Cytb Gene Research Articles

Nonsense mutation in mitochondrial Cytb gene and macrophage lipid metabolism

Nonsense mutation in mitochondrial Cytb gene and macrophage lipid metabolism

Development of colorimetric loop-mediated isothermal amplification (LAMP) assay for detecting the azoxystrobin-resistant isolates in Colletotrichum truncatum

Anthracnose, mainly caused by Colletotrichum truncatum, is a serious threat to soybean production worldwide. Azoxystrobin, a quinone outside inhibitor (QoI) fungicide, has long been frequently used in Thailand. As expected, its performance has decreased as the pathogen has developed fungicide resistance, mainly due to mutations in the target cytochrome b (cytb) gene. The substitution from glycine to alanine at codon 143 (G143A) is related to the azoxystrobin-completely resistant phenotype of C. truncatum. We thus used the G143A mutation as the reaction marker to develop a loop-mediated isothermal amplification (LAMP) method combined with hydroxynaphthol blue (HNB) for visual, simple, and rapid detection of azoxystrobin-resistant isolates of C. truncatum. Among four sets of primers designed, primer sets 1, 2, and 4 distinguished the isolate carrying the G143A mutation from wild-type isolate. Optimum conditions for the LAMP assay using primer set 4 were 63 °C for 80 min with a visible detection limit of 1 ng. Moreover, the LAMP assay was highly specific and accurate in tests of 125 isolates of C. truncatum from 13 fields in northern Thailand. This LAMP assay is highly efficient, simple, accurate for rapid field monitoring of azoxystrobin resistance in C. truncatum and implementing effective control measures.

Population genetic structure of Dermatophagoides farinae (Acari: Pyroglyphidae) from different geographic populations based on mitochondrial cytochrome b gene

Dermatophagoides farinae (Acari: Pyroglyphidae) has been regarded as an important source of indoor allergens due to its prevalence and close proximity to humans. This study serves as an introductory probe into the population genetic structure of D. farinae, for which a few related studies of population genetic structure are available. In this study, the genetic structure of seven D. farinae populations in China was investigated based on the 372 bp mitochondrial cytochrome b (cytb) gene. A total of 29 haplotypes of the 68 cytb gene sequences were detected. The network diagram could be divided into Branch I (regions north of the Yangtze River: SJZ, CZ, FY and HN) and Branch II (regions south of the Yangtze River: AQ, WX and WH). Analysis of molecular variance (AMOVA) conducted according to the results of the network diagram revealed major genetic differentiation among groups, indicating that a significant genetic structure existed among groups. The results of population genetic structure could be largely attributed to geographical isolation. The existence of gene flow in a few populations may be influenced by grain trade activities. This study may facilitate a better understanding of the population genetic structure of D. farinae and could be utilized as the foundation for the development of efficient management strategies for D. farinae.

Identification and genetic characterization of Angiostrongylus cantonensis isolated from the human eye.

Angiostrongylus cantonensis, or the rat lungworm, is the causative agent of human angiostrongyliasis associated with eosinophilic meningitis or meningoencephalitis. Additionally, this nematode can cause ocular angiostrongyliasis, though this is rare. The worm can cause permanent damage to the affected eye and sometimes even blindness. Genetic characterization of the worm from clinical samples is limited. In the present study, we investigated the genetics of A. cantonensis recovered from a patient's eye in Thailand. We sequenced two mitochondrial genes (cytochrome c oxidase subunit I, or COI, and cytochrome b, or cytb) and nuclear gene regions (66-kDa protein and internal transcribed spacer 2, or ITS2) from a fifth-stage larva of Angiostrongylus sample that was surgically removed from the human eye. All sequences of the selected nucleotide regions were highly similar (98-100%) to the sequences of A. cantonensis in the GenBank database. The maximum likelihood and neighbor-joining trees of the COI gene indicated that A. cantonensis was closely related to the AC4 haplotype, whereas the cytb and 66-kDa protein genes were closely clustered with the AC6 and Ac66-1 haplotypes, respectively. In addition, the phylogeny of the concatenated nucleotide datasets of the COI and cytb revealed that the worm was closely related to the Thai strain and strains from other countries. This study confirms the identification and genetic variation of the fifth-stage larvae of A. cantonensis recovered from a patient's eye in Thailand. Our findings are important for future research on the genetic variation of A. cantonensis that causes human angiostrongyliasis.

