CURRENT MICROBIOLOGY Vol. 45 (2002), pp. 111–114 DOI: 10.1007/s00284-001-0090-9
Current Microbiology An International Journal © Springer-Verlag New York Inc. 2002
Campylobacter fetus subspecies venerealis Surface Array Protein from Bovine Isolates in Brazil Agueda Castagna de Vargas,1 Mateus Matiuzzi Costa,1 Marilene Henning Vainstein,2 Luiz Carlos Kreutz,3 Jairo Pereira Neves4 1
Departamento de Medicina Veterina´ria Preventiva (DMVP), Centro de Cieˆncias Rurais, Universidade Federal de Santa Maria (UFSM), Santa Maria, 97105-900, RS, Brazil 2 Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil 3 Faculdade de Agronomia e Medicina Veterina´ria, Universidade de Passo Fundo (UPF), Passo Fundo, RS, Brazil 4 Departamento de Clı´nica de Grandes Animais, UFSM, Santa Maria, RS, Brazil Received: 27 August 2001 / Accepted: 4 December 2001
Abstract. The electrophoretic patterns of 31 Campylobacter fetus subspecies venerealis capsular Surface Array Protein (SAP) isolated from bovines in reproduction from different regions of Brazil were analyzed. The persistence of the bacteria in the reproductive tract of naturally infected bovines and the dynamic of SAP expression were also evaluated. Cervical mucous and prepucial aspirates from five animals naturally infected were cultured for isolation of Campylobacter fetus and the SAPs extracted from the bacteria isolated were analyzed by SDS-PAGE. Ten different patterns of SAP expression were demonstrated by the identification of proteins with molecular mass of 97, 100, 127, and 149 kDa, respectively. The most prevalent identified protein had a molecular mass of 100 kDa (41.9%). Taking into consideration the time during which the five animals were evaluated, it was possible to conclude that one of these animals persisted with the etiological agent up to 171 days. The five naturally infected bovines analyzed presented variation on their surface protein pattern during the period of this study. C. fetus subspecies venerealis persisted in the reproductive tract of naturally infected animals. In natural condition of infection C. fetus subspecies venerealis persisted in an intermittent condition and an alteration of the protein surface was shown.
Campylobacter fetus is a gram-negative, curved, spiralshaped, and microaerophilic bacteria. This bacterium has been reported causing infertility and sporadic abortion in sheep and cattle, as well as opportunistic infections in humans [2, 9, 10]. The bovine campylobacteriosis causes considerable economic losses, commonly in countries where modern reproductive practices, such as artificial insemination, are not used. The ingestion of the C. fetus subspecies fetus induces sepsis and abortion in pregnant cows due to the bacterial natural tropism for the placenta. After several months, the cow’s immune system overcomes the infection, but chronic vaginal colonization may persist for months or even years [8, 28]. The natural habitat of C. fetus subsp. venerealis is the prepuce of the male and is transmitted venereally to susceptible females where it can cause cervicitis, infertility, and infectious abortion. Correspondence to: A. Castagna de Vargas; email:
[email protected]
The ability of C. fetus to develop wide infections in animals and humans has drawn the attention to elucidate the possible role of bacterial surface molecules in disease development [11, 15, 16, 26]. McCoy et al. [19] were the first to describe C. fetus microcapsule. Moreover, in vitro and in vivo experiments established that bacterial resistance to serum activity and to phagocytosis occurs due to the presence of Surface Array Proteins (SAP) or S-Layer proteins with high molecular mass [1, 26]. Eaglesome and Garcia [12] and Blaser et al. [2] have proved that bacterial ability to express these proteins are changed after successive passages in artificial media, producing spontaneous mutants less virulent to inoculated mice. Several SAP have been purified and biochemically characterized [7, 8, 22]. A peculiar aspect of the C. fetus superficial proteins is the fact that each bacterium may produce up to three different proteins with molecular
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CURRENT MICROBIOLOGY Vol. 45 (2002)
Table 1. Electrophoretic profiles of surface array protein (SAP) from 31 C. fetus subspecies venerealis isolates from naturally infected bovines Electrophoretic Profilea
Proteins (kDa)
Number of isolates
Percentage (%)
A B C D E F G H I J
97b (100, 127)c 97 (127) 100 100 (97) 100 (127) 100, 127 127 127 (100) 149 (100, 127) 149, 127 (100)
3 2 9 1 3 1 5 3 3 1
9.7 6.5 29.0 3.2 9.7 3.2 16.1 9.7 9.7 3.2
Total
31
100
Fig. 1. Electrophoretic profile assigned for surface proteins (SAP) from C. fetus subsp. venerealis isolated from bovines. Lanes 1 to 10 shows patterns A to J, respectively. The positions of molecular weight markers (in kDa) are indicated on the left.
