Urine sampling for real-time polymerase chain reaction based diagnosis of canine leishmaniasis.

A real-time polymerase chain reaction (PCR) assay was used for quantifying Leishmania infantum DNA in urine samples from naturally infected dogs. Forty-one infected dogs were divided into 3 groups: 22 dogs showing only cutaneous signs (group 1), 12 dogs showing hematuria (group 2), and 7 dogs affected by severe nephropathy (group 3). Groups 2 and 3 dogs showed altered laboratory parameters related to an impairment of renal function. The real-time PCR analysis showed higher levels of Leishmania DNA in the lymph node aspirates from all groups of infected dogs versus those measured in their blood or urine. Interestingly, urine samples from dogs belonging to groups 2 and 3 contained a higher Leishmania DNA load than that detected in their blood. This finding suggests that a real-time PCR analysis of urine from infected dogs could be a useful and noninvasive tool for monitoring the severity of leishmaniasis.     

Diagnosis of canine leishmaniasis by a polymerase chain reaction technique

A polymerase chain reaction (PCR) technique for the diagnosis of canine leishmaniasis on bone marrow samples was developed which amplified a 120 bp DNA fragment of the Leishmania kinetoplast DNA, common to all Leishmania species. Forty-five of 46 dogs in which leishmaniasis had been diagnosed were positive with the PCR technique, whereas none of 41 healthy dogs gave a positive result. Fifteen dogs with leishmaniasis that had been treated for six months with N-methylglucamine antimoniate and allopurinol were also investigated. Seven were positive, implying that they remained infected despite the resolution of their clinical signs.     

A case of canine streptococcal meningoencephalitis diagnosed using universal bacterial polymerase chain reaction assay

A 3-year-old, spayed female, mixed-breed dog was evaluated for acute, progressive neurological disease. Analysis of cerebrospinal fluid (CSF) showed neutrophilic pleocytosis. The dog later developed liver disease, thrombocytopenia, and anemia that were presumably secondary to ceftriaxone administration. Bacterial cultures of blood, urine, and CSF were negative. However, a universal bacterial polymerase chain reaction assay of CSF identified deoxyribonucleic acid from Streptococcus spp. The dog recovered with therapy for streptococcal encephalitis.     

Quantification of MDR-1 gene expression in canine tissues by real-time reverse transcription quantitative polymerase chain reaction

MDR-1 gene product mediated multidrug resistance is thought to play a major role in the outcome of chemotherapy in some canine tumors, especially malignant lymphoma. In the present study, MDR-1 RNA expression in normal lymph node and liver tissue as well as in tumor biopsies from 23 dogs with lymphomas and two dogs with liver tumors was measured by real-time RT-quantitative PCR. MDR-1 gene expression was detected in all samples analyzed. Comparably high MDR-1 RNA levels were measured in all normal liver tissues, one of the lymphomas and a cholangiocarcinoma. MDR-1 expression levels in canine lymphomas were found to vary over a wide range with most tumors expressing relative low levels. Interestingly, gastrointestinal lymphomas expressed higher MDR-1 RNA levels than multicentric lymphomas (p = 0.03). In conclusion, real-time RT-quantitative PCR appears to be a suitable method for sensitive and quantitative determination of MDR-1 gene expression in canine normal and neoplastic tissues.     

Molecular characterization of canine parvovirus in Brazil by polymerase chain reaction assay

Canine parvovirus (CPV) was first isolated in 1978 in the USA. Analysis of CPV isolates by monoclonal antibodies and restriction enzymes have shown that after the first emergence of CPV (CPV-2) it evolved to give rise to new antigenic types, which were designated CPV type 2a and type 2b. These new types have replaced the original CPV type 2, although the proportions of each of the new antigenic types vary in different countries. In Brazil, CPV-like infections were first observed in 1979, however, there has been no information concerning the antigenic types of CPV prevailing in South America. In this study, we designed a PCR assay to type canine parvovirus strains in fecal samples collected from symptomatic dogs during 1980 through 1986 and 1990 through 1995. Our data showed that the CPV epizootic in Brazil followed the same pattern observed in the USA of emergence of CPV-2 followed by replacement by the variants CPV-2a and 2b. The predominant strain found during 1980 was CPV-2a, which was substantially replaced by CPV-2b from 1990 to 1995.     

Genotypic screening of pseudorabies virus strains for thymidine kinase deletions by use of the polymerase chain reaction.

Genetic recombination between field strains and vaccine strains of pseudorabies virus (PRV) has been suggested as a scenario that might arise from use of deletion-mutant modified-live vaccine strains, particularly those strains attenuated by deletions within the thymidine kinase (TK-) gene locus. To address this hypothesis experimentally, it is necessary to screen large numbers of PRV isolates for their TK genotype. Techniques to detect the native TK genotype are routinely used in molecular virology laboratories, but are time-consuming. We adapted the polymerase chain reaction to define the genotypic status of PRV isolates with regard to the presence or absence of deletions in the TK gene locus. Used in tandem with the existing glycoprotein-specific ELISA that discriminate between PRV-vaccinated and field strain-infected swine populations, the described technique may help to clarify whether vaccine-derived recombinants are generated under natural conditions and after normal vaccine usage.     

