Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.     

Synergistic influence of Ostertagia ostertagi and Trichostrongylus axei on Ostertagia ostertagi larval inhibition and abomasal lesions in cattle

Parasite-free 4-month-old calves were inoculated with Ostertagia ostertagi and/or Trichostrongylus axei followed 6 weeks later by increasing doses of O ostertagi for 8 weeks. Clinical signs of parasitism, fecal egg counts, and plasma pepsinogen concentrations were monitored, and gross lesions and parasite burdens were determined postmortem. Clinical signs of parasitism were not observed and weight gains were not affected in experimentally infected calves. In calves infected with O ostertagi, mean plasma pepsinogen concentrations were greater than for control calves and were diagnostically significant 4 weeks after inoculation and during the last 4 weeks of serial inoculations with O ostertagi. In calves that were given O ostertagi and T axei, abomasal pH was significantly increased, and abomasal lesions were more pronounced than in control calves or in calves inoculated with only O ostertagi or T axei. Abomasal lymph nodes were enlarged in all parasitized calves; other lymph nodes in the calves inoculated with both O ostertagi and T axei were usually smaller than in calves inoculated with only O ostertagi or T axei. Numbers of O ostertagi-inhibited larvae were small in all inoculated calves, but the percentage inhibition was significantly greater in calves inoculated with both O ostertagi and T axei. The percentage inhibition was 3.53% for the O ostertagi-inoculated calves and 7.07% for calves inoculated with both O ostertagi and T axei. These percentages indicated a synergistic effect of concurrent abomasal parasitism, whereas a synergistic effect on T axei worm burden was not observed. The low percentage of larval inhibition indicated that factors other than host resistance are involved in naturally occurring pretype II ostertagiosis.     

The susceptibility of the goat to Fasciola hepatica infections

Mixed breed goats were infected with metacercariae of Fasciola hepatica and the resulting worm burdens were quantitated after primary and secondary exposure of the goats to the parasite. Mean length and width of the parasite recovered after all primary exposures were 1.91 +/- 0.2 cm and 0.91 +/- 0.2 cm, respectively. A mean of 71.8 +/- 5.9% of the flukes were recovered from all of the primary infections. In the secondary infections, the mean length and width of the flukes from the physically smaller population was 0.88 +/- 0.27 cm and 0.53 +/- 0.19 cm, respectively. A mean of 67 +/- 6.7% of the flukes were recovered from this secondary infection. It appears that the goat is susceptible to challenge infections with F. hepatica and that its response to this infection is much like that of sheep.     

The ability of diamphenethide to control immature Fasciola hepatica in sheep at a lower than standard dose level

Groups of eight Welsh Mountain sheep were dosed with diamphenethide at the rate of 70 mg/kg bodyweight at either one, four, six or eight weeks after artificial infection with approximately 300 Fasciola hepatica metacercariae. Comparisons were made with similarly infected but undosed sheep and with sheep which were neither infected nor dosed. The good clearance of flukes up to six weeks of age (above 97 per cent on pooled data) was reflected in the plasma concentrations of the accepted liver damage marker enzymes glutamate dehydrogenase and gamma-glutamyl transpeptidase. Highly significant correlations were demonstrated between the numbers of flukes recovered, the plasma levels of these enzymes and haemoglobin and plasma albumin values. At 70 mg/kg, diamphenethide was shown to be able to control F hepatica populations of up to six weeks of age. The systematic use of diamphenethide at this dose level at intervals of up to six weeks during the period of metacercarial challenge should prevent ovine fascioliasis.     

Establishment and pathogenicity of two strains of Ostertagia ostertagi and Cooperia oncophora in calves in different locations.

An experiment was carried out simultaneously in Glasgow and in Wageningen to investigate possible differences between the local strains of Ostertagia ostertagi and Cooperia oncophora. In each location calves of the local Friesian breed were infected with 100,000 larvae of either the Glasgow or Wageningen strain of O ostertagi or C oncophora. At both locations the calves received the same diet. The Glasgow strain of O ostertagi was more pathogenic than the Wageningen strain and a larger proportion of the worm burden was found in the abomasal mucosa. The number of ova per female was greater in the Wageningen strain. For C oncophora the Wageningen strain gave rise to higher worm burdens and longer worms. Differences were also present between locations. The British Friesians had higher worm burdens of C oncophora and the worms of this species were longer in this host. Compared with the Dutch Friesians the British calves had a higher proportion of O ostertagi in the mucosa. This experiment showed how difficult it is to compare data from the literature because of differences in parasite and host strains and laboratory techniques.     

