Growth and replication of infectious bursal disease virus in the fish cell line as an experimental vaccine.

Recently, several attempts have been made to replace egg-based with cell-based vaccines to prevent and control Infectious Bursal Disease Virus (IBDV). This study aimed to evaluate a new fish cell line (M99) for culturing and replicating IBDV. After observing complete cytopathic effects (CPE) on the M99 cell line, virus titers were determined using the TCID50 test, and the presence of the virus was confirmed using an RT-PCR test. Subsequently, 135 broiler chickens (14 days old) were randomly divided into three equal groups for immune response measurements: G1: immunized with a commercial vaccine, G2: immunized with an experimental vaccine, and G3: control. Antibody responses, bursal index, and histopathological evaluations were examined on different days after immunization. Based on the results, CPE of the virus was noticeable from the first passage, becoming complete by the third passage. The infectious titer of the virus was log106.9. Antibody titer measured 21 days after immunization in both vaccinated groups were significantly differed from the control group (p < 0.05). The results obtained from examining the bursal index and histopathological evaluations showed no significant difference between the studied groups at different times. Overall, this research is the first report on the successful cultivation of infectious bursal virus on a permanent cell line of fish origin, with the advantages of tolerance to a wide temperature range (26-40 degrees Celsius). Therefore, this cell line has potential for use to attenuate, cultivate, and adapt other pathogens to cold temperatures in future studies.

Hematological and serum biochemical parameters of the captive long-legged buzzard (Buteo rufinus) in Iran.

BACKGROUND: Although normal haematological and serum biochemical values for both pet and wild birds have been published, little information is available on the haematological and serum biochemical values in long-legged buzzards (Buteo rufinus). OBJECTIVES: This is the first study that aimed to define reference values of haematological, biochemical parameters, and protein electrophoretic fractions of long-legged buzzards in Iran. METHODS: Blood samples were collected from 30 clinically healthy adult long-legged buzzards of both sexes. Hematological, biochemical parameters, and protein electrophoretic fractions were measured. The mean and standard deviations were calculated. RESULTS: Mean values for red blood cells, packed cell volume, haemoglobin, and white blood cells were 2.72 ± 0.60 ×106 /µl, 39.10 ± 3.70%, 13.45 ± 1.30 g/dl, and 3.92 ± 1.39 ×103 /µl, respectively. Mean values for biochemistry parameters were total protein 4.46 ± 1.27 g/dl, albumin 1.78 ± 0.55 g/dl, creatinine 0.54 ± 0.22 mg/dl, uric acid 7.81 ± 2.89 mg/dl, calcium 9.63 ± 2.22 mg/dl, phosphorus 4.31 ± 1.00 mg/dl, glucose 398.87 ± 96.90 mg/dl, blood urea nitrogen 10.46 ± 3.85 mg/dl, alkaline phosphatase 127.01 ± 1.46 IU/L, aspartate aminotransferase 262.22 ± 116.30 IU/L, and alanine aminotransferase 56.63 ± 27.85 IU/L. Mean values for serum protein fractions included pre-albumin, albumin, α-1 globulin, α-2 globulin, ß- globulin, and ϒ-globulin was 0.20 ± 0.09, 2.35 ± 0.67, 0.28 ± 0.13, 0.32 ± 0.07, 0.62 ± 0.24, and 0.68 ± 0.53 g/dl, respectively. CONCLUSION: The reference data presented in this study can be used as health assessment values for veterinary laboratories and clinicians when developing release criteria for rehabilitated long-legged buzzards.

Effect of prophylactic administration of vitamin C in chickens with staphylococcal septic arthritis.

