Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds.

Spotty liver disease (SLD) is a bacterial disease of chicken, causing mortalities and reduction in egg production, hence, contributing to economic loss in the poultry industry. The causative agent of SLD has only recently been identified as a novel Campylobacter species, Campylobacter hepaticus. Specific primers were designed from the hsp60 gene of Campylobacter hepaticus and PCR followed by high-resolution melt curve analysis was optimised to detect and differentiate three species of Campylobacter (Campylobacter coli, Campylobacter jejuni and Campylobacter hepaticus). The three Campylobacter species produced a distinct curve profile and was differentiated using HRM curve analysis. The potential of the PCR-HRM curve analysis was shown in the genotyping of 37 Campylobacter isolates from clinical specimens from poultry farms. PCR-HRM curve analysis of DNA extracts from bile samples or cultures from bile samples, were identified as Campylobacter hepaticus and confirmed by DNA sequencing. The DNA sequence analysis of selected samples from each of the three HRM distinctive curves patterns showed that each DNA sequence was associated with a unique melt profile. The potential of the PCR-HRM curve analysis in genotyping of Campylobacter species was also evaluated using faecal specimens from 100 wild birds. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Campylobacter species using either bacterial cultures or clinical specimens.

Introduction

Spotty liver disease (SLD) is a disease of poultry that causes mortality and reduced egg production. It mainly affects free-range laying hens. On post mortem examination there are multiple 1-2mm pale focal lesions in the liver [1]. Spotty liver disease was first reported from the Eastern states of Australia in the 1980s [2] and subsequently has been reported from Europe, North America and Africa [3-7]. Spotty liver disease is similar to avian vibrionic hepatitis (AVH) a disease which was reported in the USA and Canada between the 1950s and 1970s. The causative agent of AVH was never fully defined. Both SLD and AVH have been used to describe an acute, randomly distributed, focal necrotic hepatitis [8].

Campylobacter hepaticus (C. hepaticus) has recently been identified [9], named [10] and shown to be the cause of SLD [11]. It is fastidious, difficult to isolate and will not grow on many of the selective media used to isolate Campylobacter [9].

Spotty liver disease is recognised as a significant threat to the Australian layer industry [12]. Mortality in an affected flock can be 10% with a 10% fall in egg production [1]. The incidence of the disease appears to be increasing and this coincides with changes in the husbandry of laying hens with a greater proportion managed free-range in response to welfare concerns about cages [8].

Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) commonly colonise the gastrointestinal tract of poultry and are found in the faeces [13]. They are a major source of human food poisoning in Australia, the United States and Europe [14]. Studies of Campylobacter hepaticus have shown that it is also able to colonise the GIT and is present in the faeces of poultry [15, 16].

Confirmation of SLD by laboratory testing is important to differentiate it from other diseases such as fowl cholera and erysipelas which can have a similar history and gross pathology. Testing may also be used for SLD surveillance which could contribute to disease prevention and control. While culture of C. hepaticus from samples collected in the field is difficult [9] PCR testing has proved to be more sensitive and is able to detect C. hepaticus in bile samples, intestine content samples and faeces [15, 16].

PCR testing of clinical samples can be followed by high resolution melting (HRM) analysis where differences in curve shape and melting temperature correlate with variations in the sequence of amplicons. A major advantage of HRM analysis is that the variations in sequence can be detected without the need to sequence the amplicon [17-22]. The reagents required are relatively low cost and the technique is simple to perform [20].

The aim of this study was to develop and optimise a PCR–HRM assay suitable for clinical samples which was able to detect C. hepaticus and differentiate it from other poultry Campylobacter sp. in particular C. jejuni and C. coli.

Materials and methods

Ethics statement and sample collection

Approval for the sample collection from chicken was granted by the Charles Sturt University Animal Care and Ethics Committee (Protocol number A19046) and all experiments were performed in accordance with the relevant guidelines and regulations. Opportunistic chicken bile samples were collected from the gall bladder of 40 free-range egg layers found dead on farms or culled. Swabs of fresh faecal samples from 101 waterfowl including 66 Pacific black duck (Anas superciliosa) and 35 Australian wood duck (Chenonetta jubata) were collected from Wagga Wagg lake and lagoons and tested.

