两种人体钩虫感染性幼虫的形态鉴别

人体十二指肠钩虫和美洲钩虫感染性幼虫的形态鉴别,对钩虫病流行病学调查及人体寄生虫学教学均具有实践意义。在实际工作中,掌握口矛（咽管矛）和鞘膜横纹的特征,就可以准确鉴别两种钩虫的感染性幼虫。     

Necator americanus and Ancylostoma ceylanicum: development of protocols for dual infection in hamsters

Two-day-old baby hamsters were infected initially with the infective larvae of hamster-adapted human hookworm, Necator americanus (NaL3). After a specified period they were again infected orally with infective larvae of Ancylostoma ceylanicum (AcL3). Three weeks after the second infection they were killed and the establishment of N. americanus and A. ceylanicum was assessed. The effect of different infection levels and exposure period of N. americanus on the concurrent establishment of A. ceylanicum was also examined. An infection with 50 NaL3 percutaneously, and 3 weeks later, a second infection with 50 AcL3 orally has produced reasonably equal number of hookworms (no statistical difference in the burden of N. americanus and A. ceylanicum) in the intestine of hamsters. Thus this protocol of dual infection was found suitable to develop two species of hookworms in hamsters for anthelmintic screening.     

Polymerase chain reaction-based differential diagnosis of Ancylostoma duodenale and Necator americanus infections in humans in northern Ghana

We evaluated a two-step semi-nested polymerase chain reaction (PCR)-based approach for the specific detection of Ancylostoma duodenale DNA in human faeces. The test was used to determine to what extent this species of hookworm is present in the regions of Bolgatanga and Garu of northern Ghana. Initially, the sensitivity and specificity of the PCR were tested using a range of well-defined control samples. Subsequently, a total of 378 human faecal DNA samples from Bolgatanga and Garu were subjected to the PCR. The results were compared with those obtained using a previously established PCR for the specific detection of Necator americanus DNA in human faeces. Infection with A. duodenale was recorded in 74 (19.6%) samples and N. americanus in 278 (73.5%), of which 64 (16.9%), represented co-infections with both species. While A. duodenale was predominantly detected in the samples from Bolgatanga, infections in Garu related almost exclusively to N. americanus. The results showed that the present PCR approach is a valuable complementary tool for the diagnosis of A. duodenale infection in humans in Ghana, having implications for epidemiological studies and for the monitoring of the success of control programmes in regions in Africa.     

Molecular identification of human hookworm infections in economically disadvantaged communities in Peninsular Malaysia

Species identification of human hookworm infections among eight communities in rural areas of Peninsular Malaysia was determined during 2009-2011. Fecal samples were examined by microscopy and subsequently, the internal transcribed spacer 2 and 28S ribosomal RNA region of Necator americanus and Ancylostoma spp. were sequenced. Overall, 9.1% (58 of 634) were identified positive by microscopy for hookworm infection, and 47 (81.0%) of 58 were successfully amplified and sequenced. Sequence comparison found that N. americanus (87.2%) was the most predominant hookworm identified, followed by Ancylostoma ceylanicum (23.4%). No A. duodenale infection was detected in this study. Detection of A. ceylanicum in humans highlighted the zoonotic transmission among humans living near dogs. Thus, implementation of effective control measures for hookworm infections in future should seriously consider this zoonotic implication.     

美洲钩虫和十二指肠钩虫雄虫交合刺扫描电镜观察

本文应用扫描电镜观察美洲钩虫和十二指肠钩虫雄虫交合刺的形态结构,发现两种钩虫的交合刺均为一根呈凹槽状,另一根呈扁圆管状。由于其槽状交合刺的槽横切面的弧度不同,致使美洲钩虫的交合刺在外观上以一根为多见,而十二指肠钩虫的则以两根分开为多见。     

一株抗十二指肠钩蚴单克隆抗体的特性研究

用间接荧光抗体技术等方法观察了一株抗十二指肠钧蚴单克隆抗体杂交瘤细胞株Ｂ１２分泌的抗体（ＭｃＡｂＢ１２）特性。结果表明ＭｃＡｂＢ１２与十二指肠钧虫成虫切面的肠管、生殖器官和肌肉的基膜及排泄管出现明显的免疫反应，提示ＭｃＡｂＢ１２的“靶抗原”为分泌排泄（ＥＳ）抗原。ＳＤＳＰＡＧＥ结果提示ＭｃＡｂＢ１２具有３条蛋白区带，分子量分别为６１、５２和２１ｋＤ。ＥＬＩＢ结果显示ＭｃＡｂＢ１２能识别十二指肠钧蚴抗原，主要抗原条带的蛋白分子量为２８ｋＤ。     

