Unlocking a Mystery: Characterizing the First Appearance of Clavibacter nebraskensis in Mexican Cornfields.

The Goss's wilt and leaf blight is a disease of maize (Zea mays) caused by Clavibacter nebraskensis, which was widespread in the last several years throughout the Midwest in the United States, south in Texas, and north to Canada. The bacterium is included within the high-risk list of quarantine pathogens by many plant protection organizations and countries including Mexico. Severe blight symptoms on maize plants were found in different provinces from Coahuila and Tlaxcala, Mexico, in 2012 and 2021, respectively. Twenty bacterial isolates with morphology similar to C. nebraskensis were obtained from the diseased maize leaves. The isolates were confirmed by phenotypic tests and 16S rRNA and gyrB sequencing. Two strains were tested for pathogenicity tests on seven hybrid sweet corn cultivars available in Mexico, and the most sensitive cultivar was tested for all the strains to fulfill Koch's postulates. The phylogenetic reconstruction based on two single loci reveals a remarkable clustering of Mexican strains to American strains reported approximately 50 years ago. The presence of this pathogen represents a risk and a significant challenge for plant protection strategies in Mexico and maize diversity.

Comprehensive genetic diversity and molecular evolutionary analysis of Theileria annulata isolates based on TAMS 1 gene.

Molecular epidemiological studies related to the phylogenetic characterization of Theileria annulata are important in delineating the evolutionary history of the parasite. In the current study, the Theileria annulata (T. annulata) merozoite surface antigen 1 (TAMS 1) gene from 14 bovine isolates of T. annulata originating from semi-arid zone of northern India were amplified and sequenced. TAMS 1 gene sequences (n= 337) reported from 16 countries were subsequently analyzed for haplotype network along with genetic diversity. A total of five haplotypes out of the 14 sequenced isolates and 92 haplotypes out of 337 worldwide sequences are documented in this study. Phylogenetic and molecular evolutionary analyses based on TAMS 1 gene sequences showed that T. annulata is dissipated across different countries and numerous strains are closely linked, even though they belong to different geographical locations. The nucleotide homology between 14 isolates from northern India varied between 91.3 and 100%, whereas it was between 31.5 and 100% when sequences across the globe were compared. Haplotype 14 was recognized as most widely distributed haplotype, with 46 isolates circulating in 10 countries. Globally, negligible genetic distance (FST˂0.15) and very high gene flow (Nm˃1) was found in the five populations of the world (South Asia, East Asia, West Asia, Europe and Africa), supporting the absence of clearly defined subgroups in the phylogenetic analysis. Significant negative values of neutrality tests; Tajima's D (D) and Fu and Li's F (F) provided evidence for recent population expansion through positive selection of advantageous variations.

Molecular Prevalence and Phylogenetic Characterization of Enterocytozoon bieneusi in Sheep in the Van Region

OBJECTIVE: In this study, we aimed to determine the prevalence of Enterocytozoon bieneusi in healthy sheep in Van province using molecular techniques and to reveal genotypes of the detected isolates. METHODS: A total of 200 healthy appearance sheep comprise 38 male and 162 female, 32 preweaned, 38 postweaned lamb and 130 adult sheep from several farms in the Van region were included in the study between May and September 2021. Genomic DNA (gDNA) extractions were utilized on fecal samples collected from sheep by commercial kits, and E. bieneusi DNA was investigated by Nested polymerase chain reaction (PCR) amplifying ITS rRNA in the gDNA isolates. PCR products of the positive isolates were subjected to sequence analyze for genotyping and phylogenetic analyses of E. bieneusi. RESULTS: E. bieneusi DNA was determined in 16 out of 200 examined sheep fecal gDNA samples (8.0%) by Nested PCR. The highest E. bieneusi prevalence was determined in preweaned lambs with a rate of 18.8%. This was followed by postweaned lambs and adult sheep with a prevalence of 10.5% and 4.6%, respectively. The prevalence of the infection in males and females was 7.9% and 9.3%, respectively. All the ITS rRNA amplicons from 16 positive isolates were subjected to sequence analyses for genotyping and phylogenetic analyses. Sequence analyses revealed that all the isolates determined in sheep belonged to the BEB6 genotype and clustered in genogroup 2 of E. bieneusi with the BEB6 isolates from different hosts in several countries. CONCLUSION: Molecular epidemiological data on the prevalence of E. bieneusi in sheep in Turkey were obtained with this study and the common genotype was determined as BEB6 in the research area. The obtained data contribute to the molecular epidemiology and diversity of E. bieneusi in sheep.

