Genska karakterizacija, kloniranje i ekspresija Toll-like receptora 1 mRNA nilske tilapije (Oreochromis niloticus)

Toll-like receptors (TLRs) are the most studied group of pathogen recognition receptor categories that detects infectious agents in vertebrates. Fish TLRs exhibit clear, distinct features, structure and a larger diversity than in other vertebrates. This study focused on identifying and detecting the structure of Oreochromis niloticus (Nile tilapia) Toll- like receptor-1 (TLR1|) as a model in freshwater bony fish. The full-length cDNA sequence of Oreochromis niloticus TLR1 mRNA was cloned. Cloning and sequence analysis revealed that the complete cDNA sequence of Oreochromis niloticus TLR1 consists of 2355 base pairs and encodes a polypeptide of 785 amino acids. The molecular analysis of the amino acid sequence indicated that Oreochromis niloticus TLR1 has the standard structural features and major components of amino acids of TLR family members, and is considered an orthologue to the vertebrate TLR1, not a paralogue. The translated amino acid analysis showed 96%, 88%, 85%, and 85% degrees of identity with Zebra Mbuna, Sea bass, Damsel fish, and Clownfish, respectively; and showed 66% identity t with electric eels and 61% with starlets. Phylogenetic analysis revealed that the Nile tilapia TLR1 is closely related to Larimichthys crocea, Epinephelus coioides, and Takifugu rubripes TLR1. Oreochromis niloticus TLR1 was expressed in the kidneys, brain, spleen, intestines, muscle, liver, gills, heart and skin. Quantitative RT-PCR showed differences in the expression levels between the tested tissues. In conclusion, this study is the first report (according to our knowledge) and provides a complete molecular and functional characterization of Oreochromis niloticus toll-like receptor 1, which is considered to be functionally orthologous to TLR1 in other species models.Toll-like receptori (TLR) najviše su istraživana skupina receptora za prepoznavanje uzročnika bolesti u kralježnjaka. TLR u riba pokazuju jasna razlikovna svojstva, strukturu i veliku raznolikost u odnosu na druge kralježnjake. Ovo je istraživanje usredotočeno na identifikaciju i otkrivanje Toll-like receptora 1 (TLR1) u nilske tilapije (Oreochromis niloticus) kao predstavnika slatkovodnih riba. Klonirana je puna sekvencija cDNA TLR1 mRNA. Utvrđeno je da se kompletna sekvencija cDNA TLR1 nilske tilapije sastoji od 2355 baznih parova i kodira polipeptid od 785 aminokiselina. Molekularna analiza sekvencija aminokiselina upućuje na to da TLR1 nilske tilapije ima standardna strukturna svojstva i glavne komponente porodice TLR receptora i smatra se ortologom, ne paralogom TLR1 kralježnjaka. Analiza prevedenih aminokiselina pokazala je stupanj identičnost od 96 % s mbuna zebrom, 88 % s lubinom, 85 % s damsel ribom i 85 % s ribom klaun, dok je stupanj identičnosti s električnom jeguljom bio 66 %, a s ribom starlet 61 %. Filogenetska analiza pokazala je da je TLR1 nilske tilapije usko povezan s TLR1 vrsta Larimichthys crocea, Epinephelus coioides i Takifugu rubripes. TLR1 nilske tilapije bio je izražen u bubrezima, mozgu, slezeni, crijevima, mišiću, jetri, škrgama, srcu i na koži. Kvantitativni RT-PCR pokazao je razlike u razini ekspresije među testiranim tkivima. Prema našim podacima ovo je istraživanje prvo koje donosi kompletnu molekularnu i funkcionalnu karakterizaciju Toll-like receptora 1 nilske tilapije, te se smatra funkcionalnim ortologom TLR1 u drugih vrsta

Virulence, Resistance Profile, Antimicrobial Resistance Genes of ESBLs, XDR Escherichia coli Isolated from Ducks

