Standardization of Modified Counterimmunoelectrophoresis for Human Rabies Serological Control: Drive to One Health

Abstract

The aim of the study was to standardize the modified counterimmunoelectrophoresis test using a reference serum, converting the method into a quantitative assay. A retrospective survey was conducted on titration assays of sera from individuals vaccinated against rabies, compared to the national reference rabies antiserum. The intermediate precision of the assay was assessed, and a control chart was developed. The coefficients of variation (CV%) for the modified counterimmunoelectrophoresis test were 28.31% and 46.31% for assays with the national reference antiserum diluted to 1.0 IU/mL and 2.0 IU/mL, respectively. The results plotted in the control charts were suitable for monitoring process control. No significant difference was found between the mean results for the reference serum obtained using Combistats® software and those recorded on the result forms. The coefficients of variation observed were considered acceptable due to the high variability typical of biological assays. The use of control charts proved to be suitable for ensuring the quality of the results. The technique was successfully improved, enabling the quantitative determination of neutralizing antibody titers against rabies virus in humans, contributing to public health and rabies health surveillance in the One Health context.

Keywords:
Standardization; Modified counterimmunoelectrophoresis test; Rabies; Antibody titration; One Health.

HIGHLIGHTS

Counterimmunoelectrophoresis is a suitable method for determining rabies antibody titers.

The method was standardized and converted into quantitative.

Combistats software improves the reliability of the results.

Control charts were useful for identifying systematic issues.

The method can contribute for One Health approach in rabies surveillance.

INTRODUCTION

Rabies is a serious acute infectious disease, characterized as a progressive acute encephalitis with an almost 100% fatality rate [¹]. Due to this high lethality, rabies remains a public health problem in several countries across all continents, except Antarctica [²]. The etiological agent responsible for the infection is a neurotropic virus from the Rhabdoviridae family, Alpharhabdovirinae subfamily, Lyssavirus genus, and Rabies lyssavirus species [³].

Pan American Health Organization (PAHO) coordinates rabies control programs and surveillance in the Americas. Despite all the successful efforts, advances in controlling rabies and decreasing number of cases in the Americas, from 2010 to 2024, 378 human rabies cases were reported in the region, with 2011 recording the highest number at 73 cases. Grenada, Suriname, Panama, Belize, Costa Rica, Nicaragua, Paraguay, and El Salvador were the only countries in the Americas with no reported human cases during that period [⁴].

Animal rabies cases have become significant in Brazil and other countries in the Americas, and given the overlap of human, domestic, and wild animal populations in the same environment, it represents one health concern [⁵-¹⁰]. Invariably, a rabies infection has nearly 100% fatality rate, involving the traditional epidemiological triad of host, agent, and environment, forming an appropriate backdrop. For example, rabies virus infection has been demonstrated in marsupials in the southwestern United States and in bats in several regions of Brazil, affecting domestic animals such as cats, raising intriguing questions about the public health significance of viral spillover, adaptation, compartmentalization, and disease persistence [⁵,¹⁰,¹¹].

Rabies prophylaxis consists of preor post-exposure vaccination with inactivated anti-rabies vaccine, with or without the administration of anti-rabies serum or human rabies immunoglobulin [¹²,¹³]. Serological rabies monitoring in humans is crucial to verify prophylaxis efficacy, monitor professionals at-risk, support epidemiological studies, validate diagnostic methods, and in clinical trials of vaccines. It assesses whether an adequate immune response to vaccination occurred (>0.5 IU/mL) and gives help in deciding on booster doses [¹⁴,¹⁵].

The neutralization assays routinely used and recommended by the World Health Organization (WHO) and the World Organization for Animal Health (WOAH) for detecting and titrating rabies virus neutralizing antibodies (RVNA) are the rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test. While effective, these techniques are labor-intensive and time-consuming, requiring infectious virus and biosafety facilities [¹⁶].

Since 2018, WHO has recognized the modified counterimmunoelectrophoresis test (MCIE), developed in the 1980s [¹⁷], as a suitable method for titrating anti-rabies antibodies and evaluating rabies vaccination regimens [¹⁶]. However, incorporating quality assurance procedures-such as assay acceptance criteria-is still necessary to enhance its reliability [¹⁸]. MCIE is a qualitative assay that shows good correlation with the mouse neutralization test [¹⁷] and RFFIT [¹⁹]. The aim of this study was to test the effects of quality improvements in the method and convert it into a quantitative technique, by adopting assay acceptance criteria and process control to ensure the method’s quality, thereby facilitating result interpretation by healthcare professionals and enhancing the relevance of the method for rabies surveillance.

