The establishment and development of Haemonchus contortus in goats

Twelve goats were inoculated with 40,000 third-stage Haemonchus contortus larvae and two were killed on each of Days 4, 7, 11, 14, 18 and 21 after inoculation (DAI). The number of worms that established, and the site of development were recorded. More worms established in the fundic, than in the middle or pyloric thirds of the abomasum. Early development occurred within the mucosa; emergence into the lumen started between 7 and 11 days after infection. By 4 DAI, all worms had completed the third moult to the L4 stage. At 11 DAI the majority of the worms were adults. A mean of 13.2% of the female worms had eggs in their uteri at 18 DAI; by 21 DAI more than half of the female worms had eggs in their uteri. The development of H. contortus was essentially similar to that described in sheep.     

Distribution and pathogenicity during development of Camelostrongylus mentulatus in the abomasum of sheep

Fifteen lambs were each inoculated orally with 150 000 infective Camelostrongylus mentalutus larvae. Lambs were killed 3, 4, 8, 12, 16, 21 and 28 days after infection (DAI). Feed consumption and plasma total protein levels of the lambs killed 21 and 28 DAI did not differ significantly from those of uninfected controls. Three and 8 DAI most larvae were recovered from the fundic mocusa, but the intensity of establishment of larvae per gram of tissue was greatest at the cardia and diminished gradually toward the pylorus. Emergence of worms from the cardia and fundus occurred between 8 and 12 DAI, and a proportion of the lume-dwelling population shifted into the antral area, while the number of worms in the antral mucosa remained stable. Nodularity of the mucosa, mucous metaplasia of fundic epithelium, and vascular permeability as indicated by colloidal carbon label, were evident 3–4 DAI, and were most severe 8–12 DAI, when erosion of and inflammatory cell effusion from heavily infected mucosa was marked. pH and Na⁺ concentration in abomasal content were elevated at all times after infection, with peak values 12 DAI. Plasma pepsinogen levels in infected lambs were elevated beginning 3–12 DAI.     

Morphogenesis of Trichostrongylus rugatus and distribution during development in sheep

Morphogenesis of Trichostrongylus rugatus was examined in 16 sheep experimentally-infected with 120 000 third-stage larvae and killed 2, 4, 6, 8, 10, 12, 16 and 20 days later (DAI). Third stage larvae moulted between Days 4 and 6, and fourth stage larvae moulted on Day 10 after infection. Four sheep first passed eggs in the faeces between Days 16 and 18 after infection. Rate of growth of larvae was constant between 2 and 10 DAI followed by a period of rapid growth from 10 to 16 DAI. Major features of larval development are described. Nematodes were largely restricted to the first 6 m of gut with 71% of worms occurring in the first 3 m.     

Infection of goats with Haemonchus contortus and Trichostrongylus colubriformis: histopathology and pH changes.

Two groups of goats were experimentally infected with Haemonchus contortus and Trichostrongylus colubriformis, and killed at various days after infection (DAI). The percentage of worms that established in the abomasum and the small intestine was low. At necropsy, abomasums from infected goats had thickened walls and oedematous folds. At 7 DAI there was an initial infiltration of eosinophils and some neutrophils which tend to increase with age of infection. The mean pH of the abomasum in goats infected with H. contortus was 5.43 (range 5.3-5.7), while that of the control goats was 3.30 (range 2.8-3.7).     

Susceptibility of fourth-stage Ascaris suum larvae to fenbendazole and to host response in the pig

Fenbendazole given at the rate of 2.5 g/kg of feed for 3 days had 100% efficacy against 4th-stage Ascaris suum larvae in 8 pigs. Eight control pigs had a total of 108 A suum. In 6 pigs infected 3 times with 3rd-stage A suum larvae and treated with fenbendazole after the larvae molted to the 4th stage, the challenge exposure-derived population was decreased by 64%. Similar sequential infections in 6 pigs similarly infected, but not treated with fenbendazole, decreased the challenge exposure-derived population by 98%; however, developing and/or adult worms from the vaccinating infections were present.     