The immune response of rats to vaccination with the cDNA or protein forms of the cysteine proteinase of Fasciola hepatica

Our previous experiments have shown that intramuscular injection of Sprague-Dawley rats with a pcDNA 3.1 vector carrying cDNA encoding for a cysteine proteinase (CP) of F. hepatica may induce a high level of protection against subsequent infection with F. hepatica metacercariae (mc). The aim of the present study is to compare the immune response of Sprague-Dawley rats vaccinated intranasally with plasmid containing cDNA of CP of the fluke and intramuscularly or intraperitoneally with the recombinated enzyme protein to challenge with fluke metacercariae. In addition, protection following intranasal DNA vaccination was evaluated. Two experiments were carried out. In the first experiment rats were vaccinated twice with 50microg of cDNA containing plasmid or with 100microg protein of recombinated CP. Three weeks after the second vaccination rats were challenged orally with 25 mc. On days 0, 21, 42 and 63 after the challenge blood samples were collected for the evaluation of white blood cell, eosinophil and specific antibody responses. During the second experiment groups of five male and female rats were vaccinated twice intranasally with CPcDNA then challenged with 30 mc and dissected 5 weeks later. Results obtained in the experiments suggested that intranasal immunisation of rats with CPcDNA seems to favour a Th2 regulated antibody response. Intramuscular or intraperitoneal injections of CP protein stimulate both Th1 and Th2-dependent antibodies. Mean worm burdens found in rats vaccinated intranasally 5 or 10 weeks after the challenge were reduced by 61-75% in comparison with the challenge controls which suggests that intranasal vaccination with CPcDNA may protect hosts against F. hepatica infection.     

水牛感染肝片吸虫后肝脏组织的病理学变化

15 头水牛在人工感染条件下进行肝片吸虫感染, 慢性感染组水牛每天口服60 个囊蚴, 20 d 总共口服1 200 个囊蚴。急性感染组水牛1 次口服1 600 个囊蚴。慢性感染的水牛于感染后第26 周宰杀。急性感染的水牛分别于第10 , 13 , 16 和22 周宰杀。肝脏的组织病理学显示, 肝片吸虫幼虫可引起肝脏一系列炎症反应。急性感染后第10 周和13 周肝细胞变性、坏死, 肝小叶内淤血, 大量淋巴细胞浸润, 继而发展为肝索萎缩。急性感染后第22 周及慢性感染后第26 周, 汇管区内有大量结缔组织及新生的小胆管增生。实验表明慢性感染和急性感染肝片吸虫的水牛均可导致肝脏典型的病理学变化。     

水牛实验感染肝片吸虫后IGF-Ⅰ和β内啡肽含量变化及其对免疫功能的影响

头健康阉公水牛, ５ 头经口每日感染肝片吸虫囊蚴６０ 个／ 头, 连续２０ ｄ ; ３ 头不感染作对照。于感染前和感染后每周采集颈静脉血样１次, 共２７ 次, 分别测定血浆 Ｉ Ｇ Ｆ Ⅰ、β内啡肽 （β Ｅ Ｐ） 以及 Ｗ Ｂ Ｃ 、 Ｄ Ｃ ; Ｔ、 Ｂ 淋巴细胞比例和血清抗肝片吸虫 Ｉｇ Ｇ 水平。结果 Ｉ Ｇ Ｆ Ⅰ和β Ｅ Ｐ 分别在开始感染后第５ 周和第４ 周显著升高, 并持续到第２３ 周和第９ 周; Ｉｇ Ｇ 水平也在第４ ～２２ 周显著高于对照组, 嗜酸性粒细胞和 Ｂ 淋巴细胞比例也明显升高。结果表明肝片吸虫感染后水牛免疫功能的变化可能与 Ｉ Ｇ Ｆ Ⅰ及β Ｅ Ｐ 有关。     

Heterologous resistance to Fasciola hepatica conferred upon rats by passive transfer of serum from different breeds of sheep

Two experiments compared the protection against oral challenge with 20 metacercariae of Fasciola hepatica conferred on rats by intraperitoneal injection of serum from three breeds of sheep infected with F. hepatica (Barbados Blackbelly, St. Croix, Florida Native). Experiment 1 used serum from sheep 5-6 months of age following two infections of 250 metacercariae each, while Experiment 2 utilized serum collected from the same sheep at 10-11 months of age following either a primary (first exposure) or challenge (after two previous exposures of 250 metacercariae each) infection with 500 metacercariae. Similar numbers of flukes were recovered from rats given either immune or nonimmune (control) serum from each breed of sheep in Experiment 1. In Experiment 2, rats given serum from infected St. Croix sheep had significantly fewer flukes than rats given either control or immune serum from Barbados Blackbelly or Florida Native sheep. There was no significant correlation of fluke counts between individual serum donors (sheep) and serum recipients (rats).     

