Archives of Razi Institute
Volume:74 Issue: 2, Spring 2019

One of the most important QC tests of whole-cell pertussis vaccine (WCPV) is potency test. In this regard, mouse protection test (MPT) is the current potency method, which is associated with severe animal distress and large variability in results. The purpose of this study was to assess Pertussis Serological Potency Test (PSPT) as a serological alternative method to intracerebral challenge in MPT assay. In the current study, the potency of three experimental batches of WCPV (1, 2, and 3) and standard vaccine were compared using MPT and PSPT methods. In the MPT method, mice were immunized with tests and standard vaccines. After 2 weeks, they were intracerebrally challenged with Bordetella pertussis strain (18323). The potency was calculated via parallel line analysis based on the numbers of survivors 2 weeks after the challenge. Similar to MPT method, mice were immunized in the PSPT method and bled after 4 weeks. In the next step, sera were titrated by 18323-WCP-ELISA assay and potency values were estimated via parallel line analysis. Pearson correlation test was used to measure the strength of association between MPT and PSPT assay results. The potency values of the experimental laboratory batches 1, 2, and 3 in MPT assays were 11.14, 5.02, and 4.24 Iu/ml, whereas the obtained results of PSPT assays were 10.32, 4.11, and 3.06 Iu/ml, respectively. The correlation of MPT and PSPT results was 0.807. The findings of the present study demonstrated a significant correlation between MPT and PSPT results. The implementation of PSPT was more advantageous, compared to MPT due to its ethical approaches and less variability in results. The PSPT is a promising alternative method for intracerebral challenge. However, additional validation is needed to support the establishment of this method.

Mannheimia haemolytica is responsible for considerable economic losses to cattle, sheep, and goat industries in many parts of the world. This bacterium isone of the causative agents of shipping fever in cattle. Current vaccines against M. haemolytica are moderately efficacious since they do not provide complete protection against the disease. Production of an economic vaccine for protecting farm animals against M. haemolytica has attracted the attention of many scientists. The outer membrane proteins (OMPs) play a major role in the pathogenesis and immunogenicity of M. haemolytica. Research on M. haemolytica OMPs has shown that antibodies to a particular OMP may be important in immune protection. In the current study, the gene for M. haemolytica OMP PlpE was cloned into the expression vector pET26-b, and then expressed in Escherichia coli BL21. The expression of the protein was carried out by the induction of cultured Escherichiacoli Bl21 cells with 1mM isopropyl-β-D-thiogalactopyranoside. The recombinant PlpE was purified using Ni-NTA agarose resin, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The identity of the expressed protein was analyzed by western blotting. It was revealed that rPlpE was expressed and produced properly. To assess the immunogenicity of the recombinant protein, the purified rPlpE was used as an antigen for antibody production in goats. The observations suggested that the produced recombinant protein can be used as a antigen for developing diagnostic tests and or as a vaccine candidate.

Chlamydiae are obligate generally Gram-negative intracellular parasites with bacterial characteristics, including a cell wall, DNA, and RNA. They have a worldwide distribution in different animal species. Chlamydia felis (C. felis) is an important agent with zoonotic susceptibility often isolated from cats with chronic conjunctivitis. The aim of the present survey aimed to determine the molecular occurrence of C. felis in cats in Ahvaz, Iran. In this regard, a total of 152 cats (126 households and 26 feral) were included in the current study. After recording their history information, two swabs were taken from the oropharyngeal cavity and eye conjunctiva of the investigated cats. The extraction of DNA was followed by PCR targeting the pmp gene of C. Felis. In the next step, the positive samples were sequenced based on the Gene Bank. Out of 152 samples, 35 (23.03%) were positive using polymerase chain reaction technique (95% CI: 16.30-29.70). Regarding infection with Chlamydiosis, the obtained results showed a significant difference between cats suffering from ocular or respiratory diseases (44.64%; 25 out of 56) and the healthy ones (10.42%; 10 out of 96; P=0.01). The prevalence of infection was significantly higher in cats younger than 1 year (34.12%; 29 out of 85), compared to those older than 1 year (8.96%; 6 out of 67; P=0.02). No significant difference was noted in terms of gender (25.45% in males and 21.65% in females), breed (23.81% in DSH and 19.23% in Persian), and lifestyle (22.22% companions [28 out of 126] and 26.92% ferals [7 out of 26]; P>0.05). It can be concluded that a significant number of cats are infected with C. felis in Ahvaz. The use of molecular tests, such as PCR, has revolutionized the diagnosis of chlamydial infections.

