Testing of amplified fragment length polymorphism (AFLP) technique as a tool for molecular epidemiology of Trichinella nativa.

A total of nine Trichinella nativa isolates were compared by amplified fragment length polymorphism (AFLP). Four hundred nanograms of genomic DNA from a pool of 10--20 larvae were digested using HindIII and MseI restriction endonucleases. Of the 16 primer combinations initially tested, Hind-C and Mse-C primers showed rich polymorphism with approximately 40--90 bands in the range of 30--270 bp. Genetic similarities were estimated visually. AFLP provided discriminatory banding patterns and may therefore be used as a method for detecting variation in T. nativa populations. However, the heterogeneous patterns obtained from pooled samples emphasize the need for further development of the sampling and numerical analysis of the patterns for epidemiological and taxonomical interpretation.

Spatial variation of Trichinella prevalence in rats in Finnish waste disposal sites.

Trichinellosis is 1 of the most widespread parasitic zoonoses in the world and can be lethal to humans. Trichinella spp. are also parasites of considerable economic importance. Because rats may play a role in the transmission of trichinellosis to swine and farmed wild boar, 767 brown rats (Rattus norvegicus Berkenhout) from 13 Finnish waste disposal sites were examined for Trichinella spp. by a HCl-pepsin digestion method. Trichinella spp. were found to be a common parasite in trapped rats (overall prevalence, 19%) detected in 12 of 13 dumps. Significant differences were observed between sites in the prevalence (0-49%) of Trichinella spp. Female rats were more often and more heavily infected than males, but age was not shown to be a risk factor for trichinellosis. In addition, positive correlation was demonstrated between rat population density and prevalence. Trichinella spiralis was identified by multiplex polymerase chain reaction in 28 rats. The median density of infection was 42 (range, 0.5-6,925) larvae/ g of host tissue, but neither the occurrence nor the density of the parasite was related to the physical condition of the animal.

Extension of the species Helicobacter bilis to include the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and Finnish canine and feline flexispira strains.

The evolution and taxonomy of enterohepatic Helicobacter species with flexispira morphology were studied by a polyphasic approach including phenotypic characterization, analysis of 16S rRNA and ureB gene sequences and dot-blot DNA-DNA hybridization of whole genomic DNA. In addition, available phylogenetic data on the HSP60 gene were used in the analysis. The study included 14 Finnish canine and feline flexispira strains, the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and Helicobacter bilis ATCC 51630(T). Phenotypically, all canine and feline strains were similar to H. bilis. Analysis of 16S rRNA gene sequences of these strains revealed a similarity of 97-99.5 %. Similarity of ureB nucleotide and amino acid sequences within the studied flexispira group was 97-100 % and 99-100 %, respectively, revealing close relatedness. ureB sequences of Helicobacter hepaticus had only 64-66 % similarity to the flexispira group. The similarity to Helicobacter trogontum was 81.5-82.1 %. High levels of DNA-DNA hybridization between the strains were found in dot-blot tests. Polyphasic analysis of the phenotypic and genotypic characteristics of the Finnish flexispira strains and the reference strains of taxa 2, 3 and 8 showed that they differed from other Helicobacter species and are members of the previously described species H. bilis. In addition, canine strain F56 differed in all phylogenetic analyses from the H. bilis group and probably represents a novel Helicobacter species.

Trichinella nativa and T. spiralis induce distinguishable histopathologic and humoral responses in the raccoon dog (Nyctereutes procyonoides).

Three experimental groups of six male raccoon dogs (Nyctereutes procyonoides) each were formed by placing one of three littermates from six litters into each group. One group was inoculated with pig-origin Trichinella spiralis, the second was inoculated with raccoon dog-origin T. nativa, and the third served as a control group. The infective dose was 1,000 larvae/kg of body weight. Every third week, biopsies from M. triceps brachii were taken, and serum samples were collected for up to 12 weeks postinfection. In the early phase of the infection, cysts of both parasites were elongated cylinders that later became more spherical. However, at the end of the experiment, the cysts of T. nativa were more rounded than those of T. spiralis (mean length/width = 2.5 versus 1.5 in T. spiralis versus T. nativa, respectively). Both species accumulated a collagen-rich capsule around the nurse cell, but the capsule was thicker in T. nativa. In both parasites, the total surface area of the sagittal section of the cyst was equal. Inflammation was more intense around T. nativa cysts. Specific antibodies were recognizable 2 weeks after infection by both enzyme-linked immunosorbent assay (ELISA) and western blot. In western blots, serum from both T. nativa- and T. spiralis-infected animals recognized the same components, but reaction with the homologous antigen was stronger. The same pattern was also seen in the ELISA. Immunoreactive epitopes were localized only in internal organs and cuticula of larvae in muscle.

