Development and evaluation of a recombinant antigen-based ELISA for serodiagnosis of cattle lungworm

An optimised enzyme-linked immunosorbent assay (ELISA) for the detection of Dictyocaulus viviparous-specific antibodies was developed and evaluated following the testing of various microtitration plates and anti-bovine Ig-conjugates. Based on recombinant major sperm protein (MSP) expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli, sera collected from 112 cattle experimentally infected with D. viviparus, from 129 helminth-na?ve calves, 8 calves experimentally infected with Ostertagia ostertagi, and 2 calves infected with Cooperia oncophora were tested. ELISA results showed a calculated specificity and sensitivity as well as positive and negative predictive values of >99%. No cross-reactions with sera from calves infected with O. ostertagi or C. oncophora were observed. Lungworm-specific immunoglobulins were first detected from 28 to 35 days post-infection onwards. To differentiate between antibody-binding to the MSP-part or the GST-part of the fusion protein, additional ELISAs were performed using pure recombinant MSP or GST. Optical densities obtained from the ELISAs with the MSP showed a similar pattern to optical densities measured in the ELISAs with the fusion protein, whereas GST gave only a low background. By testing serum samples from naturally infected calves, it was found that the MSP-ELISA is positive even for sera from calves showing very low faecal larval counts. Thus, we conclude that the ELISA using the recombinant MSP-fusion protein appears to be a suitable method for routine diagnosis and epidemiological studies of cattle lungworm.     

Preliminary investigation into the usefulness of the indirect haemagglutination

Summary To give an impression of the usefulness of indirect haemagglutination (IHA) in the diagnosis of lungworm infections in cattle under practical conditions, five calves vaccinated against Dictyocaulus viviparus and five unvaccinated calves were periodically subjected to clinical, parasitological, and serological examinations over a period of seven months. All calves grazed on a lungworm-infected plot. 82% of the observations in unvaccinated calves, which were positive with respect to one or more of the used parameters, concerned IHA-positive animals which, however, showed negative results with the parasitic parameters. The titre variation of the serological examination was a further indication of the fact that the IHA detected antibodies against lungworm antigens. No indications of false positive reactions were obtained. An investigation carried out on 46 farms on the correlation between serological and clinical findings on lungworm infections revealed a positive correlation in 80% of the groups between results obtained with both methods. The authors consider that IHA offers good prospects for the diagnosis of lung-worm infections.     

Efficacy of febantel against abomasal nematodes and lungworms in cattle

The efficacy of febantel at a dosage of 5 mg/kg (45.5% paste formulation) against inhibited early 4th-stage larvae (EL4) of Ostertagia ostertagi, other nematodes of the abomasum, and Dictyocaulus viviparus was investigated in 4- to 6-month-old Holstein calves that grazed on pasture heavily contaminated with parasites from February 24 to April 1, 1986 (36 days). In Louisiana, this is the first month of a 3-month period in which increasing numbers of inhibition-prone O ostertagi larvae are acquired, and infection risk with D viviparus may remain high. Three of 4 calves that died of lungworm infection during the pasture-exposure period were necropsied. Large numbers of abomasal nematodes, including inhibited O ostertagi larvae, and large numbers of D viviparus were recovered. Twenty-five calves were randomly allotted by equal distribution of body weight to 2 groups and treated on April 4: placebo-treated calves (n = 13) and febantel-treated calves (n = 12). Equal numbers of treated and control calves were killed at 6 and 7 days, respectively, after treatment. Mean numbers of O ostertagi in control cattle were: adults, 4,931; developing 4th-stage larvae (DL4), 1,119; and inhibited EL4, 3,410. Ostertagia lyrata, Trichostrongylus axei, Haemonchus sp, and D viviparus were well distributed in nearly all control calves. Percentage reduction of O ostertagi in treated calves, when compared with controls, was: adults, 83.6%; DL4, 57.8%; and inhibited EL4, 34.8%. Percentage reductions of other species were: O lyrata, 92.6%; T axei adults, 99.3% and 4th-stage larvae (L4), 100%; Haemonchus sp adults, 66.7%, and L4, 64%; D viviparus adults 90.6%, and immature forms, 97.1%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary investigation into the usefulness of the indirect haemagglutination

Summary

To give an impression of the usefulness of indirect haemagglutination (IHA) in the diagnosis of lungworm infections in cattle under practical conditions, five calves vaccinated against Dictyocaulus viviparus and five unvaccinated calves were periodically subjected to clinical, parasitological, and serological examinations over a period of seven months.

