Serological lessons from the bovine lungworm Dictyocaulus viviparus: Antibody titre development is independent of the infection dose and reinfection shortens seropositivity.

Infections with the bovine lungworm Dictyocaulus viviparus, the causative agent of parasitic bronchitis, are accompanied by substantial economic losses due to impacts on production, clinical respiratory disease or even death of diseased cattle. To detect lungworm antibodies in cattle, an enzyme-linked immunosorbent assay (ELISA) based on recombinant major sperm protein (MSP) has been developed. However, it remained unknown whether the infection dose influences antibody levels, and how acquired immunity influences antibody level patterns during reinfections. The latter may lead to low within-herd seroprevalence and thus to negative MSP-ELISA results in examination of bulk tank milk (BTM). Thus, infection experiments with 12 different doses ranging from 10 to 3000 D. viviparus larvae were performed to assess whether the antibody response is dose-dependent. Second, the impact of reinfections on the antibody response was evaluated in infection experiments, and third, antibody patterns in dairy cows during naturally occurring reinfections were assessed in a longitudinal field study based on individual milk samples. Results of this study demonstrate that the rise in MSP antibodies during first infection is dose-independent at infection doses of 25 lungworm larvae and above. However, following reinfections the magnitude and duration of the MSP antibody response are reduced or lacking, depending on the interval to reinfection. The field study revealed short periods of seropositivity as a common pattern in dairy cows subjected to natural D. viviparus reinfections. Low within-herd seroprevalence in dairy herds can thus be a result of continuous reinfections. Low infection doses should not be a barrier to serodiagnosis of lungworm infection in first-time infected cattle.

Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle.

BACKGROUND: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs. METHODS: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI). RESULTS: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement. CONCLUSIONS: A versatile bead-based assay using fluorescence detection (xMAP technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

Evaluation of a milk ELISA for the serodiagnosis of Dictyocaulus viviparus in dairy cows.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the bovine lungworm Dictyocaulus viviparus in milk was established. This test is based on recombinant major sperm protein (MSP) as the antigen and ELISA results are expressed as optical density ratio (ODR) values. The cut-off value of the milk ELISA was determined as the arithmetic mean of negative milk samples plus three standard deviations (SD). Specificity and sensitivity were 100% and 97.5%, respectively, using either milk or serum samples as positive control to calculate the ODR. Therefore, the presented recombinant antigen-based ELISA is suitable for routine veterinary diagnosis of exposure to bovine lungworms using milk samples instead of sera. To assess the course of antibody titres following lungworm infection, milk and serum samples from experimentally infected dairy cows were collected over a period of 23-30 weeks in three trials. The milk and serum antibody titre curves showed strong Pearson correlation coefficients in all three trials (Trial 1=0.85; Trials 2 and 3=0.93). In milk D. viviparus-specific antibodies exceeded the cut-off value 30-32 days post-infection (dpi) and remained above this value until day 112-138 post-infection (pi) with an overall detection period of 79-107 days. Treatment with eprinomectin during the pre-patent period prevented larval shedding and the antibody response was eliminated; treatment during patency similarly caused a cessation of larval shedding, but had no effect on the pattern of antibody responses compared to the untreated, infected controls.

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.

Preventive vaccination of lactating and pregnant heifers against lungworm: safety and protection in three dairy herds.

A study of the safety of a vaccine against lungworm was carried out with pregnant and lactating heifers from three dairy herds with a previous history of lungworm outbreaks in adult cows. Half of the heifers were vaccinated while the other half were not. A slight temporary cough following the vaccination was only observed in one herd. No adverse effects on pregnancy or milk production were seen. All heifers were serologically and coprologically examined before the first, before and after the second immunization, 3 months after introduction to pasture and at the end of the grazing season. Serological and faecal examination of the dairy cows before introduction into pasture confirmed the presence of at least one Dictyocaulus viviparus carrier in each herd. Lungworm infection occurred in all herds during the grazing season, most prominently in the herd with the highest number of heifers. In this herd, mild coughing associated with the lungworm infection was noticed, especially in the non vaccinated heifers. No other signs or symptoms were observed. It is concluded that a vaccine against D. viviparus can be used safely in heifers, before they are introduced into the adult herd, and that this vaccine can be used as a preventive measure against lungworm outbreaks in adult cattle.

Acute phase proteins in response to Dictyocaulus viviparus infection in calves.

Three experiments were carried out to examine the acute phase response, as measured by the acute phase proteins (APP) haptoglobin, serum amyloid A (SAA) and fibrinogen, in calves infected with lungworm, Dictyocaulus vivparus. In addition, eosinophil counts were analysed. Three different dose models were used in 3 separate experiments: 1) 250 D. viviparus infective third stage larvae (L3) once daily for 2 consecutive days, II) 100 D. viviparus L3 once daily for 5 consecutive days, and III) 2000 L3 once. All 3 dose regimes induced elevated levels of haptoglobin, SAA and fibrinogen, although there was considerable variation both between and within experiments. A significant increase was observed in all 3 APP at one or several time points in experiment I and III, whereas in experiment II, the only significant elevation was observed for fibrinogen at one occasion. The eosinophil numbers were significantly elevated in all 3 experiments. The results show that lungworm infection can induce an acute phase response, which can be monitored by the selected APP. Elevated APP levels in combination with high numbers of eosinophils in an animal with respiratory disease may be used as an indicator of lung worm infection, and help the clinician to decide on treatment. However, high numbers of eosinophils and low levels of APP do not exclude a diagnosis of lungworm. Thus, lungworm infection may not be detected if measurements of APP are used to assess calf health in herds or individual animals.

