A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals

Theileria parva causes East Coast fever, an economically important disease of cattle in sub-Saharan Africa. We describe a nested polymerase chain reaction (nPCR) assay for the detection of T. parva DNA in cattle blood spotted onto filter paper using primers derived from the T. parva-specific 104-kDa antigen (p104) gene. The sensitivity of this assay was compared to a previously described p104-based PCR and also the reverse line blot (RLB) technique, using serial dilutions of blood from a calf with known T. parva piroplasm parasitaemia. The relative sensitivities of the three assays were 0.4, 1.4 and 4 parasites/μl corresponding to blood parasitaemias of 9.2 × 10⁻⁶%, 2.8 × 10⁻⁵% and 8.3 × 10⁻⁵%, respectively. The three assays were applied to samples from two calves infected with the T. parva Muguga stock. Parasite DNA was consistently detectable by the two p104 PCR assays until 48 and 82 days post-infection, respectively, and thereafter sporadically. RLB detected parasite DNA in the two infected calves until days 43 and 45. Field samples from 151 Kenyan cattle exhibited 37.7% positivity for T. parva by regular p104 PCR and 42.3% positivity using p104 nPCR. Among 169 cattle blood samples from Southern Sudan, 36% were positive for T. parva using nPCR. The nPCR assay represents a highly sensitive tool for detection and monitoring of asymptomatic carrier state infections of T. parva in the blood of cattle.     

Direct amplification of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> DNA from amoebic liver abscess pus using polymerase chain reaction

An important and serious complication of intestinal infection with Entamoeba histolytica is the involvement of the liver (hepatic amoebiasis). Hepatic amoebiasis is usually diagnosed by the clinical picture (pain in the right upper quadrant and fever), ultrasound examination and positive serology. However, none of these tests are definitive and the picture overlaps with pyogenic liver abscess caused by bacteria. It is for this reason that the feasibility of using polymerase chain reaction (PCR) for the detection of E. histolytica DNA in liver abscess pus was investigated. A comparative study was done to verify the sensitivity of ten pairs of primers specific for detecting E. histolytica in stools. Samples of liver abscess pus from 22 serology-positive patients were collected under ultrasound guidance; and these were used directly in PCR assays without any prior pre-treatment of the samples. Of the ten pairs of previously published primers tested, two pairs of primers (P1 + P2 and P11 + P12) were found to give 100% sensitivity. Based on these results, we recommend that PCR assay can be successfully used to confirm the diagnosis of amoebic liver abscess with the primers identified. Received: 12 March 2000 / Accepted: 18 April 2000     

Real-time PCR strategy and detection of bacterial agents of lymphadenitis

The aim of this study was to compare 16 S rRNA gene amplification and sequencing with a systematic real-time PCR assay screening strategy that includes all common known pathogens recovered from lymph node biopsy specimens. Lymph node biopsy samples sent to our laboratory from January 2007 to December 2008 were tested in the study. Lymph nodes were screened for the presence of any bacteria by PCR amplification and sequencing targeting the 16 S rRNA gene and also by a specific real-time PCR strategy that includes Bartonella henselae, mycobacteria, Francisella tularensis, and Tropheryma whipplei. By testing 491 lymph nodes, we found that the sensitivity of our specific real-time PCR assay strategy was significantly higher than 16 S rRNA PCR amplification and sequencing for the detection of Bartonella henselae (142 vs 98; p < 10⁻⁴), Francisella tularensis (16 vs 10, p < 10⁻⁴), and mycobacteria (8 versus 3, p < 10⁻⁴). None of the samples was positive for Tropheryma whipplei. Our study demonstrates the usefulness and specificity of a systematic real-time PCR strategy for molecular analysis of lymph node biopsy specimens and the higher sensitivity compared with standard 16 S rRNA gene amplification and sequencing.     

