Screening for infection of Trichinella in red fox (Vulpes vulpes) in Denmark.

A total of 6141 foxes (Vulpes vulpes) were examined for infection with Trichinella. The foxes were killed in Denmark during the hunting season 1995-1996 and 1997-1998; 3133 and 3008, respectively. Foxes included in the investigation came from throughout the country with the exception of the island of Bornholm. The right foreleg from each fox was submitted for investigation. The legs were stored at -20 degrees C for 3-10 months prior to examination. Following thawing, muscle tissue (10 g) from each leg was examined by trichinoscopy and by a pepsin-HCl digestion technique. In 1995-1996, three foxes were found positive corresponding to a prevalence of 0.001. Each of the infected foxes harboured an extremely low infection, i.e. about one larva per 10 g muscle tissue. It was not possible to obtain sufficient larval material for species identification. All three foxes were shot in the vicinity of a small village in the north-western part of Denmark. In 1997-1998 no Trichinella cases were found. The results, compared with previous studies, indicate that the prevalence of infection of Trichinella sp. among wild living foxes in Denmark is very low. This is further supported by the fact, that no infection of Trichinella sp. has been found in slaughtered pigs in Denmark for more than 65 years, which suggests that the infection pressure is very low. Considering the facts above we conclude that the risk of Trichinella infections is negligible in intensive indoor pig production units in Denmark whereas high local prevalence of Trichinella infections in the wildlife might constitute a serious risk for the expanding outdoor pig production.

The efficacy of two isolates of the nematode-trapping fungus Duddingtonia flagrans against Dictyocaulus viviparus larvae in faeces.

A series of experiments was carried out to examine the effects of two different isolates of the nematode-trapping fungus Duddingtonia flagrans to reduce the number of free-living larvae of the bovine lungworm, Dictyocaulus viviparus. A laboratory dose-titration assay showed that isolates CI3 and Troll A of D. flagrans significantly reduced (P < 0.05 to P < 0.001) the number of infective D. viviparus larvae in cultures at dose-levels of 6250 and 12,500 chlamydospores/g of faeces. The larval reduction capacity was significantly higher for Troll A compared to CI3 when lungworm larvae were mixed in faecal cultures with eggs of Cooperia oncophora or Ostertagia ostertagi and treated with 6250 chlamydospores/g of faeces. Both fungal isolates showed a stronger effect on gastrointestinal larvae than on lungworm larvae. Two plot trials conducted in 1996 and 1997 involved deposition of artificial faecal pats containing free-living stages of D. viviparus and C. oncophora on grass plots. Herbage around the pats was collected at regular intervals and infective larvae recovered, counted and identified. These experiments showed that both D. flagrans isolates reduced the number of gastrointestinal as well as lungworm larvae in faecal pats. During both plot trials, the transmission of C. oncophora larvae, but not D. viviparus, from faecal pats to the surrounding herbage was clearly affected by climatic conditions. After collection of faecal pats from the grass plots one month after deposition, the wet and dry weight of pats as well as organic matter content were determined. No differences were found between the fungus-treated and non-treated control pats. This indicated that the rate of degradation of faeces was not affected by the addition of the fungus.

A comparison of in vitro tests and a faecal egg count reduction test in detecting anthelmintic resistance in horse strongyles.

This study reports a comparison between faecal egg count reduction test (FECRT), egg hatch assay (EHA) and larval development assay (LDA) for detecting anthelmintic resistance in equine strongyles. Resistance to benzimidazoles was demonstrated in 33 of 42 (79%) farms tested by FECRT and in 32 (62%) of the 52 farms tested by EHA. As the reference strain used was not fully susceptible to benzimidazoles it was not possible to determine the level of resistance by LDA. Pyrantel resistance was indicated on three of 15 farms by faecal egg count reduction. Resistance was also indicated by LDA for one of these farms. In addition resistance was indicated by LDA on two more farms that were not tested by FECRT. Further testing is needed to confirm if these findings are truly indicative of resistance. Generally, correlations between the tests were poor and it was not possible to use the outcome of one test to predict the outcome of another.

