Ocjena brzog imunoenzimnog testa u otkrivanju onečišćenja mlijeka, vode i izmeta bakterijom Salmonella Typhimurium

Serology-based detection methods have more applicable characteristics than standard bacterial culture methods. The aim of this study was to evaluate a cost-efficient, rapid, sensitive and simple test to diagnose milk, water and feces contaminated with Salmonella enteric subsp. enterica serovar Typhimurium (S. Typhimurium). The method developed was based on the addition of HRP-conjugated anti-Salmonella antibodies to the sample, precipitation of the complexes formed using centrifuge, and evaluation of the peroxidase activity of the sample supernatants. Raw cow’s milk, water and cattle feces samples were spiked with seven concentrations of S. Typhimurium. The optimized test recorded an accurate volume and concentration of HRP-conjugated antibodies, the bacteria, samples, ionic compounds and the precise time of the reaction. Also, the method of the test and standard sample preparation were carefully optimized. The tests were repeated at least ten times, and the minimum level of detectable bacteria was determined for each repetition. The results obtained were compared with the conventional bacterial culture method. The results showed that in an optimal condition, conjugated anti-Salmonella antibodies interacted with the bacteria in any matrix. The developed method can detect at least 30, 30, 3×103, and 3×103 cell/mL of S. Typhimurium in water, PBS, milk and feces samples respectively, in less than 20 minutes. Also, antibody detection using this EIA can detect an antibody concentration range of 4.3-70 μg/mL. The developed method is a rapid, cost-efficient and simple assay, and can be used to detect S. Typhimurium in various samples.Serološke dijagnostičke metode znatno su primjenjivije u odnosu na standardne bakteriološke kulture. Cilj ovoga istraživanja bio je ocijeniti ekonomsku isplativost, brzinu, osjetljivost i jednostavnost testa u otkrivanju onečišćenosti mlijeka, vode i izmeta bakterijom Salmonella enteric subsp. enterica serovar Typhimurium (S. Typhimurium). Metoda se temelji na dodatku HRP-konjugiranih anti-Salmonella protutijela u uzorku, precipitaciji nastalih kompleksa centrifugom i procjeni peroksidazne aktivnosti supernatanta. Uzorci sirovog kravljeg mlijeka, vode i izmeta goveda inokulirani su sa sedam koncentracija bakterije S. Typhimurium. Primjenom optimiziranog testa zabilježeni su precizni podaci o volumenu i koncentraciji HRP-konjugiranih protutijela, bakterija, uzoraka, ionskih spojeva i vremenu reakcije. Test i priprema standardnih uzoraka pažljivo su optimizirani. Test je ponovljen najmanje deset puta i u svakom je ponavljanju određena najmanja razina detektibilnih bakterija te su rezultati uspoređeni s konvencionalnim pretragama kultura. Rezultati su pokazali da su u optimalnim uvjetima konjugirana anti-Salmonella protutijela reagirala s bakterijama u svakom tipu uzoraka. Razvijena metoda može u manje od 20 minuta otkriti barem 30 stanica/mL bakterije S. Typhimurium u vodi, 30 stanica/mL u PBS-u, 3×103 stanica/mL u mlijeku i 3×103 stanica/ mL u izmetu. Također, primjena ove metode mogla bi otkriti koncentraciju protutijela od 4,3 do 70 μg/mL. Metoda je brza, jeftina i jednostavna te može otkriti bakteriju S. Typhimurium u različitim uzorcima

Profil gena za virulenciju izolata bakterije Pasteurella multocida izdvojenih iz goveda i bivola

