Occurrence of Baylisascaris transfuga in wild populations of European brown bears (Ursus arctos) as identified by a new PCR method

The European brown bear (Ursus arctos) is a species present in limited areas of Europe and several small populations are considered endangered. This species can be affected by a range of parasites. In particular, the genus Baylisascaris is an emerging parasite of wild animals which can cause severe larva migrans syndrome in aberrant hosts, which include 100 species of birds, mammals and also humans. Baylisascaris transfuga is the species reported from bears, and with the exception of a few laboratory trials, little is known about the capacity of this species to infect other animals. Furthermore, the identification of this species has traditionally been based on light microscopy, using either morphology of the adults at necropsy or detection of the eggs in faeces, which are methods limited by the experience and the efforts of the observer. The current work was aimed at developing a specific PCR to detect the parasite directly from faecal samples of naturally infected brown bears in the field, without the need for previous flotation. Using eggs and adults of B. transfuga collected in Croatia, we first developed a PCR to detect a portion of the second internal transcribed spacer region (ITS-2) of ribosomal DNA and then applied it to bear faecal samples spiked with a known number of B. transfuga eggs. We show here for the first time that this method allows the detection of a minimum of two Baylisascaris eggs in 25mg of faecal material, thus it demonstrates a high diagnostic capacity that could be applied to evaluate the prevalence of the parasite in faecal samples from wild populations of brown bears.     

Detection of Histomonas meleagridis in turkeys cecal droppings by PCR amplification of the small subunit ribosomal DNA sequence

Histomonas meleagridis is a protozoan parasite that may cause histomoniasis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis and high mortality. Diagnosis of this disease is based on direct identification or on cultivation of the parasite. With the aim of developing more sensitive, rapid and useful tools for parasite detection, PCR that amplified a DNA target of 209 pb of the 18S rRNA gene was designed to detect the genome of H. meleagridis and to differentiate it from the genome of Tetratrichomonas gallinarum, another common protozoan parasite of fowl. The sensitivity of the test was evaluated using serial diluted samples of cultured H. meleagridis and showed positive amplification for concentrations comprised between 10 and 10(-1)parasites/ml of culture. The sensitivity for cecal droppings samples was assessed using spiked material and was comprised between 3 x 10(3) and 3 x 10(5)parasites/ml of stool. The reliability of the PCR for the detection of Histomonas infection was also evaluated by experimental infection of turkeys. Results of the PCR appeared to be in agreement with the development of the clinical signs and of the cecal lesions. The PCR developed in this study may be a useful tool in the detection and identification of H. meleagridis for rapid, routine screening as a supplement to direct identification or cultivation of the parasite.     

兽药缓释巨丸剂的研究进展及应用发展前景

目前、高效、广谱、安全的抗寄生虫药在国内外兽医临床上的越来广泛，由于防治家畜疾病的特殊需求，缓释大丸剂（Sustain-Released Bolus)在兽医临床上逐渐被重视。缓释大丸剂的应用可有效地降低了寄生虫的污染程度和寄生虫病的发生，对控制寄生虫感染和牧场净化都发挥了较好的作用。因此制剂适合于驱杀反刍动物蠕虫。缓释巨丸剂具有使动物体增重、毛产量等生产性能提高的优点，在某些情况下可以取代一般制剂。从国内兽药的供应情况看，该制剂尚不多，可作为今后研究开发的重点，也是畜牧业发展的客观需要。     

Cryptosporidium and its potential as a food-borne pathogen

Cryptosporidium species are intestinal protozoan parasites and are excreted in animal feces as stable oocysts. Cryptosporidium has now been detected in the feces of a wide range of ruminant and non-ruminant farmed animals, wild animals, domestic pets and birds and the parasite appears to be well adapted to survive and persist in feces for extended periods, ranging from several weeks to many months. Because of this persistence, these materials are important as potential vehicles of transmission within herds, farms, the water chain, the fresh food chain, and the wider environment. Appropriate handling of animal waste is necessary to control spread of this pathogen and to limit the significant risks of human infection. While water is a well-recognized vector of Cryptosporidium, it has only recently emerged that food may play a more significant role than previously realized in the transmission of the Cryptosporidium to humans. In the last 3-5 years, research efforts have been directed both at the development of suitable methods for isolation and detection of the parasite in foods and at the application of these methods to assess the prevalence and persistence of the parasite in a range of foods. Additionally, molecular subtyping methods have been used to establish the transmission routes of the parasite. This paper summarizes the general biology of Cryptosporidium and overviews the current research on C. parvum in the food chain. The risks posed by certain foods, such as salad/vegetable crops and beef, are discussed and control measures which may be useful in the farm-to-fork chain for these products are described.     

