How to make DNA count: DNA-based diagnostic tools in veterinary parasitology

Traditional methods for the diagnosis of parasitic helminth infections of livestock have a number of limitations, such as the inability to distinguish mixed-species infections, a heavy reliance on technical experience and also sub-sampling errors. Some of these limitations may be overcome through the development of rapid and accurate DNA-based tests. For example, DNA-based tests can specifically detect individual species in a mixed infection at either the larval or egg stages, in the absence of morphological differences among species. Even so, some diagnostic problems remain the same, irrespective of whether a DNA-based or traditional method is used. For example, sub-sampling errors from an aggregated distribution are likely to persist. It is proposed, however, that DNA-based diagnostic technologies offer an opportunity to expand diagnostic capabilities, and are discussed in the current review. The future introduction of DNA-based diagnostic technologies into routine diagnostic settings will also be discussed.     

Molecular genetic methods in the veterinary clinical bacteriology laboratory: current usage and future applications

In the last 5 years, numerous molecular methods have been published for the detection and characterization of bacteria in the field of veterinary medicine. PCR has been the most commonly used technology. Although not currently used for clinical veterinary diagnosis, new technologies such as liquid-phase hybridization, real-time PCR, pathogen load determination and DNA/protein microarray have been described and have many possible applications in the clinical bacteriology laboratory because of their sensitivity and efficiency. This review describes the basic principles and application of recently published DNA-based molecular techniques for the purpose of veterinary clinical bacteriological diagnosis. It covers advances in probe hybridization technology, DNA/RNA amplification techniques and other molecular detection methods, including 16S rRNA analysis for bacterial characterization and DNA microarrays for bacterial detection. The review briefly summarizes the application of molecular methods for the diagnosis of specific important bacterial infections of animals, and for other animal pathogens that are slow or difficult to isolate in the clinical bacteriology laboratory. In addition, the molecular detection of antimicrobial resistance genes and of bovine mastitis pathogens is briefly described and current commercially available tests are listed.     

干酪乳杆菌群和植物乳杆菌群的分子鉴定技术和分类方法的研究进展

乳杆菌属是乳酸菌类群中最大的一个属,在乳酸杆菌的研究和应用中,分类鉴定是其中的一个重要领域。由于乳杆菌属内许多种间的表型特征难以区别,利用生理生化实验方法往往无法进行准确鉴定。近几年,随着分子生物学的飞速发展,从分子和基因水平来认识乳酸菌的遗传结构和分类已成为可能,而采用常规的16SrDNA序列同源性分析不能将相近的植物乳杆菌群和干酪乳杆菌群分别鉴定到种及亚种的水平,目前许多新的分子鉴定技术和分类方法被用来进行相近乳酸菌的分类和鉴定。本文结合自己的试验归纳出如蛋白酶编码基因序列分析、扩增片段长度多态性分析、种间特异性PCR等九种用于区分和鉴定植物乳杆菌群和干酪乳杆菌群的分子技术和方法,对乳酸菌的多相分类学研究和工业化生产具有的重要意义。     

Zoonotic potential of Chlamydophila

The purpose of this article is to present the diseases induced in humans and animals by the different species of Chlamydophila, after providing an overview on the history of these infectious agents and their taxonomy. The route of transmission and the available methods for prevention and control in the different animal species are reviewed.     

What's new in muscle and peripheral nerve diseases?

It is likely that most neuromuscular diseases that are described in humans will have a counterpart in our companion animals. With the advent of molecular genetics and the completion of the canine and feline genomes, an ever expanding number of DNA-based tests should become available for the diagnosis of muscle and peripheral nerve diseases. Molecular testing procedures should enable us to continue to unravel the molecular basis of neuromuscular diseases for which the cause is still unknown. It is important that accurate clinical evaluations and diagnostic testing, including muscle and peripheral nerve biopsies, are performed in order to reach these goals. This review focuses on recently identified inherited neuromuscular diseases in companion animals.     

