Shedding of surface-bound antibody by adult Dictyocaulus viviparus.

Dictyocaulus viviparus-infected calves exhibit strong antibody responses to the surface of all stages of the parasite. To examine whether surface-bound antibody is released, fluorescent-labelled antibody was bound to living parasites that were incubated either at 37 degrees C, or in the presence of a metabolic inhibitor at 2 degrees C. The amount of surface-bound antibody was measured by quantitative fluorescence before and after incubation for 24 h. Loss of antibody from the parasite surface was compared in adult worms, sheathed and artificially exsheathed third-stage larvae (L3). Rapid reductions in fluorescence were observed with adult parasites maintained at 37 degrees C but this was inhibited by incubation at 2 degrees C in the presence of sodium azide. In contrast, there was no such loss from the surface of the sheathed or exsheathed L3 maintained at 37 degrees C.

Comparative sequence analysis of the intergenic spacer region of cyathostome species.

The ribosomal DNA intergenic spacer was amplified by the polymerase chain reaction from 16 cyathostome species using primers derived from conserved regions within the flanking 18S and 26S rRNA genes. This generated a 1.5-2.5 kb fragment which was sequenced from five species. The areas covering the 26S and 18S rRNA genes were more than 99% similar among the five species. Furthermore, in all species there existed a highly conserved region of approximately 380 bp at the 3' end of the intergenic spacer. Subsequently, two cyathostome-specific primers were designed to amplify a smaller, more variable region of the intergenic spacer. Eleven further species were amplified using these primers and analysis showed that sequence similarities varied from 40 to 97% between species. The sequence information obtained in this study is being used to develop a PCR-based assay for the differentiation of preparasitic stages of cyathostomes.

Protective immunisation of guinea pigs against Dictyocaulus viviparus using excretory/secretory products of adult parasites.

Parasite preparations were examined for their ability to induce protective immunity against Dictyocaulus viviparus in guinea pigs. Dunkin-Hartley strain guinea pigs were immunised with somatic extracts of adult parasites, somatic extracts of third stage larvae or excretory/secretory (ES) products from adult parasites. The groups were immunised twice with Freund's adjuvant four weeks apart and challenged with 6000 infective L3. Significant levels of protective immunity were observed only in the adult ES-immunised animals. The antibody responses of the different groups were compared following analysis by ELISA and immunoprecipitation. To examine the protective role of antibody, guinea pigs were passively immunised with serum from animals immunised with adult ES products or serum from guinea pigs exposed to experimental D. viviparus infection. Following challenge with infective L3, lung-worm burdens of these groups were significantly lower than in guinea pigs which received normal sera. The results suggest that D. viviparus adult ES products contain protective antigens and that antibody-mediated mechanisms contribute to immune protection.

The immunogenicity of the acetylcholinesterases of the cattle lungworm Dictyocaulus viviparus.

Somatic extracts and excretory/secretory (ES) products of the adult stage of the cattle nematode, Dictyocaulus viviparus, were examined for acetylcholinesterase (AChE) activity. Both were found to contain activity which had an optimum pH of 9.5, however, the adult ES products contained over 200 times more AChE activity per unit protein. Gel electrophoresis and specific enzyme staining revealed 5 migratory isoforms of AChE which were common to adult ES products and adult homogenates. Comparison of L3 with L4 and adult extracts indicated that the AChE were only produced by later developmental stages of this parasite. The antigenicity of D. viviparus AChE was demonstrated by binding to serum IgG from naturally and experimentally infected calves but the enzymes were not recognized by calves vaccinated twice with 400 Gy-irradiated larvae. This is the first report of helminth AChE release by a parasitic nematode in a pulmonary location. The presence of these enzymes in such high amounts in the ES products, along with their immunogenicity, suggests that they might have an important role to play in the immunobiology of D. viviparus in the lungs.

Immunisation of guinea pigs against Dictyocaulus viviparus using adult ES products enriched for acetylcholinesterases.