a
Classes assigned with the same electrophoretic profile. Most prevalent proteins are shown in bold. c Less prevalent proteins are shown in parenthesis. b
mass varying from 97 to 149 kDa, but usually, one of such proteins is prevalent [8, 9, 10]. The variation in SAP expression causes antigenic changes due to modifications in dominant epitopes during the bacterial persistence in the genital tract [15]. The two Campylobacter fetus subspecies are able to colonize the host epithelium chronically and to resist to the natural defense systems [28]. Fujimoto et al. [13] analyzed the relationship among SAPs of different molecular masses (98, 127, and 149 kDa), their structure and antigenicity in several clinical samples and spontaneous mutants suggesting that only the 98 and 149 kDa proteins cross react. Due to their localization on the bacterial surface and their relation with pathogenesis, Pei et al. [22] suggested these proteins as potential candidates to vaccine development. The bacterial persistence in the infected bovine genital tract may be associated with failures in the defense mechanisms against C. fetus infection. Corbeil [6] showed that during natural infections, the local immunity kills the microorganisms slowly, rendering the uterus clear of infection earlier than the vagina. This occurs since the humoral response against C. fetus is predominantly IgA in the vagina and IgG in the uterus; IgA can neutralize, but not opsonize C. fetus, however, IgG has both functions [5]. Schurig et al. [24, 25] reported that the systemic cattle immunization with dead cells results in high levels of IgG both in serum, uterine, and vaginal fluids. This induces protection against subsequent infections and eliminates chronic infections in the female reproductive tract. Therefore, the cattle vaccination has proved to be an efficient alternative to control campylobacteriosis. The present work has focused the determination of
dominant surface proteins in C. fetus subsp. venerealis isolated from bulls in Brazil, as well as to evaluate the bacterial persistence and the dynamic of the SAP patterns in C. fetus strains from naturally-infected cattle. Materials and Methods Samples. The electrophoretic patterns of surface proteins from 31 Campylobacter fetus subsp. venerealis isolates were determined. The samples, from different regions in Brazil, were prepucial aspirates from bulls, vaginal secretions, and abomasal contents from aborted fetus. The proteins were extracted soon after bacterial isolation and identification. The persistence of the bacteria in the genital tract of naturally infected cattle was analyzed and the dynamic in the patterns of SAP expression was studied from several samples of cervical secretions and prepucial aspirates periodically collected from five naturally infected bovines. The cultivation of Campylobacter fetus was in conformity to Office International des Epizoties (OIE) standards [21]. Protein extraction. The extraction was as previously described [20, 13]. In short, the isolates were grown 48 h in Mueller Hinton Agar media and suspended in 10 ml of 0.2 M glicine (pH 2.2) to a final concentration of 5 ⫻ 109 CFU/ml. After gentle vortexing (20 min) at room temperature the cells were collected by centrifugation (5.000 g, 30 min). The supernatants pH was adjusted to 7.5 with NaOH (4N) and the proteins were precipitated (overnight at 4°C) by addition of (NH4)2SO4 (5 g/10 ml). After centrifugation (5.000g for 1 h), the precipitated proteins were resuspended into 500 l 50 mM Tris-HCl pH 7.5 and stored at ⫺20°C. The protein concentration was determined by the Lowry method [18] using bovine albumin (1 mg/ml) as a standard. Electrophoresis. SDS-PAGE (7.5%) was as previously described [13]. The protein extracts (1.5–3.0 mg/ml) were mixed with an equal volume of the sample buffer and boiled for 1 minute before being loaded in the gels. After staining the gels with Coomassie blue the protein patterns were recorded in an image capture system (Kodak Digital Science 1D).
