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.

Importance of screening severe COVID-19 patients for IFN-λ1, IL-6 and anti-S1 IgG levels.

Cytokine storm is an important cause of death in COVID-19 patients. A recent clinical study showed that administration of recombinant interferon lambda 1 (IFN-λ1 or IL-29) may prevent severe COVID-19. On the other hand, IL-6 has been associated as a prognostic marker of worsening for COVID-19 patients. The objective of this study is to screen IFN-λ1, IL-6 and antibody levels in consecutive serum sample sets of COVID-19 patients. A total of 365 serum samples collected from 208 hospitalized COVID-19 patients were analyzed for IFN-λ1 and IL-6 levels as well as SARS-CoV-2 neutralizing antibodies and anti-S1 IgG antibodies. Analyses of serum samples for cytokine levels showed that IFN-λ1 (>8 pg/mL) and IL-6 (>2 pg/mL) were detected in approximately 64% and 21% patients, respectively. A decrement in IFN-λ1 levels and IL-6 levels above 35 pg/mL can be sign of clinical severity and upcoming dead. An increment in IL-6 levels wasn't detected in every COVID-19 patient but a decrement in IL-6 levels was related to clinical improvement. Importantly, the detection of IFN-λ1 level together with an increase in anti-S1 IgG antibody response were observed in clinically improved patients. Screening severe COVID-19 patients for IFN-λ1, IL-6, and anti-S1 IgG antibody levels during their hospital stay especially in intensive care units may be beneficial to monitor the clinical status and management of treatment strategies. Importantly, detection of IFN-λ1 together with protective IgG antibody response can be an indication of clinical improvement in severe COVID-19 patients and these patients may be discharged from the hospital soon.

A preliminary study to develop a lateral flow assay using recombinant GRA1 protein for the diagnosis of toxoplasmosis in stray cats.

Toxoplasma gondii is a protozoan parasite that may infect many mammals including humans. Cats are one of the main sources of infection for humans. Therefore, routine screening of cats with tests that are inexpensive, rapid, and do not require sophisticated laboratory equipment is important. In this study, a lateral flow assay (LFA) was designed to rapidly diagnose toxoplasmosis in cats. For this purpose, we selected GRA1 protein of T. gondii due to its high antigenicity in diagnostic and vaccine studies. We further analyzed the immunological properties of GRA1 protein using in silico tools. Then, we expressed and purified recombinant GRA1 (rGRA1) protein and used it during the development of LFA to detect toxoplasmosis in serum samples (n = 40) of cats. According to the results, rGRA1 protein has negative GRAVY value, high aliphatic index, alpha helix, random coil and 12 B cell epitopes. The in silico data supported the high antigenic properties of rGRA1 protein and showed that it can be a good antigen candidate for LFA. Among 30 cat positive serum samples, 27 were found positive by the LFA while seronegative sera (n = 10) were negative by the LFA. The preliminary data showed that the LFA has high sensitivity (90 %) and specificity (100 %). When we used high responsive cat sera (i.e. sera that have optical density > 0.5 with ELISA) the sensitivity value reached 100 %. These results showed that rGRA1 protein is a good candidate to develop a LFA for rapid diagnosis of toxoplasmosis in cats.

Statistical optimization and characterization of monodisperse and stable biogenic gold nanoparticle synthesis using Streptomyces sp. M137-2.

Microbial synthesis of gold nanoparticles (AuNPs), which are used in various forms with different properties in medicine, as a renewable bioresource has become increasingly important in recent years. In this study, statistical optimization of stable and monodispersed AuNPs synthesis was performed using a cell-free fermentation broth of Streptomyces sp. M137-2 and AuNPs were characterized, and their cytotoxicity was determined. The three factors determined as pH, gold salt (HAuCl4) concentration, and incubation time, which are effective in the extracellular synthesis of biogenic AuNPs, were optimized by Central Composite Design (CCD) and then UV-Vis Spectroscopy, Dynamic Light Scattering (DLS), X-Ray Diffraction (XRD), Scanning Electron Microscope (SEM), Scanning Transmission Electron Microscope (STEM), size distribution, Fourier-Transform Infrared (FT-IR) Spectroscopy, X-Ray Photoelectron Spectrophotometer (XPS) and stability analyzes of AuNPs were carried out. Optimum values of the factors were determined as pH 8, 10- 3 M HAuCl4, and 72 h incubation using Response Surface Methodology (RSM). Almost spherical AuNPs with 20-25 nm protein corona on the surface, 40-50 nm in size, monodisperse, and highly stable form were synthesized. Biogenic AuNPs were confirmed from characteristic diffraction peaks in the XRD pattern, UV-vis peak centred at 541 nm. The FT-IR results confirmed the role of Streptomyces sp. M137-2 metabolites in the reduction and stabilization of AuNPs. The cytotoxicity results also showed that AuNPs obtained using Streptomyces sp. can be used safely in medicine. This is the first report to perform statistical optimization of size-dependent biogenic AuNPs synthesis using a microorganism.

