Haemonchus contortus and Trichostrongylus colubriformis in pen-trials with Javanese thin tail sheep and Kacang cross Etawah goats.

Weight gain costs due to infection were higher in sheep than goats, 28 and 17.5%, respectively, for Trichostrongylus colubriformis and 48.7 and 32.2%, respectively, for Haemonchus contortus. The extent of bodyweight cost attributed to anorexia in sheep infected with H. contortus was higher (13.5 g/day) than in sheep infected with T. colubriformis (2.3 g/day). On the other hand, bodyweight cost due to the other pathogenic effects in sheep infected with T. colubriformis were higher (35.6 g/day) compared to sheep infected with H. contortus (10.9 g/day). A strong relationship between faecal egg count and worm count (r=0.79, P=0.006) was shown only in sheep infected with T. colubriformis. About half of the infected sheep and goats had low or zero faecal egg counts throughout the study. In about 40% the egg count rose initially but became low by weeks 10-16, whereas in about 10% counts increased progressively throughout the period of observation and these animals also had the highest numbers of worms at slaughter. Packed cell volume was reduced in sheep and goats infected with H. contortus but serum protein and haemoglobin levels were unaffected. Sheep infected with T. colubriformis had a higher level of eosinophilia after 8 weeks (18.4%) than sheep infected with H. contortus (11.4%), whereas this pattern was reversed in goats and levels were also lower (4.1 and 8.9%, respectively). There was no apparent relationship between eosinophilia and resistance to infection with H. contortus or T. colubriformis.

Increase in cellular immune responses in Onchocerca-infected cattle after treatment with the microfilaricide, milbemycin.

Lymphocytes from Onchocerca-infected steers treated with the microfilaricide, milbemycin showed increased proliferation when challenged with antigen from Dirofilaria immitis, concanavalin A, tuberculin and tetanus toxoid, compared with untreated animals. This paper confirms that Onchocerca infection induces immunosuppression to filarial and non-filarial antigens. It raises the possibility that filarial-induced immunosuppression may increase the susceptibility to mycobacterial infections and reduce the efficacy of vaccinations and strongly indicates that further research is required.

Biological control of Fasciola gigantica with Echinostoma revolutum.

A field trial was carried out in West Java to investigate the potential for control of fasciolosis of antagonism between larvae of Fasciola gigantica and Echinostoma revolutum in Lymnaea rubiginosa. The trial was undertaken in 26 farmers' irrigated rice fields, each chosen because it was adjacent to a cattle pen the effluent from which flowed into or was used as fertiliser in the rice field. Fourteen of the fields chosen at random were retained as controls and received no treatment while in 12, faeces from 5 to 15 ducks containing eggs of E. revolutum were introduced to the rice from a duck pen located over the effluent drain from the cattle pen before it emptied into the adjacent rice field, or at the site bovine faeces was added to the field as fertiliser. After harvest significantly fewer L. rubiginosa were found infected with F. gigantica in fields where duck and cattle dung entered the field together than in control fields, supporting a conclusion that this method of biological control would reduce the infectivity of rice fields fertilised with bovine dung (which are those with the highest potential for being a source of infection with F. gigantica). Positive features of using dung from ducks infected with E. revolutum to control F. gigantica are the minimum additional work and disruption to existing farming practices required to implement the scheme, the common natural infection with E. revolutum in village ducks, and effectiveness of dung from 5 to 15 ducks, a number commonly kept by farmers.

Echinococcus granulosus in northern Queensland. 2. Ecological determinants of infection in beef cattle.

OBJECTIVE: To provide information on possible ecological determinants of infection with Echinococcus granulosus in a beef pastoral area of northern Queensland. PROCEDURE: An ecological study was carried out on the prevalence and viability of infection with Echinococcus granulosus in definitive and potential intermediate hosts, and their predator prey relationships. Seven adjacent extensive beef properties 100 km south of Townsville, that included areas of savannah, open woodland and dense closed scrub, were selected for the study. Infection with E granulosus in dingoes was determined at post mortem, and in domestic dogs by examining duodenal mucus after purging with arecoline hydrobromide. Cattle, wild pigs and macropods were examined at post mortem for viable hydatid cysts. The diet of dingoes was investigated by identifying the hair of prey species found in their stomach and colon, and that of domestic dogs by questioning their owners. RESULTS: Prevalence of hydatidosis in adult cattle ranged from 41% in animals from properties with large areas of dense closed scrub, to 3% on properties with little or no scrub. Hydatid cysts were found in 21.8% of black-striped wallabies (Macropus dorsalis), 9.4% of feral pigs, 1.5% of wallaroos (Macropus robustus), and 1.4% of eastern grey kangaroos (Macropus giganteus). No rufous rat kangaroos (Aepyprymnus rufescens) or swamp wallabies (Wallabia bicolor) were infected. Most cysts in macropods were viable, whereas in pigs about half were viable and in cattle only 0.7% contained viable protoscoleces. Infection with E granulosus was detected in 76% of dingoes, whereas no infection was detected in domestic dogs in the study area. CONCLUSIONS: It was concluded that the sylvatic cycle of E granulosus in the study area was maintained mainly through predation of black-striped wallabies by dingoes, and that the verges of dense scrub were the main nidus of infection.

