Clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs

OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) assay on urine samples for diagnosis of leptospirosis in dogs. DESIGN: Prospective case study. ANIMALS: 132 dogs with clinical signs suggestive of leptospirosis and 13 healthy dogs. PROCEDURE: PCR testing was performed on urine samples to detect leptospiral DNA; results were compared with results of conventional criteria for the diagnosis of leptospirosis. RESULTS: Leptospirosis was diagnosed in 8 dogs via established criteria; all these dogs had positive results of PCR assay, including 1 dog with positive results before seroconversion developed. A positive PCR assay result was also obtained in 16 dogs that did not have a confirmed diagnosis of leptospirosis. In the 8 dogs that had a confirmed diagnosis of leptospirosis, serovars pomona (n = 3 dogs), grippotyphosa (2), canicola (2), and bratislava (1) were identified serologically. The remaining 121 dogs all had a diagnosis other than leptospirosis or were healthy. For PCR testing on urine, sensitivity was 100%, specificity was 88.3%, positive predictive value was 33%, and negative predictive value was 100%. CONCLUSIONS AND CLINICAL RELEVANCE: Positive PCR test results prior to seroconversion may have value in establishing an early diagnosis. Positive results in dogs that had signs consistent with leptospirosis despite failing to meet established criteria for leptospirosis raise questions regarding the sensitivity of serologic testing in diagnosis of leptospirosis. Serovars pomona, grippotyphosa, and canicola were most common.     

奶牛新孢子虫病PCR检测方法的建立

根据已发表的新孢子虫（N.caninum）Nc-5基因序列设计合成了1对特异性引物,建立了检测新孢子虫PCR方法。从新孢子虫ELISA检测抗体阳性奶牛血液中提取DNA,用PCR方法对新孢子虫基因组DNA进行扩增,并对扩增产物进行克隆及序列测定,证明其可靠性。结果显示,扩增的目的片段大小为350bp,扩增产物经MspⅠ酶切为218bp和132bp2条片段,与预期结果一致;序列分析表明,本试验扩增的Nc-5基因片段与GenBank发表的其他新孢子虫Nc-5基因序列同源性为99.6%～95.4%;通过敏感性、特异性、重复性试验证明,该方法具有特异、灵敏、快速、准确、可靠的优点。     

Use of a polymerase chain reaction assay to detect and differentiate two strains of Haemobartonella felis in naturally infected cats

OBJECTIVE: To develop a polymerase chain reaction (PCR) assay that detects and differentiates the Ohio strain of Haemobartonella felis (H. felis-OH) and the California strain of H. felis (H. felis-CA) and to apply the assay to blood samples from cats with and without suspected haemobartonellosis (suspect and control cats, respectively). SAMPLE POPULATION: 220 blood samples were examined; 82 were from suspect cats, and 138 were from control cats. PROCEDURE: A PCR assay was designed to detect and differentiate H. felis-OH and H. felis-CA. RESULTS: On the basis of PCR assay results, the overall prevalence of H. felis infection was 19.5% (43/220). Suspect cats (28.0%; 23/82) were significantly more likely than control cats (14.5%; 20/138) to be H. felis infected. Significantly greater numbers of suspect cats were H. felis-OH infected (12.2%, 9/82) or H. felis-OH and H. felis-CA infected (4.9%, 4/82) than control cats (0% [0/138] and 0.7% [1/138], respectively). Significantly more anemic cats were H. felis-OH infected (14.3%; 4/28) or H. felis-OH and H. felis-CA infected (7.1%; 2/28) than nonanemic cats (2.3% [3/128] and 0.8% [1/128], respectively). The PCR assay was more accurate than cytologic examination for detection of H. felis. CONCLUSIONS AND CLINICAL RELEVANCE: Haemobartonella felis infections are more common in cats than previously recognized. Haemobartonella felis-OH is apparently more pathogenic than H. felis-CA. The PCR assay is more accurate than cytologic examination for detection of H. felis infection and is an effective clinical tool for the detection and differentiation of both H. felis strains known to infect cats.     

