Fluke Burden Research Articles

Vaccination of buffaloes with Fasciola gigantica recombinant fatty acid binding protein

Recombinant fatty acid binding protein (rFABP) of Fasciola gigantica was expressed in Escherichia coli and used as vaccine in Freund's adjuvant to evaluate the level of protection induced in buffalo (Bubalus bubalis) calves. Fifteen buffalo calves were distributed to three groups of five calves each. An antigen dose of 400 mug for each of the three immunizations at 3-week intervals, and a challenge dose of 600 metacercariae was administered per calf. Levels of anti-FABP antibodies increased rapidly by 2 weeks after the first immunization and were always significantly higher in the immunized-challenged group than in the infected control group. Immunization with FABP induced both humoral and cell-mediated immune response in these animals. Vaccination showed a moderate level of protection in terms of reduced fluke burden (35.8%) and liver damage as assayed by aspartate aminotransferase and sulfhydryl group levels as well as anti-fecundity effect of the vaccine.

Prevalence of Infection with Fasciola gigantica and Its Relationship to Carcase and Liver Weights, and Fluke and Egg Counts in Slaughter Cattle and Buffaloes in Southern Mindanao, Philippines

[Extract] According to Tongson (1978), fasciolosis is the most widespread and most destructive parasitic disease of farm animals in the Philippines, with incidence rates of34.61-100% in endemic areas. In Cotabato province where rice is primarily produced, fasciolosis is considered to be common among cattle and buffaloes. This study was undertaken to describe the infection in these animals because published reports on fasciolosis in this area are lacking. The prevalence of infection and the relationship of infection to carcase and liver weights, and the number of mature and immature flukes and the faecal egg count were investigated. This information may assist in understanding the importance of the infection and the need to implement control. Differences in response to infection with Fasciola gigantica between cattle and buffaloes have been observed in previous studies (Ening Wiedosari, personal communication, 1997), where it was found that infected buffaloes did not show significant reduction in weight gain, whereas weight gain in infected Bali and Ongole cattle was significantly reduced. The fluke burden in buffaloes was also significantly lower than in Ongole cattle. Hence, responses to infection with F gigantica may vary among different breeds. This abattoir study was done to observe the responses of Philippine cattle and buffaloes to natural infection with F gigantica and to see whether these differ between species.

An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3).

A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.

Risk of gallstone disease in advanced chronic phase of fascioliasis: an experimental study in a rat model.

In Wistar rats experimentally infected with Fasciola hepatica, the association between time of infection, number of flukes, rat weight, and serum lipid levels and the risk of developing pigment stones in the main bile duct was examined using data obtained at 100, 200, 300, 400, and 500 days postinfection. Gallstone presence increased with infection time. The relative risk of gallstone disease increased when the number of flukes per rat and rat weight increased. The presence of gallstones was associated with serum high-density lipoproteins and triglyceride levels. In a multivariate analysis, the association between gallstones and rat weight disappeared after adjustment for serum lipids. The absence of an effect of rat weight independent from serum lipids suggests that serum lipids are more closely linked to gallstone pathogenesis than is overweight. The presence of gallstones was strongly associated with the number of flukes located in the bile duct. A high risk of developing gallstones may be expected in human subjects inhabiting areas where F. hepatica is highly endemic and where high egg outputs detected in humans suggest that liver fluke burdens may also be very high.

Immunization of sheep against Fasciola gigantica with glutathione S-transferase

Glutathione S-transferase in Fasciola gigantica (FgGST) was isolated by affinity chromatography, by which highly purified enzyme was obtained. FgGST on the SDS-PAGE showed three protein bands ranging 24.5–26.5 kDa. Glutathione S-transferase (GST) activity was determined by HABIG method. FgGST was evaluated as vaccine alone or in combination with either aluminum hydroxide or saponin in sheep against F. gigantica infection. ELISA was used for detection of anti-FgGST IgG. After vaccination, all sheep were challenged with 120 metacercaria of F. gigantica. The results indicated that anti-GST IgG was not elevated after challenge. All sheep were slaughtered 24–26 weeks after challenge. The results indicated that, although after second vaccination, antibody titers rose markedly in GST–Al(OH) 3 and GST–saponin groups, but declined 4 weeks after challenge. No correlation between anti-GST IgG titers and protection was observed. The highest fluke burden reduction was observed in the group vaccinated with GST–saponin (32%), but this reduction was not statistically significant in comparison with the control group.

