Campylobacter infections are a prevalent cause of diarrheal disease in humans and are the most significant zoonotic pathogens worldwide. Human campylobacteriosis is generally via ingestion of contaminated poultry products. However, based on recent studies chicken egg yolk antibody (IgY) powder has great potential to reduce the cecum load of Campylobacter jejuni (C. jejuni) in broilers. To understand the effective and economically feasible dosage, two immunization and challenge studies were conducted using 30 layer hens and 250 broiler chickens and found a scientific approach, starting with in vitro evaluations and progressing with in vivo studies confirmed. In this study it was demonstrated that specific IgY powder (SIgY), produced by immunized hens via bacterin, was highly effective in inhibiting bacterial growth and adhesion, as well as exhibiting bactericidal and agglutination properties (P < 0.05). Notably, doses of 0.5% and 1% SIgY significantly enhanced both the height and width of intestinal villi, along with improving the villus height-to-crypt depth ratio when compared to the positive control group (P < 0.05). Furthermore, medium and high doses of SIgY were effective in preserving the integrity of the intestinal epithelium, as evidenced by a reduction in crypt depth and the number of goblet cells, which serve as important markers in the immune system (P < 0.01). Additionally, analyses of cecal and liver bacterial counts in response to the 0.5% SIgY treatment revealed a significant reduction in C. jejuni counts compared to other challenged groups throughout the 28 d experiment (P < 0.01). Based on these results, it may be concluded that specific antibodies play a crucial role in maintaining the integrity of intestinal villi, support the health of the intestinal epithelium, and reduce the colonization of C. jejuni. These findings could form the basis for developing an economical and effective strategy to enhance poultry and human health in the context of C. jejuni infection.

ABSTRACT Background Pathogenic strains of spirochetes of Leptospira spp. cause a globally distributed zoonotic disease called leptospirosis. The disease has several clinical manifestations, ranging from asymptomatic and subclinical infection to fatal and severe forms. Hypothesis/objectives The aim of this study was to produce a recombinant Leptospiral immunoglobulin‐like surface protein‐A (r‐LigA) antigen of Leptospira interrogans in a prokaryotic expression system and to assess its efficacy in a mouse model. Materials and methods The optimal epitopes of the LigA protein were identified via bioinformatics studies. The pET32a+‐LigA plasmid construct was cloned into E. coli Top10‐DH5α, expressed in E. coli pLysS strains, and subjected to different IPTG concentrations at different times and temperatures. The expressed r‐LigA was purified using nickel‐affinity (Ni‐NTA) chromatography from the insoluble fraction and reassessed by SDS‐PAGE, western blotting, dot blotting, and Bradford assay. Female Balb/C mice were immunised subcutaneously with r‐LigA alone or emulsified in Freund's adjuvant and subsequently boosted at 2 and 4 weeks. Specific antibody levels were evaluated by indirect ELISA. Results Bioinformatics analysis identified the key antigenic region of LigA spanning amino acids 852 to 1210. Colony PCR and digestion confirmed the successful transformation. Induction using 0.5 mM IPTG at 30°C for 5 h was found to be optimal. Overexpression of r‐LigA under optimised conditions accumulated proteins as inclusion bodies. Purification of r‐LigA under native conditions using optimised Ni‐NTA yielded 1050 µg/mL protein and high immunogenicity by effectively stimulating the immune system in female Balb/C mice. Conclusions These findings support r‐LigA as a strong candidate for future leptospirosis diagnostic tools and subunit vaccine development.

