Cleaning and disinfection programs against Campylobacter jejuni for broiler chickens: productive performance, microbiological assessment and characterization1 Maria Fernanda de Castro Burbarelli,∗,2 Gustavo do Valle Polycarpo,§ Karoline Deliberali Lelis,∗ Carlos Alexandre Granghelli,∗ Agatha Cristina Carão de Pinho,∗ Sabrina Ribeiro Almeida Queiroz,† Andrezza Maria Fernandes,† Ricardo Luiz Moro de Souza,† Maria Estela Gaglianone Moro,† Roberto de Andrade Bordin,‡ and Ricardo de Albuquerque∗ ∗Department of Animal Nutrition and Production (VNP), Faculty of Veterinary Medicine and Animal Science, University of São Paulo (FMVZ-USP), Pirassununga, Brazil; †Department of Veterinary Medicine, Faculty of Veterinary Medicine and Animal Science, University of São Paulo (FZEA-USP), Pirassununga, Brazil; ‡Nutrition, Animal Production, Health - FATEC-SP; and §São Paulo State University (Unesp), School of Technology and Agricultural Sciences, Campus of Dracena ABSTRACT Detailed cleaning and disinfection pro- grams aims to reduce infection pressure from microor- ganisms from one flock to the next. However, stud- ies evaluating the benefits to poultry performance, the sanitary status of the facilities, and the sanitary qual- ity of the meat are rarely found. Thus, this study was designed to evaluate 2 cleaning and disinfecting pro- grams regarding their influence on productive perfor- mance, elimination of Campylobacter, and characteri- zation of Campylobacter jejuni strains when applied to broiler chickens’ facilities. Two subsequent flocks with 960 birds each were distributed into 32 pens contain- ing 30 birds each. In the first, the whole flock was inoculated with a known strain of Campylobacter je- juni in order to contaminate the environment. In the second flock, performance and microbiological evalua- tions were done, characterizing an observational study between 2 cleaning and disinfection programs, regu- lar and proposed. The regular program consisted of sweeping facilities, washing equipment and environment with water and neutral detergent. The proposed clean- ing program consisted of dry and wet cleaning, appli- cation of 2 detergents (one acid and one basic) and 2 disinfectants (250 g/L glutaraldehyde and 185 g/L formaldehyde at 0.5% and 210 g/L para-chloro-meta- cresol at 4%). Total microorganism count in the en- vironment and Campylobacter spp. identification were done for the microbiological assessment of the environ- ment and carcasses. The positive samples were submit- ted to molecular identification of Campylobacter spp. and posterior genetic sequencing of the species iden- tified as Campylobacter jejuni. The birds housed in the facilities and submitted to the proposed treatment had better performance when compared to the ones in the regular treatment, most likely because there was a smaller total microorganism count on the floor, walls, feeders and drinkers. The proposed program also re- sulted in a reduction of Campylobacter spp. on floors, drinkers and birds. Moreover, it was possible to iden- tify 6 different Campylobacter jejuni strains in the facilities. The proposed treatment resulted in a pos- itive influence on the birds’ performance and reduc- tion of environment contamination for broiler chick- ens. Key words: biosecurity, campylobacteriosis, disinfectant, health, poultry 2017 Poultry Science 96:3188–3198 http://dx.doi.org/10.3382/ps/pex153 C© The Author 2017. Published by Oxford University Press on be- half of Poultry Science Association. This is an Open Access article distributed under the terms of the Creative Commons Attribution Li- cense (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, pro- vided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. Received December 12, 2016. Accepted May 10, 2017. 1The nucleotide sequence data reported in this paper have been sub- mitted to GenBank (National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, INTRODUCTION Preventive practices that include cleaning and disin- fection are fundamental steps for biosecurity programs and are indispensable for the maintenance of high productivity of poultry flocks. The aim of cleaning is the maximum removal of or- ganic matter from facilities and equipment. Therefore, 20894 USA) nucleotide sequence database and have been assigned the accession numbers. 2Corresponding author: mfcb@usp.br 3188 D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/9/3188/3952746 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 19 August 2019 http://creativecommons.org/licenses/by/4.0/ mailto:journals.permissions@oup.com mailto:mfcb@usp.br CLEANING AND DISINFECTION PROGRAMS FOR POULTRY 3189 detergents that reduce superficial tension, act in the emulsification of lipids, have dissolving powers on min- eral residues and peptizing action on protein residues are utilized. Detergents can be alkaline, acid, or neu- tral (UGA, 2005). Alkaline detergents have high dis- solving power on organic residues; acid detergents have high dissolving power on mineral residues and some or- ganic ones; and the neutral detergents are indicated for delicate surfaces and with weakly adhered residues. The maximum efficacy of disinfection procedures is only possible on surfaces with appropriate removal of organic matter (Ward et al., 2006). Another