BACKGROUND: Heparin is a sulfated glycosaminoglycan. Blood is a common source for DNA detection in all kinds of samples, and anticoagulants such as heparin and ethylenediaminetetraacetic acid (EDTA) are used to prevent coagulation. Because heparin has a strong inhibitory effect on polymerase chain reaction (PCR), it is not used in samples that will be tracked by DNA. There are physical, chemical, and enzymatic methods to eliminate the inhibitory effect of heparin on PCR test.OBJECTIVES: First, to compare the intensity of the inhibitory effect of two anticoagulants, heparin, and EDTA, on the Real-Time PCR (qPCR), and then to investigate the impact of the heparinase enzyme present in the medium culture extract of Pseudomonas aeruginosa, on removing the inhibitory effect of heparin during the real-time PCR.METHODS: In the present study, two blood samples containing heparin and EDTA were subjected to a real-time PCR test to check the intensity of the inhibitory effect. Then, the medium culture extract of Pseudomonas aeruginosa was added to the heparinized blood sample infected with Escherichia coli bacteria in two groups with different conditions. In the first group, the DNA in the heparinized blood sample was extracted by the phenol-chloroform isoamyl alcohol method. Then, these samples were incubated with the extract of Pseudomonas aeruginosa bacteria culture medium at different hours, but in the second group, the samples were incubated at different hours before DNA extraction. Also, the DNA concentration in both groups was measured by a Nanodrop device, and finally, all samples were subjected to a real-time PCR test.RESULTS: The results of the research samples showed that although the heparinized blood sample contains more DNA concentration than the EDTA blood sample, it completely prevents genome replication. Also, incubating heparinized blood with Pseudomonas aeruginosa culture medium extract before DNA extraction for more than 24 hours removes the inhibitory effect of heparin during the real-time PCR, even at a lower cycle threshold than the EDTA-containing sample.CONCLUSIONS: The Pseudomonas aeruginosa culture medium extract may enable researchers to use heparinized blood samples for genome amplification and diagnosis without using expensive and limited commercial heparinase enzyme.

BACKGROUND: AmpC and ESBLs as mediated-plasmid extended spectrum β-lactabases are the main factors of resistance to extended-spectrum cephalosporins in enterobacteriacea especially E. coli and will follow treatment failure, high costs of treatment in human and economic losses in the poultry industry. OBJECTIVES: The purpose of this study was to screen and study the faecal E. coli isolates producing extended spectrum β-lactamases (ESBLs) and AmpC enzymes and related workers. METHODS: A total of 500 cloacal swab samples from broiler chickens and 25 rectal swab samples from workers were collected from five poultry houses around Tehran. All samples were seeded on MacConkey agar and identification of E. coli isolates were performed via biochemical tests. Antibiotic susceptibility was determined against 12 antibiotics using the disk diffusion method as recommended by the clinical and laboratory standard institute (CLSI2012). Ceftazidim / ceftazidim-clavolanic acid and cefoxitin / cefoxitin-EDTA disks were used for the detection of ESBL and AmpC phenotypes, respectively. phonetic analysis of the drug resistances was performed via SPSS software and Chi-square test. ESBL- producing E. coli screened by PCR for the presence of genes encoding beta-lactamases of TEM, CTX-M and SHV.       RESULTS: A total 467 E. coli isolates were isolated from 88.9% of the samples as 92% and 72.7% of isolates presenting MDR phenotype among chickens and workers respectively. ESBL phenotype detected in 5.5% (26) of poultry isolates while, none of the workers isolates have this phenotype. Six isolates carried both of TEM and CTX-M whereas, five and one isolates were detected only for TEM and CTX-M, respectively. Eighty-eight and nine-tenths percent of ESBL E. coli displayed AmpC phenotype. CONCLUSIONS: Since cephalosporins are not used in broilers in Iran, isolation of faecal E. coli isolates producing extended spectrum β-lactamases in broilerchickens can indicate transfer of the resistance genes via plasmids and other mobile genetic elements among Enterobacteriaceae.

