Conventional PCR Protocols Research Articles

Existencia/ausencia de los genes <i>stx2e</i> y <i>f18</i> en aislados de<i> Escherichia coli </i>de cerdos libres de diarrea

The aim of the study was to evaluate Escherichia coli isolates for the presence or absence of the f18 and stx2e genes. In total, 186 isolates obtained from piglets under 50 days of age that did not have diarrhoea at the time of sampling were studied. The animals were from four high-tech breeding farms located in the Lima region. Sampling was done in 2019 as part of a preliminary study using rectal swabs. A commercial purification kit was used for DNA extraction and a conventional PCR protocol was used for gene detection. No PCR product for stx2e or f18 could be detected in any of the E. coli isolated from these piglets.

Comparison of Molecular and Morphological Identification Methods for Anguina Seed Gall Nematodes in Oregon Grasses Grown for Seed

Oregon's grass seed industry specializes in the production of forage grasses, including annual ryegrass ( Lolium multiflorum) and orchardgrass ( Dactylis glomerata). These species are hosts of seed gall nematodes (SGNs): Anguina funesta and Anguina sp. SGNs cause yield-limiting seed galls and can also vector toxic Rathayibacter bacteria. Trade partners have strict phytosanitary regulations leading to rejection of seed lots infested with SGNs. Current best practices for SGN detection focus on postharvest seed evaluation. Methods to evaluate fields before harvest could improve risk management decisions. In this study, we evaluated timing, collection, and detection methods to generate new recommendations for SGN detection throughout the growing season. Fields of annual ryegrass (21) and orchardgrass (7) were sampled in the 2022 and 2023 growing seasons at tillering (March), flowering (May), harvest (July), and germination (November). At each time point, tillers, seed heads, or soil samples were collected. Nematodes were extracted from soil, tiller, and seed head samples using traditional nematology methods. Alternatively, SGN-specific real-time and conventional PCR protocols were evaluated on DNA extracted from tillers or seed heads. Direct enumeration of SGN from tillers with traditional nematology methods resulted in positive detections in 11 to 19% of fields, depending on sample time and year, as opposed to 33 to 44% of fields when using molecular methods. SGNs were detected in 40% of fields using both methods when evaluating seed head samples. This study indicates the utility of incorporating molecular methods for risk evaluations of SGNs and provides recommendations for accurate SGN detection throughout the growing season. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Post-mortem detection of hemoplasmas (hemotropic Mycoplasma spp.) in South American fur seal (Arctocephalus australis) sampled in Rio Grande do Sul State, southern Brazil

Hemotropic mycoplasmas are bacteria that attaches to erythrocytes surface, which some species presents zoonotic concerns. In the suborder Pinnipedia, genera Otaria and Arctocephalus are prominent in Brazil. This study investigated the occurrence of hemoplasmas in Arctocephalus sp. and Otaria flavescens found dead along the coast of a Southern Brazilian State. DNA from 135 spleen samples were extracted and subjected to conventional PCR protocols, targeting the 16 S rRNA and 23 S rRNA gene. Three (2.22 %) Arctocephalus australis were positive in the 16 S rRNA gene, and no samples amplified in the 23 S rRNA gene. Samples from this study clustered with Zalophus californianus and Arctocephalus tropicalis mycoplasmas on a Bayesian phylogenetic analysis. Genetic diversity analysis suggested distinct genotypes, indicating A. australis as a new host for hemoplasma, and also a potential putative novel hemoplasma genotype. These findings raises future awareness for pinnipeds conservation, and adds Mycoplasma spp. to be taken into consideration when clinically evaluating rescued animals.

