Bacterial strains and specimen types
Vaccine strains: Strain-19 and RB51 (B. abortus biovar 1) and Rev-1 (B. melitensis biovar 1) (CZ veterinaria S.A., Pontevedra, Spain).
References strains: 544 (B. abortus biovar 1), 16 M (B. melitensis biovar 1), ETHER (B. melitensis biovar 3), REO198 (Brucella ovis) and Brucella suis 1330 (B. suis biovar 1) were kindly supplied by Prof. Dr. J.M. Blasco, CITA Institute, Zaragoza, Spain. Additionally, S99 (B. abortus biovar 1) was kindly supplied by the Veterinary Serum and Vaccine Research Institute (VSVRI).
Field isolates: Twenty-five Egyptian field isolates were recovered from different animal species and kindly supplied by Prof. Dr. Ashraf Sayour and Prof. Dr. Waleed Shell. These field isolates were identified serologically, biochemically and via a Bruce-Ladder multiplex PCR assay (OIE, 2018). Twenty-two of these 25 isolates were Brucella melitensis biovar 3 and the other three field isolates were Brucella abortus.
Other bacteria: Yersinia enterocolitica O9, Escherichia coli O157:H7 and Salmonella serovars of Kauffmann-White Group N as cross reacting bacteria with serological tests used for the diagnosis of Brucella.
A spiked sample is prepared by adding a Brucella field isolate at a colony count equal to the LOD of this lateral flow assay to Brucella free heparinized bovine serum and grinded Brucella free bovine lymph nodes in PBS (matrix) (Ming et al, 2016). LOD is detected by an initial study which was performed to determine the detectible range (colony forming unit/LF sample= 40 μl) in the spiked specimens. These spiked samples are used in validation assay experiments to determine the selectivity of the method and identify matrix effects.
Synthesis of nanogold (40 nm): Aboelqassem ZM et al., 2022 and Frens, 1973
One milliliter of a 1% aqueous solution of HAuCl4 (Sigma-Aldrich, USA) was added to 100 mL of deionized water. The resulting mixture was boiled with adding 1.5 mL of 1% aqueous solution of sodium citrate with constant and vigorous stirring. The mixture turned red in approximately 2 minutes and the solution was continues to boil for 15 minutes, then cooled and stored at 4°C. After cooling, deionized water was added to the mixture until the volume reached 100ml. Sodium azide (0.02 %) was added to the obtained colloidal gold and stored at 4°C. The particle diameter of the obtained gold colloids was checked using transmission electron microscopy (TEM, H-7650).
Preparation of smooth Brucella lipopolysaccharide (S-LPS)
In-house-prepared smooth lipopolysaccharide (S-LPS, hot Saline Extract) was prepared from Brucella abortus S99 according to Alton et al., (1988) and Plackett et al. (1976).
Preparation of Rabbit IgG anti LPS (Aboul‑Ella et al 2023, Ishman and Berg 2018b, dos Santos et al. 1989):
1. Rabbit inoculation of LPS
For priming immunization, S-LPS emulsion antigen was prepared by adding equal volumes of complete Freund’s adjuvant (CFA, SIGMA-ALDRICH) and LPS antigens then mixed adequately using two syringes and connector for about 20 min till a milky white viscous creamy emulsion appeared and be stable and emulsion incubated for overnight testing at refrigerator temperature (6 °C). on other hand,The boostering immunization emulsion consisted of equal volumes of LPS antigens and incomplete Freund’s adjuvant (IFA, SIGMA-ALDRICH) and then mixed adequately using two syringes and connector for 20 min till reached a milky, white, viscous, creamy emulsion with stable formulation after overnight testing at refrigerator temperature (6 °C). Both types of immunization emulsions have been prepared and inoculated under aseptic techniques. Two fully mature white male New-Zealand rabbits weighing 2 kg were used as the bio-factory for LPS pAbs production. As using a fully mature animal will ensure a completely functioning immune system which is the cornerstone of antibody production. Also, rabbits were the chosen as it is characterized by their small size, relatively long life span, strong immune response, easy blood obtaining and inexpensive
2. Rabbit IgG purification:
Pooled serum collected from inoculated rabbit with Brucella S-LPS was placed on a magnetic stirrer, and then caprylic acid was slowly added drop wise at a concentration of 2.02 ml/25 ml rabbit serum while stirring at 25 °C for 30 minutes. The resulting mixtures were centrifuged at 10,000×g for 20 min, after which the supernatants were collected. The collected supernatants were dialyzed at 4 °C overnight against PBS using 12,000–14,000 Dalton molecular weight cut-off (MWCO) dialysis bags (SIGMA-ALDRICH). Finally, the concentration of immunoglobulins was obtained by measuring both the total protein and the albumin concentrations of each obtained purified antibody sample from the dialysis bags. Immunoglobulin concentrations ranged from 2 to 2.4 g/dl. A 1 mg/ml concentration was then obtained using ultrapure water dilution. Furthermore, half the volume of the separately obtained IgG antibodies against LPS was pooled together in equal ratios to form Brucella-specific polyclonal antibodies (pAbs). Agar gel precipitation test optimization was carried out for evaluating the separated polyclonal antibodies.
Preparation of IgY against LPS (Amro etal., 2018):
1. Immunization of chicken and egg collection
Five chickens of Single Comb White Leghorns that were 6 months old were inoculated S/C with 0.1 ml of 5 mg of S-LPS. Three doses were inoculated with a two weeks interval between the first and the second vaccination and one week between the second and the third vaccination. To detect the best quantities of IgY antibodies, Eggs were collected from the immunized chickens between specific time intervals of the three doses. Samples were then stored at 4 C until the extraction of the IgY from the immunized egg yolk.
