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.     

Exposure of dairy cows to nematode infections at the end of the grazing season in The Netherlands

In autumn 2000, a study was carried out on 25 dairy farms in the vicinity of Utrecht with the aim to estimate infectivity levels for nematode parasites in cows. On each farm, faecal samples were collected from 15 cows, blood samples from 5 of these and herbage samples from 2 cow pastures. Faecal examination demonstrated a variation between farms and within farms in faecal egg output with a mean number of 4 eggs/g faeces (EPG) and Ostertagia spp. and Cooperia oncophora being the dominant species. In 6 out of 21 farms examined, lungworm larvae were detected in at least 1 cow. Serum pepsinogen values and serology using ELISA's with crude adult Ostertagia, crude adult C. oncophora and a specific recombinant C. oncophora protein as antigens indicated low to moderate infection levels. Pasture infectivity levels varied between farms with again Ostertagia spp. and C. oncophora as the dominant larval types and correlated with the crude worm Ostertagia ELISA, the crude worm Cooperia ELISA and the pepsinogen values. Exposure levels were high enough to enable the possible occurrence of production losses on the majority of farms.     

Serum and abomasal antibody response of sheep to infections with Haemonchus contortus

Serum and abomasal IgA, IgG and IgM antibody response against adult worm, L3 and egg antigens of Haemonchus contortus was monitored by the ELISA technique after one or two infections with this nematode. Following the first infection, antibody levels in serum did not change materially. After administration of a challenge dose of infective larvae, antibodies of the three immunoglobulin classes in infected animals rose slightly, but this rise appeared later than the fall in the faecal egg counts. In contrast, in abomasal mucosa, IgA anti-larval antibody levels, which did not increase materially after the primary infection, rose rapidly after a transient inhibition when sheep were challenged. A close temporal relationship was observed between the rise in local anti-worm IgA antibodies and the self-cure reaction, but antibody levels fell rapidly after worm diminution. The local antibody response was thus considered to be related to immunity of sheep to H. contortus.     

Nippostrongylus brasiliensis infection in the rat: 1. Production of bile lgA and serum lgG antibodies by adult rats

Bile lgA and serum lgG antibody responses were assayed by a micro ELISA technique. Antibodies of both classes were produced by 14 days after infection against whole worm antigens and worm metabolic product antigens, by adult rats infected once with $\text{Nippostrongylus brasiliensis}$. Rats were reinfected on day 28 and titres of both classes of antibody against both antigens were greater 7 and 14 days after reinfection than after the initial infection. Bile lgA antibodies were produced against phosphorylcholine after a single infection with the parasite but no increased response occurred after reinfection. The possible significance of lgA and lgG antibody responses in relation to immunity to intestinal nematode infections is discussed.     

Immunodiagnosis of experimental Parelaphostrongylus tenuis infection in elk

Elk infected with the meningeal worm, Parelaphostrongylus tenuis (Protostrongylidae), do not consistently excrete larvae in feces, making the current method of diagnosing live animals using the Baermann fecal technique unreliable. Serological diagnosis could prove more useful in diagnosing field-infected animals but depends on the identification and availability of good quality antigen. To mimic field infections, 2 elk were inoculated with 6 infective L3 larvae of P. tenuis, and another 2 with 20 L3 larvae. Fecal samples were examined for nematode larvae using the Baermann technique and serum samples taken were tested for anti-P. tenuis antibody with ELISAs by using the excretory-secretory (ES) products of L3, and sonicated adult worms as antigens. One animal passed first-stage larvae in its feces 202 days postinoculation, but passed none thereafter. The remaining 3 inoculated animals did not pass larvae. In contrast to parasite detection, antibodies against larval ES products were detected in all animals starting from 14 to 28 days postinoculation and persisted until the termination of the experiment on day 243 in 2 animals that harbored adult worms. Antibodies against somatic antigens of the adult worm were not detected until day 56 but also persisted until the end of the experiment in the animals with adult worms. In 2 elk that had no adult worms at necropsy, anti-ES antibodies were detected transiently in both, while anti-adult worm antibodies were present transiently in one. These findings confirm the superiority of P. tenuis larval ES products over somatic adult worm antigens as serodiagnostic antigens, as previously observed in studies of infected white-tailed deer, and extend the application of the newly developed ELISA test in diagnosing and monitoring cervids experimentally infected with P. tenuis.     

