The pET30a-BcMSA1-BcSA1 recombinant plasmid was constructed by Sangon Biotech Co., Ltd. (Shanghai, China). Goat anti-Mouse IgG and mouse IgG were obtained from Arista Biologicals, Inc. (Pennsylvania, United States). The conjugate pad, sample pad, absorbent paper, and nitrocellulose membranes (NC) were obtained from Equibox Biotech Co., Ltd. (Guangdong, China), while polyvinyl chloride (PVC) sheets were supplied by Shanghai Zhuo Yue Technology Co., Ltd. (Shanghai, China). All other reagents were acquired from EtAc, Innochem Co., Ltd. (Beijing, China).

The BcMSA1 gene (KR134351) and BcSA1 gene (KR134352) of Babesia canis was designed based on the protein sequence from NCBI Gene bank. After analyzing the hydrophilicity and hydrophobicity of the protein using ExPASy-ProtScale, the BcMSA1 (1–70 aa) sequence and the BcSA1 (10–250 aa) sequence were selected from the region predicted to have a relatively high hydrophilic content for fusion. SignalP-5.0 analysis identified a secretory signal peptide in the native BcSA1 sequence (residues 1–22, including MM-GE), which was excluded from the fusion construct (BcSA1: 10–250 aa) to ensure cytosolic expression. The two proteins were linked by the flexible peptide GSGSG. Therefore, the amino acid sequence of the BcMSA1-BcSA1 recombinant fusion protein is as follows:

MMLLFALSTLVTFAFCDGENTILLSNVEFHTPVSSVKLLKEYSSNQESMAVIMMLTEMPNTSGKLTDGKVGSGSGHPHNFILIFQLLATMGNAQSTSSQENSRDGLREVLEYTNQLHNNYGSAVRKVTDKLKNEIDVYCKSTDDKGYYFANGSFGYFRKALNDSFNFRFQLLSNYNDYRKYKTRFQDTDDEAEKHVKYLKENLFDLFGTLSYMYFQCSHKCQKYNGGKWEEQSMNQSGSEVSKWLMGSNSAATDSVHFLGRDFSTSELTNIKGKELADKDRASLSDLIKYSGRGNLQHALFWMLFIGPWVDGKTGH.

The amino acid sequence of the fusion protein was converted into a nucleotide sequence and codon-optimized based on Escherichia coli preferences. The nucleotide sequence of the BcMSA1-BcSA1 fusion gene is as follows:

ATGATGCTGCTGTTCGCTCTGAGCACGCTGGTGACCGCGTTCTGCGATGGTGAAAACACCATCCTGCTGAGCAACGTGGAATTCCACACCCCGGTGAGCAGCGTGAAACTGAAAGAATACAGCAGCAACCAGGAAAGCATGGCGGTGATCATGATGCTGACCGAAATGCCGAACACCAGCGGTAAACTGACCGATGGTAAAGTTGGTTCTGGTAGCGGTCACCCGCACTACTTTATCCTGATCTTCCAGCTGCTGGCGACCATGGGTAACGCGCAGAGCACCAGCAGCCAGGAAAACAGCCGTGATGGTCTGCTGCGTGAAGTGCTGGAATACACCAACCAGCTGCACAACAACTACGGTAGCGCGGTGCGTAAAGTGACCGATAAACTGAAAAACGAAATCGACGTGTATTGCAAAAGCACCGATGACAAAGGTTACTACTTCGCGAACGGTAGCTTCGGTTATTTCCGTAAAGCGCTGAACGATAGCTTCAACTTCCGTTTCCAGCTGCTGAGCAACTACAACGATTACCGTAAATACAAAACCCGTTTCCAGGACACCGATGATGAAGCGGCAGAACACGTGAAATACCTGAAAGAAAACCTGTTCGATCTGTTTGGCACCCTGAGCTACATGTATTTCCAGTGCTCTCACAAATGCCAGAAATACAACGGTGGTAAGTGGGAAGAACAGAGCATGAACCAGAGCGGTAGCGAAAGCAAATGGCTGATGGGTAGCAGCAGCGCGGCGACTGACAGCGTGCACTTCCTGGGTCGTGATTTTAGCACCAGCGAACTGACCAACATCAAAGGTAAAGAACTGGCAGACAAAGATCGTGCGAGCCTGAGCGATCTGAAATACAGCGGTAGCGGTAACCTGCAGCACGCGCTGTTCTGGATGCTGTTCATCGGTCCGTGGGTGGATGGTAAAACCGGTCAC.

