Development and Validation of TaqMan Real-Time Quantitative PCR Assay for Improved Diagnosis of Spotty Liver Disease

Spotty liver disease (SLD) is an acute infectious disease that primarily affects cage-free and free-range commercial layer hens. However, the disease is not exclusive to these systems, as the first reported case in the United States occurred in caged layers (1). Flocks affected by SLD suffer from significant economic losses because of increased mortality and decreased egg production. One of the most important signs of SLD is a drop in egg production, which ranges from 5% to 25% (2,3,4,5). This disease is increasingly becoming one of the most common and serious health issues for the egg production industry (4,6).

When SLD first appeared in the 1950s, it was described as avian vibrionic hepatitis in the United States, Canada, and Europe (7,8). There were several attempts to isolate the causative agent at the time; it was suggested that vibrio-like bacteria, Campylobacter jejuni, Campylobacter coli, and Helicobacter pullorum may have been implicated in disease pathogenesis, but this was not experimentally verified (9,10,11). In 2015, Crawshaw et al. (2) identified a novel Campylobacter species. When they infected 4-wk-old specific-pathogen-free birds with the newly discovered Campylobacter, microscopic lesions were induced in the liver, but no gross hepatic lesions were observed. Later, Van et al. (12,13) proposed the name Campylobacter hepaticus and demonstrated that C. hepaticus was the cause of SLD by reproducing the disease. Recently, Campylobacter bilis has also been reported as a second causative agent of SLD in poultry, after being isolated from bile samples of chickens with clinical signs of SLD from several farms in Australia (14,15).

The name “spotty liver disease” describes the most prominent gross pathology that can be seen at necropsy of affected birds, characterized by numerous white- to cream-colored necrotic spots across the liver surface (1,2,16), which may be associated with perihepatitis in some cases (4,6,17). The general appearance of a SLD-diseased flock is sometimes not easily noticed when only small percentages of birds are affected in the flock (1,17,18). The role that SLD plays in disease and mortality is unclear, as gross lesions of spotty liver have been occasionally found in apparently healthy birds of affected flocks (1). In addition to C. hepaticus and C. bilis, multifocal necrotizing hepatitis can be caused by a wide range of systemic bacterial diseases such as Escherichia coli (19), Salmonella enterica, Erysipelothrix rhusiopathiae (20), Staphylococcus aureus, Clostridium perfringens (21), and Pasteurella multocida (22). Therefore, reliance on clinical signs and postmortem lesions provides little diagnostic value.

Campylobacter hepaticus is a Gram-negative organism that is slow growing after 3 to 7 days of incubation under microaerophilic conditions at 37–42 C, and it has an S-shaped cell morphology. The individual bacterium measures 0.3–0.5 μm in diameter and approximately 1–3 μm in length and has a single unsheathed flagellum on one or both of its ends (2,12). The colony morphology shows cream-colored, flat-spreading wet colonies (12). Bacterial isolation is considered to be one of the classic diagnostic tools. However, bacterial isolation of all Campylobacter species, including C. hepaticus or C. bilis, is not easy and faces multiple challenges, including the fastidious nature of the bacteria and slow growth, in addition to the requirements of selective culture media and special culture conditions (23). Furthermore, the isolation of C. hepaticus is vulnerable to be overgrown by commensals and/or other pathogens, which is due to the lack of specific isolation media and the long incubation time under microaerophilic conditions (2,12,14). This eventually leads to an increase in the number of false negatives and misdiagnosis of SLD cases. Therefore, molecular identification of C. hepaticus implicated in SLD directly from clinical samples emerged as a promising superior alternative to the traditional bacteriologic culture due to the higher sensitivity, greater specificity, and shorter turnaround time (24,25).

