Development of a rapid assay for the diagnosis of Myxobolus cerebralis in fish and oligochaetes using loop-mediated isothermal amplification

A loop-mediated isothermal amplification assay was developed for the rapid detection of Myxobolus cerebralis in both fish and oligochaete hosts. The assay was optimized to amplify parasitic DNA by incubation with Bst DNA polymerase and a set of six specially constructed primers at 65 degrees C for 60 min. The amplification products were detected visually using SYBR Green I dye which gave identical results to gel electrophoresis analysis. Parasite DNA was detected from infected oligochaetes, and from the anal fin, caudal fin, dorsal fin and operculum of clinically infected fish. This 'Myxo-LAMP' assay has a detection limit similar to that of a polymerase chain reaction assay (10(-6)), but is more rapid and only requires a water bath for amplification and is therefore practical for simple and rapid diagnosis of infected tissue.     

Diagnosis and treatment of coenurosis in sheep

Coenurosis is a disease of the central nervous system in sheep, caused by Coenurus cerebralis, the larval stage of Taenia multiceps, a tapeworm, which infests the small intestine of carnivores. In 80-90% of cases, the cyst is located in one cerebral hemisphere, whilst in 5-10% of cases, it is localised in the cerebellum; rarely it involves two sites in the brain of the affected animal. Listeriosis, louping-ill, sarcocystosis and polioencephalomalacia and brain abscessation should be considered when formulating a diagnosis of acute coenurosis. In all cases, it is essential to carefully examine the animal and not simply rely on results of ancillary tests (mainly of cerebrospinal fluid examination), as disorders other than coenurosis can be responsible for changes in the results of these tests. Treatment is based on surgical removal of the coenurus cyst after general anaesthesia of the animal; the approach has a very good success rate, especially after accurate localisation of the lesion. Despite that, many farmers may choose to slaughter those sheep fit for marketing for economic reasons and euthanise those in poor condition.     

牦牛脑多头蚴病药物治疗试验

用丙硫咪唑和吡喹酮两种驱虫药物,采用"药物增量法"和"药物联合法"对24头具有明显临床症状的脑多头蚴病例牦牛进行不同剂量治疗试验.结果表明,吡喹酮增量法治疗后症状消失时间最短,为8.5 d;丙硫咪唑增量法有效率和治愈率均为100%,吡喹酮增量法的有效率80%,治愈率70%;丙硫咪唑和吡喹酮联合法的有效率67%,治愈率50%.对照组10头脑多头蚴病牦牛均在不同时间内死亡.     

湖南省山羊脑多头蚴的线粒体nad1和nad4基因的序列测定及种系发育分析

本研究旨在阐明脑多头蚴湖南分离株线粒体烟酰胺腺嘌呤二核苷酸( NADH)脱氢酶亚单位1基因(nad1)部分序列(pnad1)和烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位4基因(nad4)部分序列(pnad4)的遗传变异情况,并用pnad1和pnad4序列重构脑多头蚴与其它带科绦虫的种群遗传关系.利用聚合酶链反应(PCR)扩增脑多头蚴的pnad1和pnad4,应用ClustalX 1.81程序对序列进行比对,再用Phylip3.67程序MP法和Mage4.0程序NJ法绘制种系发育树,并用Puzzle5.2程序构建最大似然树,同时利用DNAstar5.0中的Megalign程序进行同源性分析.结果显示所获得的pnad1和pnad4序列长度分别均为666和887 bp,湖南分离株与已知多头带绦虫位于同一分枝.由于脑多头蚴pnad1和pnad4序列种内相对保守,种间差异较大,故均可作为种间遗传变异研究的标记,从而为脑多头蚴的分子流行病学和其相关疾病的诊断奠定基础.     

