The establishment and development of Trichostrongylus colubriformis in goats

Twelve goats were inoculated with 20,000 infective larvae of Trichostrongylus colubriformis 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. Most of the worms established within the first 3 m of the small intestine. There was little relocation or loss of nematodes after establishment. The worms started to migrate from the mucosa to the lumen 11 days after infection. At 4 DAI, 63% of the worms were still at the late L3 stage; the remainder of the worm population had completed the third moult to the L4 stage. The population at 11 DAI comprised largely young adults. When 21 DAI was reached, about 57% of the female worms had eggs in their uteri.     

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).     

Evaluation of Haemonchus contortus infections in sexually intact and ovariectomized ewes

Three experiments were conducted to investigate experimentally the occurrence of periparturient nematode egg rise in ewes and the hormonal modulation of Haemonchus contortus infections. In the first experiment, fall-bred and winter-bred pregnant (n = 4 and 14, respectively) and nonpregnant (n = 5 and 29, respectively) ewes were treated with anthelmintic and were pastured together on fields that were contaminated with H contortus. Three weeks before lambing, all ewes were placed in concrete pens; fecal egg counts for the winter-bred group were obtained on alternate days. Pregnant and lactating ewes had significantly larger numbers (P less than 0.01) of H contortus eggs than did the nonpregnant controls 1 week before and after lambing. Lactating, fall-bred ewes had significantly (P less than 0.01) more adult worms in their abomasum through natural acquisition than the nonpregnant controls. In the second experiment, fall-bred and winter-bred, helminth-free, pregnant (n = 4 and 8, respectively) and nonpregnant (n = 3 and 15, respectively) ewes were inoculated on 5 alternate days, beginning 70 days after breeding with 20,000 infective H contortus larvae. The ewes were maintained on concrete pens throughout pregnancy. Fecal egg counts were significantly (P less than 0.05) greater in pregnant ewes, beginning 1 week before lambing until 1 week after lambing. Abomasums of lactating ewes from both lambing seasons yielded significantly (P less than 0.01) more adult worms at necropsy than nonpregnant ewes. In the third experiment, ewes were ovariectomized (n = 15) or sham-operated (n = 9); half of the control ewes were bred.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

THE ROLE OF INHIBITED LARVAE IN THE EPIDEMIOLOGY OF OVINE HAEMONCHOSIS

Three paddocks were contaminated with Haemonchus contortus eggs from early spring to mid summer by yearling sheep. In Paddock 1, all contamination was derived from worms that had been previously inhibited in their development. The contamination in Paddock 2 was produced only by infections recently acquired from pasture, and in Paddock 3 contamination was attributable to both sources. When the yearling sheep were replaced in mid-summer by newly weaned lambs, severe outbreaks of haemonchosis occurred on all paddocks. It was concluded that inhibited H. contortus larvae are capable of producing sufficient eggs, when they resume development, to initiate an outbreak of haemonchosis in susceptible sheep.     

Experimental infection of Black Belly and INRA 401 straight and crossbred sheep with trichostrongyle nematode parasites

Compared to INRA 401 lambs reared in France, Black Belly (BB) lambs reared in Guadeloupe (F.W.I.) were highly resistant to both primary and secondary experimental infection with Haemonchus contortus. To investigate this huge inter-breed difference, a nucleus flock of BB was constituted, and experiments were conducted to: (i) confirm this difference in lambs born in France, (ii) check whether it was similar for Trichostrongylus colubriformis and Teladorsagia circumcincta, and (iii) find out whether this difference was age-related. Forty BB lambs, 84 F1 lambs (BB siresxINRA 401 ewes) and 88 INRA 401 lambs born in two cohorts were used in an experimental design involving three host breeds, both genders and two age-groups (3.5- and 7-month-old when first infected). The limited availability of BB lambs made the study incomplete. Infection consisted of the administration of two doses of 10,000 infective larvae of one of the nematode species, separated by an anthelmintic treatment and an interval of 1 week before the second dose was administered. Fecal egg counts (FECs) were done on Days 28 and 35 after each infection; ewe lambs of the INRA 401 and F1 breeds were necropsied, the worm burden was established, the length of the female worms measured and the eggs in utero counted. For H. contortus and T. colubriformis, the FEC was lower in the BB than in the INRA 401 lambs, and the FEC found for the F1 lambs was intermediate. For T. circumcincta, only data for the F1 and INRA 401 lambs were available, and the FECs for these breeds were the same. In all three breeds, the FEC determined after the second dose was significantly lower than that found after the first dose in the ewe lambs, but not in the ram lambs. Infecting the lambs when they were 7- instead of 3.5-month-old significantly lowered egg excretion of both parasites in the F1 (P<0.0001), but not the INRA 401 lambs. Worm numbers in the F1 and in the INRA 401 ewe lambs confirmed the FEC data, many F1 lambs being free of T. colubriformis. The H. contortus female worms were shorter and had fewer eggs in utero in the F1 than in the INRA 401 lambs. A higher proportion of T. circumcincta was at the fourth larval stage in the F1 lambs. In conclusion, the BB breed is much more resistant than INRA 401 to H. contortus, with the F1 lambs being more like the BBs after the second dose. This difference was also found for T. colubriformis and, to a lesser extent, for T. circumcincta.     

