Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.     

Detection of circulating hypodermin C: an antigen capture ELISA for diagnosis of cattle grub (Diptera: Oestridae) infestations

An antigen capture assay for the detection of circulating hypodermin C was developed for diagnosis of hypodermosis. A murine monoclonal antibody to recombinant hypodermin C was raised using rapid immunization and a one-step hybridization-cloning technique. A highly reactive, specific monoclonal antibody was tested using sera spiked with known quantities of purified, native hypodermin C or with recombinant hypodermin C. Sensitivity of 96.4% and specificity of 95.6% for the antigen capture assay was assessed using a panel of sera from animals unexposed to cattle grubs and from cattle with palpation proven cattle grub infestations. Data from this panel of sera was used to establish the cut-off OD for further testing. The kinetics of circulating hypodermin C was assessed using the assay in three groups of cattle artificially infested with 50, 100 or 200 first instar Hypoderma lineatum. Antigen was first detected approximately 6 weeks after infestation. The amount of antigen detected increased in each group of animals reaching peaks at different times in each group. Levels of antigen fell quickly following arrival of grubs at the back and completion of the molt to second instar.     

Cross-transmission studies with Hypoderma lineatum de Vill. (Diptera: Oestridae): attempted infestation of goats (Capra hircus)

The potential for cross-transmission of Hypoderma lineatum from cattle to domestic goats (Capra hircus) was examined using artificial infestation techniques. Two routes of infestation, subcutaneous injection and dermal penetration, were used to expose goats to newly hatched first instars. Presence of antibodies and appearance of circulating antigen (hypodermin C) were evaluated at selected intervals for up to 40 weeks post-infestation. In addition, immunoblots against H. lineatum first-instar proteins were conducted using sera taken at 10 weeks post-infestation. Goats were palpated for the presence of developing larvae at sub-dermal sites beginning at week 30 pi. No developing larvae were palpated at any time, regardless of the route of infestation nor was circulating antigen detected in any infested goats. Antibodies were present at weeks 6 and 10 and week 27 pi in both infested groups. Immunoblots indicated all infested goats produced antibodies to first instar H. lineatum antigens. H. lineatum appears to be incapable of completing development in domestic goats although the transient appearance of ELISA detectable antibodies and the presence of bands on immunoblots suggests that at least some larvae survive long-enough to engender a humoural response. The host specificity of H. lineatum is discussed in light of the general concepts of host-parasite relationships of oestrids.     

Impact of previous infestation on dynamics of circulating hypodermin C in cattle artificially infested with Hypoderma lineatum (Diptera: Oestridae)

Four groups of cattle were artificially infested with 50 first instar Hypoderma lineatum after either a primary natural infestation or in the absence of a primary infestation. In two groups the primary infestation had been terminated by the application of either an organophosphate insecticide or a macrocyclic lactone parasiticide. Circulating hypodermin C and specific antibodies were measured for 40 weeks after the artificial infestation. Stage specific mortality of the larvae was also monitored. Previously uninfested cattle exhibited typical antibody and antigen profiles during the infestations. Antibodies were first detected on Week 7 p.i., they rose to maximum values between Weeks 24 and 25, then declined as larvae reached the back. A second peak occurred at Week 32 and antibody remained more less constant thereafter. Previously infested groups exhibited a dramatic anamnestic response by Week 3 p.i. with antibody levels peaking at Week 8. A second peak was noted between Weeks 24 and 26 p.i. after which antibody levels declined and then remained relatively stable. The dynamics of circulating hypodermin C in the previously infested cattle resembled those in the previously uninfested cattle. Mortality of first instars did not differ among the four groups. Similarly mortality of second and third instars, in the warble, did not differ although there was a tendency for higher mortality in the previously infested, untreated group. These results reinforce previous work demonstrating the development of a significant immune response during the primary infestation that is reflected in the rapid and substantial production of antibodies upon re-infestation. It is significant that a challenge model using subcutaneous injection of newly hatched first instars avoids host immune responses in the skin that result in substantial larval mortality. The current data also support the concept that migrating first instars induce significant reduction in host immune response. There is a peak of antibody production as antigen levels peak during first instar migration to the back. As larvae molt to the second instar and antigen production ceases there is a persistence of antibody which suggests release of the immune response from the suppression induced by the first instar secretory antigens.     