Currently, only a DNA-binding dye quantitative PCR (qPCR) assay has been developed for the identification of C. hepaticus from clinical samples (16). However, this assay has been shown to have low specificity due to the nonspecific binding capacity of DNA binding dye, and it requires additional melting curve analysis for result confirmation. Furthermore, the most significant limitation is the nonspecific detection of C. bilis (15). This increases the number of flocks where C. hepaticus diagnosis cannot be confirmed. Given the limitations of the current C. hepaticus qPCR and the challenges in confirming diagnosis of C. hepaticus in the laboratory, this study aimed to develop and validate a new probe-based C. hepaticus–specific qPCR assay. This new diagnostic assay was designed to be highly sensitive and specific, enabling the rapid detection of C. hepaticus associated with SLD directly from clinical samples. It is important to differentiate between SLD caused by C. hepaticus versus SLD caused by C. bilis for further epidemiologic investigation and future vaccine development specific for each pathogen. Therefore, we are currently working on development of a qPCR assay specific for C. bilis.

The glycerol kinase (GK) gene was chosen as the target for the qPCR assay specific to C. hepaticus based on previous reports demonstrating that the GK gene sequence is unique in C. hepaticus and C. bilis and absent in other Campylobacter species such as Campylobacter jejuni and Campylobacter coli (13,16). To further verify this, a BLAST analysis was performed using the GK gene sequence (accession number: NZ_CP031611.1) in the GenBank nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=MegaBlast&PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&DATABASE=n/a&QUERY=&SUBJECTS) (25). The analysis confirmed that the GK gene sequence is specific to C. hepaticus and C. bilis genomes.

To identify a specific region within the GK gene that differentiates between C. hepaticus and C. bilis, all available sequences of the GK gene for C. hepaticus (n = 36) and C. bilis (n = 6) were obtained from GenBank. The sequences were then aligned using Clustal W (26).

Primers and the TaqMan® probe were designed within the selected region of the GK gene using the PrimerQuest™ Tool (https://www.idtdna.com/PrimerQuest/Home/Index) according to the general principles of PCR primers and probe design (27,28). Primers and probe were designed based on the divergent regions in the GK gene sequences between C. hepaticus and C. bilis, specifically targeting C. hepaticus. Our designed primers and probe were checked for their specificity through in silico analysis using the National Center for Biotechnology Information (NCBI) BLASTn against the nr/nt database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=MegaBlast&PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&DBSEARCH=true&QUERY=&SUBJECTS=). Due to the low numbers of the available complete genome sequences for C. hepaticus and the absence of any complete genome for C. bilis in the nr/nt database, another in silico analysis was done against the more comprehensive NCBI whole-genome shotgun contigs database (29). The oligonucleotides were analyzed using the Oligo Analysis tool (http://www.operon.com/tools/oligo-analysis-tool.aspx) to check the possibility of secondary structure and primer dimer formation. Moreover, we used the online Integrated DNA Technologies (IDT) OligoAnalyzer 3.1 tool (https://www.idtdna.com/calc/analyzer; accessed on 16 July 2024) to estimate the stability of any secondary structure or primer dimers using the Gibbs free energy (ΔG) (30,31). All oligonucleotides (primers and probe) were synthesized by IDT (IDT, Coralville, IA). The sequences of the primers and probe for our assay are shown in Table 1.

All qPCR reactions were conducted in a Real-Time PCR System 7500 (Applied Biosystems, Carlsbad, CA). Primers and probe were mixed in a 20-μl reaction vessel containing TaqMan Fast Virus 1-step Master Mix (5 μl; Applied Biosystems), primers (0.4 μmol final concentration), probe (0.2 μmol final concentration), water (8.145 μl), and DNA template (5 μl). The following amplification conditions were adopted based on the calculated melting temperature (Tm) of the primers and probes: 50 C for 5 min; 95 C for 20 sec with optics off; and 40 cycles of 95 C for 15 sec, followed by 60 C for 60 sec with optics on.

A nontemplate negative control (PCR-grade water) and a positive control (extracted DNA) from C. hepaticus isolates, confirmed by whole genome sequencing (WGS) using an Illumina MiSeq system (Illumina, San Diego, CA), were included in each run. We spiked an exogenous internal positive control (XIPC) at a concentration of 6250 copies per reaction and used the unpublished XIPC real-time qPCR assay developed by Iowa State University, Veterinary Diagnostic Laboratory (ISU-VDL), to monitor potential PCR inhibitors. All results were analyzed using SDS 1.5.1 software (Applied Biosystems).

