Simple tests for rapid detection of canine parvovirus antigen and canine parvovirus-specific antibodies

Canine parvovirus (CPV) is the number one viral cause of enteritis, morbidity, and mortality in 8-week-old young puppies. We have developed twin assays (slide agglutination test [SAT] for CPV antigen and slide inhibition test [SIT] for CPV antibody) that are sensitive, specific, cost-effective, generic for all genotypes of CPV, and provide instant results for CPV antigen detection in feces and antibody quantification in serum. We found these assays to be useful for routine applications in kennels with large numbers of puppies at risk. The results of these assays are available in 1 min and do not require any special instrumentation. SAT-SIT technology will find applications in rapid screening of samples for other hemagglutinating emerging viruses of animals and humans (influenza virus and severe acute respiratory syndrome coronavirus).     

A rapid method for establishment of a reverse genetics system for canine parvovirus

Canine parvovirus (CPV) is an important and highly prevalent pathogen of dogs that causes acute hemorrhagic enteritis disease. Here, we describe a rapid method for the construction and characterization of a full-length infectious clone (rCPV) of CPV. Feline kidney (F81) cells were transfected with rCPV incorporating an engineered EcoR I site that served as a genetic marker. The rescued virus was indistinguishable from that of wild-type virus in its biological properties.     

One-step immunochromatography assay kit for detecting antibodies to canine parvovirus

This study was performed to determine the feasibility of using whole serum to detect antibodies to canine parvovirus (CPV) under nonlaboratory conditions and to evaluate the performance characteristics of an immunochromatography assay kit. Precise detection of levels of antibody against CPV in puppies can be used to determine a vaccination schedule, because maternal antibodies frequently result in the failure of protective vaccination, and can also be used to determine the antibody levels of infected puppies. Several methods for the titration of CPV antibodies have been reported, including the hemagglutination inhibition (HI) assay, which is considered the "gold standard." These methods, however, require intricate and time-consuming procedures. In this study, a total of 386 serum specimens were tested. Compared to the HI assay, the rapid assay had a 97.1% sensitivity and a 76.6% specificity (with a cutoff HI titer of 1:80). This single-step assay could be performed rapidly and easily without special equipment. The kit provides a reliable method for detection of anti-CPV antibody where laboratory support and personnel are limited.     

A rapid and highly predictive in vitro assay for non-steroidal anti-inflammatory agents

The inhibition of the production of malonyldialdehyde (MDA) in guinea-pig lung homogenates, incubated in the presence of 50 microM arachidonic acid and 1.4 mM adrenaline, has been exploited as a simple and reliable assay to test in vitro non-steroidal anti-inflammatory agents (NSAIA). The inhibitory potencies of a series of reference NSAIA, which correlated fairly well with in vivo anti-inflammatory activity as determined by carrageenin oedema, are herewith reported. The specificity of the assay was also evaluated by testing up to forty miscellaneous drugs: none of these significantly reduced the MDA production.     

A reliable, rapid and inexpensive two-color fluorescence assay to monitor serum cytotoxicity in xenotransplantation

Removal and/or neutralization of preformed anti-pig antibodies in non-human primate blood have been shown to prevent the hyperacute rejection of transplanted pig organs. The purpose of this study was to establish a suitable in vitro method that would allow for screening and comparison of various agents and methods potentially useful in the prevention of hyperacute rejection. The pig kidney cell line (PK15), pig aortic endothelial cell line (AG08472), and a primary culture of endothelial cells explanted from a pig aorta were incubated with either human or baboon sera. Complement-dependent cytotoxic activity of human and baboon sera was determined on all three types of pig cells using a two-color fluorescence assay and compared with the conventional 51Chromium (51Cr)-release assay. The assay was also performed on PK15 cells as a 2-chambered slide assay and compared with a microcytotoxicity assay performed in Terasaki trays. Using the microcytotoxicity assay, a 1-step assay utilizing endogenous complement was compared with a 2-step assay where rabbit complement was added. Of the three types of cells studied, PK15 cells were the most sensitive to cytotoxic injury, followed by AG cells and the primary endothelial culture. Good correlation between the 51Cr-release and the two-color fluorescence method was documented. There was good agreement between the results obtained using the 2-chambered slide method and the microcytotoxicity assay, as there was between the 1- and the 2-step assays. The 1- and 2-step assays provided information on the level and efficacy of endogenous complement. We conclude that the two-color fluorescence assay is suitable for the rapid and inexpensive screening of therapeutic interventions that might be useful in the prevention of hyperacute xenograft rejection, and that PK15 cells are suitable for use in this assay.     