Effective,appropriate and simple culture,egg hatching and cryopreserving of the nematode Cheilospirura hamulosa

1. Successful invasion by nematode parasites is associated with several factors including egg hatching at the right time in their hosts. To determine a simple and appropriate medium for culture and egg hatching of the highly pathogenic species of the Acuariidae family, Cheilospirura hamulosa were cultured in three different media. In addition the viability of C. hamulosa eggs was determined after storage in frozen infected gizzards.

2. Eggs removed from the uteri of the female worms in infected gizzards were pooled and washed in distilled water and screened under a stereo dissecting microscope. Eggs were counted and cultured in three different media, nutrient agar, normal saline 0.9% and Bearman, at room temperature. Additionally, 10 infected gizzards were kept at ?20°C for 2 and 8 months.

3. After 4–5 d there had been no growth in the nutrient agar medium, whereas 11% of the cultured eggs in the Bearman medium contained larvae 2–3 d after culturing. In 0.9% normal saline medium the two polar knobs appeared on the two poles of the eggs at 2 d post cultivation, and 74% of the eggs contained a larva on the third day. Mature larvae gradually exited from the eggs.

4. Eggs collected from female worms in gizzards frozen at ?20°C were cultured in the same three culture media at room temperature. Larvae were visible in the eggs after 2–3 d in the Bearman and 0.9% normal saline media and hatched thereafter.

5. The 0.9% normal saline medium is recommended for egg hatching and cultivation of C. hamulosa due for simplicity, efficacy and cost effectiveness. Moreover, freezing of the infected gizzards at ?20°C is proposed for long-term storage of the eggs.     

Biological parameters of Trichostrongylus colubriformis in Meriones unguiculatus

In a series of three experiments, 64 jirds, Meriones unguiculatus, were infected with 700 infective larvae of Trichostrongylus colubriformis for the study of several biological parameters of this laboratory host-parasite model. In jirds the third stage larvae of T. colubriformis were shown to reach the fourth larval stage by the Day 6 post-infection (PI). By Day 10 PI, all the worms harvested had reached the immature adult stage. All immature adult stages of T. colubriformis developed to sexually mature adult stage by Day 12 PI when the female worms showed developing eggs in their uteri. Developed eggs were observed in the uteri of females on Days 13 and 14 PI. The first eggs of T. colubriformis appeared in the faeces of jirds on Day 13 PI. The peaks of egg production were recorded between Day 21 and Day 31 PI. Immunosuppression of jirds infected with 700 L3, by administration of dexamethasone from Day 57 to Day 94 PI led to increased faecal egg count when compared to untreated controls. All the worms were located in the small intestine. The jirds of dexamethasone treated group harboured higher number of adult worms than those of the untreated group. The number of adult worms was significantly higher in the first part of the small intestine than in the three other similar parts of the small intestine. The coefficient of correlation between the faecal egg count and worm number on the day of necropsy of jirds ranged between r = 0.58 and r = 0.89. Patent infections in jirds were maintained till the end of experiment on Day 100 PI, indicating that in this host and unlike other laboratory hosts, T. colubriformis is responsible for long lasting infections similar to what happens in domestic ruminants. The results of the present study suggest that the jird is a suitable laboratory model to study various aspects of this host-parasite relationship.     

Evaluation of the phenotypic performance of a Red Maasai and Dorper double backcross resource population: indoor trickle challenge with Haemonchus contortus

