rDNA Intergenic Spacer Region Research Articles

Occurrence of Trichinella spiralis in farmed wild boars (Sus scrofa): an underrated risk in China.

Natural infection by Trichinella sp. has been reported in humans and more than 150 species of animals, especially carnivorous and omnivorous mammals. Although the presence of Trichinella sp. infection in wild boars (Sus scrofa) has been documented worldwide, limited information is known about Trichinella circulation in farmed wild boars in China. This study intends to investigate the prevalence of Trichinella sp. in farmed wild boars in China. Seven hundred and sixty-one (761) muscle samples from farmed wild boars were collected in Jilin Province of China from 2017 to 2020. The diaphragm muscles were examined by artificial digestion method. The overall prevalence of Trichinella in farmed wild boars was 0.53% [95% confidence interval (CI): 0.51–0.55]. The average parasite loading was 0.076 ± 0.025 larvae per gram (lpg), and the highest burden was 0.21 lpg in a wild boar from Fusong city. Trichinella spiralis was the only species identified by multiplex polymerase chain reaction. The 5S rDNA inter-genic spacer region of Trichinella was amplified and sequenced. The results showed that the obtained sequence (GenBank accession number: OQ725583) shared 100% identity with the T. spiralis HLJ isolate (GenBank accession number: MH289505). Since the consumption of farmed wild boars is expected to increase in the future, these findings highlight the significance of developing exclusive guidelines for the processing of slaughtered farmed wild boar meat in China.

Molecular Marker-Based Identification and Genetic Diversity Evaluation of Fusarium spp. - А Review

The Fusarium genus encompasses filamentous fungi of global significance in agriculture, ecology, and the food industry. Certain members of this genus act as plant pathogens, leading to substantial fungal infections in crucial crops, especially cereals. Many Fusarium species produce mycotoxins that are species or even strain-specific, contaminating the food chain and posing risks to both animal and human health. Identifying Fusarium species remains a formidable challenge due to the dynamic taxonomy within the ge¬nus. Traditional identification relies on biological, phylogenetic, and morphological criteria. However, mod¬ern genomic analysis techniques have revolutionized species identification, including that of Fusarium spp. Established molecular markers like the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA-ITS1), Translation elongation factor 1-α (TEF1-α), Intergenic spacer region of rDNA (ISG), and Beta-tubulin (β-tub) facilitate precise genetic identification. The “DNA barcoding” method, recognized in scientific literature, offers a meticulous approach to molecular identification and taxonomic classification of Fusarium species. Integrating traditional identification methods with contemporary DNA analysis tech-niques broadens the possibilities for discerning and establishing phylogenetic relationships among closely related mycotoxigenic species. This integrated approach greatly aids in the development of effective control and management strategies.

Plant Growth-Promoting (PGP) Traits of Endophytic Bacteria from In Vitro Cultivated Tectona grandis L.f

Tectona grandis L.f. (teak) is a tropical tree cultivated mainly due to its resistance, valuable wood and tolerance to biotic and abiotic factors. An abundant bacteria community exists in teak tissues and knowledge of the functional roles of teak endophytic bacteria, from in vitro tissue culture, is essential for improving micropropagation techniques. In this study, we isolated endophytic bacteria with plant growth-promoting (PGP) traits from two teak clones (Proteca® A3 and E4) in calli, leaves, and stems segments in a culture medium. We analyzed colony pigmentation, gram reaction, and evaluated PGP traits (phosphorous solubilization, nitrogen fixation and indol-acetic acid production) of 54 colonies from clone A3 and 50 colonies from clone E4. A total of 35 colonies of clone A3 and 42 colonies of clone E4 were capable of fixing nitrogen. Four isolated bacteria from clone A3 were capable of solubilizing phosphorous (P-Ca3(PO4)2), while no strain of E4 clone showed that capacity. Furthermore, 49 endophytic bacteria from clone A3 showed capacity to synthesize indol-acetic acid, while only 4 bacteria from the E4 clone presented that characteristic. We also identified six teak endophytic bacteria, by analysis of the 16S-23S rDNA intergenic spacer region, showing high identity with the genera Curtobacterium sp., Ochrobactrum sp., and Bacillus spp. Therefore, we demonstrate here that the abundant bacterial community existing in teak tissues, including those of in vitro cultivated plants, have PGP traits that can be further harnessed for preparation of bioformulations, for example. Our findings open the possibility for studying these isolated endophytic bacteria more closely in order to understand their association with teak growth.

