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1.  Presence of ESBL/AmpC -Producing Escherichia coli in the Broiler Production Pyramid: A Descriptive Study 
PLoS ONE  2013;8(11):e79005.
Broilers and broiler meat products are highly contaminated with extended spectrum beta-lactamase (ESBL) or plasmid-mediated AmpC beta-lactamase producing Escherichia coli and are considered to be a source for human infections. Both horizontal and vertical transmission might play a role in the presence of these strains in broilers. As not much is known about the presence of these strains in the whole production pyramid, the epidemiology of ESBL/AmpC-producing E. coli in the Dutch broiler production pyramid was examined. Cloacal swabs of Grandparent stock (GPS) birds (one−/two-days (breed A and B), 18 and 31 weeks old (breed A)), one-day old Parent stock birds (breed A and B) and broiler chickens of increasing age (breed A) were selectively cultured to detect ESBL/AmpC-producing isolates. ESBL/AmpC-producing isolates were found at all levels in the broiler production pyramid in both broiler breeds examined. Prevalence was already relatively high at the top of the broiler production pyramid. At broiler farms ESBL/AmpC producing E. coli were still present in the environment of the poultry house after cleaning and disinfection. Feed samples taken in the poultry house also became contaminated with ESBL/AmpC producing E. coli after one or more production weeks. The prevalence of ESBL/AmpC-positive birds at broiler farms increased within the first week from 0–24% to 96–100% independent of the use of antibiotics and stayed 100% until slaughter. In GPS breed A, prevalence at 2 days, 18 weeks and 31 weeks stayed below 50% except when beta-lactam antibiotics were administered. In that case prevalence increased to 100%. Interventions minimizing ESBL/AmpC contamination in broilers should focus on preventing horizontal and vertical spread, especially in relation to broiler production farms.
doi:10.1371/journal.pone.0079005
PMCID: PMC3820706  PMID: 24244401
2.  Comprehensive report of an Enterococcus cecorum infection in a broiler flock in Northern Germany 
BMC Veterinary Research  2014;10(1):311.
Background
Enterococcus cecorum is considered as an emerging pathogen in poultry and can cause substantial losses in broiler flocks. Femoral head necrosis and spondylitis were described as the main pathological changes in infected chickens. Nevertheless, little is known about the pathogenesis of Enterococcus cecorum infection in broilers. This report shows for the first time the whole course of disease over an entire growing period including repeated necropsies and subsequent microbiological investigations.
Case presentation
In a flock of 18200 broilers, a decrease in flock uniformity was detected from 14 days post hatch onwards with affected chickens showing lameness and an increase in flock mortality up to 7.22% at day 33 post hatch. In the first 3 weeks post hatch, pericarditis and hepatitis were found as the main pathological changes in 27.6% and 9.8% of the examined broilers respectively. Femoral head necrosis and vertebral osteomyelitis were detected in the last week of the growing period with 10.3% and 2.3% respectively. Heart, liver, spleen, yolk sac and vertebral column of 59 broilers with pathological changes were subjected to bacteriological analysis. Enterococcus cecorum was isolated from 23 birds (39%), the first broiler was already positive at day 3 post hatch in the yolk sac. Additionally, 9.75% of the broilers were rejected at the slaughterhouse primarily because of pathological changes. The investigated broiler cycle had by far the best footpad score compared to 7 cycles before and 4 cycles after the Enterococcus cecorum infection at the same farm.
Conclusions
Bacteraemia and generalized infection appear to be important steps in the pathogenesis of Enterococcus cecorum infection in broilers. Furthermore, this disease causes economic losses for the farmer not only due to an increase in flock mortality, but probably also through substantially higher condemnation rates at the slaughterhouse. It was speculated that the broilers were infected via the respiratory tract as this flock had lower footpad scores likely the result of drier litter. The latter may have led to higher dust concentrations and thus airborne Enterococcus cecorum.
doi:10.1186/s12917-014-0311-7
PMCID: PMC4297365  PMID: 25539747
Gallus gallus domesticus; Chicken; Broiler; Infection; Septicemia; Osteomyelitis; Pericarditis; Hepatitis; Enterococcus cecorum; Pathogenesis; Route of infection; Condemnation rate
3.  Molecular detection of infectious bronchitis and Newcastle disease viruses in broiler chickens with respiratory signs using Duplex RT-PCR 
Veterinary Research Forum  2014;5(4):319-323.
Infectious bronchitis (IB) and Newcastle disease (ND) are highly contagious and the most economically important diseases of the poultry affecting respiratory tract and causing economic losses in poultry industry throughout the world. In the present study, the simultaneous detection and differentiation of causative agents of these diseases were investigated using duplex-RT-PCR. RNA was extracted from vaccinal and reference strains of infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) and then cDNA was synthesized. Using two universal primer sets for detection of IBV and NDV, the duplex-RT-PCR was developed. In order to assess the efficiency of the developed duplex RT-PCR, a number of 12 broiler farms with the symptoms of respiratory tract infection was sampled (trachea, lung and kidney were sampled from affected birds suspicious for IBV and NDV infections). After RNA extraction from tissues and cDNA synthesis, the presence of IBV and NDV genome were investigated using duplex-PCR. The results showed that three of twelve examined broiler farms were positive for IBV and two farms were positive for NDV and IBV. The results revealed that the duplex-RT-PCR is a quick and sensitive procedure for simultaneously detecting IBV and NDV in birds with respiratory infections.
