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.

[Detection of serovars of Salmonella in artificially reared calves in Mar y Sierras Dairy Basin, Argentina]. / Detección de serovares de Salmonella en terneros de crianza artificial de la región lechera Mar y Sierras, Argentina.

Salmonellosis in calves is a bacterial disease that affects their digestive tract causing diarrhea. A cross-sectional epidemiological study was carried out with the aim of studying the prevalence of various serovars of Salmonella in calves and their relationship with diarrhea signs. The study was conducted in Mar and Sierras Dairy Basin located in Buenos Aires province, Argentina. Seven hundred and twenty six calves both with diarrhea signs or not were sampled by rectal mucosa swab in 50 dairy farms during the rearing period. Isolates identified as Salmonella spp. were classified using polyvalent and monovalent antisera against somatic, flagellar and capsule antigens (Vi). Salmonella spp. was found in 36% of the farms and serotypes were: S. Mbandaka, S. Anatum, S. Typhimurium, S. Dublin, S. Montevideo, S. Meleagridis, S. Newport, S. Seftemberg, S. subesp. 16,7:z1, S. Infantis, S. Give. A percentage of 5.5% calves was positive and calves showing diarrheal signs were 5.9 times more likely to be infected with Salmonella spp. than those having no signs. The age of positive calves ranged from the first day of life to 53; the second day being the most frequent time. In conclusion, 11 Salmonella serovars were detected in one out of 3dairy farms in Mar and Sierras Dairy Basin, and not only were these serovars associated with diarrhea signs including the presence of mucus in feces, but they were also more prevalent among calves aged up to 21 days.

Apoptosis and cell proliferation in the mouse model of embryonic death induced by Tritrichomonas foetus infection.

Bovine tritrichomonosis is a sexually transmitted disease caused by the protozoon Tritrichomonas foetus and characterised by embryonic-death and abortion. During pregnancy, the processes of cell proliferation and death play a crucial role for blastocyst implantation and the subsequent maintenance of early pregnancy, and their misbalance may lead to the abortion. In this study, we aimed to investigate whether cell proliferation and death may be altered during tritrichomonosis. For this purpose, we used pregnant BALB/c mice as an alternative experimental animal model that has successfully reproduced the infection. We analysed the immunohistochemical expression of active caspase-3 and proliferating cell nuclear (PCNA) antigens in the endometrium of infected mice. We found an increase in the number of caspase-3 positive cells in infected mice that were not pregnant at the necropsy. Besides, the number of positive proliferating cells increased in the uterine luminal epithelium of infected animals killed at 5-7 days post coitum (dpc). Pregnant infected mice killed at 8-11 dpc showed higher proliferation than control animals. We suggest that the cytopathic effect induced by T. foetus in the uteri of infected mice may induce the apoptosis of the epithelial cells and, as a result, promote a compensatory proliferative response. The information described here will be helpful to further study the pathogenesis of the bovine tritrichomonosis.

Variation in the saccharide lectin binding pattern from different isolates of Tritrichomonas foetus.

Tritrichomonas foetus (T. foetus) is the causal agent of bovine tritrichomonosis (BT), a venereal disease that causes significant economic losses in the bovine livestock industry. The structural organization of T. foetus presents a cell membrane, an undulating membrane which extends along the parasite, three anterior flagella and a recurrent posterior flagellum. The interaction between the superficial glycoconjugates of the parasite and the host cell is one of the most relevant pathogenic mechanisms. In the present study, we analyzed the saccharide pattern through lectincytochemistry of the cell membrane, undulating membrane, cytoplasm and flagella of 28 isolates of T. foetus. Lectins that labeled most of the isolates were WGA, Con-A, RCA-I, LCA, GS-II and PHA-E showing the presence of D-mannose, D-glucose, N-acetylglucosamine and sialic acid. On the other hand, no labeling was observed in any of the structures with VVA, STA, LEA, Jacalin, GS-I, SJA, PHA-L, DSA, and weak labeling was observed with DBA, PNA, SBA and UEA I, showing therefore a low expression of N-acetylgalactosamine, L-fucose and galactose. In addition, GS II labeled in a granular pattern when lectincytochemistry was positive, whereas LCA strongly labeled the membranes and weakly the cytoplasms. The labeling variations observed among the isolates analyzed in the present work, could be related to differences in the pathogenic behavior of the isolates.

