Archives of Razi Institute
Volume:75 Issue: 3, Summer 2020

Ticks are reservoir hosts of pathogenic Rickettsia in humans and domestic animals. Most pathogenic Rickettsia species belong to the spotted fever group (SFG). The present study aimed to determine the tick species infected with Rickettsia based on the genus-specific 23S ribosomal ribonucleic acid (rRNA), 16S rRNA, and citrate synthase (gltA) gene fragments. A total of 61 tick specimens were selected for molecular assay and 12 samples for sequencing. Phylogenetic analysis was conducted using neighbor-joining and Bayesian inference methods. Argas persicus, Haemaphysalis sulcata, Ha. inermis, and Hyalomma asiaticum were infected by spotted fever Rickettsia. The SFG is the main group of Rickettsia that can be detected in the three genera of ticks from Iran.

Mediterranean visceral leishmaniasis is a zoonotic disease caused by Leishmania infantum and transmitted by the bites of infected female sand flies.  Iran is one of the endemic areas of this disease. Dogs and canines are the major reservoir hosts of Leishmania infantum in the new and old world, including Iran. If visceral leishmaniasis is left untreated, it may result in a 90% mortality rate. The identification and elimination of infected dogs are efficient ways to control this disease. The diagnostic methods used to identify these animals cannot yield 100% detection. Therefore, in the present study, we used a multiepitope recombinant protein (PQ10) to distinguish between symptomatic and asymptomatic infections caused by Leishmania infantum in animal reservoirs (dogs). The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. In the following process, sequencing was performed with plasmid-specific primers and followed by the expression, optimization of expression. The purified recombinant protein was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The efficacy of recombinant PQ10 protein was evaluated by an indirect enzyme-linked immunosorbent assay (ELISA) test using 150 dog sera (25 symptomatic positive sera, 25 asymptomatic positive sera, 80 negative sera, and 20 sera of dogs with other infectious diseases). Direct agglutination test (DAT) as the standard method was used to compare and determine specificity and sensitivity. The results indicated that the sensitivity and specificity of ELISA using infected dog sera were 94% and 86%, respectively. Positive and negative predictive values for dog sera were reported as 87.03% and 93.47%, respectively. This protein was able to identify 92% of asymptomatic dogs with visceral leishmaniasis. The results showed that the recombinant protein PQ10 is able to identify positive cases of canine visceral leishmaniasis, especially asymptomatic cases.

In recent years, the H9N2 influenzavirus has been circulating widely in poultry farms causing extensive damage. The hemagglutinin (HA) genes of the two virus isolates of H9N2 subtype in specific pathogen-free chickens were studied to determine the shedding rate in the host’s oropharyngeal and cloacal routes and their genetic relationship. The sequence analysis and phylogenetic study of the samples were performed by comparing each isolate with other H9N2 isolates in the gene bank. In the present study, the chickens were inoculated with low pathogenic avian influenza virus (LPAIV) (A/Chicken/Iran/ZMT-101/1998 [H9N2]) through the intranasal route. Oropharyngeal and cloacal swabs were collected from the chickens within 1-10 days after inoculation. The rate of viral shedding was measured within the previous 10 days by the real-time reverse transcriptase polymerase chain reaction molecular technique. No clinical symptoms were observed during the experiment in the chickens. The results obtained from this technique showed that the main route of shedding for LPAIV was oropharyngeal areas (P<0.05). Both isolates had a similar proteolytic R-S-S-R sequence at the cleavage site of the HA gene and contained glutamine (Q) amino acid at position 226 of the HA receptor-binding site, indicating that these isolates were nonpathogenic. Phylogenetic analysis demonstrated that both isolates belonged to the Eurasian clade. The comparison of these isolates with other isolates in the gene bank showed that they had the greatest similarity with the isolates in clade 1 and the least homology with the isolates in clade 4.