Molecular diagnosis of infections and resistance in veterinary and human parasites

Since 1977, >2000 research papers described attempts to detect, identify and/or quantify parasites, or disease organisms carried by ecto-parasites, using DNA-based tests and 148 reviews of the topic were published. Despite this, only a few DNA-based tests for parasitic diseases are routinely available, and most of these are optional tests used occasionally in disease diagnosis. Malaria, trypanosomiasis, toxoplasmosis, leishmaniasis and cryptosporidiosis diagnosis may be assisted by DNA-based testing in some countries, but there are very few cases where the detection of veterinary parasites is assisted by DNA-based tests. The diagnoses of some bacterial (e.g. lyme disease) and viral diseases (e.g. tick borne encephalitis) which are transmitted by ecto-parasites more commonly use DNA-based tests, and research developing tests for these species makes up almost 20% of the literature. Other important uses of DNA-based tests are for epidemiological and risk assessment, quality control for food and water, forensic diagnosis and in parasite biology research. Some DNA-based tests for water-borne parasites, including Cryptosporidium and Giardia, are used in routine checks of water treatment, but forensic and food-testing applications have not been adopted in routine practice. Biological research, including epidemiological research, makes the widest use of DNA-based diagnostics, delivering enhanced understanding of parasites and guidelines for managing parasitic diseases. Despite the limited uptake of DNA-based tests to date, there is little doubt that they offer great potential to not only detect, identify and quantify parasites, but also to provide further information important for the implementation of parasite control strategies. For example, variant sequences within species of parasites and other organisms can be differentiated by tests in a manner similar to genetic testing in medicine or livestock breeding. If an association between DNA sequence and phenotype has been demonstrated, then qualities such as drug resistance, strain divergence, virulence, and origin of isolates could be inferred by DNA-based tests. No such tests are in clinical or commercial use in parasitology and few tests are available for other organisms. Why have DNA-based tests not had a bigger impact in veterinary and human medicine? To explore this question, technological, biological, economic and sociological factors must be considered. Additionally, a realistic expectation of research progress is needed. DNA-based tests could enhance parasite management in many ways, but patience, persistence and dedication will be needed to achieve this goal.     

犬常见皮肤病的日常诊护

犬皮肤病是犬临床诊治中常见病之一。犬因品种、年龄、个体、营养、生活环境等不同而引起的皮肤病有很大的差异。本文就几种常见的犬皮肤病的不同的临床症状进行了简要综述,旨在给饲养者提供一些早期的、简单的鉴别及诊断,能尽快做出相应的预防和护理措施,并及时就诊。     

犬常见皮肤病的日常诊护

犬皮肤病是犬临床诊治中常见病之一。犬因品种、年龄、个体、营养、生活环境等不同而引起的皮肤病有很大的差异。本文就几种常见的犬皮肤病的不同的临床症状进行了简要综述,旨在给饲养者提供一些早期的、简单的鉴别及诊断,能尽快做出相应的预防和护理措施,并及时就诊。     

A brief review on the laboratory diagnosis of leptospirosis

Leptospirosis is a ubiquitous zoonotic disease that may be maintained in either wild or domesticated animal species. These bacteria have been classified into serovars based on their antigenic characteristics and, more recently, into species based on genomic studies. They produce both chronic and acute infections. Chronic infections of serovars in the host species to which they have become adapted can result in long term shedding, providing a source of acute infection for other species. As clinical presentation can vary greatly, diagnosis often depends on laboratory methods. In addition to diagnostic testing, herd health monitoring and screening for international trade purposes are performed at veterinary laboratories. The test method selected varies depending on the samples available and the purpose of testing. An increasing variety of laboratory methods are being described for detection of bacteria and antibodies. In addition to classical methods such as culture, dark-field, microscopy and the microscopic agglutination test (MAT), a variety of polymerase chain reaction (PCR), indirect enzyme-linked immunosorbent assay (ELISA), competitive ELISA and other rapid serological tests have been described. This review describes the advantages and limitations of these assays together with other factors that may affect results and their interpretation, such as species variation, vaccination and antibiotic administration.     