The resulting sequence was synthesized, and subsequently inserted into the pET-30a vector, resulting in the formation of a recombinant plasmid. The pET-30a-BcMSA1-BcSA1 fusion plasmid was expressed in Escherichia coli BL21 (DE3). Expression was induced in 500 mL of Luria-Bertani (LB) medium supplemented with 50 μg/mL kanamycin at 37°C with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h. Cells were harvested by centrifugation at 5,000 × g for 10 min, resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole), and lysed by sonication (5 cycles of 30 s on, 30 s off, at 40% amplitude). The lysate was clarified by centrifugation at 12,000 × g for 20 min at 4°C. The purification was performed according to the manufacturer’s instructions (Qiagen Ni-NTA Purification System) (18). The protein was eluted with 250 mM imidazole, yielding approximately 2.5 mg/L of culture. Purified BcMSA1-BcSA1 was verified by SDS-PAGE and western blot with B. canis-positive serum, obtained from a naturally infected dog at a veterinary clinic in Shanxi Province, China, an area with known B. canis prevalence. The serum was confirmed positive by PCR targeting the 18S rRNA gene (12), with additional validation by clinical symptoms (e.g., anemia, fever) and microscopic observation of B. canis piroplasms in Giemsa-stained blood smears.

Colloidal gold particles were prepared followed as described previously (21). Briefly, 4 mL of 10% chloroauric acid to 1,000 mL of ultrapure water. After boiling on a magnetic stirrer, 6 mL of 10% trisodium citrate was gradually added under stirring. The mixture was cooled to room temperature, filtered through a 0.22-μm filter and stored at 4°C in a dark bottle. To prepare the colloidal labeled protein conjugate, 100 mL of colloidal gold solution was placed in a beaker, and the pH was adjusted in the range of 6.8, 7.2, 7.6, 8.0, 8.2, and 8.4 by slowly adding 0.2 M K₂CO₃ while stirring. The color variation of the colloidal gold solution was observed. Subsequently, the optical density (OD) of the solution was measured through scanning within the wavelength range of 400–700 nM by Agilent BioTeck Synergy H1 Multi-Mode Microplate Reader (Santa Clara, CA, United States). In addition, 2 mg of BcMSA1-BcSA1 recombinant protein dissolved in 200 μL of phosphate-buffered saline (PBS, pH 7.4) to a concentration of 10 mg/mL were added to 1 mL of colloidal gold fluid and the mixture was stirred at room temperature for 15 min. Subsequently, 1 mL of a 10% bovine serum albumin (BSA) solution was added. The mixture was then continuously stirred for an extra 15 min at room temperature. Following this, the resultant mixture underwent centrifugation at 13,363 × g for a duration of 10 min. The precipitate was resuspended in 1 mL of gold label dilution buffer (20 mM Tris, 1% BSA, 0.03% Proclin 300, pH 8.0) to obtain the colloidal gold-BcMSA1-BcSA1 fusion protein conjugate. The preparation was stored at 4°C until use.

To prepare the colloidal gold-mouse IgG conjugate, another 100 mL of the colloidal gold solution was transferred to a separate beaker. The pH was adjusted to 8.0 with 0.2 M K₂CO₃. Then, the same procedure as for the colloidal gold-BcMSA1-BcSA1 fusion protein conjugate was followed to form the colloidal gold-mouse IgG conjugate.

As shown in Figure 1, a sandwich lateral flow immunochromatographic assay was developed to detect antibodies against Babesia canis. To construct the test line (T-line) and identify the optimal antigen coating concentration, the purified BcMSA1-BcSA1 fusion protein was diluted to various concentrations (0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mg/mL) with a spotting diluent buffer consisting of 50 mM Tris, 2% sucrose, and adjusted to pH 8.5. These diluted proteins were then dispensed onto the nitrocellulose (NC) membrane. For the control line (C-line), goat anti-mouse IgG, diluted to 0.3 mg/mL using the same buffer, was applied onto the NC membrane. Afterward, the NC membrane was left to dry at 37°C 12 h.

