Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates.

Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.

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.

Vaccination against the Old World screwworm fly (Chrysomya bezziana).

Chrysomya bezziana is an endemic pest of livestock or a threat to livestock production in large areas of Africa, the Middle East, southern and south-east Asia and Australia. Its control is difficult. The feasibility of vaccinating against this pest has now been explored. In-vitro and in-vivo assays have been established. Using these assays, it has been shown that first instar larvae, third instar peritrophic membrane and cardia are all sources of material able to induce immunological reactions in sheep which lead to significant reductions in larval growth. In-vitro assays following vaccination with peritrophic membrane also show larval mortality. Taken together, these effects lead to an 82% reduction in the weight of recovered larvae in vitro and 45% reduction in vivo. Preliminary evidence suggests that the mechanism of protection may be complex.

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana.

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.

The occurrence of Trypanosoma evansi in buffaloes in Indonesia, estimated using various diagnostic tests.

The prevalence and incidence of Trypanosoma evansi infections in village buffaloes in Central Java were estimated using parasitological tests, two antigen-detection ELISAs (2G6 Ag-ELISA and Tr7 Ag-ELISA), an antibody-detection ELISA (IgG ELISA) and a card agglutination test (CATT). Of 2387 village buffaloes tested in five districts, 4 % (95 % confidence interval [CI]: 3 %, 5 %) were positive with the microhaematocrit test (MHCT), 58 % (95 % CI: 56 %, 60 %) were positive with the 2G6 Ag-ELISA and 70 % (95 % CI: 68 %, 72 %) were positive with the Tr7 Ag-ELISA. An increasing prevalence with age was found and the proportion of positive buffaloes was highest in the over 84 months-old age-group (68 %) with the 2G6 Ag-ELISA and in the 37-60 months-old age-group (78 %) with the Tr7 Ag-ELISA. Parasitaemic buffaloes were found in more than half of the villages visited. Corrected village-specific prevalence values obtained with the two Ag-ELISAs ranged from 0% to over 100%, and prevalence differed significantly (P < or = 0.0001) between villages in four of the five districts. Overall, 10% of buffaloes tested in markets were found to be parasitaemic and 39, 56 and 47 % were found positive with the 2G6 Ag-ELISA, IgG ELISA and CATT, respectively. Incidence rates varied according to the test used and ranged from 0.22 (95 % CI: 0.09, 0.44) to 0.44 (95 % CI: 0.24, 0.76), per animal-year at risk, in two villages. The results highlight the importance of using validated diagnostic tests to obtain accurate estimates of prevalence and incidence. These parameters are needed, for example in mathematical models, for the development and evaluation of different control strategies for T. evansi infections in buffaloes.

Evaluation of antigen detection and antibody detection tests for Trypanosoma evansi infections of buffaloes in Indonesia.

Two Ag-ELISAs, an IgG-specific antibody detection ELISA (IgG ELISA) and a card agglutination test (CATT) for the detection of Trypanasoma evansi infections in buffaloes in Indonesia, were compared. Diagnostic sensitivity estimates were obtained by testing sera from 139 Indonesian buffaloes which had been found to be infected by parasitological tests. Diagnostic specificity was estimated by testing sera from 263 buffaloes living in Australia. Response-operating characteristic curves were constructed, and optimal ELISA cut-off values, which minimized the number of false-negative and false-positive results, were chosen. The IgG ELISA had the highest sensitivity (89%) and the CATT had the highest specificity (100%). There was a significant difference between the sensitivities (71 and 81%), but not between the specificities (75 and 78%), of the two Ag-ELISAs. The four tests were further compared by calculation of post-test probabilities of infection for positive and negative test results using a range of prevalence values, and likelihood ratios. The results suggested that the CATT was the best test to 'rule-in' infection (i.e. the highest probability of infection in test-positive animals) and the IgG ELISA was the best test to 'rule-out' infection (i.e. the lowest probability of infection in test-negative animals).

Immune responses in Indonesian thin tail and Merino sheep during a primary infection with Fasciola gigantica: lack of a specific IgG2 antibody response is associated with increased resistance to infection in Indonesian sheep.

After a primary infection with Fasciola gigantica, the immune responses in a resistant (Indonesian thin tail) and a susceptible (Merino) breed of sheep were analysed. The number of adult flukes recovered from the livers of the Indonesian thin tail sheep were significantly lower than those found in the Merino animals. On days 8, 14 and 25 p.i., Indonesian thin tail sheep exhibited a significantly higher eosinophilia than Merino sheep, whereas neutrophilia was significantly elevated in the Indonesian thin tail sheep on days 36 and 48 p.i. Serum from both sheep breeds demonstrated IgM, IgG1 and IgE responses to F. gigantica. In contrast, the Indonesian thin tail sheep produced significantly lower levels of IgG2 antibodies relative to the high level detected in Merino sheep. The IgE response was biphasic in both sheep breeds with the first response detected by day 14 and the second response developing from days 30 to 60 p.i. Western blotting showed that a similar profile of adult fluke antigens was recognised by IgG1 and IgE antibodies in both the Indonesian thin tail and Merino sheep. The IgE response was directed to a major antigen at about 92 kDa. We postulate that IgG2 could act as a blocking antibody for protective effector responses against F. gigantica in sheep and that the Indonesian thin tail sheep, by downregulating IgG2 responses, have an enhanced capacity for killing F. gigantica in vivo.

Resistance of Indonesian thin tail sheep against Fasciola gigantica and F. hepatica.

