Abstract
Background
Giardia duodenalis is a protozoan of dogs and cats that comprises several genotypes (assemblages A–G). The common assemblages affecting small animals (C, D, F, G) are not associated with disease in humans. However, assemblages A and B can be zoonotic. Several assays can be used for detection of G. duodenalis, but there is a need for an accurate and faster detection method for G. duodenalis zoonotic assemblages. The aim of this study was to compare a beta-giardin quantitative polymerase chain reaction (PCR) (bg-qPCR) to a standard multilocus genotyping technique for detection of G. duodenalis zoonotic assemblages in dog and cat feces.
Methods
Canine and feline fecal samples submitted to a commercial laboratory (Antech Diagnostics) that tested positive for Giardia by centrifugal flotation, Giardia enzyme-linked immunosorbent assay (ELISA), and quantitative small subunit ribosomal ribonucleic acid PCR or those positive for Giardia spp. by a commercial direct fluorescent assay were included in this study (140 cases with zoonotic and non-zoonotic assemblages). The bg-qPCR assay was optimized and then all samples were assessed by both methods. Agreement among the two methods was assessed by Cohen’s kappa agreement.
Results
A total of 140 samples that were previously positive for Giardia were analyzed by both bg-qPCR and multilocus genotyping. Both bg-qPCR and multilocus genotyping identified 70 of 76 (92.10%) of assemblage A and B samples. A total of 64 (n = 40 for assemblage A and n = 24 for assemblage B) of 76 samples yielded concordant results, with both diagnostic techniques giving a Cohen’s kappa agreement value of 0.828. Non-zoonotic assemblages were amplified only by multilocus genotyping in 55 samples, and 9 samples were negative for both methods. Multilocus genotyping reported mixed infections in 23 cases and included A/D (1 dog), B/C (1 dog), B/F (1 dog; 2 cats), and C/D (18 dogs).
Conclusions
For the zoonotic assemblages, the results between multilocus genotyping and the bg-qPCR agreed for most cases. These data support the bg-qPCR as a less expensive and laborious option to determine zoonotic Giardia A/B assemblages in feces of dogs and cats.
Graphical Abstract
Keywords: Giardia duodenalis, Beta giardin, qPCR, Multilocus genotyping, Dogs, Cats
Background
Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a common protozoan in dogs and cats that also infects other mammals including humans. It comprises several genotypes (assemblages A–G). The most common assemblages affecting dogs and cats (C, D, F) are not associated with disease in humans. However, the zoonotic assemblages A and B can be detected in feces of some dogs and cats. While several assays have been developed to identify zoonotic assemblages of G. duodenalis in humans, there remains a significant need for more precise, affordable, efficient diagnostic methods with quick turnaround specifically designed to detect zoonotic assemblages of G. duodenalis in companion animals [1, 2]. Developing such methods is crucial to better understand the role of companion animals in zoonotic transmission and to implement effective public health measures for disease prevention.
A meta-analysis reported an overall prevalence of Giardia in dogs and cats of 15% and 12%, respectively [3]. However, in dogs, infection rates ranged between 1.9% and 57.9% [4]. The occurrence of G. duodenalis in cats varied between 1.3% and 27.3% [4]. The majority of the G. duodenalis isolates genotyped from dogs were dog-specific assemblages C and D [4–7]. In cats, most isolates that were genotyped were assemblage F followed by assemblage A [8].
In humans, G. duodenalis is also a very common protozoan and is estimated to cause approximately 280 million human cases of diarrhea per year [9]. Giardia duodenalis can cause a variety of clinical signs in people and companion animals, ranging from the subclinical passage of cysts in feces in some individuals to severe, acute, and potentially chronic diarrhea in others. The risk for people of acquiring Giardia infections directly from dogs and cats is relatively low; however, pets shedding potentially zoonotic assemblages contribute to environmental contamination. In addition, people with compromised immune systems could experience symptoms of longer duration or severe complications after contact with pathogens, including those carried by animals [10].
The molecular characterization of Giardia duodenalis has been conducted using several genetic markers, including small subunit ribosomal RNA (SSU rRNA), beta-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) [11–14]. However, many primers designed to amplify the SSU rRNA gene target a short and conserved fragment, which limits the ability to differentiate between G. duodenalis assemblages [15]. As a result, protein-coding genes such as gdh, bg, and tpi are more commonly used for genotyping [15].
Genotyping inconsistencies have often been reported in G. duodenalis isolates from dogs. The choice of genetic markers can influence whether isolates are identified as host-adapted, non-zoonotic assemblages (C and D) or as zoonotic assemblages (A or B), which have the potential to infect humans [12, 16].
Owing to the variability in amplification rates of different genes and the preferential amplification of assemblages by specific primers, a multilocus genotyping approach is recommended [17]. Multilocus genotyping reduces the risk of misclassification that can occur with single-locus genotyping, provides enhanced resolution, enables the detection of mixed infections, and offers a more comprehensive understanding of host-specific and zoonotic assemblages.
Despite its advantages, multilocus genotyping has limitations. These include the potential for false-negative results due to differences in gene amplification rates and its time-intensive and costly nature [18]. While multilocus genotyping is widely considered the most informative method for characterizing G. duodenalis assemblages, single-locus genotyping remains a valid alternative, particularly when targeting genes with high sequence heterogeneity, such as bg.
