| Journal of Animal Biology & Therapeutics
Received: 10 August 2026; Revised: 11 September 2026; Accepted: 17 September 2026; Published Online: 22 September 2026.
J. Anim. Biol. Ther., 2026, 1(1), 26703 | Volume 1 Issue 1 (September 2026) | DOI: https://doi.org/10.64189/abt.26703
© The Author(s) 2026
This article is licensed under Creative Commons Attribution NonCommercial 4.0 International (CC-BY-NC 4.0)
Development of a Latex Agglutination Test (LAT)
using Whole-Cell Protein for the Detection of
Mycoplasma ovipneumoniae Antibodies in Small
Ruminants
Shanmugasundaram Udhayavel,
1*
Kuppannan Sukumar,
2
Kuppusamy Senthilkumar,
3
Palani Srinivasan
4
and
Ayyasamy Elango
5
1
Department of Veterinary Microbiology, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences
University, Salem, Tamil Nadu, 636112, India
2
Department of Veterinary Microbiology, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences
University, Namakkal, Tamil Nadu, 637002, India
3
Centralised Clinical Laboratory, Madras Veterinary College, Veterinary and Animal Sciences University, Chennai, Tamil Nadu,
600007, India
4
Poultry Disease Diagnosis and Surveillance Laboratory, Veterinary College and Research Institute campus, Tamil Nadu Veterinary
and Animal Sciences University, Namakkal, Tamil Nadu, 637002, India
5
Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Salem, Tamil Nadu, 636112,
India
*Email: udhayacrivet@gmail.com (Shanmugasundaram Udhayavel)
Abstract
Mycoplasma ovipneumoniae is a major pathogen responsible for respiratory illness in sheep and goats,
particularly chronic nonprogressive pneumonia, and its presence results in substantial economic losses within
the small ruminant sector. Although diagnostic methods such as culture and polymerase chain reaction (PCR)
are routinely used, each approach has inherent limitations. There is no rapid, inexpensive and field-deployable
serological assay available for the detection of M. ovipneumoniae antibodies in small ruminants. The objective
of the present study was to develop a latex agglutination test (LAT) to detect Mycoplasma ovipneumoniae
antibodies in small ruminants at the field level. A latex agglutination test (LAT) was developed using whole-
cell protein antigen extracted from M. ovipneumoniae coupled with latex beads (0.80 µm) and tested on serum
samples from 33 PCR-positive and 77 PCR-negative animals. A comparison of the latex agglutination test
results with those of PCR revealed 93.94% sensitivity and 93.51% specificity. The developed serological assay
strongly agreed with the PCR results, with a kappa value greater than 0.81. Hence, this latex agglutination test
kit offers a convenient and rapid means of diagnosing infections in field settings and is particularly useful for
large-scale surveillance of infections in flocks.
Keywords: Latex agglutination test; Small ruminants; Mycoplasma ovipneumoniae; Antibodies; Penside diagnosis;
Serology.
1. Introduction
Sheep and goats play a vital role in the livelihoods of Indian farmers by contributing substantially to meat and
milk production and by exhibiting rapid growth rates. Consequently, they are considered valuable economic
assets. Respiratory diseases in these animals lead to reduced weight gain, increased mortality and
considerable financial losses.
[1]
Such infections arise from multiple interacting factors, with a wide range of
etiological agents contributing to the respiratory disease complex.
[2,3]
Among these, mycoplasmosis, an
emerging transboundary disease of sheep and goats, imposes heavy economic burdens on farmers and
disrupts trade in many regions.
[4-6]
Mycoplasma ovipneumoniae is among the most important pathogens
associated with chronic, nonprogressive pneumonia in small ruminants. First reported in 1963 in Scotland in
sheep with pulmonary adenomatosis, the organism has since been identified in both clinically affected and
apparently healthy animals.
[7]
Molecular detection of the organism has been reported in various parts of India:
isolates from pneumonic sheep and goats in Andhra Pradesh have been confirmed using PCR on nasal swabs,
while Santhiya et al., identified the pathogen in goats exhibiting respiratory symptoms in northern and central
Kerala.
