305 Veterinaria Italiana 2021, 57 (4), 305-310. doi: 10.12834/VetIt.2111.12262.1 Accepted: 28.04.2020 | Available on line: 31.12.2021 Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran. *Corresponding author at: Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran. E-mail: h.dastmalchi@urmia.ac.ir. Somayyeh Ahmadi, Habib Dastmalchi Saei*, Seyed Meisam Abtahi Froush and Mohammad Zavarshani Keywords Avian, Colibacillosis, Escherichia coli, Heterophil, PCR, Phylogenetic groups, Immunological activities, Iran. Summary Avian pathogenic Escherichia coli (APEC) is a major cause of colibacillosis and is associated with economic losses to the poultry production worldwide. Heterophils are the first line of immune defense of the avian host against invasive pathogens. In this study, APEC isolates from chickens with colibacillosis were assigned to phylogenetic groups and immunological activities of heterophils against these groups were examined. A total of 92 APEC isolates was obtained from 106 samples of diverse organs collected from chickens with colibacillosis from different farms in West Azerbaijan province, Iran. Isolates were assigned to phylogenetic groups based on the Clermont triplex PCR method, and immunological activities (including phagocytosis, respiratory burst and bacterial killing) of heterophils against these groups were examined. As results, the frequency of A, B1, B2 and D groups were 35.87, 44.57, 5.43 and 14.13%, respectively. In addition, opsonized Escherichia coli isolates belonging to B1 group significantly enhanced the level of respiratory burst (0.52 ± 0.02%) while the killing level of them was significantly lower than the other groups (29.40 ± 5.09%). There was no significant difference in phagocytic activity of heterophils against the phylogenetic groups. In conclusion, incomplete immune responses to B1 phylogenetic group maybe a principal cause of mortality by colibacillosis caused by this group. It is suggested to study heterophilic immune reaction against E. coli phylogenetic group for development of effective prevention strategy. Effects of different phylotypes of avian pathogenic Escherichia coli isolated from broiler chickens with colibacillosis on heterophils functional activities: an in vitro study are extra-intestinal pathogenic strains (Clermont et al. 2000). In poultry, avian pathogenic E. coli (APEC) is the main cause of, colibacillosis characterized by fever, depression and respiratory inflammation due to primary penetration of heterophils and monoconjugate phagocytes (Ramadan et  al. 2016). It has been reported that young animals are more susceptible than adults to this illness. This transient sensitivity of chickens and turkeys to infection during the first weeks of life is associated with impaired quality of innate and adaptive immune system (Hussain et  al. 2017a). Heterophils, the counterparts to the mammalian neutrophils, are the first line of defense against any microbial attack and play an indispensable role in the immune response of the avian host (Genovese et al. 2013). These cells fight against infectious diseases by phagocytosis and respiratory burst (Kogut et  al. 2012). The aims Introduction Escherichia coli is a facultative Gram negative bacterium belonging to the normal flora of the mammals and birds intestine (Katouli 2010). This organism is one of the most frequent causes of many common primary and secondary bacterial infections, but it is generally considered to be an opportunistic pathogen that can cause infections following the suppression of the host immune system (Hussain et al. 2017b). E. coli causes different clinical manifestations in birds among which the colibacillosis is the most common form causing major economic impact (Lutful Kabir 2010). Based on the Clermont triplex PCR method, E.  coli strains can be classified into four phylogenetic groups (A, B1, B2 and D), of which groups A and B1 are normal flora and group B2 and, to a lesser extent, group D 306 In vitro study of effects of APEC on heterophils functional activities Ahmadi et al. Veterinaria Italiana 2021, 57 (4), 305-310. doi: 10.12834/VetIt.2111.12262.1 Avian heterophils preparation The isolation of heterophild, their count and their viability were carried out and assessed as previously described (Andreasen and Latimer 1989, Blattes et al. 2017). In brief, the blood samples from healthy chicken were transferred to the EDTA tubes and mixed with 1% methylcellulose, in calcium and magnesium-free Hank's balanced salt, at a 1:5 ratios. The samples were centrifuged for 20 min at 25 g. The buffy coat was isolated, washed and re-suspended. Following the procedure, the samples were centrifuged on a Ficoll-Hypaque density gradient (400 g, 30 min). The mononuclear cell layer and plasma were removed, and contaminant erythrocytes were deleted by hypotonic lysis. The remaining heterophils were washed twice and counted in a Neubauer chamber. The viability of the cells was checked by Trypan blue dye exclusion. Evaluation of respiratory burst of heterophils by Nitroblue tetrazolium reduction (NBT) The suspension of opsonized