Improvement of bovine pestiviral diagnosis by the development of a cost-effective method for detecting viral RNA in fresh specimens and samples spotted in filter papers / Optimización del diagnóstico de Pestivirus bovinos mediante el desarrollo de una metodología económica para detectar ARN viral en muestras frescas y fijadas en papeles de filtro

Abstract Bovine pestiviruses are the causative agents of bovine viral diarrhea, a disease thatcauses severe economic losses in cattle. The aim of this study was to improve their diagnosisby developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set up a protocolfor collecting, shipping and preserving bovine pestiviral RNA on filter papers. The developedRT-qPCR showed high sensitivity in detecting these viruses in different matrices: viral stocks,semen and serum samples. With regard to the possibility of using the technique to test serumpools, it was possible to identify a positive serum sample within a pool containing 30 sera.In addition to evaluating the qPCR from fresh samples, the use of filter papers to sow bovinesamples was analyzed. The sampling method on two different filter papers using bovine blooddrops was a useful alternative for diagnostic purposes and allowed to preserve pestiviral RNAfor up to 12 months under refrigeration.

Resumen Los Pestivirus bovinos son los agentes causales de la diarrea viral bovina, una enfermedad que genera importantes pérdidas económicas en el ganado vacuno. El objetivo de este trabajo fue mejorar su diagnóstico mediante el desarrollo de una RT-qPCR para detectar los Pestivirus bovinos A, B y H y disenar un protocolo de recolección, envío y conservación de ARN viral en papeles de filtro. La RT-qPCR desarrollada demostró alta sensibilidad en la detección de estos virus en diferentes matrices: stock viral, suero y semen. Respecto de la posibilidad de usar la técnica para testear pools de suero, fue posible identificar un suero positivo dentro de un pool compuesto por 30 sueros. Además de evaluar la qPCR en muestras frescas, se analizó el uso de papeles de filtro para sembrar muestras de bovinos. La metodología de toma de muestras en dos tipos de papeles de filtro usando gotas de sangre fue una alternativa útil para el diagnóstico y permitió conservar ARN viral por hasta 12 meses a temperaturas de refrigeración.

Differential immune response to two Staphylococcus aureus strains with distinct adaptation genotypes after experimental intramammary infection of dairy cows.

The aim of this study was to evaluate and compare the ability of two S. aureus strains with different adaptation genotypes (low and high) to the bovine mammary gland (MG) to establish an intramammary infection (IMI) and induce an immune response after an experimental challenge in lactating cows. Two isolates (designated 806 and 5011) from bovine IMI with different genotypic profiles, harboring genes involved in adherence and biofilm production, belonging to different capsular polysaccharide (CP) type, accessory gene regulator (agr) group, pulsotype (PT) and sequence type/clonal complex (ST/CC) were selected. Strains 806 and 5011 were associated with low (nonpersistent-NP) and high (persistent-P) adaptation to the MG, respectively. Strain 806 (NP) was characterized as agr group II, cap5 positive and ST350; strain 5011 (P) agr group I, cap8 positive and CC188. Three groups of clinically healthy cows, 4 cows/treatment group, were inoculated by the intramammary route with strain 806 (NP), strain 5011 (P) and pyrogen-free saline solution. All mammary quarters challenged with strain 806 (NP) developed mild clinical mastitis between 1 and 7 d post inoculation (pi). Quarters challenged with strain 5011 (P) developed a persistent IMI; bacteria were recovered from milk from d 7 pi and up to d 56 pi. In quarters inoculated with strain 806 (NP) the inflammatory response induced was greater and earlier than the one induced by strain 5011 (P), since a somatic cell count (SCC) peak was observed at d 2 pi, while in quarters inoculated with strain 5011 (P) no variations in SCC were observed until d 4 pi reaching the maximum values at d 14 pi; indicating a lower and delayed initial inflammatory response. The highest levels of nitric oxide (NO) and lactoferrin (Lf) detected in milk from quarters inoculated with both S. aureus strains coincided with the highest SCC at the same time periods, indicating an association with the magnitude of inflammation. The high levels of IL-1ß induced by strain 806 (NP) were associated with the highest SCC detected (d 2 pi); while quarters inoculated with strain 5011 (P) showed similar IL-1ß levels to those found in control quarters. In quarters inoculated with strain 806 (NP) two peaks of IL-6 levels on d 2 and 14 pi were observed; while in quarters inoculated with strain 5011 (P) IL-6 levels were similar to those found in control quarters. The strain 806 (NP) induced a higher total IgG and IgG1 response; while strain 5011 (P) generated a higher IgG2 response (even against the heterologous strain). The present study demonstrated that S. aureus strains with different genotype and adaptability to bovine MG influence the local host immune response and the course and severity of the infectious process.