Phenotypic and genotypic approach to characterize a Trueperella pecoris strain isolated from necrotic vestibulitis of a camel (Camelus dromedarius).

The present study was designed to characterize phenotypically and genotypically a Trueperella (T.) pecoris strain isolated from necrotic vestibulitis of a 10-year-old camel (Camelus dromedarius). The species identity of T. pecoris 203/7 investigated in the present study could be confirmed by phenotypic properties and by phylogenetic analyses based on partial sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, elongation factor Tu encoding gene tuf and the target gene rpoB encoding the β-subunit of bacterial RNA polymerase. T. pecoris strain 203/7 was grouped within the genus Trueperella in the family Arcanobacteriaceae. The 16S rRNA gene analysis showed a sequence identity of 99·9% to reference strain T. pecoris DSM 111392T . The present isolate was clearly identified as T. pecoris, the most recently described species of the genus Trueperella. Strain T. pecoris 203/7 was isolated in moderate numbers from necrotic vestibulitis of the camel and could be of some importance for the infectious process. However, the investigated strain represents the first isolation of T. pecoris from a camel.

Prevalence, Enterotoxigenic Potential and Antimicrobial Resistance of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Algerian Ready to Eat Foods.

Staphylococcus aureus causes a foodborne intoxication due to the production of enterotoxins and shows antimicrobial resistance, as in the case of methicillin-resistant strains (MRSA). Herein, we analyzed 207 ready-to-eat foods collected in Algeria, reporting a S. aureus prevalence of 23.2% (48/207) and respective loads of coagulase positive staphylococci (CPS) ranging from 1.00 ± 0.5 to 5.11 ± 0.24 Log CFU/g. The 48 S. aureus isolates were widely characterized by staphylococcal enterotoxin gene (SEg)-typing and 16S-23S rDNA intergenic spacer region (ISR)-PCR, as well as by detecting tst and mecA genes, genetic determinants of toxic shock syndrome toxin-1 and methicillin resistance, respectively. We found that the S. aureus isolates belonged to seven different SEg-types harboring the following combinations of genes: (1) selW, selX; (2) egc (seG, seI, seM, seN, seO), selW, selX; (3) seA, seH, seK, seQ, selW, selX; (4) seB, selW, selX; (5) seD, selJ, seR, selW, selX; (6) seH, selW, selX, selY; and (7) seA, egc, selW, selX, while among these, 2.1% and 4.2% were tst- and mecA- (staphylococcal chromosomal cassette mec-type IV) positive, respectively. Selected strains belonging to the 12 detected ISR-types were resistant towards antimicrobials including benzylpenicillin, ofloxacin, erythromycin, lincomycin, tetracyclin, kanamycin, oxacillin, and cefoxitin; 8.3% (1/12) were confirmed as MRSA and 16.7% (2/12) were multidrug resistant. The present study shows the heterogeneity of the S. aureus population in Algerian ready-to-eat foods as for their toxigenic potential and antimicrobial resistance, shedding the light on the quality and safety related to the consume of ready-to-eat foods in Algeria.

A potential probiotic Leuconostoc mesenteroides TBE-8 for honey bee

An isolated bacterium TBE-8, was identified as Leuconostoc mesenteroides according to the sequences of 16S rDNA and the 16S–23S rDNA intergenic spacer region. The probiotic properties of the L. mesenteroides TBE-8 strain were characterized and revealed that TBE-8 could utilize various carbohydrates, exhibited high tolerance to sucrose’s osmotic pressure and acidic conditions, and could mitigate the impact of the bee pathogen Paenibacillus larvae. In addition, we found that the TBE-8 broth increased the expression of the nutrition-related genes major royal jelly protein 1 and vitellogenin in bees by approximately 1400- and 20-fold, respectively. The expression of genes encoding two antibacterial peptides, hymenoptaecin and apidaecin, in the bee abdomen was significantly increased by 17- and 7-fold in bees fed with the TBE-8 fermented broth. Furthermore, we fed four-frame bee colonies with 50% sucrose syrup containing TBE-8 and can detect the presence of approximately 2 × 106 16S rDNA copies of TBE-8 in the guts of all bees in 24 h, and the retention of TBE-8 in the bee gut for at least 5 days. These findings indicate that the L. mesenteroides TBE-8 has high potential as a bee probiotic and could enhance the health of bee colonies.

