The Effects of Nonpromotion on Achievement and Maturation in the Junior High School

The present study has dealt with the problem of the non-effects of nonpromotion--a practice which continues because many educators and parents are convinced that grade repetition will help the immature or underachieving child catch up

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke

Comparative studies of resistance on Indonesian Thin Tail (ITT) sheep, St. Croix, merino and the crossbreed of ITT and St. Croix, against the infection of Fasciola gigantica

resistance is heritable. In order to re-evaluate this evidence, 20 ITT sheep were infected with 350 metacercariae of F. gigantica and for comparison, 10 St. Croix sheep, 10 Merino sheep and 20 crossbred of ITT x St. Croix sheep were also infected with the same dose of metacercariae. The results showed that ITT sheep was highly resistant than the other breed, whereas St. Croix and Merino sheep were susceptible. 60% of the crossbred were as resistant as ITT sheep and the other 40% were as susceptible as the St. Croix sheep. Thus, it is proposed that there might be a hereditary resistance factor such as a dominant gene which inducing the mechanism of resistance in ITT sheep, and there is some indication that IgG2 might act as a blocking antibody that interferes the mechanism of resistance.   Key words : ITT sheep, Fasciola gigantica, genetic resistance, dominant gen

Serum levels of cytokines in water buffaloes experimentally infected with Fasciola gigantica

Fasciola gigantica infection in water buffaloes causes significant economic losses especially 27 in developing countries. Although modulation of the host immune response by cytokine 28 neutralization or vaccination is a promising approach to control infection with this parasite, our 29 understanding of cytokine's dynamic during F. gigantica infection is limited. To address this, 30 we quantified the levels of serum cytokines produced in water buffaloes following experimental 31 infection with F. gigantica. Five buffaloes were infected via oral gavage with 500 viable F. 32 gigantica metacercariae and blood samples were collected from buffaloes one week before 33 infection and for 13 consecutive weeks thereafter. The levels of 10 cytokines in serum samples 34 were simultaneously determined using ELISA. F. gigantica failed to elicit the production of 35 various pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-2, IL-6, IL-12, and 36 IFN-γ. On the other hand, evidence of a Th2 type response was detected, but only early in the 37 course of parasite colonization and included modest increase in the levels of IL-10 and IL-13. 38 The results also revealed suppression of the immune responses as a feature of chronic F. 39 gigantica infection in buffaloes. Taken together, F. gigantica seems to elicit a modest Th2 40 response at early stage of infection in order to downregulate harmful Th1- and Th17-type 41 inflammatory responses in experimentally infected buffaloes. The full extent of anti-F. 42 gigantica immune response and its relation to pathogenesis requires further study

Evaluation of immunogenicity and efficacy of Fasciola hepatica Tetraspanin 2 (TSP2) fused to E. coli Heat-Labile Enterotoxin B Subunit LTB adjuvant following intranasal vaccination of cattle

Fasciolosis, caused by the liver flukes Fasciola hepatica and F. gigantica, is an economically important and globally distributed zoonotic disease. Liver fluke infections in livestock cause significant losses in production and are of particular concern to regions where drug resistance is emerging. Antigens of the F. hepatica surface tegument represent promising vaccine candidates for controlling this disease. Tetraspanins are integral tegumental antigens that have shown partial protection as vaccine candidates against other trematode species. The Escherichia coli heat-labile enterotoxin’s B subunit (LTB) is a potent mucosal adjuvant capable of inducing an immune response to fused antigens. This study investigates the potential of F. hepatica tetraspanin 2 extracellular loop 2 (rFhTSP2) as a protective vaccine antigen and determines if fusion of FhTSP2 to LTB can enhance protection in cattle. Cattle were immunised subcutaneously with rFhTSP2 mixed in the Freund’s adjuvant and intranasally with rLTB-FhTSP2 in saline, accounting for equal molar ratios of tetraspanin in both groups. Vaccination with rFhTSP2 stimulated a strong specific serum IgG response, whereas there was no significant serum IgG response following rLTB-FhTSP2 intranasal vaccination. There was no substantial antigen specific serum IgA generated in all groups across the trial. Contrastingly, after the fluke challenge, a rise in antigen specific saliva IgA was observed in both vaccination groups on Day 42, with the rLTB-FhTSP2 vaccination group showing significant mucosal IgA production at Day 84. However, neither vaccine group showed a significant reduction of fluke burden nor faecal egg output. These results suggest that intranasal vaccination with rLTB-FhTSP2 does elicit a humoral mucosal response but further work is needed to evaluate if mucosal delivery of liver fluke antigens fused to LTB is a viable vaccine strategy

Evaluation of Immunogenicity and Efficacy of Fasciola hepatica Tetraspanin 2 (TSP2) Fused to E. coli Heat-Labile Enterotoxin B Subunit LTB Adjuvant Following Intranasal Vaccination of Cattle