Study of the uterine local immune response in a murine model of embryonic death due to Tritrichomonas foetus.

PROBLEM: Bovine tritrichomonosis is a sexually transmitted disease caused by Tritrichomonas foetus, characterized by conceptus loss. We developed a mouse model of tritrichomonosis to study the mechanisms involved in the embryonic death. We hypothesized that embryonic death may be due to an exacerbated maternal response to the pathogen that then affects embryo development. METHOD OF STUDY: We infected BALB/c mice with Tritrichomonas foetus and paired them after confirming active infection. We studied the expression of pro- and anti-inflammatory cytokines, markers for T regulatory and T helper 17 cells as well as haem-oxygenase-1 expression in uterine tissue by real-time RT-PCR. RESULTS: As expected, TNF-α was augmented in infected animals. IL-10 and IL-4 were also up-regulated. Treg-associated genes were higher expressed in uteri of infected group. In mice that have lost their conceptus after the infection, haem-oxygenase-1 (HO-1) mRNA levels were strongly decreased, while RORγt mRNA, a reliable marker for Th17, was augmented in uterus. CONCLUSION: A T effector response of type 1 and 17 may be involved in tritrichomonosis-related embryonic death. This alters protective mechanisms as HO-1. Increased regulatory T cells may facilitate embryonic death by promoting the persistence of infection.

Quantitative analysis of campylobacter fetus venerealis adhesion to bovine reproductive tract cell cultures / Análise quantitativa da adesão de campylobacter fetus venerealis em culturas de células do aparelho reprodutor bovino

Campylobacter fetus is the etiological agent of bovine genital campylobacteriosis, a sexually transmitted disease which is associated with reproductive losses in bovines. Campylobacter colonizes the vagina and the uterus and then infects the epithelial cells of the endometrium. The objective of this work was to develop an ex vivo model to quantify the adhesion of Campylobacter to its natural specific target cells; this is a key step for the establishment of infection and studies regarding the adherence and cytotoxicity on the natural host cells are not available. The assays were carried out by seeding Campylobacter fetus venerealis on bovine vaginal and uterine epithelial cell cultures. HeLa cells were used as control. Bacterial adhesion was corroborated by optical microscopy and determination of the percentage of adherent bacteria was performed on immunochemically-stained slides. Results are presented as percentage of cells with adherent Campylobacter and as number of bacteria per cell. In comparison to the control HeLa cells, the statistical analysis revealed that primary cultures show a higher percentage of infected cells and a lower variation of the evaluated parameters. This primary culture model might be useful for studies on cytopathogenicity and adhesion of different field strains of Campylobacter fetus.

Campylobacter fetus é o agente etiológico da campilobacteriose genital bovina, uma doença sexualmente transmissível que está associada com perdas reprodutivas em bovinos. Campylobacter coloniza a vagina e o útero e então infecta as células epiteliais do endométrio. O objetivo deste trabalho foi desenvolver um modelo ex vivo para quantificar a adesão de Campylobacter às células-alvo naturais específicas; este é um passo fundamental para o estabelecimento da infecção e estudos acerca da adesão e citotoxicidade sobre as células do hospedeiro natural não estão disponíveis. Os ensaios foram realizados a través da semeadura de Campylobacter fetus venerealis em culturas celulares epiteliais vaginais e uterinas.Células HeLa foram utilizadas como controle.A aderência bacteriana foi confirmada por microscopia óptica e a determinação da porcentagem de bactérias aderidas foi realizada em lâminas tingidas imunoquimicamente. Os resultados são apresentados como porcentagem de células com Campylobacter aderente e como o número de bactérias por células. Em comparação com as células HeLa controle, a análise estatística revelou que as culturas primárias mostram uma maior porcentagem de células infectadas e uma menor variação dos parâmetros avaliados. Este modelo de cultura primária pode ser útil para estudos sobre citopatogenicidade e adesão de diferentes cepas de campo de Campylobacter fetus.