Monoclonal antibodies to Toxoplasma gondii strain 119 identify recently isolated Danish strains as one group.

Four mAb raised against the Danish Toxoplasma gondii strain 119, were selected by screening hybridoma supernatants by indirect immunofluorescence against tachyzoites of the RH strain in order to obtain strain-restricted markers. Strain restriction extended beyond discrimination of the 119 and RH strains, as demonstrated on a further six T. gondii reference strains [BK and GT1 (group A), NTE and 561 (group B), and NED and C56 (group C)]. The bradyzoite-specific mAb, 4.3, reacted to the GT1, NTE and 561 strains, but not to the BK, NED or C56 strains. The tachyzoite-specific mAb, 4.25, reacted to all strains tested except the RH strain, while mAb 5.1 reacted to tachyzoites of strains NTE and 561, but not to those of the BK, GT1, NED or C56 strains. Monoclonal antibody 5.15 reacted with the same strain restriction as monoclonal antibody 5.1, but to bradyzoites as well as tachyzoites. A T. gondii strain collection representative for a small geographic area (Denmark) was established within a short time span from a variety of animal species. Using the mAb as typing reagents to this Danish strain collection, all 36 animal and two human strains were identified as having the same reaction pattern as strains 119, NTE and 561.

Survey of anthelmintic resistance on Danish horse farms, using 5 different methods of calculating faecal egg count reduction.

This study reports on the prevalence of anthelmintic resistance in strongyles of horses in Denmark. Of 5 methods used for the calculation of faecal egg count reduction (FECR) the method recommended by the World Association for the Advancement of Veterinary Parasitology, for the detection of resistance in sheep was the most sensitive procedure for detecting resistance. Using this method benzimidazole resistance was detected on 33 of 42 farms (79%) examined. Pyrantel was tested on 15 farms and FECR tests indicate resistance on 3 (30%) farms. On 2 farms on which resistance to pyrantel was detected resistance to benzimidazoles was also detected. On one of 16 farms examined ivermectin resistance was indicated at Day 14 but not at Day 19. On the 15 remaining farms ivermectin was effective. Due to the high prevalence of anthelmintic resistance in Danish horse herds it is recommended that tests of anthelmintic efficacy be conducted routinely to monitor the effectiveness of the strongyle control programmes.

Dung-derived biological agents associated with reduced numbers of infective larvae of equine strongyles in faecal cultures.

Two sets of dung-derived organisms from soil routinely fertilized with manure (MA) and soil chemically fertilized (CH) were cultured separately in the laboratory. Baermannized organisms from these cultures were added to 20 g of faeces from strongyle-infected horses to form three treatment groups: (i) no soil organisms; (ii) low inoculum of soil organisms containing all organisms present in a suspension of approximately 100 adult female free-living nematodes; and (iii) high inoculum containing those soil organisms present with approximately 1000 adult female free-living nematodes. Three studies were conducted using MA cultures and faeces containing 50 stronglye epg, CH cultures and faeces containing 1500 strongyle epg, and a mixture of soil organisms from the two cultures (MC) and faeces containing 600 strongyle epg. Within each study, five control cultures and 15 each of low and high inoculum cultures were prepared and incubated at 24 degrees C and 95% humidity in a climate chamber for 15 days. Parasitic and free-living nematodes were then recovered by the Baermann technique and counted. The numbers of third stage larvae were significantly lower in the high inoculum group compared to controls. The percent reductions in the number of third stage larvae for the low and high inoculum groups were 63.6% and 90.9%, 85.1% and 97.1%, 84.5% and 98.4% for MA, CH, and MC studies, respectively, indicating that mortality increased with the number of soil organisms added to cultures. Examination of the source cultures detected the presence of two species of nematophagous fungi and three genera of free-living nematodes reported to be predacious.

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.

Trichinella spiralis, T. britovi, and T. nativa: infectivity, larval distribution in muscle, and antibody response after experimental infection of pigs.