All calves grazed on a lungworm‐infected plot. 82% of the observations in unvaccinated calves, which were positive with respect to one or more of the used parameters, concerned IHA‐positive animals which, however, showed negative results with the parasitic parameters. The titre variation of the serological examination was a further indication of the fact that the IHA detected antibodies against lungworm antigens. No indications of false positive reactions were obtained.

An investigation carried out on 46 farms on the correlation between serological and clinical findings on lungworm infections revealed a positive correlation in 80% of the groups between results obtained with both methods.

The authors consider that IHA offers good prospects for the diagnosis of lung‐worm infections.     

A study of helminth parasites in culled cows from Ireland

The objective of this study was to determine the prevalence and intensity of gastrointestinal nematode, lungworm and liver fluke infection in culled cows in Ireland. Abomasa, colorectal contents and livers were collected from 30 to 68 culled beef and dairy cows during autumn 2002 and summer 2003, respectively. Ostertagia ostertagi were found in the abomasa of only three (10%) cows sampled in autumn and in 38 (57%) cows examined in summer. The majority of positive animals had low burdens of O. ostertagi but a few individuals in the group sampled during the summer had a moderate infection (5000-10,000 adult worms). A proportion of the cows in the summer group were also co-infected with small numbers of Trichostrongylus axei. Cooperia oncophora predominated in the recoveries from the larval cultures although O. ostertagi were also recovered. The overall prevalence of Dictyocaulus viviparus was 14%, based on larval identification in faecal samples. Liver fluke, or varying degrees of pathology attributable to Fasciola hepatica, were present in 65% of the livers. The results of this study extend those of previous workers, which were largely limited to dairy cows alone and which focussed on gastrointestinal nematodes and did not include simultaneous infections with lungworm and liver fluke. It was concluded, from the level of polyparasitism evident in this study, that adult cattle should be considered in preventative approaches to bovine helminthosis.     

Immunity of ivermectin treated cattle to challenge from helminth parasites in the following season

Two groups of yearling cattle which had been treated with ivermectin either three and eight, or three, eight and 13 weeks after turn out to trichostrongyle contaminated pasture in their first grazing season were exposed in the following season to natural challenge with helminth parasites. To assess their immunity to this challenge each group shared a pasture with parasite naive first season calves. No anthelmintic treatments were administered at any time during the year. Throughout the grazing period the yearlings showed normal respiratory rates, negative faecal lungworm larval counts, and, relative to the calves, low faecal trichostrongyle egg counts. All the first season calves developed patent lungworm infections and on one occasion the mean respiratory rates of each group of calves were significantly greater than those of the yearling cattle. At the end of the grazing period, from early May until late September or October 1986, the cattle were removed from pasture and together with parasite naive controls challenged with either 10 or 22 third stage larvae of Dictyocaulus viviparus/kg bodyweight and necropsied between 18 and 23 days later. Although the experimental challenge resulted in relatively heavy lungworm infection of the naive controls, none of the yearlings and only three of the 11 calves which had been at pasture were found to be infected. However, large numbers of arrested fourth stage larvae of Ostertagia ostertagi were present in all the naturally infected yearlings and calves.     

Interactions between lungworms and gastrointestinal worms in calves

Interactions between gastrointestinal worms (Ostertagia ostertagi, Cooperia oncophora) and lungworms (Dictyocaulus viviparus) in calves were studied by assessing the effect of primary infections with either group of worms on the development of homologous or heterologous challenge infections. Primary infections with lungworms resulted in some degree of resistance to challenge with gastrointestinal worms, but this resistance was lower than that found after homologous infection. Primary infections with gastrointestinal worms did not confer any resistance to challenge with lungworms. On the contrary, an indication was found of some enhancing effect of previous gastrointestinal worm infection on the establishment of lungworms. The highest degree of resistance against lungworm challenge was found where calves have been primarily infected with lungworms. Lungworm infections produced some elevation of serum pepsinogen levels. Gastrointestinal worms evoked a rise in circulating eosinophils, although this rise was smaller and occurred later than in lungworm-infected calves. Under the conditions of the experiment, the effect of 6000 infective lungworm larvae on weight gain was larger than the effect of 100,000 L3 of Ostertagia ostertagi and 100,000 L3 of Cooperia oncophora.     

