BACKGROUND: Infectious diseases and microbial antibiotic resistance are the major problems of fish farming industry annually causing remarkable losses. Apart from the economic losses caused by these infections, some of these agents are zoonotic and may be transmitted to humans.OBJECTIVES: This study was aimed to identify the common causative agents of infections in rainbow trout farms and to determine their antibiotic resistance toward some common antibiotics.METHODS: Sampling was performed during a nine-month period between March and December 2021 by visiting and inspecting rainbow trout farms and the affected fish with disease symptoms were obtained from the farmed fish in Mazandaran, Lorestan, Chaharmahal and Bakhtiari and Zanjan provinces. Bacterial culture was undertaken from anterior kidney or spleen organs and the isolated bacterial strains were identified by phenotyping, biochemical and molecular assays. Antibiotic resistance pattern was evaluated by disk diffusion method (DDM) and minimum inhibition concentration against erythromycin, oxytetracycline, florfenicol, enrofloxacin and nitrofurantoin.RESULTS: Seventy-four bacterial isolates of Gram-positive cocci or Gram-negative coccobacilli were isolated. In phenotyping, biochemical and molecular (PCR) assays Lactococcus garvieae (12 isolates, 16.2 %), Aeromonas hydrophila (9 isolates, 12.2 %), Streptococcus iniae (17 isolates, 23 %), Streptococcus agalactiae (20 isolates, 27 %), and Yersinia ruckeri (16 isolates, 21.7 %) were identified. The majority of these isolates were obtained from the fish farms in Mazandaran province. Erythromycin and oxytetracycline with 87.8 % resistance were antibiotics with the highest resistance, while enrofloxacin with 24.3 % resistance revealed the lowest level of resistance. Antibiotic resistance rates for florfenicol and nitrofurantoin were also 43.2 % and 44.4 %, respectively. The highest antibiotic resistance was detected in the bacterial isolates of Lactococcus garvieae, Aeromonas hydrophila, Streptococcus iniae, Streptococcus agalactiae and Yersinia ruckeri, respectively.CONCLUSIONS: This study shows that the spread of streptococcosis, lactococcosis, yersiniasis and Aeromonas septicemia and their frequent treatments has led to an increase in antibiotic resistance, especially against commonly used drugs such as erythromycin and oxytetracycline.

BACKGROUND: Heparin is a sulfated glycosaminoglycan. Blood is a common source for DNA detection in all kinds of samples, and anticoagulants such as heparin and ethylenediaminetetraacetic acid (EDTA) are used to prevent coagulation. Because heparin has a strong inhibitory effect on polymerase chain reaction (PCR), it is not used in samples that will be tracked by DNA. There are physical, chemical, and enzymatic methods to eliminate the inhibitory effect of heparin on PCR test.OBJECTIVES: First, to compare the intensity of the inhibitory effect of two anticoagulants, heparin, and EDTA, on the Real-Time PCR (qPCR), and then to investigate the impact of the heparinase enzyme present in the medium culture extract of Pseudomonas aeruginosa, on removing the inhibitory effect of heparin during the real-time PCR.METHODS: In the present study, two blood samples containing heparin and EDTA were subjected to a real-time PCR test to check the intensity of the inhibitory effect. Then, the medium culture extract of Pseudomonas aeruginosa was added to the heparinized blood sample infected with Escherichia coli bacteria in two groups with different conditions. In the first group, the DNA in the heparinized blood sample was extracted by the phenol-chloroform isoamyl alcohol method. Then, these samples were incubated with the extract of Pseudomonas aeruginosa bacteria culture medium at different hours, but in the second group, the samples were incubated at different hours before DNA extraction. Also, the DNA concentration in both groups was measured by a Nanodrop device, and finally, all samples were subjected to a real-time PCR test.RESULTS: The results of the research samples showed that although the heparinized blood sample contains more DNA concentration than the EDTA blood sample, it completely prevents genome replication. Also, incubating heparinized blood with Pseudomonas aeruginosa culture medium extract before DNA extraction for more than 24 hours removes the inhibitory effect of heparin during the real-time PCR, even at a lower cycle threshold than the EDTA-containing sample.CONCLUSIONS: The Pseudomonas aeruginosa culture medium extract may enable researchers to use heparinized blood samples for genome amplification and diagnosis without using expensive and limited commercial heparinase enzyme.