Molecular investigation of bovine tropical theileriosis outbreak in an organized dairy cattle farm in Madhya Pradesh, India.

The present study aimed to investigate an outbreak of Theileria annulata (T. annulata) infection in an organized dairy cattle farm in Madhya Pradesh, India, using clinical and molecular techniques. Following the deaths of two crossbred cattle in March 2021, 43 blood samples were collected from infected and apparently healthy animals and examined by blood smear and polymerase chain reaction (PCR) techniques. The blood smear examination showed that 23.25% of samples were positive for Theileria organisms, while conventional PCR targeting the 18S ribosomal RNA (18S rRNA) and T. annulata merozoite surface antigen-1 (TAMS-1) genes revealed that 32.55% of samples were positive for T. annulata. PCR targeting cytochrome b (Cytb) gene showed 46.51% of samples were positive for T. annulata. Haematological analysis confirmed clinical signs of infection in affected animals, which were treated with buparvaquone @ 2.5mg/kg body weight intramuscularly along with supportive medicine. Two 18S rRNA gene amplicons were sequenced and analysed in a phylogenetic tree and haplotype network with 54 Indian and 38 foreign sequences. The phylogenetic tree revealed two groups with a high posterior probability and bootstrap value, while the haplotype network revealed 35 haplotypes, with haplotype 1 (H1) being the most abundant and several single haplotypes clustering around it, indicating fast and widespread expansion. Genetic diversity indices and neutrality tests confirmed that the population was expanding. These studies highlight the significance of prompt and precise diagnosis and management of T. annulata outbreaks and provide insights into its evolutionary history and population dynamics of T. annulata in India, which could aid improving disease preventive and control strategies.

Coastal-inland divergence and postglacial expansion in the populations of the orchid bee Euglossa annectans

Biogeographic patterns in the southernmost portion of the Atlantic Forest (AF) are still elusive for a high number of endemic species where the main phylogeographic hypothesis in AF (e.g., CM model or AF expansion) fails to predict refuges during the Last Glacial Maximum (LGM). Euglossa annectans is an orchid bee species endemic to AF, distributed throughout the longitudinal edges of the southern portions of this biome. Combining ecological niche modelling and genetic analysis, we investigated whether the Pleistocene climate fluctuations played some role in the population structure of this species. We analysed 640 bp of the Cytb gene and eight microsatellite loci from males sampled from twelve localities along E. annectans distribution. Microsatellite markers indicated two main groups with a disjunction between inland and coastal areas. Although mitochondrial data showed no spatial structure, we found signals of a bottleneck followed by population expansion after the LGM. Pleistocene climatic models revealed a fragmented scenario with suitable areas concentrated at the coastal continental shelf and the northern-west inland areas, both in line with the genetic nuclear markers’ structure and synchronic with the mitochondrial bottleneck at the LGM. The climatic instability along the Pleistocene has played a central role in determining the phylogeographic and demographic patterns of the E. annectans, resulting in a genetic structure reported for the first time in this area.

Genetic Diversity and Phylogeography of Taenioides cirratus in Five Geographical Populations Based on Mitochondrial COI and Cytb Gene Sequences

The genetic diversity of Taenioides cirratus was investigated on the basis of mitochondrial cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) gene sequences. A total of 159 specimens collected from the Chaohu Lake (CL), Nansihu Lake (NL), Taihu Lake (TL), Pearl River (PR), and Nandu River (NR) were sequenced. The total length of the sequence was 2485 bp with 412 polymorphic sites. A total of 73 haplotypes were identified, with Hap1 being the most widely distributed. The PR and NR populations showed high genetic diversity, while the CL population showed low genetic diversity. TL and NL showed high haplotype diversity but low nucleotide diversity. The analysis of molecular variance demonstrated that the sequence variations were mainly occurred among populations. T. cirratus populations are declining, and rare alleles are present at low frequencies, as analysed using a neutral test and a mismatched distribution analysis. There was a relatively high level of genetic differentiation among the populations of the Yangtze River basin (including NL), PR, and NR (Fst > 0.15). The two similar phylogenetic trees constructed by the maximum likelihood (ML) and Bayesian inference (BI) methods presented three major lineages, of which lineage II contains haplotypes from PR and NR, lineage III contains haplotypes from CL, NL, TL, and PR, whereas lineage I contains only a portion of haplotypes from NR. Based on the neutral test, mismatch analysis, and Bayesian Skyline Plot (BSP), geological and climatic events were inferred to have played an important role in the historical dynamics of T. cirratus population. Hap1, Hap25, and Hap58 were inferred as possible ancestral haplotypes by network analysis. Our study offers an essential foundation for resource preservation and additional taxonomic clarification of T. cirratus.

Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, β-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). β-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

Universal mtDNA fragment for Cervidae barcoding species identification using phylogeny and preliminary analysis of machine learning approach

The aim of the study was to use total DNA obtained from bone material to identify species of free-living animals based on the analysis of mtDNA fragments by molecular methods using accurate bioinformatics tools Bayesian approach and the machine learning approach. In our research, we present a case study of successful species identification based on degraded samples of bone, with the use of short mtDNA fragments. For better barcoding, we used molecular and bioinformatics methods. We obtained a partial sequence of the mitochondrial cytochrome b (Cytb) gene for Capreolus capreolus, Dama dama, and Cervus elaphus, that can be used for species affiliation. The new sequences have been deposited in GenBank, enriching the existing Cervidae mtDNA base. We have also analysed the effect of barcodes on species identification from the perspective of the machine learning approach. Machine learning approaches of BLOG and WEKA were compared with distance-based (TaxonDNA) and tree-based (NJ tree) methods based on the discrimination accuracy of the single barcodes. The results indicated that BLOG and WEKAs SMO classifier and NJ tree performed better than TaxonDNA in discriminating Cervidae species, with BLOG and WEKAs SMO classifier performing the best.

Study of Local Black Iraqi Goats Genotypes for the Cytb Gene.

Goats are the earliest domesticated ruminants. The local goat, Capra hircus, is considered one of the most important animals globally to provide good livestock production under harsh environmental conditions. This study aimed to detect the genetic structures of the local Iraqi goats bred in the central and southern regions of the country and investigate the possibility of benefiting from their genetic structures to construct improvement programs for increasing the productivity of these animals. To this end, blood samples were taken from 15 domestic black goats. A total of 10 ml of each animal's blood was placed in plastic containers of 10 ml. The DNA was extracted and sent to the laboratories of Juan Ju University, People's Republic of China, to analyze the sequences of the nitrogenous bases of the Cytochrome b (Cytb) gene. The results showed the presence of a genetic morphology for a segment of 670 base pairs for all the studied samples, and 15 sequences of this strain were recorded in the gene bank under the following accession numbers (LC496353.1:1-LC496367.1:1). The sequences of the nitrogenous bases of this segment of the gene, which were registered in the gene bank of some international goat breeds, were used for comparison with the sequences of black Iraqi goats to analyze the phylogenetic tree, calculate the genetic distance, study haplotypes, and calculate neutrality. The results showed the presence of one mutation in the studied segment of the Cytb gene, with a size of 670 bp. The mutation in base 46 of the studied gene converted from the purine group to the pyrimidine group (the shift from the nitrogen leaders A<C) in all the studied samples. It led to the transformation of the amino acid Asparagine into Histidine, where 233 amino acids were obtained, dominated by the amino acid Isoleucine and Leucine, over other amino acids at a rate of 14.34% and 11.21%, respectively. The phylogenetic tree showed the existence of two main branches, one of which included the local black Iraqi goat breed, and the other included all the international breeds under comparison. It is concluded that the black Iraqi goat breed has a different origin from other breeds.

The Global Spread Pattern of Rat Lungworm Based on Mitochondrial Genetics.