Evaluation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene

OBJECTIVE: To evaluate sensitivity and specificity of a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Rhodococcus equi and differentiation of strains that contain the virulence-associated gene (vapA) from strains that do not. SAMPLE POPULATION: 187 isolates of R equi from equine and nonequine tissue and environmental specimens and 27 isolates of bacterial species genetically or morphologically similar to R equi. PROCEDURE: The multiplex PCR assay included 3 gene targets: a universal 311-bp bacterial 16S ribosomal RNA amplicon (positive internal control), a 959-bp R equi-specific target in the cholesterol oxidase gene (choE), and a 564-bp amplicon of the vapA gene. Duplicate multiplex PCR assays for these targets and confirmatory singleplex PCR assays for vapA and choE were performed for each R equi isolate. An additional PCR assay was used to examine isolates for the vapB gene. RESULTS: Results of duplicate multiplex and singleplex PCR assays were correlated in all instances, revealing high specificity and reliability (reproducibility) of the vapA multiplex assay. Of the pulmonary isolates from horses with suspected R equi pneumonia, 97.4% (76/78) yielded positive results for vapA. Seven of 50 (14%) human isolates of R equi yielded positive results for vapA. Six human R equi isolates and 1 porcine isolate yielded positive results for vapB. No isolates with vapA and vapB genes were detected. CONCLUSIONS AND CLINICAL RELEVANCE: The multiplex PCR assay is a sensitive and specific method for simultaneous confirmation of species identity and detection of the vapA gene. The assay appeared to be a useful tool for microbiologic and epidemiologic diagnosis and research.     

Distribution of Neospora caninum within the central nervous system and other tissues of six dogs with clinical neosporosis

Six cases of neosporosis in dogs were diagnosed on the basis of clinical signs, serology and immunohistochemistry. The brains and spinal cords, at several levels, and various other tissues were examined using anti-Neospora caninum serum by an immunoperoxidase technique. Parasite density varied markedly from case to case. Although found most consistently in the cerebrum, parasites were distributed throughout the central nervous system (CNS), both within the grey and white matter and within nerve roots. Clinical signs were not related to the position of parasites. Tissue cysts were found infrequently in all areas of the CNS, but not in other tissues. Parasite density was not related to the age of the dog or whether treatment had been given. However, with the exception of the only adult dog examined, more CNS parasites were found in dogs with a longer duration of illness and with higher antibody titres. Tachyzoites were present in skeletal muscles, in the muscularis of the oesophagus, in heart, lung and, less frequently, liver, and rarely in the adrenal gland, thyroid gland and uterus; no clinical signs were seen resulting from damage to these organs. Parasites were not observed in lymphoid tissue. In visceral organs, parasites were most widely distributed in peracute cases. For post mortem diagnosis of neosporosis, the CNS, particularly the cerebrum, is the optimum tissue to examine but parasites may also be found in many other tissues, especially in acute cases. Muscle biopsy of appropriate muscles (as suggested by the clinical signs) provides the possibility of a definitive pre-mortem diagnosis.     

Use of a polymerase chain reaction assay for detection of Haemobartonella canis in a dog.

A polymerase chain reaction (PCR) originally developed for detection of Haemobartonella felis in cats was successfully used for detection of H canis in an 8-year-old spayed Great Dane. The dog had been splenectomized and was undergoing immunosuppressive chemotherapy at the time of diagnosis. Sequence analysis of the 16S ribosomal RNA gene revealed that the Haemobartonella spp infecting this dog was 97% homologous to the sequence previously reported for the Ohio strain of H felis. Clinical and hematologic abnormalities as well as identification of the organisms by use of light and electron microscopy supported the diagnosis of H canis. The PCR assay used for detection of H felis may be useful for the detection of H canis in dogs prior to blood donation, splenectomy, or treatment with immunosuppressive drugs.     

