Using as reference reported sequences of TBDT in Av. paragallinarum (GenBank: WP_052716793.1, KKA99715.1, WP_051185305.1 and WP_017806794.1), we annotated the TBDT gene (GenBank: MG242130.1) on the previously reported genome of Av. paragallinarum strain 72 . The following regions were flagged on the protein sequence: the signal peptide (predicted using SignalP 4.1) and regions with an identity of 50% or higher with any protein of other avian pathogens (identified using BLASTp). Then, peptides were predicted on the unflagged regions using Bebipred 2.0 and Optimum Antigen (GenScript Laboratories, Piscataway, NJ, USA), selecting those peptides with the highest conservation and located in the outer part of the protein structure (by analyzing the 3D model). The best candidate obtained (named Ma-4) was used in the downstream analysis.
Monoclonal antibody production using a peptide
For monoclonal antibody production, the peptide (Ma-4) was synthesized and used to immunize a BalbC mouse which was provided by GenScript Laboratories. After 5 weeks, spleen cells were isolated for hybridoma production by fusion with the SP2/0 mouse myeloma line. Peptide-reactive clones were finally selected by ELISA assay and then isotyped. Antibody titers in hybridoma supernatants were calculated as the highest positive dilution against the peptide by indirect ELISA assay. The peptide synthesis, hybridoma production, clone selection, isotyping and antibody titers were performed by GenScript Laboratories. The positive clones were sent to FARVET Laboratories for monoclonal antibody production according to a previously published methodology . The hybridoma culture supernatants were used for the antibody purification steps.
The mouse was euthanized using cervical dislocation without anesthesia following the American Veterinary Medical Association (AVMA) guidelines and the terminated animal was disposed by contracted biological waste disposal company .
Chromatographic runs were conducted on a commercial prepacked affinity HiTrap rProtein A FF column (0.7 cm × 2.5 cm) (GE Healthcare, Uppsala, Sweden), which occurred at room temperature, and an AKTA Pure system (GE Healthcare). The column was equilibrated with 8 column volumes (CV) of 200 mM sodium phosphate buffer and 3 M NaCl at pH 7. The hybridoma culture supernatants were previously centrifuged for 10 min at 4 °C at 13000 x g, filtered using a 0.22 μm filter and then loaded into the column at 156 cm/h linear flow velocity using a 150 mL superloop (GE Healthcare). After washing unbound compounds with 8 CV of the equilibration buffer, the antibodies were eluted stepwise with 8 CV of 0.1 M sodium citrate at pH 3.5. Column eluates were continuously collected as 1 mL fractions in a F9-R fraction collector (GE Healthcare).
Preparation of recombinant protein
The sequence of the gene encoding the TonB-dependent transporter (TBDT) was obtained from NCBI reference sequence MG242130. The protein sequence consists of 756 amino acids with a theoretical molecular weight of 86.5 kDa. The expression of this recombinant protein was carried out in an E. coli BL21 strain, and a 6x-histidine tag was added at the amino terminus (Creative Diagnostics, Shirley, NY, USA). This recombinant protein was produced to be used as a positive control in Western blots.
Bacterial pellet, lysate and culture supernatant
The Av. paragallinarum isolates FARPER-113 (serovar B-1), FARPER-114 (serovar A-2) and FARPER-140 (serovar C-1) from an outbreak in Peru were serotyped (Page and Kume scheme), characterized and grown using methods that were previously reported . The bacteria were grown in brain-heart infusion (BHI) broth (Sigma Aldrich Co., St Louis, Missouri, USA) supplemented with 5% equine serum and 0.01% NAD (TM/SN) for 18 h under agitation at 37 °C . Later, the Av. paragallinarum cultures were centrifuged at 11000 x g for 5 min at 4 °C. The supernatants were filtered using a microfilter (0.45 μm) and stored at − 80 °C until use. The bacterial pellet was resuspended with 10 mL of phosphate-buffered saline, pH 7.4, plus Tween 20 5% (v/v) (Sigma Aldrich Co.), and incubated on ice for 30 min. Subsequently, the resuspended bacterial pellet was sonicated at an amplitude of 100 (On/Off time, 30 s/10 s) for 2 min on ice using a sonicator S-4000 (Qsonica, LLC, Newton, CT, USA) and centrifuged at 11000 x g for 30 min at 4 °C. Finally, the bacterial lysates obtained from sonication were stored at − 80 °C, the pellet was resuspended using 1 mL of 8 M urea (Sigma Aldrich Co.) and was stored at − 20 °C. Culture lysates and pellets were quantified using a Bradford assay (Merck KGaA, Darmstadt, Germany). Additionally, the pellets, lysates and supernatants of related bird bacteria cultures, such as Ornithobacterium rhinotracheale (FARPER-172), Gallibacterium anatis (FARPER-173), and Pasteurella multocida (FARPER-069) and non-related bird bacteria Bortedella bronchiseptica (FARPER-118), were locally isolated and cultivated using standard bacteriological techniques [16, 28,29,30]. For a better understanding, the serovars B-1, A-2 and C-1 obtained using Page and Kume scheme were referred as serogroups B, A and C, respectively.
