The effect of temperature and humidity on longevity of metacercariae of Fasciola gigantica.

Studies were undertaken on the viability of the metacercariae of Fasciola gigantica when stored in water at 13 degrees C for periods up to 23 weeks, exposed to the sunlight for up to 8 h or stored at a range of temperatures and humidities for up to 10 weeks. Excysted metacercariae were catergorized microscopically as viable (motile and undamaged), dubious (not motile and undamaged) or dead (visible necrosis). The infectivity of viable and dubious metacercariae and unselected reference metacercariae held in water at 7 degrees C for 20 days or longer was assessed by comparing numbers of flukes recovered from infected Merino sheep. Mean recovery rates were 54.6%, 7.2% and 37.2%, respectively, for viable, dubious and unselected metacercariae. Metacercariae immersed in water remained viable longer than those allowed to desiccate. Viability was promoted by decreasing temperature and increasing humidity. Exposure to direct sunlight killed metacercariae within 8 h. Results indicated that in lowland Indonesian irrigated rice paddies, metacercariae immersed in water are likely to survive for less than 5 weeks while those that become desiccated will survive less than 2 weeks. This information, together with the option of exposing fresh rice stalks to direct sunlight before feeding them to livestock, can assist farmers in reducing infection with F gigantica.

Variations in the survival of Fasciola gigantica eggs in bovine dung stored in the sun as opposed to the shade.

Eggs of F. gigantica were placed in dung heaps that were located in the shade or exposed to sun light, and examined at intervals for up to 14 weeks. The rate and extent of decline in viability of eggs was greater in dung exposed to sun light than in shaded dung. This difference was attributed the higher temperature in dung in sun light, owing to the effect of direct sunlight and to a higher rate of fermentation in exposed than in shaded dung. It was concluded that strategies for storing dung that would reduce the risk it poses for infecting L. rubiginosa with F. gigantica when used as fertilizer in rice fields include storing dung in sun light rather than in the shade, preferably in a thin layer to allow sunlight to heat and desiccate it, and mixing a carbohydrate with the stored dung to increase heat through fermentation.

Optimization of the procedure for counting the eggs of Fasciola gigantica in bovine faeces.

This paper describes a method for counting eggs of F. gigantica in bovine faeces that optimizes the proportion of eggs recovered and the repeatability of estimates. The method uses 3 g of faeces suspended in 0.05% Tween 20. The suspension is passed through three 6 cm diameter sieves in tandem to remove fibrous debris, with respective apertures of 1 mm, 450 microm, and either 266 or 200 microm. The filtrate is allowed to sediment for 3 min in a conical flask; the sediment is recovered, then resuspended in 200 ml of 0.05% Tween 20 and allowed to sediment. After 3 min the sediment is washed in a sieve with an aperture of 53 microm, which retains the eggs. Eggs suspended in 15 ml of 1% methylene blue are counted using a dissecting microscope. Use of Tween 20 instead of water as the suspending agent for faeces gave a significant threefold increased the proportion of eggs recovered and reduced variability between repeated counts. This method is able to detect about one-third of the eggs present. It was concluded that the high proportion of F. gigantica eggs lost may be due to the presence of hydrophobic and covalent bonds on the eggs that bind them to debris, with which they are discarded.

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.

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.

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.

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.

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.

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.

The development and validation of an antibody-ELISA to detect Trypanosoma evansi infection in cattle in Australia and Papua New Guinea.

Trypanosoma evansi is exotic to Australia and Papua New Guinea (PNG). However, it might have been introduced to Papua (Indonesia); thus, there is a risk of it entering PNG and thence Australia. Because of logistical difficulties in PNG and northern Australia, surveillance for T. evansi must rely on serological tests. The accuracy of an Ab-ELISA using a detergent extract of T. evansi and three antigen fractions purified from the detergent extract using stepwise precipitation with saturated ammonium sulphate (AS) were compared. The ELISA using the AS 40-50% fraction had greater discriminatory power compared to the ELISA using the other antigen fractions. This ELISA then was compared with two commercial tests: the Card Agglutination Test for trypanosomiasis/T. evansi (CATT) and Suratex. CATT/T. evansi at 1/4 serum dilution has higher sensitivity and the ELISA has higher specificity. There is no likely benefit in combining antibody detection tests to improve the accuracy of diagnosis. Furthermore, the combination of Suratex (which was independent of the antibody tests) with the CATT or the ELISA did not improve the sensitivity. None of the tests was sufficiently sensitive to be used confidently to determine freedom from infection in animals imported into Australia from countries where T. evansi infection is endemic.

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.

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.

The susceptibility of two species of wallaby to infection with Trypanosoma evansi.