[Phospholipid and fatty acid content of the blood of sheep infected with the nematode Dictyocaulus filaria]. / Fosfolipidy i zhirnokislotnyi sostav krovi ovets, zarazhennykh nematodami Dictyocaulus filaria.

The results of analysis of phospholipids (PL) and fatty acid content in the blood of sheep infected with the nematodes Dictyocaulus filaria are displayed. A significant increase of lysophosphatidylcholine and arachidonic acid as well as a decrease of docozagexaenic acid in PL of infected sheep have been recorded. That points out to structural and functional disorders of cellular membranes during the infection. These disorder could be used as a metabolic criterion to estimate the relationships within the host-parasite system examined.

Induction of protective immunity to Dictyocaulus viviparus in calves while under treatment with endectocides.

Three groups of five parasite-naive calves were used. The treatments were: (a) Group 1 calves were weighed on Day 0 and injected with doramectin at 200 microg/kg. From Day 1 to 19 they were dosed orally with 2000 infective larvae of Dictyocaulus viviparus. On Day 28 they were again injected with doramectin, and infected with D. viviparus larvae from Days 33 to 41. They were then left untreated until Day 81 when they were infected with 20 infective larvae of D. viviparus per kg body weight. They were killed on Day 110 and lungworms were counted; (b) Group 2 calves were immunised with oral lungworm vaccine on Days 0 and 28, and infected and slaughtered as Group 1 on Days 81 and 110, respectively; (c) Group 3 calves acted as infection controls. Blood samples were taken at Days 0, 21, 49, 77 and 110 for antibody tests to D. viviparus. At autopsy there were no significant differences between the number of lungworms from Groups 1 and 2 (Means 17.4 and 31.3, respectively); Group 1 had significantly less value than Group 3 (Mean 228) (p < 0.05). Increased antibody titres to the larval sheath of the infective larvae were observed from Groups 1 and 2, showing that the larvae in Group 1 had penetrated the intestine before being killed by the circulating anthelmintic. This experiment shows that if calves are exposed to infective larvae while under systemic endectocide cover, an immune reaction is stimulated.

Isotype-specific antibody responses to the surface-exposed antigens of adult and larval stages of Dictyocaulus viviparus in infected and vaccinated calves.

The antibody responses to the surface-exposed antigens of living larval and adult Dictyocaulus viviparus were measured by quantitative immunofluorescence using sera from calves infected with, or vaccinated against, the parasite. In infected animals, the surface of the sheath of the third-stage larvae (L3) (retained cuticle of second-stage larvae (L2)) proved highly immunogenic despite the fact that it is thought to be shed prior to parasite penetration of the host intestine. When responses to the surface of exsheathed larvae (L3 cuticle) were measured, a high level of heterophile IgM antibody was detected in the serum of animals that had not been previously exposed to the parasite and, following infection, a specific IgG response was detected against the exsheathed L3 surface. The antibody response, however, was less marked than that observed against the intact L3 sheath. Responses of patently infected animals to the adult surface showed an initial IgM response that was superseded with time by IgG1 and IgG2 responses. Vaccinated animals showed only low level responses to the surfaces of the L3 sheath, L3 cuticle and adult stages following immunisation with two doses of irradiated larvae. The immunised animals produced a strong antibody response to the larval surface antigens following challenge with infective larvae but they failed to produce antibody to the surface of adult parasites. These results show that the surfaces of all the stages of D. viviparus examined are immunogenic in infected calves and, depending on the developmental stage, infection regime, or time of infection, high levels of parasite-specific IgG1 or IgM are stimulated. It has previously been shown that significant levels of protective immunity can be obtained in naive animals following passive transfer of serum from infected calves. Thus, the antibody responses detected in the work reported here may be of relevance in protective immunity against dictyocaulosis.

Efficacy of moxidectin pour-on against nematode infections in cattle.

Three groups of eight calves, naturally infected with gastrointestinal nematodes and artificially infected with Dictyocaulus viviparus were used to evaluate the efficacy of moxidectin pour-on at dose rates of 0.35 mg/kg and 0.5 mg/kg bodyweight. With both doses the efficacy was 100 per cent against adult D viviparus, Trichostrongylus axei, Ostertagia species and Nematodirus helvetianus. It was more than 99 per cent against Ostertagia and Nematodirus species fourth stage larvae. A small number of Cooperia species were found after treatment, and for this parasite, the efficacy of moxidectin ranged from 97.6 per cent against the larval stages to 98.8 per cent against the adults. No adverse reactions to the moxidectin treatment were observed.

