Swabs Of Chickens Research Articles

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

Chicken skin virome analyzed by high-throughput sequencing shows a composition highly different from human skin.

Recent studies show that human skin at homeostasis is a complex ecosystem whose virome include circular DNA viruses, especially papillomaviruses and polyomaviruses. To determine the chicken skin virome in comparison with human skin virome, a chicken swabs pool sample from fifteen indoor healthy chickens of five genetic backgrounds was examined for the presence of DNA viruses by high-throughput sequencing (HTS). The results indicate a predominance of herpesviruses from the Mardivirus genus, coming from either vaccinal origin or presumably asymptomatic infection. Despite the high sensitivity of the HTS method used herein to detect small circular DNA viruses, we did not detect any papillomaviruses, polyomaviruses, or circoviruses, indicating that these viruses may not be resident of the chicken skin. The results suggest that the turkey herpesvirus is a resident of chicken skin in vaccinated chickens. This study indicates major differences between the skin viromes of chickens and humans. The origin of this difference remains to be further studied in relation with skin physiology, environment, or virus population dynamics.

ANTIBIOTIC RESISTANCE IN ENTEROBACTERIACEAE STRAINS ISOLATED FROM CHICKEN AND MILK SAMPLES

Antibiotic resistance and identification of strains in Enterobacteriaceae genera isolated from milk, milk products and rectal swabs of chicken was examined in this experiment. After samples collection cultivation and identification of bacterial strain was done. MALDI TOF MS Biotyper for identification of Enterobacteriaceae strains was used. For susceptibility testing disc diffusion methodology was used according by EUCAST. Results showed high level of ampicillin resistance in isolates from milk and milk samples. The highest streptomycin resistance was detected in isolates from rectal swabs of chicken. After identification, we determined that S. enterica ser. Typhimurium, which was isolated from rectal swabs of chicken showed the most multi-resistance from all identificated strains of Enterobacteriaceae. The most isolates bacterial strain was E. coli, which showed resistance against four antibiotics from rectal swabs of chicken. Also our results showed that the higher resistance level is in rectal swabs of chicken like in milk samples.

An impedance immunosensor based on low-cost microelectrodes and specific monoclonal antibodies for rapid detection of avian influenza virus H5N1 in chicken swabs.

Early screening of suspected cases is the key to control the spread of avian influenza (AI) H5N1. In our previous studies, an impedance biosensor with an interdigitated array microelectrode based biochip was developed and validated with pure AI H5 virus, but had limitations in cost and reliability of the biochip, specificity of the antibody against Asian in-field H5N1 virus and detection of H5N1 virus in real samples. The purpose of this study is to develop a low-cost impedance immunosensor for rapid detection of Asian in-field AI H5N1 virus in chicken swabs within 1h and validate it with the H5N1 virus. Specific monoclonal antibodies against AI H5N1 virus were developed by fusion of mouse myeloma cells with spleen cells isolated from an H5N1-virus-immunized mouse. Dot-ELISA analysis demonstrated that the developed antibodies had good affinity and specificity with the H5N1 virus. The microelectrodes were redesigned with compact size, fabricated using an improved wet-etching micro-fabrication process with a higher qualified production rate of 70-80%, and modified with the antibodies by the Protein A method. Equivalent circuit analysis indicated that electron transfer resistor was effective with the increase in impedance after capturing of the H5N1 viruses. Linear relationship between impedance change and logarithmic value of H5N1 virus at the concentrations from 2(-1) to 2(4) HAU/50 μl was found and the lower limit of detection was 2(-1) HAU/50 μl. No obvious interferences from non-target viruses such as H6N2, H9N2, Newcastle disease virus, and infectious bronchitis virus were found. Chicken swab tests showed that the impedance immunosensor had a comparable accuracy with real-time RT-PCR compared to viral isolation.

