Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.     

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 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.     

Validation of real-time polymerase chain reaction tests for diagnosing feline immunodeficiency virus infection in domestic cats using Bayesian latent class models

The objectives of the current study were to estimate the sensitivity and specificity of three real-time polymerase chain reaction (PCR) tests for diagnosis of feline immunodeficiency virus (FIV) infection in domestic cats, both individually and when interpreted in series with one of two serological tests, separately in populations of cats at low and high risk of being infected with FIV. One PCR test targeted the pol gene and two targeted the gag gene of FIV. For comparison, sensitivities and specificities of the individual serological tests (IDEXX SNAP(?) test and AGEN Simplify(?) test) were also estimated. The study populations consisted of domestic cats thought to be not vaccinated against FIV. Low-risk (males aged 4 years or less and females; n=128) and high-risk (males over 4 years; n=128) cats were selected from those where blood samples were submitted to a commercial clinical pathology service. Bayesian latent class models were used to obtain posterior probability distributions for sensitivity and specificity for each test, based on prior distributions obtained from three experts. Medians of the posterior sensitivity distributions for the PCR tests based on the pol gene and two regions of the gag gene tests ranged from 0.85 to 0.89, compared to 0.89-0.97 for the two serological tests. The medians of posterior specificity distributions for these PCR tests were 0.94-0.96, and 0.95-0.97 for the serological tests. In contrast, the PCR based on one region of the gag gene had lower median sensitivity. Sensitivities of combinations of these serological and PCR tests interpreted in series were low; medians of posterior sensitivity distributions ranged from 0.75 to 0.83. Relative to the low-risk population, median sensitivities in the high-risk population were lower for all tests other than the AGEN Simplify(?) test; specificities were similar in both populations. We conclude that the sensitivities of the two PCR tests based on the pol gene and two regions of the gag gene, respectively, in non-vaccinated cats are probably lower than the sensitivities of the two serological tests we assessed. We do not recommend screening cats whose FIV vaccination status is uncertain with one of these serological tests and then testing positives with one of these PCR tests because in non-vaccinates, the sensitivities of combinations of these serological and PCR tests interpreted in series are low. Assessment of the validity of these PCR assays in FIV-vaccinated cats is required.     

Molecular detection of feline hemoplasmas in feral cats in Korea

The purpose of this study was to determine if Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' exist in Korea. Three hundreds and thirty one feral cats were evaluated by using PCR assay targeting 16S rRNA gene sequence. Fourteen cats (4.2%) were positive for M. haemofelis, 34 cats (10.3%) were positive for 'Candidatus M. haemominutum' and 18 cats (5.4%) were positive for both species. Partial 16S rRNA gene sequences were closely (>98%) related to those from other countries. This is the first molecular detection of feline hemoplasmas in Korea.     

Duplex real-time polymerase chain reaction for simultaneous detection and quantification of Anaplasma marginale and Anaplasma centrale

Anaplasma marginale and Anaplasma centrale are rickettsial pathogens responsible for acute disease and mild infections, respectively, in cattle herds. A duplex real-time polymerase chain reaction (PCR) assay with probes labeled with different fluorophores was developed for simultaneous detection and quantification of A. marginale and A. centrale DNA in bovine blood samples. The assay was able to detect as few as 10(1) and 10(2) DNA copies for A. marginale and A. centrale, respectively, with optimal specificity and reproducibility. Analysis by real-time and nested PCR carried out on 54 samples previously tested by reverse line blot hybridization showed that the established duplex real-time PCR assay can detect and quantify the 2 Anaplasma spp., even if present simultaneously in the same blood samples. Such an assay could be used in pathogenesis studies on bovine acute anaplasmosis.     

The use of immunohistochemistry and the polymerase chain reaction for detection of feline leukemia virus and feline sarcoma virus in six cases of feline ocular sarcoma

Ocular sarcoma was diagnosed by light microscopic examination in enucleated globes ( n  = 4), orbital tissue biopsy ( n  = 1) and ocular evisceration contents ( n  = 1) from six cats. To determine if feline leukemia virus (FeLV) or a replication-defective FeLV, feline sarcoma virus (FeSV), was present in these ocular sarcomas, immunohistochemistry (IHC) and polymerase chain reaction (PCR) for FeLV were utilized. Immunohistochemical staining for FeLV glycoprotein 70 (gp70) was performed on all six formalin-fixed, paraffin-embedded tumors using an avidin–biotin complex technique. DNA was extracted from each specimen and a 166 bp region of the FeLV long-terminal repeat (LTR) was amplified by PCR. All tumors were composed primarily of spindle cells; two neoplasms had PAS-positive basement membrane enveloping areas of spindle cells. All tumors involved the uvea and five of six tumors showed transcleral extension, one of which invaded the optic nerve. Immunohistochemical staining for FeLV gp 70 was negative. PCR to amplify a portion of the FeLV LTR was negative. Based on these findings of these limited number of cases, FeLV/FeSV may not play a role in the tumorigenesis of feline ocular sarcomas. However, additional tumors representing all morphological subtypes should be investigated for the presence of viral antigen and DNA. It is important to determine the etiology and pathogenesis of these malignant ocular sarcomas. If the cell of origin and pathogenesis involve ocular and lenticular injury, and FeLV/FeSV is not present, then the clinical management of cases of feline ocular trauma, uveitis and glaucoma may prevent the development of this tumor.     