Ducks are possible carriers of zoonotic diseases to humans. Public health is impacted by the widespread dissemination of Enterobacteriaceae carrying extended-spectrum-lactamase (ESBL) genes. The prevalence of antimicrobial resistance in Escherichia coli isolated from Egyptian ducks, as well as the molecular characteristics of ESBLs to ESBLs genes and non-ESBLs genes, were studied.15% E. coli isolates were recovered from duck, and all of them were virulent as hemolytic and congo red positive. All ESBL-producing E. coli isolates were resistant to tetracycline, and nalidixic acid and 83.3% of isolates were also resistant to trimethoprim/sulfamethoxazole, penicillin, both ceftazidime, and cefotaxime. Recovered ESBL-producing E. coli strains harbored qnrA, tetA, sul1, blaTEM, blaCTX-M, aadA1, blaOXA-1, and blaSHV antimicrobial-resistance genes with a prevalence of 100%, 100%,83.3%, 83.3%, 83.3%, 75%, 58.3%, and 41.6%, respectively. 33.3% of the ESBL-producing E. coli isolates were MDR, and 66.7% were recognized as XDR. The extension of ESBLs- E. coli threatens public health should be carefully monitored.

Disinfectant and Multidrug-resistant Gram-negative Bacteria in Chicks

In recent years there has been a dramatic development for multidrug-resistant and disinfectant-resistant bacteria in poultry farms, to investigate the current prevalence and losses associated with these phenomena, Samples from chicks were taken from 3 poultry stations A, B, and C beside broiler hatcheries (newly hatched chicks). Newly hatched chicks were monitored for Clinical signs, postmortem examination, and performance and mortality rate for one week old. All newly hatched chicks showed the appearance of gasped chicks with low vitality and the rates of gasping increased at the farm level with increased mortality from 8.8 %- 15.5% in the first week. Postmortem examination revealed the presence of nephritis with urate deposition in the ureter, air vasculitis with the appearance of a nodule, a large gall bladder, and a cecum with a greenish color. E. coli was recorded with a high percentage 57.4% followed by S. Typhimurium 8.2% and P.aeruginousa 4.1%. Clostin showed impressive results for treating isolated bacteria from chicks, while erythromycin, spiramycin, lincomycin, oxytetracycline, bacitracin, streptomycin, followed by ampicillin, doxycycline, and gentamycin showed high resistance among isolated bacteria. Alarmingly, 88.9% (8/9) of the E. coli strains were XDR to different classes. In contrast, 11.1% (1/9) of the isolated E. coli strains were multi-drug resistant. One isolate of S. Typhimurium was PDR and another isolate was MDR. All of the P. aeurginosa strains were XDR to different classes. The previous results showed the risk of infection comes from the improper disinfection of the hatchery and how it is associated with losses in poultry farms and there is a need for the prevalence of the disinfection genes in hatchery bacteria

Molecular Characterization of Virulence Genes among MDR and XDR Avian Pathogenic E. coli

One of the most costly diseases is avian colibacillosis. Virulence genes determine E. coli pathogenicity. This study was undertaken to explore the existence of some virulence-associated genes and resistant configurations of Escherichia coli recovered from broiler chicks. Thirteen E. coli isolates were exposed to an investigation of antimicrobial susceptibility profile against 17 antimicrobial agents that exhibited the highest resistance found against amoxicillin, florfenicol, penicillin, amoxicillin clavulanate, tetracycline, meropenem, sulfamethoxazole-trimethoprim, and chloramphenicol in the percentage of 100%, 100%,100%, 92.3%, 76.9%, 69.2%, 61.5%, and 61.5%, respectively while the isolates exhibited highest sensitivity found to fosfomycin, imipenem, azetronam and ciprofloxacine in the percentage of 100%,92.3%,76.9% and 69.2%, respectively. Moreover, the thirteen E. coli isolates were exposed to the revealing of some virulence genes (iss, omp-T, hlyF, iroN, iuta, iucD, papC, cva, astA, tsh, and irp2) by polymerase chain reaction (PCR). The results showed that the percentages rates were 84.6, 76.9, 76.9, 76.9, 61.5, 53.8, 38.4, 30.7, 23, 15.3 and 15.3%, respectively. A significant correlation between most antimicrobial-resistant phenotypes and virulence genes in E. coli isolates. Antimicrobial use in chickens should be reasonable to prevent antibiotic-resistant microorganisms, according to our findings