The infectivity of Trichinella spiralis, T. nativa, and T. britovi was experimentally compared in pigs. Blood sampling was performed weekly, and muscle juices were obtained at slaughter 10 weeks after inoculation. Muscle larvae were found in all of four pigs inoculated with T. spiralis [mean 190 larvae per gram (lpg)] and in three of four pigs inoculated with T. britovi (mean 7 lpg). No larvae were found in pigs inoculated with T. nativa. For T. spiralis and T. britovi, the neck muscle (m. splenius) appears to be a predilection site in addition to the tongue, the diaphragm, and the jaw. High antibody responses were found in all experimental groups, independent of the antigen used, and even in pigs in which no muscle larvae were recovered. The strong and consistent antibody response found with meat juice indicates the usefulness of this material where a blood sample is not obtainable, e.g. meat samples from wild animals. Immunoblotting (Western blots) on slaughter sera revealed no species specificity when comparing homologous versus heterologous staining.

Clinical observations, pathology, bioassay in mice and serological response at slaughter in pigs experimentally infected with Toxoplasma gondii.

Experimental infections of a total of 47 pigs with tachyzoites of the Toxoplasma gondii RH-strain, tissue cysts of the SSI-119 and R92 strains as well as oocysts of the SSI-119 strain were performed to determine the sensitivity of an indirect IgG-ELISA, using tachyzoite lysate of the RH-strain as antigen. The infections led to a dose dependent moderate clinical affection (inappetence, fever and poor general condition). Pigs infected with 10000 oocysts or with 1/2 mouse brain containing tissue cysts of the SSI-119 strain showed a significant decrease in weight gain compared to uninoculated pigs during the first 2 weeks p.i., followed, however, by compensatory growth during the next 6 weeks. At slaughter 3 to 4 months after inoculation 39/41 (95.1%) of pigs positive by bioassay in mice were seropositive in ELISA. Tissue cysts were not demonstrable by immunohistochemistry. ELISA OD-values obtained by analysis of meat juice from heart muscle and tongue (diluted 1:40) correlated strongly with OD-values by analysis of serum (diluted 1:400) (r heart juice = 0.942; r tongue juice = 0.915). Thus, meat juice samples were shown to provide a suitable alternative to serum for serological detection of Toxoplasma infection in pigs.

The time course of the specific antibody response by various ELISAs in pigs experimentally infected with Toxoplasma gondii.

With the aim of developing routine serological tests for monitoring the Toxoplasma infection status of Danish swine herds, four ELISAs based on tachyzoite antigen were set up: (1) an indirect ELISA for IgG-antibody; (2) a blocking ELISA for antibody to the membrane antigen, P30; (3) an indirect ELISA for IgM; (4) a reverse, antibody-catching IgM-ELISA. Groups of pigs (number between 6 and 10) were inoculated with tachyzoites of the RH-strain, tissue cysts of two complete strains, or oocysts in two doses (10(3) and 10(4). All inoculations were tolerated well. Irrespective of strain and stage used for inoculation, specific IgG and anti-P30 blocking activity appeared after 1-2 weeks, with OD-values stabilizing after 3-6 weeks and persisting throughout the study period (3-4 months). Specific IgM appeared quickly, but was short-lived (approximately 2 weeks). A cut-off OD-value of 0.36 for positive seroreaction in the indirect IgG-ELISA was determined on the basis of 69 sera from four herds, investigated in the dye-test (serum dilution 1:10) and ELISA. The chosen cut-off gave optimal combined sensitivity and specificity of 0.94 and 0.92, respectively, using the dye-test as a standard. Corresponding figures for the blocking ELISA were 37% inhibition as cut-off, with sensitivity and specificity of 0.94 and 0.94, respectively. Sera from a total of 87 pigs, experimentally infected with bacteria of the genera Salmonella, Yersinia or Actinobacillus and with the parasites Isospora suis, Trichinella spiralis or Ascaris suum, in no case produced cross-reactions in the IgG-ELISA. However, 3/9 pigs inoculated with 50 000 sporocysts of Sarcocystis miescheriana gave maximal OD-readings of 0.40-0.45 during the 13-15 weeks observation period. None of the sera from heterologously infected animals produced inhibitions in the anti-P30 blocking ELISA exceeding 36%.

