One Health in Action: Operational Aspects of an Integrated Surveillance System for Zoonoses in Western Kenya

Surveillance of diseases in Kenya and elsewhere in East Africa is currently carried out by both human and animal health sectors. However, a recent evaluation highlighted the lack of integration between these sectors, leading to disease under-reporting and inefficiencies. This project aimed to develop an integrated and cost-effective surveillance and reporting system for 15 zoonotic diseases piloted in the counties of Bungoma, Busia, and Kakamega in western Kenya. Specifically, in this paper we describe the operational aspects of such a surveillance system. Interviews were carried out with key informants, and this was followed by field visits to identify sentinel sites and liaise with relevant stakeholders. Based on this information, a sampling strategy comprising 12 sentinel sites, 4 in each county, was developed. Each sentinel site comprised of a livestock market, 1–2 neighboring slaughter houses/slabs, and a hospital in the vicinity; each of the 12 sites, comprising 12 × 3 = 36 sampling locations, was visited every 4 weeks for 20 cycles. At each site, animal or patient sampling included a clinical examination and collection of blood, feces, and nasal swabs; in slaughtered animals, mesenteric lymph nodes, hydatid cysts, and flukes were also collected. At the end of each field visit, data on staff involved and challenges encountered were recorded, while biological samples were processed and tested for 15 zoonotic diseases in the field laboratory in Busia, Kenya. Public engagement sessions were held at each sentinel site to share preliminary results and provide feedback to both stakeholders and study participants. A livestock market visit lasted just over 3 h, and the most common challenge was the frequent refusals of animal owners to participate in the study. At the slaughterhouses, visits lasted just under 4 h, and challenges included poorly engaged meat inspectors or slaughter processes that were too quick for sampling. Finally, the hospital visits lasted around 4 h, and the most frequent challenges included low patients turn-out, frequent staff turn-over leading to poor institutional memory, and difficulty in obtaining patient stool samples. Our experiences have highlighted the importance of engaging with local stakeholders in the field, while also providing timely feedback through public engagement sessions, to ensure on-going compliance.

The monoclonal antibodies used in the test developed by Brandt et al. (1992) and modified by Van Kerckhoven et al. (1998) and Dorny et al. (2000) were originally prepared against antigens from T. saginata, but can not only detect viable cysticerci in bovines, but also cysticerci of T. solium in pigs and humans. The cross-reactions between antigens produced by T. solium and T. saginata metacestodes are a welcome advantage here. There are, unfortunately, also cross-reactions with antigens from T. hydatigena metacestodes (Dorny et al., 2004) and T. s. asiatica metacestodes (Fall et al., 1996; in pigs. Therefore, in regions where these parasites are endemic, the detection of T. solium cysticercosis is restricted (Dorny et al., 2001) and accurate data on the prevalence of porcine cysticercosis are not easily available or are of questionable reliability (Rajshekhar et al., 2003).

The test used for antigen detection in urine is a slightly modified test!
• General principle of the test To use this assay (see Figure), a purified antibody (the "capture" antibody) is bound to a solid phase (a polystyrene plate). Antigen is then added and allowed to complex with the bound antibody. Unbound products are then washed away; a labeled second antibody (the "detection" antibody) is allowed to bind to the antigen, thus completing the "sandwich". The assay is then quantified by measuring the amount of labeled antibody.
The intensity of the color can be measured (OD). The obtained OD's are then processed statistically to determine whether a sample is positive or not. This interpretation is based on a set of known positive and   The monoclonal antibodies are developed, produced and labeled by the Veterinary Helmintholgy Unit (VHU).
The production procedure is described in general by Harlow and Lane (1988).
• The capturing antibody The capturing antibody (B158C11A10) is used at 5 µg/ml coating buffer (pH 9.6). The quantity of monoclonal you must take to have 5 µg depends on the batch of monoclonal you are using.
We recommend that the capturing antibody be stored at -20°C.
• The detecting antibody The detecting antibody (B60H8A4) is labeled to biotin and is used at 1.25 µg/ml blocking buffer. The quantity of monoclonal you must take to have 1.25 µg depends on the batch of monoclonal you are using. We recommend that you store the antibody at +4°C. Following the manufacturer's instructions, we added 1% bovine serum albumin.
• Buffers and product preparations

Phosphate Buffered Saline (PBS)
The PBS buffer is prepared using tablets.
One tablet in 100 ml of RO-DI water yields a 100 ml PBS buffer, pH 7.3.

Trichloroacetic acid (TCA)
The solution used for the "pretreatment" of the serum samples is a 5% (W/V) solution in RO-DI water.
Example: dissolve 0.5 g of TCA crystals in 10 ml RO-DI water.

Washing buffer
The washing buffer consists of PBS with 0.05% (V/V) Tween 20.

