在藏羚羊上发现的中国第四种皮蝇

对于从可可西里藏羚羊上采集到的藏羚羊皮蝇Hypoderma sp.3龄幼虫进行了形态学观察,发现其在伪头、第10腹节上的刺、气门板形态上与中国现有记录的3种皮蝇（牛皮蝇、纹皮蝇和中华皮蝇）有着明显的区别。对藏羚羊皮蝇的线粒体COI 基因种属特异性序列研究表明,其与牛皮蝇、纹皮蝇和中华皮蝇对应序列的相似性分别为88.1％,87.8％和88.8％。据此认为采自藏羚羊的此种皮蝇可能为中国境内又一皮蝇新种或新记录种,为中国第四种皮蝇。     

Myiasis caused by Oestridae: serological and molecular diagnosis

Myiasis-causing Oestridae (bot flies) infect several animal species world-wide, from palaearctic to subtropical/tropical areas. Oestrids affect livestock production causing abortion, reduced milk production, losses in weight and fertility, poor hide quality and an impairment of the host's immune system. In the last few years much research has been carried out on the immunology of these infestations, in order to acquire efficient and reliable diagnostic serological tools; the genome of the different species of Oestridae has been studied to further their molecular identification, taxonomy and phylogenesis. The immunodiagnostic methods for many myiasis causing Oestrids have proven to be a viable alternative to the clinical parasitological examination or the post-mortem examination. Numerous serological tests have been developed for the diagnosis of bovine hypodermosis caused by Hypoderma bovis and Hypoderma lineatum, and ELISAs using larval hypodermin C as the antigen are currently used on serum, individual and pooled milk samples to detect the presence of circulating anti-Hypoderma antibodies. In Italy the best period to sample the animals is November-January, since it is in this period that the antibody kinetics of the animals reaches a peak. Recently the efficacy of the ELISA test on pasteurized milk samples has been demonstrated, allowing the diagnosis of bovine hypodermosis also in areas where there is no information on the presence of the disease and the sampling of the animals is laborious. The cross-reactivity between Przhevalskiana silenus antigens and anti-Hypoderma antibodies led to assessing the usefulness of a simple and cost-effective ELISA for the diagnosis of goat warble fly infection. In particular, it has been demonstrated that infected goats display an antibody peak in November-December in blood and milk, thus making this period suitable for sampling. Although no extensive data is available on the immunology of sheep and goat oestrosis caused by Oestrus ovis, the efficacy of ELISA has been demonstrated by correlating serological results with clinical post-mortem examinations. No immunological techniques are currently used to diagnose gasterophilosis of equids and only one study reports the efficacy of ELISA for detecting anti-Gasterophilus antibodies in infected equids. Several studies have been conducted into the molecular characterization of the mitochondrial DNA (mtDNA)--in particular of the gene encoding for the cytochrome oxidase I (COI)--for many free-living and parasitic arthropods for diagnostic, taxonomic and phylogenetic purposes. As regards Oestridae causing myiasis, the first study features a PCR-RFLP assay of the most common Italian species (i.e. H. bovis, H. lineatum, Gasterophilus intestinalis, P. silenus, O. ovis), which showed clear genetic differences among the genera examined, but no inter-specific variation between the two species of Hypoderma considered. The molecular characterization of the most variable region of the COI gene (encoding for the region from E4 to the terminal COOH) was able to clearly differentiate H. bovis and H. lineatum. The E4-COOH region of the COI gene has been characterized for 18 oestrid species and from a taxonomical point of view, molecular data confirm the morphological classification, with the examined species divided into four subfamilies. New insights have also been gained on the molecular differentiation of the most common species of Hypoderma (i.e. H. bovis, H. lineatum, Hypoderma actaeon, Hypoderma diana and Hypoderma tarandi) and, in particular, the restriction enzyme BfaI, provides a diagnostic profile that can be used to simultaneously differentiate all the species examined. The characterization of the E4-COOH COI gene and the hypervariable region of the gene encoding for the ribosomal Isu revealed the identity of Hypoderma sinense as a new species, infecting cattle and yaks in China. Finally, the molecular analysis of the same mitochondrial and ribosomal regions showed that P. silenus, Przhevalskiana aegagri and Przhevalskiana crossii are morphotypes of the same species.     