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira spp., . , often occurring in tropical and subtropical regions. Focusing on development of rapid diagnostic methods to facilitate early diagnosis and a universal vaccine are the main critical issues to overcome the burden of leptospirosis. Here, we have studied the immunogenic potential of prepared recombinant Loa22 protein (rLoa22) of local pathogenic Leptospira species in mice and its ability to induce humoral and cellular immunity, being further evaluated by analyzing the immunoglobulin G (IgG) subclasses and cytokines produced through immunization. Based on the results, mice immunized with rLoa22/adjuvant and a trivalent vaccine, induced high titers of total IgG. All immunized groups increased IgG1 almost on the same level; but, IgG2a level was significantly higher in the vaccine and rLoa22/adjuvant groups than rLoa22 alone group. Animals immunized with the vaccine produced more interleukin 4 than rLoa22/ adjuvant group. The results of evaluating interferon gamma level showed that the rLoa22/adjuvant and vaccine groups had a significant increase compared to the rLoa22 alone group. The results also demonstrated that the rLoa22 protein, in indirect enzyme-linked immunosorbent assay, was able to detect the anti- Leptospira antibodies in mice serum that can be used as a marker in assessing the seroprevalence of leptospirosis and/or in combination with other leptospiral antigens in development of an effective vaccine against leptospirosis. (c) 2024 Urmia University. All rights reserved.

Background and Objectives: Leptospirosis is a zoonotic disease caused by pathogenic Leptospira serovars. The genus Leptospira cannot differentiated by conventional techniques. However, identity determination of pathogenic serovar is pre- cious of public health problems and epidemiological studies. Pulsed-field gel electrophoresis facilitates rapid identification of Leptospires to the serovar levels. Materials and Methods: In this study, we employed PFGE to evaluate 28 Leptospira isolates, with animal, human and en- vironmental origin, obtained from Razi Vaccine and Serum Research Institute of Karaj, Iran. PFGE patterns of 28 Leptospira serovars were generated using the Not I restriction enzyme in comparison with the lambda ladder. Results: Out of 28 serovars evaluated, we identified 22 different pulsed types, designated P1- P22. Out of 22 pulse groups, 3 were found to be a common type, but others were a single Type. Groups consisting of the common type were P3, P9, P14, and P16. The results showed that the discriminatory index of PFGE by Not I enzyme was 0.99, demonstrating heterogeneous differentiation among serovar members. Conclusion: The PFGE methodology used in this study showed excellent interlaboratory report usability, rapid, reliable, enabling standardization and data sharing between laboratories.

The loa22 protein is highly conserved among pathogenic Leptospira serovars and it is expressed during both acute and chronic infections. The aim of this study was to clone and sequence of the loa22 protein-encoding gene of Leptospira serovars. In this study, 23 pathogenic Leptospira serovars and two nonpathogenic Leptospira serovars were used. These serovars were obtained from the microbial culture collection of Leptospira Reference Laboratory, Department of Microbiology, Razi Vaccine and Serum Research Institute, Karaj, Iran. Three serovars, including L. Sejroe Hardjo-bovis, L. Grippotyphosa, L. Canicola, are used in the preparation of the trivalent vaccine. The loa22 gene was amplified by specific primers and the PCR products were then purified using kit and were cloned into a pTZ57R/T vector and transformed in competent E. coli DH5α cells. The cells were then plated onto LB agar containing ampicillin and recombinant colonies subjected to colony PCR to confirm the presence of the Leptospiral gene. Positive colonies plasmid vector was isolated from cells by High Pure Plasmid Isolation Kit. The loa22 gene was detected in all 23 pathogenic serovars, while this gene was not observed in nonpathogenic L. biflexa. It was determined that the similarity percentage of the sequenced pathogenic serovars is between 95.5% and 100%. The results concluded that the loa22 gene was highly conserved among various pathogenic Leptospira serovars and can be used to develop an effective recombinant vaccine.

Background and Objectives: Leptospirosis is an infectious zoonotic disease that can result in severe complications. It is widespread, especially in hot and humid climates such as the northern region of Iran. The immune responses to leptospirosis are multifaceted. Lipl41 is an outer membrane protein that is expressed during infection and is highly conserved among pathogenic species. This makes it a good candidate for diagnosis and induction of specific immune responses. The aim of the present study was to evaluate immune responses against recombinant Lipl41 in mice. Materials and Methods: After immunizing of different groups of mice with recombinant Lipl41 (rLipl41), the levels of specific antibodies and cytokine profiles interferon-gamma/ interleukin-4 (IFN-γ/IL-4) were measured. Results: The results revealed that rLipl41 showed a significant increase in antibody levels compared with the control groups (P< 0.05). Although the level of IL-4 in the groups that received Lipl41 was similar to that in the other control groups, the IFN-γ levels showed a significant increase (P<0.05). Conclusion: It has been concluded that recombinant Lipl41 protein could strongly stimulate specific immune responses and be considered a potential candidate for vaccine development and diagnostic research.