2. Extraction of chicken IgY antibodies from immunized Single Comb White Leghorns egg yolk
This step was carried out by using Polyethylene Glycol (PEG 6000) precipitation. The eggshell was cracked very carefully and the egg yolk was transferred on a filter paper to remove the remaining egg white. The egg yolk skin membrane was cut before the yolk was poured into a 50 ml tube to measure its volume. Twice the egg yolk volume of Phosphate buffered saline (PBS) was added to the yolk tube and mixed by vortexing. A 3.5% of PEG 6000 of the total volume was added to the yolk tube, vortexed and rolled for 10 min by a rolling mixer before the tube was centrifuged for 40 minutes at 5000 rpm at 4 C. The supernatant was then poured through a folded filter paper and transferred to another tube before 8.5% PEG 6000 was added in relative to the new volume and then vortexed and centrifuged at 5000 rpm for 40 min at 4 C. The supernatant was discarded and the pellet was dissolved in 1 ml PBS using a glass stick, then was mixed by vortexing before 9 ml of PBS were added to the tube forming a final volume of 10 ml. The formed solution was then mixed with 12% PEG 6000 and then mixture was centrifuged at 5000 rpm for 40 min at 4 C. The supernatant was discarded and the pellet was dissolved in 800 ml PBS using a vortex glass and stick. Extract was finally transferred to a dialysis membrane (Cellu Sep H1, part #5050-28) to get rid of the salt. Dialysis tube was first soaked in distilled H2O for 15 min before the sample was passed through the opening end of the tube. The filled dialysis membrane was immersed in the dialysis buffer solution (10 mM SP buffer) and the sample was stirred overnight using magnetic stirrer. In the next day, the SP buffer was discarded and the sample was soaked in PBS for 3 hours. Following the soak, the sample was pipetted from the dialysis bag in 2 ml Eppendorf tubes and stored at -20 C until used.
Conjugation nanogold with rabbit IgG anti S-LPS (Sayed etal 2023):
Using 0.02M K2CO3, The colloid gold solution was adjusted to pH 8.5. With gentle stirring, 100ul rabbit IgG anti S-LPS (1mg/0.1ml of 0.05% NaCl buffer) were added drop wise to 10ml of pH-adjusted colloid gold solution. The mixture was gently mixed for 10min and then blocked using 1% (m/v) final concentration of polyethylene glycol (PEG - 20,000 kDa) followed by stirring for an additional 15 min and centrifugation for 30 min at 10,000 g. The pellets were suspended in 1ml dilution buffer [20mM Tris/HCI buffer (pH 8.2) containing 3% (w/v) sucrose, 1% (w/v) BSA and 0.02% sodium azide then stored at 4°C until used. IgY extraction was confirmed by SDS-PAGE and Western blot
Preparation of the immunochromatographic lateral flow kit (Rafik et al., 2023 and Ajaikumar et al., 2021):
1. Sample pad (Ahlstrom)
Sample pad is made of glass fiber and were saturated with PBS solution of pH 7.2 containing blockers like bovine serum albumin (1%), casein (0.1–0.5%), gelatin (0.05–0.1) and surfactants like Triton X-100 (< 0.05%) and Tween-20 (< 0.05%) and dried at 37ºC. Then it was kept under dry conditions at room temperature until used.
2. The conjugate pad (Ahlstrom):
The conjugate pad made of glass fiber and was treated with 0.1% Tween-20 for 10 min and dried at 60 °C. The prepared glass fiber was cut into sections (4cm×0.5 cm), and then saturated with 150µl of colloidal gold probe consisting of rabbit IgG anti brucella S-LPS conjugated with nanogold. The conjugate pad was dried for 1 hour at 37°C and stored under dry conditions at 4 °C until used.
3. Nitrocellulose membrane (BIODOT -XYZ-3):
Two lines were dispensed on the nitrocellulose membrane (25mm×300mm) first line was the IgY anti LPS (1.5mg/0.1ml) which was dispensed on the test (T) line (1µl per 1 cm line) and the second line was for goat antirabbit IgG immunoglobulin (1mg/0.1ml) which was dispensed on the control line C (1µl per 1 cm line) .
4. Cutting the membrane:
After that, the membrane was covered with top laminate and cut into 0.5-cm-width test strips by using an automated cutter (Guillotino Cutter GCI1800),
Interpretation of the test
This LFT is coated with two lines (a control line and a test line). If the specimen contains Brucella antigens, they will bind to the Brucella antibodies coated on the test line region (T) generating a colored line, indicating a positive result. While if Brucella antigens are not present in the samples, the test line region will not generate any colored line indicating a negative result. As a control, a colored line will always appear in the control line region (C) either in negative or positive specimens, indicating that the test procedure has been carried out in a proper manner.
In case of positive results two colored lines will appear, and a colored line should always appear in both control (C) line and in test line (T) region. A positive result indicates the detection of Brucella antigen in specimen as shown in Figure 1 (1). But in the case of negative results, a colored line appears only in the control region, while a colorless test line appears, as shown in Figure 1 (2). Both A and B are valid results. Invalid results as shown in Figure 1 (3), occur when a colored control line does not appear. Interpretation and reading of the test were performed within 5-10 minutes.
Gold standard tests
Isolation and detection of Brucella recovered from clinical specimens and its identification traditionally and by multiplex PCR assay (BRUCE-LADDER) are used in this study as gold standard tests.