Comparison of diagnostic methods for assessment of Ostertagia ostertagi exposure in Norwegian dairy herds.

BACKGROUND: The gastrointestinal nematode (GIN) Ostertagia ostertagi can cause severe disease in first season grazers (FSG) and impaired performance due to subclinical infections in adult cows. Diagnostic methods to assess exposure include faecal egg count and detection of specific antibodies using antibody-ELISAs resulting in an optical density ratio (ODR). Using the ELISA test on bulk tank milk (BTM) allows for a herd level diagnosis. Appropriate use of diagnostic methods for evaluation of O. ostertagi exposure is required to optimize herd parasite surveillance and aid in a sustainable control regime. The aim of this study was to describe the relationship between different diagnostic tests used to assess GIN exposure in Norwegian production systems. A cross-sectional field study was carried out in twenty herds in Norway in the fall of 2020. Serum and faecal samples were taken from 380 individuals, of which 181 were FSG and 199 were cows. In addition, milk was collected from every cow and one BTM sample was taken from each herd. Faecal egg counts were performed. The distribution of ODR values in individual samples within and between herds and the associations between BTM ODR and individual ODR values were described. The data were analysed using visual assessment of scatter plots, Pearson correlation coefficients and linear regression. RESULTS: A high variability of the within-herd individual ODR values in serum and milk in every herd was detected. The ODR in BTM explained a low degree of the variation in the individual serum and milk samples. When plotting the ODR results in milk or serum according to four BTM categories, the distribution of ODR values were notably different in the highest and lowest BTM categories. The correlation between individual milk and serum samples was moderate (r = 0.68), while the highest correlation (r = 0.81) was between the BTM ODR and the group average individual milk samples. CONCLUSIONS: A poor predictive ability for BTM ODR to assess individual ODR values in both FSG and cows was demonstrated. However, the study indicates that the evaluation by ELISA test on BTM to assess exposure to GIN could be useful in herds with a very high or low BTM ODR.

Development and evaluation of an indirect ELISA for detection of Teladorsagia circumcincta infection in sheep.

BACKGROUND: The gastrointestinal helminth, Teladorsagia circumcincta, is one of the major health risks and production-limiting diseases in small ruminant populations, particularly in temperate regions. With the increasing importance of disease management and recruited anthelmintic resistant types, accurate approaches are needed for the diagnosis of the infection in the host. Due to uncertain results using faecal examinations, the ELISA method was indicated for the detection of nematode antigenic materials. Despite some promising results, problems were described in terms of test specificity and cross-reactions. Therefore, this study aimed to evaluate the IgG response to worm somatic and excretory/secretory (ES) products using western blot analysis and an indirect ELISA for the detection of T. circumcincta infection in sheep. RESULTS: Based on the immuno-reactivity analysis, immunogenic fractions with molecular weights (MWs) of approximately 60, 75 and 100 kDa were detected in somatic content and two antigens of about 63 and 75 kDa in ES material. Accordingly, a specific product at 75 kDa had the strongest reaction and appeared as the most common antigenic protein. In ELISA, all the sera from the infected sheep revealed the OD rates above the calculated cut-off value with about two-fold greater average. Negative control samples were also specifically recognized with the mean OD rate of about 1/3 of the estimated cut-off value. The cross-reaction test, using rabbit anti-T. circumcincta IgG, did not show reactivity with the ES antigens of other prevalent nematodes including Haemonchus contortus, Protostrongylus rufescens and Marshallagia marshalli. In contrast, a strong positive reaction was observed with the somatic antigens of M. marshalli. CONCLUSIONS: The results of this study indicated that the indirect ELISA method using the ES content enables distinguishing the T. circumcincta infected sheep with high specificity. Those antigenic ES peptides with 63 and particularly 75 kDa MWs should be further investigated due to the potential for serological diagnostic methods and immunoprotective targets in the host.

Electrochemical enzyme-linked immunosorbent assay (e-ELISA) for parasitic nematode Ostertagia ostertagi (brown stomach worm) infections in dairy cattle.

