نشریه میکروبیولوژی دامپزشکی
سال هفدهم شماره 2 (پیاپی 43، پاییز و زمستان 1400)

Aspergillus flavus, A. parasiticus and A. nomius are the most important aflatoxin-producing species of B1, B2, G1 and G2 in food and feed. M1 and M2 Aflatoxins are the hydroxylated metabolites of B1 and B2 aflatoxins. The aim of this study was to evaluated the amount of aflatoxin B1 in animal feed and its relationship with aflatoxin M1 in raw milk. For this purpose, 20 industrial and 20 semi-industrial farms were selected and a sample of TMR ration and raw milk of each farm was sampled separately. Aflatoxin B1 in feed and Aflatoxin M1 in milk were evaluated by competitive ELISA, and the samples with values above maximum residue limit (MRL) were evaluated by high performance liquid chromatography (HPLC). The ELISA results showed that 35% of industrial farm feed and 75% of semi-industrial farm feed were contaminated with aflatoxin B1, of which 5% and 15% of industrial and semi-industrial farm had values above MRL, respectively. Also, in evaluating the amount of aflatoxin M1 in produced milk, it was found that aflatoxin M1 was observed only in the milk of farms that were positive for aflatoxin B1 and this rate was 20% and 55% in industrial and semi-industrial farms, respectively. The samples of milk with values above MRL were measured by HPLC confirmation test, which indicates that none of the samples of industrial and semi-industrial dairy production milk had higher values (0.5 ng/ml). Due to the importance of per capita milk consumption in the community diet and the dangers of aflatoxin M1 in it, extensive and accurate monitoring of milk production and supply centers as well as dairy factories is essential.

The Middle-East, known-to-be the birthplace for animal domestication, is among the few remained geographical theaters in the world where glanders-stricken solipede and human cases are still reported. In the Persian environment with some 60 years of maleination test history in its horse, mule and donkey populations, mini or midi outbreaks of glanders are seen now and then mostly in the Western and central regions. This work was aimed to characterize the phenotypic and genotypic properties of the most recently-collected isolates of Burkholderia mallei in Iran (A tiger sample and 3 horse samples). Bacterial culture, Biochemical tests, complement fixation test and Western blot, Flip 407-PCR, Bim A-PCR and also Strauss reaction tests were included in the assessment. The results obtained from experiments conducted on four isolates from Tehran, Kordan, Oshnavieh and Semirum outbreaks, displayed the expected characteristics of B. mallei. Whether our isolates of interest represent the local/regional population(s) of B. mallei, we assume further molecular epidemiology studies will enable us to address.

Newcastle disease (ND) is one of the most contagious and fatal viral diseases which infects many bird species, including the Japanese quail. This study was aimed to evaluate the effect of experimental infection with virulent Newcastle disease virus (NDV) on immune response against the virus as well as on the relative weight of bursa of Fabricius in vaccinated and unvaccinated Japanese quails. A total of 160 one-day-old quails were allocated to 4 groups, including a vaccinated and challenged group (1), an unvaccinated and challenged group (2), a vaccinated and unchallenged group (3), and an unvaccinated and unchallenged group (4). The birds were raised under the similar conditions. Vaccination was done on the 20th day with live vaccine B1 strain (Razi Vaccine and Serum Research Institute, I.R.Iran) via eye drop and the challenge by Newcastle disease virus (NDa: KP347437) was induced via eye drop on the 34th day. Blood samples were collected for serological assessments (HI) and the bursa of Fabricius was weighed after autopsy at different time periods. The first postvaccination serological change was observed in group 3 twenty-one days after vaccination while the first post-challenge serological change was detected in groups 1 and 2 one week after the challenge induction. The same three groups also showed significant increments in the serum NDV-specific antibody titer in the subsequent weekly samplings. The antibody titer in group 4 was zero throughout the experiment. Regarding the relative weight of bursa, the only statistically noticeable change was observed within group 2 where the samples taken on the 5th day were significantly heavier than those collected on the 9th and 14th days. The results of this study showed that a single vaccination of quails with B1 strain, in the absence of maternal antibody, could provide a good protection against virulent NDV. With due attention to the Newcastle disease status in Iran, at least once vaccination is recommended during the rearing period of quails.