This study investigated the protective effects of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular calcium chelator, and ethylenediaminetetraacetic acid (EDTA), which chelates calcium and most metal ions, against carbon tetrachloride (CCl₄)-induced acute hepatotoxicity in mice. Mice were treated with EGTA or EDTA at a dose of 20 (low) or 100 mg/kg (high) subcutaneously 1 h before CCl₄ administration. The mice were fasted and sacrificed 18 h after CCl₄ treatment.

O presente trabalho objetivou estudar o quadro sintomatológico, algumas variáveis bioquímicas e a resposta ao tratamento com cálcio de bovinos com hipocalcemia induzida experimentalmente. Foram utilizadas 12 novilhas distribuídas nos grupos controle (n = 5) e tratado (n = 7). Foi infundida solução de EDTA a 5% até o animal apresentar sinais clínicos de hipocalcemia, quando então era iniciado o tratamento com solução contendo cálcio, fósforo, magnésio e glicose, na dose de 1 mL/kg/PV, em 30 minutos, enquanto que o grupo controle recebia apenas solução fisiológica na mesma dose. Exame clínico e coleta de amostras sanguíneas foram realizados nos tempos T0 (basal), T1 (Fase I, caracterizada por tremores musculares), T2 (ao final da infusão com EDTA), T3 (ao final do tratamento) e T4 (24 horas após o término do experimento). Todas as novilhas mostraram diminuição temporária da concentração de cálcio total e livre, fósforo, e apresentaram quadro clássico de hipocalcemia. A taquicardia, a hipofonese e a atonia ruminal desapareceram no decorrer do tratamento, sendo observado aumento no cálcio livre e total e fósforo. O medicamento usado no tratamento dos animais foi eficaz na recuperação do quadro clínico de hipocalcemia dentro de 30 minutos, promovendo retorno das principais variáveis do perfil bioquímico aos valores basais.

O presente trabalho objetivou estudar o quadro sintomatológico, algumas variáveis bioquímicas e a resposta ao tratamento com cálcio de bovinos com hipocalcemia induzida experimentalmente. Foram utilizadas 12 novilhas distribuídas nos grupos controle (n = 5) e tratado (n = 7). Foi infundida solução de EDTA a 5% até o animal apresentar sinais clínicos de hipocalcemia, quando então era iniciado o tratamento com solução contendo cálcio, fósforo, magnésio e glicose, na dose de 1 mL/kg/PV, em 30 minutos, enquanto que o grupo controle recebia apenas solução fisiológica na mesma dose. Exame clínico e coleta de amostras sanguíneas foram realizados nos tempos T0 (basal), T1 (Fase I, caracterizada por tremores musculares), T2 (ao final da infusão com EDTA), T3 (ao final do tratamento) e T4 (24 horas após o término do experimento). Todas as novilhas mostraram diminuição temporária da concentração de cálcio total e livre, fósforo, e apresentaram quadro clássico de hipocalcemia. A taquicardia, a hipofonese e a atonia ruminal desapareceram no decorrer do tratamento, sendo observado aumento no cálcio livre e total e fósforo. O medicamento usado no tratamento dos animais foi eficaz na recuperação do quadro clínico de hipocalcemia dentro de 30 minutos, promovendo retorno das principais variáveis do perfil bioquímico aos valores basais. | The present work aims to study the clinical picture, biochemical profile and treatment response in cattle with induced hypocalcaemia. Were utilized 12 heifers randomly distributed in treated (n = 7) and control (n = 5) groups. The induction model was carried on by continuous EDTA infusion into jugular vein until the animals present clinical signs of hypocalcaemia. After that, the treated group received a calcium (Ca) solution enriched with phosphorus, magnesium and glucose with a dose of 1 mL/kg/BW in 30 minutes, meanwhile, the control group was treated with the same dose of physiologic solution. Clinical examination were performed and blood samples were obtained in times T0 (basal time), T1 (beginning of hypocalcaemia); T2 (end of EDTA infusion); T3 (end of treatment) and T4 (24 hours after the induction). All the heifers present temporary blood calcium and phosphorus reduction and demonstrated classical clinical picture of hypocalcaemia. The treated group present full clinical recovery and blood calcium and phosphorus increase. Most evident clinical signs were increasing heart beat, hypophonesis and rumenal atony. Those symptoms were reversed after calcium treatment. The solution used for treatment was efficient on clinical recovery within thirty minutes, promoting the return to basal levels of the most of biochemical's variables.