The specificity of antibody responses in cattle naturally exposed to Fasciola hepatica

Fasciola hepatica causes significant morbidity and mortality in dairy cattle in the Andean region of Cajamarca, Peru, where prevalence of infection of up to 78% has been reported. ELISA and Western blot analyses were used to characterise antibody responses in dairy cattle to adult F. hepatica to excretory-secretory (E/S), somatic (SO) and surface (SU) antigens. Three groups of dairy cattle - calves, heifers and adult cows - naturally exposed to F. hepatica in this region, were monitored every 2 months over a 2-year period. Calves, heifers and adult cows all had antibodies which recognised a 28kDa protein in the SO preparation, whereas only adult cows had antibodies that recognised a 28kDa protein in E/S products. All three groups of cattle responded to a 60-66kDa group of proteins in E/S and SU preparations and a 17kDa antigen in SO products was recognised by antibodies from cows and heifers but not calves. The total antibody response to E/S antigens measured by ELISA, increased over time in calves and remained constantly high over the 2-year period in all three groups of cattle. Slight fluctuations in the antibody response occurred in the group of heifers and cows coinciding with seasonal changes in the level of challenge.     

Dictyocaulus species: cross infection between cattle and red deer

Aim: To discover whether cross infection between red deer (Cervus elaphus) and cattle is possible with either a bovine isolate of the cattle lungworm, Dictyocaulus viviparus, or with a cervine isolate of the lungworm, Dictyocaulus eckerti which is thought to be maintained primarily in deer. Method: Twelve cattle and 12 red deer were reared parasite-free from birth. At 3-4 months of age, half of each species (n=6) were experimentally infected with D. viviparus and the other half with D. eckerti. The course of infection was monitored for 34 days, after which the animals were slaughtered and the lungs removed to assess levels of infection. Results: Faecal larval counts demonstrated that patent Dictyocaulus infections occurred in all groups. At necropsy, adult worms were found in the lungs in all groups except the cattle that were infected with D. eckerti. The largest numbers of adult worms were found in the red deer infected with D. eckerti. Conclusion: It was demonstrated that both cattle and red deer could be infected with either D. viviparus or D. eckerti. However, D. eckerti larvae that originated from deer established more successfully in deer and D. viviparus larvae that originated from cattle established more successfully in cattle.     

Observations on the epidemiology of Dictyocaulus viviparus in north west England

A five year ley pasture was used as a source of natural infection with Dictyocaulus viviparus for cattle in anthelmintic trials. Pasture larval counts, faecal larval counts of permanently grazing calves and lungworm burdens harboured by tracer calves were monitored in three grazing seasons to assess the pattern of infection. Carrier calves were introduced at the beginning of the grazing season in the first two years of the study but not in the third. In the fourth year the pasture was subdivided into two paddocks where overwintered infection with and without carrier infection were compared. A control paddock exposed to carrier infection but no overwintered infection was also monitored. Pasture larvae survived the winter but carrier infection appeared to make a larger contribution to pasture larval counts and the onset of parasitic bronchitis in susceptible calves. In the absence of grazing cattle at the end of the grazing season the concentration of D viviparus larvae on the herbage fell rapidly to undetectable levels. Discrepancies between contamination of herbage by infective D viviparus larvae and infectivity of pasture for susceptible cattle occurred in all years but were particularly marked on the third year when natural immunity appeared to influence the number of lungworms accumulating in tracer calves. Failure to recover lung worms from tracer calves cannot be regarded as an accurate indication of lungworm free pasture. In the first three years the proportion of the lungworm population which was inhibited in tracer calves was higher early and late in the grazing season and negligible in mid season. This suggests that a predisposition to inhibition in larvae which have overwintered on pasture may influence the time of onset of parasitic bronchitis in the next grazing season, but results from the fourth year did not support this hypothesis.     

Dictyocaulus viviparus seroprevalence and epidemiology in Costa Rican dairy cattle

A cross-sectional serological survey of Dictyocaulus viviparus was carried out to determine the prevalence of lungworm infections in 28 dairy cattle farms distributed in five selected areas from Costa Rica. The influence of area, farm, host (breed, age and lactation number) and ecological factors (altitude and life zones) on the presence of lungworm infection was analyzed. A sub-sample of 924 sera collected between September 1998 and July 1999 was processed by ELISA (Ceditest). A total of 162 (17.5%) animals from 26 (93.0%) farms showed antibodies against D. viviparus. The overall seroprevalence detected among areas was Poás 25.0%, Cartago 24.3%, Tilarán 22.0%, Alfaro Ruiz 12.0% and San Carlos 12.1%. Using analysis of variance no significant influence of area and host factors on D. viviparus infections was determined, whereas the variable farm within area was highly significant (p<0.001). However, altitude and life zones showed significant association to seropositive animals, when a Chi-square test was applied. In altitudes of 1000-2000 m (p<0.001) and life zones of Lower Montane moist forest and Montane moist forest (p<0.001) D. viviparus infections in bovines were significantly higher. The results obtained in this study indicate a high D. viviparus seroprevalence in the analyzed farms and that the factors farm, altitude and life zones were significantly related to lungworm infections.     