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.

Experimentally induced intravaginal Tritrichomonas foetus infection in a mouse model

A necessidade de esclarecer a relação agente-hospedeiro na tricomoníase bovina deu motivo para o uso de modelos experimentais alternativos ao bovino. O camundongo BALB/c resultou como espécie mais adequada para a infeção vaginal com Tritrichomonas foetus, requerendo uma estrogenização prévia. Visando a necessidade de um modelo experimental sem estrogenização persistente e com ciclos estrais normais, foi avaliada a instalação e persistência da infeção vaginal no camundongo BALB/c com diferentes quantidades de T. foetus, em dois grupos experimentais. O Grupo A foi tratado com 5µg de 3-benzoato de beta estradiol, para sincronizar o estro, 48 h antes da inoculação vaginal com T. foetus, e o Grupo B foi inoculado durante o estro natural. O efeito do 3-benzoato de beta estradiol decresceu gradualmente. A totalidade dos animais ciclaram regularmente após 5-7 dias pós-tratamento. Após a primeira semana pós-infecção, todos os animais foram amostrados durante 34 semanas, para avaliar o andamento da infeção vaginal e o estádio de cío. O Grupo A apresentou 93,6% de animais infetados e o Grupo B 38%. Com diferentes dosagens de T. foetus se obteve a instalação e persistência da infecção vaginal durante 34 semanas. Embora tenham-se registrados diferentes comportamentos entre cada um dos lotes dos Grupos A e B, não houveram diferenças significativas entre as doses infectantes. O tratamento com 3-benzoato de beta estradiol teve efeito favorável na instalação da infecção (P<0,0001), porém não teve influência na persistência (P= 0,1097). Em função dos resultados obtidos, apresenta-se um modelo experimental em camundongo, adequado para aprofundar estudos sobre mecanismos de patogenicidade, resposta imune, avaliação protetora de imunógenos e o efeito terapêutico de fármacos.

Immunohistochemical detection of Tritrichomonas foetus in experimentally infected mice

A necessidade de aumentar o conhecimento da patogenia da tricomoníase genital bovina (BGT) conduziu ao uso de modelos experimentais alternativos como o camundongo. Näo obstante, é necessário elucidar a dinâmica da infecçäo nesta espécie e avaliar as diferentes fases da colonizaçäo e evoluçäo das alterações patológicas. A imunohistoquímica (IHQ) oferece vantagens sobre as técnicas histoquímicas de rotina para a observaçäo do protozoário em tecidos, detritos celulares e dentro de macrófagos. O objetivo do presente trabalho foi demonstrar pelo uso de uma técnica de IHQ a presença de Tritrichomonas foetus no sistema reprodutivo de camundongos infectados. Camundongos BALB/c fêmeas foram infectados pela via intravaginal, com uma suspensäo de T. foetus, na fase de estro, detectado com citologia exfoliativa vaginal. Depois de 10 semanas, os animais foram sacrificados; útero e vagina forma fixados e processados para histologia. Alguns cortes foram corados com HE. O restante dos cortes foi processado para IHQ. Foi usado um soro policlonal imunoadsorvido anti-T. foetus. A técnica de avidina-biotina (HistoMouse, ZymedÖ) foi empregada. Os estudos histopatológicos mostraram uma dilataçäo das glândulas uterinas, presença de macrófagos no lúmen do órgäo e parte interna das glândulas endometriais. T. foetus näo foi identificado por esse método. A IHQ permitiu identificar as mesmas lesões observadas e a presença do protozoário no endométrio, nas glândulas endometriais, no lúmen uterino e dentro de neutrófilos e macrófagos. O estudo citológico em lâminas coradas por IHQ, mostrou T. foetus aderido a células epiteliais, ou dentro de macrófagos. Esta técnica demonstra ser uma ferramenta útil para o estudo da patogenia da tricomoníase genital bovina (BGT) utilizando-se o camundongo como modelo experimental.