Management practices and use of anthelmintics on dairy cattle farms in The Netherlands: results of a questionnaire survey

In December 1996, a questionnaire about farm management and parasite control measures in calves was sent to 956 randomly chosen dairy cattle farmers in The Netherlands. Another 150 farmers in the vicinity of Deventer who had vaccinated their calves in 1995 against lungworm were approached with the same questions. Our objective was to investigate the consequences on worm control of the withdrawal of the lungworm vaccine from the market for reasons of possible BSE contamination of the vaccine. Of the returned questionnaires, 411 (43%) of the `at random' group and 89 (59.3%) of the `Deventer' group were valid. The most important data with regard to the farms of the `at random' group (411) were: mean area 31.6 ha, mean number of calves 23, heifers 23 and milking cows 53. Sheep (mean 37) were present on 18.3% of the farms. With regard to management: 74.5% of the farmers turned the calves in their first year onto pasture, 25.5% kept them indoors. The average time on pasture was ca. 5 months. Rotational grazing was practised on 81.4% of the farms, on 18.6% calves were set stocked. The first pasture of the calves was mown before turn-out on 72.9% of the farms. On 48.2% of these farms, calves were always moved to mown pastures. With regard to treatments: 33.8% of the farmers vaccinated their calves against lungworm in the years 1993, 1994 and 1995. Despite the withdrawal of the vaccine from the market in 1996, 7.2% of the farmers vaccinated their calves as recommended, with two doses, and 13.1% with a single dose. At turn-out, 41.5% of the farmers gave the calves a preventive anthelmintic treatment. Of these treatments, 66.9% were sustained of pulse release long acting devices. During the grazing season, 36.6% of the farmers treated their calves. After housing, 50.3% of the farmers gave a treatment. Signs of lungworm infection were noticed on 18.6% of the farms. Of the `Deventer' group (89 farmers), 96.6% turned the calves out. Of these farmers, 86.0% had used the lungworm vaccine in 1995. In 1996, 52.7% of the farmers had vaccinated the calves: 36.5% with a single dose and 16.2% with the double dose. Of the 35 farmers who did not vaccinate in 1996, 62.9% gave a preventive treatment at turn-out. Clinical signs of lungworm infection were not observed on the 12 farms which vaccinated the calves twice. On 11% of the farms which vaccinated once and on 14% of the farms which did not vaccinate, signs of lungworm infection were observed. It is concluded that more than 80% of Dutch dairy cattle farmers take appropriate measures to control gastrointestinal nematode and lungworm infections in calves in their first grazing season by grazing on aftermath, rotational grazing on mown pastures combined or not with preventive anthelmintic treatments. However, combinations of aftermath grazing and preventive treatment occurred on 30% of the farms. This may be overprotective and may prevent sufficient build up of immunity, causing worm problems at a later age. The withdrawal of the lungworm vaccine from the market did not cause a rise in lungworm problems. Some farmers did vaccinate, despite the withdrawal. The majority used other preventive treatment measures, mainly the application of long acting boli.     

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.     

The value of a bulk-tank milk ELISA and individual serological and faecal examination for diagnosing (sub)clinical Dictyocaulus viviparus infection in dairy cows