Pasteurella multocida is responsible for numerous economically relevant diseases in domestic animals worldwide. In cattle and buffaloes the organism is associated with hemorrhagic septicemia (HS) and bovine respiratory disease (BRD). The aim of this study was to investigate twelve virulence associated genes in 22 strains of P. multocida isolated from slaughtered cattle and buffaloes. The most frequently detected genes among bovine isolates were ptfA, nanH, exbBD-tonB and oma87; whereas hgbB and toxA genes occurred less frequently. Some of the adhesions, sialidases, iron acquisition and protectin proteins occurred at considerably (P<0.05) higher frequencies in bovine isolates. The prevalence of oma87, exbBD-tonB and hgbA genes from buffaloes was significant (P<0.05), whereas the prevalence of hgbB, ompH, pfhA and toxA genes was much lower. All tested strains of P. multocida contained the sodC gene and only 22.7% of them had sodA. By using the virulence gene profiles, 12 and 21 different gene combinations were identified among the strains isolated from cattle and buffaloes, respectively, of which Profile C1 was the most common, with all strains possessing toxA. Our results indicate the presence of virulence factors (VFs) in P. multocida strains isolated from the tested cattle and buffaloes. The occurrence of these factors in apparently healthy animals could possibly indicate early infection or a contained infection which did not lead to disease. Moreover, differences in the frequency of these factors may indicate variations in the pathogenicity of the organism.Pasteurella multocida odgovorna je za mnoge gospodarski važne bolesti domaćih životinja diljem svijeta. U goveda i bivola ta je bakterija povezana s pojavom hemoragijske septikemije (HS) i respiratorne bolesti. Cilj je ovog rada bio istražiti prisutnost 12 gena odgovornih za virulenciju u 22 izolata bakterije P. multocida izdvojena iz zaklanih goveda i bivola. Najčešće dokazani geni iz goveđih izolata bili su ptfA, nanH, exbBD-tonB i oma87, dok su geni hgbB i toxA bili rjeđe dokazani. Neki od adhezina, sijalidaza, proteina koji na sebe vežu slobodno željezo i zaštitnih proteina dokazani su sa znatno većom učestalošću (P<0,05) u goveđih izolata. Prevalencija gena oma87, exbBD-tonB i hgbA bila je značajno viša (P<0,05) dok je prevalencija gena hgbB, ompH, pfhA i toxA bila niža u bivoljih izolata. Svi pretraženi izolati bakterije P. multocida sadržavali su gen sodC, a samo 22,7% njih i gen sodA. S obzirom na profil gena za virulenciju, 12 različitih kombinacija ustanovljeno je među izolatima iz goveda, a 21 kombinacija među izolatima iz bivola od kojih je profil C1 bio najčešći u izolata koji su posjedovali toxA. Rezultati naznačuju prisutnost čimbenika virulencije u izolata bakterije P. multocida izdvojenih iz pretraženih goveda i bivola. Pojava tih čimbenika virulencije u klinički zdravih životinja mogla bi značiti ranu infekciju ili infekciju koja se neće klinički očitovati. Razlika u učestalosti spomenutih čimbenika također upućuje na različitost u patogenosti izolata

Isolation, Antimicrobial Resistance, and Virulence Genes of Thermophilic Campylobacter Species from Backyard Ducks in Amol, Northern Iran

Domestic poultry are considered natural reservoirs for the transmission of Campylobacter spp., mainly C. jejuni and C. coli, to other birds and humans. This study aimed to determine the Campylobacter infection status in backyard ducks in Iran. A total of 100 cloacal swabs were obtained from apparently healthy backyard ducks in different rural areas of Amol, a city in northern Iran. Bacterial isolation was based on traditional culture procedures, and genus and species identification were performed using an mPCR. All isolates were examined for antimicrobial resistance to seven antibiotics by Kirby Bauer’s disk diffusion test. The virulence-associated genes cadF, iamA, pldA, cdtA, cdtB, cdtC, and wlaN were detected as well. Out of the 27 Campylobacter isolates recovered, 19 (70.4%) were C. coli, and 3 (11.1%) were C. jejuni. The remaining five isolates (18.5%) were not identified. All (100%) isolates showed resistance to ciprofloxacin. Most isolates were resistant to ampicillin, tetracycline, and nalidixic acid. The resistance rate to amoxicillin-clavulanic acid and erythromycin was moderate but was relatively low to gentamicin. Moreover, over two-thirds of the isolates were MDR. All virulence genes, except iamA, were variably detected. The cadF and pldA genes had the highest (92.6%) and lowest (7.4%) positivity rates, respectively. In addition, a statistically significant association was observed between Campylobacter spp. and most of the critical virulence genes (p < 0.05). Our findings imply that backyard ducks should be paid attention to as a major source of human campylobacteriosis