Lactoferrin is a multifunctional protein that has various and amazing properties with antimicrobial properties ,can be a good candidate for clinical and commercial applications. Lactoferrin was purified by ion exchange chromatography using CM sephadex C-50 and to confirm the purification, SDS-PAGE tests and the absence of dye in the presence of tetramethylbenzene were used In this study, in order to isolate lactoferrin, first the whey proteins of milk using ion exchange chromatography by Sephadex C-50 was purified and SDSPAGE test was performed to confirm the purification as well as no dye in the presence of tetramethyl benzidine. New Zealand white male rabbits were immunized with 120 µg / ml lactoferrin and using complete and incomplete Freund’s adjuvant for 6 weeks and after serum isolation were confirmed specific antibodies by dot blot and ELISA. To purify of the specific antibody, the precipitation method with 40% ammonium sulphate was used. After confirmation, it was attached to cyanogen-activated Sepharoz. The whey portion of bovine colostrum was passed through the affinity chromatographic column. In order to purify lactoferrin, it reacts to form a complex of antibody antigens, and eventually other unbound compounds are washed and removed. Purification of lactoferrin was confirmed and its concentration was calculated by Bradford test. The results showed that the immunization method was used to produce specific antibodies against Lactoferrin is a suitable method and purification of lactoferrin from bovine milk using affinity chromatography has an efficiency of 64%.

Plant extracts can provide antifungal effects on fungi, including dermatophytes. Cinnamon and turmeric, with their cinnamic aldehyde and curcumin, respectively, can act as important antifungal plants. The two fungal strains of Microsporum canis and Trichophyton mentagrophytes are among the most important causative agents of dermatophytosis. Dermatophytosis is a zoonotic and dangerous disease against human and animal health. First, we prepared two fungal strains of Microsporum canis (PTCC No:5069) and Trichophyton mentagrophytes (PTCC No:5054) as live culture from the Persian Type Culture Collection centre (PTCC). Then, for the preparation of methanolic extracts of cinnamon and turmeric, we performed Maceration method, and after that, we prepared a standard fungal suspension. For positive control, in addition to the chemical drug itraconazole, we also used positive and negative controls in the microdilution method. To expose these two extracts to these two fungal strains by microdilution method using serial agar dilution in 1640 RPMI medium and then finding the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC). The initial concentrations of the extracts used for this experiment were 500, 1000, 1500, 2000, 3000, 5000 PPM. In these experiments, we used Paired Samples Test or T-test of paired samples and SPSS software.According to the results, the effects of cinnamon and turmeric extracts on Trichophyton mentagrophytes at Minimum Inhibitory Concentration (MIC) are respectively 1250 and 750 PPM, and on Microsporum canis are respectively 125 and 250 PPM. Similarly, the effect of cinnamon and turmeric extracts on the Minimum Fungicidal Concentration (MFC) on Trichophyton mentagrophytes is 2500 and 1500 PPM, respectively, and on the Microsporum canis at both concentrations are 500 PPM. The effective concentrations of itraconazole on Microsporum canis and Trichophyton mentagrophytes were respectively 32 and 64 PPM for MIC and 64 and 128 PPM for MFC. Following the conclusion, we found that cinnamon and turmeric were effective on these two fungal strains, so that these two extracts were more effective on the fungal strain of Microsporum canis than Trichophyton mentagrophytes, but their fungicidal and inhibitory effects have been far less than those of an antifungal chemical such as Itraconazole.

Avian influenza is a contagious viral disease with economic and public health impact that affects birds, humans, and other mammals. Influenza virus belongs to the Orthomyxoviridae family and based on the difference between nucleoprotein (NP) and matrix protein (M) antigens is classified into three subgroups A, B, and C. Subgroup A is responsible for causing disease in birds. Wild aquatic birds such as ducks, geese, swans, and shorebirds are the main reservoirs of influenza viruses. Influenza viruses are shed through respiratory secretions and feces. The aim of this study was detection of avian influenza virus in aquatic birds in a bird supply center and in the bird garden of Tehran. To detect the influenza virus, 142 sample of fresh feces, cloacal and oropharyngeal were collected by using sterile swabs from 10 bird species including Domestic duck, Dalmatian pelican, Greater flamingo, Whooper swan, Mute swan, Black swan, Crested duck, Bar-headed goose, Canada goose, and Ruddy shelduck during July to September 2021. Collected samples were pooled in 50 tubes where 2 and 3 swabs include 1 sample. Samples of each tube were related to a bird or a species. All samples were immediately transferred to the laboratory with ice and tested for detection of influenza virus using the matrix gene and nucleoprotein gene by RT-PCR method. Comparing positive control and negative control, the results of all samples were negative. So, all the samples were negative. Therefore, at the time of doing this study, there was no serious risk of transmission of influenza viruses to terstal birds and to humans that visited these places. This fact could be influenced by the time of the sample collection and this could be related to low titer shedding of influenza virus in the summers. Besides, detection of no positive sample could be related to vaccination against influenza virus in the bird garden of Tehran. In this way, vaccination in aquatic birds can play an important role in reducing infection to influenza virus and improving public health.

