First Isolation and Molecular Typing of Pathogenic and Intermediate Leptospira Species from Urine of Symptomatic Dogs

Aim of this study was to evaluate, the presence and diversity of Leptospira spp. in blood and urine samples collected from 175 owned-dogs from Sardinia, Italy. After determination of leptospiral infection by microscopic agglutination test (MAT), urine from MAT-positive dogs were examined by real-time polymerase chain reaction (lipL32 rt-PCR) and then isolated by culture. In order to characterize obtained serovars, positive cultures were then subjected to 16S rRNA and secY sequencing, phylogenetic analysis and Multilocus Sequence Typing (MLST). Results showed that seven dogs (4%; 95% CI: 0–55) had Leptospira DNAs in their urine and five strains were isolated from urine cultures. The three different sequence types (ST17, ST198 and ST24) belonging to Leptospira interrogans genomospecies identified by MLST analyses in this study, confirmed that the leptospiral infection was widespread in Sardinian dogs. We also reported the first characterization of a new Leptospira spp. isolated from urine of one dog living in the study area. Whole genome sequencing and phylogenetic analysis, confirmed that this genospecies was closely related to Leptospira hovindhougenii, an intermediate Leptospira spp. with unknown pathogenicity previously isolated from a rat in Denmark. Further studies are required to clarify whether healthy dogs that shed leptospires in their urine could represent a zoonotic risk for humans in this region.

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Serological diagnosis and molecular characterization of Leptospira spp. in the blood and urine of bovine females from refrigerated slaughterhouses

Semina Ciências Agrárias ◽

10.5433/1679-0359.2018v39n3p1125 ◽

2018 ◽

Vol 39 (3) ◽

pp. 1125

Author(s):

Aliny Fernanda de Oliveira ◽

Roberta Torres Chiderolli ◽

Luciano Seraphim Gasques ◽

Arianne Peruzo Pires Gonçalves ◽

Érica Dourado Neves ◽

...

Keyword(s):

One Health ◽

Preventive Measures ◽

Agglutination Test ◽

Economic Losses ◽

Chain Reaction ◽

Serum Samples ◽

Leptospira Spp ◽

Blood And Urine Samples ◽

Polymerase Chain

Leptospirosis is an important socioeconomic disease in humans, as well as in domestic and wild animals, being caused by Leptospira spp. Bovine animals are considered reservoirs of this disease, because they intermittently disseminate the bacteria into the environment through their urine. In this way, the cattle an important source of Leptospira infection. The objective of this study was to detect Leptospira spp. antibodies and DNA in bovine females from two refrigerated slaughterhouses in the microregion of Umuarama, Paraná, Brazil. In particular, blood and urine samples from 52 crossbred bovine females older than 36 months from the two slaughterhouses were used. The microscopic agglutination test (MAT) was used to detect leptospiral antibodies, and the polymerase chain reaction (PCR) and subsequent sequencing were used to detect Leptospira DNA. The MAT yielded 22 (42.3%) serum samples considered reagent, while the nested PCR test resulted in one amplified sample (1.9%) of 289 bp. This single sample was then amplified again using primers for the SecY gene (549 bp). Sequencing of this gene characterized the bacteria as L. borgpetersenii that were similar to the serovar Hardjo of the genotype Hardjobovis. This is the first molecular confirmation of Hardjobovis-like L. borgpetersenii in the urine of crossbred bovine females older than 36 months from slaughterhouses in the microregion of Umuarama. This study’s results show that it is important to combine serological and molecular diagnosis in the detection of Leptospira spp. Therefore, both methods were used to improve our understanding of the epidemiology of this disease in bovine animals from the microregion of Umuarama. In addition, the analysis informed the subsequent adoption of preventive measures and educational One Health actions to prevent economic losses related to the herd, as well as social losses related to workers and the environment.

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Sex Determination During Inflorescence Bud Differentiation in Monoecious Pistacia chinensis Bunge

Forests ◽

10.3390/f10030202 ◽

2019 ◽

Vol 10 (3) ◽

pp. 202 ◽

Cited By ~ 1

Author(s):

Qian Bai ◽

Chenyi Zhu ◽

Xia Lei ◽

Tao Cao ◽

Shuchai Su ◽

...