Identification of porcine Pneumocystis carinii as a genetically distinct organism by DNA amplification.

DNA was amplified from lung samples from three piglets infected with Pneumocystis carinii, using oligonucleotide primers designed to the P. carinii mitochondrial large subunit ribosomal RNA gene. The nucleotide sequence of the amplification product was determined and indicated lack of sequence variation among these pig-derived P. carinii samples at this locus. The data showed that porcine P. carinii was genetically distinct from P. carinii isolated from other mammalian host species.

Toxoplasmosis in a colony of New World monkeys.

In a colony of New World monkeys five tamarins (Saguinus oedipus, Saguinus labiatus and Leontopithecus rosal. rosal.), three marmosets (Callithrix jacchus and Callithrix pygmaea) and one saki (Pithecia pithecia) died suddenly. The colony comprised 16 marmosets, 10 tamarins and three sakis. The main pathological findings were necrotic lesions in the lung, the intestine, and the liver. Histopathologically T. gondii parasites were observed in organs from the tamarins and the marmosets but not in the saki. Some considerations on epidemiology are presented. Preventive measures were directed against the bottom layer of the cages, on cockroach extermination, and on freezing of raw meat.

Nematode-trapping fungi in biological control of Dictyocaulus viviparus.

Larvae of the cattle lungworm Dictyocaulus viviparus were cultured in experimental units of 200 g cattle faeces placed in semi-transparent trays in the laboratory. In each of 4 experimental series using this experimental unit, chlamydospores (chl) of the nematode-trapping fungus Duddingtonia flagrans were admixed to half of the faecal cultures in a concentration of 50.000 chl/g. In all 4 series there was a significant reduction in the development and subsequent release of infective lungworm larvae from faecal cultures containing chlamydospores. The average reduction in larval release, caused by fungal spores, was 86%.

Effect of the nematode-trapping fungus Duddingtonia flagrans on the free-living stages of horse parasitic nematodes: a pilot study.

A plot experiment was conducted to investigate the ability of the nematode-trapping fungus Duddingtonia flagrans to reduce the transmission of infective horse strongyle larvae from deposited dung onto surrounding herbage. At three different times during the summer 1995, three groups of horses, naturally infected with large and small strongyles, were fed different doses of D. flagrans spores, while a fourth group of animals served as non-fungal controls. Faeces from all four groups of horses were deposited as artificial dung pats on a parasite-free pasture. Every second week for 8 weeks after dung deposition, a subsample of the herbage surrounding each dung pat was collected and the number of larvae on the grass determined. Also, the larval reduction capacity of the fungus was evaluated by faecal cultures set up from all groups of horses. The faecal cultures showed that a sufficient number of spores of D. flagrans survived passage through the horses alimentary tract to significantly reduce the number of developing larvae. A lower reduction of larval numbers was observed when a different batch of fungal material was used at the beginning of the season. Dry climatic conditions affected the transmission of infective larvae in all groups, resulting in low numbers of larvae on the herbage. During the rainy periods a significant reduction in the number of larvae recovered was observed around all fungal containing pats. There were no significant differences between the number of fungal spores and the level of reduction caused by the fungus.

Biological control of gastro-intestinal nematodes--facts, future, or fiction?

The potential of using fungi to prevent nematodosis caused by parasites with free-living larval stages is well documented today. In this respect Duddingtonia flagrans, a net-trapping, nematode-destroying fungus, appears to be the most promising candidate. Laboratory experiments and in-vivo studies, where fungal spores have survived passage through the gastro-intestinal tract of cattle and horses, plus field studies with cattle, horses and pigs, demonstrate significant reduction in the number of infective larvae that develop in the faecal environment. In field trials this reduction subsequently leads to reduced infectivity of herbage and also reduced worm burdens in grazing animals. A status of the present situation, primarily based upon work performed in Denmark within the last 6-8 years, plus an outlook for practical implementation of an integrated control strategy including the use of nematode-destroying fungi in the future is discussed.