Antimicrobials are generally said to the materials that are used to fight with micro-organisms to eradicate or inhibit their growth. Condition medium (condition culture medium) is secreted for proliferation and cell culture of Mesenchymal stem cells in laboratory culture medium. This compound has factors such as TGF-B, TNF, GM-SCF, KGF, PDGF, VEGF, EGF, IL-1, IL-6, IL-8, BFGF, TGF-α, IGF-1, IGF-2, MCP-1, HGF, Fibronectins and Collagen. Considering the presence of these factors and their role in the body, the aim of this study has been to investigate the effect of Condition Medium of Fetal umbilical cord. In this study 5 encoded bacterial standard isolations including Escherichia Coli, Staphylococcus, Pseudomonas, Salmonella and Listeria were investigated using MBC and MIC method and a standard encoded Candida albicans yeast using Phagocytosis test in confluence of the condition Medium of fetal umbilical cord. The results of MIC and MBC showed that Condition Medium has the effect of growth inhibition in 100% of condition medium density on Salmonella and Listeria monocytogenes but it doesn’t have any effect on growth inhibition of the rest of the bacteria. Also disc results showed the ineffectiveness the material on the growth of microorganisms except for the bacteria mentioned earlier. Phagocytosis test showed reinforcement in Phagocytosisic effect and cellular immune caused from it in adjacent samples with Condition medium cells of fetal umbilical cord. Furthermore, the macroscopic and morphologic study showed clearly that the size of Phagocytosis cells in treatment group is bigger than their size in the control group. Overall, we can say that medium condition of the stems cells of fetal umbilical cord don’t have much antibacterial and antifungal effect, and this effect was seen insignificantly in some cases such as Salmonella and Listeria monocytogenes and it can’t be a substitute for antibacterial materials.

A study was performed in the Persian capital province of Tehran to assess frequency of Para tuberculosis in its operating cattle farms. As many as 8,000 cows were screened by a homemade ELISA system. Milk specimens were collected from detected reactor to perform confirmatory PCR-16srRNA, PCR-IS900 and PCR IS1311 experiments plus bacterial culture on specific media in search for Mycobacterium avium subsp Para tuberculosis (MAP). While among the 25 ELISA positive(0.3%) animals eight cases produced positive results in PCRs(0.1%) only three appeared positive in MAP isolation(0.04%). In deduction, we assume when it comes to selection of control measures against Ptb, in an environment with presumably high prevalence of Ptb like Tehran, ELISA is a much more sensible detection system compared to the zero false-positive method of bacterial culture.

Newcastle disease (ND) is a highly contagious disease in poultry caused by the Newcastle disease virus (NDV). ND is considered one of the most important poultry diseases worldwide and may cause up to 100% mortality in infected chickens. Given the high mortality caused by NDV, the disease diagnosis and characterization of the circulating viral strains is critical. In this study, further studies have been performed to determine the molecular identity of a velogenic Newcastle disease virus genotype XIII.2.1 named RT30/2010, which is the first report of an XIII.2.1 NDV from Iran. In this study, virus purification was performed in two steps by the plaque purification method. The complete coding sequence of the NP gene was investigated to provide additional information about the nature and molecular epidemiology of RT30/2010. For this purpose, the NP gene of this virus was amplified using specific primers and cloned into PTZ57R/T vector. Rapid amplification of cDNA ends (RACE) was used to characterize the 3’ end of the virus genome, which includes the beginning region of the NP gene. Phylogenetic and genetic distance analysis of the nucleoprotein gene of RT30/2010 place the virus in a clade of Pakistani viruses of XIII.2.1. Therefore, RT30/2010 has a different origin compared to other viruses identified from Iran and is in the category of Newcastle disease viruses detected from Pakistan. The information from this study helps to further characterize the virus and makes it possible to use this virus as a challenge or a vaccine strain against XIII.2.1 viruses.