Persistence of Trichinella spiralis in rat carcasses experimentally mixed in different feed.

Trichinella spiralis infected rat carcasses were incubated for 6 weeks in several animal feeds to assess how long Trichinella can present a risk for an outbreak in contaminated feeds. In groups of 6, 24 infected target rats were placed in silage, grained barley, propionic acid-preserved feed, and also into simulated pasture conditions. Test environments were sampled after one-, 2-, 4-, and 6-week-incubations. Trichinella larvae were recovered by digestion, and their infectivity was evaluated in rats. A two-week incubation reduced the number of recovered larvae, but still after 6 weeks low numbers were isolated from all feeds except from the experimental group simulating pasture conditions. After 2 weeks storage, the larvae were infective in all storage environments. However, up to 4 weeks, they survived only in the propionic acid-fermented feed and there in small numbers with reduced reproductive capability. This indicates the possibility of farm animals to get infection from rats or other infected material being hazardously mixed with hay or other feed. If silage is stored for at least one month before use, however, the risk from this forage appears to be minimized.

Evaluation of two PCR-based techniques for molecular epidemiology in Finland, a high-endemic area with four sympatric Trichinella species.

Trichinella larvae collected from wildlife, domestic and synanthropic animals in Finland were identified to species by two molecular techniques: Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and the recently described multiplex PCR. The RAPD-PCR was very sensitive to the sub-optimal preservation muscle larvae and resulting in weak and smeared bands on the gels for such material. However, the same samples yielded easily recognizable bands in the multiplex PCR; this latter technique is then recommended for epidemiological studies, especially when the preservation of the samples is sub-optimal. For larvae in good condition the unequivocal bands obtained by multiplex was the easiest identifiable. Four species of Trichinella were identified in the material: T. spiralis, T. nativa, T. britovi, and T. pseudospiralis. Trichinella britovi is a new record for Finland, and T. pseudospiralis is a new record for Northern Europe. Mixed infections between T. britovi and T. spiralis, T. nativa and T. spiralis, and between T. britovi and T. nativa were detected; this is the first record of a mixed infection between T. spiralis and T. nativa in a naturally infected host. Raccoon dogs were the only host species from which all of the four Trichinella species were detected. Trichinella spiralis was found in both domestic animals and wildlife, but none of the sylvatic Trichinella species were detected in domestic pig.

Flavonol content varies among black currant cultivars.

Flavonoids and related plant compounds in fruits and vegetables are of particular importance as they have been found to possess antioxidant and free radical scavenging activity. The HPLC-based quantitative procedure, with improved extraction and hydrolysis, was used to analyze the content of the flavonols quercetin, myricetin, and kaempferol in 10 black currant cultivars from organic farms and in 5 cultivars from conventional farms. Myricetin was the most abundant flavonol, and its amount varied significantly among cultivars, from 8.9 to 24.5 mg x 100 g(-1) (fresh weight). The quercetin levels in black currant also varied widely among the cultivars, from 5.2 to 12.2 mg x 100 g(-1). The kaempferol levels in black currant cultivars were low, ranging from 0.9 to 2.3 mg x 100 g(-1). The sum of these major flavonols varied widely among black currant cultivars. No consistent differences in the contents of flavonols were found between the same black currant cultivars grown in organic and conventional ways. The high variability in the levels of flavonols in different cultivars offers possible avenues for identifying and selecting cultivars rich in certain flavonols for the special production of berries for industrial use.

Predilection muscles and physical condition of raccoon dogs (Nyctereutes procyonoides) experimentally infected with Trichinella spiralis and Trichinella nativa.

The predilection muscles of Trichinella spiralis and T. nativa were studied in 2 experimental groups of 6 raccoon dogs (Nyctereutes procyonoides), the third group serving as a control for clinical signs. The infection dose for both parasites was 1 larva/g body weight. After 12 weeks, the animals were euthanized and 13 sampling sites were analysed by the digestion method. Larvae were found in all sampled skeleton muscles of the infected animals, but not in the specimens from the heart or intestinal musculature. Both parasite species reproduced equally well in the raccoon dog. The median density of infection in positive tissues was 353 larvae per gram (lpg) with T. spiralis and 343 lpg with T. nativa. All the infected animals had the highest larvae numbers in the carpal flexors (M. flexor carpi ulnaris). Also tongue and eye muscles had high infection levels. There were no significant differences in the predilection sites between these 2 parasite species. Trichinellosis increased the relative amount of fat, but not the body weight in the captive raccoon dogs. Thus, Trichinella as a muscle parasite might have catabolic effect on these animals.