Rodents/Shrews and their Ectoparasites are not Associated with the Enzootic Maintenance and Transmission of Coxiella burnetii to Livestock and Humans in Puducherry, India

Objectives: “Q fever,” which is caused by Coxiella burnetii, is endemic in India. In addition to livestock, rodents have also been reported to be associated with enzootic maintenance, favoring pathogen transmission. Currently, however, no data are available on the role of rodents in “Q fever” transmission in India. Methods: A cross-sectional study was undertaken in 39 Puducherry villages to screen Coxiella burnetii in synanthropic rodents (rats and shrews) and their ectoparasites (ticks, mites, and fleas) by real-time and conventional PCR protocols targeting the pathogen specific IS 1111 and com 1 genes. Results: One hundred forty animals were trapped (107 shrews and 33 rats). The ticks, mites, and fleas infesting the rodents were identified as Rhipicephalus sanguineus, Leptotrombidium deliense and Schoengastiella spp., and Xenopsylla cheopis, respectively. PCR screening of the DNA extracted from the rodent/shrew blood samples and their ectoparasites tested negative for C. burnetii. Conclusions: Synanthropic rodents, such as rodents/shrews and their ectoparasites do not have a pivotal part in the enzootic maintenance and spread of Q fever to humans and livestock in Puducherry.

The opportunistic protist, Giardia intestinalis, occurs in gut-healthy humans in a high-income country

ABSTRACT Giardia intestinalis, a cosmopolitan gastrointestinal protist, is detected mainly in patients with clinical giardiasis in high-income countries. In contrast, there is very little information on the presence of Giardia in asymptomatic individuals. Therefore, the aim of this study was to determine the presence and prevalence of Giardia in gut-healthy volunteers in the Czech Republic and to perform a comparative evaluation of different diagnostic methods, since Giardia diagnostics is complicated. Our results confirmed that the qPCR method is the most sensitive method for detecting Giardia and revealed a prevalence of 7% (22/296) in asymptomatic individuals. In most cases, the colonization intensity ranged from 10−1–101. A conventional PCR protocol targeting the TPI gene was used to identify the assemblages. However, this protocol had limited sensitivity for Giardia amplification, effectively detecting colonization above an intensity of 104. In addition, Giardia was detected in 19% of the animals, which were closely associated with the study participants. However, due to methodological limitations, zoonotic transmission could not be clearly confirmed. Notably, contact with animals proved to be the only factor that had a significant impact on the incidence of Giardia in gut-healthy humans.

B-247 Rapid and Field-deployable Microchip-based Real-time PCR for Hepatitis B Virus DNA Detection

Abstract Background Hepatitis B is one of the most common infectious diseases with global significance, seriously causing chronic liver complications and a heavy health burden in developing countries. There is no doubt that real-time polymerase chain reaction (RT-PCR) has been a golden standard for monitoring the Hepatitis B virus (HBV) level in clinical health management. However, the conventional PCR protocol cannot fulfill the field test requirement in some resource-poor areas due to its drawbacks, such as huge size, long turnaround time, and demand for professional laboratory facilities. For solving these problems, a portable microfluidic chip-based PCR detection system was developed to enable the nucleic acid test in a flexible, rapid, and economical way. With the efficient temperature control module and advanced optical path design, this novel panel can achieve fast thermal circulation with a 30°C/s heating rate and multiple fluorescent detection with a throughput of 6 samples at a time. Methods In this study, we assessed the detection performance of the system using a commercial HBV nucleic acid detection kit. The reproducibility, limit-of-detection (LoD), and linearity were validated by reference samples with gradient concentrations. We evaluated the clinical sensitivity and specificity of the microdevice by a total of 200 extracted serum samples (100 positive and 100 negative cases). The analytical results were compared with a traditional PCR device with FDA certification. Results The variation coefficients of the microdevice for reference samples with different concentrations are 1.78%, 1.98%, and 1.50% (n = 10), respectively. The field-deployable PCR system can provide the HBV DNA assay with a detection limit of 300 IU/ml, whose reaction time was approximately 67 mins rather than the 108 mins performed by the comparator. During the establishment of the standard curve, this fast PCR test shows a strong linear correlation (R2 = 0.9919) between targeted gene concentration and dilution factor. Additionally, among the 200 clinical samples, we used the microchip-based system to rapidly complete quantitative detection for each sample with high sensitivity of 100% (100/100) and specificity of 100% (100/100), which is critically consistent with the traditional PCR method. Conclusion Hence, the point-of-care microfluidic PCR system exhibits great feasibility in offering timely, accurate, and reliable molecular diagnosis for early epidemic disease screening and therapeutic guidance of chronic infections.