Echinococcus granulosus in northern Queensland. 1. Prevalence in cattle.

OBJECTIVE: To determine the prevalence and geographical distribution of hydatidosis and investigate factors that might be expected to influence the prevalence of hydatids in cattle in Queensland north of the Tropic of Capricorn. To determine the effect of natural levels of infection on carcase weight and subsequent economic loss. PROCEDURE: An abattoir survey conducted in 1981 provided information on the distribution, prevalence and viability of hydatid cysts in cattle from all shires north of the Tropic of Capricorn in Queensland. Livers, lungs and spleens from 10,382 cattle were palpated at abattoirs in Cairns, Townsville and Rockhampton to detect hydatid cysts. Prevalence of infection in cattle in each shire was estimated from results of the abattoir study together with reports of infection in a further 22,185 cattle obtained from abattoir records. Linear modelling was used to define the effect of geographical origin, age, breed and sex on prevalence of infection. Differences in the weights of carcases between infected and non-infected cattle of the same age, sex, breed and property of origin were examined. The economic loss to the beef industry in the region surveyed was estimated. RESULTS: Cattle infected with hydatids originated almost entirely from regions to the east of the Great Dividing Range. The mean prevalence inside this zone was 28% compared with 3% in other areas. Viable protoscoleces were found in 0.7% of cysts. Geographical origin and age of the cattle were the most significant factors influencing prevalence. Infection with hydatids had no effect on carcase weight. Economic loss was limited to that associated with condemnations of organs at meat inspection, estimated to be 0.5 million dollars per annum in 1981 and 6 million dollars in 2004. The distribution of hydatids in Queensland north of the Tropic of Capricorn corresponded most closely with the distribution of small wallabies such as Macropus dorsalis (black-striped wallaby), M parryi (whiptail wallaby) and M rufogriseus (red-necked wallaby). CONCLUSIONS: It was concluded that cattle are not an important part of maintaining the life-cycle of E granulosus in Queensland north of the Tropic of Capricorn. Within the endemic zone, which is almost all to the east of the Great Dividing Range, the local pattern of bovine echinococcosis is most likely to be determined by the presence or absence of small species of wallaby such as M dorsalis, M parryi and M rufogriseus.

Experimental Trypanosoma evansi infection in the goat. I. Clinical signs and clinical pathology.

A strain of Trypanosoma evansi isolated from an equine case of surra in Mindanao, Philippines was used to infect intravenously two groups (A and B) of five male goats aged 8-10 months. Animals of groups A and B received 5000 and 50 000 trypanosomes, respectively, and five further animals (group C) served as uninfected controls. Four of the 10 infected goats died 8-78 days after inoculation. Group C goats gained weight (mean 22.8 g/day) while infected goats in groups A and B lost weight (means of 21.4 and 45.0 g/day, respectively). Parasitaemia fluctuated regularly between peaks and troughs, with repeated periods of about 6 days during which no trypanosomes were detected in the blood. Clinical signs and clinico-pathological changes in infected goats were not pathognomonic in the absence of parasites in the blood, and leucocytosis was not a reliable indicator of infection. It was concluded that in endemic areas fluctuating fever, progressive emaciation, anaemia, coughing, testicular enlargement and diarrhoea are suggestive of surra; confirmation, however, may necessitate examination of blood every few days for trypanosomes, and possibly other diagnostic tests.

Experimental Trypanosoma evansi infection in the goat. II. Pathology.