Use of the Polymerase Chain Reaction for Rapid Detection of High-Level Mupirocin Resistance in Staphylococci

 The minimum inhibitory concentrations (MICs) of mupirocin were determined by the E test (AB Biodisk, Sweden) and the agar dilution method for 107 staphylococci. The organisms consisted of 34 coagulase-negative staphylococci and 73 methicillin-resistant Staphylococcus aureus. Polymerase chain reaction (PCR) primers designed to amplify a 456 bp region of the plasmid-borne isoleucyl tRNA synthetase gene (ileS–2), responsible for high-level mupirocin resistance in staphylococci, were used on DNA preparations from these isolates. Isolates with high-level mupirocin resistance due to the ileS–2 gene should be PCR positive. There was close correlation between the E test and agar dilution MIC values, with only two strains differing by more than two serial dilutions. Most (51 of 54 strains) of the high-level resistant strains (MIC>256 μg/ml) were resistant to the highest concentration of mupirocin tested (1024 μg/ml). PCR correctly classified all but four (96%) of the isolates in accordance with the results of agar dilution. All four isolates that gave discrepant results were methicillin-resistant Staphylococcus aureus. Two of these were PCR positive, yet the MIC of mupirocin for these strains was <0.06 μg/ml; on prolonged incubation they produced halos within the inhibition zone on agar diffusion testing, suggesting that the phenotypic results may have been erroneous. One of 54 isolates for which the MIC exceeded 256 μg/ml was PCR negative when tested by the original methodology, but a 456 bp product was produced when retested using a lowered annealing temperature. One isolate for which the MIC of mupirocin was 16 μg/ml by agar dilution and 8 μg/ml by the E test was positive by PCR. PCR of the ileS–2 gene is a useful, rapid method for detecting high-level mupirocin resistance in staphylococci.     

Tomato leaf curl virus from Bangalore (ToLCV-Ban4): sequence comparison with Indian ToLCV isolates, detection in plants and insects, and vector relationships}

Summary.  Tomato leaf curl virus (ToLCV) is a whitefly (Bemisia tabaci) transmitted geminivirus (family Geminiviridae, genus Begomovirus) causing a destructive disease of tomato in many regions of India, East Asia and Australia. While ToLCV isolates from Australia and Taiwan have a single genomic component (designated DNA-A), those from Northern India have two components (DNA-A and DNA-B). The ToLCV isolates from Southern India (Bangalore) previously cloned seem to have a DNA-A-like monopartite genome. We have used degenerate DNA-A-specific PCR primers to clone the genome of a ToLCV isolate (named ToLCV-Ban4) from field-infected tomato plants growing in Bangalore, India, in 1997. Degenerate DNA-B-specific PCR primers have not allowed to amplify a putative DNA-B from infected tomato, at the time when DNA-B fragments were amplified from plants infected by known bipartite begomoviruses. The full-length 2759 nucleotide-long DNA-A-like viral genome was sequenced. Similarly to other monopartite ToLCV and TYLCV isolates, ToLCV-Ban4 contains six open reading frames, two on the virion strand and four on the complementary strand. Sequence comparisons indicated that ToLCV-Ban4 is similar to the other three isolates from Bangalore previously sequenced, and is closely related to ToLCV-Ban2 (approximately 91\% nucleotide sequence identity). Phylogenetic analysis showed that the ToLCV isolates from Bangalore constitute a group of viruses separated from those of Northern India. ToLCV-Ban4 was detected in tomato and in its whitefly vector Bemisia tabaci by one or by a combination of ELISA, Southern blot hybridization and PCR. Parameters of virus acquisition, retention and transmission by the whitefly vector were investigated in the laboratory. Single whiteflies were able to acquire ToLCV-Ban4 from infected tomato and to transmit the virus to tomato test plants, but five insects were necessary to achieve 100% transmission. Minimum acquisition access and inoculation access periods were 10 min and 20 min, respectively. A latent period of 6 h was required for B. tabaci to efficiently infect tomato test plants. Following a 24 h acquisition access period the insect retained its ability to infect tomato test plants for 12 days, but not for its entire life. In one insect/one plant inoculation tests, female whiteflies were more efficient (∼95%) than males (∼25%) in transmitting the virus. Received July 5, 1999 Accepted March 2, 2000     

Strain differences in Blastocystis isolates as detected by a single set of polymerase chain reaction primers

A total of 20 isolates of Blastocystis were characterized using a single set of polymerase chain reaction (PCR) primers. The amplification product revealed five types of pattern. All four isolates from Singapore yielded PCR products quite different from those of the local isolates. However, most of the local isolates showed a major product at either 280 or 500 bp, or both. We also suspected that the amplification product detected at 280 bp might be an indicator of the pathogenicity of this parasite. One isolate (M12) obtained from a monkey showed patterns similar to those of human isolates (10203 and KP1) and probably belongs to the same strain. The results indicate that the intraspecific or interstrain variations in these 20 Blastocystis isolates belong to 5 different patterns. The differences among isolates of the same strain revealed by the presence or absence of certain amplification products showed further intrastrain variations in this parasite. Received: 12 May 1998 / Accepted: 7 August 1998     

