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Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes are three bacterial pathogens closely associated with the bovine respiratory disease complex (BRDC). In the current study, a multiplex PCR for the simultaneous detection of these three bacteria in cultures was established. After serial optimisation, the detection limit of the method for the genomic DNA of the three bacteria was 40 pg/μl. The method could detect the genomic DNA of these three bacteria but not the genomic DNA of seven other bacterial strains. Together with the bacterial enrichment technology, the multiplex PCR could be used for detecting the three bacteria in animal tissues. This method might be valuable for speeding up laboratory diagnosis and directing the treatment of BRDC to these three bacterial pathogens.
In this study, bovine viral diarrhoea virus (BVDV) was detected and biotypically characterised in clinical samples using reverse transcriptase-polymerase chain reaction (RT-PCR). The RT-PCR technique produced two different amplicons (402 and approx. 680 bp in size) in case of the presence of both biotypes (cp and ncp) in the sample. The mixture of the biotypes as detected by RT-PCR was verified by the immunoplaque assay (IPA). Purification of biotypes was carried out by native plaque isolation and subsequent RT-PCR revealed single products (402 or approx. 680 bp in size) in each clone. The results showed that RT-PCR can be used for accurate molecular differentiation between the BVDV biotypes.
The worldwide occurrence and re-occurrence of transboundary diseases like foot-and-mouth disease or classical swine fever indicates that there is a high need for the development of powerful, robust and high-capacity new diagnostic methods, which are able to detect the causative agents before they could spread to large populations and cause tremendous losses. This article reports the experiences of a research group on the development of molecular methods for the improved diagnosis of a range of porcine viral diseases, including diseases on List A of the Office International des Epizooties (OIE). Nucleic acid hybridisation and various polymerase chain reaction (PCR) assays have been applied for routine diagnosis of a large range of viral diseases. During the last one-and-a-half decade more than 40 nested PCR assays have been developed to detect a variety of DNA and RNA viruses. False positive and negative results are avoided by the use of special tools, practices and internal controls of amplification (mimics). Recently, real-time PCR methods (TaqMan, molecular beacons, Primer-Probe Energy Transfer system) have been developed for the diagnosis of a wide range of diseases, such as foot-and-mouth disease, swine vesicular disease and vesicular stomatitis. Multiplex PCR packages have been developed for the simultaneous detection of eight important viruses of swine. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the diagnostic procedures have become rapid, robust and automated. In order to standardise the real-time PCR assays, the rules of OIE are considered. By following the five steps of OIE standardisation and validation, the new diagnostic procedures are nationally and internationally standardised and harmonised. The rapid, powerful and internationally standardised molecular diagnosis contributes to the reduction of losses caused by the transboundary viral diseases in swine populations.
The aim of this study was to compare an enzyme-linked fluorescent assay (ELFA)-based and two real-time polymerase chain reaction (PCR) methods with the results of the standard culture-based method EN ISO 6579:2002 (bacteriological standard method used in the European Union) for the detection of Salmonella spp. in raw chicken meat. Our investigations were performed on 141 poultry samples sorted from supermarkets.Relative accuracy, relative specificity and relative sensitivity were determined. According to the ISO 16140:2003 criteria for validation of alternative microbiological methods, the ELFA-based method (VIDAS ICS2 + SLM), and real-time PCR methods (TaqMan, Bax) were comparable to the reference standard method for the detection of Salmonella spp. in chicken meat. The use of these methods provide results within 48 hours with high sensitivity (100%). The TaqMan real-time PCR showed a relative specificity of 98% and both of the real-time PCR methods presented 100%.The VIDAS ICS2 + SLM and the Bax real-time PCR methods showed the highest relative accuracy (100%) and 99% in case of the TaqMan method. In conclusion, both the real-time PCR and the ELFA-based assay can be used as a rapid and user-friendly diagnostic method for detection of Salmonella spp. in chicken meat samples.