Infection and immune kinetics of Trichostrongylus axei in calves.

In one experiment, 21 calves were given daily oral immunizing inoculations of 1,000 infective larvae (L3) of Trichostrongylus axei, an abomasal nematode parasites, for 35 weeks. Calves were euthanatized in groups of three every 5 weeks to determine infection kinetics. Worm populations steadily increased through week 30, but the percentage of total inoculum that became established was about the same through week 30. At week 35, the number of worms dropped markedly. In a second experiment, 27 calves given daily oral inoculations of infective larvae were allotted to three groups comprised of three subgroups each: (A), challenge-exposed vaccinated; (B), nonchallenge-exposed vaccinated; and (C), challenge-exposed nonvaccinated. Calves of subgroups A and C were given single challenge inoculums of 200,000 L3 after the 10th, 20th, and 30th weeks. All calves were necropsied 35 days after challenge exposure. When immunity was determined from the equation: [(No. of worms in [(B + C)-A])/No. of worms in [B + C])] X 100, immunity was 35% at 10 weeks, 52% at 20 weeks, and 100% at 30 weeks.     

Lysosomal activity in enterocytes in the small intestine in piglets experimentally infected with Isospora suis

The lysosomal activity of enterocytes of the small intestine mucosa was investigated in gnotobiotic and conventional piglets experimentally infected on the first day after birth (DAB) by the oocysts of the coccidia Isospora suis. A method of the proof of beta-D-glucuronidase (EC.3.2.1.31.) activity was used to demonstrate lysosomes. The piglets were infected by different infection doses of oocysts (100,000 oocysts in gnotobiotic piglets and 200,000 oocysts in conventional piglets). In the gnotobiotic infected piglets the activity of beta-D-glucuronidase in enterocyte lysosomes was investigated in the period from day 3 to day 11 after infection (DAI) and in the infected conventional piglets in the period from day 2 to day 10 after infection. Comparing the control piglets, the group of gnotobiotic piglets at the age of 2-5 days and the group of conventional piglets at the age of 4-7 days, the higher activity of beta-D-glucuronidase was demonstrated in the lysosomes of intestinal mucosa enterocytes in the gnotobiotic control piglets (+5.30 of the average density value, Dx). In the infected gnotobiotic and conventional piglets the pattern of beta-D-glucuronidase activity was found to have three stages in the course of this infection. Two stages can be characterized by a great increase in the enzyme activity (DAI 3-9 in gnotobiotic piglets, DAI 2-3 and 7-9 in conventional piglets. The third stage, which is manifest mainly in the conventional infected piglets, is characterized by a marked decrease in the activity of beta-D-glucuronidase, reaching the level of control findings (DAI 10 and mainly 11 in gnotobiotic piglets. DAI 4-6 and 10 in conventional piglets). A topographical picture shows that the two stages of increase and the stage of beta-D-glucuronidase activity decrease occur in the whole small intestine without any predisposition defect of the enzyme in the different sections of the small intestine.     

Development of immunity to incoming radiolabelled larvae in lambs continuously infected with Trichostrongylus vitrinus

Infective third stage larvae (L3) of Trichostrongylus vitrinus were radiolabelled with 75 selenium by a method which did not affect their viability. Three groups of six-month-old lambs were infected daily with 1000 L3 for four, eight and 12 weeks, respectively. After each period, one of those groups (n = 5) and a group (n = 4) of worm-free controls were challenged with three consecutive daily doses of 1000 radiolabelled L3, killed 10 days after the first dose, and their worm burdens examined. After four weeks of continuous infection partial immunity to the establishment of challenge L3 was apparent, and by eight and 12 weeks, with the exception of one sheep, there was almost total resistance to incoming worms. Immunity was also expressed as an inhibition of the development of established worms.     