Campylobacter strains

Two C. coli and three C. jejuni isolates, including three reference strains, ATCC33559, ATCC29428 and NCT11351 were used as the controls (Table 1). The C. coli (C669) and C. jejuni (BAL172236) isolates were isolated from chicken excreta or chicken carcasses and were provided by the Birling Avian Laboratories, New South Wales, Australia. All the isolates were cultured on non-selective sheep blood agar and incubated at 42°C under microaerobic conditions (8% N2, 4% H2, 8% O2 and 5% CO2) for 72 hours. Pure cultures were then cultivated in broth medium for DNA extraction.

Table 1

List of samples, origins, mean curve peak melting points ± SD, submitted GenBank accession numbers and genotypes of samples tested using PCR- HRM curve analysis.

Sample ID	Sample Type	Origin	Culture/species	Number of times tested	Melting point (°C)	GCP±SD *	Genotype *	Genotyped by sequence /GenBank accession number
B1	chicken bile	Free range layer	Culture / C. hepaticus	6	80.2±0.2	85.5±2.4	C. hepaticus	C. hepaticus/MT682360
B2	chicken bile	Free range layer	Culture / C. hepaticus	6	80.0±0.1	91.4±2.6	C. hepaticus
B3	chicken bile	Free range layer	Culture / C. hepaticus	6	80.3±0.0	93.1±6.1	C. hepaticus
B5	chicken bile	Free range layer	Culture / C. hepaticus	6	80.1±0.4	89.7±3.4	C. hepaticus
B6	chicken bile	Free range layer	Culture / C. hepaticus	6	80.0±0.0	79.2±8.2	C. hepaticus
B7	chicken bile	Free range layer	Culture / C. hepaticus	6	80.0±0.2	87.9±5.6	C. hepaticus
C1	Chicken faeces	Free range layer	No culture	10	80.0±0.0	90.3±4.8	C. hepaticus	C. hepaticus/MT682361
C2	Chicken faeces	Free range layer	No culture	10	80.2±0.0	91.6±2.1	C. hepaticus
C3	Chicken faeces	Free range layer	No culture	10	80.1±0.1	96.1±1.5	C. hepaticus
C4	Chicken faeces	Free range layer	No culture	10	80.0±0.2	88.9±4.4	C. hepaticus
C5	Chicken faeces	Free range layer	No culture	10	80.2±0.0	93.4±6.3	C. hepaticus
F2	chicken bile	Free range layer	No culture	8	80.0±0.3	88.6±5.2	C. hepaticus	C. hepaticus/MT682362
F8-1	chicken bile	Free range layer	No culture	8	80.1±0.1	87.2±5.5	C. hepaticus	C. hepaticus/MT682364
F8-2	chicken bile	Free range layer	No culture	8	80.1±0.3	90.2±2.1	C. hepaticus
F8-3	chicken bile	Free range layer	No culture	8	80.0±0.2	89.8±5.1	C. hepaticus
F9-1	chicken bile	Free range layer	No culture	8	80.2±0.1	90.1±1.4	C. hepaticus
F9-2	chicken bile	Free range layer	No culture	8	80.3±0.0	93.6±3.2	C. hepaticus
F9-3	chicken bile	Free range layer	No culture	8	80.0±0.0	90.4±4.8	C. hepaticus
F9-4	chicken bile	Free range layer	No culture	8	80.0±0.0	94.3±2.9	C. hepaticus
PR1	chicken bile	Free range layer	No culture	5	80.0±0.0	92.9±1.2	C. hepaticus	C. hepaticus/MT682363
PR2	chicken bile	Free range layer	No culture	5	80.0±0.0	87.5±2.8	C. hepaticus
PR3	chicken bile	Free range layer	No culture	5	80.2±0.0	84.3±2.1	C. hepaticus