Keyword(s):

Sex Determination ◽

Early Stage ◽

Sex Differentiation ◽

Banding Patterns ◽

Vegetative Period ◽

Pcr Products ◽

Polymerase Chain ◽

Sex Determination Mechanisms

Pistacia chinensis Bunge is widely acknowledged to be dioecious, but rare monoecious individuals have been found. However, the origin of monoecism and the sex differentiation of different sex types remain intriguing questions. Here, sex expressions were explored by identification of sex-associated DNA markers, determination of the sex stability after grafting, and histological characterization of inflorescence bud development using anatomical analysis. The results showed that (1) although polymorphisms among individuals existed, the banding patterns of Polymerase Chain Reaction (PCR) products for different sex types on the same monoecious tree were consistent; (2) the sex expressions of grafted trees were not consistent with those of scions, indicating that monoecism probably did not originate from a stable bud mutation; and (3) both males and females underwent a bisexual period, then the stamen primordia in female buds degenerated into the second round tepals, while the pistil primordia in male buds gradually disappeared. During the sex differentiation phase, female buds were spindle-shaped, while the male buds were full teardrop-shaped, and male buds were bigger than female buds. Taken together, no sex-associated DNA marker was found, sex expressions were unstable after grafting, and the alternative sex organs appeared in the early stage of sex differentiation, suggesting that sex determination occurred during floral development instead of the early vegetative period. These results indicated that the sex expressions may be affected by environmental factors, increasing the understanding of sex determination mechanisms in P. chinensis and other species.

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Molecular Detection of Leptospira Species Serotypes in Iranian Stray Dogs

International Journal of Medical Laboratory ◽

10.18502/ijml.v6i2.1032 ◽

2019 ◽

Author(s):

Saam Torkan ◽

Hassan Momtaz

Keyword(s):

Public Health ◽

Polymerase Chain Reaction ◽

Chain Reaction ◽

Serum Samples ◽

Blood Samples ◽

Stray Dogs ◽

Wide Range ◽

Leptospira Spp ◽

Polymerase Chain ◽

Leptospira Species

Background and Aims: Leptospirosis is a spirochetal disease with public health importance globally. This disease affects a wide range of domestic and wild animals. Dogs are one of the species most sensitive to Leptospira canicola and Leptospira icterohaemorrhagiae. The present study was concluded to evaluate the prevalence rate of Leptospira species and L. canicola and L. icterohaemorrhagiae serovars in Iranian stray dogs. Materials and Methods: One-hundred and twenty blood samples were first taken from stray dogs. Then the samples were transferred to the laboratory. Sera were extracted from blood samples and genomic DNA was extracted. DNA samples were subjected to conventional polymerase chain reaction. Positive samples for Leptospira spp. were analyzed for presence of L. canicola and L. icterohaemorrhagiaeserovars using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: Nine samples out of 120 serum samples (7.5%) were positive for the flagella gene of the Leptospira spp. Prevalence of Leptospira spp. in serum samples of male and female dogs were 5.4% and 10.86%, respectively. Prevalence of L. canicola and L. icterohaemorrhagiae serovars were 55.55% and 33.33%, respectively. We found that 11.11% of samples were positive for both serovars. Two to three and 3-4 year old dogs had the highest prevalence of Leptospira spp. Conclusions: The considerable prevalence of leptospirta spp. and also their zoonotic serovars among Iranian stray dogs represented an important public health issue regarding the contact of healthy human with these dogs. Identification of infected dogs and their vaccination can inhibit the distribution of Leptospira spp.

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Characterization of a transgenic mouse line over-expressing the human ornithine decarboxylase gene

Biochemical Journal ◽

10.1042/bj2780895 ◽

1991 ◽

Vol 278 (3) ◽

pp. 895-898 ◽

Cited By ~ 24

Author(s):

M Halmekytö ◽

L Alhonen ◽

J Wahlfors ◽

R Sinervirta ◽

T Eloranta ◽

...

Keyword(s):

Transgenic Mouse ◽

Ornithine Decarboxylase ◽

Transgenic Animals ◽

Metabolizing Enzymes ◽

Transgenic Mouse Line ◽

Adenosylmethionine Decarboxylase ◽

Transgenic Lines ◽

Polymerase Chain

We have produced several transgenic mouse lines over-expressing the human ornithine decarboxylase (ODC) gene. We have now characterized one of the transgenic lines as regards the tissue accumulation of the polyamines and the activities of their metabolizing enzymes. Among the tissues analysed, the polyamine pattern was most strikingly changed in testis and brain of the transgenic animals. ODC activity was greatly enhanced in all tissues, except kidney, of the transgenic animals. The most dramatic increase, 80-fold, was found in brain of the transgenic mice. The activities of S-adenosylmethionine decarboxylase and spermidine and spermine syntheses were likewise significantly increased in testis of the transgenic animals. The activities of the enzymes involved in the back-conversion of the polyamines, namely spermidine/spermine acetyltransferase and polyamine oxidase, were similar in the transgenic and non-transgenic animals. As analysed by reverse transcriptase/polymerase chain reaction, all the six tissues of the transgenic animals expressed human-specific ODC mRNA. Determination of the half-life of testicular ODC revealed a stabilization of the enzyme in the transgenic males.