The foot-and-mouth disease virus (FMDV) with a wide variety of genomes and complicated biology is one of the infectious agents that put the lives of animals at risk. Therefore, to introduce suitable strains for vaccine production, it is essential to constantly evaluate genetic changes of circulating viruses in field. Within 2014-2015, a total of 126 clinical specimens consisting of epithelial tissue and vesicular fluid from tongue, dental pad, and hoofs suspected of FMD virus were submitted to the Reference Laboratory for FMD in Razi Vaccine and Serum Research Institute, and 86 of them were identified as FMD virus type A using sandwich Enzyme-Linked Immunosorbent Assay (ELISA). This virus was isolated from 42 samples from 16 provinces using cell culture. Firstly, the coding region that produces the main part of viral capsid was amplified by Polymerase chain reaction (PCR). This part of the genome by 800 bp length was related to the 1D gene that synthesizes the VP1 protein. The phylogenetic analysis of VP1 coding region determined two distinct genotypes with more than 15% nucleotide differences. The first cluster consisted of closely related viruses registered in the GeneBank of neighboring countries, including Afghanistan, Pakistan, and Turkey. All samples in Cluster1 were determined as relative viruses with genotype Iran-05. In-vitro serological examination indicated an antigenic relationship between Cluster 1 viruses and routine vaccine strain (A-IRN-2013). The second cluster with only two members was genetically far from earlier ones and could be considered a separate genotype. Furthermore, it was revealed that cluster 2 has not been previously reported in Iran. Genetic tracing indicated that these viruses might have been originated from circulating viruses from India.  Antigenic evaluation exhibited that this group could not be cross-protected by the routine vaccinal strain (A-IRN-2013) used during the research period.

The present study aimed to determine the seroprevalence of H9N2 influenza in broiler farms at the time of slaughter in Iran. A total of 747 birds were sampled from 74 Farms in 13 provinces within 2013-2016. The obtained sera were investigated using the hemagglutination inhibition (HI) test. Out of 74 sampled farms and 747 birds, 57 farms (77%) and 445 (59.57%) birds were reported to be seropositive. In 2013, 10 farms and 110 birds were sampled out of which three farms (29.6%) and 29 birds (30%) were seropositive. In 2014, 24 farms and 220 birds were sampled out of which 22 farms (91.6%) and 220 birds (86.6%) were positive in six provinces. In 2015, 30 farms and 278 birds were sampled out of which 5 farms (16%) and134 birds (48.2%) were positive in four provinces. Finally, in 2016, 7 farms (70%) out of 10 sampled farms and 62 birds (59%) out of 105 sampled birds were positive for H9N2 in eight provinces. The mean titer of units in 2013 was statistically lower, as compared to that in 2014 (P<0.01). In addition, the proportion of positive serum units in 2013 was statistically lower, as compared to that in 2014 (P<0.001). In general, the prevalence of H9N2 was high indicating the continuous circulation of the virus in Iran. Given the importance and impact of this virus on the poultry industry, people’s livelihood, and public health, more epidemiological studies are needed to evaluate the effectiveness of the adopted measures and methods in controlling the H9N2 virus.

The innovation of therapeutic modalities with better clinical efficacy is necessary for the treatment of patients with advanced cancers. Newcastle disease virus (NDV), an avian pathogenic virus, is one of the most promising oncolytic viruses that can replicate selectively in human cancer cells. In humans, NDV can cause transient conjunctivitis and mild flu-like symptoms. However, this virus poses no hazard to human health. The elucidation of the mechanisms of cancer cell killing by NDV is helpful for the clinical application of NDV in cancer patients. Regarding this, the present study was performed to evaluate apoptosis induction by NDV LaSota strain vaccine in human breast carcinoma cells. To this end, MCF-7 cells, a human breast adenocarcinoma cell line, were infected with NDV in vitro. Tumor cell cytotoxicity, apoptosis induction, and expression levels of apoptosis-related genes were examined in NDV-infected breast carcinoma cells. Tumor cell cytotoxicity was measured by 3-[4,5-dimethylthiazol-2yl]2,5-diphenyl-tetrazoliumbromide (MTT) assay. The induction of apoptosis was assessed by annexin V/propidium iodide staining. In addition, the expression levels of apoptosis-related genes were evaluated using real-time reverse transcription polymerase chain reaction (PCR) technique. The NDV showed cytotoxic effects on MCF-7 cells and induced apoptosis in the infected carcinoma cells. The gene expression levels of BAX, caspase-9, and caspase-3, but not BAK-1, were increased in NDV-infected cancer cells, compared to the gene expression levels in the non-infected cancer cells. These results suggest that the induction of the intrinsic pathway of apoptosis is one of the mechanisms that can contribute to cancer cell killing by NDV. Additional studies are required to investigate other probable mechanisms involved in NDV-mediated cancer cell killing.

