Sequence analysis of the VP1 gene in three very virulent infectious Iranian bursal disease virus strains

Infectious bursal disease [IBD] is a highly contagious disease of chickens caused by the infectious bursal disease virus [IBDV]. This study was conducted to characterize three IBDV strains from Iran. A reverse transcriptase-polymerase chain reaction [RT-PCR] procedure was used to amplify a 715-bp fragment of the VP1 gene from IBDV strains. Amplified VP1 fragments of the three Iranian IBDV strains were sequenced and compared with published sequences of IBDV strains from around the world, and their phylogenetic relationships were analyzed. Alignment of IBDV strains revealed 23 nucleotide differences between vvIBDV [except for IL and PT] and other non-vvIBDV strains. Two nucleotide positions, 863G and 1023A, were specific as JRMP07IR and JRMP14IR strains. All vvIBDVs differed [except for IL and PT strains] from non-vvIBDVs at aa [amino acids] positions 242E and 287A. In the three Iranian IBDV strains, aa positions 251R in both JRMP07IR and JRMP14IR, and 360L in JRMP14IR differed from those of other vvIBDVs. In phylogenetic analyses, all three Iranian strains clustered together with vvIBDVs. One Iranian strain, JRMP30IR, was more closely related to two European strains [HOL and UK661] and two south-east Asian strains [OKYM and ZJ2000]. However, the other two Iranian strains, JRMP07IR and JRMP14IR, were closer to two Turkish strains [OA/G1 and OE/G2] and a Malaysian strain [UPM94]. Further comprehensive investigations will provide researchers a better knowledge on the distribution, variability, and phylogenetic relationships of different IBDVs isolated in Iranandother parts of the world. Key words: Infectious bursal disease virus, Very virulent strains, VP1, Chicken, Iran

Characterization of Salmonella isolates from poultry sources in Iran

Salmonellosis is one of the most important zoonotic diseases throughout the world. The purpose of this study was to characterize a large collection of Salmonella isolates from different poultry sources in Iran. A total of 123 Salmonella isolates from different poultry sources were subjected to drug susceptibility test, hemolysin production, motility test, and plasmid profile [50 isolates]. Seventy-one resistance patterns were found to 29 antimicrobial agents among 123 Salmonella isolates, in which 81% of isolates were resistant to more than one antibacterial agent. The resistance patterns of 123 isolates to 10 commonly used antibacterials in Iranian poultry industry were also quite variable and included 31 patterns. Four different plasmid patterns were found among 50 Salmonella isolates. Fifty four percent of Salmonella isolates harbored one or three plasmids with approximate molecular size ranging from 2.3 to 68 kb. No plasmid was detected in 46% of isolates. A band of 68 kb size was detected in all isolates that harbored plasmid. All isolates were motile but no isolate showed hemolysin production. The frequency of resistance to antibacterial agents among avian Salmonella isolates is a major public health concern

Detection of avian reoviruses causing tenosynovitis in breeder flocks in Iran by reverse transcription-polymerase chain reaction [RT-PCR] and restriction enzyme fragment length polymorphism [RFLP]

Avian reoviruses [ARVs] are members of the Orthoreovirus genus; one of the 12 genera of the Reoviridae family. The ARVs are the cause of some important diseases in poultry such as reovirus-induced arthritis, tenosynovitis, chronic respiratory disease, and mal-absorption syndrome. In this study, the presence of ARVs in the Iranian breeder flocks was investigated through reverse transcriptionpolymerase chain reaction [RT-PCR] and restriction enzyme fragment length polymorphism [RFLP]. A total of 800 fecal swab samples were initially collected from breeder flocks [older than 45 weeks of age]. They were then sent to the laboratory in containers with PBS, and after that they were pooled and finally to 120 samples were obtained. The total RNA extracted from the pooled fecal samples were used to amplify the selected parts of the S1 [1023 bp] and S4 [437 bp] genes from the ARV field isolates using RT-PCR. The positive RT-PCR amplified products were further analyzed by RFLP using five restriction enzymes. Based on the findings, 5 samples were positive with the S1 primer and 6 samples were with the S4 one. The patterns observed after the digestion of PCR products revealed that the isolates of this study were identical to both the S1133 vaccine and standard strains. The findings suggested that the RT-PCR/RFLP analysis might be considered as a simple and rapid approach for the differentiation of ARV isolates. This study was the first molecular detection of the ARVs presence in the Iranian breeder flocks using the RTPCR amplification of the S1 and S4 genes and RFLP analysis

