Effects of feeding pasteurized waste milk or saleable milk to calves on weight, health and fecal Escherichia coli antimicrobial resistance.

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.

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.

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.

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.

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.

Efficacy of Panax ginseng extract combined with cephalexin as a dry cow therapy.

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.

Capacity of two Staphylococcus aureus strains with different adaptation genotypes to persist and induce damage in bovine mammary epithelial cells and to activate macrophages.

The aim of this study was to evaluate and compare the ability to adhere/internalize, persist, and induce damage in mammary epithelial cells (MAC-T) of two Staphylococcus aureus strains with different adaptation genotypes (low and high) to the bovine mammary gland (MG). Also, the phagocytic and bactericidal capacity induced after the interaction between macrophages, isolated from mammary secretion, of both S. aureus strains was evaluated. Two isolates (designated 806 and 5011) from bovine intramammary infection (IMI) 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). Strains 806 and 5011 were associated with low (nonpersistent-NP) and high (persistent-P) adaptation to the MG, respectively. Strain 5011 (P), agr group I, cap8 positive and strong biofilm producer showed higher capacity to adhere/internalize in MAC-T compared with strain 806 (NP), characterized as agr group II, cap5 positive and weak biofilm producer. Strain 5011(P) could be recovered from MAC-T lysates up to 72 h pi; while strain 806 (NP) could be recovered only at 4 h pi. Strain 5011 (P) showed greater capacity to induce apoptosis compared with strain 806 (NP) at 4, 24 and 48 h pi. Macrophages infected with strain 5011 (P) showed a greater phagocytic capacity and higher percentage of intracellular reactive oxygen species (ROS) production than strain 806 (NP). No viable bacteria were isolated from macrophages lysates stimulated with any of the S. aureus strains at 2, 4, 8 and 24 h pi. The knowledge of the molecular profile of the S. aureus strains causing bovine mastitis in a herd could become a tool to expose the most prevalent virulence gene patterns and advance in the elucidation of the pathogenesis of chronic mastitis.

Risk factors of S. aureus intramammary infection in pre partum dairy heifers under grazing conditions and molecular characterization of isolates from heifers and cows.

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.

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.

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.

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.

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.

Assessment of the potential utility of different regions of Streptococcus uberis adhesion molecule (SUAM) for mastitis subunit vaccine development.

Streptococcus uberis is one of the most prevalent pathogens causing clinical and subclinical mastitis worldwide. Among bacterial factors involved in intramammary infections caused by this organism, S. uberis adhesion molecule (SUAM) is one of the main virulence factors identified. This molecule is involved in S. uberis internalization to mammary epithelial cells through lactoferrin (Lf) binding. The objective of this study was to evaluate SUAM properties as a potential subunit vaccine component for prevention of S. uberis mastitis. B epitope prediction analysis of SUAM sequence was used to identify potentially immunogenic regions. Since these regions were detected all along the gene, this criterion did not allow selecting a specific region as a potential immunogen. Hence, four fractions of SUAM (-1fr, 2fr, 3fr and 4fr), comprising most of the protein, were cloned and expressed. Every fraction elicited a humoral immune response in mice as predicted by bioinformatics analysis. SUAM-1fr generated antibodies with the highest recognition ability towards SUAM native protein. Moreover, antibodies against SUAM-1fr produced the highest proportion of internalization inhibition of S. uberis to mammary epithelial cells. In conclusion, SUAM immunogenic and functionally relevant regions were identified and allowed to propose SUAM-1fr as a potential candidate for a subunit vaccine for S. uberis mastitis prevention.

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.

Short communication: Relationship between the level of bovine leukemia virus antibody and provirus in blood and milk of cows from a naturally infected herd.

We explored the relationship between the level of bovine leukemia virus antibodies and provirus load during natural infection. For that purpose, a set of 50 blood and milk paired samples were analyzed for the presence of bovine leukemia virus provirus and antibodies. Additionally, provirus load and antibody titers were measured and the relationship between these variables was investigated. Bovine leukemia provirus was detected in 59% of milk samples and a negative correlation was observed between the level of milk provirus load and milk antibody titers. By the consumption of raw milk, calves might be exposed to bovine leukemia virus favoring the perinatal transmission of this disease.

Comparison of phenotypic tests for detecting penicillin G resistance with presence of blaZ gene in Staphylococcus aureus isolated from bovine intramammary infections.

Few studies have described the relationship between genotypic and phenotypic methods for detecting penicillin resistance in Staphylococcus aureus isolated from bovine intramammary infection (IMI). Six phenotypic methods for penicillinase detection were compared with a genotypic method testing the presence of the ß-lactamase gene blaZ in Staph. aureus (n = 150) isolated from bovine IMI. Highest sensitivities and specificities were observed for disk diffusion (DD) (93 and 97.4%), minimum inhibitory concentration (MIC) (90.3 and 97.4%), Cefinase™ (85.9 and 97.4%) and Diatabs™ (85.7 and 98.7%). The estimated cut-off points estimated in the present study can be considered close to the ones indicated by CLSI (2013). The molecular detection of blaZ gene is the only method that may indicate the real or potential capacity of producing ß-lactamase in Staph. aureus. Considering that from a clinical standpoint a false negative result from a phenotypic test is the most unfavourable situation, a combination of standard DD with Diatabs™ or Cefinase™ should be performed by routine mastitis laboratories to minimise false negative results.

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.

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

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.

Immune response of heifers against a Staphylococcus aureus CP5 whole cell vaccine formulated with ISCOMATRIX™ adjuvant.

The shortcomings of Staphylococcus aureus vaccines to control bovine mastitis have been attributed to insufficient capacity of the vaccines to induce opsonizing antibodies and to stimulate cellular immune responses. Types of antigen, administration route and adjuvant used in a vaccine formulation have been identified as critical factors for the development of opsonic antibodies. Current commercially available vaccines for Staph. aureus bovine mastitis control are formulated with Al(OH)3 and oil-based adjuvants. The aim of this study was to evaluate the immune response of heifers immunized with a Staph. aureus CP5 whole cell vaccine formulated either with Al(OH)3 or ISCOMATRIX™. Twenty primigravid Holstein dairy heifers in the last trimester of gestation were immunized either with a vaccine formulated with ISCOMATRIX™ (n = 6), Al(OH)3 (n = 7), or saline solution (placebo) (n = 7). Immunization was carried out 38 and 10 d before calving. Heifers vaccinated with Staph. aureus adjuvanted with ISCOMATRIX™ responded with significantly higher levels of anti-bacterin and anti-CP5 IgG and IgG2 in sera than animals in the Al(OH)3 or control groups. Animals in the ISCOMATRIX™ group responded with significantly higher anti-bacterin specific IgG in whey than animals in the Al(OH)3 and control groups, detected from the first week post calving until 60 d of lactation. Sera from animals inoculated with Staph. aureus in ISCOMATRIX™, obtained 7 d post partum, significantly increased both the number of neutrophils ingesting bacteria and the number of bacteria being ingested by the neutrophils, compared with sera obtained from heifers vaccinated with Al(OH)3 or non-vaccinated controls. These features coupled to safety of the ISCOMATRIX™ formulation, warrant additional studies.