Monitoring Ceratomyxa shasta infection during a hatchery rearing cycle: comparison of molecular, serological and histological methods

The prevalence of Ceratomyxa shasta infection in production stocks of steelhead Oncorhynchus mykiss and cutthroat trout O. clarki was monitored using a parasite-specific polymerase chain reaction (PCR) assay. For all 4 stocks of fish followed through their 1 yr rearing cycle, C. shasta infection was detected despite their genetic resistance to the disease and the treatment of the incoming water with ozone. Infection was confirmed using serological methods and standard histological procedures, except when prevalence was low (<10%). This suggests that at the lowest infection levels PCR is more sensitive than other methodologies, and can be used as an early indicator of infection. Results of the PCR assay continued to correlate with histological and serological detection as the numbers of parasites and the lesion severity increased over the rearing cycle. For both steelhead and cutthroat trout, early infections were characterized by large numbers of parasites on the epithelial surface, but with little associated inflammation. At release as yearlings, the infection prevalence in all stocks was greater than 90 % and the inflammatory response in many fish was extensive, with tissue necrosis and mucosal damage. Although C. shasta infections no longer result in high mortality at this facility, results of this study indicate that the parasite remains a contributor to low condition indices in these fish, despite their genetic resistance and ozone disinfection of the water supply.     

Modification and optimization of PCR methods for detection of MIE region from human herpesvirus 5

Human herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.     

肺炎支原体感染鼠P1特异抗原的动态检测

通过实验动物模型探讨肺炎支原体感染动物肺泡灌洗液中特异抗原检出率的动态变化,为肺炎支原体感染的临床诊断提供理论依据。小鼠经鼻自然感染肺炎支原体,分别采集感染后不同时间点小鼠的支气管灌洗液,应用量子点标记肺炎支原体P1蛋白抗体,直接免疫荧光法检测感染鼠肺泡灌洗液中肺炎支原体P1特异抗原,同时通过PCR检测肺组织肺炎支原体DNA及肺组织病理切片观察肺部炎性变化确定小鼠感染。结果显示,感染鼠肺炎支原体特异抗原在感染后第3天检出阳性率为75%,第7天达高峰为83%,之后随病程延长,抗原检测的阳性率逐渐下降,在感染后第14、21天检出阳性率分别为58%和25%。肺炎支原体特异抗原在感染早期检出率高。应用量子点标记肺炎支原体P1蛋白抗体,直接免疫荧光法检测肺炎支原体特异抗原可应用于肺炎支原体感染的早期诊断。     

Serodiagnosis of Mycoplasma pulmonis infection in mice and rats by an enzyme-linked immunosorbent assay

Murine antibody against Mycoplasma pulmonis (Mp) was detected sensitively and specifically in experimentally and naturally infected animals by an enzyme-linked immunosorbent assay (ELISA), using urease conjugated antimurine immunoglobulin. More than 98% of the experimentally infected mice and rats exhibited positive reaction in the ELISA two or more weeks after infection, and the titer remained for a prolonged period (up to one year) after infection. However, we failed to detect antibody in the sera of one-week-postinfected animals. Mice and rats from breeding colonies were tested with the ELISA and compared with isolation of Mp from the respiratory organs. Positive reactions were shown in the ELISA using the sera from 91% of the mice and 98% of the rats from which the organisms were isolated. Conversely, 97% of the mice and 78% of the rats among Mp-free animals showed negative results in the ELISA. The sensitivity and specificity of the complement fixation test, which has been used widely for serodiagnosis of Mp-infection, were apparently lower compared to those of the ELISA. From these results, the ELISA was found to be available for the serodiagnosis of Mp-infection in mice and rats.     

Diagnosis of congenital toxoplasmosis: pre- and post-natal evaluation in Sicilian (Italy) epidemiological area. Preliminary data

To evaluate the usefulness of conventional serological methods with western blot assay (WB) in congenital toxoplasmosis diagnosis, we prospectively enrolled in a clinical and serological follow-up all pregnant women with Toxoplasma gondii infection and their offspring, referred to us from October 2004. Western blot and standard serological test were performed on sera collected from mother during pregnancy and from mother and child at birth, at postpartum month 1-3-6-9 and 12. At this point in time, 22 pregnant women and 14 infants have completed the follow-up. 4 newborns were infected and 2 had specific toxoplasmosis anomalies at the birth. In mothers without seroconversion, the WB performed during pregnancy demonstrates the highest accordance with postnatal follow-up whereas in 1 case the negative result of PCR analysis was not confirmed by postnatal observation. The detection of anti-T gondii IgG against 8 kDa accessory antigenic band and against the accessory band included between 35 and 40 kDa band in immunoblot assay was useful for diagnosis of acute phase but did not improve the evaluation of comparative postnatal profile. Althougth few infants have concluded the postnatal follow-up, the preliminary results showed a greater value of using a IgM and IgA WB test than other standard method for the early diagnosis of toxoplasmosis at birth also in child born to treated mothers. The comparative anti-T gondii IgG immunoblot profile of mother and child permitted us to reduce the time of ruling out infection in newborns born to mothers with probable or possible infection and/or when prenatal diagnosis is negative or not performed.     

Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation

Respiratory infections, including Mycoplasma pneumoniae (Mp), contribute to asthma pathobiology. To date, the mechanisms underlying the increased susceptibility of asthmatics to airway Mp infection remain unclear. Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is a recently described large airway epithelial cell-derived molecule that was predicted to exert host defense activities. However, SPLUNC1 function and regulation in an infectious or allergic milieu are still unknown. We determined host defense and anti-inflammatory functions of SPLUNC1 protein in Mp infection and the regulation of SPLUNC1 by Mp and allergic inflammation (e.g., IL-13). SPLUNC1 function was examined in Mp or human airway epithelial cell cultures by using SPLUNC1 recombinant protein, overexpression and RNA interference. Human and mouse bronchial epithelial SPLUNC1 was examined using immunostaining, Western blotting, ELISA, laser capture microdissection, and real-time PCR. Mouse models of Mp infection and allergic inflammation and air-liquid interface cultures of normal human primary bronchial epithelial cells were used to study SPLUNC1 regulation by Mp and IL-13. We found that: 1) SPLUNC1 protein decreased Mp levels and inhibited epithelial IL-8 production induced by Mp-derived lipoproteins; 2) normal human and mouse large airway epithelial cells expressed high levels of SPLUNC1; and 3) although Mp infection increased SPLUNC1, IL-13 significantly decreased SPLUNC1 expression and Mp clearance. Our results suggest that SPLUNC1 serves as a novel host defense protein against Mp and that an allergic setting markedly reduces SPLUNC1 expression, which may in part contribute to the persistent nature of bacterial infections in allergic airways.     

Diagnosis of Barmah Forest Virus Infection by a Nested Real-Time SYBR Green RT-PCR Assay

Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative) samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative) was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized.     

CD4+CD25+ T regulatory cells from FIV+ cats induce a unique anergic profile in CD8+ lymphocyte targets

Background

Detection of a retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), has recently been reported in 67% of patients with chronic fatigue syndrome. We have studied a total of 170 samples from chronic fatigue syndrome patients from two UK cohorts and 395 controls for evidence of XMRV infection by looking either for the presence of viral nucleic acids using quantitative PCR (limit of detection <16 viral copies) or for the presence of serological responses using a virus neutralisation assay.

Results

We have not identified XMRV DNA in any samples by PCR (0/299). Some serum samples showed XMRV neutralising activity (26/565) but only one of these positive sera came from a CFS patient. Most of the positive sera were also able to neutralise MLV particles pseudotyped with envelope proteins from other viruses, including vesicular stomatitis virus, indicating significant cross-reactivity in serological responses. Four positive samples were specific for XMRV.

Conclusions

No association between XMRV infection and CFS was observed in the samples tested, either by PCR or serological methodologies. The non-specific neutralisation observed in multiple serum samples suggests that it is unlikely that these responses were elicited by XMRV and highlights the danger of over-estimating XMRV frequency based on serological assays. In spite of this, we believe that the detection of neutralising activity that did not inhibit VSV-G pseudotyped MLV in at least four human serum samples indicates that XMRV infection may occur in the general population, although with currently uncertain outcomes.     

A real-time PCR diagnostic method for detection of Naegleria fowleri

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence.Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.     

Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep

Aims: To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Methods and Results: Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis‐specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1·2% (2/162) and 17·2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27·7% (45/162) of blood and 29·0% (47/162) of lymphoid tissue samples. Conclusions: The species‐specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0·01 in blood PCR, P < 0·001 in tissue PCR) and serologically negative (P < 0·001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. Significance and Impact of the Study: The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes.     

Development of enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antigens

The variant of enzyme-linked immunosorbent assay (ELISA) for detection of Mycoplasma pneumoniae (Mp) antigens in sera of patients with respiratory infections was developed. Sensitivity of detection of soluble antigens of Mp in modeling experiment varied from 1.5 to 1.0 ng/ml (on protein). Approbation of the assay was performed using 50 serum samples obtained from patients with confirmed diagnosis of respiratory mycoplasmosis. In the ELISA test Mp antigens were detected in 96% of samples. Obtained results were confirmed by testing of these serum samples and isolated from them circulating immune complexes (CICs) in immunoblotting using polyclonal antibodies labeled by horse-radish peroxidase. Mp antigenswere detected both in free state and as components of CICs. Specific reaction was observed with proteins, which molecular mass varied from 30 to 170 kDa (30, 37, 45, 56, 58, 72, 90, 130 and 170 (160) kDa). Obtained results point to appropriateness of use of developed assay for detection Mp antigens in sera of patients with respiratory infections.     

Detection of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR enzyme-linked immunosorbent assay

A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Detection of Campylobacter jejuni and Campylobacter coli in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay

A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.     