[8,9]
Similarly, M. ovipneumoniae has been detected in nasal secretions, tissues and synovial fluids of
symptomatic small ruminants in the Bengaluru region of Karnataka.
[10]
These reports emphasize the need for
accurate and prompt diagnosis to implement appropriate control measures. Although diagnostic methods
such as culture, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are
widely utilized, each has inherent drawbacks. The culture is labor intensive, requires stringent growth
conditions, and often fails because of the fastidious nature of Mycoplasma species. PCR provides superior
sensitivity and specificity but is costly, depends on advanced laboratory facilities, and is impractical for large-
scale screening in field settings. ELISA is more user friendly, yet its reliability may fluctuate because antigens
can cross-react with related Mycoplasma spp. The latex agglutination test (LAT) has been explored historically
as a rapid serodiagnostic tool. Kende reported that LAT performed comparably to tetrazolium reduction
inhibition and complement fixation tests (CFT) for detecting M. pneumoniae antibodies.
[11]
Slavik and Switzer
applied LAT to diagnose M. hyopneumoniae infection in pigs and observed persistent LAT antibody responses
for up to 48 weeks.
[12]
Rurangirwa et al., developed a polysaccharide-based LAT for diagnosing contagious
caprine pleuropneumonia (CCPP), which has a higher sensitivity than CFT does and is suitable as a pen-side
test with results available in minutes.
[13]
Subsequent applications in various regions have demonstrated the
practicality and cost-effectiveness of LAT for screening for mycoplasmal infections.
[14-17]
Despite these
advancements, no rapid, low-cost, field-ready serological assay currently exists for detecting antibodies to M.
ovipneumoniae in sheep and goats. LAT represents a promising alternative because of its simplicity,
affordability and minimal equipment requirements. However, a dedicated LAT kit for M. ovipneumoniae has
yet to be developed. Establishing such a test would enable quick pen-side screening, enhance surveillance
programs and support timely management of respiratory diseases in small ruminant populations.
2. Materials and methods
2.1 Development and standardization of the Latex Agglutination Test kit
The latex bead-sensitized whole-cell protein of Mycoplasma ovipneumoniae was prepared by the method
outlined by Sankar et al., with slight modifications.
[16]
2.2 Preparation of whole-cell protein
A reference isolate of Mycoplasma ovipneumoniae, which was isolated in our earlier study (Udhayavel et al.,)
was initially inoculated into 2 ml of Mycoplasma experience liquid medium and incubated at 37°C for three
days.
[18]
The inoculum was then transferred to 100 ml of medium and incubated at 37°C for an additional two
days. Subculturing was subsequently performed in 500 ml of Mycoplasma Experience liquid medium, which
was subsequently incubated at 37°C for three days with intermittent shaking. Afterward, the Mycoplasma
cells were harvested by centrifugation at 18,000 rpm in a cooling centrifuge for one hour. Sterility checks
were regularly conducted with each passage to prevent bacterial contamination. The cells were subsequently
washed three times in sterile PBS, resuspended in PBS, and then sonicated with constant pulses for 30
seconds, with 30-second breaks, which was repeated 19 times. The concentration of whole-cell protein was
estimated using the Bradford method.
2.3 Sensitization of latex beads with whole-cell protein
Latex beads (Sigma, 0.80 µm) were used. The latex bead suspension (10%) was washed twice by
centrifugation at 6700 × g for three minutes each time in carbonate–bicarbonate buffer. The beads were then
made into a 2% suspension with carbonate-bicarbonate buffer that was later mixed with an equal volume of
M. ovipneumoniae whole-cell protein antigen (20 µg/ml) diluted in the same buffer. This mixture was
incubated at 37°C for six hours with constant shaking at 250 × g. The sensitized beads were subsequently
centrifuged at 6700 × g for three minutes, after which the pellet was resuspended as a 2% suspension in PBS
containing 5 mg/ml bovine serum albumin (BSA; Himedia). The latex beads were then left at 37°C in a water
bath overnight. Finally, the beads were centrifuged again as before, and the pellet was resuspended in PBS
containing 0.5 mg/ml BSA.