Worm recovery, haemagglutinating antibodies and IgE-levels after immunisation against Fasciola hepatica in rats

Female inbred Hooded Lister (HL) rats were each infected with 20 metacercariae (Mc) of Fasciola hepatica. Remarkable variations between the number of flukes established in the bile ducts suggest the presence of individual, perhaps genetically controlled, differences in immune responsiveness of HL rats to F. hepatica. Serum (4 ml) from HL rats infected with 20 Mc 6 weeks prior to transfer partially protected rats against a F. hepatica challenge infection. However, 1 X 10(6) lymphoid cells originating from rats of the same age and stage of infection did not show the same protective qualities. Furthermore, attempts to immunise HL rats i.p. with either juvenile or adult excretory/secretory (ES) products, or somatic tissue antigens and AlOH3-gel as adjuvant failed. When compared to other investigations, the present results further suggest that both the adjuvant and the route of administration are crucial for the stimulation of a protective immunity to F. hepatica. Low titers and low anamnestic responses of haemagglutinating antibodies after prior immunisation with juvenile ES antigens or both juvenile ES and somatic tissue antigen suggest the occurrence of an immunosuppressive effect caused by juvenile ES products. The total serum IgE-levels in immunised groups were generally lower when compared to the challenge control group, whereas the F. hepatica ES-specific IgE-levels rose after challenge, but immediately decreased again when compared to challenge controls. These findings support the hypothesis of an immunomodulatory effect caused by the vaccination scheme.     

Age resistance in calves to Ostertagia ostertagi and Cooperia oncophora

Calves were infected repeatedly during a period of 6 weeks with Ostertagia ostertagi and Cooperia oncophora, at an age of 3, 6 or 9 months. The inoculations were performed during three periods, February-March, May-June and August-September, to account for possible seasonal effects or effects of larval batches. Observations were done on faecal egg output, antibody titres and weight gains. Calves were slaughtered for post mortem examinations 9 weeks after the start of infections. The influence of age on worm populations and egg output was significant for C. oncophora but not for O. ostertagi. The effect of season or larval batch on worm populations was significant for O. ostertagi but not for C. oncophora. The correlations between worm numbers and several other parameters found for Cooperia were strongly indicative of a process of worm expulsion taking place at the stage of infection (9 weeks after the start of infections) when post mortem examinations were done. Such correlations were absent for Ostertagia. It is concluded that within the range of ages examined here (the range to which first season grazing calves belong), there is no influence of age on Ostertagia populations but a clear effect of age on Cooperia. This difference strongly influences the total faecal egg output of grazing calves and its interpretation.     

Diagnosis of Fasciola hepatica infection in beef calves by plasma enzyme analysis

Plasma analysis for albumin, total bilirubin, and total protein values and aspartate aminotransferase (AST), arginase, and gamma-glutamyl transpeptidase (GGT) activities was used for the early and quantitative diagnosis of experimental Fasciola hepatica infections in beef calves. Calves were infected on 3 occasions with 1,000 (n = 5), 100 (n = 5), or 10 (n = 4) metacercariae for a total infective dose of 3,000, 300, or 30, respectively. Albendazole (15 mg/kg of body weight) was administered to 7 infected calves on postinfection (initial) week (PIW) 13. All calves were euthanatized and necropsied on PIW 16 for the determination of fluke infections. Plasma constituents were determined weekly. Significant (P less than 0.05) increases in AST activity occurred as early as PIW 4 and GGT activity at PIW 9, as compared with that in noninfected controls. Fluke burden-related differences were observed in GGT activity from PIW 9 onward. Increases in AST activity reflected parenchymal liver damage, whereas increases in GGT reflected hepatobiliary damage; therefore, differentiation could be made between the migratory and ductal phases of the infection. There was no correlation between arginase activity and fluke infection. As compared with fecal examination results, plasma enzyme analysis gave an earlier and semiquantitative indication of F hepatica infection in experimentally infected calves. Although increases in these plasma constituents were not definitely diagnostic of fascioliasis, useful information on the size of the fluke burden and progress of the disease process could be obtained by these methods. Plasma enzyme analyses of AST and GGT were not indicative of chemotherapeutic success or failure when calves with mature F hepatica (14 weeks old) infections were treated.     