A canine parvovirus mutant is spreading in Italy

By antigenic and genetic characterization of canine parvovirus type 2 (CPV-2) strains collected in 2001 and 2002 in Italy, it was possible to observe the spread of viruses with an unusual mutation, Glu-426, affecting a major antigenic epitope of CPV-2. Out of 67 strains analyzed, 49 (73.13%) were characterized as CPV-2a, 6 (8.95%) were characterized as CPV-2b, and 12 (17.91%) were characterized as the Glu-426 mutant.     

A rapid and reliable technique for pinealectomizing rats

Rats may be quickly and easily pinealectomized by first removing a small area of skull between the saggital and lambdoid sutures, and then extracting the unexposed pineal gland with iridectomy forceps. Little cortical or epithalamic damage results from this procedure and loss of blood is extremely slight. Operate mortality is quite low and comparable to that of animals given sham operations.     

A simple, rapid, reliable reverse haemolytic plaque assay and positive quality control test for routine use

A simple, rapid, reliable protein A reverse haemolytic plaque assay is described. Monolayers of protein A-coupled sheep red blood cells, in liquid medium, are formed in shallow 15 mm diameter chambers of the type commercially available for leucocyte migration inhibition assays. No agarose is necessary and the chambers are quick and easy to use, economical of reagents, and of a constant size and volume. Results compare favourably with those obtained using Cunningham chambers. The assay is ideal for clinical studies in which large numbers of samples are assayed daily. A positive quality control test for the protein A reverse hemolytic plaque assays is described. Spleen cells are stimulated with pokeweed mitogen to give high numbers of secreting cells. The cells are harvested on day 6 of culture and stored in aliquots at -70 degrees C. When thawed and tested in the protein A assay, these secreting cells form a sensitive and reproducible monitor of the day-to-day performance of the assay. Variation between operators and between batches of reagents may also be checked if desired, with little additional time, effort or expense.     

A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5-Mb duplication and a deletion at chromosome 17p11.2–12 encompassing the peripheral myelin protein 22 gene (PMP22), respectively. We developed a rapid and reliable detection system for duplications/deletions of the PMP22 gene based on measurement of gene copy number. The method involves amplification of a test locus with unknown copy number and a reference locus of known copy number by multiplex PCR (MP), followed by denaturing high-performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, 17 patients with HNPP, and 61 unaffected individuals were analyzed. Using the same competitive MP protocol, the measured PMP22 gene dosage revealed concordant results between DHPLC and capillary electrophoresis analysis. The results of the MP/DHPLC or the MP/capillary electrophoresis assay were all confirmed by PCR–restriction fragment length polymorphism analysis. We concluded that the MP/DHPLC assay is an efficient, accurate, and reliable technique for gene dosage determination of the PMP22 gene for CMT1A duplication and HNPP deletion. This technique further extends the application of DHPLC as an alternative method for the measurement of gene amplifications and heterozygous deletions in different genetic diseases.Chia-Yun Lin and Yi-Ning Su share the first authorship.     

A minor groove binder probe real-time PCR assay for discrimination between type 2-based vaccines and field strains of canine parvovirus

A minor groove binder (MGB) probe assay was developed to discriminate between type 2-based vaccines and field strains of canine parvovirus (CPV). Considering that most of the CPV vaccines contain the old type 2, no longer circulating in canine population, two MGB probes specific for CPV-2 and the antigenic variants (types 2a, 2b and 2c), respectively, were labeled with different fluorophores. The MGB probe assay was able to discriminate correctly between the old type and the variants, with a detection limit of 10(1) DNA copies and a good reproducibility. Quantitation of the viral DNA loads was accurate, as demonstrated by comparing the CPV DNA titres to those calculated by means of the TaqMan assay recognising all CPV types. This assay will ensure resolution of most diagnostic problems in dogs showing CPV disease shortly after CPV vaccination, although it does not discriminate between field strains and type 2b-based vaccines, recently licensed to market in some countries.     