Experimental toxoplasmosis in buffalo calves

Eight buffalo calves were inoculated with infectious oocysts of a virulent strain of Toxoplasma gondii, five with 1 × 10⁵, and the other three with 5 × 10⁵ oocysts each while two more were kept as non-inoculated controls. Infected animals developed pyrexia, anorexia, conjunctivitis and dyspnoea. The clinical symptoms, haematological changes and serological response were of a low order. The appearance of haemagglutinating antibodies at significant levels (? 1:64) was first observed at 21 days after inoculation (DAI), peaked at 42 DAI and rapidly declined after 63 DAI. The inoculated calves were killed (or died) at intervals from 11 DAI to 110 DAI. The organism could be recovered from several times of a calf that died 11 DAI, but was rapidly removed subsequently. Only the lymph nodes were infective till 32 DAI. Parasitism of the retina was demonstrated in one calf. The significance of these findings is discussed.     

Effect of Ostertagia circumcincta excretory/secretory products on gastrin release in vitro.

It has been suggested that parasite excretory/secretory (ES) products may be capable of direct stimulation of gastrin secretion and of contributing to the hypergastrinaemia typical of abomasal parasitism. Ostertagia circumcincta ES products were tested on an ovine antral mucosal preparation which had been developed for a pharmacological study of gastrin secretion in the sheep. Its responsiveness to chemical stimulation was established by stimulation with amino acids and amines: tryptophan (0.1-5 mM) and phenylalanine (10-100 mM) stimulated gastrin release (151-160 and 117-129%, respectively), whereas glycine (0.1-100 mM) was without effect; ammonium sulphate, but not sodium sulphate, stimulated gastrin release in concentrations from 1mM (122%) to 50mM (148%). ES products were prepared by incubation of exsheathed third-stage larvae (L3) or parasites recovered on Day 8 p.i. (L4), Day 12 p.i. (10% L4, 90% immature adults), Day 21 p.i. (5% L4, 30% immature adults, 65% adults), Day 22 p.i. (20% immature adults, 80% adults), Day 30 p.i. (adults) and Day 35 p.i. (adults), or a mixed-age parasite population. Worms were recovered from agar and incubated in either distilled water or Hank's balanced salt solution (HBSS) adjusted to pH 2.5, 3.5, 4.5, 5.0 or 7.4. HBSS pH 7.4 was also prepared with antibiotics, without glucose, and with antibiotics but without glucose. Survival of Day 21 and 35 worms and exsheathed L3 in water or in a series of HBSS adjusted to pH 2.5, 3.5, 4.5, 5.0 or 7.4 was assessed from the percentage of motile parasites. L3 slowly became immotile over several days except in HBSS pH 2.5, in which survival was reduced, whereas adult worms did not tolerate incubation at 37 degrees C in water or HBSS at pH 2.5, retained motility for about 2 days at pH 3.5, but survived well at pH 4.5 and above. Incubates prepared from all stages of O. circumcincta, both in media favourable and unfavourable for parasite survival, failed to stimulate consistently the secretion of gastrin by tissue from both parasite-naive and previously exposed sheep, whereas a considerable number of incubates were significantly inhibitory. The inhibitor may not be produced by the nematodes, but by contaminating abomasal or environmental microflora, as inhibitory activity was predominantly generated by prolonged incubation, it was less potent when glucose was omitted and was not present in media containing antibiotics. This study did not find evidence for a gastrin stimulant in O. circumcincta ES products, but did demonstrate the acid intolerance of adult worms and suggests that abomasal microbes may be capable of modulating the secretory activity of the host digestive tract.     