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly ( p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 ( p<0.0001) and in feces 0.76 ( p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.

Effects of parenteral administration of doramectin or a combination of ivermectin and clorsulon on control of gastrointestinal nematode and liver fluke infections and on growth performance in cattle

To compare effects of an injectable doramectin preparation with those of an injectable ivermectin-clorsulon preparation on control of gastrointestinal nematodes and liver flukes and on growth performance in cattle. Randomized complete block design. 60 crossbred calves. Calves (20/treatment group) were treated with doramectin or ivermectin-clorsulon or were not treated. Fecal samples were collected for nematode and Fasciola hepatica egg counts on day 0 and for up to 140 days after treatment. Cattle were weighed before treatment and at 28-day intervals until day 140. From day 7 through day 49, nematode egg counts for calves treated with doramectin or with ivermectin-clorsulon were significantly lower than those for untreated control calves. As the study progressed beyond day 56, the percentages of cattle with fluke eggs in their feces increased, but differences in regard to these percentages were not detected among the 3 groups. Average daily gain for the doramectin-treated cattle (0.79 kg/d [1.74 lb/d]) was significantly greater than that for the cattle treated with ivermectin-clorsuIon (0.71 kg/d [1.56 lb/d]); values for both groups were significantly greater than that for the control cattle (0.62 kg/d [1.37 lb/d]). Results suggest that doramectin had a greater impact on subclinical gastrointestinal tract parasitism in calves, as demonstrated by growth performance, than did ivermectin-clorsulon. In the Gulf Coast region of the United States, spring-born nursing beef calves may have minimal grazing exposure to F hepatica during the peak fluke transmission period; therefore, mature fluke burdens may be negligible at the beginning of the fall season.

Vaccination of sheep against Fasciola hepatica with homologous fatty acid binding proteins

The current study was designed to test the immunoprophylactic properties of native (nFh12) and recombinant (rFh15) antigens from Fasciola hepatica in sheep subsequently infected with the fluke. Thirty lambs were divided into six groups according to various patterns of immunisation and times of infection and necropsy. The antigens were emulsified in Freund’s adjuvant. Levels of specific anti-nFh12 and anti-rFh15 antibodies rose rapidly by 2 weeks after the first immunisation and were always significantly higher in immunised-infected sheep than in control-infected sheep. On completion of the trial there was no difference in fluke burden between groups vaccinated with either of the antigens and non-immunised controls. However, worm size and faecal egg counts were significantly diminished in the sheep vaccinated with either of the antigens, suggesting an anti-fecundity effect. This is the first report of experimental vaccination of sheep against F. hepatica with purified native and recombinant antigens related to fatty acid binding proteins.

Successful DNA immunisation of rats against fasciolosis

The liver fluke Fasciola hepatica contributes to great economic and health losses in the cattle industry in many countries, including Poland. Unfortunately, no vaccine against fasciolosis is commercially available. We have designed a DNA vaccine and tested it in rats. Groups of male or female rats received one intramuscular injection of 50 μg of a pcDNA 3.1 vector carrying cDNA encoding for a cysteine proteinase of F. hepatica. The plasmid was diluted in saline containing 0.05% bupivacaine. Control rats were injected with empty plasmid or not injected at all. All rats were challenged with 45 metacercariae of the fluke on day 28 of the experiment. Seven weeks after the challenge infection fluke burdens were evaluated in vaccinated and control rats. Male rats vaccinated with cysteine proteinase cDNA revealed 100% protection against F. hepatica infection. Females immunised in the same way exhibited the reduction of fluke burden by 74%.

