Seroprevalence of Neospora caninum in free living and farmed red deer (Cervus elaphus) in Poland.

Serum samples from 47 free living and 106 farmed red deer (Cervus elaphus) from the Mazurian Lake District in north-east Poland were investigated for the presence of antibodies to Neospora caninum. A modified Neospora iscom-ELISA was used for initial screening. All sera with optical density (OD) values exceeding 0.400 absorbance units were further investigated by Western blot analysis. Eighteen sera were positive in both tests. Six of these were from free living and 12 from farmed animals giving prevalence of 13 and 11%, respectively. This is the first report of N. caninum infection in farmed and free-living red deer living in the same region where neosporosis was confirmed in cattle and the first evidence of exposure to the parasite in red deer in Poland.

The comparison of different ELISA procedures in detecting anti-Trichinella IgG in human infections.

The aim of this study was to compare the performance of four different ELISA procedures in diagnosing Trichinella spp. infections in human outbreaks. The main differences between the various procedures were: antigen concentration, dilutions of human serum sample, the type of conjugate used, and time of conjugate incubation. Serum samples were collected from 23 individuals involved in an outbreak. Results obtained in the four tested ELISA procedures differed in the rate of positive results obtained for the examined outbreak. Western blot confirmed the ELISA-positive results. Reactions to the specific 41-45kDa and 55kDa bands were obtained with all positive sera.

Persistence of reactivity against the 45 k Da glycoprotein in late trichinellosis patients.

Over the years, the opinions of clinicians on the existence of the so-called chronic trichinellosis or late sequelae of infection have differed. However, the persistence of a humoral immune response against Trichinella in these late-stage patients has been confirmed using specific tests such as the competitive inhibition assay (CIA). We evaluated sera from late-stage trichinellosis patients (2--8 years from acute infection), for their reactivity against Trichinella spiralis antigens. The following tests were carried out: (i) indirect immunofluorescence assay (IFA), performed on muscle sections from mice, 30 days following synchronous infection by intramuscular injection with T. spiralis newborn larvae (NBL); (ii) enzyme immunoassay, employing a synthetic beta-tyvelose antigen conjugated to bovine serum albumin (BSA-Ag); and (iii) western blot (WB) with both an "in house" kit and a commercial kit. The results of IFA obtained by confocal laser microscopy showed that sera reacted against both surface and internal structures of L(1) larvae but at varying levels. Employing the synthetic antigen, EIA showed that 50% of sera tested were positive for the presence of specific antibodies against beta-tyvelose. By WB, all sera were reactive with the 45 k Da glycoprotein (45 gp). These data suggest that reactivity against the beta-tyvelosylated 45 gp persists even in very late stages of human trichinellosis.

Antibodies to Neospora caninum in the blood of European bison (Bison bonasus bonasus L.) living in Poland.

The prevalence of antibodies to Neospora caninum was examined in European bison (Bison bonasus bonasus L.) living in free and fenced areas in Poland. Sera of 320 European bison, different ages and sexes, from breeding areas in Poland were tested for N. caninum antibodies using ELISA test. Positive antibody responses were found in 23 bison (prevalence 7.3%). Additionally, all positive sera were tested by Western blot to verify the ELISA results. The Western blot results confirmed the presence of antibodies to Neospora tachyzoites antigens in all 23 sera tested. The antibodies were detected against a wide range of NC-1 tachyzoite antigens. The antibody responses were directed against proteins at: 9.5, 17, 21, 27, 31, 36.5, 38, 40, 43, 47, 48.5, 53.5 and 58 kDa. The most heavily stained bands had molecular weights of 9.5, 17, 27 and 58 kDa. The most important is that antibody to N. caninum was detected for the first time in sera from bison cow shot in 1988. It is the year of recognition of this protozoan parasite. Our results indicate strongly the presence of N. caninum in European bison in Poland and suggest that a sylvatic cycle of N. caninum can exist. However, further studies are needed to evaluate the existence of a sylvatic cycle of N. caninum. The study on the effect of the infection on the health status and conservation of European bison should be taken under consideration too.

Molecular identification of Trichinella spiralis and Trichinella britovi by diagnostic multiprimer large mitochondrial rRNA amplification.