Enhanced protection against the migratory phase,but defective protection against the intestinal phase of Strongyloides venezuelensis infection in cotton rats,Sigmodon hispidus

The protective capacity of the cotton rat, Sigmodon hispidus, against the migratory and intestinal phases of Strongyloides venezuelensis infection was examined. After subcutaneous infection with infective larvae (L(3)), adult worm recovery rates from male and female animals on Day 71 were only 0.10% and 0.06% of initial dose, respectively. To determine whether this enhanced protection was expressed during the migratory phase or the intestinal phase, larval recovery from the lungs of cotton rat was evaluated 3 days after subcutaneous L(3) infection. The larval recovery rate was only 0.5% of initial dose and about 40-fold lower than that from control mice. Protection in the intestine was also evaluated after intraduodenal implantation of adult worms. About 30% of implanted worms became established and worm burden remained constant until Day 28. Despite a high worm burden on Day 28, EPG was about 25-fold lower than the peak count. To evaluate expulsive capacity and monitor the cellular responses in the intestine of cotton rats, adult Nippostrongylus brasiliensis worms were implanted in addition to S. venezuelensis. Cotton rats were unable to expel adult S. venezuelensis worms, even after 21 days of observation. Although the number of mucosal mast cells increased significantly, the intraepithelial migration of mast cells was not observed. In contrast, N. brasiliensis was expelled by Day 6 in association with goblet cell hyperplasia. These results suggest that in cotton rats, the defective intestinal protection against adult S. venezuelensis worms results from dysfunction of mucosal mast cells.     

The therapeutic efficacy and prophylactic activity of doramectin against Dictyocaulus viviparus in cattle

SUMMARY Three groups of 8, 4-month-old male Jersey or Jersey-cross calves were infected with 2400 Dictyocaulus viviparus L₃ larvae and either left untreated or injected subcutaneously with 200 μg/kg doramectin 5 or 25 days after infection (DAI). Lungworms were found in all untreated cattle (geometric mean = 49) at necropsy 39 or 40 DAI. None was found in any of the treated cattle. In a second experiment, groups of 6, 8-month-old calves were untreated or injected with 200 μg/kg doramectin 28, 21 or 14 days before each calf was challenged with 2700 D viviparus larvae. Lungworms were recovered at necropsy 32 to 34 DAI. The geometric mean worm burden in the untreated cattle was 550. This was reduced by 100%, 99.5% and 94.1% in calves treated with doramectin 14, 21 or 28 days, respectively, before infection. It was concluded that doramectin is a highly effective anthelmintic against D viviparus adult or L₄ infections of cattle, and that reinfection of treated cattle will be significantly reduced for at least 28 days after treatment.     

Efficacy of milbemycin oxime against fourth-stage larvae and adults of Ancylostoma tubaeforme in experimentally infected cats

The efficacy of milbemycin oxime against fourth-stage (L4) larvae and adults of Ancylostoma tubaeforme was investigated in a trial involving 24 young domestic shorthair cats. The animals were inoculated with approximately 300 infective stage three (L3) larvae and divided into three groups. After 12 days, eight cats (group 1) were treated with medicated tablets containing 4 mg milbemycin and 10 mg praziquantel to test the efficacy against L4 larvae; eight cats in group 2 were treated with the same tablets after 33 days to test the efficacy against adult worms; and eight cats in group 3 were treated with a placebo tablet. Faecal egg counts were determined periodically in each cat and after 40 or 41 days the number of worms in each animal was determined postmortem. The egg count reduction was determined by comparing the geometric mean numbers of eggs per gram of faeces in the placebo and medicated groups, and the worm reduction by comparing the geometric mean numbers of worms. The egg count reduction was more than 99 per cent in both treated groups, while the number of worms in groups 1 and 2 were reduced by 94.7 per cent and 99.2 per cent, respectively.     

Abortion and death in goats inoculated with Sarcocystis sporocysts from coyote feces

