Diseased Chickens Research Articles

Prevalence of Clostridium perfringens infection and virulence genes molecular study in broiler chickens

Clostridium perfringens (C. perfringens) produces toxins that cause necrotic enteritis (NE) in broiler chickens resulting in sever losses in poultry industry. This study aimed to investigate the prevalence of C. perfringens in broiler chickens in Toukh, Qaliubiya governorate, Egypt. A total of 100 broilers (10-40 days old) collected from Toukh, Qaliubiya in 2017. The samples were collected from liver and intestine of both diseased and apparently healthy broilers. All samples were subjected to bacteriological examination. Clostridium Perfringens was isolated from (70%) and (22%) of diseased and apparently healthy broilers respectively. The isolates were typed by dermonecrotic test in Guinea pig and PCR. The results of dermonecrotic test from diseased chickens showed that 40 (65.6%) of the diseased isolates were type A and 8 (13.1%) were type D. But the isolates from the apparently healthy chickens were (9) type A and (6) type D. Typing of the 9 C.Perferingens isolates (diseased broilers) by PCR for toxin genes (cpa ,cpb, etx ) showed that 8 strains were positive for cpa gene (typed as type A). Detection of the virulence genes by PCR showed, pfoS and colA genes were in (8) isolates but netB gene was detected in (3) isolates only. Clostridium perfringens showed high prevalence among diseased broiler chickens with high isolation rate from intestinal sample than hepatic samples. Moreover, high percentage of C.perfringens are toxigenic type A in case of diseased or apparently healthy broiler chickens. The netB gene seems to be unnecessary for inducing necrotic enteritis in broiler chickens.

Detection of virulence factors of Mycoplasma species isolated from chicken by multiplex PCR

One hundred and sixty clinically diseased broiler local chickens collected from 22 different farms belonging to Qalubia, Dakahlia and Gharbia governorates were subjected to bacteriological examination and molecular characterization. Clinical Signs were chronic respiratory disease with respiratory manifestations,lameness, loss or reduction in egg production.Bacteriological examination showed that 18.18% of the isolates were Mycoplasma positive and showed growth in pleuropneumonia-like organism (PPLO) agar plates, with fried egg appearance when examined by stereoscopic microscope. Mycoplasma colonies were tested for antimicrobial sensitivity tests against 12 antimicrobial antibiotics and showed higher sensitivity to nitrofurantoin, gentamicin, norfloxacin, ciprofloxacin and neomycin. Intermediate sensitivity were recorded to ceftriaxone and cefotaxime and antibiotic resistance were recorded to tetracyclines, lincomycin, chloramphenicole and sulphamethoxasine. Identification of the bacterial strains of the isolates were conducted by multiplex PCR by using two primer pairs for Mycoplasma synoviae (vlhA) and Mycoplasma gallisepticum (mgc2). The amplicons expected size were 396 bp, and 300 bp for vlhA and mgc2 respectively. Only13 out of 22 farms were positive, representing 59.09 %.Moreover, the incidence rate of Mycoplasma synoviae (vlhA) and Mycoplasma gallisepticum (mgc2) were 22.72% and 13.63% respectively and 22.72% of the inspected farms showed positive results for both Mycoplasma strains.

Molecular characterization and genetic diversity of the infectious laryngotracheitis virus strains circulating in Egypt during the outbreaks of 2018 and 2019.

Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic losses in the poultry industry worldwide. In this study, ILT outbreaks were reported on 30 farms located in eight Egyptian governorates between January 2018 and May 2019. Gross examination of diseased chickens revealed congestion and hemorrhage of laryngeal and tracheal mucosa with fibrinohemorrhagic casts and/or caseous material in the lumens. Histopathological examination showed epithelial sloughing, syncytium formation, heterophilic exudation, and development of eosinophilic intranuclear inclusion bodies. Infectious laryngotracheitis virus (ILTV) antigen was detected in the tracheal epithelium, infiltrated inflammatory cells, and syncytial cells, using immunohistochemistry. PCR targeting a portion of the thymidine kinase gene was further utilized to confirm the presence of ILTV DNA. The complete coding sequences of three envelope glycoprotein genes, gG, gD, and gJ, and a partial sequence of the infected cell polypeptide 4 (ICP4) gene from samples representing all of the farms and disease outbreaks were determined. Five prototype strains with unique sequences were chosen for detailed molecular characterization. Sequence comparisons and phylogenetic analysis of the partial ICP4 gene revealed that two strains were chicken embryo origin (CEO)-vaccine-like strains, and three were tissue culture origin (TCO)-vaccine-like strains. Analysis of the gJ gene sequence indicated that all of the strains were CEO vaccine-like strains. It was predicted that the latter three strains were recombinants of CEO- and TCO-vaccine-like strains. In conclusion, immunohistochemistry coupled with multi-genomic PCR sequencing proved to be efficient for identification and typing of ILTV strains during disease outbreaks. Both CEO-vaccine-like and recombinant virus strains were circulating in Egypt during the 2018 and 2019 outbreaks.

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Salmonella Species Isolated from Different Sources with Special Reference to Biofilm Formation

The aim of this work was to study the distribution of Salmonella serovars among diseased chickens and diarrheic calves, also to determine the genotypic and phenotypic causes of resistance of Salmonella to antimicrobials. To achieve this aim, two hundred samples (100 diseased chicken samples and 100 fecal swabs from diarrheic calves) were directed to bacteriological examination according to ISO 6579: 2002 A1: 2007. Then confirmed by biochemical reactions, serotyping and molecular characterization which revealed the isolation of fourteen (14) Salmonella spp. isolates (9 from calves and 5 from chickens) with a percentage of 9% and 5% respectively. Also, Antimicrobial susceptibility testing of the total isolates was done which indicated the presence of multidrug resistance among three isolates, one isolate isolated from chickens which was S. Gallinarum while the other two isolates were S. Enteritidis and S. Typhimurium which were isolated from calves. Biofilm binding activity of the 14 Salmonella isolates showed that two isolates had strong binding activity which was multidrug resistant and 12 isolates showed moderate binding. With addition of lactic acid 2%, the binding ability had been decreased.

Virulence and resistance determinants in Pseudomonas aeruginosa isolated from pericarditis in diseased broiler chickens in Egypt.

Objective:This study was performed to probe the antimicrobial resistance and virulence genes profiling in Pseudomonas aeruginosa recovered from the cases of pericarditis in broiler chickens.Materials and methods:The samples (n = 250) collected from the cases of pericarditis in broiler chickens were bacteriologically examined. Antimicrobial susceptibility was tested by disc diffusion technique. The isolates were genotypically studied for the presence of antimicrobial resistance and virulence gene traits. Finally, the nucleotide sequence of representative resistance gene (mexR gene) and virulence genes (toxA and lasI genes) was analyzed.Results:P. aeruginosa was isolated from 45 samples (18%). Antimicrobial susceptibility testing revealed multidrug resistance in most of the recovered P. aeruginosa isolates, whereas colistin and imipenem were the furthermost in vitro-sensitive antibiotics. Antimicrobial resistance genes, such as blaCTX, fox, and mexR, were prevalent in 100%, 80%, and 100% of the isolates, respectively. PCR confirmed virulence genes such as toxA, exoY, lasB, and lasI in 100%, 60%, 80%, and 80% of the isolates, respectively. Nucleotide sequence analysis of representative resistance gene (mexR gene) and virulence genes (toxA and lasI genes) revealed a high correlation between P. aeruginosa recovered from pericarditis in broiler chickens in the present study with PAO1 (reference strain) and with other sequences published on the GenBank representing different localities worldwide.Conclusion:It could be concluded that P. aeruginosa recovered from pericarditis in broiler chickens in the current study is highly virulent bacteria, resisting most of the therapeutic agents which not only bear hazards for poultry industry but also represent a public health concern.

