Bovine genital campylobacteriosis: main features and perspectives for diagnosis and control

Campilobacteriose genital bovina: principais características e perspectivas para o diagnóstico e controle

Cláudia Balzan Rosangela Estel Ziech Letícia Trevisan Gressler Agueda Palmira Castagna de Vargas About the authors

ABSTRACT:

Bovine genital campylobacteriosis (BGC) is a venereal disease caused by Campylobacter fetus subsp. venerealis. In countries with large cattle herds, such as Brazil, where the use of natural breeding as a reproductive strategy is a common practice, BGC is considered an important cause of reproductive failure and economic losses. In these cases, the bull is the asymptomatic carrier of the bacterium and the infected females can have infertility and even abortions. The techniques for the diagnosis of C. fetus are isolation in culture medium and identification by biochemical tests, immunofluorescence, immunoenzymatic assays and molecular techniques. Disease control is based on vaccination with bacterins. This review described the epidemiology, etiology, pathogenesis, and advances in the diagnosis and control of BGC.

Key words:
Campylobacter fetus subsp. venerealis; infertility; venereal disease; beef cattle.

RESUMO:

A campilobacteriose genital bovina (CGB) é uma importante enfermidade de caráter venéreo causada por Campylobacter fetus subsp. venerealis. Em países com grandes rebanhos bovinos, como o Brasil, onde o uso da monta natural como estratégia reprodutiva é uma prática corrente, a CGB é considerada uma importante causa de falhas reprodutivas e perdas econômicas. Nestes casos, o touro é o portador assintomático da bactéria e as fêmeas infectadas podem apresentar infertilidade e até mesmo abortos. As técnicas para o diagnóstico de C. fetus são o isolamento em meio de cultura e identificação por testes bioquímicos; imunofluorescência; ensaios imunoenzimáticos e técnicas moleculares. O controle da doença é baseado em vacinação. Neste sentido, esta revisão consiste em uma abordagem sobre a epidemiologia, a etiologia, a patogenia, os avanços no diagnóstico e controle da CGB.

Palavras-chave:
Campylobacter fetus subsp. venerealis; infertilidade; doenças venéreas; bovinos de corte.

INTRODUCTION:

Bovine genital campylobacteriosis is caused by the microaerophilic bacterium Campylobacter fetus subsp. venerealis, a gram-negative rod. This is a venereal disease with worldwide distribution and high incidence in developing countries where natural breeding is widely used for bovine reproduction (MSHELIA et al., 2010MSHELIA, G. D. et al. Epidemiology of bovine venereal campylobacteriosis: Geographic distribution and recent advances in molecular diagnostic techniques. Reproduction in Domestic Animals, v.45, n.5, 2010. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/19929895 >. Accessed Sep. 28 2014. doi: 10.1111/j.1439-0531.2009.01546.x.
https://www.ncbi.nlm.nih.gov/pubmed/1992...
), such as in Brazil. The BGC was first diagnosed in Brazil in 1955 in an aborted fetus (RAMOS et al., 1983) and remains in the herds of this country to date. Also, BGC is on a list of notifiable diseases that are significant in international trade in animals or animal products, maintained by the World Organization for Animal Health (OIE, 2017OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
). Published reviews have highlighted the prevalence, epidemiology, diagnosis and/or control of BGC (ALVES et al., 2011ALVES, T. M. et al. Campilobacteriose genital bovina e tricomonose genital bovina: Epidemiologia, diagnóstico e controle. Pesquisa Veterinaria Brasileira, v.31, n.4, p.336-344, 2001. Available from: <Available from: http://www.scielo.br/pdf/pvb/v31n4/a11v31n4.pdf >. Accessed: Oct. 05, 2015. doi: 10.1590/S0100-736X2011000400011.
http://www.scielo.br/pdf/pvb/v31n4/a11v3...
; BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
; CORBEIL et al., 2003CORBEIL, L. B. et al. Vaccines against sexually transmitted diseases. Reproductive Biology and Endocrinology, v.1, n.118, p.1-6, 2003. Available from: <Available from: http://www.rbej.com/content/1/1/118 >. Accessed Oct. 10, 2016. doi: 10.1186/1477-7827-1-118.
http://www.rbej.com/content/1/1/118...
; HOFFER, 1981HOFFER, M. A. Bovine campylobacteriosis: a review. Canadian Veterinary Journal, v. 22, n. 11, p. 327-330, 1981. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1789996/ >. Accessed: Sept. 10, 2012.
https://www.ncbi.nlm.nih.gov/pmc/article...
; MICHI et al., 2016MICHI, A. N. et al. A review of sexually transmitted bovine trichomoniasis and campylobacteriosis affecting cattle reproductive health. Theriogenology, v.85, n.5, p.781-791, 2016. Available from: <Available from: http://dx.doi.org/10.1016/j.theriogenology.2015.10.037 >. Accessed: Jan. 20 2017. doi: 10.1016/j.theriogenology.2015.10.037.
http://dx.doi.org/10.1016/j.theriogenolo...
; MSHELIA et al., 2007MSHELIA, G.D. et al. Bovine venereal campylobacteriosis: an overview. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources, v.2, n.080, p.1-14, 2007. Available from: <Available from: http://www.cabi.org/cabreviews/review/20083052195 >. Accessed: Jun. 10 2012. doi: 10.1079/PAVSNNR20072080.
http://www.cabi.org/cabreviews/review/20...
; SILVEIRA et al., 2018SILVEIRA, C. S. et al. Diagnosis of Bovine Genital Campylobacteriosis in South America. Frontiers in Veterinary Science, v.5, p.1-9, 2018. Available from: <Available from: https://www.frontiersin.org/article/10.3389/fvets.2018.00321/full >. Accessed Jan. 10, 2019. doi: 10.3389/fvets.2018.00321.
https://www.frontiersin.org/article/10.3...
). Therefore, facing the woldwide impact of BGC, this review discussed the distribution, etiology, pathogenesis, C. fetus virulence factors, epidemiology, and advances in diagnosis and control of BGC, emphasizing its occurrence in Brazil.

Epidemiology and distribution

Recent data from the OIE (2019)OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
indicated the presence of BGC in Argentina, Brazil, Colombia, Uruguay, Australia, New Zealand, Ireland, France, South Africa, Iran, and Nigeria between January and June 2018. In Nigeria, approximately 520,000 cattle are affected by BGC with a direct loss of 8.5 million dollars due to abortions and low fertility rates (MSHELIA et al., 2012MSHELIA, G. D. et al. The prevalence of bovine venereal campylobacteriosis in cattle herds in the Lake Chad basin of Nigeria. Tropical Animal Health and Production, v.44, n.7, p.1487-1489, 2012. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/22318768 >. Accessed: Sept. 30, 2014. doi: 10.1007/s11250-012-0092-6.
https://www.ncbi.nlm.nih.gov/pubmed/2231...
).

In Brazil, BGC is among the most important causes of reproductive failure in beef and dairy farms which use natural breeding (VARGAS et al., 2002VARGAS, A. et al. Campylobacter fetus subspecies venerealis surface array protein from bovine isolates in Brazil. Current Microbiology, v.45, n.2, p.111-114, 2002. Available from: < Available from: https://www.ncbi.nlm.nih.gov/pubmed/12070688 >. Accessed: Oct. 05, 2012. doi: 10.1007/s00284-001-0090-9.
https://www.ncbi.nlm.nih.gov/pubmed/1207...
; LEAL et al., 2012LEAL, D. R. et al. Prevalência da campilobacteriose e da tricomonose genitais bovinas no Distrito Federal e em seu entorno. Revista Brasileira de Reprodução Animal, v.36, n.4, p.256-259, 2012. Available from: < Available from: https://www.cbra.org.br/pages/publicacoes/rbra/v36n4/p256-259%20(RB416).pdf >. Accessed: Sept. 10, 2014.
https://www.cbra.org.br/pages/publicacoe...
; MIRANDA, 2005MIRANDA, K. L. Prevalência da infecção por Campylobacter fetus em bovinos de corte do Brasil. [S.l.]: Universidade Federal de Minas Gerais, 2005. ; OLIVEIRA et al., 2015OLIVEIRA, J. M. B. et al. Prevalence and risk factors associated with bovine genital campylobacteriosis and bovine trichomonosis in the state of Pernambuco, Brazil. Tropical Animal Health and Production, v.47, n.3, p. 549-555, 2015. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/25633914 >. Accessed: Oct. 10, 2016. doi: 10.1007/s11250-015-0761-3.
https://www.ncbi.nlm.nih.gov/pubmed/2563...
; STYNEN et al., 2003STYNEN, A. P. R. et al. Campilobacteriose genital bovina em rebanhos leiteiros com problemas reprodutivos da microrregião de Varginha - Minas Gerais. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, v.55, n.6, p.766-769, 2003. Available from: < Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-09352003000600015 >. Accessed: Jun. 25, 2013. doi: 10.1590/S0102-09352003000600015.
https://www.scielo.br/scielo.php?script=...
). As shown in table 1, over the last 23 years, BGC has been reported in all regions of Brazil. There are specific prevalence studies, which usually involve a unique region of a state or diagnostic laboratory data. In Brazil, there are no official surveys, no estimates of losses caused by the disease, and no prediction of an eradication and control program. Moreover, BGC incidence remains understimated due to the absence of systematic diagnosis, which is associated with logistic issues, such as sample collection and shipment, as well as a limited number of laboratories qualified to perform BCG diagnosis (ALVES et al., 2011ALVES, T. M. et al. Campilobacteriose genital bovina e tricomonose genital bovina: Epidemiologia, diagnóstico e controle. Pesquisa Veterinaria Brasileira, v.31, n.4, p.336-344, 2001. Available from: <Available from: http://www.scielo.br/pdf/pvb/v31n4/a11v31n4.pdf >. Accessed: Oct. 05, 2015. doi: 10.1590/S0100-736X2011000400011.
http://www.scielo.br/pdf/pvb/v31n4/a11v3...
).

Table 1
Surveys reporting the prevalence of BGC in Brazil between 1995 and 2018.

The main risk factor for the spread of BGC is natural breeding (MSHELIA et al., 2012MSHELIA, G. D. et al. The prevalence of bovine venereal campylobacteriosis in cattle herds in the Lake Chad basin of Nigeria. Tropical Animal Health and Production, v.44, n.7, p.1487-1489, 2012. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/22318768 >. Accessed: Sept. 30, 2014. doi: 10.1007/s11250-012-0092-6.
https://www.ncbi.nlm.nih.gov/pubmed/2231...
), especially when bulls older than 4 years and without sanitary control for BGC are employed (HOFFER, 1981HOFFER, M. A. Bovine campylobacteriosis: a review. Canadian Veterinary Journal, v. 22, n. 11, p. 327-330, 1981. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1789996/ >. Accessed: Sept. 10, 2012.
https://www.ncbi.nlm.nih.gov/pmc/article...
; BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
). Even in farms using artificial insemination (AI), the use of bulls after AI is a very common practice (STYNEN et al., 2003STYNEN, A. P. R. et al. Campilobacteriose genital bovina em rebanhos leiteiros com problemas reprodutivos da microrregião de Varginha - Minas Gerais. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, v.55, n.6, p.766-769, 2003. Available from: < Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-09352003000600015 >. Accessed: Jun. 25, 2013. doi: 10.1590/S0102-09352003000600015.
https://www.scielo.br/scielo.php?script=...
). Another risk factor is the use of semen without appropriate antimicrobials (BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
).

