Search

One of the most essential systems applied to the eradication of bovine tuberculosis by Mycobacterium bovis is the epidemiologic surveillance of animals slaughtered in abattoir by means of inspection and sample taking of lesions compatible with tuberculosis, confirming the existence of the disease through culture and molecular detection, which takes weeks before a result can be obtained. An interesting alternative is to develop high-throughput molecular systems for the direct detection of M. bovis on biological samples. In this sense, our research has developed a molecular detection system by means of a real-time based PCR process which is applied directly to bovine biological samples and it allows to differentiate between Mycobacterium tuberculosis complex, Mycobacterium avium complex and other atypical mycobacteria that are interesting from the veterinary point of view. The sensitivity was analyzed by applying a conventional extraction system based on guanidine thiocyanate and a robotized system based on the selective magnetic capture of mycobacterial DNA. The molecular detection system showed a high specificity and a detection threshold of only two to three genomes. The sensitivity depended on the DNA extraction system being used and on the kind of lesions on which it was used; the sensitivity ranged from 61.11% for samples with non-visible lesions to 80.64% for chronic lesions, with an average sensitivity of 73.87% when using the manual extraction system and between 27.77 and 74.19% (average sensitivity 47.74%) when using the automated robotic system. In conclusion, our multiplex real-time PCR assay represents a fully controlled, high-throughput diagnostic tool for the rapid detection of Myobacterium presence directly in animal clinical specimens, which could be a practical tool in the context of bovine tuberculosis abattoir surveillance programs and granuloma submission programs.

A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases.

Staphylococcus aureus is considered as one of the leading causes of food-intoxication and on the other hand E. coli and particularly, Shiga toxin (ST) producing Escherichia coli (STEC) have emerged as important food-borne enteropathogens frequently associating serious to fatal disorders in humans and both species have shown to be resistant to a wide spectrum of antibiotics. The current study was performed to determine the prevalence of two common food-borne pathogens: Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli ) in white soft cheese being one of the most popular dairy products in Egypt. A total 150 samples of soft cheese were purchased from the market and were microbiologically tested. The S. aureus isolates were identified according to recommended biochemical tests, and polymerase chain reaction (PCR) targeting the nuc gene, as a species-specific marker gene for S. aureus. Meanwhile, E. coli isolates were identified based on biochemical tests, serological tests, haemolytic activity, and multiplex PCR. This has been done to assess capability of producing STs based on targeting stx1 and stx2 genes. Result revealed that S. aureus and E. coli were observed in 66.66% and 36% of samples, respectively. The antimicrobial resistance of isolates was phenotypically investigated against eleven different antibiotics and results showed the presence of a variable multidrug-resistance among isolates against selected antibiotics. These findings highlighted the importance of periodical screening of food-borne pathogens in artisanal products due to lack of strict hygienic measures in markets; the reason that facilitate the contamination of such artisanal products by several food-borne pathogens. [ABSTRACT FROM AUTHOR]

Background: Contagious agalactia causes significant economic losses. The aim of this study is to investigate the presence of contagious agalactia disease in cities of Isparta and Afyonkarahisar, Turkey. Methods: The study includes 45.500 animals in 220 ovine enterprises and samples were taken from those suspected of contagious agalactia disease. 202 animals in the 21 ovine enterprises comprised of 139 goats, 56 sheep, 3 kid goats, 2 goats, 2 lambs in total suspected of the disease were sampled. A total of 289 samples were collected, including 91 milk samples, 28 nasal swabs, 101 eye swabs, 8 joint fluids and 61 ear swabs. The isolates obtained after incubation were identified with polymerase chain reaction by using specific primers to assess film and spot formation, glucose fermentation, growth inhibition tests. Result: Three Mycoplasma spp. isolates obtained from 28 nasal swabs turned out to be negative for M. agalactiae after PCR analysis. Colony morphology, biochemical tests and growth inhibition tests revealed that one agent was M. arginine and the two factors were identified as M. ovipneumoniae with centerless colony morphology. The obtained results were confirmed with the polymerase chain reaction. None of the four factors causing contagious agalactia were isolated and identified. [ABSTRACT FROM AUTHOR]

