Effect of Campylobacter fetus on in vitro fertilization and embryonic development of preimplantation bovine embryos.

Campylobacter fetus is an important veterinary pathogen that causes campylobacteriosis. This disease causes decreased productivity of cattle by inducing reproductive losses. Although several virulence factors have been recognized in C. fetus, including a cytolethal distending toxin (CDT), the exact mechanism responsible for embryonic death remains unknown. The aim of this work was to evaluate the effect of C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv), and their toxin activity on the in vitro fertilization of bovine ova and early embryonic development. Two different experiments were performed. In Experiment 1, a total of 1524 cumulus-oocyte complexes (COCs) were inseminated, distributed into three groups: two of them infected with the microorganism (Cff, Cfv) and a control group. Percentages COCs cleaved were similar among groups (p = 0.1243); however, the embryonic development rate (blastocyst at day 7) in the control group was greater (p < 0.001) than those obtained in Cff and Cfv groups. In experiment 2, a total of 746 COCs were inseminated, divided into three groups: two of them treated with the bacterial-free culture filtrates to test toxin activity (Cff-CDT, Cfv-CDT) and a control group. Both cleavage and embryonic development rates were greater (p < 0.001) in the control group than those obtained in Cff-CDT and Cfv-CDT groups. This study provides evidence that both subspecies of C. fetus do not interfere with fertilization but do affect in vitro embryonic development. It is the first report on the biological effect of the CDT on bovine embryonic development.

Multidrug Resistance and Molecular Characterization of Streptococcus agalactiae Isolates From Dairy Cattle With Mastitis.

Streptococcus agalactiae is a pathogen-associated to bovine mastitis, a health disorder responsible for significant economic losses in the dairy industry. Antimicrobial therapy remains the main strategy for the control of this bacterium in dairy herds and human In order to get insight on molecular characteristics of S. agalactiae strains circulating among Argentinean cattle with mastitis, we received 1500 samples from 56 dairy farms between 2016 and 2019. We recovered 56 S. agalactiae isolates and characterized them in relation to serotypes, virulence genes, and antimicrobial susceptibility. Serotypes III and II were the most prevalent ones (46% and 41%, respectively), followed by Ia (7%). In relation to the 13 virulence genes screened in this study, the genes spb1, hylB, cylE, and PI-2b were present in all the isolates, meanwhile, bca, cpsA, and rib were detected in different frequencies, 36%, 96%, and 59%, respectively. On the other hand, bac, hvgA, lmb, PI-1, PI-2a, and scpB genes could not be detected in any of the isolates. Disk diffusion method against a panel of eight antimicrobial agents showed an important number of strains resistant simultaneously to five antibiotics. We also detected several resistance-encoding genes, tet(M), tet(O), ermB, aphA3, and lnu(B) (9%, 50%, 32%, 32%, and 5%, respectively). The results here presented are the first molecular data on S. agalactiae isolates causing bovine mastitis in Argentina and provide a foundation for the development of diagnostic, prophylactic, and therapeutic methods, including the perspective of a vaccine.

Effects of Campylobacter fetus on bull sperm quality.

Campylobacter fetus is a gram-negative, motile, spiral or S-shaped bacterium, which induces campylobacteriosis. This disease causes decrease productivity of cattle. Although considerable research has been done on the role of C. fetus on female fertility, little is known about the effect on bulls. The aim of this work was to evaluate the effect of C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv) on bull sperm quality. Samples of frozen semen (n = 29 straws) were each distributed into three groups: two of them incubated with the microorganism (Cff, Cfv) and a control group. The proportions of live spermatozoa, with functional membrane and true acrosomal reaction in control group were significantly (P < 0.01) greater than those observed in Cff and Cfv groups. However, no significant differences (P > 0.05) were found in sperm chromatin structure among treatments. In adhesion assay, proportions of spermatozoa with adhered Campylobacter were similar for both subspecies. Results confirm that Cff and Cfv have the same ability to bind in an irreversible way to bull spermatozoa and to affect sperm quality. It is proposed that adherence could be considered as the main cause of sperm alterations, and also an important step of pathogenesis and venereal transmission.

Tritrichomonas foetus: experimental infection in pregnant BALB/c mice.

Bovine genital tritrichomonosis is a venereal disease produced by the flagellate Tritrichomonas foetus. The disease is characterized by the repetition of oestrus and infertility due to embryonic or foetal death. Numerous experimental rodent models have been developed, but none of them has been applied in pregnant females. In this work, we reproduced genital tritrichomonosis in pregnant BALB/c mice. The results were analysed considering the following pregnancy phases: early, middle and final. In the infected group, embryonic loss was significantly higher and occurred in the early and middle phases, in accordance with the time of embryo death in infected bovines. In infected animals at the early phase of pregnancy there was evidence of embryonic death without inflammatory changes in the uterus, suggesting a pathogenic mechanism that does not involve direct tissue damage. In the later days, conceptus loss was associated with endometritis and changes in the decidua.

Partial purification and characterization of a trypsin-like serine protease from bovine sperm.

Sperm proteolytic activities are relevant in the enzymatic mechanism of fertilization. Several authors have suggested the presence of serine proteases other than acrosin in mice and human spermatozoa. In this work we describe the characterization of a partially purified bovine sperm serine protease BSp66 and its dimmer, BSp120. Partial purification of the monomer was performed from fresh spermatozoa, while the dimer form of the protease was obtained from cryopreserved spermatozoa. The Mr of BSp120 and BSp66 estimated by zymography and gel filtration chromatography were 120 and 66 kDa, respectively. They were positively stained by Schiff-PAS reagent for glycoproteins and they both digested synthetic peptides with basic amino acids in the P1 site. Polyclonal antibodies against acrosin or proacrosin did not cross-react neither with BSp120, nor BSp66. In addition, antibodies raised in our laboratory against BSp120 and BSp66 did not recognize acrosin or proacrosin suggesting that they are not antigenically related proteins. Also, no cross- reactivity was detected with proteins in the range of 120-66 kDa when antibodies against the proteasome were used. The cellular localization of this protease by optical immunocytochemistry using specific antibodies revealed a positive signal in the apical portion of the sperm head suggesting acrosomal or membrane localization. The evidences presented here characterize BSp66 as a trypsin-like serine protease, a putative new member of this highly redundant proteolytic system of the sperm acrosome.

Low temperature-induced dimerization of the bovine sperm serine protease, BSp66.

BSp120 and BSp66 are trypsin-like serine proteases from bovine spermatozoa. The former is active in cryopreserved sperm samples while the latter shows proteolytic activity in recently obtained fresh sperm. Both proteases are immunologically related and co-localize in the apical portion of the sperm head. In Western blots with specific antibodies, sperm samples incubated with reducing agents showed a decrease in the amount of BSp120, while BSp66 was detected with both anti-BSp120 and anti-BSp66 antibodies. BSp120 was evident in frozen intact spermatozoa after 60 days of semen cryopreservation and the kinetic of appearance of this protein was coincident with the decrease in the amount of BSp66. Identical results were obtained by freezing sperm extracts from fresh semen at -20 degrees C. Our results suggest that BSp120 results from disulfide bond-dimerization of BSp66 and that this process may be induced by temperatures below zero in both intact spermatozoa and in sperm extracts.