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Abstract Background Enterocytozoon bieneusi is the most common species found in humans. Although E. bieneusi has been investigated in humans, genotype profile of E. bieneusi is not known in Türkiye. Methods In this study, we screened E. bieneusi in patients (n = 94) with different types of malignant solid tumors by Real Time PCR and then sequenced E. bieneusi positive samples. All cancer patients were undergoing chemotherapy and had diarrhea. Moreover, as control groups, we also screened E. bieneusi in patients with diarrhea (n = 50) and without diarrhea (n = 50). Results Among all patients analyzed, 33 (17%) were found to be E. bieneusi-positive. As the patients were categorized, the molecular prevalence of E. bieneusi increased to 25.5% among cancer patients with diarrhea. However, the molecular prevalence of E. bieneusi was found to be lower in patients with presenting only diarrhea (8%) and patients without diarrhea (10%). The high molecular prevalence value detected among cancer patients with diarrhea was also statistically significant compared to other patient groups (P = 0.00112 and P = 0.0269). Among the 33 Real Time PCR positive samples, 10 of them were amplified by nested PCR and among these 10 samples, 6 of them were successfully genotyped. The phylogenetic tree showed the presence of D and Type IV which were also identified in stray cats living in İzmir in our previous study. Conclusions High molecular prevalence value indicates the importance of screening stool samples of cancer patients with diarrhea for E. bieneusi and genotyping results indicate that D and Type IV are circulating between humans and cats.

Abstract Severe acute respiratory syndrome coronavirus 2 had devastating consequences for human health. Despite the introduction of several vaccines, COVID-19 continues to pose a serious health risk due to emerging variants of concern. DNA vaccines gained importance during the pandemic due to their advantages such as induction of both arms of immune response, rapid development, stability, and safety profiles. Here, we report the immunogenicity and protective efficacy of a DNA vaccine encoding spike protein with D614G mutation (named pcoSpikeD614G) and define a large-scale production process. According to the in vitro studies, pcoSpikeD614G expressed abundant spike protein in HEK293T cells. After the administration of pcoSpikeD614G to BALB/c mice through intramuscular (IM) route and intradermal route using an electroporation device (ID + EP), it induced high level of anti-S1 IgG and neutralizing antibodies (P

Abstract 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.

Objective:Acanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples.Methods:Acanthamoeba strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL.Results:To determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%.Conclusion:It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples.

Toxoplasma gondii (T. gondii) that can infect most warm-blooded animals and humans, is an obligate intracellular apicomplexan parasite with a wide host range. About one-third of the world’s population is infected with this parasite. While toxoplasmosis progresses asymptomatically in individuals with a strong immune system, it can cause serious clinical manifestations and death in immunocompromised individuals. The parasite is transmitted to humans through the consumption of water and food contaminated with cat feces, as well as raw or undercooked animal products, congenital infection and blood/organ transplantation. Additionally, T. gondii is often observed in farm animals such as sheep and goats. Clinical manifestations and abortions caused by T. gondii in sheep and goats lead to enormous economic loss worldwide. There is a commercial vaccine against T. gondii, called Toxovax (MSD, New Zealand) that can only be used in sheep. For these reasons, there is a need for innovative T. gondii vaccine that is harmless, easily produced, which can prevent losses and be used in all living things. Advances in immunology, molecular biology, genetic, biotechnology and proteomics bring new perspectives to vaccine studies. Studies in innovative vaccine studies against T. gondii have accelerated with the discovery of new antigens by in vitro screenings, and bioinformatic analyzes, the use of various expression systems and new adjuvant types. Recombinant protein vaccines are biotechnological vaccines that are frequently preferred due to their rapid and easy production in various expression systems, availability of very and high purity products, ease of manipulation and stimulation of both cellular and humoral immune responses. Recombinant protein vaccines, developed by biotechnological methods, are promising tools for providing a protective immune response against toxoplasmosis. In this review, an overview of the parasite complex life cycle, its pathogenesis, humoral and cellular immune responses in the host, and recombinant protein vaccine studies developed against the parasite are presented.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that can infect almost all warm-blooded animals, including humans, and one-third of the global population is thought to be infected with this parasite. Infection can occur through consumption of contaminated food, contact with an infected host, or congenital transmission. While toxoplasmosis is asemptomatic in people with a healthy immune system, it can cause severe infections in people with a suppressed immune system or with immunodeficiency. In addition to causing diseases in humans, it also causes infections in livestock and may result in stillbirth and abortion in sheep and goats. There is no 100% effective medicine or vaccination against the parasite that causes major clinical symptoms and financial losses. There is a need for an effective, safe, and durable vaccine that can provide protective immunity for use in humans and animals. Vaccination studies against toxoplasmosis have gathered speed since the 1990s. Today, studies can be carried out to develop effective and safe vaccines depending on the developments in molecular biology, biotechnology, and immunology. DNA vaccines are a promising vaccine platform against toxoplasmosis because they are easy to produce, they are safe, they do not need a cold chain, and they can stimulate both humoral and cellular immune responses. This review provides an overview of the complex life cycle, pathogenesis, and epidemiology of the parasite; the immune response that develops in the host against the infection it causes; and the DNA vaccines developed against toxoplasmosis and these vaccines.