Molecular Studies on Salmonella Species Isolated From Chicken

Although many pathogens have recently received considerable attention, Salmonella remains among the leading sources of food-borne illness throughout much of the world. A poultry producer suffers losses due to Salmonella infection of the flock including loss of birds and production time. The present study was carried out to compare between the conventional bacteriological method and pre-enriched PCR assay for detection of Salmonella in chicken swabs samples The culture method can detect Salmonella in 6 samples out of 105 of examined samples under investigation with an incidence of 5.7 %, where by using PCR can detect 7 Salmonella positive samples out of 105 examined samples with an incidence of 6.7 %. Dendrogram or phylogentic tree of 5 isolated salmonella serotypes on RAPD-PCR analysis – PCR pattern similarity. The phylogentic tree was constructed applying UPGMA to matrix of similarity established from the combined data obtained by using three RAPD-PCR primers (1, 2, and primer 5) which give the best discrimination out of the six Primers used in the study. The numbers indicate the percentage of similarity among RAPD-PCR profiles based on pair wise band sharing as compared with the molecular weight of the marker (DNA ladder).. it was found that Salmonella serotypes isolated chickens and environmental samples have different degrees of genetic similarity as follow; S. anatum with S. typhimurium (52 %), S. anatum with S. derby (57 %), S. anatum with S. kentucky ( 62 %), S. anatum with S. enteritidis ( 48 %). S. typhimurium with S. derby ( 67 %), S. typhimurium with S. kentucky ( 43 %), S. typhimurium with S. enteritidis ( 38 %). S. derby with S. kentucky ( 37 %) S. derby with S. enteritidis (33 %). S. kentucky with S. enteritidis (29%). These results indicate that there are a different degrees of polymorphism between the 5 serotypes under investigation from lower degree of genetic similarity between (29%) between S. kentucky and S. enteritidis to higher degree (67 %) between S. typhimurium with S. derby.

Detection of Salmonella spp, Salmonella Enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay

The presence of Salmonella in the intestinal tract, on the chickens skin and among their feathers, may cause carcasses contamination during slaughtering and processing and possibly it is responsible by the introduction of this microorganism in the slaughterhouses. A rapid method to identify and monitor Salmonella and their sorovars in farm is becoming necessary. A pre-enriched multiplex polymerase chain reaction (m-PCR) assay employing specific primers was developed and used to detect Salmonella at the genus level and to identify the Salmonella enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica serovar Typhimurium (S. Typhimurium) in broiler chicken swab samples. The method was validated by testing DNA extract from 90 fresh culture cloacal swab samples from poultry chicken cultured in phosphate buffer peptone water at 37 °C for 18 h. The final results showed the presence of Salmonella spp. in 25% of samples, S. Enteritidis was present in 12% of the Salmonella-positive samples and S. Typhimurium in 3% of the samples. The m-PCR assay developed in this study is a specific and rapid alternative method for the identification of Salmonella spp. and allowed the observation of specific serovar contamination in the field conditions within the locations where these chickens are typically raised.

Development of an antigen-capture ELISA for the detection of avian leukosis virus p27 antigen

An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) employing monoclonal and polyclonal antibodies against p27 was developed for the detection of the avian leukosis virus (ALV). The specificity of the optimized AC-ELISA was evaluated using avian leukosis virus subgroup J (ALV-J), avian leukosis virus subgroup A (ALV-A), avian leukosis virus subgroup B (ALV-B), avian infectious bronchitis virus (IBV), Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), Fowlpox virus (FPV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV), avian influenza virus (AIV) and Escherichia coli. The only specimens that yielded a strong signal were ALV-J, ALV-A and ALV-B, indicating that this assay is suitable for the detection of ALV. The limit of detection of this assay was 1.25 ng/ml of rp27 protein and 10(1.79)TCID(50) units of HLJ09MDJ-1 (ALV-J). Moreover, this AC-ELISA can detect ALV in cloacal swabs of chickens experimentally infected as early as 12 days post-infection. The AC-ELISA detected the virus in the albumin and cloacal swabs of naturally infected chickens, and the results were confirmed by PCR, indicating that the AC-ELISA was a suitable method for the detection of ALV. This test is rapid and sensitive and could be convenient for epidemiological studies and eradication programs.

Isolation ofMycoplasma meleagridisfrom Chickens

Mycoplasma meleagridis (MM) is a major cause of disease and economic loss in turkeys. Formerly it was thought that this species was very host specific and only restricted to turkey. In this study, we report on the recovery of MM from breeding flocks of chickens located near a turkey breeding unit. Ten MM field strains were isolated (by culture on Frey broth medium) from tracheal swabs of chickens displaying clinical signs of mycoplasmosis-essentially respiratory symptoms and poor performance. Assignment of the isolated field strains to MM was confirmed by a growth inhibition assay using MM-specific polyclonal antiserum and by PCR amplification targeting the 16S rRNA sequence as well as the Mm14 sequence, a MM-species-specific DNA fragment previously identified and characterized in our laboratory. The nucleotide sequence of Mm14 proved to be highly conserved among the 10 MM field strains, indicating a common source of infection. However, on the basis of slight differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell proteins and western blot profiles, two groups of the isolated MM field strains could be distinguished. Evidence of MM infection of chickens was further provided by serology, since 13.77% (35/254) of sera proved positive to MM by either rapid serum agglutination or recombinant antigen-based enzyme-linked immunosorbent assay. In addition, sera of all chickens from which MM was isolated were positive for antibodies to MM. Collectively, the data unambiguously show that MM could infect chickens; thus, MM warrants further exploration to determine its pathogenicity in this unusual host.