In herds recently exposed to BGC, the conception rates can be 50% lower than expected (DEDIE et al., 1982 apud BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
). The main risk groups are heifers and cows newly introduced in the herd, in which clinical signs are most pronounced due to low levels of immunity (HOFFER, 1981HOFFER, M. A. Bovine campylobacteriosis: a review. Canadian Veterinary Journal, v. 22, n. 11, p. 327-330, 1981. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1789996/ >. Accessed: Sept. 10, 2012.
https://www.ncbi.nlm.nih.gov/pmc/article...
).

Etiology and pathogenesis

Microorganisms of the genus Campylobacter are gram-negative curved rods 0.2 to 0.5 μm diameter, mobile with polar flagella, oxidase positive, and with variable catalase reactions (VAN BERGEN et al., 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
; QUINN et al., 2011QUINN, P.J. et al. Campylobacter and Helicobacter species. In: _____. Veterinary microbiology and microbial disease. 2. ed. Iowa: Wiley-Blackwell, 2011. Cap.34, p.914-936. ).

Microaerophilic bacteria comprised the genus Campylobacter, which generally require oxygen concentrations between 3 and 15% and carbon dioxide concentrations from 3 to 5%. Only a few species are aerotolerant and can grow in the presence of oxygen (HOLT et al., 1994HOLT, J. G. et al. Bergey’s Manual of Determinative Bacteriology. 9 ed. ed. Philadelphia: Lippincott Williams & Wilkins, 1994. ). C. fetus as a pathogen in cattle and sheep and is subdivided into two subspecies, C. fetus subsp. fetus and C. fetus subsp. venerealis, by habitat characteristics, transmission, clinical manifestations, and laboratory phenotypic identification (VÉRON & CHATELAIN, 1973VÉRON, M. , CHATELAIN, R. Taxonomic Study of the Genus Campylobacter Sebald and Véron and Designation of the Neotype Strain for the Type Species, Campylobacter fetus (Smith and Taylor) Sebald and Veron, v.23, n.2, p.122-134, 1973. Available from: <Available from: https://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/00207713-23-2-122 >. Accessed: Jun. 20, 2012. doi: 10.1099/00207713-23-2-122.
https://ijs.microbiologyresearch.org/con...
). These subspecies are phenotypically differentiated according to HOLT et al. (1994)HOLT, J. G. et al. Bergey’s Manual of Determinative Bacteriology. 9 ed. ed. Philadelphia: Lippincott Williams & Wilkins, 1994. , with tests of tolerance to 1% glycine and production of hydrogen sulphide (H2S), with C. fetus subsp. fetus returning positive results in both assays, and C. fetus subsp. venerealis as negative. C. fetus subsp. venerealis still presents as a biovar, classified by the ability to produce H2S but not 1% glycine tolerant, and is referred to as C. fetus subsp. venerealis biovar intermedius (VÉRON & CHATELAIN, 1973VÉRON, M. , CHATELAIN, R. Taxonomic Study of the Genus Campylobacter Sebald and Véron and Designation of the Neotype Strain for the Type Species, Campylobacter fetus (Smith and Taylor) Sebald and Veron, v.23, n.2, p.122-134, 1973. Available from: <Available from: https://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/00207713-23-2-122 >. Accessed: Jun. 20, 2012. doi: 10.1099/00207713-23-2-122.
https://ijs.microbiologyresearch.org/con...
).

C. fetus subsp. fetus is commensal in the gastrointestinal tract and occasionally causes abortion in sheep and cattle (FLORENT, 1959 apud SKIRROW, 1994SKIRROW, M. B. Diseases due to Campylobacter, Helicobacter and related bacteria. Journal of comparative pathology, v.111 (2), p.113-149, 1994. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/7806700 >. Accessed: May, 25, 2014.
https://www.ncbi.nlm.nih.gov/pubmed/7806...
); whereas, C. fetus subsp. venerealis is present exclusively in the genital tract of cattle resulting in infertility and abortions, the bacterium being transmitted exclusively by the venereal route (HUM & QUINN., 1994HUM, C.; QUINN, D. K. Diagnosis of bovine venereal campylobacteriosis by ELISA. Australian Veterinary Journal, v.71, n.5, p.140-143, 1994. Available from:<Available from:https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-0813.1994.tb03367.x >. Accessed: Nov. 20, 2016. doi: 10.1111/j.1751-0813.1994.tb03367.x.
https://onlinelibrary.wiley.com/doi/pdf/...
).

Infection by C. fetus subsp. venerealis in bulls is not associated with clinical signs, changes in semen quality, or genital abnormalities (BIER et al., 1977aBIER, P. J. et al. Experimental infections with Campylobacter fetus in bulls of different ages. Veterinary Microbiology, v.2, p.13-27, 1977a. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/0378113577900281 >. Accessed: Jun. 02, 2015. doi: 10.1016/0378-1135(77)90028-1.
https://www.sciencedirect.com/science/ar...
), and the microorganism persists in the epithelial crypts of the prepuce. Transmission to females occurs during natural mating with infected bulls, or by AI with contaminated semen, when the bacterium is distributed through the mucosa of the vagina, cervix, uterus, and uterine tubes (BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
). Approximately one third of infected cows become carriers (QUINN et al., 2011QUINN, P.J. et al. Campylobacter and Helicobacter species. In: _____. Veterinary microbiology and microbial disease. 2. ed. Iowa: Wiley-Blackwell, 2011. Cap.34, p.914-936. ), and other males may be contaminated by mating with sick or carrier cows (HOFFER, 1981HOFFER, M. A. Bovine campylobacteriosis: a review. Canadian Veterinary Journal, v. 22, n. 11, p. 327-330, 1981. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1789996/ >. Accessed: Sept. 10, 2012.
https://www.ncbi.nlm.nih.gov/pmc/article...
).

C. fetus subsp. venerealis persistently colonizes the preputial cavity of the bull, preventing it from elaborating an effective immune response, and thus the animal remains as an asymptomatic carrier (VAN BERGEN, 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
). Bulls older than 4 years of age are more likely to be carriers because they have deeper crypts where microorganisms find favorable conditions and safe harbor (SAMUELSON & WINTER, 1966SAMUELSON, J.D.; WINTER, A. J. Bovine vibriosis: the nature of the carrier state in the bull. The Journal of Infectious Disease, v.116, n.5, p.581-592, 1966. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/5957264 >. Accessed: Nov. 20, 2016.
https://www.ncbi.nlm.nih.gov/pubmed/5957...
). Infection can persist for months to years in older bulls (BIER et al., 1977aBIER, P. J. et al. Experimental infections with Campylobacter fetus in bulls of different ages. Veterinary Microbiology, v.2, p.13-27, 1977a. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/0378113577900281 >. Accessed: Jun. 02, 2015. doi: 10.1016/0378-1135(77)90028-1.
https://www.sciencedirect.com/science/ar...
).

After coitus, an ascending infection is established in females, and in 12 to 14 days the microorganism is distributed from the vagina/cervix to the uterine horns (BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
; CORBEIL, 1999CORBEIL, L.B. et al. Immunization and diagnosis in bovine reprodutive tract infections. Advances in Veterinary Medicine, v.41, p.217-239, 1999. Available from: < Available from: http://www.ncbi.nlm.nih.gov/pubmed/9890019 >. Accessed Nov. 25, 2016.
http://www.ncbi.nlm.nih.gov/pubmed/98900...
; YAEGER & HOLLERY, 2007YAEGER, M. J.; HOLLERY, L. D. Bacterial Causes of Bovine Infertility and Abortion. In: YOUNGQUIST, R. S. & THRELFALL, W. R. (Org.). Current Therapy in Large Animal Theriogenology. second edition, ed. EUA: Saunders Elsevier, 2007, p. 391-393. ). Then, development of endometritis and salpingitis occur during progestational phase of the estrous cycle, when both the amount and activity of neutrophils decline (VAN BERGEN, 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
; QUINN et al., 2011QUINN, P.J. et al. Campylobacter and Helicobacter species. In: _____. Veterinary microbiology and microbial disease. 2. ed. Iowa: Wiley-Blackwell, 2011. Cap.34, p.914-936. ). C. fetus subsp. venerealis does not interfere with the process of fertilization and initial embryonic development. However, endometritis prevents implantation of the embryo (HOFFER, 1981HOFFER, M. A. Bovine campylobacteriosis: a review. Canadian Veterinary Journal, v. 22, n. 11, p. 327-330, 1981. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1789996/ >. Accessed: Sept. 10, 2012.
https://www.ncbi.nlm.nih.gov/pmc/article...
), followed by early embryonic death. After uterine invasion, in a period of 3 to 6 months, the cow can remain infertile and a source of infection (VAN BERGEN, 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
); and subsequently, acquired immunity is developed.

The predominant immunity in C. fetus infected animals is local (CORBEIL et al., 1974CORBEIL, L. B. et al. Bovine venereal vibriosis: variations in immunoglobulin class of antibodies in genital secretions and serum. Infection and Immunity, v.10, n.5, p.1084-1090, 1974. ), with reported differences between males and females. In females, antibody production in the vagina and uterus begins 40 to 60 days after infection (CORBEIL et al., 1974CORBEIL, L. B. et al. Bovine venereal vibriosis: variations in immunoglobulin class of antibodies in genital secretions and serum. Infection and Immunity, v.10, n.5, p.1084-1090, 1974. ).

In preputial secretions of healthy bulls, IgG predominates in abundance, followed by IgA (BIER et al., 1977bBIER, P. J. et al. Measurement of immunoglobulins in reproductive tract fluids of bulls. Veterinary Microbiology, v.2, n.1, p.1-11, 1977b. Available from: <Available from: https://www.sciencedirect.com/science/.../pii/037811357790027X >. Accessed Jun. 02, 2015. doi: 10.1016/0378-1135(77)90027-X.
https://www.sciencedirect.com/science/.....
), and in bulls infected with C. fetus subsp. venerealis, the serum antibody response is undetectable (VASQUEZ et al., 1983VASQUEZ, L. A. et al. Bovine genital campylobacteriosis (vibriosis): vaccination of experimentally infected bulls. American Journal of Veterinary Research, v.44, p.1553-1557, 1983. ). Infiltration of lymphocytes and subepithelial plasma cells is similar in the prepuce and penis of infected and uninfected bulls (BIER et al., 1977b; SAMUELSON & WINTER, 1966SAMUELSON, J.D.; WINTER, A. J. Bovine vibriosis: the nature of the carrier state in the bull. The Journal of Infectious Disease, v.116, n.5, p.581-592, 1966. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/5957264 >. Accessed: Nov. 20, 2016.
https://www.ncbi.nlm.nih.gov/pubmed/5957...
). In the vagina, IgA predominates, which may limit the spread of the infection; IgGs are produced in the uterus and these opsonize the pathogenic organisms, facilitating phagocytosis by neutrophils and mononuclear cells (CORBEIL, 1999CORBEIL, L.B. et al. Immunization and diagnosis in bovine reprodutive tract infections. Advances in Veterinary Medicine, v.41, p.217-239, 1999. Available from: < Available from: http://www.ncbi.nlm.nih.gov/pubmed/9890019 >. Accessed Nov. 25, 2016.
http://www.ncbi.nlm.nih.gov/pubmed/98900...
; table 2). As BGC is caused by an extracellular pathogen, it would be expected to elicit predominantly a humoral immune response (CORBEIL et al., 1974CORBEIL, L. B. et al. Bovine venereal vibriosis: variations in immunoglobulin class of antibodies in genital secretions and serum. Infection and Immunity, v.10, n.5, p.1084-1090, 1974. ), likely causing short duration of immunity to infectious agent (BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
).

Table 2
Immunity to Campylobacter fetus subsp. venerealis. Adapted from CORBEIL (1999CORBEIL, L.B. et al. Immunization and diagnosis in bovine reprodutive tract infections. Advances in Veterinary Medicine, v.41, p.217-239, 1999. Available from: < Available from: http://www.ncbi.nlm.nih.gov/pubmed/9890019 >. Accessed Nov. 25, 2016.
http://www.ncbi.nlm.nih.gov/pubmed/98900...
).