Molecular identification of Bartonella spp. and Rickettsia felis in fox fleas, Chile

Seventy-five flea pools (one to ten fleas per pool) from 51 Andean foxes (Lycalopex culpaeus) and five South American grey foxes or chillas (Lycalopex griseus) from the Mediterranean region of Chile were analyzed for the presence of DNA of Bartonella spp. and Rickettsia spp. through quantitative real-time PCR for the nouG and gltA genes, respectively. Positive samples were further characterized by conventional PCR protocols, targeting gltA and ITS genes for Bartonella, and gltA, ompA, and ompB genes for Rickettsia. Bartonella was detected in 48 % of the Pulex irritans pools (B. rochalimae in three pools, B. berkhoffii in two pools, B. henselae in one pool), and 8 % of the Ctenocephalides felis felis pools (B. rochalimae, one pool). Rickettsia was confirmed in 11 % of P. irritans pools and 92 % of the Ct. felis pools. Characterization confirmed R. felis in all sequenced Rickettsia-positive pools. All Ct. canis pools were negative. A Ct. felis pool from a wild-found domestic ferret (Mustela putorius furo) also resulted positive for R. felis. Although opportunistic, this survey provides the first description of zoonotic pathogens naturally circulating in fleas parasitizing Chilean free-living carnivores.

Phylogenetic variants of Mycoplasma hominis from pregnant women and women presenting with infertility in Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria

Background: Much controversies have been associated with the pathogenicity of Mycoplasma hominis but little has been done to unravel the mystery behind the different views. This study aimed at investigating the genetic variants abounding within M. hominis and the distribution of the virulent genes among the variants.
 Methodology: Twenty (20) M. hominis isolates from high vaginal swabs of women (11 from pregnant women and 9 from women presenting with infertility) attending the Obstetrics and Gynaecology clinics of Nnamdi Azikiwe University Teaching Hospital (NAUTH), Nnewi, Nigeria, were sequenced using 16S rRNA universal gene target for the purpose of phylogenetic analysis and epidemiological typing. The isolates were also screened for the presence of M. hominis variable adherence antigen (vaa) and p120 virulent genes using primer constructs from the respective genes in a conventional PCR protocol.
 Results: Of the 20 M. hominis vaginal isolates, 4 phylogenetic strains were detected; strain MHS43 constituted 10/20 (50.0%) [2/9 (22.2%) from infertile women and 8/11 (72.7%) from pregnant women]; strain MHBS constituted 3/20 (15%) [3/9 (33.3%) from infertile women and 0/11 (0%) from pregnant women]; strain MHSWP2 constituted 4/20 (20.0%) [3/9 (33.3%) from infertile women and 1/11 (9.1%) from pregnant women]; while strain MHKC87 constituted 3/20 (15%) [1/9 (11.1%) from infertile women and 2/11 (18.2%) from pregnant women]. Each of vaa and p120 genes was detected in 14 of 20 isolates, while 6 isolates did not carry the genes. A 2-way ANOVA test showed that none of the genes was significantly associated with a particular strain (p=0.8641).
 Conclusions: The different views regarding the pathogenicity of M. hominis may be linked to the heterogeneity within the species and lack of omogeneity in the virulent genes as witnessed both in the intra species and intra strain levels.

First Records of Possibly Human Pathogenic Rickettsia Species in Bat Ticks, Carios vespertilionis, in Sweden.

