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Marcin Weiner, Wojciech Iwaniak and Krzysztof Szulowski

Abstract

The aim of this study was the evaluation of fluorescence polarisation assay (FPA) in the diagnosis of porcine brucellosis in comparison with Rose Bengal test (RBT), serum agglutination test (SAT), complement fixation test (CFT), 2-mercaptoethanol test, and ELISA. Eight hundred seventeen sera from pigs, including 612 sera from healthy animals, seven sera from Brucella suis bv2 culture positive animals, and 198 sera classified as false positive, originated from confirmatory investigations, were used. All sera from healthy animals, negative in RBT, SAT, CFT, and ELISA were also negative in FPA. All sera positive in serological examination, originated from Brucella infected animals, were also positive in FPA. Among sera classified as false positive almost half of the samples tested (49.49%) reacted positively in FPA. The examinations confirmed the usefulness of FPA in diagnosis of porcine brucellosis, but the method, like the other tests, does not allow resolving the problem of discrimination cross-reacting from specific antibodies.

Open access

Marcin Weiner, Krzysztof Szulowski and Wojciech Iwaniak

Abstract

The paper presents the results of bacteriological and molecular investigations on the presence of Y. enterocolitica O:9 in the head, mammary and genital lymph nodes, spleen, liver, and uterus samples originating from 58 cows slaughtered due to the positive results of serological examinations for brucellosis. All samples were cultured for Brucella and Y. enterocolitica and examined in multiplex PCR assay (mPCR), in order to identify the universal 16S rRNA Brucella sp. marker and amplify the perosamine synthetase (per) gene, specific for Y. enterocolitica O:9 only. Out of 58 examined animals, in 23 cases the presence of Y. enterocolitica was demonstrated. Typical Yersinia-suspected colonies were seen after 24-48 h incubation on Cefsulodin-Irgasan- Novobiocin (CIN) Yersinia selective solid medium. The mPCR analysis confirmed the presence of predicted amplicon of 312 bp, typical for Y. enterocolitica O:9 in 20 out of 58 lymph node samples tested and similarly, as in bacteriological examination, other samples were negative. The presence of the 16S rRNA gene of Brucella, generating the amplicon of 905 bp, was not observed in any of the samples tested.

Open access

Marcin Weiner, Krzysztof Szulowski, Wojciech Iwaniak and Jolanta Złotnicka

Abstract

The aim of the study was to perform a molecular investigations for the presence of pathogenicity genotypic markers of Y. enterocolitica O:9 isolated from cattle, in which initially positive serological reactions for brucellosis were observed. Almost all isolates were ail-, ystA- and myfA-positive (n=19). On the other hand, one isolate, which harboured plasmid encoding gene yadA was ail- , ystA- and myfA-negative. The plasmid encoding yadA marker was present in half of the isolates tested. None of the examined isolates was ystB-positive. The results of the investigations revealed that the Y. enterocolitica O:9 isolates, related to false positive serological results for brucellosis, may be also potentially pathogenic for humans, due to the presence of chromosomal and plasmidencoded molecular markers.

Open access

Marcin Weiner, Wojciech Iwaniak, Krzysztof Szulowski and Monika Szymajda

Abstract

Fluorescence polarisation assay (FPA) was evaluated as a potential tool improving specificity of serological diagnosis of brucellosis in cattle and pigs. The evaluation was performed by comparing the results of FPA with the results of rose Bengal test (RBT), serum agglutination test (SAT), complement fixation test, and indirect ELISA when problematic sera, regarded as false positive, were tested. Four hundred and seventy-five sera, including 201 porcine and 274 bovine samples, reacting positively in at least one classical serological assay were used. Only six bovine sera were FPA positive (two positive in SAT and RBT and four positive in SAT only). Different situation was observed when porcine sera were examined. Out of 201 sera, 109 were also positive in FPA. The studies confirmed that in cattle FPA enables to reduce highly the number of false positive reactions for brucellosis. On the other hand, in pigs, the method is a little more specific in comparison to other methods applied.

Open access

Marcin Weiner, Maria Kubajka, Krzysztof Szulowski, Wojciech Iwaniak, Monika Krajewska and Marek Lipiec

Abstract

The paper concerns molecular study on pathogenicity markers of fourteen Y. enterocolitica O:9 isolated from pigs in which initially positive serological reactions for brucellosis were observed (n = 41), healthy pigs, which were brucellosis-negative (n = 258), and wild boars serologically negative for brucellosis (n = 209). PCR identification proved that all isolates were ail, ystA- and myfA-positive. The plasmid encoding yadA marker was detected in nine isolates that originated from pigs serologically positive or negative for brucellosis, and from one isolate of wild boar origin. Furthermore, none of the examined isolates was ystB-positive. Results of the investigations indicate that the Y. enterocolitica O:9 isolates from pigs or wild boars, regardless of whether they were serologically positive or negative for brucellosis, may also be potentially pathogenic for humans, due to the presence of chromosomal and plasmid encoded molecular markers.