Beta-giardin is part of a group of structural proteins that are considered unique to Giardia and so is a highly specific marker [19]. Several studies reported the bg gene to be more informative than the other genes used in multilocus analysis [20–22]. Therefore, we decided to base the quantitative polymerase chain reaction (qPCR) assay described here on the bg gene (bg-qPCR assay). The aims of this study were to optimize the performance of the bg-qPCR assay for detection of G. duodenalis zoonotic assemblages A and B in dog and cat feces and to compare the results with the standard multilocus genotyping technique using results from three genes.
Methods
Sample collection
Canine and feline fecal samples submitted to a commercial reference laboratory (Antech Diagnostics) that tested positive/detected for Giardia by at least one of the following methods: centrifugal flotation (ova and parasite [O&P]), Giardia enzyme-linked immunosorbent assay (ELISA), and quantitative small subunit ribosomal ribonucleic acid PCR (SSU rRNA-qPCR) were included in this study. The SSU rRNA-qPCR used primers previously described [11]. A subset of canine and feline samples that tested positive for Giardia spp. by a commercial direct fluorescent assay (Merifluor® Cryptosporidium/Giardia, Meridian Bioscience Cincinnati, Ohio) was also included.
Analytical validation of the bg-qPCR assay
The final primer set and methods of the bg-qPCR assay are proprietary and offered commercially (KeyScreen™ GI Parasite PCR, Antech Diagnostics, Inc.). The validation experiments described here were performed using previously established analytical parameters [23].
DNA extraction
Feces (150 mg) and 750 µl guanidinium thiocyanate-based lysis solution were added into a bashing bead tube containing zirconia beads (Spex Sample Prep, Metuchen, NJ, USA) and then homogenized in a Mini G (Spex SamplePrep) for 7.5 min according to the manufacturer’s recommendations. Lysates were cleared by centrifugation, and 200 µl of the supernatant was used to extract total nucleic acid in a magnetic bead-based automated extractor (KingFisher Apex, Thermo Fisher, Waltham, MA, USA). Nucleic acids were eluted in 120 µl of nuclease-free water and 5 µl used in 12 µl total volume real-time PCR reactions.
PCR design
Giardia duodenalis assemblage A- and B-specific hydrolysis probes were adapted from previously published regions within the bg gene targeting a variable region around nucleotide position 473 (KM190684 [24];). Hydrolysis probes included locked nucleic acids (IDT, Coralville) [25]. Assemblage specific multisequence alignments were used to design additional primers than previously published allowing amplification of more recently deposited sequences across the different G. duodenalis assemblages [26]. Flanking sequencing primers amplifying a 335-base pair (bp) region of the coding sequence of the bg gene allowed amplification and sequence confirmation of a larger fragment of the Giardia bg amplicon described in this study. The sequence data from G. duodenalis isolates by the bg-qPCR was compared by Basic Local Alignment Search Tool (BLAST) analysis with sequences from the nucleotide database from the GenBank (National Center for Biotechnology Information (NCBI) BLAST, National Center for Biotechnology Information, Bethesda, MD, USA).
Analytical validation was determined using synthetic positive controls designed by overlapping the real-time PCR primers by 15 nucleotides (Table 1). All qPCR reactions were run in an LC480 (Roche, Indianapolis, IN, USA) using standard cycling parameters according to the manufacturer. qPCR reactions contained KAPA3G master mix in combination with uracil–DNA–glycosylate (UNG) to prevent PCR product carry-over (Roche, Indianapolis, IN, USA) in a total volume of 12 µl.
Table 1.
Analytical validation results of qPCR, including ten-component validation using synthetic positive controls and clinical samples
| Precision CV | Analytical | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Within-run | In-between runs | ||||||||||
| Range | Slope | Efficiency | R2 | Cp | Absolute | Cp | Absolute | Sensitivity | Specificity | ||
| Requirements | > = 5 | b | 90–110% | > = 0.991 | < 3% | < 20% | < 3% | < 20% | 10 | “Confirmed by” | |
| G. duodenalis | Synthetic positive control | 5 (6*) | −3.215 | 104.7 | 0.9988 | 0.84 | 2.2 | 1.89 | 11.09 | < 10 | Gene sequencing |
| G. duodenalis assemblage A | Synthetic positive control | 5 (6*) | −3.2264 | 104.1 | 0.999 | 2.88 | 17.8 | 2.91 | 18.4 | < 10 | Gene sequencing |
| G. duodenalis assemblage B | Synthetic positive control | 5 (6*) | −3.2063 | 105.1 | 0.994 | 0.98 | 3.2 | 1.1 | 3.43 | < 10 | Gene sequencing |
6*, actual standard curve extended over six orders of magnitude
Analytical sensitivity
Synthetic nucleic acids from assemblage A and B were synthesized using solid-phase phosphoramidite chemistry (Ultramer, IDT DNA, Coralville, Iowa) and used as positive controls. Concentrations were determined by the manufacturer. These were tenfold serially diluted to determine the limit of detection (LOD). Each dilution was run in triplicate to calculate averages, standard deviations, and coefficients of variation (CVs). In addition, fecal samples that tested positive for G. duodenalis O&P and SSU rRNA-qPCR were used to quantify cysts using a commercial direct immunofluorescence assay. Two samples with high concentrations of Giardia cysts (40,000 and 50,000 cysts per gram, respectively) were serially diluted to achieve final concentrations of 4 and 5 cysts per gram. For each sample, three slides were prepared, and Giardia cysts were counted using a fluorescent microscope. Nucleic acids extracted from these preparations were then serially diluted in tenfold increments to determine the limit of detection (LOD) of the assay.
