Molecular evaluation of M2 protein of Iranian avian influenza viruses of H9N2 subtype in order to find mutations of adamantane drug resistance

Background: the H9N2 subtype of influenza A viruses is considered to be widespread in poultry industry. Adamantane is a group of antiviral agents which is effective both in prevention and treatment of influenza A virus infections. These drugs inhibit M2 protein ion channel which has role on viral replication

Objectives: the main objective of this study is to evaluate M gene of avian influenza viruses [AIVs] of H9N2 subtype in order to find adamantane drug resistance mutations

Methods: over 100 suspected samples were collected from different geographical regions of Iran during 2012-2013. Samples were injected via allantoic sac of 9-11 day-old chicken embryos. A total of 11 out of 100 were AIV. The H9N2 subtype was confirmed by specific RT-PCR. The RT-PCR was conducted for full length M gene. PCR amplified products were purified and then conducted for commercial direct sequencing. Finally, sequences were checked for possible sites of adamantane resistance mutations

Results: overall, 8 out of 11 viruses harbored the adamantine resistance-associated mutations. Of which, four viruses were isolated in 2013 and four viruses in 2012. Two different resistance- associated mutations were observed during different years

Conclusions: the present study provided clear evidence concerning resistance AIVs of H9N2 subtype that were circulating in Iranian poultry sector. This concern is always present as M segment might be introduced into human influenza viruses by reassortment phenomenon

Effects of different culture media on optimization of primary neuronal cell culture for in vitro models assay

BACKGROUND: In vitro model studies are becoming increasingly popular for experimental research designs. They include isolation and expansion of cells of a particular tissue, such as the nervous tissue which contributes to understanding the underlying mechanisms in many pathologies. It enables the scrutinization of intracellular signaling pathways responsible for cell death.

OBJECTIVES: In the literature, there are different methods for the isolation and culture of rat embryonic cortical neurons. However, this study developed a feasible, rapid and easily performable method.

METHODS: Isolation of neurons was performed without using enzymatic digestion. Primary cortical cultures neurite outgrowth and neuron numbers per field of common mediums were compared for neuronal cells isolation and expansion. In this study, three different culture mediums were considered: Medium I: Neurobasal medium, B-27 and L-glutamine; Medium II: DMEM, FBS and L-glutamine; and Medium III: DMEM/F-12, FBS and L-glutamine.

RESULTS: High survival rate and number of neurons was obtained with the current method. The best neuronal growth was achieved by Medium I, while Medium II and III had moderate effect on the neurite outgrowth.

CONCLUSIONS: Enzyme-free treatment was introduced and Medium I was used as an alternative method for optimal neuron isolation and expansion. The neuronal cultures are similar to nervous tissue in physiological aspects. Hence, Medium I is more similar to the in vivo condition compared to Mediums II and III.

Detection of cytolethal distending toxin [cdt] Genes in Campylo-bacterjejuni and Campylobacter coli isolated from the intestinal of commercial broiler chickens, turkey and quail of Iran

Campylobacter jejuni and Campylobacter coli are zoonotic bacteria which are frequently associated with human diarrhea. Sharing of the cytolethal distending toxin [cdt] genes in Campylobacter is common and is considered species specific. In this study we focused on detecting the presence of cdt gene in C. jejuni and C. coli isolated from broilers, turkeys and quails of Iran. Cecal samples were randomly collected from 240 broiler chickens, 100 meat type turkeys and 100 quails after slaughtering. We used PCR as a method for detecting cdt genes. In broilers, 93% of 58 C jejuni positive samples possessed cdt gene and in all cases the three different subunits of cdt genes were present. However, only 56% of 14 C. coli isolates in broilers had contained cdt genes, while one fourth having all three subunits present. In turkeys, around 65% of 34 C. jejuni positive samples had cdt gene present with 38% possessing all three subunits of cdt genes. But all 5 C. coli isolates had all three subunits cdt gene. In quails, 67% of 30 C. jejuni positive samples were identified by cdt gene, 20% of those possessed all three gene subunits. On the other hand, all 28 C. coli isolates of quails had cdt gene present while 36% of those held all three gene subunits. Our data is indicating the isolation, culture and cdt PCR amplification approaches in this study seemed to be efficient. However, the presence of different variation of Campylobacter cdt gene types in our sample isolates signifies the necessity of further functional gene studies to elucidate which gene type combinations result in encoding effective toxins

role of house fly [Musca domestica] in transmission of pathogenic strains of E.coli