Detection by multiplex PCR of Mycoplasma species associated with dairy cattle in Argentina / Detección por PCR multiplex de especies de Mycoplasma asociadas a ganado lecheroen Argentina

Abstract There is scarce information about the frequency and epidemiological and clinicalfeatures associated with the presence of Mycoplasma spp. in Argentine dairy herds. The objec-tives of this study were to develop a multiplex PCR for identifying M. bovis and M. canadenseand to describe the frequency of Mycoplasma spp. isolated from clinical samples submitted to adiagnostic laboratory. Of a total of 1548 samples from intramammary infections, bulk tank milkand biological fluids, 38 Mycoplasma isolates were obtained. M. bovis, M. canadense, M. cali-fornicum and M. leachii were detected by using two multiplex PCRs, confirming their presencein clinical conditions in dairy cattle. The techniques used in the present study can be usefulto broaden the knowledge about Mycoplasma infections in cattle, since the search for theseorganisms is not usually included in routine diagnoses.

Resumen Existe poca información sobre la frecuencia, así como las características epidemi-ológicas y clínicas asociadas con la presencia de Mycoplasma en los rodeos lecheros argentinos.Los objetivos de este estudio fueron desarrollar una PCR multiplex para identificar M. bovis yM. canadense y describir la frecuencia de especies de Mycoplasma aisladas de muestras clíni-cas enviadas a un laboratorio de diagnóstico. De un total de 1.548 muestras de infeccionesintramamarias, leche de tanque de frío y fluidos biológicos, se obtuvieron 38 aislamientos de Mycoplasma. Mediante 2 PCR multiplex se detectaron M. bovis, M. canadense, M. californicumy M. leachii, confirmando su presencia en síndromes clínicos en ganado lechero. Las técnicasutilizadas en el presente estudio pueden ser útiles para ampliar el conocimiento sobre las infec-ciones por Mycoplasma en bovinos, ya que la búsqueda de estos organismos no suele incluirseen los diagnósticos de rutina.

Effects of chronic Staphylococcus aureus infection on immunological parameters and functionality of macrophages isolated from bovine mammary secretions.

The aim of this study was to characterize the effects of chronic S. aureus intramammary infection (IMI) on local innate and adaptive immune response during active involution. Cows in late lactation that were either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. The levels of interleukin (IL)-1ß, IL-6 and IL-4 were significantly higher in mammary secretions of S. aureus-infected quarters compared with uninfected at d 7, 14 and 21 of involution. Lactoferrin (Lf), total IgG and S. aureus specific IgG1 levels were significantly lower in mammary secretions of infected quarters compared with uninfected during the first three weeks of involution. The amount of intracellular reactive oxygen species (ROS) produced per macrophage, was significantly higher in mammary secretions of infected quarters compared with uninfected at d 14 post drying off. Nitrite production was significantly higher in phagocytes from infected mammary secretions compared with uninfected at d 7 and 14 post drying off. Chronic S. aureus IMI altered normal secretion composition during bovine mammary gland involution. The high IL-1ß and IL-6 levels and increased functionality of macrophages in mammary secretions of infected quarters could be a result of the chronic inflammatory environment triggered by the presence of viable bacteria in mammary tissue. The lower levels of total and S. aureus specific antibodies and other immune factors in mammary secretion during this period may reduce the natural defense potential of the gland contributing to S. aureus persistence.