美洲钩虫及十二指肠钩虫细胞色素C氧化酶1基因测序

目的：比较美洲钩虫及十二指肠钩虫线粒体细胞色素Ｃ氧化酶１（ＣＯ１）基因序列,确定两种钩虫间ＣＯ１基因差异。方法：现场采集钩虫成虫样本,蛋白酶Ｋ消化抽提线粒体基因组ＤＮＡ,ＰＣＲ方法扩增ＣＯ１基因,ＤＮＡ测序及分析。结果：ＰＣＲ扩增获得约７００ｂｐ美洲钩虫及十二指肠钩虫ＣＯ１基因片段,其序列比较显示两种钩虫ＣＯ１基因同源性达８９．７％,存在特定的核苷酸差异。结论：ＣＯ１基因可作为鉴别两种人体钩虫虫种标记性基因。     

Epidemiology of human ancylostomiasis among rural villagers in Nanlin County (Zhongzhou village), Anhui Province, China: age-associated prevalence, intensity and hookworm species identification

Hookworm infection has traditionally been highly endemic to Anhui Province, China. Zhongzhou village in southwestern Anhui was identified as an endemic focus of infection caused by the hookworm Ancylostoma duodenale. This species was found to predominate over Necator americanus in ratios ranging from 35:1 based on the recovery of third-stage infective larvae (L,) to 21:1 based on the recovery of adult hookworms after anthelmintic chemotherapy. The overall prevalence of Ancylostoma infection in Zhongzhou was 33.2% with a greater prevalence among males. Unlike the age-associated prevalence patterns for N. americanus in Hainan and other southern Chinese provinces that show increasing prevalence with age and the highest prevalence among the elderly, the age-associated prevalence for A. duodenale in Zhongzhou exhibited a peak in middle-aged adults with subsequent decline. The age-associated intensity pattern exhibited a similar trend although the most of the hookworm infections were light or moderate infections as defined by quantitative egg counts.     

阿苯达唑、噻嘧啶治疗肠道线虫及控制钩虫感染回升的效果

采用阿苯达唑400mg/d×3d、400mg/d×5d和噻嘧啶1500mg/d×3d、1500mg/d×5d治疗肠道线虫感染者720例。治后半月检查,钩虫卵阴转率分别为98.6、98.6、86.2和93.5％;蛔虫卵阴转率分别为96.5、98.2、92.9和96.3％;鞭虫卵阴转率分别为86.4、89.0、68.9和67.0％。钩虫卵减少率均在98.0％以上。治后半年复查,各组钩虫阳性率均有不同程度回升,虫种由治前美洲钩虫为主,转为十二指肠钩虫为主。结果显示,回升的原因主要是十二指肠钩虫引起,而阿苯达唑400mg/d×3d和400ms/d×5d控制钩虫感染回升有明显作用。     

Expression of acquired immunity to the hookworm Ancylostoma ceylanicum in hamsters

Four experiments are described in which hamsters, initially exposed to primary infection with Ancylostoma ceylanicum , were given a homologous challenge and components of the secondary response were quantified and compared to relevant control groups. The initial establishment of the L3 larvae was not prevented in immunized hamsters but their growth was slowed and virtually all larvae were lost within a week of challenge, when the majority were still at the L4 stage of development. The loss of worms was associated with an accelerated mucosal mastocytosis and increased systemic antibody. Thus acquired immunity to hookworm larvae in this system acted on L3 and L4 stages, thereby preventing larvae from maturing in immunized animals. In contrast to primary infections, immunized hamsters responding to a challenge infection did not lose weight nor did they experience significant anaemia, because of the lack of adult worms. The secondary immune responses therefore prevented manifestation of hookworm disease among immunized-challenged animals .     

三苯双脒对钩虫超微结构的影响

应用透射电镜观察了用三七双脒在人体驱出的十二指肠钩虫和美洲钩虫的超微结构变化，结果显示三苯双脒400mg顿服对两种钩虫的体壁，肠管及卵巢的细胞膜，细胞核及多种细胞器均有明显的破坏作用。其中以对卵巢的破坏作用最为严重。     

Short report: Ancylostoma ceylanicum: exsheathment is not required for successful cryopreservation of third stage hookworm larvae

Third-stage larvae (L3) of the human hookworm parasite Ancylostoma ceylanicum were cultured from the feces of infected hamsters and frozen for up to 100 days in liquid nitrogen. Upon thawing, viable larvae were recovered and used to inoculate naive hamsters. The larvae recovered from this second group of hamsters were used to inoculate a third group of naive animals, which demonstrated that the originally frozen larvae were successfully maintained for two full generations following thawing. These data suggest that exsheathment, which has previously been reported to be essential for successful cryopreservation, is not necessary for recovery of viable, infectious A. ceylanicum L3.     