Bovine brucellosis is a widespread zoonosis caused by Brucella abortus. The disease is prevalent nationwide in Iran and is on an increasing trend among humans and livestock. The eradication of brucellosis is challenging and requires control policies at both national and regional levels. Regarding this, the aim of the current study was to evaluate if Brucella is implicated in an abortion outbreak that occurred in a dairy cattle herd, in Shahre Rey, Tehran province, Iran, after vaccination with B. abortus Iriba vaccine. The research context was a dairy cattle farm with 2,000 animals located in Shahre Rey. This farm was Brucella-free based on the results of two serological tests performed one month before vaccination. After the incidence of the first case of abortion following vaccination, serodiagnosis revealed a seropositive reaction in 30 non-pregnant cows and 19 pregnant cows that aborted later. Bacteriology and molecular typing facilitated the identification of 16 isolates of B. abortus biovar 3 from the aborted animals. None of the isolates were confirmed as B. abortus Iribavaccine strain. The results confirmed that B. abortus biovar 3 was the most prevalent biovar in the cattle of Iran. The source and time of infection in the current study were not detected most likely due to the low biosecurity level in the farm (e.g., uncontrolled introduction of the agents via humans, infected animals, semen, and vectors). In endemic countries, the serodiagnosis of brucellosis alone is not sufficient and has to be accompanied by isolation and molecular diagnosis. In addition, it is important to evaluate the presence of B. abortus in bovine semen and vectors.

Toxoid vaccines can provide protective immunity against clostridial diseases. Since the duration of the toxoid vaccine immunogenicity is short, these vaccines need to contain an adjuvant. The nanoparticles of chitosan can stimulate humoral and cell-mediated immune responses. In the present study, the effect of chitosan nanoparticles was investigated on the immunogenicity of the pentavalent clostridial toxoid vaccine containing Clostridium perfringens types D, C, and B, Clostridium septicum, as well as Clostridium novyi. Rabbits were immunized by two injections with 3-week intervals and checked clinically and through autopsy 2 weeks after the last injection. Hematological changes were investigated during immunization, including the changes of white and red blood cell counts, hemoglobin, packed cell volume, platelet, neutrophil, lymphocyte, eosinophil, basophile, monocyte, and Neut/Lymph. Biochemical factors, namely creatinine, blood urea nitrogen, glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, and albumin, were also studied. The changes in immune responses during the immunization period were investigated through indirect enzyme-linked immunosorbent assay (ELISA). The results of ELISA showed that chitosan significantly enhanced immunogenicity when accompanied with in the pentavalent clostridial toxoid vaccine. During the immunogenicity period and following that, no changes were observed in clinical behavior and internal organs after autopsy. The hematological and biochemical factors were reported with no significant pathologic changes during immunization in the control and vaccinated groups (P<0.05). The obtained findings revealed that the toxoid vaccines could not induce significant physiological changes in the body. The vaccine containing chitosan could stimulate humoral immunity 2-3 times higher than the nonchitosan vaccine. The humoral immune response was significantly duplicated due to the chitosan effect. Chitosan not only had no local or general side effects but also could be a good help with the enhancement of the immune system; therefore, it can be recommended as an appropriate safe adjuvant in the development of toxoid vaccines.