Infection of male goats aged 8-10 months with 5000 or 50 000 organisms of a Mindanao strain of Trypanosoma evansi was observed over a period of 90 days. The infection induced clinical disease which was lethal, especially at the higher dose rate. Lesions were more acute in goats that received the higher dose. Gross and microscopical changes were not pathognomonic, except in the presence of demonstrable trypanosomes. At necropsy, a combination of lymphadenopathy, splenomegaly, hepatomegaly, testicular enlargement, anaemic signs and consolidation of the anterior lobes of the lungs was suggestive of surra. Testicular changes, especially aspermia, indicated probable infertility. The cytopathology of the lungs, liver, intestine, kidneys, testes, bone marrow, brain and other organs was immunological in nature, characterized by mononuclear infiltration of interstitial tissues, with minor cellular damage and the presence of trypanosomes. B- and T- cell responses were observed in the lymphatic system, but the findings indicated immunosuppression in the lymph nodes, spleen and bone marrow during the third month after infection. Exudative inflammatory changes were mild. It is suggested that the cytopathology of most haemophilic trypanosomal infections is predominantly an immunological process.

Differences in the surface radioiodinated proteins of skin and uterine microfilariae of Onchocerca gibsoni.

Surface labeling studies using two populations of Onchocerca gibsoni microfilariae revealed important differences in major radioiodinated proteins. Small numbers of microfilariae harvested from the skin of cattle or the uteri of adult worms from skin nodules were purified, radioiodinated, solubilized and the proteins analysed by two dimensional gel electrophoresis and autoradiography. As reported previously, uterine microfilariae showed a complex profile of radioiodinated proteins, none of which appeared to be bovine albumin or immunoglobulin. In contrast, application of the same techniques to skin microfilariae demonstrated only one major labeled protein complex of approximate Mr 67 000. This protein complex was immunoprecipitated with an antiserum to bovine serum albumin. Surprisingly, fluorescence techniques failed to show bovine serum albumin on the surface of living microfilariae. Although the evidence is circumstantial at present, acquisition of host albumin (perhaps oriented in a particular way) may be a means whereby skin microfilariae evade immune effector mechanisms and, when living, generally fail to elicit inflammatory reactions in the skin of the host.

Identification and characterisation of a novel repetitive antigen from Onchocerca spp.

A novel repetitive antigen from the cattle parasite Onchocerca gibsoni was shown to be recognised by sera from humans infected with Onchocerca volvulus, Wuchereria bancroftii or Brugia malayi. The O. gibsoni protein was produced in a recombinant form, and antibodies raised to this protein used to screen cDNA libraries for O. volvulus. A series of clones were isolated which encoded repetitive regions very similar to those in O. gibsoni, but interspersed between these were longer repeating units which we have not so far found in O. gibsoni. The repetitive antigen was shown to be of high molecular weight and present only in the insoluble (membrane) fraction of O. gibsoni microfilariae. Immunofluorescence techniques demonstrated that the antigen was associated both with muscle and with specific membrane layers, including a peripheral layer which corresponds to either the outer hypodermis or an inner region of the cuticle in adult female O. gibsoni. In many respects, the proteins encoded by the O. gibsoni and O. volvulus cDNA clones resembled repetitive antigens from several distantly related eukaryotic parasites, and a possible common role in immune evasion is discussed.

Do excretory-secretory products of Onchocerca gibsoni contain phosphorylcholine attached to O-type glycans?

Excretory-secretory products (ES) of adult male Onchocerca gibsoni contain phosphorylcholine (PC). PC-ES are detected as a smear of M(r) approximately 60- approximately 200 kDa by western blotting employing a monoclonal antibody (mAb) against PC, suggesting that they are glycoproteins. Exposure of PC-ES to N-glycosidase F results in weak and inconsistent loss of binding of the mAb, indicating that unlike the situation with respect to ES of Acanthocheilonema viteae, PC is highly unlikely to be solely attached to N-type glycans. Conversely, treatment of O. gibsoni PC-ES with mild alkali, a strategy for removing O-type glycans, abolishes mAb binding. These results suggest that PC may be attached to O. gibsoni proteins mainly via O-type glycans, and raise the possibility that filarial parasites may vary with respect to their mode of attachment of PC. The implications of this with respect to the design of inhibitors of PC attachment for use as anti-filarial drugs, are discussed.

Biosynthetic radiolabelling of excretions-secretions of adult male Onchocerca gibsoni.

Maintenance of adult male worms of the bovine filarial parasite Onchocerca gibsoni in vitro, in the presence of radioactive precursors, resulted in the time-dependent excretion-secretion of radiolabelled parasite macromolecules (ES). Molecules labelled with amino acids ([35S] methionine, [3H] leucine) covered a wide range of molecular weights, whereas labelling with [3H] glucosamine produced predominantly molecules of high molecular weight. Many of the products were recognized by antibodies in two serum pools from humans infected with Onchocerca volvulus. O. gibsoni ES may therefore provide a substitute source of material for studies on the ES of the less readily available human parasite.

