Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa).

Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory-secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value=0.66) that increased to very good (k-value=0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P<0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from seropositive animals did not detect Trichinella DNA in any mainland animals, but did reveal the presence of a second larvae-positive wild boar on Gabba Island, supporting its utility as an alternative, highly sensitive method in muscle examination. The serology results suggest Australian wildlife may have been exposed to Trichinella parasites. However, because of the possibility of non-specific reactions with other parasitic infections, more work using well-defined cohorts of positive and negative samples is required. Even if the specificity of the ELISAs is proven to be low, their ability to correctly classify the small number of true positive sera in this study indicates utility in screening wild boar populations for reactive sera which can be followed up with additional testing.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife.

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.

A comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

Aerobio Growth of Listeria monocytogenes on Beef Lean and Fatty Tissue: Equations Describing the Effects of Temperature and pH.

The aerobio growth rate and the duration of the lag period were determined for Listeria monocytogenes strain Murray B growing on ground beef lean and on pieces of fatty tissue. The organism grew at 0°C on lean tissue at pH ≥ 6 and on fatty tissue. It failed to grow at 0°C on lean at pH 5.6 but did grow at 2.5°C. The effect of temperature, between 0 and 30°C, on the growth rate on fatty tissue can be described by a modified Arrhenius equation Ln (gen/h) = -205.73 + 1.2939 × 105/K -2.0298 × 107/K2, where K = °Kelvin. This equation accounted for 99.7% of the variance. The combined effect of temperature and pH on the growth rate on beef lean was described by Ln (gen/h) = - 232.64 + 1.4041 × 105/K - 2.1908 × 107K2 + 1.1586 × 102/pH - 4.0952 × 102/pH2 (variance accounted for 99.5%). For lean at about pH 5.5-5.6, this equation applied between about 2.5 and 35°C; for lean of pH 6-7, it applied between about 0 and 35°C. Though the lag period increased with decrease in temperature and pH, measured lag times were more variable than generation times, and the goodness of fit of modified Arrhenius equations to lag times was relatively poor (variance accounted for 83-92%).

Occurrence, Numbers, and Growth of Listeria monocytogenes on some Vacuum-Packaged Processed Meats.

Listeriae were detected on 93 of 175 samples of vacuum-packed processed meats obtained from retail stores. More than 1000 colony forming units of listeriae per g were found on seven of 130 samples in which the numbers of listeriae were estimated. When sliced corned-beef and ham, from four manufacturers, were inoculated with Listeria monocytogenes and vacuum-packed, the growth rate of the organism varied with the composition of the product. High residual nitrite or lowered aw reduced growth, particularly when products were stored at 0 to 5°C. As the storage temperature increased from 0 to 15°C, the growth rate of L. monocytogenes increased more rapidly than that of the other flora (lactic acid bacteria and Brochothrix thermosphacta ). Growth rates on inoculated packs were similar to rates observed for packs contaminated with L. monocytogenes during commercial production.

Detection of Listeria spp. in Meat and Environmental Samples by an Enzyme-linked Immunosorbent Assay (ELISA).

An ELISA kit (TECRA™) for the detection of Listeria spp. was evaluated for its ability to detect these organisms in naturally contaminated meat and in environmental samples from meat processing plants. Of the 170 samples examined, Listeria monocytogenes and/or L. innocua were detected in 74 by enrichment and selective plating. Testing of the enrichment broths with the ELISA kit detected 72 of these positive samples and gave 2 false-negative and 2 false-positive reactions.

Growth of Listeria monocytogenes on Vacuum-packaged Beef.

Pieces of beef striploin (400 g) were inoculated with Listeria monocytogenes strain Murray B, vacuum packaged, and stored at either 0°C or 5.3°C. Growth of the organism on the beef depended on the temperature of storage, the pH of the lean, and the type of tissue. Growth was more rapid at 5.3°C than at 0°C, and faster on striploins of high pH (6.0-6.1) than on striploins of low pH (5.5-5.7). During storage, the population of L. monocytogenes was higher on fatty tissue than on lean principally because growth occurred earlier on the fat. When low pH striploins were held at 5.3°C, listeria grew from an initial count of 2-5×103 CFU/cm2 to 3×107 CFU/cm2 in 16 d on the fat, and in 20 d, to 106 CFU/cm2 on the lean and to 5×107 CFU/ml in the purge fluid. After storage at 0°C for 76 d, the populations reached were 106 CFU/cm2 on the fat, 104 CFU/cm2 on the lean, and 3×105 CFU/ml in the purge fluid. When high pH striploins were held at 0°C for 10 weeks, listeria grew from an initial population of 150-400 CFU/cm2 to just over 106 CFU/cm2 on the fat, 2×105 CFU/cm2 on the lean, and 4×106 CFU/ml in purge fluid.