Comparison of nucleic acid-based detection of avian influenza H5N1 with virus isolation

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus was compared with viral culture in embryonated chicken eggs. Virus was isolated from blood or anal swabs of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). Viral nucleic acid was detected in blood samples by NASBA/ECL immediately prior to death, whilst nucleic acid extracted from anal swabs was detected from the day following artificial infection until death. Thus, blood and/or anal swabs are a suitable source of material for the detection of avian influenza in dead birds, but anal swabs are more suitable for detection of viral genetic material in live birds. Dilution of a known viral standard was used to determine the limit of sensitivity for both NASBA/ECL and egg culture detection methods. The NASBA/ECL method was equivalent in sensitivity to egg culture. The NASBA/ECL results agreed with egg culture data in 71/94 (75.5%) tissue samples obtained from artificially infected birds.

Identification of Campylobacter jejuni Isolates from Cloacal and Carcass Swabs of Chickens in Thailand by a 5′Nuclease Fluorogenic Polymerase Chain Reaction Assay

A rapid 5′ nuclease fluorogenic polymerase chain reaction (PCR) assay for identifying Campylobacter jejuni was applied to Campylobacter isolates from chicken cloacal and carcass swabs collected from three chicken farms and a slaughterhouse in Thailand. The primers and the probe were based on the sequence of the gyrA gene in C. jejuni. C. jejuni isolates were identified by fluorogenic PCR assay of bacterial cells directly from Campylobacter-selective agar medium. This assay allowed the identification of C. jejuni within 1 day after colonies appeared on selective media. The fluorogenic PCR assay yielded results comparable to those of the conventional test kit (kappa = 0.76) but required less time. When the two methods disagreed with regard to species identification, results were confirmed by PCR restriction fragment length polymorphism of 23S rRNA genes. In these instances, the fluorogenic PCR assay correctly identified more isolates of C. jejuni than did the conventional test kit (six of seven isolates were unidentifiable by the conventional test kit). The fluorogenic PCR assay is a rapid and specific method that outperforms the conventional test kit in the identification of C. jejuni from environmental samples.

In vivo variation of Mycoplasma gallisepticum antigen expression in experimentally infected chickens

The antigen expression profiles of Mycoplasma gallisepticum isolates obtained from tracheal swabs of chickens after aerosol-inoculation with M. gallisepticum strain R or clonal variant R E were examined in western immunoblots. A reference anti- M. gallisepticum chicken antiserum and antisera from individual infected chickens as well as monoclonal antibodies (mAbs) specific for surface proteins were used to monitor in vivo antigenic variation. mAbs 1E5 and 12D8, recognizing PvpA and p67a, recently shown to undergo high-frequency in vitro phase variation, were used for consecutive staining of colony and western immunoblots in order to distinguish between the resultant phenotypes with respect to the corresponding epitopes. Marked differences in the expression of major immunogenic proteins, including p67a, were observed between the two inocula as well as among reisolates recovered at different times of infection. Comparative western immunoblot analysis of the rapidly changing chicken serum antibody response and reisolates recovered during the course of an experimental infection with M. gallisepticum R or clonal variant R E suggest that immune modulation may have a key role in generating surface diversity. In addition, comparison of colony immunoblots of strain R inoculum and of reisolated colonies from tracheas of birds 8 days post infection indicated an in vivo selection of the PvpA +p67a − phenotype. This study established that surface antigens of M. gallisepticum are subjected in vivo to rapid alteration in their expression. This variability may function as a crucial adaptive mechanism, enabling the organism to escape from the host immune defense and to adapt to the changing host evironment at different stages of a natural infection.

Composition of the enterococcal intestinal flora of chickens in a tropical area

Enterococcus faecalis was the most frequently occurring enterococcal species in caeca and cloacal swabs of chickens in Assam, India. In chickens less than 8 weeks of age Ent. faecium, Ent. durans and Ent. avium were also found, but infrequently. Enterococcus hirae, which is potentially pathogenic in chickens of less than 1 week of age, was found only in chickens of more than 8 weeks. Except for a higher frequency of Ent. faecalis, the composition of this flora was essentially the same as that in European temperate climates.