Pathogenicity of a QX-like avian infectious bronchitis virus isolated in China

Avian infectious bronchitis is a serious and highly contagious disease caused by infectious bronchitis virus (IBV). We isolated a highly virulent IBV strain (CK/CH/JS/TAHY) from kidneys of diseased chickens. Phylogenetic analysis based on the S1 gene revealed that CK/CH/JS/TAHY clustered with the QX-like type. The S1 gene has 1,620 nucleotides and encoded a polypeptide of 540 amino acids with typical coronavirus cleavage recognition sites of HRRR. About 1-day-old specific pathogen-free White Leghorn chickens inoculated with CK/CH/JS/TAHY at 105.5 EID50 exhibited clinical signs including coughing, sneezing, nasal discharge, and tracheal vocalization accompanied by depression with 84% mortality and 100% morbidity. The kidneys of dead birds were swollen and pale and exhibited severe urate deposition. Histopathological examination revealed kidney hemorrhages, multifocal necrosis of the renal tubules and trachea with cilia loss, sloughing of epithelial cells, and edema of the lamina propria. IBV-specific antibodies appeared at 10 D post-infection. Chickens vaccinated with a CK/CH/JS/TAHY oil-emulsion vaccine showed 26.7% morbidity and 3% mortality indicating a protective effect. In conclusion, the IBV strain is a virulent avian IBV and that exhibited severe pathogenicity in chickens and is a vaccine candidate to prevent infection by Chinese QX-like nephropathogenic IBV strains.

Clostridium perfringens type A Causing Necrotic Enteritis Outbreaks among Chickens in Egypt

Undoubtedly, necrotic enteritis is identified as one of the most threatening diseases which face poultry industry in Egypt and need radical solution to avoid huge economic losses. The present work was designed for typing Clostridium perfringens isolated from diseased chickens suspected to have necrotic enteritis from different outbreaks in six Governorates in Egypt (Gharbia, Dakhalia, Sharkia, Ismailia, North Sinai and Kafr El Sheikh) during the period from August to December 2018. Intestine and liver samples were obtained from eighty six diseased broiler chickens representing 60 flocks. C. perfringens was isolated and toxigenicity of the recovered isolates was determined by Nagler's and dermonecrotic reactions. Furthermore, multiplex polymerase chain reaction (PCR) targeting alpha, beta, epsilon and iota toxins' genes was performed for result confirmation and typing of the toxigenic isolates. Sixty-six C.perfringens isolates (38.37%) were recovered from 172 intestine and liver samples and twenty isolates (30.3%) were toxigenic and typed as C. perfringens type A producing alpha toxin only. These findings established the fact that alpha toxin is the only main toxin of C. perfringens type A which is basically responsible for its pathogenicity and virulence. In addition, most of the positively toxigenic isolates were isolated from hepatic lesions (15 isolates) rather than intestinal lesion (5 isolates). In conclusion, alpha toxin is a major toxin for NE development in chickens. Genotyping of Clostridium perfringens by multiplex PCR is a useful adjunct to diagnosis of necrotic enteritis in chickens.

The occurrence of the multidrug resistance (MDR) and the prevalence of virulence genes and QACs resistance genes in E. coli isolated from environmental and avian sources

Colibacillosis is a major disease affecting poultry leads to high morbidity and mortality which causing tremendous economic losses worldwide. These economic disparities are amplified among low and middle-income where sanitation and hygiene are challenged by the increasing demand for quality sources of animal protein. With a view to investigating the prevalence of virulence genes and QACs resistance genes as well as monitoring the antibiogram of E. coli strains, a total of 368 specimens were collected from diseased broiler chickens (n = 226) and environmental sources (n = 142) at large-scale poultry farms in Ismailia Governorate, Egypt. The bacteriological examination proved that E. coli prevalence was 26.76% and 50.44% in the farm environment and diseased broilers, respectively. In tandem, the isolated E. coli strains were serogrouped, determining the most common serotypes were O78, O1:H7, O91:H21 and O126. Isolates were tested for antimicrobial susceptibility against 12 antibiotics, screened for 4 virulence genes (iss, papC, eaeA, and cfaI), and screened for 3 QACs resistance genes (qacEΔ1, qacA/B, and qacC/D). All the tested strains were positive for iss and papC genes, only 20.3% of the tested strains were positive for eaeA gene, moreover, the examined strains were negative to CFAI gene. Furthermore, all the tested strains were positive for qacEΔ1, qacA/B, and qacC/D genes. In conclusion; virulence genes (iss, papC) as well as QACs resistance genes are common in avian Pathogenic E. coli and environmental strains and are mainly associated with multi-drug resistance phenomena.

