Antimicrobial resistance of Streptococcus uberis isolated from bovine mastitis: Systematic review and meta-analysis.

Streptococcus uberis is one of the most common pathogens associated with bovine mastitis, commonly treated with antimicrobials (AM), favoring the appearance of antimicrobial resistance (AMR). The objective of this work was to determine the proportion of phenotypic AMR among S. uberis isolated worldwide from bovine intramammary infections between the years 1983-2022, and to assess the variables associated by means of a systematic review and metanalysis. Sixty articles were eligible for quantitative review. Ninety-four independent studies were obtained. The antimicrobials evaluated in more S. uberis strains were penicillin (21,987 strains), oxacillin (21,727 strains), erythromycin (20,013 strains), and ampicillin (19,354 strains). Most of the studies included in this meta-analysis were from Europe (44), followed by America (25), Africa (10), Asia (10), and Oceania (5). Among the included articles, 22 were published from 1983 to 2006, 23 from 2007 to 2012, 25 from 2013 to 2015, and the remaining 24 after 2016. Penicillin, erythromycin, and tetracycline were the antimicrobials with >25 studies. Therefore, the following analyses were performed only for these antimicrobials, presenting a high heterogeneity index (I2). The variability observed for penicillin and tetracycline was only explained, partially, by continent of origin. The variability observed for erythromycin was not explained by any of the potential explanatory variables included in this study. The S. uberis proportion of resistance to antimicrobials is highly variable and probably influenced by many factors other than those studied in this meta-analysis, where it was not possible to inform a unique average proportion of resistance.

Seroprevalence and risk factors associated with bovine Leukemia virus infection in argentine beef cattle.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, an endemic disease in dairy cattle of Argentina. However, little is known about the seroprevalence of BLV in beef cattle. In this study, we conducted a cross-sectional study including farms from thirteen provinces of Argentina. A total of 5827 bovine serum samples were collected from 76 farms and analyzed using an in-house developed enzyme-linked immunosorbent assay. Information about herd management was collected through a questionnaire, and univariate and multivariate analyses were performed to detect risk factors associated with BLV infection. Herd-level seroprevalence was 71.05%, while the mean animal-level seroprevalence was 7.23% (median = 2.69%; min = 0, max = 75). Only two provinces had no positive BLV samples. The other eleven provinces showed more than 50% of their farms infected with BLV. The multivariate model revealed that BLV prevalence was significantly associated with the use of animals raised in the same farm for cattle replacement (P = 0.005), breeding cows by natural mating with a bull (P < 0.001), and weaning calves after 6 months of age (P = 0.011). This extensive study revealed that BLV seroprevalence in Argentine beef farms has increased during the last years and allowed identifying some management practices associated with BLV prevalence. These data deserve special attention because BLV infection in beef cattle seems to lead to a dissemination pattern similar to that observed during the last decades in dairy cattle, especially considering that Argentina is the sixth beef producer in the world, with about 5% of global beef production.

A safe and effective vaccine against bovine leukemia virus.

Previous attempts to develop a vaccine against bovine leukemia virus (BLV) have not been successful because of inadequate or short-lived stimulation of all immunity components. In this study, we designed an approach based on an attenuated BLV provirus by deleting genes dispensable for infectivity but required for efficient replication. The ability of the vaccine to protect from natural BLV infection was investigated in the context of dairy productive conditions in an endemic region. The attenuated vaccine was tested in a farm in which the prevalence rose from 16.7% in young cattle at the beginning of the study to more than 90% in adult individuals. Sterilizing immunity was obtained in 28 out of 29 vaccinated heifers over a period of 48 months, demonstrating the effectiveness of the vaccine. As indicated by the antiviral antibody titers, the humoral response was slightly reduced compared to wild-type infection. After initial post-vaccination bursts, the proviral loads of the attenuated vaccine remained most frequently undetectable. During the first dairy cycle, proviral DNA was not detected by nested-PCR in milk samples from vaccinated cows. During the second dairy cycle, provirus was sporadically detected in milk of two vaccinated cows. Forty-two calves born from vaccinated cows were negative for proviral DNA but had antiviral antibodies in their peripheral blood. The attenuated strain was not transmitted to sentinels, further supporting the safety of the vaccine. Altogether, these data thus demonstrate that the vaccine against BLV is safe and effective in herd conditions characterized by a very high incidence. This cost-effective approach will thus decrease the prevalence of BLV without modification of production practices. After facing a series of challenges pertaining to effectiveness and biosafety, the vaccine is now available for further large-scale delivery. The different challenges and hurdles that were bypassed may be informative for the development of a vaccine against HTLV-1.

Antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis: Systematic review and meta-analysis.