bacteria and intracellular generation of reactive oxygen species (ROS) were measured by NBT reduction as described previously (Chen and Junger 2012, Motlagh et  al. 2015). Non-opsonized bacteria were used as control. Phagocytosis and microbial killing activities of heterophils Two tubes were used as test and control. Equal volume (0.5 ml) of heterophil suspension (5  ×  106  cell/ml) and chicken serum were mixed in the test tube in equal volume. In the control tube, 0.5 ml of HBSS buffer was mixed with chicken serum instead of heterophil suspension. Both tubes were incubated for 5 minutes at 37 °C. Then, 0.5 ml of bacterial suspension (5 × 107 cfu/ml in HBSS) added to each tube and then at this moment, zero time, 100 μl of the test and control tubes were removed and added to the new tubes. The test and control tubes were incubated at 37 °C for 90 minutes. In this stage we had two kinds of data: zero and 90 data. of the present study were first the phylogenetic typing of E.  coli isolated from poultry and then the evaluation phagocytosis, respiratory burst and bactericidal activity of heterophils against different phylogenetic groups. Materials and methods Sampling One-hundred and six samples of different organs (liver, heart, lung and spleen) were collected between 2015 and 2016 from broiler chickens with colibacillosis on multiple flocks located in different regions (Urmia, Naqadeh, and Khoy) of West Azerbaijan province, Iran. All the samples were cultured primarily in MacConkey agar at 37 °C for 24 h (MCA/105465, Merck, Darmstadt, Germany). Pink-colored colonies which were lactose positive, suspected as E.  coli on MCA were further streaked on Eosin-Methylene Blue agar (EMB/101347, EMB, Merck, Darmstadt, Germany) for purification. Isolates were then subjected to standard biochemical tests including IMViC (Indole, Methyl red, Voges-Proskauer and Citrate), urease production, H 2 S production and various sugar fermentation tests (Li et al. 2010). DNA extraction and confirmation of E. coli isolates After identification of the E.  coli isolates by conventional methods, a single colony of each isolate was cultured onto blood agar medium and incubated at 37 °C for 18-24 hours. Bacterial DNA was extracted by boiling method as described previously (Obeng et  al. 2012). Purity of extracted DNA was determined based on measurement of OD 260/280 using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Additionally, the integrity of extracted DNA was evaluated by electrophoresis in 1% agarose gel. DNA with an OD260/OD280 ratio of ≥ 1.8 was considered to be pure and used in PCR. PCR amplification of a 662-bp DNA fragment size of 23S rRNA gene was also conducted to determine any inhibitors remained from the extraction procedure by using Eco 2083 (5'-GCT TGA CAC TGA ACA TTG AG-3') and Eco 2745 (5'- GCA CTT ATC TCT TCC GCA TT-3') primers (Riffon et al. 2001). Detection of phylogenetic groups Phylogenetic group assignment of the isolates was performed using the Clermont triplex PCR method (Clermont et al. 2000). Primer sequences for phylogenetic classification are shown in Table I. Table I. Primer sequences used and size of PCR products. Primer Primer sequence (5'3') Size (bp) chuA F: GACGAACCAACGGTCAGGAT 279 R: TGCCGCCAGTACCAAAGACA yjaA F: TGAAGTGTCAGGAGACGCT 211 R: ATGGAGAATGCGTTCCTCAAC TSPE4.C2 F: GAGTAATGTCGGGGCATTCA 152 R: CGCGCCAACAAAGTATTACG 307 Ahmadi et al. In vitro study of effects of APEC on heterophils functional activities Veterinaria Italiana 2021, 57 (4), 305-310. doi: 10.12834/VetIt.2111.12262.1 groups were observed in 35.87, 44.57, 5.43 and 14.13% of the strains, respectively. Evaluation of respiratory burst, phagocytic and bacterial killing activities of heterophils The viability of heterophils was 94%. Results of Kruskal-Wallis test showed that there were statistically significant differences between different phylogenetic groups in the assessment of respiratory burst and microbial killing of heterophils (P value < 0.05), whereas no significant change in the phagocytic activity of heterophils was observed between the groups (Table II). According to our results, the mean ± standard deviation differences of the optical density in the evaluation of microbial killing activities of heterophils against B1 phylogenetic group were less than other groups (P value < 0.05). There were no significant differences between other groups. However, obtained data showed that the heterophils had the highest potential to kill the group B2 bacteria compared to bacteria in groups A and D. In contrast to microbial killing activity, the results showed that the amount of oxygen free radicals during respiratory burst was significantly higher than other groups against E. coli belonging to group B1 (P value < 0.05). In comparison between bacteria belonging to groups A, B1 and D, the group B2 showed the lowest potential to induce respiratory burst in the heterophils. Despite the differences between the groups, they weren’t significant in evaluating the phagocytic activities of heterophils against the different phylogenetic groups of E. coli. Albeit, the