Colibacillosis is an infectious disease caused by E. coli. This infection is spread by the feces and other secretions of infected animals and humans. Non-biting Muscid flies characteristically visit manure and decaying organic materials to feed and oviposit and may contribute to disease transmission. This paper reviews the role of house flies [Musca domestica] in E.coli transmission at poultry farms. In this study, 2000 house flies [Musca domestica] were collected from 2 commercial laying hen farms around Karaj. They were examined for the presence of the bacteria. In an adjacent study, laboratory-reared flies were experimentally exposed to E. coli [ATCC10536] strain and the infected samples were examined at regular intervals. The E. coli was detected in the dissected gasterointestinal tract of laboratory-exposed flies for up to 72 h. post exposure, whereas after that time the infected flies and the untreated control flies were negative. The results showed that considering the ability of mechanical vector during 72 hours, the E. coli transmission is possible in short time

[Antibody monitoring to canine distemper virus in unvaccinated rural dogs in the southern coastal region of Caspian sea]

Mass mortality of Caspian seal has occurred in recent years and canine distemper virus [CDV] has been identified as the main pathogenic agent in these events. Despite the repetition of this event and the presence of a large number of dead seals in the coastal region of the Caspian Sea, very little is known about the epidemiological role of these animals in canine distemper virus survival in this area. In this study the frequency of antibody against CDV in unvaccinated rural dogs in the southeast coastal region of the Caspian Sea [Iran] was evaluated by means of serum neutralization test. Serum samples [185] were randomly collected from rural dogs from 2008 to 2010. Totally the frequency of positive antibody reaction in animals against CDV was found to be 55.6% [103/185] in 1/32 dilution [CI%95: 47-61]. In this study no significant difference in susceptibility was observed between males and females and among different age groups. Moreover, most of the positive cases were observed during the winter. These results indicate that this virus is present in the ecosystem. Furthermore, there is evidence of previous natural exposure to CDV. This high frequency of antibody in serum samples might be because of previous contact with CDV contaminated corpse of Caspian seal. Dogs' population in rural areas is dense enough to maintain CDV in environment and rural dogs can be a reservoir of infection for urban dogs and wild carnivores

Molecular characterization of non-structural gene of H9N2 subtype of avian influenza viruses isolated from broiler chickens in Iran

The H9N2 subtype of avian influenza viruses [AIVs] have spread in Asia and Middle East countries and have become a serious threat to poultry industry in Iran. Characterization of genes of H9N2 subtype involving in pathogenicity and diagnosis are crucial in control of avian influenza outbreaks. The Nonstructural [NS] gene and its protein products [NS1 and NS2] are important as diagnostic marker, life cycle and pathogenicity of AIVs. The NS gene of five strains, isolated from 1998 to 2010, were completely sequenced and analyzed. All of the examined strains were composed of 890 nucleotides with 230 amino acids. In this regard, only two Iranian strains from GeneBank had 217 amino acids in NS 1 protein. All Iranian H9N2 strains subdivided into two distinct sublineages including I and II. Comparative analysis of NS genes of Iranian strains showed that since 2003, they might have originated from Pakistan H7N3 strains; whereas from 2008 they could be originated from Pakistan H9N2 strains. Although the low pathogenic H9N2 subtype has been permanently circulating from 1998 to the present in Iran, phylogenetic analysis of NS genes revealed that sublineage II has circulated more in poultry industry of Iran. These epidemio-logically variations could be related to vaccination pressure due to massive vaccination or NS gene reassortment in rural and backyard chickens

