Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardiaduodenalis and identification of potentially zoonotic subgenotypes

Human giardiasis, caused by the intestinal flagellate Giardia duodenalis, is considered a zoonotic infection, although the role of animals in the transmission to humans is still unclear. Molecular characterisation of cysts of human and animal origin represents an objective means to validate or reject this hypothesis. In the present work, cysts were collected in Italy from humans (n=37) and animals (dogs, one cat and calves, n=46), and were characterised by PCR amplification and sequencing of the beta-giardin gene. As expected, only Assemblages A and B were identified among human isolates. The host-specific Assemblages C and D were found in the majority of dog isolates; however, 6 dog isolates were typed as Assemblage A. The cat-specific Assemblage F has been identified in the single feline isolate available. Among calf isolates, most were typed as Assemblages A (n=12) and B (n=5), whereas the host-specific Assemblage E was rarely found (n=3). Sequence heterogeneity in the beta-giardin gene allowed a number of subgenotypes to be identified within Assemblage A (8 subgenotypes), B (6 subgenotypes), D (2 subgenotypes), and E (3 subgenotypes). Five of these subgenotypes, namely A1, A2, A3, A4 and B3, were found to be associated with infections of humans, of dogs and of calves; these data, therefore, supported the role of these animals as a source of infection for humans.     

宠物与肝包虫病

棘球绦虫寄生于犬科动物的小肠内,其幼虫棘球蚴寄生在动物和人的肝脏,该病严重危害人类和动物健康,对公共卫生造成了严重威胁。本文对包虫病的类型、危害、流行情况以及防治进行综述,希望有助于社会对该病正确的认识、深入的研究和对疾病的防治工作。     

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria‐like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed‐field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.     

Analysis of the Population Structure of Anaplasma phagocytophilum Using Multilocus Sequence Typing

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.     

Genotyping of Enterocytozoon bieneusi in Farmed Blue Foxes (Alopex lagopus) and Raccoon Dogs (Nyctereutes procyonoides) in China

Enterocytozoon bieneusi is the most common species of microsporidia found both in humans and animals. Farmed animals, particularly closely associated to humans, may play an important role of zoonotic reservoir in transmitting this disease to humans. The fur industry is a major economic component in some parts of China. To understand the prevalence, genotype variety and zoonotic risk of E. bieneusi in farmed foxes and raccoon dogs, two species of fur animals, fecal specimens of 110 blue foxes and 49 raccoon dogs from Heilongjiang and Jilin provinces in China were examined by internal transcribed spacer (ITS)-based PCR. E. bieneusi was detected in 16.4% (18/110) blue foxes and 4.1% (2/49) raccoon dogs. Altogether, four genotypes of E. bieneusi were identified, including two known genotypes D (n = 13) and EbpC (n = 5), and two novel genotypes named as CHN-F1 (n = 1) in a fox and CHN-R1 (n = 1) in a raccoon dog. Phylogenetic analysis revealed that all the four genotypes were the members of zoonotic group 1. Genotypes D and EbpC were found in humans previously. The findings of zoonotic genotypes of E. bieneusi in the foxes and raccoon dogs suggest these animals infected with E. bieneusi may pose a threat to human health.     

Increase in terminal restriction fragments of Bacteroidetes-derived 16S rRNA genes after administration of short-chain fructooligosaccharides

It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.     

Prevalence of Blastocystis in Shelter-Resident and Client-Owned Companion Animals in the US Pacific Northwest

