Effects of high hydrostatic pressure on embryonation of Ascaris suum eggs

High hydrostatic pressure processing (HPP) has been shown to be an effective non-thermal means of inactivating microorganisms from various food products. Little information is available regarding the effects of HPP on metazoan parasites. Outbreaks of food-borne disease have been associated with importation of food contaminated with fecal material. Ascaris suum is used as a surrogate model metazoan parasite for the human roundworm, Ascaris lumbricoides, to study the effects of treatments on the inactivation of eggs in sludge. The present study was conducted to determine the effects of HPP on A. suum eggs. Unembryonated A. suum eggs were subjected to 138-552 megapascals (MPa) for 10-60s in a commercial HPP unit. Embryonation was induced after HPP treatments by incubating eggs in 0.01N sulfuric acid at room temperature. After 21 days, 100 eggs were examined per treatment using a light microscope and the percent of embryonated eggs was determined. Embryonation was induced in 38-76% eggs that were subjected to 138 and 270MPa. No embryonation was observed in eggs exposed to pressures of 241MPa or more for 60s or in eggs exposed to 276MPa for 10-30s. These results indicate that HPP treatment could be used to protect contaminated food items by inactivating A. suum eggs and may also have potential in reducing food-borne illness resulting from fecal contamination.     

Antigens in perienteric fluid of Ascaris suum as detected through antibodies in pigs orally inoculated with fully embryonated eggs

Perienteric fluid (Pf) of adult Ascaris suum was fractionated by ammonium sulfate precipitation, gel filtration or DEAE-cellulose chromatography, and sucrose density gradient centrifugation. Anti-sera (anti-EE) from pigs which were inoculated orally with fully embryonated eggs (EE) of A. suum were used in an indirect radioimmunoassay (IRIA) to determine which fractions of Pf reacted positively in the analyses at the lowest protein concentration. These fractions were considered to contain more potent antigens. Comparative IRIA were performed employing antisera (anti-Pf) produced by injecting Pf into pigs. Six out of 35 fractions reacted positively at ? 0.2 μg protein when anti-EE was used in the IRIA. Twenty-two out of 35 fractions reacted positively at ? 0.2 μg protein when anti-Pf was used. Five of the 6 fractions reacting positively with anti-EE also reacted with anti-Pf. A 76 000 dalton component appears to be one of the major proteins in the 6 fractions which react positively with anti-EE while components from 10 000–138 000 daltons were present in the various fractions reacting positively with anti-Pf at ? 0.2 μg protein.     

A comparison of modifications of the McMaster method for the enumeration of Ascaris suum eggs in pig faecal samples

The comparative efficacies of seven published McMaster method modifications for faecal egg counting were evaluated on pig faecal samples containing Ascaris suum eggs. Comparisons were made as to the number of samples found to be positive by each of the methods, the total egg counts per gram (EPG) of faeces, the variations in EPG obtained in the samples examined, and the ease of use of each of the methods. Each method was evaluated after the examination of 30 samples of faeces. The positive samples were identified by counting A. suum eggs in one, two and three sections of newly designed McMaster chamber. In the present study compared methods were reported by: I-Henriksen and Aagaard [Henriksen, S.A., Aagaard, K.A., 1976. A simple flotation and McMaster method. Nord. Vet. Med. 28, 392-397]; II-Kassai [Kassai, T., 1999. Veterinary Helminthology. Butterworth-Heinemann, Oxford, 260 pp.]; III and IV-Urquhart et al. [Urquhart, G.M., Armour, J., Duncan, J.L., Dunn, A.M., Jennings, F.W., 1996. Veterinary Parasitology, 2nd ed. Blackwell Science Ltd., Oxford, UK, 307 pp.] (centrifugation and non-centrifugation methods); V and VI-Gr?nvold [Gr?nvold, J., 1991. Laboratory diagnoses of helminths common routine methods used in Denmark. In: Nansen, P., Gr?nvold, J., Bj?rn, H. (Eds.), Seminars on Parasitic Problems in Farm Animals Related to Fodder Production and Management. The Estonian Academy of Sciences, Tartu, Estonia, pp. 47-48] (salt solution, and salt and glucose solution); VII-Thienpont et al. [Thienpont, D., Rochette, F., Vanparijs, O.F.J., 1986. Diagnosing Helminthiasis by Coprological Examination. Coprological Examination, 2nd ed. Janssen Research Foundation, Beerse, Belgium, 205 pp.]. The number of positive samples by examining single section ranged from 98.9% (method I), to 51.1% (method VII). Only with methods I and II, there was a 100% positivity in two out of three of the chambers examined, and FEC obtained using these methods were significantly (p<0.01) higher comparing to remaining methods. Mean FEC varied between 243 EPG (method I) and 82 EPG (method IV). Examination of all three chambers resulted in four methods (I, II, V and VI) having 100% sensitivity, while method VII had the lowest 83.3% sensitivity. Mean FEC in this case varied between 239 EPG (method I) and 81 EPG (method IV). Based on the mean FEC for two chambers, an efficiency coefficient (EF) was calculated and equated to 1 for the highest egg count (method I) and 0.87, 0.57, 0.34, 0.53, 0.49 and 0.50 for remaining methods (II-VII), respectively. Efficiency coefficients make it possible not only to recalculate and unify results of faeces examination obtained by any method but also to interpret coproscopical examinations by other authors. Method VII was the easiest and quickest but least sensitive, and method I the most complex but most sensitive. Examining two or three sections of the McMaster chamber resulted in increased sensitivity for all methods.     

