Intramammary Administration Research Articles

Distribution of cefalexin and kanamycin in the mammary tissue following intramammary administration in lactating cow

Distribution of cefalexin and kanamycin in the mammary tissue following intramammary administration in lactating cow

Health management of ewes during pregnancy

The objectives of health management of ewes during pregnancy are as follows: (i) successful completion of pregnancy at term, (ii) birth of healthy and viable lambs, with optimal birth and potential weaning bodyweight, (iii) optimum milk production during the subsequent lactation and (iv) improved management in relation to drug residues in animal products. Knowledge of the physiological background of pregnancy in ewes: changes, mechanisms and interactions, during pregnancy is important for the overall health management of ewes during pregnancy. Health management of pregnant ewes includes diagnosis of pregnancy and evaluation of the number of foetuses borne, which will support strategies for subsequent management of the flock. Nutritional management of ewes depends upon the stage of lactation and specifically aims to (i) prevention of pregnancy toxaemia and other metabolic diseases during the peri-partum period, (ii) formation of colostrum in appropriate quantity and quality, (iii) production of lambs with normal future birth bodyweight and (iv) support of increased milk yield during the subsequent lactation. At the end of lactation, udder management of pregnant ewes includes its clinical examination, culling of ewes considered unsuitable for lactation and, possibly, the intramammary administration of antibiotics; objectives of that procedure are (i) to cure infections which have occurred during the previous lactation and (ii) to prevent development of new mammary infection during the dry period. Management of abortions includes the correct and timely diagnosis of the causative agent of the disorder, as well as the strategic administrations of chemotherapeutic agents, aiming to prevent abortions in flocks with confirmed infection with an abortifacient agent, especially if no appropriate vaccinations had been carried out before the mating season. During the final stage of pregnancy, health management of ewes includes administration of appropriate anthelmintic drugs, aiming to eliminate gastrointestinal helminthes (thus, increasing production output of ewes) and preventing the built-up of parasitic burdens in the environment (thus, reducing infection of lambs during their neonatal period). Vaccinations of pregnant ewes aim to protect these animals, as well as their offspring, especially against diseases which are a frequent cause of neonatal mortality (e.g., clostridial infections). Health management also aims to prevent the main metabolic disorders of pregnant ewes (i.e., pregnancy toxaemia and hypocalcaemia), as well as to monitor flocks for development of these disorders. Health management of pregnant ewes is completed with application of husbandry practices before the start of the lambing season. Finally, in some cases, health management may include induction and synchronisation of lambings, which is a management or therapeutic procedure.

Pharmacokinetic interaction of intramammary ceftriaxone and oral polyherbal drug (Fibrosin®) in goats

The aim of the present study was to determine pharmacokinetic interaction of ceftriaxone and polyherbal drug (Fibrosin(®)) in lactating goats following single dose intramammary administration of ceftriaxone with 1 h pre-single dose oral administration of Fibrosin(®). Pharmacokinetic interaction of ceftriaxone and Fibrosin(®) was evaluated in lactating goats following single dose intramammary administration of ceftriaxone at 50 mg/kg with 1 h pre-single dose oral administration of Fibrosin(®) (1.9 g). Estimation of ceftriaxone and its metabolite, ceftizoxime, was determined by high performance liquid chromatography. Fibrosin(®) treated goats showed a typical absorption-reabsorption phase of ceftriaxone in plasma following intramammary administration. Neither ceftriaxone nor ceftizoxime was detected in the plasma and urine of goats without Fibrosin(®) treatment, however, ceftriaxone persisted for 36 h and ceftizoxime was present from 48 h to 72 h in the plasma of Fibrosin(®) treated goats. Ceftizoxime was also available from 72 h to 360 h post-dosing in milk in the presence of Fibrosin(®) following intramammary administration of ceftriaxone suggesting the polyherbal drug played a major role in the penetration of ceftriaxone from milk to systemic circulation. Furthermore, the polyherbal drug increased the bioavailability of ceftizoxime in milk following the metabolism of ceftriaxone. Polyherbal drug (Fibrosin(®)) plays a major role in the penetration of ceftriaxone from milk to systemic circulation and may be responsible for increased bioavailability of its metabolite in the mammary gland resulting in higher concentration and longer persistence of the drug in milk.