Adjuvant action of needle-shaped BC microfibrils.

Bacterial cellulose (BC) is an unbranched biopolymer produced by microorganisms and composed of glucopyranose units linked by ß-1,4 bonds. This study investigates the adjuvant action of needle-shaped BC microfibrils (BCmFs) in vitro using bovine serum albumin (BSA) as a model antigen. BC produced by the static culture of Komagataibacter xylinus was then microparticled (1-5 µm) by acid hydrolysis and characterized using Dynamic Light Scattering and Scanning Electron Microscopy. Subsequently, Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy, cytotoxicity, TNF-α (tumour necrosis factor-alpha) and IL-6 (interleukin-6) cytokine secretion, and cellular uptake of the BCmFs-BSA conjugate on the human monocyte cell line (U937) differentiated into macrophages were performed. The microfibrils were determined to be 1-5 µm in size, needle-shaped, with a zeta potential of - 32 mV. Their conjugation with the model antigen, BSA, was demonstrated by FTIR analysis. In the cytotoxicity assay, BCmFs-BSA in macrophage cells showed high viability (over 70%). Although the highest TNF-α cytokine level (113 pg/ml) was obtained with BCmFs-BSA (Bovine serum albumin) conjugate (500 µg/ml) and was statistically significant (p = 0.0001) compared to the positive control group (BSA-aluminium hydroxide), IL-6 cytokine levels were not statistically different from those in the control group as desired. It has been shown in macrophage-differentiated U937 cells that microbially synthesized BC in the form of needle-shaped microfibrils (BCmFs) has a high cellular uptake capacity and increases the immunogenicity of the antigen. These results demonstrate for the first time that BCmFs have the potential to serve as a vaccine adjuvant.

Biotechnological Based Recombinant Protein Vaccines Developed Against Toxoplasmosis

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.

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.

An Overview of DNA Vaccines Development Studies Against Toxoplasma gondii

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.

Development of multistage recombinant protein vaccine formulations against toxoplasmosis using a new chitosan and porin based adjuvant system.

Toxoplasmosis is a global health problem affecting both human and animal populations. The lack of effective treatment makes the development of a vaccine against toxoplasmosis one of the main goals in the management of this disease. In our study, vaccine formulations containing the multistage recombinant antigens, rBAG1 + rGRA1 were developed with a combined adjuvant system consisting of chitosan and Salmonella Typhi porins in micro (MicroAS) and nanoparticulate (NanoAS) forms. BALB/c mice were immunized intraperitoneally with vaccine formulations two times at three-week intervals. Three weeks after the second vaccination, mice were challenged with 7-8 live tissue cysts of the virulent T. gondii PRU strain by oral gavage. Higher cellular uptake by macrophages and enhanced cellular (IFN-γ and I-4 in stimulated spleen cells) and humoral (IgG, IgG1, IgG2a) responses were obtained with the adjuvanted formulation, higher with microsystem when compared to that of nanosystem. Microsystem was found to stimulate Th1-polarized immune responses, whereasnon-adjuvanted antigens stimulated Th2-polarized immune response. The highest survival rate and reduction in cysts numbers and T. gondii DNA were obtained with the adjuvanted antigens.Our study showed that adjuvanted multistage recombinant vaccine systems increase theimmune response with strong protection againstT. gondii, more profoundly in microparticulate form.

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.

Immunogenicity of a xenogeneic multi-epitope HER2+ breast cancer DNA vaccine targeting the dendritic cell restricted antigen-uptake receptor DEC205.