Twenty-six C. hepaticus isolates, which are listed in Supplemental Table S1, were obtained from nine farms in the states of Iowa, Texas, Florida, and Kentucky, supplied by the Bacteriology section at ISU-VDL. These isolates were grown microaerophilically on blood agar and incubated at 37 C for 48–72 hr using established laboratory protocols. Additionally, 47 known SLD-positive clinical samples, which are listed in Supplemental Table S2, were obtained from 10 farms in the states of Iowa, Florida, Kansas, Kentucky, and Texas, supplied by ISU-VDL. To confirm the presence of C. hepaticus in these clinical samples, we utilized a DNA-binding dye qPCR assay followed by Sanger sequencing of the GK gene using the same primers (16). Eighteen clinically negative and culture-negative samples were also included, using liver homogenates and bile samples from apparently normal layer chickens with no history of SLD.

In total, 15 microorganisms were used in this study to analyze the exclusivity of the developed qPCR assay. These included six Campylobacter species other than C. hepaticus in addition to eight other bacterial isolates that are present in the gut or have been reported to cause SLD-similar lesions. All microorganisms included in this study are listed in Supplemental Table S1.

All bacterial isolates were harvested from the culture plate and resuspended in 1 ml of phosphate-buffered saline. The clinical tissue homogenates were prepared using Geno/Grinder (SPEX® SamplePrep, Metuchen, NJ), following the instructions provided by the manufacturer. Next, 100-μl aliquots of bacterial resuspension, tissue homogenate, bile samples, and cecal content were used to extract nucleic acid from the samples listed in Supplemental Tables S1 and S2. The MagMAX™ Pathogen RNA/DNA Kit was used for nucleic acid extraction, on a Kingfisher-Flex instrument (Thermo Fisher Scientific, Waltham, MA), following the instructions provided by the manufacturer. Finally, nucleic acids were eluted into 90 μl of elution buffer.

An in silico check for the specificity of the primers and probe included in this study was performed as previously described in primer and probe design section.

We conducted validation and evaluation of our qPCR assay according to the published guidelines (32,33), which are explained in the following steps:

For analytical specificity evaluation, we measured the ability of the qPCR assay to detect different isolates of our target (C. hepaticus; inclusivity) and non-target microorganisms (exclusivity). Inclusivity was evaluated by testing our assay against 26 C. hepaticus isolates listed in Supplemental Table S1. For exclusivity, we tested our assay against a panel of microorganisms that included six other Campylobacter species in addition to eight other bacterial isolates that are likely to cause SLD-similar lesions.

Sensitivity was evaluated by estimating the limit of detection (LOD), cycle threshold (C_T) cutoff value, and the limit of quantification (LOQ) of the developed assay.

The LOD is the lowest copy number that the assay can detect in the presence of the target in more than 95% of the tested replicates (34). Based on determination of the LOD, the equivalent C_T value was set as the C_T cutoff value of the assay (35). The LOQ is defined as the lowest copy number that can be reliably quantified where the standard curve maintains a linear pattern. These parameters were calculated through the following steps:

1. Construction of C. hepaticus gBlock. A gBlock is a double-stranded synthetic DNA fragment, which in this case represented our target sequence containing the forward and reverse primers and probe sequences. A gBlock of 350 bp was designed, ordered from IDT (and prepared following the IDT recommendations. Then, the DNA concentration was measured using a Qubit Fluorometric analysis double‐stranded DNA high-sensitivity scheme kit (Invitrogen™, Eugene, OR), and the copy number/μl was calculated using the following equation:

   

   where x = Qubit read (ng/μl); N = length of the insert; and 660 g/mol = average mass of 1 bp of double-stranded DNA.

