Toxoplasma gondii seroprevalence in wild boars (Sus scrofa) in Sweden and evaluation of ELISA test performance.

Toxoplasma gondii is a zoonotic protozoan parasite, infecting a wide range of warm-blooded animals. The Swedish wild boar population is expanding and increased hunting provides its meat to a growing group of consumers. We performed a spatio-temporal investigation of T. gondii seroprevalence in Swedish wild boars. An ELISA was set up and evaluated against a commercial direct agglutination test, using Bayesian latent class analysis. The ELISA sensitivity and specificity were estimated to 79% and 85%, respectively. Of 1327 serum samples, 50% were positive. Thirty-four per cent of young wild boars and 55% of adults were positive (P < 0.001). The total seroprevalence ranged from 72% in 2005 to 38% in 2011 (P < 0.001), suggesting a declining trend. The highest seroprevalence, 65%, was recorded in South Sweden. In other regions it varied from 29% in Stockholm to 46% in East Middle Sweden.

A field study on the effect of some anthelmintics on cyathostomins of horses in sweden.

The objective of the study was to investigate different aspects on the efficacy of three anthelmintics on cyathostomin nematodes of Swedish horses. A faecal egg count reduction (FECR) test was performed on 26 farms. Horses were treated orally with recommended doses of ivermectin, pyrantel pamoate or fenbendazole. Faecal samples were collected on the day of deworming and 7, 14 and 21 days later. No resistance was shown against ivermectin; the FECR was constantly >99%. The effect of pyrantel was assessed as equivocal in 6 farms 14 days after treatment; the mean FECR was 99%. As many as 72% of the fenbendazole-treated groups met the criteria for resistance; the mean FECR was 86%, ranging from 56% to 100%. A re-investigation of two farms where pyrantel resistance had been suspected clearly revealed unsatisfactory efficacy of pyrantel on one of these farms; the FECR varied from 72% to 89%. Twenty-six of the horses previously dosed with pyrantel or fenbendazole, and which still excreted >/=150 eggs per gram of faeces 14 days after treatment, were dewormed with ivermectin and fenbendazole or pyrantel in order to eliminate the remaining cyathostomins. A total of 13 cyathostomin species were identified from horses that initially received fenbendazole and seven species were identified from pyrantel-treated individuals. The egg reappearance period (ERP) following treatment with ivermectin and pyrantel was investigated on two farms. The shortest ERP after ivermectin treatment was 8 weeks and after pyrantel was 5 weeks. We conclude that no substantial reversion to benzimidazole susceptibility had taken place, although these drugs have scarcely been used (<5%) in horses for the last 10 years. Pyrantel-resistant populations of cyathostomins are present on Swedish horse farms, but the overall efficacy of pyrantel is still acceptable.

Reindeer as hosts for nematode parasites of sheep and cattle.

The reindeer husbandry range of Scandinavia overlaps with sheep, goat, and cattle pastures. The aim of this study was to determine whether reindeer are suitable hosts for ovine or bovine nematode parasites, and thus may spread these parasites into the reindeer husbandry regions. To render worm-free, twelve 4-month-old male reindeer calves, six lambs, and six bovine calves were given ivermectin at 200 microg/kg body weight. Five weeks post-treatment, six reindeer calves were each artificially dosed with 10,000 third-stage larvae (L3) of gastrointestinal nematodes derived from sheep, and an additional six reindeer with L3 derived from cattle. Lambs and bovine calves received the same dose of ovine and bovine larvae as reindeer, from the same larval source, respectively. Faecal samples collected on five occasions after the larval dosing revealed that by the fourth week, all reindeer calves, lambs, and bovine calves were infected. Animals were slaughtered on days 40 (reindeer) or 47 (lambs and bovine calves) after the larval dosing. Reindeer calves were most susceptible to L3 derived from sheep. The overall mean intensity of Haemochus contortus, Trichostrongylus axei, and Teladorsagia circumcincta, did not differ between reindeer and sheep; however, early fourth-stage larvae of H. contortus were more abundant in reindeer (p = 0.002). The establishment of bovine-derived Ostertagia ostertagi was similar in reindeer (62%) and bovine calves (57%), but larval inhibition was much higher in reindeer (91%, p < 0.001) than in cattle (31%). Very poor establishment of bovine derived Cooperia oncophora was recorded in reindeer calves (2%) compared with bovine calves (59%). These results show that young reindeer are susceptible hosts to the important gastrointestinal parasites of sheep (T. circumcincta, H. contortus) and cattle (O. ostertagi), as well as being a suitable host for T. axei.