The presence of carrier cows is due to the evasion of local immune responses, which CORBEIL (1999CORBEIL, L.B. et al. Immunization and diagnosis in bovine reprodutive tract infections. Advances in Veterinary Medicine, v.41, p.217-239, 1999. Available from: < Available from: http://www.ncbi.nlm.nih.gov/pubmed/9890019 >. Accessed Nov. 25, 2016.
http://www.ncbi.nlm.nih.gov/pubmed/98900...
) attributes to factors such as the relative lack of spontaneous IgG response in the vagina, possible blockage of vaginal IgG effects by IgA-binding microorganisms, and by variation of surface antigens of the microorganism against local immune responses. In bulls, infections are persistent and result in asymptomatic carrier animals, indicating that immunity during inflammation does not eliminate mucosal microorganisms (COBO et al., 2011COBO, E. R. et al. Immunity to infections in the lower genital tract of bulls. Journal of Reproductive Immunology, v. 89, n. 1, p. 55-61, 2011. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/21474188 >. Accessed: Nov. 25, 2016. doi: 10.1016/j.jri.2011.02.002.
https://www.ncbi.nlm.nih.gov/pubmed/2147...
). Nevertheless, according to TIZARD (2015),TIZARD, I. R. Imunidade nas superfícies corpóreas. Imunologia Veterinária. In: _____. 9.ed. Rio de Janeiro: Elsevier, 2014. Cap. 22, pp. 240-257. (Tradução).C. fetus infections are associated with the presence of several mononuclear cell types, as well as late cutaneous reactions (type IV hypersensitivity), so that cell-mediated immunity also participates in resistance to infection. Once the prolonged vaginal carrier status has been determined for C. fetus subsp. venerealis, the cow remains with the vaginal infection, whereas the uterus is free of infection (BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
). This allows for the return of the cow’s fertility in many cases, and may last up to 2 years in the absence of antigenic stimulation (CORBEIL et al., 1974CORBEIL, L. B. et al. Bovine venereal vibriosis: variations in immunoglobulin class of antibodies in genital secretions and serum. Infection and Immunity, v.10, n.5, p.1084-1090, 1974. ). In bulls, persistence of the bacteria in the lower genital tract is also attributed to evasion of immune responses (VARGAS et al., 2002VARGAS, A. et al. Campylobacter fetus subspecies venerealis surface array protein from bovine isolates in Brazil. Current Microbiology, v.45, n.2, p.111-114, 2002. Available from: < Available from: https://www.ncbi.nlm.nih.gov/pubmed/12070688 >. Accessed: Oct. 05, 2012. doi: 10.1007/s00284-001-0090-9.
https://www.ncbi.nlm.nih.gov/pubmed/1207...
; CORBEIL, 1975CORBEIL, L. B. et al. Bovine venereal vibriosis: antigenic variation of the bacterium during infection. Infection and Immunity, v.11, n.2, p.240-244, 1975. ).

Genes and virulence factors

Complete genomic sequencing of C. fetus is relatively recent. In 2006, the first complete and closed genome of C. fetus was made available by The Institute for Genomic Research (TIGR, USA, 2006), and this was the 82-40 strain isolated from an individual in the United States and later identified as C. fetus subsp. fetus, with a 1.77 Mpb genome and approximately 1,820 predicted genes. The first sequenced genome of C. fetus subsp. venerealis was published by MOOLHUIJZEN et al. (2009MOOLHUIJZEN, P. M. et al. Genomic analysis of Campylobacter fetus subspecies: Identification of candidate virulence determinants and diagnostic assay targets. BMC Microbiology, v.9, p.1-11, 2009. Available from: <Available from: https://bmcmicrobiol.biomedcentral.com/articles/10.../1471-2180-9-86 >. Accessed Dec. 10, 2014. doi: 10.1186/1471-2180-9-86.
https://bmcmicrobiol.biomedcentral.com/a...
) as a not assembled genome consisting of multiple contigs of the Azul-94 strain, which was isolated from an aborted bovine fetus in Argentina.

Since then, genomic studies of C. fetus have expanded. This can be verified by queries on databases of DNA sequences, such as GenBank®, where currently there are 74 genomes of C. fetus published, including 11 complete genomes (IRAOLA et al., 2017IRAOLA, G. et al. Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota. Nature Communications, v.8, n.1367, p.1-8, 2017. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/29118316 >. Accessed: Dec. 10, 2017. doi: 10.1038/s41467-017-01449-9.
https://www.ncbi.nlm.nih.gov/pubmed/2911...
; KIENESBERGER et al., 2014KIENESBERGER, S. et al. Comparative genome analysis of Campylobacter fetus subspecies revealed horizontally acquired genetic elements important for virulence and niche specificity. PloS One, v.9, n.1, p.1-13, 2014. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/24416416 >. Accessed: Oct. 05, 2014. doi: 10.1371/journal.pone.0085491.
https://www.ncbi.nlm.nih.gov/pubmed/2441...
; VAN DER GRAAF-VAN BLOOIS et al., 2014VAN DER GRAAF-VAN BLOOIS, L. et al. Inconsistency of phenotypic and genomic characteristics of Campylobacter fetus subspecies requires reevaluation of current diagnostics. Journal of Clinical Microbiology, v.52, n.12, p.4183-418, 2014. Available from:<Available from:https://www.ncbi.nlm.nih.gov/pubmed/25232170 >. Accessed: Jun. 20, 2015. doi: 10.1128/JCM.01837-14.
https://www.ncbi.nlm.nih.gov/pubmed/2523...
; WANG et al., 2015WANG, C. et al. Complete genome sequence of Campylobacter fetus subsp. testudinum strain Pet-3, isolated from a lizard (Hydrosaurus pustulatus). Genome Announcements, v.3, n.1, p.9-10, 2015. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335325 >. Accessed: Jan. 25, 2018. doi: 10.1128/genomeA.01420-14.
https://www.ncbi.nlm.nih.gov/pmc/article...
). Whole genome sequencing can be used to differentiate the mammal-associated C. fetus strains based on their core genomes (VAN DER GRAAF-VAN BLOOIS et al., 2014VAN DER GRAAF-VAN BLOOIS, L. et al. Inconsistency of phenotypic and genomic characteristics of Campylobacter fetus subspecies requires reevaluation of current diagnostics. Journal of Clinical Microbiology, v.52, n.12, p.4183-418, 2014. Available from:<Available from:https://www.ncbi.nlm.nih.gov/pubmed/25232170 >. Accessed: Jun. 20, 2015. doi: 10.1128/JCM.01837-14.
https://www.ncbi.nlm.nih.gov/pubmed/2523...
) and provides data on strains prevalent worldwide. Thus, genomic research in C. fetus is very important in providing new information about the species and its subspecies, to enable scientific advances regarding their origins and hosts, and for the correct identification of strains.

Current research on species of Campylobacter concentrates on total genomic analysis for the identification of virulence genes and characteristics that contribute to the pathogenicity differences among subspecies (FOUTS et al., 2005FOUTS, D. E. et al. Major structural differences and novel potential virulence mechanisms from the genomes of multiple Campylobacter species. PLoS Biology, v.3, n.1, 2005. Available from: < Available from: http://www.europepmc.org/abstract/med/15660156 >. Accessed: Jan. 17, 2015. doi: 10.1371/journal.pbio.0030015.
http://www.europepmc.org/abstract/med/15...
; MOOLHUIJZEN et al., 2009MOOLHUIJZEN, P. M. et al. Genomic analysis of Campylobacter fetus subspecies: Identification of candidate virulence determinants and diagnostic assay targets. BMC Microbiology, v.9, p.1-11, 2009. Available from: <Available from: https://bmcmicrobiol.biomedcentral.com/articles/10.../1471-2180-9-86 >. Accessed Dec. 10, 2014. doi: 10.1186/1471-2180-9-86.
https://bmcmicrobiol.biomedcentral.com/a...
; ALI et al., 2012ALI, A. et al. Campylobacter fetus subspecies: Comparative genomics and prediction of potential virulence targets. Gene,v. 508, n.2, p.145-156, 2012. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/22890137 >. Accessed: Jan. 25, 2018. doi: 10.1016/j.gene.2012.07.070.
https://www.ncbi.nlm.nih.gov/pubmed/2289...
; VAN DER GRAAF-VAN BLOOIS et al., 2016VAN DER GRAAF-VAN BLOOIS, L. et al. Campylobacter fetus subspecies contain conserved type IV secretion systems on multiple genomic islands and plasmids. PLoS ONE, v. 11, n. 4, p. 1-15, 2016. Available from: <Available from: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152832 >. Accessed: May, 30, 2017. doi: 10.1371/journal.pone.0152832.
http://journals.plos.org/plosone/article...
; IRAOLA et al., 2017IRAOLA, G. et al. Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota. Nature Communications, v.8, n.1367, p.1-8, 2017. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/29118316 >. Accessed: Dec. 10, 2017. doi: 10.1038/s41467-017-01449-9.
https://www.ncbi.nlm.nih.gov/pubmed/2911...
; GILBERT et al., 2018GILBERT, M. J. et al. Homologous recombination between genetically divergent Campylobacter fetus lineages supports host-associated speciation. Genome Biology and Evolution, v.10, n.3, p.716-722, 2018. Available from: <Available from: https://www.ncbi.nlm.nih >. Accessed Jan. 10 2019. doi: 10.1093/gbe/evy048.
https://www.ncbi.nlm.nih...
). In the C. fetus subespecies these virulence factors were clearly identified as classes of genes encoding proteins (MOOLHUIJZEN et al., 2009MOOLHUIJZEN, P. M. et al. Genomic analysis of Campylobacter fetus subspecies: Identification of candidate virulence determinants and diagnostic assay targets. BMC Microbiology, v.9, p.1-11, 2009. Available from: <Available from: https://bmcmicrobiol.biomedcentral.com/articles/10.../1471-2180-9-86 >. Accessed Dec. 10, 2014. doi: 10.1186/1471-2180-9-86.
https://bmcmicrobiol.biomedcentral.com/a...
), such as surface polypeptides involved in bacterial adherence, motility, toxin production and resistance, and regulatory and secretion systems.

Linkage between bacterial pathogens and epithelial cells is a prerequisite for invasion of host cells and subsequent translocation to the deeper layers of the mucosa. The spiral shape of the cell and the corkscrew-like motility conferred by the flagella of C. fetus are needed to colonize and cross the mucus barrier that covers the vaginal epithelium (SPRENGER et al., 2012SPRENGER, H. et al. So close and yet so far - Molecular microbiology of Campylobacter fetus subspecies. European Journal of Microbiology and Immunology, v.2, n.1, p.66-75, 2012. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933992 >. Accessed: Jan. 25, 2013. doi: 10.1556/EuJMI.2.2012.1.10.
https://www.ncbi.nlm.nih.gov/pmc/article...
). In addition, the flagellum is an important adhesin of C. jejuni and may have a similar function in C. fetus. Genomes of the two subspecies of C. fetus harbor homologs of adhesin PEB1, which is an outer membrane protein (OMP) and important in adherence to epithelial cells (SPRENGER et al., 2012SPRENGER, H. et al. So close and yet so far - Molecular microbiology of Campylobacter fetus subspecies. European Journal of Microbiology and Immunology, v.2, n.1, p.66-75, 2012. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933992 >. Accessed: Jan. 25, 2013. doi: 10.1556/EuJMI.2.2012.1.10.
https://www.ncbi.nlm.nih.gov/pmc/article...
). Therefore, motility and adhesion capacity of host cells play fundamental roles in the diseases caused by bacteria belonging to the genus Campylobacter.