Domestic dogs and cats are commonly infected with a variety of protozoan enteric parasites, including Blastocystis spp. In addition, there is growing interest in Blastocystis as a potential enteric pathogen, and the possible role of domestic and in-contact animals as reservoirs for human infection. Domestic animals in shelter environments are commonly recognized to be at higher risk for carriage of enteropathogens. The purpose of this study was to determine the frequency of infection of shelter-resident and client-owned domestic dogs and cats with Blastocystis spp in the Pacific Northwest region of the USA. Fecal samples were collected from 103 shelter-resident dogs, 105 shelter-resident cats, 51 client-owned dogs and 52 client-owned cats. Blastocystis were detected and subtypes assigned using a nested PCR based on small subunit ribosomal DNA sequences. Shelter-resident animals were significantly more likely to test positive for Blastocystis (P<0.05 for dogs, P = 0.009 for cats). Sequence analysis indicated that shelter-resident animals were carrying a variety of Blastocystis subtypes. No relationship was seen between Blastocystis carriage and the presence of gastrointestinal disease signs in either dogs or cats. These data suggest that, as previously reported for other enteric pathogens, shelter-resident companion animals are a higher risk for carriage of Blastocystis spp. The lack of relationship between Blastocystis carriage and intestinal disease in shelter-resident animals suggests that this organism is unlikely to be a major enteric pathogen in these species.     

基于毛细管电泳的快速检测七种人冠状病毒技术方法的建立

近来,一种新型冠状病毒(SARS-CoV-2)引发的COVID-19突发疫情,给全球公众健康和社会经济构成严重威胁。SARS-CoV-2成为继人冠状病毒229E(Human coronavirus 229E,HCoV-229E)、人冠状病毒OC43(Human coronavirus OC43,HCoV-OC43)、严重急性呼吸综合征冠状病毒(Severe acute respiratory syndrome coronavirus,SARS-CoV)、人冠状病毒NL63(Human coronavirus NL63,HCoV-NL63)、人冠状病毒HKU1(Human coronavirus HKU1,HCoV-HKU1)和中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)后第七种感染人类的冠状病毒。本研究以高分辨毛细管电泳技术为基础,针对七种人冠状病毒基因保守区分别设计特异性引物对,经常规PCR扩增后,通过具备单碱基差异分辨率的毛细管电泳分析,实现快速检测七种人冠状病毒的目标。通过构建基于毛细管电泳的人冠状病毒分子靶标,实现同时快速精准鉴定七种人冠状病毒的目的。本研究建立的人冠状病毒毛细管电泳快速检测技术方法具有极高灵敏性和精确性,分辨率高而且特异性好,操作简便成本低廉,为人冠状病毒的临床诊断、口岸快速检测等提供了新的技术支持。     

Detection of Leishmania infantum DNA in the non-parasitized lung of dogs with visceral leishmaniasis

Background

Despite the very low or absent parasitism in the lungs, the interstitial pneumonitis is a common lesion found in humans and dogs with visceral leishmaniasis. The lung is a neglected organ in the study of dogs and humans with visceral leishmaniasis, but interstitial pneumonitis represents an important lesion characterized by thickening of the alveolar septum due to fibrosis and inflammatory exudate, and its pathogenesis is still uncertain. In this study, the polymerase chain reaction (PCR) was used to detect Leishmania infantum in paraffin-embedded lung biopsies from naturally infected dogs from an endemic area in Minas Gerais State, Brazil; PCR was compared to histological and immunohistochemical techniques for detecting Leishmania.

Results

Eighteen dogs in which leishmaniasis had been diagnosed by serological tests - indirect immunofluorescence assay (IFAT), enzyme-linked immunosorbent assay (ELISA) and complement fixation tests (CFT) - were classified as asymptomatic, oligosymptomatic or symptomatic. Nine of the 18 dogs studied had a positive PCR (50%) but parasites were not detected by histopathological and immunocytochemistry methods.

Conclusions

These data indicate that PCR on DNA extracted from paraffin-embedded tissue is a valuable method for detecting Leishmania infantum parasites in lungs of naturally infected dogs, despite the apparent absence of parasites from standard HE (hematoxylin and eosin) stained slides and of labeled parasites from immunocytochemical preparations.