Effect of ivermectin treatment on anti-hypodermin C titers of Asturiana cattle naturally infected with Hypoderma lineatum

The collagenase (hypodermin C) from soluble crude extracts of Hypoderma lineatum 1st-instar larvae was purified by reverse-phase HPLC and used in a new indirect ELISA test. This pure protein had several advantages over the use of crude larval extracts allowing a much better discrimination between infested and non-infested cattle. The anti-hypodermin C titers of 19 Asturiana cattle were estimated over the course of a natural H. lineatum infestation cycle, in which the effect of ivermectin treatment was also investigated. The results showed differences in the onset and ending of the infestation with respect to those described for other European countries. The ivermectin treatment proved to be very effective and treated animals had relatively low anticollagenase titers.     

The Prevalence of Bovine Hypodermosis in Kars Province,Turkey

A total of 1276 cattle from 76 farms in 8 villages were examined by palpation of the skin for evidence of hypodermosis. The effects of age, sex, breed, and colour of cattle on hypodermosis were investigated. The overall prevalence was 31.9%. The mean number of warble fly larvae on infested animals was 8.46. Hypodermosis infestations were encountered in all the villages visited. Within a herd, the intensity of infestion decreased with the age of cattle. Infestation rates of warble fly larvae were lower in native cattle, in females, and in piebald animals.     

Stage specific mortality and humoral immune responses during pulse and trickle infestations of the common cattle grub, Hypoderma lineatum (Diptera: Oestridae)

Stage specific survival of larvae and pupae of the common cattle grub, Hypoderma lineatum de Villers, was compared in cattle artificially infested with larvae delivered by subcutaneous injection as a single infestation of 100 larvae, in trickle infestations of either two doses of 50 larvae, separated by 15 days, or in four doses of 25 larvae, separated by 9, 6, and 6 days, respectively. The duration of the migratory and 'warble' phases were also compared between treatments. The kinetics of the antibody response was monitored throughout the larval development by ELISA. No difference was noted in the level of antibodies among the three treatments. Mortality of first-instars differed among the infestation groups with mortality in the single pulse infestation group相似文献   

Efficacy of injectable doramectin in the therapy and control of Dermatobia hominis infestations in Latin America

Three studies were conducted in Latin America, one in Brazil, one in Venezuela and one in Argentina, using a common protocol to investigate the efficacy of a single subcutaneous injection of doramectin at 200 μg kg⁻¹ (1 ml per 50 kg) for the treatment and control of Dermatobia hominis infestations in cattle raised under commercial conditions. In each study, two groups of 20 animals each were allocated on the basis of D. hominis nodules present 24 h before treatment to a treated group (T1), or to a control group (T2) which received saline solution at 1 ml per 50 kg of live weight. All cattle were injected in the mid-dorsal cervical region and examined on treatment day and 2, 7, 15 and 30 days post-treatment (p.t.). At each observation day the number of D. hominis nodules was counted and the viability of the larvae inside them was assessed and recorded. Treatment with doramectin was 100% effective in eliminating D. hominis larvae and in controlling the re-establishment of the parasite under field conditions of continuous reinfestation. During the first 48 h p.t., dead larvae of different ages were found outside or partially outside the nodules in animals of the doramectin groups. After 48 h p.t., no live larvae were found inside existing nodules, nor did new nodules develop in animals of the doramectin groups in any of the three studies. When compared with nodule counts in the control group on the same observation day or with parasite burdens on the same animal before treatment, doramectin-treated animals began to eliminate parasitic nodules at 48 h p.t. Efficacy reached 100% at 7 days p.t. and remained at 100% on subsequent observation days (P<0.05). None of the doramectin-treated animals exhibited any clinical signs of adverse reaction to medication.     

Antigenicity and immunogenicity of Hypoderma lineatum soluble proteins in the bovine host

Protein species found in soluble crude extracts of Hypoderma lineatum (common cattle grub) 1st-instar larvae (HL1) were separated by non-denaturing and denaturing polyacrylamide gel electrophoresis (PAGE) and analyzed for antigenicity by Western blotting using serum from H. lineatum-infested and vaccinated cattle. All HL1 proteins resolved by non-denaturing PAGE were found to be antigenic in the infested bovine host. Treatment of the proteins with sodium dodecyl sulfate and 2-mercaptoethanol destroyed the ability of hypodermin B and the Peak 2 proteins from DEAE-ion exchange HPLC to be bound by antibody. The principal proteins, hypodermin A and hypodermin C (collagenase), appear to be the most immunogenic of the larval proteins. Although having similar amino acid composition, hypodermin A did not appear to share an antigenic epitope with the most prevalent protein, hypodermin C. These results may allow for the selection of proteins to be used in vaccine trials and studies of protective immunological mechanisms associated with acquired resistance to H. lineatum infestation in the bovine host.     