A genomic island encoding a type IV secretion system integrated into the genome of C. fetus subsp. venerealis was identified, and it was believed that this genomic island was a determinant for the tropism of this subspecies in the bovine genital tract (GORKIEWICZ et al., 2010GORKIEWICZ, G. et al. A genomic island defines subspecies-specific virulence features of the host-adapted pathogen Campylobacter fetus subsp. venerealis. Journal of Bacteriology, v.192, n.2, p.502-517, 2010. Available from: <Available from: https://www.jb.asm.org/content/192/2/502.long >. Accessed: Oct. 17, 2014. doi: 10.1128/JB.00803-09.
https://www.jb.asm.org/content/192/2/50...
). However, phylogenetic analyses between genomes of strains of the two subspecies of C. fetus showed that this coding region involving the type IV secretion system exists in both subspecies as an element acquired from different donors and contains fic (n = 4) and virD4 (n = 10) genes (VAN DER GRAAF-VAN BLOOIS et al., 2016VAN DER GRAAF-VAN BLOOIS, L. et al. Campylobacter fetus subspecies contain conserved type IV secretion systems on multiple genomic islands and plasmids. PLoS ONE, v. 11, n. 4, p. 1-15, 2016. Available from: <Available from: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152832 >. Accessed: May, 30, 2017. doi: 10.1371/journal.pone.0152832.
http://journals.plos.org/plosone/article...
). In other research, SPRENGER et al. (2017)SPRENGER, H. et al. Fic proteins of Campylobacter fetus subsp. venerealis form a network of functional toxin-antitoxin systems. Frontiers in Microbiology, v.8, p.1-17, 2017. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/29089929 >. Accessed: Jun. 20, 2018. doi: 10.3389/fmicb.2017.01965.
https://www.ncbi.nlm.nih.gov/pubmed/2908...
have demonstrated that fic genes predominate and are strongly conserved in C. fetus subsp. venerealis, and Fic proteins are related to changes from the normal to the static metabolic states, which assists in the maintenance of the bacterium for long periods in the host, even under stressful conditions.

Several bacteria, including Escherichia coli and Shigella spp., produce Cytolethal distending toxin (CDT). Among Campylobacter strains, the cdt gene cluster is distributed universally and is well conserved, particularly in C. jejuni, C. coli, and C. fetus (ASAKURA et al., 2007ASAKURA, M. et al. Comparative analysis of cytolethal distending toxin (cdt) genes among Campylobacter jejuni, C. coli and C. fetus strains. Microbial Pathogenesis, v.42, n.5-6, p. 174-183, 2007. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/17353111 >. Accessed: Jun. 10, 2018. doi: 10.1016/j.micpath.2007.01.005.
https://www.ncbi.nlm.nih.gov/pubmed/1735...
). In C. jejuni, CDT protein is recognized for causing damage to the DNA of the host and tissue necrosis (JOENS et al., 2011JOENS, L. A. et al. Veterinary microbiology and microbial disease. Second ed. Sussex, UK: Blackwell Science Ltd, 2011. ), presenting as a potential virulence factor also in C. fetus.

The lipopolysaccharide (LPS) of C. fetus is typical of gram-negative bacteria. It has a lipid fraction, denominated lipid A, and a polysaccharide fraction, with a central oligosaccharide and an “O” antigen (MORAN et al., 1996MORAN, A. P. et al. Biological characterization of Campylobacter fetus lipopolysaccharides. FEMS Immunology and Medical Microbiology, v.15, n.1, p.43-50, 1996. Available from: <Available from: https://academic.oup.com/femspd/article/15/1/43/544167 >. Accessed: Jun. 10, 2012. doi: 10.1016/0928-8244(96)00043-0.
https://academic.oup.com/femspd/article/...
). In C. fetus, lipid A has low biological activity compared to other Enterobacteriaceae family members, and this is associated with persistent colonization of the host (BLASER & ENGBERG, 2008BLASER, M.J.; ENGBERG ,J. Clinical aspects of Campylobacter jejuni and Campylobacter coli infections. In: Nachamkin I., Blaser J.M., editors. Campylobacter. 3rd ed. American Society for Microbiology (ASM) Press; Washington DC, USA: 2008. pp. 99-121.). Variation in O antigen is the basis of a serotyping scheme for C. fetus, and two main serotypes, denominated A and B, are recognized (VAN BERGEN et al., 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
; DWORKIN et al., 1995aDWORKIN, J. et al. Segmental conservation of sapA sequences in type B Campylobacter fetus cells. The Journal of Biological Chemistry, v.270, n.25, p.15093-15101, 1995a. Available from: <Available from: http://www.jbc.org/content/270/25/15093.full.pdf >. Accessed: Jun. 10, 2012. doi: 10.1074/jbc.270.25.15093.
http://www.jbc.org/content/270/25/15093....
; PEREZ-PEREZ et al., 1986PEREZ-PEREZ, G. I. et al. Lipopolysaccharide Structures of Campylobacter fetus. Infection and Immunity, v. 51, n. 1, p. 209-212, 1986. Available from: <Available from: https://iai.asm.org/content/iai/51/1/209.full.pdf >. Accessed: Jun. 10, 2012.
https://iai.asm.org/content/iai/51/1/209...
; TU et al., 2004TU, Z.C. et al. Conservation and diversity of sap homologues and their organization among Campylobacter fetus isolates. Infection and Immunity, v.72, n.3, p.1715-1724, 2004. Available from: < Available from: https://www.ncbi.nlm.nih.gov/pubmed/14977980 >. Accesses: Jun. 10, 2013. doi: 10.1128/IAI.72.3.1715-1724.2004.
https://www.ncbi.nlm.nih.gov/pubmed/1497...
). C. fetus subsp. fetus contains serotypes A and B, whereas C. fetus subsp. venerealis has only serotype A.

C. fetus has a protein structure covering the outer membrane. MCCOY et al. (1975MCCOY, E. C. et al. Superficial antigens of Campylobacter (Vibrio) fetus: characterization of an antiphagocytic component. Infection and Immunity, v.11, n.3, p.517-525, 1975. ) were the first to describe the presence of a microcapsule called the S layer, formed by protein subunit arrays known as surface array proteins (SAPs), and recognized as important in the pathogenesis of BGC (MCCOY et al., 1975MCCOY, E. C. et al. Superficial antigens of Campylobacter (Vibrio) fetus: characterization of an antiphagocytic component. Infection and Immunity, v.11, n.3, p.517-525, 1975. ; FAGAN & FAIRWEATHER, 2014FAGAN, R. P.; FAIRWEATHER, N. F. Biogenesis and functions of bacterial S-layers. Nature Reviews Microbiology, v.12, n.3, p.211-222, 2014. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/24509785 >. Accessed: Feb. 10, 2015. doi: 10.1038/nrmicro3213.
https://www.ncbi.nlm.nih.gov/pubmed/2450...
; WINTER et al., 1978WINTER, A. J. et al. Microcapsule of Campylobacter fetus: Chemical and physical characterization. Infection and Immunity, v.22, n.3, p.963-971, 1978.) because, these SAPs are responsible for the resistance of this microorganism to bactericidal and phagocytic activities of the host (MCCOY et al., 1975MCCOY, E. C. et al. Superficial antigens of Campylobacter (Vibrio) fetus: characterization of an antiphagocytic component. Infection and Immunity, v.11, n.3, p.517-525, 1975. ; RAY et al., 2000RAY, K. C. et al. Campylobacter fetus sap inversion occurs in the absence of RecA function. Infection and Immunity, v. 68, n. 10, p. 5663-5667, 2000. Available from: <Available from: https://iai.asm.org/content/68/10/5663 >. Accessed: Apr. 17, 2013. 10.1128/IAI.68.10.5663-5667.2000. doi: 10.1128/IAI.68.10.5663-5667.2000.
https://iai.asm.org/content/68/10/5663...
). Their superficial location suggested that they may be an important mediator in interactions with the host (BLASER & GOTSCHLICH, 1990BLASER, M. J.; GOTSCHLICH, E. C. Surface Array Protein of Campylobacter fetus. The Journal of Biological Chemistry, v.265, n.24, p.14529-14535, 1990. Available from: <Available from: http://www.jbc.org/content/265/24/14529.full.pdf >. Accessed Jun. 02, 2012.
http://www.jbc.org/content/265/24/14529....
), and in persistence of the pathogen in the reproductive tract (CORBEIL et al., 1975CORBEIL, L. B. et al. Bovine venereal vibriosis: antigenic variation of the bacterium during infection. Infection and Immunity, v.11, n.2, p.240-244, 1975. ).

A particular aspect of the surface proteins in C. fetus is the fact that a single bacterium can produce up to three proteins, with molecular mass ranging from 97 to 149 kDa and usually one of these being dominant (DUBREUIL et al., 1990DUBREUIL, J. D. et al. Antigenic differences among Campylobacter fetus S-layer proteins. Journal of Bacteriology, v.172, n.9, p.5035-5043, 1990. Available from: <Available from: http://jb.asm.org/content/172/9/5035.long >. Accessed Aug. 20, 2012. doi: 10.1128/jb.172.9.5035-5043.1990.
http://jb.asm.org/content/172/9/5035.lon...
; DWORKIN et al., 1995aDWORKIN, J. et al. Segmental conservation of sapA sequences in type B Campylobacter fetus cells. The Journal of Biological Chemistry, v.270, n.25, p.15093-15101, 1995a. Available from: <Available from: http://www.jbc.org/content/270/25/15093.full.pdf >. Accessed: Jun. 10, 2012. doi: 10.1074/jbc.270.25.15093.
http://www.jbc.org/content/270/25/15093....
; DWORKIN et al., 1995bDWORKIN, J. et al. A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the conserved N terminus of a family of silent and divergent homologs. Journal of Bacteriology, v.177, n.7, p.1734-1741, 1995b. Available from: <Available from: https://jb.asm.org/content/177/7/1734.full.pdf >. Accessed Aug. 05, 2012. doi: 10.1128/jb.177.7.1734-1741.1995.
https://jb.asm.org/content/177/7/1734.fu...
; FUJIMOTO, 1991FUJIMOTO, S. et al. Correlation between molecular size of the surface array protein and morphology and antigenicity of the Campylobacter fetus S layer. Infection and Immunity, v.59, n.6, p.2017-2022, 1991. Available from: < http://www.ncbi.nlm.nih.gov/pubmed/2037362>. Accessed Sept. 17, 2012. ; PEI et al., 1988PEI, Z. et al. Purification and Characterization of a Family of High Molecular weight surface-array protein from Campylobacter fetus. The journal of Biological chemistry, v.263, n.13, p.6416-6420, 1988. Available from: <Available from: http://www.jbc.org/content/263/13/6416.long >. Accessed: Jun. 15, 2012.
http://www.jbc.org/content/263/13/6416.l...
, VARGAS et al., 2002VARGAS, A. et al. Campylobacter fetus subspecies venerealis surface array protein from bovine isolates in Brazil. Current Microbiology, v.45, n.2, p.111-114, 2002. Available from: < Available from: https://www.ncbi.nlm.nih.gov/pubmed/12070688 >. Accessed: Oct. 05, 2012. doi: 10.1007/s00284-001-0090-9.
https://www.ncbi.nlm.nih.gov/pubmed/1207...
). During infection, extensive high frequency chromosomal rearrangements occurred in DNA, and this results in the modification of dominant epitopes during persistence of the bacteria in the genital tract (GARCIA et al., 1995GARCIA, M. M. et al. Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs. Journal of Bacteriology, v.177, n.8, p.1976-1980, 1995. Available from: < Available from: https://www.jb.asm.org/content/177/8/1976.abstract >. Accessed Aug. 30, 2012. doi: 10.1128/jb.177.8.1976-1980.1995.
https://www.jb.asm.org/content/177/8/197...
; WANG et al., 1993WANG, E. et al. Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus. Journal of Bacteriology, v.175, n.16, p.4979-4984, 1993. Available from: <Available from: https://jb.asm.org/content/175/16/4979.abstract >. Accessed: Sept. 20, 2012. doi: 10.1128/jb.175.16.4979-4984.1993.
https://jb.asm.org/content/175/16/4979.a...
). Thus, this evidence showed the importance of surface proteins for C. fetus subsp. venerealis; and consequently, for the maintenance of BGC infection. Because of this, such proteins are potential candidates for the development of serological diagnostic methods, and according to PEI et al. (1988)PEI, Z. et al. Purification and Characterization of a Family of High Molecular weight surface-array protein from Campylobacter fetus. The journal of Biological chemistry, v.263, n.13, p.6416-6420, 1988. Available from: <Available from: http://www.jbc.org/content/263/13/6416.long >. Accessed: Jun. 15, 2012.
http://www.jbc.org/content/263/13/6416.l...
, for use in vaccines.