Development of a polymerase chain reaction (PCR) test for the detection of virulent forms of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a marine bacterium and some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Use of randomly amplified polymorphic DNA (RAPD)-PCR produced a unique 600 bp amplicon (band Y) in the majority of clinical isolates and rarely in environmental isolates tested. The DNA from band Y was cloned and sequenced and found to code for an outer membrane protein (OMP). Two polymerase chain reaction (PCR) primers were designed to specifically amplify a 200 bp unique sequence from presumptive virulent strains (PCR-OMP). The virulence of 23 clinical and 32 environmental isolates was assessed in cytotoxicity tests by treatment of Caco-2 cells with extracellular products (ECPs). All but two of the clinical isolates (91%) were positive for the 200 bp PCR-OMP and their ECPs produced a significantly higher (p < 0.05) lactate dehydrogenase (LDH) release (mean 72.88%) than the ECPs of environmental isolates (mean 15.3%) with the exception of one environmental isolate that produced the 200 bp amplicon. A positive 200 bp PCR-OMP is strongly correlated with virulence, as determined by the cytotoxicity assay, and identified virulent forms better than current PCR tests for tdh, trh or T3SS2.     

Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis

Summary. In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 × 10² plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (T_ms): 89.23 ± 0.27 °C for velogenic strains, 90.17 ± 0.35 °C for pigeon mesogenic strains, 91.25 ± 0.14 °C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.     

Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups

Summary.  Seventy-eight bovine viral diarrhoea viruses (BVDV) recently collected in Austria, France, Hungary, Italy, Slovakia, Spain and UK were genetically typed in the 5′-untranslated (5′UTR) and autoprotease (N^pro) regions of the pestivirus genome. Seventy-six of the isolates were BVDV-1 and two French isolates were of the BVDV-2 genotype. Phylogenetic analysis of the 5′UTR (245 nt), including additional BVDV-1 sequences from USA, Canada, Germany, New Zealand, Mozambique and Sweden, taken from GenBank and from our previous works, indicated that these viruses were clustered not only into the two generally accepted groups (BVDV-1a –“NADL like” and BVDV-1b –“Osloss like”), but altogether into 11 phylogenetic groups. Similar clustering was observed with N^pro region sequences (385 nt) and the highest bootstrap values (over 95%) were obtained by phylogeny combining 5′UTR and N^pro sequences. Some associations between the genetic grouping and the origin of the isolates were apparent, probably reflecting historical trade contacts. Considering the variability of isolates it is recommended that diagnostic PCR primers should be re-examined to ensure coverage of all BVDV-1 groups. The genogroups were less clearly differentiated by monoclonal antibody typing, suggesting significant antigenic similarities within the BVDV-1 genotype. Received March 2, 2000 Accepted June 23, 2000     

Cross-protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and RT-PCR and nested PCR for their detection