Phylogenetic characterization and analysis of Sarcocystis buffalonis.

Despite its frequent presence in buffaloes, Sarcocystis buffalonis remains as one of the most under studied parasite. In the present study, isolates of S. buffalonis from,Mathura, Uttar Pradesh India were characterized for 18S rRNA (MF595842-MF595844), cox 1 (MG792800-MG792802), 28S rRNA (MH793418-MH793420) and ITS 1 (MH793421-MH793423) genes. Analysis revealed multiple haplotypes for each individual gene viz., 18S rRNA (three haplotypes), cox 1 (two haplotypes), 28S rRNA (two haplotypes) and ITS 1 (single haplotype). The studied Indian sequences showed variable homologies for individual gene loci viz., 18S rRNA (99.3-99.9%); cox 1 (99.8-100.0%); 28S rRNA (99.9-100.0%) and ITS 1 (100.0%) The phylogenetic association between S. buffalonis and closely related Sarcocystis spp. infecting buffaloes and cattle was delineated. All these gene loci placed S. buffalonis close to S. hirsuta. The study has generated A vital phylogenetic data about this erstwhile neglected parasite.

Impact of soil inorganic nitrogen on bacterial phylogeny in estuarine intertidal zones: a study of nitrogen metabolism.

Here we investigated the potential impacts of soil inorganic nitrogen (SIN) content on the phylogenetic characteristics and ecological functions of soil bacterial communities in estuarine intertidal zones in China, aiming to comprehend the response mechanism of soil microorganisms to variations in SIN content within estuarine wetlands. Our results show that SIN in estuarine areas has a significant spatiotemporal variation on spatial and seasonal scales, in this study and is significantly associated with the phylogenetic diversity and phylogenetic turnover of soil bacterial communities. In addition, the results of the metagenomic analysis showed that the relative abundance of nitrogen-cycling functional genes in bacterial communities did not differ significantly in sampling sites and seasons, and weakly correlated with SIN content. Further, the results based on structural equation modeling (SEM) analysis showed that SIN directly and significantly regulated the phylogenetic characteristics of bacterial communities, thereby indirectly affecting the potential of bacterial nitrogen metabolism. This study emphasizes the key influence of SIN variations on the phylogenetic dissimilarity in soil bacterial communities. Moreover, although there was a weak direct relationship between the functional characteristics of the bacterial nitrogen metabolism and SIN content, the spatiotemporal variation of bacterial nitrogen metabolic potential may be indirectly regulated by SIN content by influencing the phylogenetic diversity in bacterial communities. Our study unravels the pivotal mechanisms through which SIN content influences bacterial communities, thereby offering novel insights into the microbial intricacies governing nitrogen metabolism within estuaries.

An outbreak of a novel recombinant Coxsackievirus A4 in a kindergarten, Shandong province, China, 2021.

In 2021, twenty children exhibiting influenza-like illnesses were reported from a kindergarten in Shandong Province, China. Eleven genomes of Coxsackievirus A4 (CV-A4) were obtained from the pediatric cases, sharing <93% genome sequence identities with known CV-A4 strains. Further analyses suggested potential genetic recombination in the P3 region of the novel strains.

Isolation, Characterization, and Drug Sensitivity of Mycobacterium tuberculosis in Captive Sloth Bears (Melursus ursinus): Unnatural Habitat With Human Environment May Predispose Sloth Bears to Tuberculosis.