Copyright of Archives of Razi Institute is the property of Institut Razi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A survey to determine the prevalence of Mycobacterium bovis in wild mammals in Portugal was conducted by testing samples from hunted animals and those found dead between 2009 and 2013. In this study, we investigated 2116 wild mammals. Post-mortem examinations were performed, and tissues were collected from wild mammals representing 8 families and 11 different species, with a total of 393 animals analysed. Cultures were performed, and acid-fast isolates were identified by PCR. Tissues were also screened for Mycobacterium bovis by directly extracting DNA and testing for the Mycobacterium bovis-specific sequences. Mycobacterium bovis prevalence was 26.9% (95% CI: 22.8-31.5%). Mycobacterium bovis was recorded in 106 of the 393 studied species: prevalence by species were 26.9% (95% CI: 16.8-40.2%) in red foxes, 20.0% (95% CI: 7.0-45.2%) in Egyptian mongooses, 21.4% (95% CI: 16.2-27.7%) in wild boar and 38.3% (95% CI: 29.9-47.4%) in red deer. Mycobacterium bovis infection was detected in six of eight taxonomic families. For some species, the small sample sizes obtained were a reflection of their restricted range and low abundance, making estimates of infection prevalence very difficult (1 beech marten of 4; 1 Eurasian otter of 3; 2 common genet of 3). Infection was not detected in European badgers, hedgehog, wild rabbits and hare. The results of this study confirm the presence of Mycobacterium bovis infection in wild carnivores in Portugal. [ABSTRACT FROM AUTHOR]

Mycobacterium microti has recently been described as the causative agent of tuberculosis-like lesions in wild boar ( Sus scrofa), a reservoir specie of Mycobacterium tuberculosis complex ( MTBC) in some European Mediterranean ecosystem. Through a five-year survey on tuberculosis in free-living wild boars, the epidemiological trend of M. microti infections and the host and population risk factors linked with its occurrence were described. Retropharyngeal and mandibular lymph nodes of 3041 hunted wild boars from six different districts were macroscopically inspected. The sex and age of each animal were registered, as well as the animal abundance in each district. Lesions compatible with tuberculosis (190) were collected and analysed using a gyrB PCR- RFLP assay. M. microti was identified directly in 99 tissue samples (Prev = 3.26%; 95% CI: 2.67-3.97%), while neither Mycobacterium bovis, nor other members of the MTBC were detected. The probability of being M. microti positive showed spatio-temporal variability, with 26% of increase of risk of being infected for each year. Moreover, a positive effect of wild boar abundance and age on the prevalence was detected. The generalized increase in the European wild boar population, coupled with its sensitivity to M. microti infection, poses a future concern for the identification and management of MTBC members in wild boar. [ABSTRACT FROM AUTHOR]

The article presents a study which discusses the direct molecular typing of Brucella organisms through variable number tandem repeat (VNTR) analysis. The study analyzes bovine lymph node extractions using an in-house real-time PCR assay and non-vaccine B. melitensis was identified in the extractions. It also says that analysis of the reproducibility of the VNTR patterns generated by DNA extractions from milk samples suggests VNTR is not always applicable to all samples.

Contagious agalactia (CA) is one of the major animal health problems in small ruminants. It has economic effect and is caused by Mycoplasma agalactiae, the 'classic' etiological agent in sheep and goats. The significance of the different Mycoplasma spp causing CA varies depending on the geographic area. This study conducted between 2012 and 2013 on 189 small ruminants with CA signs in the west of Iran, an area where CA is endemic. All samples (milk, synovial fluid, ear swabs, conjunctival swabs and nasal swabs) were examined by PCR method. Mycoplasma spp was detected in 76.2% and M. agalactiae isolated from 16% of positive samples. M. agalactiae were isolated from 7 conjunctival swabs, 15 milk samples and one synovial fluid sample. Results showed that M. agalactiae was found to be the non-main cause of CA in small ruminants in Iran. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing E. coli (STEC) is an important foodborne pathogen causing diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome in humans. STEC is an implicated in the vast majority of outbreaks, widely via consumption of STEC contaminated beef, as important vehicle of transmission of this organism to human. The E. coli O157:H7 serotype is traditionally identified by serological identification of the somatic antigen (O157) and structural flagella (H7). In this study, the bacteria were identified as STEC serotype O157:H7 with three primer pairs that amplified fragments of secD, rfbE and fliC genes in PCR assays. These primer pairs specifically amplified different sizes of target genes: a 244bp region of the E. coli diagnostic marker gene (secD); a 317bp region of the O157 lipopolysacharide (LPS) gene (rfbE); and a 381bp region of the H7 flagellin gene (fliC). The singleplex, duplex and triplex PCR assay developed in this study have a sensitivity limit at 2.8 x 10³, 2.8 x 105 and 2.8 x 107 CFU/ml of E. coli O157:H7, respectively. Sensitivity to detect trace amount of E. coli O157:H7 DNA was reduced as the number of primer used was increased for competing to the same DNA template. [ABSTRACT FROM AUTHOR]