A 3-year field evaluation of pasture rotation and supplementary feeding to control parasite infection in first-season grazing cattle--effects on animal performance.

To evaluate non-chemical strategies to control pasture-borne parasites in first-season grazing (FSG) cattle, a 3-year grazing trial was conducted during 2002-2004 on naturally infected pastures on a commercial beef cattle farm in Sweden. A uniform pasture was divided in 4 equal 2 ha paddocks onto each of which 10, 5-9 months old dairy breed steer calves were allocated at turn-out in May each year. Two strategies were evaluated: (1) turn-out onto pasture which had been grazed the previous year by second-season grazing (SSG) steers, followed by a move to aftermath in mid-July (RT) and (2) supplementation with concentrate and roughage for 4 weeks from turn-out (FD). Comparisons were made with an untreated (UT), and an anthelmintic treated control group (DO). Animal parasitology and performance were monitored monthly throughout the 20 weeks grazing period. Additional sampling occasions were performed on day 9 (for coccidia) and 10 weeks after turn-out (mid-July). Due to clinical parasitic gastro-enteritis (PGE), salvage treatments were performed on all animals in group FD approximately 7 weeks after turn-out in 2003 and of three animals in group UT 5 weeks after turn-out in 2004. In 2003, the geometric mean oocyst excretion 9 days after turn-out was approximately 150,000 opg of mainly Eimeria alabamensis in group FD, and in 2004 approximately 180,000 opg in group UT. Apart from the DO group, geometric mean faecal egg counts (FEC) were between 80 and 400 epg 4 weeks after turn-out. Mean serum pepsinogen concentrations (SPC) of approximately 3.6 U tyrosine were recorded in the FD and UT groups from late August 2002. In 2003 and 2004, mean concentrations in these groups were between 4.1 and 7.2 U tyrosine 8 weeks after turn-out. By the end of the three grazing seasons the average weight gain difference compared to the DO group was for FD -29, -38 and -5 kg and for RT -4, -21 and +14 kg, and compared to the UT group -18, +2 and +22 for FD and +7, +19 and +41 kg for group RT. In conclusion, the rotation control strategy showed promising results, whereas the strategic feeding was poor from a parasite control standpoint.

Serological diagnosis of Neospora caninum infection.

Since the first isolation of the apicomplexan parasite Neospora caninum, a range of serological assays have been developed for use in dogs, cattle and a variety of other potential host species. The tests include the indirect fluorescent antibody test, the direct agglutination test and different enzyme-linked immunosorbent assays. This article reviews the principles and properties of the available tests which are discussed in relation to different applications.

Oral Neospora caninum inoculation of neonatal calves.

Four calves born to cows seronegative for Neospora caninum were dosed orally within 6 h after birth with tachyzoites of the bovine N. caninum Nc-SweB1 isolate added to colostrum. Two of the calves were dosed via stomach tube and two by feeding bottle. The latter two calves showed transient fever and passed blood-stained diarrhoea 1-2 weeks after inoculation. From 5 weeks after inoculation they developed a significant antibody response which remained high until the calves were euthanised and necropsied at 15 and 19 weeks after inoculation, respectively. The two calves inoculated by stomach tube showed no clinical signs and they remained seronegative throughout the study. At necropsy of the seropositive calves, no pathological lesions were seen, and parasites were not detected by immunohistochemistry. Neospora caninum was not re-isolated in cell culture from the brains of the seropositive calves; however, N. caninum DNA was detected in brain from both of them by PCR. The data suggest that oral infection of N. caninum via colostrum might be a possible route of vertical transmission in newborn calves, in addition to transplacental infection.