We describe the isolation, molecular characterization, and drug sensitivity of Mycobacterium tuberculosis recovered from lung tissues of four rescued captive sloth bears (Melursus ursinus) at Bannerghatta Biological Park (BBP), Bangalore, India. These bears had lived most of their life with humans in circus companies. They were rescued and housed in the Bear Rescue Center (BRC) of BBP. Upon rescue, they showed signs of unthriftiness, chronic debility, and failed to respond to symptomatic treatments. Over the period of the next 12-14 months, the four sloth bears died and the post-mortem examination revealed nodular lesions in the lungs that showed the presence of acid-fast bacilli. Polymerase chain reaction (PCR), culture, and nucleotide sequencing confirmed the bacilli as Mycobacterium tuberculosis. Histopathology of the lungs revealed characteristic granulomatous reaction with caseation. We determined the sensitivity of these isolates to rifampicin and isoniazid drugs by a WHO approved test, Line Probe Assay (LPA) using Genotype MTBDRplus VER 2.0. We discuss the role of unnatural habitat with the human environment in predisposing captive sloth bears for tuberculosis (TB). In the absence of any other reliable ante-mortem diagnostic test, this study recommends the use of LPA for early detection of TB in captive wild animals, which will help in taking necessary steps to prevent its further spread to animal caretakers and other susceptible animals in captivity.

Molecular, Phenotypical, and Host-Range Characterization of Robbsia andropogonis Strains Isolated from Bougainvillea spp. in Mexico.

Characteristic leaf spot and blight symptoms caused by Robbsia andropogonis on bougainvillea plants were found in three locations in different provinces of Mexico from 2019 to 2020. Eleven bacterial isolates with morphology similar to R. andropogonis were obtained from the diseased bougainvillea leaves. The isolates were confirmed as R. andropogonis by phenotypic tests and 16S rRNA, rpoD, and gyrB gene sequencing. In addition to bougainvillea, the strains were pathogenic to 10 agriculturally significant crops, including maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), coffee (Coffea arabiga), carnation (Dianthus caryophilus), Mexican lime (Citrus × aurantifolia), common bean (Phaseolus vulgaris), broadbeans (Vicia faba), and pea (Pisum sativum), but not runner bean (Phaseolus coccineus). The haplotypes network reveals the genetic variability among Mexican strains and its phylogeographic relationship with Japan, the U.S.A., and China. The presence of this pathogen represents a challenge for plant protection strategies in Mexico.

Genetic Dissection of Phosphorus Use Efficiency in a Maize Association Population under Two P Levels in the Field.

Phosphorus (P) deficiency is an important challenge the world faces while having to increase crop yields. It is therefore necessary to select maize (Zea may L.) genotypes with high phosphorus use efficiency (PUE). Here, we extensively analyzed the biomass, grain yield, and PUE-related traits of 359 maize inbred lines grown under both low-P and normal-P conditions. A significant decrease in grain yield per plant and biomass, an increase in PUE under low-P condition, as well as significant correlations between the two treatments were observed. In a genome-wide association study, 49, 53, and 48 candidate genes were identified for eleven traits under low-P, normal-P conditions, and in low-P tolerance index (phenotype under low-P divided by phenotype under normal-P condition) datasets, respectively. Several gene ontology pathways were enriched for the genes identified under low-P condition. In addition, seven key genes related to phosphate transporter or stress response were molecularly characterized. Further analyses uncovered the favorable haplotype for several core genes, which is less prevalent in modern lines but often enriched in a specific subpopulation. Collectively, our research provides progress in the genetic dissection and molecular characterization of PUE in maize.

Molecular Characterization of Human Papillomavirus Type 159 (HPV159).

Human papillomavirus type 159 (HPV159) was identified in an anal swab sample and preliminarily genetically characterized by our group in 2012. Here we present a detailed molecular in silico analysis that showed that the HPV159 viral genome is 7443 bp in length and divided into five early and two late genes, with conserved functional domains and motifs, and a non-coding long control region (LCR) with significant regulatory sequences that allow the virus to complete its life cycle and infect novel host cells. HPV159, clustering into the cutaneotropic Betapapillomavirus (Beta-PV) genus, is phylogenetically most similar to HPV9, forming an individual phylogenetic group in the viral species Beta-2. After testing a large representative collection of clinical samples with HPV159 type-specific RT-PCR, in addition to the anal canal from which the first HPV159 isolate was obtained, HPV159 was further detected in other muco-cutaneous (4/181, 2.2%), mucosal (22/764, 2.9%), and cutaneous (14/554, 2.5%) clinical samples, suggesting its extensive tissue tropism. However, because very low HPV159 viral loads were estimated in the majority of positive samples, it seemed that HPV159 mainly caused clinically insignificant infections of the skin and mucosa. Using newly developed, highly sensitive HPV159-specific nested PCRs, two additional HPV159 LCR viral variants were identified. Nevertheless, all HPV159 mutations were demonstrated outside important functional domains of the LCR, suggesting that the HPV159 viral variants were most probably not pathogenically different. This complete molecular characterization of HPV159 enhances our knowledge of the genome characteristics, tissue tropism, and phylogenetic diversity of Beta-PVs that infect humans.