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.     

Establishment of Ascaris suum in the pig: development of immunity following a single primary infection

Development of immunity after a single primary infection of Ascaris suum in pigs was investigated with regard to the worm population dynamics of a superimposed A. suum infection, host immune response and gross liver pathological changes. Group A was given a primary infection of 60,000 infective A. suum eggs and group B was left uninfected. Four weeks later both groups A and B were inoculated with 1,000 A. suum eggs, and subgroups were slaughtered 7, 14 and 21 days post challenge infection (p.c.i.). An uninfected control group C was slaughtered on day 21 p.c.i. The challenge worm recovery in group A was reduced compared to group B by 12%, 50% and 75% on day 7, 14 and 21 days p.c.i., respectively. In both groups was the expulsion of worms initiated between day 14 and 21 p.c.i. However, in group A the worms were recovered more posteriorly in the small intestine and 21 days p.c.i. the mean worm length was significantly shorter than in group B (p = 0.01). The results above were associated with significantly higher (p < 0.05) antibody response and higher eosinophil counts in group A compared to group B. The present results suggest that the larval growth and survival of a challenge infection are decreased, probably due to higher antibody and eosinophil attack during the migratory phase.     

Efficacy of nitroscanate against naturally acquired infection with Ancylostoma caninum, Dipylidium caninum, and Trichuris vulpis in dogs

Eighteen dogs with naturally acquired helminth infections were used to evaluate the efficacy of nitroscanate against Ancylostoma caninum, Dipylidium caninum, and Trichuris vulpis. Approximately 15 minutes before treatment, the dogs were given 100 to 200 g of canned dog food. Ten dogs were treated with nitroscanate (50 mg/kg of body weight, PO), and 8 dogs were given placebo tablets PO. The dogs were euthanatized and necropsied 10 days after treatment and helminths were recovered from the small intestine and cecum. On the basis of the number of worms recovered from treated dogs vs the number recovered from control dogs, we determined the efficacy of nitroscanate to be 99.6% against A caninum, 99.8% against D caninum, and 0% against T vulpis.     

In vitro evaluation of the ovistatic and ovicidal effect of the cosmopolitan filamentous fungi isolated from soil on Ascaris suum eggs

The ovicidal activity of seven fungal strains: Acremonium alabamense, Alternaria chlamydospora, Cladosporium herbarum, Fusarium solani, Paecilomyces variotii, Paecilomyces viridis and Penicillium verruculosum isolated from urban soil samples from Poland was determined in vitro. The fungal mycelium was co-cultured with Ascaris suum eggs on plates with 2% water–agar for 28 days. Eggs exposed and unexposed (control) to fungal mycelium were observed weekly by light microscopy and the percentage of malformed eggs were determined. The eggs were classified according to following parameters: type 1 – biochemical and physiological effect without morphological damage to the eggshell; type 2 – lytic effect with morphological alteration of the eggshell and embryo; type 3 – lytic effect with morphological alteration of eggshell and embryo with hyphal penetration and internal egg colonization. All examined species of fungi extended embryogenesis, but the retardation of embryonic development was varied and depended on the species. A. alabamense, A. chlamydospora and P. verruculosum exhibited very high inhibitory activity on A. suum egg development. The fungus-exposed eggs revealed morphological alternations in all stages of embryogenesis. Isolates of F. solani, P. variotii and P. viridis showed hyphal penetration and internal colonization of A. suum eggs (type 3 effect). No appressoria were produced and simple hyphal penetrations were most commonly observed. A. alabamense and P. verruculosum demonstrated morphological destruction, with eggshell destruction. The remaining fungi showed type 1 effect. The results demonstrated that examined strains of F. solani, P. variotii and P. viridis may be considered to be potential limiting factors of parasitic geohelminth populations.     

