مجله تحقیقات دامپزشکی
سال هفتاد و هشتم شماره 3 (پاییز 1402)

Toxocara canis C-type lectin (T. canis-CTL) is the main protein part of the secretory-excretory product secreted by Toxocara canis infective larvae. T. canis-CTL can stimulate immune response-mediated regulatory T lymphocytes, increase the FOXP3+ cells population, and reduce severe inflammatory responses. T. canis-CTL is a promising candidate for immune modulation in some autoimmune diseases, deserving further investigation.

The current research aimed to purify the recombinant T. canis-CTL under denaturation and native condition to increase exploration and maintain biological activity.

The expression vector, pET32a was constructed with the partial sequence 660bp of T. canis-CTL and expressed in E. coli BL21 (DE3). T. canis-CTL protein expression was induced by IPTG (1 mM) at 37°C after 6 h. In this study, different buffers were used for cell explosion and recombinant protein solubilization, including lysis buffer with urea (8 M, pH=8) and lysozyme enzyme as well as lysis buffer with Imidazole (0.01 M) and lysozyme enzyme, and previous buffers in addition to sonication. The effect of these buffers was evaluated in bacterial cells explosion, using Gram-staining and microscopic examination. Recombinant T. canis-CTL protein was extracted and purified under denaturation and native condition using Ni-NTA affinity chromatography by agarose and sepharose resin. A New Zealand male rabbit was immunized with the recombinant protein to evaluate the bioactivity of the protein. 

Lysis buffer with urea (8 M, pH=8) and lysozyme enzyme, in addition to sonication, provided acceptable results, and an additional amount of recombinant T. canis-CTL protein was secreted in the buffer. Protein purification under denaturation conditions with Ni-NTA agarose affinity chromatography also provides further recombinant protein. Most of the induction of recombinant T. canis-CTL with 41 KDa molecular weight was collected 6 h after induction at 37°C. Dot-blot results illustrate the brown dot, which showed a 1:500 titer of specific IgG polyclonal antibody has developed in the sera of rabbits immunized with T. canis-CTL recombinant protein.

The denaturation condition did not affect the biological activity of the T. canis-CTL recombinant protein and can recover a further amount of recombinant proteins.

Lyme disease is caused by a gram-negative spirochete bacterium called “Borrelia burgdorferi” and can be transmitted by the bite of infected ticks to humans and animals. This disease has a global geographic distribution Dogs can be infected by the bite of the Ixodes ricinus tick.

This study aims to detect the prevalence of Borrelia burgdorferi in guard dogs in Isfahan, Iran.

In this study, blood samples were collected from 97 guard dogs with an average age of 3.5 years in Isfahan city and analyzed by the blood smear method and the nested polymerase chain reaction method (molecular study). The data were statistically analyzed in SPSS software (version 24) using Fisher's exact test and chi square.

Twelve samples (12.37 %) were found molecularly positive, which were for 10 male dogs (10.30 %) and two female dogs (2.06 %). There was no evidence of the presence of bacteria in the blood smear and hematological changes were not found in complete blood count tests (CBC Test) performed on infected samples. There was a significant difference in the levels of infection between the age groups of 1 to 2 years (P=0.029) and between this age group (1 to 2 years) and the age group >3 years (P=0.032. (There was a significant difference in infection levels between dogs with different gender.

The prevalence of Borrelia burgdorferi in dogs of Isfahan City is relatively high. Due to the ease of transmission of this pathogen between humans and animals, special attention should be paid for its control and timely detection.

Many toxic elements enter human food in different ways by various industries which can put people's lives in danger. Heavy metals can be rarely removed from the body after absorption and deposition in tissues, which can lead to diseases and complications in the body. Mercury is one of the heavy metals that can posion people after consumption of contaminated seafood. The measurement of pollutants such as mercury that present in aquatic animals and environment is one of challenges for humans.

This study aims to measure the amount of mercury accumulation in the edible tissue of whiteleg shrimp (Litopenaeus vannamei) found in farms of Bushehr Province in Iran.

In this research, 70 whiteleg shrimps were collected during four sampling stages in July, August, September and October for two consecutive years. Total mercury level was measured by the cold vapor method.

The level of mercury was 0-0.009 mg/kg of body weight, while the recommended limit for mercury is 0.1 mg/kg according to the WHO. The microscopic study on tissue sections did not show any histopathological changes.

The mercury level in the edible tissue of whiteleg shrimps in Bushehr province is much lower than the recommded level and does not pose any danger to residents and consumers.

Numerous studies have investigated the positive effect of chitosan and nano-chitosan loaded clinoptilolite on growth performance, digestive enzyme activity, and intestinal histomorphology in different fish species. However, there are no data evaluating the potential effect of the composites on the fish stomach.

In the current study, the effects of chitosan and nano-chitosan loaded clinoptilolite on histological features and pepsin activity in the rainbow trout stomach were considered.

Chitosan and nano-chitosan loaded clinoptilolite were synthesized, and then two hundred and forty fish (~27.75 g) fish were distributed in eight groups each in three replicates. Ten days after adaptation, the fish were fed with eight diets, including control diet (CTR), clinpotilolite (T1), chitosan composites (T2, T3, T4), and nano-chitosan composites (T5, T6, T7) for 70 days. Afterward, all fish in each tank were anesthetized in clove extract (50 μl/l), and tissue samples were obtained for pepsin activity (n= 5) and histological assay (n = 5).

The groups administrated with nanochitosan composites showed the highest pepsin activity (P˂0.05). Additionally, histological examinations exhibited a higher epithelial height, increased mucosal density, and oxynticopeptic cells hypertrophy in fish fed composites compared to the CTR group (P˂0.05). Meanwhile, nanochitosan composite administration could cause higher reaction of secreted granules to periodic acid–Schiff (PAS) staining.