fundamental step is disinfection, which aims to reduce infection pressure from microorganism de- struction as well as the transmission of pathogens from one flock to the next (UGA, 2005; Tokach et al., 2012). There are a great variety of active ingredients utilized as disinfectants in poultry production such as formalde- hyde and glutaraldehyde, which are bactericides, spori- cides, and fungicides. Their activity is due to the alkyla- tion of sulfidryl, hydroxyl, carboxyl, and amino groups of microorganisms, altering their DNA, RNA, and pro- tein synthesis. There is also peracetic acid acting as a bactericide by attacking the lipid membrane, DNA, and other cell components through toxic free radicals that a disinfectant produces (Dvorak, 2008). However, cresols have bactericide and viricidal action on the protoplasm of bacterial cells, causing denaturation and protein pre- cipitation (Spinosa et al., 2006). Production environments with high contamination levels have a direct influence on increased mortality, and/or indirect influence on uniformity and decreased broiler performance (Ristow, 2008; Renaudeau, 2009). Thus, cleaning and disinfection can have a positive influences on the increase in birds’ productive per- formance, mainly in environments where there is a sanitary challenge (Burbarelli et al., 2015). Besides guaranteeing high productivity, these practices are fun- damental to ensure the quality of poultry products, making them appropriate for human consumption. The occurrence of diseases in humans, transmitted by poultry products, can be related to the birds’ con- tamination during their life in production facilities, and Campylobacter is one of the causative agents of disease (Shane et al., 1986; Stern, 1992; Smith et al., 2008). The contamination of chickens, and consequently of car- casses, is a reason to pay attention to the poultry pro- ductive chain (Evans and Sayers, 2000). In the field, one of the goals is the decrease of pathogen coloniza- tion in the birds’ intestinal tract since the horizontal transmission of the pathogen is more efficient (Newell and Fearnley, 2003). However, studies simultaneously evaluating efficiency of cleaning and disinfection protocols, benefits on poul- try performance, sanitary status of the facilities and sanitary quality of the meat are rarely found. Thus, the objective of this study was to evaluate 2 cleaning and disinfection programs regarding their effectiveness on broilers’ productive performance and on the elimination and characterization of Campylobacter jejuni strains in environments that had been previously contaminated with Campylobacter jejuni. MATERIAL AND METHODS Birds, Installations, and Experimental Scheme The experimental protocol was approved by the ethics committee for animal experimentation of the Fac- ulty of Veterinary Medicine and Animal Science, Uni- versity of São Paulo, protocol 3025/2013. A total of 1,920 day-old male Cobb 500 broiler chicks were divided into 2 subsequent flocks with 960 birds each. In both flocks, the birds were distributed into 32 pens containing 30 birds each. This work was an observational study study between 2 cleaning and disin- fection programs: a Regular treatment and a Proposed one. The floor was covered with new rice hulls litter and provided with a tubular feeder and bell drinkers. The housing density was 10 birds per m2, with average initial weight of 45.5 g ± 0.763 g. The interval between the 2 flocks was 8 d. The poultry house had average area of 500 m2 (con- sidering structure, ceiling, curtains, internal and exter- nal parts, paving, flooring, and walls) which was utilized for the analysis and calculation of the cleaning and dis- infection program. The poultry house had an internal room that divided it into 2 halves, guaranteeing the isolation of each experimental group. The diets were formulated with corn and soybean bran according to Rostagno et al. (2011) and provided ad libitum. There was no addition of growth-promoting antibiotics in pre-mixtures. The chicks received chlori- nated drinking water at a concentration of 2.0 ppm. The first-raised broiler group house experiment was designed to create a sanitary challenge by inoculat- ing the birds with Campylobacter jejuni. Performance and microbiological evaluations were done in the second flock. Sanitary Challenge In the first housing, on d 11, the chicks were in- oculated with standard Campylobacter jejuni (ATCC 33560) strains through an oral probe to deposit 1 mL of inoculum consisting of liquid BHI culture medium and 105 UFC/mL of Campylobacter jejuni, considered a high dose by Chaveerach et al. (2004). Cleaning and Disinfection Programs Litter and all the equipment were removed from the poultry house to carry out the treatments. In both pro- grams, approximately 0.4 L of diluted solution per m2 was used for detergents as well as for disinfectants. The D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/9/3188/3952746 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 19 August 2019 3190 BURBARELLI ET AL. water utilized in the treatments had chlorination at 2.0 ppm. The Regular treatment was applied in 16 pens and consisted of sweeping facilities, washing equipment (feeder, drinkers, buckets, boots) with neutral deter- gent, wetting and