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

BackgroundThe 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 presentationThe 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.ConclusionsBoth 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.

Human Outbreak of Trichinellosis Caused by Trichinella papuae Nematodes, Central Kampong Thom Province, Cambodia.

In September 2017, a severe trichinellosis outbreak occurred in Cambodia after persons consumed raw wild pig meat; 33 persons were infected and 8 died. We collected and analyzed the medical records for 25 patients. Clinical signs and symptoms included myalgia, facial or peripheral edema, asthenia, and fever. We observed increased levels of creatine phosphokinase and aspartate aminotransferase­, as well as eosinophilia. Histopathologic examination of muscle biopsy specimens showed nonencapsulated Trichinella larvae. A Trichinella excretory/secretory antigen ELISA identified Trichinella IgM and IgG. Biopsy samples were digested and larvae were isolated and counted. PCR for the 5S rDNA intergenic spacer region and a multiplex PCR, followed by sequencing identified the parasite as Trichinella papuae. This species was identified in Papua New Guinea during 1999 and in several outbreaks in humans in Thailand. Thus, we identified T. papuae nematodes in humans in Cambodia.

Potential probiotic of Lactobacillus strains isolated from the intestinal tracts of pigs and feces of dogs with antibacterial activity against multidrug-resistant pathogenic bacteria.

The occurrence of multidrug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRAB), extended-spectrum β-lactamase (ESBL) Escherichia coli, and Pseudomonas aeruginosa, has become a serious problem in animals and public. The objective of this study was to identify and isolate lactic acid bacterial (LAB) strains from the intestinal tracts of pigs and feces of dogs and then characterize them as potential probiotics with antimicrobial activity against multidrug-resistant pathogenic bacteria. In a preliminary isolation screening, 45 of 1167 isolated LAB strains were found to have anti-S. aureus ATCC 27,735 activity. Using 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR) sequences, five of these isolates were further identified as Lactobacillus animalis 30a-2, Lactobacillus reuteri 4-12E, Weissella cibaria C34, Lactococcus lactis 5-12H, and Lactococcus lactis 6-3H. Antimicrobial substance assays suggest that the L. lactis 5-12H, L. lactis 6-3H, L. animalis 30a-2, L. reuteri 4-12E, and W. cibaria C34 strains might produce bacteriocins and hydrogen peroxide (H2O2) as antimicrobial substances. The L. animalis 30a-2 and W. cibaria C34 strains were further characterized for probiotic properties and shown to have high acid and bile salt tolerance. Additionally, they have broad antimicrobial spectra, and can significantly repress the growth of all of the tested strains of MRSA isolates, some MDRAB, ESBL E. coli, and P. aeruginosa isolates, along with food-borne pathogenic bacteria such as Bacillus cereus ATCC 11778, Listeria monocytogens ATCC 19111, Salmonella spp., Shigella spp., and Yersinia enterocolitica BCRC 12986. This is the first report of H2O2-producing L. animalis 30a-2 and W. cibaria C34 isolated from the intestinal tracts of pigs and feces of dogs that have good antimicrobial activity against multidrug-resistant and food-borne pathogenic bacteria and have excellent probiotic properties.

The Population of Fusarium oxysporum f. sp. lactucae in California and Arizona.

Fusarium wilt of lettuce, caused by Fusarium oxysporum f. sp. lactucae, is now found in all major lettuce producing regions in California and Arizona. The population structure of F. oxysporum f. sp. lactucae in California and Arizona was characterized based on somatic compatibility and sequences of the translation elongation factor 1-α gene (EF-1α) and rDNA intergenic spacer region (IGS). In this study, 170 isolates were tested for somatic compatibility based on heterokaryon formation, using complementary nitrate nonutilizing (nit) mutants. Five subgroups (A to E) of somatic compatibility group 0300 were identified. Isolates associated with the same subgroup had a strong complementation reaction, whereas reactions between isolates of different subgroups were weak or delayed. An isolate from the first known infestation of Fusarium wilt of lettuce in California was associated with subgroup A, which predominated among isolates in our collection. Isolates representative of each subgroup were confirmed to be associated with race 1, based on the reaction of differential lettuce cultivars. It is possible that somatic compatibility subgroups B to E of F. oxysporum f. sp. lactucae were derived from subgroup A, as a consequence of somatic mutations affecting compatibility. If so, subgroups of F. oxysporum f. sp. lactucae may represent an intermediate step in divergence that will lead to clearly separable compatibility groups. Sequences of EF-1α and IGS were both identical for 58 isolates of F. oxysporum f. sp. lactucae that represented all somatic compatibility subgroups and locations from which isolates were obtained, indicating that subgroups were derived from the same clonal lineage (VCG 0300).