heterophils showed more potential to uptake the group D bacteria compared to other groups (Table III). Discussion Avian colibacillosis caused by avian pathogenic E. coli (APEC) is one of the important disease of poultry in terms of economic losses throughout the world (Guabiraba and Schouler 2015). There are evidences suggesting a meaningful relationship between pathogenicity of APEC and their phylogenetic Zero and 90 data were related to zero time (control tube: C0, testing tube: T0) and 90 min (control tube: C90, testing tube: T90) after incubation, respectively. Of note, the viability of heterophili along the experiment was checked during experimental set up. Fortunately, no significant change was found. Ten ml of sterile distilled water was added into each tube in order to lyse the heterophils. The mixture was stirred a few times slowly until the tube contents were thoroughly mixed. One hundred µl of current mixture were removed and transferred to 96 wells plate. For each dilution, five wells were considered and then 20 μl of MTT solution (5 mg/mL, Sigma - Aldrich, USA) were added to each well. Then it was incubated for 20 min at 37 °C. After incubation, 150  μl of DMSO solution were added to each well to dissolve Formasone crystals. The results of optical densities (OD) were read at 492 nm wave lenght (BioTek ELX800, USA). The bactericidal activity (percentage of killing) was calculated by the formula at the end of process (Mehrzad et al. 2009). The test and the control tubes which had been incubated for 90 min were centrifuged at 300 g for 10 min. One hundred μl of supernatant (containing no phagocytize bacteria) were added to the five wells of 96-wells plate. The plate was incubated for 20 min at 37 °C after adding 20 μl of MTT to each well. One hundred fifty μl of DMSO were added to each well in the next step and then the data were read at 492 nm wave lenght. Percentage of phagocytosis was calculated using the formula (Gargan et  al. 1993, Fijalkowski et al. 2012, Mehrzad et al. 2009). Data statistical analysis Data analysis was done by SPSS statistical software, version 18. The results of groups were analyzed statistically by Kruskal-Wallis test. Also, Mann-Whitney U-test was used to compare differences between two groups. P value less than 0.05 was considered significant. Results E. coli detection A total of 92 E.  coli isolates were detected from broiler chicken with colibacillosis by biochemical tests as well as amplification of the 23S rRNA gene fragment. Phylogenetic classification of E. coli isolates All E.  coli isolates were classified to one of the four main phylogenetic groups, with A, B1, B2 and D Table II. The details of Kruskal-Wallis test of optical density of respiratory burst, microbial killing and phagocytic activities of heterophils. Respiratory burst Microbial Killing Phagocytosis Chi-Square 22.55 19.64 7.56 df 3 3 3 Asymp. Sig.* 0.00 0.00 0.06 *P value < 0.05 308 In vitro study of effects of APEC on heterophils functional activities Ahmadi et al. Veterinaria Italiana 2021, 57 (4), 305-310. doi: 10.12834/VetIt.2111.12262.1 against E.  coli. In that study, E.  coli isolates were categorized based on some special factors such as O, fimbriae and K antigens. Finally, they reported that the presence of fimbriae and O78 antigens were effective in protecting E. coli against heterophils and macrophages phagocytosis (Mellata et al. 2003). As results, E. coli isolates belonging to group B1 were the most stimulating group in secretion of oxygen radicals by heterophils. This could be attributed to differences in bacterial cell surface antigens among studied phylogenetic groups. Study carried out by Ondrackova and colleagues showed that O149 strain of Enterotoxigenic E. coli (ETEC) was more efficiently associated with the neutrophils and induced a more intensive respiratory burst compared to the O147 strain of ETEC. They suggested that this difference might be due to the presence of different types of fimbriae and other factors such as flagella. Moreover, induction of higher respiratory burst by B1 phylogenetic group might be a reason for considerable economic losses due to this group which is the most common group in the studied region. It has been shown that the accumulation of inflammatory exudates in the airbag of chicken lung following the excessive release of oxygen radicals by heterophils due to much more respiratory burst is one of the main causes of chick death and as a consequence the high mortality rate of colibacillosis (Kemmett et  al. 2014). The strong induction of respiratory burst is probably due to the strong presence of pattern recognition receptors such as Toll-like receptors (TLRs) and mannose recognition receptors in B1 group (Genovese et  al. 2013). Interestingly, despite the strong induction of phagocytosis and respiratory burst in group B1, the bacterial death was defective by heterophils. This may be due to the presence of strong anti-free radical agents in group B1. For example yjaA gene encoding protein has been reported to have an important role against oxygen radicals as an anti-oxidant (Bonacorsi et  al. 2000). In previous studies, it has been proven that APEC can cut down the antimicrobial activity of heterophils (Mellata et al. 2003, Qureshi 2003). There was no significant difference in phagocytic activity of heterophils, indicating their high phagocytic ability against different phylogenetic groups of APEC likely due to the broad array of phagocytic receptors. Avian heterophils have been shown to express various combinations of pattern recognition receptors (PRR), including Toll-like receptors (TLRs), scavenger receptors, dectin-1, and mannose receptors that allow them to recognize and respond to foreign microbial invaders (Genovese et  al. 2013). Other speculation could be the lack of difference in strategies used by tested APEC isolates to avoid phagocytic engulfment. In this regard the role for K1 capsule, O78 antigen, P fimbriae, and the groups (Jaureguy et  al. 2008, Moulin-Schouleur et  al. 2007). In the current study, phylogenetic analysis revealed that APEC isolates segregated mainly in phylogenetic groups B1 (41/92; 44.57%) and A (33/92; 35.87%), indicating that intestinal strains are an important cause of colibacillosis in the West Azerbaijan, Iran. An high prevalence of phylogenetic groups A and B1 strains has also been reported elsewhere in Iran from colibacillosis cases (Alizade et  al. 2017, Ghanbarpour et  al. 2011) as well as among verotoxigenic E.  coli (VTEC) isolates from the faeces of healthy sheep and broiler chicken in Northwestern Iran (Saei and Zavarshani 2018). Resistance to ciprofloxacin, norfloxacin, co-trimoxazole, nalidixic acid and cefoperazone/ sulbactam is especially prevalent among group B1 isolates and group A. Antibiotic selection pressure may explain why most of the drug resistant strains of E.  coli belong to the phylogenetic groups A and B1 (Obeng et  al. 2012). Similar results have also been reported by Kazemnia and colleagues (Kazemnia et  al. 2014). Other speculation could be the derivation of pathogenic E.  coli strains from commensal strains by the acquisition of virulence factors (Duriez et al. 2001). In a study carried out on distribution of virulence genes among E. coli isolates from bovine mastitis in Iran, isolates belonging to the phylogenetic group B1 exhibited higher prevalence of various virulence genes in comparison to groups A and D (Ghanbarpour and Oswald 2010). Following the identification of pathogenic agents by heterophils, immune responses are initiated and eventually the pathogen is eliminated. In this regard, it has been shown that heterophils play a significant role in early host defense against bacterial agents such as E. coli in poultry (Ariaans et al. 2008, Gomis et  al. 2003). In the present study, we found a significant difference in functional activities of heterophils against different phylogenetic groups of APEC from broiler chicken with colibacillosis. This could be attributed to differences in genetic backgrounds, virulence factors as well as antigenicity among phylogroups of APEC. In a study carried out by Mellata and colleagues, phagocytic activities of chicken heterophils were evaluated Table III. Comparison of optical density of respiratory burst, microbial killing and phagocytic activities of heterophils (Mean ± SD) between two groups. Group Respiratory burst (Optical Density) Microbial killing (%) Phagocytosis (%) A 0.41 ± 0.03 41.10 ± 6.11 58.37 ± 13.80 B1 0.52 ± 0.02a 29.40 ± 5.09a 78.06 ± 25.08 B2 0.40 ± 0.05 43.05 ± 5.84 66.36 ± 19.21 D 0.43 ± 0.03 39.26 ± 4.96 85.54 ± 27.13 aSignificant differences (p < 0.05). 309 Ahmadi et al. In vitro study of effects of APEC on heterophils functional activities Veterinaria Italiana 2021, 57 (4), 305-310. doi: 10.12834/VetIt.2111.12262.1 influenced by bacterial genetic, but also affected by the host genetic. Therefore, the identification of the dominant phylogenetic groups of E. coli in a region is crucial in order to develop several strategies such as vaccination against colibacillosis. It is hoped that our results help us and other researchers to choose appropriate ways to prevent and treat colibacillosis by identifying the sensitivity of heterophils to different groups of E. coli. Grant support This work was supported by the research fund of Urmia University, Urmia, Iran. 0-min region in initial avoidance of phagocytosis has been demonstrated (Mellata et al. 2003). Further studies on distribution of various virulence factors among different phylogenetic groups of APEC and their interaction with heterophils will provide important insight into some of the mechanisms that they employ to avoid uptake and survive elimination by avian heterophils and cause clinical disease. This study, for the first time, indicated that there were significant differences in microbial killing and respiratory burst activities of heterophils against different phylogenetic groups of E. coli without any significant difference in phagocytosis. However, it should be noted that immune responses are not only Alizade H., Ghanbarpour R., Jajarami M. & Askari A. 2017. 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