washing the environment (floors, walls, ceiling, curtains) with water and neutral deter- gent, and subsequently drying the environment and fa- cilities. The Proposed treatment was also applied in 16 pens and started with cleaning and disinfecting the wa- ter supply system: washing the water reservoir and next applying 100 g/kg peracetic acid and 80 g/kg Benzyl- (C12-C16) chloro-alkyl dimethyl ammonium at 0.5%. The product was added to the water reservoir that was posteriorly drained by the water supply system, pro- viding contact with the whole supply system, and kept like that for 12 h, and then drained through the trig- gers of the bell drinkers. Dry cleaning was done by re- moving bedding and sweeping the facilities followed by wet cleaning of the house and posterior with pressur- ized water. All equipment utilized in the poultry house (feeders, drinkers, buckets, boots, trays and other uten- sils) were washed under pressurized water. Next, al- kaline and acid detergents in solution at 4% were ap- plied on all internal and external surfaces (ceiling, walls, flooring, curtains), objects and equipment, followed by rinsing under pressurized water. Disinfectants were only applied after the environment and the equipment were partially dry, without water accumulation. The first uti- lized disinfectant consisted of 250 g/L glutaraldehyde and 185 g/L formaldehyde at 0.5% and was applied on all surfaces and equipment. The second one, composed of 210 g/L para-chloro-meta-cresol at 4%, was applied only on the floor and walls up to 0.5 m of height. A knapsack sprayer was utilized for both applications. After finishing the treatments, new bedding was dis- tributed in the pens, the equipment was relocated, and the second group of day-old chicks was distributed in the rearing pens. Productive Performance The birds were weighed on d 1, 7, 21, 35, and 42 of the experiment for performance analysis. The mea- sured response variables in each pen were: body weight gain (BWG), feed intake (FI), feed: gain ratio (F:G), viability (VB), and productive efficiency index (PEI). Microbiological Evaluation Surface swabs of the floor, wall, drinkers, and feeders were done for total count and evaluation of Campy- lobacter spp. presence from surfaces of 2 cm × 5 cm totaling a 10 cm2 area. Samplings of 200 mL of the birds’ drinking water from the bell drinker tap of 5 pens in each treatment were collected. Besides the men- tioned points, swabs of 5 birds’ cloaca per program were swabbed for the evaluation of Campylobacter spp. pres- ence. The swabs were stored in tubes containing pep- tone water at 0.1% to maintain colony viability. The total microorganism count was done 24 h be- fore the cleaning and disinfection procedures and 48 h after them. Plate count agar (PCA) was utilized in previously solidified plates with sowing on the surface as described by Evancho et al. (2001). The evaluation regarding the presence of Campy- lobacter spp. was done in the first house before and af- ter inoculation. In the second flock, the evaluation was done when the birds were 2, 11, and 42 d old. More- over, harvesting was done right before slaughter, after feathering and after chiller. In each point, 10 carcasses of each experimental group were used. Direct isolation of Campylobacter spp. was done with inoculation of 0.1 mL of peptone water solution at 0.1% in petri dishes containing mCCDA (CM739, Ox- oid, Hampshire, England) culture medium with selec- tive supplement (SR155, Oxoid Hampshire, England) where inoculation of 0.1 mL of solution obtained from samples was done. Then they were incubated in an en- vironment whose microaerophilic atmosphere was mod- ified with 5% of O2, 10% CO2 and 85% N2, at 42◦C for 48 h in jars for special atmospheres (Probac do Brasil, São Paulo, Brazil). From the positive samples, 1 to 3 possible Campylobacter spp. colonies were randomly se- lected and submitted to Gram staining to differenti- ate S bacilli from spiral ones. Oxidase and catalase as- says were also carried out. The colonies with compatible characteristics to Campylobacter spp. morphology were collected for posterior PCR assay. Molecular Identification PCR testing was performed to distinguish Campy- lobacter species in positive samples from the microbio- logical culture. Five typical colonies from the positive samples of Campylobacter spp. were harvested from the same sampling point. The colonies were submitted to bacterial DNA extraction through an adapted thermal shock technique by Fang and Hedin (2003). Multiplex PCR technique was utilized, based on Klena et al. (2004). Primer pairs of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis were utilized for the am- plification of DNA fragments found in each of the cited species. The specific primers presented IpxA as the target gene. For Campylobacter coli, lpxAColi and lpxARKK2m primers were used; for Campylobacter je- juni, lpxAJej and lpxARKK2m; for Campylobacter lari, lpxALari and lpxARKK2m; and for Campylobacter up- saliensis, lpxAUps and lpxARKK2m. The amplifications were carried out in 25 μL of solu- tion containing 1.25 μL of reaction buffer 5× Colorless GoTaq Flexi Buffer, 2 μL of MgCl2, 0.5 μL of dNTP, 1 μL of each primer (lpxAColi, lpxAJej, lpxALari, lpx- AUps), 3 μL of lpxARKK2m primer, 0.25 U