The Soprano pipistrelle bat, Pipistrellus pygmaeus, is a common species in large parts of Sweden. Many of its natural habitats are near human habitations. This creates opportunities for ticks infesting these bats to encounter humans and possibly transmit zoonotic pathogens by tick bites. The bats are often infested with Carios vespertilionis, a tick species that, in addition to bats, has been recorded to bite humans on occasion. This study aimed to investigate if C. vespertilionis acts as a reservoir for Anaplasma phagocytophilum, Neoehrlichia mikurensis, Tick-borne encephalitis virus, and species of Babesia and Rickettsia and to improve currently used conventional PCR protocols for molecular species determination of Rickettsia spp. Ninety-two C. vespertilionis ticks were collected from underneath a bat-box harbouring P. pygmaeus. Pathogen-specific PCR assays showed that 58.4% were positive for Rickettsia spp. and negative for the other pathogens analysed. Phylogenetic analyses indicate that the species belong to R. parkeri, R. conorii, R. slovaca, R. sibirica subsp. mongolotimonae, R. rickettsii, and a hitherto uncultured Rickettsia sp. Several of these species are considered pathogenic to humans. Given the ecology and behaviour of C. vespertilionis, it may be a vector of these rickettsiae among bats and occasionally humans. To determine the Rickettsia species with certainty, and to determine if C. vespertilionis may be a reservoir and vector of the Rickettsia spp., further studies are needed.

Polyphasic study of phytopathogenic bacterial strains associated with deep bark canker of walnut in Serbia revealed a new species, Brenneria izbisi sp. nov.

Serious outbreaks of walnut deep bark canker were observed on young walnut trees (Juglans regia L.) in two localities in the northern part of Serbia during 2020. From the symptomatic walnut tissues, two types of bacterial colonies were isolated, predominantly, light cream, circular and smooth colonies, as well as small, yellowish, mucoid and convex ones. PCR analysis and phenotypic assays suggested that the former group belongs to Brenneria spp., while the latter isolates were identified as Xanthomonas arboricola pv. juglandis. Within the Brenneria group, two strains were identified as Brenneria nigrifluens, while other 15 strains did not belong to any Brenneria species described so far. Therefore, we selected four representative strains of the unknown Brenneria sp. and subjected them to polyphasic analysis. As expected, in a phylogenetic tree based on partial 16S rDNA sequences, four novel strains grouped with other Brenneria representatives, and showed close phylogenetic relationship to Brenneria salicis. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, gyrB, infB and rpoB housekeeping genes and core-genome phylogeny indicated that the studied strains form a novel and a clearly separate Brenneria lineage. Overall genome relatedness indices showed that they represent a new Brenneria species. The new species can be differentiated from the other Brenneria spp. infecting walnut and closely related B. salicis strains based on phenotypic characteristics, as well. Moreover, the pathogenicity tests on two-year-old walnut plants proved the ability of strains to cause necrosis and longitudinal black lesions and cracks on the trunk and branches of walnut trees. Overall, polyphasic characterization showed that the studied strains isolated from walnut with symptoms of deep bark canker represent a novel species of the genus Brenneria for which the name Brenneria izbisi sp. nov. is proposed. The type strain of B. izbisi is KBI 423T (= CFBP 9035T = LMG 32479T). To facilitate rapid identification of newly described species, a conventional PCR protocol and primers targeting the putative gene hrpP, were developed. Further study should reveal the potential role of each pathogen isolated from symptomatic walnut in disease development as well as possible interaction between them.

Poisson distribution-based conventional PCR protocol for quantification of pathogenic fungi in maize

Currents study aims to optimize and apply a conventional PCR protocol based on digital assay principle for quantification of pathogenic fungi in maize. Conventional PCR based on Poisson distribution is an effective and technically simple method for quantitative analysis of plant pathogens. It doesn’t require specific reagents or sophisticated laboratory equipment. Unlike qPCR, no standard curve is needed for quantifying the number of copies per sample. It permitted to quantify several toxigenic and non-toxigenic fungal pathogens in maize plants and reveal genome impact on the quantity of fungal DNA in host organism. Overall, fungal quantity was lowest in ‘MK01’ and ‘KU123’ and highest in ‘CP137’. High quantity of fungal DNA was also computed for ‘CP148’. For ‘B73’ the fungal DNA quantity was in between the same values for ‘KU123’ and ‘CP148’.