Comparative scanning electron microscopy of third-instar Hypoderma spp. (Diptera: Oestridae)

Scanning electron microscope study of third-instar larvae of four species of Hypoderma revealed differences among species in the pattern of spination, spine morphology and morphology of the spiracular plates. These observations identify characters that enable the differentiation of Hypoderma actaeon and H. diana, parasitizing red deer ( Cervus elaphus ) in Europe, and provide additional characters for differentiating H. bovis and H. lineatum parasitizing cattle.     

Shared epitopes between the soluble proteins of Hypoderma lineatum and Hypoderma bovis first instars.

Cattle are known to acquire immunological resistance to hypodermyiasis by repeated exposure to both species of cattle grubs, Hypoderma lineatum (Villers) and Hypoderma bovis (L.). Vaccination of cattle with purified proteins of H. lineatum, particularly hypodermin A, is known to protect cattle against hypodermyiasis by this species. The development of a protective recombinant vaccine against both species using hypodermin A isolated from H. lineatum would require that immunological epitopes be shared by complementary proteins in H. bovis. The purpose of this study was to investigate the soluble proteins of H. bovis first-instars for shared epitopes with H. lineatum. Soluble H. lineatum and H. bovis first-instar larval proteins were resolved by nondenaturing polyacrylamide electrophoresis, blotted onto nitrocellulose paper, and probed with selected polyclonal cow, polyclonal rabbit, and mouse monoclonal antisera. Considerable cross-reactivity was demonstrated by antibodies in the serum of an H. lineatum-infested cow as 6 of 10 resolved H. bovis proteins were bound by the antibodies. The most common shared epitope(s) was associated with hypodermin C, a collagenolytic protease. Hypodermin A shared epitope(s) were noted on 1 prominent H. bovis band (HB1-2). Hypodermin B, a prominent protein in H. lineatum, did not appear to share epitopes with H. bovis proteins. Shared epitopes between H. bovis proteins and hypodermins A and C of H. lineatum would suggest that cross-protection of cattle against H. bovis can be expected by vaccination with recombinant proteins of H. lineatum.     

Species identification of Hypoderma affecting domestic and wild ruminants by morphological and molecular characterization

Cuticular structures and the sequence of the cytochrome oxidase I gene were compared for Hypoderma bovis (Linnaeus), Hypoderma lineatum (De Villers), Hypoderma actaeon Brauer, Hypoderma diana Brauer and Hypoderma tarandi (Linnaeus) (Diptera, Oestridae). Third-stage larvae of each species were examined by scanning electron microscopy revealing differences among species in the pattern and morphology of spines on the cephalic and thoracic segments, by spine patterns on the tenth abdominal segment, and by morphology of the spiracular plates. The morphological approach was supported by the molecular characterization of the most variable region of the cytochrome oxidase I (COI) gene of these species, which was amplified by polymerase chain reaction and analysed. Amplicons were digested with the unique restriction enzyme, BfaI, providing diagnostic profiles able to simultaneously differentiate all Hypoderma species examined. These findings confirm the utility of morphological characters for differentiating the most common Hypoderma larvae and reconfirm the power of the COI gene for studying insect identification and systematics.     