The wild boar is an important reservoir of bovine tuberculosis ( bTB) in south-western Spain. Some risk factors such as wild boar density or age have been associated with the presence of high prevalences of bTB in wild boar. However, the influence of other risk factors such as co-infections with other pathogens has not yet been studied. This work aims to assess the influence of porcine circovirus type 2 ( PCV-2) infection on bTB prevalence and bTB lesional patterns observed in wild boar. The presence of bTB-like lesions was evaluated in 551 hunted wild boar from 11 different game estates in south-western Spain, with a known history of bTB. Tuberculosis prevalences in each estate were calculated based on the percentage of animals found with bTB-like lesions. The percentage of animals with generalized bTB lesional patterns ( bTB lesions in more than one organ) was also assessed. The prevalence of PCV-2 was studied in each estate using a specific PCR assay. The relationship between PCV-2 and bTB prevalences and between PCV-2 infections and the presence of generalized lesional patterns in wild boar were analysed. A statistical relationship between the prevalences of bTB and PCV-2 was found, with bTB prevalences being higher in estates where prevalences of PCV-2 were high. On the other hand, animals infected with PCV-2 were more likely to develop a generalized lesional pattern. Porcine circovirus type 2 prevalences seem to be associated with prevalences of bTB in wild boar. PCV-2 infection may aggravate the development and severity of bTB, favouring the presence of generalized lesional patterns and raising the risk of contagion in these estates. The implementation of sanitary measures that focus on the control of PCV-2 infection may be necessary as a preliminary measure in bTB control programmes for wild boar. [ABSTRACT FROM AUTHOR]

The present study was conducted to investigate the presence of Escherichia coli O157: H7 in food samples of animal origin and to detect its virulence genes by immunomagnetic separation technique and multiplex PCR ( mPCR). A total of 500 samples (consisting of diced meat, minced meat, burger, raw cow's milk and raw cow's milk cheese) were analysed. Escherichia coli O157: H7 was detected in 5 (1%) of 500 analysed samples including two diced meat, one minced meat and two raw-milk cheese. None of the burger samples tested contained E. coli O157: H7. Three isolates obtained from minced and diced meat were found to carry stx 1, stx 2, hlyA and eaeA genes whereas two isolates from raw-milk cheese were found to harbour the stx 1, eaeA and hlyA genes. The results of this study suggest that raw meat and raw-milk cheese tested could pose public health problems in consumers with regard to their virulence factors. Significance and Impact of the Study Escherichia coli ( E. coli) O157:H7 is an important human pathogen. Escherichia coli 0157:H7 infections have been associated with consumption of uncooked meat and meat products, as well as unpasteurized dairy products. This study demonstrated that without specific tests for E. coli virulence factors raw meat and raw-milk cheese could pose public health problems to Turkish consumers. [ABSTRACT FROM AUTHOR]

Recent studies on outbreaks of Candida showed an increased incidence of bloodstream infections in neonatal intensive care units (NICUs) caused by C. parapsilosis species, highlighting the need for the proper identification and epidemiology of these species. Several systems are available for molecular epidemiological and taxonomic studies of fungal infections: pulsed-field gel electrophoresis (PFGE) represents the gold standard for typing, but is also one of the most lengthy and expensive, while simple sequence repeats (SSRs) is based on polymerase chain reaction (PCR) amplification and is, therefore, faster. Only recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify and type microorganisms involved in nosocomial outbreaks. In our study, 19 strains of C. parapsilosis isolated from the blood cultures of neonates admitted to the University Hospital Federico II were genotyped by the amplification of eight SSR markers and by MALDI-TOF MS. Electrophoretic and spectrometric profile results were compared in order to identify similarities among the isolates and to study microevolutionary changes in the C. parapsilosis population. The discriminatory power and the unweighted pair group method with arithmetic mean (UPGMA) dendrograms generated were compared in order to evaluate the correlation of the groups established by the analysis of the clusters by both methods. Both methods were rapid and effective in highlighting identical strains and studying microevolutionary changes in the population. Our study evidenced that mass spectroscopy is a useful technique not only for the identification but also for monitoring the spread of strains, which is critical to control nosocomial infections. [ABSTRACT FROM AUTHOR]