几种兽用合成抗菌药基于抗体快速检测技术研究进展

合成抗菌药在预防、治疗动物疾病等方面应用十分广泛,但不合理使用造成的耐药及残留问题日益严重,因此对兽用合成抗菌药的残留检测势在必行,而基于抗体的快速检测技术具有简便、灵敏度高、成本低等优点,发展前景十分广阔。作者综述了近年来针对磺胺类、氟喹诺酮类、喹噁啉类、硝基呋喃类、硝基咪唑类5大类兽用合成抗菌药物的抗体检测技术最新研究趋势,旨在促进残留快速检测技术的发展。     

Research Progress on Antibodies Rapid Detection Technology for Veterinary Synthetic Antibacterial Agents

Synthetic antibacterial agents are widely used in prevention and treatment of animal diseases. However, the irrational drugs use has caused severe drug residues issues and bacteria resistance.Therefore,it is particularly important to develop the rapid detection technology.The antibodies rapid detection technology has broad prospects because of its high sensitivity, simplicity, and low cost. The author reviewed the latest research on antibodies preparation and rapid detection methods for sulfonamides,fluoroquinolones, quinoxalines, nitrofurans and nitroimidazoles. It is great significant for the development of antibody rapid detection methods on the veterinary drug residues.     

Quantification of Ehrlichia ruminantium by real time PCR

Ehrlichia ruminantium (ER) is the causative agent of Heartwater, one of the most common tick-borne diseases affecting ruminants in African countries and West Indies. Although ER can be used as an inactivated vaccine for wild and domestic animals, there are currently no easy and reliable methods for the quantification of this obligate intracellular bacterium. This report describes the development of a SYBR Green I based real time PCR protocol for the quantification of ER for vaccine production purposes. The method was validated for four ER strains. The external-standard-based PCR protocol developed has a large dynamic quantitative range allowing accurate ER measurement in samples containing from 10(2) to 10(8) gene copies; the method is also reproducible and precise, with intra- and inter-assay coefficients below 5%. The detection limits were validated for samples collected from bovine aortic endothelial cell culture bulks, which are commonly used to produce the ER vaccine. In contrast to the methods based upon protein content, no interference from the host cells in ER quantification was observed. Furthermore, the extended applicability of the new technique was demonstrated by monitoring ER production in cell culture thus rendering it a valuable tool to ensure consistency between vaccine lots and to evaluate optimal vaccine dosage.     

Public health aspects of dirofilariasis in the United States

Coin lesions in the human lung present significant differential diagnostic problems to the physician. There are at least 20 known causes of such lesions, including neoplastic lesions, infectious diseases, and granulomas. The human medical literature contains many misconceptions about the life cycle of Dirofilaria immitis, including the method of entry of the infective-stage larvae and the development of the young adult worm. These misconceptions have obscured the recognition of the clinical presentation of pulmonary dirofilariasis and the potential for D. immitis to lodge in many other areas of the human body besides the lung. Exposure to infective larvae of D. immitis is more common in humans than is currently recognized. Reported cases in humans reflect the prevalence in the canine population in areas of the United States. The veterinary literature provides compelling evidence that D. immitis is a vascular parasite, not an intracardiac one. Its presence in the right ventricle is a post-mortem artifact, because it has never been shown to be there by echocardiography or angiography in a living dog, even though these techniques have demonstrated adult D. immitis in the pulmonary, femoral, and hepatic arteries; posterior vena cava; and right atrium of live dogs. Physicians have taken the name "heartworm" literally, believing that the worm lives in the heart and only after it dies does it embolize to the pulmonary artery. However, the coin lesion is spherical in shape, not pyramidal, as embolic infarcts to the lung in humans are known to be. The coin lesion is an end-stage result of the parasite's death in the vascular bed of the lungs and the stimulation of a pneumonitis followed by granuloma formation. This pneumonitis phase of human pulmonary dirofilariasis is often not recognized by the radiologist because of the way pneumonitis is diagnosed and treated and because the developing nodule is obscured by the lung inflammation. Serologic methods for use in humans are needed for clinical evaluations of patients with pneumonitis living in highly enzootic D. immitis regions. As well, epidemiological surveys are needed to determine the real extent of this zoonotic infection.     

