Since 1989, Piscirickettsia salmonis, the causal agent of piscirickettsiosis, has killed millions of farmed salrnonids 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 s k i injunes 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.

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 TCID 50 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. KEY WORDS: Piscirickettsia salmonis · Pathogenesis · Fish disease · Coho salmonResale or republication not permitted without written consent of the publisher

Piscirickettsia salmonis is a pathogenic bacterial agent causing septicaemic disease in salmon. Since its isolation in Chile in 1989, P. salmonis has continually produced high mortality rates in salmon farms. Little information exists regarding the mechanisms of vertical transmission of this pathogen. Experimental vertical transmission was established in the present study by inoculation of male and female rainbow trout broodstock with P. salmonis. The bacterium was subsequently detected using indirect immunofluorescence in milt and coelomic fluid of the majority of inoculated broodstock (14/15). Bacteria were detected in the fry when 1 or both parents were inoculated, although none of the infected fry presented signs of the disease. P. salmonis was also detected in progeny obtained through fertilisation ova from non-inoculated females incubated in a medium containing a bacterial suspension, demonstrating transmission during the process of fertilisation. Ova infected in vitro were examined at sample periods from 30 s to 60 min using scanning electron microscopy. This demonstrated that the bacterium attaches to the ova by means of membrane extensions, structures which we have called 'piscirickettsial attachment complex' (PAC) and which would allow later penetration into the ovum.

Periodontitis is a host-mediated bacterial disease that affects the tooth attachment apparatus. Metalloproteinase-8 (MMP-8), a validated biomarker, could aid in clinical diagnosis. This study aimed to evaluate the diagnostic performance of active (a) MMP-8 immunotest versus total (t) MMP-8 ELISA for quantitative real-time diagnosis and assessment of periodontitis severity at the site level. Gingival crevicular fluid (GCF) was sampled from 30 healthy, 42 mild, and 59 severe periodontitis sites from thirty-one volunteers. MMP-8 concentrations were determined by time-resolved immunofluorometric assay (IFMA) and enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using the STATA package. Both active and total MMP-8-based methods discriminated among sites according to periodontal diagnosis and severity, with a positive correlation between the two tests (p < 0.001). (a) MMP-8 models showed the best performance in receiver operating characteristic (ROC) curves to discriminate between healthy and periodontitis sites (area under the curve [AUC] = 0.89), while (t) MMP-8 demonstrated a high diagnostic precision in the detection of mild from severe periodontitis sites (AUC ≥ 0.80). The use of (a) MMP-8 and (t) MMP-8 could represent a useful adjunctive tool for periodontitis diagnosis and severity. These results support the applicability of new point-of-care methods in the monitoring of high-risk periodontal patients.

Periodontitis is a chronic inflammatory disease with a multifactorial etiology. We investigated whether human major histocompatibility complex (MHC) polymorphisms (6p21.3) are associated with periodontal parameters. Parogene 1 population samples (n ‫؍‬ 169) were analyzed with 13,245 single nucleotide polymorphisms (SNPs) of the MHC region. Eighteen selected SNPs (P < 0.001) were replicated in Parogene 2 population samples (n ‫؍‬ 339) and the Health 2000 Survey (n ‫؍‬ 1,420). All subjects had a detailed clinical and radiographic oral health examination. Serum lymphotoxin-␣ (LTA) concentrations were measured in the Parogene populations, and the protein was detected in inflamed periodontal tissue. In the Parogene 1 population, 10 SNPs were associated with periodontal parameters. The strongest associations emerged from the parameters bleeding on probing (BOP) and a probing pocket depth (PPD) of >6 mm with the genes BAT1, NFKBIL1, and LTA. Six SNPs, rs11796, rs3130059, rs2239527, rs2071591, rs909253, and rs1041981 (r 2 , >0.92), constituted a risk haplotype. In the Parogene 1 population, the haplotype had the strongest association with the parameter BOP, a PPD of >6 mm, and severe periodontitis with odds ratios (95% confidence intervals) of 2.63 (2.21 to 3.20), 2.90 (2.37 to 3.52), and 3.10 (1.63 to 5.98), respectively. These results were replicated in the other two populations. High serum LTA concentrations in the Parogene population were associated with the periodontitis risk alleles of the LTA SNPs (rs909253 and rs1041981) of the haplotype. In addition, the protein was expressed in inflamed gingival connective tissue. We identified a novel BAT1-NFKBIL1-LTA haplotype as a significant contributor to the risk of periodontitis. The genetic polymorphisms in the MHC class III region may be functionally important in periodontitis susceptibility.