A sensitive electrochemical immunoassay (e-ELISA) has been developed for the detection of the gastrointestinal parasitic nematode Ostertagia ostertagi (brown stomach worm) in infected and control serum samples. An antigen-indirect immunoassay format was employed to detect the presence of O. ostertagi antibodies, coupled with an anti-species monoclonal horseradish peroxidase (HRP) conjugate. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and TMB (3,3',5,5'-tetramethylbenzidine/hydrogen peroxide) were investigated as both chromogenic visualising reagents for optical ELISA and electroactive substrates for electrochemical ELISA in the HRP catalysed oxidation reaction. Coulometry was applied for the detection of O. ostertagi antibodies (via TMB electrochemistry) and compared with the commercial optical ELISA (ABTS based SVANOVIR® O. ostertagi-Ab ELISA kit). Cost-effective in-house sensors were designed and fabricated using polyester and chemical adhesive materials with the aid of stencil printing and laser machining techniques. The performance of the electrochemical ELISA and sensor was evaluated by investigating redox mediators (ABTS vs. TMB), stop solutions (sodium dodecyl sulfate vs. sulfuric acid) and incubation times (150 min vs. 70 min vs. 25 min). For a total assay incubation time of 70 minutes, the TMB/H2SO4 based e-ELISA was able to differentiate between positive (P) and negative (N) control serum samples, with a P/N70 control ratio 1.6 times higher than that of optical ELISA (TMB/H2SO4 combination) and 2.9 times higher than that of the commercial ELISA kit (ABTS/SDS combination). Furthermore, the e-ELISA approach is quicker and required only 25 min (total incubation time) with even better response (P/N25 = 14.7), which is approximately 4-fold higher than the optical immunoassay (P/N25 = 3.8). The proposed e-ELISA is specific (selective Ab-Ag interactions) and highly sensitive - capable of detecting up to 16-fold dilutions of a positive control serum sample. The electrochemical ELISA approach has the potential for rapid sample screening in a portable, disposable format, contributing to the quest for effective prevention and control of parasitic Ostertagia ostertagi infections in cattle.

Droplet digital polymerase chain reaction (ddPCR) as a novel method for absolute quantification of major gastrointestinal nematodes in sheep.

In this paper, we present for the first time a new tool, based on Droplet Digital™ Polymerase Chain Reaction (ddPCR), for absolute quantification of key genera of gastrointestinal (GI) nematode parasites of grazing livestock. Four combinations of primers/probe sets targeting the internal transcribed spacer region 2 (ITS2) of the ribosomal RNA gene array were designed using the Primer3 software, following in silico analysis of nucleotide sequences from nematodes of interest downloaded from common databases. The amplified regions include both a universal region for detection of any strongylid gastrointestinal parasite and three different genus specific regions, making it possible to differentiate between the most important GI nematodes of sheep in Sweden: Haemonchus, Teladorsagia and Trichostrongylus. Analysis of samples containing serial dilutions and different mixtures of genomic DNA extracted from different species of adult worms proved useful in assessment of different threshold settings with the QuantaSoft software. Analysis of template DNA from these worms indicated that ddPCR is a viable choice for detection and absolute quantification of the different genera and also in samples with multiple species. Interpretation of the ddPCR results was straightforward and choice of analytical approach had little influence on the final results. Thus, the results obtained in the different analytical approaches seemed to be robust and the concentrations determined were uniform. Furthermore, the linear range of the Haemonchus ddPCR assay was similar to that of real-time PCR (qPCR). Taken together, our data confirm the suitability of ddPCR for detection and absolute quantification of three major sheep pathogens when tested on larval cultures from pooled ovine faeces. The results also indicate that ddPCR can be a useful complement to applications based on conventional egg counting methods such as the faecal egg reduction test (FECRT).

Do the changes in the behaviours of cattle during parasitism with Ostertagia ostertagi have a potential diagnostic value?

