Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 10⁶ CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.     

In situ coagulation and transection of the ovarian pedicle: An alternative to laparoscopic ovariectomy in juvenile horses

The feasibility of leaving the ovaries within the peritoneal cavity after laparoscopic coagulation and transection of the ovarian pedicle was assessed in the juvenile horse. Elective ovariectomy was performed on 10 quarter horses, aged 4 to 5 mo, with the fillies in a Trendelenburg position. The mesovarium was isolated, and multiple coagulation and transection cycles were performed until all ovarian attachments had been severed. The ovaries were dropped within the abdomen, and hemostasis of the transected mesovarium was evaluated before closure. The mean surgical time was 33 min (range, 23 to 48 min). Ten weeks after surgery the fillies were humanely euthanized. At postmortem examination, the ovary location within the abdomen was noted. In 1 horse, there was an abdominal adhesion; viscera had been punctured during insufflation. Of the 20 ovaries, 4 were free-floating within the abdominal cavity; the other 16 were enveloped in the free portion of the greater omentum in the cranioventral abdomen. Histologic examination of the ovaries was performed to assess follicle cell viability. In both the free-floating and the attached ovaries, the deep blood vessels and all examined follicular structures were necrotic and partially mineralized. Laparoscopic electrosurgical transection of the ovarian pedicle without removal of the ovaries should be considered an alternative to other ovariectomy techniques that may be performed in young female horses.     

Co‐culture of sea cucumber Holothuria scabra and red seaweed Kappaphycus striatum

Commercially valuable sea cucumbers are potential co‐culture species in tropical lagoon environments, where they may be integrated into established aquaculture areas used for seaweed farming. In the current study, wild‐caught juvenile sea cucumbers, Holothuria scabra, and red seaweed Kappaphycus striatum were co‐cultured on Zanzibar, United Republic of Tanzania. Sea cucumbers (97 g ± 31 SD, n = 52) were cultured in mesh enclosures at initial cage stocking densities of 124 ± 21 SD and 218 ± 16 SD g m^?2 under seaweed culture lines. Over 83 days, individual growth rate (1.6 g d^?1 ± 0.2 SD) of sea cucumbers at low stocking density was significantly higher (χ² = 8.292, d.f. = 1, P = 0.004) than at high‐stocking density (0.9 g d^?1 ± 0.1 SD). Seaweed individual growth rates [6.27 (±0.3 SE) g d^?1] were highest in co‐culture with sea cucumber at low density but did not differ significantly from high sea cucumber density or seaweed monoculture treatments (χ² = 3.0885, d.f. = 2, P = 0.2135). Seaweed growth varied significantly (χ² = 35.6, d.f. = 2, P < 0.0001) with sampling period, with the final sampling period resulting in the highest growth rate. Growth performance for seaweed and sea cucumbers (χ² = 3.089, d.f. = 2, P = 0.21 and χ² = 0.08, d.f. = 1, P = 0.777 respectively), did not differ significantly between monoculture and co‐culture treatments, yet growth in co‐culture was comparable with that reported for existing commercial monoculture. Results indicate H. scabra is a highly viable candidate species for lagoon co‐culture with seaweed. Co‐culture offers a more efficient use of limited coastal space over monoculture and is recommended as a potential coastal livelihood option for lagoon farmers in tropical regions.     

The macrophage chemotactic activity of Edwardsiella tarda extracellular products

The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from Nile tilapia, Oreochromis niloticus, 5 days following squalene injection. Non-purified ECP derived from both isolates stimulated predominantly chemokinetic migration of macrophages. Additionally, the ECP were semi-purified by high pressure liquid chromatography. The FL6-60 parent ECP yielded higher molecular weight components than did the ECP from the RET-04 mutant. The chemotactic activity of the macrophages for both the FL6-60 parent and RET-04 mutant semi-purified ECP was increased over the non-purified ECP and overall migration was primarily chemotactic. Exposure to ECP derived from virulent and less virulent E. tarda isolates promoted chemokinetic movement of macrophages that may be involved in inflammatory responses of Nile tilapia to E. tarda infection.     

Cutaneous antibodies from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis (Ich) may induce apoptosis of Ich theronts

This study explored the existence of apoptosis (programmed cell death) in Ichthyophthirius multifiliis Fouquet (Ich) theronts and determined the effect of cutaneous antibodies in skin culture fluid from fish immune to Ich on theront apoptosis. Apoptosis was detected in theronts and was clearly distinguished by fluorescent microscopy after staining with acridine orange and propidium iodide. The apoptotic theronts showed characteristic chromatin condensation and nuclear fragments containing chromatin pieces. The externalization of phosphatidylserine on the plasma membrane of apoptotic theronts was detected with fluorescein isothiocyanate-conjugated annexin using flow cytometry. Theront apoptosis was induced using the skin culture fluid from fish immune to Ich, which contained cutaneous antibodies against Ich. The highest apoptosis appeared in theronts exposed to immune skin culture fluid at a 1:10 dilution, compared with those at 1:20 and 1:40 dilutions. A direct correlation was noted between the percentage of apoptotic theronts and exposure duration to immune skin culture fluid. The study indicated that antibody reaction with theronts (immobilization) played an important role in theront apoptosis, but it could not be excluded that other components released from the excised skin had effects on theronts.