Redescription of Neospora caninum and its differentiation from related coccidia.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Experimental porcine neosporosis.

Six gilts were inoculated intramuscularly with 2.5x10(6) tachyzoites of Neospora caninum on three different days of gestation to study the pathogenic effect of Neospora infection in pigs, including possible transplacental transmission. The gilts were euthanized 59, 30, and 9/10 days postinoculation (p.i.), corresponding to days 107, 102/106 and 110/111 of pregnancy. With the exception of one animal (euthanized day 9 p.i.) all gilts seroconverted as measured by the indirect, fluorescent antibody test (IFAT). Neosporosis with multifocal intralobular necrotizing hepatitis was seen in the two gilts inoculated 9/10 days before euthanasia. The uterus of one gilt inoculated 59 days before euthanasia revealed granulomatous and focal necrotizing endometritis with a corresponding multifocal necrosis of the trophoblasts of two fetuses. Transplacental neosporosis was indicated in the two fetuses by strongly elevated Neospora IFAT titres in pleural fluid and by the presence of multifocal necrotizing encephalitis and hepatitis together with non-suppurative myocarditis, pneumonitis, nephritis and hepatitis. Furthermore, N. caninum was re-isolated in cell culture from one of these fetuses. A third fetus from the same gilt revealed only disseminated, pinpoint necroses in the liver. Immunohistochemically, N. caninum tachyzoites were detected in association with histopathological changes in the liver and the endometrium of the gilts, and in the brain, liver, and allantochorion of the three fetuses.

The phylogeny of Neospora caninum and Toxoplasma gondii based on ribosomal RNA sequences.

Neospora caninum is a newly described cyst-forming coccidium which is the cause of severe neurological disease in dogs. The parasite is morphologically similar to Toxoplasma gondii, but the two species can be differentiated serologically. In order to define the phylogenetic position of N. caninum, we have determined 16S-like rRNA sequences from three members of the family of Sarcocystidae: N. caninum, T. gondii, and Sarcocystis fusiformis. The 16S-like rRNA genes from the three parasites were amplified by polymerase chain reaction and the sequences were determined by direct solid-phase sequencing. The sequences derived were computer aligned with several other 16S-like rRNA sequences from protozoan parasites to construct phylogenetic trees. The study confirmed that N. caninum should be classified as a member of the family Sarcocystidae. However, because of the close relationship to T. gondii it seems questionable that N. caninum should be placed in a new genus.

Infectivity of Toxoplasma gondii in mutton following curing, smoking, freezing or microwave cooking.

To investigate the effects of curing with sodium chloride and sucrose, low temperature smoking, freezing at -20 degrees C, and cooking in a microwave oven, respectively, on the infectivity of Toxoplasma gondii encysted in mutton, meat from three experimentally and one naturally infected sheep was used. Samples of meat prepared accordingly as well as untreated, raw meat from each animal were assayed by mouse inoculation. Infective T. gondii was isolated from untreated samples from all animals used, but in no case from cured, smoked or frozen meat. However, in two of four steaks processed in a microwave oven, according to a standard household recipe, the parasite remained infective, possibly due to uneven heating of the meat.

Effects of temperature and humidity on oviposition, molting, and longevity of Dermanyssus gallinae (Acari: Dermanyssidae).

The juvenile development and survival of Dermanyssus gallinae (De Geer) kept in vitro at different temperatures and humidity were investigated to obtain biological baseline data for a Swedish population. Individual females, eggs, larvae, and protonymphs were observed with regard to egg production, duration of various stages, and longevity when kept at different temperatures and relative humidities. Female mites laid eggs at temperatures between 5 and 45 degrees C with the highest numbers laid at 20 degrees C and 70% RH, but development to larvae and protonymphs was only observed at temperatures ranging from 20 to 25 degrees C. The average duration of oviposition varied from 1.0 to 3.2 d within the temperature range 20-45 degrees C but was gradually increased to 28 d at 5 degrees C. Specimens survived for up to 9 mo without access to food when kept in the temperature range of 5-25 degrees C. Temperatures > 45 degrees C and at -20 degrees C were found to be lethal. Longevity was similar for females and protonymphs kept at 30 and 45% RH, but it was enhanced at 70 and 90% RH for protonymphs. This study showed that D. gallinae can survive for a long time without feeding if the microclimate is suitable, but it does not thrive at low relative humidities and at temperature extremes. This indicates that changing of the abiotic conditions in infested poultry houses could be a possible measure to reduce mite populations.