Probe-Based Real-Time qPCR Assays for a Reliable Differentiation of Capripox Virus Species.

Outbreaks of the three capripox virus species, namely lumpy skin disease virus, sheeppox virus, and goatpox virus, severely affect animal health and both national and international economies. Therefore, the World Organization for Animal Health (OIE) classified them as notifiable diseases. Until now, discrimination of capripox virus species was possible by using different conventional PCR protocols. However, more sophisticated probe-based real-time qPCR systems addressing this issue are, to our knowledge, still missing. In the present study, we developed several duplex qPCR assays consisting of different types of fluorescence-labelled probes that are highly sensitive and show a high analytical specificity. Finally, our assays were combined with already published diagnostic methods to a diagnostic workflow that enables time-saving, reliable, and robust detection, differentiation, and characterization of capripox virus isolates.

Molecular investigation of zoonotic intracellular bacteria in Chilean bats

Intracellular pathogens were investigated for the first time in 55 Chilean bats belonging to six species. Using a conventional PCR protocol targeting a fragment of the ITS region, 21 bats (38 %) were positive for DNA of Bartonella sp. Molecular characterization of fragments of the gltA, rpoB and fstZ genes and subsequent phylogenetic analysis indicated the presence of diverse genotypes related to Bartonella from bats worldwide. DNA from C. burnetii was investigated using a real-time PCR (qPCR) protocol targeting the IS1111 gene and yielded positive results for 5 individuals (9%), being the first report of C. burnetii in wildlife in Chile. All bats were negative for Rickettsia sp., evaluated by qPCR for the gltA gene, confirming that bats do not act as important reservoirs for Rickettsia. This preliminary survey calls for more comprehensive studies on the epidemiology of these agents, including larger sample sizes, the evaluation of potential transmission routes and spillover potential.

A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples.

The genus Anaplasma (Rickettsiales: Anaplasmataceae), which includes the species Anaplasma capra, Anaplasma bovis, Anaplasma ovis, and Anaplasma phagocytophilum, is responsible for a wide variety of infections in both human and veterinary health worldwide. Multiple infections with these four Anaplasma pathogens have been reported in many cases. We introduce a novel multiplex PCR for the simultaneous detection of A. capra, A. bovis, A. ovis, and A. phagocytophilum, based on species-specific primers against the groEL (A. capra and A. bovis), msp4 (A. ovis), and 16S rRNA (A. phagocytophilum) genes. To verify the specificity of the PCR reactions, we evaluated four sets of primers to analyze samples containing different blood pathogens. The sensitivity of the multiplex PCR was evaluated by amplifying 10-fold dilutions of total genomic DNA extracted from sheep blood infected with A. capra, A. bovis, A. ovis, or A. phagocytophilum. The reproducibility of the assay was evaluated by testing 10-fold dilutions of total genomic DNA extracted from sheep blood infected with these pathogens from 100 to 10–3 ng/μL per reaction in triplicate on three different days. A total of 175 field blood DNA samples were used to evaluate the reproducibility of multiplex PCR compared with the simplex PCRs. PCR primers used in this study were confirmed to be 100% species-specific using blood pathogens previously identified by other methods. The lower limit of detection of the multiplex PCR with good repeatability enabled the detection of A. capra, A. bovis, A. ovis and A. phagocytophilum at concentrations of 3 × 10–5, 5 × 10–7, 2 × 10–5, and 7 × 10–7 ng/μL, respectively. There was no significant difference between conventional and multiplex PCR protocols used to detect the four Anaplasma species (P > 0.05). The results of the multiplex PCR revealed that the A. capra groEL gene, the A. bovis groEL gene, the A. ovis msp4 gene, and the A. phagocytophilum 16S rRNA gene were reliable target genes for species identification in clinical isolates, being specific for each of the four target Anaplasma species. Our study provides an effective, sensitive, specific, and accurate tool for the rapid differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in sheep and goats.