Fasciolosis, caused by the liver flukes Fasciola hepatica and F. gigantica, is an economically important and globally distributed zoonotic disease. Liver fluke infections in livestock cause significant losses in production and are of particular concern to regions where drug resistance is emerging. Antigens of the F. hepatica surface tegument represent promising vaccine candidates for controlling this disease. Tetraspanins are integral tegumental antigens that have shown partial protection as vaccine candidates against other trematode species. The Escherichia coli heat-labile enterotoxin’s B subunit (LTB) is a potent mucosal adjuvant capable of inducing an immune response to fused antigens. This study investigates the potential of F. hepatica tetraspanin 2 extracellular loop 2 (rFhTSP2) as a protective vaccine antigen and determines if fusion of FhTSP2 to LTB can enhance protection in cattle. Cattle were immunised subcutaneously with rFhTSP2 mixed in the Freund’s adjuvant and intranasally with rLTB-FhTSP2 in saline, accounting for equal molar ratios of tetraspanin in both groups. Vaccination with rFhTSP2 stimulated a strong specific serum IgG response, whereas there was no significant serum IgG response following rLTB-FhTSP2 intranasal vaccination. There was no substantial antigen specific serum IgA generated in all groups across the trial. Contrastingly, after the fluke challenge, a rise in antigen specific saliva IgA was observed in both vaccination groups on Day 42, with the rLTB-FhTSP2 vaccination group showing significant mucosal IgA production at Day 84. However, neither vaccine group showed a significant reduction of fluke burden nor faecal egg output. These results suggest that intranasal vaccination with rLTB-FhTSP2 does elicit a humoral mucosal response but further work is needed to evaluate if mucosal delivery of liver fluke antigens fused to LTB is a viable vaccine strategy

Evaluation of LAMP for Fasciola hepatica detection from faecal samples of experimentally and naturally infected cattle

Fasciola hepatica causes liver fluke disease in production animals and humans worldwide. Faecal egg counts (FEC) are the most common diagnostic tool for the diagnosis of liver fluke disease. However, FEC has low sensitivity and is often unreliable for the detection of patent infection. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of Fasciola hepatica infection, with the aim of increased sensitivity and making it suitable for on-farm application. LAMP was initially conducted under laboratory conditions, optimised to enable visual detection using calcein dye. DNA extraction based on bead-beating was developed to enable on-farm application. LAMP results were compared to FEC and polymerase chain reaction (PCR). Under laboratory conditions, LAMP was conducted using two incubation methods: a conventional PCR thermocycler and a field-deployable LAMP instrument. When compared to a ‘rigorous’ FEC protocol consisting of multiple counts using a comparatively large volume of faeces and with infection confirmed post-mortem, LAMP was highly sensitive and specific (using silica membrane DNA extraction sensitivity 88 %, specificity 100 %; using sieving and beat-beating DNA extraction sensitivity 98.9 %, specificity 100 %). When applied on-farm, LAMP was compared to conventional FEC, which suggested high sensitivity but low specificity (sensitivity 97 %, specificity 37.5 %). However, further analysis, comparing field LAMP results to laboratory PCR, suggested that the low specificity was likely the outcome of the inability of conventional FEC to detect all true F. hepatica positive samples. Based on the high sensitivity and specificity of LAMP compared to a ‘rigorous’ FEC protocol and its ability to be used in field settings, the study demonstrates the potential of LAMP for diagnosing F. hepatica infection in agriculture. © 2024 The Author

In vitro studies: The role of immunological cells in Indonesian thin tail sheep in the killing of the liver fluke, Fasciola gigantica

Previous studies have shown that Indonesian Thin Tail (ET) sheep exhibit high resistance to challenge with Fasciola gigantica when compared with Merino sheep, and this resistance is expressed in early infection. In order to study the role of the immune system in this resistance to ET sheep, in vitro studies were undertaken in the laboratory. In vitro study to confirm the ability of immune cells from ET sheep in the killing of F. gigantica larvae has been done by incubating immune cells and F. gigantica larvae together with immune sera or normal sera. The viability of the larvae was observed over a period 3 days incubation by observing their motility. The results showed that the cells isolated from F. gigantica- challenged ET sheep in the presence of immune sera from ET were able to kill 70% of the larvae. In contrast, cells from infected Merino were unable to kill a significant number of F. gigantica using the same sera source. It seems that the cytotoxicity was dependent on the presence of immune sera and ET peritoneal cells, suggesting the potential role of an antibody-dependent cell cytotoxic (ADCC) mechanism in the resistant ET sheep.Key words: In vitro, Fasciola gigantica, peritoneal cell, sheep gigantica

A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field

The responses of eosinophil and packed cell volume (PCV) on sheep infected with Fasciola gigantica

The responses of eosinophil and packed cell volume (PCV) values were verified in infected sheep, in order to identify whether these parameters could be used to predict the flukes burden and their correlation with breed resistance. Fifteen Indonesian thin tail sheep (ET), 9 Merino sheep and 148 backcross sheep generated from mating of Merino sheep and F1 sheep (Merino X ET cross) were infected with 300 metacercariae of Fasciola gigantica. The blood samples were collected every 2 weeks by using EDTA venoject tubes in order to determine the amount of eosinophils and the PCV value. After 14 weeks of infection all of sheep were killed and the liver was collected in order to determine the number of flukes. The results showed that the amount of eosinophils increased 2 weeks after infection and reached the peak at week 4 after infection. The average of eosinophils in ET appeared higher than the other 2 breeds (Merino was the lowest and the backcross was in between). The correlation between the number of flukes recovered from the liver and the eosinophil counts were positive in ET and Merino, but negative in the backcross sheep. The PCV values remained constant along the trial, except at week 14 after infection; the PCV values were slightly decreased in backcross sheep and Merino sheep, but not in ET sheep. The correlation between number of flukes in the liver and the PCV values were negative in all breeds of sheep. These results suggested that the eosinophilic and PCV’s response of ET were higher compared to backcross and Merino sheep, thus that responses were thought to be associated with the resistant phenomenon.   Key words: Fasciolosis, eosinophil, PCV, shee