Rev. Ciencias Veterinarias, Vol. 43, N° 2, [1-9], E-ISSN: 2215-4507, julio-diciembre, 2025
DOI: https://doi.org/10.15359/rcv.43-2.3
URL: http://www.revistas.una.ac.cr/index.php/veterinaria/index
Direct immunofluorescence test for Campylobacter fetus subsp. venerealis in breeding bulls of Costa Rica
Prueba de inmunofluorescencia directa para Campylobacter fetus subsp. venerealis en toros reproductores de Costa Rica
Prova de imunofluorescência direta para Campylobacter fetus subsp. venerealis em touros reprodutores da Costa Rica
Leonel Navarro 1
, Andrea Urbina2, Jorge Chacón 1
1 Universidad Nacional (UNA), School of Veterinary Medicine, Research Program on Applied Animal Andrology, Heredia, Costa Rica. leonel.navarro.rojas@una.cr, 
https://orcid.org/0000-0003-0532-3995, jorge.chacon.calderon@una.cr, https://orcid.org/0000-0003-2432-1717
2 Universidad Nacional (UNA) School of Veterinary Medicine, Zoonosis Laboratory, Heredia, Costa Rica.
andreaurbina3@gmail.com, https://orcid.org/0000-0002-9001-761X
Corresponding author: leonel.navarro.rojas@una.cr
Received: May 8, 2023 Corrected: October 12, 2025 Accepted: October 17, 2025
Bovine genital campylobacteriosis (BGC) is a venereal infectious disease caused by a Gram-negative bacillus Campylobacter fetus subspecies venerealis (Cfv) that impairs the reproductive efficiency in cattle herds. The clinical diagnosis of BGC is based on low fertility in the breeding herd; however, laboratory confirmation is required. Although there is data on the disease being distributed worldwide, particularly in cattle herds where natural mating is the primary breeding system, data from Costa Rica is unavailable. This study aimed to detect the presence of Campylobacter fetus subsp. venerealis (Cfv) in scrapings collected from the preputial mucosa of breeding bulls allocated in the North Huetar and North Pacific regions of Costa Rica using direct immunofluorescence (DIF). There was no data regarding reproductive problems on any of the 157 participating farms. Scrapings of the preputial mucosa were collected using a plastic rod-type aspirator-scraper introduced into the preputial cavity, stored in 1% phosphate-buffered formalin, and subsequently analyzed by DIF. All the bulls tested yielded negative for the pathogen. The possible causes related to this result are discussed in this article.
Keywords: Venereal diseases, Campylobacter fetus subsp. venerealis, direct immunofluorescence.
La campilobacteriosis genital bovina (BGC) es una enfermedad infecciosa venérea causada por el bacilo Gram negativo Campylobacter fetus subespecie venerealis (Cfv) que afecta la eficiencia reproductiva en los hatos bovinos. El diagnóstico clínico de BGC se realiza con base en el análisis de la tasa de fertilidad en el hato y debe confirmarse por medios laboratoriales. Si bien la distribución de la enfermedad es mundial, especialmente en hatos bovinos donde la monta natural es el principal sistema de crianza, se carece de datos sobre la prevalencia de este agente en Costa Rica. El objetivo de este estudio fue diagnosticar la presencia de Campylobacter fetus (Cf) subespecie venerealis (Cfv) en raspados de la mucosa prepucial de sementales distribuidos en las regiones Huetar Norte y Pacífico Norte mediante el uso de inmunofluorescencia directa (DIF). No se obtuvieron datos sobre problemas reproductivos en ninguna de las 157 granjas participantes. Se recogieron raspados de la mucosa prepucial utilizando un aspirador-raspador de plástico en forma de varilla que se introdujo en la cavidad prepucial y se almacenaron en formalina tamponada con fosfato al 1% para luego analizarlos mediante DIF. Todos los toros analizados en el presente estudio fueron diagnosticados como negativos para este patógeno, resultado que se discute en este artículo.
Palabras claves: Enfermedades venéreas, Campylobacter fetus subsp. venerealis, inmunofluorescencia directa.
O objetivo deste estudo foi diagnosticar a presença de Campylobacter fetus (Cf) em raspados da mucosa prepucial de reprodutores distribuídos nas regiões Huetar Norte e Pacífico Norte mediante o uso de imunofluorescência direta (DIF). A campilobacteriose genital bovina (BGC) é uma doença infecciosa venérea que afeta a eficiência reprodutiva dos rebanhos bovinos. Ela é causada pelo bacilo Gram negativo Campylobacter fetus subespécie venerealis (Cfv). Embora a distribuição da doença seja mundial, especialmente em rebanhos bovinos onde a monta natural é o principal sistema de criação, carece-se de dados sobre a prevalência desse agente na Costa Rica. O diagnóstico clínico de BGC é realizado com base na análise da taxa de fertilidade do rebanho e deve ser confirmado por meios laboratoriais. Todos os touros analisados no presente estudo foram diagnosticados como negativos para esse patógeno, resultado que é discutido neste artigo.