Leptospirosis is a multi-faceted global disease, which causes great economic and health losses to the livestock and human population of our country each year. The northern provinces of Iran are among the rainy and humid regions of Iran, and the prevalence of waterborne diseases such as leptospirosis is high in these regions. In ruminants, cattle are more susceptible to leptospirosis than small ruminants. The purpose of this study was to determine the serological prevalence of leptospirosis in slaughtered cows in Guilan province using the standard microscopic agglutination test (MAT). For this purpose, 300 blood samples were collected from slaughtered cows in Guilan province during the spring and summer of 2023. After separating the serum and sending them to the leptospirosis research laboratory, all the samples were tested by MAT method using five serotypes of Leptospira Grippotyphosa, Pomona, Icterohaemorrhagiae, Canicola and Hardjo. The results of this study showed that 62 samples (20.66 percent) had anti-leptospiral antibodies against one of the above-mentioned 5 serotypes. The highest titer was related to Hardjo serotype with 37 samples (59.6 percent) and the lowest was related to Icterohaemorrhagiae serotype with 5 cases (8.06 percent). Titration of positive samples showed that 59 samples (86.8 percent) had a titer of 1:100 to 1:200 and 9 samples (13.2 percent) had a titer of 1:400. In conclusion, this study revealed that infection with L. interrogans is highly prevalent in slaughterhouse cows of Guilan province and the active presence of the cause of this disease in the province.

Iranian men are at risk of developing gastrointestinal cancer caused by H. pylori. It is very imperative to find effective methods to control this bacterium as there are currently no very effective treatments for it. Honey has been shown to have antimicrobial properties against various pathogens. This study analyzed 15 honey samples from A. florea bees, collected from different floral and geographical origins, for their antimicrobial efficacy against H. pylori. Using atomic absorption measurements, the honey samples were also tested for their phenolic and flavonoid content, protein concentration, and mineral content. Antioxidant activity was determined using the FRAP, DPPH, and ABTS methods. The antibacterial activity of honey samples was investigated both in-vitro and in-vivo in the gastrointestinal tract of mice. Statistical analysis revealed a significant positive correlation between antioxidant activity and antibacterial activity. All honey samples showed antimicrobial activity in-vitro, among which jujube honey from Bushehr exhibiting the highest activity. Differences in antioxidant and antimicrobial activities were likely due to the flora of the plants and the geographic region from which the honey was harvested. Based on these results, A. florea honey may be used in the prevention and treatment of H. pylori-associated infections and inflammation of the gastrointestinal tract. This feature can be applied to the control of Helicobacter pylori along with other available measures.

Background: Leptospirosis, as an emerging global public health problem, is a widespread zoonosis and substantial infectious disease caused by pathogenic Leptospira spp. The diagnosis of the disease remains a challenge due to its non-specific clinical symptoms. The Lsa21 protein is one of the leptospiral proteins that has extracellular matrix-binding properties that are expressed during the infection of pathogenic Leptospira serovars. Objectives: The study aimed to assess the presence of the lsa21 gene in Leptospira serovars. Materials and Methods: The study was conducted on 22 pathogenic Leptospira serovars and non-pathogenic Leptospira serovars, obtained from the Reference Laboratory for Leptospira, Razi Vaccine, and Serum Research Institute, Karaj, Iran. DNA Leptospira serovars were extracted and amplified by the polymerase chain reaction (PCR) using specific primers. Results: An approximately 540-bp DNA fragment from the DNA of all pathogenic Leptospira serovars was amplified by the PCR not found in non-pathogenic serovars. Conclusion: Molecular detection of pathogenic leptospires based on the lsa21 gene can be utilized for laboratory diagnosis and could be a good candidate for developing vaccines against leptospirosis.

