Differentiation of Setaria digitata and Setaria labiatopapillosa using molecular markers

5S rRNA intergenic regions of Setaria digitata and Setaria labiatopapillosa were PCR amplified with primers designed from the 5S rRNA gene of Brugia malayi. The ladder-like banding patterns obtained for the amplifications were distinctly different for the two species. Four amplified products were cloned into the pBS vector and completely sequenced. DNA clones from two individual samples of S. digitata, Sd4 and Sd6, showed 97% sequence homology to each other. All sequenced clones showed the presence of the spliced leader (SL) RNA gene with a 22 nucleotide spliced leader sequence. The phylogenetic tree constructed using these data and the 5S rRNA intergenic regions of several other filarial nematodes showed the Setaria species sharing a branch with Dirofilaria. RAPD-PCR analyses identified 107 bands of which 86 were polymorphic (80%). A dendrogram constructed for S. digitata and S. labiatopapillosa separated the two species into two distinct clusters. The polymorphic loci identified by the RAPD-PCR analyses can be studied further to develop species-specific probes/PCR primers for the identification of each species.     

水貂病毒性肠炎巢式PCR,PCR-RFLP联合诊断方法的建立

根据水貂肠炎细小病毒的VP2基因序列设计4条特异性引物,建立了检测水貂肠炎细小病毒的巢式PCR方法。采用一次扩增的敏感性来检测5个TCID50/mL的细小病毒MEVB株,二次扩增的敏感性来检测0.05个TCID50/mL的细小病毒MEVB株。同时,利用该方法第一轮PCR产物酶切,能够区分犬细小病毒与水貂肠炎细小病毒,具有重要的实用价值和应用的前景。     

Identification of Borrelia burgdorferi genospecies inducing Lyme disease in dogs from Western Poland

Canine Lyme borreliosis may be caused by three Borrelia burgdorferi sensu lato genospecies. The prevalence of infection by Borrelia species was determined by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with the enzyme Fsp4H I in the blood of dogs naturally infested by ticks in an endemic region of Poland. Blood samples were collected from 98 dogs of various breeds, delivered to the Veterinary Clinic in Szczecin (northwestern Poland) for various reasons. Nested PCR revealed the presence of DNA characteristic of only 1 genospecies, i.e. B. burgdorferi sensu stricto (s.s.), in all PCR-positive samples. Digestion of PCR products from a fragment of the fla gene amplified with primers FLA1 and FLA2 gave only one band pattern consistent with the pattern obtained from sequence analysis of the fla gene from a reference isolate of B. burgdorferi s.s. GeHo (X15660) from GenBank.     

Detection of Ehrlichia platys in dogs in Australia

OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.     

New view on the age-specificity of pig Cryptosporidium by species-specific primers for distinguishing Cryptosporidium suis and Cryptosporidium pig genotype II

Two species of Cryptosporidium are commonly identified in pigs: Cryptosporidium suis and Cryptosporidium pig genotype II. Detection of Cryptosporidium spp. is routinely based on molecular methods such as polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) or gene sequencing. However, most of these methods are hampered by low sensitivity to mixed infections. As a solution of this problem, novel species-specific primers were designed and tested in the present study. Sensitivity of our primers was identical to genus-specific primers, but more (1:48) mixed infections were detected using these species-specific primers on 477 DNA samples originating from naturally infected pigs of different age categories. Our results show differences in age-dependent susceptibility of pigs to the infection. Whereas C. suis was found in all tested categories of pigs (1-12 week of age and sows), Cryptosporidium pig genotype II was recorded only in animals older than 6 week of age. Usage of species-specific primers could help to better the understanding of epidemiology of pig specific Cryptosporidium spp. and its occurrence, which, on the basis of our results, is underestimated.     