2. Generation of standard curves. Tenfold serial dilutions (1 × 10⁸ to 1 × 10⁰ copies/μl) of the gBlock were prepared followed by three twofold serial dilutions from the last positive dilution to generate the standard curve. The LOD was estimated using the average C_T value for each dilution from four independent runs consisting of four replicates. To create linear equations with R² values for our target, the average C_T values were plotted against log₁₀ of tenfold serial dilutions of the construct (copy number/μl).

   The following parameters were estimated using the generated standard curve:

   • Coefficient of determination (R²): R² is the square of the Pearson correlation coefficient (r). It evaluates the linearity of the data and gives an indication of the consistency of serial dilutions/pipetting errors.

   • qPCR efficiency (%): This value indicates the fraction of target molecules doubled in one PCR cycle. It is estimated from the slope of the standard curve according to the following equation: Efficiency = [10(−1/slope) − 1] × 100, using the ThermoFisher Scientific qPCR Efficiency Calculator tool (https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/qpcr-efficiency-calculator.html).

   • Dynamic range: This is the range of the standard curve's highest and lowest detectable copy numbers when acceptable linearity (R² ≥ 0.98) and efficiency (between 90% and 110%) are observed.

3. Diagnostic specificity and diagnostic sensitivity (%). The diagnostic specificity (%) and diagnostic sensitivity (%) of each assay were calculated using the following formulae (36):

   
   and

   

   where TN = true negative, TP = true positive, FN = false negative, and FP = false positive.

4. Repeatability (intra-assay variation) and reproducibility (interassay variation). Every single qPCR run for each dilution of tenfold serial dilutions was tested four times (repeatability) in four independent runs (reproducibility). To assess the precision of each assay, both the standard deviation (SD) and the coefficient of variability (CV%) of the average C_T were calculated. CV% was estimated by dividing the SD by the average C_T values obtained for each tenfold serial dilution for repeatability, and this was calculated for the four runs to evaluate reproducibility. All validation runs were conducted on different days.

A univariate linear regression model was built using the lm function in R (37) to characterize the association between sample type (liver homogenate, bile, and cecal content) and C_T values. Post-hoc pairwise comparisons of the least square means were performed using the Tukey honestly significant difference test, with statistical significance evaluated at the 5% level.

We selected the GK gene as the target for the C. hepaticus–specific qPCR assay because the BLAST analysis of the GK gene between C. hepaticus and C. bilis revealed a high percentage of identity, which aligns with previous reports indicating that the GK gene shares 90.55% identity between C. hepaticus and C. bilis (15). Regions showing sequence divergence between the two species were selected for the design of C. hepaticus–specific PCR primers and probes.

BLAST analysis against NCBI BLAST nr/nt and whole-genome shotgun contigs databases confirmed the specificity of the target gene GK (1488 bp in length) to C. hepaticus and C. bilis only. We designed the forward primer with a high GC clamp and two mismatches at the last four nucleotides at the 3′ prime, allowing a perfect match with C. hepaticus but not with C. bilis. While the probe was designed to be conserved across all available C. hepaticus strains, incorporating a total of five mismatches with C. bilis ultimately increased the assay's specificity for C. hepaticus and not C. bilis. The forward and reverse primers were designed to amplify a 77-bp segment from nt number 1,216,706 to 1,216,782 (numbering according to accession number NZ_CP031611.1) (Table 1). The probe was designed to anneal six nucleotides away from the reverse primer. The difference in Tm of the forward and reverse primers within each assay was less than 1 C. The probes showed Tm (4 C) higher than the primers.

The in silico analysis through BLAST revealed that the designed primers and probe are highly specific to C. hepaticus. The query coverage and the maximum identity of the oligonucleotides were all 100% only to C. hepaticus sequences.

As regards the formation of primer dimers (self- or heterodimer) or primer/probe dimers, results showed no significant secondary structures observed, with a ΔG value below the acceptable threshold of −9 kcal/mol.

Our developed qPCR assay showed 100% specificity to all 26 isolates of C. hepaticus with no cross-reactivity with any other tested microorganisms, nor with C. bilis isolates listed in Supplemental Table S1.