Antimicrobial resistance and molecular characterization of pathogenic E. coli isolated from chickens

Forty eight clinically diseased broiler chickens from different (n=18) farms from Beni Suef and El-Fayoum Governorates were subjected to euthanasia and post mortem examination. Lesions include airsaculitis, pericarditis and perihepatitis. Bacteriological examination showed that 22.9% of isolates were E. coli positive. Serogrouping of isolates revealed O125, O112, O91, O157, O115 and O25 % of each serogroups with an incidence rate of 18.2%, 9.1%, 9.1%, 9.1%, 9.1%, and 9.1%, respectively and four strains were untyped. Antimicrobial sensitivity tests against 17 antimicrobials showed that the most common resistance patterns were against penicillin, lincomycin, oxytetracycline, clindamycin, amoxycillin and erythromycin followed by nalidix acid and trimethoprim. On the other hand, the most potent antimicrobials were colistin sulphate, gentamycin, doxycycline and ceftriaxone followed by enrofloxacin, ciprofloxacin, norfloxacin, chloramphenicol and lastly ampicillin. PCR showed that all isolates had B-lactam resistant gene (blaTEM) and tetracycline resistance gene A (tetA) but only 18 % have quinolones resistance gene A (qnrA).

Genomic analysis on broiler-associated Clostridium perfringens strains and exploratory caecal microbiome investigation reveals key factors linked to poultry necrotic enteritis

BackgroundClostridium perfringens is a key pathogen in poultry-associated necrotic enteritis (NE). To date there are limited Whole Genome Sequencing based studies describing broiler-associated C. perfringens in healthy and diseased birds. Moreover, changes in the caecal microbiome during NE is currently not well characterised. Thus, the aim of this present study was to investigate C. perfringens virulence factors linked to health and diseased chickens, including identifying putative caecal microbiota signatures associated with NE.ResultsWe analysed 88 broiler chicken C. perfringens genomes (representing 66 publicly available genomes and 22 newly sequenced genomes) using different phylogenomics approaches and identified a potential hypervirulent and globally-distributed clone spanning 20-year time-frame (1993–2013). These isolates harbored a greater number of virulence genes (including toxin and collagen adhesin genes) when compared to other isolates. Further genomic analysis indicated exclusive and overabundant presence of important NE-linked toxin genes including netB and tpeL in NE-associated broiler isolates. Secondary virulence genes including pfoA, cpb2, and collagen adhesin genes cna, cnaA and cnaD were also enriched in the NE-linked C. perfringens genomes. Moreover, an environmental isolate obtained from farm animal feeds was found to encode netB, suggesting potential reservoirs of NetB-positive C. perfringens strains (toxinotype G). We also analysed caecal samples from a small sub-set of 11 diseased and healthy broilers for exploratory microbiome investigation using 16S rRNA amplicon sequencing, which indicated a significant and positive correlation in genus Clostridium within the wider microbiota of those broilers diagnosed with NE, alongside reductions in beneficial microbiota members.ConclusionsThese data indicate a positive association of virulence genes including netB, pfoA, cpb2, tpeL and cna variants linked to NE-linked isolates. Potential global dissemination of specific hypervirulent lineage, coupled with distinctive microbiome profiles, highlights the need for further investigations, which will require a large worldwide sample collection from healthy and NE-associated birds.

Characterization of fowl adenovirus serotype 4 circulating in chickens in China

Outbreaks of fowl adenovirus (FAdV) has resulted in huge economic losses in poultry industry in China since 2015. This study detected the pathogens from diseased chickens and determined that fowl adenovirus serotype 4 (FAdV-4) and co-infection of immunosuppressive pathogens were the causes of the outbreaks. Phylogenetic analysis results indicated that these pandemic strains originated from previously FAdV-4 predecessor in China and had obtain gene mutations that might contribute to enhanced pathogenicity of these strains. Compared with early strains, the pathogenicity of novel FAdV-4 strains significantly increased, which led to systemic infections and injuries to multiple organs in the infected chickens. Our study could provide useful information for understanding of the FAdV-4 and favorable theory basis for clinical prevention and control of the disease.