We investigated the magnitude of temporal changes in activity, posture and feeding behaviour of cattle infected with Ostertagia ostertagi, and their reversal after treatment with an anthelmintic. Twenty-six, 3-month-old, Holstein-Friesian bulls were allocated to one of three treatment groups. Bulls in two of those (groups P and PA) received 100,000 larvae on three occasions (Days 0, 7 and 14) and the remaining animals served as controls (C). The PA group also received an anthelmintic on Day 31. Parasite eggs appeared in the faeces of P and PA bulls from Day 17; from approximately the same time blood pepsinogen levels increased and body weight (BW) gain decreased (P<0.001). The reduction in BW gain persisted until Day 45 for P animals only. There was a decrease in the number of steps taken for P and PA animals, as well as lying and standing episode frequency, by 41 and 44% respectively (P<0.001) from Day 21 onwards. The average lying and standing episode duration increased by 52 and 55% respectively (P<0.001) from the same time in P and PA compared to C bulls. In addition, meal frequency showed a tendency to decrease for P animals only (P=0.039) from Day 39, and this was the only aspect of feeding behaviour affected by parasitism. All behaviours, returned to control levels within a week of anthelmintic drenching of PA bulls, apart from the number of steps taken. Although BW gain and pepsinogen also started to recover after drenching, these had not returned to control levels by Day 45. The magnitude of the changes in activity, and standing and lying episode frequency and duration suggest that these might have a diagnostic value, especially as all can now be monitored by automated means. However, these behaviours did not show the rapid changes we expected before parasitism manifested clinically and following recovery.

Comparison of three methods for gastrointestinal nematode diagnosis determination in grazing dairy cattle in relation to milk production.

Development of resistance to anthelmintic drugs has motivated the search for diagnostic methods to identify animals for targeted selective treatments. We compared three methods for the diagnosis of nematode infection in relation to milk production in a fully grazing dairy herd of 150 cows in the humid Pampa (Argentina). Animals had feces, blood and milk sampled during the first postpartum month for EPG, pepsinogen and anti-Ostertagia antibody determination, respectively. With the results obtained two groups of cows, divided in high and low parasite burden, were conformed for each method, and milk production was then compared between groups. When cows were separated by the EPG method (EPG=0 (N=106) vs. EPG>0 (N=44)) a difference of nearly 800 l of milk per cow per lactation was found (P<0.05). On the other hand, milk production between groups separated by Pepsinogen (mUtyr ≤ 1000 vs. mUtyr > 1000) or by anti-Ostertagia (ODR ≤ 0.5 vs. ODR > 0.5) results did not differ. Interestingly, proportion of cows in each group differed between methods (P<0.0001), and the anti-Ostertagia method yielded significantly more cows in the high index group compared to results using the EPG or Pepsinogen method. No correlations were found between parasite indexes determined by the different methods. High parasite burden estimation found may be ascribed to the production system, fully grazing all year round, and to the sampling time, at the beginning of lactation with cows in negative energy balance and depressed immunity. The fact that the cows were born and reared outside, on pasture with continuous nematode larvae exposure, may also account for the results obtained. In conclusion, EPG counting during the first postpartum month may be a useful tool for the diagnosis of production impairment induced by high nematode burden in adult grazing dairy cows. The anthelmintic treatment of only the EPG-positive recently calved cows would improve milk production, while reducing selective pressure on nematode population for the development of resistance.

A longitudinal survey of anti-Ostertagia ostertagi antibody levels in individual and bulk tank milk in two dairy herds in Normandy.

The Ostertagia-specific antibody levels in milk were monitored in 2 dairy herds to investigate seasonal variations and the relationship between individual and bulk tank milk antibody levels. Bulk tank and individual milk samples from all lactating animals were collected over a 1-year period at weekly and monthly intervals, respectively. The Ostertagia-specific antibody levels were measured with an indirect ELISA and the test results were expressed as optical density ratios (ODR). A clear seasonal pattern that followed the expected intake of infectious larvae was observed in the individual and bulk tank milk antibody levels of both herds. Within each herd, there was a large variation in the individual ODRs. This variation remained large when the distribution of individual ODRs was plotted according to high and low bulk tank milk ODR categories. The results suggest that the effect of seasonal variations on cut-off levels that predict production responses after anthelmintic control, needs to be assessed.

Associations between anti-Fasciola hepatica antibody levels in bulk-tank milk samples and production parameters in dairy herds.