     

Immunochemical detection of oxidatively damaged DNA

Oxidatively damaged DNA is implicated in various diseases, including neurodegenerative disorders, cancer, diabetes, cardiovascular and inflammatory diseases as well as aging. Several methods have been developed to detect oxidatively damaged DNA. They include chromatographic techniques, the Comet assay, (32)P-postlabelling and immunochemical methods that use antibodies to detect oxidized lesions. In this review, we discuss the detection of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG), the most abundant oxidized nucleoside. This lesion is frequently used as a marker of exposure to oxidants, including environmental pollutants, as well as a potential marker of disease progression. We concentrate on studies published between the years 2000 and 2011 that used enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry to detect 8-oxodG in humans, laboratory animals and in cell lines. Oxidative damage observed in these organisms resulted from disease, exposure to environmental pollutants or from in vitro treatment with various chemical and physical factors.     

Canine cystinuria: polymorphism in the canine<Emphasis Type="Italic"> SLC3A1</Emphasis> gene and identification of a nonsense mutation in cystinuric Newfoundland dogs

Cystinuria is an inherited renal and intestinal disease characterized by defective amino acid reabsorption and cystine urolithiasis. Different forms of the disease, designated type I and non-type I in cystinuric humans, can be distinguished clinically and biochemically, and have been associated with mutations in the SLC3A1 (rBAT) and SLC7A9 genes, respectively. Type I cystinuria is the most common form and is inherited as an autosomal recessive trait in humans. Cystinuria has been recognized in more than 60 breeds of dogs and a severe form, resembling type I cystinuria, has been characterized in the Newfoundland breed. Here we report the cloning and sequencing of the canine SLC3A1 cDNA and gene, and the identification of a nonsense mutation in exon 2 of the gene in cystinuric Newfoundland dogs. A mutation-specific test was developed for the diagnosis and control of cystinuria in Newfoundland dogs. In cystinuric dogs of six other breeds, either heterozygosity at the SLC3A1 locus or lack of mutations in the coding region of the SLC3A1 gene were observed, indicating that cystinuria is genetically heterogeneous in dogs, as it is in humans. The canine homologue of human type I cystinuria provides the opportunity to use a large animal model to investigate molecular approaches for the treatment of cystinuria and other renal tubular diseases.     

A real time PCR assay for the detection and quantitation of Mycobacterium avium subsp. paratuberculosis using SYBR Green and the Light Cycler

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, and may also contribute to the onset and development of Crohn's disease in humans. Due to its reported heat resistance, isolation from pasteurised milk and the potential for transmission of MAP through environmental sources, rapid detection is imperative. Here, we present a rapid real time PCR assay for MAP that can simultaneously detect and quantify this organism in the laboratory using SYBR Green and the Lightcycler (LC). This method uses the highly characterised and specific P90, P91 primers, which amplify a 400-bp region of the IS900 element. Using quantified standard DNA, this assay can accurately detect as few as 20 copies (equivalent to approximately 1.5 organisms). The method is effective using a variety of templates including isolated MAP DNA, pure colonies or liquid culture sources, and can also work in the presence of contaminating bacteria. A useful application of this assay is the ability to accurately and rapidly quantitate the number of MAP cells in liquid culture as determined by comparison to previously enumerated standards.     

Current and future uses of real-time polymerase chain reaction and microarrays in the study of intestinal microbiota, and probiotic use and effectiveness

Probiotics are defined as live microorganisms that confer a health benefit to the host when administered in adequate amounts. In addition to human health benefits, probiotics can improve various aspects of growth and performance in livestock and poultry, as well as control undesirable microorganisms in food animals. Studies indicate that probiotics can prevent or treat certain conditions, including atopic disease in infants, food allergy, infection after surgery, acute diarrhea, and symptoms associated with irritable bowel syndrome. Understanding the complete mechanism, effectiveness, and potential use of probiotics is limited by the availability and sensitivity of current methods (i.e., culturing techniques). In recent years, real-time polymerase chain reaction (PCR) and microarrays have become prominent and promising methods to examine quantitative changes of specific members of the microbial community and the influence of probiotics on the structure and function of human and animal intestinal ecosystems. Culture-independent studies have established that only a fraction of organisms present in feces are cultivable, therefore, results obtained by cultivation are limited. Conversely, in-depth knowledge of microbial genomes has enabled real-time PCR and microarrays to be more sensitive and has resulted in precise methods for comprehensive analysis of the complex gut microbiota. Additionally, these technologies can assess the influence of intestinal microorganisms on host metabolism, nutrient status, and disease. This paper reviews method technologies and applications of real-time PCR and microarray assays as they relate to the effect and use of probiotics on the intestinal microbiota and gastrointestinal disease.     