[Virulence determination of Iranian Escherichia coli isolates from poultry in day-old chicks using a lethality test]

Colibacillosis due to Escherichia coli is one of the most important infectious diseases in poultry. While the influence of E. coli virulence factor in birds may differ due to interactions with other influential factors, the sole presence of such factors in E. coli does not determine its pathogenicity. Therefore, it is necessary to test the pathogenic capability of E. coli isolates on susceptible birds. The purpose of this study was to determine the virulence of three E. coli isolates from colibacillosis and compare their effects on day-old chicks using a lethality test. Seventy 1-day-old chicks were divided into 3 treatment groups of 20 chicks and 2 control groups of 5 chicks. Each treatment group was assigned to one E. coli isolate and divided into 4 sub-groups of 5 chicks. The overnight broth culture of each E. coli isolate was washed with PBS three times and diluted. Then, 0.5 ml of undiluted culture [tilde10[9] CFU/mL], and the dilutions of 0.1, 0.01, and 0.001 of each culture were injected subcutaneously to each of the 5 birds in each subgroup behind the neck. One group of 5 birds was injected by PBS while negative control group did not receive anything. The chicks were monitored every 12 hours for 6 days. The dead chicks during the course of the experiment and all survived ones were necropsied and samples were taken from hearts and livers for bacteriological culture. Virulence of each E. coli isolate was evaluated based on a scoring system developed on death time, gross pathological observations, and bacteriological findings. All E. coli isolates of this study were capable of causing mortalities and producing the lesions typical of colibacillosis. There were no significant differences among the three E. coli isolates in their in vivo virulence abilities but the difference between each of the three E. coli isolates and each of the two control groups was significant [p

Bacterial contamination of dead-in-shell embryos in ostrich hatcheries and antimicrobial resistance patterns of isolated Escherichia coli

The bacterial contamination of fertile eggs is the most common cause of embryonic death in ostrich hatchery units leading to financial loss in ostrich industry. The aim of this research was to investigate the bacterial contamination status, with emphasis on Escherichia coli, of ostrich hatcheries and the antimicrobial resistance profile of isolated Escherichia coli. A total of 120 ostrich eggs with dead embryos, at weekly intervals, were collected from three ostrich hatcheries. The dead embryos were sent to laboratory and samples were collected aseptically from different organs. Bacterial detection and identification were performed by using standard bacteriological and biochemical techniques. Antimicrobial susceptibility test was carried out by agar disk diffusion method against 27 antimicrobial agents. Different types of bacteria were isolated from 56 eggs [46.7%]. Twenty-four ostrich eggs were shown to carry E. coli. In some eggs, in addition to yolk sac, E. coli was also isolated from meconium, liver, or heart blood which increased the total number of E. coli isolates to 32. All E. coli isolates were susceptible to trimethoprim + sulphamethoxazole, danofloxacin, and flumequine, whereas all were resistant to carbenicillin and erythromycin. Resistance to other agents was variable. Multi-drug resistance pattern was found among all E. coli isolates and included 2 to 12 drugs. Thirty-two E. coli isolates generated 30 different resistance profiles against 27 antimicrobial drugs. This was the first comprehensive report regarding the bacterial, particularly Escherichia coli, contamination of dead-in-shell ostrich embryos and antimicrobial resistance status of the Escherichia coli isolates from ostrich eggs in Iran