Investigation of congenital tremor associated with Classical swine fever virus genotype 2.2 in an organized pig farm in north-eastern India.

The present study describes an outbreak of Classical swine fever (CSF) in an organized pig farm followed by an episode of CSF virus (CSFV) associated congenital tremors in piglets. The outbreak was recorded in a newly procured herd of Hampshire pigs housed adjacent to the existing pigs of the farm. The recorded CSF outbreak caused a mortality of 100% in the newly procured and 54.28% in the existing herd. As the disease subsides, the clinically recovered boars were served naturally with Tamworth gilts. Though, the sows farrowed on usual gestation period, litters born to each sow showed congenital tremors and eventually died within 24 h of birth. Necropsy analysis of affected piglets was indicative of CSFV infection and was further confirmed using RT-PCR signifying a transplacental infection. The CSFV strains from the initial outbreak and post outbreak episode of congenital tremors were successfully isolated in PK-15 cells and detected in indirect FAT and RT-PCR. Phylogenetic analysis based on E2 gene and 5'NTR of CSFV grouped the isolates within the genotype 2.2 and revealed close resemblance with previously reported Indian isolates of CSFV genotype 2.2 origin. To the best of our knowledge, this is the first report of CSFV induced congenital form reported from India under natural conditions.

Associative Genetic Diversity of RoTat 1.2 VSG in Different Trypanosoma evansi Isolates.

BACKGROUND: Numerous phylogenetic markers have been tested over a period of time for delineating evolutionary history of haemoflagellate-Trypanosoma evansi. PURPOSE: To find out the associative genetic diversity, within the various isolates of T. evansi across the globe, based on RoTat 1.2 VSG gene. METHODS: A total of 5 equine isolates of T. evansi from Northern India were characterized. PCR products were sequenced and sequences were compared with available sequences across India and world. Phylogenetic tree was constructed based on maximum parsimony (MP) method with the tree-bisection-regrafting (TBR) algorithm. RESULTS: Indian isolates formed multiple clades with two haplotypes. The present isolates showed 99.49-100.00% nucleotide homology within themselves. On broader line, Indian isolates were found to be closer to Egyptian isolates than the African counterparts. Few of the Indian isolates showed marked resemblance with a particular Egyptian isolate than with their Indian counter parts. Another remarkable finding is the close association of equine isolates from India with other equine isolates and their clear divergence from isolates of T. evansi affecting other hosts from India and abroad. CONCLUSION: Vast genetic divergence was seen between the isolates suggesting of multiple distinct lineages of T. evansi amongst the Indian livestock. Interestingly, variations in sequences were seen based on the host range of isolates. The findings are very important from molecular evolutionary point of view.

Prevalence and Bayesian Phylogenetics of Enteroviruses Derived From Environmental Surveillance Around Polio Vaccine Switch Period in Shandong Province, China.

We present the results of environmental surveillance for poliovirus (PV) and non-poliovirus (NPEV) around the switch from trivalent to bivalent oral polio-vaccine (OPV) which occurred in China in May 2016. Sewage samples were collected in Jinan and Linyi city from 2015 to 2017. Enterovirus (EV) isolation, VP1 amplification, Sanger sequencing, and phylogenetic analyses were performed. Among105 sewage samples (36 in Jinan and 69 in Linyi), 101 were positive for EV, with 74.3% (78/105) PV-positive samples and 90.5% (95/105) NPEV-positive samples. A total of 893 EV isolates were obtained, including 326 (36.5%) PVs and 567 (63.5%) NPEVs. Echovirus (E) -11 was the most common serotype out of 18 detected NPEV types (120/567), followed by E-3 (75/567) and E-6 (74/567). PV2 vanished and PV3 came to be the ascendant PV type in sewage after May 2016. Eight PV isolates were judged as pre-vaccine-derived poliovirus (pre-VDPV) and no VDPV or wild PV isolates were monitored. Bayesian phylogenetics demonstrated global E-11 originated in 1876 and evolved with the estimated rate of 4.63 × 10-3 nucleotide substitutions per site per year (s/s/y). Multiple circulating clusters that originated at different times were coexisting in Shandong province. The most recently common ancestor of global coxsackievirus B5 could date back to 1867, at the evolutionary rate of 3.95 × 10-3 s/s/y. In conclusion, our study described the changes of PVs and NPEVs around the polio vaccine switch period and provided meaningful global molecular epidemiological data for further studies of EV-related diseases among the population.