PMCID: PMC4299999  PMID: 25610585
Duplex-RT-PCR; Infectious bronchitis virus; Newcastle disease virus
4.  Epidemiology of Campylobacter spp. at two Dutch broiler farms. 
Epidemiology and Infection  1995;114(3):413-421.
Broiler flocks on two Dutch poultry farms were screened weekly for the presence of campylobacter in fresh caecal droppings during eight consecutive production cycles. Hatchery and fresh litter samples were taken at the start of each new cycle. Water, feed, insects, and faeces of domestic animals, present on the farms were also included in the sampling. Penner serotyping of isolates was used to identify epidemiological factors that contribute to campylobacter colonization in the broiler flocks. Generally, broiler flocks became colonized with campylobacter at about 3-4 weeks of age with isolation percentages of 100%, and stayed colonized up to slaughter. A similar pattern of serotypes was found within the various broiler houses on one farm during one production cycle. New flocks generally showed also a new pattern of serotypes. Most serotypes isolated from the laying hens, pigs, sheep and cattle were different from those isolated from the broilers at the same time. Campylobacter serotypes from darkling beetles inside the broiler houses were identical to the ones isolated from the broilers. No campylobacter was isolated from any of the hatchery, water, feed or fresh litter samples. Conclusive evidence of transmission routes was not found, but results certainly point towards horizontal transmission from the environment. Horizontal transmission from one broiler flock to the next one via a persistent contamination within the broiler house, as well as vertical transmission from breeder flocks via the hatchery to progeny, did not seem to be very likely.
PMCID: PMC2271305  PMID: 7781729
5.  Infectious bronchitis virus in Jordanian chickens: Seroprevalence and detection 
Infectious bronchitis (IB) is one of the most important viral diseases of poultry; it causes major economic losses to the poultry industry. This study was conducted to investigate the prevalence of infectious bronchitis virus (IBV) in commercial chicken flocks in Jordan. Serum samples from 70 commercial chicken flocks (40 broilers, 18 layers, and 12 broiler breeders) free from respiratory disease were collected and screened for the presence of Massachusetts-41 (M-41), D274, and 4/91 strain antigens of IBV by using the hemagglutination inhibition (HI) test. In addition, 51 commercial chicken flocks (25 broilers, 15 layers, and 11 broiler breeders) suffering from respiratory disease were tested for the presence of IBV, using the reverse-transcription polymerase chain reaction. Overall, 92.9% of the flocks free from respiratory disease were seropositive for antibodies to the M-41 strain, whereas 90% and 61.4% of the flocks were seropositive for antibodies to the 4/91 and D274 strains, respectively. Infectious bronchitis virus nucleic acid was detected in 16 broiler (64%), 8 layer (53%), and 6 broiler breeder (54.54%) flocks affected with respiratory disease. This study clearly demonstrates that several strains of IBV are present in poultry flocks in Jordan. Future work should include the isolation and serotyping of IBV in the region, so that a suitable vaccination program, using the common field serotypes as vaccines, can be adopted to protect against IBV-caused disease. Furthermore, farmers need to be educated about the clinical signs of IB and the importance of IBV.
PMCID: PMC2603658  PMID: 19337618
6.  Modifications of Gait as Predictors of Natural Osteoarthritis Progression in STR/Ort Mice 
Objective
Osteoarthritis (OA) is a common chronic disease for which disease-modifying therapies are not currently available. Studies to seek new targets for slowing the progress of OA rely on mouse models, but these do not allow for longitudinal monitoring of disease development. This study was undertaken to determine whether gait can be used to measure disease severity in the STR/Ort mouse model of spontaneous OA and whether gait changes are related to OA joint pain.
Methods
Gait was monitored using a treadmill-based video system. Correlations between OA severity and gait at 3 treadmill speeds were assessed in STR/Ort mice. Gait and pain behaviors of STR/Ort mice and control CBA mice were analyzed longitudinally, with monthly assessments.
Results
The best speed to identify paw area changes associated with OA severity in STR/Ort mice was found to be 17 cm · seconds−1. Paw area was modified with age in CBA and STR/Ort mice, but this began earlier in STR/Ort mice and correlated with the onset of OA at 20 weeks of age. In addition, task noncompliance appeared at 20 weeks. Surprisingly, STR/Ort mice did not show any signs of pain with OA development, even when treated with the opioid antagonist naloxone, but did exhibit normal pain behaviors in response to complete Freund's adjuvant–induced arthritis.