The efficacy of ivermectin against larvae of the screw-worm fly (Chrysomya bezziana)

An in vitro technique for screening systemic insecticides against larvae of the screw-worm fly, Chrysomya bezziana is described. Susceptibilities of screw-worm larvae of different ages to ivermectin (MK-933) were determined. Based on 24 h larval mortality, the LD50 of 1-,2-,3-,4- and 5-day larvae was 0.01, 0.02, 0.03, 0.2 and 0.4 ppm of ivermectin. LD50 based on adult emergence following treatment of 4- and 5-day larvae was 0.02 and 0.05 ppm. The LD99.9 for 4-day larvae based on 24 h larval mortality and adult emergence was 11.0 and 0.15 ppm respectively and for 5-day larvae, was 44.3 and 0.4 ppm respectively. Pen and field trials with cattle infested with screw-worm fly demonstrated the potential of ivermectin as a systemic insecticide. Dosages of 50, 100 and 200 micrograms/kg, of ivermectin administered subcutaneously to experimentally infested cattle gave complete control for 6, 12 and 14 days respectively. Ivermectin at 200 micrograms/kg caused 100% mortality of screw-worm larvae up to 2 days old at the time of treatment with 70, 64 and 21% mortality of 3-, 4- and 5-day old larvae at the time of treatment. The residual protection from a single dose of 200 micrograms/kg was 16 to 20 days. When bull calves were treated with ivermectin at a dose of 200 micrograms/kg at the time of castration and branding, none of the 77 treated animals sustained a screw-worm strike in the scrotal area compared with 47 strikes (44%) in the 106 control cattle.     

Detection of IgM antibodies against Babesia bovis in cattle

The diagnosis of acute babesiosis by direct examination of blood smears has some limitations and the indirect serological methods currently in use are designed for detection of IgG, which may not be detectable at an early stage of infection. There is a need, therefore, for rapid and reliable procedures to diagnose acute infections. An ELISA system using a crude antigenic preparation of Babesia bovis was standardized for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Serum samples of cattle imported from tick-free areas collected before and during an immunization process were used to validate the tests. The specificity was 94% and sensitivity 100%. Specific IgM antibodies against B. bovis first appeared on the 11th day post-inoculation (p.i.) in animals infested with Boophilus microplus ticks and on the 19th day p.i. in animals which had been inoculated with infected blood. Antibody titers decreased after Day 33; however, all animals remained positive until the end of the experiment (124 days).     

Enhanced resistance to cattle grub infestation (Hypoderma lineatum de Vill.) in calves immunized with purified hypodermin A, B and C plus monophosphoryl lipid A (MPL).

The influence of an antigen-specific cellular and humoral immune response, stimulated by immunization, on survival of a challenge infestation of Hypoderma lineatum was investigated. Calves immunized with a purified combination of hypodermin A, B and C plus monophosphoryl lipid A (MPL) developed a strong antigen-specific cellular immune response by completion of the immunization schedule which persisted to 12 weeks post-infestation. Responsiveness of peripheral blood lymphocytes to the mitogens concanavalin A and pokeweed was also elevated at 4 and 12 weeks post-infestation. Western blot analysis at the time of maximum grub counts demonstrated that immunized calves responded to hypodermin A, B and C while those receiving only MPL or infested controls responded only to hypodermin B and C. The antigen-specific antibody response as measured by ELISA at maximum grub count was significantly higher in vaccinated calves than in infested controls while the response in calves receiving only immunostimulator was also significantly elevated. Immunized (antigen plus MPL) calves produced 5.0 +/- 6.9 grubs per animal which successfully pupated while those receiving MPL alone produced 16.4 +/- 6.1 and infested controls produced 32.2 +/- 10.9 grubs per animal.     

Studies on naturally acquired immunity to African ticks. II. Observations on cattle exposed to Rhipicephalus appendiculatus under varying periods of repeated infestations.

Groups of Friesian cattle were infested repeatedly with Rhipicephalus appendiculatus at weekly intervals (eight infestations) and at intervals of 3 weeks (seven infestations) and 6 weeks (seven infestations). The engorgement weight of adults, nymphs and larvae decreased significantly initially, from the second infestation, but rose significantly at later infestations. This rise was more dramatic with larval instars than with any other. A new method for quantifying the resistance status of animals was introduced; this method showed that the resistance acquired by cattle during 6-weekly reinfestations was more stable than that acquired by cattle during weekly and 3-weekly reinfestations. The criteria for assessing natural resistance to ticks was reappraised. These are classified into those serving long- and short-term purposes, and those which have direct and indirect effects. Criteria which could serve long- and short-term purposes include a reduced number of engorged ticks, an increased number of dead male and female ticks, a decreased number of ticks which fed above the critical engorgement weight and an increased number which fed within the pre-mating weight and decreased the number of copulations. The criterion of reduced engorgement weight can serve the short-term purpose only. A new manifestation of resistance to ticks, i.e. decreased moulting weights of nymphs and adults, together with the criterion of decreased egg hatchability, were classified as indirect effects because they were manifested after the ticks had dropped off the resistant cattle.     