The adult ES products of Dictyocaulus viviparus are a source of protective antigens against challenge in the guinea pig laboratory model. High levels of acetylcholinesterase (AChE) activity are present in these products and these enzymes are immunogenic in infected cattle. Here, the potential role of these enzymes in protective immunity was investigated using a fraction enriched for AChE to immunise guinea pigs. The antibody response stimulated by immunisation with AChE-enriched ES products and the worm burdens obtained following challenge with infective larvae were compared with those in animals immunised with whole ES products and challenge controls. The AChE-enriched preparation stimulated high levels of enzyme-specific antibody in immunised animals, which was not the case for those which received unfractionaed ES products. Worm burdens of guinea pigs which received the AChE-enriched fraction were significantly lower than those obtained in adjuvant controls. The animals which received the unfractionated ES products were not significantly protected against challenge. These results suggest that AChEs may be potential candidates for incorporation in a sub-unit vaccine against D. viviparus.

Natural history study of pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is a well-characterized autosomal dominant dwarfing condition. A great deal of information is available about orthopedic complications, but little is known about extraskeletal complications in adulthood. This study was undertaken to delineate the natural history of PSACH at all ages. Seventy-nine individuals responded to an extensive questionnaire that included information about deformities, operations, general health, chronic diseases, and reproduction. PSACH individuals were ascertained through the University of Texas Medical Genetics patient population, a genetic linkage study, and the social organization, Little People of America. The results show that PSACH individuals with a family history do not have a distinct or more severe phenotype than new mutation cases. There were not differences in the number of orthopedic complications, operations, or number of offspring between these two groups. Less than half of affected adults reported having total hip replacement surgery, which was less common than previously reported. Extraskeletal complications were generally uncommon. There were four cases of cancers in 41 individuals queried. Premature osteoarthritis was the major health problem for PSACH individuals. PSACH individuals are generally healthy but have problems associated with debilitating osteoarthritis.

A longitudinal study of local and peripheral isotype/subclass antibodies in Dictyocaulus viviparus-infected calves.

Although infection with the bovine lungworm, Dictyocaulus viviparus, stimulates high levels of resistance, the mechanisms involved in immunity to this parasite remain poorly understood. In an attempt to address the possible role of antibody in protective immunity, a longitudinal study was carried out in which the levels of both local and peripheral parasite-specific IgG, IgG1, IgG2, IgM and IgA were measured by ELISA. Five calves were infected orally with ten third stage larvae per kilogram on days 0, 65 and 112. Three challenge controls remained uninfected until day 112. Peripheral responses were measured in serum collected weekly and local responses were measured in bronchoalveolar lavage fluid (BALF) collected pre-infection and on two occasions after each infection. After the secondary infection, there were increases in respiratory rates in all the calves, but four of five calves had no first stage larvae (L1) in their faeces, suggesting that the parasites reached the lungs but did not develop to patency. Respiratory rates remained within normal limits after the tertiary infection and there were no parasites in the lungs at postmortem. Locally, in BALF, levels of all the antibody isotypes/subclasses increased after the primary infection, then again after the secondary infection. The highest levels of antibody were detected after the tertiary infection, when the calves were fully immune. In contrast, serum antibody levels increased from day 21 after primary infection and rose again after secondary infection, but thereafter slowly declined, with no increases after tertiary infection. Our findings suggest that the local antibody response was important in the immune response to D. viviparus infections in calves.

Isotype-specific antibody responses to the surface-exposed antigens of adult and larval stages of Dictyocaulus viviparus in infected and vaccinated calves.