An in vitro larval motility assay to determine anthelmintic sensitivity for human hookworm and Strongyloides species

With the implementation of programs to control lymphatic filariasis and soil-transmitted helminths using broad spectrum anthelmintics, including albendazole and ivermectin, there is a need to develop an in vitro assay for detection of drug resistance. This report describes an in vitro assay for measuring the effects of ivermectin and benzimidazoles on the motility of larvae of the hookworm species Ancylostoma ceylanicum, A. caninum, and Necator americanus, and Strongyloides species including Strongyloides stercoralis, and S. ratti. A dose-response relationship was demonstrated with each of the parasite species, with distinct differences observed between the various species. In pilot field testing of the assay with N. americanus larvae recovered from human fecal samples, a dose-response relationship was observed with ivermectin. While the assay has demonstrated the ability to determine drug responsiveness, its usefulness in resistance detection will require correlation with the clinical outcome among individuals infected with parasite strains showing different drug sensitivities.     

十二指肠钩虫巨噬细胞迁移抑制因子基因的克隆和表达

目的克隆、表达十二指肠钩虫巨噬细胞迁移抑制因子(MIF)AduMIF-1基因。方法设计、合成特异引物,以十二肠钩虫成虫cDNA为模板,通过PCR扩增AduMIF-1基因。将获得的AduMIF-1编码序列克隆至原核表达载体pET32a,构建重组表达质粒pET32a/AduMIF-1。重组表达质粒转入大肠埃希菌BL21(DE3)中,IPTG诱导表达并分离纯化重组AduMIF-1。结果成功扩增到AduMIF-1全长编码序列,完整阅读框长度为360bp,编码119个氨基酸。构建了重组表达质粒pET32a/AduMIF-1,经IPTG诱导表达和分离、纯化,获得了重组AduMIF-1,融和蛋白分子质量单位约为33ku。结论本研究从十二指肠钩虫中分离到MIF基因,并成功进行了重组表达、分离与纯化,为进一步研究AduMIF-1的生物学功能奠定了基础。     

Lectin binding to distinguish cell types in fixed atherosclerotic arteries

In order to assess the possible utility of lectin binding to identify the cellular components of fixed arterial lesions we studied lectin binding in experimental rabbit and monkey vessels, as well as in human atherosclerotic arteries obtained at surgery. The avidin-biotin-peroxidase technique was used to localize the binding of the following biotinylated lectins: Concanavalin A (Con A), Dolicho biflorus agglutinin (DBA), soybean agglutinin (SBA), peanut agglutinin (PNA), Phaseolus vulgaris agglutinin (PHA), Ricinus communis agglutinin (RCA), wheat germ agglutinin (WGA), and Ulex europaeus agglutinin (UEA). PHA demonstrated specific cytoplasmic staining of macrophages in rabbit, monkey, and human tissues and differentiated macrophages from other cell types in atherosclerotic lesions. When morphometric comparisons were made between lesion PHA staining and another macrophage marker, acid lipase, very similar results were obtained. Con A, RCA, and WGA stained macrophages intensely and differentiated them from other cell types in normal reticuloendothelial tissues and lesions, but also stained smooth muscle cells and endothelial cells when these cells developed lipid vacuoles. UEA stained the endothelium of vasa vasorum consistently in human arteries, but staining of artery lumen endothelium was variable. Endothelial cells of rabbit or monkey vessels did not stain with UEA. DBA, PNA, and SBA did not consistently stain any cellular structures in arteries. PHA was found to be an excellent marker to differentiate and quantify macrophages in glutaraldehyde or formalin-fixed, paraffin-embedded experimental and human atherosclerotic lesions. Con A, RCA and WGA merit further detailed study in conjunction with other histochemical tests as possible markers of functional changes in arterial cells during lesion development.     