Summary.  A 1-step RT-PCR assay, targeting a 730 bp fragment of the nucleocapsid (N) gene of bovine coronavirus (BCV), and a nested PCR assay, targeting a 407 bp fragment of the N gene, were developed to detect BCV in nasal swab and fecal samples of calves experimentally exposed to BCV. Both 1-step RT-PCR and nested PCR recognized cell culture passaged isolates of 10 bovine respiratory coronavirus (BRCV), 5 calf diarrhea (CD) and 8 winter dysentery (WD) strains of BCV, but not transmissible gastroenteritis coronavirus or bovine rotavirus. The sensitivity of the 1-step RT-PCR and nested PCR was compared to that of an antigen-capture ELISA. The lowest detection limit of the 1-step RT-PCR and nested PCR as determined by using tenfold serial dilutions of the BRCV 255 and 440 strains in BCV negative nasal swab suspensions from preexposure gnotobiotic calves was 2 × 10⁴ and 2 × 10² TCID₅₀/0.1 ml for each strain, respectively. The lowest detection limit of the antigen-capture ELISA as determined by using the same serially diluted samples was 1 × 10⁶ TCID₅₀/0.1 ml for each strain. Therefore, the 1-step RT-PCR and nested PCR assays were 50 and 5000 times, respectively more sensitive than the antigen-capture ELISA to detect BRCV in nasal swab suspensions. To investigate in vivo cross-protection between the BRCV and CD or WD strains of BCV and to detect nasal and fecal shedding of BCV using the 1-step RT-PCR, nested PCR and antigen-capture ELISA, 6 colostrum-deprived and two gnotobiotic calves were inoculated with a BRCV, a CD or a WD strain of BCV and then challenged 3–4 weeks later with either BRCV, CD or WD strains of BCV. All calves developed diarrhea after inoculation and BCV antigen (ELISA) or RNA (RT-PCR) was detected in the diarrheic fecal samples or the corresponding nasal swab samples. In addition, low amounts of BCV were also detected only by nested PCR in the fecal and nasal swab samples before and after diarrhea. No respiratory clinical signs were observed during the entire experimental period, but elevated rectal temperatures were detected during diarrhea in the BCV-inoculated calves. All calves recovered from infection with the BRCV, CD, or WD strains of BCV were protected from BCV-associated diarrhea after challenge exposure with either a heterologous or homologous strain of BCV. However, all calves challenged with heterologous BCV strains showed subclinical BCV infection evident by detection of nasal and fecal shedding of BCV RNA detected only by nested PCR. Such results confirm field and experimental data documenting reinfection of the respiratory and enteric tracts of cattle, suggesting that, in closed herds, respiratory or enteric tract reinfections may constitute a source of BCV transmissible to cows (WD) or neonatal or feedlot calves. In addition, the present 1-step RT-PCR and nested PCR assays were highly sensitive to detect BCV in nasal swab and fecal specimens. Therefore, these assays should be useful to diagnose BCV infections in calves and adult cows. Received Septemper 21, 2000 Accepted January 17, 2001     

Symbiosis of Mycoplasma hominis in Trichomonas vaginalis may link metronidazole resistance in vitro

Fourteen of 28 Trichomonas vaginalis isolates collected from patients in Guangzhou, China from 2003 to 2004 were found to be naturally infected with Mycoplasma hominis, as determined by PCR using specific primers. In vitro metronidazole sensitivity assay of the 28 isolates revealed four displaying low susceptibility [minimum lethal concentration (MLC)=∼13–25 μg/ml] and another four displaying high resistance (MLC=50–100 μg/ml). The overwhelming majority of these resistant isolates (7/8) were mycoplasma-infected. The mean of MLCs of mycoplasma-infected isolates is ∼10-fold higher than the mean of noninfected isolates (p=0.029). Sequence analyses of PCR-amplified small subunit–large subunit rRNA interspacer regions (ITS1/5.8S/ITS2) revealed that 23 of the 28 samples are identical, the remaining five being separable into two groups, each with a single point mutation. These internal transcribed spacer sequence variants are associated neither with mycoplasma infection nor with drug resistance. In contrast, random amplified polymorphic DNA analyses of DNAs using 10 different primers showed that the drug-resistant isolates are clustered together in association with mycoplasma infection, albeit more loosely. Taken together, the results obtained from this study suggest that in vitro metronidazole resistance of T. vaginalis is related to mycoplasma infection of this protozoan.     

Detection and quantification of hepatitis B virus DNA by SYBR green real-time polymerase chain reaction

A single-round real-time polymerase chain reaction (PCR) assay based on SYBR green dye technology for the detection and quantification of hepatitis B virus (HBV) DNA in serum was evaluated and compared with a qualitative nested PCR and the Cobas Amplicor HBV Monitor assay (Roche Molecular Diagnostics, Milan, Italy). The performance of the real-time PCR assay was evaluated in a routine clinical laboratory setting with a total of 212 clinical specimens. The sensitivity of the real-time PCR corresponded to 31 IU/ml (70 copies/ml), and comparison with the qualitative nested PCR showed significant concordance for 94% of samples. The linear curve over 7 log units, spanning 10³–10⁹ IU/ml (2.28 × 10³ to 2.28 × 10⁹ copies/ml), was observed in the quantitative determination. The interexperimental variability coefficient of the assay ranged from 0.22 to 0.39 and the intraexperimental variability coefficient from 0.24 to 0.41. By excluding values outside of the dynamic ranges of both tests, the HBV Monitor and the real-time PCR gave an agreement within ±1 log unit for 90% of samples, while those for the remaining 10% were found to be above 1 log unit but less than 1.5 log units. When the results inside and outside the dynamic range of the HBV Monitor were examined, 90% of the results were in agreement. In conclusion, the real-time PCR based on SYBR green technology proved suitable for routine diagnostic purposes, showing good sensitivity, high specificity, high reproducibility, and good linearity over a broad dynamic range of quantification.     