To test the value of a recently developed bulk-tank milk (BTM) ELISA for diagnosing (sub)clinical Dictyocaulus viviparus infection in lactating dairy herds under field conditions, bulk milk samples were collected from farms with or without clinical symptoms suspected to be caused by lungworm infection. Results of the BTM ELISA were compared against individual examinations for lungworm larvae in faeces and lungworm antibodies in serum from up to 20 heifers (parity 1) and up to 20 cows (parity ≥ 2) on the same farms. This also allowed, for the first time, to examine the value of individual faecal and serological examinations in the diagnosis of (sub)clinical lungworm infections. In total, 33 farms participated. Of these, 16 reported clinical symptoms possibly related to lungworm infection (defined as a suspected positive clinical status or CS(+)) and 17 reported having no such symptoms (CS(-)). In total, 503 heifers and 649 cows were sampled. Of all faeces samples positive for lungworm larvae, 94 were from heifers (18.9% of all heifers) and 75 from cows (11.7% of all cows) (P<0.001). Of all sera positive for lungworm antibodies, 130 were from heifers (26.1% of all heifers) and 113 from cows (17.5% of all cows) (P<0.001). Of the CS(-) farms 41% had at least one heifer or cow shedding larvae and 71% had at least one seropositive heifer or cow. Of the CS(+) farms this was 81% and 94%, respectively. There were only 4 farms, all CS(-), where none of the animals were found shedding larvae and all animals tested seronegative. This implies that on 76% of the CS(-) farms lungworm infection circulated unnoticed. On all CS(+) farms the suspicion that lungworm caused the respiratory symptoms was confirmed by the individual faecal and serological examinations, whereas the BTM ELISA confirmed presence of lungworm on half of the CS(+) farms. The latter in particular occurred on farms with the more severe outbreaks. Overall, of 32 available BTM samples 10 tested positive (8 of 15 CS(+) and 2 of 17 CS(-) farms). For diagnosing suspected lungworm disease it was concluded that testing a BTM sample might suffice in case of moderate to severe outbreaks. However, in case of a mild outbreak with just a few animals coughing, examining individual animals has to be preferred over testing a BTM sample. The likelihood to detect lungworm infection is higher if heifers are sampled compared to cows. Sensitivity of the BTM ELISA was 35.7% if the presence of at least one seropositive and/or one larvae shedding animal in the herd was used to define lungworm positive farms. On average, at least 30% of the herd had to be seropositive before the BTM ELISA was found positive for lungworm antibodies. Results indicate that the BTM ELISA in its current form does not appear to be suitable for surveys on the prevalence of lungworm presence on farms. However, this BTM ELISA might be used in large-scale surveys to detect, for instance, annual changes in percentage positive farms, as long as it is recognized that positivity is more closely related to incidence of lungworm disease than to prevalence of lungworm infection.     

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.     

Measurement of antibody in serum and genital fluids of bulls by ELISA after vaccination and challenge with Tritrichomonas foetus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to Tritrichomonas foetus using both whole cell antigen (WCA) and membrane protein antigen (MPA). The test was used to detect specific antibody in serum, preputial washings and seminal plasma samples from 7 adult bulls which were vaccinated subcutaneously on 3 occasions with a membrane protein vaccine against T. foetus var brisbane in an oil adjuvant, and from 4 unvaccinated control animals. One month after administration of the third dose of vaccine, vaccinated and control bulls were repeatedly challenged with the live vaccine strain of the T. foetus. A steady increase in serum antibody titre was detected after each inoculation of vaccine when both antigens were used in the ELISA. However, MPA was more sensitive. After challenge, vaccinated bulls developed an increased titre. No specific antibody was detected in control bulls, except in one bull after challenge in which seroconversion was detected. The serum antibody titres of both groups of animals were also measured with the microagglutination test which proved less sensitive than the ELISA. Antibody titres to both antigens, although lower than in serum, were detected in the seminal plasma of vaccinated animals. The control bulls remained non-responsive. No antibody was detected by ELISA in preputial washings from either control or vaccinated bulls prior to challenge. Post-challenge, some of the vaccinated bulls were responsive with both antigens whereas the control bulls remained negative.     