Persistent activity of doramectin and ivermectin against Ascaris suum in experimentally infected pigs.

A study was conducted to investigate the persistent nematocidal activity of two avermectins against experimentally-induced infections of Ascaris suum in swine. Seventy-two nematode-free cross-bred pigs of similar bodyweight were randomly allotted to nine treatment groups of eight pigs each. Eight of the groups were treated with injectable solutions containing 300 microg of doramectin/kg (IM) or 300 microg of ivermectin/kg (SC) either 0 (same day), 7, 14, or 21 days prior to an oral challenge of 50000 embryonated A. suum eggs. The ninth group (control) was challenged in parallel without any avermectin treatment. At 41 or 42 days after challenge, pigs were euthanatized and adult and larval stages of A. suum were collected from the gastrointestinal tract of each pig and counted. Both avermectins significantly (P < 0.0002) reduced nematode counts when given on the day of challenge (0 days prior), and the efficacy was 100% and 97.5% for doramectin and ivermectin, respectively. Doramectin given 7 days prior to challenge significantly (P < 0.0001) reduced nematode counts, and the efficacy was 98.4%. For all other avermectin-treatment groups, nematode counts were not significantly reduced compared to those in control pigs. These data indicated that anthelmintic activity of ivermectin against A. suum persisted for less than 7 days and the activity of doramectin persisted for more than 7, but less than 14 days.     

重组猪蛔虫抗菌肽cecropin P1在毕赤酵母中的表达及抑菌活性

根据已发表的蛔虫抗菌肽cecropin P1基因编码序列设计引物,采用RT-PCR方法从猪肠道蛔虫中扩增出cecropin P1基因,并将其插入到酵母分泌型表达载体pPIC9K中α-因子信号肽下游的SnaBⅠ和NotⅠ位点之间,构建成cecropin P1基因的酵母分泌型表达载体pPIC9K/cecropin P1。载体经SalⅠ酶切线性化,电转化到组氨酸缺陷型的酵母宿主菌GS115中,然后利用以葡萄糖为碳源的培养基（MD）和以甲醇为碳源的培养基（MM）筛选出组氨酸His＋型和甲醇利用正型（Mut＋）酵母重组体,再经G418加压筛选得到高拷贝cecropin P1基因的重组酵母。经PCR检测证明cecropin P1基因已整合到毕赤酵母的染色体中。重组酵母经摇瓶发酵培养和甲醇诱导,通过Tricine-SDS-PAGE检测,重组酵母能够分泌表达重组cecropin P1,同时表达的cecropin P1对大肠杆菌和金黄色葡萄球菌均有明显的抑菌活性。     

Production and distribution of immunoglobulin-bearing cells in the intestine of young pigs infected with Ascaris suum

Pigs of 10 days-1 month old were orally infected with eggs of Ascaris suum at different rates and inoculation schedules. Histological sections from various parts of the small intestines were prepared to observe the production and localization of immunoglobulin-bearing cells. Fluorescent antibody and immunoperoxidase staining methods were used to determine the number of IgM-, IgA- and IgG-producing plasma cells in the intestinal lamina propria. Significant increases in immunoglobulin-bearing cells were observed in those pigs which received single inoculations of A. suum eggs. Pigs infected every 2,4,8 and 10 days with 10,000-20,000 embryonated eggs showed numerical increases in IgM-bearing cells. Increases in IgA-bearing cells were noted in pigs which received the higher number of eggs every 8-10 days. Higher concentrations of IgA- and IgM-bearing cells were observed in the jejunal mucosa of infected pigs as compared to those in the duodenum and ileum.     

