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.

Trichinellosis in farmed wild boar: meat inspection findings and seroprevalence.

A reflection of highly prevalent endemic wildlife trichinellosis is seen in wild boar farming in Finland. During the last five years, 0.7% (15/2265) of wild boars undergoing official meat inspection have been determined to be Trichinella-positive. These findings originate from six different farms. In Finland, T. spiralis and T. pseudospiralis have been discovered in meat inspection of wild boars. ELISA showed 11 out of 99 serum samples (11%) as having specific antibodies for T. spiralis crude antigen. Positive samples were from three out of the thirteen farms from which the sera were available. Most of the positive serum samples (8/11) originated from a farm where trichinellosis was also revealed in meat inspection, the other two seropositive farms were without previous Trichinella records. Over the last few decades, no reports have been made of human trichinellosis acquired in Finland. This indicates both efficient meat inspection as well as public awareness of high-risk foodstuff.

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.

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.

Experimental trichinellosis in reindeer.

Six female reindeer calves were inoculated intraruminally with various doses of Trichinella muscle larvae. Four calves were inoculated with T. nativa, receiving 15,000 (n = 1), 5,000 (1), and 2,500 (2) larvae each. Two calves were inoculated with 5,000 T. spiralis larvae each. Blood samples were collected twice per week for total white blood cell (WBC) and differential counts and for serology using enzyme-linked immunosorbent assay (ELISA) based on T. spiralis excretory-secretory antigen. On day 56, the calves were slaughtered and muscle samples were examined according to the standard digestion method for Trichinella larvae. Blood samples were also collected twice a week from 4 uninoculated, but otherwise similar, reindeer calves corralled separately. Both the total WBC and eosinophil counts of the inoculated animals were, on average, higher during the experimental period. All the inoculated calves seroconverted, showing an increase in the optical density (OD) in the ELISA starting between day 23 and day 27 postinoculation. Very few muscle larvae (<0.08 larvae/g [lpg]) were to be found from the animals inoculated with T. nativa, but about 4 and 6 lpg were recovered from the masseter muscles of those inoculated with T. spiralis.

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.