Scanning electron microscopy of the posterior spiracles of cattle grubs Hypoderma bovis and Hypoderma lineatum

Posterior spiracles of newly hatched first instar larvae of Hypoderma bovis (L.) and H. lineatum (DeVill.) consist of two pairs of spiracular openings. Each pair is surrounded by a rima bearing three spines. Posterior spiracles of second instar larvae are composed of a pair of medial ecdysial scars bounded laterally by spiracular plates. H. bovis spiracular plates have twenty-nine to forty openings, each surrounded by a slightly raised rima. H. lineatum spiracular plates have eighteen to twenty-five openings. Spiracular openings lead to posterior felt chambers which are connected to a common anterior felt chamber filled with a meshlike network. In third instar H. bovis each medial ecdysial scar is surrounded by a strongly concave spiracular plate. Spiracular openings are surrounded by slightly raised rima. Most rimae bear a spine. Spiracular plates of H. lineatum are flat and rimae are without spines. Each spiracular opening leads to a posterior felt chamber, several of which are confluent with a larger anterior felt chamber. Anterior felt chambers open into the dorsal longitudinal tracheal trunk. Felt chambers in third instar larvae are also filled with a complex mesh.     

Early detection of cattle grub (Hypoderma lineatum and H.bovis) (Diptera, Oestridae) using ELISA

An enzyme-linked immunosorbent assay (ELISA) to detect cattle grub-infested animals in the autumn in Alberta has been developed. Antibody to Hypoderma lineatum de Vill. was detectable as early as 6 weeks post-infestation in artificially infested steers. Peak antibody concentrations preceded the peak in maximum 'apparent' grub numbers which occurred between 37 and 43 weeks after infestation. Natural infestations with H. lineatum and H. bovis L., ranging from one to ninety-two grubs per calf, were readily detected by November and the incidence of false positives in uninfested calves was only 5%. Low level (1-4 grubs) infestations of H. bovis only became detectable in February with peak antibody concentrations occurring at the end of April. Prevalence of cattle grub infestations in southern Alberta was shown to be 37% during this study.     

Immunization and Parenteral Chemotherapy for the Control of Cattle Grubs Hypoderma Lineatum

Immunization with two types of warble larvae antigens A and B, the latter treated with tannic acid, and injections of dimethoate, an organic phosphate, were tried for the control of prehypodermic larvae of Hypoderma lineatum De Vill., and H. bovis L., in range calves. In test groups of 20 calves, given intramuscularly, antigen A had no effect, but combined treatment with antigens A and B reduced the number of H. bovis L., larvae by 81 per cent (P<.001), and proved as effective as dimethoate subcutaneously or intramuscularly. H. lineatum De Vill., did not respond to any treatment. Antigen B and dimethoate were free from harmful effects on the host, but antigen A caused anaphylaxis and irritation at the site of injection.     

Toxicity of Alpholate to Cattle

Although Apholate is used as a sexual sterilant of both sexes of houseflies (Musca domestica L.) it can not be used for ;systemic' chemosterilization of the prehypodermic larvae of the warble flies Hypoderma bovis (L.) and H. lineatum (De Vill.) because of its high toxicity to 4- to 5-month-old calves. An intramuscular dose of 2.5 mg./Kg. killed the calves in 5 to 7 days. The pathognomonic clinical signs were impaction of the rumen, anorexia, depression, and general weakness. The hematopoietic system was affected. There was marked leukocytopenia characterized by lymphocytopenia within 24 hours of Apholate injections.     

Hypodermin B, a trypsin-related enzyme from the insect Hypoderma lineatum. Comparison with hypodermin A and Hypoderma collagenase, two serine proteinases from the same source

Hypodermin B, a serine proteinase with a molecular weight of 23000, was purified to homogeneity from the larvae Hypoderma lineatum. It is stoichiometrically inhibited by diisopropylfluorophosphate and fully inactivated by N-tosyllysine chloromethyl ketone and soya bean and bovine pancreatic trypsin inhibitors. N-Tosylphenylalanine chloromethyl ketone and ovomucoid are without effect on its activity. Hypodermin B hydrolyses both amide and ester substrates of trypsin but does not display any chymotryptic activity on synthetic substrates. Its specificity on the B chain of insulin is slightly broader than that of bovine trypsin. Its amino acid composition and N-terminal sequence suggest structural homology with serine proteinases of the trypsin family and with two other serine proteinases, hypodermin A and Hypoderma collagenase, previously isolated from the same larvae. Hypodermins A and B are very similar with respect to their inhibition and specificity, they differ however strongly from Hypoderma collagenase.     