Study on small ruminant lungworms in northeastern Ethiopia

A cross-sectional study of lungworm infection was carried out with the aim of determining the prevalence of lungworm infection of small ruminants and identifying the species of the respiratory helminthes circulating in six districts of northeastern Ethiopia: Debresina, Legambo, Habru, Kalu, Chaffa-Dawi and Artumana-Fersejelle. Faecal and postmortem examination were conducted from 1162 and 104 animals, respectively. An overall infection rate of 53.6% and 66.3% was found by faecal and postmortem examinations, respectively. Significant difference (p<0.05) was found between areas of different altitude with an infection rate of 30.4%, 32.5% and 71.3% at low, medium and high altitude areas, respectively. Prevalence on the different months was insignificant (p>0.05). The prevalence of Dictyocaulus filaria and Muellerius capillaris infection showed a significant difference (p<0.05) between young, adult and old age groups. The prevalence of D. filaria decreases and that of M. capillaris increase with increasing age of the animal. Animals under relatively good management system have been found less affected with significant difference (p>0.05) than their counterparts at relatively poor management system. The infection rate between male and female animals showed significant difference (p<0.05) with prevalence rate of 44.4% and 59.3%, respectively. A significant difference between sheep and goats was also noted with infection rate of 24.4% and 50.7%, respectively. Monthly worm burden of D. filaria infection showed significant difference (p>0.05) while it was insignificant in case of M. capillaris. Due to its impact on production, emphasis should be given for the control and prevention of lungworm infection in highland areas.     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fag) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fag and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

Identification and isolation of a specific antigen with diagnostic potential from Dictyocaulus viviparus.

The purpose of the investigation was to isolate and identify a specific antigen of Dictyocaulus viviparus that can be used to diagnose lungworm infections in cattle. Somatic, excretion and secretion antigens of adult D. viviparus and somatic antigens of L3 larvae were examined in an indirect enzyme-linked immunosorbent assay (ELISA) to determine whether they cross-reacted with sera collected from calves with mono-infections of Fasciola hepatica. Ostertagia ostertagi, Ascaris suum, or Cooperia oncophora. Serum samples containing antibodies directed against F. hepatica, A. suum, and O. ostertagi cross-reacted with somatic antigens of adult D. viviparus; these sera cross-reacted less with excretion and secretion antigens. When somatic antigens of adult D. viviparus were analysed in a Western blot, a 17-kDa protein that did not react with the heterologous sera was detected. This protein was isolated by ultrafiltration and anion chromatography. Sera collected from calves infected with D. viviparus was tested in indirect ELISAs with either somatic antigens of adult D. viviparus or with a low molecular antigen fraction of this preparation containing the 17-kDa protein. The extinction values that were measured in both assays correlated well. We conclude that the 17-kDa protein isolated from somatic antigens of adult D. viviparus may be useful in developing an improved immunoassay to diagnose lungworm infections in cattle.     

Comparison of two serum and bulk-tank milk ELISAs for diagnosing natural (sub)clinical Dictyocaulus viviparus infection in dairy cows

Lungworm antibody ELISAs developed in Germany (DE) and The Netherlands (NL) were compared using four sets of serum (S) and bulk-tank milk (BTM) samples from adult dairy cows. The samples originated from 37 farms with or without a suspected clinical lungworm infection during August–October 2010 (Dataset 1), from cows excreting lungworm larvae or not during August–October 2010 (n = 59) or May–June 2011 (n = 164) (Dataset 2), from 305 farms in a national survey during October 2010 (Dataset 3), and 14 zero-grazing farms during February–April 2011 (Dataset 4).During August–October 2010, covering the second half of the grazing season, the NL-S and NL-BTM ELISA outperformed the DE-S and DE-BTM ELISAs in terms of sensitivity. For at least the NL-S and DE-S ELISA the opposite was found during May–June 2011, covering the end of the winter housing period and the early grazing season. Of the 305 farms in the survey 62.6% were found positive with the NL-BTM ELISA, whereas 2.6% was found positive with the DE-BTM ELISA. ODR values for the zero-grazing farms indicated that a cut-off value of 30% for the DE-BTM ELISA might be more appropriate than the currently used 41%. Results suggest that the NL ELISAs also respond to lungworm antigens other than Major Sperm Protein as used in the DE ELISAs.It is concluded that the generally higher sensitivity of the NL-BTM ELISA makes it better suited for large-scale prevalence studies and herd health monitoring programmes than the DE-BTM ELISA, positivity of which is more associated with the presence of clinical lungworm infection. Reducing the cut-off value of the DE-BTM ELISA from the original 49.3% to the current 41% or the possibly more appropriate 30% increased its sensitivity for detecting lungworm infections, but did not lead to similar sensitivity estimates as found for the NL-BTM ELISA.     