[ survey of the effect of Cuminum cyminum L. essential oil on the growth of Bacillus cereus in a food model system]

Bacillus cereus is a gram positive and spore-forming bacteria that often associated with food products such as meat, vegetables, soup, rice, and milk and other dairy products. 1 - 20% of total outbreaks of food intoxication in the world are caused by Bacillus cereus. There is an increasing interest in the use of plant-derived antimicrobial compounds as natural preservatives for foods; such as Cuminum cyminum L. essential oil that inhibition of the growth of several pathogens by it has been reported in various articles. The objective of this survey is evaluation of antibacterial effect of Cuminum cyminum L. essential oil on Bacillus cereus in a food model. The essential oil of Cuminum cyminum plant was obtained by steam-distillation and analyzed by GC/MS. Then, effects of different concentrations of this essential oil [0, 50, 150, 300 and 450ppm] on growth of Bacillus cereus were evaluated in commercial barely soup in two storage temperature [10 and 25°C] at 12 intervals [0, 1, 2, 3, 4, 5,6, 9, 12, 15, 18 and 21 days]. The logarithm of Bacillus cereus was significantly decreased by 300 and 450ppm EO concentration incubated at 10°C storage temperature and 450 ppm EO concentration incubated at 25°C storage temperature [p < 0.01]. The results showed, the potential inhibitory effects of the Cuminum cyminum L. essential oil on Bacillus cereus in a food model [commercial barely soup]

Isolation of chlamydophila psittaci from pet birds in Iran

Avian chlamydiosis is one of the most important infectious diseases of birds. Despite the rapid growth of exotic bird populations in Iran, there is little or no information on the specific infections that these types of birds carry. In this study, conventional isolation methods were used in cell culture to study occurrence of infection in pet birds. Samples from the conjunctiva, choana, and cloaca and/or droppings were provided from 17 birds of different species. The samples were used to infect McCoy cell culture to isolate Chlamydophila psittaci. The inoculated cells were fixed, stained by Giemsa, mounted on slides using Entellan_ and observed by light microscope for the presence of typical chlamydial inclusion bodies. Chlamydophila psittaci was isolated from four birds including a ring-necked parakeet [Psittacula krameri], an Alexandrine parakeet [Psittacula eupatria], an African grey parrot [Psittacus erithacus], and a Timneh grey parrot [Psittacus erithacus timneh]. All negative cultures were passaged a further two times. To the best of our knowledge; the report represents the first isolation of chlamydia from birds in Iran

Relationship between in vitro antimicrobial sensitivity of bovine subclinical mastitis isolates and treatment outcome in lactating dairy cows

The objective of the present study was to determine whether there was an association between in the in vitro antimicrobial sensitivity test results of subclinical mastitis pathogens and bacteriological cure following intramammary treatment using a combination of nafcillin, penicillin, and streptomycin [NPS]. Eighty-six intramammary pathogens from 43 cows were examined in this study. Most intramammary infections were due to coagulase-negative staphylococci, coliforms, and environmental streptococci. The antibiotic sensitivity to NPS was determined using the Kirby-Bauer disc diffusion method. Bacteriological cure rates for sensitive, intermediate, and resistant isolates in the standard treatment group [3 intramammary infusions of NPS once daily] were 84.44, 88.9, and 100%, respectively. These figures in the extended treatment group [6 intramammary infusions of NPS once daily] were 100% for the 3 categories. Bacteriological cure was not associated with the sensitivity test result. Based on this study, Kirby-Bauer sensitivity test results were not useful as predictors of the bacteriological outcome of subclinical mastitis treated with intramammary NPS

Conventional and extended intramammary therapy of persistent subclinical mastitis using nafcillin-pencillin-dihydrostreptomycin in lactating dairy cattle