Analytical specificity
Nucleic acids were extracted from samples that tested positive for Cystoisospora spp., Ancylostoma spp., Cryptosporidium canis, Cryptosporidium felis, Dipylidium caninum, Toxoplasma gondii, Neospora caninum, Tritrichomonas blagburni, Toxocara canis, Toxocara cati, and Trichuris vulpis at either fecal zinc sulfate-based centrifugation and flotation, ELISA, or qPCR for each of the parasites (Table 2). These qPCR tests were confirmed for analytical specificity by using outside sequencing primers with conventional Sanger sequencing and BLAST analysis (NCBI BLAST, National Center for Biotechnology Information, Bethesda, MD, USA) as previously described [30]. The extracted nucleic acid was then assessed using Giardia bg qPCR.
Table 2.
Specificities of SSU rRNA and beta-giardin primer and probe sets using characterized parasite DNA samples from clinical samples
| Detection by qPCR | |||||
|---|---|---|---|---|---|
| G. duodenalis | G. duodenalis beta giardin | G. duodenalis beta giardin | |||
| Parasite | Animal | Confirmation method | SSU rRNA | Assemblage A | Assemblage B |
| Giardia duodenalis | Dog* | ELISA, qPCR, multilocus sequencing | Positive | Positive | Negative |
| Ancylostoma caninum | Dog | O&P, qPCR, sequencing | Negative | Negative | Negative |
| Toxocara canis | Dog | O&P, qPCR, sequencing | Negative | Negative | Negative |
| Toxocara cati | Cat | O&P, qPCR, sequencing | Negative | Negative | Negative |
| Trichuris vulpis | Dog | O&P, qPCR, sequencing | Negative | Negative | Negative |
| Cryptosporidium canis | Dog | qPCR, sequencing | Negative | Negative | Negative |
| Cryptosporidium felis | Cat | qPCR, sequencing | Negative | Negative | Negative |
| Toxoplasma gondii | Cat | qPCR, sequencing | Negative | Negative | Negative |
| Neospora caninum | Cattle | qPCR, sequencing | Negative | Negative | Negative |
| Tritrichomonas blagburni | Cat | qPCR, sequencing | Negative | Negative | Negative |
| Dipylidium caninum | Cat | O&P, qPCR, sequencing | Negative | Negative | Negative |
| Cystoisospora spp. | Dog | O&P, qPCR, sequencing | Negative | Negative | Negative |
*Sample no. 4z from Table 3
Multilocus genotyping method
The G. duodenalis-nested PCR assays and the sequencing of the amplified genes gdh, bg, tpi generic, and tpi dog-specific were performed using published protocols with some modifications [6, 12–14, 27]. In the gdh protocol, 24 µl per reaction of a commercial master mix (Hot Start Qiagen Master Mix, Qiagen, Germantown, Maryland, USA) and 24 µl of water were used as a replacement for the master mix described in the original publication. Furthermore, 2 µl of genomic DNA were included per reaction, instead of 1 µl, as specified in the original protocol [12].
For the bg protocol, 24 µl per reaction of the same commercial master mix (Hot Start Qiagen Master Mix, Qiagen, Germantown, Maryland, USA) and 24 µl of water were used, replacing the master mix described in the original publication [14].
In the tpi protocol, the annealing temperatures for the first and second amplification reactions were adjusted to 51 °C and 55 °C, respectively, compared with the 50 °C annealing temperature described in the original protocol [13]. In addition, the total reaction volume was reduced to 50 µl, whereas the original protocol specified a volume of 100 µl [13]. The thermal protocols of the gdh and bg assays were identical to those described in the original methods.
DNA sequences were analyzed in forward direction using an ABI3100 Genetic Analyzer (Applied Biosystems, Foster City, California). The sequence data from G. duodenalis isolates by the three genes was compared by BLAST analysis with sequences from the nucleotide database from the GenBank (NCBI BLASTn, National Center for Biotechnology Information, Bethesda, MD, USA). Sequences were also aligned with reference strains of assemblage A sub-assemblages to confirm sub-assemblage identity [4]. Representatives of the nucleotide sequences generated in this study were placed in GenBank under the accession numbers OQ434171–OQ434188.
Methods of analysis/assay comparisons
Cohen’s kappa was used to determine the level of agreement between the bg-qPCR and multilocus genotyping (MLG) results for assemblage A and B. Values range from 0.01 to 0.20: none to slight agreement to 0.21–0.40: fair agreement, 0.41–0.60: moderate agreement, 0.61–0.80: substantial agreement, and 0.81–1.00: almost perfect agreement. For the calculation of Cohen’s kappa, samples were classified in five groups (sort key presented in Table 3):
1 (Sort key 1): A/B bg-qPCR positive/A/B MLG positive;
2 (Sort key 2): A/B bg-qPCR positive/A/B MLG negative or assemblages CDFG;
3 (Sort key 3): A/B bg-qPCR negative/A/B MLG positive;
4 (Sort key 4): A/B bg-qPCR negative/assemblages CDF MLG positive, and
5 (Sort key 5): A/B bg-qPCR negative/A/B MLG negative.
Table 3.