OBJECTIVE: To determine the susceptibility of the agile wallaby (Macropus agilis) and the dusky pademelon (Thylogale brunil) to infection with Trypanosoma evansi. METHOD: Two agile wallabies and three dusky pademelons were experimentally infected with between 5 x 10(4) and 10 x 10(4) T evansi from a cryopreserved stabilate isolated from an indonesian buffalo. Animals were observed twice daily for clinical signs and blood was collected every 3 days to determine parasitaemia. Necropsy was conducted on animals that died or were euthanised when in extremis and representative tissue sections examined. RESULTS: All wallabies developed a high parasitaemia by 6 days after infection, which persisted until death or euthanasia in extremis, between days 8 and 61. Clinical signs included anorexia, weakness and ataxia. Anaemia occurred in one wallaby that survived for 61 days. Gross pathological changes varied between animals. They included pericarditis, serous atrophy of fat, splenomegaly, ulcerative gastritis and enteritis. Histological changes were characterised by a mononuclear cell infiltration of the connective tissue of most organs with little cellular destruction. Striking lesions were seen in the choroid, heart, stomach and small intestine. CONCLUSION: Agile wallabies and pademelons are highly susceptible to infection with T evansi. Wallabies, therefore, have the potential to spread T evansi within New Guinea and Australia if infection is introduced. Mortality is likely to be high thereby acting as an indicator of recent introduction. Histological changes seen in wallabies infected with T evansi are diagnostic for infections occurring in Australia and Papua New Guinea.

Helper T cell and antibody responses to infection of CBA mice with Babesia microti.

Helper T cell cytokine and antibody responses were investigated in mice after infection with Babesia microti (King strain). Infection of CBA mice with 106 parasitized erythrocytes resulted in the development of a transitory high parasitaemia which peaked 14 days post infection (DPI), and was resolved at 24 DPI. Th1 responses were activated predominately during the acute phase (6-18 DPI) whereas Th2 responses predominated during the recovery phase (14-28 DPI) as detected by the reverse transcriptase polymerase chain reaction. Increased expression of Th1 cytokines was first detected at 6 DPI (IL-2) and 8 DPI (IFN-gamma) and their peak levels were reached at 12 DPI. After the peak levels were reached, they progressively declined and fell to baseline levels (22 DPI). Increased expression of Th2 cytokines (IL-4 and IL-10) first appeared at 14 DPI, peaked at 20 DPI and Th2 cytokine levels were elevated till the end of the study (28 DPI). Levels of serum IFN-gamma detected by a sandwich ELISA correlated well with IFN-gamma gene expression and were detectable at 8-18 DPI. IgM against B. microti was first detected in serum by ELISA at 4 DPI, and peaked at 10 DPI. The levels of IgM subsequently declined but remained positive at low titre till the end of study. IgG against B. microti was first detected at 8 DPI and peak levels were reached at 24 DPI and remained at that level until the end of study. The results of the present study show that Th1 cytokines predominated in the early inflammatory response and might be involved in control of levels of acute parasitaemia whereas the Th2-associated responses, including expression of IL-4 and IL-10 and the production of parasite-specific IgG, might be the functional means for the reduction and clearance of the parasite from the body. It was concluded that an effective vaccine against Babesia spp. should be designed to induce Th1 responses to maintain the parasitaemia at unfulminating levels and also maintain Th2 responses to clear the parasite from the body.

Surveys in Papua New Guinea to detect the presence of Trypanosoma evansi infection.

OBJECTIVE: To confirm serological evidence that Trypanosoma evansi is present in Papua New Guinea. DESIGN: Three surveys were undertaken in PNG during 1997/1998. Animals were selected for sampling on the basis of convenience. Samples of blood were examined for the presence of T evansi by the haematocrit centrifugation technique (HCT) and mouse inoculation test (MI). Sera were tested in the field using the card agglutination test for trypanosomiasis/T evansi (CATT). Bovine sera were tested at James Cook University using an antibody-detection ELISA (Ab-ELISA). Results from testing bovine sera with the Ab-ELISA and sera from wallabies with the CATT were analysed using FreeCalc to determine the probability that animals in these populations were infected with T evansi. RESULTS: A total of 545 serum samples were collected, during the three surveys of which 39 cattle, two pig and three agile wallaby samples were positive with the CATT. All bovine sera collected were negative when tested with an Ab-ELISA. T evansi was not isolated using the HCT or the MI from any of these animals. CONCLUSION: Based on the Ab-ELISA results it was concluded that T evansi infection was not present in cattle in villages around Balimo at a minimum expected prevalence of 10% (P < 0.05) and, based on the CATT results, that infection was not present in wallabies on the Bula plain at a minimum expected prevalence of 10% (P < 0.1). These results indicate that it is unlikely that T evansi is endemic in PNG.

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.