Primer aislamiento de Mycoplasma leachii asociado a enfermedad en terneros de tambo en Argentina / First report of Mycoplasma leachii isolation associated with disease in dairy calves in Argentina

There are few reports about the isolation of Mycoplasma species associated with cattle disease in Argentina. In this work we describe the detection of Mycoplasma leachii associated with disease in dairy calves in Santa Fe Province, Argentina. Samples obtained from a 4 day-old dairy calf suffering from polyarthritis and from two other calves, one with arthritis and the other one with a mandibular abscess, were subjected to microbiological culture. Classical culture and generic PCR confirmed the presence of Mycoplasma spp. The spacer region between the 16S and 23S ribosomal RNA gene from the first isolate was amplified and sequenced. The sequence obtained showed 99% identity with M. leachii. A PCR was developed to amplify a specific fragment of the 16S-23S ITS region corresponding to M. leachii, which allowed to identify the isolates associated with disease in calves.

Existen pocos informes acerca del aislamiento de especies de Mycoplasma asociadas con enfermedades del ganado en Argentina. En esta comunicación se describe el aislamiento de Mycoplasma leachii asociado a enfermedad en terneros de tambo en la provincia de Santa Fe, Argentina. Se obtuvieron muestras de un ternero de 4 días de vida con poliartritis, de un ternero con artritis y uno con un absceso mandibular. A partir del cultivo clásico se detectó la presencia de Mycoplasma, lo cual fue confirmado por PCR genérica. Se amplificó y secuenció la región ITS 16S-23S a partir del primer aislamiento, mostrando una identidad del 99% con Mycoplasma leachii. Se desarrolló una PCR para amplificar un fragmento específico de la región ITS 16S-23S correspondiente a M. leachii, que permitió identificar los aislamientos asociados con enfermedad en terneros.

Panax ginseng extract reduces Staphylococcus aureus internalization into bovine mammary epithelial cells but does not affect macrophages phagocytic activity.

Panax ginseng extract (PGe) has been shown to possess immunomodulatory effects in healthy dairy cows at drying off and to trigger an adequate immune response to protect from an experimental intramammary infection (IMI) with Staphylococcus aureus in a murine model. S. aureus is one of the major pathogens isolated from bovine IMI; being capable to invade and survive within mammary epithelial cells. However, the precise mechanism by which PGe interacts with bovine mammary epithelial cells (MAC-T) and bovine macrophages in the course of a S. aureus infection remains unclear. We evaluated the effect of PGe on MAC-T cytokine response and on the internalization of S. aureus into MAC-T. In addition, we evaluated the effect of PGe on the phagocytic activity of macrophages isolated from bovine mammary secretions. Results shown that MAC-T cells TLR4 and NF-κB mRNA expression was not affected by PGe at all evaluated times. IL-6 mRNA expression and protein level and IL-4 protein level were significantly induced in MAC-T treated with 3 mg/ml of PGe. PGe at 3 mg/ml reduced significantly the internalization of two S. aureus strains in MAC-T. In addition, PGe did not affect the percentage of phagocytosis and the NO and ROS production of macrophages co-cultured with two strains of S. aureus. These results, obtained in in vitro models together with those obtained in in vivo previous studies carried out in bovines and mice can contribute to improve the understanding of the effects of PGe following inoculation in bovine mammary glands.

Characterization of immune response in Staphylococcus aureus chronically infected bovine mammary glands during active involution.