MATERIAL AND METHODS

This study conducted a reanalysis with the aim of standardizing the MCIE using samples from a retrospective review of assays performed in 2016. A direct comparison with neutralization methods such as RFFIT and FAVN was not carried out.

MCIE Reagents

Antigens (AG) from two batches were prepared by intracerebral inoculation of Challenge Virus Standard 37 (CVS-37) strain in suckling mice. It was produced in a 40% (w/v) suspension and, before standardization, should present a titer of 10⁶ LD50/30µL in weaned mice. AG remains stable for up to 24 months, but it is recommended to perform its titration every six months. The indicator serum (IS) was produced by immunizing rabbits and had its titer determined by the reciprocal of the highest dilution which presents a precipitation line, as described by Diaz and WHO [¹⁵,¹⁷]. The national reference standard of equine anti-rabies serum, batch BR011 (RS), containing 560 IU of antibodies against the rabies virus per ampoule, diluted at 1 and 2 IU/mL, was produced by the Butantan Institute in São Paulo, standardized and established by the National Institute for Quality Control in Health (INCQS) - Fiocruz, Brazil.

Samples

A total of 169 MCIE assays conducted in 2016 were analyzed, using the RS to determine the RVNA titer expressed in IU/mL. For this study, 573 serum samples from individuals receiving preor post-exposure rabies prophylaxis were analyzed. No invalid assays occurred during the study period, and no samples were excluded for technical reasons. The data were obtained from the database of the Virology Laboratory of the Municipal Health Department of Rio de Janeiro, Brazil.

MCIE Standardization

Calculation of Results of Titration in IU/mL

To calculate the results in IU/mL, the following mathematical model was used:

$$\text{RVNA}=\text{RDA}/\text{RDSR}\times \text{PDSR}$$

Where: RVNA is the rabies virus neutralizing antibody titer; RDA is the reciprocal of the highest dilution of the test sample that neutralized the AG; RDSR is the reciprocal of the highest dilution of the RS that completely neutralized the AG; PDSR is the pre-dilution of the RS in IU/mL.

The original results were rechecked against the MCIE manual calculations recorded in the worksheets for each test serum sample, verifying any inconsistencies in readings or calculations. New RVNA titers were recalculated using Combistats® software to verify analytical precision.

Statistical Analysis

The D’Agostino & Pearson, Shapiro-Wilk, and Kolmogorov-Smirnov tests were performed to assess whether data followed a normal distribution. To compare the mean titration results recorded on the assay sheets with those calculated by the Combistats® software, the non-parametric Wilcoxon signed-rank test was used. For the recalculation of results in the software, the sigmoid curves model (4-PL, ln dose) was used, employing a completely randomized design and the probit method for estimating values using linearizing transformation.

Evaluation of Intermediate Precision

The assay’s measurement dispersion was evaluated by calculating the coefficient of variation (CV%) of the RS results at concentrations of 1.0 IU/mL and 2.0 IU/mL.

Control Chart

The performance of the assay over time was monitored using an individual value control chart (x-chart) for the RS. From the first 20 results, the mean, standard deviation (SD), and warning (mean ± 2 SD) and control limits (mean ± 3 SD) were calculated, following international guidelines [¹⁶,²⁰]. If the distribution does not follow normality, the control chart will be constructed for process control purposes [²¹].

This research used secondary data with no human or animal involvement. Authorization for data collection was granted by the Technical Coordination of the General Coordination of Innovation, Projects, Research, and Sanitary Education of IVISA Rio.

RESULTS

A total of 169 MCIE assay runs were identified, comprising 573 serum samples from individuals vaccinated against rabies, received by the Laboratory of Virology from the Municipal Health Department of Rio de Janeiro during 2016. All assays followed the MCIE described previously [¹⁷], with adaptations for standardization and titration of the IS, AG, and RS [¹⁶,²²,²³].

Adoption of the National Reference Anti-Rabies Serum and Application of the Mathematical Model

During the visual re-analysis were found in 19 (3.32%) inconsistences of the 573 test sample results, showing discrepancies between the manual calculations recorded in the worksheets and those generated by the software. The results were recalculated and the new values were used for statistical analysis.

Evaluation of MCIE Intermediate Precision

The RVNA titration results in IU/mL did not follow a normal distribution, even after logarithmic transformation (Figure 1 and Table 1).