PR4	chicken bile	Free range layer	No culture	5	80.3±0.1	72.5±1.1	C. hepaticus
PR5	chicken bile	Free range layer	No culture	5	80.4±0.1	87.4±2.8	C. hepaticus
PR6	chicken bile	Free range layer	No culture	5	80.4±0.1	82.4±4.1	C. hepaticus
PR7	chicken bile	Free range layer	No culture	5	80.4±0.1	89.2±2.4	C. hepaticus
PR8	chicken bile	Free range layer	No culture	5	80.3±0.2	78.5±5.1	C. hepaticus
PRC1	chicken bile	Free range layer	No culture	3	80.5±0.4	76.2±2.1	C. hepaticus	C. hepaticus/MT682365
PRC4	chicken bile	Free range layer	No culture	3	79.5±0.1	85.3±3.2	C. hepaticus
PRC5	chicken bile	Free range layer	No culture	3	80.3±0.2	73.7±7.2	C. hepaticus
PRC6	chicken bile	Free range layer	No culture	3	79.9±0.3	75.2±4.4	C. hepaticus
PRC8	chicken bile	Free range layer	No culture	3	80.1±0.2	77.5±8.3	C. hepaticus
PRC10	chicken bile	Free range layer	No culture	3	79.9±0.0	74.3±6.2	C. hepaticus
PRC11	chicken bile	Free range layer	No culture	3	80.3±0.1	72.0±0.2	C. hepaticus
PRC12	chicken bile	Free range layer	No culture	3	80.4±0.2	77.9±2.1	C. hepaticus
PRC13	chicken bile	Free range layer	No culture	3	80.5±0.1	93.0±5.2	C. hepaticus
UN1	chicken bile	Free range layer	No culture	3	80.4±0.1	90.3±2.7	C. hepaticus
UN2	chicken bile	Free range layer	No culture	3	80.4±0.1	85.6±3.8	C. hepaticus
0283–1	chicken bile	Free range layer	No culture	3	80.3±0.4	74.5±4.8	C. hepaticus
0283–2	chicken bile	Free range layer	No culture	3	80.2±0.2	76.3±5.4	C. hepaticus
PBD-13	faeces	Pacific Black duck	No culture	3	81.5±0.06	0.04±0.0	Variation	C. canadensis/MW269513
PBD-14	faeces	Pacific Black duck	No culture	3	81.6±0.04	0.00±0.0	Variation	C. canadensis/MW269514
ATCC29428	Reference strain	Human faeces	Culture / C. jejuni	12	81.7±0.0	0.19±0.3	Variation	C. jejuni /MT682368
ATCC33559	Reference strain	Pig faeces	Culture / C. coli	12	80.5±0.0	56.1±5.1	Variation	C. coli /MT682367
NCTC11351	Reference strain	Bovine faeces	Culture / C. jejuni	12	82.0±0.3	0.0±0.1	Variation
C669	Chicken dropping	Broiler	Culture / C. coli	12	80.7±0.1	51.7±5.4	Variation
BAL172236	Chicken carcass	Broiler	Culture / C. jejuni	12	81.8±0.1	0.3±0.1	Variation
HV10	chicken bile	Free range layer	Culture / C. hepaticus	16	80.3±0.1	98.3±1.1	C. hepaticus	C. hepaticus/MT682366

* when C. hepaticus was used as reference genotype using a cut-off value of ≥72.

The field isolates were obtained from bile and faecal specimens collected at post mortem examination from the gall bladder and large intestine of chickens, respectively. The bile and faecal specimens were collected into 5 mL sterile tubes which were stored in zip lock bags, on ice in a thermally insulated container until received at the laboratory where they were stored at -20°C prior to DNA extraction. All isolates are listed in Table 1 with source details of Campylobacter species.