Antimicrobial susceptibility of one thousend bacterial isolates to five antibacterial agents commonly used in the Iranian poultry industry

Different susceptibility rates of pathogenic bacteria to antimicrobial agents are considered major factors in the choice of drugs and the success of treatments. Concerns have been raised regarding the emergence of antimicrobial resistance among pathogenic bacteria that may result in unpredictable antimicrobial susceptibilities and therapy failure. The purpose of this investigation was to determine the antimicrobial susceptibility of 1,000 bacterial isolates to five antibacterial agents commonly used in the Iranian poultry industry. From July 2008 to June 2009, the antimicrobial susceptibility of 1,000 bacterial isolates to five antibacterial agents was tested. These agents that are commonly used in the Iranian poultry industry include colistin, doxycycline, enrofloxacin, florfenicol, and sulfamethoxazole + trimethoprime. The data were provided by 19 laboratories in eight Iranian provinces. The bacterial species belonged mainly to Escherichia coli and Salmonella spp. Of all tested samples, 55.5% were resistant to colistin, 61.5% to doxycycline, 41.5% to enrofloxacin, 34.5% to florfenicol, and 65.5% to sulfamethoxazole+trimethoprime. The findings of this survey represent the high frequency of resistance to antimicrobial agents commonly used in the Iranian poultry industry. They also highlight the need for the implementation of a national monitoring program for antimicrobial resistance and for a rational use of antimicrobial drugs

Mixed cutaneous round cells tumor in a cock [Gallus domesticus]: a case report

Cutaneous round cell tumors have been classified as mast cell tumor [MCT], histiocytoma [HCT], lymphosarcoma, undifferentiated round cell tumors and occasionally rhabdomyosarcoma in veterinary medicine. An adult cock [Gallus domesticus] showing a large solitary integument mass raised on dorso bilateral of cervical part, extending to intercapsular and cranial mid part of the back was referred to the birds clinic of the faculty of veterinary medicine at the university of Tehran. Microscopic examination revealed sheeted cells with large, round to oval nuclei with each one containing one or more prominent nucleoli with scant cytoplasm. The myofibrils of the neck were degenerated by aggressive tumor cells. The condition was differentiated from other round cell tumors by electron microscope, histochemical staining, as well as the application of a large panel of antibodies. Polymerase chain reaction failed to confirm the involvement of both Marek's disease virus and avian leukosis virus subgroup-J. It was concluded that the tumor cells were consistent with both B-cell lymphocytes and histiocytes that unusually covered the entire dermal layer of dorsal neck skin. This unusual cutaneous lymphoma was named as lymphoblastic histiocytoma

[Pathogenicity indices of Newcastle disease viruses isolated from Iranian poultry flocks in Iran]

Newcastle disease [ND] is caused by serotype I of avian paramyxoviruses. The ND virus [NDV] strains are conveniently grouped as velogenic, mesogenic, lentogenic, and nonpathogenic-intestinal pathotypes. The purpose of this study was to determine the pathogenicity indices of the isolated NDVs from poultry flocks in Iran. Samples were provided from poultry flocks in different provinces of Iran and prepared for NDV isolation. From many isolated NDVs, 12 isolates belonged to 10 provinces with highly populated poultry farms which were selected for this study. A clone for each of these virus isolates was generated using limiting dilution procedure. Then, the mean death time [MDT], intracerebral pathogenicity index [ICPD, and intravenous pathogenicity index [IVPI] were determined for each virus clone and compared with those of standard virulent strains such as Hertz 33.56 and Texas GB. The results showed that the pathogenicity indices of the NDVs in the present study ranged from 41.6 - 60 hr for MDT, 1.76 - 1.91 for ICPI, and 2.68- 2.88 for IVpI indicate which the velogenic type of our viruses. The findings of this study suggested that the very virulent NDVs currently circulating in Iranian poultry flocks are close to and even more virulent than standard virulent NDVs. Isolation, identification, pathotype determination, and molecular characterization of Iranian NDVs may help authorities to make right decisions to reduce the risks posing the Iranian poultry industry