Staphylococcus aureus, one of the main contagious mastitis pathogens worldwide, is characterized for causing chronic intramammary infections that respond poorly to antimicrobial therapy, disseminating within the herd leading to high economic losses. The aim of this study was to determine the prevalence of phenotypic resistance to antimicrobial agents among S. aureus collected worldwide in the context of bovine intramammary infections between the years 1969-2020. A systematic review was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). One hundred and fifty-five articles were eligible for quantitative review. Most of studies included in this meta-analysis were from Europe (88), followed by Asia (56), Latin America (39), Africa (32), North America (26), and Oceania (8). The highest overall prevalence of resistant S. aureus was against penicillin (pestimate 0.451, CI95 % 0.415-0.487), followed by clindamycin, erythromycin, and gentamycin (p-estimate = 0.149, 0.085, and 0.069, respectively). Ceftiofur and cephalotin presented the lowest overall prevalence of antimicrobial resistance (AMR, p-estimate = 0.020 and 0.015, respectively). The AMR to almost all the antimicrobials evaluated presented an increasing pattern over time, more apparent from 2009 onwards. The antimicrobials with a higher increase in their AMR prevalence over time were clindamycin, gentamycin, and oxacillin. Africa, Asia and Latin America were the continents with higher AMR to most compounds included in this study. No differences in AMR were detected regarding the clinical origin of the isolates (subclinical vs clinical mastitis) for almost all antibiotics evaluated. Differences in the method for testing AMR (disc diffusion method vs minimum inhibitory concentration) and type of study design for monitoring AMR were detected underscoring the importance of these variables as critical factors to enable comparisons for evaluating emergence of AMR.

Infección experimental en ovinos con el virus de leucosis bovina: dosis mínima de células BLV-FLK y virus libre de células, y actividad neutralizante de anticuerpos naturales / Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies

Abstract Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Resumen El virus de la leucosis bovina (bovine leukemia virus [BLV]) es un importante agente patógeno del ganado que causa importantes pérdidas económicas en todo el mundo, especialmente en los rodeos lecheros. El uso de modelos animales proporciona información valiosa sobre la patogénesis de las infecciones virales. Se realizaron infecciones experimentales en ovejas usando sangre de bovinos infectados con BLV, clones moleculares de BLV infecciosos o células derivadas de tumores. La línea celular Fetal Lamb Kidney, persistentemente infectada con el BLV (FLK-BLV), es uno de los cultivos a largo plazo más utilizados para la producción permanente de virus. Las células FLK-BLV o las partículas virales obtenidas del sobrenadante del cultivo libre de células podrían usarse como fuente de provirus o de virus para infectar experimentalmente ovejas. En este trabajo, nuestro objetivo fue determinar la cantidad mínima de células FLK-BLV o de sobrenadante libre de células que contiene BLV necesaria para producir infección en ovejas. También evaluamos la cantidad de anticuerpos bovinos anti-BLV necesaria para neutralizar la infección. Observamos que las dos ovejas inoculadas experimentalmente con 5000 células FLK-BLV se infectaron, y que una de las dos ovejas que recibieron 500 células FLK-BLV se infectó. Ninguno de los animales inoculados con 50 células FLK-BLV mostró evidencia de infección. El sobrenadante FLK-BLV libre de células demostró ser infectivo en ovejas hasta la dilución 1:1000. Los anticuerpos BLV específicos mostraron actividad neutralizante, ya que ninguna de las ovejas se infectó. Por el contrario, los animales que recibieron un suero BLV negativo mostraron signos de infección por BLV. Estos resultados contribuyen a la optimización de un bioensayo en ovejas útil para caracterizar la infección por BLV.

BLV: lessons on vaccine development.

Vaccination against retroviruses is a challenge because of their ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is almost impossible once infection is established, the primary goal is to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. In this review, we discuss the different issues that we faced during the development of an efficient vaccine against bovine leukemia virus (BLV). We summarize the historical failures of inactivated vaccines, the efficacy and safety of a live-attenuated vaccine and the economical constraints of further industrial development.

Liposomal and CpG-ODN formulation elicits strong humoral immune responses to recombinant Staphylococcus aureus antigens in heifer calves.

Bovine mastitis caused by Staphylococcus aureus is a serious problem in dairy production and effective immunoprophylaxis is an unmet goal so far. The objective of this work was to assess the humoral immune response of heifer calves against two recombinant S. aureus antigens: Clumping factor A (ClfA) and Fibronectin Binding Protein A (FnBPA), formulated with a novel adjuvant based on cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODN). Six groups of 6-8 months old heifer calves received three doses biweekly of antigens, formulated with Al(OH)3, liposomes, CpG-ODN or Lip + CpG-ODN. Animals also received a fourth dose after a year (day 410) and a booster before calving. The administration of Al(OH)3+FnBPA/ClfA and Lip + FnBPA/ClfA + CpG-ODN induced the highest specific IgG levels, after the first 3 doses and induced a fast increase of antibodies after the fourth dose. All the formulations stimulated the production of specific IgG1, after the third and fourth dose. Specific IgG2 for both proteins was only stimulated after the fourth dose by Lip + FnBPA/ClfA + CpG-ODN. Pre-calving immunisation with Lip + FnBPA/ClfA + CpG-ODN led to the highest IgG levels during the calving period and to the production of the IgG2 subclass. The formulation was also able to stimulate the highest antibody levels in milk, 30 and 45 days after pre-calving booster. The combination of liposomes and CpG-ODN as adjuvant for a subunit vaccine, together with the immunisation schedule described, induced a strong humoral immune response with production of specific IgG2. The formulation demonstrated to induce immune memory allowing the application of a single pre-calving booster to maintain high antibody levels throughout the period of increased susceptibility to intramammary infections.

Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies.

Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.