First report of a novel 108 bp deletion and five novel SNPs in PRNP gene of stray cats and in silico analysis of their possible relation with feline spongiform encephalopathy.

Prion diseases are fatal neurodegenerative diseases affecting humans and animals. A relationship between variations in the prion gene of some species and susceptibility to prion diseases has been detected. However, variations in the prion protein of cats that have close contact with humans and their effect on prion protein are not well-known. Therefore, this study aimed to investigate the variations of prion protein-encoding gene (PRNP gene) in stray cats and to evaluate variants detected in terms of genetic factors associated with susceptibility or resistance to feline spongiform encephalopathy using bioinformatics tools. For this, cat DNA samples were amplified by a PCR targeting PRNP gene and then sequenced to reveal the variations. Finally, the effects of variants on prion protein were predicted by bioinformatics tools. According to the obtained results, a novel 108 bp deletion and nine SNPs were detected. Among SNPs, five (c314A>G, c.454T>A, c.579G>C, c.642G>C and c.672G>C) were detected for the first time in this study. Bioinformatics findings showed that c.579G>C (Q193H), c.454T>A (Y152N) and c.457G>A (E153K) variants have deleterious effects on prion protein and c.579G>C (Q193H) has high amyloid propensities. This study demonstrates prion protein variants of stray cats and their deleterious effects on prion protein for the first time.

Molecular characterization of Anaplasma ovis Msp4 protein in strains isolated from ticks in Turkey: A multi-epitope synthetic vaccine antigen design against Anaplasma ovis using immunoinformatic tools.

Tick-borne pathogens increasingly threaten animal and human health as well as cause great economic loss in the livestock industry. Among these pathogens, Anaplasma ovis causing a decrease in meat and milk yield is frequently detected in sheep in many countries including Turkey. This study aimed to reveal potential vaccine candidate epitopes in Msp4 protein using sequence data from Anaplasma ovis isolates and then to design a multi-epitope protein to be used in vaccine formulations against Anaplasma ovis. For this purpose, Msp4 gene was sequenced from Anaplasma ovis isolates (n:6) detected in ticks collected from sheep in Turkey and the sequence data was compared with previous sequences from different countries in order to detect the variations of Msp4 gene/protein. Potential vaccine candidate and diagnostic epitopes were predicted using various immunoinformatics tools. Among the discovered vaccine candidate epitopes, antigenic and conserved were selected, and then a multi-epitope protein was designed. The designed vaccine protein was tested for the assessment of TLR-2, IgG, and IFN-g responses by molecular docking and immune simulation analyses. Among the discovered epitopes, EVASEGSGVM and YQFTPEISLV epitopes with properties of high antigenicity, non-allergenicity, and non-toxicity were proposed to be used for Anaplasma ovis in further serodiagnostic and vaccine studies.

In silico discovery of diagnostic/vaccine candidate antigenic epitopes and a multi-epitope peptide vaccine (NaeVac) design for the brain-eating amoeba Naegleria fowleri causing human meningitis.

Naegleria fowleri, the brain-eating amoeba, is a free-living amoeboflagellate with three different life cycles (trophozoite, flagellated, and cyst) that lives in a variety of habitats around the world including warm freshwater and soil. It causes a disease called naegleriasis leading meningitis and primary amoebic meningoencephalitis (PAM) in humans. N. fowleri is transmitted through contaminated water sources such as insufficiently chlorinated swimming pool water or contaminated tap water, and swimmers are at risk. N. fowleri is found all over the world, and most infections were reported in both developed and developing countries with high mortality rates and serious clinical findings. Until now, there is no FDA approved vaccine and early diagnosis is urgent against this pathogen. In this study, by analyzing the N. fowleri vaccine candidate proteins (Mp2CL5, Nfa1, Nf314, proNP-A and proNP-B), it was aimed to discover diagnostic/vaccine candidate epitopes and to design a multi-epitope peptide vaccine against this pathogen. After the in silico evaluation, three prominent diagnostic/vaccine candidate epitopes (EAKDSK, LLPHIRILVY, and FYAKLLPHIRILVYS) with the highest antigenicities were discovered and a potentially highly immunogenic/antigenic multi-epitope peptide vaccine (NaeVac) was designed against the brain-eating amoeba N. fowleri causing human meningitis.

Comparative molecular epidemiology, subtype distribution, and zoonotic potential of Blastocystis sp. in Equus animals (horses, donkeys, and mules) in northwestern Iran.