BACKGROUND: Septic arthritis (SA) due to Staphylococcus aureus is a major cause of lameness in poultry with improper response to antimicrobial therapy. OBJECTIVES: The study evaluates the effect of prophylactic administration of vitamin C on SA induced by methicillin resistant S. aureus in chickens. METHODS: One hundred and twenty chickens were randomly assigned into four groups: I. Negative control (NC), II. Positive control (PC) with SA induced at the age of 35 days by intra articular injection of S. aureus. III. Vehicle control (VC) and IV. Arthritic vitamin C-treated (VitC) group (15 g/100 L of drinking water from day 25 to the end of the experiment). Samplings were performed on day 44 (sampling 1) and day 54 (sampling 2) of age. RESULTS: Arthritic birds showed an obvious decrease in body weight with severe clinical arthritis and lameness which were not significantly affected by vitamin C administration at both samplings. Moreover, marked increase in serum malondialdehyde (MDA) concentration of the PC group was observed in sampling 1. Administration of vitamin C successfully reduced MDA concentration at both samplings. In sampling 2, birds in the VitC group showed significantly higher total antioxidant capacity (TAC) than NC birds (p < 0.05). Interleukin-6 concentration in synovial fluid of chickens remained statistically similar among groups in both samplings, while histopathological changes were ameliorated in the VitC group in sampling 2. CONCLUSIONS: Prophylactic administration of vitamin C especially for relatively longer period can ameliorate oxidative stress and histopathological changes due to staphylococcal arthritis in chickens, although it is not associated with a significant effect on clinical manifestations of the disease.

Effect of prophylactic vitamin C administration on the efficiency of florfenicol or sulfadiazine-trimethoprim antimicrobial therapy in chickens with staphylococcal arthritis.

Septic arthritis (SA) in chickens shows improper response to antibacterial therapy. This study evaluates the effect of prophylactic vitamin C administration on the efficiency of sulfadiazine-trimethoprim (SDT) or florfenicol (FF) in broilers with experimental SA. Broilers (210) were randomly allocated into 7 equal groups: (I) negative control (NC) (normal birds); (II) positive control (PC) arthritic birds by injection of Staphylococcus aureus in tibiotarsal joint at the age of 35 days; (III) vehicle control (injected with sterile medium); (IV) arthritic FF-treated (20 mg/kg/day); (V) arthritic vitamin C + FF-treated (as above + vitamin C at 15 g/100L of D.W. from day 25 of age); (VI) arthritic SDT-treated (35 mg/kg/day); (VII) arthritic vitamin C + SDT-treated. Antibacterial therapy started at day 39 of age and lasted for 5 days. Samplings were performed at the age of 44 and 54 days. A long lasting SA with severe fibrinoheterophilic synovitis and reduced body weights developed in PC broilers as compared to NC group (p < 0.05). Oxidative stress was present at sampling 1. Arthritis was not reflected in IL-6 levels of synovial fluid of PC group. None of the antibacterials resulted in completely successful treatment. Vitamin C did not appreciably improve lameness and arthritis scores, although it decreased lipid peroxidation and improved weights of FF treated-arthritic birds. For SDT-treated birds, vitamin C only ameliorated histopathological changes. In conclusion, except for improving body weight in FF-treated birds, prophylactic administration of vitamin C is not associated with improvements in clinical outcome of antimicrobial therapy of broilers with SA, although it ameliorates oxidative stress and some histopathological changes.

High incidence of multidrug resistance and class 1 and 2 integrons in Escherichia coli isolated from broiler chickens in South of Iran.

The objective was to investigate the multidrug resistance and presence of class 1 and 2 integrons in 300 Escherichia coli isolates obtained from 20 broiler farms during three rearing periods (one-day-old chicks, thirty-day-old chickens, and one day before slaughter) in Fars, South Iran. Results showed that 81.00%, 82.00%, and 85.00% of isolates were multidrug-resistant on the first day, thirty-day-old chickens, and one day before slaughter, respectively. Multidrug-resistant E. coli isolates were further examined for the presence of class 1 and 2 integrons using PCR assay. The existence of class 1 integron-integrase gene (intI1) was confirmed in 68.40%, 72.70%, and 60.90% of multidrug-resistant isolates from stage 1, stage 2, and stage 3 of the rearing period, respectively. The frequency of class 2 integron-integrase gene (intI2) during the first to the third stage of sampling was 2.60%, 25.50%, and 30.40%. Also, sequence analysis of the cassette arrays within class 1 integron revealed the presence of the genes associated with resistance for trimethoprim (dfrA), streptomycin (aadA), erythromycin (ereA), and orfF genes. The results revealed that percentages of antimicrobial resistance in E. coli isolates were significantly higher in the middle and end stages of the rearing period. In conclusion, widespread dissemination of class 1 integrons in all three stages and rising trends of class 2 integrons existence in E. coli isolates during the rearing period of broiler chickens could exacerbate the spread of resistance factors among bacteria in the poultry industry. Future research is needed to clarify its implication for human health.