Recombinase polymerase amplification–nucleic acid lateral flow immunoassays for Newcastle disease virus and infectious bronchitis virus detection

Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) are two poultry pathogens affecting the respiratory tract of chickens, and cause major economic losses in the industry. Rapid detection of these viruses is crucial to inform implementation of appropriate control measures. The objective of our study is developing a simple, rapid and field applicable recombinase polymerase amplification (RPA)–nucleic acid lateral flow (NALF) immunoassay for detection of NDV and IBV. Isothermal amplification of the matrix protein (M) gene of NDV and the nucleoprotein (N) gene of IBV was implemented via recombinase polymerase amplification at 38 °C for 40 min and 20 min, respectively using modified labeled primers. NALF device used in this study utilizes antibodies for detection of labeled RPA amplicons. The results revealed that RPA-NALF immunoassays can detect both viruses after 40 min at 38 °C and only NDV after 20 min. The limit of detection (LOD) was 10 genomic copies/RPA reaction. The assays results on clinical samples collected from diseased chicken farms demonstrated a good performance in comparison with quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). The assays established in this study can facilitate rapid, on-site molecular diagnosis of suspected cases of ND and IB viral infections as the results can be detected by the naked eye without the need for measuring fluorescence. Furthermore, the NALF device could be adapted to detect other infectious agents.

Virulence and Antimicrobial Characteristics of Escherichia Coli Isolated from Diseased Chickens in China and Algeria

To reveal and compare the prevalence of pathotypes and virulence genes, as well as antimicrobial resistance and genotyping of poultry E. coli isolates from China and Algeria. Pathotype and seven virulence genes were tested by PCR, susceptibility to antimicrobials was evaluated using broth microdilution method, and genotyping was analyzed by PFGE. Six isolates were identified as pathogenic E. coli. Virulence gene testing showed that the frequency of ompT, iss, fimC, iroN, hlyF and iutA was high in the isolates from Shandong, Shanxi, Jiangsu and Xinjiang province, while stx2 was detected in only two isolates from Shandong, and stx2, iss, fimC, hlyF and iutA could not be detected in Tibet isolates. Importantly, nearly all isolates from Algeria carried seven virulence genes. Drug resistance testing of 141 strains showed that 98.2% (109/111) of the isolates from China and all isolates (30/30) from Algeria resist to more than three classes of antimicrobials. The PFGE genotyping of 157 isolates yielded 134 types, demonstrated a high level of genetic diversity among these isolates. Thus, the poultry E. coli from both China and Algeria exhibited either high frequencies of antimicrobial resistance or high rates of virulence genes carrying.

Epidemiological investigation of fowl adenovirus infections in poultry in China during 2015\u20132018

BackgroundFowl adenoviruses (FAdVs) are associated with many diseases, resulting in huge economic losses to the poultry industry worldwide. Since 2015, outbreaks of FAdV infections with high mortality rates have been reported in China. A continued surveillance of FAdVs contributes to understand the epidemiology of the viruses.ResultsWe isolated 155 FAdV strains from diseased chickens from poultry in China between 2015 and 2018. PCR analysis determined that 123 samples were FAdV species C, 27 were FAdV species E, and five contained two different FAdV strains. The phylogenetic analysis demonstrates that these sequences of hexon regions were clustered into three distinct serotypes: FAdV-4 (79.4%, 123/155), FAdV-8a (13.5%, 21/155) and FAdV-8b (3.9%, 6/155), of which FAdV-4 was the dominant serotype in China.ConclusionsThe characterization of newly prevalent FAdV strains provides valuable information for the development of an effective control strategy for FAdV infections in chickens.