Conclusion
The present results identify an animal model in which OA severity and OA pain can be studied in isolation from one another. The findings suggest that paw area and treadmill noncompliance may be useful tools to longitudinally monitor nonpainful OA development in STR/Ort mice. This will help in providing a noninvasive means of assessing new therapies to slow the progression of OA.
doi:10.1002/art.38616
PMCID: PMC4271671  PMID: 24623711
7.  Effects of immediate modified feeding on infantile gastroenteritis. 
BACKGROUND: Standard treatment of infants who are dehydrated as a result of acute gastroenteritis is to administer oral rehydration therapy (ORT). Traditionally, food has been withdrawn for 24-48 h, but there is no conclusive evidence that this is of any real benefit to the patient. Immediate modified feeding, in which an infant on ORT is not starved but administered a limited diet, may have benefits in the treatment of gastroenteritis, especially in children who are nutritionally compromised before they develop the illness. AIM: A pilot study was carried out to investigate the effects of giving infants suffering from acute gastroenteritis a limited modified diet in conjunction with ORT. METHOD: Infants recruited into the study by their general practitioner or by a research doctor in the hospital casualty unit of Bristol Children's Hospital were randomly allocated to receive ORT with or without immediate modified feeding. The duration of diarrhoea, weight change, and incidence of vomiting and lactose intolerance were measured in both treatment groups, and the results were compared. RESULTS: Of the infants studied, 27 received ORT and immediate modified feeding, and 32 ORT alone. The duration of diarrhoea, and incidence of vomiting or lactose intolerance were no greater in the group receiving immediate modified feeding. Patients who received ORT and immediate modified feeding appeared to gain more weight than the infants who were starved for 24-48 h, but this difference was not statistically significant. CONCLUSION: Immediate modified feeding is safe and effective, and may have nutritional advantages over traditional ORT with starvation. A similar but multicentre study using unmodified diet, i.e. child's normal diet, is being carried out by a working group of The European Society of Paediatrics, Gastroenterology and Nutrition (ESPGAN).
PMCID: PMC1239579  PMID: 8731625
8.  Risk Factors and Characteristics of Low Pathogenic Avian Influenza Virus Isolated from Commercial Poultry in Tunisia 
PLoS ONE  2013;8(1):e53524.
Objective
Estimate the seroprevalence of influenza A virus in various commercial poultry farms and evaluate specific risk factors as well as analyze their genetic nature using molecular assays.
Materials and Methods
This report summarizes the findings of a national survey realized from October 2010 to May 2011 on 800 flocks in 20 governorates. Serum samples were screened for the presence of specific influenza virus antibodies using cELISA test. Additionally, swab samples were tested by real time and conventional RT-PCR and compared with results obtained by others assays. Phylogenetic and genetic analyses of the glycoproteins were established for some strains.
Results
Out of the 800 chicken and turkey flocks tested by cELISA, 223 showed positive anti-NP antibodies (28.7%, 95% CI: 25.6–32.1). Significantly higher seroprevalence was found among the coastal areas compared to inland and during the autumn and winter. Broiler flocks showed significantly lower seroprevalence than layers and broiler breeders. The influenza virus infection prevalence increased after the laying phase among layer flocks. In addition, AIV seropositivity was significantly associated with low biosecurity measures. The Ag EIA and rRT-PCR tests revealed significantly higher numbers of AI positive samples as compared to cell cultures or egg inoculation. All new strains were subtyped as H9N2 by real time and conventional RT-PCR. Drift mutations, addition or deletion of glycosylation sites were likely to have occurred in the HA and NA glycoproteins of Tunisian strains resulting in multiple new amino acid substitutions. This fact may reflect different evolutionary pressures affecting these glycoproteins. The role of these newly detected substitutions should be tested.
Conclusion
Our findings highlight the potential risk of AIV to avian health. Strict enforcement of biosecurity measures and possible vaccination of all poultry flocks with continuous monitoring of poultry stations may ensure reduction of AIV prevalence and avoid emergence of more pathogenic strains.
doi:10.1371/journal.pone.0053524
PMCID: PMC3543454  PMID: 23326449
9.  Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995-2004 
Virology Journal  2010;7:137.
Background
Avian influenza virus (AIV) infections have caused heavy economic losses to the poultry industry in Pakistan as well as numerous other regions worldwide. The first introduction of H7N3 AIV to Pakistan occurred during 1995, since then H7N3, H9N2 and H5N1 AIVs have each been sporadically isolated. This report evaluates the genetic origin of the H7N3 viruses from Pakistan collected 1995-2004 and how they disseminated within the country. To accomplish this we produced whole genome sequences for 6 H7N3 viruses and data for the HA and NA genes of an additional 7 isolates. All available sequence from H7N3 AIV from Pakistan was included in the analysis.
Results
Phylogenetic analysis revealed that there were two introductions of H7 into Pakistan and one N3 introduction. Only one of the H7 introductions appears to have become established in poultry in Pakistan, while the other was isolated from two separate outbreaks 6 years apart. The data also shows that reassortment has occurred between H7N3 and H9N2 viruses in the field, likely during co-infection of poultry. Also, with the exception of these few reassortant isolates, all 8 genes in the predominant H7N3 virus lineage have evolved to be phylogenetically distinct.