of GoTaq D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/9/3188/3952746 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 19 August 2019 CLEANING AND DISINFECTION PROGRAMS FOR POULTRY 3191 DNA Polymerase (Promega, Madison, WI, USA), 11 μL of nuclease-free water, and 3 μL of DNA. For the amplifications, a thermocycler was utilized, programmed for an initial denaturation cycle at 94◦C for 2 min., followed by 40 cycles with denaturation at 94◦C, 1 min; annealing at 52◦C, 1 min; extension at 72◦C, 1 min., and final extension at 72◦C, 5 min. The PCR products were analyzed by electrophoresis in agarose gel at 3%, stained with SybrGold (Invitrogen, Karlsruhe, Germany) (0.1 μL/mL) and visualized in a UV trans-illuminator (BioAgency, São Paulo, Brazil). The product sizes were determined by comparing elec- trophoretic migration standard of a 100-pb molecular size marker (GE Healthcare, USA). Nuclease-free water was utilized as negative con- trol whereas a strain of Campylobacter jejuni (ATCC 33560), the same utilized for environment contamina- tion, was used as positive control. The samples with electrophoretic migration standard compatible to the positive control were sent to genomic sequencing. Genomic Sequencing, Nucleotide Sequence Alignment and Phylogenetic Analysis DNA fragments were extracted from agarose gel for samples with band size compatible to Campylobacter jejuni, and QIAquick Gel Extraction Kit (Qiagen, USA) was utilized after PCR re-amplification following the manufacturer’s recommendation. The sequencing reactions were done utilizing BigDye R© Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies, USA) containing AmpliTaq DNA Polymerase, accord- ing to the manufacturer’s specifications, and reactions for sense and anti-sense primers were carried out using an automated sequencer, 3730 DNA Analyzer (Applied Biosystems, Life Technologies, USA). The search for consensus sequences generated by the program CAP 3 Contig (Huang and Madan, 1999) and edited by BioEdit 7.0.9 (Hall, 1999) was done by BLAST program version 2.0 (Altschul et al., 1997). The editing and multiple alignment of obtained nucleotide sequences as well as others deposited in GeneBank (Table 1) were done by ClustalW program version 1.4 (Thompson et al., 1997), implemented in BioEdit Se- quence Alignment Editor version 7.0.9 (Hall, 1999), utilizing default parameters. Distance matrices, given in percentages of similarity/identity, between the nu- cleotide sequences were calculated through MatGAT program, version 2.0 (Campanella et al., 2003), using global alignment algorithm. Phylogenetic reconstructions for sequences of 213 nu- cleotides, related to lpxA gene were done through max- imum likelihood algorithm and Jukes and Cantor (JC) substitution model with nodal bootstrap support for 1000 pseudo-replicates, utilizing MEGA 5.0 program (Tamura et al., 2013). For the reconstructions, other Table 1. Utilized nucleotide sequences of Campylobacter spp. for phylogenetic reconstruction with genotype, name, origin and respective access numbers in GenBank. Genbank Access Species Isolate Origin Number Campylobacter jejuni NCTC 11168 United Kingdom AL111168 RM 3668 California AY531515 F 38011 Arizona AY531520 KLC2851 N. Zeland AY531522 RM 3664 California AY531519 Campylobacter coli RM 1878 AY531493 RM 1858 AY531495 RM 1865 AY531494 RM 1857 AY531496 WA 27 N. Zeland AY531510 RM 3232 AY531504 RM 1896 USA AY531492 Campylobacter Lari RM 3659 United Kingdom AY531477 RM 2825 Canada AY531479 RM 2824 United Kingdom AY598984 RM 2819 Canada AY531485 RM 2822 AY531482 RM 2100 USA AY531474 RM 2823 Canada AY531481 RM 1890 AY531476 Campylobacter Upsaliensis RM 3195 South Africa AY531473 RM 2093 AY598987 RM 2089 AY531472 RM 1488 Canada AY531471 Helicobacter hepaticus ATCC 51449 USA DN202995 sequences of Campylobacter spp. deposited in GenBank were used as shown in Table 1. Statistical Analysis Data were analyzed by Statistical Analysis System (SAS Institute, 2012). Normality of studentized resid- ual was verified by Shapiro-Wilk’s Test PROC UNI- VARIATE (SAS Institute, 2012) and the variances compared by Levene’s test. The data that did not meet these requirements were submitted to logarith- mic transformation. The original or transformed data were submitted to analysis of variance utilizing PROC MIXED (SAS Institute, 2012). The occurrence fre- quency of Campylobacter was analyzed by Chi-square test through PROC FREQ (SAS Institute, 2012). The utilized level of significance was 5% of probability. RESULTS There was no significant effect of treatments in the initial periods (1 to 7, 1 to 21, and 1 to 35 d). However, during the total housing period (1 to 42 d) there was a significant effect of the Proposed treatment, where the birds exhibited greater BWG, FI, F:G, and PEI (Table 2). In the total microorganism count, counts were similar before on the floor, wall, drinkers, feeders, and water (Table 3). After the procedures, there was a differ- ence between the treatments, showing that the small- est counts were found in water, feeders, walls, and floor D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/9/3188/3952746 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 19 August 2019 3192 BURBARELLI ET AL. Table 2. Performance results obtained in the second