Toward practical, DNA-based diagnostic methods for parasitic nematodes of livestock--bionomic and biotechnological implications

Parasitic nematodes of livestock have a major economic impact worldwide. In spite of the health problems caused by nematodes and advances toward the development of vaccines and new therapeutic agents against some of them, relatively limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of parasitic nematodes of livestock are central to their effective control, particularly given the current, serious problems with anthelmintic resistance in nematode populations. Traditional diagnostic techniques have considerable limitations, and there have been some advances toward the development of molecular-diagnostic tools. This article provides a brief account of the significance of parasitic nematodes (order Strongylida), reviews the techniques that have been evaluated or used for diagnosis and describes developments in polymerase chain reaction (PCR)-based methods for the specific diagnosis of nematode infection/s and the genetic characterisation of the causative agents. The advances made in recent years provide a solid foundation for the development of practical, highly sensitive and specific diagnostic tools for epidemiological investigations and for use in control programmes.     

DNA technological progress toward advanced diagnostic tools to support human hookworm control

Blood-feeding hookworms are parasitic nematodes of major human health importance. Currently, it is estimated that 740 million people are infected worldwide, and more than 80 million of them are severely affected clinically by hookworm disease. In spite of the health problems caused and the advances toward the development of vaccines against some hookworms, limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of hookworms is central to their effective control. While traditional diagnostic methods have considerable limitations, there has been some progress toward the development of molecular-diagnostic tools. The present article provides a brief background on hookworm disease of humans, reviews the main methods that have been used for diagnosis and describes progress in establishing polymerase chain reaction (PCR)-based methods for the specific diagnosis of hookworm infection and the genetic characterisation of the causative agents. This progress provides a foundation for the rapid development of practical, highly sensitive and specific diagnostic and analytical tools to be used in improved hookworm prevention and control programmes.     

A new diagnostic approach to fast-track and increase the accessibility of gastrointestinal nematode identification from faeces: FECPAKG2 egg nemabiome metabarcoding

Effective gastrointestinal nematode management in livestock industries is becoming increasingly difficult due to the rise of anthelmintic resistance and changes in the temporal and geographical distribution of major gastrointestinal nematodes. Underpinning the response to these challenges is the need for a fast-tracked diagnostic identification technique, making it easier for livestock producers to make informed gastrointestinal nematode management decisions. The traditional ‘gold-standard’ approach, larval culture followed by morphological differentiation, is laborious and potentially inaccurate. We developed a new diagnostic approach to identify gastrointestinal nematodes that integrates a remote-location digital faecal egg count platform, FECPAK^G2, with internal transcribed spacer 2 (ITS2) nemabiome metabarcoding. The technique involves a quick and simple protocol to harvest concentrated strongyle eggs from the FECPAK^G2 cassette utilising a repurposed pipette tip, followed by DNA isolation and Illumina next generation amplicon sequencing. The gastrointestinal nematode compositions and alpha diversity generated by our FECPAK^G2 egg nemabiome metabarcoding approach was not significantly different to traditional morphological larval differentiation and nemabiome metabarcoding of larval and faecal samples. We demonstrated that storing FECPAK^G2 harvested eggs in either DNA isolation lysis buffer or 80% ethanol (v/v) had no impact on gastrointestinal nematode identification outcomes for at least 60 days; enabling the transport of biological samples from their remote origins to a molecular diagnostic facility for nemabiome metabarcoding, in the absence of a cold chain. We discovered that sustained gastrointestinal nematode egg embryonation in the lysis buffer storage solution lead to higher yields of DNA compared with ethanol-stored gastrointestinal nematode eggs or faeces with gastrointestinal nematode eggs. Taking advantage of an already well-established platform such as FECPAK^G2, and providing the livestock producers that use it with the option to identify gastrointestinal nematodesin their samples and contribute to large-scale gastrointestinal nematode distribution and/or anthelmintic resistance surveys, is an important future direction for the FECPAK^G2 egg nemabiome metabarcoding approach.     