Six F(1) Dorper (D) x Red Maasai (R) rams were mated to both D and R ewes to produce backcross lambs. These six double backcross resource families are being analysed to identify quantitative trait loci that may be controlling resistance to gastro-intestinal (GI) nematode parasites, mainly Haemonchus contortus. After assessing the phenotypic performance of the lambs following exposure to natural infections, the surviving lambs were drenched and moved indoors for an artificial challenge with H. contortus along with straightbred D and R lambs. A total of 1317 lambs were included in the analyses consisting of 523 3/4D, 580 3/4R, 87 D and 127 R. The D lambs were significantly and consistently heavier than R lambs and 3/4D were significantly heavier than the 3/4R lambs. The difference between the backcrosses was about half of that between the straightbreds. Resistance was assessed in terms of faecal egg counts (FEC) and total worm counts (TWC) at necropsy while packed cell volume (PCV) was used to assess resilience to weekly oral doses of 2500 infective larvae of H. contortus. No significant breed differences were observed for log transformed FEC (LFEC). A significant breed difference in PCV was recorded. The backcrosses had the higher values and while no differences were observed between the straightbreds, 3/4D had significantly higher PCV than the 3/4R. Despite the absence of breed differences in FEC the R and the 3/4R had significantly fewer worms than the D and the 3/4D. The D had significantly longer worms than the R and the 3/4D had significantly longer worms than the 3/4R. Worms recovered from D had more eggs than those recovered from R. Similarly worms from 3/4D contained more eggs than those from 3/4R. Thus, on a breed basis the breed with more worms had longer worms. In contrast, when, in a small part of the experiment two doses of larvae were used to check for any breed by dose interactions, worms from the low dose (and hence fewer worms) animals were longer. We postulate that in fast growing hosts like the D, worms also have a better potential for growth and reproduction than in hosts that have less potential for growth. The overall correlation coefficient between PCV and LFEC was -0.67 while that between LFEC and LTWC was 0.72. From these results it is clear that the R do not respond to the artificial challenge the way they do to natural infection suggesting that phenotyping of R and R cross lambs for purposes of selecting those that are resistant or susceptible is best done under natural challenge.     

捻转血矛线虫虫卵的大量分离纯化

笔者采用虫体取卵和孵化后感染性幼虫直接在培养瓶瓶盖内收集的方法,获得了大量纯净单一的感染性幼虫,并且感染绵羊成功。再对感染绵羊直肠采粪收集虫卵,就可收集到大量纯种单一虫卵,进行三期幼虫的大量孵化。该试验解决了在寄生性线虫分子生物学研究中需要大量单一虫种的纯化分离问题。     

The role of preparation technique, culture media and incubation time for embryonation of Heterakis gallinarum eggs

The importance of preparation technique, culture media and incubation time in the embryonation of the infective egg stages of the intestinal nematode parasite Heterakis gallinarum was studied. Mature H. gallinarum worms were isolated from the caeca of infected chickens and separated by sex. In a first experiment intact female worms were kept for the development of their eggs in four different media (0.5% formalin, 2% formalin, 0.1 N sulphuric acid, 0.1% potassium dichromate) and incubated under constant temperature (20-22 degrees C) for 2, 4, 6 or 8 weeks. Afterwards the body of the worms were ruptured and the numbers of unembryonated and embryonated eggs were determined using a McMaster egg counting chamber, and the percentage of embryonated eggs was calculated. After 8 weeks of incubation in 0.5% formalin, 0.1 N sulphuric acid or 0.1% potassium dichromate 27.6%, 26.7% and 29.4% of the eggs, respectively, embryonated into third stage larvae (p > 0.05). In contrast, incubation in 2% formalin resulted in an embryonation of 18.6% only (p < 0.05). In a second experiment H. gallinarum eggs were directly harvested from worm uteri and cultivated afterwards in different media (2% formalin, 0.1 N sulphuric acid, 0.1% potassium dichromate) at 20 to 22 degrees C for 6 weeks. An incubation of isolated eggs in 2.0% formalin or 0.1% potassium dichromate during 6 weeks resulted in a significantly higher percentage of embryonation in comparison to the incubation of intact worms (first experiment). The results suggest that preparation technique, media and time of incubation has an essential influence on the development rate of H. gallinarum eggs.     

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.     

Immunization of pigs against Ascaris suum by sequential experimental infections terminated with fenbendazole during larval migration

Three experimental infections of weaning pigs with 2000 embryonated Ascaris suum eggs each, 11 days apart, followed each time by fenbendazole treatment, produced a significant host response when compared with similar infected or uninfected control pigs as assessed by response to a subsequent challenge with 100 embryonated A. suum eggs. The response elicited from pigs treated with fenbendazole on either 2, 3, and 4 days or, 6, 7, and 8 days after each experimental infection was expressed as a reduction in the number of pigs with A. suum, in the number of worms per pig, in the weight of male and female worms, and in the length of male and female worms. No differences in average daily weight gain, feed-conversion efficiency or histology of lungs and liver were noted among the 4 treatment groups.     

Ascaris lumbricowes in lambs

Extract

A number of large nematodes identified as Ascaris lumhricoides were recovered from the small intestines of a line of lambs slaughtered in December, 1963. These parasites had grown to the usual size of mature A. lumbricoides recovered from pigs, and the uteri of female worms were full of eggs.     