Analysis of ECG in ventricular fibrillation in man and animals based on chaos theory

The methods of the chaos theory were used to estimate the degree of irregularity of ventricular fibrillation in human and experimental animals. To verify the hypothesis that the degree of chaos depends on the species of the living organisms, the parameters characterizing the degrees of irregularity of ventricular fibrillation were estimated and compared. The comparative analysis was performed using 32 fragments of electrocardiographic records from five patients with sudden ventricular fibrillation bouts and 215 episodes of induced fibrillation in 17 animals. It was shown that fibrillation in human and animals has a different degree of regularity and different values of the chaotic component. The highest values of chaos were recorded in dogs, the lowest degree of chaos was observed in human. Rabbits and rats are intermediate, between dogs and humans. The fractuality of the structure-function organization of myocardium is discussed.     

A simple duplex-PCR protocol for routine diagnosis and follow up of canine leishmaniasis

The introduction of PCR has efficiently improved diagnosis of canine leishmaniasis. In order to provide a robust, efficient and reliable diagnostic method, a duplex PCR assay targeting the Leishmania infantum kDNA minicircle and the canine GAPDH gene as inner control was designed. Sensitivity of the assay reached 0.15 parasites/ml blood. Development, testing and application of this system on a group of 10 dogs during therapy administration (60 days) are also described. Six dogs (out of eight that have been showing a positive PCR result on peripheral blood during the study) were tested negative at day 62, indicating a reduction of parasitaemia at the end of the treatment period. All the animals had a positive PCR on lymph node aspirate both at the beginning and at the end of treatment. These findings seem to suggest that, in order to test therapy efficacy, PCR on whole blood could be a useful assay in dogs that have a positive PCR at the beginning of the treatment, while PCR positivity on lymph nodes lasts longer than the observation period during therapy administration. The presence of the GAPDH inner control band efficiently contributed to prevent false negatives, by highlighting samples affected by haemoglobin inhibition or inappropriate DNA isolation.     

Novel non-rodent models of kidney disease

Kidney disease in the 21(st) century affects increasing numbers of individuals. We continue to be challenged by our lack of understanding of the pathophysiology of acute and chronic renal disease including genetic diseases involving the kidney. Rodent knockout animals or inbred strains have greatly contributed to our understanding of many monogenetic and complex diseases. Non-rodent animal models of disease have become more attractive since genomic data has become available for a variety of organisms that offer distinct advantages over mice and rats for ease in conducting high-throughput chemical or mutagenesis screens. It is thus timely to examine the physiology and pathophysiology of the kidney or kidney equivalents in these organisms to evaluate their relevance as models for human disease. In addition to organisms whose small size and accessibility facilitate large scale screening approaches, larger animals at the other end of the spectrum offer unique physiological advantages in both size equivalency to humans as well as, in some cases, physiological and pathophysiological responses that closely mimic those of humans. Here we review a selected number of non-rodent experimental models of kidney diseases, focusing on recent advances in the use of the worm Caenorhabditis elegans, the fruitfly Drosophila melanogaster, the zebrafish Danio rerio, the little skate Leucoraja erinacea, the MGH miniature swine, merino cross sheep, and the cow Bos taurus to study kidney disease.     

Real-time PCR assays for monitoring benzimidazole resistance-associated mutations in Ancylostoma caninum

Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infection has led to drug resistance that often renders whole populations of livestock unresponsive to treatment. Therefore, it is important to detect mutations associated with drug resistance before it becomes clinically manifest. To monitor developing drug resistance against benzimidazoles (BZ), we developed real-time PCR assays and applied them to analyse the beta-tubulin isotype-1 gene of the hookworm Ancylostoma caninum, an important parasite of dogs. Previously, we developed PCR assays to monitor codon positions 167 and 200. Here, we describe an assay which is able to detect resistance alleles in codon 198. These real-time PCR assays were subsequently applied to screen hookworm specimens recovered from dogs in Georgia. No elevated levels of polymorphisms at the investigated loci were found, suggesting that selection for resistance in the tested samples did not occur.     