Identification of TBDT protein using Western blot
Bacterial lysate, pellet and culture supernatant samples were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane using an e-blot device (GenScript Laboratories). The membranes were blocked with Azure Chemi Blot clocking buffer (Azure Biosystems, Dublin, CA, USA) with shaking for 1 h at room temperature. Membranes were washed three times for 10 min with phosphate-buffered saline (PBS)-Tween 20 and were incubated with purified monoclonal antibodies (0.4 μg/mL). Membranes were washed three times with phosphate-buffered saline-Tween 20. The immune reaction was performed with peroxidase-labelled goat IgG anti-mouse antibody (GenScript Laboratories) using radiance as a substrate and revealed by a CCD camera (Azure Biosystems). For bacterial lysates and pellets, 10-fold dilutions were prepared with the previously quantified samples (500, 50, 5, 0.5, 0.05 and 0.005 μg/mL). For bacterial culture supernatants, 2-fold dilutions were prepared with the supernatant (1:2, 1:4, 1:8 and 1:16) in PBS at pH 7.0. For recombinant TBDT protein, 500 ng was used. For molecular weight estimation of the studied proteins, we used a pre-stained molecular weight marker that ranged from 26,600 to 180,000 Da (Sigma Aldrich Co.) and a molecular weight marker that ranged from 30,000 to 120,000 Da (GenScript Laboratories).
Reactivity of monoclonal antibodies using indirect enzyme-linked immunosorbent assay (ELISA)
The 96-well microplate (Greiner Bio-One GmbH, Kremsmünster, Austria) was coated with 100 μL of antigen. The antigens used were bacterial lysates (103–107 CFU/mL) that were diluted with buffer carbonate/bicarbonate, pH 9.5, and supernatants from bacterial culture (dilutions from 1:10 to 1:100,000). All antigens except bacterial lysates were diluted in PBS at pH 7.4. Microplates were incubated at 4 °C overnight. Microplates were washed once with 150 μL of wash buffer (PBS + 0.05% Tween 20), blocked for 1 h at room temperature with 100 μL of blocking buffer (PBS + 0.5% BSA) and washed three times with 150 μL of washing buffer. One-hundred microliters purified monoclonal antibodies (5 μg/mL) were added to the plates and incubated for 1 h at 37 °C. Microplates were washed three times with 150 μL of washing buffer. Then, 100 μL of horseradish peroxidase conjugated goat anti-mouse IgG H&L (IgG-HRP) (Abcam, Cambridge, MA, USA) was added, and the microplates were incubated for 1 h at 37 °C. The microplates were washed four times using 150 μL of washing buffer; then, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) ELISA substrate (high-sensitivity) (Abcam) was added, and the microplates were incubated at room temperature for 30 min. Finally, the reaction was stopped using 100 μL of 2 N H2SO4, and the microplates were read at 450 nm using an EON microplate reader instrument (Biotek, Winooski, VT, USA).
Assembly of a prototype lateral flow test (LFT)
All possible combinations of antibodies were evaluated in order to determine which antibody pair was the most suitable for test development. All antibodies were tested as capture and detection reagents. Thus, all antibodies were conjugated to gold nanoparticles and used for the test. The detection antibody was immobilized at the Test (T) line region, and an anti-mouse IgG antibody was immobilized at the Control (C) line region of a nitrocellulose membrane. Briefly, two red lines in C and T regions meant a positive result, and only one red line in the C region meant a negative result for Av. paragallinarum. No band in the C region meant an invalid LFT.
