Use of ELISA to assess lungworm infection in calves

The enzyme-linked immunosorbent assay (ELISA) technique was applied to the detection of lungworm infections in calves. In experimentally infected animals different responses to larval and adult worm antigens were observed. The response to adult worm antigens was delayed in vaccinated animals when infection occurred by the gradual uptake of infective larvae from contaminated pasture. A serological survey in The Netherlands demonstrated a high incidence of lungworm infection in both vaccinated and unvaccinated herds. There was a good correlation between anti-adult worm and anti-larval ELISA-titres. ELISA appeared to be a useful technique for assessing the level of lungworm infection in a herd.     

Clinical signs following experimental lungworm infection and natural bovine respiratory syncytial virus infection in calves

Similar clinical signs have been reported in calves infected either by Dictyocaulus viviparus or bovine respiratory syncytial virus. Three experiments were carried out to establish the clinical picture and the course of the disease in animals with these infections. The clinical signs of calves infected with lungworm included coughing, nasal discharge, tachypnoea, abdominal breathing and pyrexia, and auscultation of their lungs revealed increased bronchial sounds. Similar signs were also observed after infection with bovine respiratory syncytial virus, but the signs were more acute and resolved more rapidly than in animals infected with lungworm larvae. Calves infected with lungworm had more serious clinical signs after infection with bovine respiratory syncytial virus than calves, which were not infected with lungworm.     

Increased establishment of lungworms after exposure to a combined infection of Ostertagia ostertagi and Cooperia oncophora

Three-month-old calves were infected three times weekly during a 5-week period with Cooperia oncophora, Ostertagia ostertagi or a combination of these two species. For each type of infection two dose levels were applied. In addition one group of calves was kept uninfected. After removal of the primary infection by anthelmintic treatment all calves were challenged with lungworm larvae and slaughtered 5 weeks later. The groups receiving either C. oncophora or O. ostertagi as a monospecific infection did not differ from the na?ve controls. The group receiving the combination of both species differed significantly from the other groups, the establishment of the lungworms being 177%, and the faecal excretion of larvae being 325% of that of the other groups.     

Evaluation of the efficacy of doramectin against an artificial infection of Dictyocaulus viviparus in calves

Three groups of eight Friesian calves, reared parasite-free, were experimentally infected with 1000 infective larvae of Dictyocaulus viviparus. Two groups were injected subcutaneously with 1% doramectin at 0.2 mg/kg body weight, one group 5 days after infection and the other 25 days after infection. A third group served as untreated controls. Faecal samples were examined for lungworm larvae on days 28, 32, 33, 34 and 35 after infection; the calves were killed and necropsied 39 or 40 days after infection and any lungworms present recovered and counted. Doramectin proved 100% effective against both 5-day-old and mature D. viviparus infections.     

Dermal cellular responses of helminth-free and Ostertagia ostertagi-infected calves to intradermal injections of soluble extracts from O. ostertagi L3 larvae

Twelve calves were raised helminth-free until 9 weeks of age when six were orally inoculated with 100,000 Ostertagia ostertagi infective stage larvae (L3). Three uninfected and three experimentally infected calves received intradermal injections of sterile saline and soluble larval extract (SLE) from O. ostertagi L3 with a protein concentration ranging from 1 to 200 micrograms ml-1. Biopsies were performed 48 h post-injection. A kinetic study was performed on the remaining six calves, three infected and three uninfected, using a 100 micrograms ml-1 concentration of SLE and taking biopsies 1, 4, 8, 12, 24, and 72 h post-injection at both the saline and SLE-injected sites. All calves had an immediate wheal and increase in skin thickness at the SLE-injected sites. The numbers of eosinophils infiltrating SLE-injected sites as compared to saline-injected sites were significant in both uninfected and infected calves, but the infected calves had significant numbers to a wider range of SLE concentrations and had significantly higher numbers than uninfected calves in the kinetic study. Infected calves also had significant numbers of basophils in the dose response study at concentrations of 5 and 100 micrograms ml-1 SLE. Neutrophil infiltration was similar in both groups and was significant at SLE-injected sites early in the kinetic study. Detectable mast cells were decreased in SLE-injected sites of infected animals and perivascular accumulation of mononuclear and some polymorphonuclear cells was observed in the deep dermis of infected animals.     