Efficacy of a pour-on formulation of doramectin against lice, mites, and grubs of cattle

OBJECTIVE: To determine effectiveness of a pour-on formulation of doramectin against Damalinia bovis, Haematopinus eurysternus, Linognathus vituli, Solenopotes capillatus, Chorioptes bovis, Sarcoptes scabiei, Hypoderma bovis, and Hypoderma lineatum. ANIMALS: Cattle of various ages with naturally acquired or artificial infestations with 1 or more species of lice, mites, or grubs. PROCEDURE: In 10 louse and 6 mite studies, cattle were treated with doramectin (500 microg/kg, topically) on day 0, and parasite counts were performed approximately weekly from days 0 to 35. In 6 grub studies, cattle expected to harbor Hypoderma spp were treated before emergence of warbles. After warbles began to emerge, they were counted every 2 weeks, and grubs were collected and identified by species. RESULTS: Burdens of D bovis, H eurystemus, L vituli, and S capillatus on doramectin-treated cattle were 0 by 28 days after treatment. Burdens of C bovis and S scabiei decreased to 0 in naturally infested cattle and approximately 0 in artificially infested cattle by day 14 to 15. In grub studies, 107 of 136 control cattle had warbles, whereas 2 of 136 doramectin-treated cattle had 1 warble each, which represented a cure rate of 98.5%. CONCLUSION AND CLINICAL RELEVANCE: One topical application of doramectin was highly efficacious against common species of lice, mites, and grubs known to affect performance, health, and appearance of cattle.     

Efficacy of a long-acting formulation of ivermectin against Psoroptes ovis (Hering, 1838) on cattle

A study was conducted in cattle experimentally infested with Psoroptes ovis to compare the prophylactic control against P. ovis provided by a long-acting injectable formulation of ivermectin to that of a commercially available injectable formulation of doramectin. Thirty Holstein steers were used. Animals were allocated by restricted randomization based on Day 0 body weight, forming six replicates of five animals each. Within each replicate, one animal was randomly allocated to one of the following treatment groups, with ivermectin and doramectin administered subcutaneously where indicated: (1) untreated controls; (2) ivermectin long-acting injectable (LAI) 630 mcg/kg, 56 days before challenge; (3) ivermectin LAI 630 mcg/kg, 42 days before challenge; (4) ivermectin LAI 630 mcg/kg, 35 days before challenge; or (5) doramectin 200 mcg/kg, 35 days before challenge. Animals were housed in individual pens 1 week prior to treatment. All animals were experimentally infested with P. ovis mites in the area between the shoulders, on the same day. Live mites were counted in scrapings from mange lesions at 2 sites on each animal 14, 21 and 28 days after challenge. Live mites were found in 33, 67 and 83% of the untreated controls on each respective evaluation. No P. ovis mites were found in steers treated with ivermectin LAI. Those animals showed lower (P < 0.05) mite counts than untreated controls on evaluations conducted 21 and 28 days after challenge. These results indicate that the ivermectin long-acting injectable formulation prevents induced infestations by P. ovis for at least 56 days after treatment. Doramectin injectable formulation, used at 200 mcg/kg, did not have a prophylactic effect 35 days after treatment.     

The effect of the bont tick (Amblyomma hebraeum) on the weight gain of Africander steers

The effects of the bont tick Amblyomma hebraeum on the productivity of cattle need to be quantified in order to design economically optimal control programs. Liveweight gains (LWGs) of three groups of Africander steers, maintained in the same pasture and exposed to zero, medium or high numbers of larvae, nymphs and adults of the tick, were measured. Larvae and nymphs had no significant effect on LWG but adults had a large, statistically significant effect (P less than 0.05). The tick-free group had an average LWG of 20 kg more than the heavily infested group after 3.5 months. No mortality was recorded from ticks or tick-borne diseases during the experiment. There was no relationship between the number of engorging adult female ticks counted and loss of LWG of individual animals. The latter suggests that exposure to ticks as well as engorgement by female ticks causes losses. The loss per adult female that completed engorgement was estimated, by relating the LWGs of individual animals to their tick infestations, to be 4 +/- 2 g per adult female tick. An alternative estimate, made by comparing the average LWG and tick infestations of each treatment group, was equal to 10 +/- 4 g. This latter estimate includes the effect of challenge (and rejection) as well as tick feeding and so was accepted as giving the best estimate. Screw-worm fly (Chrysomya bezziana) struck an average of 7.5% of the cattle infested with ticks in any week but the effects on the productivity of the cattle were minimized by immediate treatment. There was a significant, positive correlation between the incidence of screw-worm fly strike and the numbers of adult ticks counted on the experimental animals. The results provide data for calculating losses caused by A. hebraeum in different parts of its geographical range.     