Lungworm disease in dairy cattle: symptoms,diagnosis, and pathogenesis on the basis of four case reports

Clinical lungworm disease appears to occur frequently in Dutch dairy herds. Because the clinical diagnosis is difficult to make in adult cattle, the clinical diagnosis, laboratory diagnosis, differential diagnosis, therapy, and prevention are discussed in this article. In addition, four cases of lungworm disease in adult cattle are presented. The main clinical complaints were coughing, decreased milk production, and weight loss. Several lactating cows died in one herd. The disease history of four herds revealed that introduction of susceptible cows or heifers to herds with cows with subclinical patent lungworm infections had resulted in a pasture infection, leading to clinical problems in both the newly introduced and 'resident' cows of the herd. Further history analysis of the fourth herd revealed that re-introduction of lungworm infection by newly purchased cows in a lungworm free herd resulted in clinical lungworm problems in adult and young animals. The fourth case led to the conclusion that lungworm infection must have been re-introduced by cows purchased from another farm.     

19s and 7s antibody response of Mastomys natalensis in experimental filarial (Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi, B. pahangi) infections

Specific total antibody (ab), 19s and 7s ab levels in the serum of M. natalensis were investigated after infection with L. carinii, A. viteae, B. malayi and B. pahangi for period of about 500 days p.i., using ELISA (homologous adult antigen) and indirect immunofluorescence tests (IIFT: homologous adult and microfilariae antigen). Total ab levels in L. carinii infected animals rose moderately during prepatency Maximum levels occurred during patency. The response during prepatency was stronger in A. viteae and Brugia spp. infected hosts. Lateron ab levels increased continuously in Brugia infections; in A. viteae infection they decreased with decreasing parasitaemia. 19s abs were stimulated during prepatency and at the beginning of patency, or were found at moderate levels throughout to period of investigation (Brugia infections). 7s abs predominated beginning at the period of late prepatency (IIFT) or at the beginning of patency (ELISA). The time courses of 7s abs corresponded to those of total abs. As obvious by IIFT (adult worm antigen) total and 19s titres were higher against cuticle antigens, egg shell antigens and intrauterine amorphous material than against antigens located in the hypodermis and musculature. 7s abs showed best reactivity with cuticle antigens. Using microfilarial antigens 19s abs reacted predominantly with cuticle antigens whereas 7s abs often showed higher titres against antigens which were localized within the larvae than against cuticle antigens.     

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 the persistent efficacy of the injectable and pour-on formulations of doramectin against artificially-induced infection with Dictyocaulus viviparus in cattle

The persistent efficacy of the injectable and topical formulations of doramectin was compared against experimental challenges with infective larvae of Dictyocaulus viviparus in two separate studies. Four groups of 10 randomly-assigned calves, negative for lungworm larvae by the Baermann technique, were used in each study. Calves were treated subcutaneously in the midline of the neck or poured down the midline of the back with saline (1 ml/50 kg. injection: 1 ml/10 kg. pour-on) on Day 0 or doramectin (200 microg/kg = 1 ml/50 kg. injection: 500 microg/kg = 1 ml/10 kg. pour-on) on Day 0, 7, or 14. Two additional calves from the same pool of animals were randomly assigned as larval-viability monitors and received no treatment. Calves were inoculated daily with a gavage of approximately 100 larvae of D. viviparus from days 35 to 49 for the injectable study and days 28 to 42 for the pour-on study. The two larval viability monitor calves received approximately 3000 infective larvae in the same manner on Day 49 or 42 for the injectable and pour-on studies, respectively. Equal numbers of calves from each treatment group as well as the larval viability monitor calves were necropsied on days 14 and 15 after the last lungworm inoculation to enumerate the worm burden. The worms recovered were quantified and identified. For each study, geometric mean worm recoveries for each treatment group were back transformed from the natural log-transformed data (worm count +1) and were used to estimate percentage reduction. Doramectin injectable solution was 100.0% efficacious against lungworms for up to 49 days and the pour-on formulation was 100.0%, 93.1% and 81.5% effective in reducing lungworm infection resulting from challenge infection for up to 28, 35, and 42 days post-treatment, respectively.     

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.