A dipstick immunoassay using a recombinant antigen for the rapid diagnosis of bovine dictyocaulosis.

An immunoblot based dipstick test for the serodiagnosis of infections with Dictyocaulus viviparus using a recombinant antigen was evaluated. Lungworm infections in cattle could be detected with more than 99 per cent specificity and more than 99 per cent sensitivity between days 30 and 85 after experimental infection. Using ready-to-use recombinant antigen labelled filter strips, results could be obtained within 90 minutes of blood sampling. The dipstick test could be a rapid alternative method to the time-consuming faecal examination routinely used for the diagnosis of bovine dictyocaulosis.

Effect of bronchodilator and intravascular oxygen releaser on the course of Dictyocaulus filaria infection in lambs.

The effect of a bronchodilator (or ciprenaline sulphate) and intravascular oxygen releaser (sodium dihydrogen orthophosphate) on the host in experimental Dictyocaulus filaria infection was studied. Fifteen male lambs of Dorset-Muzaffarnagri breed, aged 4-6 months, were divided into four groups of four (infected bronchodilator), four (infected i.v. O2 releaser), four (infected untreated controls) and three (uninfected controls). The administration of i.v. O2 releaser helped in increasing the length of useful patency, estimated total larval production and survival rate of D. filaria producer lambs. The administration of I.V. O2 releaser and bronchodilator helped in efficiently restoring the altered values of blood pH, haemoglobin, packed cell volume, erythrocyte sedimentation rate and osmotic fragility of erythrocytes to near normal levels. However, the blood clotting time and level of lactate dehydrogenase activity remained altered and followed a course typical of ovine dictyocauliosis.

Osmotic fragility of sheep erythrocytes and alterations in their membrane constituents in Dictyocaulus filaria infection.

Alterations in the sheep erythrocyte membrane constituents during the course of Dictyocaulus filaria infection were studied in 4-6 month old Nali lambs. During the acute course of infection, plasma cholesterol, membrane cholesterol, cholesterol: phospholipid ratio and acetylcholinesterase activity fell significantly when compared with uninfected controls. The onset of the fall in the values of these parameters was observed 1-2 weeks prior to an increase in the osmotic fragility of erythrocytes. The altered values persistently remained at sub-normal levels during the chronic stage of the infection. However, the membrane proteins and phospholipids of sheep erythrocytes remained unaffected during the entire period of study. The clinico-parasitological picture of the disease, as judged by the clinical course of disease, faecal larval output and necropsy worm recovery, was typical of ovine dictyocauliosis.

Effect of Dictyocaulus filaria infection on the osmotic fragility of sheep erythrocytes.

The osmotic fragility of erythrocytes in lambs experimentally infected with Dictyocaulus filaria was studied weekly for 71 weeks. In acute infection, the erythrocytic fragility increased from the third week of infection onwards, reached its peak by the eleventh week and declined thereafter. However, in the chronic immune-carrier stage, this increase in the fragility did not return to normal until the end of the experiment. This enhanced fragility showed a positive correlation with the faecal larval count, worm burden and the extent of lung damage in lambs.

Preliminary observations on the effect of Dictyocaulus filaria on the blood clotting time of sheep.

The whole blood clotting time progressively increased from the second to the eighth week in lambs receiving a primary infection of Dictyocaulus filaria larvae. However, in vaccinated lambs it remained unaffected. Levamisole hydrochloride was ineffective when given 4 days after infection but restored the blood clotting time to near normal soon after treatment when it was given 30 days after infection.

The residual effect of treatment with ivermectin after experimental reinfection with nematodes in calves.

The residual effect of treatment with ivermectin after experimental reinfection in calves was tested. Twenty-four calves were divided into 6 groups of 4 calves each. All calves received a primary infection of 50,000 larvae of both Ostertagia ostertagi and Cooperia oncophora and 1000 Dictyocaulus viviparus larvae. Calves of group 1 remained untreated, and all other calves were treated 21 days after primary infection (0.2 mg/kg injected subcutaneously). Calves of groups 1 and 2 were slaughtered 7 days later. Calves of groups 3-6 were reinfected with the same number of larvae 3 days, 1, 3 and 6 weeks after treatment respectively. Slaughter was 21 days after reinfection. Based on post-mortem worm counts the efficacy of ivermectin after primary infection was 99.7% for O. ostertagi, 95.1% for C. oncophora and 100% for D. viviparus. A residual effect was present for at least one week, but could not be observed 3 weeks after treatment.

Cell adherence to larvae of Dictyocaulus viviparus in vitro.

Bovine eosinophils survived for up to 48 hours in vitro in a medium of undiluted bovine serum and became adherent to the surface of Dictyocaulus viviparus larvae earlier than other cells found in peripheral blood. Cell adherence was associated with a heat labile factor in normal bovine serum and a heat stable factor in hyperimmune serum. A factor associated with leucocytes in vitro appeared to cause larval immobility. Cells from calves treated with levamisole behaved identically to those from other sources. It is suggested that eosinophils are an important element in host defence against D viviparus infection.