An immunophenotypic study of canine leukemias and preliminary assessment of clonality by polymerase chain reaction.

There is a relative lack of information in the veterinary literature regarding the immunophenotypes present in canine leukemias. Utilizing a panel of thirty monoclonal antibodies, canine leukemias were assessed by flow cytometry alone or by flow cytometry in combination with immunocytochemical staining of smears. Canine chronic lymphocytic leukemia (CLL) occurred in older dogs (mean age 9.75 years; range 1.5-15 years; n = 73 cases). Blood lymphocyte counts ranged from 15,000 to 1,600,000/microl. Surprisingly, 73% of CLL cases involved proliferation of T lymphocytes (CD3+), and 54% of CLL cases had large granular lymphocyte (LGL) morphology. LGL CLL's were almost exclusively proliferation's of T cells that expressed CD8 and the leukointegrin alphaDbeta2 and more frequently expressed T cell receptor (TCR) alphabeta (69%) than TCRgammadelta (31%). The non-LGL T cell CLL cases (19% of CLL) involved proliferation of TCRalphabeta T cells in which no consistent pattern of CD4 or CD8 expression was found. B cell CLL, based on expression of CD2 or CD79a, comprised 26% of canine CLL cases. These results are in marked contrast to people where greater than 95% of CLL cases involve proliferation of B lymphocytes. Thirty eight (38) acute leukemias were also immunophenotyped. The majority (55%) of these leukemias had a phenotype most consistent with a myeloid origin. Acute LGL leukemias were also observed (7/38), although less commonly than the CLL counterpart. CD34 expression was common in acute, non-LGL leukemias of dogs, both myeloid and lymphoid. In some circumstances, it can be difficult to differentiate a reactive (polyclonal) lymphoid proliferation from a neoplastic (monoclonal) one. Therefore, as an adjunct to phenotypic studies, we have developed a polymerase chain reaction (PCR) based test for assessment of clonality in T cell proliferations. The test amplifies the junction of the variable gamma (Vgamma) and joining gamma (Jgamma) gene segments region of the TCR gamma genes. Preliminary data indicates that our test is effective and is capable of differentiating a neoplastic from a reactive lymphoproliferative process.     

Discrimination between six species of canine microfilariae by a single polymerase chain reaction

Canine dirofilariasis caused by Dirofilaria immitis is usually diagnosed by specific antigen testing and/or identification of microfilariae. However, D. immitis and at least six other filariae can produce canine microfilaremias with negative heartworm antigen tests. Discriminating these can be of clinical importance. To resolve discordant diagnoses by two diagnostic laboratories in an antigen-negative, microfilaremic dog recently imported into the US from Europe we developed a simple molecular method of identifying different microfilariae, and subsequently validated our method against six different filariae known to infect dogs by amplifying ribosomal DNA spacer sequences by polymerase chain reaction using common and species-specific primers, and sequencing the products to confirm the genotype of the filariae. We identified the filaria in this dog as D. repens. This is the first case of D. repens infection in the United States. Additionally, we examined microfilariae from five additional antigen-negative, microfilaremic dogs and successfully identified the infecting parasite in each case. Our diagnoses differed from the initial morphological diagnosis in three of these cases, demonstrating the inaccuracy of morphological diagnosis. In each case, microfilariae identified morphologically as A. reconditum were identified as D. immitis by molecular methods. Finally, we demonstrated that our PCR method should amplify DNA from at least two additional filariae (Onchocerca and Mansonella), suggesting that this method may be suitable for genotyping all members of the family Onchocercidae.     

A polymerase chain reaction assay for the detection of Leptospira spp. in bovine semen.

A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene. METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay. RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 104 cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%. CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

A multiplex polymerase chain reaction for discriminating Erysipelothrix rhusiopathiae from Erysipelothrix tonsillarum.

Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, and it causes great economic losses in Japan and worldwide. In meat inspection, it is very important to distinguish E. rhusiopathiae from other bacteria showing similar clinical signs of disease or similar bacterial characteristics. To distinguish E. rhusiopathiae from Erysipelothrix tonsillarum, 2 polymerase chain reaction (PCR) systems were combined. The primer sets ERY-1F and ERY-2R were designed to amplify 2210 base pairs (bp) of nucleotide sequence specific for E. rhusiopathiae chromosomal DNA, and the primer sets MO101 and ERS-1R were designed to amplify 719 bp of nucleotide sequence including a highly conserved region of genus Erysipelothrix 16S rRNA. Two fragments were amplified when E. rhusiopathiae was used as the PCR template using the primer sets, whereas a single fragment was amplified when E. tonsillarum was used as the template. No fragments were amplified when nucleic acid from other bacteria that cause clinical signs similar to swine erysipelas were used as the template. Moreover, 5 specimens collected from postinspected swine carcasses were diagnosed as E. rhusiopathiae using the PCR described in this study, in agreement with results of microbiological tests for the genus Erysipelothrix, whereas negative samples were negative both in conventional bacterial tests and by PCR. The detection limit of multiplex PCR ranged from 10(2) to 10(4) colony forming units per reaction tube for positive samples. These results suggest that this method is useful for screening of swine erysipelas in meat inspection centers.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

Evaluation of the Brucella abortus species-specific polymerase chain reaction assay, an improved version of the Brucella AMOS polymerase chain reaction assay for cattle.

In a blind test, 344 samples representing 80 bacterial isolates were analyzed by the Brucella abortus species-specific polymerase chain reaction (BaSS PCR) assay for the identification and discrimination of B. abortus field strains (wild-type biovars 1, 2, and 4) from 1) B. abortus vaccine strains, 2) other Brucella species, and 3) non-Brucella bacteria. Identical samples were tested in 2 laboratories. Half the samples were fully viable, and half were bacteria that had been killed by methanol fixation. The results in 1 laboratory correctly identified 100% of the samples, resulting in a predictive value of 100% for all categories and 100% sensitivity and specificity under the prescribed conditions. The second laboratory misidentified 31 samples, resulting in a range of 66.7-100% sensitivity, 93.2-99.7% specificity, and 77.3-98.2% predictive values depending on the category. There was no significant difference in viable versus fixed bacteria for either laboratory. Subsequent review of the protocol indicated that contamination was the likely cause of 26 of the 31 erroneous identifications. The results show that the BaSS PCR assay has the potential to be a very reliable screening tool for B. abortus identification. However, the data also provide a cautionary reminder of the importance of preventing contamination in diagnostic PCR.     

Development of a polymerase chain reaction test for Entamoeba invadens.

Entamoeba invadens is a protozoal parasite of reptiles that causes colitis, abscesses of liver and other organs, and sometimes acute death. It is generally considered a commensal of chelonians but has also been implicated as a cause of colitis, diarrhea, and death in gopher (Gopherus polyphemus) and leopard (Geochelone pardalis) tortoises. Diagnosis of E. invadens is currently by detection of trophozoites and/or cysts upon direct fecal examination. However, definitive diagnosis of E. invadens has been difficult due to the very similar morphology of nonpathogenic Entamoeba spp., including E. ranarum, E. insolita, E. barreti, and E. terrapinae. Definitive speciation of Entamoeba spp. is important to avoid misdiagnosis or overtreatment for nonpathogenic protozoa. It is also important for consideration of mixed species reptile collections to avoid exposing snakes and lizards to E. invadens. In this study, we developed polymerase chain reaction (PCR) primers for E. invadens, E. ranarum, E. terrapinae, and E. insolita and conducted PCR amplification of purified DNA from cell cultures, as well as purified DNA from reptile stool samples with E. invadens trophozoites added. As a result of this study, a naturally occurring infection of E. invadens was confirmed in a giant South American river turtle (Podocnemis expansa). This study has developed successful PCR primers for four species of Entamoeba and demonstrates that PCR is a promising diagnostic tool for the definitive identification of E. invadens.     

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     

检测和鉴别犬瘟热病毒强、弱毒株的复合反转录-套式聚合酶链式反应(RT-nPCR)的建立及初步应用

根据GenBank上登录的犬瘟热病毒（Canine distemper virus，CDV）基因组全序列，选择CDV强、弱毒株间有区别保守区设计了一对通用引物P1和P4，并在该对引物跨越区域的内部设计了CDV强毒株特异性引物P2及弱毒株特异性引物P3，用引物P1／P4进行RT—PCR，然后用引物P2／P3／P4进行复合套式PCR，建立了一种能区分CDV强、弱毒株的复合反转录-套式聚合酶链式反应（RT—nPCR）的鉴别诊断方法。应用该方法从CDV强、弱毒株的基因组中分别扩增出了大小为247bp和177bp的特异性片段，从两种病毒基因组混合物中扩增出了大小为247bp和177bp的两条特异性片段，与犬细小病毒、犬腺病毒、犬冠状病毒、狂犬病病毒、新城疫病毒的细胞培养物以及正常细胞对照组进行复合RT—nPCR扩增时均为阴性。对从黑龙江省和吉林省采集的20份疑似CDV病料进行的检测结果表明，有15份类似CDV强毒，5份类似CDV弱毒。本研究建立的复合RT—nPCR可以有效检测CDV感染，能够将强、弱毒株区分开，可用于临床快速检测、流行病学监测以及追踪疫苗免疫效果等。