Innate and adaptive resistance of Indonesian Thin Tail sheep to liver fluke: a comparative analysis of Fasciola gigantica and Fasciola hepatica infection.

In the current study, three independent trials directly compared Fasciola gigantica and Fasciola hepatica infection of ITT sheep. In all trials, F. hepatica infection resulted in higher worm burden recoveries and greater physiological damage to ITT sheep. Developmental differences of the two Fasciola species were also observed during the first twelve weeks of a primary infection, where the migration and growth of F. hepatica was more rapid than F. gigantica. Various immunological blood parameters were measured and indicated similar kinetics in the humoral and cellular responses during the time course of infection with each Fasciola species. In contrast to F. hepatica infection, we demonstrate an innate and adaptive comparative ability of ITT sheep to resist the early stages of infection with F. gigantica infection. Unraveling the mechanisms leading to this differential resistance may potentially lead to new methods for the control of fasciolosis and other human liver flukes.

International survey of Cronobacter sakazakii and other Cronobacter spp. in follow up formulas and infant foods.

A coordinated survey for Cronobacter and related organisms in powdered infant formula, follow up formula and infant foods was undertaken by 8 laboratories in 7 countries in recognition of and in response to the data needs identified in an FAO/WHO call for data in order to develop global risk management guidance for these products. The products (domestic and imported) were purchased from the local market and were categorised according to their principle ingredients. A total of 290 products were analysed using a standardised procedure of pre-enrichment in 225 ml Buffered Peptone Water (BPW), followed by enrichment in Enterobacteriaceae Enrichment (EE) broth, plating on the chromogenic Cronobacter Druggan-Forsythe-Iversen (DFI) agar and presumptive identification with ID 32 E. Presumptive Cronobacter isolates were identified using 16S rRNA gene sequence analysis. Aerobic plate counts (APC) of the products were also determined on nutrient agar. Fourteen samples had APC>10(5) cfu/g, 3 of which contained probiotic cultures. C. sakazakii was isolated from 27 products; 3/91 (3%) follow up formulas (as defined by Codex Alimentarius Commission), and 24/199 (12%) infant foods and drinks. Hence C. sakazakii was less prevalent in follow up formula than other foods given to infants over the same age range. A range of other bacteria were also isolated from follow up formulas, including Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, Citrobacter freundii, and Serratia ficaria. There was significant variation in the reconstitution instructions for follow up formulas. These included using water at temperatures which would enable bacterial growth. Additionally, the definition of follow up formula varied between countries.

Detection of coproantigens by sandwich ELISA in sheep experimentally infected with Fasciola gigantica.

Two monoclonal antibodies to Fasciola gigantica excretory/secretory (ES) antigens were used in a sandwich ELISA for the detection of Fasciola antigens in faeces of 9 sheep experimentally infected with 300 metacercariae of F. gigantica. The detection of coproantigens was found in four of the seven sheep within 5 weeks of infection, and within 7 weeks of infection coproantigens were detected in all seven of the sheep. This technique was compared to an indirect ELISA for the detection of anti-Fasciola ES antigen specific antibodies in serum. The anti-F. gigantica antibodies were detected within 3 weeks of infection in all of the infected sheep, suggesting a greater sensitivity to detect early infections. However, following anthelmintic treatment and removal of parasites, the anti-Fasciola antibody levels still remained high for at least 6 weeks when the study was terminated. In contrast, the levels of coproantigens were no longer detected in the faeces within 2 weeks of anthelmintic treatment. This study demonstrates that our sandwich ELISA for the detection of Fasciola coproantigens is able to detect immature fluke infections and more importantly, was able to detect patent infection of fasciolosis.

Studies on Streptococcus agalactiae isolated from bovine mastitis in Indonesia.

All 83 bacterial strains isolated from seven farms in three areas of the island of Java in Indonesia investigated in the present study could be identified as Streptococcus agalactiae. Identification was performed by cultural, biochemical and serological properties and by polymerase chain reaction amplification of species-specific parts of the gene encoding the 16S rRNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor (cfb) gene. All isolates were unpigmented. almost all of the isolates had the serotype pattern II/X. Despite these similarities a macrorestriction analysis of the chromosomal DNA of the bacteria revealed no significant homologies of the DNA-fingerprints of the S. agalactiae from the various areas. This last finding might possibly indicate that a single ancestral unpigmented serotype II/X S. agalactiae clone was responsible for the mastitis situation on Java and had evolved separately in the various farms and regions.

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.

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.

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.