Breast cancer was ranked first in global cancer incidence in 2020, and HER2 overexpression in breast cancer accounts for 20-30% of breast cancer patients. Current therapeutic strategies increase the survival rate, but resistance to them occurs frequently, and there is an urgent need to develop novel treatments such as DNA vaccines which can induce a specific and long-lasting immune response against HER2 antigens. To enhance the immunogenicity of DNA vaccines, dendritic cells (DCs) can be targeted using multi-epitope proteins that provide accurate immune focusing. For this purpose, we generated a DNA vaccine encoding a fusion protein composed of 1) in silico discovered antigenic epitopes of human and rat HER2 proteins (MeHer2) and 2) a single-chain antibody fragment (ScFv) specific for the DC-restricted antigen-uptake receptor DEC205 (ScFvDEC). The xenogeneic multi-epitope DNA vaccine (pMeHer2) encodes three only T-cell epitopes, two only B-cell epitopes, and two T and B cell epitopes, and pScFvDEC-MeHer2 vaccine additionally encodes ScFvDEC introduced at the N terminus of the MeHer2. Then, mouse groups were immunized with pScFvDEC-MeHer2, pMeHer2, pScFvDEC, pEmpty, and PBS to determine the elicited immune response. pScFvDEC-MeHer2 vaccinated mice showed a strong IgG response (P < 0.0001) and pScFvDEC-MeHer2 induced a significant IgG2a increase (P < 0.01). The percentages of both IFN-γ secreting CD4 and CD8 T cells were higher in mice immunized with pScFvDEC-MeHer2 compared with the pMeHer2. pScFvDEC-MeHer2 and pMeHer2 secreted significantly higher levels of extracellular IFN-γ compared with to control groups (P < 0.0001). In addition, the IFN-γ level of the pScFvDEC-MeHer2 vaccine group was approximately two times higher than the pMeHer2 group (P < 0.0001). Overall, this study identified the pScFvDECMeHer2 construct as a potential DNA vaccine candidate, supporting further studies to be conducted on HER2+ animal models.

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.

Rapid detection of Toxoplasma gondii DNA in cat feces using colorimetric loop-mediated isothermal amplification (LAMP) assays targeting RE and B1 genes.

Toxoplasma gondii is an obligate protozoan parasite that can infect mammals and birds. Cats are the definitive host of T. gondii and have a very important role in transmission of toxoplasmosis due to the shedding of millions of unsporulated oocysts, that become infective in the environment. Since cats play a major key role in the epidemiology of toxoplasmosis, rapid and accurate diagnosis of infected cats has utmost importance. In this study, we developed a novel colorimetric loop mediated isothermal amplification (LAMP) assay detecting T. gondii RE gene and modified a previously developed colorimetric LAMP assay targeting B1 gene to detect T. gondii DNA in cat feces for the first time. The analytical sensitivity of colorimetric LAMP assays was determined using plasmid controls. The clinical sensitivities of both colorimetric LAMPs were determined using cat fecal DNA samples that were confirmed to be positive by two different real-time PCRs in our previous study. According to the results, analytical sensitivities of both assays were 1 copy plasmid/reaction. Using real-time PCR as a reference method, sensitivities of colorimetric LAMP assays targeting RE and B1 genes were 100% and 97.56% whereas specificities of both assays were 100%. Overall, the colorimetric LAMP RE assay developed in this study brings an advantage in the diagnosis of T. gondii in cat fecal samples since it has higher sensitivity, does not need for experienced personnel, and can be applied in basic laboratories or in the field.

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.

GRA8 DNA vaccine formulations protect against chronic toxoplasmosis.