Escherichia coli is the causative agent of colibacillosis in poultry leading to heavy economic losses and increased antibacterial use in poultry flocks with a consequence of increased drug resistance. In order to reduce drug resistance, as a global problem, and to establish better health among humans, animals and environment, a periodical monitoring of drug resistance rate is required. The aims of this study were to investigate the resistance of Escherichia coli isolates to different antimicrobial agents and also to detect florfenicol (cfr, fexA, floR) and colistin (mcr-1) resistance genes among isolates. One hundred Escherichia coli isolates originated from typical lesions of colibacillosis were collected from poultry referred to a private laboratory in Tehran and University bacterial collection and the drug resistance of isolates to 16 antimicrobial agents including ampicillin, neomycin, gentamicin, enrofloxacin, flumequine, difloxacin, chloramphenicol, florfenicol, Fosbac, erythromycin, colistin, tetracycline, oxytetracycline, trimethoprime+sulfa, linco-spectin and doxycycline was determined by using disk diffusion method. Then, the chromosomal and plasmid DNA were extracted and florfenicol (cfr, fexA, floR) and colistin (mcr-1) resistance genes were detected among all isolates by using polymerase chain reaction test (PCR). Based on antimicrobial susceptibility test, the highest resistance was observed to erythromycin, doxycycline and tetracycline and the lowest resistance rate was found to linco-spectin, gentamicin and Fosbac. All isolates were resistant to at least one and 10% of isolates were resistant to at least 12 antimicrobial agents. The 16% of the isolates belonged to one identical pattern and 35% of isolates each belonged to a separate pattern. Among 85 tested isolates, 40 and 52.94% of the isolates showed floR gene on their plasmid and chromosomal locations, respectively. However, no isolate was positive for fexA, cfr and mcr-1 resistance genes. The findings of this study demonstrated the high frequency of resistance to commonly used antimicrobial agents among E. coli isolated from colibacillosis. Detection of resistance genes increases the researchers’ knowledge on the epidemiology of drug resistance. This information indicates the necessity for implementation of the right management programs for poultry farms and rational antimicrobial therapy besides periodic antimicrobial susceptibility monitoring.

The proliferation of antibiotic-resistant microorganisms in wounds has grown efforts to synthesis the antibacterial nanostructured compounds for overcoming on antibiotic-resistant microorganisms. The aim of the current study is to investigate the antibacterial and anti-fungal activities of laterite nanoparticles (Ltt-NPs), silver/laterite (Ag/Ltt-NCs) and silver/laterite/chitosan nanocomposites (Ag/Ltt/Chn-NCs) by analyzing minimum inhibitory concentration, minimum bactericidal concentration and killing-time of Candida albicans fungus and Acinetobacter baumannii, Streptococcus pyogenesis, Escherichia coli and Proteus mirabilis bacteria. We found that, where 5.0 µg/ml of Ag/Ltt-NCs are efficient against Acinetobacter baumannii and Streptococcus pyogenesis, 2.5 µg/ml of Ag/Ltt/Chn-NCs has a favorable efficiency against Escherichia coli and Proteus mirabilis. No significant difference was observed between the antibacterial activity of Ag/Ltt-NCs and Ag/Ltt/Chn-NCs (p=1.00). In addition, Ltt-NPs showed a poor antibacterial activity compared to the Ag/Ltt-NCs and Ag/Ltt/Chn-NCs. Moreover, we found that neither nanocomposite do not show antifungal activity. Our results show that the highest antibacterial activity of the nanocomposites occurs between 6-24 hours. In sum, the prepared nanocomposites show

The aim of this study was to investigate the probiotic characteristics of the isolated lactic acid bacteria (LAB) in coagulated cow's milk including antimicrobial activities, acid and bile tolerance, fermentation profile, hemolytic properties, acidification and coagulation collected from some villages of Tehran province were examined. Among the 13 milk samples collected, 7 isolates were identified as lactic acid bacteria based on phenotypic and biochemical tests. In order to determine the biochemical results, their DNA was extracted and amplified by PCR with specific primer and then sequenced by PCR products and they were recorded in the NCBI database. These results definitely confirmed that the isolated strains belong to lactobacilli. The results showed all isolated LABs were positive for hemolytic activity. LABs A and C showed beta hemolysis. LAB E showed the highest resistance to acidic environments, whereas all the our isolates were resistant to bile conditions. The supernatant obtained from lactic acid bacteria had an effect on Escherichia coli and a non-growth halo was created. But it had no effect on staph and fungi. all LAB isolates exhibited the ability of acid production and coagulation at 30°C. In contrast, none of the LAB isolates presented coagulation potential at 30 °C. Based on this study results, LAB E and C isolated from coagulated cow's milk could be considered as probiotic microorganisms considering the observed properties. They can be purified and used for the production of probiotic dairy products.