Use of a polymerase chain reaction assay to detect bovine leukosis virus in dairy cattle

OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) assay in detecting bovine leukosis virus (BLV) in adult dairy cows. DESIGN: Prospective study. ANIMALS: 223 adult dairy cows. PROCEDURE: Cows were tested for BLV status by use of an ELISA and a PCR assay. Sensitivity, specificity, predictive values of positive and negative tests, and the percentage of cows correctly classified by PCR assay were calculated. Ninety-five percent confidence intervals were calculated for sensitivity and specificity. RESULTS: Sensitivity and specificity were 0.672 and 1.00, respectively. Prevalence of BLV in this herd was 0.807. Predictive value of a positive test was 1.00, and predictive value of a negative test was 0.421. The percentage of cows correctly classified by PCR assay was 73.5%. CONCLUSIONS AND CLINICAL RELEVANCE: A positive PCR assay result provided definitive evidence that a cow was infected with BLV. Sensitivity and negative predictive value for PCR assay were low. Consequently, PCR assay alone is unreliable for routine detection of BLV in herds with high prevalence of the disease.     

A multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis

Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 µL of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes.     

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.     

Use of a polymerase chain reaction assay to detect infection with Eperythrozoon wenyoni in cattle.

OBJECTIVE: To determine whether a polymerase chain reaction (PCR) assay could be used to detect Eperythrozoon wenyoni in the blood of cattle. DESIGN: Prospective study. ANIMALS: 95 cattle from various herds in Alabama and Georgia and 96 bulls enrolled in Auburn University's Alabama Beef Cattle Improvement Association Bull Test program. PROCEDURE: Blood samples were collected by means of venipuncture of the median caudal vein and submitted for a CBC and PCR assay. Blood smears were made immediately after blood collection and examined by means of light microscopy. RESULTS: Three of 95 cattle from herds in Alabama and Georgia and 5 of 96 bulls enrolled in the Bull Test program had positive PCR assay results. Organisms were seen in blood smears from only 5 of these 8 animals. Organisms were not seen in blood smears from any animals for which results of the PCR assay were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a PCR assay may be an effective method for detecting E wenyoni infection in cattle and that the PCR assay may be a more sensitive test than evaluation of blood smears.     

Accuracy of polymerase chain reaction assays for diagnosis of feline immunodeficiency virus infection in cats

OBJECTIVE: To determine the sensitivity, specificity, and overall diagnostic accuracy of polymerase chain reaction (PCR) assays offered by commercial diagnostic laboratories for diagnosis of FIV infection in cats. DESIGN: Prospective clinical trial. ANIMALS: 124 cats. PROCEDURE: Blood was collected from cats that were neither infected with nor vaccinated against FIV, uninfected cats that were vaccinated with a licensed FIV vaccine, and cats experimentally and naturally infected with FIV representing subtypes A, B, and C. Coded blood samples were submitted to 3 laboratories in the United States and Canada offering PCR assays for diagnosis of FIV infection to veterinary practitioners. All laboratories tested fresh blood samples, and 1 laboratory also tested samples submitted as dried blood smears. The FIV infection status in all cats was confirmed by virus isolation. Sensitivity, specificity, and correct results were calculated for each PCR assay. RESULTS: Sensitivity ranged from 41% to 93%. Specificity ranged from 81% to 100% in unvaccinated cats and 44% to 95% in cats vaccinated against FIV. Correct results were obtained in 58% to 90% of 124 cats tested. All tests misidentified both uninfected and infected cats. False-positive results by all laboratories were higher in cats vaccinated against FIV than in unvaccinated cats, suggesting that vaccination interferes with the performance or interpretation of PCR assays used for diagnosis of FIV infection. CONCLUSIONS AND CLINICAL RELEVANCE: PCR assays used for diagnosis of FIV infection presently marketed to veterinary practitioners in North America vary significantly in diagnostic accuracy and did not resolve the diagnostic dilemma resulting from vaccination of cats against FIV.     

Development and application of a multiplex polymerase chain reaction for avian respiratory agents

A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at I and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group.     