The induction of chromosomal aberrations and SCE by visible light in combination with dyes. I. The effect of hypoxia during light exposure in unsynchronized Chinese hamster ovary cells, sensitized with acridines.

A comparison has been made between the ability of different acridine compounds to act as sensitizers for visible light (400-700 nm) induced chromosomal aberrations and sister-chromatid exchanges (SCE) in unsynchronized Chinese hamster ovary (CHO) cells. Cells were treated for 20 min with acridines (0.1-5.0 microgram/ml), washed free of excess dye and subsequently exposed to visible light (2 x 40 W/8 W m-2) either in air or in nitrogen for 5-15 min. The 4 acridines tested, proved to be effective sensitizers for the induction of both chromosomal aberrations and SCE by visible light. The most pronounced effect was observed when the light exposure of the fluorochrome-pretreated cells was performed in air. Hypoxic conditions during light exposure reduced the effect dramatically, especially in the case of induced chromosomal aberrations. The order of efficiency for the induction of both chromosomal aberrations and SCE was acridine orange greater than acridine yellow greater than proflavine greater than 3,6-diamino-10-methylacridine. The results are discussed in terms of S-independent versus S-dependent mechanisms for inducing chromosomal alterations and the potential involvement of oxygen-derived free radicals in this process.

The induction of chromosomal aberrations and SCEs by visible light in combination with dyes. II. Cell cycle dependence, and the effect of hydroxyl radical scavengers during light exposure in cultures of Chinese hamster ovary cells sensitized with acridine orange.

Chinese hamster ovary (CHO) cells were synchronized by mitotic shake-off, treated with the fluorochrome acridine orange (AO; 0.5 micrograms/ml), washed free of excess dye and subsequently exposed to visible light (2 X 40 W/8 Wm-2). The light exposure was performed on cells in the G1, G1/S, S or G2 phase of the cell cycle. AO + light induced high frequencies of aberration in the S phase and even higher in the G1 phase. The aberrations observed were all of the chromatid type. The chromosome-type aberrations (dicentrics, rings) obtained when cells in the G1 phase were exposed to X-rays were not found after corresponding treatments with AO + light. With the exception of an increased frequency of gaps, no chromosomal aberrations were induced in G2-phase cells. Sister-chromatid exchanges were efficiently produced by the photodynamic system in the G1, G1/S and S phase of the cell cycle. In other experiments, AO-treated unsynchronized CHO cells were exposed to light in the presence of the hydroxyl radical scavengers mannitol (100 mM) and 5-dimethyl thiourea (100 mM). In parallel experiments these scavengers were found to reduce markedly the chromosome breaking effects by X-rays but had no influence on the photodynamic induction of chromosomal alterations. The results presented show that the visible light-induced chromosomal alterations in CHO cells sensitized with the fluorochrome AO are obtained by an S-dependent mechanism. Furthermore, the results indicate that the hydroxyl free radical does not play a major role in the production of chromosomal alterations by AO + light.

The induction and repair of DNA damage detected by the DNA precipitation assay in Chinese hamster ovary cells treated with acridine orange + visible light.