Tartós SARS-CoV-2-PCR-pozitivitáshoz társuló, lokoregionálisan előrehaladott emlődaganat komplex onkológiai kezelése
Complex oncologic therapy for loco-regionally advanced breast cancer associated with long-lasting SARS-CoV-2 PCR-positivity
Összefoglaló. A COVID–19 mortalitását a súlyos társbetegségek, közöttük bizonyos daganatos betegségek is növelik. Immunszuppresszív hatásuk miatt felmerülhet a citotoxikus kezelések rizikónövelő hatása is. Ugyanakkor az onkológiai terápia megszakítása vagy halasztása, különösen az agresszívebb, kiterjedtebb és fiatalkorban jelentkező daganatok esetében ronthatja a kórjóslatot. Egy 39 éves nőbeteg esetét ismertetjük. A járvány során késlekedve felismert, lokoregionálisan kiterjedt emlődaganat miatt primer szisztémás kemoterápiában részesült. A kezelés 5. ciklusa során enyhe légúti tünetek kapcsán, az onkológiai ambulancián SARS-CoV-2-fertőzése igazolódott. Kemoterápiás kezelését felfüggesztettük. A diagnózistól számított 3. napon tünetmentessé vált, ám SARS-CoV-2-PCR-pozitivitása még a 43. napon is fennállt. A 19. napon hormongátló kezelést indítottunk. Az 51. napon mastectomia és axillaris block dissectio történt. A 82. napon a megszakított kemoterápiát a hormongátló kezelés leállítását követően G-CSF-profilaxis mellett újraindítottuk. A kezelés során fertőzéses szövődményt nem észleltünk. Kemoterápia és műtét SARS-CoV-2-fertőzött, tünetmentes daganatos betegnél szövődménymentesen végezhető elhúzódó virológiai pozitivitás esetén, felszabadító vizsgálat nélkül is. A daganatos betegek koronavírus-fertőzése esetén az onkológiai protokolltól történő eltérés egyénre szabott optimalizálásával és a multidiszciplináris team szorosabb együttműködésével az infektológiai és az onkológiai kockázat együttes alacsonyan tartása is megvalósítható. Orv Hetil. 2021; 162(16): 611–614.
Summary. Mortality of COVID-19 is increased when certain co-morbidities, among others advanced malignancies are present. Deleterious effect of cytotoxic therapy, related to its immunosuppressive effect, may also be hypothesised. However, postponing or cancelling oncologic treatment, especially in younger patients with advanced and more aggressive tumors may worsen the prognosis. The case of a 39-year-old female patient is presented, who was diagnosed with loco-regionally advanced breast cancer during the pandemic. Primary systemic chemotherapy was started. The patient presented with acute respiratory tract symptoms during the fifth cycle and subsequently SARS-CoV-2 infection was diagnosed. Chemotherapy was cancelled. Symptoms resolved in three days after diagnosis. SARS-CoV-2 PCR remained positive up to day 43. Antihormonal therapy was introduced on day 19 and she underwent mastectomy with axillary lymph node dissection on day 51. Chemotherapy was reset postoperatively on day 82 with prophylactic G-CSF protection. No adverse event was observed throughout the treatment. Cytotoxic chemotherapy and surgery can be successfully delivered in breast cancer patients with prolonged asymptomatic SARS-CoV-2 PCR positivity, even without negative swab result. Individual optimisation of the therapy may require deviations from standard protocols. Closer multidisciplinary cooperation may contribute to the minimisation of both oncologic and infectious risks. Orv Hetil. 2021; 162(16): 611–614.