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9–99.7%) and 98.1% (CI: 95.3–99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3–4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.     

Pathophysiology of Haemonchus placei infection in calves.

This study was conducted to investigate the pathophysiology of Haemonchus placei infection in Friesian calves. Seven calves were divided into two groups, three uninfected calves (control group) and four infected animals. The latter group were infected orally with 500 H. placei larvae kg-1 body weight. Five weeks after infection they were all housed in metabolic crates and injected with 125I-bovine albumin. 51Cr-red cells and 59Fe-transferrin, to study albumin metabolism, erythrokinetics and ferrokinetics. The results showed that there was a significant reduction in the mean haematocrit values and reduced weight gains in the infected calves compared with the controls. There was also a change in the distribution of albumin from the extravascular to the intravascular pool and a significant increase in the plasma and blood volumes of infected calves although the blood and albumin loss via the gastrointestinal tract recorded in this study was similar in both groups.     

Comparison of a levamisole sustained-release bolus and ivermectin treatment to prevent bovine lungworm infection

The efficacy of a levamisole sustained-release bolus to prevent parasitic bronchitis in calves in their first grazing season was compared to ivermectin treatment at three, eight and thirteen weeks after turn out. Contamination of the pasture was established by experimentally infected seeder calves. Twenty calves were split into two groups. Ten calves of one group received a bolus at the start of the experiment. In the other group the calves were treated with ivermectin at 21, 56 and 91 days. Two principal calves from each group were killed during the experiment to study histopathological changes. Pairs of tracer calves were introduced on both pastures at intervals of four weeks throughout the grazing period. The permanent calves were challenged with lungworm larvae at housing and slaughtered four weeks later. Both systems prevented parasitic bronchitis. Larval output was completely reduced in the ivermectin-treated calves while all bolus-treated calves excreted larvae at certain times. The highest group average was 4 larvae per gram faeces. Eosinophilia, ELISA-titres and histopathological changes confirmed the differences in larval uptake. Challenge infection was not successful in either group and no worms were found at slaughter. Weight gain was significantly different at housing in favour of the ivermectin-treated calves, but after challenge this was reduced due to a higher weight gain in the bolus-treated calves. The practical consequences of the results have been discussed.     

Effects of Fasciola gigantica infection on growth and nutrient utilisation of buffalo calves

The effects of Fasciola gigantica infection on bodyweight gain, dry matter intake, digestibility of nutrients and feed conversion efficiency in buffalo calves were investigated. Nine male buffalo calves of the Murrah breed, aged 12 to 15 months with a mean (se) bodyweight of 166 (12.5) kg, were randomly assigned to groups of five (group 1) and four (group 2). The animals in group 1 were given 1000 viable, mature metacercariae of F gigantica orally, while the animals in group 2 served as uninfected controls. They were stall fed on diets containing a concentrate mixture and ad libitum wheat straw and were maintained by standard management practices for a period of 165 days after infection. The average daily liveweight gain of the infected animals was 110.6 g, compared with 439.4 g in the uninfected controls, and was associated with the appearance and establishment of immature flukes in hepatic bile ducts. The feed conversion efficiency declined significantly (P<0.01) from 41 days after infection and was lowest at the end of the experiment. F gigantica infection did not influence the digestibility of the nutrients. The impaired feed conversion efficiency was mainly due to a reduction in dry matter intake due to inappetence.     

Immune modulation by Ostertagia ostertagi and the effects of diet

IgG1 antibody responses to Ostertagia ostertagi third stage larvae (L3) and the third party antigen, keyhole limpet haemocyanin (KLH), and faecal egg counts were determined in calves infected with a single dose of O. ostertagi and in uninfected, pair-fed calves. The infected and uninfected calves were given diets either high (H) or low (L) in protein and energy. The diets were within the normal range of husbandry practice in the UK. IgG1 antibody responses to L3 antigen were significantly greater from 6 weeks post-infection in infected calves given the L diet than in infected calves given the H diet (P less than 0.05). The effects of diet and infection on anti-KLH IgG1 responses were independent of each other. IgG1 responses to KLH were decreased by infection and by the L diet compared with the H diet.     