The photodynamic effect of the dye acridine orange (AO) in combination with visible light (400-700 nm) was studied in Chinese hamster ovary (CHO) cells, the endpoints investigated being induction, as well as repair, of DNA strand breaks. Cells were treated for 20 min with AO (0.1-3.0 micrograms/ml), washed free of excess dye and subsequently exposed to low doses of visible light (2 x 40 W/8 W/m2) for 5-15 min. AO proved to be an efficient sensitizer for light-induced DNA strand breaks, detected with the DNA precipitation assay, and expressed as percentage of DNA precipitated. The induction of breaks was linear up to 0.5 micrograms/ml AO + 10 min of light, which corresponds to 55% precipitated DNA, and was dependent on the concentration of AO as well as on the dose of light delivered. As a comparison, 18 Gy of X-rays was required to yield an equivalent amount of induced DNA strand breaks. The rejoining of the light-induced DNA strand breaks was studied by incubating the AO-sensitized cells for 30-120 min at 37 degrees C directly after light exposure. A fast recover of 67-91% of the damage (compared to initial damage, recovery time = 0, and dependent on the concentration of AO) was observed during the first 30 min of incubation. However, a significant amount of DNA damage remained after 2 h of recovery. These remaining, long-lived lesions might be involved in the photoinduced and acridine-sensitized chromosomal aberrations and sister-chromatid exchanges (SCE). The significance of these observations is discussed in relation to AO-sensitized and photoinduced DNA damage and chromosomal alterations.

Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of men occupationally exposed to styrene in a plastic-boat factory.

Workers in a Swedish factory making boats from plastics reinforced with glass fibre are exposed to a variety of chemicals, including styrene which is mutagenic after metabolic activation. The concentration of styrene in the air was measured in the breathing zones of workers occupied with various processes in boat making. Samples of air were taken 6 times during the years 1973-1978. The total exposure to styrene for the workers during this period was calculated and expressed as the average concentration in mg per m3 air during an 8-h workshift multiplied by the number of years of employment. A low-dose group (mean = 137 mg x m-3) and a high-dose group)mean - 1204 mg x m-3) were identified. Blood samples were taken in 1978 from workers belonging to the exposed groups and from a matched control group of employees in the same factory not exposed to styrene. Lymphocytes were cultured and examined for chromosomal aberrations and sister-chromatid exchanges. Exposed workers had a significantly (p less than 0.001) higher number of chromosomal aberrations (36 persons, mean = 7.9 aberrations/100 cells) compared with employees in the control group (37 persons, mean = 3.2 aberrations/100 cells). There was no significant difference between the mean values of the number of chromosomal aberrations between the highly exposed and the less exposed groups. But in the less exposed group there was an increase in the frequency of chromosomal aberrations with increasing exposure to styrene (r = 0.576). In the highly exposed group this dose response was not observed (r = 0.231). For the frequency of sister-chromatid exchanges (SCE) a slight (p less than 0.05) increase was found in the styrene-exposed group (20 persons, mean = 8.4 SCE/cell). The control group (21 persons) had a mean value of 7.5 SCE/cell. Again there was no difference between the highly exposed and the less exposed groups. Other environmental factors that may have clastogenic effects were studied, but multiple regression analysis failed to show a candidate responsible for the increase in chromosomal abnormalities in the exposed group.

Evaluation of a solid-phase immunoassay (DIG-ELISA) for the serodiagnosis of ovine toxoplasmosis.

The antibody response to Toxoplasma gondii was studied in 5 experimentally infected sheep by means of the indirect fluorescent antibody test (IFAT) and the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA). A significant increase in the antibody levels was registered by both methods as early as one week after subcutaneous inoculation with the RH strain of T.gondii. Maximal antibody levels were recorded 4 to 8 weeks after the inoculation. Analysis of a total of 135 sera obtained during different stages of experimental or natural toxoplasma infection revealed close agreement between the results of the two methods used. The expediency of regular DIG-ELISA as well as of a modified procedure for the direct analysis of blood sampled onto filter paper discs was also demonstrated. There was a close correlation between the results obtained by analysis of serum and of blood adsorbed onto filter paper.

Application of an indirect carbon immunoassay (CIA) for the rapid diagnosis of antibody to Toxoplasma gondii in sheep.

