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.     

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.     

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.     

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.     

A third species of Hypoderma (Diptera: Oestridae) affecting cattle and yaks in China: molecular and morphological evidence

Cattle and yak hypodermosis in China is caused by Hypoderma bovis and H. lineatum, with a prevalence reaching up to 98-100% of the animals and maximum intensities exceeding 400 warbles for each animal. A third species, H. sinense, is also considered by Chinese researchers to affect livestock. The molecular characterization of the most variable region of the mitochondrial cytochrome oxidase I gene and of the ribosomal 28S gene has been performed for the third-stage larvae collected from cattle and yaks in China and identified (on the basis of the spinulation on the ventral side of the 10th segment) as H. bovis, H. lineatum, and H. sinense. Amplicons were digested with the HinfI and BfaI restriction enzymes, which provided diagnostic profiles to simultaneously differentiate the 3 Hypoderma species. Third-stage larvae of H. sinense were also examined by scanning electron microscopy, which revealed proper morphological characteristics different from those of H. bovis and H. lineatum. The molecular and morphological evidence herein reported support the existence of a third species of Hypoderma affecting cattle and yaks in China, and the results provide new tools for unequivocal identification of this species and present key components for the evaluation of its endogenous cycle and pathogenicity in animals and humans.     

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 lysate from bovine erythrocytes infected with Babesia bovis. Analysis of antigens and a report on their immunogenicity when polymerized with glutaraldehyde

A group of Babesia bovis antigens obtained by a lengthy biochemical procedure involving disruption of infected erythrocytes has previously been shown to be highly protective. This study shows that these antigens can be found in a simple lysate of infected erythrocytes. The antigens have been characterized by gel filtration and nitrocellulose transfer and consist of a wide spectrum of molecular sizes. Some of the antigens exist in complex form and are easily dissociated. The lysate was polymerized with glutaraldehyde and injected per se into four splenectomized calves. All the calves produced antibody to B. bovis but did not produce erythrocytic isoantibodies. The vaccinated calves and a control group of four splenectomized calves were challenged with virulent B. bovis. Statistically, the vaccinated group differed significantly in parasitaemia, temperature change and pathophysiological parameters from the control group. All of the control group died whereas two of the vaccinated group survived infection.     

Uptake of bovine IgG by first instars of the common cattle grub, Hypoderma lineatum

The midgut of first instar Hypoderma lineatum (De Vill.) (Diptera: Oestridae) was examined for the presence of bovine immunoglobulin using immuno-gold staining in conjunction with image analysis. Newly hatched larvae, unexposed to fluids or tissues of the host, showed little or no deposit of colloidal gold particles. Midgut lumen, microvillar border, epithelial cell bodies and basement membrane of first instars, 6-7 months of age, recovered from the host oesophagus, all showed colloidal gold staining. This indicates that immuno-reactive portions of bovine immunoglobulin survive exposure to extracorporeal larval enzymes, as well as transit of the midgut, and pass through epithelial cells to enter the larval haemocoel.     

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。     

乐果对家蚕生殖腺及氧化应激反应的影响

【目的】农药施加不当导致环境污染严重,已成为蚕桑业发展面临的紧迫问题。本研究旨在评估大田喷洒有机磷农药乐果污染间作桑园可能导致对家蚕Bombyx mori正常发育的影响,以分析乐果对家蚕生殖腺的毒理损伤影响。【方法】以文献报道的乐果对家蚕幼虫的LD₅₀为400 mg/L作为参考依据,给家蚕5龄幼虫添食不同浓度（0, 50, 100, 200和400 mg/L）乐果溶液浸叶处理的桑叶,测定乐果处理后5龄幼虫茧质和体重,雌、雄幼虫生殖腺中乐果残留含量和H2O2含量,抗氧化酶（SOD和CAT）活性及其mRNA表达。【结果】结果显示,乐果处理后家蚕5龄幼虫茧质和体重以及雌、雄幼虫生殖腺中乐果残留含量均具有浓度 效应关系。100~200 mg/L乐果浓度添食下,雌雄幼虫生殖腺中H₂O₂含量与对照（清水）相比均显著上升。随着添食浓度的增加,SOD和CAT活性都出现先降低后升高的趋势,但均低于对照组。sod和cat的mRNA表达水平与相应的酶活性具有正相关性。通过HE染色以及生精囊的体外培养,观察到在乐果处理后雌、雄幼虫生殖腺的形态结构发生变化,表现为形态畸形,细胞内空泡增大,生殖细胞数目相对减少,且随浓度的增加,生殖腺损伤越严重。【结论】本研究结果说明,采用乐果对家蚕的LD₅₀以下浓度溶液浸叶添食于5龄幼虫,对家蚕生殖腺发育有毒理损伤的影响,这为从蚕业生产角度全面禁止大田使用乐果和加强农业生产区划管理提供了依据。     

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.     

