Characterization of the Campylobacter fetus subsp. venerealis Adhesion to Bovine Sperm Cells/ Caracterizacion de la Adhesion de Campylobacter fetus subsp. venerealis a Espermatozoides Bovinos.
Successful mammalian reproduction requires that sperm migrate through a long and convoluted female reproductive tract before reaching oocytes. For many years, fertility studies have focused on biochemical and physiological requirements of sperm (Barth, 1995). There are a large number of microorganisms that propagate for the seminal route. The pathogen can adhere to the sperm in the tail and/ or the head, or can infect the various components of the seminal fluid as sperms, seminal plasma and leukocytes, among others) (Bielanski & Dubuc, 1993; Bielanski et al, 1994; Bielanski & Dubuc, 1994; Bielanski & Loewen, 1994; Bielanski et al, 2004). Many of the existing studies on the association pathogen-sperm are not conclusive because, the detection of this union is complex and in some in some cases uncertain (Casadevall & Pirofski, 2000) as in the case of Campylobacter fetus (Bielanski et al, 1994).
In the project of ex vivo model for the study of the pathogenesis of the bovine genital campylobacteriosis (Chiapparrone et al., 2008; Chiapparrone et al., 2009; Chiapparrone et al., 2011; Chiapparrone et al., 2014) intends to describe the behavior of a strain of Campylobacter fetus subsp. venerealis (C. fetus subsp. venerealis) bacteria of preputial smegma of bull in its interaction with sperm cattle by light microscopy and scanning electron microscopy (Fletcher & Floodgate, 1973).
MATERIAL AND METHOD
Bacterial strain. The C. fetus subsp. venerealis strain was isolated in beef bovine of Tandil - Argentina, from preputial tract of Aberdeen Angus bull and identified using phenotypic test and PCR. The strain was grown for 48 h at 37[degrees]C incubated under microaerophilic conditions which correspond to the logarithmic growth phase. All experiments were performed with C. fetus subsp. venerealis-1 strain at a concentration of 1x[10.sup.9] cells/mL.
Sperm cells. We used frozen bovine semen pooled from fertile bulls for artificial insemination. For interaction purposes, straws of frozen semen were thawed by immersion in a water bath at 37[degrees]C for 30 s, centrifuged and the entire medium removed. The cells were washed once in PBS and transferred to Minimum Essential Medium Eagle (MEM) culture medium (M0643 Sigma-Aldrich) supplemented with 10 % fetal bovine serum.
Viability tests were based on visual morphology and motility under dark field microscopy, each sperm cell was categorized as belonging to one of four motility categories (rapid progressive, slow progressive, non-progressive and immotile) and viability staining with Nigrosin-Eosin stain (Rodriguez-Martinez, 2013).
Adherence assay. For interaction analysis, suspensions of sperm cells were counted in a Neubauer chamber and exposed to C. fetus subsp. venerealis-1 in a cell ratio of 1:1 bacteria-sperm cells, for 3 h at 37[degrees]C incubated under 5 % C[O.sub.2]. For control experiments, the same bacteria and sperm cells free were used. The bacterial motility can be determined under dark field microscopy.
Giemsa stain. Sperm cells were fixed with acetone at 4[degrees]C and stained with Giemsa 10 %. Bacterial adhesion was observed and quantified by optical microscopy.
Scanning electron microscopy. After checking the adhesion by Giemsa staining, innoculated cell cultures were processed according to Electron Microscopy Laboratory protocol of the College of Sciences, Universidad Nacional de Mar del Plata (UNMdP). Briefly, the sperm cells were fixed with 3 % glutaraldehyde in sodium cacodylate buffer and washed with 0.1 M sodium cacodylate buffer. After dehydration with ethanol they were exposed to hexamethyldisilazane over- night, and then gold and palladium metallizing (Denton Vacuum Desk II). The samples were observed with scanning electron microscope (Jeol JSM-6460 LV). The system used was an EDAX Genesis XM4--Sys 60, equipped with Multichannel Analyzer EDAX mod EDAM IV, Sapphire Si (Li) detector and super ultra-thin Window of Be, and EDAX Genesis version 5.11 software.