Molecular typing of avian Escherichia coli isolates by enterobacterial repetitive intergenic consensus sequencespolymerase chain reaction [ERIC-PCR]

Colibacillosis is one of the most economically important diseases of poultry worldwide. This study was conducted to examine the clonal relatedness and typing of 95 avian Escherichia coli isolates by ERIC-PCR.. Sixty-three E. coli isolates from two common manifestations of colibacillosis [yolk sac infection and colisepticemia] and 32 isolates from feces of apparently healthy broilers were provided. The PCR amplification reactions were performed in duplicate for all isolates. The molecular weight of the observed bands on gel electrophoresis ranged from 232 bp to 2690 bp. Sixty-five fingerprinting patterns were observed among 95 isolates on the basis of molecular weights and the number of bands. The numbers of 20, 22, and 23 fingerprinting patterns were found among isolates from yolk sac infection, colisepticemia, and feces, respectively. Among different fingerprinting patterns, the number of produced bands differed from 2 to 11. No identical pattern was observed among isolates of three sources. Isolates showing similar patterns in each source group belonged to a single farm. However, a few isolates that had been isolated from different farms also showed similar fingerprinting patterns. In conclusion, this study showed a high degree of polymorphism among E. coli isolates originated from different poultry sources when the respective bacterial genomes were analyzed by the ERIC-PCR and that no specific genotypes were responsible for different manifestations of colibacillosis

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

[ impact of the routes of live vaccine administration against infectious bursal disease on mounting antibody response in broiler chickens]

Infectious bursal disease [IBD] is one of the most important viral poultry diseases. To prevent the disease it is required that there be maternal and active immunity prior to and after three weeks of age. Live vaccines are usually used to immunize the broiler flocks. In addition to the type of vaccine, the route of vaccination, also, has effects on mounting an immune response. In this study, we administered a single dose vaccination of an intermediate IBD vaccine strain at 21 days of age via five routes including subcutaneous [SC], intramuscular [IM], drinking water, eye drop, and course spray. The impact of the vaccination route on mounting antibody response was evaluated by a commercial ELISA kit [IDEXX]. Antibody response was mounted by all routes. The highest antibody titer in the last two sampling turns belonged to birds in the group vaccinated by the SC route, but this difference was not statistically significant [p>0.05] when compared to those of other vaccinated groups. In addition to the highest antibody titer, the highest bursal/body weight ratio and body weight were observed in birds of the SC-vaccinated group. It was found that the groups vaccinated by injection, SC or IM, were the only groups that achieved to a protective level of antibody titer in the last turn of sampling. It was concluded that a single dose injection of an intermediate IBD vaccine, via SC route, is able to induce higher antibody response, and improve bursal health and performance of chickens as compared with those vaccinated via drinking water

Salmonella infections in poultry flocks in the vicinity of Tehran

The purpose of this study was to survey infections with Salmonella spp. in poultry flocks in the vicinity of Tehran and to determine the most frequent serogroups and serotypes implicated. Twenty-eight samples of pullet, layer, and broiler flocks were randomly collected [n=1463], including freshly dropped feces from live birds or visceral organs from dead birds. In most flocks, 60 samples were taken and 10 fecal samples of each were pooled. Standard cultural methods were used for Salmonella spp. isolation. The slide agglutination or tube agglutination tests were performed using Salmonella somatic O poly A-S antisera, and different somatic O monovalent or flagellar H monovalent antisera. Thirty-one Salmonella isolates were recovered from 1,463 samples. Nine broiler flocks out of 14 [64.2%] and one layer flock out of 11 [9%] were positive for Salmonella spp. but all pullet flocks were negative. One isolate was obtained from the layer flock and the other 30 Salmonella isolates were obtained from broiler flocks. The slide agglutination test determined that all isolates belonged to one of the serogroups from A to S. The frequency of serogroups among 30 broiler isolates was found to be 76.6% and 13.3% for groups C and D, respectively. Three [10%] of the broiler isolates and one layer isolate did not belong to any of the A to D serogroups. All group D isolates were found to be Salmonella enteritidis. This study showed a high incidence of Salmonella in broilers. Infection of broilers with Salmonella spp. poses a high risk to public health