Chemoprophylaxis and immunity to parasitic bronchitis in cattle — a field experiment comparing topical ivermectin and an oxfendazole intraruminal device

Seeder calves infected with Dictyocaulus viviparus were used to contaminate a field divided into three similar paddocks. Twenty-four autumn-born calves were allocated to three matched groups; one group was given topical ivermectin treatments (0.5 mg/kg) at 3, 8 and 13 weeks after turnout (Day 0); each member of a second group was given an oxfendazole pulse-release intraruminal device (OPRB) at turnout; while a third group was kept as untreated controls to monitor the natural epidemiological pattern of events. Severe pasteurella pneumonia exacerbated by lungworm infection occurred in the controls after Day 24. Two died and repeated doses of antibiotic and anthelmintic therapy were necessary to save the remainder. Clinical signs were much milder in the ivermectin and OPRB groups and resolved with only a single dose of antibiotic. The OPRB group excreted some lungworm larvae at this time, but none was detected in the faeces of the ivermectin group during the grazing season. At housing, five calves from each group and four lungworm-naive calves were challenged with D. viviparus larvae. The infection became patent in all challenge-control calves, but no larvae were passed by any of the trial animals. Post-mortem worm-counts revealed percentage takes for the challenge controls, trial controls, ivermectin and OPRB groups of 16.7, 0.01, 0.9 and 0.2, respectively. All trial groups had therefore developed a substantial immunity.     

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.     

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.     

Experimental infections with Hyostrongylus rubidus and the effects on performance of growing pigs

Pigs infected with Hyostrongylus rubidus at the rate of 550 larvae kg-1 body weight followed 15 days later with 220 larvae kg-1 body weight gained less weight (P less than 0.010) than uninfected control pigs. Feed efficiency (feed/gain) was 8% better (P greater than 0.10) in control than in infected pigs. Peak H. rubidus eggs per gram counts (EPG) occurred 22 days after each infection of pigs. H. rubidus EPG at necropsy were correlated with total number of adults recovered and with female/male ratio. High EPG were associated with H. rubidus populations composed of approximately equal numbers of males and females. Digestion trials consisted of pigs infected with 335 larvae kg-1 body weight compared to uninfected controls. Control pigs had higher (P less than 0.05) crude protein digestion coefficients, excreted less (P less than 0.05) N in feces and had a higher (P less than 0.05) N balance than infected pigs.     

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.     

A model for study of lungworm (Dictyocaulus sp.) and gastrointestinal nematode infection in young red deer (Cervus elaphus)

A model of sub-clinical parasitism in young red deer, using concurrent trickle infections of lungworm (Dictyocaulus sp.) and mixed gastro-intestinal (GI) nematodes of deer-origin was evaluated. 20 parasite-free deer calves were artificially reared indoors from 4 days of age. A further five calves were naturally reared on pasture with their dams, treated with anthelmintic and brought indoors at 3-4 months. At 4-4.5 months of age they were individually housed and allocated to five groups (n=5). Groups were dosed 3 x per week, for 9 weeks with 0, 100 and 500, 200 and 1000 (2 groups), 400 and 2000 infective larvae of lungworm and mixed GI nematodes, respectively, cultured from deer faeces. Liveweight and voluntary feed intake measurements and faecal and blood samples were taken weekly. In the fourth week following cessation of trickle infection, deer were euthanased and lung and GI nematodes recovered. Both lungworm and GI nematode infections became patent at Week 4 of infection. Maximum group arithmetic mean faecal egg counts were 100-190 epg. Maximum group arithmetic mean faecal lungworm larval counts were 58-123 lpg. Group arithmetic mean nematode counts at slaughter ranged from 439-806 for GI nematodes and 31-73 for lungworm, respectively. Despite low nematode counts, reduced liveweight gain, voluntary feed intake and serum albumin concentration, elevated serum pepsinogen, gastrin and globulin concentrations and elevated peripheral eosinophil counts and slight haemoconcentration, but no clinical signs, were observed. The reduction in liveweight gain was related to the reduction in voluntary feed intake (r2=0.83; p<0.088). Naturally-reared deer had similar liveweight gains, voluntary feed intake and nematode counts to artificially-reared deer. Thus, methods of infection to produce concurrent sub-clinical lungworm and GI nematode burdens for study of sub-clinical parasitism in young deer have been defined.     