Serine/threonine phosphatases in socioeconomically important parasitic nematodes—Prospects as novel drug targets?

Little is known about the fundamental biology of parasitic nematodes (= roundworms) that cause serious diseases, affecting literally billions of animals and humans worldwide. Unlocking the biology of these neglected pathogens using modern technologies will yield crucial and profound knowledge of their molecular biology, and could lead to new treatment and control strategies. Supported by studies in the free-living nematode, Caenorhabditis elegans, some recent investigations have provided improved insights into selected protein phosphatases (PPs) of economically important parasitic nematodes (Strongylida). In the present article, we review this progress and assess the potential of serine/threonine phosphatase (STP) genes and/or their products as targets for new nematocidal drugs. Current information indicates that some small molecules, known to specifically inhibit PPs, might be developed as nematocides. For instance, some cantharidin analogues are known to display exquisite PP-inhibitor activity, which indicates that some of them could be designed and tailored to specifically inhibit selected STPs of nematodes. This information provides prospects for the discovery of an entirely novel class of nematocides, which is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock, and has the potential to lead to significant biotechnological outcomes.     

Molecular approaches for studying ascaridoid nematodes with zoonotic potential, with an emphasis on Toxocara species

Species-specific identification of ascaridoid nematodes at any developmental stage is a prerequisite for detailed investigation of the life cycles, systematics and epidemiology of this important group, and is also crucial for the diagnosis of associated infections. The morphological identification of some species and/or their larval stages can, however, present considerable difficulty. Recently, PCR-based methods, using genetic markers in the internal transcribed spacers (ITS) of ribosomal DNA, have been shown to provide reliable alternatives to more traditional methods for the specific identification of nematodes. This article provides an account of recent research on the development of PCR-based methods (utilizing ITS sequences) for the specific identification of ascaridoid nematodes of zoonotic potential, for the diagnosis of infections, and for the analysis of genetic variation within and among individual nematodes and their populations. Prospects for using these diagnostic and analytical tools to investigate epidemiological and population genetic questions relating to ascaridoid parasites are also discussed.     

To treat or not to treat: diagnostic thresholds in subclinical helminth infections of cattle

Helminth infections of cattle place significant burdens on livestock production and farm economic efficiency. Heavy infections are relatively easy to detect and treat with anthelmintics. However, subclinical infections have major but often hidden impacts on animals, necessitating more refined diagnostics to detect them and ideally inform farmers about the likely impact of anthelmintic treatment on animal and herd performance. Here, we review recent advances in diagnosing three major cattle helminth infections – gastrointestinal nematodes (GINs), liver flukes, and lungworms – and the search for subclinical infection thresholds to guide treatment decisions. Combining refined diagnostic thresholds with farm-specific information on grazing systems and animal history enables farmers to tailor helminth treatments to specific epidemiological circumstances, thereby limiting anthelmintic resistance (AR) and boosting agricultural efficiency and food security.     

High resolution electrocardiography

Over the past decade, significant advances were made in the research, diagnosis, and treatment of cardiovascular diseases. Such progress was in every sphere of cardiology that includes non-invasive, minimally invasive, and invasive technologies. Interpretive electrocardiography, cardiac pacemakers, cardiac stents, and angioplasty are some areas where the progress has been significant. Non-invasive methods of diagnosis of cardiac disorders involve digital recording of cardiac signals at the body surface (chest) and subsequent computerized analysis. Such methods and instruments provide a vital first step to the diagnosis of the heart without involving surgical procedures. One such non-invasive field is High Resolution Electrocardiography (HRECG). A high-resolution electrocardiogram detects very low amplitude signals in the ventricles called 'Late Potentials' in patients with abnormal heart conditions. A standard electrocardiogram cannot detect these signals. The presence of late potentials is widely accepted to have prognostic significance in patients after Acute Myocardial Infarction (AMI) High Resolution Electrocardiography enhances the diagnostic capabilities of ECGs. This article describes the principles involved in HRECG and the techniques that are employed to derive such superior diagnostic capabilities. The use of these techniques may lead to more discoveries in the causes of cardiac disorders and improved drug discoveries to combat such conditions.     