Our primary objective was to determine the relationships between Fasciola-specific antibody levels in bulk-tank milk and measures of productivity to estimate economic losses that are associated with Fasciola infections. A bulk-tank milk sample was collected in March 2004 from 1105 dairy herds in Flanders and the antibody levels against Fasciola hepatica (ODRf) and Ostertagia ostertagi (ODRo) were determined. The association of ODRf with four production parameters (milk yield, milk-protein %, milk-fat % and inter-calving interval) was assessed by multivariable linear-regression models. Production data were available for 463 out of the 1105 herds sampled. An increase in ODRf from the 25% quantile (0.428) to the 75% quantile (1.064) was associated with a decrease in the annual average milk yield of 0.7kg/(cowday) (P=0.002), with a decrease in the average milk-fat % of 0.06% (P<0.001) and with an increase of the mean inter-calving interval of 4.7 days (P=0.03). No significant relationship was found with the average milk-protein %. When the relationships of ODRf and ODRo with milk yield were tested simultaneously, we saw an additive rather than synergistic effect of concurrent infections.

The effect of an experimentally induced acute mastitis on the test results of an Ostertagia ostertagi milk ELISA.

Antibody levels against Ostertagia ostertagi in the milk are a promising parameter to identify dairy cows or herds with production losses due to gastrointestinal nematodes. However, milk antibody levels can be influenced by udder-related factors. In this study, the effect of an experimentally induced mastitis on the test results (ODR) of a milk O. ostertagi ELISA was investigated. Twenty-five cows that were naturally infected with gastrointestinal nematodes, were inoculated in their left udder quarters with Escherichia coli P4:O32 and quarter milk samples were collected at several intervals from 24 h before until 144 h after experimental infection. The effect of the contribution of milk from one or more infected quarters on the bulk tank milk ODR was estimated, based on a titration experiment. The mean O. ostertagi antibody levels of the infected udder quarters were significantly (P < 0.001) higher than those of the uninfected udder quarters at each sampling time post-infection. The largest difference was observed at 24 h post-infection with a mean difference of 0.251 ODR (95%CI: 0.172; 0.330). There was also a significant increase (P < 0.001) in total IgG levels with the largest difference being observed at 24 h post-infection. Highly significant (P < 0.005) correlation coefficients were observed between O. ostertagi ODR, total IgG ODR, Na+ and Cl- ion concentration and log transformed somatic cell counts at 24 h post-infection. The results demonstrate that an acute mastitis causes a flow of specific and non-specific antibodies from serum to milk with a subsequent increase in the O. ostertagi ODR values. The effect of the contribution of milk from infected quarters on the bulk tank milk O. ostertagi ODR was estimated to be minor if the relative number of infected quarters is small (< 3%).

Assessing the agreement between Ostertagia ostertagi ELISA tests performed using the crude adult antigen and the adult and larval stage 4 excretory/secretory antigens.

The objective of this study was to determine the agreement between ELISA tests conducted using three O. ostertagia antigens: crude adult worm, larval stage 4 (L4) excretory/secretory (ES) and adult ES. This study was carried out on 289 Holstein cows from five herds in Prince Edward Island and one herd in Nova Scotia. Composite milk samples of these cows were collected (between May and September 2002) from the respective provincial laboratories and sent to the Atlantic Veterinary College where each sample was tested for antibodies to O. Ostertagi using an indirect microtitre ELISA test. Results were expressed as optical density ratio (ODR) values. Each milk sample was tested with three ELISA tests, with each test using a different O. ostertagi antigen. There was a slight rise in ODR values of both adult antigens, between May and August, with higher values obtained using the adult ES antigen. L4 ES ODR values were generally higher than those for both adult antigens during the study period, except for May. There was a more dramatic rise in L4 ES ODR values between May and August. Rises in ODR in May and end of July coincided with periods of mass maturation of L4 to adult worms. The results of the study showed that the concordance correlation coefficient (CCC) between tests performed using both ES and the crude antigens were low (crude adult versus adult ES=0.31, crude adult versus L4 ES=0.30). The highest CCC was observed between tests done using both ES antigens (CCC=0.56). Generally, the study results suggest that the antibody response (detectable by the ELISA) is mainly directed against ES antigens (especially L4) than the crude adult worm antigen.

Evaluation of the stability of Ostertagia ostertagi ELISA microtitre plates over time using cow milk samples.