Effects of the chitin synthesis inhibitor diflubenzuron on development of Ascaris suum and Haemonchus contortus

The potential of the chitin synthesis inhibitor diflubenzuron (DFB) to alter the development of the parasitic nematodes (Ascaris suum and Haemonchus contortus was investigated. DFB given orally (10 mg kg-1 per day for 30 days) to sheep inoculated with H. contortus infective larvae did not prevent the establishment of adults or affect fecal egg output. However, there was a significant (greater than 90%) decrease in the number of infective larvae recovered from fecal cultures derived from lambs harboring H. contortus adults that were treated with DFB. DFB did not affect egg hatching. Oral administration (10 mg kg-1 per day for 20 days) of DFB to swine harboring adult A. suum adults had no effect on the adult worm burden or on egg morphology, but eggs removed from worms obtained from DFB-treated swine contained less chitin than eggs removed from untreated control swine. DFB also inhibited chitin synthesis in vitro in the isolated reproductive tract of A. suum adults. These results indicate that DFB at high doses can inhibit the subsequent development of H. contortus larvae in the feces. Since H. contortus larvae lack chitin, DFB may act on these larvae by a mechanism independent of a direct effect on chitin synthesis.     

捻转血矛线虫重组H11抗原山羊免疫保护性试验

以重组H11-1、H11-2及H11-1与H11-2混合物分别免疫山羊后,ELISA检测发现血清抗体均在首次免疫后14 d达到较高水平,在第2次免疫后10 d达到高峰。用10 000只捻转血矛线虫第3期幼虫攻击山羊后,对不同免疫组山羊排出虫卵数、虫卵孵化率和成虫数的统计分析结果表明,免疫组与对照组间差异不显著,但免疫组雌虫数显著少于对照组,这对防治捻转血矛线虫病可能会起一定的作用。     

The effects of amount, timing and distribution of simulated rainfall on the development of Haemonchus contortus to the infective larval stage

Three studies were undertaken to determine the effects of amount, timing and distribution of simulated rainfall on the developmental success of Haemonchus contortus. Faeces containing H. contortus eggs were deposited onto pasture plots under a rainfall-activated retractable roof which eliminated incident rainfall. In October (spring) 2004 and January (summer) 2005, the effects of amount (6, 12, 18 or 24 mm) and timing (1, 4, 8 or 15 days post-faecal deposition) of a single simulated rainfall event was investigated via manual application of water to plots. More H. contortus pre-infective larvae (L1 and L2) developed under the d 1 simulated rainfall treatment than later treatments. There was no effect of rainfall amount on development in either experiment, and negligible development to infective larvae (L3). In February (summer) 2006, the effects of amount (12, 24 or 32 mm) and distribution (single event or three smaller but equal split events over 32 h) of simulated rainfall events was investigated with water applied via sprinkler. In this experiment L3 were recovered from the herbage in one-third of the plots harvested, however recovery was low (0.08% of eggs deposited) and there were no treatment effects. Recovery of L1 and L2 from faeces increased with simulated rainfall amount at d 4, and more L1 and L2 were recovered from the split distribution treatment at d 4. The results indicate that moisture conditions soon after faecal deposition are key determinants of H. contortus development success, with significant penalties on development when simulated rainfall was applied 7 days or more post-deposition, and when the duration of simulated rainfall was short. High rates of evaporation during both summer experiments resulted in rapid drying of the micro-environment and this appears to have limited development to L3.     

Transfer of adult Strongylus vulgaris via stomach tube

Patent infections with Strongylus vulgaris were established in 6 of 8 helminth-free ponies given 41 to 101 adult worms via nasogastric tube. The parasites were removed from the cecum and ventral colon and transferred within 1 to 2 hours of the death of the donor horses. Eggs were found in the feces of the recipients in 2 or 3 days; egg counts reached maximum, 28 eggs per gram of feces, at 4 weeks after ponies were inoculated. In 6 ponies euthanatized 3 to 7 weeks after parasitic transfers were done, 28% of the inoculated worms were found alive at necropsy. A 7th pony was maintained as a donor for establishing infections for chemotherapy trials and, although never passing more than 6 eggs per gram of feces, shed infective larvae over a period of 2 years.     

Embryonation and infectivity of Ascaris suum eggs. A comparison of eggs collected from worm uteri with eggs isolated from pig faeces

Ascaris suum eggs were collected from pig faeces or dissected from worms obtained from the same pigs. Eggs from the two sources were allowed to embryonate in 0.1 N H2SO4, in 1% buffered formalin or in tap water. The embryonation of the sulphuric acid and water cultures occurred at the same speed, while the formalin cultures developed slightly more slowly. By experimental inoculation of helminth-free pigs and subsequent counting of white spots in the livers and larvae in the lungs day 7 p.i., the infectivity of eggs dissected from worm uteri and embryonated in sulphuric acid (a normal laboratory procedure) was compared with that of eggs collected from faeces and embryonated in water (i.e. more naturally developed eggs). The results suggest that the two types of eggs were equally infective. For this reason the common practice of using Ascaris eggs dissected from worms for experimental infections might be acceptable.     

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。