Eosinophilic meningitis due to rat lungworm, Angiostrongylus cantonensis, is a global public health concern. Human cases and outbreaks have occurred in the new endemic areas, including South America and Spain. The growing genetic data of A. cantonensis provides a unique opportunity to explore the global spread pattern of the parasite. Eight more mitochondrial (mt) genomes were sequenced by the present study. The phylogeny of A. cantonensis by Bayesian inference showed six clades (I-VI) determined by network analysis. A total of 554 mt genomes or fragments, which represented 1472 specimens of rat lungworms globally, were used in the present study. We characterized the gene types by mapping a variety of mt gene fragments to the known complete mt genomes. Six more clades (I2, II2, III2, V2, VII and VIII) were determined by network analysis in the phylogenies of cox1 and cytb genes. The global distribution of gene types was visualized. It was found that the haplotype diversity of A. cantonensis in Southeast and East Asia was significantly higher than that in other regions. The majority (78/81) of samples beyond Southeast and East Asia belongs to Clade II. The new world showed a higher diversity of Clade II in contrast with the Pacific. We speculate that rat lungworm was introduced from Southeast Asia rather than the Pacific. Therefore, systematic research should be conducted on rat lungworm at a global level in order to reveal the scenarios of spread.

Non-invasive investigation of Polychromophilus parasite infections in bat populations in Serbia using bat flies

BackgroundHaemosporidian parasites of the genus Polychromophilus infect bats worldwide. They are vectored by obligate ectoparasitic bat flies of the family Nycteribiidae. Despite their global distribution, only five Polychromophilus morphospecies have been described to date. The two predominant species, Polychromophilus melanipherus and Polychromophilus murinus, are broadly distributed and mainly infect miniopterid and vespertilionid bats, respectively. In areas where species from different bat families aggregate together, the infection dynamics and ability of either Polychromophilus species to infect other host families is poorly characterized.MethodsWe collected 215 bat flies from two bat species, Miniopterus schreibersii and Rhinolophus ferrumequinum, which sometimes form mixed clusters in Serbia. Miniopterus schreibersii is known to be frequently infected with P. melanipherus, whereas R. ferrumequinum has been observed to be incidentally infected with both Polychromophilus species. All flies were screened for Polychromophilus infections using a PCR targeting the haemosporidian cytb gene. Positive samples were subsequently sequenced for 579 bp of cytochrome b (cytb) and 945 bp of cytochrome oxidase subunit 1 (cox1).ResultsPolychromophilus melanipherus DNA was detected at six out of nine sampling locations and in all three examined bat fly species collected from M. schreibersii (Nycteribia schmidlii, n = 21; Penicillidia conspicua, n = 8; Penicillidia dufourii, n = 3). Four and five haplotypes were found for cytb and cox1, respectively. Evidence for multiple Polychromophilus haplotypes was found in 15 individual flies. These results point to a high diversity of P. melanipherus parasites in Miniopterus hosts and efficient transmission throughout the study area. A single Phthiridium biarticulatum bat fly collected from R. ferrumequinum screened positive for P. melanipherus, but only yielded a partial cox1 sequence fragment. Nevertheless, this result suggests that secondary hosts (both bat and fly species) are regularly confronted with this parasite.ConclusionsThe results of this study provide new insights into the prevalence and distribution of Polychromophilus parasites in European bats and their nycteribiid vectors. The use of bat flies for the non-invasive investigation of Polychromophilus infections in bat populations has proven to be efficient and thus represents an alternative for large-scale studies of infections in bat populations without the need to invasively collect blood from bats.Graphical

Bioinformatic Analysis of the Cytb Gene to Estimate the Domestication Potential of Mahseer Fish Genus Tor spp.

The purpose of this study is to estimate the population of fish of the genus Tor as a source of proper domestication. Bioinformatic analysis was carried out by extracting data from the NCBI gene bank and alignment of the Cytb gene sequences using the ClustalW program to further analyze the nucleotide characteristics using MEGA software, determining genetic variation using the dnasp5 software, and using median joining analysis to reveal the relationship between Tor fish haplotypes from different locations. The results obtained from this study show that the haplotype diversity values of fish of the genus Tor spp. in the domesticated farm population and the natural population are still high, namely 0.9 and 0.8, respectively, as well as the occurrence of haplotype groupings based on other population origins. Domestication of Tor genus fish can be sourced from domesticated farms, and the wild is still ideal because the haplotype diversity is still high.

Reassessment of species delimitation using nuclear markers in three lentic-breeding salamanders from the Chugoku District of Japan (Amphibia: Caudata: Hynobiidae).