Surface antigens of male worms and microfilariae of Onchocerca gibsoni.

Living adult males and microfilariae of the cattle filarial parasite Onchocerca gibsoni were externally labelled with radioactive iodine using the iodogen and Bolton-Hunter procedures. Characterization of labelled surface proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed clear cut differences in the two life cycle stages. In addition, the two radiolabelling procedures yielded some differences in the profiles of radiolabelled surface proteins for both adults and microfilariae. Immunoprecipitation analysis revealed a number of labelled antigens recognized by antibodies in human onchocerciasis serum pools, thereby demonstrating the usefulness of O. gibsoni as a model in Onchocerca volvulus vaccine studies. The reactivity of microfilarial antigens extended to antibodies from other human nematode infections, whereas male surface antigens, particularly those of low molecular weight, were Onchocerca specific. This indicates that O. gibsoni can provide a convenient source of specific diagnostic antigen.

Molecular variation in Onchocerca spp.

Over the past two decades there has been an upsurge of interest in defining morphological, immunological, biochemical, biological and genetic differences between species of Onchocerca to provide solutions to practical problems associated with finding models and epidemiological tools to assist with control of human onchocerciasis. The information gathered has confirmed the close relationship between species of Onchocerca and provided highly sensitive and specific probes to distinguish species and even strains of the same species. It has also identified pathways, especially using sequences from common DNA repeat units, that may lead to a better understanding of the progression of divergence of species of this genus than has previously been possible.

The major radioiodinated cuticular antigens of Onchocerca gibsoni microfilariae are neither species nor onchocerca specific.

The possible role of microfilarial surface (cuticular) antigens in immuno-diagnosis of human filarial infections has been assessed using microfilariae (mf) of the cattle parasite Onchocerca gibsoni. A Triton X-100 extract of 125I-labeled O. gibsoni mf was reacted with a panel of sera from humans infected with Onchocerca volvulus, Wuchereria bancrofti and Schistosoma japonicum as well as sera from uninfected controls. Results of these immunoprecipitations indicated that sera from humans infected with O. volvulus or W bancrofti contain antibody specificities recognising certain of the radioiodinated cuticular proteins of O. gibsoni mf. Two-dimensional gel analysis and subsequent autoradiography of these immunoprecipitates showed that 8 radioiodinated proteins recognised by sera from calves injected with O. gibsoni mf were also immunoprecipitated by sera from humans infected with either O. volvulus or W. bancrofti. Thus there appear to be no major radioiodinated cuticular antigens of O. gibsoni mf which are species or onchocerca specific.

Identification of radioiodinated cuticular proteins and antigens of Onchocerca gibsoni microfilariae.

The filarial parasite of cattle, Onchocerca gibsoni, has been used to establish procedures of antigen identification with a view of applying these techniques to studies on human filarial parasites. Emphasis has been placed on methods suitable for use with small numbers of parasites. Microfilariae (mf) of O. gibsoni were extracted from nodular worms, purified and 125I-labeled using IODO-GEN in solid-phase. Radioactivity was shown to be confined to the cuticle of sectioned mf using the technique of electronmicroscope autoradiography. Radiolabeled mf were analysed by two-dimensional gel electrophoresis. Autoradiographs of 125I-labeled proteins of O. gibsoni mf were relatively complex, there being at least 32 proteins ranging in molecular weights from 20,000 to 120,000 and displaying considerable charge heterogeneity. Evidence was obtained that at least the major serum proteins of the host, albumin or immunoglobulin, were not absorbed on the surface of these uterine mf and detectable in the labeled surface protein patterns. Sera from infected cattle immunoprecipitated 5 labeled proteins from a Triton X-100 extract of 125I-labeled mf. Sera from either of two calves which had been given multiple injections of mf subcutaneously, and which had no detectable skin mf, recognised 6 additional proteins in this extract as well as 3 of the proteins recognised by sera from infected cattle.

Inhibition of growth of cultured Babesia microti by serum and macrophages in the presence or absence of T cells.

Serum and macrophages from the acute-phase (days 12-14 p.i.) and recovery-phase (days 23-25 p.i.) of infection of mice with Babesia microti were analyzed for their ability to inhibit the in vitro growth of B. microti in the presence or absence of T cells. Recovery-phase serum was inhibitory to the growth of B. microti, whereas, acute-phase serum had no inhibitory effects. Both acute- and recovery-phase macrophages inhibited B. microti growth. The co-culture of acute- but not recovery-phase T cells with macrophages from uninfected control mice was inhibitory to the growth of B. microti. Growth of B. microti was also inhibited in cultures containing macrophages from uninfected control mice plus culture supernatant fluid from acute-phase but not recovery-phase T cells. The supernatant fluid from B. microti cultures with acute-phase T cells contained IFN-gamma detected by a sandwich ELISA, whereas cultures with control T cells or recovery phase T cells did not. Results of the present study suggest the likelihood of a protective role against B. microti in mice for antibody which appeared in recovery-phase serum and for macrophages activated by IFN-gamma from acute-phase T cells.