The anthelmintic efficacy of netobimin against experimental infections of Fasciola hepatica in sheep

Netobimin (coded SCH 32481, Schering Corporation), a new broad-spectrum anthelmintic having both fasciolicidal and nematocidal properties was evaluated for efficacy against mature Fasciola hepatica infections in sheep. The trial was conducted with 30 cross-bred spring lambs, each experimentally infected with 250 F. hepatica metacercariae. A single treatment of netobimin was administered at 17 weeks post-infection (PI) by oral drench at 7.5 or 20 mg kg-1 body weight while 10 animals remained as untreated controls. At necropsy (either 1 or 2 weeks post-treatment), the mean number of adult flukes recovered from the control, 7.5 and 20 mg kg-1 groups were 94.7, 35.9 and 8.8, respectively. The resulting efficacies were 62% (P less than or equal to 0.05) and 90.7% (P less than or equal to 0.01), respectively. No clinical signs of fascioliasis were noted in any sheep during the trial. No signs of toxicosis nor any adverse reactions to the drug were observed.     

Serological responses of cattle after treatment and during natural re-infection with Fasciola hepatica, as measured with a dot-ELISA system

The dot-ELISA reaction was used to study the dynamics of IgG titers in cattle naturally infected with Fasciola hepatica after anthelmintic treatment and during reinfection. Excretion/secretion products (ES) of the parasite were used as antigens for the dot-ELISA. IgG antibodies were no longer detectable by dot-ELISA, 4-6 months after nine animals received the first of three weekly doses of triclabendazole (15 mg kg(-1)) and were then maintained on a pasture free of F. hepatica metacercariae. Six fluke-free cattle began shedding F. hepatica eggs 3-6 months after grazing a pasture contaminated with metacercariae of the parasite. A detectable increase in dot-ELISA IgG antibody levels was observed 2-4 weeks after natural reinfection by grazing a similar pasture contaminated with F. hepatica metacercariae. The usefulness of the dot-ELISA system to diagnose chronic infection by serology is complicated by previous treatment against the parasite. It is concluded that the ES antigens can be useful to detect early infection of cattle with F. hepatica in a dot-ELISA system     

The effect of Taenia hydatigena infection on existing and concurrent infections of Fasciola hepatica in sheep

Sheep given a primary infection of Fasciola hepatica were challenged 18 weeks later with Taenia hydatigena or F hepatica, or both parasites together, or were not challenged. At the same time, control sheep were infected with T hydatigena and/or F hepatica separately or concurrently. All sheep were killed seven weeks after challenge and the number of cysts and flukes counted. Challenge infection with T hydatigena did not affect the numbers of flukes recovered from either primary or challenge F hepatica infections. On the other hand, the numbers of cysticerci were reduced in sheep previously infected with F hepatica but not in those given T hydatigena and F hepatica concurrently.     

Responses of Fasciola hepatica infected sheep to various infection levels

The response to Fasciola hepatica was studied in sheep infected with 5, 30, 150 metacercariae. The animals were necropsied 12 weeks post-infection (p-i) for counting and measuring flukes. Cellular and humoral responses were detected by peripheral eosinophil count, peripheral blood lymphocyte proliferation with excretory-secretory products (FhESP) and ELISA. All sheep were infected at necropsy except one sheep which was infected with 5 metacercariae. Mean parasitic intensities were 40%, 44% and 27% of the infection dose in sheep infected with 5, 30, 150 metacercariae respectively. FhESP-specific lymphocyte responses of the 3 infected groups were significantly enhanced in weeks 3 and 4 p-i (p < 0.05). The kinetics of the specific humoral response were similar for the 3 infected groups but the antibody level was significantly lower in animals infected with 5 metacercariae than in the 2 other infected groups from week 5 p-i to week 12 p-i (p < 0.05). Peripheral eosinophil count was significantly enhanced (p < 0.05) in infected groups. The numbers of peripheral eosinophils were significantly different between the 3 infected groups in week 3, 4 and 6 p-i and were related to infection level. These results confirm that sheep are highly susceptible to F. hepatica infection, even when infection pressure is very low. Peripheral eosinophilia was dependent of the infection level. The immune response was similar in sheep infected with various numbers of flukes.     