Toxoplasma gondii has a very wide host range and infects all warm-blooded animals including humans. The disease causes great economic losses both in animals and humans. Vaccination is the most effective approach to fight against toxoplasmosis however an effective vaccine has not been developed yet. In the present study, GRA8 protein of T. gondii that showed high immunogenicity in our previous microarray screening study was used to develop a DNA vaccine using pcDNA 3.3 vector for the first time. In order to increase the potency of the DNA vaccine, 10 times lower amount of GRA8 DNA vaccine was combined with molecular adjuvant CpG and formulated into a commercial liposome (pcDNA3.3-GRA8+CpG+Escort). Mice were vaccinated intramuscularly two times at three-week intervals and challenged orally with the T. gondii PRU strain tissue cysts. The humoral immune response was determined by Western Blot and ELISA. The cellular immune response was analyzed by flow cytometry, cytokine ELISA and MTT assay. Among the vaccine groups, pcDNA3.3-GRA8 and pcDNA3.3-GRA8+CpG+Escort induced strong IgG response compared to controls (P < 0.001). The IgG1 and IgG2a responses showed a balanced Th1-Th2 polarization. The ratio of CD4+ and CD8+ T lymphocytes secreting IFN-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.01). The amount of tissue cysts in the group of mice vaccinated with pcDNA3.3-GRA8 decreased significantly compared to control groups (P < 0.0001). In the group vaccinated with pcDNA3.3-GRA8+CpG+Escort, the amount of tissue cysts also decreased significantly compared to PBS (P = 0.0086) and Empty plasmid+CpG+Escort (P = 0.0007) groups. This study showed for the first time that pcDNA 3.3. vector encoding GRA8 with or without CpG and Liposome can induce strong cellular and humoral immune responses and confer strong protection against mouse model of chronic toxoplasmosis.

Development of a novel recombinant ELISA for the detection of Crimean-Congo hemorrhagic fever virus IgG antibodies.

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 < 0.0001) compared to rNP (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.

Investigation of the role of stray cats for transmission of toxoplasmosis to humans and animals living in Izmir, Turkey.

INTRODUCTION: Toxoplasma gondii is a protozoan parasite that has a widespread distribution among mammalians and birds. One of the reasons for the high prevalence may be due to ingesting oocyst disseminated by stray cats' feces. In Turkey, most of the citizens are closely associated with stray cats and they love to pet and feed them on the streets. In this study, we aimed to determine the prevalence of T. gondii DNA in feces of stray cats living in Izmir, Turkey in order to identify the transmission potential to humans and other animals. METHODOLOGY: Feces and blood samples of 465 stray cats were investigated for the presence of T. gondii oocysts by microscopy and for the presence of T. gondii DNA by two real time PCR methods. Furthermore, serum samples were analyzed for anti-T. gondii IgG antibodies using an ELISA. RESULTS: Oocysts were detected in 0.43% of the stray cats by microscopy. T. gondii DNA was detected in 14.37% of the stray cats' feces samples. The seroprevalence rate was 37.84%. In the feces and/or blood PCR positive group, 35.89% of them were seropositive. Among the 176 seropositive cats, T. gondii DNA was detected in feces of 27 cats (15.34%). CONCLUSIONS: This study first time showed the inter relation of T. gondii DNA in feces and blood samples and seropositivity. In sum, over 14% of the stray cats living outdoor may have an important role in transmission of toxoplasmosis to humans in Izmir as well as to other animals.

Toxoplasma gondii destroys Her2/Neu-expressing mammary cancer cells in vitro using a continuous feed medium approach.

INTRODUCTION: Toxoplasma gondii is an opportunistic protozoan and can be grown using several human cell lines. Breast cancer is the second leading cause of cancer death in women. Her2/Neu-expressing mammary cancer cell lines called TUBO can be grown in vitro. In recent years, protozoan parasites have become popular means of use in cancer therapy research. In this study, we analyzed whether T. gondii tachyzoites can destroy TUBO cells using a novel continuous feed medium approach. METHODOLOGY: Two sets of flasks (each containing four groups) containing TUBO cells were inoculated with T. gondii Ankara strain tachyzoites. First set containing 5×106 TUBO cells were inoculated with TUBO-tachyzoite ratios of 1:2, 1:1, 2:1, and 4:1 and second set containing 1×106 TUBO cells were inoculated with TUBO-tachyzoite ratios of 10:1, 100:1, 1000:1, and 2000:1. Thereafter, culture supernatants were harvested at various days until TUBO cells were destroyed and tachyzoites were counted. RESULTS: In the first and second sets of flasks, TUBO cells were destroyed between days 8 to 12 and 12 to 25, respectively. In addition, the amount of tachyzoites increased 7- 43 and 595 to 112500 times in the first and second set of flasks, respectively. CONCLUSIONS: These results show that T. gondii tachyzoites successfully destroy Her2/Neu-expressing mammary cancer cells using a continuous feed medium approach. Although this idea may be too premature for the moment, the approach defined herein may support future researchers investigating the relationship between cancer and parasites which can make important progress toward saving cancer patient lives.