A strategic dosing scheme for the control of fasciolosis in cattle and sheep in Ireland

This study examined the effectiveness of a strategic dosing scheme in lowering the incidence of fasciolosis on a mixed dry-stock farm and in maintaining the reduced incidence following a reduction in dosing intensity. Two neighbouring farms with a history of chronic fluke disease were selected, the strategic dosing scheme being implemented on one (the trial farm) while the other (the control farm) continued to treat according to its normal practice. The strategic dosing scheme was designed to suppress the faecal egg output of Fasciola hepatica at critical times of the year in order to limit infection of the intermediate host snail population and thus reduce the subsequent contamination of the pasture with metacercariae. On the trial farm cattle and sheep were treated three times per year for the first 2 years at approximately 8 week intervals, starting in March of each year. A fourth treatment was given when the cattle were housed and out-wintered sheep received an additional treatment in January. In Years 3 and 4 the dosing intensity was reduced. By the end of Year 2, data from faecal egg counts, tracer-sheep fluke burdens and snail infection levels indicated that the treatment strategy had succeeded in suppressing the fluke population and eliminating the occurrence of clinical fasciolosis. The decrease in dosing intensity in Years 3 and 4 maintained both stock and snail infections at low levels and there was no re-emergence of the disease.

Cell-mediated immune response in calves to single-dose, trickle, and challenge infections with Fasciola hepatica

A peripheral blood mononuclear cell (PBMC) proliferation assay was used to study the cell-mediated immune response in eight calves experimentally infected with Fasciola hepatica. Hypersensitivity-related eosinophil and mast-cell responses were also assessed. The primary infection of 500 metacercariae was administered either as a single-dose or as a trickle infection over a 4-week period. Calves were challenge-infected 4 months later with 100 metacercariae and slaughtered 24 weeks postprimary infection. Skin eosinophil counts (SEC) were determined prior to infection on the basis of the intradermal reaction (IDR) to phytohaemagglutinin (PHA). These counts correlated negatively with the mean fluke length but not with the fluke burden found at necropsy. At the end of the experiment, non-specific (PHA) and specific (excretory-secretory parasite products, FhESAg, and whole-worm extract, FhSomAg) immediate type hypersensitivity IDR were elicited in contrast to delayed type hypersensitivity (DTH) responses. The SEC correlated with blood eosinophilia but not with parasite parameters. These findings suggest that the eosinophil response does not correlate clearly with the development of resistance to F. hepatica infection in cattle. A specific mononuclear cell response to FhSomAg was detectable as early as 7 days after infection in both infected groups, being significantly higher during the very early migratory phase of the juveniles in the single-dose infected calves than in the trickle infected calves. This response remained significantly higher in infected groups than in the control group throughout the experiment. Challenge elicited a significant proliferative response, less pronounced than after primary infection. No production of gamma-interferon (INF-γ) was recorded 3 weeks after challenge. At necropsy, the mean number of flukes recovered was similar in both infected groups, suggesting that the rate at which the infection is administrated has no effect on protective immunity. Hepatic lesions, similar in both infected groups, were characterised by marked eosinophil and mast-cell infiltration. Liver biopsies were performed and their diagnostic value is discussed. All results suggest that F. hepatica infection predominantly induces a Type-2 response in cattle, and that this response has little protective effect.

Immune responses of cattle to experimental anti-Fasciola hepaticavaccines

Fasciola hepaticainfection of cattle and sheep is an important cause of clinical disease and production losses, and is controlled at present by a combination of chemotherapy and management measures. However, the prospects for the control ofF. hepaticainfection by vaccination are good, and we have previously shown substantial protection of cattle against experimental challenge infection following immunisation with a combination of the purified fluke-derived enzymes cathepsin L1 (CATL1), cathepsin L2 (CATL2) and fluke-derived Hb fraction (FHB). This and other recent studies have also demonstrated fundamental differences between protective and non-protective immune responses to liver fluke infection. In this present study we have further analysed the response of animals to liver fluke challenge following experimental vaccination. Calves were vaccinated with eitherCATL2 plusFHB, orCATL1 plusCATL2. Partial protection against challenge infection was achieved in both vaccinated groups, with the greatest level of protection (55 per cent reduction in fluke burdens) recorded in the group vaccinated withCATL1 plusCATL2. This latter group also showed the greater level of lymphocyte proliferation and the greater production of γ-INF in response to stimulation with fluke antigen in vitro following challenge. These results are significant in our attempts to characterise the elements within the immune response to vaccination which are protective.