In vitro sensitivity of Blastocystis hominis to garlic, ginger, white cumin, and black pepper used in diet

To determine the growth pattern and in vitro susceptibility of Blastocystis hominis to metronidazole (MTZ), garlic, ginger, white cumin, and black pepper. Stool specimens were collected from 16 irritable bowel syndrome (IBS) and 10 controls between July–November 2010. Stool microscopy and culture for B. hominis was performed. Drug susceptibility assays was done using 0.01 and 0.1 mg/ml of MTZ, garlic, ginger, white cumin, and black pepper. Effect was assessed on B. hominis culture after 48 h. Stool DNA was extracted using stool DNA extraction kit (Qiagen) and polymerase chain reaction (PCR) done using subtype-specific sequence-tagged-site primers. B. hominis genotype 3 and coinfection of 1 and 3 tended to grow well in culture compared to isolated type 1 infection. Exposed to MTZ at a concentration of 0.01 mg/ml, 38% (6/16) B. hominis from IBS did not grow in culture compared to 100% (10/10) of B. hominis from control (p = 0.001). When they were exposed to MTZ at 0.1 mg/ml, 56% (9/16) B. hominis from IBS did not grow in cultures compared to 100% (10/10) from control (p = 0.01). Forty-four percent (7/16) B. hominis from IBS did not grow in culture compared to 100% (10/10) B. hominis from control when exposed to garlic at a concentration of 0.01 mg/ml (p = 0.003) and following exposure to garlic at 0.1 mg/ml, 38% (6/16) B. hominis from IBS did not grow in cultures compared to 100% (10/10) from control (p = 0.001). B. hominis isolates from IBS had a cell count of 6,625 at a MTZ concentration of 0.01 mg/ml that reduced to 1,250 as MTZ concentration was increased to 0.1 mg/ml (p = 0.08). B. hominis from IBS with a mean cell count of 3 × 10⁵ at baseline decreased to 1 × 10⁴ when exposed to garlic at 0.01 mg/ml (p < 0.001) and to 1 × 10³ (p < 0.001) when garlic was 0.1 mg/ml. B. hominis from IBS cell count decreased to 1 × 10⁵ when exposed to white cumin at 0.01 mg/ml (p = 0.01) and to 1 × 10⁵ (p < 0.001) when white cumin was 0.1 mg/ml. Exposed to black pepper at 0.1 mg/ml, cell count of B. hominis from IBS decreased to 1 × 10⁵ (p = 0.01). B. hominis from IBS decreased to 1.3 × 10⁵ exposed to ginger at 0.01 mg/ml (p = 0.001). B. hominis isolates were mostly genotypes 3, type 1 and 3 coinfection, and non-typeable B. hominis isolates. B. hominis isolates from IBS mostly genotype 1 demonstrated an increased sensitivity to garlic at 0.01 mg/ml with a B. hominis cell count of 3,714 compared to 6,142 when exposed to 0.01 mg/ml of MTZ. However, this sensitivity did not increase as garlic concentration was increased to 0.1 mg/ml, for B. hominis cell count was 6,000 compared to 1,428 as MTZ was increased to 0.1 mg/ml.     

Diagnosis of Wuchereria bancrofti infection by the polymerase chain reaction employing patients' sputum

A preliminary evaluation of the diagnostic potential of a polymerase chain reaction (PCR) assay using diurnally collected sputum from bancroftian filariasis patients is described. A new set of PCR primers amplifying a 254-bp-long sequence termed AccI, derived from a long dispersed repeated sequence and SspI primers previously employed for PCR-based diagnosis were employed in this study with similar results. Of the 34 sputum samples from patients, 32 (94%) were PCR positive. Of the 18 patients with low to high microfilaremia (21–1560 microfilariae/ml), 16 (88.8%) were PCR positive. Of the remaining 16 patients, 6 with very low microfilaremia (2–6 microfilariae/ml) and 10 without microfilaremia, all (100%) were PCR positive. Two PCR-positive cases among the 13 endemic normal individuals tested (15.4%) may represent cases of occult filariasis. PCR amplification was also demonstrated with one PCR-positive sputum aliquot when mixed with 14 sputum aliquots from uninfected (PCR-negative) individuals. The potential diagnostic merits of the sputum-PCR assay are discussed. Received: 18 March 1999 / Accepted: 29 April 1999     