Comparison of bg-qPCR and multilocus genotyping results
| Sample ID | Species | bg-qPCR | Multilocus genotyping genes | Original classification | Sort key | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| BG | GDH | TPI—generic primers | TPI—dog-specific primers | Assemblage classification multilocus | ||||||
| 1z | Canine | A | AI | A | A | Negative | A | A | 1 | |
| 2z | Canine | A | AI | A | A | Negative | A | A | 1 | |
| 4z | Canine | A | A | A | A | Negative | A | A | 1 | |
| 5z | Canine | B | B | B | C | Negative | B/C | B | 1 | |
| 6z | Canine | B | B | B | B | Negative | B | B | 1 | |
| 9z | Canine | B | B | BIII | BIII | Negative | B | B | 1 | |
| 10z | Canine | B | B | BIII | B | B | B | B | 1 | |
| 11z | Canine | B | B | B | BIII | BIII | B | B | 1 | |
| 12z | Canine | B | F | B | BIII | Negative | F/B | B | 1 | |
| 13z | Canine | B | B | BIV | BIII | Negative | B | B | 1 | |
| 8122 | Canine | A | AI | A | A | AI | A | A | 1 | |
| 8089 | Canine | A | A | Negative | Negative | Negative | A | A | 1 | |
| CSU 5 | Canine | A | A | A | A | Negative | A | A | 1 | |
| CSU 7 | Canine | A | Negative | Negative | A | Negative | A | A | 1 | |
| CSU 8 | Canine | B | B | B | B | B | B | B | 1 | |
| CSU 11 | Canine | A | A | Negative | AI | Negative | A | A | 1 | |
| CSU 12 | Canine | B | Negative | BIV | B | B | B | B | 1 | |
| CSU 13 | Canine | A | A | A | A | Negative | A | A | 1 | |
| CSU 17 | Canine | A | A | A | AI | A | A | A | 1 | |
| CSU 19 | Canine | A | A | A | AI | AI | A | A | 1 | |
| CSU 29 | Canine | B | BIII | BIV | B | Negative | B | B | 1 | |
| CSU 30 | Canine | B | BIV | Negative | B | Negative | B | B | 1 | |
| CSU 35 | Canine | A | A | A | AI | Negative | A | A | 1 | |
| CSU 39 | Canine | A | A | A | AI | AI | A | A | 1 | |
| CSU 40 | Canine | B | B | BIV | B | B | B | B | 1 | |
| CSU 44 | Canine | A | Negative | A | A | A | A | A | 1 | |
| CSU 45 | Canine | A | A | A | AI | Negative | A | A | 1 | |
| CSU 49 | Canine | A | Negative | A | AI | AI | A | A | 1 | |
| 8z | Canine | B | G | G | Negative | Negative | G | G | 2 | |
| CSU 26 | Canine | A | Negative | Negative | Negative | Negative | Negative | Negative | 2 | |
| CSU 27 | Canine | A | Negative | Negative | Negative | Negative | Negative | Negative | 2 | |
| CSU 2 | Canine | A/B undetected | A | D | Negative | D | A/D | A/D | 3 | |
| CSU 6 | Canine | A/B undetected | Negative | A | A | Negative | A | A | 3 | |
| CSU 10 | Canine | A/B undetected | BIV | BIV | B | Negative | B | B | 3 | |
| CSU 64 | Canine | A/B undetected | BIII | B | Negative | Negative | B | B | 3 | |
| 14nz | Canine | A/B undetected | C | C | Negative | C | C | Negative | 4 | |
| 15nz | Canine | A/B undetected | C | C | C | C | C | Negative | 4 | |
| 16nz | Canine | A/B undetected | C | C | C | C | C | Negative | 4 | |
| 17nz | Canine | A/B undetected | C | C | Negative | C | C | Negative | 4 | |
| 18nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 19nz | Canine | A/B undetected | C | C | C | C | C | Negative | 4 | |
| 22nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 23nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 24nz | Canine | A/B undetected | D | D | C | D | D/C | Negative | 4 | |
| 25nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 26nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 27nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 28nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 29nz | Canine | A/B undetected | C | C | Negative | C | C | Negative | 4 | |
| 30nz | Canine | A/B undetected | C | C | C | D | C/D | Negative | 4 | |
| 31nz | Canine | A/B undetected | D | Negative | Negative | D | D | Negative | 4 | |
| 32nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 33nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 34nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 35nz | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 36nz | Canine | A/B undetected | C | C | C | C | C | Negative | 4 | |
| 6113 | Canine | A/B undetected | C | C | Negative | Negative | C | Negative | 4 | |
| 6114 | Canine | A/B undetected | C | C | Negative | C | C | Negative | 4 | |
| 6143 | Canine | A/B undetected | C | C | Negative | D | C/D | Negative | 4 | |
| 6144 | Canine | A/B undetected | C | C | Negative | D | C/D | Negative | 4 | |
| 6137 | Canine | A/B undetected | C | C | Negative | D | C/D | Negative | 4 | |
| 6145 | Canine | A/B undetected | D | D | Negative | C | D/C | Negative | 4 | |
| 6146 | Canine | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 6147 | Canine | A/B undetected | D | D | Negative | C | D/C | Negative | 4 | |
| 6140 | Canine | A/B undetected | C | C | Negative | C | C | Negative | 4 | |
| 6268 | Canine | A/B undetected | D | Negative | Negative | Negative | D | Negative | 4 | |
| 6269 | Canine | A/B undetected | D | D | Negative | C | D/C | Negative | 4 | |
| 6282 | Canine | A/B undetected | D | D | Negative | Negative | D | Negative | 4 | |
| 6284 | Canine | A/B undetected | D | Negative | Negative | C | C/D | Negative | 4 | |
| 6285 | Canine | A/B undetected | D | D | Negative | C | D/C | Negative | 4 | |
| 6286 | Canine | A/B undetected | C | Negative | Negative | C | C | Negative | 4 | |
| 6786 | Canine | A/B undetected | C | D | Negative | D | C/D | Negative | 4 | |