poul- try house - environment submitted to cleaning and disinfection programs. Cleaning and disinfection Variable Common Proposed SEM Probability 1-7 d BW gain(g)1 120 128 0.002 0.054 FI (g) 131 136 0.001 0.202 F:G 1.096 1.081 0.006 0.344 VB (%) 99.79 99.38 0.191 0.300 1-21 d BW gain(g) 800 802 0.006 0.865 FI (g) 1493 1504 0.009 0.546 F:G 1.869 1.851 0.010 0.517 VB (%) 99.12 98.90 0.280 0.702 1-35 d BW gain(g) 2126 2141 0.019 0.196 FI (g) 3746 3836 0.027 0.106 F:G 1.810 1.781 0.011 0.270 VB (%) 98.13 97.93 0.468 0.830 1-42 d BW gain(g) 2447 2610 0.025 0.002 FI (g) 4760 4903 0.033 0.035 F:G 1.958 1.880 0.018 0.050 VB(%) 96.26 95.68 0.715 0.691 PEI 312.0 346.5 5.656 0.001 1Body weight (BW), feed intake (FI), Feed:gain ratio (FG), Viability (VB), Productive Efficiency Index (PEI) PEI = (BW × viability/age% × F:G) × 100. Table 3. Total microorganism count before and after cleaning and disinfection procedures. Cleaning and Disinfection Sampling point Common Proposed SEM p∗ Before cleaning and disinfection Water 5.06 4.34 0.290 0.234 Drinker 6.28 5.56 0.228 0.118 Feeder 4.36 4.68 0.192 0.451 Wall 4.64 5.20 0.297 0.381 Floor 4.94 4.85 0.246 0.881 After cleaning and disinfection Water 5.75 2.31 0.664 0.014 Drinker 1.56 0.70 0.380 0.282 Feeder 3.42 0.67 0.563 0.004 Wall 3.10 0.97 0.453 0.008 Floor 3.86 0.14 0.610 <.0001 Data expressed in ufc log10/10 cm2 for all variables except for water (ufc log10/ml) ∗Significance p < 0.05. with the Proposed program. The drinkers from different treatments were not different (Table 3). Table 4 shows the occurrence frequencies of Campy- lobacter spp. There was no difference in the sampled points before and after inoculation. After cleaning and disinfection, there was a smaller occurrence of Campy- lobacter spp. in drinkers and floors with the Proposed program. The birds housed in the facilities after the Proposed treatment also had less Campylobacter spp. verified by cloaca swabs 7 d after housing. There were no differences in the occurrence of Campylobacter spp. at 42 d old and at slaughter time. Table 4. Campylobacter spp. frequency in samples collected throughout the experimental period. Sampling point Common Proposed p∗ Before inoculation Drinker 30% (3/10) 50% (5/10) 0.113 Water 30% (3/10) 30% (3/10) 1.000 Bird 40% (4/10) 30% (3/10) 0.490 Floor 20% (2/10) 10% (1/10) 0.490 After inoculation Drinker 30% (3/10) 50% (5/10) 0.113 Water 30% (3/10) 40% (4/10) 0.113 Bird 20% 92/10) 40% (4/10) 0.196 Floor 10% (1/10) 0% (0/10) 0.291 Wall 10% (1/10) 0% (0/10) 0.291 Feeder 10% (1/10) 0% (0/10) 0.291 After cleaning and disinfection Drinker 30% (3/10) 0% (0/10) 0.038 Water 10% (1/10) 10% (1/10) 1.000 Bird 30% (3/10) 0% (0/10) 0.038 Floor 30% (3/10) 0% (0/10) 0.038 42 d Drinker 40% (4/10) 30% (3/10) 0.490 Water 40% (4/10) 20% (2/10) 0.525 Bird 30% (3/10) 50% (5/10) 0.291 Floor 10% (1/10) 10% (1/10) 1.000 Wall 10% (1/10) 10% (1/10) 1.000 Slaughter Live birds 7,5% (3/40) 20% (8/40) 0.076 Feathering 20% (4/20) 5% (1/20) 0.121 Chiller 10% (2/20) 10% (2/20) 1.000 ∗Chi-square test with significance level of 5% (P < 0.05). After the contamination frequency analysis, the pos- itive samples were submitted to PCR and genomic se- quencing, totaling 54 samples. The sequencing results confirmed Campylobacter jejuni for approximately 43% (23/54) of the samples. Besides the samples identified as Campylobacter jejuni, other enterobacteria were found after sequencing, such as Enterobacter cloacae, Enter- obacter asburiae, and E. coli. Table 5 presents the percentages of similarity (un- der the diagonal) and identity (over the diagonal) of nucleotide sequences found among the selected and sequenced samples and the reference sequences for Campylobacter deposited in GenBank (Table 3). The high similarity level among the samples obtained in this study can be observed, varying from 99.1 to 100%. Moreover, all the samples have a high similarity level of 98.6% with the standard ATCC 33560 strain. The same occurs with the identity among the samples, 99.1 to 100% among samples and 98.6% with standard strain. When the samples (Table 6) were compared to other species of Campylobacter, there was smaller similarity and identity, which ranged from 69 to 86.9% for both. When compared to Helicobacter hepaticus, the values were 53.5% for similarity and 52.8% for identity. Figure 1 illustrates the cladogram obtained in phy- logenetic nucleotide reconstruction for Campylobacter sequences. The sequenced samples in this study were grouped into 2 subclades of Campylobacter jejuni. The D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/9/3188/3952746 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 19 August 2019 CLEANING AND DISINFECTION PROGRAMS FOR POULTRY 3193 Table 5. Comparison of similarity (under diagonal) and identity (over diagonal) percentages of nucleotide sequences of 331pb fragments of Campylobacter jejuni IpxA gene, among 6 samples detected, posteriorly sequenced, by multiplex PCR and sequences of other Campylobacter recovered in GenBank. Similarity/Identity (%) BR597 BR212 BR1241 BR1236 BR1206 BR1037 RM3668 RM3664 NCTC11168 KLC2851 F38011 ATCC33560 RM2825 RM1878 RM3195 ATCC51449 Isolated Cjej Cjej Cjej Cjej Cjej Cjej Cjej Cjej Cjej Cjej Cjej Cjej Clari Ccoli Cups Hhepaticus BR597Cjej – 100.0 99.1 99.1 99.1 