Field efficacy of flumethrin pour-on against Psoroptes ovis in cattle.

The field efficacy of pour-on formulation of flumethrin 1 per cent was assessed in cattle naturally infested with Psoroptes ovis. In a finishing unit, 18 infested animals were selected, left untreated for one month and examined twice to monitor the progress of the infestation. They were treated twice 10 days apart with a pour-on formulation of flumethrin 1 per cent (2 mg/kg bodyweight). Skin samples were collected from the animals on days 0, 7, 14, 28 and 42 and examined for the presence of live mites. The clinical condition of the cattle was evaluated at the beginning and at the end of the trial using a standardised chart. Bodyweights were recorded on days -32, 0, 28 and 50. In addition the 65 cows in a calving unit were given the same treatment regimen and its efficacy was evaluated on days 0, 7, 14 and 28 in 11 P ovis infested animals. On the day of treatment most of the infested animals had a severe pyodermatitis. From day 7 onwards no live mites were found in the treated animals and there was a marked clinical improvement. The percentage efficacy on day 28 was 100 per cent in both units. In the finishing animals treatment was associated with a marked improvement in daily weight gains between day 0 and day 28.     

Transmission of Babesia spp by the cattle tick (Boophilus microplus) to cattle treated with injectable or pour-on formulations of ivermectin and moxidectin

OBJECTIVE: To assess the efficacy of ivermectin and moxidectin to prevent transmission of Babesia bovis and Babesia bigemina by Boophilus microplus to cattle under conditions of relatively intense experimental challenge. DESIGN: Naive Bos taurus calves were treated with either pour-on or injectable formulations of either ivermectin or moxidectin and then exposed to larvae of B microplus infected with B bovis or larvae or adults of B microplus infected with B bigemina. One calf was used for each combination of haemoparasite, B microplus life stage, drug and application route. PROCEDURE: Groups of calves were treated with the test drugs in either pour-on or injectable formulation and then infested with B microplus larvae infected with B bovis or B bigemina. B bigemina infected adult male ticks grown on an untreated calf were later transferred to a fourth group of animals. Infections were monitored via peripheral blood smears to determine haemoparasite transmission. RESULTS: Cattle treated with either pour-on or injectable formulations of ivermectin and moxidectin became infected with B bovis after infestation with infected larvae. Similarly, larvae infected with B bigemina survived to the nymphal stage to transmit the haemoparasite to animals treated with each drug preparation. Cattle treated with pour-on formulations of ivermectin and moxidectin then infested with adult male ticks infected with B bigemina did not become infected with B bigemina whereas those treated with the injectable formulations of ivermectin and moxidectin did show a parasitaemia. CONCLUSIONS: Injectable or pour-on formulations of ivermectin and moxidectin do not prevent transmission of Babesia to cattle by B microplus. Use of these drugs can therefore not be recommended as a primary means of protecting susceptible cattle from the risk of Babesia infection.     

Differentiation of resistance in cattle to larval Hypoderma lineatum

Cattle were successively infested for 4 years with larval cattle grubs, Hypoderma lineatum, to demonstrate the acquisition of resistance. The effects of host age and dietary-induced state with Vitamin A on susceptibility to infestation and their interactions with effects from previous infestations were also determined.Cattle acquired resistance to infestation after a single infestation. This resistance was not affected by host age for at least 4 years, was not affected by a deficiency of vitamin A, and was effective in the skin and connective tissues of the host. The effects of this acquired resistance were highly variable in degree, were produced primarily during the first 90 days of infestation and increased after repeated provocation to provide nearly complete protection in some animals.Calves and yearlings had innate resistance that decreased with age, was localized in the skin, was impaired by a deficiency of Vitamin A, and resulted in a mean loss of 65–75% of the larvae in an infestation dose of up to 150.The combined effects of the 2 types of resistance reduced the grub-burden in cattle throughout their life.