Scanning electron microscopic study of third-instar warbles in yak in China

Scanning electron microscopic observations were performed to compare the structures of third-instar of three species of warbles (Hypoderma bovis, H. lineatum and H. sinense) that infest yak in China. The heads of the warbles consisted of mouths and opercular sutures with spinal bands between them. We could not detect any morphological differences in the shape of the warble's heads among these three species. The spiracular plate of H. bovis was strongly concaved and had numerous spines at the spiracular opening rims. The H. lineatum had flat spiracular plates and no spines on the spiracular plates. In the case of H. sinense the spiracular plate was as flat as that of H. lineatum but had small sized and fewer spines on the spiracular plates. At the ventral side on the tenth segment of the warble body of H. bovis the spinal band was absent. Although H. lineatum had only one band at the posterior border on the tenth ventral segment, H. sinense had two bands of spines at the anterior and posterior border on the tenth segment.     

青海省藏羊感染皮蝇蛆调查及其虫种鉴定

为了解青海省藏羊感染皮蝇蛆状况及感染种类,本研究在3个地区调查了476只藏羊,对2株感染藏羊皮蝇蛆和4株感染牦牛皮蝇蛆的线粒体CO1基因种属特异性序列进行比较研究。结果表明:14只羊感染了皮蝇蛆,感染率为2.9%,总瘤胞数为27个,平均感染强度为1.9个;并扩增其长度为688 bp的序列,克隆后测序,将其序列与GenBank中的牛皮蝇、纹皮蝇蛆和中华皮蝇蛆序列进行聚类分析,结果显示青海省感染藏羊的皮蝇蛆与感染牦牛的果洛株、民和和黄南株在线粒体CO1基因片段聚类分析进化树和同源性比较中,与牛皮蝇位于同一个进化树分支上,基因片段同源性大于99%,为牛皮蝇;感染牦牛的海晏株和贵南株在线粒体CO1基因片段聚类分析进化树和同源性比较中,与中华皮蝇位于同一个进化分支上,基因片段同源性大于99%,为中华皮蝇。几个皮蝇蛆分离株种内变异率小于1%,种间变异率大于8%。     

Molecular differentiation of Hypoderma bovis and Hypoderma lineatum (Diptera,Oestridae) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

The most variable region of the cytochrome oxidase I (COI) gene of Hypoderma bovis(1) and Hypoderma lineatum(2) (Diptera, Oestridae) was amplified by PCR and the amplicons were sequenced and analysed. PCR products were digested with three restriction enzymes, namely BfaI, HinfI and TaqI, providing informative profiles. H. bovis and H. lineatum sequences revealed an inter-specific variation rate of 8.5%, and an intra-specific variation rate of 0.87 and 0.29%, respectively. The results showed that the COI gene region examined was useful for the differentiation of H. bovis and H. lineatum and that a PCR-RFLP assay is a practical tool for their identification, offering additional diagnostic and epidemiological instruments for the study of cattle grub infestation.     

Immunization of cattle against hypodermatosis (Hypoderma lineatum (deVill.) and H. bovis (L.)) using H. lineatum antigens

The feasibility of immunizing calves against Hypoderma lineatum and H. bovis with a crude H. lineatum larval extract or culture-derived antigen was investigated. Larval survival was followed through pupation. Grub appearance in the backs of both of the immunized groups was found to be 50% of that in the control groups. First appearance of H. bovis was significantly retarded in both immunized groups; however, H. lineatum was unaffected. Duration of H. bovis dropping was also shorter in animals receiving crude extract than in controls while duration of dropping of H. lineatum was not influenced by immunization. Survival of H. lineatum larvae to pupation was significantly reduced in animals immunized with crude larval extract, but not in those receiving culture-derived antigen. H. bovis survival was reduced by both treatments.     

Bovine hypodermosis in north-eastern Algeria: prevalence and intensity of infestation]

Between 1989 and 1997 different factors related to warble fly infestation (prevalence, intensity, climate, breed) were studied in north-eastern Algeria. Mean prevalence during the period was 76%. This figure was obtained from a cattle market survey of four different regions (Jijel, Constantine, Guelma, and El Tarf). One-hundred percent of the herds were found to be infested in El Tarf and 61% of animals were infested. Within a herd, the intensity of infestation decreased with the age of the cattle. The mean number of warbles per animal decreased as reinfestations occurred. The peak of the emergence of warbles observed between January and March was progressively delayed in the older animals. Two species of Hypoderma were present in cattle in Algeria (H. bovis and H. lineatum) with a predominance of H. lineatum (63%) in the semi-arid area (Batna) and a majority of H. bovis (75%) in humid area (Jijel). Warbles appeared earlier in Batna (October) than in Jijel (November). The Prim'Holstein breed was more susceptible to the warble fly infestation than Montbeliard or local breeds. The implementation of a control programme for hypodermosis in Algeria is urgent. Control can be achieved through a mass treatment of cattle. This approach must take into account the factors described in this study which influence the epidemiology of the disease.     