Detection of feline immunodeficiency proviral DNA in peripheral blood lymphocytes by the polymerase chain reaction.

Feline immunodeficiency virus (FIV) proviral DNA was detected by the polymerase chain reaction method (PCR). PCR products were detected by gel electrophoresis and ethidium bromide staining. The P-10, P-15 and P-24 regions of the gag gene of FIV were chosen as the target sequences for amplification, and three primer pairs were prepared. The PCR products subjected to amplification with each primer pair were found to possess sites of digestion by a restriction enzyme, as hypothesized. They did not react with feline leukemia virus (FeLV)-infected or feline syncytium-forming virus (FeSFV)-infected cell-derived DNA, and specifically amplified FIV-infected cell-derived DNA. FIV proviral DNA was detected by the PCR method with either primer pair (one-step amplification: single PCR) in DNA derived from peripheral blood lymphocytes (PBL) from 7 of 12 FIV antibody-positive cats. When PCR products in each of the 12 cats were subjected to a second amplification using the same primer pair (two-step amplification: double PCR), FIV proviral DNA was detected in all of the cats. When PBL samples collected from three cats that were negative and three that were positive in the single PCR were cultured for a few weeks in the presence of interleukin 2, FIV proviral DNA was detected in all six cats by the single PCR method. The results suggest that either the use of cultured PBL as the sample or the performance of the double PCR method enables simple and specific detection of FIV proviral DNA in PBL.     

An immunohistochemical and polymerase chain reaction evaluation of feline plasmacytic pododermatitis

Sections of 14 skin biopsies of cats with plasmacytic pododermatitis and a clinical follow-up of 12-36 months were stained with a polyclonal anti-Mycobacterium bovis (Bacille Calmette-Guerin = BCG) antibody cross-reactive to a broad spectrum of fungi and bacteria. All sections were negative for organisms within the actual footpad tissue with the anti-BCG antibody stains. Polymerase chain reaction (PCR) assays that amplify the DNA of Bartonella spp., Ehrlichia spp., Anaplasma phagocytophilum, Chlamydophila felis, Mycoplasma spp., Toxoplasma gondii, and feline herpesvirus 1 (FHV-1) were applied to tissue digests. DNA of those pathogens assessed was not amplified from tissue.     

Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi

OBJECTIVE: To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. SAMPLE POPULATION: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. PROCEDURE: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. RESULTS: The QPCR assay detected the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R. equi/mL and accurately quantitated virulent R. equi to 10(3) CFUs/mL of fluid. The assay was highly specific for detection of the vapA gene of virulent R. equi and was more sensitive than standard polymerase chain reaction for detection of R. equi in tracheobronchial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R. equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity of the QPCR method in detection of virulent R. equi should facilitate rapid and accurate diagnosis of R. equi pneumonia in foals.     

Rapid detection of Salmonella from poultry by real-time polymerase chain reaction with fluorescent hybridization probes

Detection of Salmonella by bacteriologic methods is known to be time consuming. Therefore, we have developed a real-time probe-specific polymerase chain reaction (PCR) to rapidly detect Salmonella invA gene-based PCR products from chicken feces and carcasses by a fluorescence resonance energy transfer assay. The sensitivity and the specificity of this system were determined as 3 colony-forming units ml(-1) and 100%, respectively. Overnight tetrathionate broth enrichment cultures of chicken feces and carcass samples were used in template preparation for PCR. Also, a standard bacteriology was performed (National Poultry Improvement Plan-U.S. Department of Agriculture, Bacteriological Analytical Manual-Food and Drug Administration Center for Food Safety and Applied Nutrition) for confirmation. Seventy-two cloacal swab, 147 intestine, and 50 carcass (neck) samples were examined. Thirteen (8.8%) and 25 (17%) of the intestinal samples were found to harbor Salmonella by bacteriology and PCR, respectively. Forty-five of 50 (90%) carcass samples were Salmonella positive by both methods. Salmonella was not detected from cloacal swab samples. Results indicate that this assay has the potential for use in routine monitoring and detection of Salmonella in infected flocks and carcasses.     

Effects of temperature and time in transit on polymerase chain reaction detection of feline herpesvirus DNA.