鉴别牛早期胚胎性别PCR方法引物的设计与筛选

根据牛Y-染色体特异重复序列、睾丸特异蛋白基因以及性别决定基因序列设计合成5对公牛Y-染色体特异引物，依据牛骨胳肌α肌动蛋白前体基因和微卫星DNA序列设计合成4对牛DNA特异引物(内标引物)。单重PcR扩增牛基因组DNA，筛选出4对牛Y-染色体特异引物和1对牛DNA特异内标引物。将不同的Y-染色体特异引物与内标引物组合，多重PCR扩增牛基因组DNA、已知性别的牛成纤维细胞和克隆胚胎，筛选出2个可用于牛早期胚胎性别鉴别的PCR引物组合：B34／A12和B78／A12。     

Species identification of trichomonads and associated coinfections in dogs with diarrhea and suspected trichomonosis

Trichomonads have been infrequently reported in the feces of dogs where their pathogenicity remains uncertain. It is currently unknown whether Tritrichomonas foetus or Pentatrichomonas hominis is identified more commonly in dogs with trichomonosis or how often these infections are accompanied by concurrent enteric infectious agents. The objective of this study was to determine the identity of trichomonads present in a series of 38 unsolicited canine diarrheic fecal samples submitted for T. foetus diagnostic polymerase chain reaction (PCR) testing between 2007 and 2010. We also examined each fecal sample for an association of trichomonosis with concurrent infection using a convenient real-time PCR panel for nine gastrointestinal pathogens. P. hominis, T. foetus, or both were identified by PCR in feces of 17, 1, and 1 dogs respectively. Feces from the remaining 19 dogs were PCR negative for T. foetus, P. hominis and using broader-spectrum Trichomonadida primers. The total number and specific identities of concurrent enteropathogens identified did not differ between fecal samples from dogs that were or were not identified by PCR as infected with trichomonads. These results suggest that P. hominis infection is more frequently identified than T. foetus infection in diarrheic dogs with trichomonosis and that concurrent enteropathogen infection is common in this population.     

Fecal polymerase chain reaction with 16S ribosomal RNA primers can detect the presence of gastrointestinal Helicobacter in dogs

Questions about pathogenesis and therapy for Helicobacter infections in dogs could be answered with a simple, noninvasive, sensitive, and specific diagnostic test. We hypothesized that a fecal polymerase chain reaction (PCR) assay would detect Helicobacter and could be useful for assessing therapeutic responses. Paired gastric biopsies and fecal samples were obtained from 39 random source dogs (group 1). Gastric biopsies from each of these dogs had histologic evidence of gastric spiral bacteria, and paired gastric tissue and fecal samples produced a 375-base pair (bp) product when amplified by PCR with Helicobacter-specific primers. Specificity of the PCR product was confirmed by detection of expected 60-, 119-, and 196-bp products following Hinfl digestion. Direct sequencing of amplicons from paired PCR products from gastric biopsy and fecal samples from 8 group I dogs showed that gastric products had the highest homologies with known gastric Helicobacter species, whereas fecal products had the highest homologies with intestinal species. Healthy mixed-breed dogs (group II; n = 8) with histologically confirmed spiral bacteria infection were treated with a 21-day course of metronidazole, amoxicillin, and famotidine. Fecal samples were collected from group II dogs twice before and within 3 days of completion of treatment. The PCR results correctly identified 15/16 pretreatment samples as positive: 1 pretreatment sample was negative. PCR results identified 8/8 posttreatment samples as Helicobacter negative. Fecal PCR is a useful test for detecting Helicobacter infection in dogs. This assay may be useful as a screening test for infection and could be used to address questions relevant to pathogenesis and therapy.     

A prevalence survey and risk analysis of filariosis in dogs from the Mt. Vesuvius area of southern Italy.

A dog microfilariae prevalence and risk factor survey was conducted in 51 contiguous municipalities of the Mt. Vesuvius area (Campania region, southern Italy) in order to add data to the limited epidemiological information available regarding filarial worms in this zone. Between May 1999 and June 2000, blood samples were collected from 351 asymptomatic dogs. Blood samples were examined using a modified Knott's technique and histochemical staining in order to count and identify microfilariae. The results were subjected to statistical analysis and choroplethic municipal maps (MMs) were drawn by a geographical information system (GIS) software. Microfilariae were detected in 63 of the 351 dogs surveyed, constituting a total filarial prevalence of 17.9%. In particular, 56 dogs (15.9%) showed only microfilariae of Dipetalonema reconditum; three dogs (0.8%) only microfilariae of Dirofilaria repens; two dogs (0.6%) microfilariae of both D. reconditum and D. repens and two dogs (0.6%) microfilariae of both Dirofilaria immitis and D. repens. High D. reconditum prevalence was associated with hunting practice, masculine gender and older dogs. There was also a tendency to find high prevalence in dogs sampled in the afternoon.In conclusion, the presence of microfilariae of D. reconditum in 92% of microfilaraemic dogs indicates that this filarial worm was the predominant filarial species in dogs in the Mt. Vesuvius area.In addition, the general trends of the MMs showed that D. immitis and D. repens were present only in a few municipalities, whereas D. reconditum was widely and homogeneously spread throughout the entire study area.     