Evaluation of the sensitivity of PCR methods for the detection of extraneous agents and comparison with in vivo testing

In this study the sensitivity of polymerase chain reaction (PCR) methods for the detection of Newcastle disease virus (NDV), avian reovirus (ARV), avian influenza virus (AIV) and avian infectious bronchitis virus (IBV) was compared to the sensitivity of the corresponding serological tests described in the European Pharmacopoeia (Ph. Eur.). For this purpose, serial 10-fold dilutions of the respective inactivated vaccines were prepared and groups of SPF chickens were vaccinated with a double dose of the vaccine dilutions. After a period of 21 days, the animals were revaccinated with a single dose. Two weeks later, serum samples from each animal were tested for antibodies using an Idexx enzyme linked immunosorbent assay (ELISA). In parallel, samples of the diluted vaccines were tested by PCR. It was found that the sensitivity of the four PCR tests is comparable to or even slightly better than that of the corresponding serological tests. Thus these PCR tests fulfil the sensitivity requirements of the Ph. Eur. and could be used as alternative tests for the detection of extraneous agents in final batches of inactivated vaccines.     

Laboratory diagnosis of Epstein-Barr virus infection

Laboratory confirmation of EBV infection requires proper methods and schema of investigation adequate to aim of diagnostic procedure. In paper the results of routine diagnostic tests of EBV infection performed in Department of Virology NIH in 2005-2006 years was included and also, evaluation of usefulness of different laboratory methods was done. Based on results of ELISA tests 10,7% routine investigated subjects was classified as primary EBV infection, 20,1% was seronegative, 7,4% was classified as reactivation of latent infection and serological markers in 45,6% subjects pointed past EBV infection. Positive result of PCR method was obtain in 11,2% samples subjected of routine laboratory investigation. Comparison of specific and non-specific serological methods results (ELISA versus tests of heterophile antibodies) showed the high percentage of false negative results in children tested by non-specific tests. PCR results in serum samples from patients with primary infection (confirmed by serological tests) were positive in 15% cases only. Based on analyzed results it could be stated that reliable confirmation of infectious mononucleosis, as primary EBV infection, is detection of specific IgM antibodies and in case of heterophile antibodies tests the possibility of false negative results, mainly in children, must be taken into account. The most proper samples for PCR method are whole blood, sections of tissue or cells from swabs.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR

Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7–30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.     

Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin

Two commercial serological kits (Oxoid BCET-RPLA and Tecra VIA) and a Chinese hamster ovary (CHO) cell cytotonicity assay for the detection of Bacillus cereus diarrhoeal enterotoxin were compared. Eleven B. cereus strains and one enterotoxigenic B. thuringiensis strain were evaluated. Both kits and the CHO cell assay yielded positive toxin responses for cell-free culture filtrates from eight out of 11 diarrhoeal enterotoxigenic strains. An emetic enterotoxin producing strain was negative with all three assays. Two B. cereus strains were negative using the BCET-RPLA kit, but positive with the Tecra VIA kit and CHO cell assay. The BCET-RPLA indicated significant levels of enterotoxin after samples were boiled, whereas the CHO cell and Tecra assays were negative. Overall, the cell culture assay was the most sensitive. However, the Tecra VIA kit provided similar results and was better suited for the rapid detection of B. cereus diarrhoeal enterotoxin.     

Advantage of PCR for detecting low amounts of HBV DNA in patients' sera

Serum hepatitis B virus DNA (HBV DNA) is now the most important and reliable marker for monitoring viral replication. Quantitative detection of HBV DNA in serum is based on a commercial standardized solution hybridization assay (Genostics). In this work, we studied the sensitivity and specificity of this method, in comparison with the polymerase chain reaction (PCR) technique, for low-value HBV DNA serum samples. Fifty-four patients with or without HBV serological markers were divided into 4 groups according to their HBV DNA values.Genomic amplication was found to affect 2 conserved regions of the viral genome, the S and C regions. Samples with an HBV DNA concentration equal to or greater than 1.5 pg/ml were considered positive in the “Genostics” test. A total of 38% of patients considered negative in the quantitative assay (< 1.5 pg/ml) were found to be positive for HBV DNA in serum after PCR. Only 26% of patients with an HBV DNA concentration of between 1.5 and 10 pg/ml in the Genostics test had PCR-detectable viral DNA in serum. Some 56% of patients with HBV DNA values between 10 and 20 pg/ml were found to be positive after amplification. All patients whose HBV DNA values were above 20 pg/ml had PCR-detectable viral DNA in serum.Our PCR results suggest that the positive limit level of the Genostics test has to be re-evaluated. Indeed, for low values of HBV DNA (under 20 pg/ml and especially under 10 pg/ml), it is not possible to conclude about the positivity from the quantitative assay, and results have to be estimated according to the clinical and serological status of the patients. Moreover, PCR can be falsely negative because of methodological problems.Nevertheless, this study confirms that PCR does enable detection of the viral genome in HBV-seronegative patients and in “old” and “cured” HBV-infection marker carriers.