The antibody responses to the surface-exposed antigens of living larval and adult Dictyocaulus viviparus were measured by quantitative immunofluorescence using sera from calves infected with, or vaccinated against, the parasite. In infected animals, the surface of the sheath of the third-stage larvae (L3) (retained cuticle of second-stage larvae (L2)) proved highly immunogenic despite the fact that it is thought to be shed prior to parasite penetration of the host intestine. When responses to the surface of exsheathed larvae (L3 cuticle) were measured, a high level of heterophile IgM antibody was detected in the serum of animals that had not been previously exposed to the parasite and, following infection, a specific IgG response was detected against the exsheathed L3 surface. The antibody response, however, was less marked than that observed against the intact L3 sheath. Responses of patently infected animals to the adult surface showed an initial IgM response that was superseded with time by IgG1 and IgG2 responses. Vaccinated animals showed only low level responses to the surfaces of the L3 sheath, L3 cuticle and adult stages following immunisation with two doses of irradiated larvae. The immunised animals produced a strong antibody response to the larval surface antigens following challenge with infective larvae but they failed to produce antibody to the surface of adult parasites. These results show that the surfaces of all the stages of D. viviparus examined are immunogenic in infected calves and, depending on the developmental stage, infection regime, or time of infection, high levels of parasite-specific IgG1 or IgM are stimulated. It has previously been shown that significant levels of protective immunity can be obtained in naive animals following passive transfer of serum from infected calves. Thus, the antibody responses detected in the work reported here may be of relevance in protective immunity against dictyocaulosis.

Molecular diagnosis of parasitic nematodes.

There is an essential requirement for highly sensitive tools that will differentiate nematode parasites of animals and plants to the species level. For studying host range, genetic variation, virulence and resistance, the availability of well defined populations is vital. Many nematode species cannot be identified with certainty using traditional morphological or morphometric techniques. This is particularly the case for the more accessible developmental stages that, depending on the particular group concerned, live as eggs and larvae in the environment or as micro-filariae that circulate in the blood or inhabit the skin. Morphological identification of these stages requires specialized expertise and is extremely time consuming. Immunological assays have their place in nematode identification but they do not discriminate between current and previous infections, an essential requirement in many epidemiological and prevalence studies. In addition to being highly sensitive, DNA-based methods of detection define present over past infection and are not dependent on the parasite stage. Many types of methodology are available for the detection and definition of nematode DNA. This paper reviews these methods citing examples that have been used with success in the laboratory as well as the field.

Vaccine development and diagnostics of Dictyocaulus viviparus.

Parasitic bronchitis is a serious disease of cattle and is caused by the nematode, Dictyocaulus viviparus. For over 30 years, a radiation-attenuated larval vaccine has been used for prevention of this disease. This vaccine has been used with considerable success in the UK and parts of Western Europe, however, it has several disadvantages. It has a short shelf-life and the vaccine has to be produced annually necessitating the use of donor calves. Following vaccination, calves must receive further boosting from natural challenge to maintain protective immunity. Sales of the irradiated larval vaccine have decreased dramatically since the 1970s. This is thought to be due to increased reliance of farmers on anthelmintic programmes to control lungworm infection. It is possible that, under certain circumstances, these programmes do not allow sufficient parasite exposure to stimulate protective immunity to further Dictyocaulus challenge. This is borne out by the recent documented increase in the number of outbreaks of parasitic bronchitis in the UK. A stable vaccine against D. viviparus that is capable of stimulating a more prolonged immunity would be beneficial. Recent research has been directed at identification and isolation of components thought to be involved in parasite survival in the host and examination of their potential as vaccine candidates. One of these components is acetylcholinesterase (AChE), an enzyme secreted by adult worms. This review describes the development of the secreted AChE as a vaccine candidate, as well as documenting recent developments in the immunodiagnosis of D. viviparus.

Proteinases released in vitro by the parasitic stages of Teladorsagia circumcincta, an ovine abomasal nematode.