99.1 99.1 98.6 99.1 99.1 99.5 98.6 76.1 86.4 77.0 52.8 BR212Cjej 100.0 – 99.1 99.1 99.1 99.1 99.1 98.6 99.1 99.1 99.5 98.6 76.1 86.4 77.0 52.8 BR1241Cjej 99.1 99.1 – 100.0 100.0 100.0 100.0 98.6 99.1 99.1 99.5 98.6 76.5 86.4 77.0 52.8 BR1236Cjej 99.1 99.1 100.0 – 100.0 100.0 100.0 98.6 99.1 99.1 99.5 98.6 76.5 86.4 77.0 52.8 BR1206Cjej 99.1 99.1 100.0 100.0 – 100.0 100.0 98.6 99.1 99.1 99.5 98.6 76.5 86.4 77.0 52.8 BR1037Cjej 99.1 99.1 100.0 100.0 100.0 – 100.0 98.6 99.1 99.1 99.5 98.6 76.5 86.4 77.0 52.8 RM3668Cjej 99.1 99.1 100.0 100.0 100.0 100.0 – 98.6 99.1 99.1 99.5 98.6 76.5 86.4 77.0 52.8 RM3664Cjej 98.6 98.6 98.6 98.6 98.6 98.6 98.6 – 99.5 99.5 99.1 100.0 76.5 86.9 77.0 54.2 NCTC11168Cjej 99.1 99.1 99.1 99.1 99.1 99.1 99.1 99.5 – 100.0 99.5 99.5 77.0 86.4 77.5 53.7 KLC2851Cjej 99.1 99.1 99.1 99.1 99.1 99.1 99.1 99.5 100.0 – 99.5 99.5 77.0 86.4 77.5 53.7 F38011Cjej 99.5 99.5 99.5 99.5 99.5 99.5 99.5 99.1 99.5 99.5 – 99.1 76.5 86.9 77.5 53.2 ATCC33560Cjej 98.6 98.6 98.6 98.6 98.6 98.6 98.6 100.0 99.5 99.5 99.1 – 76.5 86.9 77.0 54.2 RM2825Clari 76.1 76.1 76.5 76.5 76.5 76.5 76.5 76.5 77.0 77.0 76.5 76.5 – 71.4 71.2 53.7 RM1878Ccoli 86.4 86.4 86.4 86.4 86.4 86.4 86.4 86.9 86.4 86.4 86.9 86.9 71.4 – 76.5 50.7 RM3195Cups 77.0 77.0 77.0 77.0 77.0 77.0 77.0 77.0 77.5 77.5 77.5 77.0 71.8 76.5 – 54.6 ATCC51449Hhep 53.5 53.5 53.5 53.5 53.5 53.5 53.5 54.9 54.5 54.5 54.0 54.9 54.5 51.2 55.4 – Table 6. Legends of sequenced samples utilized to build the phylogenetic tree. Gene Bank Identification Sampling point Accession n◦ BR212Cjej Bird 12 –initial environment KY321332 BR597 Cjej Bird 3 –After inoculation KY321333 BR1241Cjej Bird - 42 d Proposed Program KY321334 BR1236Cjej Bird 30–42 d Common Program KY321335 BR1037Cjej Bird 1 - Feathering Proposed Program KY321336 BR1206Cjej Bird 2 Chiller - Proposed Program KY321337 samples and the standard ATCC 33560 strain are part of the same monophyletic clade. The bootstrap number of samples varied from 62 to 72%. DISCUSSION High bacterial populations are responsible for a de- crease in broiler chickens’ performance (Payne et al., 2005). Thus, cleaning and disinfection practices have positive effects on broilers’ performance (Sharma, 2010) and on prevention of disease (Cozad and Jones, 2003; Newel et al., 2011); however, there are few studies that directly relate cleaning and disinfection to poultry’s performance characteristics. The positive effects pre- sented in this study corroborate the ones by Burbarelli et al. (2015), who observed an improvement in broil- ers’ productive performance and reduction in microor- ganism count with a detailed cleaning and disinfection program for a flock with reutilized bedding. Bragg and Plumstead (2003) and Ka-Oud et al. (2008) also found beneficial effects of cleaning and disinfection such as greater final weight and lower mortality rate. In poultry, the satisfactory performance expression is related to intestinal health (Mayorka et al., 2002); therefore, the microbiota balance is an important factor for productivity. Approximately 20% of ingested crude energy is spent on the maintenance of the intestinal epithelium. In addition, the reduction in nutrient ab- sorption has negative influences on F:G, carcass yield, and production cost (Hoerr, 2001). Cleaning and disin- fection practices are responsible for reduction of envi- ronment infection pressure (Sesti et al., 1998), favoring the intestinal microbiota balance, increasing nutrient absorption and consequently resulting in better expres- sion of broiler chickens’ genetic potential. The decrease in total microorganism count in equip- ment and facilities as a result of the Proposed treatment is in accordance to the studies by Luyckx et al. (2015), who observed a greater decrease in total microorganism count when a wet phase was included in the environ- ment. This seems to be related to the easy organic mat- ter removal of microorganisms with high-pressure wash- ing (Grezzi, 2008). Organic matter removal seems to be fundamental to cleaning and disinfection programs be- cause their residues are able to decrease the action of disinfectants (Stringfellow et al., 2009; Chima et al., 2012; Luyckx et al., 2017), resulting in greater microor- ganism counts even after disinfection. Fewer positive samples of Campylobacter spp. were observed from drinkers, floor, and birds after the Pro- posed program, corroborating Van de Gissen et al. (1998) and Newell and Fearnley (2003) regarding the reduction capacity and elimination of Campylobacter spp. from the environment; however, that contradicts Bouwknegt et al. (2004), who did not find any effect of this type of treatment on facilities for broiler chickens. Although it did not differ between the analyzed groups, the occurrence of Campylobacter spp. in the birds’ drinking water even after the Proposed program deserves attention. The parts of the water provision sys- tem of broiler chickens’ houses were a location of biofilm formation due to constant water contact (Araújo et al., 2011). Bacteria such as Campylobacter frequently are associated with biofilm (Shi and Szu, 2009). Because it is a gram-negative bacterium, Campylobacter is more D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/9/3188/3952746 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 