Apoptosis inhibition of Atlantic salmon (Salmo salar) peritoneal macrophages by Piscirickettsia salmonis.

To improve the understanding of the piscirickettsiosis pathogenesis, the in vivo apoptosis modulation of peritoneal macrophages and lymphocytes was studied in juvenile Salmo salar intraperitoneally injected with Piscirickettsia salmonis. Five fish were sampled at post-exposure days 1, 5, 8 (preclinical), 20 (clinical) and 40 (post-clinical period of the disease), and the leucocytes of their coelomic washings were analysed by flow cytometry (using the JC-1 cationic dye), TUNEL and cytology to detect apoptotic cells. A selective and temporal pattern of apoptosis modulation by P. salmonis infection was observed. Apoptosis in lymphocytes was not affected, whereas it was inhibited in macrophages but only during the preclinical stage of the induced piscirickettsiosis. Hence, it is postulated that P. salmonis inhibits macrophage apoptosis at the beginning of the disease development to survive, multiply and probably be transported inside these phagocytes; once this process is complete, macrophage apoptosis is no longer inhibited, thus facilitating the exit of the bacteria from the infected cells for continuing their life cycle.

Evidence of exotoxin secretion of Piscirickettsia salmonis, the causative agent of piscirickettsiosis.

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial-free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE-214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.

Suicide attempt by self-poisoning: characteristics of suicide attempters seen at the Emergency Room at the University Hospital of the West Indies.

OBJECTIVE: To document the characteristics of self-poisoning suicide attempters who were brought to the University Hospital of the West Indies (UHWI) Emergency Room and to outline the type of drug used in the attempt. METHOD: This was a retrospective study conducted over the period 2005-2009. Data were gathered from patients' case records, log books and the hospital records using a questionnaire developed for this study. The questionnaire examined demographics, parameters of the drug ingested, patient's disposition, and reasons for attempt, final outcome and the type of discharge of patients who reported to the UHWI Emergency Room due to a suicide attempt by self-poisoning. RESULTS: Over the five-year period, 127 cases of suicide attempt by self-poisoning were reported. Significantly more females than males presented to the hospital due to self-poisoning (3:1, chi2 = 33.37; p < 0.001). Of this amount, 96 cases (75.6%) were females and 31 (24.4%) were males. The age group most recorded was 16-30 years (70.8%). The most common reason for the suicide attempt was an interpersonal conflict (52%). The drug category most often used in self-poisoning was analgesics (52%) with acetaminophens being the most common (26.2%). CONCLUSION: These findings are consistent with global suicide trends and indicate an urgent need to develop and implement national preventative and treatment measures for groups known to be at risk of suicidal attempts.

Infectious haemolytic anaemia causes jaundice outbreaks in seawater-cultured coho salmon, Oncorhynchus kisutch (Walbaum), in Chile.

In the last 9 years, epizootics of an icterus condition has affected coho salmon, Oncorhynchus kisutch (Walbaum), reared in seawater cages in southern regions of Chile. At necropsy, fish from field cases exhibited signs of jaundice accompanied by pale light-brown livers and dark spleens. Histopathological and haematological results indicated that these fish presented haemolytic anaemia. After microbiological examination no bacterial or viral agents could be identified as aetiological agents of this disease. In an infectivity trial, coho salmon, Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), were inoculated intraperitoneally with a filtrate of an organ homogenate (0.45 microm) from a diseased coho salmon and held for 60 days in tanks supplied with fresh water. The disease was only reproduced in coho salmon in which mortalities, beginning at day 23 post-inoculation (p.i.), reached a cumulative value of 24% at day 27 p.i. This condition was transmitted to non-inoculated cohabiting coho salmon suggesting that it is a waterborne disease. Thus, this icteric condition is caused by an infectious form of haemolytic anaemia, probably of viral aetiology, and coho salmon are more susceptible than either Atlantic salmon or rainbow trout.

Experimental infection of coho salmon Oncorhynchus kisutch by exposure of skin, gills and intestine with Piscirickettsia salmonis.

Piscirickettsiosis pathogenesis was examined using some tissues as entry portals of Piscirickettsia salmonis in coho salmon. Juvenile fish, weighing approximately 8.4 g, were used in this trial. Inocula were prepared using the strain SLGO-95 of P. salmonis. The micro-organism was cultured in the CHSE-214 cell line as described by Fryer et al. (1990) and doses containing 10(4.7) and 10(3.7) TCID50 were prepared. Each dose was used to infect the fish via skin, gills and intestine. Skin and gills were exposed by calibrated drops, and the intestine by an intubation through the anal opening. Some fish were injected intraperitoneally with the same P. salmonis doses, as positive virulence controls. Sham-inoculated fish for each of the tested routes were also included as negative controls. Piscirickettsiosis was experimentally reproduced with all the inoculation methods. Cumulative mortalities and survival analyses showed that the most effective entry portal was skin followed by intestinal intubation and finally by gill infection.

Routes of entry of Piscirickettsia salmonis in rainbow trout Oncorhynchus mykiss.

Since 1989, Piscirickettsia salmonis, the causal agent of piscirickettsiosis, has killed millions of farmed salmonids each year in southern Chile. The portal of entry for the pathogen was investigated by use of selected experimental infections in juvenile rainbow trout (12 g). The methods used were intraperitoneal injection, subcutaneous injection, patch contact on skin, patch contact on gills, intestinal intubation and gastric intubation. Cumulative mortalities at Day 33 post-inoculation were 98, 100, 52, 24, 24, and 2%, respectively. It was shown that intact skin and gills could be penetrated by P. salmonis. The high mortality obtained in subcutaneously injected fish indicated that skin injuries could facilitate the invasion of this pathogen. Results suggested that the main entry sites are through the skin and gills and that the oral route may not be the normal method by which P. salmonis initiates infection of salmonids.

Immunization with bacterial antigens: piscirickettsiosis.

Piscirickettsiosis is a septicaemic disease of salmonid fish caused by the obligated intracellular rickettsia, Piscirickettsia salmonis. This disease was first reported in 1989 in salmon cultured in sea water netpens in southern Chile where it is still a major problem causing high mortality among cultured salmonids. In recent years related agents have been reported in farmed salmonids from Ireland, Canada and Norway. Mortality, however, at these locations has been reported to be low. Because of the recent description of piscirickettsiosis and its aetiological agent, knowledge about the immune response of fish against this organism is limited. At present, there is only one paper in the literature dealing with this subject. To standardise challenge methods for testing the efficacy of vaccination, lethal dose 50% and infectivity dose 50% were determined for coho salmon (Oncorhynchus kisutch) and rainbow trout (O. mykiss) using intraperitoneal (i.p.) injections of P. salmonis. Experiments using bath challenge methods failed to reproduce the disease using rainbow trout although low levels of infection in their tissues were found. In a field trial, using formalin killed bacterins injected i.p. into pre-smolt coho salmon, the fish were naturally challenged by placing them in sea water where endemic piscirickettsiosis occurred. The results showed that some of the vaccinated fish groups experienced lower cumulative mortality than the non-vaccinated control group (X < 0.05), suggesting an immunoprotective response in these animals. A trial was also conducted with formalin-killed bacterins in rainbow trout using different antigen concentrations with and without booster injections. Fish were challenged by IP injection of P. salmonis. Vaccinated fish showed less mortality than their respective infected control. Unfortunately the challenge was not strong enough because mortality in the infected control fish was low (20%). Antibody levels measured by radio-immuno-assay increased until day 40 post vaccination. The highest levels of antibody were obtained in the sera of fish vaccinated with concentrated antigen using booster injections.