Palavras-chave: Doenças venéreas, Campylobacter fetus subsp. venerealis, imunofluorescência direta.
Campylobacter fetus (Cf) is a Gram-negative spiral bacillus that includes two subspecies, Campylobacter fetus subsp. venerealis (Cfv) and Campylobacter fetus subsp. fetus (Cff) (Eaglesome & Garcia, 1992; Van Bergen et al., 2005). The subspecies venerealis is the etiological agent of bovine genital campylobacteriosis (BGC), a primary venereal disease transmitted exclusively through venereal contact (Peter, 1997; Campero et al., 2021). According to the World Organization of Animal Health (OIE, 2018), the disease seriously impairs the fertility in cattle herds infected with the agent, and a negative status of Cfv in bulls is mandatory for their international semen trading. Bovine genital campylobacteriosis has a worldwide distribution and is responsible for reproductive losses (Mederos et al., 2022; Silveira et al., 2018) and reduced reproductive efficiency in cattle herds. Among the main risk factors for BGC are natural mating as the main breeding system, bulls older than 4 years, and the absence of sanitary control for this venereal disease (Balzan et al., 2020; Michi et al., 2015). Prevalence of BGC is uneven, with rates ranging between 8% to 72% (Lage & Leite, 2000; Mshelia et al., 2010; Pellegrin et al., 2002; Repiso et al., 2005). Additionally, there are few laboratories for BGC diagnosis, thus limiting the precise estimation and comparison of epidemiological reports (Balzan et al., 2020).
Even though some topical and other pharmacological treatments have been reported in the literature (Kimsey et al., 1980; Maclaren & Wright, 1977), it is known that infected bulls are unable to eliminate the agent and must be consequently culled from the breeding livestock once the clinical diagnosis is confirmed by laboratory methods (Balzan et al., 2020; BonDurant, 1997). Infected bulls are lifelong carriers of Cfv which remains in the epithelium folds of the preputial cavity in asymptomatic sires (BonDurant, 1997). For this reason, they become the targets for the diagnosis of the agent (Silveira et al., 2018).
Infected bulls show no histopathological changes in their reproductive organs, nor is semen quality affected (Campero et al., 2017; Clark, 1971). On the contrary, decreased fertility, characterized by early embryo losses and abortions, is common in infected cows (Anderson, 2007; Campero et al., 2017; Hum et al., 1994).
Bacteriological culture of Campylobacter fetus from preputial scrapings and further biochemical identification is considered the gold standard for laboratory diagnosis of BGC (OIE, 2018). However, it is difficult to achieve due to the microaerophilic and particular growth requirements of this agent (Balzan et al., 2020; Quinn et al., 2011). Immunoenzymatic and agglutination tests have also been developed, showing low sensitivity or no discrimination between the two species of C. fetus (Hum et al., 1994; Silveira et al., 2018). In recent years, several molecular methods among them the PCR, have been developed, but they are time consuming, laborious to perform, expensive, and impractical for routine use (Chaban et al., 2012; Gard, 2016; Van der Graaf-van Bloois et al., 2014).
Direct immunofluorescence (DIF) is the recommended method by the OIE for sires dedicated to international semen trading (Hum et al., 1994; OIE, 2018). The test is quicker and cheaper compared to molecular techniques. Sensitivity and specificity of DIF have been reported around 92.5% and 88.8% respectively (Campero et al., 2017; Figueireido et al., 2002). The method is widely used in most diagnostic laboratories in South America as the single diagnostic test for BGC (Silveira et al., 2018). This study aimed to detect the Cfv in bulls from cattle herds dedicated to dairy, dual purpose, and beef production located in the Northern regions of the country using DIF.
The study was carried out from 2016 to 2018 in 157 cattle farms located in the North Huetar (NH) and North Pacific (NP) regions of Costa Rica, which hold the largest bovine population in the country (333566 and 281500 heads respectively) (Instituto Nacional de Estadística y Censos [INEC], 2015).