Trichinella parasites with different epidemiological features still occur in Europe and four species of genus Trichinella have been identified: T. spiralis, T. britovi, T. nativa and T. pseudospiralis. Until now, two of them, T. spiralis and T. britovi, have been identified in Poland. In our studies we selected sequence coding for large mitochondrial rRNA (mt LrDNA) as a genetic marker and developed a sensitive LrDNA multiprimer PCR assay allowing for rapid identification of T. spiralis and T. britovi, parasites present in wild and domestic animals in Poland.

[Changes in the immune response of BALB/c mice coinfected with Heligmosomoides polygyrus and Trichinella spiralis]. / Zmiana odpowiedzi immunologicznej u myszy BALB/c w koinwazji Heligmosomoides polygyrus i Trichinella spiralis.

The influence of Heligmosomoides polygyrus on infection with Trichinella spiralis was studied in BALB/c mice. Mice coinfected with T. spiralis and previously given H. polygyrus harboured both nematode species till day 34. The number of T. spiralis muscle larvae was greater in mice coinfected with H. polygyrus/T. spiralis or T. spiralis/H. polygyrus than after infection with T. spiralis alone. Infection with H. polygyrus did not enhance eosinophil and IL-5 levels induced by T. spiralis. Additionally, the production of IgG1 specific to L1 T. spiralis was inhibited by co-infection. Changes in the levels of IFN-gamma and IgG2a implicated a disturbance in Th2 cell activation during protective response and resulted in the greater number of T. spiralis muscle larvae in coinfected mice.

[Some aspects of the immune response of Polish Wrzosówka sheep against gastrointestinal nematodes infection]. / Wybrane parametry odpowiedzi immunologicznej owiec rasy Wrzosówka przeciw inwazji nicieni zoladkowo-jelitowych.

The paper presents different aspects of natural gastrointestinal nematode infection in this primitive, prolific Polish Wrzosówka breed of sheep. The study was carried out on the flock located at the farm run by Warsaw Agricultural University at Zelazna (central Poland) in 1995-1998. Egg counts per gram of faeces, larval differentiation, total white blood cell counts, blood eosinophil counts, interleukin-5 concentration, lymphocyte blastogenic activity and IgG immune response were discussed. The egg counts were overdispersed and followed a different pattern each grazing season. The larvae obtained from faecal culture belonged to 6 categories: Trichostrongylus spp., Haemonchus contortus, Teladorsagia spp., Cooperia spp., Chabertia ovina/Oesophagostomum spp. and Nematodirus spp. However there were no discernible patterns in the composition of nematode population over time. Data analyses have shown that egg counts, eosinophil counts, IL-5 concentration and IgG level in different months each year were repeatable. The results obtained in two divergent groups of sheep, chosen for consistently low or high faecal egg counts have been presented also. Polish Wrzosówka breed of sheep is under conservation breeding program based mainly on the markers of breed standard. The results obtained in this study revealed that some parasitological, haematological and immunological parameters should be added to this program.

Relationships among peripheral eosinophilia, eosinophil peroxidase activity, interleukin-5 concentration and faecal nematode egg count during natural, mixed gastrointestinal nematode infection.

A longitudinal study of sheep, naturally infected with gastrointestinal helminths, was carried out to quantify the relationships among eosinophilia, eosinophil peroxidase activity, interleukin-5 level and faecal egg counts. Faecal egg counts, peripheral eosinophilia and peroxidase activity per eosinophil were moderately repeatable but interleukin-5 concentrations had a remarkably high repeatability. Animals with higher concentrations of interleukin-5 had greater peripheral eosinophilia and those animals with higher numbers of eosinophils had lower faecal egg counts. These associations were statistically significant but quite weak. Variation in interleukin-5 levels does not appear to be responsible for most of the variation in eosinophil responses in outbred sheep.

Trichinella spiralis: the infectivity of synchronous newborn larvae of different ages inoculated intraocularly.