Isolation, identification, and antimicrobial susceptibility of Clostridium perfringens isolates from acute necrotic enteritis of broiler chickens

The aim of this study was to isolate, identify and determine the antimicrobial susceptibility of Clostridium perfringens [CP] isolates from acute necrotic enteritis of broiler chickens. All broiler carcasses diagnosed as necrotic enteritis [NE] were sampled, subjected to microbial tests and 40 isolates were identified according to standard procedures. The antimicrobial susceptibility of CP isolates to 20 antibacterial agents was then determined. The results show widespread resistance among CP isolates. The most frequent resistance was observed to neomycin sulfate [87.5%], and then to lincomycin and tetracycline [both 80%]. No isolate was resistant to chloramphenicol and the least frequency of resistance was observed to vancomycin [10%], sulfamethoxazole+trimethoprim [17.5%], and penicillin [20%]. All isolates were multiple drug resistant types. There were 39 resistant patterns among the CP isolates, 95% of which were distributed in 38 resistant patterns. These multiple and variable resistance patterns observed among the CP isolates, even among different isolates from one farm, demonstrate a challenge for veterinarians in the field to choose the correct compound to combat the occurrence of NE

[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

Psittacine beak and feather disease in Iran, molecular and histopathologic detection

Psittacine beak and feather disease [PBFD] is a major viral disease in wild and captive psittaciformes all around the world. The disease was suspected in a 7 years old lesser sulphur-crested cockatoos [Cacatua sulphured] with a minor feather loss at the back of neck and head. The bird was comprehensively examined by macroscopic pathology, histopathology and polymerase chain reaction [PCR]. Marked intracellular edema of the keratinocytes and necrosis were evident in histopathological observation of dystrophic feather follicles. Numerous macrophages with cytoplasmic inclusions [botryoid] and Prevasculitis were also present in the dermis. Histopathologically, the feather lesions and inclusions were typical of PBFD. The presence of psittacine beak and feather disease virus [BFDV] DNA was confirmed by PCR. This is the first documented report of the occurrence of the PBFD in Iran

[Rapid differentiation between very virulent and classical infectious bursal disease viruses isolated in Iran by RT-PCR/REA]

This study was conducted to characterize infectious bursal disease virus [IBDV] isolates collected from different parts of Iran during 2005-2006. Pooled bursal samples from 49 broiler and layer pullet flocks suspected to IBD infection were collected and processed. A reverse transcriptase-polymerase chain reaction [RT PCR] procedure was used to amplify a VP2 gene fragment [743 bp] from IBDV field isolates. Amplified VP2 fragments were further characterized by two restriction enzymes, BspMI and Sad. From 49 field samples, 37 [75.5%] samples were IBDV positive by RT PCR. Digestion with two restriction enzymes, BspMI and SacI, showed patterns compatible with very virulent IBDV and classical IBDV strains in 34 [91.9%] and 3 [8.1%] IBDV-positive samples, respectively. The restriction enzyme analysis of this study was comparable to that of other isolates and reference strains with available nucleotide sequence data in the GenBank. The procedure followed in this study is a useful method to rapidly differentiate the very virulent IBDV and classical IBDV isolates

[Distribution of sefA gene among Salmonella enteritidis isolates from poultry sources and potential as diagnostic and epidemiological tools]