BACKGROUND: Clostridium perfringens (C. perfringens) is an anaerobic Gram-positive bacillus with spores, whose biotype A is responsible for a variety of diseases, including intestinal inflammation, bloody diarrhea, and gas gangrene, and hemorrhagic bowel syndrome. Genetic variety can explain the bacteria’s phenotypic diversity, geographic distribution, host specificity, pathogenicity, antibiotic resistance, and virulence. A molecular method using the pattern of DNA bands classifies bacteria based on the size of fragments produced by enzymatic digestion of the genome.OBJECTIVES: This study aims to standardize the polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) method in identifying the genetic diversity of C. perfringens biotype A isolates.METHODS: The genomic DNA of the investigated strains was extracted, and the complete sequence of the alpha toxin gene locus was synthesized using specific primers designed by PCR technique. Enzymatic cleavage of the synthesized amplicons was performed with the Mse l restriction enzyme, and the resulting fragments were separated by electrophoresis and analyzed by ImageJ and NTSYSPC software.RESULTS: The findings showed that the alpha toxin gene locus sequence may change and is not conserved. In this research, 4 different patterns were identified based on enzymatic cleavage. Mutations in this locus can lead to diversity in C. perfringens biotype A and the creation of new strains.CONCLUSIONS: The results of this research showed that the alpha toxin gene locus could be considered a DNA molecular marker in C. perfringens, and the PCR-RFLP technique can be used as a tool for typing this bacterium and estimating the phylogenetic relationships through comparative studies of nucleotide sequences.

BACKGROUND: 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.OBJECTIVES: This study aims to detect the prevalence of Borrelia burgdorferi in guard dogs in Isfahan, Iran.METHODS: 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.RESULTS: 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.CONCLUSIONS: 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.

BACKGROUND: 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.OBJECTIVES: The current study aims to design a recombinant chimera protein HBHA-Omp28 as a vaccine against Salmonella typhimurium.METHODS: 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.RESULTS: 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 (+).CONCLUSIONS: The designed recombinant chimera can be an appropriate vaccine against salmonella bacteria.

BACKGROUND Agricultural pesticides can cause environmental pollution and damage to aquatic organisms. Bensulfuron-methyl is a widely used herbicide in agricultural fields, especially rice fields. Despite the solubility of Bensulfuron-methyl in water and its entry into aquatic environments, limited research has been conducted on the toxicity of this herbicide in aquatic organisms.OBJECTIVES: This study aims to investigate the effects of chronic toxicity of Bensulfuron-methyl in common carp (Cyprinus carpio).METHODS: The fish were divided into four groups. Group 1 was considered as a control, and groups 2, 3, and 4 were exposed to 10 %, 20 %, and 30 % of the 96 h lethal concentration 50 of Bensulfuron-methyl equal to 0, 0.162, 0.324 and 0.486 g/L.  After 21 days, blood samples, serum levels, and liver tissue of fishes were analyzed.RESULTS: The number of white blood cells increased in groups 2 and 3 (received 0.162 and 0.324 g/L Bensulfuron-methyl) compared to group 1, while a significant decrease was observed in group 4 (received 0.486 g/L Bensulfuron-methyl) compared to other groups. The number of red blood cells, the amount of hemoglobin, and the percentage of hematocrit in groups 3 and 4 showed a significant decrease compared to other groups, and the values of mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were not significantly different in any groups. The amount of total serum protein in groups 3 and 4 decreased significantly compared to the control group. Serum glucose showed a significant increase in groups 3 and 4 compared to other groups. The values for aspartate aminotransferase, alanine transaminase, and alkaline phosphatase enzymes showed an increasing trend with the increase of Bensulfuron-methyl concentration. The most liver tissue damage was observed in group 4, which included hyperemia, hepatocyte vacuolar degeneration, edematous cell infiltration, bile duct hyperplasia, and hepatic necrosis.CONCLUSIONS: The increase in the concentration of Bensulfuron-methyl can cause liver tissue damage and changes in hematological and serum biochemical markers in common carp.

BACKGROUND: The H9N2 avian influenza is one of the most important viral diseases of poultry in Asian countries. Continuous mutations in the hemagglutinin (HA) protein as the main viral antigen make interpreting the HA-based serology test results difficult.OBJECTIVES: In this experimental study, the ability of polyclonal antibodies against the conserved motif of HA protein in detecting heterologous H9N2 isolates was evaluated.METHODS: Based on the bioinformatics data, a conserved motif of HA protein was selected and expressed in the pET-28a(+) vector. The recombinant peptide was mixed with an oil adjuvant and injected into specified pathogen free (SPF) chickens to produce the polyclonal antiserum. Similarly, antiserum from the inactivated virus was prepared. The efficiency of the prepared antisera was evaluated in the cross hemagglutination inhibition (HI) test using the homologous and heterologous H9N2 viruses.RESULTS: The average titer of HI antibody against homologous and heterologous viruses using the polyclonal antiserum was equal to five. This titer was estimated to be seven for the homologous virus and much lower for heterologous viruses with inactivated virus antiserum. The difference was statistically significant.CONCLUSIONS: According to the evaluations, this antiserum has a suitable efficiency in identifying different isolates of the H9N2 virus, and compared to the usual antiserum, it detects different antigens of a subtype more accurately. The resulting polyclonal antiserum does not contain non-specific inhibitors, which may cause problems interpreting serological findings. The results showed that polyclonal antiserum could identify H9N2 viruses isolated in different years by HI test; however, its validation requires testing with more viruses.