Table 1
Assessment of normality using the D’Agostino & Pearson, Shapiro-Wilk, and Kolmogorov-Smirnov tests of the results in IU/mL of rabies antibody titrations and their respective logarithmic transformations by MCIE.

Statistical Data	IU/mL		Logarithmic transformation
	Assay Sheets	Software	Assay Sheets	Software
Number of results	573	573	573	573
Minimum	0.1	0.1	-1	-1
Maximum	33	33	1.52	1.52
Mean	5.353	5.39	0.4199	0.4281
Standard deviation	5.814	5.808	0.6276	0.6228
D'Agostino & Pearson test
K2 1	262.9	261.9	63.6	67.37
p	< 0.0001	< 0.0001	< 0.0001	< 0.0001
Passed normality test (alpha=0.05)?	No	No	No	No
Shapiro-Wilk test
W 2	0.7613	0.7631	0.8918	0.8893
p	< 0.0001	< 0.0001	< 0.0001	< 0.0001
Passed normality test (alpha=0.05)?	No	No	No	No
Kolmogorov-Smirnov test
KS 3 distance	0.2709	0.2682	0.1834	0.1829
P	< 0.0001	< 0.0001	< 0.0001	< 0.0001
Passed normality test (alpha=0.05)?	No	No	No	No

• ¹
  K2 statistic;
• ²
  W statistic;
• ³
  KS satistic.

Figure 1
Histograms of the results of rabies antibody titrations by MCIE in IU/mL from the assay sheets and the results obtained by the CombiStats® software, and their corresponding logarithmic transformations. (a) Assay sheets histogram results; (b) Software results; (c) Assay sheets logarithmic results; (d) Software logarithmic results.

The Wilcoxon signed-rank test in the comparison of the mean titration values from the assay forms and Combistats® software results demonstrated no statistically significant difference between the sample result means (p = 0.0691).

The intermediate precision analysis of MCIE using RS results of 169 independent assays showed CV% values of 28.31% for six runs with RS at 1.0 IU/mL and 46.31% for 163 runs with RS at 2.0 IU/mL. The assays were performed on different days, with three antigen lots and the same operator (Table 2).

Table 2
Inter-assay variances and coefficients of variation of the logarithms of the Reference Serum diluted to 1.0 IU/mL and 2.0 IU/mL for the MCIE assays.

Statistical Data	ED50 Ref 1 1.0 IU/mL	ED50 Ref 2.0 IU/mL
Mean	9.423333	7.752515723
Variance	7.119022	12.89406663
SD2	2.66815	3.59083091
Inter CV%3	0.283143	0.463182667

• ¹
  Reference serum effective dose 50%,
• ²
  Standard deviation,
• ³
  Inter-assay coefficient of variation. N = Six assays of ED50 Ref 1.0 IU/mL; 163 assays of ED50 Ref 2.0 IU/mL.

Likewise, the mean effective doses 50% (ED50) results of RS did not follow a normal distribution, even after logarithmic transformation (Figure 2).

Figure 2
Histogram of the results of the logarithms of the ED50 of the Reference Serum. Ten trials were excluded, six due to the Reference Serum being diluted to 1.0 IU/mL and four due to the inability of the Combistats® software to calculate the ED50 of the standard.

Even with the non-normality of the data, two control charts were constructed for pragmatic process monitoring, based on the logarithmic ED50 values of the RS. The analysis revealed two distinct regions, indicating a change in the process mean from trial number 70 onwards. The distribution of results showed a notable change in the process, marked by a large number of consecutive points below the mean, which persisted until the end of the observed period (Figure 3).

Figure 3
Control chart of the ED50 of RS diluted 2.0 IU/mL by modified counterimmunoelectrophoresis test. (a) Assays number 1 to 70; (b) Assays number 71 to 163 - the first point of this chart indicates the change in behavior, corresponding to the introduction of the new batch of antigen.

DISCUSSION

Serological tests such as Counterimmunoelectrophoresis test (CIE) have primarily been used in epidemiological surveillance, studies of vaccine efficacy, and monitoring individuals exposed to infectious agents. It is widely employed in the diagnosis of various diseases and has proven to be an important tool in public health helping in the identification of infections and monitoring of immune responses [²⁴,²⁵]. The use of MCIE as an alternative to other serological tests that require suitable facilities for animal maintenance or cell culture can be of great value, as exemplified by the success observed in Brazil and other Latin American countries. This technique has provided support for one health actions in controlling human rabies in vaccinated individuals through serological monitoring.