Characterisation of gastrointestinal helminths and their impact in commercial small-scale chicken flocks in the Mekong Delta of Vietnam

Commercial small-scale chicken farms managed as all-in-all-out but operating with low standards of hygiene/biosecurity are increasingly common in Vietnam. These conditions facilitate the transmission of gastrointestinal helminths. However, there are no published data on helminths in these systems. We aimed (1) to determine the prevalence/burden of gastrointestinal helminths in small-scale commercial flocks in the Mekong Delta region and (2) to investigate the association between worm burdens and birds’ weight and disease status. Randomly selected chickens (n = 120) from ‘normal’ flocks were investigated at the end of their production cycle (~ 18 weeks), as well as 90 chickens from ‘diseased’ flocks with signs of respiratory and/or severe disease. The gastrointestinal tract of chickens was dissected and all visible helminths were identified and counted. A total of 54.2% and 54.4% normal and diseased chickens contained helminths. Among colonised birds, the diseased ones harboured a higher mass of helminth worms than normal (healthy) birds (3.8 ± SD 8.6 g vs. 1.9 ± SD 6.3 g, respectively). Eight species were identified, including nematodes (Ascaridia galli, Cheilospirura hamulosa and Heterakis gallinarum), cestodes (Hymenolepis, Raillietina cesticillus, Raillietina echinobothrida, Raillietina tetragona,) and one trematode (Echinostomatidae). Heterakis gallinarum was the most prevalent helminth (43.3% and 42.2% in normal and sick chickens, respectively), followed by A. galli (26.7% and 41.1%). Colonised chickens weighed 101.5 g less than non-colonised birds. Colonisation was higher during the rainy months (May–November) for both H. gallinarum and A. galli. Anthelminthic usage was not associated with reduced helminth burdens. We recommend upgrading cleaning and disinfection and limiting access to ranging areas to control helminths in small-scale chicken flocks.

Pathogenicity of a fowl adenovirus serotype 4 isolated from chickens associated with hydropericardium-hepatitis syndrome in China

Hydropericardium-hepatitis syndrome (HHS) is characterized by pericardial effusion and hepatitis and causes huge economic losses in the poultry industry in China. In this study, a strain of fowl adenoviruses (FAdV-4) (GX-1) was isolated from liver samples of diseased chickens with HHS. Phylogenetic analysis based on complete genome gene revealed that GX-1 clustered with the C-type fowl adenovirus and was serotyped as FAdV-4. Pathogenicity testing showed that the GX-1 strain caused 100% mortality in 10-day-old specific pathogen-free chickens at a dose of 104 tissue culture infective doses (TCID50) within 3 d post-infection. A viral dose of 103 TCID50 resulted in a 16% survival rate before day 9 and at 102 TCID50 an 80% rate before day 6. At necropsy, livers from infected chickens were swollen and yellow brown with necrotic foci. The hearts were flabby with amber-colored and jelly-like fluid in the pericardial sacs. The kidneys were swollen and congested. Histologically eosinophilic intranuclear inclusion body could be seen in the hepatic cell. The result of histopathological examination also revealed that heart muscle fibers were fractured with extensive congestion and hemorrhaging. Other tissues like kidney, bursa of Fabricius, thymus, and spleen were observed degeneration and necrosis. Virus-specific antibodies appeared in serum beginning at day 14 and reached statistically significant levels at 21, 28, 35, and 42 dpi (P < 0.001). In conclusion, we identified a highly virulent FAdV-4 virus as causative agent of the HHS outbreak reported here. The FAdV-4 GX-1 strain will be valuable for vaccine evaluation and development to prevent and reduce the spread of HHS in the poultry industry.

Isolation and identification of Marek’s disease virus (MDV) from feather follicle epithelium and internal organs of diseased chickens in Dakahlia Governorate, Egypt

In the present study, a total of 16 samples including feather follicle epithelium, ovary, spleen and kidney (4 samples for each organ) were collected from diseased chicken flocks suspected to be infected with Marek’s disease virus (MDV) at Dakahlia Governorate, Egypt during the period from October 2016 to October 2017. Each sample was pooled randomly from three to five birds (90 to 360 days old). The isolation of the suspected virus from the collected samples was carried out via chorioallantoic membranes (CAMs) of 12 days old embryonated chicken eggs (ECEs). Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard MDV. MDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Molecular identification of virus was carried out by conventional polymerase chain reaction (PCR) and real- time PCR in four selected samples. The results revealed that 14 samples (87.5%) including 4 (100%) samples from feather follicle epithelium, ovary and kidney and 2 (50%) samples from spleen, showed positive results in virus isolation after 3rd passage. The positive results percentage by AGPT for field samples were 50% (8 out of 16 samples), while after the 3rd passage in ECEs were 37.5% (6 out of 16 samples) and the positive results percentage by IFAT for field samples were 62.5% (10 out of 16 samples), while after the 3rd passage in ECEs were 81.25 % (13 out of 16 samples). Viral nucleic acid was detected in all selected samples by conventional and real- time PCR. The results indicate that feather follicle epithelium is the best organ for MDV detection. IFAT is superior over AGPT in virus detection. Conventional and real - time PCR could be efficiently used for molecular detection of the virus.