Sequencing and phylogenetic characterization of a novel Polerovirus from Nicotiana tabacum.

In this study, we reported the complete genome of a novel Polerovirus, named Tobacco yellow virus (TYV), which can be transmitted by Myzus persicae. TYV had a single-stranded RNA genome of 5735 nucleotides in length and contained six putative open reading frames (ORFs). Phylogenetic analysis with whole genome nucleotide sequences and amino acid sequences deduced from the conserved domain of the RNA-dependent RNA polymerase, clustered TYV with Potato leafroll virus from the genus Polerovirus with high bootstrap values. However, TYV clustered with Brassica yellow virus using amino acid sequences deduced from the conserved domain of the coat protein. Taken together with the identities between ORFs in TYV and related ORFs in species from Polerovirus, our results strongly suggested TYV is a novel species of the genus Polerovirus.

Sequencing and phylogenetic characterization of a novel RNA virus genome from Harmonia axyridis.

As a natural predator of many insect pests on its native Asian range, Harmonia axyridis remains amongst the insects whose pathogenic or beneficial microorganisms are yet to be studied. The genome nucleotide (nt) and amino acid sequences of open reading frames (ORFs) of the novel RNA virus were identified. Neighbor-joining (NJ) were constructed using MEGA7 software packages with nt sequences and conserved amino acid sequences of predicted RNA-dependent RNA polymerase (RdRp).The complete genome of a novel virus named Harmonia axyridis virus 1 was determined by RNA-seq and rapid amplification of cDNA ends from H. axyridis, which had a single-stranded RNA genome of 8868 nts in length and contains two putative ORFs. ORF1 encodes a polypeptide of 2182 amino acids, which contained conserved domains for 2 picornavirus-like capsid proteins and one RNA helicase. ORF2 encodes a polypeptide of 655 amino acids, which contained 1 RdRp domain. Phylogenetic analysis of whole genome nt sequences and RdRp deduced amino acid sequences suggested that the virus clustered with several unclassified Hubei picorna-like virus. To our knowledge, this is the first full annotated genome of a novel member of the unclassified group of RNA viruses, infecting H. axyridis in natural field conditions.

Field investigation and phylogenetic characterization of orf virus (ORFV) circulating in small ruminants and Pseudocowpoxvirus (PCPV) in dromedary camels of eastern Sudan.