MCIE was developed as a qualitative screening test to detect specific antibodies against the rabies virus. Primarily identifying IgG anti-viral glycoprotein antibodies, which play a crucial role in the body's defense against infection [²⁶]. It is described as a sensitive, simple, low-cost, and relatively quick procedure, making it a useful technique for rapid and cost-effective serological monitoring [¹⁶,²²,²⁶].

Previous research has demonstrated the high agreement between MCIE and RFFIT [¹⁹,²⁷-²⁹], which also infers its agreement with FAVN [³⁰]. Here, it was also demonstrated that MCIE can be used as an alternative to evaluate rabies vaccination, as previously recognized [¹⁶]. The method has proven suitable to detect the minimum acceptable level of antibodies (0.5 IU/mL) [³¹]. When compared with the standard techniques, MCIE presented good sensitivity and specificity for estimating antibodies against rabies virus [¹⁹,²⁹,³²]. The CV% values obtained in this research demonstrated adequate intermediate precision, and the use of the control chart, supports the MCIE technique described in this study as a quantitative method.

As previously identified, although MCIE is a simple technique to perform, the production of viral antigen and other biological materials and reagents used are complex steps that require careful standardization [²²]. Special attention is crucial to all stages of the technique, including production and standardization of AG, production and establishment of the working dilution of IS, as well as validation of each electrophoretic run and interpretation of the results. This allows for evaluation of the assay's performance and identification of the need for decision-making regarding preventive and/or corrective actions [¹⁶,²²].

Although this study did not perform a comparison with standard techniques, literature describes that MCIE provides results in a short period of time (4 h vs. 24-48 h) and is therefore useful for clinical practice [²⁷,³¹,³³-³⁵]. Furthermore, the technique does not use a standard dose of viable virus or a cell culture system to reveal viral neutralization, components that introduce great variability in antibody titration results and involve safety issues due to the handling of infectious material, requiring special high-containment laboratory facilities. The same applies to cell line maintenance system and media, which must be regularly monitored for adventitious agents, which increases the costs of laboratory implementation of these techniques. Several studies have reported other disadvantages of the standard techniques, especially regarding the reading of microscopic fluorescence, which is tedious and requires experienced and highly trained technicians to perform [³⁶-³⁹]. A detail that can be critical and has already been identified in vaccinated animals with low response is that RFFIT and FAVN can produce false-positive results and false-negative results when used in separate [³⁷]. This was not identified in this study using MCIE in human sera. Therefore, these tests are time-consuming, require experience, are expensive, and are generally performed in reference laboratories and are therefore not widely available. In contrast, MCIE has proven to be simple to perform and operationalize, which can help in decision-making regarding its implementation in public health laboratories [⁴⁰].

Adherence to quality control (QC) procedures, such as the use of control samples (e.g., RS), is essential to ensure analytical accuracy and precision, as supported by various studies [³²,⁴¹]. Compliance with QC goes beyond legislation, encompassing quality standards [⁴²] that standardize procedures and minimize operational errors. This practice enhances the reliability of results and safeguards patients, especially in tests for high-fatality diseases like rabies [³²].

As recommended [⁴³], official reference chemicals or biological standards must be used, even for qualitative methods. Reference materials should be traceable, whenever possible, to systems like the International System of Units or higher-order certified reference materials, such as WHO/NIBSC primary international standards. Their use provides essential traceability, enabling analysts to demonstrate result precision, calibrate instruments and methods, and monitor laboratory performance, as proposed in this study [⁴³].

Recent literature produced few specific studies to guide the formulation of public policies for controlling diseases such as rabies [⁴⁵,⁴⁶]. The adoption of the national reference serum standard proved effective in determining the antibody titer of the 573 samples analyzed by MCIE. Calibration of the entire analytical process, comparing test sample results with those from the reference material (RS), was implemented in electrophoretic runs, with the RS undergoing the same full analytical process as the test samples [⁴⁴]. This introduced a reliability parameter into the results, enhancing the method’s credibility and enabling future relevant comparisons across laboratories [⁴⁷]. Using a reference standard introduced internal control improving quality assurance.

Although serological tests are generally considered qualitative - indicating the presence or absence of antibodies [⁴⁸] - it was possible to adapt MCIE as a quantitative method, enabling the expression of equivalent values in IU/mL relative to the RS tested alongside serum from vaccinated individuals. This will make it easier for healthcare professionals to interpret the results. As demonstrated in a study designed to evaluate rabies prevention policies, performing serological monitoring using this tool will allow them to reconsider the need for booster dose policies [⁴⁵].