Cardiovascular biomarkers in body fluids: progress and prospects in optical sensors

Cardiovascular diseases (CVD) are the major causative factors for high mortality and morbidity in developing and developed nations. The biomarker detection plays a crucial role in the early diagnosis of several non-infectious and life-threatening diseases like CVD and many cancers, which in turn will help in more successful therapy, reducing the mortality rate. Biomarkers have diagnostic, prognostic and therapeutic significances. The search for novel biomarkers using proteomics, bio-sensing, micro-fluidics, and spectroscopic techniques with good sensitivity and specificity for CVD is progressing rapidly at present, in addition to the use of gold standard biomarkers like troponin. This review is dealing with the current progress and prospects in biomarker research for the diagnosis of cardiovascular diseases.Expert opinion.Fast diagnosis of cardiovascular diseases (CVDs) can help to provide rapid medical intervention, which can affect the patient’s short and long-term health. Identification and detection of proper biomarkers for early diagnosis are crucial for successful therapy and prognosis of CVDs. The present review discusses the analysis of clinical samples such as whole blood, blood serum, and other body fluids using techniques like high-performance liquid chromatography-LASER/LED-induced fluorescence, Raman spectroscopy, mainly, optical methods, combined with nanotechnology and micro-fluidic technologies, to probe patterns of multiple markers (marker signatures) as compared to conventional techniques.     

Anthelmintic resistance: the state of play revisited

Helminthosis is one of the major constraints in the successful wool and mutton industry throughout the world. Anthelmintic Resistance (AR) is said to have been established when previously effective drug ceases to kill exposed parasitic population at the therapeutically recommended dosages. Anthelmintic resistance is almost cosmopolitan in distribution and it has been reported in almost all species of domestic animals and even in some parasites of human beings. Some of the most important species of parasites of small ruminants in which AR has been reported include: Haemonchus spp., Trichostrongylus spp. Teladorsagia spp., Cooperia spp. Nematodirus spp., and Oesophagostomum spp. All the major groups of anthelmintics have been reported for development of variable degrees of resistance in different species of gastrointestinal nematodes. This paper describes the global scenario of prevalence and methods used for detection of AR in small ruminants. Different mechanisms and contributory factors for the development of AR are discussed. Various options and alternate strategies for the control and/or delay in the onset of AR are suggested in the light of available information.     

珊瑚病原微生物鉴定及其分子诊断技术进展

珊瑚礁生态系统是热带海洋最突出、最具代表性的生态系统,具有极高的生态价值和经济价值,然而由珊瑚疾病引起的珊瑚礁退化已经成为珊瑚礁生态系统的主要威胁之一。许多微生物(主要包括细菌、真菌、病毒)被认为与珊瑚疾病发生密切相关,确定珊瑚疾病的病原并建立其快速诊断的方法是开展珊瑚疾病流行病学调查和制定防控措施的必由之路。本文主要综述珊瑚疾病的病原微生物及其分子诊断技术的研究进展。     