Conclusions
Although rigorous control measures have been implemented in commercial poultry in Pakistan, AIV is sporadically transmitted to poultry and among the different poultry industry compartments (broilers, broiler breeders, table egg layers). Since there is one primary H7 lineage which persists and that has reassorted with the H9N2 AIV in poultry, it suggests that there is a reservoir with some link commercial poultry. On a general level, this offers insight into the molecular ecology of AIV in poultry where the virus has persisted despite vaccination and biosecurity. This data also illustrates the importance of sustained surveillance for AIVs in poultry.
doi:10.1186/1743-422X-7-137
PMCID: PMC2901269  PMID: 20576101
10.  Molecular quantitation of H9N2 avian influenza virus in various organs of broiler chickens using TaqMan real time PCR 
During the past decade, H9N2 low pathogenic avian influenza virus (LPAI) has caused considerable economic loss due to decreased production, increased mortality and the cost of vaccination in Iranian poultry industry. Because of widespread occurrence of this disease and the virus potential to mutate to highly-pathogenic (HP) form and transmission to humans, it is, therefore, imperative to understand the pathogenesis and properties of these viruses. In this study, a two step TaqMan real time PCR assay was performed for the quantitation of A/chicken/Iran/772/1998(H9N2) virus in various organs of broiler chickens at different days post inoculation (DPI). Forty 5-week-old commercial broiler chickens were inoculated with the virus. Five chickens were randomly selected on days 1, 3, 6 and 9 PI. Their trachea, lungs, spleen, kidneys, pancreas, blood and faeces were collected for virus detection. A PCR test was performed and the positive samples were used for quantitative real time PCR assay. The result of RT-PCR assay showed the presence of the virus in trachea (40%, 33%), lungs (20%, 66.6%) and spleen (20%, 50%) of infected chickens on days 3 and 6 PI, respectively. The virus was also detected in the kidneys of inoculated chickens on 3 (40%), 6 (60%) and 9 (100%) DPI. In faecal samples the virus was only detected on day 6 PI (83.3%). The molecular quantitation of AIV showed that the AIV titre in the trachea, lungs and spleen of chickens at 3 DPI is lower than the AIV titre at 6 DPI in these organs. The highest titre was observed in the faeces. The AIV titre in all organs of the birds which died at 6 DPI was higher than those of the same organs in the other experimental birds.
PMCID: PMC2702075  PMID: 19565023
Avian influenza; H9N2; virus quantitation; TaqMan real-time PCR
11.  Effect of organic tomato (Lycopersicon esculentum) extract on the genotoxicity of doxorubicin in the Drosophila wing spot test 
Genetics and Molecular Biology  2009;32(1):133-137.
The consumption of organic tomatoes (ORTs) reduces the risk of harmful effects to humans and the environment caused by exposure to toxic agrochemicals. In this study, we used the somatic mutation and recombination test (SMART) of wing spots in Drosophila melanogaster to evaluate the genotoxicity of ORT and the effect of cotreatment with ORT on the genotoxicity of Doxorubicin® (DXR, a cancer chemotherapeutic agent) that is mediated by free radical formation. Standard (ST) cross larvae were treated chronically with solutions containing 25%, 50% or 100% of an aqueous extract of ORT, in the absence and presence of DXR (0.125 mg/mL), and the number of mutant spots on the wings of emergent flies was counted. ORT alone was not genotoxic but enhanced the toxicity of DXR when administered concomitantly with DXR. The ORT-enhanced frequency of spots induced by DXR may have resulted from the interaction of ORT with the enzymatic systems that catalyze the metabolic detoxification of this drug.
doi:10.1590/S1415-47572009005000008
PMCID: PMC3032965  PMID: 21637658
antioxidants; co-genotoxicity; Drosophila melanogaster; organic tomato; SMART
12.  Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli on Flies at Poultry Farms 
In the Netherlands, extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli bacteria are highly prevalent in poultry, and chicken meat has been implicated as a source of ESBL-producing E. coli present in the human population. The current study describes the isolation of ESBL-producing E. coli from house flies and blow flies caught at two poultry farms, offering a potential alternative route of transmission of ESBL-producing E. coli from poultry to humans. Overall, 87 flies were analyzed in 19 pools. ESBL-producing E. coli bacteria were detected in two fly pools (10.5%): a pool of three blow flies from a broiler farm and a pool of eight house flies from a laying-hen farm. From each positive fly pool, six isolates were characterized and compared with isolates obtained from manure (n = 53) sampled at both farms and rinse water (n = 10) from the broiler farm. Among six fly isolates from the broiler farm, four different types were detected with respect to phylogenetic group, sequence type (ST), and ESBL genotype: A0/ST3519/SHV-12, A1/ST10/SHV-12, A1/ST58/SHV-12, and B1/ST448/CTX-M-1. These types, as well as six additional types, were also present in manure and/or rinse water at the same farm. At the laying-hen farm, all fly and manure isolates were identical, carrying blaTEM-52 in an A1/ST48 genetic background. The data imply that flies acquire ESBL-producing E. coli at poultry farms, warranting further evaluation of the contribution of flies to dissemination of ESBL-producing E. coli in the community.
doi:10.1128/AEM.02616-13
PMCID: PMC3910986  PMID: 24162567
13.  Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers 
BMC Genomics  2012;13:278.
