Trend of Changes in the Titer of Antibody against Avian Influenza Virus H9n2 during Raising Period in Vaccinated and Unvaccinated Broiler Farms in Qazvin Province, Iran: A Cohort Study.

Avian influenza virus (AIV) H9N2 is endemic in Iran and its large-scale circulation in the poultry industry of the country is devastating. This virus was first reported in the industrial poultry populations of Iran in July 1998. Some of the published studies showed that inactivated avian influenza (AI) vaccines are capable of inducing an immune response and providing protection against morbidity and mortality in different countries (Vasfi et al., 2002; Tavakkoli et al., 2011). Low pathogenicity avian influenza subtype H9N2 virus has been reported to have a zoonotic potential and widespread distribution in Iran. Therefore, water-in-oil emulsion vaccines are employed to control the disease in chickens (Nili and Asasi, 2003). This cohort study was conducted during July 2016-November 2017 in broiler chicken farms of Qazvin province, Iran to investigate the serological change trends in broiler chickens in this region. Level of immunity against the H9N2 virus was evaluated by hemagglutination inhibition assay. Fifteen farms out of thirty enrolled units used AI H9N2 killed vaccines. The minimum of mean antibody titers (MATs) was 4.54-2.42 and the maximum of MATs was 4.54+2.42 on day 3. In addition, the minimum and maximum MATs on day 50 were 0.4-0.64 and 0.4+0.064, respectively. The transfer rate of H9N2 AIV antibodies from the serum of breeders to the serum of chickens was calculated as 60.35% in our study. A significant difference was revealed between the maternal mean antibody titers (MMATs) and the MATs on day 3 (P<0.001). In addition, the difference between the MATs on day 3 and the MATs on day 10 was found to be significant (P<0.01). Moreover, MATs were significantly different between the vaccinated and unvaccinated herds on day 40 (P<0.05), while no significant difference was observed on days 3, 10, 20, and 30 (P>0.05). According to the results of this study, antibody titers in the vaccinated farms did not reach the protective level until the end of the rearing period. Most of the unvaccinated herds experienced a spurt in antibody titers due to exposure to the virus. Consequently, biosecurity measures must be implemented more seriously and strictly in broiler farms.

Molecular characterization of haemagglutinin-neuraminidase gene among virulent Newcastle disease viruses isolated in Iran.

BACKGROUND: Virulent Newcastle disease virus (vNDV) causes great economic losses to the poultry industry throughout the world. Despite the endemicity of Newcastle disease (ND) and occurrence of recurrent outbreaks, the nature and genetic features of circulating NDV strains in Iran are largely unknown. Aims: This study was conducted to characterize 13 NDV isolates obtained from different outbreaks in various regions of Iran during 1999-2000 by sequencing and phylogenetic analysis of complete coding sequences of haemagglutinin-neuraminidase (HN) gene. METHODS: All isolates were analyzed based on the previously determined in vivo pathogenicity indices and amino acid (aa) sequences of fusion (F) protein cleavage site (FPCS). RESULTS: Phylogenetic analysis based on the HN gene coding region revealed a very close relationship of these viruses with the recently defined genotype XIII, and more specifically, subgenotype XIIIa viruses. Analysis of HN gene nucleotide (nt) sequences revealed that all studied isolates encode for a protein length of 571 aa and there is no C-terminal extension on HN aa sequences. Sequence analysis revealed multiple aa residue substitutions at antigenic sites or neutralizing epitopes on the HN glycoprotein of studied viruses compared with commonly used vaccinal strains. CONCLUSION: In this study, molecular characterization of vNDV isolates, obtained from commercial poultry farms in Iran, were conducted through complete sequencing and analysis of HN gene. Isolation and molecular characterization of further NDV isolates from other parts of Iran and from neighboring countries in the region will be helpful to identify the nature and origin of indigenous viruses.

Co-circulation of genetically distinct groups of avian paramyxovirus type 1 in pigeon Newcastle disease in Iran.