Livestock production and animal health in Sicily, Italy

In Sicily, as in other Mediterranean areas, livestock represents one of the most important resources for the island economy. This sector involves more than 16,000 farms of cattle and 10,000 farms of sheep and goats (respectively 6% and 15% of national production) which are actually increasing their number. Most livestock in Sicily is owned by small holders and pastoralists. Regional production of milk feeds some industries, which involve a large and increasing occupational area. Due to its peculiar geographic aspect Sicily is constituted by hill areas with sporadic grasses, therefore extensive grazing methods represent an ancient, traditional practice for using poor lands. For the control of infectious diseases Veterinary Services (VS) are based on the public regional network that is coordinated by the Ministry of Health in Rome. Even if Sicilian VS had to solve many constraints related to traditional "pastoral management system", to the lack of any sanitary background of this sector, to unknown data on the numbers of the whole livestock, the effort done in the last years has allowed to monitor for brucellosis, bovine leukemia virus (BLV), bovine TBC, swine vescicular disease (MVS) and other diseases for the majority of the farms. Tick-borne diseases (TBDs) still represent a serious impediment to the improvement of meat and milk production in the region. Every year, several outbreaks related to Theileria, Babesia or Anaplasma infections in cattle are recorded. Imported breeds pay the most expensive tribute often with a mortality rate of 100%. In the last five years more than 170 outbreaks of TBDs have been notified by our Institute although the pathogen prevalence and economical impact in the Sicilian livestock is still unknown. The outdoor grazing of the animals, far away from observation, a subclinical course of the disease, can in fact create difficulties in discovering infection and therefore the therapy is often too late. New diagnostic and control methods (PCR, vaccination) are being developed at the Istituto Zooprofilattico Sperimentale of Sicily in cooperation with other national and international laboratories (such as the University of Utrecht).     

昆虫病原线虫资源概况和分类技术进展

昆虫病原线虫是具有重要潜在应用价值的害虫生物防治资源,主要包括斯氏线虫科（Steinernematidae）的斯氏线虫属Steinernema与新斯氏线虫属Neosteinernema线虫和异小杆线虫科（Heterorhabditidae）的异小杆线虫属Heterorhabditis线虫。近10年来,分子生物学方法与传统的形态学方法相结合应用到线虫的鉴定与分类,昆虫病原线虫的分类进入稳定与发展时期,越来越多的新种或品系被发现及应用于生物防治。目前已描述的昆虫病原线虫种类达65种,其中斯氏线虫属52种,新斯氏线虫属1种,异小杆线虫属12种。本文整理列出了迄今报道的昆虫病原线虫种类及其来源,并综述了昆虫病原线虫分类现状以及鉴定与分类方法上的研究进展,重点阐述了分子生物学技术在昆虫病原线虫鉴定与分类的应用状况。     

Nematophagous fungi used for the biological control of gastrointestinal nematodes in livestock and administration routes

The control of gastrointestinal nematodes relies at present mostly on antihelmintic treatments using synthetic molecules. This approach, however, has led to the appearance of resistance to some types of antihelmintics which, together with the need to cut down on the use of chemicals, has fostered the development of other control methods, such as biological control, which is the use of living organisms that are naturally antagonistic to an unwanted species. Among the natural enemies of nematode parasitic larvae is the microfungus Duddingtonia flagrans. Research has shown the ability of this fungus to reduce the number of nematode larvae in faeces, the ability of its chlamydospores to survive the passage through the gastrointestinal tract of livestock and, moreover, to keep its germinative ability, thus facilitating the development of formulations. The present review looks at the species currently used and the different ways of administering already tested nematophagous fungi.     

Seasonal variation in prevalence of gastrointestinal helminthoses in cattle of New Alluvial zone of West Bengal,India

Gastrointestinal parasitism is one of the major constraints for profitable livestock production. The present study was conducted to determine the prevalence and seasonal variation in gastrointestinal helminthic infections in cattle of New Alluvial zone of West Bengal. A total 2520 faecal samples of cattle were collected from five different places of NAZ of West Bengal during the study period i.e. March 2014 to February 2015. The faecal samples were examined by standard sedimentation and floatation technique. The overall prevalence of gastrointestinal parasites was recorded as 55.71% in cattle of NAZ and Paramphistome (40.64%) was recorded as the predominant parasite followed by Strongyle group of nematodes (8.25%) and Fasciola (3.33%). Moniezia was the only cestode parasite recorded with an overall prevalence of 1.97%. Statistically significant (p < 0.05) variation was observed in the prevalence of gastrointestinal helminths among the various seasons. Monsoon season has significantly (p < 0.05) higher prevalence of gastrointestinal helminthes (64.28%) followed by winter (57.14%) and lowest in summer (45.24%). Prevalence of Paramphistome (47.62%) and Strongyle (9.52) were also significantly (p < 0.05) higher monsoon compared to other seasons. The epidemiological knowledge/information gathered in the present study would be exploited for evolving strategic control method against gastrointestinal parasitic infections in cattle.     

