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The aim of this study was to compare the effects of feeding pasteurized waste milk or saleable milk to calves on weight, health and emergence of antimicrobial resistance in Escherichia coli strains isolated from those calves. An experimental study under field conditions on a commercial pasture-based Argentinian dairy farm was carried out. Forty Holstein calves were assigned randomly to either pasteurized waste milk (PWM) or non-pasteurized saleable milk (SM). The antimicrobial agents (AM) used on the farm, both to treat or prevent diseases, were recorded. The passive immunity level, calf live weight, AM presence in milk, clinical examination of calves, and E. coli isolation and identification, were performed. A total of 258 E. coli strains were isolated from fecal samples (132 isolates from SM calves and 126 from PWM calves at six sampling times). All E. coli isolated were used to perform AM susceptibility tests (disc diffusion and agar dilution). No differences were observed between groups in health parameters, average daily gain or prevalence of resistant E. coli strains to any AM evaluated throughout the study. Peaks of trimethoprim, sulfamethoxazole and enrofloxacin minimum inhibitory concentration (MIC) were observed at 30 d in E. coli from both groups of calves, whilst additional peaks to tetracyclin and ampicillin were observed only in SM calves. All MIC apart from gentamicin decreased at 75 and 90 d of age (during the weaning period). Gentamicin MIC behaved differently, having no peaks and increasing at 90 d only in PWM group. In conclusion, we found no evidence that emergence of antibiotic resistance is related to the consumption of pasteurized waste milk.

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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.

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Bovine pestiviruses are the causative agents of bovine viral diarrhea, a disease that causes severe economic losses in cattle. The aim of this study was to improve their diagnosis by developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set up a protocol for collecting, shipping and preserving bovine pestiviral RNA on filter papers. The developed RT-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 serum pools, 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 bovine samples was analyzed. The sampling method on two different filter papers using bovine blood drops was a useful alternative for diagnostic purposes and allowed to preserve pestiviral RNA for up to 12 months under refrigeration.

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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.

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There is scarce information about the frequency and epidemiological and clinical features associated with the presence of Mycoplasma spp. in Argentine dairy herds. The objectives of this study were to develop a multiplex PCR for identifying M.bovis and M.canadense and to describe the frequency of Mycoplasma spp. isolated from clinical samples submitted to a diagnostic laboratory. Of a total of 1548 samples from intramammary infections, bulk tank milk and biological fluids, 38 Mycoplasma isolates were obtained. M. bovis, M. canadense, M.californicum and M.leachii were detected by using two multiplex PCRs, confirming their presence in clinical conditions in dairy cattle. The techniques used in the present study can be useful to broaden the knowledge about Mycoplasma infections in cattle, since the search for these organisms is not usually included in routine diagnoses.

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The aim of this study was to characterize the protein expression of matrix metalloproteinase-2 (MMP-2) and -- 9 and their inhibitors (TIMP-1 and -2) in mammary tissue of dairy cows with naturally occurring chronic S. aureus intramammary infections (IMI) during active involution. Moreover, the gelatinolytic activity of MMP-2 and -9 in mammary secretions was evaluated. Cows in late lactation that were either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. Protein expression of MMP-2 and -9 in mammary tissues was significantly higher in S. aureus-infected than uninfected quarters at day 14 and 21 of involution. Protein expression of TIMP-1 and -2 was significantly higher in S. aureus-infected than uninfected quarters at day 7, 14 and 21 of involution. The MMP-2/TIMP-1, MMP-2/TIMP-2, MMP-9/TIMP-1 and MMP-9/TIMP-2 ratios were significantly higher in S. aureus-infected compared with uninfected quarters at day 14 of involution. The MMP-2 activity was significantly higher in mammary secretions from S. aureus-infected compared with uninfected quarters at day 1, 2, 7 and 14 of involution. The MMP-9 activity was significantly higher in mammary secretions from infected quarters compared with uninfected quarters at day 7, 14 and 21 of involution. The increased expression of MMP-2 and -9 in mammary tissue as well as the high levels of activity observed in mammary secretion from infected quarters compared with uninfected quarters during active involution, strongly suggests that these gelatinases could contribute to degradation of mammary tissue components during chronic S. aureus IMI. The MMPs/TIMPs imbalance could lead to greater proteolysis and potentially more damage to mammary tissue in S. aureus-infected quarters.

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Our objective was to evaluate the efficacy of intramammary administration, at drying-off, of a Panax ginseng extract (PGe) combined with cephalexin (Ceph) on the post-calving bacteriological cure rate of pre-existing intramammary infections (IMI) and on the occurrence of new IMI during the dry period. In addition, milk yield and somatic cell count (SCC) in the post-treatment lactation were evaluated. One hundred and eight late-lactation cows were randomly divided into two experimental groups and were treated at drying-off with Ceph alone or PGe combined with Ceph.Cure rates for IMI present at drying-off were similar for both treatments (OR = 0.95, 95% CI = 0.33-2.74). Cure rates for Staphylococcus aureus were lower (OR = 15.4, 95% CI = 1.66-142.52) in quarters treated with PGe + Ceph than in those treated with Ceph alone. Intramammary infusion of PGe + Ceph at drying-off had no effect on preventing new dry period IMI (OR = 0.75, 95% CI = 0.38-1.51), compared with infusion of Ceph alone. Milk production and SCC in the ensuing lactation were not affected by PGe + Ceph treatment. In conclusion, addition of PGe to dry cow therapy did not show any advantage over the use of dry cow therapy alone.

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The aims of the research reported here were to identify potential risk factors associated with the presence of Staphylococcus aureus intramammary infection (IMI) in pre partum dairy heifers on 17 dairy farms from three provinces of Argentina and to characterize, at molecular level, isolates from those heifers and lactating cows from two selected herds. A total of 1474 heifers and 4878 lactating cows were studied. The prevalence of Staphylococcus aureus IMI in the heifers, heifers at quarter level and lactating cow mammary quarters was 14.41, 4.82, and 14.65%, respectively. Univariate analysis showed the key variables associated with S. aureus IMI presence in the heifers were: S. aureus IMI prevalence in cows of the lactating herd, the time calves stayed with their dam after birth, the calf rearing system, the place of rearing (own farm or other dairy farm) and fly control on the farm. None of the variables included in the multivariable analysis was associated with the presence of S. aureus IMI in the pre partum heifers, probably due to low variability among management practices used by the farms for rearing the heifer calves. At the molecular level, S. aureus isolates were grouped into three main PFGE clusters and several genotypes within the clusters. Isolates from mammary secretion of pre partum heifers and milk of lactating cows comprised different PFGE clusters in both herds, although two exceptions occurred. The absence of gene fnbpB, which codifies for a virulence factor protein involved in cell invasion by S. aureus, was significantly more frequent in pre partum heifer secretion isolates than in isolates from lactating cow milk. These results suggest that, under these management conditions, isolates from mammary secretions of pre partum heifers do not originate from the milk of lactating cows, but rather other sources to which the heifer is exposed.

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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.

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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.

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