A total of 500 fecal samples were collected from Equus animals in six different cities (Ardabil, Namin, Nir, Meshginshahr, Germi, and Khalkhal) of Ardabil Province, northwestern Iran, with 200 samples from horses, 200 from donkeys, and 100 from mules. Of the horse samples, 100 were from racing horses under special monitoring and care, while the remaining 100 were from non-racing horses, including those used for herding or in rural areas. All fecal samples were examined for the presence of Blastocystis sp. using PCR amplification of the SSU rRNA gene's barcode region after DNA extraction. The molecular prevalence of Blastocystis infection in Equus animals was 7.6% (38/500). Blastocystis was more common in horses [11.5% (23/200)] than in donkeys [5.5% (11/200)] and mules [4% (4/100)] (P > 0.05). Compared to racing horses [3% (3/100)], non-racing/rural horses [20% (20/100)] exhibited a substantially higher prevalence of Blastocystis (P < 0.05). The prevalence of Blastocystis in diarrheal samples and younger animals was remarkably higher than in formed samples and older animals, respectively (P < 0.05). No significant difference in Blastocystis infection prevalence was found between the genders of examined animals (P > 0.05). In Equus animals, 38 Blastocystis isolates included eight STs: ST10 [31.6% (12/38)], ST1 [21.1% (8/38)], ST2 [15.8% (6/38)], ST3 [10.5% (4/38)], ST4 [7.9% (3/38)], ST7 [5.2% (2/38)], ST14 [5.2% (2/38)], and ST6 [2.6% (1/38)]. These results suggest that Equus animals act as a proper reservoir for numerous Blastocystis STs, consequently playing a crucial part in the spread of this protozoan infection to humans, animals, and water reservoirs.

Genetic characterization of Bartonella henselae samples isolated from stray cats by multi-locus sequence typing.

BACKGROUND: Bartonella henselae is one of the most commonly identified Bartonella species associated with several human diseases. Although B. henselae was detected in humans and cats in Turkey, they have not been genotyped previously. Therefore, this study aimed to genotype B. henselae samples (n = 44) isolated from stray cats using the multi-locus sequence typing (MLST) method. For this aim, eight different housekeeping markers were amplified by nested PCR and then sequenced to reveal sequence types (STs) of B. henselae samples. RESULTS: Allelic profiles obtained from 40 B. henselae isolates (90.9%) were compatible with available allelic profiles in the MLST online database. However, allelic profiles obtained from the remaining 4 B. henselae isolates (9.1%) were incompatible with the database. Among B. henselae isolates with compatible allelic profiles, 5 different STs including ST1, ST5, ST9, ST35 and ST36 were identified according to the B. henselae MLST online database. ST35 was the most prevalent ST with a prevalence rate of 29.5% (13/44), followed by ST36 with a prevalence rate of 22.7% (10/44). In addition, ST5 (16%, 7/44) and ST9 (18.2%, 8/44) were also among the prevalent STs. The prevalence of ST1 was 4.5% (2/44). For B. henselae isolates with incompatible allelic profiles, we recommended a new ST called ST38. CONCLUSION: The present study genotyped B. henselae samples isolated from stray cats in Turkey for the first time and ST1, ST5, ST9, ST35, and ST36 as well as a new sequence type named ST38 were identified among these B. henselae isolates.

Molecular prevalence and genetic diversity of Hepatozoon spp. in stray cats of Izmir, Türkiye.

Hepatozoon spp. are an apicomplexan protozoan parasites that infect vertebrates including mammals, marsupials, birds, reptiles, and amphibians. Among Hepatozoon species, H. canis and H. felis are causative agents of hepatozoonosis in dogs and cats, respectively and have veterinary importance. This study aimed to determine the prevalence of Hepatozoon spp. in stray cats living in Izmir and investigate genetic diversity among positive samples. To achieve this aim, the prevalence of Hepatozoon spp. 18S rRNA gene was screened by PCR in DNA samples extracted from blood samples of stray cats (n = 1012). Then, Hepatozoon-positive samples were sequenced and the generated data were used for species identification, phylogenetic and haplotype analyses. According to the results, among the samples screened, 2.37 % (24/1012) of them were found to be Hepatozoon-positive, and of these positive samples, 18 (18/24; 75 %) were successfully sequenced. BLAST and phylogenetic analyses revealed that all of these samples were H. felis. Also, phylogenetic analysis showed that H. felis samples were genotype I. Within H. felis samples isolated from cats living in different countries/regions, 9 haplotypes were detected and among these haplotypes, H-1 was found to be prevalent (n = 20 H. felis isolates). In conclusion, this study showed that the prevalence of Hepatozoon spp. was low in stray cats analyzed. Also, H. felis genotype I was predominant in comparison to other Hepatozoon species.