Selection of Farms and Sample Size
The selection criteria for the farms to be included in the survey were their consent to participate in the study and to practice natural breeding in the livestock herd. Consequently, all the bulls tested had previous sexual experience at the time of sampling. There were no previously available reports from the farms surveyed regarding reproductive problems such as abortions, or irregular cycling. The calculation for a minimal sample (n=317) was determined for an estimated population in the region under study of 25,500 sires (Livestock bovine census, INEC, 2015), considering a 0.02 margin of error, with a confidence interval of 95% and 5% expected prevalence per bull, based on the latest report for other primary venereal diseases in the same geographical region (Pérez et al., 1992). It is remarkable that the minimal animal sample size was exceeded by 187 individuals, with 504 bulls tested randomly distributed in 157 farms (average 2,26 bulls per farm).
Data Survey and Sample Collection
Information regarding productive and reproductive management on the farms, bulls’ age and breed, number of animals per herd, cow to bull ratio, was recorded. For the collection of the scrapings of the preputial mucosa, each sire was restrained in a chute to minimize their stress, always considering ethical animal welfare. The hair around the preputial orifice was cleared out and the area was cleaned with a sterile piece of gauze up to approximately 5 cm deep in the cavity (BonDurant, 1985). The scrape of the preputial mucosa was collected using a plastic rod-type aspirator-scraper (Laboratorio Azul, Argentina), which was introduced deeply into the preputial cavity. Once the sample was collected, the scraper tip was introduced into a conical tube containing 1 % phosphate buffered formalin (Garcia et al., 2021; Silveira et al., 2018). Subsequently, each tube was labelled, identified, and transported at room temperature to the Zoonosis Laboratory (Universidad Nacional, Heredia-Costa Rica) for DIF.
Direct Immunofluorescent Antibody Test for the Diagnosis of Cfv
Detection of Cfv was carried out using a direct fluorescent antibody test according to the protocol of the Instituto Nacional de Tecnología Agropecuaria (INTA, Balcarce-Argentina). The collected scraping suspended in buffered formalin solution was centrifuged at 500 g for 15 minutes, and subsequently, the supernatant was transferred and centrifuged again at 15,000 g. Twenty µl of each sediment were placed on immunofluorescent slides. At the same time, positive and negative control strains of Cfv and of Campylobacter sputorum subsp. bubulus respectively (INTA, Balcarce-Argentina), were placed on each slide. After fixing the samples, the fluorescent conjugate IgG anti Cfv was added and incubated for 30 minutes at 37˚C. The slides were then washed, air-dried and observed under fluorescence microscopy (Olympus, BX51, Japan) at 100x. Positive and negative bulls were associated with sample slides showing fluorescent typical spirilla with a green-lemon and red color fluorescence, respectively (Figuereido et al., 2002; OIE, 2018).
Seventy-nine (50.2%) and 78 (49.8%) out of the 157 farms sampled were located in the NP and NH regions of Costa Rica, respectively. The characteristics according to type of production system are described in the table 1.
Table 1. Characteristics of the 157 farms according to production system
|
Production system |
Farms (n) |
Bulls (n) |
Farms using records (%) |
Farms with veterinary support (%) |
Farms using only natural mating (%) |
|
Dairy |
87 |
148 |
95.4 |
83.9 |
54.0 |
|
Dual purpose |
38 |
150 |
86.8 |
47.4 |
73.7 |
|
Beef |
32 |
206 |
81.3 |
50 |
62.5 |
|
TOTAL |
157 |
504 |
Regarding reproductive practices, 74.5% of farms (n=117) reported practicing continuous mating, while seasonal and guided accounted for 14% (n=22) and 11.5% (n=18) respectively. In relation to other reproductive practices, 8.3% (n=13/157) of the farmers reported the habit of loaning bulls between farms. Moreover, 35.0% (n=55/157) stated they purchased non-virgin sires. In addition, 11.5% used to buy breeding replacement bulls or heifers at commercial livestock auctions (n=18/157).
From the 504 bulls tested the average was 2.26 bulls by farm and the average number of cattle heads per herd and cows per bull was 172.8±164.1 and 53.1±46.0, respectively. The average bull´s age was 50.6±21.8 months (range 18-144), with 52.4% being older than 4 years. In addition, the proportion of sires disclosed by genotype was 42.9% (n=216) Bos taurus, 32.9% (n=166) Bos indicus and 24.2% (n=122) B. indicus x B. taurus. At the time of sampling, 209 bulls (41.5%) had been under sexual rest for at least 15 days, compared to 295 (58.5%) that were breeding.
In relation to the sire system, the 504 bulls were classified into 3 categories as shown in Table 2.