The purpose of this study was to report methods currently recommended by commercial laboratories for collecting, shipping, and processing of samples for feline herpesvirus type 1 (FHV-1) testing using polymerase chain reaction (PCR) and to determine the effect of temperature and time on the ability of 1 PCR method to detect FHV-1 DNA in experimental and clinical samples. Eleven laboratories offering FHV-1 PCR were surveyed. There was notable variation in sample types and shipping conditions recommended and PCR protocols used by these laboratories. Subsequently, using a single PCR method, FHV-1 DNA was detected in samples exposed to various temperatures within the laboratory. Finally, FHV-1 PCR was performed on paired clinical samples collected from 25 cats and shipped at ambient temperatures via US Postal Service (USPS) or with an ice pack via a courier. Samples sent by USPS were exposed to significantly longer transit time and arrived at significantly higher temperature than did samples sent by courier. Despite this, all sample pairs yielded concordant results when tested for FHV-1 DNA using this PCR method. Although it may not be necessary for samples collected for detection of FHV-1 DNA using this PCR method to be shipped under the most expedient or temperature-controlled conditions, this should be verified for a variety of PCR assays and sample types.     

Development of quantitative real-time polymerase chain reaction for duck enteritis virus DNA

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, we introduce a quantitative real-time polymerase chain reaction (PCR) assay for DEV DNA using TaqMan technology and a two-step protocol. It was confirmed to be rapid, sensitive, and specific for DEV detection. The primers and probe were designed and directed to the DNA polymerase gene of DEV. The method will provide a valuable tool for rapid laboratory diagnosis of DEV infection. By virtue of its high-throughput format and its ability to accurately quantify the viral DNA, the method may be useful for large epidemiological surveys and clarification of pathogenesis, such as latency and reactivation of the virus.     

Bovine leukaemia proviral DNA detection in cattle using the polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent in enzootic bovine leukosis a disease occurring worldwide. This virus is normally detected by the agar gel immunodiffusion or ELISA assays which rely on the appearance of antibodies to a major surface protein of the virus, gp51, present in the serum of infected cattle. We have used the polymerase chain reaction, which depends on the amplification of specific DNA sequences as a sensitive assay for the detection of BLV. It was possible to detect proviral DNA in 100 pg of tumour DNA from an infected host using agarose gel electrophoresis followed by ethidium bromide staining. The sensitivity of the assay was increased by two log orders when hybridization analysis, using a BLV proviral DNA probe, was used in combination with amplification of the DNA. Proviral DNA was detected in both lymphocytic and tumour DNA and at all stages of infection in cattle.     

Use of pooled samples for the detection of Salmonella in feces by polymerase chain reaction.

Many epidemiological studies of Salmonella rely on conventional bacteriological culture methods to detect Salmonella in fecal samples. These culture-based methods are inefficient for epidemiological studies in populations with a low prevalence of Salmonella. The objective of this study was to optimize a protocol that uses pooled Salmonella enrichment broth cultures of bovine feces and polymerase chain reaction (PCR) for the detection of the invA gene of Salmonella in feces. In one field trial, 196 animals were sampled, and all samples were tested by culture, invA PCR on individual samples, invA PCR on pools of 5 samples, and BAX PCR on individual samples. All assays showed a high agreement on individual samples (kappa > or = 0.75). The invA PCR was run on each of 40 pools and detected 19 of 22 culture-positive pools. In another field trial, 152 samples were taken from 4 dairies, and the invA PCR was performed on pools of 5 samples in addition to bacteriological culture of individual samples. Salmonella was detected in 5 of the 32 pools (7 total positive samples) by both PCR and culture. One pool was PCR-positive but culture-negative. Pooling did not dramatically affect the performance of the invA PCR; most of the culture-positive samples were detected, including all of the samples when there were 4 or more Salmonella colonies on the agar plate. Based on these field trials, invA PCR on pooled samples appears to be an efficient method of Salmonella detection as long as Salmonella loads are not extremely low.     

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.     

A real-time polymerase chain reaction assay for the detection of the myxozoan parasite Henneguya ictaluri in channel catfish

Proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya ictaluri, is the most prevalent parasitic infection affecting commercial channel catfish (Ictalurus punctatus) aquaculture. There are currently no effective chemotherapeutic or biological control measures for PGD, which often peaks during the spring and fall when water temperatures are between 16-25 degrees C. The current diagnostic techniques of gross examination of gill clip wet mounts and histopathology are subject to false-negatives during the early stages of infection, and the quantifiable nature of end-point polymerase chain reaction (PCR) is subjective. Consequently, a rapid and more sensitive quantitative real-time PCR assay was developed for the detection of H. ictaluri during the early stages of infection in channel catfish. A 23 base-pair TaqMan probe was designed based on previously published H. ictaluri PCR protocols. The sensitivity of the assay was the equivalent of a single H. ictaluri actinospore, and in a pond challenge study, quantitative real-time PCR proved to be more sensitive than gross examination, microscopic examination of gill clip wet mounts, and histopathologic examination of gill tissue sections. Future applications of this assay will focus on developing methodologies to be used in conjunction with current pond-monitoring protocols to evaluate potential treatments and better manage this significant seasonal disease.