Applicability evaluation of mtDNA based molecular identification in mosquito species/subspecies/biotypes collected from Thessaloniki, Greece.

The genus Culex, containing many described species, plays a role as a vector for diseases of medical and veterinary importance worldwide. Among these species, Culex pipiens is one of the most widespread mosquitoes and is classified into two biological forms (biotypes), named as Culex pipiens pipiens and Culex pipiens molestus. Due to similar morphological structure between these biotypes, morphological identification is inadequate. Thus, molecular methods have been developed and are considered more reliable, some of which are based on analyses of mitochondrial DNA. The aim of the present study was to evaluate the applicability and reliability of mtDNA based molecular identification methodologies. Initially, mosquito specimens (n = 100) collected from Thessaloniki, Greece were morphologically analyzed. Then, mitochondrial cox1 sequencing and PCR-RFLP methods were used to confirm the morphological identification results as well as to discriminate species and subspecies/biotype of Culex pipiens complex. According to morphological identification results, Culex pipiens complex (n = 92), Culex modestus (n = 6) and Culex theileri (n = 2) were detected. Using mtDNA sequencing, all Culex modestus and Culex theileri samples were confirmed whereas 86 of Culex pipiens complex were detected as Culex pipiens but surprisingly the remaining six of them were detected as Culex quinquefasciatus. Among Culex pipiens specimens, PCR-RFLP detected that frequency of Culex pipiens pipiens (85%; 85/100) was very high compared to Culex pipiens molestus (1%, 1/100). In conclusion, this study shows the necessity of use of molecular methods beside morphological methods for especially specimens detected as Culex pipiens. Also, it was shown that mtDNA PCR-RFLP methodology represents a well-established alternative for Culex biotypes identification.

Investigation of the genetic diversity and flea-borne pathogens in Ctenocephalides felis samples collected from goats in Izmir and Sanliurfa provinces of Turkey.

The cat flea "Ctenocephalides felis" has veterinary and medical importance since it is a vector for numerous important pathogens. In this study, a total of 249 flea samples were collected from goats bred in eight different farms (located in Izmir and Sanliurfa provinces of Turkey) and morphologically identified under microscopy. Later, the genetic diversity was investigated in 117 of C. felis samples that were morphologically identified by sequencing the mitochondrial cox1 gene, followed by phylogenetic tree, haplotype, genetic differentiation and gene flow analyses. In addition, Rickettsia spp. and Bartonella spp. which are zoonoses were screened in 27 pools comprising 249 flea samples by PCR. The phylogenetic tree showed that 117 flea samples were clustered in Clade 1 together with isolates from Australia, New Zealand, the Czech Republic, and India. Four haplotypes (haplotypes I, II, III and IV) were detected within the C. felis species. The most prevalent haplotype was haplotype I (57/117; 48.7 %). Among the population of flea samples in Izmir and Sanliurfa, the Fst and Nm values were 0.16261 and 2.57, respectively, indicating a moderate genetic differentiation and high gene flow. Rickettsia spp. was detected in four of C. felis pool samples whereas Bartonella spp. was detected in 25 of them. BLAST analysis identified R. raoultii as well as B. henselae and B. elizabethae. In conclusion, the findings showed that C. felis samples collected from goats in Turkey were classified within Clade 1 representing four different haplotypes with a moderate genetic diversity for the first time. Also, R. raoultii, B. henselae and B. elizabethae were demonstrated for the first time in cat flea samples collected in Turkey.

Molecular prevalence and genetic diversity of Bartonella spp. in stray cats of Izmir, Turkey.