Pathophysiological and parasitological studies on Ostertagia ostertagi infections in calves

Friesian calves given a low level infection of the abomasal parasite Ostertagia ostertagi over a six week period displayed a mild diarrhoea with high faecal egg counts and elevated plasma pepsinogen values. At necropsy on day 23 abomasal lesions characteristic of ostertagiasis were widespread. At 42 and 84 days oedema and congestion were also prominent. Total worm burdens on days 23 and 42 were similar but a marked decrease had occurred by day 84. Feed digestibility and nitrogen economy were not markedly affected but radioisotopic measurements demonstrated an increase in albumin disappearance and catabolic rates, and plasma faecal clearance during the course of the infection. Prior administration of a morantel sustained release bolus to a group of similarly infected calves reduced the total worm burdens to less than 50 per cent of those recorded in the infected calves on days 23 and 42 and this fell to 3 per cent on day 84. Abomasal damage and the adverse pathophysiological changes associated with infection were prevented in this group.     

THE EFFECT OF PRIOR INFECTION WITH FASCIOLA HEPATICA ON THE RESISTANCE OF SHEEP TO THE SAME PARASITE

The development of flukes that resulted from a challenge dose of 175 F. hepatica metacercariae was compared in 4 groups of sheep that were maintained under grazing conditions on fluke-free pasture. One group had been previously uninfected and all subgroups of each of the other 3 groups had been exposed to one of a range of previous doses of metacercariae. The preliminary infections of 2 of the 3 groups had been terminated respectively after 7 and 14 weeks. The third previously infected group had received its preliminary infection as 2 doses of metacercariae, 7 weeks apart. The latter infection had been terminated after 14 weeks. No appreciable differences in the mean numbers, length, prepatent period or fecundity of flukes, established as a result of the challenge dose of metacercariae, were detected between the control group and the 3 previously infected groups. It was concluded that under the conditions of the present experiment no evidence was detected that would suggest that previous infection with F. hepatica conferred any significant resistance to a future challenge.     

Effect of Ostertagia ostertagi on lamb performance and cross resistance to O circumcincta

Fifteen worm-free lambs (two-and-a-half to three months or four to four-and-a-half months old) were infected with 3500 or 4000 Ostertagia ostertagi larvae on five days each week for six weeks, and their performance compared to that of controls. Eleven lambs were killed after eight weeks and four were challenged with O circumcincta to determine whether any cross resistance had developed. A feature of the O ostertagi infection was the considerable variation in response. Overall liveweight gain was lowered by 24 per cent in the two-and-a-half to three-month-old infected lambs, mainly due to reductions of 27 to 40 per cent in four of the seven lambs. There was no consistent effect in the older lambs. The worm populations consisted mainly of early fourth stage larvae and developing worms, but a small percentage reached sexual maturity and these produced a low faecal egg count (1 to 63 eggs per gram). Numerous intraluminal refractive crystals were present in the gut of both adult worms and developing stages, possibly reflecting degenerative changes. Hypertrophy of the abomasal mucosa with patchy loss of differentiation was a feature of the infection, and in four lambs serum pepsinogen concentrations were elevated. Exposure to O ostertagi did confer some protection against challenge with O circumcincta in that worm counts were reduced to about 60 per cent of those in controls, although no increase was observed in the numbers of arrested larvae. The successful passage of O ostertagi through young lambs could be important in mixed or alternate grazing systems by providing a reservoir of infection for the alternate host.     

Experimentally induced ostertagiosis in rabbits inoculated with Ostertagia ostertagi of bovine origin

Weanling specific-pathogen-free rabbits were orally inoculated with 50,000 Ostertagia ostertagi 3rd-stage infective larvae cultured from bovine feces and were killed 42 days after inoculation. Two preliminary trials were done, using conventionally reared weanling rabbits inoculated with 2,500, 5,000, and 50,000 O ostertagi 3rd-stage larvae and dexamathasone in 1 of the trials; the rabbits were killed 14 and 28 days after inoculation. Fecal egg counts were monitored, and worm burdens were determined. The pH of the gastric contents was measured at necropsy. Recoveries of the parasite from the gastric mucosa comprised early 4th-stage larvae and larvae that had developed beyond the early 4th stage. The maximum worm burden established at 42 days after inoculation was 1,668 immature nematodes or 3.34% of the inoculum. Gross and microscopic gastric lesions were present. Gross nodular mucosal elevations, which tended to be confluent in the cardiac region, were observed. Microscopic changes were principally mucosal hyperplasia, focal lymphocyte accumulation with moderate glandular dilation, and slight variable eosinophil and plasma cell accumulation. The pathologic changes had characteristics similar to those in cattle infected with O ostertagi and other conditions characterized by gastric mucosal hyperplasia.