To assess measurement dispersion, assay precision was calculated as recommended by ISO 5725-3 [⁴⁸], using the same method in the same laboratory with identical or standard samples. This allowed evaluation of the degree of agreement across multiple determinations of the same sample by calculating the CV% between assays using RS at 1.0 and 2.0 IU/mL [⁵⁰]. According to Gomes [⁵¹], the CV% for RS at 1.0 IU/mL would be classified as high, and for RS at 2.0 IU/mL as heterogeneous, since it exceeded 30%. The lack of robustness of moment-based measures like the mean and SD in the presence of outliers is well known [⁵²]. While these CV% ranges apply mainly to chemical analyses, WHO states that biological assays may present a variability higher than 50%. For anti-rabies vaccines, up to 400% variation is accepted, as specified by the European and Brazilian pharmacopoeias [¹⁸,⁵³]. Thus, the values obtained in this study are justified by the high variability of bioassays [⁵⁴].

The Wilcoxon test showed no statistically significant differences between the mean values of the results from assay worksheets and those generated by Combistats®. However, using computational software allows for faster, more reliable result generation, minimizing the risk of errors - as evidenced by inconsistencies in readings and calculations during the study - and reduces subjectivity in result interpretation [⁵⁵].

Researchers have shown that test performance should be monitored using control charts for both AG and RS, as they are critical reagents [¹⁶]. In this study, only a control chart for RS was created, since AG is not titrated during electrophoretic runs, but only during standardization, as described in previous studies [¹⁶,²²,³³].

Lacking QC in MCIE runs could lead to false negatives, for example, failing to detect seroconversion. This could burden the public health system, since anti-rabies vaccination in Brazil is provided free of charge through the Unified Health System (SUS). Beyond financial costs, unnecessary stimulation of vaccinated individual’s immune systems must be considered, as suggested by Moreira and coauthors [⁵⁶].

To build the control chart, only results from RS diluted to 2.0 IU/mL were used, as the number of results at 1.0 IU/mL was insufficient for such statistical analysis, per WHO [¹⁶,¹⁸] and ISO [²⁰]. In the first 70 assays, the process was shown to be under control. The chart then indicated that from the 71st assay onward, the process appeared out of control. During this period, the AG batch was replaced, and the working dilution was changed. Two separate control charts were constructed - one for assays 1-70 and another for 71-163 (Figure 3). Systematic causes like these should be identified, allowing for interventions or corrections, as recommended by WHO [¹⁶], Queiroz and coauthors [²²], and Díaz [³³]. As reported in other studies [⁵⁷,⁵⁸], numerous other variation sources exist, including instrument calibration and maintenance, operational procedures, reagent storage conditions, and environmental factors [⁵⁹]. These were not addressed in this study. As described by others, serological assays for infectious diseases exhibit normal lot-to-lot variation, as observed here [⁴⁸]. If deviations occur, it may be necessary to retitrate standards, as advised by WHO [¹⁶], Díaz [¹⁹], and Queiroz and coauthors [²²].

Dimech [⁶⁰] emphasized the importance of results being comparable between laboratories and reproducible over time. However, analysis of the control chart of RS ED50 values indicated a period of process instability, suggesting the model used was inadequate. Moreover, applying standard antibody testing procedures and control chart rules like Westgard or Western Electric - used to detect non-random variation - may not be appropriate for serology, leading to waste [²¹,⁴⁸]. Serological assays are essentially qualitative and involve biological and polyclonal analytes, unlike the inert analytes used in clinical biochemistry. Thus, standardization principles from biochemistry are not directly transferable to serology [⁴⁸,⁶⁰].

While QC guidelines were developed for clinical biochemistry, they don’t directly apply to serology for infectious diseases, as the results don’t follow a normal distribution. As such, applying rules like Westgard’s may lead to false rejections. Dimech, Karakaltsas, and Vincini highlight this concern, as do other authors who argue that normality should not be a prerequisite for control chart use. In biochemistry, standardization allows traceability and comparability between lots, but in serology, analyte and assay component variability limits this equivalence-requiring distinct approaches [²¹].

The following control rules were applied to assess whether the process was under control: the occurrence of a point beyond the upper or lower control limits, nine consecutive points on the same side of the mean, six consecutive ascending or descending points crossing the mean line, and two out of three consecutive points beyond the upper or lower warning limits, to demonstrate whether there were trends in the results that require investigation or supervision [²⁰].