Comparative gastric anatomy of Cricetomys gambianus and Saccostomus campestris (Cricetomyinae) in relation to Mystromys albicaudatus (Cricetinae)

The gastric anatomy of two African cricetomyines is described and compared with that of the only African cricetine. The stomach of C. gambianus is more specialized than that of S. campestris and shows many parallels with that of M. albicaudatus. Both cricetomyines possess an oesophageal groove system which is absent from the cricetine, while C. gambianus and M. albicaudatus have forestomach papillae supporting vast colonies of symbiotic bacteria that are not found in S. campestris. Specializations in gastric anatomy are discussed in relation to phylogeny, using taxonomy to distinguish between apomorphic and plesiomorphic characters, convergence and divergence. Complex gastric adaptations can be explained only by increased digestive efficiency, while the symbiotic association with numerous autochthonous bacteria implies-revolutionary adaptation.     

Applications of DNA amplification techniques in veterinary diagnostics

An overview of the principles of the polymerase chain reaction, ligase chain reaction, self-sustained sequence replication and Q replicase is given. The application of these methods for the diagnosis of veterinary infectious and hereditary diseases as well as for other diagnostic purposes is discussed and comprehensive tables of reported assays are provided. Specific areas where these DNA-based amplification methods provide substantial advantages over traditional approaches are also highlighted. With regard to PCR-based assays for the detection of viral pathogens, this article is an update of a previous review by Belák and Ballagi-Pordány (1993).These two authors contributed equally to this review and are listed in alphabetical order.     

布鲁菌进化和分类学研究进展

布鲁菌属革兰氏阴性兼性胞内寄生菌,能感染多种宿主动物和人。该属可分为6个典型种,包括羊种、牛种、猪种、沙林鼠种、绵羊附睾种以及犬种布鲁菌等。此分类是基于其致病性以及宿主偏好性的差异划分。尽管6个种通过传统表型试验能区分,但布鲁菌种内采用DNA-DNA杂交证明DNA同源性高度一致（相似性大于90%）。因此有人提议布鲁菌由单一种组成,即布鲁菌属中只有羊种布鲁菌,其他种都是羊种菌的生物亚型之一。然而基于其他分子技术的基因分型表明其DNA多态性表现明显,说明目前对这个种的分型还是比较准确。而最近分离的海洋种布鲁氏菌分离株（鳍型和鲸型）采用传统分型标准和一些特异的分子标记也证明这种分型比较正确。本文对目前布鲁菌种属进化和分类学进行综述,希望对研究其进化和分类有所帮助。     

Bartonella,bats and bugs: A review

Ecological, immunological, and epidemiological factors enable bats to transmit an increasingly recognized spectrum of zoonotic agents, and bartonellae are among those emerging pathogens identified in bats and their arthropod ectoparasites. Current data reveal a multifaceted disease ecology where diverse host species distributed around the world interact with a number of Bartonella spp. and several potential vectors. This review summarizes the methods and findings of studies conducted since 2005 to illustrate that Bartonella bacteremia varies by bat species, location, and other potential variables, such as diet with a very high prevalence in hematophagous bats. Among bat families, Bartonella prevalence ranged from 7.3% among Nycteridae to 54.4% in Miniopteridae. Further research can build on these current data to better determine risk factors associated with Bartonella infection in bat populations and the role of their ectoparasites in transmission.     

Serologic and molecular characterization of tickborne pathogens in lions (Panthera leo) from the Fasano Safari Park, Italy.

Lions (Panthera leo) are an endangered species threatened by illegal hunting, habitat loss, and infectious diseases. Little is known about the tick-borne pathogens that infect lions and could contribute to population declines. The objective of this study was to characterize Rickettsia spp., Anaplasma phagocytophilum, and Coxiella burnetii infections in 10 lions from the Fasano Safari Park in Italy by serology, polymerase chain reaction, and sequence analysis. Although animals did not show clinical signs of tick-borne diseases, evidence of infection with C. burnetii, spotted fever group Rickettsia sp., and A. phagocytophilum were found in 50%, 20%, and 10% of the lions, respectively. One of the lions tested positive for all three pathogens. This study is the first report of molecular evidence of infection with C. burnetii, Rickettsia sp., and A. phagocytophilum in lions and provides evidence that these felids become infected and serve as hosts for tick-transmitted bacteria.     