The objective of this study was to assess the stability of ELISA plates prepared with one of three blocking agents and used with one of two conjugates at various time intervals after preparation of the plates. Two of the blocking agents used were commercially available: one termed stabilgaurd (stab) and one manufactured by SVANOVA Biotek AB Inc. (svan). The third blocking agent used was bovine serum albumin (bsa). A polyclonal rabbit anti-bovine IgG (poly) and an anti-bovine IgG monoclonal (mono) conjugate were used. Eighteen composite individual cow milk samples collected late in lactation (200-400 days in milk) were used in this study. An indirect microtitre plate ELISA that used the Ostertagia ostertagi antigen was used to quantify antibodies against the parasite, present in the milk samples. Each of six blocking agent/conjugate combinations (called systems) were used to test 18 milk sub-samples at 1, 4 and 24 weeks after blocking the plates. Plates blocked with stab and svan were kept at room temperature and an additional set were incubated at 37 degrees C so as to mimic long term storage (about 1 year) and tested only once at 4 weeks. Those blocked with bsa were frozen at -20 degrees C. Concordance correlation coefficients (CCC) and reproducibility were used to assess the agreement between test results conducted on the same milk sample at the various test-times using a particular system. Generally, there was good agreement between tests conducted at different times for all systems. However, the svan-mono and bsa-poly systems had the best agreement with overall CCC values of 96% and 93%, respectively. The svan-poly system had the lowest CCC of 75%. The CCC and reproducibility ranked the systems in a similar way. The high CCC between tests done using plates kept at room temperature and ones incubated at 37 degrees C, suggested that plates would be stable up to a year after blocking. The storage of plates blocked with svan and stab agents under room temperature, makes them more convenient to use and transport relative to bsa-blocked plates that have to be frozen.

Assessment of the repeatability of a milk Ostertagia ostertagi ELISA and effects of sample preparation.

An indirect Ostertagia ostertagi ELISA on milk is a promising diagnostic tool in bovine parasitology. Interpretation of the test results requires a good knowledge of the test characteristics. In this study, border effects, the repeatability of the ELISA and the effect of different factors such as storage, skimming and freeze-thaw cycles of the milk samples were investigated. The border effects trial showed that significant border effects can occur. The repeatability trial was conducted over 3 days. An alternative graphical technique to assess the repeatability over a large number of ELISA plates measured over different days was developed. From these graphs, it was obvious that the ODR values obtained on the third day were deviating from the values on the first and second day. On the third day, also abnormal control values were observed. When the control values were normal, 94% of the variability was explained by the milk sample and 6% by assay variability. The expected 95% range of the difference of 2 ODR readings of the same sample on the same plate and the same sample on different plates was -0.14 to 0.14 and -0.16 to 0.16. No extra variability was observed when samples were tested on a different day, however these results are based on the measurement of 2 days. Storage for 2-4 days at 4 degrees C, using whole milk instead of skimmed milk and up to 2 extra freeze-thaw cycles of the milk samples did not significantly affect the test results.

Copro-antigen capture ELISA for the detection of Teladorsagia (Ostertagia) circumcincta in sheep: improvement of specificity by heat treatment.

A copro-antigen capture ELISA for the detection of Teladorsagia circumcincta infection in sheep was developed and evaluated. Experiments with faeces from worm-free sheep, that had been spiked with known concentrations of excretory-secretory (E-S) antigen indicated that a positive signal was generated with 180 ng of E-S/ml. A nested design, based on 8 infected and 8 worm-free sheep, was employed to assess the stages during sample preparation contributing to variation in signal from the assay. This showed that 87% of the variance in the optical density readings (ODs) was directly explained by infection status. Variation between individual sheep within infection groups, and between samples at various stages in the assay, collectively accounted for the remaining variance. Initial evaluation of specificity using faeces from animals carrying a range of monospecific infections indicated cross-reactivity with Haemonchus contortus and Nematodirus spathiger. However, by treating the supernatant from faeces for 20 min at 100 degrees C, the cross-reactive signal was eliminated whilst the specific signal was largely preserved. Heat treatment of faeces from 12 non-infected sheep, 12 sheep with T. circumcincta and 6 with H. contortus resulted in sensitivity being increased from 66.7 to 85.7%, and specificity from 62.5 to 87.5%. OD values showed a significant positive relationship with adult worm burdens, although at low infection intensities there was some overlap between infected and worm-free animals. We discuss the application of CC-ELISAs in facilitating selective chemotherapy of sheep, as a means of avoiding the development of anthelmintic resistance in pastoral regions where sheep are farmed on a large scale.