Mouse IgG and BcMSA1-BcSA1 fusion protein were conjugated with colloidal gold. The conjugates were applied to the conjugate pad and dried at 37°C for 4 h. After drying, the assembled nitrocellulose membrane, conjugate pad, sample pad, and absorbent paper were cut into 3-mm strips (Figure 1) and mounted on a PVC backing. The optimal T-line antigen coating concentration of the immunochromatographic strip was determined by comparing the color intensity of the T- and C-lines.

The B. canis standard-positive serum was serially diluted with 0.01 mol/L PBS at ratios of 1:2, 1:4, 1:8, and 1:16. Equal volumes of the diluted standard-positive and standard-negative sera were applied to the test pads to assess the sensitivity of the immunochromatographic strip. Subsequently, 100 μL of each prepared serum sample was dispensed onto the sample pad. After incubation at room temperature for 10 min, images were captured for analysis. After incubation at room temperature for 10 min, band intensities were evaluated visually by three independent observers blinded to the sample status to minimize observer bias.

To assess the specificity of the strip, a series of tests was conducted using sera from dogs with common diseases caused by Theileria, Toxoplasma gondii (T. gondii), Ancylostoma caninum (A. caninum), Eimeria canis, canine distemper virus (CDV), and canine parvovirus (CPV) infection. These sera were collected from naturally infected dogs at veterinary clinics in Shanxi Province, China, between January 2023 and March 2024. Infections were confirmed using commercial diagnostic kits and/or pathogen-specific PCR (22–25). As described above, 100 μL of each serum sample was tested following the identical procedure.

To evaluate the stability of the CGIA strip, strips were stored at room temperature (25°C) in sealed plastic bags with desiccants for up to 18 months. They were used to test negative and positive serum samples after 6, 12, and 18 months of storage. For each of the negative and positive groups, three replicates were tested. Their results were then compared in terms of color intensity.

A total of 72 canine serum samples were collected from veterinary clinics in Shanxi Province, China, between January 2023 and March 2024, with owner consent and ethical approval from the Shanxi Agricultural University Animal Ethics Committee (Approval No. SXAU-2022-013). Parasitological tests, including microscopic examination of Giemsa-stained blood smears for Babesia canis piroplasms (13), and clinical assessments noting symptoms such as anemia, fever, or lethargy in positive cases, were performed to support PCR results. This assessment involved testing the samples to evaluate the CGIA strip’s sensitivity, specificity, and concordance with the InBios B. canis ELISA kit, as detailed in subsection 2.5. No formal power calculation was performed due to the preliminary nature of this study; however, the sample size aligns with similar initial validations of rapid diagnostic tests (26). The commercial ELISA kit (InBios International, Inc., United States) designed to detect Babesia canis-specific antibodies in canine serum and colloidal gold test strips were tested in conjunction, the agreement between the CGIA strip and ELISA was evaluated using Cohen’s Kappa coefficient (27). Cohen’s Kappa was calculated using a 2 × 2 contingency table comparing positive and negative results, with the formula: k = P o − P e 1 − P e , where P_o is the observed agreement and P_e is the expected agreement by chance (27). The sensitivity was calculated as follows: [(true positive)/(true positive + false positive) × 100%], while the specificity was calculated as follows: [(true negative)/(true negative + false negative) × 100%] (26). All animal-based experimental procedures were conducted in accordance with the Guidelines of the Animal Ethical Committee.

The BcMSA1 (Babesia canis Merozoite Surface Antigen 1) and BcSA1 (Babesia canis Secreted Antigen 1) proteins are the major immunogenic proteins of Babesia canis, which can trigger and initiate the immune response of the host immune system. Fusing the main antigenic epitopes of BcMSA1 and BcSA1 proteins for antibody detection can not only enhance the diagnostic sensitivity, but also reduce cross-reactions with other pathogens. After analyzing the hydrophilicity and hydrophobicity of the proteins, the BcMSA1 (1–70 aa) sequence was selected for fusion from the regions predicted to have a relatively high hydrophilic content (Figure 2A). Similarly, after hydrophilicity and hydrophobicity analysis, the BcSA1 (10–250 aa) sequence was chosen for fusion from the regions with a predicted high hydrophilic content (Figure 2A). The two proteins were linked by the flexible peptide GSGSG. After codon optimization, the sequence was cloned into an Escherichia coli vector for expression. A Ni-NTA agarose column was used to purify the His-tagged protein, yielding approximately 2.5 mg/L of culture, as described in subsection 2.2. SDS-PAGE and western blot analysis with Babesia canis-positive serum confirmed the successful expression and purification of the BcMSA1-BcSA1 fusion protein, with a molecular weight of approximately 37 kDa, consistent with the predicted size of the His-tagged fusion protein (Figure 2B).