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 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P 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.

Detection of Treponema denticola in Chronic Periodontitis by Quantitative Real-Time Polymerase Chain Reaction.

Chronic periodontitis constitutes a significant public health issue, particularly in China. Treponema denticola is one of the bacterial species critically involved in the development of this disease. Therefore, an effort was made in this study to design a technique for isolation of DNA from gingival fluid and detection of T. denticola genes by PCR methodology. For this purpose, samples were collected from 30 patients with severe periodontitis and 20 patients with mild periodontitis. A group of 50 healthy individuals served as a control. Following the isolation of DNA from the gingival fluid by magnetic microbeads, the material was analyzed for the presence of 16S rRNA by conventional and quantitative real-time PCR protocols. This newly developed methodology identified the presence of T. denticola in all samples from periodontitis patients. Quantitative analysis of copy numbers demonstrated that the bacterial count was highest in the severe periodontitis group and intermediate in the mild periodontitis group. The smallest number of bacteria were present in healthy controls. Besides being rapid, accurate and specific, the proposed method eliminates the need for anaerobic bacterial cultures, making it applicable in a typical clinical setting.

Hemotropic mycoplasmas in bats captured near human settlements in Nigeria

The presence of DNA of hemotropic mycoplasmas (hemoplasmas) was investigated for the first time in bats in Africa. Blood samples from 90 bats captured within or near human settlements in nine study areas from five states in Nigeria belonging to six genera of the families Pteropodidae, Rhinolophidae, and Molossidae were analyzed using conventional PCR protocol targeting a 391 bp part of the 16S rRNA gene. Of these, 32 samples (35 %) resulted positive. Eight nucleotide sequence types were identified, which were assigned to five genotypes showing between 93–99 % similarity with hemoplasmas from bats and/or rodents from other parts of the world, and/or Candidatus Mycoplasma haemohominis from a human patient. Network analysis showed genetic structure of hemoplasma sequences among bat species, but some sequences were shared among bats of different taxonomic groups and distant study areas. Further characterization of the samples using a protocol targeting ∼1200 bp of the 16S rRNA gene resulted in seven sequences that confirmed the results of the screening protocol. Hemoplasmas in Nigerian bats are prevalent, widely distributed and genetically diverse. The zoonotic risk to local inhabitants should not be neglected, due to the high similarity of some of the retrieved sequences with the human pathogen C. M. haemohominis.

Molecular detection of Hepatozoon spp. in non-hematophagous bats in Brazil

Even though Hepatozoon spp. has been molecularly detected in several wild animals in Brazil, there is no report on the occurrence of Hepatozoon spp. DNA in bats in Brazil. This study aimed at detecting Hepatozoon, in addition to ectoparasites, in non-hematophagous bats sampled in central-western Brazil using blood smears, hematoxylin-eosin (HE)-staining liver/spleen preparations and molecular and phylogenetic techniques. A total of 135 spleen, 127 liver, and 133 blood samples were collected from 135 non-hematophagous bats from 12 different species in two different sites in Campo Grande, Mato Grosso do Sul state, in the Brazilian Cerrado region. Spleen and blood DNA samples were submitted to two conventional PCR protocols for Hepatozoon spp. based on 18S rRNA. No Hepatozoon spp. gamonts or meronts were observed in blood smears and HE-stained-liver preparations, respectively. While none of the spleen samples was positive for Hepatozoon spp. in the PCR assays, 5 (3 %) blood samples contained 18S rRNA Hepatozoon DNA, including 2/37 (5 %) Artibeus lituratus, 2/32 (6 %) A. planirostris, and 1/23 (4 %) Platyrrhinus lineatus. Out of 5 bats positive for Hepatozoon spp., 3 were parasitized by either Macronyssidae/Spinturnicidae mites or Streblidae flies. BLAST analysis showed that the sequences detected in bats had >99 % identity with Hepatozoon sequences detected in amphibians and reptiles from Brazil, including Hepatozoon caimani detected in Caiman crocodilus. The phylogenetic inferences estimated by the Maximum Likelihood and Bayesian methods clustered the Hepatozoon sequences detected in Brazilian bats with those detected in reptiles and amphibians.