Animal models of human microsporidial infections

Two new models have been described for Enterocytozoon bieneusi, non-human primates and immuno-suppressed gnotobiotic pigs, but there still is no successful cell culture system. The intestinal xenograft system holds promise as an animal model for Encephalitozoon intestinalis. Encephalitozoon hellem is easily propagated in mice, and also may be an important cause of spontaneous disease of psittacine birds. Encephalitozoon cuniculi occurs spontaneously in a wide variety of animals and can be induced experimentally in athymic mice. This is a useful experimental system and animal model, but the infection is relatively rare in man. Mammalian microsporidioses first were recognized as spontaneous diseases of animals that later confounded studies intended to elucidate the nature of diseases of humans. Much was learned about both experimental and spontaneous animal microsporidial infections that subsequently has been applied to the human diseases. In addition, new diseases have appeared, in both animals and humans, for which models are being developed. Since there are now animal models for almost all the known human microsporidioses, information on pathogenesis, host defenses, and effective treatments may become available soon. The microsporidioses provide a good example of the value of comparative pathology. Dr. Payne: Joe Payne. How much accidental infection has occurred with adjacent laboratory animals? Dr. Shadduck: A hard question. The organisms are thought to spread horizontally, and there is some pretty good evidence for that in rabbits. One assumes that this also is the explanation for the occurrence in infected kennels. Horizontal transmission probably occurs via contaminated urine, at least in the case of rabbits and dogs. Experimentally, horizontal transmission has been difficult to demonstrate in mice. Relative to the danger in people, I don't know how to answer that. I have always treated this as one of those things where you should be careful, but you shouldn't get paranoid. So, we have handled infected cell cultures and animals as if they were potentially infectious for man, but not as if they were something as hot as the human AIDS virus, for example. With the increasing number of reports in humans, I think it is clear that one would never want anybody who was at risk of being immunocompromised to work with these organisms. Dr. Fenkel: Are there other questions? Dr. Mysore: How do the parasites spread within the infected hosts? Dr. Shadduck: The usual answer is hematogenously via infected macrophages, but data that actually support that statement are rare. One does see infected macrophages in tissues, so it is not unreasonable to think that some of them escape and lodge in other tissues. But that has never actually been formally demonstrated. Dr. Nakeeb: Is E. bieneusi a human pathogen? Dr. Shadduck: The answer depends on which paper you read and what approach the authors took. There are papers in which the authors argue that the organism is not a cause of clinical disease in AIDS patients, but the general belief today is that the parasite does cause diarrhea and enteritis. I think the evidence for pathogenicity is quite strong for the various species of the Encephalitozoon, based on the severity and distribution of the lesions.     

Prevalence and distribution of Clostridium difficile PCR ribotypes in cats and dogs from animal shelters in Thuringia,Germany

Clostridium difficile is an important cause of nosocomial diarrhoea in humans. Pet animals and livestock are discussed as potential natural reservoirs and sources of infection. In this study faecal samples from dogs and cats were collected at 10 animal shelters in Thuringia, Germany. C. difficile was isolated from 9 out of 165 (5.5%) canine and 5 out of 135 (3.7%) feline samples. Five PCR ribotypes (010, 014/020, 039, 045, SLO 066) were identified. PCR ribotypes 010 and 014/020 were detected in more than one shelter and PCR ribotypes 014/020 and 045 were isolated from dogs and cats. MLVA profiles of strains of a PCR ribotype from one shelter were identical or closely related, while strains of the same PCR ribotype from different shelters showed significant differences. This study shows that dogs and cats kept in animal shelters are a reservoir of C. difficile PCR ribotypes which can infect also humans.