BACKGROUND: Bartonella spp. are vector-borne pathogens that cause zoonotic infections in humans. One of the most well-known of these is cat-scratch disease caused by Bartonella henselae and Bartonella clarridgeiae, with cats being the major reservoir for these two bacteria. Izmir, Turkey is home to many stray cats, but their potential role as a reservoir for the transmission of Bartonella to humans has not been investigated yet. Therefore, the aim of this study was to investigate the prevalence of Bartonella species and their genetic diversity in stray cats living in Izmir. METHODS: Molecular prevalence of Bartonella spp. in stray cats (n = 1012) was investigated using a PCR method targeting the 16S-23S internal transcribed spacer gene (ITS), species identification was performed by sequencing and genetic diversity was evaluated by haplotype analysis. RESULTS: Analysis of the DNA extracted from 1012 blood samples collected from stray cats revealed that 122 samples were Bartonella-positive, which is a molecular prevalence of 12.05% (122/1012; 95% confidence interval [CI] 10.1-14.2%). Among the Bartonella-positive specimens, 100 (100/122; 81.96%) were successfully sequenced, and B. henselae (45/100; 45%), B. clarridgeiae (29/100; 29%) and Bartonella koehlerae (26/100; 26%) were identified by BLAST and phylogenetic analyses. High genetic diversity was detected in B. clarridgeiae with 19 haplotypes, followed by B. henselae (14 haplotypes) and B. koehlerae (8 haplotypes). CONCLUSIONS: This comprehensive study analyzing a large number of samples collected from stray cats showed that Bartonella species are an important source of infection to humans living in Izmir. In addition, high genetic diversity was detected within each Bartonella species.

Molecular investigation of Blastocystis sp. and its subtypes in cancer patients under chemotherapy in Aegean region, Turkey.

Blastocystis sp. is a common enteric protist found in humans and many other animals. Although the clinical relevance of Blastocystis sp. is currently fully unknown for humans, the prevalence of Blastocystis and subtypes are investigated in immunocompetent individuals presenting with symptoms like diarrhea or immunocompromised individuals including cancer patients. In this comprehensive study, the prevalence of Blastocystis sp. and subtypes were investigated in patients (n=94) with different types of malignant solid tumors using PCR targeting SSU rDNA gene and sequencing. All patients were undergoing chemotherapy and had diarrhea. According to obtained results, 46 patients were found to be Blastocystis positive and the molecular prevalence was detected as 48.9%. Among the positive specimens, 43 (43/46; 93.5%) of them were successfully subtyped. ST4 was the most predominant subtype and detected in 24 (55.8%) patients, followed by ST1 (11 patients, 25.6%) and ST3 (8 patients, 18.6%). In the colon cancer group, which had the highest number of patients, Blastocystis sp. was detected with a higher prevalence rate of 61.5% compared with the prevalence rate (48.9%) of all patients. Interestingly, ST3 was not detected in any of this patient group in contrast to ST4 and ST1. In conclusion, high prevalence of the Blastocystis in the immunocompromised patient groups shows the susceptibility of this patient group against any other infectious agents.

Molecular prevalence of Blastocystis sp. and subtype diversity in fecal samples collected from cattle in dairy farms in Turkey.

Close contact with infected animals is one of the main risk factors for zoonotic transmission of enteric protozoan parasite Blastocystis and thus, several animal species are being screened for the detection of the zoonotic subtypes. For this purpose, 22 fecal samples were collected from healthy cattle aged > 2 months and 39 fecal samples were also collected from cattle (aged <2 months) which are treated with TMP-SMX due to diarrhea. Later, Blastocystis sp. and subtypes were investigated by a PCR targeting the SSU rRNA gene and subsequently by sequencing. Among the 22 fecal samples collected from healthy cattle, Blastocystis was detected in 12 of them with a prevalence rate of 54.5 %. Among Blastocystis-positive samples, five different subtypes (ST3, ST5, ST10, ST12, and ST13) were detected. The predominant subtype was ST10 (allele 152) with a prevalence rate of 50 % (6/12). In the other group treated with TMP-SMX due to diarrhea, Blastocystis was detected in only one (2.56 %;1/39) fecal sample and its subtype was ST1 (allele 2). High prevalence of Blastocystis as well as predominance of ST10 (allele 152) were detected in healthy cattle. The identification of zoonotic ST1, ST3, ST5 and ST12 subtypes among the detected subtypes with a high prevalence (46.1 %; 6/13) showed the importance of cattle as a source for transmission of Blastocystis to humans. It was observed that the efficiency of TMP-SMX on the clearance of Blastocystis in cattle was very strong. Moreover, to our knowledge, this is the first study detecting Blastocystis ST13 subtype in the cattle.