Of the sperm cells 78 % were alive, before bacteria - sperm interaction. This number reduced to 56 % in the third hour, when bacteria and sperm cells were under interaction. Sperm that were white (unstained) were classified as alive, and those that showed any pink or red coloration were classified as dead, with the sole exception for sperm with a slight pink or red appearance restricted to the neck region ('leaky necks'), which were assessed as live. Motile bacteria were observed during the next 3 h.
By scanning electron microscopy sperm cell treated with C. fetus subsp. venerealis showed adhesion and alterations in the bovine spermatozoa. All the superficial structures of spermatozoa such as head (66 %), mid piece, neck, and tail (34 %) were involved in bacterial adhesion.
The capacity of adherence to different parts of the sperm, the number of adherent C. fetus subsp. venerealis and the characteristics of interaction were analyzed. In the images, the presence of one or more C. fetus subsp. venerealis adhered by the apical body, mainly to the sperm head (Figs 1, 2 and 3), was observed and confirmed by routine colorations (Fig. 4). In others cases, it was possible to determine the presence of bacterial shapes in an extracellular matrix, adhered to the external membrane of the head and tail of sperm, grouping with biofilm characteristics (Figs. 5 and 6).
C. fetus subsp. venerealis is one bacteria that cause infertility in cows (Campero, 2002; Catena, 2002; Catena, 2003). However, there is no documented information about Campylobacter effect on sperm cells viability (Bhavsar & Kapadnis, 2006; Brenner et al., 2005; de Melo & Pechere, 1990; Graham & MacDonald, 1988; Joens et al., 2010) and there is no complete agreement on the detrimental role of bacteria presence in the semen.
The ability of bacteria to bind to host cells is important as it represents an early event in the creation establishment in vivo microorganism - cell relationship (Casadevall & Pirofski, 2000; Benchimol et al., 2008; Chiapparrone et al., 2014). Sometimes, such binding process is also a requirement for pathogenicity. Understanding the capability of adherence of C. fetus subsp. venerealis to the host cell (Chiapparrone, 2011) is significant for a thorough knowledge of the initial steps involved in the pathogenesis of bovine genital campylobacteriosis.
The light microscopy and scanning electron microscopy allowed us to characterize some aspects of the interaction of C. fetus subsp. venerealis and bovine sperm while preserving the cellular and bacterial structure.
Cagnoli, Claudia. She holds a fellowship from Agencia Nacional de Promocion Cientifica y Tecnologica. Ministerio de Ciencia y Tecnologia. Republica Argentina.
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Maria L. Chiapparrone
Area de Enfermedades Infecciosas.
Departamento de Sanidad Animal y Medicina Preventiva
Facultad de Ciencias Veterinarias
Paraje Arroyo Seco S/N
Maria L. Chiapparrone *; Pedro Soto * & Maria Catena *
Laboratorio de Microbiologia Clinica y Experimental, NACT. SAMP, Unidad Ejecutora de CONICET (CIVETAN). Facultad de Ciencias Veterinarias. Universidad Nacional del Centro de la Provincia de Buenos Aires, Argentina.
Caption: Fig. 1. Campylobacter fetus subsp. venerealis adhered to head of sperm cells for the apical end. Scanning electron microscopy. 5500X.
Caption: Fig. 2. Campylobacter fetus subsp. venerealis adhered to head of sperm cells for the apical end. Scanning electron microscopy. 6000X.
Caption: Fig. 3. Campylobacter fetus subsp. venerealis adhered to head and tail of sperm cells for the apical end. Scanning electron microscopy. 4000X.
Caption: Fig. 4. Campylobacter fetus subsp. venerealis adhered to head of sperm cells. Giemsa stain. 100X.
Caption: Fig. 5. Observation of more than one Campylobacter fetus subsp. venerealis adhered to sperm cells. Scanning electron microscopy. 8500X.
Caption: Fig. 6. Observation of grouping of Campylobacter fetus subsp. venerealis adhered to head of sperm cells. Scanning electron microscopy. 7000X.