Pigeons are considered as one of the major natural reservoirs in the epidemiology of Newcastle disease (ND). In this study, the partial sequence of fusion protein gene of 17 pigeon-origin ND viruses (NDVs) isolated during 2012-2013 in Iran was analysed. Since the studied isolates showed F0 protein cleavage sites compatible with velogenic NDVs, all were considered as virulent NDVs. Two isolates carried 112RRQKRF117 as the cleavage site motif, whereas the rest demonstrated 112KRQKRF117 motif which just recently has been reported among Iranian virulent NDVs. Phylogenetic analysis divided all these diverse isolates in two distinct clusters within class II genotype VI. Based on the partial fusion protein gene sequence, 15 out of 17 isolates showed the highest genetic identity to subgenotype VIb/2 and the other two isolates were placed in a distinct genetic group of genotype VI. Based on recent findings, at least two different sublineages of genotype VI are causing the ND outbreaks in the pigeon population and are circulating simultaneously along with virulent NDVs of genotype VII in various species in Iran. The continuing circulation of a diverse group of virulent NDVs as an enzootic in widespread species such as pigeon can cause outbreaks in commercial poultry flocks and also failure in controlling programmes. Therefore, the constant monitoring and awareness of the virus characteristics should be considered in controlling programmes against ND in Iran.

PCR-based diagnosis, molecular characterization and detection of atypical strains of avian Chlamydia psittaci in companion and wild birds.

Chlamydiosis is one of the most important infectious diseases of birds. In this study, 253 clinical samples were taken from 27 bird species belonging to seven orders. Thirty-two (12.6%) samples were positive for Chlamydia psittaci major outer membrane gene (ompA) DNA by a nested polymerase chain reaction (PCR). Twelve nested PCR-positive specimens were typed by ompA gene-based PCR-restricted fragment length polymorphism, using CTU/CTL primers and AluI restriction enzyme. Four restriction patterns were identified, including genotype A (two specimens from an African grey parrot [Psittacus erithacus] and a lorikeet [Trichoglossus haematodus]), genotype B (two specimens from a rock dove [Columbia livia] and a canary [Serinus canaria]), a third new restriction pattern (six specimens from African grey parrots), and a fourth new restriction pattern (two specimens from a ring-necked parakeet [Psittacula krameri] and an Alexandrine parakeet [Psittacula eupatria]). The third and the fourth restriction patterns are suggested to be provisional genotypes I and J, respectively. Partial sequencing of the ompA gene of seven specimens completely correlated with the results of PCR-restricted fragment length polymorphism and confirmed the presence of genotypes A and B and the two new provisional genotypes I and J. The two new genotypes have the closest identity with C. psittaci genotype F and Chlamydia abortus, respectively. From an evolutionary perspective, both new genotypes, particularly genotype J, are intermediate between the two species, C. psittaci and C. abortus.

Molecular detection and characterization of cpb2 gene in Clostridium perfringens isolates from healthy and diseased chickens

Clostridium perfringens is an important pathogen in both human and veterinary medicine. Necrotic enteritis (NE) is the most clinically dramatic bacterial enteric disease of poultry induced by C. perfringens. The pathogenicity of this bacterium is associated with the production of extracellular toxins produced by some of its strains, such as beta2 toxin. The exact role of beta2 toxin in NE pathogenesis is still controversial. In the present study, C. perfringens isolates from healthy and diseased poultry flocks from different parts of Iran were analyzed by PCR assay to determine the presence of all variants of the beta2 toxin gene (cpb2). The products of two positive cpb2 PCR reactions were sequenced, compared to each other and to the cpb2 sequences published in GenBank (by multiple alignment and phylogenetic analysis). The current work represents the first study of cpb2 in poultry C. perfringens isolates in Asia, and reports the highest percentage of cpb2-positive isolates in both apparently healthy chickens (97.7%) and those afflicted with NE (94.4 %). The sequenced isolates were classified as atypical. This study did not show a direct correlation between NE occurrence and cpb2 presence.(AU)

Isolation and characterization of a very virulent Infectious bursal disease virus from turkey.