19 August 2019 3194 BURBARELLI ET AL. Figure 1. Cladogram representing phylogenetic reconstruction based on nucleotide sequence alignment referring to the amplification of a 340 pb fragment of Campylobacter IpxA gene. resistant to the action of disinfectants (Dahl et al., 1989), through a complex enzymatic system of resis- tance to oxidative stress, and among the enzymes of this system are superoxide dismutase, catalase and cy- tochrome C peroxidase (Atack and Kelly, 2009). The disinfectants utilized in the water system disinfection were peracetic acid and benzalkonium chloride, the for- mer is an oxidant agent and the latter a quaternary ammonium compound. Besides the bacterial resistance to the active ingre- dient we used, biofilm represents an additional resis- tance to bacteria (Chapman, 2003), because it is able to form a protection through its compounds, making oxidant compounds be inactivated even before getting in contact with the microorganisms (Chen and Stewart, 1996). Efficiency reduction of peracetic acid, an oxidant agent, was also observed by Trachoo and Frank (2002) against Campylobacter in the presence of biofilms. The D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/9/3188/3952746 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 19 August 2019 CLEANING AND DISINFECTION PROGRAMS FOR POULTRY 3195 same authors also observed that quaternary ammonium compounds such as benzalkonium chloride have their ef- ficiency affected as well. These factors can be related to re-colonization of facilities by Campylobacter after the Proposed treatment when the birds are 42 d old. The absence of Campylobacter spp. in the other sam- ples right after the Proposed treatment of cleaning and disinfection may be related to low resistance of Campy- lobacter spp. to glutaraldehyde and formaldehyde as found by Wang et al. (1983) and Gutiérrez-Mart́ın et al. (2011). At 42 d old, there was no difference between the con- tamination frequency of Campylobacter for both treat- ments, which can be related to the high dissemination capacity of these bacteria, as Knudsen, et al. (2006) ob- served in their studies. Contamination by Campylobac- ter was found in samples from the environment, consid- ered negative for Campylobacter spp. as also reported by Van de Gissen et al. (1998). When investigating the contamination origin of those samples, the same au- thors found the bacteria in insects and staff shoes, sug- gesting that even the facilities that were previously free from Campylobacter spp. can be contaminated during the birds’ stay due to external sources. Overall, the absence of sanitizing procedures can be considered a Campylobacter spp. contamination risk factor for broiler chickens (Evans and Sayers, 2000; Newell and Fearnley, 2003; Bouwknegt et al., 2004; McDowell et al., 2008; Newell et al., 2011), but even with efficient strict programs of cleaning, disinfection and biosafety, this bacterium may enter the facilities and colonize the birds (Van de Gissen et al., 1998). There is a noteworthy relation between the rear- ing environment contamination and Campylobacter spp. presence in broiler chickens’ carcasses because facili- ties with Campylobacter-positive birds generate posi- tive carcasses (Herman et al., 2003). Elvers et al. (2011) found little changes in the profile of the strains found in carcasses from flocks that were positive for Campy- lobacter, indicating that if the contaminated flock has reached the slaughter plant, there will be little or no influence on the sanitary quality improvement of those carcasses. Although no significant difference was found in the contamination frequency of 42-day-old birds and car- casses in different slaughter points between the evalu- ated treatments, it is important to point out that clean- ing and disinfection must be adopted to reduce the risk of campylobacteriosis risk in consumers of poultry prod- ucts (Van de Gissen et al., 1998; Gibbens et al., 2001; Vandeplas et al., 2008; Meunier et al., 2015). In positive samples of Campylobacter submitted to PCR analyses and posterior genetic sequencing, it was possible to detect Campylobacter jejuni, and Enterobac- ter cloacae, Enterobacter asburiae and E. coli, which should have had their growth inhibited by the utiliza- tion of selective supplement mCCDA culture medium. Chon et al. (2011) observed low sensitivity and selectiv- ity to this medium, mainly when the sample microflora was abundant. Bolton et al. (1996) found 13% of con- tamination of this medium when evaluating samples of human feces. The utilized multiplex PCR was based on the methodology proposed by Klena et al. (2004), which uses lpxA gene in species differentiation of Campy- lobacter spp. (C. jejuni, C. coli, C. lari, and C. up- saliensis) that codifies Lpxa enzyme, the initial step of lipid A production, an essential molecule of LPS system found in bacteria of Campylobacter genus. This gene, found in several gram-negative bacteria (Weckesser and Mayer, 1988), was identified in Neisseria meningitidis (Odegaard et al., 1997), Pseudomonas aeruginosa (Dot- son et al., 1998), Enterobacter asburiae (Osei Sekyere et al., 2016), Enterobacter cloacae (Mcgann et al., 2015) and Escherichia fergusonii (Touchon et al., 2009). The identification of Enterobacter