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Molecular Typing of Pathogenic Leptospira Species Isolated from Wild Mammal Reservoirs in Sardinia

Animals ◽

10.3390/ani11041109 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1109

Author(s):

Ivana Piredda ◽

Maria Nicoletta Ponti ◽

Bruna Palmas ◽

Malgorzata Noworol ◽

Aureliana Pedditzi ◽

...

Keyword(s):

Phylogenetic Analyses ◽

Wild Mammal ◽

Pathogenic Leptospira ◽

Wild Mammals ◽

Strategic Plans ◽

Polymerase Chain ◽

And Control ◽

Sequence Types ◽

Leptospira Species ◽

Wild Hosts

Leptospirosis is a global zoonosis caused by pathogenic species of Leptospira that infect a large spectrum of domestic and wild animals. This study is the first molecular identification, characterization, and phylogeny of Leptospira strains with veterinary and zoonotic impact in Sardinian wild hosts. All samples collected were cultured and analyzed by multiplex real time polymerase chain reaction (qPCR). Sequencing, phylogenetic analyses (based on rrs and secY sequences), and Multilocus Sequence Typing (MLST) based on the analysis of seven concatenated loci were also performed. Results revealed the detection of Leptospira DNA and cultured isolates in 21% and 4% of the samples examined, respectively. Sequence analysis of Leptospira positive samples highlighted the presence of the interrogans and borgpetersenii genospecies that grouped in strongly supported monophyletic clades. MLST analyses identified six different Sequence Types (ST) that clustered in two monophyletic groups specific for Leptospirainterrogans, and L. borgpetersenii. This study provided about the prevalence of leptospires in wild mammals in Sardinia, and increased our knowledge of this pathogen on the island. Monitoring Leptospira strains circulating in Sardinia will help clinicians and veterinarians develop strategic plans for the prevention and control of leptospiral infections.

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Differences in Virulence and Genomic Features of Strains of ‘Candidatus Phytoplasma mali’, the Apple Proliferation Agent

Phytopathology ◽

10.1094/phyto-97-8-0964 ◽

2007 ◽

Vol 97 (8) ◽

pp. 964-970 ◽

Cited By ~ 46

Author(s):

Erich Seemüller ◽

Bernd Schneider

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Blot Hybridization ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Southern Blot Hybridization ◽

Apple Proliferation ◽

Candidatus Phytoplasma Mali ◽

Polymerase Chain

Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with ‘Candidatus Phytoplasma mali’ were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52°C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in ‘Ca. P. mali’. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.

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HPA-10wb (Laa): Genetic Determination of a New Platelet-Specific Alloantigen on Glycoprotein IIIa and Its Expression in COS-7 Cells

Blood ◽

10.1182/blood.v89.7.2422 ◽

1997 ◽

Vol 89 (7) ◽

pp. 2422-2428 ◽

Cited By ~ 51

Author(s):

O. Peyruchaud ◽

F. Bourre ◽

M.-C. Morel-Kopp ◽

D. Reviron ◽

P. Mercier ◽

...

Keyword(s):

Platelet Destruction ◽

Genetic Determination ◽

Fibrinogen Receptor ◽

Glycoprotein Iiia ◽

Polymerase Chain ◽

Heterodimeric Complex ◽

Alloimmune Thrombocytopenia ◽

Conformation Polymorphism

Abstract The heterodimeric complex glycoprotein (GP)IIb-IIIa, the fibrinogen receptor of platelets, carries numerous alloantigen systems. These polymorphisms are responsible for the immune response after transfusion or during pregnancy. In the latter case, the mother develops an antibody against an epitope present on fetal platelets, and this results in platelet destruction in the fetus. In this report, we describe the molecular characterization of a new alloantigen (Laa) on GPIIIa responsible for neonatal alloimmune thrombocytopenia (NAIT). Using polymerase chain reaction (PCR)–singlestrand conformation polymorphism (SSCP) and DNA sequencing, we found a point mutation (G to A) in a heterozygous state on the GPIIIa gene leading to amino acid substitution Arg to Gln at position 62 of the mature protein. Transient expression of GPIIb-IIIa complexes in Cos-7 cells using wild-type or mutated GPIIIa cDNA allowed us to demonstrate that this mutation was responsible for expression of the Laa epitope.