Presence of immunoglobulins and antigens in serum, lung and small intestine in Ascaris suum infected and immunised pigs

The immunodetection of local Ascaris suum antigens and local and systemic antibodies were analysed in pigs reinfected with eggs or immunized with the 14, 42 and 97 kilodalton (kDa) fractions from A. suum. Twenty-one Iberian pigs were divided in 7 groups of 3 pigs. Groups 1 and 2 were uninfected and challenge control groups, respectively. Groups 3 and 4 were infected weekly with increasing doses of A. suum eggs and Group 4 was additionally treated with pyrantel pamoate. Groups 5, 6 and 7 were immunised with the 14, 42 or 97 kDa fractions from adult worms, respectively. Groups 2-7 were challenged with 10,000 infective eggs. Animals of Groups 3 and 4 showed a pulmonary granulomatous reaction with moderate number of eosinophils and leukocytes, while Groups 5-7 presented higher number of cells, especially in animals immunized with the 42 kDa fraction. These immunized groups presented abundant deposition of Ascaris body fluid (BF) and body wall (BW) antigens as well as the 14 and 42 kDa fractions in the pulmonary and intestinal tissues, while lower deposition of antigens was observed in animals of Groups 3 and 4. The immunized pigs of Groups 5 and 6 showed the highest systemic IgG titres in serum and these antibodies were negatively correlated with the number of larvae recovered in the lungs, suggesting that the IgG response may have a protective function against the ascariosis. The highest concentrations of IgA-bearing cells were observed in animals of Groups 3 and 4 compared to the immunised pigs (Groups 5-7), suggesting that local IgA production may be involved in the protection against migrating larvae. The main localisations of IgA-bearing cells were the bronchial and peribronchial areas of lungs and the lamina propia of duodenum. Low numbers of local IgG-bearing cells were observed in all animals and no IgM-bearing cells were detected in the local tissues.     

西北部分地区猪蛔虫rDNAITS基因序列测定及遗传变异分析

为了摸清西北部分地区猪蛔虫内转录间隔区(ITS)的遗传变异特点,扩增了猪蛔虫ITS基因,进行测序,依据GenBank公布的ITS序列将测序结果截为ITS1、5.8S及ITS2三段,分析比对各段序列的差异性。结果显示,所有样品ITS序列长约1 000bp,其中ITS1、5.8S和ITS2的长度分别为450bp~453bp、159bp、272bp,种内差异分别为0~0.2%、0、0。基于ITS1的种系发育分析表明,23个猪蛔虫样品均位于同一分支,而与贝蛔属蛔虫分属两个不同分支。ITS1序列不能作为区分西北不同地区猪蛔虫的种内分子标记,但可以作为区分蛔属蛔虫与贝蛔属蛔虫的种间分子标记,研究结果为猪蛔虫的鉴定和分子流行病学调查提供了基础资料。     

苏云金芽胞杆菌伴胞晶体毒素对小鼠体内猪蛔虫三期幼虫的作用及其免疫组织化学定位

将培养好的感染性猪蛔虫卵以每只5×103个虫卵感染小鼠,于次日以不同方式(灌胃、肌肉注射、静脉注射和腹腔注射)注入苏云金芽胞杆菌伴胞晶体蛋白(insecticidal crystal proteins,Icps),对照组静脉注射BSA(小牛血清白蛋白),每只0.5mL,连续3d。小鼠于第6天剖杀,肝脏分离幼虫,计算减虫率。取肝脏,以4%多聚甲醛固定48h,制作石蜡切片,应用免疫组织化学SABC法定位蛔虫幼虫体内的Icps。研究结果显示,静脉注射晶体蛋白毒素效果最好,减虫率达到72.7%;肌肉注射、灌胃、腹腔注射效果次之,分别为19.1%、14.6%、14.36%。免疫组织化学研究显示,Icps结合到虫体的体表及肠道。     

Serum levels of gastrin, insulin and glucagon as possible factors of anorexia in pigs infected once with Ascaris suum

In order to determine possible mediators for development of anorexia in pigs infected with Ascaris suum, serum levels of gastrin, insulin and glucagon were measured. After a single high oral dose of 100,000-200,000 embryonated eggs the serum levels of gastrin and insulin in the infected pigs did not significantly differ from those in controls. Serum glucagon levels in the infected groups, however, were lower than those in controls and the difference was more evident 24 days postinoculation and later.     