The aim of this study was to characterize the immune response in Staphylococcus aureus chronically infected bovine mammary glands during active involution. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic naturally acquired S. aureus intramammary infections (IMI) were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical analysis were taken. Protein expression of toll-like receptor 2 (TLR2) and TLR4 was significantly higher in S. aureus-infected quarters than in uninfected controls at the three involution stages studied. Protein expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α and IL-17 was significantly affected by IMI; being higher in S. aureus-infected than uninfected quarters during all evaluated stages. In S. aureus-infected and uninfected quarters protein expression of lactoferrin increased from day 7-14 of involution, decreasing significantly to day 21 in mammary quarters with chronic infections. The number of monocytes-macrophages was significantly higher in S. aureus-infected than in uninfected control quarters at 7 and 21 d of involution. The number of T lymphocytes was significantly higher in S. aureus-infected than in uninfected quarters at 7 and 14 d of involution while the number of B lymphocytes was significantly higher in S. aureus-infected than in uninfected quarters during all evaluated stages, showing a progressive increase as involution advanced. These results demonstrated a sustained and exacerbated innate and adaptive immune response during chronic S. aureus IMI, playing a critical role in the infection control during active involution.

Proliferation-apoptosis balance in Staphylococcus aureus chronically infected bovine mammary glands during involution.

The objective of this study was to determine whether Staphylococcus aureus chronic intramammary infection (IMI) influences expression of proteins related to regulation of proliferation and apoptosis processes and proliferation/apoptosis index during active involution in bovine mammary gland. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical analysis were taken. Protein expression of Bcl-2, Bax, Fas and active caspase-3 in mammary tissue was significantly affected by chronic S. aureus IMI, all showing increased immunoexpression in S. aureus-infected quarters at all involution stages. The percentage of apoptotic cells was increased by IMI in both mammary parenchyma and stroma, and the percentage of parenchymal and stromal cell proliferation was also increased. The proliferation/apoptosis ratio was significantly increased by IMI only in stromal cells. This imbalance to favour proliferation in S. aureus-infected mammary quarters could be one of the underlying causes that induce aberrant involution with permanence of nonsecretory tissue and increase of stromal components.

Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells.

Staphylococcus aureus is one of the most prevalent pathogens isolated from bovine mastitis, causing chronic intramammary infections (IMI) that limit profitable dairying. The course of infection is often associated with factors both related to the host and the bacterium. Aims of this study were to select S. aureus isolates from bovine IMI with different genotypic profiles harboring genes involved in adherence and biofilm production, to determine the behavior of these strains in contact with bovine mammary epithelial cells (MAC-T) and the expression of those genes during bacterial-cell early interactions. The genetic diversity of 20 S. aureus strains that were isolated from milk samples taken from cows with persistent-P and non-persistent-NP IMI was high, discriminated into 13 fingerprint groups. The occurrence of genes coding for S. aureus surface proteins (clfA, clfB, fnbA, fnbB, fib, cna) and biofilm formation (icaA, icaD, icaC, bap) and in vitro biofilm-forming ability was not related to strain clinical origin (NP or P). Internalization of S. aureus into MAC-T cells was strain-dependent and internalized bacteria overexpressed adherence and biofilm-forming genes compared with those that remained in the supernatant of co-cultures; particularly those genes encoding FnBPs and IcaD. Strains yielding highest invasion percentages were those able to overexpress fnBP, irrespectively of the presence of other evaluated genes. Strains from NP IMI showed a greater multiplication capacity in vitro compared with strains from P IMI. These results provide new insights about S. aureus differential gene expression of adhesion-internalization factors during early interaction with mammary epithelial cells.

Genotipificación y estudio de los genes pauA y sua de aislamientos de Streptococcus uberis de mastitis bovina / Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6 % concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8 % (134/137) and 94.9 % (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95 % and 100 % with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis.