Structural study on the active site of the collagenase from Hypoderma lineatum

The collagenase from the larvae Hypoderma lineatum is a serine proteinase sequentially related to the trypsin family. The tryptic peptide containing the serine residue of the active site, labelled with [3H] diisopropylfluorophosphate was isolated and determined to be Ser-Pro-Cys-Phe-Gly-Asp-Ser-Gly-Gly-Pro-(Phe-Ser)-Lys. It is highly conservative with respect to the corresponding peptide in other serine proteinases related to trypsin.     

Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies: cross-reactivity and antibody kinetics in naturally infested goats.

To evaluate the cross-reactivity between Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies six protocols with different concentrations of antigen and different dilutions of sera and conjugate were applied. The highest cross-reaction between the H. lineatum antigen and the anti-P. silenus antibodies is given by 2 micrograms/ml of antigen concentration, 1:400 of serum and 1:10,000 conjugate dilution. The study on the kinetic development of antibodies in goats naturally infested by P. silenus and the natural course of infestation pointed out the existence of a good correlation between individual antibody kinetics and the natural evolution of the cycle of infestation. The highest antibody concentration may be registered from October through November, coinciding with the end of the migration fo the larvae inside the animal's body. In our condition, this period can be considered as a favorable sampling period for immunodiagnosis and immunoepidemiological studies of goat warble fly infestation.     

Type and parameters of common warble fly (Hypoderma bovis) larval distribution in cattle herds in different parts of a geographic range

The method of statystical analysis of the distribution of the 2nd and 3d instar larvae of Hypoderma bovis in 54 herds (8120 heads) of cattle from Czechoslovakia and in 20 herds (1809 heads) from Mongolia has shown that in most cases the negative binomial distribution models the distribution of larvae with sufficient reliability and that the variability in parameters of the distribution is stable independent on the parts of the area.     

中国南方两个革菌新种：中国丝皮革菌和棉絮丝皮革菌（丝皮革菌科,多孔菌目）

依据形态学和分子系统学研究结果,描述了产自中国云南省的丝皮革菌属2个新种,即中国丝皮革菌Hyphoderma sinense和棉絮丝皮革菌H. floccosum。中国丝皮革菌以光滑子实层体表面、被结晶和念珠状的囊状体及圆柱形至腊肠状的担孢子（8-11.5×3-5μm）为主要识别特征。棉絮丝皮革菌具有棉絮状至粉状子实层体表面、管状和分隔状囊状体及椭圆形至腊肠状担孢子。对新种的ITS和nLSU片段进行了测序和分析,采用最大似然法、最大简约法和贝叶斯推断法对研究样本的ITS+nLSU nrRNA基因序列进行系统发育分析,结果显示两个新种隶属于丝皮革菌属,其中,中国丝皮革菌与变形丝皮革菌聚在一起;棉絮丝皮革菌与松生丝皮革菌、丝皮革菌和拟丝皮革菌聚为一类。本研究提供了两新种的详细描述、线条图、生态照片及与相似种的区别,同时编写了我国丝皮革菌属23个种的检索表。     

Specificity of the collagenase from the insect Hypoderma lineatum

Specificity of the collagenase from the larvae Hypoderma lineatum, a serine protease related to trypsin, has been investigated by using native collagen and non-collagenous substrates. At 25 degrees C and neutral pH the degradation of collagen by the larval enzyme in solution results in a 52% loss of specific viscosity, without loss of helicity. Electron microscopy of segment-long-spacing crystallites of the digest shows the occurrence of one cleavage region between bands 41 and 44 whereas Edman degradation indicates several cleavage loci in this region. Hypoderma collagenase differs from proteinases I and II from the crab Uca pugilator, which catalyse cleavages in multiple regions of the collagen molecule, and also from vertebrate collagenases, which cleave collagen only between residues 775 and 776. Apart of specific action on collagen, Hypoderma collagenase degrades the oxidized chain B of insulin; the major cleavage occurs at the Leu15-Tyr16 bond followed by two minor cleavages at the Arg22-Gly23 and Lys29-Ala30 bonds. The larval enzyme has no action on synthetic peptide substrates of trypsin or chymotrypsin.     