Evaluation of the repeatability of a crude adult indirect Ostertagia ostertagi ELISA and methods of expressing test results.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Ostertagia ostertagi using a crude adult worm antigen was evaluated using serum and milk samples from adult cows, as well as from bulk tank milk. Within and between plate repeatabilities were determined. In addition, the effects of factors such as antigen batch, freezing, preserving of the samples and somatic cell counts (SCCs) of the samples were evaluated. Raw optical densities (ODs) and normalized values were compared using the concordance correlation coefficient (CCC), the coefficient of variation (CV), Bland-Altman plots (BA). Based on raw OD values, there was a high repeatability within a plate (CCC approximately 0.96 and CV<10%). Repeatability between plates was evaluated following normalization of OD values by four methods. Computing normalized values as (OD-Nt)/(Pst-Nt), gave the most repeatable results, with the CCC being approximately 0.95 and the CV approximately 11%. When the OD values were higher than 1.2 and 0.3 for the positive and the negative controls, respectively, none of the normalization methods evaluated provided highly repeatable results and it was necessary to repeat the test. Two batches of the crude antigen preparation were evaluated for repeatability, and no difference was found (CCC=0.96). The use of preservative (bronopol) did not affect test results, nor did freezing the samples for up to 8 months. A significant positive relationship between ELISA OD for milk samples and SCC score was found. Therefore, the use of composite milk samples, which have less variable SCC than samples taken from each quarter, would be more suitable when the udder health status is unknown. The analytical methods used to evaluate repeatability provided a practical way to select among normalization procedures.

Development of a copro-antigen capture ELISA for detecting Ostertagia ostertagi infections in cattle.

The present study reports on the development of a copro-antigen capture ELISA for detecting Ostertagia ostertagi infections in cattle. The ELISA was based on polyclonal rabbit antibodies, which recognize O. ostertagi excretory/secretory antigens (ES). ES antigens are released by the metabolic active stages of the parasite in the abomasum, and passed in the faeces of the host. The detection limit of pure ES material was 30 ng ml(-1) in sample buffer and 125 ng ml(-1) in faecal extract. The test was evaluated using a follow up from six artificially infected calves. Elevated levels of Ostertagia coproantigens could be measured from 21 days after infection, indicating that only the presence of adult parasites can be detected. To evaluate the capacity of the assay to measure levels of infection, three groups of cattle were tested: 38 artificially infected calves, 17 naturally infected first grazing season calves and 16 naturally infected adult dairy cows. Optical densities were significantly correlated to the worm burdens of the animals and the ELISA had an overall sensitivity of 91% and a specificity of 45%. The test gave negative readings for faeces of animals carrying patent mono-infections with Cooperia oncophora.

Identification and semi-quantitation of Ostertagia ostertagi eggs by enzymatic amplification of ITS-1 sequences.

A region within the first internal transcribed spacer (ITS-1) of the ribosomal DNA repeat of Ostertagia ostertagi has been identified that is 408 base pairs (bp) in length and is comprised of a 2 x 204 bp repeat. Universal polymerase chain reaction (PCR) primers which span this region, as well as a portion of the 5.8S rDNA, generate a 1011 bp fragment using genomic DNA from O. ostertagi. However, these same primers generate only a 600 bp (approximate) fragment using DNA from Haemonchus contortus, Cooperia oncophora and Oesophagostomum radiatum, as well as other species within the genus Ostertagia. When DNA samples derived from adult parasites of the different genera were mixed and simultaneously amplified, the O. ostertagi component could be identified within the mixed DNA populations. Furthermore, a correlation was observed between relative fluorescence intensities of the 1011 bp and the 600 bp PCR fragments and the percentage of O. ostertagi DNA within a mixture of parasite DNAs. A similar high correlation was obtained between the percentage of O. ostertagi DNA and percent O. ostertagi eggs in feces containing eggs of other nematode genera. This resulted in the generation of a protocol that can determine the percentage of O. ostertagi eggs within a mixed population of gastrointestinal nematode eggs. Results indicate a detection equivalent to 0.05 eggs.