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.     

Response of dorper and red Maasai lambs to trickle Haemonchus contortus infections

Six-month-old red Maasai lambs were more resistant than Dorper lambs to repeated infections at one to two week intervals with 1000 Haemonchus contortus infective larvae. Resistance after infection was assessed by means of faecal egg counts, packed cell volumes, eosinophil counts, total serum protein concentrations and mortality rates. The weight gains of the infected animals were only marginally lower than those of their uninfected controls, most probably because of their significantly higher feed consumption, and evidently the infected lambs were not utilising all of the extra feed for growth. This absence of anorexia in spite of the infection was probably due to the palatability of the high protein diet fed to the lambs.     

Effect of forage legumes containing condensed tannins on lungworm (Dictyocaulus sp.) and gastrointestinal parasitism in young red deer (Cervus elaphus)

To investigate the effect of feeding forage legumes containing condensed tannins (CT) on internal parasitism, red deer calves were fed either lucerne (Medicago sativa; 0.1 per cent CT), birdsfoot trefoil (Lotus corniculatus; 1.9 per cent CT) or sulla (Hedysarum coronarium; 3.5 per cent CT) and trickle-infected with deer-origin gastrointestinal nematode and lungworm (Dictyocaulus sp.) larvae for 5 weeks, then slaughtered at 7 weeks. There was a significant negative linear relationship between dietary CT concentration and abomasal nematode burdens. No significant differences in faecal egg counts, lungworm burdens or voluntary feed intake were found. Deer fed sulla had higher liveweight gain, carcass weight and carcass dressing-out percentage, higher serum total protein and albumin concentration and lower serum gastrin concentration and faecal lungworm larval count, compared with lucerne-fed deer. Inclusion of sulla in diets for young red deer may reduce the impact of internal parasites and/or reduce the dependence on anthelmintic treatment.     

Seroprevalence of Dictyocaulus viviparus in first grazing season calves in Sweden

A serological survey was carried out to determine the prevalence and geographical distribution of Dictyocaulus viviparus in calves after their first grazing season in Sweden. A total of 754 animals from 76 randomly selected herds in seven geographical regions were examined between September 24 and December 19, 2001. To get an indication about the geographical distribution of the infection 41 herds with beef-suckler calves were investigated. On each farm, blood was collected from 8 to 10 animals after an average of 26 +/- 24 days post-housing to determine specific IgG1 levels against a possible lungworm sperm antigen that is highly specific against patent infections of D. viviparus. We also investigated the seroprevalence of lungworm infection in relation to cattle management. In one region additional samples were analysed from 35 herds either with: (a) beef-suckling calves that were dewormed at housing, (b) untreated organically raised dairy calves, and finally from conventionally raised dairy calves either, (c) with or, (d) without a prophylactic anthelmintic treatment programme against gastrointestinal parasites on pasture. A questionnaire was used to obtain information about herd size and management, including measures to control nematode parasites on the farm. A total of 86 (11.8%) out of 754 animals had antibodies against D. viviparus, and at least one infected individual was detected in 30 (39.5%) of the 76 herds examined. Lungworm infected animals were found throughout the country and there was no significant differences between regions, although in southern and southwestern Sweden 70.0% of the herds were infected. Furthermore, there were no major differences in the seroprevalence in relation to management. Between 40.0 and 44.4% of the herds were infected irrespective of management, with the exception of calves from organic herds where no seropositive samples were found (0%). This result is in contrast to previous findings of lungworms in Sweden, and indicates that the parasite status on organic farms is diverse.