with an unusual organization. This transposon bears a catB3 gene (chloramphenicol resistance) that has not been detected yet in A. salmonicida subsp. salmonicida. The plasmid is transferable by conjugation into Aeromonas hydrophila, but not into Escherichia coli. Based on PCR analysis and genomic sequencing (Illumina and PacBio), we determined that the transposon is unstable in A. salmonicida subsp. salmonicida Strain SHY15-2939, but it is stable in A. hydrophila trans-conjugants, which explains the chloramphenicol resistance variability observed in SHY15-2939. These results suggest that this bacterium is likely not the most appropriate host for this plasmid. The presence of pAsa-2939 in A. salmonicida subsp. salmonicida also strengthens the reservoir role of this bacterium for antibiotic resistance genes, even those that resist antibiotics not used in aquaculture in Québec, such as chloramphenicol.

Expansion of the pRAS3 Plasmid Family in Aeromonas salmonicida subsp. salmonicida and Growing Evidence of Interspecies Connections for These Plasmids.

Aeromonas salmonicida subsp. salmonicida is a pathogenic bacterium responsible for furunculosis in salmonids. Following an outbreak of furunculosis, the infection can be treated with antibiotics, but it is common to observe ineffective treatment due to antibiotic resistance. This bacterium has a wide variety of plasmids responsible for this resistance. Among them, pRAS3 carries a tetracycline resistance gene. Several variants of this plasmid have been discovered over the years (pRAS3-3432 and pRAS3.1 to 3.4). During the present study, two new variants of the plasmid pRAS3 were identified (pRAS3.5 and pRAS3-3759) in strains of A. salmonicida subsp. salmonicida. Plasmid pRAS3-3759, which has been found in many strains from the same region over the past three years, has an additional genetic element identical to one found in pRAS3-3432. This genetic element was also found in Chlamydia suis, a swine pathogen. In this study, we analyzed the bacteria's resistance to tetracycline, the number of copies of the plasmids, and the growth of the strains that carry five of the pRAS3 variants (pRAS3.3 to 3.5, pRAS3-3432, and pRAS3-3759). The results show no particular trend despite the differences between the plasmids, except for the resistance to tetracycline when analyzed in an isogenic background. Blast analysis also revealed the presence of pRAS3 plasmids in other bacterial species, which suggests that this plasmid family has widely spread. This study once again highlights the ability of A. salmonicida subsp. salmonicida to adapt to furunculosis antibiotic treatments, and the still-growing family of pRAS3 plasmids.

A mouse model of human mucopolysaccharidosis IX exhibits osteoarthritis.

Hyaluronidases are endoglycosidases that hydrolyze hyaluronan (HA), an abundant component of the extracellular matrix of vertebrate connective tissues. Six human hyaluronidase-related genes have been identified to date. Mutations in one of these genes cause a deficiency of hyaluronidase 1 (HYAL1) resulting in a lysosomal storage disorder, mucopolysaccharidosis (MPS) IX. We have characterized a mouse model of MPS IX and compared its phenotype with the human disease. The targeted Hyal1 allele in this model had a neomycin resistance cassette in exon 2 that replaced 753 bp of the coding region containing the predicted enzyme active site. As a result, Hyal1(-/-) animals had no detectable wild-type Hyal1 transcript, protein or serum activity. Hyal1 null animals were viable, fertile and showed no gross abnormalities at 1 year and 8 months of age. Histological studies of the knee joint showed a loss of proteoglycans occurring as early as 3 months that progressed with age. An increased number of chondrocytes displaying intense pericellular and/or cytoplasmic HA staining were detected in the epiphyseal and articular cartilage of null mice, demonstrating an accumulation of HA. Elevations of HA were not detected in the serum or non-skeletal tissues, indicating that osteoarthritis is the key disease feature in a Hyal1 deficiency. Hyal3 expression was elevated in Hyal1 null mice, suggesting that Hyal3 may compensate in HA degradation in non-skeletal tissues. Overall, the murine MPS IX model displays the key features of the human disease.

Cytokine and chemokine gene expression of IL-1beta stimulated equine articular chondrocytes.

OBJECTIVE: To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. STUDY DESIGN: In vitro experiment using equine chondrocyte cultures. SAMPLE POPULATION: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). METHODS: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and ubiquitin (house keeping gene) mRNA expression were investigated by real-time RT-PCR. RESULTS: IL-1beta, IL-6, and IL-8 mRNA were expressed in unstimulated chondrocytes from macroscopically normal joints and were significantly up-regulated after stimulation (5/5 horses). IL-4 mRNA was not detected in any samples (0/5 horses). TNF-alpha mRNA, by comparison, was expressed in 2/5 unstimulated samples and in all stimulated samples but a considerable sample variation in response to IL-1beta stimulation was observed. CONCLUSIONS: Equine chondrocytes express mRNA for several proinflammatory cytokines and chemokines and IL-1beta modulates their expression. CLINICAL RELEVANCE: Chondrocytes express proinflammatory cytokines and chemokines capable of modulating a local inflammatory cascade in articular cartilage, which could potentially lead to focal degradation and osteoarthritis.

Evaluation of coexpression of microsomal prostaglandin E synthase-1 and cyclooxygenase-2 in interleukin-1-stimulated equine articular chondrocytes.

OBJECTIVE: To characterize expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) and regulation of prostaglandin E2 (PGE2) production by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage from the metacarpophalangeal joints of 7 adult horses. PROCEDURE: Equine chondrocyte monolayer cultures were stimulated with different concentrations (2.5, 5, 10, and 20 ng/mL) of recombinant human interleukin-1beta (rhIL-1beta) for 24 hours and then with rhIL-1beta (5 ng/mL) for 3, 6, 9, 12, and 24 hours. Concentration of PGE2 in the media was measured via radioimmunoassay. Total RNA was extracted from harvested chondrocytes, and regulation of COX-2 and mPGES-1 mRNA was studied via reverse transcriptase-polymerase chain reaction assay and Southern blot analysis with equine-specific probes. Western blot analyses were performed on cellular extracts to characterize expression of COX-2 and mPGES-1 protein. RESULTS: Stimulation with 5, 10, and 20 ng of rhIL-1beta/mL caused a significant increase in PGE2 concentrations in the culture media, and incubation of cells with rhIL-1beta (5 ng/mL) for 6 to 24 hours increased PGE2 production significantly. The increase in prostaglandin production was associated with an induction of COX-2 and mPGES-1 transcripts. There also was an rhIL-1beta-dependent induction in COX-2 and mPGES-1 protein expression. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, results indicated that the rhIL-1beta-dependent increase in PGE2 production in equine chondrocytes in monolayer culture was associated with coordinated upregulation of COX-2 and mPGES-1 expression. The pathophysiologic consequences of upregulated COX-2 and mPGES-1 expression and of PGE2 synthesis in rhIL-1beta-stimulated equine chondrocytes remain to be elucidated.