High resistance of Indonesian thin tail (ITT) sheep against Fasciola gigantica has been confirmed. Naive ITT sheep had only 17% of the number of mature parasites collected from control St. Croix sheep. In contrast, the level of resistance of ITT sheep against F. hepatica was the same as that of the low resistance Merino breed after both primary and secondary infections. It is suggested that resistance of ITT sheep against F. gigantica was manifested in two phases. The major phase appeared to be specific to the ITT sheep : F. gigantica relationship, acting against immature parasites in both naive and previously exposed hosts and could be innate or acquired. The second phase appeared to be specific to F. gigantica, and was acquired, because it killed young adult parasites only after secondary infection. F. gigantica from ITT sheep were heavier than those from the St. Croix sheep, possible because feeding was impaired by more extensive liver damage in the latter. Approximately 45 F. gigantica killed 25-kg sheep before and during migration from the liver parenchyma into the bile ducts. Death was caused by haemorrhages into the liver parenchyma, bile ducts and peritoneum.

Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle.

Vaccine trials were conducted in Brahman cross cattle evaluating the efficacy of 4 native antigens purified from adult Fasciola gigantica flukes, and 1 recombinant F. gigantica antigen, as vaccines against tropical fasciolosis. The antigens tested were native glutathione S-transferase, cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli, and were formulated in 1 or more of several adjuvants (Quil A, Squalene Montanide 80, MF59-100, Auspharm, NAGO, polylactoglycolide microspheres, Algammulin, DEAE, Freund's). Vaccination induced low, moderate or high antibody titres to the various antigens which were dependent on the adjuvant. Low but significant reductions in fluke burdens (31%, P < 0.026) and fluke wet weight (36%, P < 0.041) were only observed in cattle vaccinated with the native FABP in Freund's adjuvant. There was no correlation between total antibody titres to FABP and protection. The protection observed in cattle vaccinated with native FABP of F. gigantica supports the notion that this class of proteins is a useful target for protection of animals against Fasciola and extends the efficacy of FABPs to the tropical liver fluke. This is the first report of vaccination of cattle against F. gigantica with a purified protein.

Efficacy of Cymelarsan in Friesian Holstein calves infected with Trypanosoma evansi.

Two studies on the efficacy of mel Cy (Cymelarsan, Rhone Merieux, France) for the treatment of cattle infected with Trypanosoma evansi were carried out with groups of 5 Friesian Holstein calves infected with an Indonesian stock of T. evansi and treated 14 days after infection. In the first study 3 groups were injected subcutaneously with Cymelarsan at dose rates of 0.125, 0.25 and 0.50 mg/kg and in the second study 2 i/m at 0.50 and 0.75 mg/kg. The response to treatment was monitored parasitologically by daily microhaematocrit centrifugation technique and weekly mouse inoculation. Enzyme linked immunosorbent assays were used to monitor trypanosomal antibodies and trypanosomal antigens in serum samples collected weekly. Relapse infections occurred in all the groups given the drug s/c whilst all the animals treated i/m remained parasitologically negative up to 80 days after treatment. Results from serological assays, however, suggested the possible persistence of trypanosome infection in the animals treated at a dose rate of 0.50 mg/kg i/m although trypanosomes could not be demonstrated parasitologically. A dose rate of 0.75 mg/kg administered i/m is recommended, therefore, for the treatment of T. evansi infection in Friesian Holstein cattle.

Experimental infection of Friesian Holstein calves with an Indonesian isolate of Trypanosoma evansi.

The effect of a single, primary, artificial infection with Trypanosoma evansi was studied in nine Friesian Holstein calves. Body temperature, packed cell volume (PCV) and parasitaemia measurements were obtained daily from each of the infected calves for up to 90 days after parasites were injected, body weights were monitored weekly. T. evansi infection had a marked depressive effect on PCV profiles and the rate of body weight gain.

Trypanosoma evansi infection in worked and unworked buffaloes (Bubalus bubalis) in Indonesia.

The effect of controlled amounts of exercise on the outcome of Trypanosoma evansi infection was studied in groups of swamp buffaloes (Bubalus bubalis) experimentally infected with T. evansi. Daily body temperature, packed cell volume (PCV) and parasitaemia measurements were obtained from each animal for up to 110 days after infection. Exercise did not appear to exacerbate the effect of T. evansi infection in that similar temperature, PCV and parasitaemia profiles were obtained with both exercised and rested animals. Trypanosoma evansi infection, however, had a marked effect on temperature and PCV profiles, both of which could adversely affect an infected animal's work output and work tolerance.

Trypanosoma evansi infection in cattle, buffaloes and horses in Indonesia.

Cattle, buffaloes and horses in several areas of Indonesia were examined for evidence of infection with Trypanosoma evansi by the microhaematocrit centrifugation technique (MHCT) and an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to T. evansi. Evidence of infection was found in animals at each sampling site although differences were seen in prevalence rates between sites. Prevalence rates in buffalo were usually higher than in cattle in the same area while in horses they were much lower than in cattle or buffalo. An age-dependent prevalence rate was seen in buffalo and cattle with the highest rates seen in animals older than 2 years. These results concur with the view that T. evansi infection is widespread throughout most of the livestock-producing areas of Indonesia. The apparent lack of any obvious disease owing to T. evansi infection in the sampled animals suggests that a form of stability exists in most endemic areas which serves to ameliorate the effect of T. evansi infection and has an immunological basis linked to the parasite's limited antigenic diversity.