Este estudio pretendió determinar la relación clonal entre 137 aislamientos de S. uberis obtenidos de leche de bovinos con mastitis clínica o subclínica en la Argentina, como así también la prevalencia y la conservación de los genes sua y PauA entre dichos aislamientos. Esta información es crítica para el diseño racional de una vacuna que prevenga la mastitis bovina por S. uberis. Los aislamientos se tipificaron molecularmente por amplificación al azar del ADN polimórfico (RAPD) y mediante electroforesis de campos pulsados (PFGE). Los 137 aislamientos mostraron 61 pulsotipos mediante PFGE y 25 tipos de RAPD diferentes. Los índices de Simpson calculados fueron 0,983 por PFGE y 0,941 por RAPD; esto evidencia el elevado poder discriminatorio de ambas técnicas. El análisis de la relación entre pares de aislamientos mostró un 92,6 % de concordancia entre ambas técnicas, lo que indica que cualquier par de aislamientos que fue distinguido por un método tendió a ser distinguido por el otro. La prevalencia de los genes sua y puaA fue del 97,8 % (134/137) y 94,9 % (130/137), respectivamente. Las secuencias de nucleótidos y de aminoácidos codificados por los genes sua y pauA de los 20 aislamientos de S. uberis seleccionados sobre la base de su tipo de PFGE y RAPD y origen geográfico tuvieron un porcentaje de identidad de entre 95 % y 100 % con respecto a todas las secuencias de referencia registradas en GenBank. Estos resultados demuestran que, a pesar de la diversidad clonal de S. uberis, los genes sua y pauA son prevalentes y están altamente conservados y deberían ser incluidos en futuros estudios de vacunas para prevenir mastitis bovina causada por S. uberis.

Aspectos de la respuesta inmune innata en las infecciones intramamarias causadas por Staphylococcus aureus en bovinos / Aspects of the innate immune response to intramammary Staphylococcus aureus infections in cattle

Staphylococcus aureus es el principal agente causante de mastitis bovina en Argentina y en el mundo. Esta bacteria ocasiona infecciones crónicas que generan importantes pérdidas a los productores y la industria lechera. El objetivo de este artículo es caracterizar los mecanismos que intervienen en la infección causada por S. aureus en la glándula mamaria bovina, evaluando dos aspectos diferentes del proceso infeccioso: por un lado, lo vinculado con la respuesta inmune innata por parte del hospedador, y por otro, la capacidad de la bacteria para evadir el sistema inmune e interactuar con diferentes tipos celulares. La exploración de la interacción de S. aureus con el sistema inmune de la glándula mamaria bovina permitirá identificar blancos para delinear nuevas alternativas preventivas o curativas, que contribuyan a evitar o eliminar las infecciones causadas por este organismo

Staphylococcus aureus is the pathogen most frequently isolated from bovine mastitis worldwide, causing chronic intramammary infections that limit profitable dairying. The objective of this article is to characterize the mechanisms involved in S. aureus mammary gland infections considering two different aspects of the infectious process; on the one hand, the aspects involved in the host innate immune response and on the other hand, the capacity of this organism to evade the immune system and interact with different cell types. The exploration of S. aureus interactions with the immune response of bovine mammary gland will help identify targets to outline new preventive or curative alternatives for intramammary infections caused by this organism

Functional role of antibodies generated in heifers through immunization with Staphylococcus aureus vaccines in invasion and phagocytosis assays.

A successful Staphylococcus aureus vaccine should elicit a long-term antibody response that prevents establishment of the infection. The aim of the present study was to evaluate the functional role of antibodies raised against different S. aureus CP5 vaccines in invasion to bovine mammary epithelial cells (MAC-T) and phagocytosis by bovine milk macrophages in vitro. Sera and whey from cows immunized with a whole-cell S. aureus CP5 vaccine adjuvanted with Al(OH)3 or with ISCOM Matrix, significantly reduced internalization of S. aureus in MAC-T cells without significant differences between both groups. The effect of antibodies generated by a S. aureus whole-cell and a lysate vaccine formulated with ISCOM Matrix was also evaluated. Sera and whey from both immunized groups significantly reduced S. aureus internalization in MAC-T cells without significant differences between both groups. Whey antibodies against whole-cell and lysate vaccines were also able to inhibit internalization in MAC-T cells of a heterologous S. aureus strain. In addition, sera from animals vaccinated with S. aureus lysate or bacterin promoted milk macrophage phagocytosis. These results provide an insight into the potential mechanisms by which these vaccines can afford protection to the mammary gland against S. aureus intramammary infection.