The LOD of C. hepaticus in our assay was 5 copies/μl with a C_T cutoff value equal to 34.01. As our assay could not maintain the linearity on the standard curve to the LOD, the LOQ was limited to 10 copies/μl, as shown in Fig. 1 and Table 2.

Plotting average C_T values from four independent runs against log ₁₀ of tenfold serial dilutions (10⁸–10¹ copies/μl) generated a linear equation with an R² equal to 1, as shown in Table 3. Based on the standard curve slope represented in Supplemental Figure S1, the qPCR efficiency of our assay was 95.15%. Our assay showed a wide dynamic range from C_T 8.4 to C_T 32.6, as shown in Table 3.

Our assay showed diagnostic sensitivity and specificity equal to 100%. It detected 47 true known positive SLD clinical samples (liver, bile, and cecal content) and 26 C. hepaticus isolates, with no cross-reactivity against clinical samples from apparently normal birds or any other tested microorganisms listed in Supplemental Tables S1 and S2.

The intra-assay variation (repeatability) to their LOQ was evaluated in terms of C_T coefficient of variation (%CV), which ranged from 0.10 to 1.56 among the different runs. The %CV representing the interassay variation (reproducibility) ranged from 0.25 to 0.94, as shown in Table 3. These values reveal good repeatability and acceptable reproducibility of the developed qPCR assay (where %CV less than 10% is acceptable for intra-assay variability, and 15% is acceptable for interassay variability; 38).

Comparison among C_T values from different sample types (liver homogenate, bile, and cecal content) showed that the tested bile samples had significantly (p < 0.0001) lower C_T values compared to liver homogenate samples. C_T values obtained from bile samples were also much lower than those from the cecal contents. Our study suggests that bile samples are the most sensitive for C. hepaticus detection by the newly developed qPCR assay, as shown in Table 4 and Fig. 2.

SLD has emerged in the United States since the 1950s as one of the important differentials of multifocal necrotizing hepatitis leading to reduced egg production in egg-laying flocks (39,40,41). The causative agent of the disease has been identified as C. hepaticus and, more recently, as C. bilis as well. The first fundamental step for implementing prevention, control, and management strategies for any infectious disease is accurate and rapid diagnosis. However, the current available laboratory diagnostic tools for detection of C. hepaticus cannot achieve that target. Bacterial isolation and identification of the causative agent represent the gold standard for laboratory diagnosis of SLD. However, bacterial isolation of Campylobacter species is difficult to achieve due to its fastidious, slow-growing nature, requirement of selective culture media, and special culture conditions (23).

Molecular identification of Campylobacter species from clinical samples complements the traditional bacteriologic isolation. Currently, a DNA-binding dye qPCR assay is available for identifying C. hepaticus from clinical samples (16). However, this assay has several limitations. Most notably, it cannot distinguish between C. hepaticus and the newly identified C. bilis in clinical samples. Additionally, it lacks specificity and may bind nonspecifically to any double-stranded DNA (16,42). From a diagnostic perspective, probe-based qPCR offers a superior diagnostic alternative, which has gained popularity due to its speed, high sensitivity, and greater specificity (24,25), leading this technology to constitute the most popular diagnostic assays in veterinary diagnostic laboratories. Therefore, we aimed in this study to develop and validate a specific probe-based qPCR assay to enhance diagnosis of SLD from clinical samples.

The GK gene was selected as a target for our newly developed assay because it has been reported to have unique sequences for both C. hepaticus and C. bilis and could be used to differentiate these two species from other Campylobacter species such as C. jejuni and C. coli (16). This was confirmed by conducting BLAST searches for the gene against the NCBI database (43). We initially set out to design two different PCR assays, one specific for C. hepaticus and the other specific for C. bilis, using the same target gene, which is the GK gene. However, we were successful in designing a specific assay only for C. hepaticus and not for C. bilis. This was due to the limited genetic differences within the GK gene between the two species that allowed for the design of only one specific assay. This could be resolved by conducting genomic comparisons to identify other genetic targets that are unique to C. bilis and by designing a second assay that is specific only for this bacterium. However, this was beyond the scope of this study; therefore, we decided to present the C. hepaticus–specific assay in this current article and consider the development of the C. bilis assay for future research. Nevertheless, accurate diagnosis of C. hepaticus apart from C. bilis will be helpful for flocks with potential mixed infections or flocks currently using or considering the use of autogenous vaccines.