| 6782 | Canine | A/B undetected | C | C | Negative | D | C/D | Negative | 4 | |
| 6783 | Canine | A/B undetected | C | Negative | Negative | D | C/D | Negative | 4 | |
| 6785 | Canine | A/B undetected | D | C | Negative | C | D/C | Negative | 4 | |
| 6901 | Canine | A/B undetected | C | C | Negative | D | C/D | Negative | 4 | |
| 6905 | Canine | A/B undetected | D | Negative | Negative | Negative | D | Negative | 4 | |
| CSU 62 | Canine | A/B undetected | F | Negative | Negative | Negative | F | Negative | 4 | |
| CSU 63 | Canine | A/B undetected | F | Negative | Negative | Negative | F | Negative | 4 | |
| 8095 | Canine | A/B undetected | D | D | Negative | C | D/C | Negative | 4 | |
| 8125 | Canine | A/B undetected | C | C | C | D | C/D | Negative | 4 | |
| 6149 | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 6134 | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 6912 | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 6897 | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 6898 | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 6906 | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 6907 | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 7998A | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 8106 | Canine | A/B undetected | Negative | Negative | Negative | Negative | Negative | Negative | 5 | |
| 3z | Feline | A | A | AI | AI | Negative | A | A | 1 | |
| 7z | Feline | B | B | B | BIII | Negative | B | B | 1 | |
| 7125 | Feline | A | A | A | A | Negative | A | A | 1 | |
| 7126 | Feline | A | A | A | A | Negative | A | A | 1 | |
| 7146 | Feline | A | A | A | A | Negative | A | A | 1 | |
| 7168 | Feline | B | B | BIV | BIII, BIV | Negative | B | B | 1 | |
| 7203 | Feline | B | B | BIV | B | Negative | B | B | 1 | |
| CSU 1 | Feline | A | A | A | Negative | Negative | A | A | 1 | |
| CSU 3 | Feline | A | A | AI | AI | Negative | A | A | 1 | |
| CSU 4 | Feline | A | AI | A | AI | AI | A | A | 1 | |
| CSU 9 | Feline | A | A | A | AI | Negative | A | A | 1 | |
| CSU 14 | Feline | A | A | A | A | A | A | A | 1 | |
| CSU 15 | Feline | B | F | BIV | F | BIII | B | B | 1 | |
| CSU 16 | Feline | A | A | A | A | AI | A | A | 1 | |
| CSU 18 | Feline | B | BIII | BIV | B | Negative | B | B | 1 | |
| CSU 20 | Feline | A | A | AI | AI | AI | A | A | 1 | |
| CSU 21 | Feline | A | A | A | AI | AI | A | A | 1 | |
| CSU 22 | Feline | B | BIII | BIV | B | Negative | B | B | 1 | |
| CSU 23 | Feline | A | AI | A | A | AI | A | A | 1 | |
| CSU 24 | Feline | B | BIII | BIV | B | Negative | B | B | 1 | |
| CSU 25 | Feline | B | BIII | Negative | B | Negative | B | B | 1 | |
| CSU 28 | Feline | A | A | A | Negative | Negative | A | A | 1 | |
| CSU 31 | Feline | A | A | Negative | AI | AI | A | A | 1 | |
| CSU 32 | Feline | B | BIII | BIV | B | Negative | B | B | 1 | |
| CSU 33 | Feline | A | A | Negative | Negative | Negative | A | A | 1 | |
| CSU 34 | Feline | A | A | A | AI | AI | A | A | 1 | |
| CSU 36 | Feline | A | AI | A | AI | AI | A | A | 1 | |
| CSU 37 | Feline | A | A | AI | AI | Negative | A | A | 1 | |
| CSU 38 | Feline | A | A | A | AI | Negative | A | A | 1 | |
| CSU 41 | Feline | B | BIII | BIV | B | B | B | B | 1 | |
| CSU 42 | Feline | B | BIII | BIV | B | B | B | B | 1 | |
| CSU 43 | Feline | A | A | A | AI | AI | A | A | 1 | |
| CSU 46 | Feline | A | AI | A | A | Negative | A | A | 1 | |
| CSU 47 | Feline | A | Negative | A | AI | AI | A | A | 1 | |
| CSU 48 | Feline | A | Negative | A | AI | AI | A | A | 1 | |
| CSU 50 | Feline | B | B | BIV | B | B | B | B | 1 | |
| 7097 | Feline | B | Negative | Negative | Negative | Negative | Negative | Negative | 2 | |
| 7166 | Feline | B | Negative | Negative | Negative | Negative | Negative | Negative | 2 | |
| 7182 | Feline | A | Negative | Negative | Negative | Negative | Negative | Negative | 2 | |
| 37nz | Feline | A/B undetected | F | F | BIII | Negative | F/B | B | 3 | |
| CSU 52 | Feline | A/B undetected | B | BIV | B | Negative | B | B | 3 | |
| 20nz | Feline | A/B undetected | F | F | Negative | Negative | F | Negative | 4 | |
| 21nz | Feline | A/B undetected | F | F | Negative | Negative | F | Negative | 4 | |
| 8130 | Feline | A/B undetected | D | D | Negative | C | D/C | Negative | 4 | |
| 8131 | Feline | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 8132 | Feline | A/B undetected | D | Negative | Negative | Negative | D | Negative | 4 | |
| 8133 | Feline | A/B undetected | D | D | Negative | D | D | Negative | 4 | |
| 8134 | Feline | A/B undetected | D | D | Negative | Negative | D | Negative | 4 | |
| CSU 51 | Feline | A/B undetected | F | F | F | Negative | F | Negative | 4 | |
| CSU 55 | Feline | A/B undetected | F | Negative | Negative | Negative | F | Negative | 4 | |
Reference: original classification: SSU rRNA-qPCR result. Sort key 1: A/B bg-qPCR positive/A/B MLG positive; 2: A/B bg- qPCR positive/A/B MLG negative or assemblages CDFG; 3: A/B bg-qPCR negative/A/B MLG positive; 4: A/B bg-qPCR negative/assemblages CDF MLG, and 5: A/B bg-qPCR negative/A/B MLG negative
In addition, diagnostic sensitivity, diagnostic specificity, positive predictive value (PPV), and negative predictive value (NPV) of the bg-qPCR assay in comparison with the MLG were calculated.