Babesia bovis: analysis of and preliminary vaccination studies with a defined infected erythrocyte membrane binding antigen.

Murine monoclonal antibodies (MAB) were produced against the 'beta' fraction of Babesia bovis. A MAB, W11C5, selected on the criterion of its staining of erythrocytes infected with B. bovis, was purified. The antigen identified by MAB W11C5 was extracted from B. bovis infected erythrocytes by affinity chromatography and used in a vaccination trial to test its vaccine efficacy against homologous B. bovis infection in splenectomized calves. The vaccinated group showed significantly different parasitaemias from the control group and it was concluded that the B. bovis antigen 11C5 induced a protective immune response when used as a vaccine. This antigen should be synthesized using recombinant DNA techniques to determine its efficacy and suitability as a commercial vaccine against B. bovis infection.     

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.     

Histochemical localization and analysis of blood group-related antigens in human pancreas using immunostaining with monoclonal antibodies and exoglycosidase digestion

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.     

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.     

Stage-specific antigens of Nematospiroides dubius Baylis, 1926 (Nematoda: Heligmosomides)

Antigens were identified from Nematospiroides dubius recovered from outbred Quackenbush mice between 4 and 10 days postinoculation (PI). Parasite surface proteins were radioiodinated and extracts of the whole worms were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and reacted with normal and immune mouse sera followed by an avidin-biotin-peroxidase assay. Antigens ranged between 250,000 and 20,000 molecular weight (MW). A major surface antigen, 60,000 MW, which appeared to be a complex of different antigens, and a 250,000-MW internal antigen were found on fourth-stage (L4) and fifth-stage (L5) larvae 5-10 days PI but not earlier. A group of minor surface antigens (24,000-30,000 MW) were also expressed as larvae molted from L4 to L5, 6 and 7 days PI, but they differed from antigens of similar MW expressed by adult worms. An antigen, 45,000 MW, was detected in worms 5-10 days PI, but it was only expressed on the surface of L5 worms 9 and 10 days PI. We suggest that the antigen(s) common to adults and larvae may account for protective immunity.     

A common high molecular weight antigen of Babesia bovis isolates from Mexico

Cattle from an area of Mexico endemic with Babesia bovis infections have a dominant antibody response to a 152kDa antigen of the Tamaulipas strain of B. bovis. A mAb termed PB/5, showing a specific reactivity to this 152kDa antigen in Western blots, was identified. The mAb which reacted with the blunt end of B. bovis in an indirect fluorescent antibody test also reacted to a 152kDa antigen in two other isolates (Nuevo Leon and Yucatan), and a 175kDa antigen in the Huasteca B. bovis isolate from Mexico. Polyclonal monospecific sera from a calf inoculated with mAb-affinity purified 152kDa antigen (Tamaulipas strain) identified B. bovis by the indirect fluorescent antibody test and two antigens of B. bovis (65kDa and 152kDa) in Western blot. Since the epitope reacting to the mAb PB/5 is conserved, this antigen provides a basis for developing a diagnostic test or an immunogen.     

Moxidectin evaluation against Solenoptes capillatus (Anoplura: Linognathidae), Bovicola bovis (Mallophaga: trichodectidae), and Musca autumnalis (Diptera: Muscidae) on cattle.

Slow release formulations of 375, 750, and 1,125 mg (AI) in 50-g boluses and a subcutaneous injectable formulation (0.2 mg AI/kg body wt) of moxidectin (CL301423) were tested for the control of the little blue cattle louse, Solenoptes capillatus (Enderlein), and the cattle biting louse, Bovicola bovis (L). S. capillatus populations were reduced 4 wk after treatment and complete control was observed 6 wk after treatment in groups treated with boluses, B. bovis were first observed at 3 wk and continued to increase throughout the 14-wk test period. These were experimental boluses and future boluses may perform differently. Subcutaneous injections of moxidectin gave complete control of S. capillatus for a 27-d test period. Feces from animals treated with boluses were tested with face fly larvae, Musca autumnalis De Geer, to demonstrate fecal activity of moxidectin. Larval mortality in these groups ranged from 90 to 30% from 2 d to 10 wk after treatment.