Specificity evaluation of the prototype LFT
Evaluation of specificity was carried out using culture lysates and supernatants of related and non-related bird bacterial cultures that were prepared as described above. All culture lysates were diluted until reaching 1 × 107 CFU/mL, from which 100 μL was mixed with 100 μL of diluent buffer. Finally, 100 μL was added to the sample port of the LFT.
Additionally, cell cultures were infected with avian respiratory viruses in order to evaluate LFT specificity. Cell lines such as chicken hepatocellular carcinoma cell line (LMH), chicken fibroblast cell line (DF-1) and African green monkey kidney cell line (VERO) were seeded at 1 × 106 cells/mL with Dulbecco’s modified Eagle medium (DMEM) F12 (HyClone, Logan, UT, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS) in a 75 cm2 flask and cultivated in 5% CO2 at 37 °C until reaching 70% confluency. These cell lines were infected with infectious laryngotracheitis virus (VFAR-043 strain) , which was initially adapted to the LMH cell culture following a previously reported method , Newcastle disease virus (LaSota strain)  or avian metapneumovirus (SHS-FAR strain)  at 0.02 multiplicity of infection (MOI). The infected cells were cultivated until a cytopathic effect was observed. Then, the supernatants were collected and centrifuged at 3000 g for 5 min at room temperature. Finally, the supernatants with viruses were quantified using plate assay and expressed in plaque-forming units (PFU)/mL. The quantified supernatants were diluted until reaching 1 × 106 PFU/mL, and 100 μL was mixed with 100 μL of diluent buffer and added to the sample port.
Limit of detection of the prototype LFT
The limit of detection was performed using Av. paragallinarum culture lysates from FARPER-140 (serogroup C). Colony count was performed following a previously reported method . A bacterial culture (50 mL) was quantified (1 × 106 CFU/mL) and centrifuged at 11000 x g for 5 min at 4 °C. The bacterial pellet was resuspended with 5 mL of phosphate-buffered saline, pH 7.4, plus 5% Tween 20 (v/v) (Sigma Aldrich Co.), incubated on ice for 30 min and sonicated based on factors mentioned above in order to obtain 1 × 107 CFU/mL. The lysates were serially diluted at 10-fold from 1 × 107 to 1 × 101 CFU/mL in diluent buffer. The lowest positive dilution was considered the limit of detection of the test.
Chicken sample evaluation using the prototype LFT, PCR and bacterial isolation
Thirty-five chickens suspected of having infectious coryza were sent from farms located in the south of Peru (cities: Arequipa and Ica) to FARVET laboratories, after 2 days of clinical sign onset, for routine evaluations. Chicken nasal mucus was obtained by exerting pressure on the nasal sinuses. The nasal mucus was taken using a 2-mL transfer pipette and mixed with 200 μL of diluent buffer in a 1.5-mL microtube until reaching a homogeneous mixture. From this mixture, 100 μL was taken to perform the LFT, and 100 μL was used for the PCR assays. The homogeneous mixture was added to the sample port of the LFT, and the results were assessed visually after 30 min. PCR assays based on the HPG-2 gene  were used for detection, and a multiplex PCR (mPCR) based on the HMTp210 gene  was only used for chicken nasal mucus typing. Bacterial isolation was performed using bird heads according to a previous report .
All chickens were euthanized using cervical dislocation without anesthesia following the American Veterinary Medical Association (AVMA) guidelines  and the disposal of dead animals was performed according to the Peruvian Regulation .
All quantitative data were analyzed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Indirect ELISA assays performed for duplicate and optical density (O.D.) values were represented as the mean and standard deviation (SD). The cutoff was calculated as the mean O.D. value +2SD. The O.D. value used for cutoff calculation was obtained from the 1:10,000 dilution for supernatants and 1 × 104 CFU/mL for lysates. The O.D. values obtained above the cutoff were considered positive, and those below the cutoff were negative. Two-way ANOVAs with Tukey’s post hoc test were used for serogroup comparison analysis (p < 0.05).
Two-by-two contingency tables were created to analyze associated values among the three methods of detection. The following indicators for the prototype LFT were calculated using bacterial isolation and PCR as reference methods: sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV) and kappa index (K). Each indicator was calculated using a 95% confidence interval. Kappa index (agreement level) was denoted using Landis and Koch values , where 0 is poor, 0.01–0.2 is slight, 0.21–0.4 is fair, 0.41–0.6 is moderate, 0.61–0.8 is substantial and 0.81–1.0 is almost perfect.