Cefquinome sulfate behavior after intramammary administration in healthy and infected cows

Maintenance of adequate drug concentration at the site of infection is an important problem in mastitis antibiotic therapy, and the efficacy of intramammary β-lactams can be optimized by maintaining the drug concentration at the site of infection above the minimum inhibitory concentration (MIC) as long as possible. The most important pharmacokinetic and pharmacodynamic parameter for efficacy evaluation is time during which drug concentrations exceed the MIC (t>MIC). In this study, we assessed the pharmacokinetic profile of cefquinome (CFQ) after repeated intramammary administration in healthy cows and cows subclinically infected with Staphylococcus aureus as well as the MIC of Staph. aureus field strains. In addition, the degree of drug passage was investigated from udder to bloodstream by measuring systemic drug absorption in healthy and infected animals. Cefquinome concentrations were quantified by HPLC (UV-visible detection) in milk samples collected from quarters and from blood serum samples. The systemic drug absorption was negligible in healthy and subclinically infected animals (maximum concentration 0.09±0.02 and 0.1±0.01μg/mL in healthy and subclinically infected animals, respectively). The MIC90 value for CFQ in Staph. aureus field strains (n=20) was 0.24μg/mL. The pharmacokinetic and pharmacodynamic evaluation, determined by t>MIC, showed an equal persistence of CFQ in all quarters, indicating an equivalent activity of the drug regardless of the pathological status of the udder. Moreover, with literature data regarding CFQ MIC, the t>MIC has been calculated for other bacterial species.

Hot topic: Milk and plasma disposition of thymol following intramammary administration of a phytoceutical mastitis treatment

Despite the recent growth of the organic dairy industry, organic producers and veterinarians have limited information when choosing mastitis treatments for animals in organic dairy production. Organic producers commonly administer homeopathic or other plant-based products without having research evaluating the efficacy of these products and using estimated or no withholding times to treat mastitis and other health problems in their herds. In this pilot study, we attempted to identify several active ingredients of Phyto-Mast (Penn Dutch Cow Care, Narvon, PA), a plant-based mastitis treatment used on organic dairy farms, and to quantify the product residue in milk and plasma after intramammary administration. We developed an assay to quantify thymol (one of the active ingredients in Phyto-Mast) in milk and plasma using gas chromatography and mass spectrometry (GC-MS). Thymol is a volatile aromatic compound with antiinflammatory properties. As a model for dairy cows, 5 healthy, lactating alpine dairy goats were given 5mL of Phyto-Mast per udder half. For 10 d following treatment, we analyzed blood and milk samples for thymol residues using GC-MS. The GC-MS assay was very sensitive for thymol detection, to a concentration of 0.01 μg/mL in plasma. Using thymol as a marker, Phyto-Mast was detectable and quantifiable in plasma beginning with the 15-min posttreatment sample, but was no longer detectable in the 4-h posttreatment sample. Thymol residues were only detected in the 12-h posttreatment milk sample. An inflammatory response was not evident in the udder following phytoceutical administration. Although this study provides information about the elimination of thymol, the product contains several other active chemicals, which may have different pharmacokinetic behaviors. Further analysis and additional study animals will help to determine a milk withholding time for Phyto-Mast. Given the recent growth of the organic dairy industry, understanding the pharmacokinetics of therapeutics used in organic production and developing accurate withholding recommendations will help to ensure milk safety.

Elimination of erythromycin in milk after intramammary administration in cows with specific mastitis: relation to dose, milking frequency and udder health

Elimination of erythromycin in milk following intramammary therapy of specific mastitis in cows was studied. Five cows received therapy in one quarter (G1), and eight in two quarters with five milked twice (G2) and three thrice a day (G3). Dose infused was 300 mg/quarter 12 h × 5 times. The drug concentrations in milk were determined using microbial assay technique with Micrococcus luteus as the test organism. Considerable variations occurred in the excretion of drug; levels for treated quarters being 8.25 to 37.61 μg/ml at first milking that declined rapidly at 24 h and no drug activity was observed beyond 36 h post treatment. In total, about 6-25% of the last infused dose appeared in the milk. Drug crossed to 1/15 quarter (G1), 6/10 quarters (G2) and all the six untreated quarters (G3). Crossover levels were significantly higher in mastitic quarters and for G3 cows, but duration of excretion remained same in all cases. It seems that crossover of erythromycin to untreated quarters is related to the udder health and dose infused.