In this study, livestock herders in eastern Sudan were interviewed through structured questionnaire involved 14046 animals in 151 herds (87 camel herds, 51 sheep and 13 goats) from June to September of 2016 in Showak area of Gadarif State to get some epidemiological information on contagious ecthyma (CE) infection. 102 suspected cases of CE were investigated (38 sheep, 22 goats and 42 camels) by a second questionnaire focusing on age and sex of affected animals beside number and localization of the lesions. Representative tissue samples of scab lesion scrapings were collected from a total of 36 suspected sheep, goats and camels for DNA extraction to identify PPV by quantitative real-time PCR and gel-based PCR, then a PCR protocol was used to obtain DNA fragment of B2L gene from six DNAs (2 from each animal species) for sequencing. Phylogenetic tree based on nucleotide sequences was constructed and all data were analyzed statistically. Obtained result has shown morbidity rate of 23.8% and a case fatality rate of 4.7 % in overall investigated animals resulting in a significant economic loss. Within individual herd, the morbidity rate varied from 5.6 to 42.8%, while the case fatality rate ranged between 0 and 33.3%. Camels accounted for the highest case fatality rate with 6.5% compared to sheep and goats which their rates were 2.8% and 1.3%, respectively. 93% of the affected animals were young less than one-year-old. The prevalence of CE was high in the rainy season compared to winter and summer. Out of 36 scab materials collected from sheep, goats, and camels, 24 gave positive specific amplification in real-time PCR and 21 in the gel-based PCR. DNA sequencing confirmed the PCR results. All sequences had a high G + C content of 62.6-63.9%. A BLAST search also revealed that the studied sheep PPV (SPPV) isolates shared 99.08% nucleotide sequence intragroup identity, 96.88-97.27% identity with the goat PPV (GPPV) isolates and together they belong to the Orf virus (ORFV) species, while the camel PPV (CPPV) isolates are close to the Pseudocowpoxvirus (PCPV) species of the PPV genus and share 92.51-93.62 % identity with the GPPV isolates. In conclusion the present study demonstrated that the gross lesion produced by PPV in sheep, goats and camels is generally similar, yet the PPVs circulating in eastern Sudan in camels (PCPV) are genetically distinct from those affecting sheep and goats (ORFV). Contagious ecthyma in eastern Sudan causes significant morbidities and mortalities and control measures, guided by the results of this investigation ought to be implemented.

Molecular Prevalence and Phylogenetic Characterization of Enterocytozoon bieneusi in Healthy Cattle

Objective: In this study, it was aimed to determine the molecular prevalence and genotypes of Enterocytozoon in healthy cattle. Methods: Fecal samples were collected from 50 cattle in Sivas between October 2017 and March 2018 and genomic DNA (gDNA) isolations were performed. gDNA isolates were processed by Nested PCR specifically amplifying ITS rRNA gene region to identify E. bieneusi. ITS rRNA region of E. bieneusi positive isolates were sequenced for genotyping and phylogenetic analyzes. Obtained sequences were assembled with appropriative genetic software, then phylogenetic relationships were revealed. Results: According to Nested PCR analyses, 29 (19.3%) out of totally examined samples were found positive for E. bieneusi. As a result of the sequence analyses, five distinct genotypes were determined. The most frequent genotype ERUSS1 and the other ERUSS2-4 genotypes were characterized as close to each other, which was reported for the first time in the world. Two isolates were determined in N genotype that was reported from cattle in Germany and were more different from the other genotypes. Phylogenetic analysis revealed that all the genotypes characterized in the study belonged to the genogroup 2. Conclusion: First molecular epidemiological data on E. bieneusi in cattle from Turkey were obtained with this study.

Relationships of Bradyrhizobium strains nodulating three Algerian Genista species.

The Mediterranean world is the cradle for the diversification of a large number of plant species, including legumes belonging to the Tribe Genisteae. Nodule bacteria from three species of Genista legumes indigenous to northwestern Africa (G. ferox, G. numidica, G. tricuspidata) were sampled across a 150km region of Algeria in order to investigate symbiotic relationships. Partial 23S rRNA sequences from 107 isolates indicated that Bradyrhizobium was the predominant symbiont genus (96% of isolates), with the remainder belonging to Rhizobium or Mesorhizobium. A multilocus sequence analysis on 46 Bradyrhizobium strains using seven housekeeping (HK) genes showed that strains were differentiated into multiple clades with affinities to seven species: B. canariense (17 isolates), B. japonicum (2), B. ottawaense (2), B. cytisi/B. rifense (9), 'B. valentinum' (5), and B. algeriense (11). Extensive discordance between the HK gene phylogeny and a tree for four loci in the symbiosis island (SI) region implied that horizontal transfer of SI loci has been common. Cases of close symbiont relationship across pairs of legumes hosts were evident, with 33% of isolates having as their closest relative a strain sampled from a different Genista species. Nevertheless, tree permutation tests also showed that there was substantial host-related phylogenetic clustering. Thus, each of the three Genista hosts utilized a measurably different array of bacterial lineages.

Development and field evaluation of a species-specific mt-COI targeted SYBR-Green Real Time PCR for detection and quantification of Haemonchus contortus in cattle in Turkey.