Characterization of <i>Escherichia coli</i> isolated from samples of different biological and environmental sources

Escherichia coli from 10 different biological and environmental sources were isolated and characterized in the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh during the period from January to May 2007. A total of 100 samples, 10 from each of human feces and urine, rectal swab of cattle, sheep and goat, cloacal swab of chicken, duck and pigeon, drain sewage and soil were collected aseptically and subjected to primary isolation by propagating in nutrient broth followed by culture on different agar media. Gram's staining and hanging drop techniques were also performed. Biochemical properties of the isolates were studied and reaction in TSI agar slant was also observed. Pathogenicity of 10 representative E. coli isolates, one from each source were determined by lethality assay in 12 day-old embryonated eggs, in day-old chicks and in day-old suckling mice models. E. coli was isolated successfully from all the samples. All the E. coli isolates were found to produce bright pink colonies on MacConkey agar, yellowish green colonies surrounded by an intense yellow green zone on BG agar and characteristic metallic sheen colonies on the EMB agar. In case of E. coli isolated from cattle, slight variation in colony character on EMB agar was observed showing greenish red colonies with faint metallic sheen. In Gram's staining technique, all the isolates were pink coloured, small rod shaped Gram negative bacilli and in the hanging drop technique they were motile. Reactions in TSI agar slant revealed yellow slant and butt with gas but no hydrogen sulphide production. Almost all the E. coli isolates fermented dextrose, maltose, lactose, sucrose and mannitol with the production of both acid and gas except E. coli isolated from drain sewage which did not ferment maltose and isolates from pigeon showed less production of acid and gas during sucrose fermentation. The results of Catalase, MR and indole test of the E. coli isolates were positive but V-P test was negative. In the embryo lethality assay, E. coli isolates from chicken, pigeon, duck, human urine, cattle, sheep and goats were virulent causing 33.33-100% death of the embryo except isolates from human faeces and drain sewage which were moderately virulent and that from soil which was avirulent. E. coli isolate of chicken origin found to be more virulent which caused 100% death of the embryos. Most of the embryos died between day-1 and day-2 PI. Chick lethality assay indicated that all the E. coli isolates were virulent as the mortality rate was more than 50%. In mice lethality assay, all the E. coli isolates were in the killer group causing cent percent death of mice within 10 to 42 h following inoculation. Among these three lethality assay models, avian embryo lethality assay was found to be most suitable to discriminate between virulent and avirulent isolates compared to day-old chick lethality assay and day-old suckling mice lethality assay where inconsistent results were observed. In conclusion, our result showed that E. coli isolated from different biological and environmental sources were found to be varied in virulence and avian embryo lethality assay was assumed to be the best model for discriminating virulent and avirulent E. coli. Key words: Escherichia coli, pathogenicity, human, cattle, poultry, soil DOI = 10.3329/bjvm.v5i1.1305 Bangl. J. Vet. Med. (2007). 5 (1 & 2): 25-32The PDF for this article was corrected and replaced on 9/11/2009.

Studies on Cytopathogenic Avian Enteroviruses II. Influence of Age on Virus Excretion and Incidence of Certain Serotypes in a Colony of Chicks

Enteroviruses producing cytopathogenic changes in chicken kidney tissue have been isolated by a number of workers from the feces or rectal swabs of chickens and found to occur in up to 15 distinct serological groups on the basis of serum neutralization tests (1,2,3,7,9). It was further observed in examination of birds of various ages that percentage of isolation was definitely correlated with age but not with season (2,3,7,9). Similar results have been reported for bovine enteroviruses (4). Burke et al. (3) examined fecal samples from 10 chickens weekly between 4 weeks and 8 weeks of age and isolated at least two types of enteric viruses. The incidence of infection with different serotypes in different flocks of chickens has been very variable (7). In routine examination of fecal samples from apparently healthy offspring, different strains of ECHO viruses have been isolated (5,8). A study was undertaken to establish the correlation of age with virus excretion by examining the monthly fecal samples from 34 chickens from one month of age up to eleven months and to classify the isolates serologically on the basis of serum neutralization tests.

Studies on Cytopathogenic Avian Enteroviruses. I. Their Isolation and Serological Classification

Enders et al. (6) were the first to evaluate in vitro the usefulness of tissues other than nervous tissues for the propagation of polio virus. Since then, enteric cytopathogenic agents have been isolated by a number of workers using monolayers of kidney tissue culture from animals and man. Kalter (7) and Bindrich (1), in their reviews, enumerated the isolation of enteric cytopathogenic orphan viruses from monkey, ox, swine, poultry, cat, and rodents. Since these viruses were not associated with diseases, they were termed orphan viruses. In analogy with the ECHO viruses in man, cytopathogenic viral agents isolated from the intestinal tract of chickens were termed entero-cytopathogenic avian orphan (ECAO) viruses. Available literature is scant on the isolation of viruses from the intestinal tract of chickens, as compared with species of animals and man. The problem has recently been attacked by Burke et al. (2), who reported the isolation of 45 cytopathogenic agents from rectal swabs of chickens. They also observed that these agents belong to at leasit 2 serological groups. Similarly, Taylor and Calnek (12) reported the isolation of 45 enterocytopathogenic agents and grouped them into 15 dis~tinct antigenic types. Sarma and Pomeroy (9) isolated two identical viruses from the cecal tonsils of chickens by inoculation and serial