New advances in molecular epizootiology of canine hematic protozoa from Venezuela, Thailand and Spain

The prevalence of hematozoan infections (Hepatozoon canis and Babesia sp., particularly Babesia canis vogeli) in canids from Venezuela, Thailand and Spain was studied by amplification and sequencing of the 18S rRNA gene. H. canis infections caused simultaneously by two different isolates were confirmed by RFLP analysis in samples from all the geographic regions studied. In Venezuela, blood samples from 134 dogs were surveyed. Babesia infections were found in 2.24% of the dogs. Comparison of sequences of the 18S rRNA gene indicated that protozoan isolates were genetically identical to B. canis vogeli from Japan and Brazil. H. canis infected 44.77 per cent of the dogs. A representative sample of Venezuelan H. canis isolates (21.6% of PCR-positives) was sequenced. Many of them showed 18S rRNA gene sequences identical to H. canis Spain 2, albeit two less frequent genotypes were found in the sample studied. In Thailand, 20 dogs were analyzed. No infections caused by Babesia were diagnosed, whereas 30 per cent of the dogs were positive to hematozoan infection. Two protozoa isolates showing 99.7-100% identity to H. canis Spain 2 were found. In Spain, 250 dogs were studied. B. canis vogeli infected 0.01% of the animals. The sequence of the 18S rRNA gene in Spanish isolates of this protozoa was closely related to those previously deposited in GenBank (> 99% identity). Finally, 20 red foxes were screened for hematozoans employing semi-nested PCR and primers designed to detect Babesia/Theileria. Fifty percent of the foxes were positive to Theileria annae. In addition, it was found that the PCR assay was able as well to detect Hepatozoon infections. Thirty five percent of the foxes were infected with two different H. canis isolates showing 99.8-100% identity to Curupira 1 from Brazil.     

应用DPO-PCR方法特异性检测志贺氏菌

In this study, a dual-priming oligonucleotide (DPO)-based PCR method for specific detection of Shigella was established using ipaH gene of Shigella as the target gene. The results showed that detection sensitivity of the DPO-PCR method was 1.65×10² CFU/mL. Compared with conventional PCR primers, the DPO primers were easily designed, which simplified the design procedure of PCR primers. DPO primers were not sensitive to annealing temperature, which could efficiently amplify the target gene in the annealing temperature range from 50 to 70 ℃. Moreover, due to the special structure of DPO primers, it had a higher specificity than conventional PCR primers, and none nonspecific amplifications were produced in reaction. In the practical application, tests on 133 samples including frozen/fresh meat, fruits and vegetables, fresh milk, eggs and cooked food by the DPO-PCR method showed that 15 samples were Shigella positive, which were in accordance with the testing results using GB method (GB/T 4789.5-2012), showing good practicality and reliability. The DPO-PCR method provided a new tool for fast and accurate detection of Shigella.     

Detection of Anaplasma platys and Babesia canis vogeli and their impact on platelet numbers in free-roaming dogs associated with remote Aboriginal communities in Australia