Brucellosis is a zoonotic infection that is associated with fever in humans and abortion in animals. The agent of this disease is a facultative intracellular gram-negative coccobacillus called Brucella. There are six classic species, including B. abortus, B. melitensis, B. suis, B. canis, B. neotomae, and B. ovis.  In recent years, four new species have been reported, including Brucella ceti, B. microti, B. pinnipedialis, and B. inopinata. Human disease causes hygienic and economic losses, including inactivity of workforces in the community and high cost of treatment. The disease also causes catastrophic losses in the livestock industry. There is no effective vaccine against human brucellosis. Hence, attempts to prevent human infection with Brucella are focused on preventative measures, including control of infection in livestock, which lead to a reduction in its incidence in humans. The common methods for diagnosis of this disease are serologic methods including Rose Bengal, Wright -2 ME and the ring test. B. abortus strain S99 is used to produce these diagnostic antigens. The production of these antigens requires the presence of a well-characterized seed with full identity. The aim of this work was confirmation of the identity of B. abortus S99 by phage typing, AMOS and multiplex PCR techniques. Therefore, it is essential to carry out the identification of the strains used as seed for the production of the brucellosis diagnostic antigens. In this project, B. abortus strain 99 was supplied by the bacterial collection of the Brucellosis Department of Razi Vaccine and Serum Research Institute. Then, the main aim of the present study was the confirmation of the seed identity by doing the tests through the standard phage typing method, AMOS PCR and multiplex PCR (Brucladder) methods. Results were in support of the identity of the studied strain, and the molecular methods could also be used as the sensitive approaches for validation of antigenic seed.

Scorpion venom is the richest source of peptide toxins with high levels of specific interactions with different ion-channel membrane proteins. The present study involved the amplification and sequencing of a 310-bp cDNA fragment encoding a beta-like neurotoxin active on sodium ion-channel from the venom glands of scorpion Androctonus crassicauda belonging to the Buthidae family using reverse transcription polymerase chain reaction (RT-PCR) technique. The amplified complementary DNA (cDNA) fragment had a coding sequence of 240 bp. The deduced precursor open-reading frame was composed of 80 amino acid residues contain a signal peptide of 22 amino acid residues, followed by a mature toxin of 58 amino acids. It had a molecular mass of 6.84 kDa and isoelectric point of 4.58. The sequence similarity search revealed several matches with the scorpion toxin-like domain of toxin-3 superfamily with a homology range of 35- 75%. Multiple alignments and secondary structure prediction demonstrated that the toxin peptide deduced from the amplified cDNA was related to the long-chain neurotoxins in size but stabilized by three disulfide bridges instead of four. The level of difference implies that the corresponding genes have originated from a common ancestor. This level of difference may also confirm an evolutionary link between the ‘short-chain’ and ‘long-chain’ toxins. The analysis showed one major segment within this neurotoxin with maximal hydrophilicity which was predicted to be antigenic by inducing an antibody response.

Foot and mouth disease (FMD) is a contagious animal disease that causes irreparable damage to the economy of countries, including Iran in which this disease is a native one. Among the ways to combat FMD are vaccination and slaughter. Due to the specific situation of Iran, it is not possible to kill infected animals. Therefore, vaccination is the most important way to fight this disease. Serum neutralization test (SNT) and enzyme-linked immunosorbent assay (ELISA) are two main methods to evaluate the safety and calculate antibody titer.  In this study, an indirect ELISA test was developed based on the coating of a complete viral particle (140s) which made it possible to determine antibody.  In addition, serotype and viral type were determined without the need for time-consuming and complex molecular tasks, including gene expression. Moreover, in case of a new epidemic, a new epidemic condition can be detected using a serum antibody method. However, the coating of the complete viral particle leads to virus purification as well as the conjugated anti-immunoglobulin antibody testing of the same animal. In this study, the SNT was used as a gold standard test to determine the serum antibody level and compare its results with indirect ELISA method to determine the sensitivity and specificity of the indirect ELISA.  To measure the anti-virus antibody rate of FMD (type A2013) through receiver operating characteristic analysis with 100% sensitivity and the specificity of 90%, the routine formulas were utilized using 100 % and 82%sensitivity and specificity, respectively. In this study, the cutoff value for the optical density was obtained at 0.3 and there was a significant difference between the vaccinated animals and the unvaccinated ones in terms of antibody level against the A2013 type. This indicates the correctness of the test and the accurate and proportional antibody detection against the understudy viral types of FMD.