2.4 Latex Agglutination Test kit
A volume of 25 µl of suspected serum was placed onto a clean glass slide. Similarly, 25 µl of the prepared
sensitized latex beads coated with Mycoplasma ovipneumoniae whole-cell antigen was placed adjacent to the
serum. The serum samples collected from the animals whose PCR results were positive and negative in our
earlier study (Udhayavel et al.,) and the antigen were mixed using a stirring rod to form a circular area
approximately 1.5 cm in diameter.
[18]
The glass plate was gently rocked for three minutes.
2.5 Assessment of the diagnostic characteristics of the developed Latex Agglutination Test kit
A latex agglutination test was performed on serum samples collected from 33 PCR-positive and 77 PCR-
negative animals. The results of the latex agglutination test were compared with those of the PCR assay to
determine indices such as sensitivity, specificity and Cohen’s kappa coefficient (k) statistics, with PCR used as
the gold standard. MedCalc’s diagnostic test evaluation calculator was used. The strength of agreement
determined by Cohen’s kappa coefficient (k) was interpreted using standard benchmarks: k ≤ 0 indicates no
agreement, 0.01–0.20 slight, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, and 0.81–1.00
indicates almost perfect agreement.
3. Results
A latex agglutination test was performed on serum samples collected from 33 Mycoplasma ovipneumoniae-
specific PCR-positive and 77 PCR-negative animals. Among the 110 corresponding serum samples tested, 31
showed agglutination when the latex agglutination kit was used (Fig. 1). Two nasal swab samples tested
positive by PCR, but the corresponding serum samples did not show agglutination in the LAT region. Similarly,
five nasal swabs were negative according to PCR, but when the corresponding serum samples were tested,
they were positive according to the latex agglutination test. Therefore, among the 110 serum samples
screened, 36 (32.73%) tested positive by the latex agglutination test. The results are shown in Table 1.
Fig. 1: Latex agglutination test for the detection of Mycoplasma ovipneumoniae antibodies
*Note: Agglutination with Latex beads sensitized with Mycoplasma ovipneumoniae whole-cell protein)
Table 1: Comparative evaluation of the latex agglutination test with PCR
PCR
Positive
Negative
Total
Latex agglutination test
Positive
31 (a)
5 (b)
36 (a+b)
Negative
2 (c)
72 (d)
74 (c+d)
Total
33 (a+c)
77 (b+d)
110
*Note: a - true positive, b- false positive, c- false negative, d- true negative
3.1 Assessment of the diagnostic characteristics of the developed Latex Agglutination Test kit
The sensitivity and specificity of the latex agglutination test compared with those of PCR were 93.94 and
93.51%, respectively. Kappa statistics, which were used to analyze the agreement between the two tests,
revealed a score of 0.852, indicating a high level of agreement (Table 2).
Table 2: Diagnostic characteristics of the latex agglutination test
Formula
Value
[a/(a + c)] × 100
93.94% (95% CI: 79.82%–99.34%)
[d/(b + d)] × 100
93.51% (95% CI: 85.50%–97.85%)
k= a+d-P/1-P
0.852 ± 0.054 (SE)
Fisher's Exact Test
p < 0.0001
*Note: An association is considered highly statistically significant when p < 0.05.
4. Discussion
The plate agglutination test primarily detects animals in the early (acute) stage of the disease.
[19]
The plate
agglutination test requires larger bacterial cells to form visible aggregates. By attaching Mycoplasma cells to
carrier particles such as latex, the mass of the cells can be increased, and the specificity of the Mycoplasma
surface antigens can be maintained.
[20]
The latex agglutination test is a simple macroagglutination test that
combines sensitivity with affordability and ease of use under field conditions, eliminating the need for
specialized training or equipment. In this study, a latex agglutination test (LAT) was performed using latex
beads (0.8 µm diameter) coated with sonicated Mycoplasma ovipneumoniae whole-cell protein. Ramadass et
al., employed latex beads with a diameter of 0.8 µm for an agglutination test and reported that the test results
were influenced by the size of the latex particles.