Factores de virulencia de Staphylococcus aureus asociados con infecciones mamarias en bovinos: relevancia y rol como agentes inmunógenos / [Virulence factors of Staphylococcus aureus associated with intramammary infections in cows: relevance and role as immunogens].

Staphylococcus aureus is the most prevalent mastitis pathogen in Argentina and worldwide. Lack of effectiveness of traditional control measures based on milking hygiene and antibiotic therapy against this organism has led to the development of alternatives directed to prevent the disease. Among them, the manipulation of host immune mechanisms through vaccination has been explored. The identification of virulence factors able to stimulate host immune defenses is key to developing a rational vaccine. S. aureus has multiple virulence factors that interact with the host at different stages of an intramammary infection. The use of some of these factors as immunogens has been shown to elicit protective responses in the host. The structure, function, and use as immunogens of S. aureus virulence factors considered to be relevant at different stages of intrammamary infections caused by this organism are reviewed in this article.

Association between milking practices and psychrotrophic bacterial counts in bulk tank milk / Asociación entre prácticas de ordeño y recuento de organismos psicrótrofos en leche de tanque de frío

The objective of this work was to determine on-farm risk factors for psychrotrophic bacterial counts in bulk tank milk from dairy farms in Argentina. Raw milk samples from bulk tanks of 27 dairy farms were examined for total psychrotrophic counts (TPC), proteolytic psychrotrophic counts (PPC) and lipolytic psychrotrophic counts (LPC) (dependent or outcome variables). A survey recording infrastructure conditions, milking equipment and milking management (independent variables) was performed. Bivariate association proofs and logistic regression analyses were used to determine association between independent variables and psychrotrophic bacterial counts. Milk cooled in plate heat exchangers or barrel tanks were 16.39 and 10.52 times more likely to yield TPC and PPC above the standard established for high quality milk compared with milk cooled in bulk tanks, respectively. Periodic cleaning of cooling tanks (3 times a week or daily) was associated with lower TPC (approximately 1.5 log CFU/ml) than weekly cleaning frequency and farms where milkers did not wash their hands during milking time were 7.81 times more likely to have higher PPC. No association was found between LPC and any of the independent variables. The only variable associated with TPC and PPC in a logistic regression model was the refrigeration system used on the farm. Dairy farms that possessed bulk milk cooling tanks yielded the lowest bacterial counts. Results of this study highlight the importance of both the type of cooling system used on the farm and its adequate hygienic maintenance for obtaining low pshychrotrophic counts at dairy farm.

El objetivo del trabajo fue determinar los factores de riesgo para altos recuentos de organismos psicrótrofos en leche de tanques de tambos de la Argentina. Se examinaron muestras de leche cruda de tanques de frío de 27 tambos, y se realizó el recuento de organismos psicrótrofos totales (PT), de psicrótrofos proteolíticos (PP) y de psicrótrofos lipolíticos (PL) (variables dependientes). Se realizó una encuesta para registrar las condiciones de infraestructura, el equipo de ordeño y las prácticas de ordeño (variables independientes). Se utilizaron pruebas bivariadas de asociación y regresión logística para determinar la asociación entre las variables independientes y los recuentos de organismos psicrótrofos. La leche enfriada en sistemas de placas de intercambio o tanques tipo cuba tuvo una probabilidad mayor de dar recuentos elevados de PT y PP (16,39 y 10,52) comparada con la enfriada en tanques tipo "panza fría". La limpieza periódica del equipo de frío (3 veces por semana o diariamente) se asoció con bajos recuentos de PT (aproximadamente 1,5 log de UFC/ml). Los tambos cuyos ordeñadores no se higienizaban las manos durante el ordeño tuvieron una probabilidad 7,81 veces mayor de tener recuentos elevados de PP. No se encontró asociación entre el recuento de PL y las variables independientes. La única variable asociada con los recuentos de PT y PP en el modelo de regresión logística fue el sistema de enfriamiento utilizado en el tambo. El tipo de sistema de refrigeración usado y su adecuado mantenimiento higiénico son importantes para la obtención de leche con baja carga de organismos psicrótrofos en el tambo.

Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms

The aim of this study was to investigate the phenotypic and genotypic characteristics of Streptococcus uberis isolated from subclinical mastitis (SCM) cases, and to examine the possible association between both characteristics. A total of 32 S. uberis were isolated from 772 quarter milk samples (SCM > 250,000 cells/ml) collected from 195 cows selected randomly from 18 dairy farms located in Argentina. The S. uberis strains were characterized phenotypically by the presence of virulence factors as plasminogen activator factor (PAF), hyaluronidase (HYA), capsule (CAP) and CAMP factor, and were further characterized genotypically by pulsed-field gel electrophoresis (PFGE). S. uberis strains expressed plasminogen activator factor, hyaluronidase or capsule (65.5 %, 56.3 %, 59.4 %, respectively), but only 25 % of isolates were CAMP factor positive. Thirteen different virulence profiles were identified on the basis of the combination of virulence factors. Eighteen PFGE patterns with 90% of similarity were identified among 32 S. uberis. A great diversity of virulence profiles and PFGE patterns were present among dairy farms. S. uberis strains with the same PFGE pattern showed different virulence profiles. Bovine S. uberis strains causing SCM included in the present study showed heterogeneity in regard to their phenotypic and genotypic characteristics, and the PFGE patterns are not associated with the virulence profiles.

Caracterización fenotípica y genotípica de Streptococcus uberis aislados de mastitis bovina subclínica en tambos de Argentina. El objetivo de este estudio fue investigar las características fenotípicas y genotípicas de Streptococcus uberis aislados de casos de mastitis subclínica (MSC) y examinar la posible asociación entre ambas características. Un total de 32 cepas de S. uberis fueron aisladas de 772 muestras de leche de cuartos mamarios (MSC > 25 0000 células/ml) colectadas de 195 vacas seleccionadas al azar pertenecientes a 18 tambos lecheros localizados en Argentina. Las cepas de S. uberis fueron caracterizadas fenotípicamente sobre la base de la presencia de factores de virulencia tales como el factor activador del plasminógeno (FAP), la hialuronidasa (HIA), la cápsula (CAP) y el factor CAMP. Además, fueron caracterizadas genotípicamente por electroforesis de campos pulsados (PFGE). Las cepas de S. uberis expresaron el factor activador del plasminógeno, la hialuronidasa o la cápsula (65,5 %, 56,3 % y 59,4 %, respectivamente), pero solo el 25 % fueron CAMP positivas. Sobre la base de la combinación de los factores de virulencia se identificaron 13 perfiles de virulencia diferentes. Asimismo, se identificaron 18 patrones de PFGE con un 90 % de similitud entre las 32 cepas de S. uberis. Se presentó una gran diversidad de perfiles de virulencia y patrones de PFGE entre los tambos. Cepas con el mismo patrón de PFGE presentaron perfiles de virulencia diferentes. Las cepas de S. uberis causantes de MSC en bovinos incluidas en el presente estudio mostraron heterogeneidad con respecto a sus características fenotípicas y genotípicas, y los patrones de PFGE no estuvieron asociados con los perfiles de virulencia.