Clinical features of experimental trichinellosis in the raccoon dog (Nyctereutes procyonoides).

Three groups of six raccoon dogs (Nyctereutes procyonoides) were provided for the experiment: the first group was infected with pig-origin Trichinella spiralis, the second with raccoon dog-origin Trichinella nativa, and the third served as controls. Infection dose for both parasite species was 1000 larvae/kg of body weight, which led to intense final infection. Clinical signs, haematology and serum biochemistry with repeated blood samples were monitored up to 12 weeks post-infection. The most significant findings were a short-term eosinophilia in peripheral blood from the end of the first week post-infection until the end of the third week, loss of weight, and mild anaemia. In the early phase of the infection, the animals had gastrointestinal signs, loss of appetite and diarrhoea. No specific differences in clinical findings could be noticed between the groups infected with T. nativa and T. spiralis. In contrast to the symptoms reported in human outbreaks, fever was not observed in any of the infected animals and serum levels of muscle-specific enzymes did not change. No acute-phase response was observed in the enteral or parental phase of the infection. These findings indicate that because Trichinella spp. are very well adapted to the raccoon dog, it thus, could serve as the most crucial reservoir animal for sylvatic trichinellosis in Finland.

An outbreak of trichinellosis in farmed wild boar in Finland.

Nine farmed wild boar out of 25 slaughtered from a single farm were condemned at meat inspection because of trichinellosis. With RAPD-PCR, Trichinella spiralis was identified in all positive wild boar. Out of the available serum samples (n=7), all wild boar which had failed the meat inspection showed seroconversion in ELISA and Western blotting, as did one additional animal which had passed the inspection. The animals became infected during an invasion of rats from an improperly closed dump near the farm. Unfortunately, by the time trichinellosis was discovered in the wild boar, the invasion had already been brought under control; thus, no samples from rats were available. However, having lived through the rat invasion was shown to be a risk factor for trichinellosis in wild boar (relative risk, RR=6.3). In wildlife samples from surrounding areas, sylvatic trichinellosis was found to be very common (74%; n= 19 red foxes). Intriguingly, the prevalent species in trichinella-positive foxes differed from that in wild boar, Trichinella nativa and T. spiralis being found in 12 foxes and in one fox, respectively.

Reproducibility and validity of a food frequency questionnaire in a case-control study on breast cancer.

A 110-item food frequency questionnaire was tested among 152 community controls of the Kuopio Breast Cancer Study. They completed the questionnaire twice and kept two 7-day diet records at 3-month intervals. The intraclass correlations for nutrients varied from 0.49 (thiamine with supplements) to 0.81 (lactose), and for foods from 0.52 (poultry) to 0.84 (alcoholic drinks). The Pearson correlations between the first food frequency questionnaire and the 14-day diet records, after adjustment for energy, varied for nutrients from 0.18 (thiamine without supplements) to 0.80 (alcohol), and for foods from 0.30 (inner organs) to 0.90 (coffee). Comparison of quintile classification between the two methods is reasonably accurate when observed restrictions concerning some nutrients and foods are taken into account. A low association (r = 0.12) was observed between toenail selenium and dietary selenium intake, indicating the difficulty of estimating selenium intake in the Finnish diet.

Craniofacial and dental characteristics of Silver-Russell syndrome.

We found significant differences in a craniometric, cephalometric, and dental study of 19 Silver-Russell syndrome patients (13 without growth hormone treatment) with appropriate controls. Although head circumference was normal for age, head length was increased, while cranial and facial widths and facial heights were reduced. Posterior facial height, posterior cranial base length, cranial base height, and mandibular body size were significantly smaller than in healthy children of the same height. Articulatory speech disorders were common. Enamel defects pointed to an early prenatal insult. Delayed dental age and small mandibular and cranial base dimensions support the possibility of physiological growth hormone deficiency in many Silver-Russell syndrome children; however, facial soft tissue structures were strikingly different from those observed in classical growth hormone deficiency.