Background and Objectives:One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. The study aims is to achieve the optimum conditions for high expression of recombinant Loa22 (rLoa22) protein. Materials and Methods:Complete coding sequence of loa22 gene sub-cloned into a pET32a (+) expression vector. BL21 competent E. coli (pLysS) used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers. Then expression conditions optimized by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration. Results:SDS-PAGE analysis showed that high production of rLoa22 protein obtained when post induction incubation, IPTG concentration, and duration of induction were 37°C, 0.1 M and 5 h in 2×TY medium respectively. The purification of rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Conclusion:The findings of the study led to high production of pure Loa22 protein, which can form the basis for future investigation on the design of rapid diagnostic tests and more effective vaccine candidates for leptospirosis.

Leptospirosis is an emerging infectious zoonotic disease caused by pathogenic Leptospira. The disease is more prevalent among farmers in hot and humid areas of Iran. Lack of clear clinical signs have impeded the diagnosis of leptospirosis. In this study, we attempted to produce a recombinant LipL21 protein of Leptospira based on a dominant pattern of Iranian isolates and to evaluate it in ELISA test. One hundred and sixty-two complete sequences of LipL21 available in GenBank until January 1, 2019 were compared. One dominant LipL21 protein pattern was selected. The codon optimised sequence was cloned into the pET32a+ expression vector. Trx-LipL21 fusion protein was induced, purified and confirmed by 10% SDS-PAGE followed Coomassie blue staining and immune blotting. For evaluation of effectiveness of rLip21 in ELISA test, 200 µg rLip21 with Montanide ISA70 adjuvant was injected subcutaneously in rabbits three times. Results showed that rLipL21 protein was highly expressed in 2YT media in presence of 0.1 mM IPTG after 16 hours incubation at 37 oC. Recombinant protein was purified 36 mg per liter using affinity batch formation method by Ni-NTA resin. ELISA with micro plate coated with 250 ng rLipL21 protein demonstrated prominently diffe­rences between test and control groups (P<0.01). The rLipL21 protein produced large amounts of antibodies in the rabbit. The protein was also able to detect high levels of antibody in animals immu­nised with Leptospira vaccine. The rLipL21 might be a good candidate for diagnosis and evaluation of antibody levels against Leptospira.

Background & Aim: Diphtheria is an acute vaccine-controlled respiratory disease caused by the grampositive bacterium Corynebacterium diphtheria.Since the preparation, purification, and detoxification of the vaccine against this disease involves many and complex steps, the present study aimed reduction of time of various stages of toxoid purification.Materials & Methods: For this purpose, three factors were first optimized for the concentration of iron in the culture medium, incubation temperature and rotation speed to produce more toxins.In each case, the experiment was carried out in triplicate and Lf and Kf values were tested to evaluate toxin production.After toxin inactivation and production of the toxoid, step reductions were performed to purify the toxoid.For this, ultrafiltration, precipitation, dialysis and chromatography with Sephadex G-25 column are used.In step reduction conditions, dialysis, chromatography or both of them were removed in the first, second and third cases, respectively.The results of each method were evaluated by SDS-PAGE and the quantitative data was analyzed using Minitab 18 (p‹0.05). Results:The results for optimizing the culture medium showed that highest amounts of toxin were produced at an iron concentration of 1 mg/l, a temperature of 35 °C, and a rotation speed of 200 rpm(p‹0.05).In the reduction experiments related to toxoid purification steps, the best results were obtained in a series of purified samples with ultrafiltration, precipitation and column chromatography steps.Conclusion: Since time and cost are two key factors in industrial production, toxoid production for the diphtheria vaccine can be produced at a lower cost and in less time by eliminating the two-week dialysis phase.

Salmonella is a zoonotic bacterium that is considered to be one of the most common causes of foodborne infections worldwide. Bearing in mind the genes involved in its virulence, identifying these genes can enable experts to better understand bacterial pathogenicity, which could subsequently help develop more efficient means to control and prevent infections. This study aimed to analyze stn, sipB, and sopB genes in various Salmonella serovars. To carry out this study, 103 Salmonella serovars were extracted from livestock, poultry, and humans from existing samples at the Department of Microbiology of the Razi Serum and Vaccine Research Institute in Karaj, Iran. These samples were cultured in selection and differential media, and their serovars were identified using specific antibodies based on Kaufman-White Tables. Utilizing PCR and specific primers, stn, sopB, and sipB genes were detected among these serovars. In this investigation, the most common human serovars were Salmonella paratyphi A, Salmonella paratyphi B, and Salmonella enteritidis; the most common serovars among livestock consisted of Salmonella dublin and Salmonella typhimurium and the most common Salmonella serovars among poultry consisted of Salmonella infantis and Salmonella enteritidis. The results of PCR on stn, sipB, and sopB genes demonstrated segments with 617bp, 875 bp, and 220 bp on agar gel, respectively. Based on the obtained findings, stn, sipB, and sopB genes were detected in 96.11%, 99.02%, and 98.05% of Salmonella serovars, respectively. Considering the fact that the aforementioned genes play significant roles in bacterial virulence, they can be used to develop diagnostic ELISA kits and recombinant vaccines.

Leptospirosis is a serious zoonotic infection and the most prevalence disease is in the tropical and subtropical region. The definitive diagnosis of Leptospirosis, caused by spirochetes of the genus Leptospira infection is already using culture methods, serological tests such as the microscopic agglutination test (MAT) and molecular detection methods (PCR) are possible. In this study, we used multiplex PCR method for detection of pathogenic and non - pathogenic Leptospira based on lipL32 and 16S rRNA genes. All serovars were obtained from the Leptospira Reference Laboratory of Microbiology Department, Razi Vaccine and Serum Research Institute, Karaj, Iran. The PCR product for the lipL32 and 16S rRNA genes was 272 bp and 240 bp respectively. The sensitivity amplification for the multiplex assay was 10-6 pg / μl for 16S rRNA gene and 10-4 pg / μl for lipL32 gene. The sensitivity for multiplex PCR was 10-3 pg / μl. The results supported the idea that multiplex PCR can be used to detect Leptospira samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much easily than conventional methodologies. Due to the slow growth of Leptospira and the importance of time in diagnosis, molecular methods such as PCR are suggested.

Background and Aim: Leptospirosis is one of the most common zoonotic diseases worldwide, occurring mostly in tropical, subtropical, temperate, and humid regions with heavy rainfall. It is important to diagnose this condition correctly and promptly. Loa22 is an outer membrane protein exposed to the surface in some Leptospira serovars. The purpose of this study was to determine the presence of the gene encoding Loa22 protein in Leptospira interrogans serovars. Materials and Methods: The present study was conducted on 23 pathogenic leptospira serovars and two non-pathogenic leptospira serovars. These serovars were prepared from the Reference Laboratory for Leptospira, Department of Microbiology, Razi Vaccine, and Serum Research Institute, Karaj, Iran. After genomic DNA extraction using the standard phenol-chloroform method, loa22 gene was amplified by specific primers. Results: PCR was performed on the loa22 gene by producing a 671-bp fragment. The results showed that the loa22 gene was present in all 23 pathogenic leptospira serovars but not in the non-pathogenic L. biflexa . The specificity of tested primers was confirmed as well. Conclusion: The loa22 gene is a specific gene for pathogenic leptospira serovars that is not found in saprophytic serovars, so it is suggested that this gene be used to detect leptospira pathogenic serovars.

Salmonellosis, among poultry infectious diseases, not only imposes economic losses in the field of poultry breeding but also is considered a zoonotic disease. This study aimed to investigate the presence of invA, sivH, and agfA virulence genes in Salmonella species. The present study was conducted on 30 Salmonella strains. Samples were cultured on selective and differential media, and afterward, the isolates were serotyped using specific antisera based on the Kauffman-White table. Subsequently, the samples were analyzed to detect invA, sivH, and agfA genes by polymerase chain reaction technique. The results indicated that 30 (100%) isolates had invA and agfA virulence genes and 28 (93.33%) isolates had a sivH virulence gene. The highest frequency of serotypes was related to Salmonella infantis. Among the studied serotypes, Salmonella uno and Salmonella O35 lacked the sivH virulence gene, unlike other serotypes. The findings of this study could pave the way for Salmonella monitoring and be used as a pattern to detect Salmonella bacteria-bearing genes encoding invasion and fimbria.