OBJECTIVE: To detect Anaplasma platys and Babesia canis vogeli infection, using polymerase chain reaction (PCR)-based assays, in free-roaming dogs associated with eight Aboriginal communities in remote areas of Australia and to determine the impact of infection through the assessment of platelet numbers. PROCEDURES: Blood samples from 215 dogs were screened by PCR for A platys and B canis vogeli using established genus-specific DNA primers for the 16S and 18S rRNA genes respectively. Both A platys DNA and B canis vogeli DNA were confirmed from the screening PCR either by sequencing or by the use of species-specific primers. Peripheral blood films from 92 of the 215 dogs were used to estimate platelet numbers through an indirect method. RESULTS: Of 215 dogs, 69 (32%) were positive for A platys, 22 (10%) for B canis vogeli and 24 (11%) for both. The two organisms were detected singularly and as coinfection in all communities. For the 92 dogs in which peripheral blood films were examined, the mean estimated platelet counts for the non-infected dogs was 318 x 10(9)/L, those infected with A platys alone was 256 x 10(9)/L, those with B canis vogeli alone was 276 x 10(9)/L and those infected with both parasites was 169 x 10(9)/L. In young dogs, infection produced significantly decreased mean platelet counts when compared to uninfected dogs. Thrombocytopenia (< 200 x 10(9)/L) was detected in 18 (51%) dogs infected with A platys alone, 3 (33%) dogs infected with B canis vogeli alone, 13 (72%) dogs coinfected, and 8 (27%) uninfected dogs. CONCLUSIONS: A platys and B canis vogeli infection, either singularly or together, was widespread in free roaming dogs associated with remote Aboriginal communities in the Northern Territory and north-western New South Wales. Moreover, both A platys and B canis vogeli infections were associated with a reduction in mean platelet numbers in dog populations, particularly in young dogs. The fact that 51% of dogs infected with A platys alone and 72% dogs coinfected were thrombocytopenic compared to 27% of uninfected dogs suggests that the organism alone or in combination with B canis vogeli has the potential to cause thrombocytopenia and perhaps contribute to a clinical bleeding disorder in infected dogs.     

鸡传染性贫血病毒套式PCR检测方法的建立

本试验根据GenBank中登录的禽传染性贫血病毒(CAV)基因序列,设计合成2对引物,外引物的扩增片段大小为485 bp,内引物的扩增片段大小为297 bp,建立了适合CAV快速检测的套式PCR方法(nested PCR),并采用该方法对CAV阳性毒株及临床病料进行了检测。结果显示,该方法能扩增到297 bp的条带,禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、禽网状内皮增生病病毒、减蛋综合征病毒、禽呼肠孤病毒、马立克氏病病毒的扩增结果均为阴性。该方法第1步扩增的敏感性是100 pg,第2步PCR扩增的敏感性是1 fg,敏感性提高了10⁵倍。本研究建立的CAV套式PCR方法具有敏感性高、重复性好、特异性强等优点,可用于CAV的临床诊断和分子流行病学调查等。     

用PCR技术鉴定犬传染性肝炎病毒强、弱毒株的研究

根据Genebank中发表的犬传染性肝炎病毒 (ICHV)标准强毒株Glaxo和人工驯化的弱毒疫苗株CLL的保守序列间大小不同 ,按照引物设计的原则 ,设计合成了一对通用引物。该对引物可由ICHV强毒株扩增出 569bp的片段 ,而弱毒株可扩增出 2 4 4bp的片段。对PCR产物分别进行电泳、酶切和测序 ,证明PCR产物片段大小、酶切位点和核苷酸序列与设计的产物完全一致。正常DK细胞上清和犬传染性喉气管炎病毒 (CAV 2 )强、弱毒株细胞培养物均不能被该引物扩增 ,说明具有良好的特异性。其检测到的病毒量分别为 1 5TCID50 、 31TCID50 、说明该技术具有很高的敏感性。可用于ICHV强、弱毒株的鉴定和ICH的诊断     

Establishment and Application of a Nested PCR Assay for Detection of Fowl Adenovirus Group Ⅰ

According to the sequence of hexon gene of fowl adenovirus groupⅠ(FAVⅠ) strain published in GenBank,two pairs of primers were designed and synthesized.The outer primers amplified a fragment of 475 bp in length, and the inner primers amplification fragment was 237 bp in length. A nested PCR assay for rapid detection of FAVⅠ was established.A specific 237 bp fragment was amplified from DNA templates of FAVⅠstrain,but no bands were amplified with templates extracted respectively from avian influenza virus (AIV) subtype H9,Newcastle disease virus (NDV), infectious bursal disease virus (IBDV),duck plague virus (DPV), reticuloendotheliosis (REV), avian reovirus (ARV), Marek's disease virus (MDV). Sensitivity of the 1st and 2nd amplifications by the nested PCR assay were 100 pg and 1 fg,respectively.The sensitivite of the 2nd amplifications increased by 10⁵ times.The results showed that the nested PCR was specific,sensitive,rapid,accurate,and could be used as a routine assay for the detection of FAVⅠ.This method had good reproducibility, specificity and sensitivity, and might detect low content FAVⅠ accurately and rapidly. This method could be used as a method for the diagnosis and detection of clinical cases,and molecular epidemiological investigation of FAVⅠ.     