Research Progress Towards the Liquid Chip Technology in Detection of Animal Infectious Diseases

Liquid chip technology is a novel biomolecular detection technology which integrates laser technology,flow cytometry,digital signal processing and traditional chemical technology.It is widely used in various immunological analysis and nucleic acid detection.Single and mutiplex analysis are supported,with protein and nucleic acid targets detected in a variety of detection methods in high throughput manner.It has advantages of high throughput,easy operation,wide range of application,good repeatability,high specificity,less sample needed,high sensitivity and stablility,and low cost.Therefore,they are gradually replacing the traditional detection and quantitative pathogen methods,such as Real-time fluorescent quantitative nucleic acid amplification detection system (qPCR),enzyme-linked immunosorbent assay (ELISA) and other detection methods.Animal infectious diseases seriously endanger the development of the breeding industry,futhermore,some zoonoses such as highly pathogenic avian influenza also pose a serious threat to human health.An efficient and sensitive diagnostic system will help to screen a large number of samples during the outbreak of infectious diseases and prevent the spread of infection.The development of liquid chip technology provides a new platform for high-throughput detection and disease prevention.In this review,the principle and advantages of liquid chip technology are briefly described.The research progress of liquid chip technology in the detection of animal infectious diseases,including pigs diseases,poultry diseases,rabbit diseases,dog diseases,rodent diseases and other animal infectious diseases are summarized.We believe that in the future,this technology will become an important analytical and testing tool in clinical diagnosis,basic research,new drug development,judicial identification,food health supervision,biological weapons prevention and other fields.The development of this technology will greatly promote the research and development of life science.     

液相芯片技术在动物疫病检测中的研究进展

液相芯片技术是整合了激光技术、流式细胞仪、数字信号处理和传统化学技术的一种新型生物分子检测技术,目前广泛应用于各种免疫分析和核酸检测中。液相芯片技术支持单重和多重分析,可在多种测定方法中对蛋白质和核酸靶标进行高通量检测,具有高通量、操作简单、适用范围广、重复性好、特异性高、所需样品量少、更灵敏稳定、成本低等优点,正逐渐代替传统检测和定量病原体的工具,如实时荧光定量PCR扩增检测系统（qPCR）、酶联免疫吸附试验（ELISA）等检测方法。动物传染病严重危害着养殖业健康发展,一些诸如高致病性禽流感的人畜共患病对人类健康造成了严重威胁。高效灵敏的诊断系统将有助于在传染病暴发期间筛选大量样本,防止感染扩散。液相芯片技术的发展为高通量检测和疾病的预防提供了新的平台。作者简要概述了液相芯片技术的原理和优点,重点阐述了液相芯片技术在检测动物疫病,包括猪病、禽病、兔病、犬病、啮齿类动物和其他动物疫病方面的研究进展。相信今后液相芯片技术会成为临床诊断、基础研究、新药开发、司法鉴定、食品卫生监督、生物武器防范等领域的一项重要分析检测技术,该技术的发展将大大推动生命科学研究与进步。     

Rapid detection of porcine reproductive and respiratory syndrome viral nucleic acid in blood using a fluorimeter based PCR method

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus recognised world wide as an important cause of reproductive failure and pneumonia in pigs. American and European strains of PRRSV, differentiated antigenically and genomically, have been reported. PRRSV infections are currently diagnosed using serology, virus isolation and/or immunocytochemistry. In order to overcome various drawbacks associated with these techniques, conventional, block-based RT-PCR methods for the detection of PRRSV nucleic acid in clinical samples have been described. These methods require gel electrophoresis for analysis of PCR products and present high risk of DNA carry-over contamination between the samples tested. We describe the detection of PRRSV RNA in serum samples and in blood impregnated filter disks (FDs), obtained from experimentally inoculated pigs, using a closed-tube, fluorimeter-based PCR assay. The assay eliminates the use of gel electrophoresis, and is as sensitive and specific as the conventional block-based PCR assay, detecting positive samples as early as 1 day post-inoculation. We also report a rapid fluorimeter based PCR method for differentiating American and European strains of PRRSV.     