Evaluation of a micro method for the routine determination of serum pepsinogen in cattle.

Estimation of serum pepsinogen concentration in cattle is used to aid the detection of clinical or subclinical infections with the abomasal nematode Ostertagia ostertagi. An inexpensive, simple micro method for the routine determination of pepsinogen concentration in bovine serum samples is described which is based on the hydrolysing effect of serum on buffered bovine albumin substrate. Comparison of this assay with a macro method, based on the same principle, gave almost identical results in the range of 0 to 10 Units tyrosine. The reproducibility of the assay was shown to be very satisfactory, intra-assay and day-to-day coefficients of variations were less than 4.7 and 8 per cent, respectively.

Local and plasma antibody responses to the parasitic larval stages of the abomasal nematode Ostertagia circumcincta.

Ovine isotype-specific antibody responses to the parasitic larval stages of the abomasal nematode Ostertagia circumcincta were measured in a simple, indirect enzyme-linked immunosorbent assay. Analysis of variance of replicate tests showed that the assay was very reliable. There was substantial variation among individual sheep in their IgA and IgG1 responses even though the sheep had been matched for breed, age and sex, were born on the same farm, were reared identically and had the same history of exposure and challenge with O. circumcincta. The local IgA responses to a somatic extract of fourth-stage larvae were very similar to responses to excretory-secretory products of fourth-stage larvae. The responses to third stage larvae were correlated with the responses to fourth stage larvae. There was a negative correlation between parasite-specific plasma IgG1 and parasite-specific plasma IgA responses. There was only a moderate association between IgA responses in the mucus and the plasma. Therefore, antibody responses measured in plasma cannot be easily extrapolated to antibody responses in the abomasal mucus.

Indirect gene diagnoses for complex (multifactorial) diseases--a review.

The analysis of multifactorial diseases requires the efficient investigation of large numbers of (gene) loci and patient (family) samples. Since simple repetitive DNA markers are dispersed all over the chromosomes, molecular techniques employing these tools render most conventional screening procedures obsolete. Examples of tumors, autoimmune diseases and infections are presented to validate concepts of indirect gene diagnoses via simple, tandemly arranged, repetitive DNA sequences. The salient advantages of microsatellite technologies vs. those of multilocus DNA fingerprinting are weighed.

Ostertagia ostertagi: changes in lymphoid populations in the local lymphoid tissues after primary or secondary infection.

The purpose of this study was to begin to define the changes in the local lymphoid tissues that accompany Ostertagia ostertagi infection in naive and immunized calves. Abomasal lymph nodes were taken from calves beginning as early as 2 days post-infection. Phenotypic changes in the resulting lymphocyte populations were assessed by flow cytometry utilizing monoclonal antibodies specific for the cell surface determinates CD2, CD4, and CD8. Changes in antigen specificity were determined by limiting dilution analysis utilizing antigen derived from fourth-stage O. ostertagi. Primary infection of naive calves caused a rapid 30-40% decrease in the percentage of T cells in the abomasal lymph nodes. This decrease in T cell percentage was due to a decrease in cells bearing the CD4 marker, a marker usually associated with helper T cells. Immunized calves were able to maintain normal T cell percentages of 50-60% for the first 5 weeks of infection. Immunization greatly increased the total number of Ostertagia-specific T cells in the abomasal lymph nodes owing to a marked increase in the size of the lymph nodes. Challenge infection of naive and immunized calves caused an increase in the frequency of parasite-specific T cells in both groups, but the increase was more rapid in the previously immunized calves. Within 5 weeks of infection, Ostertagia-specific cells could not be detected in the abomasal lymph nodes. These results indicate that the critical time period for expansion and regulation of Ostertagia-specific T cells in infected calves is early in the infection at a time that coincides with larval development. In addition, previous exposure to parasite antigens appears to result in more rapid responses and in the maintenance of normal ratios of T cell subpopulations in the draining lymphoid tissues.