Molecular prevalence and subtyping of Cryptosporidium spp. in fecal samples collected from stray cats in Izmir, Turkey.

BACKGROUND: Cryptosporidium spp. are obligate intracellular apicomplexan parasites transmitted to humans and other animals by contaminated water, food, or direct contact. They mainly cause gastrointestinal symptoms, although subclinical infections are also common. Cats are primarily infected by host-adapted Cryptosporidium felis while C. parvum and C. muris have also been detected in some cases. In this study, the molecular prevalence of Cryptosporidium spp. was investigated by screening 399 fecal samples collected from stray cats using nested PCR targeting the 18S rRNA gene for the first time in Turkey. Additionally, Cryptosporidium PCR-positive samples were genotyped by nested PCR- restriction fragment length polymorphism (RFLP), and subsequently, amplicons of 18S SSU rRNA were sequenced. They were further subtyped by amplification and sequencing of the gp60 gene. RESULTS: Among fecal samples screened, 12 of them (3%) were found to be Cryptosporidium-positive, and according to RFLP and sequencing of 18S rRNA gene, all positive samples were identified as C. felis. Subtyping analyses at the gp60 gene showed that C. felis isolates belonged to the XIXa subtype family, which are closely related to human subtypes of the parasite. CONCLUSIONS: The results of this study are important in terms of indicating the potential role of stray cats for transmission of Cryptosporidium spp. to humans or other animals. Also, the presence of XIXa, which is the dominant subtype family of C. felis in cats and humans was shown for the first time in stray cats of Izmir, Turkey.

Development of a new serotyping ELISA for Toxoplasma gondii type II, type III and Africa 1 lineages using in silico peptide discovery methods, well categorized feline and human outbreak serum samples.

BACKGROUND: Discovery of new Toxoplasma gondii serotyping epitopes is important due to reports showing the influence of genotype on the severity of toxoplasmosis. In Turkey, genotypes belonging to type II, type III and Africa 1 lineages were mainly detected. The present study focused on to find out epitopes with high discriminative capacity to serotype these genotypes using well characterized strains isolated from Turkey. METHODS: To meet this objective, GRA6 and GRA7 genes were sequenced from strains belonging to the type II, III and Africa 1 lineages, and B cell epitopes inside these sequences were predicted by Bcepred and additional docking analysis was performed with B cell receptor. Based on these analyses, 22 peptides harboring lineage specific epitopes were synthesized. Then, the serotyping potency of these peptides was tested using peptide ELISA and well categorized serum samples collected from stray cats infected with genotypes of the different lineages type II (n:9), III (n:1) and Africa 1 (n:1). As a result of peptide-ELISA, a serotyping schema was constructed with peptides that show high discriminative capacity and this assay was validated by sera collected from humans after an outbreak (n:30) and mother/newborn pair sera (n:3). Later, the validated serotyping schema was used to serotype a larger group of human (n:38) and cat (n:24) sera. RESULTS: Among 22 peptides, GRA6II/c, GRA7III/d, and GRA6 Africa 1/b epitopes have shown discriminative capacity. During the validation of peptide-ELISA, the serotype of toxoplasmosis outbreak and mother/newborn cases were detected to be serotype II. Moreover, the analyses in a larger group showed that serotype II was prevalent in humans and stray cats. CONCLUSIONS: Overall, the results showed that the serotyping schema could be successfully used to serotype T. gondii infections caused by type II, III and Africa 1 genotype.

Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor.

BACKGROUND: Cryptosporidium spp. is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp. can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp. in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp. prevalence was determined using Ziehl-Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp. in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl-Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl-Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None.

Molecular investigation of bacterial and protozoal pathogens in ticks collected from different hosts in Turkey.