Background
Avian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited.
Results
Total RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher’s exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection.
Conclusions
A comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122–1, 122–2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.
doi:10.1186/1471-2164-13-278
PMCID: PMC3496578  PMID: 22726614
Chicken; miRNA; AIV; Deep sequencing; Microarray
14.  The Toxicological Impacts of the Fusarium Mycotoxin, Deoxynivalenol, in Poultry Flocks with Special Reference to Immunotoxicity 
Toxins  2013;5(5):912-925.
Deoxynivalenol (DON) is a common Fusarium toxin in poultry feed. Chickens are more resistant to the adverse impacts of deoxynivalenol (DON) compared to other species. In general, the acute form of DON mycotoxicosis rarely occurs in poultry flocks under normal conditions. However, if diets contain low levels of DON (less than 5 mg DON/kg diet), lower productivity, impaired immunity and higher susceptibility to infectious diseases can occur. The molecular mechanism of action of DON has not been completely understood. A significant influence of DON in chickens is the impairment of immunological functions. It was known that low doses of DON elevated the serum IgA levels and affected both cell-mediated and humoral immunity in animals. DON is shown to suppress the antibody response to infectious bronchitis vaccine (IBV) and to Newcastle disease virus (NDV) in broilers (10 mg DON/kg feed) and laying hens (3.5 to 14 mg of DON/kg feed), respectively. Moreover, DON (10 mg DON/kg feed) decreased tumor necrosis factor alpha (TNF-α) in the plasma of broilers. DON can severely affect the immune system and, due to its negative impact on performance and productivity, can eventually result in high economic losses to poultry producers. The present review highlights the impacts of DON intoxication on cell mediated immunity, humoral immunity, gut immunity, immune organs and pro-inflammatory cytokines in chickens.
doi:10.3390/toxins5050912
PMCID: PMC3709269  PMID: 23628787
deoxynivalenol; Fusarium mycotoxin; immune responses; gut immunity; cytokines; poultry
15.  Dissemination and tracking of Salmonella spp. in integrated broiler operation 
Journal of Veterinary Science  2007;8(2):155-161.
Controlling Salmonella in integrated broiler operation is complicated because there are numerous potential sources of Salmonella contamination, including chicks, feed, rodents, wild poultry operations, and the processing plant. The objective of this study was to investigate the distribution of Salmonella through all phases of two integrated broiler operations and to determine the key areas related to the control of all known sources of infection. Two different Salmonella serotypes were observed at integrated broiler chicken company A. S. enteritidis, the predominant company A isolate, was consistently found in the breeder farm, hatcheries, broiler farms, and chicken slaughterhouse. At company B, a total of six different serotypes, S. heidelberg, S. senftenberg, S. enteritidis, S. blockley, S. gallinarum, and S. virchow, were detected. Although S. heidelberg was not found in the broiler farms, it was consistently found in the breeder farm, hatcheries, and chicken slaughterhouse. In addition, S. enteritidis was found in the hatcheries, broiler farm, and chicken slaughterhouse. In order to obtain the genetic clonality, 22 S. enteritidis isolates were digested with XbaI and analyzed by pulsed-field gel electrohporesis (PFGE). A difference in the PFGE pattern was found to be related to the origin of the integrated broiler operation. These data support the critical need to control Salmonella in breeder farms and hatcheries, and demonstrate important points related to the control of infection in large-scale poultry operations of Korea.
doi:10.4142/jvs.2007.8.2.155
PMCID: PMC2872714  PMID: 17519569
broiler; operation; Salmonella spp. slaughterhouse
16.  S1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in Egypt 
Virology Journal  2006;3:78.
Background
Infectious bronchitis is highly contagious and constitutes one of the most common and difficult poultry diseases to control. IBV is endemic in probably all countries that raise chickens. It exists as dozens of serotypes/genotypes. Only a few amino acid differences in the S1 protein of vaccine and challenge strains of IBV may result in poor protection. Tropism of IBV includes the respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and the kidney.
Results
Infectious bronchitis virus (IBV) strain closely related to Massachusetts (Mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. The isolate was serologically identified by Dot-ELISA and further characterized by RT-PCR then genotyped using S1 gene sequence analysis. Alignment of the S1 sequence of the isolate with 16 IBV strains revealed high homology to isolates related to Mass serotype. Inoculation with the strain reproduced the disease in experimental 1-day-old chickens and resulted in 20% mortality, severe renal and moderate respiratory distresses. Marked histopathological changes in both kidney and trachea were observed in experimentally infected chickens. A protection study using the H120 live attenuated vaccine showed low protection rate in spite of high S1 sequence homology (97%). Protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as IBV antigens detection by immunofluorescent antibody technique in kidney sections.
Conclusion
Periodical evaluation of cross-protective capabilities of IBV vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of IBV.
doi:10.1186/1743-422X-3-78
PMCID: PMC1592083  PMID: 16987422
17.  Molecular Epidemiology of Campylobacter Isolates from Poultry Production Units in Southern Ireland 
PLoS ONE  2011;6(12):e28490.