Molecular Typing of Virulence and Antimicrobial Resistance Genes with Mutation Tracking of gyrA Gene of Fluoroquinolone-Resistant Strains of Campylobacter Isolated from Broiler Chickens

 Campylobacter is the most common bacterial cause of gastroenteritis globally. A total of 182 fully identified strains of Campylobacter species (42 C. coli and 140 C. jejuni) collected from 6 broiler farms were subjected to studying the antimicrobial resistance pattern and molecular typing of virulence (cadF, ctdA, dnaJ waaC,iam, and fla) and antimicrobial resistance genes (blaOXA-61, gyrA, tetA, tetO, and IR) with sequencing of gyrA region of one strain of fluroquinolones resistant C. coli and C. jejuni. The identified isolates were highly resistant to erythromycin and sulfamethoxazole-trimethoprim. Furthermore, both meropenem and imipenem were effective against the recovered isolates. The tested C. jejuni and C. coli strains had 100%, 83.3%, and 83.3% prevalence of cadF, ctdA, and dnaJ virulence genes, respectively, while waaC, iam, and fla genes couldn’t be detected. The blaOXA-61 resistance gene was found in all of the Campylobacter spp. examined. Furthermore, gyrA, tetA, tetO, and IR resistance genes were found in 100%, 83.3%, 83.3%, and 66.7% of the retrieved C. jejuni strains, respectively.  Likewise, resistance genes were found in 83.3%, 83.3%, 83.3%, and 66.7% of the retrieved C. coli strains, respectively. Approximately 58% (7/12) of the Campylobacter spp. recovered were MDR. Furthermore, 50% (3/6) of the C. jejuni strains recovered were MDR, while 66.7% (4/6) of the C. coli isolates recovered were MDR with MARI(0.22-0.55). For detection of mutations of the gyrA gene, the sequence data of two isolates (C. jejuni and C.coli) were analyzed against the reference sequence on the gene bank where the C. jejuni strain had six mutations, while the C. coli strain had twenty-three. The current findings suggest that MDR Campylobacter strains in poultry may be able to transmit highly virulent Campylobacter as a foodborne pathogen

A First Report of Molecular Typing, Virulence Traits, and Phenotypic and Genotypic Resistance Patterns of Newly Emerging XDR and MDR <i>Aeromonas veronii</i> in <i>Mugil seheli</i>

Aeromonas veronii is associated with substantial economic losses in the fish industry and with food-borne illness in humans. This study aimed to determine the prevalence, antibiogram profiles, sequence analysis, virulence and antimicrobial resistance genes, and pathogenicity of A. veronii recovered from Mugil seheli. A total of 80 fish were randomly gathered from various private farms in Suez Province, Egypt. Subsequently, samples were subjected to clinical, post-mortem, and bacteriological examinations. The retrieved isolates were tested for sequence analysis, antibiogram profile, pathogenicity, and PCR detection of virulence and resistance genes. The prevalence of A. veronii in the examined M. seheli was 22.5 % (18/80). The phylogenetic analyses revealed that the tested A. veronii strains shared high genetic similarity with other A. veronii strains from India, UK, and China. Using PCR it was revealed that the retrieved A. veronii isolates harbored the aerA, alt, ser, ompAII, act, ahp, and nuc virulence genes with prevalence of 100%, 82.9%, 61.7%, 55.3%, 44.7%, 36.17%, and 29.8%, respectively. Our findings revealed that 29.8% (14/47) of the retrieved A. veronii strains were XDR to nine antimicrobial classes and carried blaTEM, blaCTX-M, blaSHV,tetA, aadA1, and sul1 resistance genes. Likewise, 19.1% (9/47) of the obtained A. veronii strains were MDR to eight classes and possessed blaTEM, blaCTX-M, blaSHV,tetA, aadA1, and sul1 genes. The pathogenicity testing indicated that the mortality rates positively correlated with the prevalence of virulence-determinant genes. To our knowledge, this is the first report to reveal the occurrence of XDR and MDR A. veronii in M. seheli, an emergence that represents a risk to public health. Emerging XDR and MDR A. veronii in M. seheli frequently harbored aerA, alt, ser, ompAII, and act virulence genes, and blaTEM, sul1, tetA, blaCTX-M, blaSHV, and aadA1 resistance genes