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S-23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated. The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6. Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372P being the highest producer of SED in milk found to date, producing as much as ca. 47,300ng/mL and 49,200ng/mL of SED, after 24 and 48h of incubation in milk at 37°C, respectively. S. aureus 372P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers' health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.

Concordance of Giardia duodenalis assemblages determined by different PCR methodologies in three observational studies in Cuba

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index=1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates.

BIOLOGICAL SOLUBILIZATION OF SOME METALS BY A NEW ACIDITHIOBACILLUS SPECIES ISOLATED FROM A MODERATE SULFUR HOT SPRING

A chemolithotrophic bacterium was isolated from sulfur hot spring. According to phenotypic traits and 16-23S rDNA intergenic spacer region analysis, the isolate was identified and named as Acidithiobacillus sp. MR39, which was a gram-negative, rod- shape and non-motile bacterium. The strain was able to grow in a synthetic liquid medium supplemented with the mineral ore as the source of energy. The optimum conditions were found to be within initial pH range of 2.0-2.5, at 34±1˚C and with shaking at 120 rpm. The bacterium had a remarkable potential for mineralization of 88% iron, 75% copper, 59% zinc, 59% nickel and 40% cobalt upon their growth in the liquid media. After adapting the bacterial cells to copper ions in 100 mM for 5-day incubation, biorecovery of Cu increased about 10% comparing to unadapted cells that are able to dissolve approximately 15% of total cu of mineral concentrate. Considering the finding in this study, the strain MR39 offers a great prospect for in situ extraction of metals from various ores along with other indigenous bacteria that can grow under ambient conditions.

5S Ribosomal DNA of Distantly Related Quercus Species: Molecular Organization and Taxonomic Application

The genus Quercus (oak) is a widespread and economically important genus of tree plants. The intrageneric taxonomy of this group remains controversial due to widely distributed interspecific hybridization and convergent similarity of morphological traits. Further progress in this problem requires application of molecular methods. Taking into account that the comparison of 5S rDNA was successfully used in the molecular taxonomy of plants, we cloned and sequenced this genomic region for representatives of three taxo-nomically distant Quercus species: Q. acutissima (sect. Cerris) and Q. glauca (sect. Cyclobalanopsis) from East Asia and Q. texana (sect. Lobatae) from North America. We also identified 5S rDNA in the genome sequences available in the Genbank database for North American species Q. lobata (sect. Quercus) and for the related genus Castanea. It was found that the 5S rDNA repeated units demonstrate a high level of intragenomic sequence similarity in representatives of genus Quercus. The potential external elements of the 5S rDNA promoter differ from those in other families of dicotyledonous plants. The results of the comparative sequence analysis of the 5S rDNA intergenic spacer region support the existing taxonomy of the genus and indicate the isolated position of section Cyclobalanopsis, which can be considered as a separate subgenus.

Intraspecific genetic variation in Trichinella spiralis and Trichinella britovi populations circulating in different geographical regions of Poland.

Trichinella spiralis and Trichinella britovi are species of nematodes which are responsible for the majority of Trichinella infections in the world and the most prevalent in Poland. The most abundant species – T. spiralis, is considered to be more genetically homogeneous in Europe than T. britovi. The aim of the present study was to determine the genetic variability in T. spiralis and T. britovi populations based on nuclear 5S rDNA intergenic spacer region (5S rDNA) and cytochrome c oxidase 1 (COX1) gene sequences. For the study, 55 isolates of T. spiralis and 50 isolates of T. britovi isolated from wild boars, pigs, brown rat and a red fox were analyzed. Based on the analysis of both genes, the genetic variability within populations of T. spiralis and T. britovi differed. In T. spiralis, two single nucleotide polymorphisms (SNPs) were observed in the 612 bp 5S rDNA gene fragment, and one SNP was detected in the 700 bp COX1 gene fragment. In T. britovi, 17 single nucleotide variations (SNVs) were detected in the 5S rDNA gene fragment (among them 16 SNPs), while COX1 sequence analysis revealed the occurrence of 20 SNVs between the sequences tested (among them 19 SNPs).For the majority of T. spiralis isolates the investigated larvae presented uniform haplotypes. In contrast, most of the isolates of T. britovi consisted of larvae of different haplotypes. Geographical analysis showed that each region exhibited different haplotype composition and richness. Warmińsko-Mazurskie and Zachodniopomorskie regions were the richest in haplotypes (15 and 16 haplotypes, respectively). We used heatmaps showing a characteristic pattern for each region graphically. This may allow to differentiate regions based on the occurrence of particular haplotypes. Furthermore, a PCA analysis on the SNP level yielded biplots that show that certain haplotypes/genotypes are associated with (clusters of) regions.