Hynobius akiensis sensu lato has recently been split into three species based on short sequence analyses of cyt-b gene of mtDNA and without data of nuclear DNA, and strange sympatric distribution in some areas has been indicated in two species. We analyzed nuclear DNA marker (SNPs) and complete sequence of cyt-b in H. akiensis sensu lato to reassess species delimitation and genetic introgression among species. As a result, we found two lineages with discordant mitochondrial and nuclear DNA in some areas. Of H. akiensis sensu lato, each of the two contains the type locality of two species recently reported (H. sumidai and H. geiyoensis), and the use of these names has been previously advocated. However, their sympatric distribution was rejected based on nuclear DNA data, which we consider is more reliable than mtDNA. We thus clarify geographic boundary of these two species and revise the species delimitations.

Genetic characterization of fish, Cynoglossus lingua Hamilton, 1822 found in Chandipur Coast of Odisha using COI and Cytochrome b genes

Aim: In this study we characterized the fish Cynoglossus lingua collected from Chandipur Coast of Odisha using the mtDNA markers cytochrome c oxidase I (COI) and cytochrome b (Cyt-b) genes. Methodology: Samples of flat fish were collected from Balaramgadi fish landing center, Chandipur sea beach, Odisha coast. The collected specimens were initially identified based on the morphology using standard taxonomic keys. Molecular characterization was accomplished through PCR amplification of COI and Cyt-b genes. The sequences were subjected to BLAST analysis and aligned using Clustal W for homology mapping. Phylogenetic status was inferred by the neighbor-joining method using MEGA v7.0. Results: The taxonomic keys and the COI and Cyt-b gene sequences identified the specimens as C. lingua. The COI and Cyt-b gene sequences showed 98% and 99% sequence similarity with C. lingua isolate FMU.CIFRI.FF-7 COI gene (GenBank accession: MN889532.1) and the C. lingua isolate FM.CIFRI.CYTB.FF-7 Cyt-b gene (GenBank accession MT362541.1), respectively. Interpretation: The study supports the reliability of combining classical taxonomic approaches with molecular analysis using COI and Cyt-b genes for the identification of C. lingua. Key words: Cynoglossus lingua, COI gene, Cytochrome b gene, DNA barcoding, Molecular taxonomy

Inhibitory Effect and Control Efficacy of Picoxystrobin against Neopestalotiopsis clavispora, Causing Vine Tea Leaf Blight

Vine tea (Ampelopsis grossedentata) is a traditional herb widely consumed in southwestern China that possesses paramount potential for human health. In 2021, the outbreak of typical leaf blight disease was observed in almost all vine tea plantations in Zhangjiajie of Hunan province, resulting in significant economic losses of vine tea production. In this study, we identified Neopestalotiopsis clavispora as the causal agent of vine tea leaf blight via its morphological characteristics and molecular identification. The sensitivity distribution of N. clavispora isolates to picoxystrobin were determined based on mycelial growth and spore germination inhibition assays. The EC50 values for mycelial growth ranged from 0.0062 to 0.0658 µg/mL, with a mean of 0.0282 ± 0.0148 µg/mL. The EC50 values for spore germination ranged from 0.0014 to 0.0099 µg/mL, and the mean value was 0.0048 ± 0.0022 µg/mL. Picoxystrobin increased fungal cell membrane permeability, but inhibited fungal ATP biosynthesis. Moreover, picoxystrobin exhibited good in planta control efficacy on vine tea leaves. Three picoxystrobin-resistant mutants were obtained in the current study, but no mutations were detected in the N. clavispora Cytb gene. Competitive ability assays showed that the conidium production and pathogenicity of all picoxystrobin-resistant mutants decreased as compared to their progenitors, indicating that picoxystrobin-resistant mutants suffer fitness penalty. These findings provide important evidence for picoxystrobin in vine tea leaf blight management and increase understanding of the resistance mechanism of picoxystrobin against N. clavispora.