This study aimed to identify the sources and routes of transmission of Campylobacter in intensively reared poultry farms in the Republic of Ireland. Breeder flocks and their corresponding broilers housed in three growing facilities were screened for the presence of Campylobacter species from November 2006 through September 2007. All breeder flocks tested positive for Campylobacter species (with C. jejuni and C. coli being identified). Similarly, all broiler flocks also tested positive for Campylobacter by the end of the rearing period. Faecal and environmental samples were analyzed at regular intervals throughout the rearing period of each broiler flock. Campylobacter was not detected in the disinfected house, or in one-day old broiler chicks. Campylobacter jejuni was isolated from environmental samples including air, water puddles, adjacent broiler flocks and soil. A representative subset of isolates from each farm was selected for further characterization using flaA-SVR sub-typing and multi-locus sequence typing (MLST) to determine if same-species isolates from different sources were indistinguishable or not. Results obtained suggest that no evidence of vertical transmission existed and that adequate cleaning/disinfection of broiler houses contributed to the prevention of carryover and cross-contamination. Nonetheless, the environment appears to be a potential source of Campylobacter. The population structure of Campylobacter isolates from broiler farms in Southern Ireland was diverse and weakly clonal.
doi:10.1371/journal.pone.0028490
PMCID: PMC3232229  PMID: 22163024
18.  Genomic Diversity of Campylobacter coli and Campylobacter jejuni Isolates Recovered from Free-Range Broiler Farms and Comparison with Isolates of Various Origins†  
Applied and Environmental Microbiology  2005;71(10):6216-6227.
In many industrialized countries, the incidence of campylobacteriosis exceeds that of salmonellosis. Campylobacter bacteria are transmitted to humans mainly in food, especially poultry meat products. Total prevention of Campylobacter colonization in broiler flocks is the best way to reduce (or eliminate) the contamination of poultry products. The aim of this study was to establish the sources and routes of contamination of broilers at the farm level. Molecular typing methods (DNA macrorestriction pulsed-field gel electrophoresis and analysis of gene polymorphism by PCR-restriction fragment length polymorphism) were used to characterize isolates collected from seven broiler farms. The relative genomic diversity of Campylobacter coli and Campylobacter jejuni was determined. Analysis of the similarity among 116 defined genotypes was used to determine clusters within the two species. Furthermore, evidence of recombination suggested that there were genomic rearrangements within the Campylobacter populations. Recovery of related clusters from different broiler farms showed that some Campylobacter strains might be specifically adapted to poultry. Analysis of the Campylobacter cluster distribution on three broiler farms showed that soil in the area around the poultry house was a potential source of Campylobacter contamination. The broilers were infected by Campylobacter spp. between days 15 and 36 during rearing, and the type of contamination changed during the rearing period. A study of the effect of sanitary barriers showed that the chickens stayed Campylobacter spp. free until they had access to the open area. They were then rapidly colonized by the Campylobacter strains isolated from the soil.
doi:10.1128/AEM.71.10.6216-6227.2005
PMCID: PMC1266014  PMID: 16204541
19.  Darkling Beetles (Alphitobius diaperinus) and Their Larvae as Potential Vectors for the Transfer of Campylobacter jejuni and Salmonella enterica Serovar Paratyphi B Variant Java between Successive Broiler Flocks ▿  
Applied and Environmental Microbiology  2008;74(22):6887-6891.
Broiler flocks often become infected with Campylobacter and Salmonella, and the exact contamination routes are still not fully understood. Insects like darkling beetles and their larvae may play a role in transfer of the pathogens between consecutive cycles. In this study, several groups of beetles and their larvae were artificially contaminated with a mixture of Salmonella enterica serovar Paratyphi B Variant Java and three C. jejuni strains and kept for different time intervals before they were fed to individually housed chicks. Most inoculated insects were positive for Salmonella and Campylobacter just before they were fed to the chicks. However, Campylobacter could not be isolated from insects that were kept for 1 week before they were used to mimic an empty week between rearing cycles. All broilers fed insects that were inoculated with pathogens on the day of feeding showed colonization with Campylobacter and Salmonella at levels of 50 to 100%. Transfer of both pathogens by groups of insects that were kept for 1 week before feeding to the chicks was also observed, but at lower levels. Naturally contaminated insects that were collected at a commercial broiler farm colonized broilers at low levels as well. In conclusion, the fact that Salmonella and Campylobacter can be transmitted via beetles and their larvae to flocks in successive rearing cycles indicates that there should be intensive control programs for exclusion of these insects from broiler houses.
doi:10.1128/AEM.00451-08
PMCID: PMC2583492  PMID: 18791034
20.  Molecular Characterization of Motile Serovars of Salmonella enterica from Breeder and Commercial Broiler Poultry Farms in Bangladesh 
PLoS ONE  2013;8(3):e57811.