Cefoperazone sodium preparation behavior after intramammary administration in healthy and infected cows

Selection of the antimicrobial agent and maintenance of adequate drug concentrations at the site of infection are the most relevant problems in mastitis antibiotic therapy. Intramammary drug efficacy can be maximized by keeping drug concentrations at the site of infection above the minimum inhibitory concentration (MIC) as long as possible; the most important pharmacokinetic and pharmacodynamic (PK/PD) measure for efficacy evaluation is time during which drug concentrations exceed the MIC (t>MIC). To evaluate this measure, the PK profile of cefoperazone (CFP) after single intramammary administration in healthy and subclinical infected Staphylococcus aureus cows and the MIC of Staph. aureus field strains were assessed. In addition, the degree of drug passage from udder to bloodstream was investigated by measuring systemic drug absorption in healthy and infected animals. Cefoperazone concentrations were quantified by HPLC in quarter milk samples and blood serum samples. Systemic drug absorption was negligible in healthy animals (0.020±0.006 μg/mL serum at 4h), whereas it was higher in infected animals (0.102±0.079 μg/mL at 4h and 0.025 μg/mL at 24h), probably due to the damage of epithelial cell junctions caused by subclinical infections. The MIC90 value for CFP in Staph. aureus field strains (n=24) was 0.64 μg/mL. The PK/PD evaluation, determined by t>MIC, showed a longer persistence of CFP in infected quarters than in healthy ones (mean residence time was 8.37±1.51 vs. 11.42±5.74h in September and 2.07±0.43 vs. 3.31±0.91h in October), with a t>MIC of 45±6h for infected quarters versus 38±5h for healthy quarters different only in October. This could mean a prolonged time in which microorganisms are exposed to drug activity and thus, a greater efficacy of the drug.

Linum usitatissimum (linseed/flaxseed) fixed oil: antimicrobial activity and efficacy in bovine mastitis

In vitro antimicrobial activity and in vivo therapeutic efficacy of L. usitatissimum (linseed/flaxseed) fixed oil in bovine mastitis were investigated. In vitro antimicrobial activity of L. usitatissimum fixed oil was evaluated against a number of microorganisms by disc diffusion method and MIC determination. The in vivo efficacy of the oil was evaluated in nine mastitis-affected cows divided into three groups (three in each group), following once-a-day intramammary infusion of oil, cefoperazone or an oil-cefoperazone combination for 7 days and by monitoring the California mastitis test score, somatic cell count and microbial count in milk samples. The in vitro antimicrobial activity of the oil against Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli was comparable to that of cefoperazone while the antimicrobial activity against Enterococcus faecalis, Micrococcus luteus and Candida albicans, was greater than that of cefoperazone. In the in vivo study, the oil exhibited significant reduction in the California mastitis test score and somatic cell count in milk samples from infected udders following 7 days of intramammary administration suggesting its anti-inflammatory effect. The microbial count in milk samples was also reduced significantly following oil treatment. The effects were comparable to the treatment by cefoperazone (Mastiwock) alone or in combination with the oil. Apparently, the anti-inflammatory and antimicrobial properties of the oil contribute to its therapeutic efficacy in mastitis; the oil could be used as an alternative treatment for bovine mastitis The results suggest possible therapeutic potential of L. usitatissimum fixed oil in bovine mastitis.

Florfenicol concentrations in milk of lactating cows postreated by intramuscular or intramammary routes

Objetivo. Determinar la concentración de florfenicol en leche bovina después del tratamiento intramuscular o intramamario, para establecer el tiempo de retiro, la eficacia terapéutica, y su influencia sobre la producción láctea. Materiales y métodos. Se seleccionaron doce vacas Holstein en lactancia de la hacienda de Universidad de Antioquia (Colombia). Se asignaron al azar al grupo control (n=6) o al grupo florfenicol (n=6), al cual se le administró 20 mg/kg de florfenicol por vía intramamaria e intramuscular con 15 días de diferencia entre tratamientos. Resultados. La Tmax y Cmax para la vía intramuscular fueron 6 horas y 2.86 mg/L respectivamente. La Tmax y Cmax para la vía intramamaria, fueron estimados como 0 hora y aproximadamente 20000 mg/L respectivamente por extrapolación de la línea de regresión. La fase de eliminación del florfenicol en leche, tuvo una vida media de eliminación (t½) promedio de 19.8 horas y 4.9 horas para la vía intramuscular e intramamaria respectivamente. Cuando la concentración inhibitoria minima (C.I.M) del florfenicol para Stahphylococcus aureus, fue usada como el valor de referencia, la eficacia terapéutica sólo se alcanzó por la vía intramamaria. No existió diferencia estadística significativa entre las producciones lácteas de las vacas tratadas y no tratadas. Conclusiones. De acuerdo con estos resultados, el tiempo de retiro postratamiento no debe ser menor de 3 días para la administración intramamaria y al menos 7 días para la administración intramuscular. La eficacia terapéutica sólo se alcanzó por la vía intramamaria. Además no existió diferencia estadística significativa en la producción láctea de las vacas tratadas con respecto a las no tratadas.

