Identification and Characterization of Arcanobacterium canis from Companion Animals in Germany and The United Kingdom

Arcanobacterium canis is a novel species of the Arcanobacterium most closely related to A. haemolyticum. This study aims to characterize two A. canis isolates recovered from companion animals, specifically the claw of a cat and a vaginal swab from a dog. This study used real-time PCR to characterize A. canis isolated from companion animals. Two isolates of A. canis were recovered from purulent material from the claw of an 11-year-old cat in Germany and a vaginal swab of a dog in the United Kingdom. The samples were characterized phenotypically and genotypically. Both isolates were analyzed using culture methods, biochemical analysis, MALDI-TOF MS, real-time PCR amplification and sequencing of the 16S rRNA gene, and rpoB, gap, and tuf genes. The findings showed that the isolates P5197-15 and M214-96-1 obtained from companion animals were successfully characterized and confirmed to species level by real-time PCR amplification and sequencing of the 16S rRNA gene, as well as the genes of rpoB, gap, and tuf. This study seeks to comprehensively understand the characteristics of A. canis isolates obtained from companion animals. Such knowledge is essential for accurate diagnosis, treatment, and control of infections caused by this pathogen in veterinary medicine. Additionally, it contributes to the broader understanding of the genetic diversity and characteristics of A. canis, which can have implications for public health and animal well-being.</p

Combined Loop-Mediated Isothermal Amplification Assays for Rapid Detection and One-Step Differentiation of Campylobacter jejuni and Campylobacter coli in Meat Products

A loop-mediated isothermal amplification (LAMP) assay system was established, allowing rplD gene-based simultaneous detection of Campylobacter jejuni and Campylobacter coli in enriched meat products. Additionally, one-step differentiation of target species on agar plates was enabled by cdtC gene- and gyrA gene-based duplex LAMP. Both the rplD and cdtC–gyrA LAMP assays amplified the target sequences in all 62 C. jejuni and 27 C. coli strains used for determining inclusivity and revealed 100% exclusivity toward 85 tested non-target species. Throughout the entire experiments, C. jejuni and C. coli strains were 100% distinguishable by melting curves of cdtC and gyrA LAMP products. After 24-h enrichment, the rplD LAMP assay reliably detected initial inoculation levels of 10–100 CFU/g in artificially contaminated minced meat. Investigation of naturally contaminated meat samples revealed a diagnostic accuracy of 95% toward real-time PCR and 94.1% toward the standard culture method applying the 24-h incubation period. Diagnostic sensitivity and specificity, and positive and negative predictive values were 89.8, 100, 100, and 91.2%, respectively, when measured against real-time PCR, and 89.6, 98.1, 97.7, and 91.2%, respectively, when measured against the standard culture method. After 48-h enrichment, the detection limit of the rplD LAMP assay improved to initial inoculation levels of 1–10 CFU/g in artificially contaminated minced meat. Applying the 48-h incubation period on naturally contaminated meat samples resulted in 100% concordant results between rplD LAMP, real-time PCR, and the standard culture method. The established LAMP assay system was proved to be suitable for rapid meat sample screening. Furthermore, it constitutes a promising tool for investigating other Campylobacter sources and could therefore make a valuable contribution to protect consumers from foodborne illness

Microbiological quality assessment of meat and dairy products from small-scale factories in european Side of Turkey

The present study was conducted to investigate the microbiological quality and safety of 67 meat and 112 dairy product samples manufactured by small-scale factories at the European side of Turkey. The results revealed that the bacteriological quality of the meat and dairy products tested was poor as Enterobacteriaceae, Escherichia coli, Staphylococcus aureus and mold counts were found in 76.1 %, 47.8 %, 25.4 % and 50.7 % meat product samples, respectively, and in 67.0 %, 50.0 %, 2.8 % and 54.5 % dairy product samples respectively. Concerning food safety, E. coli 0157 and Listeria monocytogenes were detected by real time PCR assay in 10.5 % and 10.5 % meat product samples, and 5.4 % and 6.3 % dairy product samples, respectively. These results indicated a generally poor microbiological quality of a broad variety of products and the existence of foodborne pathogens in these products highlighted serious health issues. Therefore, in order to increase microbiological safety and quality of products, manufactured in small-scale factories of Turkey, we recommend improving and questioning existing HACCP concepts and conducting a monitoring system as a control of success.WOS:0004263376000042-s2.0-8506482714

Evaluation of enterotoxin gene expression and enterotoxin production capacity of the probiotic strain Bacillus toyonensis BCT-7112T.

