Monitoring of local CD8ß-expressing cell populations during Eimeria tenella infection of naïve and immune chickens.

The purpose of this study was to monitor abundance and activation of local CD8ß-expressing T-cell populations during Eimeria tenella infections of naïve chickens and chickens immune by previous infections. Chickens were infected with E. tenella up to three times. Caecal T-cell receptor (TCR) γ/δ-CD8ß+ cells (cytotoxic T lymphocytes; CTL) and TCRγ/δ+CD8ß+ cells were characterized with respect to activation markers (blast transformation, CD25 and cell surface CD107a). Cells were also induced to degranulate in vitro as a measure of activation potential. Major findings included a prominent long-lasting, up to 6 weeks, increase in the proportion of CTL among caecal CD45+ cells in the later stages after primary E. tenella infection. These CTL also showed clear signs of activation, that is blast transformation and increased in vitro induced degranulation. At second and third E. tenella infection, chickens showed strong protective immunity but discrete signs of cellular activation were observed, for example increased in vitro induced degranulation of CTL. Thus, primary E. tenella infection induced clear recruitment and activation of local CTL. Upon subsequent infections of strongly immune chickens cellular changes were less prominent, possibly due to lower overall numbers of cells being activated because of the severe restriction of parasite replication.

Expression of perforin, granzyme A and Fas ligand mRNA in caecal tissues upon Eimeria tenella infection of naïve and immune chickens.

Cytotoxic cells of the immune system may kill infected or transformed host cells via the perforin/granzyme or the Fas ligand (FasL) pathways. The purpose of this study was to determine mRNA expression of perforin, granzyme A and FasL in Eimeria tenella-infected tissues at primary infection and infection of immune chickens as an indirect measure of cytotoxic cell activity. Chickens were rendered immune by repeated E. tenella infections, which were manifested as an absence of clinical signs or pathological lesions and significantly reduced oocyst production upon challenge infection. During primary E. tenella infection, perforin, granzyme A and FasL mRNA expression in caecal tissue was significantly increased at 10 days after infection, compared to uninfected birds. In contrast, at infection of immune birds, perforin and granzyme A mRNA expression in caecal tissue was significantly increased during the early stages of E. tenella challenge infection, days 1-4, which coincided with a substantial reduction of parasite replication in these birds. These results indicate the activation of cytotoxic pathways in immune birds and support a role for cytotoxic T cells in the protection against Eimeria infections.

Coccidian oöcysts as type-specimens: long-term storage in aqueous potassium dichromate solution preserves DNA.

Preservation of the exogenous oöcyst stage of coccidian parasites (phylum Apicomplexa N.D. Levine, 1970) as type-specimens of newly described species has long been problematical. Conventional fixatives have proved unsatisfactory, and compromises such as embedding oöcysts in resin or photographing them are not entirely appropriate for various reasons. As an alternative, chilled potassium dichromate solution (normally used in the laboratory to prevent putrefaction of temporary preparations of live oöcysts) has been tested as a long-term preservative of sporulated oöcysts of Eimeria brunetti P.P. Levine, 1942, E. maxima Tyzzer, 1929, E. mitis Tyzzer, 1929, E. necatrix Johnson, 1930, E. praecox Johnson, 1930 and E. tenella (Railliet & Lucet, 1891) (suborder Eimeriorina Léger, 1911; family Eimeriidae Minchin, 1903). Oöcysts from faeces of chickens Gallus gallus (Linnaeus) were placed in 2.5% w/v aqueous potassium dichromate solution (PDS) and stored in the dark at 4 +/- 2 degrees C. After 23 years in storage, oöcysts of each species were administered orally to chickens and failed to initiate infections, indicating that the oöcysts were dead. Nevertheless, after about 24 years, DNA was still recoverable from the oöcysts, and the original species identifications made by classic parasitological methods were confirmed by polymerase chain reaction assays. Furthermore, after almost 25 years, microscopical examination revealed that the walls and internal structures remained well preserved in 83-98% of the oöcysts of the six species investigated. Hence, PDS is potentially suitable for the long-term preservation of sporulated coccidian oöcysts as type-specimens for taxonomic purposes. The samples used in this study are now in the care of the Natural History Museum, London, UK. It is recommended that they be monitored in like manner, by suitably qualified scientists, at intervals of about 5 years to assess their state of preservation and the recoverability of DNA. Enough material is available to monitor it until it is at least 100 years old.