Evolutionary transition-transversion bias by the example of the cytb gene of palearctic Muridae (Rodentia) and Vespertilionidae (Chiroptera)

A comparative analysis of the features of the transition-transversion bias of the nucleotide sequence of the cytb gene in microbats (Vespertilionidae, Chiroptera) and mice (Muridae, Rodentia) shows both general regularities and certain family-level features. A common feature of the two families is the fact of the sharp predominance of transitions over transversions at the early stages of the evolutionary process, followed by the equalization of the ts/tv-displace- ment at the species and genus levels of divergences, as well as the fact that the increase in the frequency of transitions in phyletic lineages is gradual, and the transition is intermittent. At the same time, the levels of spontaneous mutations and evolutionary drift, as well as the rate of ts/tv drift compensation, are specific to families. These circumstances do not make it possible to obtain comparable estimates of divergence in different phyla and cause insurmountable difficulties in creating a universal formula for molecular clocks.

Phylogenics of the genus Glossogobius in the Mekong Delta based on the mitochondrial cytochrome b (cytb) gene

The Glossogobius species play an essential role in food supply and are distributed widely from the marine to freshwater, especially in the Mekong Delta, Vietnam (VMD). Some of their morphometrics and meristics are observed to vary with species and sampling sites. Therefore, the present study aims to verify if their mitochondrial Cytochrome b (Cytb) gene, one of the popular gene sequences used in fish phylogenic variation assessment, varies with species and sampling sites in the VMD. The Cytb gene size was 1300 bp for GcytbH/GcytbL primer pair and 1045 bp for GluMuq1-F/Mixcyto937-2R. The genetic distances within and among these three fish species groups were 0–11%. The Cytb gene sequences’ similarity between this study and the NCBI database was 85.84–100%. The Glossogobius specimens were observed to disperse in small branches of the phylogenetic tree with a low K2P value, suggesting that the Cytb genetic diversity may be low among species.

Detection of Salmonidae ingredient using mini-DNA barcoding in conjunction with a rapid visual inspection method

Using species outside Salmonidae to prepare salmon products has been widely revealed as a new type of fraud in China and other countries with less stringent labeling stipulations, highlighting the necessity to establish proper methods for rapid screening of Salmonidae ingredient. The present work selected mitochondrial Cytb gene as the target, and developed a novel mini-DNA barcoding assay to identify the salmonids ingredient in processed fish products. Results showed that the novel method is highly specific for salmonids detection and only the seven common salmonids species were successfully amplified. With the inclusion of HRM analysis, we can make an accurate differentiation of Salar salar, Oncorhynchus mykiss, and the other wild-caught Oncorhynchus species, which are deep in the scandal of species mislabeling. Moreover, a closed-tube visual detection can also be achieved by the preaddition of HNB (hydroxyl naphthol blue) and SYTO 9 in the reaction mixture, allowing an easy and rapid way to distinguish positive from negative. Finally, the novel assay was successfully applied on commercial products and the absence of salmonids ingredient indicated mislabeling with 4 commercial products. Overall, mini-DNA barcoding is a robust tool for fish species identification and the novel method is effective in screening Salmonidae ingredient.

Screening of Domestic Cats from North-Eastern Hungary for Hepatozoon felis and Cytauxzoon europaeus That Cause Infections in Local Wildcat Populations.

Among vector-borne protozoa Hepatozoon felis and Cytauxzoon europaeus are considered emerging species in felids in Europe. To investigate the presence of these two protozoa 127 domestic cats and 4 wildcats were screened by PCRs targeting the 18S rRNA gene of Hepatozoon spp. and piroplasms, as well as the cytb gene of Cytauxzoon spp. The samples were collected inside and outside a region of Hungary, where both protozoan groups are endemic in wildcats. Among domestic cats, one proved to be infected with H. felis. Furthermore, spleen samples of four wildcats were also examined, among which three tested positive for H. felis, and one had co-infection with C. europaeus. Importantly, H. felis from the co-infected wildcat belonged to genogroup II, similarly to H. felis from the positive domestic cat. Based on phylogenetic evidence, this genogroup probably represents a separate species from genogroup I of H. felis, which was hitherto reported from Mediterranean countries in Europe. The two other wildcats also harbored H. felis from genogroup I. Neither Hepatozoon nor Cytauxzoon infections were detected outside the recently discovered endemic region. In conclusion, this study demonstrates for the first time in Europe that H. felis from genogroup II may emerge in free-roaming domestic cats in regions where this protozoan parasite is endemic in wildcats.