This study was conducted to detect the presence of sefA gene among Salmonella Enteritidis isolates from poultry sources by polymerase chain reaction [PCR] and evaluate its potential as diagnostic and epidemiological tools. Thirty Salmonella isolates from poultry sources: broilers, broiler breeders, layers, hatcheries, and poultry abattoirs were investigated. Upper and forward primers were constructed based on the published sequence of the sefA gene that encodes the SEF14 fimbrial subunit [fimbrin]. The size of target product was 526 bp. To confirm the specificity, the PCR products were digested with BamHI restriction enzyme that divides the product to two segments of 186 and 340 bp. The PCR reaction was set up as described in the previous literature. All Salmonella Enteritidis isolates showed the presence of 526 bp product. None of isolates belonging to serogroups B and C were positive for the 526 bp fragment. The restriction enzyme BamHl divided each 526 bp product into two fragments of 186 and 340 bp. This pattern was demonstrated for all Salmonella Enteritidis isolates. The results of the present study showed that the sefA gene carries a high potential to be used as a diagnostic and an epidemiological tool for Salmonella Enteritidis

Detection of common bacteria implicated in bovine mastitis in bulk tank milk by polymerase chain reaction

This study was conducted to detect the common bacteria implicated in bovine mastitis in bulk tank milk by polymerase chain reaction [PCR]. Forty-four milk samples from bulk tank milk were obtained and submitted to our laboratory. To detect Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, Streptococcus uberis, and Streptococcus parauberis, two sets of universal primers were used. The PCR reaction was set up as described in previous literature. Using universal primers, PCR amplification results demonstrated 28 positive samples [63.6%] of 44. The percentages of positive samples for farms with production rates of < 3, 3-10, and 10 < tones were 48, 85, and 100%, respectively. In the present study, we concluded that using universal primers, a simplex PCR is able to detect common important bacteria implicated in bovine mastitis

Experimental induction of necrotic enteritis in broiler chickens by clostridium perfringens isolates from the outbreaks in Iran

To establish an stable challenge system for induction of necrotic enteritis [NE] in broiler chickens, different potential factors and predisposing conditions were imposed in 6 separate different experiments using 525 day-old chicks in total. Despite the presence of some predisposing factors and using 4 isolates of Clostridium perfringens [CP] from acute and severe NE outbreaks as challenge bacteria, the disease was not successfully reproduced in the first 4 experiments. In the 5th and 6th experiments, the predisposing conditions were changed and each bird was challenged with 3 ml [3_10[8] CFU/ml] of CP live culture via oral route and also the feed mixed with CP suspension 2 times per day and for 5 consecutive days. Clinical signs and mortalities, and lesions associated with NE were observed in the later two experiments. This study showed that by stressful nutritional and management procedures such as increased stocking density and high levels of wheat and fish meal; induction of relative immunosuppression; using sufficient CP concentration and appropriate ways of its multiplication; and applying challenge bacteria isolated from acute outbreaks, NE may be experimentally induced in broiler chickens

Cholangiocarcinoma in a broiler chicken

Twenty carcasses from a small flock of 5000 four-weeks old broiler chickens were submitted to a poultry disease diagnostic clinic in Tehran. At necropsy, 19 carcasses showed the typical lesions of colibacillosis such as pericarditis, prihepatitis and airsaculitis. One case did not show any gross lesion of colibacillosis or chronic respiratory disease [CRD] but the liver had an abnormal size containing a cystic part at the base of right lobe filled with a whitish fluid. In bacteriological culture, proteus was isolated from liver. Histopathological examination revealed multicentric bile duct hyperplasia and cholangiocarcinoma in the liver. Neoplastic cells effaced hepatocellular architecture. Histological examination of the neoplastic areas in the liver revealed cholangiocarcinoma [bile duct carcinoma]. This appears to be the first reported case of cholangiocarcinoma in birds in Iran