Generalist nematodes dominate the nemabiome of roe deer in sympatry with sheep at a regional level

The growth of livestock farming and the recent expansion of wild ungulate populations in Europe favor opportunities for direct and/or indirect cross-transmission of pathogens. Comparatively few studies have investigated the epidemiology of gastro-intestinal nematode parasites, an ubiquitous and important community of parasites of ungulates, at the wildlife/livestock interface. In this study, we aimed to assess the influence of livestock proximity on the gastrointestinal nematode community of roe deer in a rural landscape located in southern France. Using ITS-2 rDNA nemabiome metabarcoding on fecal larvae, we analysed the gastrointestinal nematode communities of roe deer and sheep. In addition, we investigated Haemonchus contortus nad4 mtDNA diversity to specifically test parasite circulation among domestic and wild host populations. The dominant gastrointestinal nematode species found in both the roe deer and sheep were generalist species commonly found in small ruminant livestock (e.g. H. contortus), whereas the more specialised wild cervid nematode species (e.g. Ostertagia leptospicularis) were only present at low frequencies. This is in marked contrast with previous studies that found the nemabiomes of wild cervid populations to be dominated by cervid specialist nematode species. In addition, the lack of genetic structure of the nad4 mtDNA of H. contortus populations between host species suggests circulation of gastrointestinal nematodes between roe deer and sheep. The risk of contact with livestock only has a small influence on the nemabiome of roe deer, suggesting the parasite population of roe deer has been displaced by generalist livestock parasites due to many decades of sheep farming, not only for deer grazing close to pastures, but also at a larger regional scale. We also observed some seasonal variation in the nemabiome composition of roe deer. Overall, our results demonstrate significant exchange of gastrointestinal nematodes between domestic and wild ungulates, with generalist species spilling over from domestic ungulates dominating wild cervid parasite communities.     

Trichostrongylus colubriformis larvae induce necrosis and release of IL33 from intestinal epithelial cells in vitro: implications for gastrointestinal nematode vaccine design

Gastrointestinal nematodes represent a major production problem for ruminant livestock. Enhancing immunity to gastrointestinal nematodes through vaccination is desirable but mechanistic understanding of initial host responses that facilitate gastrointestinal nematode protective immunity is limited. We hypothesise that gastrointestinal nematode invasion induces mucosal epithelium damage and alarmin (e.g. IL33) release, thereby contributing to initiation of protective gastrointestinal nematode immunity. To test this, an in vitro air-liquid interface human HT-29 epithelial cell-Trichostrongylus colubriformis co-culture system was developed. Exsheathed L3 T. colubriformis exhibited both sinusoidal and burrowing motions in the co-culture system. Burrowing parasites, but not ivermectin-paralysed larvae, induced necrotic death of epithelial cells (annexin V(+)/propidium iodide(+)/caspase 3/7(-)). Microscopy confirmed that larvae consumed labelled necrotic epithelial cell contents. Trichostrongylus colubriformis larvae and their post-exsheathment antigens (excretory/secretory products) significantly induced IL33 mRNA expression in the epithelial cells. Immunoblot confirmed that IL33 was released from epithelial cells due to the damage caused by motile larvae. Exposure of HT-29 cells to alum or Sigma proprietary adjuvants induced significant epithelial cell IL33 mRNA expression without inducing cellular necrosis. Hence, the intracellular contents were not released externally where they might exert alarmin activity and this may limit their ability to trigger a protective anti-gastrointestinal nematode response. We conclude that T. colubriformis motion at the infection site induces intestinal epithelial cell necrosis which facilitates the release of intracellular contents, including IL33, and may be fundamental to the initiation of an appropriate host response to gastrointestinal nematodes. Our co-culture model is useful for studying initial epithelial cell-parasite interactions without conducting expensive animal trials.     