To identify the optimal pH for labeling the colloidal gold solution, diverse volumes of K₂CO₃ solution were introduced into 1 mL of the identical colloidal gold solution awaiting labeling. The color alterations of the colloidal gold solution were monitored, and the absorption peak curve was generated by scanning the solution within the optical density (OD) spectrum of 400–700 nm. Results showed that evident color changes in the colloidal gold solution took place when the pH reached between 8.8 and 9.4. Moreover, the highest peak on the absorption peak curve was detected at a pH of 9.4 (Figure 3A). Consequently, the ideal pH for labeling the colloidal gold solution was established as 9.4.

The purified BcMSA1-BcSA1 fusion protein was diluted to various concentrations (0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mg/mL) with a spotting diluent buffer and then dispensed onto the nitrocellulose (NC) membrane to construct T-line. As shown in Figure 3B, the results indicated that when the NC membrane was coated with BcMSA1-BcSA1 fusion protein at concentrations of 0.2 to 0.6 mg/mL, no distinct bands were observed in the T-line. Even at a concentration of 0.7 mg/mL, only a faint band could be seen in the T-line. However, at a concentration of 0.8 mg/mL, a clear band appeared in the T-line. Therefore, the optimal antigen coating concentration for the T-line is 0.8 mg/mL.

To evaluate the sensitivity of the immunochromatographic strips, the Babesia canis-positive canine serum were serially diluted at ratios of 1:2, 1:4, 1:8, and 1:16 using PBS (Figure 4). The diluted positive serum samples, along with negative serum controls, were applied to the test strips following the established protocol. Visual inspection revealed that distinct T-lines were consistently observed for dilutions up to 1:8. Conversely, at a dilution of 1:16, the T-lines exhibited significant blurring and reduced intensity, rendering them difficult to interpret. Based on these findings, the limit of detection for the developed strips for Babesia canis-positive canine serum was determined to be 1:8, indicating the lowest dilution at which a reliable positive signal can be detected.

The specificity of the immunochromatographic strips was systematically evaluated using canine positive serum reactive to a panel of relevant canine pathogens, including Theileria spp., T. gondii, A. caninum, Eimeria canis, CDV, and CPV. In accordance with the standardized testing protocol, each serum sample was applied to the test strips, and the resulting reactions were carefully observed and documented. Results demonstrated that only sera containing antibodies against Babesia canis yielded positive results, whereas antiserum positive for other pathogens showed negative outcomes (Figure 5). The C-line in the B. canis sample appeared brighter, likely due to moderate antibody binding at the T-line, allowing sufficient unbound gold-labeled BcMSA1-BcSA1 conjugate to reach the C-line, or due to sample matrix effects enhancing conjugate flow. These findings indicate that the immunochromatographic strip exhibits high specificity for Babesia canis and lacks cross-reactivity with other canine pathogens.

Following extensive validation, the colloidal gold test strip developed in this study exhibited excellent specificity and accuracy, effectively detecting both strong and weak positive samples. Additionally, the strip demonstrated outstanding reproducibility when after stored at room temperature for up to 18 months (Figure 6).

Furthermore, a total of 72 clinical canine serum samples were collected for analysis. To assess the presence of Babesia canis infection, a commercially available ELISA kit (InBios International, Inc., United States) was utilized to screen these samples. The agreement between the CGIA strip and ELISA was evaluated using Cohen’s Kappa coefficient. The Kappa coefficient was calculated based on the 72 canine serum samples tested, where 15 samples were positive and 55 were negative by both CGIA and ELISA, and two samples were positive by ELISA but negative by CGIA. Using the formula for Cohen’s Kappa:

where P_o is the observed agreement (70/72 = 0.972) and P_e is the expected agreement by chance, yielding a value of 0.935 (95% CI: 0.839–1.000), indicating almost perfect agreement (27). The sensitivity and specificity of the colloidal gold test strip were 84 and 93.6% between the colloidal gold test strips developed herein and the commercial ELISA kit. This finding strongly indicates that the immunochromatographic strips developed in this study exhibit excellent performance in clinical detection, suggesting their potential utility as a reliable diagnostic tool for Babesia canis infection in canines.