Oligonucleotides of the developed qPCR assay showed optimum design with the absence of any significant primer dimer formation or primer-probe heterodimer formation. The newly designed qPCR assay showed high specificity to C. hepaticus, and it was able to inclusively detect different C. hepaticus isolates with no cross-reactivity to C. bilis as the primers and probe were constructed based on detection of the region of divergence in GK gene sequences of the two species. The newly developed assay also showed no cross-reactivity to other Campylobacter species or any other tested microorganisms that could be present in the intestinal tract or that can cause lesions similar to SLD, such as Escherichia coli, Salmonella enterica, Erysipelothrix rhusiopathiae, Staphylococcus aureus, Clostridium perfringens, Pasteurella multocida and Enterococcus faecium. The LOD for the newly developed assay was 5 copies/μl, revealing high analytical sensitivity. This is in accordance with guidelines, which state that positive results should be detected in 95% of tested replicates (34). The coefficient of determination (R²) is used mainly to assess the linearity of the data (44), in addition to detecting variability among the assay replicates. An R² threshold value ≥0.98 is essential to indicate a good qPCR assay. The linearity of our developed qPCR assay, reflected by R², was maintained (R² = 1) over eight logs of dynamic range. The efficiency of PCR is a crucial indicator for evaluating the performance of a qPCR assay (45). Ideally, the qPCR efficiency should be 100%, which indicates doubling of PCR product with each amplification cycle (46). However, practically, the efficiency parameter has been reported to fall within the range of 90% to 110% (33,47). The developed qPCR showed efficiency of 95.15%, which falls within the acceptable limit for an efficient qPCR assay.

Previous studies have demonstrated that C. hepaticus can be isolated from the liver and bile samples of infected birds (2,12), and its colonization has also been observed in the small intestine and ceca (13). In this study, the results indicated that bile samples were the most effective for detecting C. hepaticus, as evidenced by the statistically lower C_T values compared to those from liver homogenate and cecal contents. For a qPCR assay to be used as a dependable and reliable diagnostic test, it needs to show a good degree of both repeatability and reproducibility. Our developed qPCR assay exhibited a high level of both repeatability and reproducibility. This signifies its strength and indicates its validity as a reliable diagnostic tool for SLD caused by C. hepaticus.

In conclusion, this study successfully developed a novel TaqMan qPCR assay for the efficient and sensitive identification and quantification of C. hepaticus in clinical samples. The assay demonstrated 100% specificity in detecting C. hepaticus from both isolates and clinical samples, making it a valuable diagnostic tool for enhancing the diagnosis and control of SLD in layer flocks. While this qPCR assay significantly improves our diagnostic capabilities for C. hepaticus, further research is needed to develop and validate a diagnostic assay for C. bilis, another important causative agent of SLD.

Supplemental data associated with this article can be found at https://doi.org/10.1637/aviandiseases-D-24-00066.s1.

We acknowledge Dr. Orhan Sahin and Dr. Qijing Zhang for providing us with Campylobacter isolates.

Abbreviations:

C_T =

cycle threshold;

CV =

coefficient of variation;

D-SN =

diagnostic sensitivity;

D-SP =

diagnostic specificity;

FN =

false negative;

FP =

false positive;

GK gene =

glycerol kinase gene;

IDT =

Integrated DNA Technologies;

ISU-VDL =

Iowa State University, Veterinary Diagnostic Laboratory;

LOD =

limit of detection;

LOQ =

limit of quantification;

NCBI =

National Center for Biotechnology Information;

qPCR =

real-time quantitative PCR;

SLD =

spotty liver disease;

Tm =

melting temperature;

TN =

true negative;

TP =

true positive;

WGS =

whole genome sequencing;

XIPC =

internal positive control