多功能磁共振造影剂研究进展

磁共振成像技术因对人体无创、任意方向断层扫描三维图像且分辨率较高、提供形态与功能两方面诊断评价等突出优点,成为了临床上用于疾病诊断的重要手段之一。临床上使用磁共振造影剂可以提高成像的分辨率和灵敏度,提高图像质量,增强对比度和可读性。但是,各种成像技术由于实现原理不同,具有各自的优势和缺陷,靠传统单一的诊断模式无法提供疾病的全面信息,因而在对各种复杂疾病进行诊断时会受到一定的限制。因此,将磁共振成像与其他成像技术如CT成像、超声成像等联合起来使用,则可以达到优势互补的效果,能为疾病的临床诊断提供更快捷精确的信息,同时可将磁共振成像与各种治疗方式结合在一起,即开发基于磁共振成像的诊断治疗一体化试剂,以实现对疾病的即时治疗和实时监控。本文主要介绍了磁共振成像造影剂的原理和种类,并且综述了目前国内外在基于磁共振成像的多功能造影剂/诊疗制剂这一领域的研究进展,最后就未来可能的研究方向进行了展望。     

上消化道造影和CT扫描诊断胃部肿瘤的应用价值比较

目的:比较上消化道造影和CT扫描诊断胃部肿瘤的临床应用价值。方法:选取我院胃外科收治的胃部肿瘤82例,随机分为两组,其中对照组41例,采用上消化道造影技术诊断;实验组41例,采用螺旋CT扫描,进行胃部肿瘤诊断。对比两组检查方式的诊断结果与病理诊断的符合率。结果:1进行螺旋CT扫描诊断与病理诊断符合率(95.12%,39/41)明显高于上消化道造影技术诊断符合率(80.49%,33/41),差异有统计学意义(P0.05);2淋巴结转移的敏感性和特异性比较中,螺旋CT明显高于上消化道造影技术,差异有统计学意义(P0.05)。结论:上消化道造影和CT扫描在诊断胃部肿瘤的临床应用中,上消化道造影技术是早期胃部肿瘤的首选检查方法,但是螺旋CT对胃部肿瘤正确诊断率要高,螺旋CT对淋巴结转移的敏感性和特异性较高,能够给确定胃部肿瘤分期提供依据,值得临床推广。     

PCR为基础的分子技术检测沃尔巴克氏体的研究进展

沃尔巴克氏体（Wolbachia）是广泛分布于节肢动物生殖组织内的一类共生细菌。这些细菌通过卵的细胞质传播并参与多种调控其寄主生殖活动的机制。由于Wolbachia的重大科学意义和Wolbachia在生物防治和遗传工程领域的潜在用途,近年来迅速成为国际生物学研究的热点。其中,Wolbachia检测技术,尤其是以PCR为基础的分子检测技术的进步是Wolbachia研究蓬勃发展的重要技术基础。本文对Wolbachia的研究及其分子检测技术进行介绍,旨在促进我国Wolbachia研究的起步和快速发展。 Abstract:Wolbachia is a common and widespread group of bacteria found in reproductive tissues of arthropods． These bacteria are transmitted through the cytoplasm of eggs and have evolved various mechanisms for manipulating reproduction of their hosts．Because of important relationship with some major biological issues and implication of biological control and genetic engineering,the study of Wolbachia has under gone an explosive growth in recent years.A tremendous progress has been made based on the advancing molecular diagnostic technique of Wolbachia.In this paper,those molecular diagnostic techniques are detailed and it is aimed to be the primer for the research of Wolbachia in China.     

Plant pathogen diagnostics: immunological and nucleic acid-based approaches

Accurate disease diagnosis and precise identification of any pathogens involved is an essential prerequisite for understanding plant diseases and controlling them effectively. Traditional methods of identifying plant pathogens can be slow and inconclusive, and this has prompted the search for alternative diagnostic techniques. Here, we review recent developments in antibody-based and nucleic acid-based diagnostic methods, and their applications in plant pathology. We also highlight some new developments in diagnostics that are likely to make an impact in the future.