Open access

Monika Krajewska, Agnieszka Czujkowska, Marcin Weiner, Marek Lipiec and Krzysztof Szulowski

Abstract

The paper describes avian tuberculosis in a captive bred cassowary. A two-and-a-half-year-old bird was obtained by a Polish zoo in 2010 from the Netherlands under conditions compliant with the recommendations of the European Association of Zoos and Aquaria. Despite being of small size for the age, the bird appeared healthy and showed no signs of the disease until the day when it was found recumbent in its pen. Later on it was euthanised due to lack of treatment possibilities. Pathological changes typical of avian tuberculosis were found in the liver and spleen. Mycobacterium avium ssp. avium was cultured from both organs.

Open access

Marcin Weiner, Hanna Różańska, Maria Kubajka, Krzysztof Szulowski, Monika Krajewska and Bernard Wasiński

Abstract

The aim of study was the preliminary evaluation of the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum ß-lactamases (ESBL) - producing Escherichia coli in 650 milk and inflammatory secretions from cows with clinical or subclinical mastitis. One millilitre of the sample was added to Mueller-Hinton broth supplemented with 6.5% NaCl, Tryptone Soya Broth with cefoxitin and aztreonam, and then to MRSA ID agar. Presumptive MRSA colonies were analysed for the presence of mecA gene. Parallel to MRSA identification, the samples were incubated in buffered peptone water, lauryl tryptose broth and McConkey agar supplemented with cefotaxim for ESBL-producing E. coli isolation. These bacteria were identified using API Rapid 32 E and the ability of ESBL production was initially established using disc test D68C and confirmed by MIC technique using Sensititre ESBL plates. The primers (blaCTX, blaTEM, blaSHV, and blaCMY-2-group) for the detection of some of the genes encoding ESBL production were used. The 45 strains of S. aureus with mecA gene and 41 strains of E. coli with blaTEM gene were detected.

Open access

Monika Krajewska-Wędzina, Anna Zabost, Ewa Augustynowicz-Kopeć, Marcin Weiner and Krzysztof Szulowski

Abstract

Introduction: Tuberculosis is a highly infectious disease affecting humans and animals. It is caused by the Mycobacterium tuberculosis complex (MTBC) – Mycobacterium bovis and Mycobacterium caprae, which are aetiological factors of bovine tuberculosis (bTB). In Poland, the bTB eradication programme exists. Animals diagnosed with tuberculosis are in the majority of cases not treated, but removed from their herd and then sanitary slaughtered.

Material and Methods: In total, 134 MTBC strains isolated from cattle in Poland were subjected to microbiological analysis. The resistance phenotype was tested for first-line antimycobacterial drugs used in tuberculosis treatment in humans: streptomycin, isoniazid, rifampicin, ethambutol, and pyrazinamide. The strains were isolated from tissues collected post mortem, so the test for drug resistance fulfilled only epidemiological criterion.

Results: The analysis of drug-resistance of MTBC strains revealed that strains classified as M. bovis were susceptible to 4 antimycobacterial drugs: isoniazid, rifampicin, streptomycin, and ethambutol, and resistant to pyrazynamide. The strains classified as M. caprae were sensitive to all tested drugs.

Conclusion: The results indicate that despite enormously dynamic changes in mycobacterial phenotype, Polish strains of MTBC isolated from cattle have not acquired environmental resistance. The strains classified as M. bovis are characterised by natural resistance to pyrazinamide, which is typical for this species.

Open access

Agnieszka Kędrak-Jabłońska, Sylwia Budniak, Anna Szczawińska, Monika Reksa, Marek Krupa and Krzysztof Szulowski

Abstract

The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes. Five strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes, which confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.

Open access

Agnieszka Kędrak-Jabłońska, Sylwia Budniak, Marek Krupa, Anna Szczawińska, Monika Reksa, Krzysztof Szulowski and Wojciech Iwaniak

Abstract

Introduction: The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes in biological samples of the liver, brain, and blood.

Material and Methods: Five strains of L. monocytogenes and single strains of each species L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification the hlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging to Listeria spp. and L. monocytogenes were conducted.

Results: The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes which confirm their belonging to Listeria spp. and L. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products.

Conclusion: Both real-time PCR methods for the detection of Listeria spp. and L. monocytogenes in biological samples demonstrated a significant sensitivity and high specificity.