Isospora orlovi infection in suckling dromedary camel calves (Camelus dromedarius) in Kenya.

Outbreaks of isosporosis in young suckling dromedary camel calves (Camelus dromedarius) in Dubai, UAE and in Kenya were recently described. In the former outbreak the pathogen was shown to be Isospora orlovi by morphological features and was later characterized molecularly. In the present study, we have made a longitudinal investigation of 159 suckling dromedary calves < or =12 weeks of age belonging to 8 ranched camel herds (M1) in Northern Kenya. The study was carried out during 18 months. In three of the herds frequent samples were taken irregularly every 1-6 weeks. All calves < or =12 weeks of age present in the respective herds were sampled during the visits. In addition, 91 calves of the same age group but belonging to 42 pastoral herds (M2) in Northern Kenya were point sampled at convenience. Faecal samples from each calf were taken and the faeces were investigated for coccidia. Samples found with coccidian oocysts were suspended in a 2% potassium dichromate solution. Isospora sp. was identified and samples with relatively high numbers of Isospora sp. were analysed molecularly. The SSU rRNA gene and internal transcribed spacer 1 (ITS1) were amplified with primers complementary to conserved regions of the SSU rRNA gene in eukaryotes as well as a conserved part of the 5.8S rRNA gene of Eimeria. A relatively high number of the calves exhibited diarrhoea, 30.2% and 41.8% in the M1 and M2 herds, respectively. Isospora sp. was only found in diarrhoeic calves or in calves convalescent from recent scouring periods. No calf >8 weeks of age was found to be excreting Isospora sp. The parasite was only found in calves < or =4 weeks of age in the M1 herds and in the M2 herds in calves <8 weeks of age. Of the M1 and M2 calves exhibiting diarrhoea, 20.8% and 26.3% excreted Isospora sp., respectively. Morphologically the Isospora sp. was similar to I. orlovi and sequence analysis of the SSU rRNA gene from four Kenyan isolates (unfortunately only from the pastoral herds, M2) and ITS 1 segments from three of the isolates from Kenya and one from Dubai, confirmed that the Isospora isolates belonged to the species I. orlovi, and that the sequences were similar to the Dubai isolates.

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.

Effect of betaine supplement on broiler performance during an experimental coccidial infection.

A trial was conducted to investigate the effects of betaine as a feed supplement, given singly and in combination with the ionophore coccidiostat narasin, on broiler performance during an experimental coccidial infection. Five hundred and sixty female Ross broiler chickens were kept in floor pens and given a wheat-based diet. At 10 d of age, 420 chickens were individually inoculated with a mixture of Swedish chicken Eimeria isolates containing E. acervtulina, E. praecox, E. maxima, and E. tenella. Remaining birds were kept as uninoculated controls. The effects of betaine (0 or 1.0 g/kg) and narasin (0 or 70 ppm) added to the basal diet were evaluated. Overall, betaine as a single feed supplement improved live weight by 5.7, 5.4, and 5.6% at 22, 29, and 36 d, respectively, but had no positive effect in combination with narasin. A longer withdrawal period of the coccidiostat (10 vs 5 d) did not affect live weight, but significantly increased feed intake by 9.6% and feed conversion ratio by 12.6%, irrespective of betaine supplement. Inoculated birds had a 10% lower live weight than uninoculated chickens. Performance of uninoculated birds was similar to that of inoculated birds treated with narasin, except at 7 d after inoculation, when live weights of uninoculated birds were significantly higher. Chickens given coccidiostat had less Clostridium perfringens in their ceca, but the prevalence was not altered by betaine supplement. There was no difference in intestinal lesion scores between inoculated chickens given coccidiostat or not, despite the better performance of chickens given coccidiostat. Betaine did not affect Eimeria oocyst output or intestinal lesion scores.

Sporulation of Eimeria maxima oocysts in litter with different moisture contents.