BACKGROUND: The emergence of tick-borne disease is increasing because of the effects of the temperature rise driven by global warming. In Turkey, 19 pathogens transmitted by ticks to humans and animals have been reported. Based on this, this study aimed to investigate tick-borne pathogens including Hepatozoon spp., Theileria spp., Babesia spp., Anaplasma spp., Borrelia spp., and Bartonella spp. in tick samples (n = 110) collected from different hosts (dogs, cats, cattle, goats, sheep, and turtles) by molecular methods. METHODS: To meet this objective, ticks were identified morphologically at the genus level by microscopy; after DNA isolation, each tick sample was identified at the species level using the molecular method. Involved pathogens were then investigated by PCR method. RESULTS: Seven different tick species were identified including Rhipicephalus sanguineus, R. turanicus, R. bursa, Hyalomma marginatum, H. anatolicum, H. aegyptium, and Haemaphysalis erinacei. Among the analyzed ticks, Hepatozoon spp., Theileria spp., Babesia spp., and Anaplasma spp. were detected at rates of 6.36%, 16.3%, 1.81%, and 6.36%, respectively while Borrelia spp. and Bartonella spp. were not detected. Hepatozoon spp. was detected in R. sanguineus ticks while Theileria spp., Babesia spp., and Anaplasma spp. were detected in R. turanicus and H. marginatum. According to the results of sequence analyses applied for pathogen positive samples, Hepatozoon canis, Theileria ovis, Babesia caballi, and Anaplasma ovis were identified. CONCLUSION: Theileria ovis and Anaplasma ovis were detected for the first time to our knowledge in H. marginatum and R. turanicus collected from Turkey, respectively. Also, B. caballi was detected for the first time to our knowledge in ticks in Turkey.

Molecular characterization of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene and frequency of blood types in stray cats of Izmir, Turkey.

BACKGROUND: Cytidine monophospho-n-acetylneuraminic acid hydroxylase (CMAH) gene associated with blood groups in cats encodes CMAH enzyme that converts Neu5Ac to Neu5Gc. Although variations in CMAH gene of pedigree cats have been revealed, the presence/lack of them in non-pedigree stray cats is unknown. Therefore, the present study aimed to investigate the variations in CMAH gene and the quantity of Neu5Ac and Neu5Gc on erythrocytes of non-pedigree stray cats (n:12) living in Izmir, Turkey. Also, the frequency of blood types was determined in 76 stray cats including 12 cats that were used for CMAH and Neu5A/Neu5Gc analysis. RESULTS: In total, 14 SNPs were detected in 5'UTR as well as in exon 2, 4, 9, 10, 11 and 12 of CMAH gene. Among these SNPs, -495 C > T in 5'UTR was detected for the first time as heterozygous in type A and AB cats, and homozygous and heterozygous in type B cats. The remaining 13 that have been detected in previous studies were also found as homozygous or heterozygous. Both Neu5Gc and Neu5Ac were detected in type A and AB cats. In type B cats, only Neu5Ac was detected. Among two type AB cats, the level of Neu5Ac was found higher in cat carrying heterozygous form (T/C) of 1392T > C. The prevalence of type B cats (67.1 %) was higher than others. CONCLUSIONS: The presence of a new SNP as well as previous SNPs indicates that more variations can be found in stray cats with a more comprehensive study in the future. Also, the high prevalence of type B cats demonstrates the possible risk of neonatal isoerythrolysis among stray cats living in Izmir, Turkey.

In silico discovery of antigenic proteins and epitopes of SARS-CoV-2 for the development of a vaccine or a diagnostic approach for COVID-19.

In the genome of SARS-CoV-2, the 5'-terminus encodes a polyprotein, which is further cleaved into 15 non-structural proteins whereas the 3' terminus encodes four structural proteins and eight accessory proteins. Among these 27 proteins, the present study aimed to discover likely antigenic proteins and epitopes to be used for the development of a vaccine or serodiagnostic assay using an in silico approach. For this purpose, after the full genome analysis of SARS-CoV-2 Wuhan isolate and variant proteins that are detected frequently, surface proteins including spike, envelope, and membrane proteins as well as proteins with signal peptide were determined as probable vaccine candidates whereas the remaining were considered as possible antigens to be used during the development of serodiagnostic assays. According to results obtained, among 27 proteins, 26 of them were predicted as probable antigen. In 26 proteins, spike protein was selected as the best vaccine candidate because of having a signal peptide, negative GRAVY value, one transmembrane helix, moderate aliphatic index, a big molecular weight, a long-estimated half-life, beta wrap motifs as well as having stable, soluble and non-allergic features. In addition, orf7a, orf8, and nsp-10 proteins with signal peptide were considered as potential vaccine candidates. Nucleocapsid protein and a highly antigenic GGDGKMKD epitope were identified as ideal antigens to be used in the development of serodiagnostic assays. Moreover, considering MHC-I alleles, highly antigenic KLNDLCFTNV and ITLCFTLKRK epitopes can be used to develop an epitope-based peptide vaccine.