Isolation and identification of Marek’s disease virus (MDV) from feather follicle epithelium and internal organs of diseased chickens in Dakahlia Governorate, Egypt

In the present study, a total of 16 samples including feather follicle epithelium, ovary, spleen and kidney (4 samples for each organ) were collected from diseased chicken flocks suspected to be infected with Marek’s disease virus (MDV) at Dakahlia Governorate, Egypt during the period from October 2016 to October 2017. Each sample was pooled randomly from three to five birds (90 to 360 days old). The isolation of the suspected virus from the collected samples was carried out via chorioallantoic membranes (CAMs) of 12 days old embryonated chicken eggs (ECEs). Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard MDV. MDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Molecular identification of virus was carried out by conventional polymerase chain reaction (PCR) and real- time PCR in four selected samples. The results revealed that 14 samples (87.5%) including 4 (100%) samples from feather follicle epithelium, ovary and kidney and 2 (50%) samples from spleen, showed positive results in virus isolation after 3rd passage. The positive results percentage by AGPT for field samples were 50% (8 out of 16 samples), while after the 3rd passage in ECEs were 37.5% (6 out of 16 samples) and the positive results percentage by IFAT for field samples were 62.5% (10 out of 16 samples), while after the 3rd passage in ECEs were 81.25 % (13 out of 16 samples). Viral nucleic acid was detected in all selected samples by conventional and real- time PCR. The results indicate that feather follicle epithelium is the best organ for MDV detection. IFAT is superior over AGPT in virus detection. Conventional and real - time PCR could be efficiently used for molecular detection of the virus.

Adaptation of a Mice Doppler Echocardiography Platform to Measure Cardiac Flow Velocities for Embryonic Chicken and Adult Zebrafish.

Ultrasonography is the most widely used imaging technique in cardiovascular medicine. In this technique, a piezoelectric crystal produces, sends, and receives high frequency ultrasound waves to the body to create an image of internal organs. It enables practical real time visualization in a non-invasive manner, making the modality especially useful to image dynamic cardiac structures. In the last few decades, echocardiography has been applied to in vivo cardiac disease models, mainly to rodents. While clinical echocardiography platforms can be used for relatively large animals such as pigs and rats, specialized systems are needed for smaller species. Theoretically, as the size of the imaged sample decreases, the frequency of the ultrasound transducer needed to image the sample increases. There are multiple modes of echocardiography imaging. In Doppler mode, erythrocytes blood flow velocities are measured from the frequency shift of the sent ultrasound waves compared to received echoes. Recorded data are then used to calculate cardiac function parameters such as cardiac output, as well as the hemodynamic shear stress levels in the heart and blood vessels. The multi-mode (i.e., b-mode, m-mode, Pulsed Doppler, Tissue Doppler, etc.) small animal ultrasound systems in the market can be used for most in vivo cardiac disease models including mice, embryonic chick and zebrafish. These systems are also associated with significant costs. Alternatively, there are more economical single-mode echocardiography platforms. However, these are originally built for mice studies and they need to be tested and evaluated for smaller experimental models. We recently adapted a mice Doppler echocardiography system to measure cardiac flow velocities for adult zebrafish and embryonic chicken. We successfully assessed cardiac function and hemodynamic shear stress for normal as well as for diseased embryonic chicken and zebrafish. In this paper, we will present our detailed protocols for Doppler flow measurements and further cardiac function analysis on these models using the setup. The protocols will involve detailed steps for animal stabilization, probe orientation for specific measurements, data acquisition, and data analysis. We believe this information will help cardiac researchers to establish similar echocardiography platforms in their labs in a practical and economical manner.