Ten 75- to 105-day-pregnant does each were inoculated orally within 1 million (2 does), 10,000 (4 does), or 1,000 (4 does) sporocysts of Sarcocystis from coyote feces. Two does not inoculated with sporocysts served as controls. The 2 does inoculated with 1 million sporocysts died from acute sarcocystosis 21 and 22 days after inoculation (DAI), and each had 2 dead fetuses. The 4 does inoculated with 10,000 sporocysts were ill 19 to 33 DAI but survived; 1 aborted at 33 DAI, 1 had a live kid that died within 2 hours of birth 31 DAI, 1 aborted 2 dead fetuses 23 DAI, and 1 had a normal kid 56 DAI. The 4 does inoculated with 1,000 sporocysts and the 2 control does remained clinically normal and had normal kids. Does and their offspring were killed within 24 hours of parturition, and their tissues were examined histologically and microbiologically. Meronts of Sarcocystis were found in the maternal placenta of does inoculated with 1 million sporocysts. Sarcocystis was not found in the placenta, fetuses, or tissues of kids from does inoculated with 10,000 or 1,000 sporocysts, or from control does. Other abortifacient agents were not found in the placenta, fetuses, or kids from any does.     

Morphometric analysis of the activity of alkaline phosphatase in the small intestine of piglets infected with the coccidium Isospora suis

Gnotobiotical one-day-old piglets were infected with 100,000 Isospora suis coccidia oocysts, and were immediately killed. In piglets killed on the 3rd to 11th day after infection (DAI), the morphometric analysis of alkaline phosphatase activity was performed in the area of the microvillus zone of small intestine. In the control 2-7 days old animals, the small intestine was not equally supplied with alkaline phosphatase. In duodenum the activity reached 88.37 per cent of the active length of absorbent surface (% LAac), in the middle jejunum 95.98 per cent LAac, in the dorsal jejunum 78.63% LAac and the ileum 90.55 % LAac. The width of the active area was more balanced and ranged from 5.003 microM in the ileum to 6.129 microM in the dorsal jejunum. In infected gnotobiotical piglets the lowest activity was found out on the 3rd to 4th DAI, with a greater decline on the 9th day after infection. The range from 25.99 to 40.50 per cent LAac with minimum in the duodenum and maximum in the ileum was observed on the 3rd DAI. In the middle and dorsal ileum the activity was nearly equal (28.34 and 27.69 per cent LAac). nI the dorsal jejunum a moderate increasing was up to 47.13% LAac on the 4th DAI, with the exception of the ileum, where the activity of alkaline phosphatase decreased to 24.96% LAac. On the 9th DAI the activity of alkaline phosphatase was nearly equal in the whole small intestine (from 55.70 to 60.01% LAac) with the maximum in the middle jejunum. In the width of the reaction product a direct dependence on the total activity of alkaline phosphatase was evident only in the segment of the middle and dorsal jejunum and ileum, but merely on the period of the 3rd to 4th DAI. The lowest values were measured in the middle jejunum (0.982 micron on the 3rd DAI and 0.709 micron on the 4th DAI). No dependence was observed between the total activity and the reaction product in the middle jejunum (0.982 micron on the 3rd DAI and 0.709 micron on the 4th DAI), there was no general stabilisation of the activity of alkaline phosphatase.     

Effect of repeated oxfendazole treatments on small strongyle infections in Shetland ponies

Overwintering of horse cyathostomes as inhibited third stage larvae (L3) and the effect of repeated oxfendazole (OFZ) treatment on strongyle infections were studied in an experiment with two groups of three Shetland ponies. Both groups were grazed together from May 28 to November 11, 1986 and subsequently housed. Treatments with 10 mg OFZ kg-1 were given on May 26, July 1 and July 28 and again one week before each group was necropsied in December and April, respectively. Worm populations of both groups were dominated by inhibited early L3. The proportion of fourth stage larvae (L4) was significantly higher in April than in December. The faecal egg counts showed that the OFZ treatment in May was more effective than those in July. This resulted in high pasture larval counts from the end of August onwards. The final treatments revealed a low efficacy against L3 and L4 but not against adult worms.     