Filtering Myxobolus cerebralis Triactinomyxons from contaminated water using rapid sand filtration

Rapid sand filtration was explored as a means of removing Triactinomyxon actinospores (Tams), the waterborne infective stage of the salmonid parasite Myxobolus cerebralis that causes whirling disease, from contaminated water. Preliminarily, a batch of sand was sieved to create 12 size ranges from 180 to 2000 μm. These individual ranges were tested for their efficacy of removing Tams through sand beds either 2 cm or 4 cm deep. The critical size at which no Tams passed through the sand bed was 300 μm at 2 cm depth and 425 μm for 4 cm bed depth. Additional tests evaluated the passage of Tams through filter beds comprised of sand that had all particles smaller than 180 μm removed. With this sand, 0.2±0.5% of Tams passed through a 2 cm bed, and 0.0±0.0% with a 4 cm sand bed. Based on these preliminary results, small (61 cm×15 cm) rapid sand filters were placed in-line with aquaria containing rainbow trout fry. The sand bed depth was 10 cm under which lay 10 cm of aquarium gravel. Four treatments were (1) negative control, (2) positive control, (3) sand of >180 μm, (4) sand of >300 μm. Tams were regularly introduced to the rearing systems above the sand filters. After 60 days, clinical signs of whirling behavior and black tails were seen among the positive controls. A polymerase chain reaction assay for Myxobolus cerebralis 1 month after exposure proved negative for negative controls and the >180 μm group, whereas 10% of the >300 μm group and 71% of the positive controls were infected. Results from the PCR assay at the study’s conclusion indicated the negative controls and >180 μm group were still disease free. All positive control fish were infected, and 49% of >300 μm fish were infected. These results were mirrored by those obtained from a pepsin–trypsin digest assay, except one fish among the >180 μm group was found to be infected. These results demonstrate that sand filtration may be a viable option in treating hatchery water supplies that are contaminated with whirling disease.     

脑多头蚴病研究进展

脑多头蚴病是由多头带绦虫的中绦期幼虫脑多头蚴寄生于绵羊、山羊、黄牛、牦牛等有蹄动物的脑及脊髓中引起的一种人兽共患寄生虫病,人可因误食虫卵而感染此病。该病在宿主出现典型临床症状后如果得不到治疗,多以死亡为转归,给畜牧业生产造成了巨大的经济损失。论文就脑多头蚴病的病原学、生活史、流行病学、临床症状、诊断、治疗及免疫预防等方面的研究进展进行了综述。     

Coenurus cerebralis Infection in Ethiopian Highland Sheep: Incidence and Observations on Pathogenesis and Clinical Signs

An investigation was carried out at Debre Berhan, Ethiopia, between 1996 and 1997, into the epidemiology of coenurosis in Menz and Horro breeds of sheep. A total of 37 heads from clinically sick and 183 heads from apparently healthy sheep were examined post mortem for the presence of the cystic larvae of Taenia multiceps, of which 37 and 5 heads, respectively, contained 1 to 8 coenurus cysts (diameter 0.8 to 6.5 cm). The bladder worms were located in the cerebral hemisphere in 96% of the cases (43% and 57% for left and right, respectively), 4% being in the cerebellum. Prediction of cyst locations based on the direction of circling and head deviation had a 62% success rate. A retrospective study generated from the health record book at the ILRI Debre Berhan Station in 1992–1996 (199 Horro and 174 Menz) revealed that the incidence of coenurosis ranged from 2.3% to 4.5%. There was no significant breed difference in the incidence. The age of the affected sheep ranged from 4 to 96 months, with a mean of 19.3 months, and 72% of the cases were within the range of 6 to 24 months. In a complementary survey on necropsied stray dogs, 8 out of 17 were positive for Taenia spp. Both studies confirm the endemicity of coenurosis at the ILRI Debre Berhan Research Station. Appropriate strategies for the control of coenurosis are suggested.     

动物脑多头蚴病研究进展

脑多头蚴病是由多头绦虫的幼虫寄生于家畜所引起的疾病,人也可感染,但相对较少。该病通常对动物是致命的,给世界许多地区的养殖业造成了巨大的经济损失。论文主要介绍了不同药物对该病的疗效,以及脑多头蚴不同抗原成分免疫原性、分子分类和相关抗原基因的研究进展。     

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.     

斑点免疫金渗滤试验检测绵羊脑多头蚴病的研究

为了寻找一种快速、简便诊断绵羊脑多头蚴病的新方法，应用自行配制的胶体金探针标记SPA，建立了斑点免疫金渗滤试验（DIGFA）。该法同ELISA具有良好的相符性。将抗原、待检血清滴加于膜上进行反应，数分钟内即可用肉眼进行结果判定。本法具有操作简单、反应快速、敏感性强、重复性好、不需要贵重仪器等优点，适合于基层推广应用。