In the MLG, the assemblage of each sample was determined on the basis of the genotyping results of one or more genes. When results were not in agreement, all the assemblages detected by the different genes were assigned to that sample and were considered mixed infections.
Results
Sample collection
A total of 140 fecal samples from dogs (n = 90, 64.29%) and cats (n = 50, 35.71%) that tested positive/detected for Giardia spp. by at least one of the following methods: centrifugal flotation (ova and parasite, O&P), Giardia ELISA (TechLab Giardia II, Thermo Fisher Scientific, Waltham, Massachusetts), and quantitative small subunit ribosomal ribonucleic acid PCR (SSU rRNA-qPCR) were included in this study [11]. The sampling distribution was: Antech Diagnostics (n = 84) and the Center for Companion Animal Studies (n = 56).
Analytical validation of the bg-qPCR assay
Analytical sensitivity: Amplification of serial dilutions showed that this assay detected 10 copies per 5 µl of synthetic fragment DNA reproducibility (Table 1). The standard curve displayed good linearity between 1 × 101 and 1 × 107 copies per 5 µl. This bg-qPCR assay was performed with an efficiency of 104.7% and R2 values were greater than 0.99 (Fig. 1). This bg-qPCR had good repeatability with intra- and inter-assay coefficients of variation less than 1.1%. When using enumerated cysts to obtain limit of detection information, the lowest detection limit obtained was 8.9 cysts per gram of feces.
Fig. 1.
Detection of analytical sensitivity for the bg-qPCR
Analytical specificity: The bg-qPCR assay amplified DNA from Giardia but not the other parasites tested (Table 2).
Multilocus genotyping results
The multilocus genotyping method detected 126 of 140 positive samples, for a detection rate of 90% (Table 3). Of the 126 positive samples, assemblages A or B were amplified from 70 samples and 56 corresponded to non-A/B assemblages. The primer set with the highest number of positive results was the bg gene followed by the gdh gene. Both tpi-generic and tpi dog-specific primers showed the lowest number of positive results. Among the 41 assemblage A isolates, sub-assemblage AI was identified in 7, 4, and 21 isolates on the basis of the bg, gdh, and tpi genes, respectively. Molecular typing of assemblage B using BLAST analysis identified two sub-assemblages, BIII and BIV. Of the 25 assemblage B isolates, sub-assemblage BIII was identified in 9, 2, and 6 isolates on the basis of the bg, gdh, and tpi genes, respectively. Sub-assemblage BIV was identified in 2 and 6 isolates on the basis of the bg and gdh genes.
Method of analysis/assays comparison
When the results of multilocus genotyping and the bg-qPCR were compared for detection of zoonotic assemblages, the results showed a Cohen’s kappa value of 0.828. When results of the bg-qPCR assay were compared with those of the MLG for detection of zoonotic assemblages, the diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were all 91.47%.
Non-zoonotic assemblages were amplified only by MLG in 55 samples (Table 3). Multilocus genotyping reported mixed infections in 23 cases and included A/D (1 dog), B/C (1 dog), B/F (1 dog; 2 cats), and C/D (18 dogs) (Table 3). The discrepancies between bg-qPCR and MLG are displayed in Table 4.
Table 4.
Discordance between bg-qPCR and multilocus genotyping results
| Multilocus genotyping genes | |||||||
|---|---|---|---|---|---|---|---|
| Sample ID | Species | bg-qPCR | BG | GDH | TPI—generic primers | TPI—dog specific primers | Assemblage classification multilocus |
| 7166 | Feline | B | Negative | Negative | Negative | Negative | Negative |
| 7182 | Feline | A | Negative | Negative | Negative | Negative | Negative |
| CSU 2 | Canine | Negative | AI | D | Negative | D | Mixed A, D |
| CSU 6 | Canine | Negative | Negative | AI | A | Negative | A |
| CSU 10 | Canine | Negative | BIV, B | BIV | B | Negative | B |
| CSU 26 | Canine | A | Negative | Negative | Negative | Negative | Negative |
| CSU 27 | Canine | A | Negative | Negative | Negative | Negative | Negative |
| 37nz | Feline | Negative | F | F | BIII | Negative | Mixed B, F |
| CSU 64 | Canine | Negative | B, BIII | B | Negative | Negative | B |
| CSU 52 | Feline | Negative | B | BIV | B | Negative | B |
Discussion
The aim of this study was to validate a bg-qPCR for detection of G. duodenalis assemblages with zoonotic potential A and B in dog and cat fecal samples. The bg-qPCR assay described in this manuscript was demonstrated to be a highly accurate assay in identifying G. duodenalis assemblages A and B. The analytical validation confirmed that the assemblage A and B bg-qPCR assay amplified with high efficiency (104.1% and 105.1%, respectively) over at least a five-log standard curve, providing a wide dynamic range to quantify Giardia parasite burden in clinical samples (Fig. 1).