Development of loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of ovine theileriosis in China

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid detection method in which the target deoxyribonucleic acid (DNA) can be efficiently amplified with high specificity and sensitivity under isothermal conditions using a set of either four or six specific primers. In this study, we have identified a conserved sequence for Theileria luwenshuni (UTRlu8) and for T. uilenbergi (UTRu6) suitable for designing a set of six primers for the simultaneous detection by LAMP of these pathogens causing theileriosis in sheep and goats in China. LAMP was performed at 63°C, and the amplified DNA was detectable within 15 min. The specificity of the reaction was confirmed through EcoRI restriction enzyme digestion analysis and sequencing. The assay was proven sensitive since specific amplification was obtained from 0.1 pg DNA of T. luwenshuni or T. uilenbergi. The LAMP assay was evaluated by testing 86 field samples in comparison to the reverse line blot method, showing a sensitivity and specificity of 66.0% and 97.4%, respectively. These results indicate that the LAMP assay is rapid and simple to run, cost effective, sensitive, and specific and has potential usefulness for application in diagnostics of and epidemiological studies on T. luwenshuni and T. uilenbergi infection of small ruminants.     

Release of canine parvovirus from endocytic vesicles

Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A(2) like domain in N-terminus of VP1. In this study we characterized the role of PLA(2) activity on CPV entry process. PLA(2) activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA(2) inhibitors inhibited the viral proliferation suggesting that PLA(2) activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA(2) activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A(1), brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A(1), brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA(2) activity of the virus. These results suggest that parvoviral PLA(2) activity is essential for productive infection and presumably utilized in membrane penetration process of the virus, but CPV also needs other pH-dependent changes or factors to be released to the cytoplasm from endocytic vesicles.     

A latex agglutination test for the detection of canine parvovirus and corresponding antibodies

Latex particles coated with rat monoclonal antibodies directed against a canine parvovirus are agglutinated by the virus antigens. In this study, the reaction was quantitated by a technique which had been described as immunoassay by particles counting. The method is as sensitive as a radioimmunoassay with the same antibody, but has the advantages of simplicity and safety. It allows the detection of parvoviral antigens to 4 ng/ml. Incubation of these antigens with specific antibodies resulted in inhibition of the agglutination reaction. By this procedure the presence of antibodies against canine parvovirus can be detected to a concentration of 150 ng/ml. The system can be easily automated, thus increasing reproducibility. This latex agglutination technique can be performed as a slide test; and although this method is 4-5 times less sensitive, it could be useful in field studies, for the detection of the viral antigens and the corresponding antibodies.     

Characterization of a nonhemagglutinating mutant of canine parvovirus

A nonhemagglutinating mutant of a 1978 isolate of canine parvovirus (CPV) was derived after repeated passages in the NLFK feline kidney cell line. The mutant CPV was antigenically indistinguishable from wild-type virus when tested with 82 monoclonal antibodies, and it replicated in cat and dog cell lines in culture. Sequences of the VP-1 and VP-2 genes revealed two nucleotide and two predicted amino acid sequence differences at 77 and 88 genome map units in the mutant compared to hemagglutinating viruses. One or both of those two mutations must determine the difference in the ability of the virus to agglutinate erythrocytes.     

A rapid microneutralization assay for cytomegalovirus

A rapid, simple and reproducible microneutralization test for human cytomegalovirus is described. The results can be read in 1-2 days and the neutralization titer detected in human and guinea pig sera and in monoclonal antibody-containing supernatants is consistent with that derived by the plaque-reduction neutralization test.     

A rapid assay method for cephalosporins

A simple, rapid assay for cephalosporins is described. The method is based on the inhibition by cephalosporins of the fermentation of glucose or inositol by a strain of Providence resistant to aminoglycoside antibiotics.The method gives answers which are as accurate as those obtained by standard agar diffusion techniques within four hours, and utilizes skills and resources readily available in most routine bacteriology departments. Results are not affected by gentamicin or kanamycin concurrently administered to the patient.This method will be of value in helping to monitor cephalosporin therapy in patients with serious sepsis, especially those with impaired renal function, and may help in elucidating and preventing the problem of nephrotoxicity associated with cephalosporin administration.     

Study of canine parvovirus polypeptides by immunoblot analysis

Summary The immunoblot analysis of canine parvovirus (CPV) polypeptides with 47 rat monoclonal antibodies (RH) has revealed four different types of reaction. Many of these antibodies do not recognize the electroblotted proteins. However, among these monoclonals that do react, 12 recognize all three viral polypeptides (VP 67, VP 70 and VP 85), 3 recognize preferentially the VP 85 and one is exclusively directed against VP 70. Thus, three antigenic regions are proposed which correspond to domains of the CPV polypeptides. In addition, these results together with evidence for the biological activities of the monoclonals suggest that the C and N terminals of VP 85 are exposed at the surface of the viral particle.