Trichinella spiralis infection results in the transformation of muscle cells into a new, non-muscular cell called the nurse cell, and the nurse cell-muscle larva complex is finally created. To investigate whether T. spiralis infectivity is NBL age-dependent, five groups of synchronous newborn larvae (sNBL) were obtained at 1, 9, 24, 48, and 72 h of age and were inoculated into mice by intravenous injection into the retro-orbital venous plexus. When both "young" groups of sNBL (1 and 9 h old) were injected, the highest number of larvae were capable of infecting the muscle cells. The highest infectivity of 80.0 % was observed for 9-h-old sNBL. In older sNBL the infectivity gradually decreased; thus, for 72-h-old sNBL the lowest level - 0.1% - was detected. Therefore, an "age limit" for NBL infectivity in the present study was precisely determined.

Trichinella spiralis: in vitro cytotoxicity of peritoneal cells against synchronous newborn larvae of different age.

Two different immune sera obtained from mice infected with muscle larvae (ML) of Trichinella spiralis (oral infection) or injected with 20-h-old newborn larvae (NBL) via retro-orbital venous plexus (intraocular injection) were compared in antibody-dependent cellular cytotoxicity test. Four synchronous stages of NBL, 0-2 h old, 22 24 h old, 46-48 h old and 6 days old (sNBL) were used to study susceptibility of larvae to the cytotoxic reaction of peritoneal cells. Peritoneal cells adhered to and destroyed sNBL of T. spiralis only in the presence of both immune sera. Living sNBL, living sNBL covered by peritoneal cells and dead sNBL were observed in all experimental groups. The lowest percentage of dead sNBL was detected in the 22- to 24-h-old groups. A dramatic increase in mortality was observed in older groups. A greater susceptibility of sNBL to serum obtained from mice infected per os with ML was observed. Serum obtained after oral infection showed higher levels of IgG1 isotype specific to ML than serum obtained after ocular injection.

The repeatability of faecal egg counts in Polish Wrzosówka sheep.

The number of nematode eggs in the faeces was estimated in Polish Wrzosówka sheep, in the spring and autumn of 1993 and again in 1994. The sheep had been naturally infected. The dominant species were Haemonchus contortus and Teladorsagia circumcincta, but Trichostrongylus spp., Cooperia curticei, Nematodirus spp. and Chabertia ovina were also present. Anthelmintics were not used. Egg counts were skewed, with a range of 0-4100 eggs g-1 (EPG); most sheep had egg counts below 100 EPG. Egg counts were approximately four times higher in spring than in autumn. Repeatability values within a season were all significant and positive. The repeatability of egg counts between seasons was estimated from the correlation between the mean transformed value in spring and in autumn, and was 0.52 in 1993 (P < 0.001) and 0.41 in 1994 (P < 0.05). The results show that animals with higher than average values in spring are likely to have higher than average values in autumn, and suggest that similar mechanisms regulated egg counts in both seasons even though egg counts were much lower in autumn.

Propionibacterium granulosum activities in rats during the muscle phase of Trichinella spiralis invasion.

Immune response of Wistar rats, infected with 4000 L1 T. spiralis and treated with P. granulosum during the muscle phase of nematode invasion were measured. The increase of spleen mass was observed in all groups infected and exposed to P. granulosum. Intraperitoneal injection of bacteria results in higher level of T lymphocytes and activated neutrophils. The level of inhibition of macrophages migration was depended on relation to the time and doses of injection. In non-specific stimulated animals there were not statistically significant changes in the level of specific IgG1 antibodies determined by ELISA, against the crude extract of infective larvae of T. spiralis. The reduction of intensity of nematode invasion during the muscle phase was not observed in rats after P. granulosum treatment.

[Development of early larval forms of Moniezia expansa under laboratory conditions]. / Rozwój wczesnych form larwalnych Moniezia expansa w warunkach laboratoryjnych.

Three species of oribatid mites: Scheloribates latipes, Pergalumna nervosa and Ceratozetes sp. were experimentally infected with Moniezia expansa eggs or oncospheres. The intermediate hosts were kept under constant laboratory conditions at 27 degrees C and 80% relative humidity. Three species of oribatid mites became infected and completely developed cestode cysticercoids were found. The early part of life cycle of M. expansa was studied in S. latipes. The mites were examined on 20th, 24th and 29th day after cestode oncosphera invasion. A fully formed cysticercoid of M. expansa was observed on 29th day after infection. The mean of intensity of infection was 1-5 cysticercoids per mite. The infected and living oribatid mites could be kept under laboratory conditions for 7 months. The cysticercoids which had been recovered from S. latipes after this time were able to infect sheep.