Efficacy of Cydectin moxidectin 1% injectable against experimental infections of Dictyocaulus viviparus and Bunostomum phlebotomum superimposed on natural gastrointestinal infections in calves.

Twenty male Holstein calves averaging 105 kg in weight and naturally infected with gastrointestinal nematodes and small numbers of lungworm and hookworm, were given experimental infections with the two latter species to provide adult and larval stages for anthelmintic evaluation. Following random allotment, one group of 10 calves was injected subcutaneously with moxidectin at a dosage of 0.2 mg kg-1 of body weight. A second group of 10 was injected subcutaneously with unmedicated blank vehicle at a dosage of 1 ml per 50 kg of body weight. Fecal samples were examined before treatment and at 7 and 13 days after treatment. The 20 calves were necropsied for worm recovery at 13 and 14 days after treatment. All calves were positive for lungworm and hookworm on the treatment date. Treatment was 100% effective in elimination of hookworm eggs and lungworm larvae and 99.9% in reducing total egg counts at both 7 and 13 days after treatment. Moxidectin was 100% effective (P less than 0.01) in eliminating the following 11 species of nematodes. Dictyocaulus viviparus mature and immature adults (E5), Bunostomum phlebotomum adults and L4, Ostertagia ostertagi adults and early L4, Ostertagia lyrata adult males, Haemonchus placei adults. Trichostrongylus axei adults, Cooperia spp., including Cooperia punctata, Cooperia spatulata, and Cooperia pectinata adults, Oesophagostomum radiatum adults and Trichuris discolor adults. No adverse reactions to moxidectin treatment were observed.     

A comparison of interactions between vaccination against Dictyocaulus viviparus and anthelmintic suppression in immunised and unimmunised yearling cattle

Twenty parasite-naive calves aged approximately four months were divided randomly into four groups of five. Two groups were treated with oral lungworm vaccine. One immunised group plus another non-immunised group were put out to graze on May 1 on a pasture known to be contaminated with Dictyocaulus viviparus infective larvae during the previous autumn. All the calves both indoor and outdoor were treated with ivermectin at three, eight and 13 weeks after the first groups started to graze and again at housing at the end of September. After the winter housing period on April 27 of the following year all the calves were given an artificial challenge of D viviparus infective larvae at the rate of 15 larvae per kg bodyweight, and the clinical and parasitological reactions monitored. All the calves which had been vaccinated or exposed to field infection during year 1 reacted strongly in ELISAs using antigen prepared from fourth stage D viviparus larvae but much less strongly in similar tests using adult derived antigen. Clinically those calves exposed to previous field infections were less severely affected than the housed calves, although parasitologically all three groups with prior exposure to D viviparus appeared to have a similar functional level of immunity to the challenge infection in comparison to the unexposed calves of the same age.     

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle.

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.     

Experimental infections with Dictyocaulus viviparus in vaccinated and unvaccinated red deer

Four of eight red deer calves which had been artificially reared and were lungworm free were vaccinated with bovine lungworm oral vaccine when eight weeks old; the other four were not vaccinated. Three of each category were challenged daily with 500 Dictyocaulus viviparus infective stage larvae per kg liveweight for 17 days when six months old while one in each category was left as an unchallenged control. The effects of challenge were monitored and all challenged deer and one control were killed for post mortem assessment. Challenge with D viviparus was associated with reduced food intakes and weight gains but vaccinated calves were less affected than unvaccinated ones. The reaction of the alveolar tissue of red deer lung to D viviparus was mild in vaccinated and unvaccinated animals and differed from that of bovine lung in that alveolar epithelialisation was limited and hyaline membrane formation and interstitial emphysema were not seen. The disease was most evident in and around airways and was less in vaccinated calves. It was concluded that young red deer are tolerant to D viviparus but will readily acquire infection.     

Comparison of vaccination and ivermectin treatment in the prevention of bovine lungworm

Three groups of calves were put out to graze on separate paddocks within a field known to be infected with Dictyocaulus viviparus and were also given a small initial trickle infection of the parasite. The first group were untreated controls, the second were immunised with live irradiated lungworm vaccine and the third were injected three times with ivermectin; the injections taking place after they had grazed for three, eight and 13 weeks. The subsequent infections of D viviparus were estimated by grazing a series of parasite-free tracer calves in the paddocks used by each group. The first group of such calves grazed from July 17 until August 7, the second from August 22 to September 29. During the first half of the grazing season all the untreated and three of the six immunised calves were observed to excrete D viviparus larvae, in contrast to none of the ivermectin-treated group. As a result all the tracer calves on the areas occupied by the untreated and immunised calves became infected with the parasite whereas only one worm was found in one of the 10 tracer calves grazing the area occupied by the ivermectin-treated calves.     