Contaminated poultry and poultry products are a major source of motile Salmonellae for human salmonellosis worldwide. Local circulation of any motile Salmonella serovar in poultry has a wider public health impact beyond its source of origin for being dispersed elsewhere through poultry trades or human travels. To investigate the status of motile Salmonella serovars in breeder farms in Bangladesh, multiple flocks of two breeder farms were observed for a period of six months. In addition, a cross-sectional survey was carried out to determine the prevalence and serovar distribution of motile Salmonella by randomly selecting 100 commercial broiler poultry farms. Five pooled faecal samples representing an entire housed flock of breeders or broilers were screened for presence of motile Salmonella following conventional bacteriological procedures. The Salmonella isolates obtained were subsequently serotyped, and characterized by plasmid profiling and pulsed-field gel electrophoresis (PFGE). The results revealed that both the breeder farms were positive with three Salmonella serovars: S. Virchow, S. Paratyphi B var Java (S. Java) and S. Enteritidis. Eleven of the 100 broiler farms investigated were positive for motile Salmonella, giving a farm-level prevalence of 11% (95% confidence interval 5–17%). S. Virchow and S. Kentucky were the two predominant serovars isolated from the broiler farms. The PFGE genotyping demonstrated that the isolates belonging to the same serovars were closely related due to variation in only 1–4 bands. All the S. Virchow and S. Java isolates, irrespective of breeder or broiler farm origin, were plasmid-free, except for one S. Virchow isolate from a broiler farm that harboured a 9.7 kb-sized plasmid. The S. Kentucky isolates belonged to three plasmid profiles having plasmids of four different sizes, ranging from 2.7 to 109 kb. This is the first report of any motile Salmonella serovars from breeder and commercial broiler poultry farms in Bangladesh.
doi:10.1371/journal.pone.0057811
PMCID: PMC3590279  PMID: 23483931
21.  Passive immunization against highly pathogenic Avian Influenza Virus (AIV) strain H7N3 with antiserum generated from viral polypeptides protect poultry birds from lethal viral infection 
Virology Journal  2008;5:144.
Our studies were aimed at developing a vaccination strategy that could provide protection against highly pathogenic avian influenza virus (AIV), H7N3 or its variants outbreaks. A purified viral stock of highly pathogenic H7N3 isolate was lysed to isolate viral proteins by electrophresing on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by their elution from gel through trituration in phosphate buffered saline (PBS). Overall, five isolated viral polypeptides/proteins upon characterization were used to prepare hyperimmune monovalent serum against respective polypeptides independently and a mixture of all five in poultry birds, and specificity confirmation of each antiserum through dot blot and Western blotting. Antiserum generated from various group birds was pooled and evaluated in 2-week old broiler chicken, for its protection against viral challenge. To evaluate in-vivo protection of each antiserum against viral challenges, six groups of 2-week old broiler chicken were injected with antiserum and a seventh control group received normal saline. Each group was exposed to purified highly pathogenic AIV H7N3 strain at a dose 105 embryo lethal dose (ELD50). We observed that nucleoprotein (NP) antiserum significantly protected birds from viral infection induced morbidity, mortality and lowered viral shedding compared with antiserum from individual viral proteins or mixed polypeptides/proteins inclusive of NP component. The capability of individual viral polypeptide specific antisera to protect against viral challenges in decreasing order was nucleoprotein (NP) > hemagglutinin (HA) > neuraminidase (NA) > viral proteins mix > viral polymerase (PM) > non-structural proteins (NS). Our data provide proof of concept for potential utilization of passive immunization in protecting poultry industry during infection outbreaks. Furthermore conserved nature of avian NP makes it an ideal candidate to produce antiserum protective against viral infection.
doi:10.1186/1743-422X-5-144
PMCID: PMC2627827  PMID: 19040734
22.  Immune Response of Broiler Chickens Fed Diets Supplemented with Different Level of Chromium Methionine under Heat Stress Conditions 
The objectives of this study were to investigate the immune responses of broiler chickens fed diets supplemented with different level of chromium methionine (CrMet) in heat stress (HS) condition. Two hundred and eighty eight male broiler chickens (Ross 308) were allocated to four treatment groups (supplementation with 0, 200, 400 or 800 ppb Cr in the form of CrMet) in a completely randomized design. The experiment was conducted at heat stressed condition and all birds were kept under temperature of 33±2°C. Antibody titers against Newcastle disease virus (NDV) and infectious bronchitis virus (IBV), heterophil to lymphocyte ratios (H/L), and concentration of plasma cortisol (CPC) were measured at 21 and 42 d. At 42 days of age two birds were chosen randomly from each replicate, slaughtered, spleen and bursa of Fabricius were collected, weighed and expressed as a percentage of live body weight. Antibody titers against NDV and IBV at 21 and 42 days of age in broiler fed supplemental CrMet were higher than in broiler chickens fed control diet (p<0.05). CPC level in broiler chickens fed CrMet were significantly (p<0.05) decreased. Increases in lymphocyte counts and consequently a decrease in heterophil to lymphocyte ratios in broiler chickens fed 800 ppb Cr were observed at 21 and 42 d. Supplementation with CrMet had no significant effect on lymphoid organs of broilers. The results suggest that dietary CrMet supplementation at a level of 800 ppb can improve some immune responses of broiler chickens under heat stress conditions.
doi:10.5713/ajas.2011.11217
PMCID: PMC4093138  PMID: 25049559
Broiler; Chromium Methionine; Immune Response; Heat Stress
23.  OVARIAN RESERVE TESTS AND THEIR UTILITY IN PREDICTING RESPONSE TO CONTROLLED OVARIAN STIMULATION IN RHESUS MONKEYS 
American journal of primatology  2010;72(8):672-680.