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.     

纹皮蝇Hypodermin C基因原核表达及间接ELISA检测方法的建立

为建立牛皮蝇蛆病的抗体检测方法,本研究从纹皮蝇I期幼虫中提取总RNA,采用RT-PCR扩增了Hypodermin C(HC)基因.将该基因片段插入到原核表达载体pET-30a(+)中进行原核表达.获得了31ku以包涵体形式表达的重组HC蛋白.Western blot结果表明表达产物能够与阳性牛血清IgG特异性结合,具有良好的抗原活性.以纯化的重组HC作为包被抗原建立了牛皮蝇蛆病间接ELISA诊断方法,实验确定抗原最佳包被浓度为15.63 μg/mL,血清最佳稀释度为1:80,阳性标准判定为待检血清OD值＞0.256.所建立的诊断方法具有较好的特异性、敏感性和可重复性.应用该检测方法对230份临床血清进行检测,阳性率为20.6％.研究结果表明该间接ELISA方法是一种有效的牛皮蝇蛆病血清学检测方法.     

Enzyme-linked immunosorbent assay for the detection of antibodies to Hypoderma bovis in cattle

An adaptation of the enzyme-linked immunosorbent assay has been shown to detect antibodies in the circulation of cattle infected with Hypoderma bovis. Strong positive reactions were obtained from all infected cattle, even those which harboured only one larva. A strong cross reaction was observed in sera taken from cattle infected with H lineatum, but not in cattle infected with Fasciola hepatica or Ostertagia ostertagi.     

Toxicity of Sevin (1-Naphthyl-n-Methyl-Carbamate) To Cattle Grubs, Lice, and Cattle

Sevin, a cholinergic insecticide, was used for the control of cattle lice Haemotopinus eurysternus and Bovicolar bovis and cattle grubs Hypoderma lineatum and H. bovis. Partial or complete spraying of the animal body with 0.5 per cent Sevin was equally effective for louse control and one application killed the lice and prevented reinfestation in `carrier' cows for 17-20 weeks.

The dermal or parenteral administration of Sevin to the host animal had no larvicidal effect on cattle grubs migrating in its body. Similarly, the sprays of Sevin had no lethal effect on grubs encysted in the back of cattle.

     

Efficacy of moxidectin injectable and pour-on formulations in a pilot control program against bovine hypodermosis in Southern Italy

Bovine hypodermosis is a myiasis caused by Hypoderma bovis and Hypoderma lineatum (Diptera, Oestridae) larvae, which has a severe economic impact on the livestock industry. Though myiasis is widespread throughout Italy, no nationwide eradication program has ever been planned, unlike in other European Countries. With a view to setting up a national control program, a pilot study was carried out in Southern Italy on 9939 cattle bred in an area with a high prevalence of cattle hypodermosis, using moxidectin 0.5% pour-on (Cydectin, Fort Dodge) and 1% injectable (Cydectin, Fort Dodge) formulations. At the recommended dosage, moxidectin displayed efficacy levels of 99.9% in the pour-on and 100% in the injectable formulation, whereas the microdose (1 mg per head regardless of body weight) was less effective (65.7%). This trial contributed to a significant reduction in infestation rates in the study area and represented the first step through which a national program for eradicating warble fly infestation in Italy.     

Antigen-specific lymphocyte proliferative responses in vaccinated and Hypoderma lineatum-infested calves.

Cattle infested with the common cattle grub, Hypoderma lineatum (Villers) develop specific humoral antibodies and a cellular immune reaction, defined by delayed-type hypersensitivity, to purified H. lineatum proteins. This investigation was designed to study the antigen-specific bovine lymphocyte response to hypodermin A (HyA), a serine protease of larval first-instar H. lineatum. Calves were vaccinated with either native or denatured HyA, and challenge-infested with H. lineatum. The kinetic development of a cellular immune response to HyA was monitored during vaccination and infestation. The HyA-specific responses were highly variable and weak during vaccination and infestation. Although HyA-specific lymphocyte blastogenic responses were observed, no correlation was noted between the magnitude of antigen-specific, peripheral lymphocyte proliferation and larval mortality. In striking contrast to responses observed during infestation, intense HyA-specific lymphocyte responses were observed with 3 calves 6 months after recovery from infestation. In addition, those responses were further heightened by a 250 micrograms booster injection of pure HyA.     