The objective of the present study was to compare the efficacy of conventional and extended intrammamary [IMM] therapy of persistent subclinical mastitis in lactating dairy cattle using nafcillin-penicillin-dihydrostreptomycin combination [NPD]. Sixty-five dairy cows with 126 infected quarters were enrolled in the study. Infected cows were allocated randomly to 1 of 3 different treatment regimens: [1] conventional group: NPD administered IMM 3 times at 24-h intervals [20 infected cows, 43 intramammary infections [IMI]], [2] extended group: NPD administered IMM 6 times at 24-h intervals [23 cows, 43 IMI], and [3] untreated control group [22 cows, 40 IMI]. The overall bacteriological cure [BC] rates for subclinical IMI were 86.04%, 100%, and 20% for the conventional, extended and the control groups, respectively; indicating a higher BC rate [P<0.0001] for the treated groups than the control group. Significant difference [P=0.029] concerning the BC rate was also observed between the extended and the conventional groups. Significant difference [P=0.0021] in somatic cell count [SCC] was detected between the extended and the conventional groups. Significant difference [P=0.0021] in somatic cell count [SCC] was detected between the extended and the control group. Fat percentage increased in the conventional [P=0.029] and in the extended [P<0.0001] groups, and protein percentage increased only in the extended group [P=0.0016]. There was no significant difference in posttreatment milk production between the groups [P>0.05]. Results of this study indicate that NPD therapy was effective in eliminating subclinical IMI in lactating dairy cows, and that extended therapy enhanced BC rate and reduced SCC

Relationship between in vitro susceptibility of bovine subclinical mastitis isolates and bacteriological outcome of intramammary treatment with cefquinome

The objective of the present study was to determine whether there was an association between the in vitro antimicrobial susceptibility test results of subclinical mastitis pathogens and bacteriological outcomes of intramammary treatments using cefquinome. A total of 110 intramammary pathogens from 51 cows were assessed in this study. Most intramammary infections were due to coagulase-negative staphylococci, environmental streptococci, and coliforms. The antimicrobial susceptibility to cefquinome was determined using the Kirby-Bauer disc diffusion method. Bacteriological cure rates for the sensitive, intermediate, and resistant isolates in the standard treatment group [three intramammary infusions of 75 mg cefquinome at 16 h intervals] were 82.4%, 90%, and 87.5%, respectively. These figures in the extended treatment group [six intramammary infusions of 75 mg cefquinome at 16 h intervals] were 83.3%, 100%, and 100%, respectively. Treatment outcomes were not associated with the results of sensitivity tests in the standard group. However, in the extended group, the probability of a bacteriological cure was lower in quarters from which cefquinome-sensitive pathogens were isolated than the quarters from which intermediate or resistant pathogens were isolated. Based on this study, the Kirby-Bauer susceptibility test result is a poor predictor for the bacteriological cure of subclinical mastitis treated with intramammary cefquinome

effect of sodium arsenite in induction of protein stress in chicken embryo model

Arsenic is one of the most important current environmental toxicants. Arsenic is one of the biggest protein stress inducer in several organs and systems. One of the basic and sensitive criteria for following protein stress is assessing carbonyl and thiol groups of proteins. Therefore, we assessed protein stress that produced by sodium arsenite in chicken embryos by measuring carbonyl and thiol proteins. After 4 days of incubation, 36 fertilized eggs were candled. The eggs that had alive embryos received a single injection of 0.1 and 0.5 ppm arsenite sodium in two separate groups of 12 eggs and the rest 12 [control group] received 0.5 ml saline into the yolk sac. After 20 days of incubation, teratogenicity and external defects in embryos were investigated, one ml of embryo blood was analyzed for assaying protein thiol and carbonyl as well. Data were analyzed by SPSS [version 16] with ANOVA test [tukey]. The mean of carbonyl protein was in 0.1 ppm group 0.835, 0.5 ppm group 0.844 and control group 0.804 and this change was significant and dose dependent. In addition, the mean of thiol protein was in 0.1 ppm group 0.053, 0.5 ppm group 0.014 and control group 0.054 and this change was also significant and dose dependent. The carbonyl and thiol protein alterations in serum of embryos exposed to arsenite sodium, suggest the embryotoxicity of this agent induction of plasma carbonyl and thiol protein stress