福建漳州蕉区人群钩虫感染情况的调查

用改良加藤法和试管滤纸培养法,对漳州市郊区蕉园社员进行粪检,共检查2069人,其中钩虫感染者1402例,其感染率为67.8％。人群的钩虫感染有随年龄增长而增高的趋势,而70岁以上年龄组的感染率有所下降,这可能与参加劳动多少有关。926例钩虫幼虫培养结果,单纯十二指肠钩虫占67％,美洲钩虫占13％,两种钩虫混合感染占20％。十二指肠钩虫与美洲钩虫之比为2.6:1。表明漳州郊区系以十二指肠钩虫为主的两种钩虫混合感染地区。     

Incidence and risk factors of hookworm infection in a rural community of central Thailand

A cohort study to identify incidence and risk factors of hookworm infection was conducted in a rural community, central Thailand from November 2005 to February 2007. Stool specimens were examined for hookworm eggs using wet preparation, Kato thick smear, and water-ethyl acetate sedimentation technique. The incidence rate of hookworm infection was 7.5/100 person-years. The independent risk factors for acquiring hookworm infection were barefoot walking (incidence rate ratio [IRR] = 4.2, 95% confidence interval [CI] = 1.2-14.5) and raising buffaloes around the house (IRR = 4.8, 95% CI = 1.9-11.8). Sequencing of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the ribosomal RNA gene were performed for identifying species of hookworm. Necator americanus was the most common hookworm identified in this population. Ancylostoma duodenale and A. ceylanicum were also detected. Our data suggest transmission of both human and animal hookworms in this community. Thus, prevention and control strategies of hookworm infection should cover both human and animal infection.     

Vaccination with neutrophil inhibitory factor reduces the fecundity of the hookworm Ancylostoma ceylanicum

Neutrophil inhibitory factor (NIF), a protein isolated from hookworms of the genus Ancylostoma, inhibits CD11b/18-dependent leucocyte function, binding to the I domain of CD11b. Historically, NIF was serendipitously isolated from whole worm extracts during a search for novel antihaemostatic agents, and little is known of its source or biological significance to the parasite. NIF has also been identified as a possible hookworm vaccine candidate. Ancylostoma ceylanicum recombinant NIF, expressed in its active form in Pichia pastoris, was purified and its functional activity confirmed using neutrophil adhesion assays and confirmatory immunoassay. Recombinant NIF was subsequently used in vaccination trials in the A. ceylanicum-hamster model system for human hookworm infection. Vaccinated and challenged animals were not protected in terms of worm burden or haematocrit values, despite the presence of high levels of specific antibody against NIF. However, adult worms resident in vaccinated animals showed a significant reduction in fecundity (85.8% by day 21 postinfection), indicating a degree of protection against subsequent transmission by vaccination. These data indicate that targeted vaccination with recombinant subunit material, derived from a known and effective immune suppressant secreted by the parasite, may offer partial protection against the transmission of hookworm infection. Furthermore, we can also report that a biological activity characteristic of NIF is detectable in the secretions of A. ceylanicum using two complementary bioassays. Complete neutralization of this secreted activity by vaccination in combination with other vaccine candidates may result in improved protection against A. ceylanicum infection.     

Selective primary health care: strategies for control of disease in the developing world. XVII. Hookworm infection and anemia

Approximately one-quarter of the world's population is infected with either Ancylostoma duodenale or Necator americanus. Humans transmit the hookworm by fecal contamination of the soil. The eggs hatch and the larvae may survive several months in warm, damp soil. Humans are infected when the larvae penetrate the skin and migrate through the body to the intestines, where the median survival time of the hookworm is one year. The loss of red blood cells into the gut is proportional to the worm load. Symptoms result from iron deficiency anemia and protein loss caused by a heavy worm burden. The peak age-specific prevalence of infection occurs in adult life, but only a small proportion of those infected ever become symptomatic. Possible control measures include chemotherapy to reduce the intensity of infection and the transmission potential and sanitation.     

Short report: an agar plate method for culturing hookworm larvae: analysis of growth kinetics and infectivity compared with standard coproculture techniques

An agar plate (AP) method has been developed for culturing infectious larvae of the hookworm Ancylostoma ceylanicum. The third-stage larvae reared using the AP method displayed similar morphology to those cultured using Baermann or Harada-Mori coproculture techniques. The yield of viable larvae from the AP method (50%) was comparable to that of the Baermann (47%), and both were superior to Harada-Mori (2.1%). Third-stage larvae cultured by the AP method established patent infection in naturally permissive laboratory hosts, although the yield of adult worms was reduced compared with animals infected with L3 obtained by Baermann culture. The AP method is useful for defining growth requirements for hookworm development, as well as characterizing the effects of bacterially expressed compounds on hookworm larvae in vivo.