Efficacy of ivermectin in a topical formulation against induced gastrointestinal and pulmonary nematode infections, and naturally acquired grubs and lice in cattle

Efficacy of ivermectin in a topical formulation was evaluated in cattle against adult gastrointestinal and pulmonary nematode infections (experiment 1, n = 24), the 2nd- and 3rd-stage larvae of Hypoderma spp (experiment 2, n = 12), and the biting louse Damalinia bovis (experiment 3, n = 12). Nematode infections were induced and grubs and lice were naturally acquired. Treatments consisted of a single dose of ivermectin in a topical formulation of 200, 500, or 1,000 micrograms/kg of body weight in experiment 1 or 500 micrograms/kg in experiments 2 and 3. At 1,000 micrograms/kg, ivermectin was 100% effective against Ostertagia ostertagi, Trichostrongylus colubriformis, Oesophagostomum radiatum, Nematodirus helvetianus, Haemonchus placei, and Dictyocaulus viviparus and was greater than 99% effective against Cooperia oncophora, C punctata, and T axei. At 500 micrograms/kg, the efficacy was 100% against C oncophora, C punctata, O ostertagi, T axei, Oes radiatum, N helvetianus, Haem placei, and Dict viviparus and greater than 99% against T colubriformis. At 200 micrograms/kg, the efficacy was 100% against Oes radiatum, Haem placei, and Dict viviparus, greater than 99% for O ostertagi, 96% for C oncophora, 86% for C punctata, 90% for T axei, 85% for T colubriformis, and 71% for N helvetianus. At 500 micrograms/kg, ivermectin was highly effective against the grubs Hypoderma bovis and H lineatum and eliminated the louse Damalinia bovis.     

Lymphocyte responsiveness in cattle previously infested and uninfested with Hypoderma lineatum (de Vill.) and H. bovis (L.) (Diptera: Oestridae)

Cattle exposed to their third consecutive warble (Hypoderma lineatum and H. bovis) infestation had significantly reduced apparent and accumulative grub populations and produced significantly fewer grubs than animals exposed to their first infestation. These resistant animals had enhanced reactivity to mitogens (concanavalin A (con A) and pokeweed) upon reinfestation and a strong antigen-specific response at 2 months post-infestation. Their responsiveness to con A and antigen was also enhanced at 7 months post-infestation when grubs appeared in the backs, and the response to antigen continued at 10 months post-infestation. Responsiveness to mitogens in previously uninfested animals was similar to the control response throughout the infestation apart from the suppression of the con A response at 7 months post-infestation. These animals finally responded to antigen at 10 months post-infestation. These results suggest that acquired resistance to hypodermosis has a cellular basis with participation of both B- and T-cell components.     

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.     

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.     

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.     

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.     

Presence of Hypoderma lineatum stage I larvae in the esophagus of cattle slaughtered in Chihuahua, Chih., Mexico

In order to detect the presence of Hypoderma lineatum stage I larvae within the esophagus of cattle slaughtered in Chihuahua, Chihuahua, Mexico, a total of five samplings were carried out between July and November 2000. In each instance, a random sample was taken from 10% of the animals slaughtered in a single work shift in each of the two slaughterhouses included in this study. The esophagus were cut longitudinally in order to carry out visual inspection and detect the presence of H. lineatum stage I larvae in the submucosa. The larvae were separated and counted. We identified the presence of H. lineatum stage I larvae in the esophagus for all sampling dates, nevertheless, within the last sampling only one esophagus had them. For all sampling dates the prevalence ranged between 11 and 33%; the latter corresponded to the sampling in October. A total of 287 esophagus was inspected of which 54 were positive with one or more larvae (19%); 233 larvae were obtained from these cases. The number of larvae recovered per sampling ranged from 46 to 74 between July and October, the highest number was found in September's sampling. The largest amount of stage I larvae per esophagus was 22 in the months of July and August. Larvae were always located in the submucosa of the esophagus and all were oriented longitudinally.     

内蒙古自治区牛皮蝇蛆病的流行与防治

内蒙古自治区牛皮蝇蛆病主要分布于中、东部的锡盟、呼盟、通辽市等地区.侵袭牛的皮蝇主要有两个种,即纹皮蝇(Hypoderma lineatum)和牛皮蝇(H.bovis),纹皮蝇较多,占70%～80%.感染率一般在50%以上,多为80%～90%,不少地区高达100%.感染强度40～70个瘤包,最高达770个.牛皮蝇蛆病给内蒙古自治区的畜牧业生产造成非常严重的损失.四十余年来,未曾间断过对该病的防治,目前其方法主要采用倍硫磷注射或浇泼,收到一定效果,但并未得到安全控制.     

Serodiagnosis of goat warble fly infestation by Przhevalskiana silenus with a commercial ELISA kit.

Cross-reactivity between Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies has been demonstrated by an ELISA technique. To evaluate the applicability of a commercial ELISA kit for the immunodiagnosis of goat warble fly infestation, different dilutions of serum and conjugate were tested, the development of antibody to P silenus in naturally infested goats was studied, and the results were compared with an ELISA technique using an antigen extracted from the first instar larvae of H lineatum. The best results were obtained with a serum dilution of 1:50; with both techniques the highest antibody concentration were recorded in October, November and December. In view of the confirmed cross-reactivity between H lineatum antigen and anti-P silenus antibodies, and the simplicity and rapidity of the assay, the commercial ELISA kit can be considered as a useful tool for the diagnosis of goat warble fly infestation.