Haemonchus contortus is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. We developed a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the mitochondrial cytochrome oxidase subunit I (mt-COI) gene, and evaluated this technique in the detection of H. contortus infections in cattle in Central Anatolia Region of Turkey. The newly developed qPCR assay successfully discriminated H. contortus from other GIN species infecting cattle in the specificity evaluations, with a specific melt peak of 77.5 °C. Our results revealed the efficient amplification of the proposed target COI region within the range of plasmid copies, from 2 × 106 to 2 × 101 per µl, with 96.9 % efficiency, R² value of 0.999, and a slope of -3.398. Among the 920 cattle fecal samples from the field, 58 samples (6.3 %) were positive with qPCR assay, whereas 45 samples (4.9 %) were positive, as determined by larval culture, suggesting the utility of SYBR Green qPCR. Phylogenetic characterization of the partial COI gene of H. contortus isolates was also evaluated for 100 eggs and third stage larvae recovered from positive cattle faecal samples, which were verified with the qPCR assay prior to analyses. COI sequences were classified into three haplotypes (THC1 to THC3) with intraspecific nucleotide differences of 0.50 to 0.76 %. Phylogenetic analyses revealed that the haplotypes grouped with H. contortus isolates from several countries in a monophyletic cluster, with evidence of at least two main haplogroups. Overall, the SYBR Green qPCR assay was highly specific and sensitive, suggesting that it can be used for screening of H. contortus infections in livestock populations in epidemiological studies and the control of this important parasite.

Morphological, molecular and phylogenetic characterization of Borrelia theileri in Rhipicephalus microplus / Caracterização morfológica, molecular e filogenética de Borrelia theileri em Rhipicephalus microplus

Abstract This study aimed to perform a morphological, molecular and phylogenetic characterization of Borrelia theileri obtained from infected Rhipicephalus microplus in Brazil. Fifty engorged R. microplus females from cattle in the municipality of Seropédica, Rio de Janeiro, were analyzed for spirochetes by hemolymph smear. Macerated eggs and positive ticks, as well as blood from the bovine infested by these ticks, were analyzed the glpQ, flaB and hpt genes by PCR. The PCR products were purified and sequenced for analysis and construction of a phylogenetic tree. Only 2% (1/50) of the ticks generated a positive result by both smear and PCR. The spiral forms (n = 50) had (media ± SD) a mean length of 19.17 ± 4.12 µm, diameter of 0.2935 ± 0.0469 and number of turns 8.44 ± 2.59. Sequence alignments of the three evaluated genes exhibited 98% similarity to B. theileri isolates, occurring in a clade highly related to B. theileri strain KAT. Egg maceration samples were positive for the three evaluated genes, whereas bovine blood was negative by PCR. This is the most detailed characterization of B. theileri in the Americas to-date, presenting morphological, molecular and phylogenetic data, including the transovarial transmission of the spirochete in the host tick.

Resumo O estudo teve como objetivo realizar a caracterização morfológica, molecular e filogenética de Borrelia theileri obtida de Rhipicephalus microplus naturalmente infectado em bovino no estado do Rio de Janeiro, Brasil. Um total de 50 fêmeas de R. microplus ingurgitadas foram analisadas para espiroquetas por meio de esfregaço de hemolinfa. Ovos macerados e carrapatos, assim como sangue de bovinos infectados por esses carrapatos, foram analisados os genes glpQ, flaB e hpt por PCR. Os produtos de PCR foram purificados e sequenciados para análise e construção de uma árvore filogenética. Apenas 2% (1/50) dos carrapatos geraram um resultado positivo tanto pelo esfregaço como pela PCR. As formas espirais (n = 50) apresentaram (média ± DP) comprimento médio de 19,17 ± 4,12, diâmetro de 0,2935 ± 0,0469 e número de voltas de 8,44 ± 2,59. Os alinhamentos das sequências dos três genes avaliados exibiram 98% de similaridade aos isolados de B. theileri, ocorrendo em um clado altamente relacionado à linhagem de B. theileri KAT. As amostras de maceração de ovos foram positivas para os três genes avaliados, enquanto o sangue bovino foi negativo pela PCR. Esta é a mais completa caracterização de B. theileri nas Américas, apresentando dados morfológicos, moleculares e filogenéticos, incluindo a transmissão transovarial da espiroqueta no carrapato hospedeiro.