Molecular Typing of Virulence and Antimicrobial Resistance Genes with Mutation Tracking of gyrA Gene of Fluoroquinolone-Resistant Strains of Campylobacter Isolated from Broiler Chickens

 Campylobacter is the most common bacterial cause of gastroenteritis globally. A total of 182 fully identified strains of Campylobacter species (42 C. coli and 140 C. jejuni) collected from 6 broiler farms were subjected to studying the antimicrobial resistance pattern and molecular typing of virulence (cadF, ctdA, dnaJ waaC,iam, and fla) and antimicrobial resistance genes (blaOXA-61, gyrA, tetA, tetO, and IR) with sequencing of gyrA region of one strain of fluroquinolones resistant C. coli and C. jejuni. The identified isolates were highly resistant to erythromycin and sulfamethoxazole-trimethoprim. Furthermore, both meropenem and imipenem were effective against the recovered isolates. The tested C. jejuni and C. coli strains had 100%, 83.3%, and 83.3% prevalence of cadF, ctdA, and dnaJ virulence genes, respectively, while waaC, iam, and fla genes couldn’t be detected. The blaOXA-61 resistance gene was found in all of the Campylobacter spp. examined. Furthermore, gyrA, tetA, tetO, and IR resistance genes were found in 100%, 83.3%, 83.3%, and 66.7% of the retrieved C. jejuni strains, respectively.  Likewise, resistance genes were found in 83.3%, 83.3%, 83.3%, and 66.7% of the retrieved C. coli strains, respectively. Approximately 58% (7/12) of the Campylobacter spp. recovered were MDR. Furthermore, 50% (3/6) of the C. jejuni strains recovered were MDR, while 66.7% (4/6) of the C. coli isolates recovered were MDR with MARI(0.22-0.55). For detection of mutations of the gyrA gene, the sequence data of two isolates (C. jejuni and C.coli) were analyzed against the reference sequence on the gene bank where the C. jejuni strain had six mutations, while the C. coli strain had twenty-three. The current findings suggest that MDR Campylobacter strains in poultry may be able to transmit highly virulent Campylobacter as a foodborne pathogen

Efficacy and Safety of Ceftiofur for Treating Serious Respiratory Diseases in Cattle: Clinical, Histopathological, and Microbiological Assessments

Bovine respiratory disease (BRD) is one of the most serious diseases counted for economic loss and extensive usage of antibiotics in cattle. Ceftiofur, a third-generation cephalosporin antibiotic, has been approved for use in cattle in the United States. This study was done to investigate the clinical effect of ceftiofur on calves as well as its efficacy and safety for treating BRD. Thirty Holstein calves from a dairy farm were divided into three groups. Group I served as a health control group. Group II consisted of healthy animals while Group III comprised calves clinically diagnosed with BRD. Both groups II and III received a single subcutaneous injection of ceftiofur (2mg/kg B.W) in the ear. All groups were clinically evaluated at day 0, 7, and 14 after drug administration for illness score, body weight, body gain, feed intake, body temperature, depression score, discharges, ear and coughing score. Clinical illness score showed clear signs of BRD (elevation of body temperature and depression). Nasal and ocular discharges were recorded and ranked. Significant increases in ear and coughing score were observed in diseased calves