A novel PCR protocol for detection and differentiation of neuropathogenic and non-neuropathogenic equid alphaherpesvirus 1.

Equid alphaherpesvirus 1 (EHV-1) infections can have a major impact on the horse industry and equine welfare by causing abortion or respiratory or neurologic disease. A single nucleotide polymorphism (A2254→G2254) in open reading frame (ORF) 30, encoding the catalytic subunit of the DNA polymerase, has been shown to be a strong predictive marker for neuropathogenicity. Given that a previously established real-time PCR (rtPCR) protocol yielded unsatisfactory results concerning determination of the EHV-1 genotype, we developed and evaluated a new conventional PCR protocol enabling identification of the genotype by sequencing and restriction enzyme analysis (REA). Thirty samples from horses with signs typical for EHV-1 infection were tested by rtPCR and our new conventional PCR. The results showed that compared to rtPCR, the conventional PCR protocol combined with sequencing and REA was more reliable concerning unambiguous determination of the EHV-1 genotype. Results of our new assay confirmed previous findings, according to which the non-neuropathogenic genotype A2254 is predominantly found in animals with fever, respiratory signs, and abortions or perinatal mortality, whereas the neuropathogenic genotype G2254 is primarily detected in animals suffering from neurologic disease. In some samples, results pointed towards coinfection with both genotypes. Further studies are required in order to elucidate the significance of infections with genotype A2254 and G2254 in neurologic and non-neurologic cases, respectively.

Novel Ehrlichia sp. detected in Magellanic penguins (Sphenicus magellanicus) and in the seabird tick Ixodes uriae from Magdalena Island, southern Chile

Ehrlichia spp. are obligatory intracellular microorganisms that infect hematopoietic, endothelial or blood cells of mammals. Ticks are the only vectors of these agents in nature. To date, the role of birds and their associated ticks as reservoirs of ehrlichiae remains almost unexplored. In this study, we performed a molecular screening for bacteria of Anaplasmataceae family in samples of spleen (n = 72) and lung (n = 17), recovered from 72 carcasses of Magellanic penguins (Spheniscus magellanicus) in Brazil and Chile. One apparently unengorged tick (Ixodes uriae) was also collected while wandering upon one of the carcasses and submitted to molecular analyses as well. Through conventional and nested PCR protocols three genes (16S rRNA, dsb and groEL) of a new Ehrlichia sp. were partially characterized upon organs of three penguins and in the tick coming from Magdalena Island (Chile). First matches after BLASTn comparisons showed that our sequences share 99.4% (16S rRNA), 94.6% (groEL) and 79.3% (dsb) of identity with “Candidatus Ehrlichia ornithorhynchi”, Ehrlichia sp. NS101 and Ehrlichia canis CCZ, respectively. Matrixes of genetic distance including other representatives of the Ehrlichia genus point a 99.4%, 94.0%, and 80.0% of identity with 16S rRNA, groEL and dsb genes from Ehrlichia sp. It25, Ehrlichia sp. NS101, and Ehrlichia chaffeensis San Louis, respectively. A Bayesian phylogenetic analysis of Anaplasmataceae 16S rRNA gene places the detected Ehrlichia sp. into a group with Ehrlichia sp. BAT and Ehrlichia sp. Natal. Although depicting different topologies, Bayesian unrooted phylogenetic trees constructed for groEL and dsb genes position this Ehrlichia sp. into well-supported branches, which reinforces the finding of a new taxon. For the moment, any pathogenic effect of this new Ehrlichia sp. on penguins is still unknown. However, this fact becomes important to assess from a conservation point of view since populations of Magellanic penguins are currently threatened and in an ongoing decrease.