Staphylococcus aureus is a gram-positive coccus that, in specific conditions, is able to generate various diseases. By secreting different enterotoxins, this bacterium prepares the settings to attack the host; among these, enterotoxins A and B play the most important roles in food poisoning. This study was performed to trace the genes coding enterotoxins A and B in Staphylococcus aureus isolated from the clinic and poultry slaughterhouse. In addition, the present study analyzed the relation between the prevalence of these genes and resistance to erythromycin and tetracycline. This study was performed from October 2015 to December 2016. A total of 200 samples of noses and cloaca from broiler poultry farms in Ilam, Iran, were collected, including 150 samples from the slaughterhouse and 50 samples from the clinic isolated for separating Staphylococcus aureus. After bacterial culture and confirmation of biochemical tests, the samples were evaluated for the identification of Staphylococcus aureus and the resistance pattern to antibiotics regarding the presence of femA, tets, ermb, sea, and seb genes using the disk diffusion method and polymerase chain reaction test. Out of 200 tested samples, 112 strains of Staphylococcus aureus (56%) were identified from which 91 and 21 strains were associated with the poultry slaughterhouse and clinic, respectively, and all the samples were identified using biochemical tests. After the detection of femA gene as an exclusive gene for the identification of Staphylococcus aureus strain, 100 strains (50%) were confirmed to be contaminated with this bacterium. Out of 100 strains, 46%, 14%, and 5% possessed the genes coding enterotoxin A, the genes coding enterotoxin B, and both genes, respectively. The results of antibiotic tests showed that 85% and 86% of the examined strains were resistant to erythromycin and tetracycline, respectively. In the present study, the analysis performed using QuickCalcs software showed that the strains resistant to these two antibiotics possessing the sea gene were more prevalent than those possessing seb genes in the samples isolated from the poultry slaughterhouse. This comparison revealed that during the short period of broiler poultry farms growth, resistant strains were able to proliferate sea gene among the herd, and its prevalence increased until reaching into the slaughterhouse. This study showed that the relation between the genes resistant to erythromycin and tetracycline and the sea gene was close and significant.

Cytomegalovirus (CMV) is a type of herpes virus. This virus is one of the most common causes of congenital and prenatal infections. CMV infection in pregnant women, especially in the first trimester, may lead to congenital abnormalities in newborns. The prevalence of CMV infection in developed countries is approximately 40%, and in developing countries, this prevalence may be up to 100%. Because there is no information available related to the seroepidemiological patterns of this infection in different cities of Alborz province, Iran, this study was conducted. This seroprevalence study was based on sera collected from adults who were referred to health care providers or Medical Diagnostic Laboratory Centers (MDLS) in Alborz province, Iran, from 2011 to 2015 for different purposes. Using ELISA (IgG), a retrospective serological survey of CMV antibodies in serum samples was performed in a non-immunized community. Frozen sera from 2001 individuals who were randomly selected by a cluster sampling technique were collected from spring 2013 to winter 2015. Seroprevalence was stratified by age (>1 to <50 years). Mathematical techniques were used to determine whether there is a relationship between CMV seroprevalence and age, sex, and level of education in this community. Data were analyzed using Fisher’s exact test and the χ2 test using SPSS software version 11.5. The mean age of seropositive individuals varied between 15 and 50 years. CMV IgG was found in 1813 (91%) of 2001 individuals. In total, 188 individuals (9%) were negative for CMV IgG. There were significant relationships between seropositivity (CMV IgG) and age, sex, level of education, and level of antibody titer by sex. As in other developing countries, the prevalence of CMV infection in adults in Alborz province is high. Since CMV infection is prevalent and there are potential abnormalities associated with it, we strongly recommend the expansion of preventive measures and the establishment of programs to inform at-risk populations, especially vulnerable populations such as transplant recipients, immunocompromised patients, and school children on how to prevent this infection and its associated consequences.