BACKGROUND: Cadmium is a heavy metal harmful to animals and humans. Exposure to it causes inflammation, apoptosis, or necrosis in numerous tissues, including the adrenal.OBJECTIVES: The present research investigates the effect of cadmium toxicity on the expression of genes involved in inflammation and fibrosis. Inflammation increases the rate of parenchymal cell death, and fibrosis will only fill the place of dead cells without being able to perform the function of the primary parenchyma.METHODS: In this research, cadmium chloride with a concentration of 20 mg/kg was added to the diet of ten mice in two groups of five. On the 30th day of the study, the adrenal glands were quickly sent to the laboratory. The expression of NF-kB/MAPK, hematoxylin, eosin tissue staining, and immunohistochemistry (CD163) were performed.RESULTS: The inflammation mentioned in others’ research can also be associated with the activation of the nuclear factor kappa (NF-kB) pathway. NF-κB gene products initiate mitogen-activated protein kinase (MAPK) and p38 pathways. Previous studies indicate that MAPK induces necrosis or apoptosis in tissues. In histopathology, dense and possibly pyknosis nuclei are more common in the cadmium group. The higher expression of the CD163 molecule in the cadmium group reveals the beginning of the fibrosis process after chronic inflammation.CONCLUSIONS: This report provides more basic data to investigate the mechanism of adrenal damage in cadmium poisoning. Cadmium causes the death of cells by affecting the inflammatory pathways. Additionally, the stimulation of the fibrosis process causes greater irreparable damage to the damaged tissue of the adrenal gland.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis is the cause of a common disease in dairy herds. Early diagnosis of paratuberculosis infection can improve Johne’s disease control programs.OBJECTIVES: This study aimed to compare the sensitivity, and specificity to methods; blood serum ELISA and stool Nested-PCR for the detection of Mycobacterium avium subspecies paratuberculosis infection in dairy cattle.METHODS: A commercial ELISA kit was used to perform the absorbed ELISA test, which was conducted after exposing serum samples to Mycobacterium phlei antigens to limit cross-reactions. Nested-PCR test was performed using nucleotide sequences related to specific MAP gene fragments, i.e. IS900.RESULTS: As a result of the ELISA antibodies kit, out of the total 2203 serum samples, 112 samples were positive (5.08 %) and 2091 samples were negative (94.92 %). The results of Nested-PCR tests of rectal feces showed that out of 59 cows with the positive results in serum ELISA, 47 (79.66 %) samples were positive and 12 (20.34 %) samples were negative. Moreover, out of 31 cattle with a negative result on the ELISA test, 15 (48.38%) and 16 cattle (51.62 %) had positive and negative results, respectively, on the nested PCR tests of the feces samples.CONCLUSIONS: Due to the low sensitivity of PCR compared to ELISA, the positive and negative predictive values, and the accuracy of ELISA test, as well as the high cost and time-consuming nature of PCR and the need for more and more complex facilities than ELISA, the authors concluded that ELISA is a more suitable method for screening and epidemiological studies than PCR.

BACKGROUND: Horses are economically and emotionally valuable animals in various activities, especially sports. Thus, paying attention to their limb's health and conformation is vital. One of the most common diseases in the limbs of horses is laminitis. Horses with this condition suffer from lameness because it affects laminar tissue. In addition to clinical signs, radiographic criteria are essential for identifying this disease.OBJECTIVES: It is predicted that examining the effectiveness of quantitative radiographic criteria of the hoof can be helpful in the diagnosis of laminitis. Therefore, in this study, five quantitatively effective factors were investigated before and after hoof trimming to determine the changes in the radiographic diagnosis of laminitis.METHODS: A total of 11 clinically healthy horses were used in the current study. Using Marco DICOM Viewer software, lateral and dorsopalmar radiographs from the hoofs of both forelimbs were evaluated for the diagnosis of laminitis using effective quantitative criteria. Using SPSS version 24, paired T-tests were used to analyze quantitative data. P≤0.05 was considered significant.RESULTS: According to the results of this study, there were no significant differences between the right and left forelimbs after hoof trimming. On the other hand, significant differences were observed in the four following criteria: dorsal thickness between the dorsal surface of the third phalanx and the dorsal surface of the hoof, the angle between the dorsal surface of the third phalanx and the dorsal surface of the hoof, sole thickness, and the ratio of the third phalanx dorsal surface thickness to its maximum length in each forelimb before and after hoof trimming.CONCLUSIONS: During the radiographic examination, the hoof should be positioned in a standard way to diagnose laminitis accurately. However, if the hoof is not trimmed or not trimmed properly, it can interfere with laminitis diagnosis.