DNA extraction

Total genomic DNA was extracted from bile specimens and bacterial cultures using Wizard® SV Genomic DNA purification system (Promega, Australia). The Quick-DNA Faecal/soil Microbe Miniprep Kit (Zymo Research, USA) was used to extract DNA from faecal samples, according to the manufacturer’s instructions. Faecal swab samples of the same species of wild birds were pooled in groups of five before DNA extraction. All extracted DNA was quantified using the Nanodrop 2000 (Thermo Fisher Scientific, Australia). The concentration of each DNA sample was adjusted to 5 ng/μl for subsequent PCR or stored at -20°C until use.

PCR amplification

The hsp60 gene was used to design the PCR primers (hsp60-F 5’- AAGAAATATTACAGCAGGAG-3’ and hsp60-R 5’- GCATACCTTCAACAACATT-3’). The hsp60 gene has been successfully used in PCR for detection of enteric bacteria and different species of Campylobacter [23-25]. The PCR amplification was performed in 25 μl reaction volume on a Rotor-Gene™ 6000 thermal cycler (Qiagen, Melbourne, Australia). PCR amplification was performed in 25 μl reaction volume and the reaction mixture contained 3 μl of extracted genomic DNA, 25 μM of each primer, 1.5 mM MgCl2, 1250 μM of each dNTP, 5 μM SYTO 9 green fluorescent nucleic acid stain (Invitrogen, Australia), 5 x GoTaq Green Flexi Reaction Buffer and 1 U of Go Taq DNA Polymerase (Promega, Australia). The optimal PCR conditions were initial denaturation at 96°C for 3 min, 35 cycles of 96°C for 30 s, 55°C for 30 s and 72°C for 30 s, and a final extension of 72°C for 5 min. In each set of PCR, HV10 C. hepaticus genomic DNA and distilled water were included as positive and negative controls, respectively.

High-resolution melt curve analysis

On completion of PCR, HRM curve analysis was performed. The PCR products were subjected to 0.5°C/s ramping between 50°C and 90°C. All samples were tested in triplicate and their melting profiles were analysed using Rotor-Gene 1.7.27 software and the HRM algorithm provided. Normalisation regions of 76–77°C and 83.5–84.5°C were applied for analysis.

The HV10 isolate (C. hepaticus) was set as a ‘genotype’ (GenBank accession CP031611), and the average HRM genotype confidence percentage (GCP) (the value attributed to each isolate compared with the genotype, with a value of 100% indicating an exact match) for the replicates was predicted by the software. The GCPs for C. hepaticus known isolates were averaged and the standard deviation (SD) calculated and used to establish the GCP range for the C. hepaticus cut-off point. The cut-off point was applied in HRM analysis to evaluate the differentiation power of the assay to discriminate the Campylobacter spp.

Sequencing and nucleotide sequence analysis of PCR amplicons

Selected PCR amplicons (B1, C1, F2, PR1, PRC1, UN1, ATCC29428, ATCC33559 and HV10) were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Australia) according to the manufacturer’s instructions. Purified amplicons were sequenced in both directions with the same primers as used for PCR by Australian Genome Research Facility Ltd (AGRF Ltd., Brisbane, Australia). The sequences were analysed using Clustal W [26] and DNASTAR (Meg Align) software. GenBank accession numbers were assigned to the nucleotide sequences of the Campylobacter isolates and reference strains (Table 1).

Detection limit of the assay

The detection limit of the assay on DNA extracted from the bile sample was determined using dilutions of quantified C. hepaticus DNA. DNA extracted from C. hepaticus was serially diluted 10-fold from 1 ng to 10−8 ng. Each DNA dilution was tested in PCR. To evaluate the specificity of the assay, DNA was extracted from seven bacterial strains of genetically similar genera (Klebsiella, Pseudomonas, Enterobacter, Staphylococcus, streptococci, E. coli and Pasteurella) and tested in PCR-HRM.

Results

Gel electrophoresis demonstrated that the PCR generated only amplicons of expected size of 269 bp and non-specific DNA amplification was not observed.