Diagnosis of BGC

The subspecies of C. fetus showed different adaptations to host tissues. However, at the genetic level they are practically indistinguishable (MOOLHUIJZEN et al., 2009MOOLHUIJZEN, P. M. et al. Genomic analysis of Campylobacter fetus subspecies: Identification of candidate virulence determinants and diagnostic assay targets. BMC Microbiology, v.9, p.1-11, 2009. Available from: <Available from: https://bmcmicrobiol.biomedcentral.com/articles/10.../1471-2180-9-86 >. Accessed Dec. 10, 2014. doi: 10.1186/1471-2180-9-86.
https://bmcmicrobiol.biomedcentral.com/a...
). Sequence alignment reveals that the genotypes of C. fetus subspecies are highly homologous, with 92.9% average identity (KIENESBERGER et al., 2014KIENESBERGER, S. et al. Comparative genome analysis of Campylobacter fetus subspecies revealed horizontally acquired genetic elements important for virulence and niche specificity. PloS One, v.9, n.1, p.1-13, 2014. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/24416416 >. Accessed: Oct. 05, 2014. doi: 10.1371/journal.pone.0085491.
https://www.ncbi.nlm.nih.gov/pubmed/2441...
).

The materials for laboratory diagnosis of BGC from suspect animals include samples of prepucial smegma and semen in bulls, and female specimens of cervicovaginal mucus (OIE, 2017OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
). In addition, samples of aborted fetuses and placenta can be collected. The diagnosis of BGC is at the herd level, so it is not considered for isolated cases, but an epidemiological profile of the herd based on the history of clinical manifestations compatible with this disease and analysis of reproductive rates, to be confirmed with laboratory assays.

In females, cervicovaginal mucus can be collected with an insemination pipette or absorbent pad (OIE, 2017; STYNEN et al., 2003STYNEN, A. P. R. et al. Campilobacteriose genital bovina em rebanhos leiteiros com problemas reprodutivos da microrregião de Varginha - Minas Gerais. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, v.55, n.6, p.766-769, 2003. Available from: < Available from: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-09352003000600015 >. Accessed: Jun. 25, 2013. doi: 10.1590/S0102-09352003000600015.
https://www.scielo.br/scielo.php?script=...
). Preputial samples from bulls can be obtained by scraping or washing with sterile saline (OIE, 2017OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
). An important aspect to be observed is the maintenance of bulls in sexual rest for 7 to 15 days before harvesting the material to increase the sensitivity of the diagnosis (SKIRROW & BONDURANT, 1988SKIRROW, S.Z.; BONDURANT, R. H. Bovine trichomoniasis. Veterinary Bull, v. 58, p. 591-603, 1988. ). Moreover, the OIE (2017)OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
indicated the use of transport media to maintain viability in samples that will not be processed in the laboratory on the same day of collection. Enriched and selective transport media (Lander, Foley and Clark, for example) are suitable for transporting the samples, and when these media are not available, the collected material should be placed in a sterile recipient container (temperature 4-10 °C) and protected from light.

Techniques for the diagnosis of C. fetus involve the isolation and identification of the infectious agent, immunofluorescence, immunoenzymatic tests, and molecular identification (OIE, 2017). The use of two combined techniques to obtain a result is preferred to give reliability to the result of the diagnosis, but several laboratories do not apply or do not perform the techniques correctly (VAN BERGEN et al., 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
).

Isolation and identification of C. fetus

The isolation and identification of C. fetus by microbiological culture is considered the standard and confirmatory test for diagnosis of infection (BROOKS et al., 2004BROOKS, B. W. et al. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. Veterinary Microbiology, v.103, n.1-2, p.77-84, 2004. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0378113504002640 >. Accessed. Dec. 10, 2014. doi: 10.1016/j.vetmic.2004.07.008.
http://www.sciencedirect.com/science/art...
) and is a suitable assay for certifying individual animals prior to movement (OIE, 2017OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
). However, the same depends on the sample quality, the way it is sent to the laboratory, and the viability of the microorganisms. Campylobacter species are microaerophilic and C. fetus grows fastidiously, requiring strictly atmospheric conditions for cultivation, including selective and enrichment media (QUINN et al., 2011QUINN, P.J. et al. Campylobacter and Helicobacter species. In: _____. Veterinary microbiology and microbial disease. 2. ed. Iowa: Wiley-Blackwell, 2011. Cap.34, p.914-936. ).

The discrimination of subspecies isolates is based on colony morphology on blood agar plates, certain biochemical properties, and antimicrobial susceptibility (QUINN et al., 2011QUINN, P.J. et al. Campylobacter and Helicobacter species. In: _____. Veterinary microbiology and microbial disease. 2. ed. Iowa: Wiley-Blackwell, 2011. Cap.34, p.914-936. ). Gram staining, catalase test, and oxidase activity are the assays most frequently used in diagnostic laboratories, followed by motility testing (VAN BERGEN et al., 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
). In addition, the differentiation of C. fetus subspecies is carried out mainly by antimicrobial susceptibility tests using cefalotin and nalidixic acid (HOLT et al., 1994HOLT, J. G. et al. Bergey’s Manual of Determinative Bacteriology. 9 ed. ed. Philadelphia: Lippincott Williams & Wilkins, 1994. ), and by the evaluation of biochemical characteristics. Moreover, growth at 25°C and 42°C in the presence of 3.5% sodium chloride, H2S production, and 1% glycine tolerance testing (HOLT et al., 1994HOLT, J. G. et al. Bergey’s Manual of Determinative Bacteriology. 9 ed. ed. Philadelphia: Lippincott Williams & Wilkins, 1994. ) are used, the latter being the standard assay for subspecies differentiation (VAN BERGEN et al., 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
).

However, researchers report doubts concerning differentiation by the phenotypic tests mentioned above (VAN DER GRAAF-VAN BLOOIS et al., 2014VAN DER GRAAF-VAN BLOOIS, L. et al. Inconsistency of phenotypic and genomic characteristics of Campylobacter fetus subspecies requires reevaluation of current diagnostics. Journal of Clinical Microbiology, v.52, n.12, p.4183-418, 2014. Available from:<Available from:https://www.ncbi.nlm.nih.gov/pubmed/25232170 >. Accessed: Jun. 20, 2015. doi: 10.1128/JCM.01837-14.
https://www.ncbi.nlm.nih.gov/pubmed/2523...
). First, C. fetus subsp. venerealis biovar intermedius is able to produce H2S (a characteristic previously attributed only to C. fetus subsp. fetus). In terms of glycine tolerance, CHANG & OGG (1970CHANG, W.; OGG, J. E. Transduction in Vibrio fetus. American Journal of Veterinary Research, v.31, p.919-924, 1970. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/5442454 >. Accessed: Nov. 10, 2016.
https://www.ncbi.nlm.nih.gov/pubmed/5442...
) have shown that this characteristic can be influenced by transduction of this phenotypic characteristic by bacteriophages. In addition, antimicrobial resistance can be acquired through transduction or mutations (CHANG & OGG, 1970CHANG, W.; OGG, J. E. Transduction in Vibrio fetus. American Journal of Veterinary Research, v.31, p.919-924, 1970. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/5442454 >. Accessed: Nov. 10, 2016.
https://www.ncbi.nlm.nih.gov/pubmed/5442...
; SALAMA et al., 1992SALAMA, S. M. et al. Differentiation of the subspecies of Campylobacter fetus by genomic sizing. International Journal of Systematic Bacteriology, v.42, n.3, p.446-450, 1992. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/1354478 >. Accessed: Nov. 5, 2012. doi: 10.1099/00207713-42-3-446.
https://www.ncbi.nlm.nih.gov/pubmed/1354...
).

The procedures for isolation and identification of C. fetus are laborious and require special culture media and atmospheric conditions, restricting the number of laboratories able to carry out diagnosis of this microorganism. In addition, inconsistencies between phenotypic and genomic characteristics of C. fetus samples revealed in recent research (VAN DER GRAAF-VAN BLOOIS et al., 2014VAN DER GRAAF-VAN BLOOIS, L. et al. Inconsistency of phenotypic and genomic characteristics of Campylobacter fetus subspecies requires reevaluation of current diagnostics. Journal of Clinical Microbiology, v.52, n.12, p.4183-418, 2014. Available from:<Available from:https://www.ncbi.nlm.nih.gov/pubmed/25232170 >. Accessed: Jun. 20, 2015. doi: 10.1128/JCM.01837-14.
https://www.ncbi.nlm.nih.gov/pubmed/2523...
) have stimulated a critical evaluation of the clinical relevance of identification of C. fetus subspecies using phenotypic tests. Therefore, some techniques such as fluorescence antibody test (FAT) and polymerase chain reaction (PCR) are also used for the detection of BGC, combined with microbiological isolation (VAN BERGEN et al. , 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
).

In Brazil, only one published research report used microbiological isolation in combination with another technique. The study of ROCHA et al. (2009ROCHA, F. S. et al. Investigação de Campylobacter fetus e Tritrichomonas foetus na mucosa prepucial de touros da região do Médio Paraíba, RJ. Ciência Rural, v.39, n.5, p.1587-1590, 2009. Available from: <Available from: https://www.scielo.br/pdf/cr/v39n5/a213cr1128.pdf >. Accessed: Nov. 20, 2016. doi: 10.1590/S0103-84782009005000103.
https://www.scielo.br/pdf/cr/v39n5/a213c...
) investigated the presence of C. fetus in samples from bulls on dairy and beef farms in the region of the Médio Paraíba, state of Rio de Janeiro and used microbiological isolation combined with direct immunofluorescence (DIF) testing. By DIF, ROCHA et al. (2009)ROCHA, F. S. et al. Investigação de Campylobacter fetus e Tritrichomonas foetus na mucosa prepucial de touros da região do Médio Paraíba, RJ. Ciência Rural, v.39, n.5, p.1587-1590, 2009. Available from: <Available from: https://www.scielo.br/pdf/cr/v39n5/a213cr1128.pdf >. Accessed: Nov. 20, 2016. doi: 10.1590/S0103-84782009005000103.
https://www.scielo.br/pdf/cr/v39n5/a213c...
confirmed the presence of C. fetus in 35.9% (14/30) of samples, and in the microbiological culture and biochemical tests, obtained 10.3% (4/30) positivity for C. fetus subsp. venerealis (Table 1).