Genospecies of Borrelia burgdorferi sensu lato detected in 16 mammal species and questing ticks from northern Europe

Lyme borreliosis is the most common vector-borne zoonosis in the northern hemisphere, and the pathogens causing Lyme borreliosis have distinct, incompletely described transmission cycles involving multiple host groups. The mammal community in Fennoscandia differs from continental Europe, and we have limited data on potential competent and incompetent hosts of the different genospecies of Borrelia burgdorferi sensu lato (sl) at the northern distribution ranges where Lyme borreliosis is emerging. We used qPCR to determine presence of B. burgdorferi sl in tissue samples (ear) from 16 mammalian species and questing ticks from Norway, and we sequenced the 5S–23 S rDNA intergenic spacer region to determine genospecies from 1449 qPCR-positive isolates obtaining 423 sequences. All infections coming from small rodents and shrews were linked to the genospecies B. afzelii, while B. burgdorferi sensu stricto (ss) was only found in red squirrels (Sciurus vulgaris). Red squirrels were also infected with B. afzelii and B. garinii. There was no evidence of B. burgdorferi sl infection in moose (Alces alces), red deer (Cervus elaphus) or roe deer (Capreolus capreolus), confirming the role of cervids as incompetent hosts. In infected questing ticks in the two western counties, B. afzelii (67% and 75%) dominated over B. garinii (27% and 21%) and with only a few recorded B. burgdorferi ss and B. valaisiana. B. burgdorferi ss were more common in adult ticks than in nymphs, consistent with a reservoir in squirrels. Our study identifies potential competent hosts for the different genospecies, which is key to understand transmission cycles at high latitudes of Europe.

Characterization and Differentiation of Chicken Mycoplasma Isolates Using 16S-23S Intergenic Spacer Region Sequencing

ABSTRACT The objective of this study was to identify the species and characterize the genetic relationships among mycoplasma isolates from commercial layer hen flocks using 16S-23S rDNA intergenic spacer region (IGSR) sequencing. Twenty-one isolates were obtained from samples collected from commercial layer flocks in four Brazilian states: Sao Paulo, Minas Gerais, Rio de Janeiro and Espirito Santo. The isolates were recovered from the Sao Paulo, Rio de Janeiro and Espirito Santo states. Eleven isolates were originated from tracheal swabs, five from shell gland swabs and five from ovary fragment collection. The 16S-23S rDNA IGSR of isolates were amplified by PCR, and the obtained products were subsequently sequenced. The consensus of each isolate was compared to the available sequences using Nucleotide BLAST® to determine the mycoplasma species. A phylogenetic analysis of the Mycoplasma gallisepticum (MG) sequences was performed. Pairwise analyses showed homologies of 99% to 100% with the previously characterized sequences listed in GenBank®. Four Mycoplasma gallinaceum were isolated from three flocks and seven M. pullorum isolates were obtained from a single flock. The other 10 isolates were all identified as MG and were obtained from four flocks. The 16S-23S rDNA IGSR sequencing was a good method to identify Mycoplasma species isolated from field samples, providing fast and reliable results at relatively low costs. The results were also satisfactory for the single-locus sequence typing of MG isolates.

Further characteristics of Arcanobacterium pinnipediorum DSM 28752T and Arcanobacterium wilhelmae DSM 102162T, two novel species of genus Arcanobacterium.