Differentiation of C. hepaticus, C. coli and C. jejuni strains using conventional and normalised HRM curve analysis

The three Campylobacter species, (ATCC33559 C. coli, ATCC29428 C. jejuni and HV10 C. hepaticus) each produced one peak in conventional melt curve and distinctive normalised curves (Fig 1a and 1b). The mean melting points and SD of C. coli, C. jejuni and C. hepaticus were 80.50 ±0.00 °C, 81.65±0.00 °C and 80.25±0.14 °C, respectively.

Fig 1

Conventional and normalised melt curve analysis of three Campylobacter reference strains.

(a) Conventional and (b) normalised HRM curve analysis of PCR (hsp60 gene) amplicons.

Non-subjective differentiation of Campylobacter species using GCP values and a mathematical calculation

The HRM curve analysis produced a distinct profile for C. hepaticus positive isolates. The positive control C. hepaticus (HV10) produced one peak and field samples (samples 1–40) also produced one peak at 80.15–80.40°C. The C. jejuni samples (ATCC29428, NCTC11351 and BAL172236) produced a single peak at 81.75–82.0°C, and the C. coli samples (ATCC33559 and C699) at 80.65–80.75°C (Table 1). The negative control samples did not produce a peak. The conventional and normalised curves produced by C. coli samples was close to the curves range produced by the C. hepaticus isolates (Fig 2a and 2b).

Fig 2

Conventional and normalised melt curve analysis of Campylobacter strains and isolates.

a) Conventional and b) normalised melt curve analysis of PCR amplicons.

Using GCPs of all C. hepaticus isolates, a cut-off value was generated as a mathematical model to assess the relationship of the isolates without requiring visual interpretation by the operator. The mean GCP of all C. hepaticus specimens was 92.4 and the mean SD was 4.1. The value of 5SD was subtracted from the average GCP to determine a cut-off point. A cut off point value of 71.9 was determined for C. hepaticus genotypes. Thus, the GCP range of the C. hepaticus isolates was determined to be 71.9–100 and was used for detection of all Campylobacter isolates. When C. hepaticus reference strain (VH10) was used as the reference genotype with a cut off value of 71.9, all C. hepaticus isolates produced a GCP (72–93) higher than 71.9 and genotyped as C. hepaticus. All C. jejuni and C. coli isolates also generated GCPs between 0.0–0.6 and 40.3–63.1, respectively, which were less than cut-off point (71.9) and therefore were automatically genotyped as ‘variation’. When the C. hepaticus was used as reference genotype, the highest GCP values of C. coli and C. jejuni were 63.1 and 0.6, respectively. Therefore, the GCP gap between C. hepaticus and C. coli was about 9 and between C. hepaticus and C. jejuni was 71. To show the differentiation power of PCR-HRM in discriminating between C. hepaticus and C. coli and C. jejuni, the mean GCP values of each Campylobacter isolate was plotted in a dot plot which shows the GCP gap when C. hepaticus (HV10) is used as the reference genotype (Fig 3). The PCR-HRM had a higher discrimination power in differentiating between C. hepaticus and C. jejuni isolates.

Fig 3

Comparison of the distribution of GCPs from C. jejuni and C. coli isolates by individual value plot when C. hepaticus (VH10) was used as reference genotype.

Detection of minor variations in hsp60 nucleotide sequence by the newly developed PCR-HRM curve analysis technique

To confirm that the differentiation of Campylobacter species by HRM curve analysis was related to variations in the nucleotide sequences of the amplicons, nucleotide sequences of amplicons from each distinct curve profile were determined and compared. The PCR amplicon for C hepaticus, C. jejuni and C. coli reference strains was 269 bp in length. One of C. hepaticus isolates (F8-1) had two substitutions (T to G and G to A) at positions 259 and 260, respectively. C. coli and C. jejuni strains each had 25 and 27 nucleotide substitutions when compared with C. hepaticus HV10 (Fig 4).

Fig 4

Alignment of nucleotide sequences (hsp60 gene) of selected Campylobacter isolates using CLUSTAL W.