[21]
In this study, carbonate–bicarbonate buffer was used to
dilute the latex particles during the sensitization of the beads. This approach aligns with the findings of
Ramadass et al., who also identified this buffer as suitable for latex bead sensitization.
[14]
Sankar et al., also
developed LAT based on whole-cell protein antigens to detect Mycoplasma gallisepticum antibodies from
chicken serum in Kerala.
[16]
In the current study, the results of LAT were compared with those of PCR. Among
the 110 serum samples tested, 31 showed agglutination in the LAT region. Two false negative and five false
positive results were obtained. Hence, the seropositivity of M. ovipneumoniae antibodies in the latex
agglutination test was 32.73% (36/110). The sensitivity and specificity of the newly developed LAT kit were
determined to be 93.94% and 93.51%, respectively. Furthermore, the calculated Cohen’s kappa coefficient
was 0.852, which falls within the 0.81–1.00 range. This statistically demonstrates an almost perfect
agreement with the reference PCR gold standard, confirming the diagnostic robustness and reliability of the
assay. This finding is in accordance with the findings of Sankar et al. (2013), who reported a sensitivity and
specificity of 95.24% and 93.33%, respectively.
[16]
Hence, this agglutination-based kit offers a convenient and
rapid means of diagnosing infections in field settings and is particularly useful for large-scale surveillance of
infections in flocks rather than individual testing.
5. Conclusion
The latex agglutination test (LAT) developed in the present study demonstrated good diagnostic performance
for the detection of Mycoplasma ovipneumoniae antibodies in small ruminants. The test was simple, rapid and
easy to perform, with minimal technical requirements, making it suitable for routine screening of sheep and
goat flocks. The satisfactory sensitivity and specificity observed in the study indicate that the LAT can serve as
a useful preliminary serological screening tool for identifying animals exposed to M. ovipneumoniae. Its
simplicity and rapid visual interpretation also offer advantages for application under field conditions,
particularly in areas where sophisticated laboratory facilities are not readily available.
Acknowledgement
The authors thank the Tamil Nadu Veterinary and Animal Sciences University, Chennai, India.
CRediT Author Contribution Statement
Shanmugasundaram Udhayavel: Investigation, Data Curation, Formal Analysis, Writing – original draft.
Kuppannan Sukumar: Conceptualization, Methodology. Kuppusamy Senthilkumar: Writing – review &
editing. Palani Srinivasan: Writing – review & editing. Ayyasamy Elango: Writing – review & editing. All
authors have read and approved the final version of the manuscript for publication and agree to be
accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part
of the work are appropriately investigated and resolved.
Funding Declaration
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-
profit sectors.
Institutional Review Board Statement
The serum samples utilized in this study were archival specimens obtained during a previous diagnostic field
surveillance study [Ref. 18]. The original sampling was restricted to non-invasive nasal swabs from clinically
ill field animals and post-mortem tissue from slaughterhouses, adhering to national animal welfare practices
for routine veterinary diagnostics. For the current study, no new animals were handled, and the work was
strictly restricted to in vitro laboratory analysis of these existing archival sera.
Informed Consent Statement
Prior verbal informed consent was obtained from the respective livestock owners during field sampling in the
previous study.
Consent to Publish Statement
Not applicable.
Data Availability Statement
The datasets generated and/or analyzed during the current study that support the findings are available from
the corresponding author upon reasonable request.
Conflict of Interest
Ayyasamy Elango serves as the Editorial Board Member and is a co-author of this manuscript. To ensure a
rigorous and unbiased peer-review process, he was not involved in any stage start from editorial evaluation,
peer review process and final publication decision. The handling of this manuscript was managed
independently by another editorial board member. The other authors declare no competing interests.
Artificial Intelligence (AI) Use Disclosure
The authors declare that artificial intelligence (AI)-assisted tools were used only for language refinement,
grammar improvement, and manuscript structuring purposes during the preparation of this work. All
technical content, experimental implementation, results, and interpretations were independently developed
and verified by the authors.
Supporting Information
Not applicable.
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