Liver pathology and immune response in experimental Fasciola hepatica infections of goats

The relationship between the immune response and the pathogenesis of the disease was studied in different primary and secondary experimental Fasciola hepatica infections of goats. The establishment of the infection, measured as percentage of recovered flukes at the necropsy, was similar in primarily and secondarily infected animals (between 19.7% and 24.3%), but the hepatic damage was much more severe in secondarily infected goats, as revealed by the levels of serum hepatic enzymes GGT and LDH. Primary infection evolves to chronic fasciolosis that did not induce the development of resistance, since goats were highly susceptible to secondary infection, showing severe acute and chronic hepatic lesions that led to the death of some animals in each group. The immune response to the infection was proved by the production of specific IgG antibodies to ESP of F. hepatica and the involvement of CD3 + T lymphocytes and lambda IgG + plasma cells in the hepatic infiltrate. Secondary infection did not induce any difference in either IgG response or in the cellular composition of the infiltrate of hepatic lesions, although this was much more extended. However, neither antibodies nor cell-mediated response were protective: there was no correlation between IgG levels and fluke burden and there was no evidence of cell-mediated killing of the parasite. This suggests the existence of some immune evasion mechanisms in goat infection with F. hepatica. The parasite may depress the local inflammatory and immune response, as suggested by the scarcity of CD3 + T cells in the infiltrate surrounding acute migratory tunnels. Moreover, in secondary infected goats can be suspected an immunological damage of the liver, since a very severe infiltrate of immune cells replaced wide areas of hepatic parenchyma and an immune-mediated damage of hepatocytes could occur.

T cell subset involvement in immune responses to Fasciola hepatica infection in cattle.

The lymphocyte response to F. hepatica during a primary infection in cattle was analysed to define the role of T cell subsets in the immune response. Blood lymphocytes were isolated from eight cattle infected with F. hepatica via trickle infection over a ten-day period and from two non-infected controls. CD4+, CD8+ and gamma delta + T cells were depleted from whole lymphocyte populations by magnetic bead depletion. Lymphocytes from infected animals demonstrated a transient, but marked elevation in responsiveness to F. hepatica antigen between weeks 3 and 8 post-infection. Responses were attenuated by depletion of CD4+ and CD8+ T cells during this period. Depletion of gamma delta + T cells attenuated antigen responses at one time point only, and at an earlier stage post-infection than when alpha beta + T cells were depleted. Responses to antigen correlated positively with both hepatic fluke burden and with the degree of hepatic damage. This suggests that the cellular immune response was not protective. Antigen responses in gamma delta + T cell-depleted populations were also associated with post-mortem fluke burden and with hepatic damage. This suggests that gamma delta + T cells are involved in down regulating alpha beta + lymphocytes which may have a role in a non-protective or immunopathological immune responses.

Use of excretory/secretory antigens in a competition test to follow the kinetics of infection by Fasciola hepatica in cattle

Eight 16–18-month-old Charolais heifers were experimentally infected with Fasciola hepatica. An antigen competition assay was used to follow the kinetics of the infection and was compared to antibody tires and serum liver enzymes. The antigen competition assay was able to detect the presence of infection as soon as 6 days after the start of the experimental infection which is considerably sooner than other methods. Consequently, this assay would be useful in diagnosing fasciolosis early in the prepatent period. The animals were slaughtered at the end of the experiment, the livers recovered and post-mortem fluke burdens determined. However, only serum liver enzyme levels gave any indication of the intensity of infection in the different animals.

Correlation of specific antibody titre and avidity with protection in cattle immunized against Fasciola hepatica

Cattle produce specific serum antibody mainly of the IgG1 isotype in response to infection with the liver fluke, Fasciola hepatica. In these animals a positive correlation between fluke-specific serum IgG1 levels and fluke-burden in non-immunized infected animals was observed. In contrast, immunization of cattle with a combination of the fluke-derived antigens cathepsin L2 (CL2) and fluke haemoglobin (FHb) in Freund's complete/incomplete adjuvant ( FCA FIA ) induced a· specific antibody response involving IgG2, as well as IgG1. These immunized animals also exhibited very high (72%) levels of protection against a subsequent challenge infection. When the vaccine was administered in FIA alone the specific antibody response, while still involving IgG1 and IgG2, was of lower magnitude (10-fold and 100-fold, respectively) and no significant reduction in fluke burden was observed following challenge. Nevertheless, in these animals, a strong IgG2 response was associated with low fluke burdens. These results provide further evidence of the non-protective nature of specific immune responses in cattle following F. hepatica infection, and demonstrate that vaccination can induce a qualitatively different, and protective, response.