Beta giardin, gdh, and tpi genes are commonly used for genotyping to the assemblage- and sub-assemblage-level G. duodenalis [4]. All these assays are reported to be highly specific at detecting G. duodenalis assemblages [4]. We chose the bg as the target gene for the single-gene bg-qPCR since giardin genes are unique to Giardia protozoa. Giardins are structural proteins located at the ventral disk of the trophozoites [28]. The bg gene includes conserved regions within assemblages with variable domains in between assemblages; these regions have been targeted using locked-nucleic acid hydrolysis probes to have the highest possible specificity for assemblages A and B [25].
The specificities of the primer and probe sets were examined by performing bg-qPCR assays with a panel of DNA samples from different gastrointestinal parasites of dogs and cats (Table 2). Our results agree with those of other researchers in which no cross-amplification of various gastrointestinal parasites was detected [29]. These samples were confirmed either by gene sequencing using flanking primers, by using established SSU rRNA-qPCR [30], and by multilocus sequencing analysis.
Regarding the analytical sensitivity of the bg-qPCR, the standard curves confirmed a reproducible analytical sensitivity of five molecules per PCR reaction for assemblage A and B (Fig. 1, Table 1). The detection limit of the bg-qPCR was 8.9 cysts per gram of feces. The detection limit of a previous bg-qPCR assay was performed by spiking Giardia DNA into Giardia-negative samples [26]. In the study mentioned, Giardia-negative PCR samples were spiked with Giardia DNA with dilutions that ranged from 1 to 1000 Giardia cysts, and DNA was amplified at every concentration [26]. We believe, however, that spiking fecal samples with Giardia cysts provides a more accurate representation of the protozoal burden in clinical diagnostic specimens.
Furthermore, shedding dynamics of Giardia cysts by cats may fluctuate from undetectable to concentrations greater than 1,000,000 cysts per gram of feces [31, 32]. Similarly, shedding Giardia cysts in dogs may also vary; young dogs shed an average of 2000 cysts per gram of feces, and in one study, the mean cyst count per gram of feces for all infected dogs was 705.8 [33]. The reported range in another study was between 26 and 114,486 cysts per gram of feces [34]. Therefore, we are confident that the detection limit of the bg-qPCR presented in this study is adequate to detect the low numbers of Giardia cysts shed by cats and dogs.
Both bg-qPCR and multilocus genotyping identified 70 of 76 (92.10%) of the A and B assemblages. The Cohen’s kappa value was 0.828, which indicates almost perfect agreement between the two methods (Table 4). The high Cohen’s kappa value and the similar detection rates demonstrate that the bg-qPCR method is a reliable alternative to the multilocus genotyping for identifying G. duodenalis potentially zoonotic assemblages A and B. The fact that the assay is simpler, less expensive, and more rapid are additional benefits.
A meta-analysis reported the prevalence of Giardia spp. infections in cats and dogs to be 12% and 15%, respectively [3]. Dogs and cats with giardiasis can occur without clinical signs or have acute or chronic diarrhea [3]. In general, the transmission of dog-specific assemblages is predominant, particularly when dogs are in close contact such as crowded kennels [15]. In contrast, in household dogs, with minimal interaction with other dogs, the frequency of dog-to-dog transmission could be lower and infections with potentially zoonotic assemblages in dogs could be more frequent [15].
In this study, dogs carried more D (n = 16) than C (n = 11) Giardia assemblages, when counting concordant assemblage identification using the multilocus genotyping PCR tests. Dogs also carried slightly more A (n = 20) than B (n = 15) assemblages. Two dogs carried F assemblages, most likely explained by coprophagic behavior. In cats, more A (n = 24) than B (n = 14) assemblages were detected. In addition, assemblage F was detected in four cats and four were identified as assemblage D. Assemblage D has occasionally been detected in cats using molecular surveys in cats and is considered a spill-over phenomenon in mixed environments [35].
Dog-specific assemblages C and D are predominant in dogs; however, assemblages A and B have been commonly detected in cats and dogs [4, 35]. Assemblage A was the dominant assemblage in dogs in studies from USA, China, Japan, Germany, Brazil, in dogs from indigenous communities Canada, and Jamaica [36–43].
Multilocus characterization of assemblage A identified three major sub-assemblages (AI, AII, and AIII) with distinct subtypes at individual loci [44]. In cats and dogs, sub-assemblage AI, considered to be host specific to animals, was the predominant subtype but was also reported in humans [15, 40, 45–49]. While sub-assemblage AII is primarily associated with human infections, it has also been detected in dogs and cats [50–53]. Similarly, sub-assemblage AIII, typically linked to wild ruminants, has also been reported in cats and dogs [54].
Our study identified 40 isolates as assemblage A using the BLASTn algorithm (Table 3). However, subtyping resolution varied significantly. While some samples were successfully classified at the sub-assemblage level, others could only be confirmed at the broader assemblage level. This limitation often arose because many samples exhibited an identical percentage of homology to reference sequences from more than one sub-assemblage simultaneously, including sub-assemblage AII that is predominant in humans.
While BLASTn is an excellent tool for initial sequence screening, the high genetic diversity and significant allelic sequence heterozygosity characteristic of Giardia make definitive sub-assemblage assignment challenging. Therefore, we performed a secondary subtyping of assemblage A by aligning our sequences with reference strains representing each sub-assemblage [4]. The detection of sub-assemblage AI was an expected finding, given its frequent association with animal hosts. To ensure the highest confidence in classification, we only assigned sub-assemblage identification to isolates that showed 100% homology to the reference strains [4]. Despite the inherent limitations in sub-assemblage classification methodologies, the bg-qPCR assay described herein successfully detected the prevalent Giardia sub-assemblages reported across both human and companion animal hosts (Table 3).