Dientamoeba fragilis in swine population: a preliminary investigation

Dientamoeba fragilis, a protozoan with worldwide distribution is considered to be responsible for enteric disease in humans. A wide spectrum of clinical symptoms including; diarrhoea (acute or prolonged), flatulence, abdominal pains and other unspecific bowel symptoms have been ascribed to this parasite. Asymptomatic infection has also been reported. Dientamoeba fragilis is as its name indicates an extremely delicate protozoon and only the trophozoite has ever been demonstrated in stool samples. The definitive diagnosis of this infection is based on demonstration in permanently stained stool samples. In Italy examination of ova and parasite (O&P) samples are not currently performed. This protozoan is extremely difficult to cultivate but molecular techniques such as the Polymerase Chain Reaction offer promise as a means of diagnosing infection. The epidemiology of Dientamoebiasis is not clear. This paper will present preliminary results from a study looking for the parasite's presence in swine faeces. The possible role of pigs as a reservoir of infection was studied; 121 faecal samples from breeding and fattening pigs were examined using a Giemsa permanent stain. Dientamoeba fragilis was found in 53 (43.8%) of the stool samples examined.     

A review of mycoplasma diagnostics in cattle

Mycoplasma species have a global distribution causing serious diseases in cattle worldwide including mastitis, arthritis, pneumonia, otitis media and reproductive disorders. Mycoplasma species are typically highly contagious, are capable of causing severe disease, and are difficult infections to resolve requiring rapid and accurate diagnosis to prevent and control disease outbreaks. This review discusses the development and use of different diagnostic methods to identify Mycoplasma species relevant to cattle, with a particular focus on Mycoplasma bovis. Traditionally, the identification and diagnosis of mycoplasma has been performed via microbial culture. More recently, the use of polymerase chain reaction to detect Mycoplasma species from various bovine samples has increased. Polymerase chain reaction has a higher efficiency, specificity, and sensitivity for laboratory diagnosis when compared with conventional culture‐based methods. Several tools are now available for typing Mycoplasma spp. isolates, allowing for genetic characterization in disease outbreak investigations. Serological diagnosis through the use of indirect ELISA allows the detection of antimycoplasma antibodies in sera and milk, with their use demonstrated on individual animal samples as well as BTM samples. While each testing method has strengths and limitations, their combined use provides complementary information, which when interpreted in conjunction with clinical signs and herd history, facilitates pathogen detection, and characterization of the disease status of cattle populations.     

Emerging perspectives in the research of bovine babesiosis and anaplasmosis

The Babesia bovis and B. bigemina apicomplexan protozoa in conjunction with the rickettsia Anaplasma marginale are intraerythrocytic pathogens that are responsible for the most prevalent and costly tick borne diseases (TBD's) of cattle worldwide. These organisms are historically associated as they can cause clinically related hemolytic diseases in cattle, are all transmitted by Rhiphicephallus (Boophilus) ticks, and share an uncanny ability to evade the immune systems of the vertebrate hosts, causing persistent disease. In addition, acute babesiosis and anaplasmosis can be prevented quite effectively by combining tick control and vaccination with living attenuated organisms. However these methods of control have numerous limitations and improved approaches are needed. Importantly, immunizations of cattle with inactivated experimental Babesia and Anaplasma vaccines can elicit variable degrees of protection, indicating the feasibility for the development of inactivated or subunit vaccines. A new research toolbox that includes full genome sequencing combined with the improved ability to genetically modify the organisms is enhancing our understanding of their biology. An emerging paradigm is the use of recently developed Babesia and Anaplasma transfection methods for functional gene characterizations and for vaccine development. Promising recently identified subunit vaccine candidates are also emerging, including babesial proteases, putative rhoptry, microneme, and sexual stage antigens, as well as subdominant, conserved, A. marginale outer membrane major surface proteins. However, significant knowledge gaps on the role of key parasite molecules involved in cell invasion, adhesion, asexual and sexual reproduction, tick transmission, and evasion of the immune system, remain. A better understanding of the biology of these organisms and the protective immune responses will positively contribute toward the goal of developing improved immunological and pharmacological interventions against these elusive pathogens that are responsible for the most devastating TBD's of cattle. Importantly, the currently available research toolbox provides basic research instruments for helping close current knowledge gaps which will aid the design and production of effective vaccines and alternative pharmacological interventions.     

Chicken anaemia virus—a glimpse of the future?

This paper uses background information about chicken anaemia virus as a guide to how the study and control of virus diseases of poultry may develop in the future. It is predicted that ‘new’ viruses will be discovered in poultry, many of which will be difficult to grow in vitro and whose pathogenicity may appear uncertain. When new diseases/syndromes arise in the future, it should be a priority activity to define their pathology. The limitations of currently available virus diagnostic methods are highlighted. The possibility of vaccinating against economically important subclinical disease is discussed, as is the use of recently developed technologies in differentiating virus strains and in developing new vaccines.     