A survey on Dictyocaulus viviparus antibodies in bulk milk of dairy herds in Northern Germany

Parasitic bronchitis caused by the bovine lungworm, Dictyocaulus viviparus, occurs worldwide in temperate areas. The parasite is found predominantly in calves and heifers, but dairy cattle can suffer from lungworms when they become infected for the first time or if they have lost immunity due to lack of exposure to lungworm larvae during the grazing season. The present study was performed to determine the D. viviparus bulk milk antibody prevalence in dairy herds in the East Frisian region of northwestern Germany, Lower Saxony, by analysing bulk milk samples collected in January (860 samples), September (866 samples) and November (860 samples) 2008, thereby representing 906 dairy farms. These samples were tested for antibodies against D. viviparus by a milk ELISA. This test detects patent infections only since it is based on recombinant major sperm protein as antigen. While in January 12.8% of dairy farms were positive for D. viviparus antibodies, the bulk milk samples collected in September and November revealed 6.9% and 6.6% positive dairy herds. From the 906 dairy farms included in the study, 191 (21.1%) tested positive at least once for antibodies against lungworm. From 810 dairy farms from which bulk milk samples were obtained during all three samplings, 146 (18.0%) farms were positive at one sampling date, 27 (3.3%) at two, and 4 (0.5%) on all three sampling dates. The majority of the farms represented in the study belonged to four districts of East Frisia, which showed no significant difference in the proportion of positive dairy farms.     

Persistent anthelmintic activity of ivermectin in cattle

Two studies are described which demonstrate the persistent activity of ivermectin injected subcutaneously into cattle at 200 micrograms/kg in preventing the establishment of induced infections with the gastrointestinal parasites Ostertagia ostertagi and Cooperia oncophora and the lungworm Dictyocaulus viviparus. These results indicated a reduction in mean worm count compared with the control group for O ostertagi of more than 99, 45 and 94 per cent with a seven, 14 or 21 day interval between treatment with ivermectin and the administration of infective larvae, respectively, in trial 1 and more than 99, more than 99 and 99 per cent at seven, 10 or 14 days, respectively, in trial 2. Corresponding values against C oncophora were 99, 0 and 45 per cent at seven, 14 and 21 days in trial 1 and more than 99, 84 and 31 per cent at seven, 10 and 14 days in trial 2. Against D viviparus, reduction in counts were more than 99, 98 and more than 99 per cent at seven, 14 and 21 days, respectively, in trial 1 and 100, 100 and 100 per cent at seven, 10 and 14 days, respectively, in trial 2. The relevance of these results to the build-up of infective larvae on pasture and infection in cattle is discussed.     

Efficacy of abamectin against natural infections of gastrointestinal nematodes and lungworm of cattle with special emphasis on inhibited, early fourth stage larvae of Ostertagia ostertagi.

The anthelmintic efficacy of abamectin (avermectin B1) was evaluated against gastrointestinal nematodes, including Ostertagia ostertagi inhibited larvae and lungworm, in yearling crossbred beef heifers during late spring. The calves were grazed on contaminated pasture for 10 weeks and then held under conditions free of nematode infection for 3 weeks prior to allotment and treatment on 5 June. Thirteen calves were randomly assigned to two groups of six by restricted randomization on body weights; the extra lightest calf was assigned to the non-treated control group. Group 1 calves were treated with abamectin at 200 micrograms kg-1 body weight by s.c. injection and Group 2 calves were not treated; all were killed at 14 days after treatment. Ostertagia ostertagi was present in all controls; arithmetic mean numbers of adults, developing fourth stage larvae (L4) and inhibited EL4 were 7683, 605 and 36,102, respectively. Other nematode genera present in controls in sufficient numbers for the experiment were Haemonchus placei adults, Trichostrongylus axei adults, Cooperia spp. adults, Oesophagostomum radiatum adults, Bunostomum phlebotomum adults, Dictyocaulus viviparus adults and E5 (immature adults). Abamectin was highly effective (consistently greater than 99% efficacy and P less than 0.05) in removing all nematodes present in treated calves as represented in non-treated controls, including the primary target of Ostertagia ostertagi inhibited EL4. The lowest efficacy was 93.8%, against D. viviparus E5.     

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.