Evaluation the latex agglutination test for detection of the causative agents of bacterial mastitis in milk samples

Mastitis is the main disease in dairy farms that causes serious losses. The early diagnosis and specific treatment can prevent the spread of the disease and the economic losses. The aim of this study was to evaluate the accuracy of the latex agglutination assay for detection of the main bacterial mastitis agents. The antiserum against Staphylococcus aureus, Trueperella pyogenes, Streptococcus agalactiae and Escherichia coli were prepared from immunized rabbits. The couplings of antibodies to latex particles were optimized and after that, the limit of detection (LOD) of latex agglutination test was evaluated for detection of the mentioned bacteria. The detection limit for the Streptococcus agalactiae, E. coli, Staphylococcus aureus and T. pyogenes were respectively 1.3×103, 2×107, 1.58×104 and 5.4×104 colony-forming unit per each milliliter of the bacterial suspensions. The prepared latex test has more sensitivity in the setting of phosphate buffered saline than in contaminated milk samples. This method can be used for the fast detection of the mentioned bacteria in bacterial cultures and milk samples. The latex agglutination test could be evaluated as a fast, cost benefit, and practical method in dairy farms

Isolation, Molecular Detection, and Risk Factors of Campylobacter Infection From Companion Dogs

Background: Campylobacter is an organism that is usually associated with diarrhea in pet animals and humans, as well as other domestic, wild, and laboratory animals. Objective: The aim of the present survey was the isolation, molecular detection, and risk factors of Campylobacter infection from companion dogs referred to the Veterinary Hospital of Ahvaz district, the South-West of Iran. Materials and Methods: Rectal swabs were examined by culture and polymerase chain reaction (PCR) methods from 122 companion dogs (52 diarrheic and 70 clinically healthy). Several risk factors were reviewed, including age, gender, breed, nutrition status, and lifestyle. Results: The results showed that only five samples (4.1%) were positive for Campylobacter spp. in the culture method. Campylobacter spp. was detected in 18 out of 122 dogs by the PCR, yielding an overall prevalence of 14.8%. The most prevalent species of Campylobacter among the referred dogs were C. coli (38.89%) and C. jejuni (33.33%). A lower prevalence was found for C. upsaliensis (11.11%) and C. lari (5.55%). Concurrent infections were observed in two cases of C. upsaliensis + C. lari (5.55%) and C. coli + C. lari (5.55%). No significant difference was noted between healthy (11.43%) and diarrheic (19.23%) dogs (P&gt;0.05). Eventually, age, gender, breed, nutrition status, and lifestyle had no significant effect on Campylobacter infection (P&gt;0.05). Conclusion: Although the prevalence of Campylobacter was moderate in the dog population of Ahvaz district, these bacteria can constitute a public health hazard because of the frequent presence of Campylobacter species in the feces. </jats:p

Evaluation of a rapid enzyme immunoassay test for diagnosis of contamination of milk, water and feces to Salmonella Typhimurium

Serology-based detection methods have more applicable characteristics than standard bacterial culture methods. The aim of this study was to evaluate a cost-efficient, rapid, sensitive and simple test to diagnose milk, water and feces contaminated with Salmonella enteric subsp. enterica serovar Typhimurium (S. Typhimurium). The method developed was based on the addition of HRP-conjugated anti-Salmonella antibodies to the sample, precipitation of the complexes formed using centrifuge, and evaluation of the peroxidase activity of the sample supernatants. Raw cow’s milk, water and cattle feces samples were spiked with seven concentrations of S. Typhimurium. The optimized test recorded an accurate volume and concentration of HRP-conjugated antibodies, the bacteria, samples, ionic compounds and the precise time of the reaction. Also, the method of the test and standard sample preparation were carefully optimized. The tests were repeated at least ten times, and the minimum level of detectable bacteria was determined for each repetition. The results obtained were compared with the conventional bacterial culture method. The results showed that in an optimal condition, conjugated anti-Salmonella antibodies interacted with the bacteria in any matrix. The developed method can detect at least 30, 30, 3×103, and 3×103 cell/mL of S. Typhimurium in water, PBS, milk and feces samples respectively, in less than 20 minutes. Also, antibody detection using this EIA can detect an antibody concentration range of 4.3-70 µg/mL. The developed method is a rapid, cost-efficient and simple assay, and can be used to detect S. Typhimurium in various samples.</jats:p