羊痘的研究进展

羊痘病毒能引起山羊痘、绵羊痘和牛的结节性疹块病。羊痘是以发热、全身性的皮肤损伤、痘疹和淋巴结病变为特征。羊痘病毒是所有动物痘病毒中最为重要的一种，严重影响养羊业和国际贸易的发展。本文从病原学、流行病学、诊断和预防控制等方面对羊痘做一综述。虽然此病从临床症状和宿主特异性上很容易做出诊断，但进一步的实验室确诊还是必要的，现已有多种检测方法和诊断试剂。控制该病最有效的方法还是使用疫苗对易感动物进行免疫接种。     

The feline oral microbiome: A provisional 16S rRNA gene based taxonomy with full-length reference sequences

The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species.The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria.Clone libraries were produced using “universal” and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees.The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as “Propionibacterium sp. feline oral taxon FOT-327” is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.     

营养物质对生物自由基产生及清除影响的研究进展

本文主要就营养物质对生物自由基产生及清除的影响进行了综述,并阐述了生物自由基的种类和生物学功能及其产生和清除的途径。     

Actinobacillus species and their role in animal disease

Actinobacillus species are Gram-negative bacteria responsible for several quite distinct disease conditions of animals. The natural habitat of the organisms is primarily the upper respiratory tract and oral cavity. A. lignieresii is the cause of actinomycosis (wooden tongue) in cattle: a sporadic, insidiously-developing granulomatous infection. In sharp contrast is A. pleuropneumoniae which is responsible for a rapidly spreading often fatal pneumonia, common among intensively reared pigs. Detailed investigation of this organism has provided a much clearer picture of the bacterial factors involved in causing disease. A. equuli similarly causes a potent septicaemia in the neonatal foal; growing apparently unrestricted once infection occurs. Other members of the genus induce characteristic pathogenesis in their preferred host, with one, A. actinomycetemcomitans, being a cause of human periodontal disease. This article reviews recent understanding of the taxonomy and bacteriology of the organisms, and the aetiology, pathogenicity, diagnosis and control of animal disease caused by Actinobacillus species.     

PCR identification of Leishmania in diagnosis and control of canine Leishmaniasis

Leishmaniases are endemic in many countries, mainly in rural areas. In Brazil, Leishmania infection is responsible for many cases of Leishmaniases, including recent reports in urban regions. Despite their sensitivity, traditional serological and parasitological methods for detecting Leishmaniases have proven inadequate for species discrimination. This study aimed to identify Leishmania species in biological samples by a fast methodology, avoiding "in vitro" cultivation. Knowledge of the Leishmania species is an important tool in regions where both New World visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are prevalent. As these new foci appear in areas not traditionally endemic for VL, the main problem is to distinguish between true autochthonous infections and infections acquired in other well-known endemic areas. Since, domestic dogs are known to be the main VL and CL reservoir, they are regularly investigated in endemic areas to prevent, principally, severe and often fatal VL in humans. However, several infected dogs present no clinical signs or clinical signs similar to other canine diseases. Here, we evaluated the ability of PCR to diagnose VL and distinguish L. (L.) chagasi from other Leishmania species in domestic dogs. Samples from 114 dogs from 30 cities (Sao Paulo, Brazil) were divided into two groups: 44 symptomatic and 70 asymptomatic. They were assayed by parasitological methods (culture and microscopic examination) and PCR to determine L. (L.) chagasi, L. (V.) braziliensis; and in some cases, Leishmania spp. Parasitological tests and PCR-L. chagasi were concordant in 105 samples (92%). VL was confirmed in 49 dogs, while 56 had negative results. Of the 114 samples, 9 had discordant results, but were further tested by PCR-Leishmania spp. with positive results. VL was also confirmed in 4 dogs having negative parasitological tests and positive PCR-L. chagasi. Consequently, this PCR was positive for 100% (53/49) of dogs with parasites detected in parasitological tests. Also, PCR demonstrated high specificity detecting 61 dogs negative for VL. Leishmania infection was negative in 56 dogs, and 5 with positive culture and PCR-Leishmania spp. had CL since they were positive in PCR-L. braziliensis. This study shows the importance of including PCR in diagnosis of Leishmaniases by differential diagnosis contributing to the surveillance and control of VL programs.