Reduced Performance Due to Adenoviral Gizzard Erosion in 16-Day-Old Commercial Broiler Chickens in Iran, Confirmed Experimentally.

Adenoviral gizzard erosion (AGE) in broilers is an emerging infectious disease with negative impact on flock productivity. Despite of known primary etiological role of fowl adenovirus serotype 1 (FAdV-1) in AGE, there are a limited number of field reports worldwide, possibly because the disease is less noticeable and clinically difficult to assess. The present study documents an outbreak of AGE in 16-day-old broiler chickens on a farm in the north of Iran and the reproduction of the disease in an experimental setting. In the field, a sudden onset of mortality was noticed in affected broilers resulting in 6% total mortality and decreased weight gain leading to approximately 1-week delay to reach the target slaughter weight. Necropsy findings in dead broilers revealed black colored content in crop, proventriculus and gizzard together with severe gizzard erosions characterized by multiple black-brown areas of variable size in the koilin layer and mucosal inflammation. Microscopic examination revealed necrotizing ventriculitis marked with severe dissociation of koilin layer and degeneration of glandular epithelium with infiltration of mononuclear inflammatory cells. FAdV-1 was isolated from affected gizzards. Phylogenetic analysis of the hexon loop-1 (L1) sequence of the isolated virus showed 100% identity with pathogenic FAdV-1 strains previously reported from broiler chickens with AGE. Subsequently, an in vivo study infecting day-old commercial layer chickens with the field isolate demonstrated characteristic lesions and histopathological changes of AGE together with decreased weight gain in the infected birds. For the first time, the progress of a natural outbreak of AGE in Iran is described and experimental reproduction of the disease is demonstrated. The findings highlight the economic impact of the disease for regional poultry production due to mortality and impaired weight gain of the affected broilers.

Comparative Effects of Dexamethasone and Meloxicam on Magnitude of the Acute Inflammatory Response Induced by Escherichia coli Lipopolysaccharide in Broiler Chickens.

PURPOSE: Dexamethasone has been widely used to treat acute inflammatory diseases and endotoxic shocks in animal models. Meloxicam is one of the most commonly used anti-inflammatory agents in avian species. However, little is known about the effects of dexamethasone and meloxicam on lipopolysaccharide (LPS)-induced acute inflammatory response in birds. In the present study, LPS-challenged broiler chickens were used to investigate the comparative protective effects of meloxicam and dexamethasone on LPS-induced acute inflammatory responses. METHODS: Lipopolysaccharide (LPS)-induced acute lung injury (ALI) histopathological scores, selected serum acute phase reactants, inflammatory mediators, and gangliosides were evaluated in broiler chickens inoculated with E. coli LPS and simultaneously treated with two doses of meloxicam (0.5 and 2 mg/kg BW) and dexamethasone (2 and 4 mg/kg BW). RESULTS: LPS-induced ALI scores were not significantly different between the meloxicam-treated, dexamethasone-treated, and untreated positive control groups at 4 hours after LPS inoculation. Interleukin-6 concentrations were also statistically the same among the positive control, dexamethasone-treated, and meloxicam-treated groups at 3 and 12 hours after LPS inoculation. However, these anti-inflammatory drugs reduced adenosine deaminase, ceruloplasmin, lipid-bound sialic acid, protein-bound sialic acid, and total sialic acid in LPS-inoculated broiler chickens at 12, 24, and 48 hours after LPS inoculation in a drug- and dose-dependent manner. Ovotransferrin concentrations were not significantly different between positive control and treatment groups at 12 hours after LPS inoculation. However, twenty-four hours after LPS inoculation, all the treated groups, except the one treated with 0.5 mg/kg meloxicam, showed significantly lower concentrations of ovotransferrin as compared with the positive control group. CONCLUSION: Our results showed that dexamethasone was more effective than meloxicam in inhibiting the LPS-induced response in broiler chickens by diminishing the serum levels of adenosine deaminase, ceruloplasmin, and gangliosides.