Induction of traps by Ostertagia ostertagi larvae, chlamydospore production and growth rate in the nematode-trapping fungus Duddingtonia flagrans.

Biological control of parasitic nematodes of domestic animals can be achieved by feeding host animals chlamydospores of the nematode-trapping fungus Duddingtonia flagrans. In the host faeces, D. flagrans develop traps that may catch nematode larvae. In experiments on agar, D. flagrans had a growth rate between 15 and 60 mm/week at temperatures between 20 and 30 degrees C. The presence of nematodes induces the fungus to produce traps. The rate of trap formation in D. flagrans has an optimum at 30 degrees C, producing 700-800 traps/cm2/2 days, when induced by 20 nematodes/cm2 on agar. Approaching 10 and 35 degrees C the ability to produce traps is gradually reduced. The response of chlamydospore production on agar to changes in temperature is the same as that for trap formation. On agar, at 10, 20 and 30 degrees C D. flagrans loses its trap inducibility after 2-3 weeks. During the ageing process, increasing numbers of chlamydospores are produced up to a certain limit. The time for reaching maximum chlamydospore concentration coincided with the time for loss of induction potential. The implications of these results in relation to biological control in faeces are discussed.

Pneumocystis carinii from pigs and humans are antigenically distinct.

The antigens of Pneumocystis carinii cysts isolated from pigs and humans were compared by the Western immunoblotting technique. Convalescent pig serum reacted with two antigens (approximately 78 kDa and 32.5 kDa) of porcine P. carinii cysts, whereas convalescent serum from humans did not react with porcine P. carinii cyst antigens. The results indicate that porcine and human P. carinii cysts are antigenically distinct.

Biological control. Aspects of biological control--with special reference to arthropods, protozoans and helminths of domesticated animals.

Biological control describes situations in which a living antagonist (a predator, parasite, parasitoid or a pathogen) is distributed by man to lower pest (parasite) populations to acceptable sub-clinical densities or to keep the population at a non-harmful level. Ideally, biological control has no negative effects on the environment, whereas chemical control is not always so harmless. Laboratory and field observations have revealed many organisms, such as viruses, bacteria, fungi, protozoans, turbellarians, nematodes, earthworms, tardigrades, insects, copepods and mites as antagonists to parasitic arthropods, protozoans and helminths of domesticated animals. However, only very few of these antagonists have shown promising qualities as biological control agents within veterinary science. The lack of success should be linked to the lack of knowledge about complex natural biological systems and the antagonists that may be found there. This situation has restricted the interest of industry in developing biological products. In the future, however, industry may become more interested in biological control considering the increasing problems with parasite resistance to drugs in combination with the increasing cost of developing new chemical products, and because of increasing public concern about chemical residues in animal products and in the environment.

The capacity of the fungus Duddingtonia flagrans to prevent strongyle infections in foals on pasture.

A field trial was conducted to evaluate the potential of the nematode-destroying fungus Duddingtonia flagrans to control free-living stages of horse strongyles. In late Spring 2 groups of horses (yearlings) with mixed infections of strongyles were allowed to contaminate 2 equal-sized pastures. One of the groups (F) received a daily dose of D. flagrans mixed in a feed supplement, while the other (C) received a similar amount of supplement without fungus. During a 3-month contamination period strongyle egg counts in faeces and number of infective strongyle larvae harvested from faecal cultures were determined. Grass samples were collected fortnightly. After the contamination period the yearlings were removed and 2 groups of young tracer foals (TF and TC) grazed the fungus and control pastures respectively for 4 weeks, housed for another 15 weeks and then killed to determine their worm burdens. The number of larvae in cultures from group TF was significantly lower than that in TC and herbage infectivity was reduced to a very low level on the pasture grazed by horses fed fungi. The number of Strongylus vulgaris and Strongylus edentatus larvae was also significantly lowered in group TF. Cyathostome larvae recovered from the mucosa of the ventral and dorsal colon and from the caecum were significantly lowered in group TF foals. Also, the number of strongyles found in the gut contents of group TF foals were significantly reduced in the dorsal colon, but numbers of worms in the ventral colon and in the caecum were similar to those of the controls.