Infectious bursal disease (IBD) is a highly contagious disease of chickens caused by Infectious bursal disease virus (IBDV). In turkeys, however, infection with classical virulent IBDV strains lead only to subclinical forms of the disease. We attempted to isolate IBDV from the bursa of turkey and characterize it. Amplification of a 743-bp fragment of VP2 gene by RT-PCR and restriction analysis of the product showed a pattern compatible with very virulent IBDV (vvIBDV). Comparison of the sequence of this isolate with those of other IBDVs and phylogenetic analysis confirmed very virulent nature of the isolate. This is the first report on the isolation of vvIBDV from turkey in Iran.

Molecular characterization of Iranian infectious bursal disease viruses.

This study was conducted to characterize nine infectious bursal disease virus (IBDV) isolates from Iran. A reverse transcriptase-polymerase chain reaction (RT-PCR) procedure was used to amplify a 743-bp fragment of the VP2 gene hypervariable region from IBDV field isolates. Amplified VP2 fragments of the nine IBDV isolates were sequenced and compared with published sequences of IBDV strains from Iran and around the world, and their phylogenetic relationships were analyzed. Three isolates demonstrated close relation to classical attenuated strains of IBDVs, and six isolates showed sequences common in European and Asian strains of very virulent IBDVs (vvIBDVs). Four nucleotide changes--802A, 934A, 940A, and 1366A--were common in all Iranian vvIBDVs except in one isolate. Amino acid sequences of three Iranian vvIBDVs were 100% identical and resembled vvIBDV strains from European (UK661), Asian (HK46, GZ96), and Iranian origins (IR01, SDH1). Some unique amino acid substitutions after major hydrophilic peak A in Iranian vvIBDV field isolates were observed: 231S-L, 231S-P, and 233N-K. Phylogenetic analysis showed that the Iranian vvIBDVs were closely related to European and Asian vvIBDVs. Further comprehensive investigations will provide more information on the distribution, variability, and phylogenetic relationships of different IBDVs isolated in Iran and other parts of the world.

Safety, immunogenicity, and efficacy of two Escherichia coli cya crp mutants as vaccines for broilers.

Attenuated derivatives (delta cya delta crp mutants) of an O2 and an O78 avian septicemic Escherichia coli strain were used to immunize broiler chickens by spray to determine the safety, immunogenicity, and efficacy of the derivatives in single- and double-dose regimens. In the safety and immunogenicity studies, groups of 10 chickens were vaccinated by spray (droplet size approximately 20 microm) with the parent E. coli, the mutant organisms, or phosphate-buffered saline (PBS) at 14 days of age and euthanatised 21 days later. There was no deaths or gross pathologic finding in any of the chickens immunized with the vaccine strains. Compared with the levels in chickens exposed to PBS, there were significantly higher levels of immunoglobulin (Ig) G antibody in serum and air sac washings and of IgA antibody in air sac washings in response to the virulent parent strains than to the vaccine strains. In efficacy studies, chickens were immunized with the O2 or the O78 vaccine strain or PBS at day 14 and with the O2 vaccine strain or PBS at days 10 and 14 and challenged with the parent strain 10 days after the last vaccination. There was no significant difference in local IgA and IgG and serum IgG responses between vaccinated and control groups. Chickens vaccinated with the O2 strain, but not the O78 strain, had significantly lower air sac lesion scores compared with those of the unvaccinated groups in both single- and double-dose regimens. We conclude that the mutant O2 strain provided moderate protection against airsacculitis.

Experimental Escherichia coli respiratory infection in broilers.