In vitro and in vivo activity of cinnamaldehyde against Eimeria kofoidi in chukar partridge (Alectoris chukar).

One hundred and twenty one-day-old chukar partridges were randomly divided into eight groups which received diets with different supplementations. There were four unchallenged groups. One group received salinomycin (50 ppm), two groups received cinnamaldehyde (CINN) (100 and 200 mg/kg of diet), and another one received only the basal diet from the 1st to the 31st day. There were also four corresponding groups orally challenged by 3 × 105Eimeria kofoidi sporulated oocysts at the 21st day. Three samplings were done at the 24th, 26th, and 31st days of rearing for pathological and biochemical assessments. Fecal samples were daily taken to check the pattern of oocyst shedding from the 26th to 31st day. The body weight of birds was measured at 21st and 31st days. Along with the in vivo experiment, an in vitro sporulation inhibition test was carried out. The in vitro results showed that CINN decreased sporulation rate at 1 and 0.5 mg/ml. In vivo, it was found that CINN did not prevent the oocyst shedding. Furthermore, the histopathological findings revealed that CINN and salinomycin had no effect on infection establishment. However, our findings showed that CINN (200 mg/kg of diet) could enhance the body weight and improve antioxidant status. Although our results did not support the in vivo anticoccidial activity of CINN, it had a promising potential to improve antioxidant status and body weight in the chukar partridge.

Effect of Newcastle disease and infectious bronchitis live vaccines on the immune system and production parameters of experimentally infected broiler chickens with H9N2 avian influenza.

H9N2 Avian influenza (AI) is an infectious disease which considered to have low pathogenic virulence, but in the case of coinfection with other pathogens it has the potential to become a major threat to the poultry industry. Infectious bronchitis (IB) and Newcastle diseases (ND) are other common problems to the poultry industry, which there are an extensive vaccination program against these viral pathogens. To investigate the effects of administration of infectious bronchitis and Newcastle disease live vaccines (IBLVs and NDLVs) in the presence of H9N2 AI infection on the immune system and some production parameters, 180 one-day-old broiler chicks were randomly allocated into six groups with different vaccination programs including H120 IBLV, 4/91 IBLV, B1 NDLV and LaSota NDLV. At the age of 20 days, all birds of the experimental groups except the negative control group, were inoculated intra-nasally (at dose of 106 EID50) with H9N2 AIV. After the inoculation, gross and microscopic lesions of the immune organs, serological changes and some production parameters were examined. The findings of this study showed that coinfection of H9N2 AI with NDLVs exacerbated the gross and microscopic injuries in the immune organs; especially the bursa of Fabricius. LaSota + AIV group had the most severe lesion in the bursa of Fabricius, spleen and thymus. Furthermore, the birds of LaSota + AIV group consumed the least amount of feed and water and their final body weight were significantly (P ≤ 0.05) lower in comparison with the other groups. Interestingly, in the context of this experiment both 4/91 and H120 IB live vaccines enhanced the HI antibody titers against H9N2 AIV, but the 4/91 showed the most significant (P ≤ 0.05) increase compared to the other experimental groups.

Production, purification, and evaluation of quail immunoglobulin Y against Salmonella typhimurium and Salmonella enteritidis.

Salmonella species have been the major foodborne problems in food production systems, with Salmonella enterica serovars typhimurium (S. typhimurium) and enteritidis (S. enteritidis) being among the more common isolates. The oral administration of chicken egg yolk specific antibodies (IgYs) has been established as an efficient alternative for treatment and prevention of gastrointestinal pathogens including Salmonella. The present study was aimed to investigate the possible production of specific IgYs against Salmonella typhimurium and Salmonella enteritidis in quail egg yolks. Salmonella spp.-free female Japanese quails (Coturnix coturnix japonica) were intramuscularly immunized with formalin or heat-inactivated Salmonella immunogens (1.0 × 109 CFU/mL) emulsified with Freund adjuvants. Egg yolk IgYs were purified using ammonium sulfate precipitation method. Anti-Salmonella IgYs titer and specificity were determined using enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Salmonella specific IgYs detected in the immunized quails were significantly higher than those of the control group, which confirmed the immunization procedure. Specific IgYs against S. typhimurium and S. enteritidis were identified in both groups immunized with heat or formalin-inactivated immunogens. However, formalin-inactivated immunogens induced relatively higher immune responses over the heat-inactivated ones. Quail anti-Salmonella IgYs showed a high specificity to their corresponding immunogens, with moderate cross-reactivity to other members of Enterobacteriaceae family. Quail can be regarded as a valuable and inexpensive source for producing large-scale of specific antibodies that can be used for immunodiagnostic and immunotherapeutic purposes.