Blocking buffer
The blocking buffer consists of washing buffer + 1% (V/V) of Newborn Calf Serum (NBCS).
Example: 49.5 ml of PBS-Tween 20 + 0.5 ml of NBCS Note that the NBCS has to be heat inactivated before use. To do this, you must put the serum at 56°C for 30 minutes (in a waterbath for example).

Coating buffer
The coating buffer is prepared using powder-filled capsules.
One tablet in 100 ml of RO-DI water yields a 0.05 M carbonate/bicarbonate buffer, pH 9.6.
Alternative: Carefully add HCl or NaOH until pH to 9.6 is reached.
Adjust volume to 250 ml with RO-DI water Adjust pH to 10 by adding either HCl or NaOH.
Once the pH is set, adjust the volume to 500 ml with RO-DI water.

Phosphate citrate buffer
The Phosphate-citrate buffer is prepared using tablets.
1 tablet in 100 ml of RO-DI water to obtain a 0.05M Phosphate citrate buffer, pH 5.0.

Sulfuric acid (H2SO4)
The acid we use comes in cartridges. Take the contents of a cartridge and add RO-DI water up to 250 ml. This gives you 250 ml of H2SO4 4N.
• Recommended storage temperature and storage time for buffers and products The storage time of Streptavidin-HRP depends on the storage temperature.

Sample for analysis
• "Pretreatment" of the samples Aim of the pretreatment: 1. Break down immune complexes to obtain free circulating antigen.

Reduce cross-reactions with sera of i.a. individuals infected with Trypanosoma.
To do this, the samples are mixed with an equal volume of trichloroacetic acid (TCA).
This breaks down the immune complexes. The samples are then neutralised (= bring pH from low the neutral) with a neutralisation buffer.
• Incubate for 20 min. at ambient temperature.
• Mix again by vortexing.
• Neutralise mixture by adding 75 or 150 µl (for negative controls or other samples, respectively) of the supernatant into the eppendorfs with the same amount of neutralisation buffer. This results in a final dilution of 1/4 of your sample(s).
Important note: When using positive control sample K504, you must "pre-dilute" the sample before doing the TCA treatment. This gives the sample a final dilution of 1/640 as opposed to a 1/4 dilution for a normal sample.

• Controls
Please adopt the plate layout as it is shown in the table.
Note that positive controls (+1 & +2) and the unknown samples (? 1 to ? 40) are 2wells/ sample, while the negative controls (-1 to -8) are 1 well/sample. • TCA and H2SO4 may cause severe burns, so wear protective gloves and avoid release to the environment. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
• OPD may cause discomfort after skin contact and there is limited evidence of a carcinogenic effect, so wear protective gloves and avoid release to the environment.

Method
Check to make sure all reagents and products are prepared and/or available. If you have a lot of samples to test, start with the pretreatment of the samples (as described in section 1.3). If not, you can start with the coating of the plate and do the treatment during the incubation time of the coating and the blocking step.
Note: The below procedure should be done quickly. A multichannel pipet can be used to expedite the process.

Quality control
There is a conjugate and substrate control on each plate (CC en SC, respectively). Non-specific reactions between the plate, coating/blocking and conjugate are intercepted by the conjugate control. The quality of the substrate (e.g. by influence of light) can be traced by the substrate control. Both controls need to be negative (below cut-off value).
The table below shows the contents of the SC and CC controls for each step of the ELISA assay. If desired, you can contact the VHU for a set of positive and negative reference samples to evaluate the assay in your lab.
• Consistency between the wells One of the most important aspects of any assay is consistency and standardization of conditions as this will affect the reproducibility and accuracy of your results.
Therefor (automatic) multichannel pipets and plate washers provide more consistent and faster results, as well as higher throughput. Certainly in this ELISA the use of a multichannel pipet is very important if you work with full multiwell plates because of the short incubations. Further calibrations of all pipettors on a regular base are necessary, or there can be significant variation in the results.
• Technical validation • The eight negative reference samples can be checked by means of the following formula: 100 X SD/MEAN < 100 • The unknown samples can be evaluated also with the above formula.
• The positive reference samples are used to avoid mistakes during the test. The fact that they give a positive result suffices.
• Interpretation of the results • All positive and unknown samples are done in duplicate. Check if the 2 wells containing the same sample give roughly the same OD. If this is ok, you then calculate the average OD for every sample.
average OD = [OD well 1 + OD well 2 ]/2 • The cut off is calculated based on the OD's of the negative samples using a variation of the students test (Sokal and Rohlf, 1981). Once the cut off has been determined, it can be used to calculated a ratio. Ratio = average OD/cut off • When the ratio is > 1, the sample is considered positive with a certainty of 99.9 %.
• There is a Microsoft Office Excel file available to assist with these calculations.
Please contact the VHU for more information.
Ç 5 Reporting and Archiving