Sub-assemblages have been identified in humans, companion animals, and wildlife and are considered to have zoonotic potential [15]. Sub-assemblage BIII was predominant at the bg gene, whereas sub-assemblage BIV was predominant at the gdh gene (Table 3). Assemblage B is more polymorphic than assemblage A, and the genotyping of the three genes revealed multiple subtypes at each of the three common genotyping loci [4]. Owing to a lack of reliable discriminatory power in the bg, gdh, and tpi gene sequences, phylogenetic analysis is unable to distinguish between isolates. Consequently, sub-assemblages within assemblage B cannot be reliably identified using MLG [4]. Owing to the absence of G. duodenalis assemblage B reference sequences, sequence comparisons were using the BLASTn function only.
Although the detection of assemblage B in dogs has been infrequently documented [15], it has been reported in various studies conducted globally [55]. Several studies detected both dog-specific and zoonotic assemblages with a predominance of the zoonotic genotypes [20, 39, 51, 56–58]. The presence of assemblage A and B in dogs is likely due to cross-species transmission between humans or wildlife animals and dogs. Cats are mainly infected with cat-specific assemblage F followed by assemblage A [4]. In a study from Canada, all the eight samples typed as assemblage A and, among them, sub-assemblage AI was predominant [59]. On the basis of a meta-analysis, the prevalence of different G. duodenalis assemblages in cats was reported as follows: assemblage F was the most common at 55.8%, followed by assemblage A at 38.7%, assemblage D at 8.9%, assemblage C at 3.1%, assemblage E at 2.9%, and assemblage B at 2.8% [8].
Discordant results among assemblages can be explained by preferential amplification of the multilocus primers for certain assemblages or lack thereof [5, 27]. In general, however, the MLG approach is recommended for assemblage and sub-assemblage determination. In this study, the multilocus genotyping approach failed to amplify Giardia DNA in five samples, a finding potentially attributable to the high sensitivity of the bg-qPCR assay in comparison to conventional PCR. Conversely, the bg-qPCR assay failed to amplify DNA from three samples. We believe that the failure to amplify DNA in these three samples can be attributed to the high specificity of the qPCR locked-nucleic acid probe and potential mismatches leading to false negative results. In such situations, the probe failed to anneal owing to sequence variations, including single-nucleotide polymorphisms. Among these three samples, one sample, identified as a mixed infection of assemblages A and D by the multilocus approach, was negative by the bg-qPCR assay. This could also be due to the predominance of assemblage D DNA, which may have outcompeted with the amplification of assemblage A. The use of whole-genome sequencing analysis has recognized the existence of allelic sequence heterogeneity (ash) in single trophozoites and cysts of assemblage B at the genotyping loci (gdh, bg, tpi). The presence of ash could be due to genetic recombination, that could also be facilitated by the high occurrence of infections with mixed assemblages. This finding stresses the need for more in-depth deep sequencing of G. duodenalis zoonotic assemblages.
The bg-qPCR assay successfully amplified potentially zoonotic G. duodenalis assemblages and sub-assemblages while showing no amplification of cat- and dog-specific assemblages. This is supported by the assay’s high diagnostic sensitivity and specificity, along with an almost perfect percentage of agreement with the multilocus genotyping method, which indicates that the bg-qPCR assay is a highly accurate tool for identifying the zoonotic Giardia assemblages A and B. Consequently, the bg-qPCR assay is recommended for ruling out the presence of potentially zoonotic strains. The high positive and negative predictive values provide valuable guidance for practitioners in the risk assessment of a Giardia infection in a dog or a cat.
The assemblage A- and B-specific bg-qPCR assays have broader applications beyond their current use. These assays could serve as a valuable tool for detecting these potentially zoonotic assemblages in fecal samples from other animal species, thereby expanding our understanding of their prevalence and distribution within a One Health framework. The assay could also be included in a multipathogen detection PCR panel to support the risk analysis of Giardia-infected pets as part of the clinical diagnostic workup.
Conclusions
The bg-qPCR assay reliably identified the potentially zoonotic Giardia assemblages A and B, offering a new rapid, single-gene, qPCR-based method for assessing zoonotic risk. By reliably identifying the zoonotic assemblages A and B, which are known to infect both humans and animals, this bg-qPCR assay directly contributes to a One Health approach by providing a critical tool for disease surveillance and risk assessment at the human–animal interface.
Acknowledgements
We thank Troy Goddu for critical review of the manuscript.
Author contributions
Drs. Leutenegger, Lappin, and Scorza designed the study. Dr. Leutenegger, Lozoya, Tereski, and Loo performed the qPCR, while Dr. Scorza conducted the multilocus genotyping. Dr. Scorza and Dr. Leutenegger wrote the first draft of the manuscript and shared it with the other co-authors for their comments. Dr. Jimenez Castro: data analysis, manuscript writing, and review. Lozoya, Tereski, and Loo: Sample collection and handling, extractions, qPCR analysis, sequencing protocols, data collection, and manuscript review.
Funding
This study was supported by Antech Diagnostics, Loveland, Colorado, and the Center for Companion Animals Studies, Colorado State University. http:/csuvets.colostate.edu/companion/index.htm.
Data availability
Data supporting the main conclusions of this study are included in the manuscript.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
All authors agreed to the final manuscript, figure, and tables.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Andrea V. Scorza, Christian M. Leutenegger, and Michael R. Lappin contributed equally to this work.
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Associated Data
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Data Availability Statement
Data supporting the main conclusions of this study are included in the manuscript.