This study determined optimal conditions for experimental reproduction of colibacillosis by aerosol administration of avian pathogenic Escherichia coli to 2-to-4-wk-old broiler chickens. The basic model for reproducing disease was intranasal administration of approximately 10(4) mean embryo infectious dose of infectious bronchitis virus (IBV) followed by aerosol administration of an 02 or an 078 strain of E. coli in a Horsfall unit (100 ml of a suspension of 10(9) colony-forming units/ml over 40 min). Scores were assigned to groups of infected chickens on the basis of deaths; frequency and severity of lesions in the air sacs, liver and heart; and recovery of the challenge E. coli 6 days post-E. coli infection. An interval of 4 days between the IBV and E. coli challenges was best whether the chickens received the IBV at 8 or 20 days of age. Typically, 50%-80% of the chickens developed airsacculitis and 0 to 29% of the chickens developed pericarditis or perihepatitis, with little or no mortality. Escherichia coli alone resulted in no deaths and 0 to 20% airsacculitis, but these percentages increased to 0 to 5% and 52%-60% when the E. coli aerosol was administered through a cone-shaped chamber. Administration of IBV alone failed to induce lesions. Recovery of the challenge E. coli from chickens did not correlate well with lesions. On the basis of these data, administration of IBV to 20-day-old chickens followed 4 days later by exposure to an avian pathogenic E. coli reproduces avian colibacillosis with the low mortality, high percentage of airsacculitis, and low percentage of septicemic lesions characteristic of the conditions seen in the natural disease.

Construction and characterization of avian Escherichia coli cya crp mutants.

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.

Escherichia coli cellulitis: experimental infections in broiler chickens.

The objectives of this study were to evaluate the role of trauma to the skin in development of Escherichia coli cellulitis and to compare the abilities of three cellulitis isolates (O78, O115, O21,83), one airsacculitis isolate (untypable) and one fecal isolate (O86) of E. coli to induce cellulitis in broiler chickens. Forty-eight 4-week-old commercial broiler chickens were housed in groups of six in eight battery cages. For five groups, the skin on the left side of the abdominal region of chickens was traumatized by scratching with a 22-gauge needle, then contaminated with a swab dipped in a broth culture of one of the five E. coli isolates. For chickens in the remaining three groups, an avian cellulitis culture (O115, O21,83) or sterile broth was applied to intact skin. The experiment was duplicated. All birds were euthanatized 10-13 days postinoculation. No lesion developed in chickens in which the skin had not been traumatized. Among the traumatized birds, cellulitis isolates induced characteristic lesions of cellulitis in 86% of the birds, whereas airsacculitis and fecal isolates induced lesions in 42% and 8% of birds, respectively. Severe or moderate gross pathologic changes were found in 86% and microscopic pathologic changes were found in 88% of birds inoculated with cellulitis isolates; the corresponding percentages for the airsacculitis isolate were 25% and 17%. This study demonstrated that trauma to the skin is necessary for initiating disease and that strains of E. coli of serotypes epidemiologically associated with cellulitis are highly virulent in experimental infection.

Characteristics of Escherichia coli isolates from avian cellulitis.

The purpose of this study was to characterize Escherichia coli isolates from avian cellulitis, particularly with respect to the occurrence of potential virulence factors. At slaughter, five broilers with lesions of cellulitis were selected from each of 20 farms from among the broilers processed. One hundred E. coli isolates from the lesions were characterized with respect to serotype; biotype; drug susceptibility; plasmid profile; ability to produce aerobactin, colicin, colicin V, and hemolysin; and cytotoxicity for Vero cells and chicken fibroblasts. The same properties were determined from a collection of 25 E. coli from the feces of chickens. Serotyping showed that, among the cellulitis isolates, 23 belonged to O group 78, 14 belonged to O2, eight belonged to O115, and seven belonged to O(21.83); 25 were untypable. Isolates from a single farm typically belonged to three to five O groups. More than half of the fecal isolates were untypable, and the rest were distributed among seven O groups. Biotype, drug-resistance pattern, and plasmid profile could not be used as markers of avian cellulitis E. coli. No plasmid was detected in 12% of cellulitis isolates and 48% of fecal isolates. No isolate was hemolytic or showed cytotoxic effects. Aerobactin was produced by 90% of cellulitis isolates, and colicin was produced by 85% of these isolates; the corresponding percentages for the fecal isolates were 16% and 40%. Production of colicin V was detected in 21% of cellulitis isolates and 24% of fecal isolates.(ABSTRACT TRUNCATED AT 250 WORDS)