Copyright of Kafkas Universitesi Veteriner Fakultesi Dergisi is the property of University of Kafkas, Faculty of Veterinary Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Shiga toxin-producing E. coli carrying the stx1 and/or stx2 genes can cause multi-symptomatic illness in humans. A variety of terrestrial and aquatic environmental reservoirs of stx have been described. Culture based detection of microbes in deer species have found a low percentage of samples that have tested positive for Stx-producing microbes, suggesting that while deer may contain these microbes, their overall abundance in deer is low. In this study, quantitative PCR (qPCR) was utilized to test for the presence of stx genes in white-tailed deer fecal matter in western Pennsylvania. In this culture independent screening, nearly half of the samples tested positive for the stx2 gene, with a bias towards samples that were concentrated with stx2. This study, while limited in scope, suggests that deer may be a greater reservoir for stx than was previously thought. [ABSTRACT FROM AUTHOR]

The article offers information on the use of IS6110_T, real time polymerase chain reaction (PCR) assay technique, for the testing of mycobacteria specimens submitted at the National Veterinary Services Laboratory in the U.S. It is suggested that the use PCR for the diagnosis of mycobacterium bovis, complements culture technique. The study establishes that IS6110_T technique is compatible with the diagnostic protocols of the U.S.

Forty three (n=43) genomic DNA of Escherichia coli (11 isolates from eggs and 32 isolates from imported beef meats) were characterized by shiga toxin 1 (stx1), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random ampliied polymorphic DNA-PCR (RAPD-PCR) analyses. In the shiga toxin 1 (stx1) gene detection with primer stx 1F (5'-TTCTTCGGTATCCTATTCCC-3') and stx 1R (5'- CTGTCACAGTAACAACCGT-3'), 9 E. coli of beef meats isolates were positive toward sxt1 gene. The results of the ERIC-PCR and RAPD-PCR were analyzed using GelCompar II software. ERIC-PCR with primer ERIC1 (5'-CACTTAGGGGTCCTCGAATGTA -3') and ERIC2 (5'-AAGTAAGTGACTGGGGTGAGCG-3') discriminated the E. coli into 6 clusters and 10 single isolates at 80% similarity. RAPD-PCR with primer Gen8 and Gen9, produced 10 clusters and 15 single isolates and 12 clusters and 14 single isolates of 80%, respectively. These results demonstrated that both ERIC-PCR and RAPD-PCR are useful and suitable tools for molecular typing of those isolates examined. [ABSTRACT FROM AUTHOR]

The presence of major virulence factors of enterohaemorrhagic Escherichia coli (EHEC; stx1, stx2, eae, Ehly) were determined among isolates from 158 diarrhoeic calves by multiplex polymerase chain reaction (PCR). Strains positive for virulence factors were subjected to serotype specific PCR assays for O157:H7 and O111 antigens. Additionally, serogroups were determined by three monovalent antisera for O26, O111 and O157 somatic antigens and enterohaemolysin production were also shown phenotypically. Thirteen (8.2%) calves carried strains positive for one or more of the virulence factors tested, and eleven (6.9%) calves harboured the shiga toxin-producing strains ( stx1 or stx2) . stx1 was detected in eight (5%) and stx2 in three (1.9%) calves. eae and Ehly were observed in the same frequency (6.3%) and were detected in parallel. Of the 13 virulence-positive strains, the predominant genotype was ( stx1/ eae/ Ehly) at 53.8%. None of the EHEC in this study belonged to O157:H7 or O111 serotypes, but four strains (30.7%) belonged to the O26 serogroup. The results show the possible role of stx1/ eae in calf diarrhoea and the particular importance of O26 EHEC. Calves can also act as a reservoir for EHEC and in the transmission of the disease to humans. [ABSTRACT FROM AUTHOR]