International standards are not effective for calibrating immunoassays on different testing platforms. The complexity of this scenario increases when the same QC sample is tested over time, as performed in this study, and the introduction of new reagent batches results in a change in the reactivity of the QCs, as observed in the control chart developed from the RS ED50. In this situation, the mean of the results obtained with previous reagent batches, used to define the QC acceptance limits, becomes incompatible with the new batches, leading to the rejection of the quality controls. This inconsistency can lead to difficulties in interpreting results for laboratories that apply Westgard Rules, as indicated by Dimech [⁶⁰], since these guidelines do not specify which approach should be adopted when changes in reagent batches impact the reactivity of the assays. For this reason, a second chart was constructed as a new region.

Another factor that may explain the control chart behavior is that although the results are individual, they can form a pattern when grouped, showing unique location, dispersion, and shape, as described by Thomé, Souza, and Caten [⁶¹]. Chart interpretation revealed a shift in the central mean, characterizing a new region and suggesting that process became out of control. When this happens, laboratories often redefine the limits using a new data set. However, this ignores the root cause - replacement of critical reagents like AG in MCIE. This was observed in the study and, as suggested by Dimech and coauthors [⁴⁸] and Dimech & Vincini [⁵⁴], may lead to clinically significant changes affecting MCIE results.

An alternative way to assess RS result distribution is to determine whether titer changes occurred. No titer changes greater than one dilution was observed in this study, as indicated by the Food and Agriculture Organization. According to this organization, the RS titer tested on the same day with the same AG preparation and protocol may vary. A one-dilution difference (equivalent to one log₂ unit) is acceptable due to random error and biological response variability. If the RS titer differs by more than one dilution from a central value - which did not occur in this study - the test is invalid, and a new AG lot must be prepared and the test repeated [⁶²].

Alternatively, non-parametric control charts for non-normal data can be used as suggested by other authors [⁶³-⁶⁵] requiring further studies for future improvements.

Although the process was found to be out of control during a certain period even with the CV% values, there was no risk of releasing incorrect results for patients who had not adequately responded to prophylaxis. When the AG is standardized in MCIE and the working dilution is established for electrophoretic runs, it is calibrated against RS diluted to 1 IU/mL. Thus, any sample that completely neutralized the antigen at the lowest test dilution (1:2) indicates that the patient showed seroconversion at this dilution and had at least a titer of 0.5 IU/mL, which is the WHO-recommended threshold for humoral immune response to vaccination [¹⁵].

CONCLUSION

Based on the results obtained, it was possible to refine the MCIE for determining rabies antibody titers in IU/mL, effectively converting it into a quantitative method. It was demonstrated that the mean results for the reference serum obtained through Combistats® software and those from MCIE result sheets did not show significant differences. Nevertheless, using the software enhances the reliability of the IU/mL titers, facilitates result interpretation and reporting, and reduces the likelihood of analysis inconsistencies. The CV% values found in this study were considered acceptable due to the high expected variability in biological assays. The use of control charts as a process control and analysis tool for evaluating MCIE quality was proven useful for identifying systematic issues, such as changes in critical reagent lots. The application of the proposed model increased the reliability of results from a qualitative technique, proving valuable for confirming the serological immune response of individuals vaccinated against rabies. This constituted a significant contribution to public health and rabies surveillance, enhancing the One Health approach in this context.

• Funding:
  This research received no external funding.
• Institutional Review Board Statement:
  Not applicable.
• Informed Consent Statement:
  Not applicable.

Acknowledgments:

To Alessandra Medeiros Lopes Endres for English support, to professor Márcio José de Figueiredo (in memoriam) and the Virology Unit of the Jorge Vaitsman Municipal Veterinary Hospital for their collaboration.

Use of Generative Artificial Intelligence:

The authors declare that large language models and other generative artificial intelligence (AI) or AI-assisted technologies cannot be credited as authors and have not been listed as authors of this paper.

The author declare that did not use the artificial intelligence.

Data Availability Statement:

Research data are only available upon request for corresponding author.

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• Editor-in-Chief:
  Paulo Vitor Farago

• Associate Editor:
  Paulo Vitor Farago

Publication Dates

• Publication in this collection
  17 July 2026
• Date of issue
  2026

History

• Received
  31 July 2025
• Accepted
  13 Jan 2026