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Identification and Molecular Characterization of Eimeria tenella based on EtMic5 Gene in Pakistan

Pakistan Veterinary Journal ◽

10.29261/pakvetj/2020.063 ◽

2020 ◽

Vol 40 (04) ◽

pp. 443-448

Author(s):

Sayyed Raza Ali Shahid

Keyword(s):

Phylogenetic Analysis ◽

Molecular Characterization ◽

High Frequency ◽

Rna Extraction ◽

Evolutionary Relationship ◽

Eimeria Species ◽

Eimeria Tenella ◽

Pcr Products ◽

Polymerase Chain

The aim of this study was to investigate the molecular characterization and phylogenetic analysis of microneme gene 5 of Eimeria tenella (EtMic5) from Pakistan to confirm its evolutionary relationship among different Eimeria species. Birds were reared and infected with Eimeria tenella oocysts. Postmortem of birds revealed the presence of lesions within intestinal caeca. Oocysts were collected, sporulated and used for RNA extraction. RNA was converted to cDNA and analyzed for EtMic5 gene using polymerase chain reaction (PCR). PCR products were confirmed through gel electrophoresis and the samples positive for EtMic5 gene were cleared using PCR cleanup process. EtMic5 gene was partially sequenced from Macrogen® laboratory Korea. Phylogenetic analysis revealed that the sequence is similar to all those previously reported in other parts of the world. The nucleotide sequence was deposited in GenBank and the assigned accession number is MT684461. The outcomes of this investigation indicate the presence of high frequency of Eimeria tenella infection in Pakistan

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Molecular Characterization of Corynebacterium pseudotuberculosis Isolated over a 15-Year Period in Switzerland

Veterinary Sciences ◽

10.3390/vetsci8080151 ◽

2021 ◽

Vol 8 (8) ◽

pp. 151

Author(s):

Jessica Schlicher ◽

Sarah Schmitt ◽

Marc J. A. Stevens ◽

Roger Stephan ◽

Giovanni Ghielmetti

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analysis ◽

Chronic Disease ◽

Economic Impact ◽

Molecular Characterization ◽

Geographical Origin ◽

Toxin Gene ◽

Caseous Lymphadenitis ◽

Sequence Types

Corynebacteriumpseudotuberculosis biovar Ovis is the etiological agent of the contagious and chronic disease caseous lymphadenitis (CLA) in sheep and goats. The economic impact of CLA in Switzerland remains largely unknown, and the transmission modalities, as well as the genetic diversity of circulating strains, are poorly understood. This work presents further characterization data for 215 C. pseudotuberculosis isolates from sheep, goats and a dromedary originating from Switzerland and the Principality of Liechtenstein, collected over a 15-year period. The isolates were classified into the two biovars Ovis and Equi, analyzed for the presence of the diphtheria-like toxin gene and characterized using MLSA. All sheep and goat isolates were classified as C. pseudotuberculosis biovar Ovis. The isolate from a dromedary was classified as biovar Equi. No isolates harboring the diphtheria-like toxin gene were detected. Phylogenetic analysis of the concatenated sequences of four genes revealed the existence of 24 clusters. There was no correlation between MLSA sequence types, year of isolation and the geographical origin of the isolates. These findings confirm the presence of several MLSA sequence types in the study area and over a 15-year period. Moreover, no sheep- and goat-specific MLSA sequence types were found.

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Molecular Identification of Leptospira interrogans in Naturally Infected Cows from a Rural Property in the Border Region - Case Report

Semina Ciências Agrárias ◽

10.5433/1679-0359.2020v41n4p1433 ◽

2020 ◽

Vol 41 (4) ◽

pp. 1433

Author(s):

Giovani Batista Pastre ◽

Isabela Carvalho dos Santos ◽

Robson Michael Delai ◽

Edinalva Madalena de Almeida Mota ◽

Lidiane Nunes Barbosa ◽

...

Keyword(s):

Production Efficiency ◽

Dairy Farm ◽

Border Region ◽

Antibody Detection ◽

Chain Reaction ◽

Border Regions ◽

Leptospira Spp ◽

Blood And Urine Samples ◽

Polymerase Chain ◽

Serum Agglutination

The aim of this study was to investigate the antibodies and DNA of Leptospira spp. isolated from infected cattle in a small rural dairy farm in a border region between Brazil and Paraguay. Blood and urine samples were collected from 50 Holstein cows aged between 1 and 15 years. The diagnostic tests performed were microscopic serum agglutination for antibody detection and polymerase chain reaction for Leptospira spp. detection. Out of the samples analyzed, 48% were MAT positive with titers ranging from 100 to 400, and the most prevalent antibody was to the serovar Hardjo. One serum sample was amplified to 549 bp for the sec y gene, and sequencing identified it as L. interrogans. This is the first report from northwestern Paraná (PR) State of L. interrogans identification in naturally infected milk cattle. Thus, based on these results, to enhance production efficiency, new serological and molecular studies on dairy cattle from border regions are required to characterize the epidemiology of possible genotypes and their consequences in affected herds.

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