The aim of this study was to determine if the sporulation of Eimeria maxima oocysts was affected by the moisture content of the litter. Fresh feces were collected from chickens experimentally infected with E. maxima. The feces were mixed with dried wood shavings and different amounts of water to obtain final moisture contents of 16, 42, and 62% and a final oocyst concentration of 5,000 per g of mixture. The samples were kept at 23 C and 75% relative humidity and were thoroughly aerated every 12 h. Oocysts kept under ordinary laboratory sporulation conditions in 2% wt/vol aqueous potassium dichromate at 27 C were used as a standard for optimal sporulation. The proportion of sporulated oocysts was determined microscopically every 12 h. Sporulation of E. maxima was most efficient under the driest conditions studied (16% moisture content), and poorest in the samples with the highest moisture content (62%). Even though the differences may not have resulted from a direct effect of humidity on the oocysts, but more likely resulted from limited oxygen in the moister substrates, it is clear that sporulation is not favored by moist litter.

Clinical picture and antibody response to experimental Sarcoptes scabiei var. vulpes infection in red foxes (Vulpes vulpes).

Three red foxes (Vulpes vulpes) were experimentally infected with Sarcoptes scabiei isolated from a naturally infected wild red fox. A fourth red fox served as a control. The first signs of sarcoptic mange became evident on the 31st day post infection (dpi). The signs gradually increased thereafter and between dpi 49 and 77 characteristic lesions of hyperkeratosis developed. Two of the infected foxes developed severe sarcoptic mange, and one of these animals died on dpi 121. The third fox developed a chronic hyperkeratotic lesion on its back, at the site where the mites had been applied. On dpi 127 the surviving foxes were treated systemically with ivermectin, and within 4 weeks the skin lesions had healed except on the pinnae of one animal. Antibodies to S. scabiei var. vulpes were demonstrated in the infected foxes by an ELISA with which seroconversion was seen around 4 weeks post infection (wpi). Western blot analysis of sequential sera of the infected animals demonstrated antibody activity consistently after the 2nd wpi. The fourth, non-infected, fox did not show any skin lesions throughout the experimental period nor any specific antibodies to S. scabiei var. vulpes.

Comparison between effects of standard feed and whole wheat supplemented diet on experimental Eimeria tenella and Eimeria maxima infections in broiler chickens.

The effects of experimental infections with Eimeria tenella (Experiment 1, n = 144) or E. maxima (Experiment 2, n = 216) in broiler chickens fed whole wheat, with or without access to grit, as compared to a standard pelleted feed were studied. Inclusion of whole wheat was gradually increased up to 30% at 3 weeks of age. Grit was given separately. The chickens were kept on litter in a parasite-free environment with free access to water and feed. At 3 weeks of age half the number of chickens were individually inoculated with 500 sporulated oocysts of E. tenella (Experiment 1) or 3,000 sporulated oocysts of Eimeria maxima (Experiment 2), and the remaining birds were kept separate as uninfected controls. Neither coccidiostats nor growth enhancers were used. Oocyst concentration was determined from each group separately. Intestinal lesions were scored on 6 birds per feed regime 7 d postinoculation, and on the remaining birds at slaughter. Diet had no significant effect or bird performance during infection. However, there was an indication that the E. maxima infection had more negative effect on weight gain in birds given standard feed than in those given whole wheat supplement, but the difference was not significant (p < 0.09). The number of oocysts shed or mean intestinal lesion scores did not differ between diets in either experiment. In both experiments, the number of Clostridium perfringens was higher in the caeca of inoculated birds, but there were no differences between diets.

Comparison between a live, attenuated anticoccidial vaccine and an anticoccidial ionophore, on performance of broilers raised with or without a growth promoter, in an initially Eimeria-free environment.