cloacae, Enter- obacter asburiae, and E. coli can be related to the pres- ence of lpxA gene in these bacteria and there is also the possibility of genetic information exchange between the environmental microbiota bacteria through plasmids, transposons and gene insertion sequences. Fouts et al. (2005) when studying the genome of some Campylobac- ter strains, found plasmids involved in the transfer and secretion of virulence factors, sequences of chromosome and plasmid DNA insertion. As the utilized gene, lpxA, belongs to LPS virulence factor of Campylobacter, there is the probability of lat- eral genetic transfer occurrence, which makes it possi- ble that the primers utilized in multiplex PCR reaction, specific for Campylobacter species (jejuni, coli, lari, and upsaliensis), have interacted with similar sequences, but with other bacteria. Moffat et al. (2011) when studying Acinetobacter baumannii, a bacterium that possess lpxA gene, found a gene insertion element of this same gene, showing that bacteria that have it can perform lateral genetic trans- fers. In this same study, the studied insertion sequence was related to resistance to antibiotics against Acineto- bacter baumannii, which means a serious public health problem. In Campylobacter, LPS composition, codified by sev- eral genes, including lpxA, is also related to resistance to antibiotics (Van Mourik et al., 2010). Thus, it is im- portant to point out that as reported for A. baumannii (Potron et al., 2015), Campylobacter can have an im- portant role in the dissemination of genes resistant to antibiotic against other gram-negative microorganisms when they may transfer genes laterally. Evaluating the phylogenetic tree and the similar- ity and identity table, it is possible to verify that the sample strains are very similar among them- selves, are very close to one another in cladogram for lpxA gene, and have high similarity and identity in- dices. When comparing the samples to the standard strains, there was a slight distancing in the cladogram and lower similarity and specificity indices, showing that it is small despite the differences among these strains. D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/9/3188/3952746 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 19 August 2019 3196 BURBARELLI ET AL. The high level of similarity and specificity found in this study are in accordance with Lucien (2012) when evaluating the tuf gene of these bacteria, with similar- ity and identity varying from 98 and 99% of Campy- lobacter jejuni samples with standard ATCC 33560 strain. In the present study, it was possible to ob- serve 4 distinct clades for the phylogenetic recon- struction of Campylobacter spp, one for each stud- ied species of Campylobacter, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campy- lobacter upsaliensis, similarly to Kärenlampi et al. (2004), Klena et al. (2004) Hill et al. (2006) and Lucien (2012) when studying groEL gene, lpxA gene, tuf gene, and cpn60 gene, respectively. Our results showed that the strains of Campylobac- ter jejuni circulating the assessed experimental environ- ment are very similar to the ones that could be found in different countries and types of samples, making it possible to observe that even with a great diversity of Campylobacter jejuni strains, they have genetic simi- larity among themselves, making the utilized method- ology reliable for their identification and phylogenetic reconstruction. Although the recovery of standard ATCC 33560 strains in the samples was expected, no sample could be identified after sequencing. This result can be re- lated to a reduced adaptation to the environment and competition with the other Campylobacter jejuni, in- hibiting a possible growth of standard ATCC 33560 strain. Cawthraw et al. (1996) reported the fragility of bacteria from in vitro cultures, since the labora- tory conditions in which they are submitted differ from the in vivo environment, reducing their resistance, ad- hesion factors, motility, and virulence (Ringoir and Korolik, 2003). There is also the possibility of genomic rearrangement occurrence, insertions, deletions, or mu- tations of Campylobacter jejuni DNA as described by Wassenaar et al. (1998), Hänninen et al. (1999), and De Boer et al. (2002), resulting in modifications of these same factors. CONCLUSION The Proposed program shows greater efficiency in the total environmental microorganism count reduction and the capacity to eliminate Campylobacter from the floor and drinkers of facilities. With the adoption of the Proposed treatment, it is possible to obtain better per- formance of the birds. Both programs do not influence the occurrence frequency of Campylobacter in the facil- ities and birds at 42 d old and at slaughter time. It was possible to identify 6 different strains of Campylobac- ter jejuni, which occupy the same phylogenetic clade, and become effectively differentiated with high values of nodal support associated with other species of Campy- lobacter to which they were compared. ACKNOWLEDGMENTS The authors are grateful to FAPESP for providing the graduate scholarship (2013/02457-8) to carry out this study. REFERENCES Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. 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