J亚群禽白血病病毒套式PCR检测方法的建立

本试验根据GenBank中登录的禽白血病病毒(ALV)基因组序列,设计合成了2对引物,外部引物的扩增片段大小为478 bp,内部引物的扩增片段大小为314 bp,建立了适合ALV快速检测的套式PCR方法(nested-PCR)。采用该方法对ALV毒株进行了检测,试验结果表明,能扩增到314 bp的条带,禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒、禽网状内皮增生病病毒、禽呼肠孤病毒、马立克氏病病毒的扩增结果均为阴性。该方法第1次扩增的敏感性是100 pg,第2次扩增的敏感性是1 fg,第2次比第1次扩增的敏感性高10⁵倍。所建立的套式PCR方法具有敏感性高、重复性好、特异性强等优点,可用于禽白血病病毒(ALV)的临床诊断和分子流行病学调查等。     

The occurrence of Dirofilaria immitis in dogs in South Australia

Heart, lung and samples of blood from 230 dogs were examined for infections of filarial parasites. Dirofilaria immitis worms and microfilariae were detected in one dog. Blood samples from a further 1428 dogs were examined for microfilariae and 22 were found to be infected. Eighteen dogs were infected with D immitis microfilariae and four with Dipetolonema reconditum microfilariae. The histories were available for 18 of the dogs infected with heartworm and only seven dogs had not travelled outside South Australia. It was concluded that heartworm infection was endemic in South Australia but the apparent prevalence was only about 1%.     

Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay

The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP-based identification procedures of 17 different field isolates agreed with those obtained by conventional methods.     

Cultivation and molecular identification of Ehrlichia canis and Ehrlichia chaffeensis from a naturally co-infected dog in Venezuela

Background: Diagnosis of canine ehrlichiosis in Venezuela is normally performed by examination of buffy coat smears (BCS). Characteristic inclusion bodies are frequently observed in leukocytes and platelets from dogs with clinical signs of the disease. Objective: The purpose of this study was to investigate the co-infection of a dog with Ehrlichia canis and E hrlichia chaffeensis using microbiological and molecular techniques. Methods: Primary cultures of monocytes from a dog showing signs of ehrlichiosis were performed. Ehrlichial inclusions in blood cells were demonstrated by BCS and in cultured cell smears with direct immunofluorescence and Dip Quick staining. Nested PCR analysis was performed with DNA from blood samples and cultures, using primers specific for E. canis and E. chaffeensis. The amplified DNA fragments were sequenced to confirm the specificity of the amplifications. Results: The BCS of the naturally infected dog contained intracellular morulae. Ehrlichial inclusions were observed 9 days after inoculation of the primary cultures. After 3 passages with monocytes from a healthy dog, 65% of infected cells, and cells with >60 morulae were observed. A healthy female German Shepherd dog, seronegative for E. canis and E. chaffeensis antigens and without contact to ticks, was inoculated with an infected culture. The animal developed signs of canine monocytic ehrlichiosis and became seropositive. Nested PCR results and sequencing of amplified DNA fragments demonstrated the simultaneous presence of E. canis and E. chaffeensis in both dogs. Conclusions: This is the first report of E. chaffeensis in dogs in South America. This organism was previously identified in dogs by PCR only in the United States.     

The first report on detection of canine Acantocheilonema reconditum in Poland and the associated diagnostic problems

Acanthoheilonema reconditum was found during monitoring dogs living in the vicinity of Warsaw, for Dirofilaria spp. infection. The microfilaremia in blood was at first detected by microscopy and then molecular tests for distinct filarial markers were performed. PCR product sequencing confirmed that the microfilaria detected in two dogs were A. reconditum. These are the first two cases of canine acanthocheilonemiasis detected in Poland.