Evaluation of a diagnostic monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of a 26- to 28-kd Fasciola hepatica coproantigen in cattle

Abstract Objective To develop a monoclonal antibody-based capture ELISA for detection of a 26- to 28-kd coproantigen of Fasciola hepatica in the feces of infected cattle. Animals 27 crossbred yearling calves, 2 New Zealand White rabbits. Procedure A capture ELISA that uses a previously described monoclonal antibody (MAB) M2D5/D5F10 was developed. The MAB was used to capture the antigen from the feces, and hyperimmune rabbit serum raised against the purified 26- to 28-kd glycoprotein was used to detect the coproantigen. This test was used for the detection of the antigen in the feces of 27 experimentally infected calves with known numbers of flukes. Fecal specimens obtained before infection from the same calves were used as negative controls. Results The assay results identified all calves infected with more than 10 flukes at necropsy, and as little as 300 pg of coproantigen/ml of fecal supernatant was detected. The assay results correlated well with the number of flukes, suggesting that it is possible to estimate fluke burden. Infections as early as 6 weeks’ duration were detected, before flukes mature to adults and start to shed eggs. Conclusions In experimentally infected calves, the coproantigen capture ELISA was more sensitive and easier to perform than microscopic examination for the diagnosis of F hepatica infection; moreover, 6-week-old prepatent infections were detectable. Clinical Relevance This capture ELISA containing an F hepatica 26- to 28-kd coproantigen is a quantitative assay that is more sensitive than fecal egg counting. In addition, the assay is rapid, easy to perform and lends itself well to large numbers of samples. Because it is antigen based, the ELISA may be useful for diagnosis of F hepatica infection in other species, including human beings. (Am J Vet Res 1998;59:533–537)

The susceptibility differences of buffalo and Ongole calves against trickle infection with Fasciola gigantica

A pen trial was carried out in order to determine the susceptibility differences of a trickle infection with Fasciola gigantica in buffalo and Ongole calves. Treated animals were infected orally with 15 metacercariae of F. gigantica twice weekly for 32 weeks and were slaughtered at 36 weeks. The results showed that buffalo calves had significantly lower fluke burdens than Ongole calves (P<0 .01) . All of the infected Ongole calves had fasciola eggs in their faeces 18 weeks after the commencement of infection. In contrast, eggs were detected only in 3 out of 7 infected buffalo calves at week 20, in 4 at week 28 and in 6 at week 30. Faeces of the seventh buffalo remained free of eggs until week 36. Rates of growth were reduced by 25%and 10,3% in infected Ongole and buffalo calves respectively . Circulating blood eosinophilia ofboth hosts, ahallmark of helminth infections, increased following infection, but values in buffaloes was greater than Ongole calves mainly in week 4 and 8 after infection (P<0.01). These results might be concluded that the susceptibility of buffalo calves to trickle infection with F. gigantica was lower compared to Ongole calves. Keywords : Fasciola gigantica; susceptibility, buffalo calves, Ongole calves

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.

Protection of cattle against Fasciola hepatica infection by vaccination with glutathione S-transferase

Glutathione S-transferase (GST) from the liver fluke Fasciola hepatica was assessed as a vaccine immunogen in cattle in a number of immunological adjuvants. Significant reductions in fluke burdens (49–69%) were only observed in cattle vaccinated with GST in Quil Alsqualene Montanide® (SM) and PLG microspheres in SM but there was no correlation between anti-GST IgG titres and protection. In separate experiments, animals vaccinated with GST in Quil A/SM were still significantly protected (48%, P<0.05) 6 months after boosting and no significant differences in protection were seen when the metacercarial challenge was given over 1 month instead of as a single bolus. Inhibition of GST enzyme activity in vitro by cattle antisera did not correlate with reduced fluke burdens.