Tetracycline Resistance Genes in Campylobacter jejuni and C. coli Isolated From Poultry Carcasses.

BACKGROUND: Campylobacter is one of the leading bacterial species causing foodborne illnesses in humans. Antimicrobial agents have been extensively used for treatment of Campylobacter infections; but in the recent years, both animal and human isolates of this bacterium have shown resistance to several antibiotics such as tetracycline. OBJECTIVES: The aim of this study was to investigate the presence of genetic determinants of tetracycline resistance in Campylobacter spp. recovered from poultry carcasses in Shiraz, Iran. MATERIALS AND METHODS: Eighty-three thermophilic Campylobacter spp. Isolates were first identified based on multiplex polymerase chain reaction (PCR) and then screened for presence of tetracycline resistance genes (tet (A), tet (B), tet (O) and te (S)) by PCR. RESULTS: The overall prevalence of Campylobacter jejuni and C. coli among the examined isolates was 51.8% and 48.2%, respectively. Tetracycline resistance genes of tet (B) and tet (S) were not seen among these Campylobacter spp. Isolates, whereas the most common tet gene identified was tet (O), found in 83.1% (69/83) of all the isolates. The tet (O) gene sequence comparison between C. jejuni and C. coli showed 100% similarity and these sequences (JX853721and JX853722) were also identical to the homologous sequences of other strains of Campylobacter spp. existing in the GenBank databases. In addition, tet (A) was found in 18% (15/83) of Campylobacter spp. isolates. To our knowledge, this represents the first report of tet (A) in Campylobacter spp. There was 100% homology between the sequences of tet (A) from this study (JX891463 and JX891464) and the tet (A) sequences mentioned for other bacteria in the GenBank databases. CONCLUSIONS: The high prevalence of tet (O) resistance gene along with new detection of tet (A) resistance gene in Campylobacter spp. isolated from poultry carcasses revealed an extensive tetracycline resistance among Campylobacter isolates from poultry in Iran. It emphasized the need for cautious use of tetracycline in poultry production to decrease the extension of tetracycline-resistant Campylobacter spp.

Rapid detection of Escherichia coli gyrA and parC mutants in one-day-old broiler chicks in Iran.

Vertical and consequently horizontal transmission of quinolone and fluoroquinolone resistant Escherichia coli clones following hatch in chickens enables a massive amplification of these clones into a large population. The aim of this study was to determine the antibiotic resistance and susceptibility of Iranian E. coli isolates (n=105) from one-day-old chicks to fluoroquinolones and the relation of this resistance with mutations in gyrA and parC genes using PCR-RFLP. For the first time, EcoRV restriction enzyme was used for rapid mutation screening in parC (Ser80Ile). The results showed that the low level of Minimum Inhibitory Concentration (MIC) for ciprofloxacin (0.25-4µg ml-1) and enrofloxacin (0.25-4µg ml-1) corresponded to a single mutation in gyrA, while intermediary to high level of MIC for ciprofloxacin (8 --> 64 µg ml-1) and enrofloxacin (16 --> 64 µg ml-1) were related to 2 mutations in gyrA or 3 mutations, 2 in gyrA and 1 in parC. There was a strong positive correlation (R = 0.93, P < 0.001) between MIC levels of enrofloxacin and ciprofloxacin among these isolates. The article concludes by stressing that the rising incidence of enrofloxacin resistant E. coli isolates from chicken sources may increase the potential risk of ciprofloxacin resistant E. coli acquisition by humans.