Table 2. Classification of 504 according to sire system
|
Sire system |
Bulls (n) |
% |
|
Single sire system |
335 |
66.5 |
|
Multiple sire system |
161 |
31.9 |
|
Sire rotation |
8 |
1.6 |
|
Total |
504 |
100 |
After examining the 504 preputial scrapings, no positive cases of bulls infected with bovine genital campylobacteriosis were diagnosed by DIF.
To our knowledge, there are no previous reports about laboratory detection of Cfv in Costa Rica. In our opinion, this may be due to the lack of a systematic diagnostic sampling and laboratory capabilities as mentioned by Balzan et al. (2020). To date, there are no documented reports of the disease in the Central American region either. The diagnosis of Cfv in the present study was based on DIF which is recommended by the OIE as a suitable diagnostic method in BGC control programs (OIE, 2018). It is widely used in several endemic countries of South America (Campero et al., 2017; Marcellino et al., 2015) due to being rapid, low-cost and easy to perform (Silveira et al., 2018). A reduction in test sensitivity has been associated with the sampling method, the quality of the sample, and the observer’s experience (Silveira et al., 2018). The interpretation of the slides may present a challenge for the observer since cellular debris, contaminating particles, and artifacts make the observation of the agent difficult specially when it is in low concentration. A well trained and experienced observer is necessary in order to assure reliable interpretation of the test (Figueireido et al., 2002; Marcellino et al., 2015) as was done in the present study. A recent evaluation of DIF demonstrated a sensitivity and specificity of 79% and 100%, respectively. The positive predictive value (PPV) was of 100% and the negative predictive value (NPV) was of 76% with no cross reactions with 15 bacteria normally found in preputial bull microbiota. Moreover, an 83% concordance between bacteriological culture and DIF was demonstrated (Garcia et al., 2021). The results obtained in the present study probably reflect the real status of BGC in the country. The DIF uses polyclonal antibodies against Cfv conjugated to fluoresceine isothiocyanate, and in clinical samples, it does not discriminate between Cff and Cfv; however, both agents impact the reproductive health of cattle (Campero et al., 2017). To increase the sensitivity of the test, bulls should be kept in sexual rest for 15 days before sampling, and three samples at intervals of 15 days should be taken to assure a reliable diagnosis of the disease (Silveira et al., 2018). It has also been recommended that at least two consecutive scrapings per bull should be collected and analyzed before diagnosing an animal as negative (Garcia et al., 2021).
It must be considered that in our study, only 41,5% of sires had at least two weeks of sexual rest at the time of sampling and only a single scraping was taken from each bull. On the other hand, our results may have been influenced by the improved productive and reproductive practices in the tested farms. For example, 88% of the scrutinized farms reported the current use of productive records and routinely received herd health veterinary assistance. Practices such as these, can contribute to diminishing the risk of spreading infections of venereal agents such as Cfv. In addition, the average heads per herd in our study (172.8±164) was low compared to other reports, which suggest an increased risk for BCG in herds with 1,000 or more animals (Filho et al., 2018; Mardones et al., 2008). The absence of positive bulls should be considered with caution and should not be pondered as indicating that Costa Rican cattle herds are free from the disease.
The use of two combined techniques is recommended to increase reliability of the results (Balzan et al., 2020). It seems that there is consensus that PCR-based molecular techniques and protocols still need to be validated and harmonized to reduce disparity of results and enhance their applicability in field work and in diagnostic laboratories (Silva et al., 2020; Silveira et al., 2018; Waldner et al., 2017). Further investigations are needed in the country, implementing microbiological culture to determine more accurately the current status of the disease. In addition, including a greater proportion of farms with known impaired reproductive indexes, and a more random selection of them would be necessary to determine the precise status of this agent in the country.
We appreciate the support by Dr. Carlos Campero (INTA, Balcarce-Argentina) through his valuable comments and providing the controls for the diagnosis of the agent studied. Also, we are grateful to Dora Beatriz Cano for his technical assistance (INTA, Balcarce-Argentina). This study received financial support from the Project UEPS-CRIOSEMEN (SIA-UNA 020-05), Consejo Nacional para Investigaciones Científicas y Tecnológicas (CONICIT) and Ministerio de Ciencia, Tecnología y Telecomunicaciones (MICITT), Costa Rica.
The authors declare no conflict of interest.
Authors Contribution
Navarro L. Sample collection and Laboratorial Diagnosis, Data collection, Manuscript Edition.
Urbina A: Experimental design, Laboratorial Diagnosis, Manuscript Edition.
Chacón J. Experimental design, Sample collectionand Laboratorial Diagnosis, Data collection, Manuscript Edition.
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