捻转血矛线虫ES24抗原基因的克隆与表达特性分析

根据捻转血矛线虫ES24抗原基因序列(U64793.1)设计1对特异性引物,用RT-PCR方法扩增出大小约为670 bp的DNA片段。将该DNA片段克隆到pMD18-T载体后进行序列测定和分析,结果发现该基因与GenBank中已知的捻转血矛线虫24 ku ES抗原基因的相似性达96%～98%。将该基因的开放阅读框插入pET28a(+)载体中,获得原核表达质粒pET28/ES24,并转化大肠杆菌BL21。重组细菌用IPTG诱导,经SDS-PAGE分析,结果表明该基因获得了表达,重组蛋白分子量大小约为25 ku。用实时荧光定量PCR技术对该基因在捻转血矛线虫的虫卵、第3期幼虫、雌虫和雄虫等不同发育阶段、不同性别虫体内的表达情况进行了定量分析,结果显示ES24基因在雄性成虫中表达量最高,雌虫和虫卵其次,在第3期幼虫中表达最低。     

In vitro development of cyathostomin larvae from the third stage larvae to the fourth stage: morphologic characterization, effects of refrigeration, and species-specific patterns

A mixed population of equine cyathostomin (Nematoda, Strongyloidea) infective third stage larvae (L3) was cultured in vitro using a cell-free medium. Some L3 were cultured immediately after Baermann collection from fecal cultures, while others were kept in water at 4 °C for 7 days before initiating the in vitro cultures. Cultures were examined daily for viability. At days 2, 7, 14 and 21 larvae were collected for identification of developmental stage and morphological changes, using both light and scanning electron microscopy. Larvae were classified as early L3 (EL3), developing L3 (DL3), late L3 (LL3) and fourth stage larvae (L4) on the basis of morphological features. Viability remained high throughout the entire study period in cultures of both non-refrigerated (84.7%) and refrigerated (77.4%) larvae. However, viability of the non-refrigerated was significantly greater from 7 through 21 days of culture. Significant differences were also observed in the percentage of DL3 between the non-refrigerated and refrigerated larval cultures by day 7. The highest percentage of DL3 larvae (22.5%) was reached at the end of study in those larvae that were not previously refrigerated. The data suggests that prior refrigeration decreases viability and slows L3 development. At day 21 LL3 larvae were only a small percentage of the DL3: 6.9 and 5% in non-refrigerated and refrigerated cultures, respectively. Few of these larvae freed themselves from the L3 cuticle and moulted to L4 stage. Characteristics of individual species in vitro developmental patterns were determined by the molecular identification of individual larvae in pools of larvae randomly collected at days 0 and 21. Seven species (Coronocyclus coronatus, Cylicostephanus goldi, Cylicostephanus longibursatus, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus ashworthi, Petrovinema poculatum) were identified in the day 0 pool. The greatest tendency to develop in vitro was shown by the genus Cylicostephanus with the species C. goldi and C. longibursatus that developed to the LL3-L4 stages. C. nassatus, C. ashworthi and C. coronatus did not progress in their development beyond the EL3 stage, while no apparent signs of development were registered for C. catinatum.     

Effects of doxycycline on early infections of Dirofilaria immitis in dogs

The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L(3) and L(4) of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L(3) and 200 B. pahangi L(3) by SC injection. Dogs received doxycycline on Days 0-29 (Group 1); Days 40-69 (Group 2); or Days 65-94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218-222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0-36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25-41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218-222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.     

The response of Nigerian West African Dwarf goats to experimental infections with Haemonchus contortus

One option for controlling haemonchosis in warm pastoral regions is improvement of resistance by selective breeding. Variation in acquired immunity to H. contortus and immunological correlates of infection were studied in West African Dwarf (WAD) goats. Following exposure to 5000 L3, 63 per cent of the inoculum established but 77 per cent of established worms were expelled by week 5. All infected animals were anaemic (day 14). When exposed to 2000L3, 36 per cent of the inoculum was still present (day 35) with no loss by day 49. Persisting primary infection worms survived a superimposed challenge (day 35), but their growth was slowed and resistance to challenge was significant. Most goats showed eosinophilia and parasite-specific IgG responses to primary infection, but only eosinophilia increased after challenge. No consistent associations were found between parasite burden and any immunological measures of infection, but parasite egg counts showed considerable variation. Overall, our results suggest that resistant genotypes exist among the WAD goat population.