Isolation, identification, antibiotic resistance profile and molecular analysis of Ornithobacterium rhinotracheal isolates from turkeys

Abstract Background Ornithobacterium rhinotracheal (ORT) infects numerous birds, particularly chickens and turkeys. ORT is an emerging bacterial pathogen of global concern in the poultry industry. As ORT is rapidly spreading throughout commercial poultry, it requires intensive studies of its epidemiology, diagnostic procedures, molecular typing, virulence genes and antimicrobial resistance. Objectives The present study was conducted in isolation and identification of ORT from slaughtered turkeys. Methods Cleft palate swabs of 200 were collected from slaughtered turkeys and cultured on blood agar. ORT was characterized using biochemical tests and PCR targeting the ORT 16S rRNA gene. Virulence genes of isolates were determined targeting adenylate kinase (adk), copA and virulence‐associated protein D (vapD) genes. Additionally, diversity of ORT isolates was performed by enterobacterial repetitive intergenic consensus (ERIC) and RAPD PCR. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. Results ORT was identified in 23 (11.5%) samples using both the biochemical tests and PCR. The result of detecting virulence genes showed that all the isolates (23: 100%) had the adk gene, whereas two (8.7%) isolates had the copA gene, and seven (30.43%) isolates had the vapD gene. Molecular typing of isolates revealed 21 different patterns by RAPD PCR assay using M13 primer and 20 distinct patterns by ERIC PCR test. Both ERIC and RAPD PCR were distinctive methods for investigating the genetic diversity of ORT isolates. The antibiotic resistance test showed that 18 (78.26%) isolates were resistant to gentamicin, amikacin, cefazolin, streptomycin and penicillin. All isolates (100%) were resistant to cloxacillin and fosfomycin. Conclusions This study showed the prevalence of ORT in turkey and high resistance of this bacterium to many common veterinary antibiotics. Moreover, both ERIC and RAPD PCR are distinctive methods for investigating the genetic diversity of ORT isolates. These data may help monitor antibiotic resistance and typing of ORT in epidemiological studies and serve as the foundation for designing region‐specific vaccines for future use

Rapid detection of Ornithobacterium rhinotracheale infection by a latex agglutination test in turkeys

Ornithobacterium rhinotracheale (ORT) is a gram-negative pathogen recognized as one of the causative agents of respiratory diseases in chickens and turkeys. This study aimed to design and evaluate the diagnostic validity of the latex agglutination test for ORT detection. A Western blot test was conducted to assess the various serotypes present in the bacterial isolates, as well as the immunogenicity of their lipopolysaccharide. Following the design and optimization of the ELISA and latex agglutination tests using prepared mixed cell membrane antigens from the different isolates, a serological evaluation of the infection was performed on a total of 244 turkey serum samples, both with and without respiratory symptoms.The protein profiles of the isolated ORT samples revealed equal expression of proteins with molecular weights of 75 kDa and 100 kDa. The collected sera exhibited common antibodies that reacted with antigens weighing 48 kDa, 75 kDa and 100 kDa in Western blot analysis. The sensitivity, specificity and similarity of the latex agglutination test, in comparison to ELISA, were 87.94, 90.29 and 88.93 %, respectively. The developed latex agglutination test resulted in a positive reaction at the presence of a minimum antibody concentration equal to 308.63 μg/mL. The results indicate that the latex agglutination test is effective for the rapid diagnosis and control of Ornithobacterium infection in turkeys