Molecular Identification of Salmonella Strains Isolated from Livestock in Alborz Province and Their Serotyping

Background: Leptospirosis is one of the most common zoonotic diseases, which is caused by a Spiral shaped bacterium called Leptospira. The recommended diagnosis method is to perform microscopic agglutination test (MAT) which is both hazardous, due to using live bacteria, and time-consuming. As a result, many attempts have been recently made to develop other serological methods, such as ELISA. Objective: This study aimed to develop an indirect ELISA method using leptospiral whole antigens for diagnosing pathogenic Leptospira. Materials and Methods: In this study, four pathogenic serovars of Leptospira were used and cultured in selective culture medium. The cultured bacteria were sonicated and the extracted antigens were used as captured antigen in ELISA method. A total of 74 samples from bovine suspected to leptospirosis and 43 samples from healthy animals were examined by MAT method. Results: According to the study results, 42 samples (56.7%) out of 74 suspected ones were found positive while 32 ones (43.2%) were determined negative by MAT analysis. All of the 43 negative control samples were found negative after performing MAT. The sensitivity and specificity of ELISA, compared to those of MAT, were measured as 87.5% and 84.2%, respectively. Conclusion: Taking into account the high sensitivity and appropriate specificity of the developed indirect ELISA method, it was recommended that ELISA be employed as an accurate method for early and rapid diagnosis of bovine leptospirosis.

Leptospirosis is a zoonotic disease with global importance, and the animals are the source of transmission of this disease through shedding in their urine. Accordingly, it is essential to conduct epidemiological studies of leptospirosis in order to diagnose this disease in dogs and reduce the risk of transmission to humans. This study aimed to perform a seroepidemiological analysis of Leptospiral infection in stray dogs in Alborz, Iran, using the Microscopic Agglutination Test (MAT). In total, 110 blood samples were collected from stray dogs to detect the antibodies against leptospira interrogans serovarsby the MAT. The prevalence rate of positive MAT tests in stray dogs was estimated at 21.84%. The following protocol confirmed that the most common titers were 1:200 (50%) and 1:400 (25%). In addition, the most prevalent Leptospira serovars were L. Canicola (33.33%), and the lowest belonged to L. Pomona (4.1%). Moreover, no significant difference was observed between the age and gender of the dogs regarding their MAT titer (P>0.05). The results also showed a high prevalence of leptospirosis in stray dogs of Koohsar in Alborz province, Iran. Since Leptospirosis is a zoonosis disease, it should be studied continuously in humans and animals, especially dogs.

Pathogenic species of Leptospira lead to a zoonotic disease called leptospirosis, which is spread worldwide. A major topic of investigation is to detect the antigens that induce an immune response and to utilize them in diagnostic kits or vaccine development. The outer membrane proteins (OMPs) of Leptospira are potential candidates for this purpose. LipI41 is an OMP that is conserved among pathogenic Leptospira. The aim of this study was to express and purify the Lip141 recombinant protein in Iranian isolates. All collected Lip141 protein sequences were compared and analyzed using bioinformatics tools from NCBI databases. Complete codon sequences of the Iranian pattern of LipI41 recombinant protein were codon optimized and sub-cloned into a pET32a+ expression vector, and transformed into Escherichia coli BL21 (DE3). Optimal expression of recombinant LipI41 (47kDa) was achieved post-induction with IPTG within the inclusion body. It was then purified by denaturation using serial concentrations of urea, and the recombinant protein was confirmed by western blot. In this study, sufficient amounts of Lip141 were expressed and purified to be used for the development of a diagnostic kit and subunit vaccine.