Evaluation of an antibody-ELISA using five crude antigen preparations for the diagnosis of Trypanosoma evansi infection in cattle.

Attempts were made to improve the accuracy of an antibody-detection ELISA for the detection of Trypanosoma evansi infection in cattle by improving the method of preparation of the crude antigen used. An IgG-ELISA was performed with five different antigen preparations: crude soluble antigen, soluble and insoluble fractions of crude antigen treated with 0.1% formalin and whole formalin-fixed trypanosomes treated with either trypsin or 2-mercaptoethanol. An IgM-ELISA using crude soluble antigen was also performed. Each ELISA was evaluated using serum from 44 Indonesian cattle infected with T. evansi and 262 uninfected cattle from Australia. There was no significant difference between the sensitivity or specificity of the IgG-ELISA using each of the five antigens. The IgM-ELISA using a crude untreated lysate was significantly less sensitive (p<0.05) than the IgG-ELISA using the same antigen, trypsin-treated antigen or the 0.1% formalin-treated soluble antigen (68, 64 and 64%, respectively). These results show that these modifications to the method of producing crude antigens for the Ab-ELISA does not improve the accuracy of diagnosis of T. evansi infection in cattle.

High resistance to experimental infection with Fasciola gigantica in Javanese thin-tailed sheep.

Innate resistance of Javanese thin-tailed sheep to Fasciola gigantica was investigated in animals infected with single doses of 150 or 500 metacercariae and killed 4, 8, 12 or 16 weeks after infection. Infected and non-infected sheep had similar values for packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin concentration, serum glutamate dehydrogenase, serum gamma glutamyl transferase and serum aspartate transferase throughout the trial, except for one animal infected with 500 metacercariae from which the highest recovery of flukes (55) was made. This animal developed pathologically altered values from 12 weeks post infection, coincident with the period of greatest hepatic haemorrhage and destruction of hepatic tissue by migrating flukes and their entry into bile ducts. However, values were altered much less than those reported in other sheep given as few as 200 metacercaria of F. gigantica. Both susceptibility to infection with F. gigantica, as indicated by percentage take of metacercariae and the severity of pathological changes were low in this study in comparison with reports involving other breeds of sheep infected with this parasite. These findings support the conclusion that Javanese thin-tailed sheep have a high innate resistance to F. gigantica.

A geographic information systems model for mapping risk of fasciolosis in cattle and buffaloes in Cambodia.

A geographic information systems (GIS) model for mapping the risk of fasciolosis in cattle and buffaloes was developed for the Kingdom of Cambodia using determinants of inundation, proximity to rivers, land use, slope, elevation, and the density of cattle and buffaloes. Determinants were subjectively weighted according to their perceived relative importance before combining them to produce a risk-map of fasciolosis. The model estimates that 28% of Cambodia is potentially at risk of fasciolosis with areas of high and moderate risk concentrated in southern and central Cambodia. The estimates of risk reflect the actual prevalence of fasciolosis in most districts surveyed, suggesting that the epidemiological determinants and weightings used to produce the model were appropriate. These results will be progressively refined as more detailed field surveys are completed to fully validate the model.

Evaluation and improvement of parasitological tests for Trypanosoma evansi infection.

Research was undertaken to critically evaluate parasitological tests for the detection of Trypanosoma evansi in blood. The relative sensitivity of mouse inoculation (MI), the haematocrit centrifugation technique (HCT) and a modified miniature anion-exchange centrifugation technique (MAECT) were compared using blood and buffy coat. The effect that storage of blood prior to inoculation into mice has on the reliability of the MI test was also evaluated. The tests may be ranked in increasing order of sensitivity: HCT, MAECT with whole blood, MI with whole blood, MAECT with buffy coat and MI with buffy coat. The latter was able to detect 1.25 T. evansi per 4ml of blood. The reliability of the MI test was not reduced with storage of blood containing at least 25 T. evansi per ml for up to 21h prior to inoculation into mice. These results demonstrate that sensitivity of the MI and MAECT are increased approximately 10-fold through the use of buffy coat in place of whole blood. Although, the MI is marginally more sensitive MAECT is better suited to field use.