Background In the absence of a functional dermatophyte-specific polymerase chain reaction (PCR), current diagnosis of dermatophytoses, which constitute the commonest communicable diseases worldwide, relies on microscopy and culture. This combination of techniques is time-consuming and notoriously low in sensitivity. Objectives Recent dermatophyte gene sequence records were used to design a real-time PCR assay for detection and identification of dermatophytes in clinical specimens in less than 24 h. Patients and methods Two assays based on amplification of ribosomal internal transcribed spacer regions and on the use of probes specific to relevant species and species-complexes were designed, optimised and clinically evaluated. One assay was for detecting the Trichophyton mentagrophytes species complex plus T. tonsurans and T. violaceum. The second assayed for the T. rubrum species complex, Microsporum canis and M. audouinii. Results The analytical sensitivity of both assays was 0·1 pg DNA per reaction, corresponding to 2·5–3·3 genomes per sample. The protocol was clinically evaluated over 6 months by testing 92 skin, nail and hair specimens from 67 patients with suspected dermatophytosis. Real-time PCR detected and correctly identified the causal agent in specimens from which T. rubrum, T. interdigitale, M. audouinii or T. violaceum grew in culture, and also identified a dermatophyte species in an additional seven specimens that were negative in microscopy and culture. Conclusions This highly sensitive assay also proved to have high positive and negative predictive values (95·7% and 100%), facilitating the accurate, rapid diagnosis conducive to targeted rather than empirical therapy for dermatophytoses. [ABSTRACT FROM AUTHOR]

A 750-bp internal fragment of the alkaline serine protease gene ( asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5′- and 3′- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD50 of 0.25 μg protein g−1 body weight. [ABSTRACT FROM AUTHOR]

Abstract: This study was carried out to evaluate the role of wild artiodactyls as reservoirs of Escherichia coli O157:H7 for livestock and humans. Retroanal mucosal swabs samples from 206 red deer (Cervus elaphus), 20 roe deer (Capreolus capreolus), 6 fallow deer (Dama dama) and 11 mouflon (Ovis musimon), collected during the hunting season (autumn–winter) in South-western Spain, were screened. Samples were pre-enriched in modified buffered peptone water, concentrated by an immunomagnetic separation technique and cultured onto selective cefixime tellurite sorbitol MacConkey agar. Polymerase chain reaction (PCR) was used to detect the presence of genes coding O157 and H7 antigens and the virulence factors verocytotoxin, intimin and enterohaemolysin. Three E. coli O157:H7 isolates were obtained from red deer (1.5%). Two of them showed inability to ferment sorbitol and lack of β-d-glucuronidase (GUD) activity, however, the other strain investigated was an atypical sorbitol-fermenting E. coli O157:H7 with GUD+ activity. This is the first report pointing to red deer as a reservoir of E. coli O157:H7 in Spain. [Copyright &y& Elsevier]

Simple Summary: This study targeted a small epidemiological area of a selected wildlife-livestock-human interface in Selangor to detect important veterinary and public health mycobacteria in free-ranging wild boar (Sus scrofa) and wild macaques (Macaca fascicularis) using a combination of diagnostic methods, tuberculosis-like lesion (TBLL) detection and nucleic acids detection by conventional and molecular analyses. Conventional PCR on wild boar tissues showed that 75% (9/12) of the lymph node samples were positive for Mycobacterium bovis (95% CI: 46.8–91.1). For macaques, 33.3% (10/30) were positive for Mycobacterium avium (95% CI: 19.2–51.2). Wild animals are considered reservoirs, contributing to the transmission of emerging zoonotic diseases such as tuberculosis (TB). A cross-sectional study was conducted by opportunistic sampling from fresh carcasses of free-ranging wild boar (n = 30), and free-ranging wild macaques (n = 42). Stained smears from these tissues were tested for acid-fast bacilli (AFB) with Ziehl–Neelsen staining. Mycobacterial culture was conducted using Lowenstein–Jensen media and Middlebrook 7H11 agar media. Polymerase chain reaction (PCR) was performed through the detection of the 16S rRNA gene, with multiple sets of primers for the detection of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC). In wild boars, 30% (9/30; 95% Confidence Interval: 16.7–47.9%) of examined samples showed gross tuberculosis-like lesions (TBLLs). Multiple nodular lesions that were necrotic/miliary with cavitation were found in the submandibular lymph nodes, tonsils, lungs, kidney and liver, while single nodular lesions were found in the mediastinal lymph nodes, spleen and mesenteric lymph nodes. Conventional PCR on the submandibular lymphoid tissues of wild boar (nine samples with TBLLs and three non-TBLL samples) showed that 75% (9/12) were positive for Mycobacterium bovis (95% CI: 46.8–91.1), and 91% (CI: 64.6–98.5) were positive for Mycobacterium avium. For macaques, 33.3% (10/30) were positive for M. avium (95% CI: 19.2–51.2) but negative for MTBC. [ABSTRACT FROM AUTHOR]