Identical nucleotides and deletions are shown by ‘.’ and ‘-’, respectively.

Sensitivity and specificity of the assay

The concentration of DNA extracted from C. hepaticus positive control was measured using NanoDrop 2000 (ThermoFisher Scientific, Australia) and serial 10-fold dilutions were prepared. Each dilution was tested in PCR and 10 μl of each amplicon was run on 1.5% of agarose gel electrophoresis. The DNA sample was detectable up to 10−3 ng of C. hepaticus DNA (S1 Fig). None of seven bacterial strains (Klebsiella, Pseudomonas, Enterobacter, Staphylococcus, streptococci, E. coli and Pasteurella) produced a curve and remained negative using the PCR-HRM. The specificity of the assay was 100% as confirmed with DNA sequencing of Campylobacter species used in this study.

Evaluation of PCR-HRM assay for differentiation of Campylobacter spp. in wild waterfowl

Out of 20 pooled samples (each pooled sample consisted of five faecal specimens of a particular wild duck species), two samples from Pacific black duck (PBD-13 and PBD-14) were found positive in PCR-HRM. Both samples produced similar melt curves (81.5±0.06) which were higher than melting points of C. coli (80.5±0.00), C. jejuni (81.2±0.02) and C. hepaticus (79.9±0.04) and therefore, generated distinct normalised curves (Fig 5). The amplicons of 272 bp were detected in the two groups of samples (both from Pacific black ducks) with identical nucleotide sequences (Fig 6). Sequence comparisons using BLAST showed 87.1% sequence similarity with Campylobacter canadensis (C. canadensis) and 85.8% similarity with C. coli. The phylogenetic analysis showed that Campylobacter species detected in wild waterfowls were genetically closer to C. canadensis when compared with C. coli, C. jejuni and C. hepaticus in their hsp60 gene (Fig 6).

Fig 5

Conventional and normalised melt curve analysis of Campylobacter species.

a) Conventional and b) normalised melt curve analysis of PCR amplicons from Campylobacter isolates detected in wild birds. All campylobacter spp. produced a single peak at different temperature. Campylobacter isolates from wild birds (PBD-13 and PBD-14) had melting points higher than C. coli, C. jejuni and C. hepaticus.

Fig 6

Phylogenetic analysis of hsp60 gene sequences (269–272 bp) from Campylobacter species isolated from chickens (F8-1, PR1, PRC8, PRC1, F2, C1 and B1) and wild birds (PBD-13 and PBD-14).

Sequences were compared with those of Campylobacter reference strains. The tree was constructed using the Neighbour-joining method (DNASTAR software).

Discussion

Spotty liver disease, caused by the bacteria C. hepaticus, is a disease of significance to the poultry industry, and may continue to increase in prevalence as the layer industry moves away from cages to free range and barn husbandry systems. Diagnosis of SLD can be difficult in the field as the majority of birds in a flock will appear to be in good health, with sick birds showing only mild depression or found dead [12]. To limit the spread of disease and to treat affected birds, the identification of the disease-causing pathogen needs to be rapid.

This study describes the development and assessment of a PCR with HRM curve analysis for the detection of C. hepaticus. Molecular tests are a more rapid method for detection of bacterial pathogens than traditional culture methods particularly for slow growing bacteria such as C. hepaticus. A PCR has been developed and optimised to detect C. hepaticus [16], and also a multiplex PCR has been developed which could simultaneously and specifically identify the presence of C. jejuni, C. coli and C. hepaticus [27]. While PCR and multiplex PCR are suitable for diagnosis of C. hepaticus, running samples on agarose gel electrophoresis is required in both methods to reveal the results which takes about one hour to conclude. While pre-poured agarose gels or buffer-free electrophoresis can save time, still loading samples and running the gel and obtain photographic record of the results is a time-consuming exercise. The use of HRM curve analysis is a relatively new technology which provides further automation by eliminating the need for gel electrophoresis and provides faster analytics to identify the genotype of samples in less than 15 minutes when compared to gel electrophoresis and saves time to pour, load and run the gel and therefore reduces time and labour-cost. The use of PCR-HRM curve analysis as a rapid genotyping method compared with PCR and gel electrophoresis has been well documented before [21, 28]. A multiplex-PCR and HRM curve analysis for differentiation of C. jejuni and C. coli suitable for use with poultry faecal or carcass swab specimens has also been reported [14].