An experiment was carried out to study the effects of vaccination with Paracox, a live, attenuated vaccine against avian coccidiosis, on broilers isolated from extraneous Eimeria parasites. The study involved 3200 broiler chickens raised in floor pens similar to commercial conditions, but in an initially Eimeria-free environment. Forty percent of the chickens were vaccinated at 3 days of age and given either a basal unmedicated feed or a feed supplemented with the feed antibiotic virginiamycin. Unvaccinated birds were given either the basal feed or feed supplemented either with virginiamycin or the anticoccidial ionophore narasin. At slaughter at 36 days of age vaccinated birds had a lower live weight than non-vaccinated birds. The difference was 4.6% in unmedicated, and 6.0% in virginiamycin medicated chickens. Feed conversion ratio at slaughter was 2.5% higher for unmedicated vaccinated birds, and 1.3% higher for virginiamycin medicated vaccinated birds, compared to respective non-vaccinated groups. There was no significant difference in overall performance of unvaccinated birds given narasin as compared to virginiamycin. At 10 days post vaccination vaccinated birds had a higher number of Clostridium perfringens in the caeca, but there was no difference thereafter. Throughout the experiment, caecal clostridial counts were considerably higher in vaccinated unmedicated birds than in unvaccinated birds given narasin. The number of oocysts shed in the vaccinated groups was very low, but during a subsequent challenge with E. maxima and E. tenella the birds' immunity was found to be satisfactory.

Equine herpes virus 1 (EHV-1) in liver, spleen, and lung as demonstrated by immunohistology and electron microscopy.

Ten aborted foals, diagnosed as infected with Equine Herpes Virus 1 (EHV-1) on histopathological criteria, were examined for the presence of EHV-1 using immunohistology as the investigative instrument. The primary reagent was an antiserum specific for viral envelope glycoproteins. Immunohistology localised EHV-1 to areas of liver necrosis and to the cytoplasm of infected Kupffer cells and hepatocytes. Cytoplasmic immunolabelling was also prominent in reticular cells of the red pulp of the spleen and in intact and degenerated bronchiolar epithelium. Cytoplasmic immunolabelling was seen in morphologically unchanged cells and in cells containing intranuclear inclusion bodies. Three aborted foetuses with no histological signs of EHV-1 infection were negative when immunostained for EHV-1. Detection by electron microscopy of EHV-1 virions confirmed the EHV-1 specificity of the immunolabelling procedure.

Regional assignments of NP and MPI on chromosome 7 in pig, Sus scrofa.

Fibroblasts from a pig with a spontaneous reciprocal translocation involving chromosome 7, were used to prepare a set of pig-mouse somatic cell hybrids. The isozyme analysis strongly indicated that in pigs, the NP (nucleoside phosphorylase) gene is located on the distal part of the long arm of chromosome 7, (q21----qter), while the MPI (mannose phosphate isomerase) gene is in the region q21----pter. This confirmed previously reported chromosomal assignment of these genes in pigs and that this synteny has been evolutionarily conserved in several different animal species.

Identification of seven Eimeria species in Swedish domestic fowl.

The aim of the study, conducted during the period 1992 to 1996, was to identify the Eimeria species present in Swedish chickens. All samples, including litter, faeces and guts from dead birds submitted for coccidial diagnosis, were obtained from farms where no live coccidiosis vaccines had ever been used. Identification of the different species was based on the criteria of oocyst morphology, location and characteristics of intestinal lesions, morphology of parasite endogenous stages, prepaient time and isoenzyme electrophoresis profiles of glucose phosphate isomerase. All seven Eimeria species of the domestic fowl were identified, namely E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella. Furthermore, Swedish monospecific isolates of E. maxima, E. necatrix and E. tenella were established.

Eimeria infections in litter-based, high stocking density systems for loose-housed laying hens in Sweden.

1. Coccidiosis, caused by different Eimeria species, is believed to be a more prominent problem in loose-housed layers kept on litter than in battery cages. In this study, the impact and development of Eimeria infections were investigated in layers kept in litter-based, high stocking density systems for loose-housed hens. 2. Layers from 57 flocks on 26 farms were followed by necropsy of a representative sample of birds that died or had to be culled. Coccidiosis was diagnosed in 11 flocks (19.3%) from 9 (31%) of the farms. The outbreaks occurred when the birds were 19 to 32 weeks old. E. maxima was identified in 6 and E. tenella in 3 of the outbreaks. 3. Sixteen of the flocks were also monitored with faecal and litter samples collected at regular intervals. Oocysts were detected in samples from all these flocks. The pattern of oocyst excretion was similar in most of the flocks, with maximum counts at 4 to 8 weeks after introduction to the laying house. There was no significant correlation between the levels of oocysts in faeces and clinical coccidiosis. 4. Raising pullets without any coccidiostat, to increase their chance to develop immunity against coccidia, was not found to decrease the risk of coccidiosis during the production period when compared to the practice of giving amprolium and ethopabate during the rearing period.