Monoclonal antibody technology in the development of vaccines for livestock parasites

Vaccination against animal parasites offers an alternative to chemotherapy for the control of losses due to morbidity and mortality. However, only a few vaccines are currently available, and these are based on controlled infections with living parasites. Further advancement in the development of defined vaccines against parasite infections has been hindered by incomplete knowledge of the immunological relationship between the host and the parasite. The advent of monoclonal antibody technology has provided a powerful new tool for the identification and isolation of parasite antigens. Exploitation of this technique in veterinary parasitology has greatly facilitated progress toward the development of vaccines against several animal parasites.     

Research Progress on Microbiological Detection Methods for Residual Antibacterials in Animal Food

Antibacterials are used for the treatment or prevention of animal diseases by direct administration or feed additives. The inappropriate use of antibacterials in aquaculture inevitably lead to excessive residue of antibacterials in animal food. Microbiological methods are the earliest methods for the detection of antibacterials residues. Compared with other detection methods, microbiological methods are widely used for detecting residual antibacterials in livestock and poultry, milk and others animal food because it is easy to operate, low cost and high throughput. The purpose of this paper is to review the development history of microbiological methods and its application status for the detection of residual antibacterials in milk, feeding livestock and poultry, eggs, aquatic products and honey, which provides a theoretical and practical basis for the subsequent development of new microbiological methods. This review also makes a prospect of the development of microbiological methods.     

Conjugation and Characterization of Latex Particles with Toxoplasma gondii–specific Immunoglobulin Y Antibodies for Diagnostic Aim and Evaluation Efficiency in In Vitro Culture

Toxoplasma gondii is a parasite that causes severe health problems in the world. Toxoplasmosis, an infection caused by T. gondii, leads to high risk of mortality in patients with immunodeficiency, transplantation, and cancer. Besides that, it causes miscarriages in pregnancy, various abnormalities such as hydrocephalus in infants and congenital diseases. Because the clinical indication of the disease is not specific, it is confused with many diseases, and this leads to the necessity of directly detecting the presence of the toxoplasmosis. Therefore, various diagnostic assays are needed for the diagnosis of the disease. Amongs them, latex agglutination assay is widely used for the detection of specific antibodies or antigens in samples. Latex particles are coated with immunogenic molecules (antigens) to detect antibodies in the blood or used to identify antigens when coated with specific antibodies. In both, aggregation of latex particles results in agglutination. Monoclonal antibodies are often used in latex agglutination assay as in other diagnostic methods. However, monoclonal antibodies can be produced in low quantities at a high cost. Besides, to produce monoclonal antibodies, an experienced staff, a well-equipped cell culture laboratory, a long period of time, and a burdened budget are needed. In recent years, as an alternative to monoclonal antibodies, immunoglobulin Y (IgY) antibodies, which are obtained from chicken eggs, and specifically produced against desired antigenic constructs, have become quite attractive in terms of both low cost and abundant production without requiring infrastructure. In contrast, the latex assay based on IgY antibodies for use in the diagnosis of T. gondii has not been developed. This study aimed to conjugate T. gondii-specific IgY antibodies to latex particles, characterize the particles by Fourier transform infrared spectroscopy, scanning electron microscopy, and spectroscopic methods, and finally demonstrate the interaction with T.gondii parasites in culture with scanning electron microscopy analysis.     

动物源性食品中抗菌药残留的微生物学检测技术研究进展

抗菌药通过直接用药或用作饲料添加剂投入养殖业中,用于治疗或预防动物疾病。养殖业不当使用抗菌药,不可避免地造成抗菌药在动物源性食品中残留过量。微生物学法是最早用于抗菌药残留检测的方法,较其他检测方法而言,微生物学法操作简单、成本低、高通量,广泛应用于牛奶、畜禽组织等动物源性食品中抗菌药残留快速筛选检测,为后期的化学分析确证法节约大量成本。文章主要综述了微生物学法的发展历程及微生物学法在牛奶、可食性畜禽组织、鸡蛋、水产品、蜂蜜等动物源性食品中抗菌药残留检测的应用情况,为后续研制新微生物学法提供理论依据和实践基础,并对未来的微生物学法的发展做出展望。