This study assessed the use of bile specimens collected from dead or culled birds at post mortem examination for diagnostic purposes. Campylobacter spp. are recognised as bile resistant [29] and the level of C. hepaticus detected in the bile of hens has been shown to be considerably higher than in the liver [16], therefore bile was selected as the preferred sample for use in this study, however, a few faecal samples were also included in this study.

Targeting hsp60 gene in PCR for detection and differentiation of enteric bacterial pathogens including Campylobacter species has been reported to be a highly specific and reproducible approach [25, 30]. The specificity of this test was assessed by the inclusion of reference isolates of the two common Campylobacter spp, C. coli and C. jejuni, as well as C. hepaticus negative and positive field samples. This PCR with HRM curve analysis was able to correctly detect C. hepaticus, and differentiate C. hepaticus from C. coli and C. jejuni. The sensitivity of the test was determined using serial 10-fold dilutions of the C. hepaticus reference sample with the sensitivity determined to be 10−3-ng of C. hepaticus DNA. The sensitivity of PCR and culture in detection of Campylobacter species has been reported to be very similar [31].

The positive samples in PCR-HRM which were collected from wild Pacific black duck were genetically 87.1% similar to C. canadensis which has been reported in captive whooping cranes (Grus Americana) in Canada [32]. While partial hsp60 gene sequences of wild bird samples (PBD-13 and PBD-14) show similarity with C. canadensis, full genome sequence of these samples is required for confirmation of the species. Among different Campylobacter species, C. coli, C. jejuni, C. lari and C. hepaticus are commonly associated with birds [8, 32–35]. While C. coli, C. lari and C. jejuni have also been reported in waterfowl, C. jejuni has been found more prevalent in faeces of waterfowl [36]. There is limited information on the biology of C. canadensis and its role in human health.

Banowary, Dang [14] concluded that PCR with HRM curve analysis is a powerful and valuable tool to study the nucleotide diversity of amplicons between tested specimens. The test enables detection of multiple species in the same test and is suitable for automation which makes this method a powerful diagnostic screening tool. There is also a reduction in the risk of cross-contamination as the PCR with HRM uses closed tubes, reducing the risk of DNA contamination of controls and samples [18].

This PCR-HRM curve analysis is suitable for the testing of birds suspected to have died due to SLD. This test would also be suitable for epidemiological studies at the processing plant, where a large volume of samples could be collected and tested to assess the prevalence of SLD on farms and regions. There is still considerable research required to understand the spread and pathogenicity of C. hepaticus and the test developed in this study could be useful to study disease prevalence. The PCR-HRM curve analysis showed both specificity and sensitivity for detection of C. hepaticus. This assay represents a rapid and sensitive diagnostic tool for assisting in the diagnosis of SLD in poultry.

Sensitivity of the C. hepaticus PCR (10× serial dilutions) gel electrophoresis image.

Lane M: DNA ladder, Lane 1–9: C. hepaticus DNA at 1 ng, 10−1 ng, 10−2 ng, 10−3 ng, 10−4 ng, 10−5 ng, 10−6 ng, 10−7 ng and 10−8 ng concentrations.

(TIF)

Click here for additional data file.

31 Mar 2021

PONE-D-21-00269

Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds

PLOS ONE

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In your Methods section, please provide additional details regarding the chicken and wild birds, including the number and species, used in your study and ensure you have described the source. For more information regarding PLOS' policy on materials sharing and reporting, see https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-materials.

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We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PONE-D-21-00269: Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds

The study evaluated high-resolution melt curve analysis to differentiate Campylobacter hepaticus from Campylobacter coli and Campylobacter jejuni. However, the authors have not conducted any other method comparison study to call the overall method “rapid.” Therefore, this rapid differentiation can be changed or removed or else should add about facts on time duration and justify accordingly.