For routine diagnostic activities of the Laboratory of Bacteriology of the Universidade Federal of Santa Maria, microbiological culture combined with PCR was established (HUM et al., 1997HUM, S. et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. BMC microbiology, v.75, n.11, p.827-831, 1997. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-0813.1997.tb15665.x >. Accessed: Oct. 26, 2012. doi: 10.1111/j.1751-0813.1997.tb15665.x.
https://onlinelibrary.wiley.com/doi/full...
). From 2011 to 2018, 261 samples of beef and dairy cattle from 43 breeding farms in Rio Grande do Sul state were analyzed. Prepural aspirate (n = 147), cervical mucus (n = 108) and fetal abomasal content (n = 6) were analyzed. The presence of samples positive for C. fetus in this period was 2.72% (4/147) in bulls, 14.82% (16/108) in cows, and 16.66% (1/6) in fetuses. For farms, 23.25% (10/43) had at least one C. fetus positive animal in 42.86% (9/21) of the municipalities analyzed (BALZAN, unpublished data).

Fluorescent Antibody Test (FAT)

Immunofluorescence tests can be applied for direct diagnosis of microorganism in samples, or to confirm the identification of microorganisms after isolation (VAN BERGEN et al., 2005aVAN BERGEN, M. A. P. et al. Global detection and identification of Campylobacter fetus subsp. venerealis. Revue Scientifique Et Technique-Office International Des Epizooties, v.24, n.3, p.1017-1026, 2005a. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16642772 >. Accessed: Oct. 05, 2015. doi: 10.20506/rst.24.3.1629.
https://www.ncbi.nlm.nih.gov/pubmed/1664...
; OIE, 2017OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
). FATs are generally used as a screening test for preputial samples (CIPOLLA et al., 2001CIPOLLA, A. et al. Campylobacter fetus diagnosis: Direct immunofluorescence comparing chicken IgY and rabbit IgG conjugates. Altex-Alternativen Zu Tierexperimenten, v.18, n.3, p.165-170, 2001. Available from:< Available from:https://www.ncbi.nlm.nih.gov/pubmed/11565050 >. Accessed: Sept. 15, 2012.
https://www.ncbi.nlm.nih.gov/pubmed/1156...
).

In a study by FIGUEIREDO et al. (2002FIGUEIREDO, J. F. et al. Evaluation of direct fluorescent antibody test for the diagnosis of Bovine Genital Campylobacteriosis. Revista Latinoamericana de Microbiologia, v.44, n.3-4, p.118-123, 2002. Available from: < http://www.ncbi.nlm.nih.gov/pubmed/17063594>. Accessed. Nov. 12, 2014.
https://doi.org/http://www.ncbi.nlm.nih....
), the direct FAT (DFAT) demonstrated good detection limits (100 CFU/mL) in preputial washes, with 92.59% sensitivity and 88.89% specificity. These results demonstrated the use of DFATs as an important support technique for the control of BGC. In Argentina, due to the implementation of a national health plan to control venereal diseases in cattle from 1983, there are approximately 30 laboratories that mainly use DFAT assays to diagnose BGC from bull samples (CIPOLLA et al., 2001CIPOLLA, A. et al. Campylobacter fetus diagnosis: Direct immunofluorescence comparing chicken IgY and rabbit IgG conjugates. Altex-Alternativen Zu Tierexperimenten, v.18, n.3, p.165-170, 2001. Available from:< Available from:https://www.ncbi.nlm.nih.gov/pubmed/11565050 >. Accessed: Sept. 15, 2012.
https://www.ncbi.nlm.nih.gov/pubmed/1156...
). This health plan was successful in controlling the disease, given the reduction in the percentage of BGC in herds of beef cattle from approximately 50% in 1983 to 15-18% in the period 1997-1999 (CIPOLLA et al., 2001CIPOLLA, A. et al. Campylobacter fetus diagnosis: Direct immunofluorescence comparing chicken IgY and rabbit IgG conjugates. Altex-Alternativen Zu Tierexperimenten, v.18, n.3, p.165-170, 2001. Available from:< Available from:https://www.ncbi.nlm.nih.gov/pubmed/11565050 >. Accessed: Sept. 15, 2012.
https://www.ncbi.nlm.nih.gov/pubmed/1156...
).

The immunofluorescence test was not widely evaluated and the reported problems were false-positive results due to non-specific fluorescence and inability to differentiate C. fetus subspecies (SILVEIRA et al., 2018SILVEIRA, C. S. et al. Diagnosis of Bovine Genital Campylobacteriosis in South America. Frontiers in Veterinary Science, v.5, p.1-9, 2018. Available from: <Available from: https://www.frontiersin.org/article/10.3389/fvets.2018.00321/full >. Accessed Jan. 10, 2019. doi: 10.3389/fvets.2018.00321.
https://www.frontiersin.org/article/10.3...
), with low availability of conjugate. In addition, technicians must be trained and experienced so that performance of the test is not impaired by subjectivity (FIGUEIREDO et al., 2002FIGUEIREDO, J. F. et al. Evaluation of direct fluorescent antibody test for the diagnosis of Bovine Genital Campylobacteriosis. Revista Latinoamericana de Microbiologia, v.44, n.3-4, p.118-123, 2002. Available from: < http://www.ncbi.nlm.nih.gov/pubmed/17063594>. Accessed. Nov. 12, 2014.
https://doi.org/http://www.ncbi.nlm.nih....
).

In Brazil, DFAT was widely used in surveys of the occurrence of C. fetus in several regions (table 1) until 2009 and obtained high rates of positivity. Nowadays, the commercial unavailability of conjugates makes the use of the technique restricted to certain research laboratories producing these inputs.

Molecular identification of C. fetus

The amplified fragment length polymorphism (AFLP) (WAGENAAR et al., 2001WAGENAAR, J. A. et al. Comparative Study Using Amplified Fragment Length Polymorphism Fingerprinting, PCR Genotyping, and Phenotyping To Differentiate Campylobacter fetus Strains Isolated from Animals. Journal of Clinical Microbiology, v.39, n.6, p.2283-2286, 2001. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/11376071 >. Accessed: Nov. 10, 2012. doi: 10.1128/JCM.39.6.2283-2286.2001.
https://www.ncbi.nlm.nih.gov/pubmed/1137...
) and multilocus sequence typing (MLST) (VAN BERGEN et al., 2005cVAN BERGEN, M.A.P. et al. Clonal Nature of Campylobacter fetus as Defined by Multilocus Sequence Typing. Society, v.43, n.12, p.5888-5898, 2005c. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16333072 >. Accessed: Jan. 20, 2016. doi: 10.1128/JCM.43.12.5888.
https://www.ncbi.nlm.nih.gov/pubmed/1633...
) methods were firstly recommended to differentiate the two subspecies, fetus and venerealis, but these tests are laborious, impractical for routine use and, according to VAN DER GRAAF-VAN BLOOIS et al. (2013VAN DER GRAAF-VAN BLOOIS, L. et al. Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay. Journal of Microbiological Methods, v.95, n.1, p.93-97, 2013. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/23810970 >. Accessed: Feb. 10, 2015. doi: 10.1016/j.mimet.2013.06.005.
https://www.ncbi.nlm.nih.gov/pubmed/2381...
) and IRAOLA et al. (2017IRAOLA, G. et al. Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota. Nature Communications, v.8, n.1367, p.1-8, 2017. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/29118316 >. Accessed: Dec. 10, 2017. doi: 10.1038/s41467-017-01449-9.
https://www.ncbi.nlm.nih.gov/pubmed/2911...
), are not fully reliable for C. fetus subspecies differentiation.

A practical platform for BGC diagnosis is the PCR technique, since this assay is rapid, simple, and reliable. Several PCR assays have been developed to identify C. fetus (ABRIL et al., 2007ABRIL, C. et al. Discovery of insertion element IS Cfe1: A new tool for Campylobacter fetus subspecies differentiation. Clinical Microbiology and Infection, v.13, n.10, p.993-1000, 2007. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/17697006 >. Accessed: Nov. 12, 2016. doi: 10.1111/j.1469-0691.2007.01787.x.
https://www.ncbi.nlm.nih.gov/pubmed/1769...
; VAN BERGEN et al., 2005bVAN BERGEN, M. A. P. et al. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies. Journal of Medical Microbiology, v.54, n.12, p.1217-1224, 2005b. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/%0A16278437 %0A >. Accessed: Oct. 05, 2015. doi: 10.1099/jmm.0.46186-0.
https://www.ncbi.nlm.nih.gov/pubmed/%0A1...
; HUM et al., 1997HUM, S. et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. BMC microbiology, v.75, n.11, p.827-831, 1997. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-0813.1997.tb15665.x >. Accessed: Oct. 26, 2012. doi: 10.1111/j.1751-0813.1997.tb15665.x.
https://onlinelibrary.wiley.com/doi/full...
; MCMILLEN et al., 2006MCMILLEN, L. et al. Comparison of culture and a novel 5′ Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle. Journal of Clinical Microbiology, v.44, n.3, p.938-945, 2006. Available from: <Available from: https://jcm.asm.org/content/44/3/938.full >. Accessed: Sept. 02, 2015. doi: 10.1128/JCM.44.3.938-945.2006.
https://jcm.asm.org/content/44/3/938.ful...
; TU et al., 2005TU, Z. C. et al. Genetic divergence of Campylobacter fetus strains of mammal and reptile origins. Journal of Clinical Microbiology, v.43, n.7, p.3334-3340, 2005. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16000457 >. Accessed: Sept. 30, 2012. doi: 10.1128/JCM.43.7.3334-3340.2005.
https://www.ncbi.nlm.nih.gov/pubmed/1600...
; WANG et al., 2002WANG, G. et al. Colony Multiplex PCR Assay for Identification and Differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis and C. fetus subsp. fetus. Journal of Clinical Microbiology, v.40, n.12, p.4744-4747, 2002. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC154608/pdf/0600.pdf >. Accessed: Oct. 05, 2013. doi: 10.1128/JCM.40.12.4744.
https://www.ncbi.nlm.nih.gov/pmc/article...
). The evaluation of these several PCRs (VAN DER GRAAF-VAN BLOOIS et al., 2013VAN DER GRAAF-VAN BLOOIS, L. et al. Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay. Journal of Microbiological Methods, v.95, n.1, p.93-97, 2013. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/23810970 >. Accessed: Feb. 10, 2015. doi: 10.1016/j.mimet.2013.06.005.
https://www.ncbi.nlm.nih.gov/pubmed/2381...
) showed that only those of HUM et al. (1997) HUM, S. et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. BMC microbiology, v.75, n.11, p.827-831, 1997. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-0813.1997.tb15665.x >. Accessed: Oct. 26, 2012. doi: 10.1111/j.1751-0813.1997.tb15665.x.
https://onlinelibrary.wiley.com/doi/full...
and ABRIL et al. (2007)ABRIL, C. et al. Discovery of insertion element IS Cfe1: A new tool for Campylobacter fetus subspecies differentiation. Clinical Microbiology and Infection, v.13, n.10, p.993-1000, 2007. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/17697006 >. Accessed: Nov. 12, 2016. doi: 10.1111/j.1469-0691.2007.01787.x.
https://www.ncbi.nlm.nih.gov/pubmed/1769...
are valid for identification of C. fetus in terms of sensitivity and specificity; however, both techniques require the isolation of bacteria or culture of samples in medium for enrichment for DNA extraction.