Abstract 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.

Abstract 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 İzmir, Turkey.

Abstract 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.

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.

Abstract 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.

Abstract Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral infection caused by Crimean-Congo hemorrhagic fever virus (CCHFV). Serological screening of CCHF is important and current ELISA use antigens prepared from virus which is expensive due to requirement of high bio-containment facilities. In this study, we aimed to develop a new recombinant ELISA. For this purpose, CCHFV genome were expressed as 13 proteins in E. coli and among them abundantly purified recombinant Nucleocapsid protein (rNP) and Mucin-like variable domain (rMLD) were used as antigen in ELISA (Rec-ELISA). Rec-ELISA using rNP, rMLD and a combination of both (rNP/rMLD) were probed with acute (n = 64; collected between days 1 and 7 after onset of symptoms), convalescent (n = 35; collected 8 days after onset of symptoms), consecutive sera (n = 25) of confirmed CCHF cases and control sera (n = 43). The sensitivity and specificity of Rec-ELISA using rNP/rMLD were 73% and 98% in acute cases and 97% and 98% in convalescent cases. The median interquartile absorbance value to discriminate the acute and convalescent phases of CCHF was significantly higher with ELISA using rNP/rMLD (P 0.05) and rMLD (P = 0.001). These results indicate that the Rec-ELISA using rNP/rMLD may be very useful to diagnose convalescent CCHF cases especially in field studies.

Abstract Background Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. Toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. A vaccine against T. gondii is required to prevent consequences of the infection. The aim of this study is to generate a multivalent recombinant protein vaccine against T. gondii. Methods 49 previously discovered antigenic proteins of T gondii were evaluated by their expression level in E. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. Based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites. Results In mice vaccinated with hexavalent vaccine, strong total IgG (P

Abstract Background Toxoplasma gondii is an opportunistic protozoan parasite that can infect all warm-blooded animals including humans and cause serious clinical manifestations. Toxoplasmosis can be diagnosed using histological, serological, and molecular methods. In this study, we aimed to detect T. gondii RE gene in various human samples by in house and commercial real time polymerase chain reactions. Methods A total of 38 suspected cases of toxoplasmosis [peripheral blood (n:12), amnion fluid (n:11), tissue (n:9), cerebrospinal fluid (n:5), and intraocular fluid (n:1)] were included to the study. An in house and a commercial RT-PCR were applied to investigate the T. gondii RE gene in these samples. Results The compatibility rate of the two tests was 94.7% (37/38). When the commercial RT-PCR kit was taken as reference, the sensitivity and specificity of in house RT-PCR test was 87.5 and 100%. When the in house RT-PCR test was taken as reference, the commercial RT-PCR kit has 100% sensitivity and 96.8% specificity. Incompatibility was detected in only in a buffy coat sample with high protein content. Conclusions Both the commercial and in house RT-PCR tests can be used to investigate T. gondii RE gene in various clinical specimens with their high sensitivity and specificity. In house RT-PCR assay can be favorable due to cost savings compared to using the commercial test.