The aim of the present study was to evaluate the safety of the probiotic strain Bacillus toyonensis BCT-7112T (active ingredient of Toyocerin) in relation to the enterotoxins haemolysin BL (Hbl) and the non-haemolytic enterotoxin (Nhe) by performing a quantitative reverse transcription (RT) real-time polymerase chain reaction (PCR) and a Western blot assay. The expression levels of the enterotoxin genes hblA, hblD, nheA, nheB and nheC, determined by means of RT real-time PCR in B. toyonensis, were lower than those in B. cereus reference strains. No expression of hblC was detected. The Western blot assays of native and 25-fold concentrated supernatants from B. toyonensis, using monoclonal antibodies directed against the Hbl component L1 and the Nhe component NheB, showed weak bands. The NheC component was not detected in the native supernatant, but weakly in the 25-fold concentrated supernatant. According to the results of the present study, the enterotoxin expression and protein levels of B. toyonensis BCT-7112T were absent or clearly lower compared to the B. cereus reference strains. Thus, their ability to form functional enterotoxins can also be considered to be lower or unlikely compared to the B. cereus reference strains. This experimental approach can be implemented when studying the health and safety as well as harmlessness of probiotic microorganisms

Determination of Seasonal Distribution of Aflatoxin M-1 Level in Cheese Production

The aim of this study was to determine the Aflatoxin M-1 (AFM(1)) quantities in raw milk samples and cheese samples produced from the same lot. The samples were obtained from six dairy plants in four different seasons. AFM(1) amounts of the samples were assessed by a Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) device with fluorescence detector, using a preliminary immune affinity column (IAC) for post-column derivatization and these values were compared with legal limits. Average of AFM(1) values of raw milk samples were determined to be 41.9 ng/l, 31.3 ng/l, 68.5 ng/l and 92.0 ng/I in spring, summer, autumn, and winter, respectively. 54.2 % of the investigated milk samples exceeded legal limits. AFM(1) concentrations were higher during the autumn and winter. Average values of AFM(1) in cheese samples were determined to be 92.7 ng/kg, 72.3 ng/kg, 190.6 ng/kg and 255.8 ng/kg in spring, summer, autumn, and winter, respectively. AFM(1) was at levels of 1.25 to 5.18-fold higher than those presented in the raw milk used for the cheese production. Based on the admissible limit, 16.7 % of the cheese samples were shown to have exceeded this valid maximum limit value. Consequently, it was found that seasonal change had a significant effect on the amount of AFM(1).Kirklareli UniversityKirklareli University [KLU-BAP-27]This work was supported by Kirklareli University Scientific Research Projects Coordinator (Project No: KLU-BAP-27)WOS:0004597485000032-s2.0-8506486681

Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat.

Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes

Anisakid nematode species identification in harbour porpoises (Phocoena phocoena) from the North Sea, Baltic Sea and North Atlantic using RFLP analysis.