Chemical and enzymatic characterization of the collagenase from the insect Hypoderma lineatum.

The collagenase from the larvae Hypoderma lineatum, with a molecular weight of 24 000 and isoelectric point of 4.1, was obtained in homogeneous form by ion-exchange chromatography. It is stoichiometrically inhibited by diisopropylfluorophosphate. On the other hand it is unaffected by ethylenediaminetetraacetate, p-chloromercuribenzoate, dithiothreitol, N-tosyllysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone and ovomucoid trypsin inhibitor. The enzyme which degrades native collagen in its helical parts, has a specific activity on thermally reconstituted collagen fibrils of 150 micrograms collagen degraded x min-1 x (mg enzyme)-1 at 37 degrees C. It hydrolyses casein but has no esterolytic activity characteristic of trypsin, chymotrypsin nor elastase. It has no action on the synthetic peptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-L-glycyl-L-prolyl-D-arginine. The amino acid composition of Hypoderma collagenase indicates a distinct similarity with the serine proteinases of the trypsin family and with another athropode serine collagenase, that of the fiddler crab Uca pugilator. This suggests that eucaryotic collagenases with digestive rather than morphogenic function represent a new category of members of the trypsin family.     

Immunological responses of rabbits artificially infested with the cattle grubs Hypoderma bovis (L.) and H. Lineatum (De Vill.) (Diptera: Oestridae)

Boulard Chantal and Weintraub J. 1973. Immunological responses of rabbits artificially infested with the cattle grubs Hypoderma bovis (L.) and H. lineatum (De Vill.) (Diptera : Oestridae). International Journal for Parasitology3: 379–386. Immunological responses against first-instar larvae of Hypoderma bovis (L.) and H. lineatum (De Vill.) were traced in artificially infested rabbits in which larvae grew normally in the first instar but did not survive to the second instar. Passive haemagglutination demonstrated a rapid rise in antibody titres during the first 2 months to maximum levels that persisted until about 200 days of the infestation. Immunoelectrophoretic analyses of the sera demonstrated that larval metabolic products were more important than breakdown products of dead larvae in stimulating the production of antibodies. Larval collagenase, which seemed to induce the most significant immune response, was inhibited by homologous antibodies in the serum of a hyperimmunized rabbit. The implications of these results for infestations in the normal bovine host were discussed, with emphasis on the need to identify antigenic fractions other than collagenase, which by itself had not produced total control of larvae in previous vaccination tests.     

The immunosuppression mechanism of hypodermin A on complement

Hypodermin A (HA), a serine protease secreted by first-instar larvae of Hypoderma lineatum (Diptera: Oestridae) is associated with inflammatory and the specific immune responses in cattle hosts. In the present study, the cDNA sequence of HA was synthesized, and found to have fifteen amino acids which differed from the sequence available in GenBank. We then examined the association between recombinant HA and guinea-pig complement component 3 (C3) through a co-immunoprecipitation assay. Cos7 cells stably expressing HA were generated, and were found to be more resistant to lysis by guinea-pig C3 than the controls. HA was also able to degrade the C6 and C5b-9 of guinea-pig C3. The presumed DNA binding site of HA with guinea-pig C3 was detected by an electrophoretic mobility shift assay (EMSA). In contrast, after stable transfection, mHA was unable to reduce the amount of C3 or to inhibit its cytotoxicity, while HA could degrade guinea-pig C3 and inhibit the complement pathway. The findings suggest that recombinant HA could serve as an immunosuppressive agent against organ rejection after xenotransplantation.