The purpose of this study was to compare the effects of using different levels of flaxseed in diets on the reproductive performance of estrous-synchronized Baluchi ewes (para 3). Diets contained either basal diet (control) or different levels of extruded flaxseed (2%, 5%, 7%, 10%, and 12%) and were fed from lambing to 60 days after lambing. The ewe estrus cycles were synchronized using controlled internal drug release (CIDR) for 14 days starting from day 16 of fat supplementation. The rams were introduced 24 h after CIDR removal. The ewes fed control diets had the highest mean dry matter intake (1,800±35 g) which was declined with the increase of flaxseed levels. The experimental diets exerted no effects of urea concentration in blood plasma. However, plasma glucose concentration was lower (p <0.05) in the ewes fed the control diet and 2% flaxseed, compared to those in other groups. Nonetheless, there was no difference among the ewes fed 5%, 7%, 10%, and 12% flaxseed in terms of plasma glucose concentrations (p <0.05). The ewes fed 2% flaxseed had the highest level of plasma triglyceride concentration among other groups. In addition, the control group had the lowest level of plasma total cholesterol and low-density lipoproteins concentration in comparison to other groups (p <0.05). However, plasma nonesterified fatty acids and β-hydroxybutyrate concentrations were similar among the groups (P>0.05). The mean interval between CIDR removal and the exhibition of estrus ranged from 30 to 40 h with the shortest interval being recorded in the ewes fed 12% flaxseed (p <0.05). The control group had the lowest number of follicles on estrus day among other groups (p <0.05). Furthermore, the ewes fed 10% and 12% flaxseed had the highest ovulation, pregnancy, and lambing rates, compared to other groups (p <0.05). In conclusion, the findings revealed that feeding the ewes with 10% and 12% flaxseed resulted in the improvement of reproductive performance.

The Androctonus crassicuda is the most diverse scorpion species in the family of Buthidae, which is endemic to Khuzestan province, Iran. Investigation of the relationship of species by means of a molecular study of specimens is one of the new approaches due to the limitations of the morphological approaches. In the current study, the analysis was based on 32 morphological characteristics of A. crassicuda native to southwest Iran. Moreover, the DNA sequencing of two mitochondrial markers, namely cytochrome oxidase subunit I and 12sRNA loci was performed, and the phylogenetic tree was constructed using maximum likelihood method with 1000 replications using MEGA software (version 7). Based on the results of the phylogenetic tree, A. crassicuda was classified into a monophyletic group. However, the genetic diversity of this species populations was not significant (0.001). The highest and lowest genetic distance of A. crassicuda was compared with the reports obtained in Urmia and west Azerbaijan, Iran. There was a clear divergence between the A. crassicuda isolated from northern and southern areas of Iran. This study showed the importance of geographical and climate features of the region and genetic distance among the populations. The phylogenetic analysis of Androctonus species from other regions showed the highest and lowest genetic distance with A. gonneti (Morocco) and A. amoreuxi (Portugal), respectively. The comparison of the morphological characteristics and morphometric results revealed that metasoma characteristics are important in the identification of A. crassicuda. The results of the analysis of the morphometric values of A. crassicuda were mainly compatible with the phylogenetic trees and supported the traditional morphological classification, thereby presenting a clearly definition of the genera of Androctonus species.

Cardiovascular disease is one of the most common causes of morbidity and mortality in the world. Atherosclerosis is an inflammatory process, and serum C-reactive protein (CRP) is an acute-phase protein rising in response to inflammation. Serum iron (Fe) is one of the essential metals for the human body. Inflammation and infection are characterized by changes in Fe metabolism. Since atherosclerosis is an inflammatory process, changes in CRP and serum Fe levels are expected. However, the distribution of the disease in the coronary arteries is important for mortality and morbidity. The distribution of the disease can be determined by the syntax score. This study included 407 patients with a mean age of 56.4±10.7 years. The majority of the patients were male (51.4%). In this study, 53 and 354 patients had critical and no critical lesions, respectively. According to the baseline coronary angiograms, the syntax score was calculated in all patients. The laboratory variables, including hemoglobin levels, blood glucose, creatinine, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, Fe, and CRP were also evaluated in this study. Regarding the laboratory parameters of all groups, the mean CRP levels, Fe levels, and syntax score were estimated at 0.75±1.8 mg/dl, 80.4±27.5 mg/dl, and 1.5±4.8, respectively. Furthermore, a high syntax score correlated with Fe and CRP levels. Based on the findings of the present study, elevated serum Fe and CRP concentrations were associated with increased syntax score and atherosclerosis severity.