The newly described type strains Arcanobacterium pinnipediorum DSM 28752T and Arcanobacterium wilhelmae DSM 102162T, initially isolated from an anal swab of a harbor seal (Sammra et al. Int J Syst Evol Microbiol 65:4539-4543, 2015) and the genital tract of a rhinoceros (Sammra et al. Int J Syst Evol Microbiol 67:2093-2097, 2017), could be further characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Fourier transform infrared (FT-IR) spectroscopy and by sequencing the genomic targets 16S-23S rDNA intergenic spacer region (ISR) and the genes rpoB, gap, and tuf. The two strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of both species remained unclear. However, the detection of specific spectra by MALDI-TOF MS and by FT-IR spectroscopy and the presented genotypic approaches might help to identify A. pinnipediorum and A. wilhelmae in the future and might elucidate the role these two species play in infections of animals.

Near infrared hyperspectral images and pattern recognition techniques used to identify etiological agents of cotton anthracnose and ramulosis

Hyperspectral imaging near infrared (HSI-NIR) has the potential to be used as a non-destructive approach for the analysis of new microbiological matrices of agriculture interest. This article describes a new method for accurately and rapidly classifying the etiological agents Colletotrichum gossypii (CG) and C. gossypii var. cephalosporioides (CGC) grown in a culture medium, using scattering reflectance HSI-NIR and multivariate pattern recognition analysis. Five strains of CG and 46 strains of CGC were used. CG and CGC strains were grown on Czapek-agar medium at 25 °C under a 12-hour photoperiod for 15 days. Molecular identification was performed as a reference for the CG and CGC classes by polymerase chain reaction of the intergenic spacer region of rDNA. The scattering coefficient µs and the absorption coefficient µa were obtained, which resulted in a µs value for CG of 1.37 × 1019 and for CGC of 5.83 × 10–11. These results showed that the use of the standard normal variate was no longer essential and reduced the spectral range from 1000–2500 nm to 1000–1381 nm. The results evidenced two type II errors for the CG 457-2 and CGC 39 samples in the soft independent modelling model of the analogy model. There were no classification errors using the algorithm of the successive projections for variable selection in linear discriminant analysis (SPA-LDA). A parallel validation of the results obtained with SPA-LDA was performed using a box plot analysis with the 11 variables selected by SPA, in which there were no outliers for the HSI-NIR models. The new HSI-NIR and SPA-LDA procedures for the classification of CG and CGC etiological agents are noted for their greater analytical speed, accuracy, simplicity, lower cost and non-destructive nature.

Comparison of four polymerase chain reaction assays for the detection of Brucella spp. in clinical samples from dogs.

Aim:This study aimed to compare the sensitivity (S), specificity (Sp), and positive likelihood ratios (LR+) of four polymerase chain reaction (PCR) assays for the detection of Brucella spp. in dog’s clinical samples.Materials and Methods:A total of 595 samples of whole blood, urine, and genital fluids were evaluated between October 2014 and November 2016. To compare PCR assays, the gold standard was defined using a combination of different serological and microbiological test. Bacterial isolation from urine and blood cultures was carried out. Serological methods such as rapid slide agglutination test, indirect enzyme-linked immunosorbent assay, agar gel immunodiffusion test, and buffered plate antigen test were performed. Four genes were evaluated: (i) The gene coding for the BCSP31 protein, (ii) the ribosomal gene coding for the 16S-23S intergenic spacer region, (iii) the gene coding for porins omp2a/omp2b, and (iv) the gene coding for the insertion sequence IS711.Results:The results obtained were as follows: (1) For the primers that amplify the gene coding for the BCSP31 protein: S: 45.64% (confidence interval [CI] 39.81-51.46), Sp: 95.62% (CI 93.13-98.12), and LR+: 10.43 (CI 6.04-18); (2) for the primers that amplify the ribosomal gene of the 16S-23S rDNA intergenic spacer region: S: 69.80% (CI 64.42-75.18), Sp: 95.62 % (CI 93.13-98.12), and LR+: 11.52 (CI 7.31-18.13); (3) for the primers that amplify the omp2a and omp2b genes: S: 39.26% (CI 33.55-44.97), Sp: 97.31% (CI 95.30-99.32), and LR+ 14.58 (CI 7.25-29.29); and (4) for the primers that amplify the insertion sequence IS711: S: 22.82% (CI 17.89 - 27.75), Sp: 99.66% (CI 98.84-100), and LR+ 67.77 (CI 9.47-484.89).Conclusion:We concluded that the gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples.