Evaluation the efficacy of oral immunization of broiler chickens with a recombinant Lactobacillus casei vaccine vector expressing the Carboxy-terminal fragment of α-toxin from Clostridium perfringens.

BACKGROUND: Clostridium perfringens (C. perfringens) is a serious anaerobic enteric pathogen causing necrotic enteritis (NE) in broiler chickens. Following the ban on antibiotics as growth promoters in animal feedstuffs, there has been a remarkable rise in occurrence of NE which resulted in considering alternative approaches, particularly vaccination. The objective of this work was to evaluate the recombinant Lactobacillus casei (L. casei) expressing the C-terminal domain of α-toxin from C. perfringens as a potential probiotic-based vaccine candidate to immunize the broiler chickens against NE. RESULTS: The broiler chickens immunized orally with recombinant vaccine strain were significantly protected against experimental NE challenge, and developed specific serum anti-α antibodies. Additionally, the immunized birds showed higher body weight gains compared with control groups during the challenge experiment. CONCLUSIONS: The current study showed that oral immunization of broiler chickens with a safe probiotic-based vector vaccine expressing α-toxin from C. perfringens could provide protective immunity against NE in birds.

Targeted gene inactivation in Salmonella Typhi by CRISPR/Cas9-assisted homologous recombination.

BACKGROUND: Targeted gene inactivation (TGI) is a widely used technique for the study of genes' functions. There are many different methods for TGI, however, most of them are so complicated and time-consuming. New promising genetic engineering tools are developing for this purpose. In the present study, for the first time we disrupted a virulence gene from Salmonella enterica serovar Typhi (S. Typhi), located in the bacterial chromosome using CRISPR/Cas9 system and homology directed repair (HDR). METHODS: For this aim, pCas9 plasmid containing Cas9 enzyme and required proteins for homology directed recombination was transferred to S. Typhi by electroporation. On the other hand, a specific guide RNA (gRNA) was designed using CRISPOR online tool. Synthetic gRNA was cloned into pTargetF plasmid. Also, a DNA fragment (HDR fragment) was designed to incorporate into the bacterial chromosome following the cleavage of the bacterial genome by Cas9 enzyme. pTargetF containing gRNA and HDR fragment were co-transferred to S. Typhi containing pcas9 plasmid. The transformed bacteria were screened for recombination using PCR, restriction digestion and sequencing. RESULTS: The results of PCR, restriction digestion and sequencing showed the successful recombination of S. Typhi, in which the gidA gene is disrupted. CONCLUSION: In the present study we aimed to develop a rapid and robust method for targeted gene inactivation in a bacterial species, S. Typhi. This procedure can be exploited for disruption of other Salmonella as well as other bacteria's genes.

Development of a diagnostic indirect ELISA test for detection of Brucella antibody using recombinant outer membrane protein 16 kDa (rOMP16).

Brucellosis is considered as one of the important global zoonotic diseases that causes medical as well as economic problems especially in tropical countries. The illness has no specific pathognomonic signs; therefore, the rapid and accurate diagnosis of the disease has a very important role in preventing the Brucella spillover and treatment. The purpose of this study was to design a new indirect ELISA test for detection of human brucellosis based on using recombinant Brucella abortus outer membrane protein 16 kDa (rOMP16) as an antigen. OMP16 gene of B. abortus was initially synthesized and cloned in pET-21d vector and then expressed in Escherichia coli cells. The expression was confirmed by the SDS-PAGE, western blotting and dot blotting. The purified protein was coated in ELISA plates and an indirect ELISA was performed on 70 human serum samples. The results were evaluated with a commercial IgG ELISA kit and Rose Bengal plate agglutination tests as reference tests. Diagnostic performance of designed OMP16 ELISA test in comparison with Rose Bengal plate test revealed 100% of sensitivity, 95.00% of specificity and good Fleiss kappa agreement, whereas, where it was compared to commercial ELISA kit, it revealed very good kappa agreement with 100% of sensitivity and 100% of specificity in cut-off value of 0.13. It was concluded that OMP 16 kDa could be acceptable alternative antigen for detecting Brucella IgG antibody with high accuracy.

Lactobacillus casei displaying Clostridium perfringens NetB antigen protects chickens against necrotic enteritis.

Necrotic enteritis is a serious economical disease of poultry caused by Clostridium perfringens. NetB toxin of Clostridium perfringens is considered the causative agent of necrotic enteritis. Following the withdrawal of in-feed antibiotic growth promoters, there has been an urgent need to develop alternative approaches such as vaccination. Currently, there are no commercially available vaccines to control necrotic enteritis especially in broiler chickens as the target population. In the present study, we constructed a recombinant Lactobacillus casei strain expressing NetB protein of C. perfringens on the cell surface and used this probiotic-based vaccine strain to immunize broiler chickens orally against experimental induction of necrotic enteritis. The birds immunized with the oral vaccine strain were significantly protected against necrotic enteritis challenge and developed strong serum anti-NetB antibody responses to NetB protein. Furthermore, the immunized birds showed higher body weight gains during the challenge experiment compared with control birds. This study showed, for the first time, that a probiotic-based vector vaccine could be a promising vaccine candidate to provide protection against necrotic enteritis in broiler chickens. KEYPOINTS: • The probiotic L. casei carrying pT1NX-netB plasmid displayed NetB antigen on the cell surface. • The LC-NetB vaccine strain induced high anti-toxin antibody response in broiler chickens. • The LC-NetB vector vaccine provided significant protection against experimental NE challenge.

Immunogenicity of a recombinant Lactobacillus casei, surface-expressed H151P mutant of Clostridium perfringens epsilon toxin and its protective responses in BALB/c mice.

Epsilon toxin (Etx) is the most important virulence factor of type D C. perfringens in ruminants. The recombinant vaccines can be used against Etx intoxication. This study aimed to investigate the humoral immune responses of mice against a recombinant Lactobacillus casei which surface-expressed H151P mutant of Etx (L. casei-ε) after oral and parenteral immunization routes. The protective immunity was determined by challenge with trypsin-activated Etx. Higher humoral immune responses were seen in parenterally vaccinated mice with Freund's-adjuvanted L. casei-ε than non-adjuvanted and negative controls (P<0.05). In the oral immunized mice, L. casei-ε displayed a significant difference in IgG titres compared with the negative controls. Challenge results showed full protection of oral immunized mice against one and two MLDs, and partial protection against 10 MLD of the trypsin-activated Etx, whereas, the parenteral immunized mice only induced 75 % of protection against one MLD. This may be related to the appropriate immunity responses by L. casei-ε at the mucosal surfaces, which highlights the role of the oral immunization. Thus, L. casei-ε can be considered as an oral vaccine candidate against Etx intoxication and enterotoxaemia.

Development of a double-recombinant antibody sandwich ELISA for quantitative detection of epsilon toxoid concentration in inactivated Clostridium perfringens vaccines.

BACKGROUND: Epsilon toxin (ETX) causes a commonly fatal enterotoxemia in domestic animals. Also, ETX causes serious economic losses to animal husbandry. In this study, we selected several clones against ETX using repertoires displayed on filamentous phage. Anti-ETX specific clones were enriched by binding to immobilized antigen, followed by elution and re-propagation of phage. After multiple rounds of binding selection, ELISA analysis showed that most isolated clones had high affinity and specificity for ETX. RESULTS: Two recombinant monoclonal antibodies against ETX were isolated by phage display technology. B1 phage VH antibody isolated from DAb library and G2 soluble scFv antibody isolated from Tomlinson I + J libraries have been applied as the capture and detection antibodies for developing an ETX sandwich ELISA test, respectively. CONCLUSIONS: Designed ETX sandwich ELISA could be a valuable tool for quantitative detection of ETX in inactivated commercial vaccines against enterotoxemia.

Electrochemical immunosensor for determination of Staphylococcus aureus bacteria by IgY immobilized on glassy carbon electrode with electrodeposited gold nanoparticles.

A new ultrasensitive immunosensor is proposed based on the covalently attached anti-protein A antibody (IgY) on deposited gold nanoparticle (AuNP)-modified glassy carbon electrode (GCE) for the electrochemical measurement of Staphylococcus aureus (S. aureus). Chicken IgY as a capture antibody provides highly selective and specific binding to the target bacteria and selectively captures the S. aureus in its three-dimensional space. Due to that it can eliminate the interference from protein G-producing Streptococcus. In addition, the electron-transfer characteristic of [Fe(CN)6]4-/3- is hindered by this combination; as it is reflected on the electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) curves. The proposed immunosensor displays a wide linear dynamic range from 10 to 107 CFU mL-1 with a detection limit of 3.3 CFU mL-1 with RSD 3.0%. It is capable to accurately determine S. aureus in milk and human blood serum as a complex matrix sample with satisfactory recovery of ∼ 97-103%. The immunosensor also displays high selectivity over other bacteria and acceptable stability. Presumably, our study can be regarded as the first one to report chicken IgY in order to detect S. aureus based on an electrochemical method.Graphical abstract.

Application of the Leishmania infantum 21-kDa recombinant protein for the development of an immunochromatographic test.

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania infantum, is a systemic parasitic disease and presents a global health problem which can be fatal if not diagnosed and treated. Dogs are the main hosts and provide reservoirs for the transmission of the disease to humans. METHODS: In this study, the gene encoding a 21-kDa protein was cloned and expressed as a fusion protein in Escherichia coli strain BL21 (DE3) for developing a rapid immunochromatographic test (ICT) to identify infected dogs. The expression of the recombinant 21-kDa protein (r21) was investigated using SDS-PAGE and Western blot methods. The purified r21-kDa protein was spotted onto ICT strips and tested by sera from experimentally infected, naturally infected and uninfected dogs. RESULTS: The SDS-PAGE and Western blot methods showed the successful expression of r21-kDa protein. The ICT strip test revealed that the r21-kDa protein was detected by the sera of experimentally and naturally infected dogs. The specificity tests also confirmed no cross-reactivity with animals infected with Trypanosoma cruzi, Toxoplasma gondii and Ehrlichia canis. CONCLUSIONS: Based on these findings, the new r21-kDa protein may be a suitable target for developing a new simple, specific and rapid serological method to detect VL in infected dogs.

Scanning electron microscopy of Onchocerca fasciata (Filarioidea: Onchocercidae) adults, microfilariae and eggs with notes on histopathological findings in camels.

BACKGROUND: Onchocerca fasciata is a prevalent filarial species in camelids of Asia and Africa forming nodules in the skin of dromedary and Bactrian camels. In spite of recent advances in the biology and epidemiology of this nematode species, a relatively scant number of studies have focussed on the morphology of this parasite. The main objective of this study was to describe morphological characteristics of adults, microfilariae and eggs of O. fasciata by scanning electron microscopy (SEM), staining and histology. METHODS: From April 2016 to March 2017 dromedary camels (n = 456) were inspected for infection with O. fasciata in a slaughterhouse in Kerman (south of Iran). Adult worms in nodules were isolated by digestion of nodules in collagenase and used for SEM. Skin nodules were also fixed, sectioned and stained with hematoxylin and eosin for histopathology. Skin microfilariae that were isolated from tissues surrounding the nodules were confirmed as O. fasciata by sequencing of the cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes and used for SEM and Giemsa staining. RESULTS: Single or multiple O. fasciata nodules (1.2-2.2 cm in diameter and 507-845 mg in weight) were found in 30.3% of the examined camels. SEM analysis helped identify 18 papillae in the caudal region of the male. Discontinuous longitudinal cuticular crests were observed in the posterior region of the male. In female nematodes, the ridges had a rounded shape with a height/width ratio of 7/16 in longitudinal sections. Unsheathed skin microfilariae with a rounded anterior extremity measured 210.7 × 2.5 µm on average. Developed eggs containing microfilariae measured 35.9 × 31.0 µm and their smooth shell surface had characteristic tongue-like appendages. In addition to inflammatory reactions surrounding the parasites, accumulation of intracellular ceroid pigment, golden-yellow to brown in colour, was observed within macrophages upon histopathological examination. CONCLUSIONS: We found longitudinal crests on the surface of the posterior region of the male nematode. Measurements of the main morphological features of microfilariae and eggs, and the shape index of ridges (height/width) in female nematodes are described for the first time.

High protection of mice against Brucella abortus by oral immunization with recombinant probiotic Lactobacillus casei vector vaccine, expressing the outer membrane protein OMP19 of Brucella species.

Brucellosis is a zoonotic disease threatening the public health and hindering the trade of animals and their products, which has a negative impact on the economic development of a country. Vaccination is the most effective way to control brucellosis. The recombinant vector vaccines are promising candidates for immunization in humans and animals. In this study, the gene encoding OMP19 antigen was primarily amplified and cloned into an expression vector called pT1NX, and then transformed to L. casei cell via electroporation technique. The expression was confirmed using specific antibody against the recombinant protein via immunological screening tests such as western blot and immunofluorescence assay. Finally, recombinant L. casei was orally fed to mice and the results were further recorded, indicating that the mice group which received OMP19 through L. casei based vaccine represented a very good general and mucosal immune responses protective against challenges with virulent B. abortus 544 strain compared with negative control recipient groups. Therefore, the vaccine produced in this research plan can be a very good candidate for protection against brucellosis.

Immunological properties of the SLLTEVET epitope of Influenza A virus in multiple display on filamentous M13 phage.

Small peptides require large carriers to stimulate the humoral immune system. The filamentous phages, such as M13, have been proposed as vectors for expressing and carrying these peptides on their capsid surface. M2e 2-9 (SLLTEVET) residues of the transmembrane protein M2 of Influenza A virus are conserved and considered as a suitable target for immunization against a wide range of Influenza A virus strains. Here, M2e (2-9) sequence of Influenza A virus was fused to the N-terminus of the major coat protein gpVIII of M13 phage and was used to immunize broiler chickens. The results showed that the SLLTEVET peptide on the surface of M13 phage was expressed, the hybrid phage was immunogenic and produced specific antibodies against M2e (2-9) in broiler chickens.

Detection of Brucella abortus by immunofluorescence assay using anti outer membrane protein of 19 kDa antibody.

BACKGROUND: Brucellosis in humans is one of the most prevalent zoonotic diseases around the world with more than 500,000 new cases per year. It is a weakening disease that requires long-term antibiotic treatment, often resulting in permanent and disabling consequences. Outer membrane proteins (OMPs) of Brucella, which are non-lipopolysaccharide (LPS) antigens, have been used for the diagnostic kits of brucellosis and vaccine design. OBJECTIVES: The aim of this study was to identify Brucella abortus with an immunofluorescence (IF) test using an antibody against recombinant outer membrane protein (OMP) of 19 kDa of this bacterium. MATERIAL AND METHODS: The OMP19 gene of Brucella spp. was synthesized, cloned and expressed in Escherichia coli cells. The OMP19 protein was purified by metal chelate affinity chromatography and subsequently used for the immunization of rabbits to produce a polyclonal antibody. Then, this antibody was conjugated to fluorescein isothiocyanate (FITC) and used for the detection of Brucella by an IF test. Also, the sensitivity and specificity of this antibody for the diagnosis of clinical isolates was calculated. RESULTS: Outer membrane protein 19 was expressed well and reacted with a commercial antiserum against His-tag in an immunoblot assay. Polyclonal antibodies obtained from the serum of rabbits immunized with the purified protein showed strong reactivity in the enzyme-linked immunosorbent assay (ELISA). Moreover, the polyclonal antibody conjugated to FITC was able to properly identify Brucella abortus. Sensitivity and specificity of this IF test in comparison with a polymerase chain reaction (PCR) assay was 84.2% and 50%, respectively. CONCLUSIONS: This high-titer antibody could potentially be valuable for the specific diagnostic test of brucellosis.

A Recombinant Probiotic, Lactobacillus casei, Expressing the Clostridium perfringens α-toxoid, as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis.

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa 247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.

Recombinant M2e-HA2 fusion protein induced immunity responses against intranasally administered H9N2 influenza virus.

Influenza is a highly contagious respiratory tract disease and is considered a serious community health problem. Influenza viruses possess multiple conserved epitopes which are used for designing universal vaccines. To this aim, the gene coding for N-terminal part of M2e (SLLTEVET) and HA2 (GLFGAIAGF), was synthesized, linked by a (Gly4Ser)4 peptide linker, and cloned into pGS-21a vector. Afterwards, the construct was transferred into E. coli BL21 (DE3) cells to produce the designed antigenic protein called M2e-HA2. Immunization of mice with these peptides significantly induced humoral immune responses against the influenza virus. Three weeks after the last booster, mice were inoculated intranasally with 1 × 106 EID50 of H9N2 virus. The results indicated that the recombinant M2e-HA2 fusion protein could protect mice against H9N2 virus.

Orally administered recombinant Lactobacillus casei vector vaccine expressing ß-toxoid of Clostridium perfringens that induced protective immunity responses.

Clostridium perfringens types B and C cause enteritis and enterotoxemia in animals. The conventional vaccine production systems need time-consuming detoxification and difficult quality control steps. In this study, a modified ß-toxoid gene was synthesized, cloned into the pT1NX vector, and electroporated into Lactobacillus casei competent cells to yield L. casei-ß recombinant strain. Surface expression of the recombinant ß-toxoid was evaluated by ELISA and confirmed by immunofluorescence microscopy. Vaccinated BALB/c mice with L. casei-ß induced potent humoral and cell-mediated immune responses that were protective against lethal challenges with 100 MLD/mL of the ß-toxin. Safety and efficacy of the recombinant clone was evaluated and the presumptive toxicity of L. casei-ß was studied by toxicity test and histopathological findings, which were the same as negative controls. Our results support the use of L. casei as a live oral vector vaccine, and that the recombinant L. casei-ß is a potential candidate for being used in the control of enterotoxemia diseases caused by C. perfringens types B and C.

A comparison of methods for extracting plasmids from a difficult to lyse bacterium: Lactobacillus casei.

There are few practical protocols to extract efficient plasmid DNA from the difficult-to-lyse bacterium, Lactobacillus casei. This is related to production of a large amount of exopolysaccharide coat and its special physiological characteristics. In this study, we optimized a protocol to extract efficient plasmid DNA from a recombinant L. casei strain. Different extraction methods were evaluated in three classes of conventional, kit-based, and combined protocols. The quantity and quality of the extracted plasmid DNA were determined by spectrophotometry, agarose gel electrophoresis, and PCR. Results revealed that the yield of the extracted plasmids differed for each protocol and conventional protocols showed higher plasmid yields. We suggested an effective, inexpensive protocol to extract plasmid DNA from the recombinant L. casei for downstream biological processes.

First serological study of equine hydatidosis in Iran.

Hydatidosis, is an important worldwide zoonotic disease caused by larval stages (metacestodes) of tapeworm parasites of the genus Echinococcus. The objective of the present study was to determine the seroprevalence of equine hydatidosis in Iran by latex agglutination test. This study also served to correlate sex and age with mentioned results in cases. Therefore, 193 serum samples were collected from clinically healthy horses at 9 race clubs in Kerman, Yazd and Golestan provinces, Iran. According to the results, antibodies against hydatidosis were detected in 6 sera (3.11 %) among 193 samples. Results showed two male and four female horses were sero-positive against hydatidosis. In conclusion, present study shows that antibodies against hydatidosis have been detected in Iran equine population. Therefore, it seems that Iranian horse clubs should improve their management and health levels to increase their proficiencies.

Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid.

Clostridium perfringens type D infects ruminants and causes the enterotoxemia disease by ε-toxin. A mutated ε-toxin gene lacking toxicity was designed, synthesized, and cloned into the pT1NX vector and electroporated into Lactobacillus casei competent cells to yield LC-pT1NX-ε recombinant strain. BALB/c mice, immunized orally with this strain, highly induced mucosal, humoral, and cell-mediated immune responses and developed a protection against 200 MLD/ml of the activated ε-toxin. This study showed that the LC-pT1NX-ε could be a promising vaccine candidate against the enterotoxemia disease.

Prevalence and risk factor of Q fever among veterinary students in Iran.

Q fever, caused by Coxiella burnetii, is a zoonosis with a worldwide distribution. This study is aimed to determine seroprevalence of Q fever and to identify the correlation between 8 risk factors for Q fever among students at Faculty of Veterinary Medicine in the study in Iran. In the present study, 121 blood samples (serum) were taken from students and tested using indirect diagnostic ELISA kit. The data were analyzed using descriptive statistics and 95% confidence interval, Chi-square statistical test, and logistic regression. Results showed that 34.7% were positive from all the serum samples. Results of the regression test showed that correlation only between age (P-Value = 0.038) and sex (in women; P-Value = 0.05, OR = 2.22 95% CI = [1.00 - 4.90]) with positive serum titer of acute Q fever. According to the results, high seroprevalence of Q fever was observed among the veterinary students. This problem can be solved by taking more careful preventive measures against this disease in the training centers and veterinary students.

Production and characterization of monoclonal antibodies against aflatoxin B1.

In this article, we embarked on production of mouse monoclonal antibodies against aflatoxin B1 which is the most commonly occurring fungal toxin in food and feed products. After immunization and fusion with myloma cells, two stable clones (A218 and B319) were selected. Isotyping showed that these monoclonal antibodies (mAbs) were IgG2b with kappa light chains. The affinity of A218 and B319 clons were 5×10(11) M(-1) and 6×10(9) M(-1), respectively. Competitive indirect ELISA results indicated these mAbs had complete (100%) cross-reaction with four major types of aflatoxins: B1, B2, G1, and G2. These mAbs could be used for immunoassay measurement of aflatoxins with high affinity and low detection limits.