Abstract Background Toxoplasma gondii is an obligate intracellular protozoan parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations in immune compromised humans. The parasite has also been recently linked to behavioral diseases in humans and other mammalian hosts. New antigens are being evaluated to develop a diagnostic kit for the diagnosis of acute infection or a protective vaccine. Methods In this study, we have focused on the discovery of new antigenic proteins from T. gondii genomic data using a high throughput protein microarray screening. To date, microarrays containing > 2870 candidate exon products of T. gondii have been probed with sera collected from patients with toxoplasmosis. Here, the protein microarrays are probed with well-characterized serum samples from animal models administered orally with oocysts or tissue cysts. The aim was to discover the antigens that overlap in the mouse profile with human antibody profiles published previously. For this, a reactive antigen list of 240 antigens recognized by murine IgG and IgM was identified using pooled sera from orally infected mice. Results Analyses of screening data have identified plenty of antigens and showed strong immunogenicity in both mouse and human antibody profiles. Among them, ROP1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA14, MIC1, MIC2 and MAG1 have shown strong immunogenicity and used as antigen in development of vaccines or serological diagnostic assays in previous studies. Conclusion In addition to the above findings, ROP6, MIC12, SRS29A and SRS13 have shown strong immunogenicity but have not been tested in development of a diagnostic assay or a vaccine model yet.

Toxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype(s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of İzmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondii strains within type II and III lineage have close relation with strains previously isolated from stray cats in İzmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation.

Toxoplasma gondii causes congenital toxoplasmosis in newborns resulting with fetal anomalies. Determining the initiation time of infection is very important for pregnant women and current serological assays have drawbacks in distinguishing the recently acute toxoplasmosis. Diagnosis of recently acute infection may be improved by using stage specific antigens in serological assays. In the present study, the diagnostic value of sporozoite specific SporoSAG, bradyzoite specific BAG1 proteins and GRA1 protein expressed by all forms of the parasite have been evaluated ELISA using sera systematically collected from mice administered orally with tissue cyst and oocysts. The anti-SporoSAG IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 1, 10, and 15 (P

Currently, some Toxoplasma gondii genotypes are being associated with serious clinical presentations. A recent report showing the Africa 1 genotype in two local congenital toxoplasmosis cases acquired in Turkey formed the basis of this study because atypical Africa 1 genotype is most frequently detected in animals and patients from sub-Saharan Africa. Since stray cats are considered as the linkage between wild life and urban life in T. gondii transmission, the present study aimed to isolate and characterize T. gondii strains circulating in stray cats of İzmir (Western Turkey). A secondary objective was to determine toxoplasmosis seroprevalence in this cat population. Tissues obtained from 100 deceased stray cats were bioassayed and isolated strains were genotyped using 15 microsatellite markers. In addition, toxoplasmosis seroprevalence was analyzed in 1121 cat sera collected from several large veterinary clinics in İzmir. Among the 22 isolates, 19 were Type II (86.3%), two were Type III (9%) and one was Africa 1 genotype (4.5%). The overall seropositivity rates in cats were 42-48% and 33.4-34.4% according to IFA and ELISA, respectively. Seroprevalence in deceased cats was significantly higher than in healthy cats (P = 0.0033). Finding both the major clonal Type II lineage together with the Type III lineage also found in Middle East, and an atypical genotype, Africa 1 appears consistent with the specific geographic location of Turkey between three continents and raises the possibility of transportation of these strains between continents through trade routes or long distance migratory birds. In addition, the first large study of toxoplasma seroprevalence in a stray cat population was also reported. The relatively high seropositivity rates and the variety of T. gondii genotypes confirm the local stray cat population as a risk factor for human toxoplasmosis in İzmir.