The effect of continuous in-feed medication with thiophanate on Ascaris suum and Oesophagostomum spp. egg output in young pigs

The full potential of anthelmintics now available for single dose treatment is not achieved because the devising system for worm control in piglets/weaners is not efficiently applicable in practice. Therefore an in-feed medication programme for growing young pigs, allowing only one feed lot to be handled by the farmer, was tested in two studies. Study A Feed containing 0.0225% thiophanate was continuously fed almost ad lib. to piglets from birth right up to about 25 kg body weight when ready for fattening. This control measure effectively prevented A. suum and Oesophagostomum from becoming established during the whole pre-fattening period, thus allowing "worm-free" weaners to be produced. -33% of animals receiving unmedicated feed harboured mature Oesophagostomum already at an age of 63 days when first examined. Three out of 97 unmedicated pigs were then A. suum egg-count positive. At the same time all medicated pigs, except one with a low Oesophagostomum egg output, were egg-count negative. All medicated were still egg-count negative at 23-29 days after the withdrawal of the feed. About 30% of unmedicated pigs were then shedding eggs of A. suum and Oesophagostomum respectively. At 45-49 days after the withdrawal of the medicated feed 8% of previously medicated pigs and 43% of unmedicated pigs were A. suum egg-count positive. The corresponding figures for Oesophagostomum egg-count positive pigs were 6% and 40% respectively. The acquisition of worm infections by previously medicated pigs most probably was made in the fattening unit after the withdrawal of the thiophanate medicated feed. Study B In this study it was further substantiated that in-feed medication of pigs with thiophanate prevents A. suum from becoming established. All treated pigs were A. suum egg-count negative at Day 43 and 46 after the withdrawal of the medicated feed whereas about 62% of untreated control pigs were shedding A. suum eggs at the same time. This finding justify the proposal that the in-feed medication performed prevented larval migration. Furthermore it was shown that the in-feed medication must proceed right up to the transfer of piglets to the fattening unit in order to achieve its full potential. Farrowing pens may be heavily contaminated with infective Oesophagostomum larvae at the end of the pre-fattening period resulting in sudden and heavy nodular worm infections after the withdrawal of the medicated feed.     

使君子提取物对驱除猪蛔虫效果研究及对猪主要生理生化指标的影响

云南使君子提取物作为实验用药,对感染猪蛔虫的动物进行了药效实验研究。结果使君子提取物给药最佳剂量为0．05mg／kg。粪检虫卵数计数表明,每克粪便虫卵数与用药前相比存在差异显著性（P〈0．05）。所检测的血液生化指标在给药前后均没有出现显著性的变化（P〉0．05）。从而可初步推断使君子提取物对猪的肝脏、肾脏、心脏和肌肉等组织器官没有产生明显的影响。使君子提取物对猪蛔虫有较好的驱虫效果,且毒副作用小。     

A comparison of the efficacy of two ivermectin formulations against larval and adult Ascaris suum and Oesophagostomum dentatum in experimentally infected pigs

A study was conducted to evaluate and compare the efficacy of two injectable formulations of ivermectin (IVM-1 and IVM-2) at a dose rate of 0.3 mg/kg bodyweight versus placebo in the treatment and control of larval and adult stages of Ascaris suum and Oesophagostomum spp. in experimentally infected pigs. Seventy helminth free pigs were allocated on a liveweight basis to 7 groups each comprising 10 pigs (A-G). Group A served as an untreated control group. Groups B and C were used to investigate the efficacy of both formulations against adult stages of A. suum and Oesophagostomum spp., Groups D and E for efficacy against larval stages of A. suum and Groups F and G for efficacy against larval stages of Oesophagostomum spp. Pigs of groups A, B, C, D and E were infected on Day-0 with 1000 infective A. suum eggs each. Infective larvae of Oesophagostomum spp. (10,000/pig) were given on Day-0 to pigs of Groups F and G and on Day-21 to pigs of Groups A, B and C. Treatment was given to pigs of Group A (saline as placebo) on Day-7 and -28, IVM-1 to pigs of Group F on Day-7, pigs of Group D on Day-14 and pigs of Group B on Day-49. IVM-2 was given to pigs of Group G on Day-7, Group E on Day-28 and Group C on Day-49. Pigs of Groups F and G were sacrificed on Day-28, pigs of Groups A, D and E on Day-49 and pigs of Groups B and C on Day-56. Post mortem worm counts showed the following efficacies: (IVM-1) against larval A. suum 100%, against adult A. suum 94.4%, against larval Oesophagostomum spp. 52.0% and against adult Oesophagostomum spp. 83.0%. (IVM-2) against larval A. suum 100%, against adult A. suum 90.3%, against larval Oesophagostomum spp. 94.0% and against adult Oesophagostomum spp. 94.7%.