Carbon immunoassay (CIA), a novel indirect rapid test for Toxoplasma antibody in sheep, was compared with indirect fluorescent antibody assay (IFA). CIA relies on the adherence of carbon particles of India-ink to rabbit immunoglobulin G.l Carbon labelled anti-sheep rabbit IgG was used for the detection of sheep antibody when attached to tachyzoites of Toxoplasma gondii. The result was read in an ordinary light microscope and there was a clearcut difference between negative and positive reactions. Out of a total of 97 sheep sera tested, 15 sera were negative in both tests and 12 were negative in CIA but showed low positive titres in IFA. The remaining 70 sera were positive in both tests but the titres were usually about 3 dilution steps lower when investigated with CIA as compared to IFA.

An IgG avidity ELISA to discriminate between recent and chronic Neospora caninum infection.

The avidity of IgG antibodies directed to Neospora caninum was measured using an IgG avidity enzyme-linked immunosorbent assay (ELISA) employing N. caninum proteins incorporated into immunostimulating complexes as antigen. In this ELISA, low-affinity antibodies were eluted by adding an incubation step with urea after the serum incubation. The antibody titers obtained with and without incubation with urea were then used to calculate the IgG avidity values. Analysis of sequential sera collected from experimentally infected calves revealed that the avidity increased during the course of infection. Three weeks after infection, the IgG avidity was 9-18%, and 24 weeks later it had increased to 58-76%. Cattle naturally infected for more than 6 months all had avidities >50%. The results in this study, however preliminary, indicate that the IgG avidity ELISA can be used to discriminate between recent and chronic N. caninum infections and may therefore be a valuable complement to IgG assays in epidemiologic studies of N. caninum infection in cattle.

Eimeria alabamensis infection as a cause of diarrhoea in calves at pasture.

Large numbers of oocysts of Eimeria alabamensis have been found in the faeces of calves suffering from diarrhoea shortly after being turned out to pasture. To investigate the source and clinical significance of this coccidial infection, the numbers of oocysts excreted, the consistency of the faeces and the growth rates of four groups of 12 calves were compared. Group I calves were kept indoors and their diet was unchanged, Group II calves were turned out onto a previously ungrazed pasture, Group III calves were turned out onto a permanent pasture and Group IV calves were kept indoors and fed cut grass from a previously ungrazed field. Eight days after the animals were turned out there was an almost 1000-fold increase in the numbers of oocysts in the faeces of Group III calves, the dominant species being E. alabamensis, but there were only minor fluctuations in the numbers of oocysts excreted by the other groups. It was therefore concluded that the source of the infection was oocysts that had overwintered on the permanent pasture. Most of the calves in Group III developed watery diarrhoea 5 days after turnout, but there was only a slight softening of the faeces of the calves in Groups II and IV at about the same time. The faeces of the calves in Group I was of firm consistency throughout the trial. The calves in Group III lost 18 kg during the 24 day period following turnout, whereas the calves in the other groups gained between 6 and 18 kg.

Schistosoma mansoni antibodies in tissue specimens demonstrated by a diffusion-in-gel ELISA technique.

A modification of the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) for analysis of antibodies in tissue fragments has been elaborated. In this technique, tissue specimens are placed on top of a thin gel layer covering a plastic surface coated with an antigen preparation. The gel thus serves as a medium through which diffusion of antibodies contained in tissue samples may occur. Specimens from skin, lung, liver, kidney, heart and skeletal muscle as well as blood serum and blood sampled onto filter paper were successfully used for analysis of the antibody response to two different antigens (soluble adult worm antigen and soluble egg antigen) during experimental infection of mice with the helminth parasite Schistosoma mansoni. It was concluded that skin and lung tissue reflected the serum antibody response more closely than the other tissues tested, although zone sizes were slightly smaller than those registered for corresponding serum samples. The DIG-ELISA reaction zone size was, as expected, shown to be dependent on the weight (size) of the tissue specimen. The variability of the modified technique was comparable to that found for analysis of serum by the conventional DIG-ELISA technique. It is concluded that the described technique may be used for serological analysis when blood samples cannot be easily obtained, e.g. at examination of animal carcasses or single organs.