Harbour porpoises (Phocoena phocoena) are the only native cetacean species in the German North and Baltic Seas and the final host of Anisakis (A.) simplex, which infects their first and second gastric compartments and may cause chronic ulcerative gastritis. Anisakis simplex belongs to the family Anisakidae (Ascaridoidea, Rhabditida) as well as the phocine gastric nematode species Pseudoterranova (P.) decipiens and Contracaecum (C.) osculatum. These nematode species are the main causative agents for the zoonosis anisakidosis. The taxonomy of these genus with life cycles including crustaceans and commercially important fish is complex because of the formation of sibling species. Little is known about anisakid species infecting porpoises in the study area. Mature nematodes and larval stages are often identifiable only by molecular methods due to high morphological and genetic similarity. The restriction fragment length polymorphism (RFLP) method is an alternative to sequencing and was applied to identify anisakid nematodes found in harbour porpoises from the North Sea, Baltic Sea and North Atlantic to species level for the first time. In the study areas, five gastric nematodes from different harbour porpoise hosts were selected to be investigated with restriction enzymes HinfI, RsaI and HaeIII, which were able to differentiate several anisakid nematode species by characteristic banding patterns. Anisakis simplex s. s. was the dominant species found in the North Sea and Baltic porpoises, identified by all three restriction enzymes. Additionally, a hybrid of A. simplex s. s. and A. pegreffii was determined by HinfI in the North Sea samples. Within the North Atlantic specimens, A. simplex s. s., P. decipiens s. s. and Hysterothylacium (H.) aduncum were identified by all enzymes. This demonstrates the value of the RFLP method and the chosen restriction enzymes for the species identification of a broad variety of anisakid nematodes affecting the health of marine mammals

Anisakid nematode species identification in harbour porpoises (Phocoena phocoena) from the North Sea, Baltic Sea and North Atlantic using RFLP analysis

Harbour porpoises (Phocoena phocoena) are the only native cetacean species in the German North and Baltic Seas and the final host of Anisakis (A.) simplex, which infects their first and second gastric compartments and may cause chronic ulcerative gastritis. Anisakis simplex belongs to the family Anisakidae (Ascaridoidea, Rhabditida) as well as the phocine gastric nematode species Pseudoterranova (P.) decipiens and Contracaecum (C.) osculatum. These nematode species are the main causative agents for the zoonosis anisakidosis. The taxonomy of these genus with life cycles including crustaceans and commercially important fish is complex because of the formation of sibling species. Little is known about anisakid species infecting porpoises in the study area. Mature nematodes and larval stages are often identifiable only by molecular methods due to high morphological and genetic similarity. The restriction fragment length polymorphism (RFLP) method is an alternative to sequencing and was applied to identify anisakid nematodes found in harbour porpoises from the North Sea, Baltic Sea and North Atlantic to species level for the first time. In the study areas, five gastric nematodes from different harbour porpoise hosts were selected to be investigated with restriction enzymes HinfI, RsaI and HaeIII, which were able to differentiate several anisakid nematode species by characteristic banding patterns. Anisakis simplex s. s. was the dominant species found in the North Sea and Baltic porpoises, identified by all three restriction enzymes. Additionally, a hybrid of A. simplex s. s. and A. pegreffii was determined by HinfI in the North Sea samples. Within the North Atlantic specimens, A. simplex s. s., P. decipiens s. s. and Hysterothylacium (H.) aduncum were identified by all enzymes. This demonstrates the value of the RFLP method and the chosen restriction enzymes for the species identification of a broad variety of anisakid nematodes affecting the health of marine mammals

Studies on Trueperella pyogenes isolated from an okapi (Okapia johnstoni) and a royal python (Python regius)

BACKGROUND: The present study was designed to characterize phenotypically and genotypically two Trueperella pyogenes strains isolated from an okapi (Okapia johnstoni) and a royal python (Python regius). CASE PRESENTATION: The species identity could be confirmed by phenotypic properties, by MALDI-TOF MS analysis and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. Furthermore, sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region (ISR), the target genes rpoB encoding the β-subunit of bacterial RNA polymerase, tuf encoding elongation factor tu and plo encoding the putative virulence factor pyolysin allowed the identification of both T. pyogenes isolates at species level. CONCLUSIONS: Both strains could be clearly identified as T. pyogenes. The T. pyogenes strain isolated in high number from the vaginal discharge of an okapi seems to be of importance for the infectious process; the T. pyogenes strain from the royal python could be isolated from an apparently non-infectious process. However, both strains represent the first isolation of T. pyogenes from these animal species