Proteinases released during in vitro maintenance of third (L3) and fourth larval stage (L4) and adult Teladorsagia circumcincta (formerly Ostertagia circumcincta), an ovine abomasal nematode parasite, were characterized on the basis of pH optima, molecular size and specific proteinase inhibitor sensitivity. Enzyme activity was maximal at alkaline pH and stage-specific release was demonstrated. Proteinases released by the adult parasite degraded a variety of protein substrates including plasminogen, albumin and haemoglobin, in a pH-dependent manner. At alkaline pH fibrinogen degradation was restricted to the alpha and beta peptide chains although the gamma peptide chain was also degraded at acidic pH. Inhibitor sensitivity studies indicated that degradation was predominantly due to metalloproteinases although aspartyl proteinase activity was indicated at acidic pH.

Heterogeneity in the recognition of Ostertagia circumcincta antigens by serum antibody from mature, infected sheep.

The recognition of parasite molecules from third-stage and adult Ostertagia circumcincta by serum antibody was studied in a group of matched, mature Scottish Blackface sheep that had been naturally and then deliberately infected. A total of 20 molecules was recognized in somatic extracts from third-stage larvae and 31 molecules in somatic extracts from adult parasites. However, no sheep recognized all immunogenic molecules and no molecule was recognized by all sheep. There was no obvious relationship between recognition of any parasite antigen and polymorphism at class I loci or at the DRBI class II locus of the major histocompatibility complex in these outbred animals. Only 15 molecules from third-stage larvae were present at a frequency suitable for statistical analysis and recognition of three of these 15 molecules was associated with differences in worm burdens. Recognition of two of five molecules from adult parasites was associated with differences in worm length. These results indicate that variation in the recognition of specific, identifiable parasite molecules may be partly responsible for variation among sheep in resistance to O. circumcincta.

Genetic control of the antibody repertoire against excretory/secretory products and acetylcholinesterases of Dictyocaulus viviparus.

Outbred Dunkin-Hartley and inbred strain 2 and strain 13 guinea pigs were immunized with Dictyocaulus viviparus adult ES products prior to challenge with third stage larvae. Antibody responses of the three strains to adult ES products and the acetylcholinesterase (AChE) isoforms which they contain were examined. Using immunoprecipitation and ELISA, it was observed that responses in the three strains to adult ES products were distinct: considerable heterogeneity in the antibody repertoire was observed between outbred Dunkin-Hartley animals, with only slight variation occurring amongst the inbred individuals. Responses to the AChE isoforms were heterogeneous amongst individual outbred guinea pigs but were more consistent in inbred strain 2 and 13 animals in which strain-specific patterns of recognition were observed. Previous studies with nematode infections have indicated a role for the major histocompatibility complex in determining the nature and level of the immune response. As the inbred strains bear different alleles at the Class II region but are identical at the Class I region, the differences observed are likely to be due to genes mapping to the Class II locus. This is therefore the first report of genetic restriction of the antibody repertoire to secreted AChEs of a parasitic nematode.

Teratoma with trisomy 16 in a baboon (Papio hamadryas).

A teratoma was found during a planned cesarean section in a 10-year-old primigravida baboon. This teratoma had a female sex chromosome complement and trisomy for chromosome 16. This is the first report of a teratoma in a baboon and the first report of a chromosomal abnormality in a nonhuman primate teratoma. It is also the first case in a nonhuman primate to address the mechanism of origin. Through the use of genetic markers from human chromosomes 5, 8 and 17, the origin of the teratoma was shown to be most consistent with failure of meiosis II or endoreduplication in a mature ovum, while the trisomy for chromosome 16 originated after the formation of the tumor.

A baboon (Papio hamadryas) with an isochromosome for the long arm of the X.

A 5.5-yr-old female baboon was evaluated for sexual immaturity. She was small for her age and had normal external female genitalia. However, she lacked cyclical perineal turgescence and displayed atypical coloration of the perineal skin. Laparoscopy revealed a small uterus and absence of both ovaries. Cytogenetic analysis revealed a 42,X,i(X)(q10) karyotype. DNA analysis using loci DXS1683, which maps to Xp22.1, and DXS297, which maps to Xq27.3, was consistent with inheritance of the normal X chromosome from the dam and formation of the isochromosome Xq from the paternal X.