Further, multiplex PCR assays (HUM et al., 1997HUM, S. et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. BMC microbiology, v.75, n.11, p.827-831, 1997. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-0813.1997.tb15665.x >. Accessed: Oct. 26, 2012. doi: 10.1111/j.1751-0813.1997.tb15665.x.
https://onlinelibrary.wiley.com/doi/full...
) and real-time PCR with specific probes (MCMILLEN et al., 2006MCMILLEN, L. et al. Comparison of culture and a novel 5′ Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle. Journal of Clinical Microbiology, v.44, n.3, p.938-945, 2006. Available from: <Available from: https://jcm.asm.org/content/44/3/938.full >. Accessed: Sept. 02, 2015. doi: 10.1128/JCM.44.3.938-945.2006.
https://jcm.asm.org/content/44/3/938.ful...
), are able to identify C. fetus to the species level only. The OIE (2017) OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
recommendation is that a PCR assay to identify C. fetus isolates reliably to subspecies level is not available and that researchers need to be careful and critical when publishing results at the (unreliable) subspecies level.

CHABAN et al. (2012CHABAN, B. et al. Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples. Canadian Journal of Veterinary Research, v.76, n.3, p.166-173, 2012. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384278/ >. Accessed: Dec. 17, 2014.
https://www.ncbi.nlm.nih.gov/pmc/article...
) used the conventional PCR primers of HUM et al. (1997HUM, S. et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. BMC microbiology, v.75, n.11, p.827-831, 1997. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-0813.1997.tb15665.x >. Accessed: Oct. 26, 2012. doi: 10.1111/j.1751-0813.1997.tb15665.x.
https://onlinelibrary.wiley.com/doi/full...
) applied to a quantitative real-time PCR platform for direct processing of preputial samples, aiming to improve the original assay, which had low analytical sensitivity. The test developed proved to be sensitive and low cost; however, the processing of preputial samples for direct detection is as laborious as for conventional PCR, or for isolation of bacteria.

To allow differentiation of C. fetus subspecies, MCGOLDRICK et al. (2013MCGOLDRICK, A. et al. Real Time PCR to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis. Journal of Microbiological Methods, v.94, n.3, p.199-204, 2013. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/23811208 >. Accessed: Jun. 20 2016. doi: 10.1016/j.mimet.2013.06.014.
https://www.ncbi.nlm.nih.gov/pubmed/2381...
) adapted some PCR methods already published (CASADÉMONT et al., 1998CASADÉMONT, I. et al. Cloning of a sapB homologue (sapB2) encoding a putative 112-kDa Campylobacter fetus S-layer protein and its use for identification and molecular genotyping. FEMS Immunology and Medical Microbiology, v.21, n.4, p.269-281, 1998. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/9752999 >. Accessed: Jun. 10, 2012. doi: 10.1016/S0928-8244(98)00081-9.
https://www.ncbi.nlm.nih.gov/pubmed/9752...
; HUM et al., 1997HUM, S. et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. BMC microbiology, v.75, n.11, p.827-831, 1997. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-0813.1997.tb15665.x >. Accessed: Oct. 26, 2012. doi: 10.1111/j.1751-0813.1997.tb15665.x.
https://onlinelibrary.wiley.com/doi/full...
) for quantitative PCR (qPCR), and reported a sensitivity of 98.7% and specificity of 99.8% for C. fetus subsp. venerealis identification, and this technique has been approved for routine subspecies characterization when there are doubts in the OIE (2017) OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
recommended trials. VAN DER GRAAF-VAN BLOOIS et al. (2013VAN DER GRAAF-VAN BLOOIS, L. et al. Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay. Journal of Microbiological Methods, v.95, n.1, p.93-97, 2013. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/23810970 >. Accessed: Feb. 10, 2015. doi: 10.1016/j.mimet.2013.06.005.
https://www.ncbi.nlm.nih.gov/pubmed/2381...
) developed a qPCR assay with 100% sensitivity and 100% specificity for the detection of C. fetus, but did not meet the objective of differentiating the subspecies of C. fetus.

Recently, papers published in Brazil (Table 1) used the conventional PCR technique described by HUM et al. (1997HUM, S. et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. BMC microbiology, v.75, n.11, p.827-831, 1997. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-0813.1997.tb15665.x >. Accessed: Oct. 26, 2012. doi: 10.1111/j.1751-0813.1997.tb15665.x.
https://onlinelibrary.wiley.com/doi/full...
). Some of this research (FILHO et al., 2018; NASCIMENTO et al., 2018; OLIVEIRA et al., 2015OLIVEIRA, J. M. B. et al. Prevalence and risk factors associated with bovine genital campylobacteriosis and bovine trichomonosis in the state of Pernambuco, Brazil. Tropical Animal Health and Production, v.47, n.3, p. 549-555, 2015. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/25633914 >. Accessed: Oct. 10, 2016. doi: 10.1007/s11250-015-0761-3.
https://www.ncbi.nlm.nih.gov/pubmed/2563...
) used only a pair of primers specific for C. fetus subsp. venerealis identification (VENS - parA gene), which is not recommended. As evaluated by VAN DER GRAAF-VAN BLOOIS et al. (2013VAN DER GRAAF-VAN BLOOIS, L. et al. Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay. Journal of Microbiological Methods, v.95, n.1, p.93-97, 2013. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/23810970 >. Accessed: Feb. 10, 2015. doi: 10.1016/j.mimet.2013.06.005.
https://www.ncbi.nlm.nih.gov/pubmed/2381...
), the technique of HUM et al. (1997)HUM, S. et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. BMC microbiology, v.75, n.11, p.827-831, 1997. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-0813.1997.tb15665.x >. Accessed: Oct. 26, 2012. doi: 10.1111/j.1751-0813.1997.tb15665.x.
https://onlinelibrary.wiley.com/doi/full...
has 100% sensitivity and 100% specificity for the detection of C. fetus, and 58% sensitivity and 83% specificity for C. fetus subsp. venerealis, so it is reliable for species identification only.

Therefore, a critical evaluation of publications regarding available PCR techniques has been recommended for implementation of such techniques in a laboratory for the diagnosis of Campylobacter fetus.

Enzyme-linked immunosorbent assay (ELISA)

In the diagnosis of BGC, ELISA could be used for evaluating herd immunity, but is not acceptable for diagnosis of the infection in individual animals, and cannot differentiate between infections caused by the two subspecies of C. fetus that causes disease in ruminants (OIE, 2017OIE (WORLD ORGANISATION FOR ANIMAL HEALTH). Chapter 2.4.4 Bovine Genital Campylobacteriosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2017. Available from: <http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.04_BGC.pdf>. Accessed Jan. 20, 2018.
http://www.oie.int/fileadmin/Home/eng/He...
). In Brazil, currently, reports on the use of ELISA to diagnose BGC are unavailable.

Previous studies to improve BGC diagnosis have used ELISA to detect IgA in samples such as cervical mucus, preputial washings, and contents of aborted fetuses (DEVENISH et al., 2005DEVENISH, J. et al. Validation of a monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus. Clinical Diagnostics and Laboratory Immunology, v.12, n.11, p.1261-1268, 2005. Available from: <Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16275938 >. Accessed Oct. 15 2016. doi: 10.1128/CDLI.12.11.1261-1268.2005.
http://www.ncbi.nlm.nih.gov/entrez/query...
; HEWSON et al., 1985HEWSON P.I. et al. An enzyme-linked immunosorbent assay for the detection of antibodies to C. fetus in bovine vaginal mucus. Research in Veterinary Science, v.38, p.41-45, 1985. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/3975480 >. Accessed: Oct. 15 2014.
https://www.ncbi.nlm.nih.gov/pubmed/3975...
; MSHELIA et al., 2010MSHELIA, G. D. et al. Epidemiology of bovine venereal campylobacteriosis: Geographic distribution and recent advances in molecular diagnostic techniques. Reproduction in Domestic Animals, v.45, n.5, 2010. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/19929895 >. Accessed Sep. 28 2014. doi: 10.1111/j.1439-0531.2009.01546.x.
https://www.ncbi.nlm.nih.gov/pubmed/1992...
; PELLEGRIN et al., 2011PELLEGRIN, A. O. et al. The use of enzyme-linked immunosorbent assay and immunoblotting for the detection of Campylobacter fetus immunoglobulins in the cervico-vaginal mucus of female cattle. Pesquisa Veterinária Brasileira, v.31, n.3, p.247-254, 2011. Available from: <Available from: https://remvt.cirad.fr/revue/notice_fr.php?dk=516885 >. Accessed: Oct. 22, 2012. doi: 10.1590/S0100-736X2011000300011.
https://remvt.cirad.fr/revue/notice_fr.p...
; HUM & QUINN, 1994HUM, C.; QUINN, D. K. Diagnosis of bovine venereal campylobacteriosis by ELISA. Australian Veterinary Journal, v.71, n.5, p.140-143, 1994. Available from:<Available from:https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-0813.1994.tb03367.x >. Accessed: Nov. 20, 2016. doi: 10.1111/j.1751-0813.1994.tb03367.x.
https://onlinelibrary.wiley.com/doi/pdf/...
). These IgA antibodies were chosen because they persist for longer and their concentration remains constant in the genital tract for many months (HUM & QUINN, 1994HUM, C.; QUINN, D. K. Diagnosis of bovine venereal campylobacteriosis by ELISA. Australian Veterinary Journal, v.71, n.5, p.140-143, 1994. Available from:<Available from:https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-0813.1994.tb03367.x >. Accessed: Nov. 20, 2016. doi: 10.1111/j.1751-0813.1994.tb03367.x.
https://onlinelibrary.wiley.com/doi/pdf/...
). However, problems with sensitivity and specificity (false-positive/-negative) were reported by HUM & QUINN (1994)HUM, C.; QUINN, D. K. Diagnosis of bovine venereal campylobacteriosis by ELISA. Australian Veterinary Journal, v.71, n.5, p.140-143, 1994. Available from:<Available from:https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-0813.1994.tb03367.x >. Accessed: Nov. 20, 2016. doi: 10.1111/j.1751-0813.1994.tb03367.x.
https://onlinelibrary.wiley.com/doi/pdf/...
in an ELISA for IgA detection, in which the vaginal mucus of bovine females with suspicted C. fetus infection was used.

BROOKS et al. (2004BROOKS, B. W. et al. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. Veterinary Microbiology, v.103, n.1-2, p.77-84, 2004. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0378113504002640 >. Accessed. Dec. 10, 2014. doi: 10.1016/j.vetmic.2004.07.008.
http://www.sciencedirect.com/science/art...
) and DEVENISH et al. (2005DEVENISH, J. et al. Validation of a monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus. Clinical Diagnostics and Laboratory Immunology, v.12, n.11, p.1261-1268, 2005. Available from: <Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16275938 >. Accessed Oct. 15 2016. doi: 10.1128/CDLI.12.11.1261-1268.2005.
http://www.ncbi.nlm.nih.gov/entrez/query...
) used monoclonal antibodies against C. fetus LPS antigen in capture ELISA, but did not obtain a satisfactory detection limit (105 to 107 CFU/mL), making this technique using monoclonal antibodies viable only after a period of enrichment of the sample for 4 to 5 days. Antigens used to sensitize plaques were whole bacteria or obtained by acid extraction from C. fetus cultures using glycine buffer. However, these samples are not easy to obtain and there are false-positive results. The main problems with ELISA techniques developed are limitations in sensitivity and/or specificity.

ZHAO et al. (2010ZHAO, H. et al. Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle. Research in Veterinary Science, v.88, n.3, p.446-451, 2010. Available from: <Available from: http://linkinghub.elsevier.com/retrieve/pii/S0034528809002690 >. Accessed: Jan. 20, 2013. doi: 10.1016/j.rvsc.2009.11.013.
http://linkinghub.elsevier.com/retrieve/...
) developed and evaluated a highly specific (94.3%) and sensitive (88.6%) indirect ELISA for the detection of IgG antibodies against C. fetus in bovine sera by validating as antigen the recombinant proteins SapA-N and SapA-C. These researchers were successful in choosing the N-terminal region of SapA protein from a field strain, noting their immunodominance and the presence of multiple antigenic epitopes, as reported by WANG et al. (1993WANG, E. et al. Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus. Journal of Bacteriology, v.175, n.16, p.4979-4984, 1993. Available from: <Available from: https://jb.asm.org/content/175/16/4979.abstract >. Accessed: Sept. 20, 2012. doi: 10.1128/jb.175.16.4979-4984.1993.
https://jb.asm.org/content/175/16/4979.a...
).