Clostridium perfringens Associated with Foodborne Infections of Animal Origins: Insights into Prevalence, Antimicrobial Resistance, Toxin Genes Profiles, and Toxinotypes

Several food-poisoning outbreaks have been attributed to Clostridium perfringens (C. perfringens) worldwide. Despite that, this crisis was discussed in a few studies, and additional studies are urgently needed in this field. Therefore, we sought to highlight the prevalence, antimicrobial resistance, toxin profiles, and toxinotypes of C. perfringens isolates. In this study, 50 C. perfringens isolates obtained from 450 different animal origin samples (beef, chicken meat, and raw milk) were identified by phenotypic and genotypic methods. The antimicrobial susceptibility results were surprising, as most of the isolates (74%) showed multidrug-resistant (MDR) patterns. The phenotypic resistance to tetracycline, lincomycin, enrofloxacin, cefoxitin/ampicillin, and erythromycin was confirmed by the PCR detections of tet, lnu, qnr, bla, and erm(B) genes, respectively. In contrast to the toxinotypes C and E, toxinotype A prevailed (54%) among our isolates. Additionally, we found that the genes for C. perfringens enterotoxin (cpe) and C. perfringens beta2 toxin (cpb2) were distributed among the tested isolates with high prevalence rates (70 and 64%, respectively). Our findings confirmed that the C. perfringens foodborne crisis has been worsened by the evolution of MDR strains, which became the prominent phenotypes. Furthermore, we were not able to obtain a fixed association between the toxinotypes and antimicrobial resistance patterns

Resistance profiles, virulence and antimicrobial resistance genes of XDR S. Enteritidis and S. Typhimurium

Abstract Avian salmonellosis is concomitant with high financial crises in the poultry industry as well as food-borne illness in man. The present study is designed to investigate the emergence of Salmonella Enteritidis and Salmonella Typhimurium in diseased broilers, resistance profiles, and monitoring virulence and antibiotic resistance genes. Consequently, 450 samples (cloacal swabs, liver, and spleen) were collected from 150 diseased birds from different farms in Giza Governorate, Egypt. Subsequently, the bacteriological examination was done. Afterward, the obtained Salmonella isolates were tested for serogrouping, antibiogram, PCR monitoring of virulence (invA, stn, hilA, and pefA), and antimicrobial resistance genes (bla TEM, bla CTX-M, bla NDM, ermA, sul1, tetA, and aadA1). The total prevalence of Salmonella in the examined diseased broilers was 9.3%, and the highest prevalence was noticed in cloacal swabs. Among the recovered Salmonella isolates (n = 35), 20 serovars were recognized as S. Enteritidis and 15 serovars were identified as S. Typhimurium. Almost 60% of the retrieved S. Enteritidis serovars were extensively drug-resistant (XDR) to seven antimicrobial classes and inherited sul1, bla TEM, tetA, bla CTX-M, ereA, and aadA1 genes. Likewise, 25% of the recovered S. Enteritidis serovars were multidrug-resistant (MDR) to six classes and have sul1, bla TEM, tetA, bla CTX-M, and ereA resistance genes. Also, 66.7% of the retrieved S. Typhimurium serovars were XDR to seven classes and have sul1, bla TEM, tetA, bla CTX-M, ereA, and aadA1 genes. Succinctly, this report underlined the reemergence of XDR S. Typhimurium and S. Enteritidis in broiler chickens. Meropenem and norfloxacin exposed a hopeful antimicrobial activity toward the re-emerging XDR S. Typhimurium and S. Enteritidis in broilers. Moreover, the recurrence of these XDR Salmonella strains poses a potential public health threat