Classical swine fever is a highly contagious, notifiable disease of pigs and wild boars listed by the World Organisation for Animal Health (OIE). Therefore, methods employed in the diagnosis of CSF should be fast, sensitive and specific. The aim of this study was optimisation of the reverse transcription reaction to increase the sensitivity of real-time RT-PCR for the detection of classical swine fever virus, the aetiological agent of the disease. The efficiency of reverse transcription reaction was compared including a range of reverse transcriptases, thermal conditions and priming methods based on results obtained in the following realtime PCR. Depending on catalysis and the priming method used in the study a significant diversity of results was observed. The best efficacy of reverse transcription was obtained using SuperScript II reverse transcriptase and priming with random nonamers and reverse, gene-specific primer. This combination improved the sensitivity of RT-PCR nearly 1000 times as compared to the method with AMV reverse transcriptase coupled with random hexamers. In summary, this study has demonstrated that the optimisation of reverse transcription can contribute to a higher sensitivity of RT-PCR diagnostic methods.
Atotal of 44 Pseudomonas aeruginosa clinical strains were studied by random amplified polymorphic DNA PCR. (RAPD)-PCR analysis determined the presence of 15 genotypes, with the most frequent genotype A detected in 27.3% of the strains. It was observed that clonally related strains were isolated from patients within the same ward and among different wards of two hospitals. The collection of P. aeruginosa was also screened in microtiter plates made of polystyrene to test for their ability to form a biofilm on an abiotic surface. Generally most of the strains (88.6%) showed a significant ability to form biofilm. We found a correlation between twitching motility activity and between biofilm production and source of isolation of strains. No correlation was observed between P. aeruginosa strain genotypes and biofilm formation, as well as source and place of isolation.
Footrot is widely considered the most severe and most common foot pathology in small ruminants. This study tested the ability of a molecular typing system based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay of the 16S rRNA gene to discriminate between the strict anaerobe genera most commonly isolated from footrot ( Bacteroides, Dichelobacter, Fusobacterium, Porphyromonas and Prevotella ) in goats in Extremadura (Spain), with a view to facilitating identification for diagnostic purposes and thus providing a useful tool for future epidemiological studies. Although the electrophoretic patterns obtained with the enzyme Tru 1I were more readily interpreted, and may thus be the best initial option, results may be confirmed by a second enzyme ( Rsa I). The PCR-RFLP assay of the 16S rRNA gene may therefore prove a useful addition to conventional biochemical identification techniques, providing taxonomic information at genus level.
In order to investigate haemotropic Mycoplasma (formerly Eperythrozoon) infection of goats, blood samples and blood-sucking lice (Linognathus stenopsis) were collected in two goat herds. DNA was extracted from 20 blood samples and from 49 lice allocated to six pools according to host individuals. Haemoplasma infection was detected in four goats by real-time PCR. From the sample with the highest bacterial load the simultaneous presence of M. ovis and ‘Candidatus M. haemoovis’ was demonstrated by cloning and sequencing. Louse pools were haemoplasma negative, including those from bacteraemic animals. However, not only were Anaplasma inclusion bodies seen in blood smears from goats, but relevant PCR-positivity was also detected among lice. This is the first report of a molecular investigation on caprine haemoplasmas, including analysis of their bloodsucking lice. In summary, goats are susceptible to both molecularly characterised ovine haemoplasmas. On the other hand, goat sucking lice (L. stenopsis) do not appear to be potential vectors of these agents.
Anaplasma phagocytophilum is a tick-borne rickettsial pathogen responsible for granulocytic anaplasmosis in mammalian hosts including humans. Wild animals may play an important role in the epidemiology of this disease. The aim of this study was to estimate the prevalence of infection with A. phagocytophilum among wildlife in Slovenia. Serum samples (n = 376) from the most important game species [red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa), chamois (Rupicapra rupicapra) and brown bear (Ursus arctos)] were examined by A. phagocytophilum-specific indirect fluorescent-antibody assay (IFA) and wild boar spleen samples (n = 160) were tested by polymerase chain reaction (PCR). A. phagocytophilum-specific antibodies were found in 72% of sera and A. phagocytophilum DNA was present in 6.2% of spleens. The data indicate that A. phagocytophilum is present and widespread in Slovenian game animals and that game species are involved in the natural life cycle of A. phagocytophilum.