Results and Discussion The patterns of the surface proteins (SAP) expression from 31 Campylobacter fetus subsp. venerealis isolates
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A. Castagna de Vargas et al.: C. fetus Surface Array Protein Table 2. Persistence and electrophoretic profile of surface proteins (SAP) from C. fetus subsp. venerealis isolated from naturally infected bovines Animal/Sex #338/female #313/female #503/female #144/female #441/male
Electrophoretic profile / Sampling time Ha (day 1)b I (day 1) C (day 1) E (day 1) F (day 1)
(⫺)c (day 16) E (day 75) (⫺) (day 136) B (day 40) G (day 49)
(⫺) (day 99) G (day 111) G (day 171) (⫺) (day 61) Nc
C (day 122) (⫺) (day 151) NCd NC NC
a
Capital letters represent the electrophoretic profile assigned for SAP in Table 1 and Fig. 1. First sample collected for culture. c (⫺): Negative Campylobacter culture. d NC: Sample not collected. b
obtained by SDS-PAGE are shown in Table 1 and Fig. 1. The analysis allowed the identification of ten different patterns of SAP expression according to the presence of the 97, 100, 127, and 149 kDa proteins. The most prevalent profile was assigned as pattern C (29.0%). Thirteen samples (41.9%) presented a 100 kDa dominant protein in their profiles being these samples assigned as those with pattern C (9), D (1), E (3), or F (1) (Table 1). The proteins with 127, 97, and 149 kDa were less prevalent corresponding to 25.8, 16.2, and 9.7%, respectively. The results obtained are in agreement with other groups that reported the presence of surface proteins around 97, 127, and 149 kDa in C. fetus [8, 22]. In the majority of the protein patterns obtained (A, B, D, E, H, I, J) other SAPs were expressed with less evidence associated with the dominant protein (Table 1 and Fig. 1). These surface proteins may be synthesized in association with the dominant proteins, reflecting a high frequency of DNA rearrangement producing a mixed population or an alternative locus with low level of protein expression [2, 27]. Garcia et al. [15] showed the variation in SAP expression from inoculated cattle and they verified that some of these proteins became less evident after the second experimental week. The results of the isolation and the dynamic in the electrophoretic patterns of the surface proteins in C. fetus subsp. venerealis samples from naturally infected bovine are presented in Table 2. The analysis of the results suggests the persistence of the bacteria in the infected animals reproductive tract up to 171 days. Epidemiological studies and management practices (removal of bulls from herd) may allow us to suggest that the infection could have occurred four months before the first sample was collected. Cipolla et al. [3] showed that C. fetus subsp. venerealis colonizes and persists in experimentally infected females, being recovered up to 90 days, suggesting that it can also occur in natural conditions. The frequency of the spontaneous elimination or re-
isolation of C. fetus from bovine reproductive tract is not completely understood, although the immune response may play an important role [15]. Garcia et al. [14] reported that although IgA is not a good opsonizing agent it neutralizes the microorganism reducing its access to the uterus, facilitating the carrier status development. Two animals with positive cultures turned negative and positive again in later cultures. This may be explained by intermittence in bacterial elimination, also demonstrated by Wesley and Bryner [29] or by the low sensitivity of the diagnostic methods used, due to the fastidious growth characteristics of C. fetus. The intermittence in C. fetus elimination observed in naturallyinfected females can be supported by the estral cycle phase when the samples were collected. According to Garcia et al. [14], successful bacterial isolation occurs in periods close to the estrus phase. This fact shows the importance of several samples isolation from clinical materials to obtain a real negative diagnostic of C. fetus infection. All C. fetus subsp. venerealis isolates, from bovines with persistent infections, display variations in the electrophoretic patterns of SAP after different samplings. The variation in SAP expression to protect C. fetus against complement mediated killing and antibody mediated clearance, through changes in antigenic determinants has been reported [3, 5, 15, 26]. Wang et al. [28] and Garcia et al. [15], using monoclonal antibodies, demonstrated that the persistent colonization of the bovine genital tract is associated with spontaneous variation in the SAP production, antigenic changes, and evasion of the host immune system. Corbeil et al. [5] reported that C. fetus subsp. venerealis recovered from experimentally infected cattle, presented surface proteins serologically different from those found in the original bacteria kept in artificial media. The authors suggest that in persistent infections the bacteria change superficial epitopes to evade the host defenses.
114 The mechanism to explain the variation on SAP expression in C. fetus has been addressed in several studies. The genetic analysis of the SAP codifying genes has shown that variation in protein expression can be induced by both spontaneous deletions or DNA rearrangement of the promoter region [26]. Garcia et al. [15] proved that the antigenic variation of C. fetus occurs with high recombination frequency among the homologue copies of the SAP gene in the bacterial chromosome. However, Ray et al. [23] reported that although the main DNA rearrangement mechanism is dependent of recombinases, alternative processes can occur. The C. fetus subsp. venerealis persistence in bulls, originated mainly by chronically infected cows, hampers the disease control. Our results show substantial alteration on SAP patterns during the course of infection; this is critical if effective vaccination programs are to be used as an alternative to reduce bovine campylobacteriosis in infected herds. ACKNOWLEDGMENTS We are thankful to Dra. Eliana Scarcelli Pinheiro from the Instituto Biolo´ gico de Sa˜ o Paulo and Silvia Ladeira from the Universidade Federal de Pelotas for providing some C. fetus isolates for analysis. Part of this research has been financed by the Conselho Nacional de Pesquisa e Desenvolvimento Tecnolo´ gico—CNPq grant 521049/01-6 and Fundac¸ a˜ o de Apoio a Pesquisa do Estado do Rio Grande do Sul— Fapergs grant 99/60070-5 and 99/60071-1.
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