Presence of enterotoxigenic Staphylococcus aureus in bulk tank milk from Argentine dairy farms / Presencia de Staphylococcus aureus enterotoxigénico en leche de tanque de frío de tambos de Argentina

Staphylococcus aureus is the most prevalent bovine mastitis pathogen in Argentina. The ability of this organism to produce enterotoxins is linked to staphylococcal food poisoning. Staphylococcal enterotoxins are low molecular weight proteins, highly resistant to heat and proteolytic enzyme activity. The aim of this study was to determine the ability to produce enterotoxins and types of enterotoxins A through E produced among 94 S. aureus isolated from bulk tank milk in Argentina by enzyme-linked immunosorbent assay. Eleven isolates (11.7 %) produced enterotoxins. Seven of them (7.4 %) produced enterotoxin C, two (2.1 %) enterotoxin D, one (1.1 %) enterotoxin B and one (1.1 %) enterotoxins C-D-E. None of the isolates produced enterotoxins A or E alone. Since presence of staphylococcal enterotoxins constitute a potential risk to public health, these findings underscore the need to control S. aureus bovine mastitis and to limit bacterial multiplication in bulk tank milk.

Staphylococcus aureus es el patógeno causante de mastitis más prevalente en Argentina. Las enterotoxinas producidas por este organismo constituyen una de las causas más importantes de intoxicación alimentaria en seres humanos. Las enterotoxinas estafilocócicas son proteínas de bajo peso molecular, termoestables y resistentes a enzimas proteolíticas. El objetivo del presente trabajo fue determinar por enzimoinmunoensayo la presencia de enterotoxinas A-E y establecer su tipo en 94 aislamientos de Staphylococcus aureus obtenidos de leche de tanque de frío de tambos de Argentina. Se identificaron 11 % aislamientos enterotoxigénicos (11,7 %); siete (7,4 %) produjeron enterotoxina C, dos produjeron enterotoxina D (2,1 %), uno produjo enterotoxina B (1,1 %) y uno produjo enterotoxinas C-D-E (1,1 %). No se detectaron aislamientos que produjeran enterotoxinas A o E solamente. Estos hallazgos indican la necesidad de implementar un eficaz control de la mastitis bovina para disminuir la presencia de S. aureus en leche de tanque y evitar riesgos potenciales para la salud pública.

Intramammary inoculation of Panax ginseng extract in cows at drying off enhances early mammary involution.

This study was designed to evaluate the effects of a single intramammary infusion of Panax ginseng extract on cell proliferation and death mechanism in bovine mammary gland during early involution. Eight mammary quarters from six non-pregnant cows in late lactation were infused with 10 ml of ginseng solution (3 mg/ml), six quarters were treated with 10 ml of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Animals included in the three groups were slaughtered 7 d after inoculation and samples for histological analysis were taken. Morphometric analysis showed a significant increase in percentages of mammary tissue area occupied by stroma in ginseng-treated quarters compared with controls. A significant increase of immunostained area for bax protein and active caspase-3 was observed in ginseng-treated quarters compared with controls, whereas no differences were observed for bcl-2 immunostaining. Expression of bax mRNA was significantly higher in ginseng-treated quarters than in controls. The bax/bcl-2 ratio indicated a significant predominance of bax over bcl-2 mRNA expression in ginseng-treated quarters compared with controls. The rise of epithelial and stromal cell apoptosis in situ by TUNEL was more marked in quarters treated with ginseng than in controls. Ginseng inoculation had no effect on the number of epithelial and stromal proliferating cells labelled with Ki-67 antibody. Ratio of apoptotic to proliferating cells was higher in quarters treated with ginseng compared with controls, indicating a net loss of cells in parenchymal components. Also, the intramammary inoculation of ginseng extract at drying off increased the rate of mammary cell apoptosis without inhibiting cell proliferation. Taken together, these changes are indicative of mammary regression enhancement during early involution.