Intestinal digesta viscosity decreases during coccidial infection in broilers.

1. The effect of intestinal digesta viscosity on bird performance in chickens with coccidiosis was compared to those without coccidiosis. 2. Six hundred chicks were divided into five groups: one control group was fed a basal maize/soyabean-based diet and the other groups were fed the basal diet supplemented with 2, 4, 6 or 8 g carboxymethyl cellulose (CMC) per kg of feed. At 14 d of age half the birds were individually inoculated with sporulated oocysts of Eimeria acervulina and Eimeria praecox. 3. Intestinal digesta viscosity increased with increasing inclusion of CMC. This effect was considerably less pronounced in inoculated than in non-inoculated birds. 4. There was a significant negative effect on live weight gain and feed conversion ratio (FCR) with increasing CMC inclusion in non-inoculated birds, but in inoculated birds there was no clear relation between CMC inclusion and performance. Neither intestinal lesion scores, nor numbers of Clostridium pefringens in the caeca, were significantly affected by CMC inclusion. 5. Across all diets inoculation impaired growth rate by 9% and FCR by 8%, but did not affect the amount of C. perfringens in the caeca.

Further data on chromosomal assignments of pig enzyme loci LDHA, LDHB, MPI, PEPB and PGM1, using somatic cell hybrids.

Clear interspecies differentiation between the chromosomes in pig-mouse somatic cell hybrids was achieved by using the THA-technique for the cytogenetic analysis. The assignments of LDHB and MPI to pig chromosomes nos 5 and 7 respectively, reported previously, were confirmed by analysis of 34 hybrid clones. The LDHA, PEPB and PGM1 genes were assigned to pig chromosomes nos 2, 5 and 6 respectively. Both LDHB and PEPB were indicated to be located on the long arm, except the most proximal part, of pig chromosome no. 5. The proposed synteny between LDHB and PEPB in pigs is in accordance with the synteny observed between these two loci in several other mammalian species.

PCR identification of chicken Eimeria: a simplified read-out.

A polymerase chain reaction (PCR) assay, based on the amplification of internal transcribed spacer 1 (ITS1) regions of ribosomal DNA, was developed for the chicken coccidian species Eimeria maxima, E. mitis and E. praecox. Thus, taking into account our previous work, a complete set of ITS1-based, species-specific primers for the detection and discrimination of all seven Eimeria species that infect the domestic fowl is now available. ITS1 primers for each of these seven species of Eimeria were also used as capture probes in a paper chromatography assay (PACHA). The addition of PACHA to the PCR assay provided a faster, more simplified read-out compared to staining of amplified bands in an agarose gel with ethidium bromide.

Development of a diagnostic PCR assay for the detection and discrimination of four pathogenic .Eimeria species of the chicken.

We describe a polymerase chain reaction (PCR)-based assay for the detection, identification and differentiation of pathogenic species of .Eimeria in poultry. The internal transcribed spacer 1 (ITS1) regions of ribosomal DNA (rDNA) from .Eimeria acervulina, E. brunetti, E. necatrix and .E. tenella were sequenced and regions of unique sequences identified. Four pairs of oligonucleotide primers, each designed to amplify the ITS1 region of a single .Eimeria species, were synthesised for use in the PCR assay. In tests on purified genomic DNA from all seven species of .Eimeria that infect the chicken, each of the four primer pairs amplified the ITS1 region from only their respective target species. The robustness of the approach was further demonstrated by the amplification of specific DNA fragments from tissues of experimentally infected animals and from oocysts recovered from field samples. We conclude that the ITS1 regions of .Eimeria species contain sufficient inter-specific sequence variation to enable the selection of primers that can be applied in PCR analyses to detect and differentiate between species. In future work they may provide excellent markers for epidemiological studies.