The study does not include any experiment to find out the specificity of the assay. Also, the inclusion of other Campylobacter species and different related genus is important to assess the possibility of false-positive results.

In conclusion, “….rapid and sensitive diagnostic tool….” This can be rephrased or possibly soften appropriately

Change the figure no. to maintain the order

Lines 204-205: Rephrase this sentence

**********

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Reviewer #1: No

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7 Apr 2021

Academic Editor

Dear Prof. Iddya Karunasagar,

Thank you for your correspondence on 1st April 2021, regarding our manuscript Re: PONE-D-21-00269 (Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds).

We are delighted that the manuscript is received well by the reviewers and we thank them for their constructive comments. We have answered reviewers’ comments and carefully revised our manuscript based on the reviewers’ feedback. The revisions in the manuscript are highlighted in track changes.

We have also provided detailed responses to reviewers’ comments and concerns in a point-by-point manner, with their comments in red font. We also provided gel image as Supporting Information File.

We appreciate your time and effort during the evaluation process of our manuscript, and we look forward to hearing your editorial decision.

Kind regards

Seyed Ali Ghorashi

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

The manuscript was re-formatted based on PLOS ONE’s style.

2. In your Methods section, please provide additional details regarding the chicken and wild birds, including the number and species, used in your study and ensure you have described the source. For more information regarding PLOS' policy on materials sharing and reporting, see https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-materials.

Required information including the number of tested chickens, wild birds, species and location of collected samples were added to the text lines 87-90.

3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

The institutional permit for sample collection was added in the text lines 84-86.

4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

Gel image was provided as supporting information (S1 Fig) and the rest of figures were re-numbered accordingly.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

Done.

Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

Done.

6. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

The related phrase was removed from the text (line 179-180).

________________________________________

5. Review Comments to the Author

Reviewer #1: PONE-D-21-00269: Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds

The study evaluated high-resolution melt curve analysis to differentiate Campylobacter hepaticus from Campylobacter coli and Campylobacter jejuni. However, the authors have not conducted any other method comparison study to call the overall method “rapid.” Therefore, this rapid differentiation can be changed or removed or else should add about facts on time duration and justify accordingly.

To address this comment, additional text has been added to the discussion lines 245-249 and 253-256 and provided references that compared the time required to complete PCR-HRM with PCR gel electrophoresis (Thomsen et al., 2012 and Sarker et al., 2014) that confirms PCR-HRM is rapid when compared with PCR gel electrophoresis.

The study does not include any experiment to find out the specificity of the assay. Also, the inclusion of other Campylobacter species and different related genus is important to assess the possibility of false-positive results.

To evaluate the specificity of the assay, DNA was extracted from seven bacterial strains of genetically similar genera (Klebsiella, Pseudomonas, Enterobacter, Staphylococcus, streptococci, E. coli and Pasteurella) and tested in PCR-HRM to address this comment. This is now added to the text in lines 154-157 and 215-218 of new version of manuscript.

In conclusion, “….rapid and sensitive diagnostic tool….” This can be rephrased or possibly soften appropriately

This comment is addressed above (first comment).

Change the figure no. to maintain the order

One figure (gel image- Fig 4) was provided as supporting information file and the rest of figures were re-numbered accordingly to maintain the order in the text.

Lines 204-205: Rephrase this sentence

To address this comment, the sentence was revised in lines 211-212 of new version of manuscript.

________________________________________

Submitted filename: Response to reviewers Comments.docx

Click here for additional data file.

26 Apr 2021

Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds

PONE-D-21-00269R1

Dear Dr. Ghorashi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Iddya Karunasagar

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have addressed the reviewer comments satisfactorily.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

3 May 2021

PONE-D-21-00269R1

Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds

Dear Dr. Ghorashi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Iddya Karunasagar

Academic Editor

PLOS ONE