Effect of systemic antibacterial administration during prepartum period on coagulase negative Staphylococcal intramammary infection in Holstein Heifers

A total of 183 coagulase negative Staphylococcal infected quarters and 64 uninfected quarters were randomly allotted to 4 experimental groups: infected quarters that did not receive any treatment [positive control group; n = 60]; infected quarters that received Tylosin [n = 61] or Cefquinome [n = 62] and also uninfected quarters that did not receive any treatment were considered as negative control group [n = 64]. Treated heifers received Tylosin or Cefquinome 10 to 14 days before the expected calving date. The bacteriological cure rate based on sampling obtained 3 days after calving was significantly higher [P<0.01] in mammary glands treated with systemic Tylosin [85.3%] than in the positive control group and mammary glands treated with systemic Cefquinome [69.4%]. At the same time Tylosin treated heifers had significantly lower [P<0.01] somatic cell count scores compared to the positive control and Cefquinome groups. With the exception of one heifer that calved 8 days earlier than the expected calving date, there was no detectable antibiotic residues in the milk of the treated heifers on the 3rd day of lactation

[Study the effects of different levels of hull-less barley on caecal micro flora of broiler chicks]

This study was conducted to investigate the effects of diets containing different levels of hull-less barley on caecal microflora of broiler chicks. Two hundred and forty one-day old male broiler chicks [Arbor Acres strain] were used in a completely randomized design with 4 tratments, and 3 replicates were allocated to each treatment. Different levels of hull-less barley in diets had not significant effects on total bacterial count, E.coli, Clostridia and Lactobacillus population in caeca at starter period [21 days old], but at 49 days of age, caecal Lactobacilus population increased significantly by increasing the levels of hull-less barley in diets[p<0.05]. Based on the results of the present study, it can be stated that inclusion of hull-less barley in diets, changes the caecal microflora of broiler chicks. These components reduced pathogenic bacteria [Clostridia] and increased the usefull bacteria [Lactobacillus]

[Serologic profile of salmonella enteritidis in poultry flocks of Iran]

Salmonella Enteritidis [SE] is frequently isolated from poultry and humans. Chicken meat and egg are two important sources of SE infection for humans. This study was conducted to detect the SE infection in Iranian poultry farms using serological methods. A number of 8208 serum samples were provided from 171 poultry flocks [pullet, commercial layer, broiler breeder, layer breeder, grandparent breeder, and broiler] and were analyzed by rapid slide agglutination test [RSA] and ELISA. All samples were negative in RSA but 45% 1 of samples [from 112 flocks] contained anti-SE antibody in ELISA. The titres ranged 0.476-0.590, which were at low level and showed the value of ELISA vs. RSA. The results indicated that the poultry flocks had been infected with SE at least once during the course of production. This is the first comprehensive study, at this sample size, in Iran regarding the serologic profile of SE in poultry flocks and its findings are very important for poultry industry and the control strategy for SE infection

Bacterial causative agents of conjunctivitis and keratoconjunctivitis in cattle, sheep and goat and survey of pathogenicity and immunogenicity of Moraxella species

Bacterial culture of eye secretion from bovine and ovine infected eyes was done during a period of three years [1992-1994]. Moraxella bovis [M. bovis] in bovine and Branhamella ovis [B.ovis] in ovine cases were the most isolates. M. bovis was isolated from 72 of a total of 132 bovine cases [54.54%]. The other isolations were staphylococcus sp., streptoccus sp. Pseudomonas aeroginosa and non pathogenic moraxella sp. In sheep and goat B ovis was isolate from 7 cases of a total of 17 [41%] and two cases were non pathogenic moraxella sp. This observation indicates the high incidence of pathogenicity of B. ovis in these animals however the mechanism of bacterial infection is still unknown. Since Chlamydia and rickettsiae are tought to be responsible for primary conjunctivitis two methods of staining were employed to observe these organisms in eye epithelial cell: 1] Macchiavello staining and 2] modified Ziehl Nelson staining. Only two cases of cattle infected eyes showed chlamidial bodies with second staining method