Molecular investigation of Rickettsia spp. and Francisella tularensis in ticks from three provinces of Turkey.

Ticks are obligate hematophagous ectoparasites as well as mechanical and biological vectors of a wide variety of microbial pathogens. To date, 19 tick-borne diseases have been reported from Turkey. In this study, ticks collected from Aydin, Izmir and Sanliurfa provinces of Turkey were identified using morphological and molecular methods. After the presence of bacterial DNA was checked, Rickettsia spp. and Francisella tularensis were investigated in bacterial DNA-positive tick specimens by PCR. Furthermore, amplicons belonging to tick specimens and positive bacterial samples were sequenced and processed for BLAST, alignment and phylogenetic analysis. As a result, seven tick species were identified: Rhipicephalus sanguineus, Rh. bursa, Rh. turanicus, Hyalomma marginatum, Hy. aegyptium, Hy. anatolicum and Haemaphysalis erinacei. Fifty-five tick specimens tested positive for bacterial DNA and among them, rickettsial DNA was found in five ticks (infection rate = 9.1%) belonging to Hy. marginatum, Hy. aegyptium, Rh. bursa and Rh. turanicus. Of the five Rickettsia-positive ticks, three contained Rickettsia aeschlimannii, one Ri. massiliae and one an unidentified Rickettsia sp. No Francisella tularensis DNA was detected. Sequence analysis of the ompB gene indicated two novel single nucleotide polymorphisms (SNP) in two different Ri. aeschlimannii strains and two novel SNPs as well as a novel insertion (GACGGT) were found in Rickettsia sp. This study indicated the presence of polymorphic Rickettsia species in ticks from Turkey.

High frequency of knockdown resistance mutations in the para gene of cat flea (Ctenocephalides felis) samples collected from goats.

Fleas are ectoparasites of mammals and birds. In livestock such as sheep and goat, flea bites cause many clinical signs. Several types of insecticides including pyrethroids are used to struggle against fleas. The widespread use of these insecticides causes an increase in the number of resistant individuals in flea populations. T929V and L1014F mutations corresponding to pyrethroid resistance have been found in the para gene of cat fleas. We aimed to investigate T929V and L1014F mutations in flea samples (n:162) collected from goats in seven different farms where cypermethrin, a synthetic pyrethroid, had been used intensively. To achieve this aim, collected flea samples were morphologically identified under a stereo microscope and DNA isolation was conducted by HotSHOT method. Later, a bi-PASA targeting the para gene was applied to identify both mutations in corresponding samples. According to the results obtained, all fleas were Ctenocephalides felis. Frequencies of T929V and L1014F mutations in fleas were 92.6% (150/162) and 95.7% (155/162), respectively. In conclusion, the frequency of mutations related to pyrethroid resistance was very high in the fleas collected from all the farms and it was thought that the high frequency of these mutations can be attributed to intensive use of pyrethroids.

Do Toxoplasma gondii apicoplast proteins have antigenic potential? An in silico study.

Toxoplasma gondii, one of the extensively studied Apicomplexan parasites, is prevalent worldwide in animals and humans. Apart from its nuclear genome, T. gondii contains an apicoplast genome in 35 kb length which is originated from a secondary endosymbiotic event. In this study, we aimed to investigate the antigenic potential of apicoplast genome encoded proteins (n:28) of T. gondii using in silico analysis. For this purpose, proteins were primarily predicted to reveal antigenic probability and then, several bioinformatics analyses were applied for all predicted antigenic apicoplast proteins to analyze physico-chemical parameters, subcellular localization and transmembrane domain. Also, further prediction analyses including structural, B cell and MHC-I/II epitope sites as well as post-translational modifications were performed for antigenic proteins that have a signal peptide or a high antigenicity value. Of the 28 apicoplast proteins, 19 were predicted as probable antigen. Among antigenic proteins, ribosomal protein S5, L11 and S2 were predicted to have signal peptide whereas ribosomal protein L36 and S17 were predicted to have a significantly high antigenicity value (P < 0.05). In addition, ribosomal protein S5, L11, S2, L36 and S17 were predicted to have a lot of epitopes which have low IC50 and percentile rank value indicating a strong binding among epitopes and MHC-I/II alleles, and post-translational modifications such as N-linked glycosylation, acetylation and phosphorylation. To the best of authors' knowledge this is the first study to show the antigenic potential and other properties of apicoplast-derived proteins of T. gondii.