Controlled ovarian stimulation (COS) is an alternative to natural breeding in nonhuman primates; however, these protocols are costly with no guarantee of success. Toward the objective of predicting COS outcome in rhesus monkeys, the current study evaluated three clinically used ovarian reserve tests (ORTs): day 3 (d3) follicle-stimulating hormone (FSH) with d3 inhibin B (INHB), the clomiphene citrate challenge test (CCCT), and the exogenous FSH Ovarian Reserve Test (EFORT). A COS was also performed and response was classified as either successful (COS+) or unsuccessful (COS−) and retrospectively compared to ORT predictions. FSH and INHB were assessed for best hormonal index in conjunction with the aforementioned tests. INHB was consistently more accurate than FSH in all ORTs used. Overall, a modified version of the CCCT using INHB values yielded the best percentage of correct predictions. This is the first report of ORT evaluation in rhesus monkeys and may provide a useful diagnostic test prior to costly follicle stimulations, as well as predicting the onset of menopause.
doi:10.1002/ajp.20823
PMCID: PMC2909193  PMID: 20336797
inhibin; ovarian reserve; follicular response; peri-menopause; hormone predictors of fertility
24.  En Face OCT Imaging for the Diagnosis of Outer Retinal Tubulations in Age-Related Macular Degeneration 
Journal of Ophthalmology  2012;2012:542417.
Purpose. “En face” is an emerging imaging technique derived from spectral domain optical coherence tomography (OCT). It produces frontal sections of retinal layers, also called “C-scan OCT.” Outer retinal tubulations (ORTs) in age-related macular degeneration (AMD) are a recent finding evidenced by spectral-domain OCT. The aim of this study is to characterize the morphology of ORT according to the form of AMD, using “en-face” spectral domain OCT. Methods. “En face” OCT imaging was prospectively performed in 26 consecutive eyes with AMD that also had ORT. Results. There were 15 neovascular, 8 atrophic, and 3 eyes with a mixed (fibrotic and atrophic) form of AMD. Among the neovascular group, the most frequent tubulation pattern on “en-face” OCT was a branching network emanating from a fibrovascular scar; we term this pattern as “pseudodendritic.” It did not require treatment when observed as an isolated finding. In all cases of atrophic AMD, the tubular network was located at the edge of the geographic atrophy area, and formed a “perilesional” pattern. Six atrophic cases showed tubular invaginations inside this area. Conclusion. “En face” OCT is a valuable technique in the diagnosis and followup of macular disease. It revealed the main characteristic patterns of ORT associated with neovascular and atrophic AMD.
doi:10.1155/2012/542417
PMCID: PMC3437289  PMID: 22970349
25.  Population genetic structure of Ascaridia galli re-emerging in non-caged laying hens 
Parasites & Vectors  2012;5:97.
Background
The poultry roundworm Ascaridia galli has reappeared in hens kept for egg production in Sweden after having been almost absent a decade ago. Today this is a frequent intestinal nematode parasite in non-caged laying hens. The aim of this study was to investigate the genetic diversity (Fst) in A. galli collected from different poultry production sites in southern Sweden, to identify possible common routes of colonization.
Methods
Adult parasites (n = 153) from 10 farms, including both broiler breeder parents and laying hens, were investigated by amplified restriction fragment length polymorphism analysis (AFLP). Worms from a Danish laying hen farm were also included for comparison. Most of the farms were represented by worms from a single host, but on two farms multiple samples from different hosts were assessed in order to study flock variation.
Results
A total of 97 fragments (loci) were amplified among which 81% were variable alleles. The average genetic diversity was 0.13 (range = 0.09-0.38), which is comparable to other AFLP studies on nematodes of human and veterinary importance. Within-farm variation showed that worms harboured by a single hen in a flock covered most of the A. galli genetic variation within the same flock (Fst = 0.01 and 0.03 for two farms). Between-farm analysis showed a moderate population genetic structure (Fst = 0.13), along with a low mutational rate but high gene flow between different farms, and absence of strong genetic selection. Network analysis showed repeated genetic patterns among the farms, with most worms on each farm clustering together as supported by high re-allocation rates.
Conclusions
The investigated A. galli populations were not strongly differentiated, indicating that they have undergone a genetic bottlenecking and subsequent drift. This supports the view that the investigated farms have been recently colonized, and that new flocks are reinfected upon arrival with a stationary infection.
doi:10.1186/1756-3305-5-97
PMCID: PMC3403953  PMID: 22607623
AFLP; Ascaridia galli; Nematoda; Parasite infection; Population genetics; Network analysis

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