The findings demonstrated the potential application of chitosan and nano-chitosan loaded clinoptilolite composites for improvements in the histomorphology and pepsin activity of the rainbow trout stomach, resulting in higher growth performance.

The effect of nanoherbal substances in the treatment of infectious diseases before mating or during pregnancy must be evaluated. Saponin has adverse effects on a pregnant mother and her fetal through complex mechanisms.

This study aims to investigate the toxic effects of saponin encapsulated by ferritin nanoparticles (Nanosaponin) on fetal lung development in female mice with Streptococcus pneumonia.

Extraction of crude saponin from licorice roots was done by the powder heating and using 20 % ethanol and diethyl ether. Then, n-butanol and 5 % sodium chloride solution were added to the aqueous layer. The mice were divided into five groups of 10 including control, pneumonia, pneumonia treated with ferritin, pneumonia treated with saponin, and pneumonia treated with nanosaponin. Then, two groups of untreated pregnant pneumonia and pregnant pneumonia treated with nanosaponin were separated. In this study, tumor necrosis factor alpha (IFN-γ), heparin-binding epidermal growth factor-like growth factor (HB-EGF), Methyl-CpG binding domain 2 (MBD-2), and kruppel like factor 2 (KLF-2) genes were evaluated in the maternal lungs, and TNF-α level was evaluated using ELISA method in the fetal lungs. Maternal tissue, uterus tissue, and fetal lung were examined by hematoxylin and eosin staining, and maternal body tissue was examined by trichromason staining and Oxidative stress parameters were investigated.

Nano saponin decreased the expression of IFN-γ (P<0.05) (P<0.01), MBD-2 (P<0.05) and HB-EGF  genes, KLF-2 protein level and TNF-α levels in fetal lung.The thickness of uterine myometrium and blood flow of endometrium increased the number of live embryos and the rate of successful pregnancy. In addition, Nano saponin effectively improved the oxidative stress response in the fetus without any harmful effect on the mother's liver tissue.

The nanosaponin has no toxic effects on the sudy organs of the mother and fetus. It has therapeutic effects on the fetal lung development process after the infection of mother with Streptococcus pneumonia.

Ischemia-reperfusion injury (IRI) can induce major changes in the function of different organs, including the liver. Studies have indicated that ischemic post-conditioning (HIPO) can protect the tissues against ischemia-reperfusion injury.

To investigate the antioxidant and anti-inflammatory effects of ischemia post-conditioning against the IRI of the rat liver through four 30-second cycles of alternating ischemia and reperfusion, before 24-hour persistent reperfusion.

Fifteen rats were randomly divided into 3 groups 1) operation control group, 2) ischemia-reperfusion (IR) group whose liver was exposed to 60-miute ischemia of by 24-hour reperfusion and 3) ischemic post-conditioning (IR+IPO) group underwent the same procedure as the second group except that before persistent reperfusion, the rats were subject to post-conditioning by four 30-second cycles of alternating ischemia and reperfusion. The changes induced by IRI and the antioxidant and anti-inflammatory effects of ischemic post-conditioning were assessed by the serum level of IL-6 using the ELISA method, malondialdehyde (MDA), and total antioxidant capacity (TAC). 

Ischemic post-conditioning potentiated antioxidant effects and reduced the inflammation caused by the IR in the liver. The serum level of IL-6 reduced from 394.4±126.4 to 124.4±29.07 pg/ml (post-conditioning group), and the tissue MDA reduced from 431.4±76.53 to 207.2±25.77 nmol/g) compared to the IR group. The data revealed that the levels of the indices returned almost to the level of the operation control group (P<0.001). Additionally, the total antioxidant capacity of the liver significantly improved (P<0.01) from 11.58±1.87 (in the IR group) to 17.53±2.51 mmol/mg (in the IR+IPO group).

The study demonstrates that the protective effect of ischemic post-conditioning against IR-induced injury may be mediated through decreasing inflammation and improving antioxidant activities.

Salmonellosis is a dangerous disease that can threaten the health of humans and animals. This disease can lead to economic losses annually; therefore, many studies have been conducted to prevent this disease.

The current study aims to design a recombinant chimera protein HBHA-Omp28 as a vaccine against Salmonella typhimurium.

The nucleotide and amino acid sequences of Omp28 and HBHA proteins were first extracted from the NCBI database. Then, the recombinant chimera of HBHA-Omp28 was bioinformatically assembled using a rigid linker. Epitope prediction of T and B cells, antigenicity, allergenicity, and physicochemical features assessments of HBHA-Omp28 were done using Immune Epitope Database (IEDB), ABCpred, VaxiJen, AllerTOP and ProtParam online servers, respectively. To assess the secondary and tertiary structures, the Self-Optimized Prediction Method with Alignment (SOPMA) and the Iterative Threading ASSEmbly Refinement (I-TASSER) server were used, respectively. Molecular docking between recombinant chimera and TLR4/MD2 receptor was assessed by ClusPro server. Finally, after codon optimization of nucleotide sequence of recombinant chimera to express in Escherichia Coli k-12 strain, the cloning of recombinant chimera in pET21-a (+) vector was examined.

The designed recombinant chimera was classified as an antigenic and non-allergenic protein with molecular weight of 34.19 kDa. According to the results of molecular docking study, the HBHA-Omp28 protein was able to bind to TLR4/MD2 receptor using 9 hydrogen bonds. The results of cloning study demonstrated that HBHA-Omp28 successfully cloned into pET21-a (+).

The designed recombinant chimera can be an appropriate vaccine against salmonella bacteria.