Efficacy of <Emphasis Type="Italic">Eimeria tenella</Emphasis> rhomboid-like protein as a subunit vaccine in protective immunity against homologous challenge

The immune responses and protective efficacy against homologous challenge in chickens elicited by recombinant proteins of a rhomboid-like gene (ETRHO1) from Eimeria tenella was investigated in the present study. When chickens were immunized with the recombinant rhomboid antigen, specific antibody was generated by ELISA assay. In comparison with the PBS group, the expression levels of interleukin-2, interferon-γ, as well as the percentages of CD4⁺ and CD8⁺ cells in the group immunized with the recombinant rhomboid proteins were significantly increased (p < 0.01, p < 0.05, and p < 0.05, respectively). These results suggest that rhomboid was capable of eliciting humoral and cell-mediated immunity response in birds. Challenge experiments demonstrated that the recombinant rhomboid protein could provide chickens with a protection rate around 77.3%. Numbers of oocysts and cecal lesion from chickens in the group immunized with recombinant rhomboid proteins decreased significantly, and the body weight increased significantly when compared with chickens in the PBS group (p < 0.05). These results suggested that the recombinant rhomboid antigen was able to impart partial protection against homologous challenge in chicken and could be a potential candidate for an E. tenella vaccine development.     

Improved detection of canine Angiostrongylus vasorum infection using real-time PCR and indirect ELISA

This study reports the development of a real-time PCR assay and an indirect ELISA to improve on current detection of canine Angiostrongylus vasorum infection. A highly specific fluorescent probe-based, real-time PCR assay was developed to target the A. vasorum second internal transcribed spacer region and detected DNA in EDTA blood, lung tissue, broncho-alveolar larvage fluid, endotracheal mucus, pharyngeal swabs and faecal samples. PCR was fast (∼1 h), highly efficient when using EDTA blood samples, consistently detected a single molecule of parasite DNA and did not amplify DNA from other parasitic nematodes or definitive host species. An indirect ELISA was also developed using the soluble protein fraction from adult A. vasorum worms. Some cross-reactive antigen recognition was observed when tested against sera from dogs infected with Crenosoma vulpis (n = 8), Toxocara canis (n = 5) and Dirofilaria immitis (n = 5). This was largely overcome by setting the cut-off for a positive result at an appropriately high level. Field evaluation of the real-time PCR and ELISA was conducted by testing sera and EDTA blood from dogs with suspected A. vasorum infection (n = 148) and compared with the Baermann's larval migration test in faeces. Thirty-one dogs were positive by at least one test. Of these, 20 (65%) were detected by the Baermann method, 18 (58%) by blood PCR, 24 (77%) by ELISA and 28 (90%) by blood PCR and ELISA together. Combined testing using real-time PCR and ELISA therefore improved the detection rate of A. vasorum infection and holds promise for improved clinical diagnosis and epidemiological investigation.     

RFLP mapping of the whole genome of ten viral isolates representative of different biological groups of potato virus Y

Summary.  Ten PVY isolates representative of four PVY groups (Y^N, Y^NTN, Y^N-W, Y ^O), differing by their ability to induce reactions of vein necrosis on tobacco and tuber necrosis on potato, were studied in order to research the regions of the viral genome involved in these necrosis phenomena. The whole genome of these isolates was amplified in two fragments (4 063 and 5 670 nucleotides) and was subjected to a restriction fragment length polymorphism (RFLP) study. In the first 4 063 nucleotides of the PVY genome, a phenetic analysis of RFLP data resulted in a clustering of our PVY isolates into three groups: PVY^N isolates (group A); PVY^NTN and PVY^N-W isolates (group B) and PVY^O isolates (group C). In the last 5 670 nucleotides, two groups were found: PVY^N and PVY^NTN isolates (group D) and PVY^O and PVY^N-W isolates (group E). From this clustering and the necrosing properties known for theseisolates, the tobacco necrosis determinants seem more likely located inthe 5′ than in the 3′ half part of the viral RNA, whereas it would be theopposite situation for the determinants of the necrosis on potato tubers.Moreover a recombination event seemed to have occurred in the genome ofthe PVY^N-W isolates. Received February 11, 1998 Accepted June 25, 1998