Species diagnosis of a tissue using the polymerase chain reaction

The unambiguous identification of a biological specimen can deliver invaluable evidence to solve criminal cases. In this case the origin of a heart had to be clarified. Using the polymerase chain reaction technique and species-specific primer pairs for two genes it was clearly shown that this tissue was not from a human but from a pig.     

Clinical application of a polymerase chain reaction assay in the diagnosis of pneumonia caused by Rhodococcus equi in a horse

Diagnosis of pneumonia caused by Rhodococcus equi can be made more rapidly by use of a polymerase chain reaction (PCR) assay than by use of conventional bacteriologic culture techniques. Use of a PCR assay aids in the differentiation between virulent and avirulent strains of R equi, and the assay may be used to identify R equi in feces and soil of breeding farms.     

CSFV、PRV和PRRSV多重PCR检测方法的建立及其应用

由猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)和猪繁殖与呼吸综合征病毒(PRRSV)引起的繁殖障碍性疫病在中国的不断暴发,增加了猪的发病率和死亡率。为了建立同时检测这3种病毒的多重PCR方法,本研究根据GenBank中这3种病毒的参考基因序列设计引物,并对反应中的影响因素进行优化,建立了同时检测CSFV、PRV和PRRSV的多重PCR方法。该方法扩增的基因片段大小分别为570(CSFV),232 (PRRSV)和173bp(PRV)。敏感性和特异性试验结果显示,该方法对3种病毒的核酸最低检出量分别为23.88(CSFV),13.10(PRRSV)和14.60pg(PRV),对大肠杆菌(Ec.oli)、沙门菌(Salmonella)、猪乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的检测结果均为阴性。对2015年5月至2016年1月收集的168份临床样本检测结果显示,CSFV阳性率为24.4%,PRRSV阳性率为21.4%,提示河北省该段时间内引起猪繁殖障碍性疫病的主要病原为CSFV和PRRSV。经多重PCR检测为PRRSV、PRV或CSFV阳性的临床样本,再次使用商品化的试剂盒进行检测,符合率为100.0%。这些结果表明,本试验建立的多重PCR方法省时、高效,可用于PRRSV、PRV和CSFV感染的临床诊断。     

Use of anti-coronavirus antibody testing of cerebrospinal fluid for diagnosis of feline infectious peritonitis involving the central nervous system in cats

OBJECTIVE: To assess the use of measuring anti-coronavirus IgG in CSF for the diagnosis of feline infectious peritonitis (FIP) involving the CNS in cats. DESIGN: Prospective study. SAMPLE POPULATION: CSF and serum samples from 67 cats. PROCEDURES: CSF and serum samples were allocated into 4 groups: cats with FIP involving the CNS (n = 10), cats with FIP not involving the CNS (13), cats with CNS disorders caused by diseases other than FIP (29), and cats with diseases other than FIP and not involving the CNS (15). Cerebrospinal fluid was evaluated for concentrations of erythrocytes, leukocytes, and total protein. Anti-coronavirus IgG was measured in CSF and serum by indirect immunofluorescence assay. RESULTS: CSF IgG (range of titers, 1:32 to 1:4,096) was detected in 12 cats, including 6 cats with neurologic manifestation of FIP, 4 cats with FIP not involving the CNS, and 2 cats with brain tumors. Cerebrospinal fluid IgG was detected only in cats with correspondingly high serum IgG titers (range, 1:4,096 to 1:16,384) and was positively correlated with serum IgG titers (r = 0.652; P < 0.01), but not with any other CSF parameter. Blood contamination of CSF resulted in < or = 333 erythrocytes/microL in cats with CSF IgG. CONCLUSIONS AND CLINICAL RELEVANCE: The correlation between serum and CSF IgG and the fact that CSF IgG was detected only in strongly seropositive cats suggested that CSF anti-coronavirus IgG was derived from blood. Measurement of anti-coronavirus IgG in CSF was of equivocal clinical use.