Laboratory animal models are an important part of test design. Certain conditions such as microbial contamination in diets of these models could affect the results of experiments. One of the most important routes that predispose to contamination is generated through feeding of laboratory animals. This study aimed to show the effect of gamma irradiation in reducing bacteria concentrations, crude nutrient content, and concentrations of some minerals and trace elements in laboratory animal diets. Large-sized pellets with 10–15 mm diameter (commonly used for rats and hamsters) and small-sized pellets with 3–5 mm diameter (used for rabbits and guinea pigs) along with skimmed milk powder (SMP) as a food additive were exposed to gamma irradiation with different doses ranging from 3 to 30 kGy. The total microbial contamination and any possible changes in some mineral nutrient composition and the crude nutrient content were determined pre- and post-irradiation. Our data revealed that 25 kGy in pelleted diets and 18 kGy in SKM had superior effects in the reduction of bacterial contamination with little change in crude nutrient content and minerals and trace elements in nutrient requirements of laboratory animals. According to the results, gamma irradiation had minimal effects on crude nutrient content and the concentrations of some minerals and trace elements of laboratory animal diets, and it also eliminated bacterial and fungal contamination load. By using gamma irradiation, this method could yield a favorable outcome in controlling microbial contamination of animal diets.

Hydatidosis is an important zoonosis caused by a parasitic tapeworm, namely Echinococcus granulosus. This infection is distributed worldwide and affects the health as well as economic loss in both humans and animals. In most cases, the disease needs chemotherapy with or without surgery. Conventional drugs have some major problems, including drug complications, harmful side effects, and also progressive resistance. According to the importance of biological productions as alternative medicine, a large number of studies confirmed that whole venom and many peptide ingredients of the scorpion venom have various different medical benefits, including antimicrobial properties, due to the mechanism of blocking gated ion channel. In this study, the venom peptides of Mesobuthus eupeus scorpionwere purified using gel filtration chromatography and subsequently ion exchange chromatography, followed by the determination of the molecular weights of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure. After collecting the hydatid cysts fluids from the liver of infected sheep, protoscolices were derived, washed, and encountered to the whole venom as well as eight different fractions of toxin 30, 60, 120, and 240 min after the exposure. In the next step, the viability of protoscolices was determined by eosin staining. The obtained results revealed that a venom fraction under 10 kDa killed all protoscolices after 30 min. Moreover, it was found that the scolicidal activity of fractions increases according to the time of exposure. As a result, it can be concluded that M. epeus venom peptides under its LD50 (1/2 LD50) can properly and quickly destroy the protoscolices of hydatid cysts at the level of applied concentrations and such components are good alternatives to treat hydatidosis.

Vasectomy in laboratory animals is a crucial step in the production of surrogate female mice. The surrogate mothers play a key role in successful embryo transfer, most important steps for the production of transgenic animal models, investigation of the preimplantation embryo development, and revitalization of cryopreserved strains. Abdominal and scrotal surgeries are common surgical procedures used in routine veterinary practice to produce vasectomized males. Two different surgical practice, namely electrosurgery and cold surgical practice, have been used as common techniques in operating rooms. Based on current knowledge, there is no published “technical note” as a detailed and step by step guideline to describe vasectomy using an electrosurgery machine (i.e., Bovie machine) in laboratory animal research and breeding facilities.The common problem during the laboratory animal surgery would be animal mortalities as a consequence of profound bleeding. The use of Bovie machine leads to the prevention of profound bleeding during the surgical practice.

Three families of venomous snakes exist in Iran including Viperidae, Elapidae, and Hydrophidae. Viperidae family is the only family with a widespread distribution. Saw-scaled vipers are important poisonous snakes in Asia and Africa. This name is given to this snake due to the presence of obliquely keeled and serrated lateral body scales. Distribution of this genera is mostly reported in the central and southern regions of Iran. This genus has four main clades: the Echis carinatus, E. coloratus, E. ocellatus, and E. pyramidum. Design pattern in Echis species plays an important role in camouflage and variety of habitat. In the present report, we investigated a specimen from the eastern region of Iran; we examined 25 specimens of Echis that were collected from the eastern region of our country. Among them, only one specimen with a different pattern was found compared with the other 24 specimens by surveying meristic, mensural, and design pattern characters using valid key identifiers. The similarities between the specific Echis with a different pattern and other 24 specimens were also studied and compared. The results of this investigation clearly showed that although the pattern of the lateral white line and block on dorsal body of the specific Echis snake was different, since the meristic and mensural characters were similar to other Echis snakes it can be concluded that this specimen is not a different species; the difference in these patterns may be due to a minor genetic mutation of that specimen. It is the first case report of Echis carinatus sochureki Stemmler, 1969 from Iran with a different pattern.