Differential diagnosis

Other agents transmitted by sexual contact cause reproductive problems in cattle herds and should be considered for a correct diagnosis. The diagnosis of reproductive disease is mostly performed with serum samples, but when herd history reveals infertility, samples such as preputial smegma, semen, fetal fluid, placenta, and vaginal discharge can provide a definitive diagnosis (GIVENS, 2006GIVENS, M. D. A clinical, evidence-based approach to infectious causes of infertility in beef cattle. Theriogenology, v. 66, p. 648-654, 2006. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16725187 >. Accessed: Oct. 12, 2012. doi: 10.1016/j.theriogenology.2006.04.021.
https://www.ncbi.nlm.nih.gov/pubmed/1672...
).

The protozoan Tritrichomonas fetus, responsible for bovine genital trichomoniasis, causes disease with aspects similar to BGC and should be investigated as a differential diagnosis (BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
). Trichomoniasis is usually diagnosed using culture and/or PCR, and the sample types are the same for BGC diagnosis (MICHI et al., 2016MICHI, A. N. et al. A review of sexually transmitted bovine trichomoniasis and campylobacteriosis affecting cattle reproductive health. Theriogenology, v.85, n.5, p.781-791, 2016. Available from: <Available from: http://dx.doi.org/10.1016/j.theriogenology.2015.10.037 >. Accessed: Jan. 20 2017. doi: 10.1016/j.theriogenology.2015.10.037.
http://dx.doi.org/10.1016/j.theriogenolo...
). In addition, BONDURANT (2005)BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
and GIVENS (2018GIVENS, M. D. Review: Risks of disease transmission through semen in cattle. Animal, v.12, n.s1, p.s165-s171, 2018. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/29665869 >. Accessed: Jan. 10, 2019. doi: 10.1017/S1751731118000708.
https://www.ncbi.nlm.nih.gov/pubmed/2966...
) cite Haemophilus somnus, Ureaplasma, and other Mycoplasmas and in some special conditions, Leptospira spp., Brucella abortus, and viral diseases such as Bovine Viral Diarrhea (BVD) and Infectious Bovine Rhinotracheitis (IBR) causing infertility and/or abortions in cattle herds. In these cases, clinical signs and epidemiological characteristics should be taken into account, along with the results of laboratory tests (GIVENS, 2006GIVENS, M. D. Review: Risks of disease transmission through semen in cattle. Animal, v.12, n.s1, p.s165-s171, 2018. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/29665869 >. Accessed: Jan. 10, 2019. doi: 10.1017/S1751731118000708.
https://www.ncbi.nlm.nih.gov/pubmed/2966...
).

Another microorganism very similar to Campylobacter in microbiological cultures is Arcobacter sp. (ETONSI, 2013ETONSI, M. A. Studies on Arcobacter species, their isolation and pathogenicity. [S.l.]: Heriot-Watt University, United Kingdon, 2013. Available from: < Available from: https://www.ros.hw.ac.uk/bitstream/handle/.../EtonsiMA_%0A0813_sls.pdf%0A >. Accessed: Vov. 21, 2016.
https://www.ros.hw.ac.uk/bitstream/handl...
). This species growth in aerobiosis and it is enough to undo the mistake, as well as the use of selective broth to enrichment can eliminate this bacterium. The Arcobacter sp.; although, isolated from bovine abortion cases (FERNÁNDEZ et al., 1995FERNÁNDEZ, H. et al. Toxigenic and invasive capacities: possible pathogenic mechanisms in Arcobacter cryaerophilus. Memórias do Instituto Oswaldo Cruz, vol.90, n.5, 1995. Available from: <Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02761995000500018 >. Accessed: Jan. 20, 2015. doi: 10.1590/S0074-02761995000500018.
http://www.scielo.br/scielo.php?script=s...
; NEILL et al., 1985NEILL, S. D. et al. Taxonomic Position of Campylobacter cvyaevophila sp. nov. International Journal of Systematic Bacteriology, v.35, n.3, p.342-356, 1985. Available from: <Available from: https://ijs.microbiologyresearch.org/content/.../10.../00207713-35-3-342 >. Accessed Jan. 15, 2015.
https://ijs.microbiologyresearch.org/con...
), was also recovered from preputial bovine washes, and from vaginal swabs of cows without observable reproductive problems (KABEYA et al., 2004KABEYA, H. et al. Prevalence of Arcobacter species in retail meats and antimicrobial susceptibility of the isolates in Japan. International Journal of Food Microbiology, v.90, n.3, p.303-308, 2004. Available from: < Available from: https://www.sciencedirect.com/science/article/pii/S0168160503003222 >. Accessed: Jan. 12, 2015. doi: 10.1016/S0168-1605(03)00322-2.
https://www.sciencedirect.com/science/ar...
). Therefore, in situations of microbiological culture of samples from animals suspected of BGC infection, Arcobacter spp. should be included for differential diagnosis.

Prevention and control

The control of the disease in herds is carried out with the implementation of AI programs, avoiding the use of bulls (BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
), and mainly with the introduction of vaccination in bulls and cows. The practice of discarding bulls bearing C. fetus subsp. venerealis, according to PELLEGRIN et al. (2002PELLEGRIN, A.O. et al. Bovine Genital Campylobacteriosis in Pantanal, State of Mato Grosso do Sul, Brazil. Revue d’élevage et de médecine vétérinaire des pays tropicaux, v.55, n.3, p.169-173, 2002. Available from: <Available from: https://revues.cirad.fr/index.php/REMVT/article/view/9820 >. Accessed: Aug. 15, 2014.
https://revues.cirad.fr/index.php/REMVT/...
), as well as the implementation of a limited breding season (60 to 90 days), discarding of nonpregnant females at the end of the breeding season, and sexual rest for 3 to 4 cycles for recovery of females, are also measures recommended for BGC control.

Vaccination is quite effective in preventing the recurrence of estrus and abortion caused by C. fetus subsp. venerealis, according to BONDURANT (2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
). Commercial vaccines used for the control and prophylaxis of BGC are composed of bacterins with adjuvants, given as subcutaneous or intramuscular injections. There are reports of good efficacy when administered to females, inducing at least partial protection against experimental genital infection (COBO et al., 2004COBO, E. R. et al. Immunization in heifers with dual vaccines containing Tritrichomonas foetus and Campylobacter fetus antigens using systemic and mucosal routes. Theriogenology, v.62, n.8, p.1367-1382, 2004. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/ S0165037811000404 >. Accessed: Dec. 17, 2014. doi: 10.1016/j.theriogenology.2003.12.034.
http://www.sciencedirect.com/science/art...
). In herds infected with C. fetus subsp. venerealis, reproductive age females should be vaccinated annually, approximately 30 days before the start of breeding (BONDURANT, 2005BONDURANT, R. H. Venereal diseases of cattle: Natural history, diagnosis, and the role of vaccines in their control. Veterinary Clinics of North America - Food Animal Practice, v.21, n.2, p.383-408, 2005. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0749072005000320 >. Accessed Nov. 10, 2012. doi: 10.1016/j.cvfa.2005.03.002.
http://www.sciencedirect.com/science/art...
). In the case of first vaccination, heifers and cows should receive two doses, at approximately 60 and 30 days, respectively, before the beginning of the breeding season. Vaccination alone is often not considered sufficient to manage an outbreak of BGC, and in bulls it is necessary to test and cull positive animals (ERICKSON et al., 2017ERICKSON, N. E. N. et al. Evaluation of long-acting oxytetracycline and a commercial monovalent vaccine for the control of Campylobacter fetus subsp. venerealis infectionin beef bulls. Canadian Veterinary Journal, v.58, n.10, p.1051-1058, 2017. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5603938/ >. Accessed: Nov. 10, 2018.
https://www.ncbi.nlm.nih.gov/pmc/article...
).

When comparing the efficacy of 10 commercial vaccines for the prevention of abortion following exposure to C. fetus, BRYNER et al. (1988) reported 0 to 89% in efficiency. These authors reported deficiencies in immune responses to some commercial vaccines, suggesting that the percentage of protection that a vaccine offers is directly proportional to the bacterial mass used, and that immunogenicity of the samples used in the vaccine or type of adjuvant used may arise.

BGC positive cows and bulls can be treated. Cows are not usually treated, and after a few months, can eliminate the infection and return to estrus. Decisions regarding the treatment of bulls should be evaluated taking into consideration the age and value of the animal, since treatment incurs some costs and the animals will need to be managed for several days. There are reports of the use of topical and systemic antimicrobial treatments, with variable results. The tested antimicrobials included streptomycin, dimetridazole chlorhydrate, and oxytetracycline (CAMPERO et al., 1993CAMPERO, C. M. et al. Tratamientos sistemicos en toros con infeccion genital a Campylobacter fetus subsp. fetus. Veterinaria Argentina, v. 10, p. 303-309, 1993.; ERICKSON et al., 2017ERICKSON, N. E. N. et al. Evaluation of long-acting oxytetracycline and a commercial monovalent vaccine for the control of Campylobacter fetus subsp. venerealis infectionin beef bulls. Canadian Veterinary Journal, v.58, n.10, p.1051-1058, 2017. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5603938/ >. Accessed: Nov. 10, 2018.
https://www.ncbi.nlm.nih.gov/pmc/article...
). ERICKSON et al. (2017) ERICKSON, N. E. N. et al. Evaluation of long-acting oxytetracycline and a commercial monovalent vaccine for the control of Campylobacter fetus subsp. venerealis infectionin beef bulls. Canadian Veterinary Journal, v.58, n.10, p.1051-1058, 2017. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5603938/ >. Accessed: Nov. 10, 2018.
https://www.ncbi.nlm.nih.gov/pmc/article...
tested a combination of antimicrobial treatment with injectable oxytetracycline and vaccination with two doses of a monovalent oil-based C. fetus subsp. venerealis commercial vaccine in BGC-positive bulls. Two treatments with long-acting oxytetracycline at label doses and a commercial monovalent bacterin did not eliminate BGC in all study bulls. Therefore, this is not recommended as an effective management strategy. However, due to the antimicrobial resistance reported in the last decades and because of legislation in some countries, treatment must be carried out observing the legislation surrounding antimicrobial use in livestock production, and on the recommendation of a veterinarian.

CONCLUSIONS:

BGC is a relevant disease because of its clinical presentation, which causes large economic losses. Published prevalence studies and a survey carried out involving diagnosis of the disease in LABAC/UFSM affirmed that the disease affects cattle herds in several regions of Brazil, and that the losses are underestimated. BGC diagnosis is laborious due to the culture characteristics of Campylobacter and the limitations of other techniques. In addition, there are no projections for disease control and eradication programs at either regional or national levels. Further research aimed to optimize the diagnosis of BGC using molecular techniques could facilitate the establishment of results regarding occurrence and; consequently, taking preventive measures at herd level. Finally, the use of combined diagnostic methods is essential to ensure reliable results.

ACKNOWLEDGEMENTS

This study was finnanced in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (finance code 001 and scholarship 88881.135832/2016-01/PDSE) and by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).

REFERENCES

  • CR-2019-0272.R2

Publication Dates

  • Publication in this collection
    3 Apr 2020
  • Date of issue
    2020

History

  • Received
    05 Apr 2019
  • Accepted
    24 Nov 2019
  • Reviewed
    10 Feb 2020
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