Tissue distribution of cloxacillin after intramammary administration in the isolated perfused bovine udder

BackgroundVarious intramammary suspensions containing cloxacillin benzathine are registered for use in cattle as antibiotics for intramammary use at drying off. To ensure antibacterial efficacy, the glandular tissue concentration of an antimicrobial agent must be sufficient. Since the possibilities to measure concentrations in the different areas of the glandular tissue in vivo are very limited, it was the aim of the present study to examine the distribution of cloxacillin in vitro using the isolated perfused bovine udder.MethodsMammary glands taken at slaughter from healthy lactating cows were perfused in vitro with warmed and gassed Tyrode solution. 600 mg cloxacillin benzathine were administered as Orbenin Extra Dry Cow by the intramammary route to six front and rear quarters each. Samples of glandular tissue - at different distances from and vertical to the teat right up to the udder base - were gathered from the treated quarters after 6 h. Perfusate was also sampled before and hourly after treatment for 6 h. The cloxacillin content of the tissue samples and perfusate samples was analysed by high performance liquid chromatography.ResultsThe concentration of cloxacillin in the glandular tissue of front quarters measured 6 h after administration tended to decrease with increasing vertical distance from the teat. The decrease pattern of the concentration was not quite clear in rear quarters. A considerable variation in the tissue concentrations of cloxacillin was obvious, which reflects in vivo conditions. The concentrations measured in the perfusate samples were below the limit of quantification at all time points, indicating limited absorption of the antibiotic from the glandular tissue.ConclusionAfter intramammary administration of the dry off product containing cloxacillin benzathine concentrations of more than 0.5 μg/g (MIC) were reached in all regions of the front and rear quarters.

Relationship between results of in vitro susceptibility tests and outcomes following treatment with pirlimycin hydrochloride in cows with subclinical mastitis associated with gram-positive pathogens

To determine the association between results of in vitro antimicrobial susceptibility tests and outcomes in cows that received intramammary treatment with pirlimycin hydrochloride for subclinical mastitis associated with gram-positive pathogens. Case-control study. 132 dairy cows (178 mammary glands with subclinical mastitis caused by 194 pathogen isolates). Cows with positive results for a California mastitis test (CMT) were assigned to receive 50 mg of pirlimycin via intramammary administration into each CMT-positive mammary gland every 24 hours for 2 consecutive days or no treatment. Duplicate milk samples were collected before treatment and approximately 21 days later. Target pathogens included coagulase-negative Staphylococcus spp (n = 118 isolates), Streptococcus spp (28), Staphylococcus aureus (7), and other gram-positive cocci (30). Antimicrobial susceptibilities were determined via broth microdilution. Overall treatment success rate was 66% (128/194) for both groups. In vitro resistance to pirlimycin ranged from 0% (0/7 isolates of S aureus) to 50% (13/26 isolates of other gram-positive cocci). For the treated group, 62 of 94 (66%) target pathogens were classified as treatment successes and 32 (34%) were classified as failures. Similarly for the control group, 66 of 100 (66%) target pathogens were classified as treatment successes, whereas 34 (34%) were classified as failures. Many target pathogens from cows with subclinical mastitis were eliminated without treatment, and treatment with pirlimycin did not improve the treatment success rate. Results of in vitro antimicrobial susceptibility tests were not useful as predictors of treatment success following intramammary treatment with pirlimycin.

Effect of endotoxic mastitis on epithelial cell numbers in the milk of dairy cows

To measure epithelial cell percentages and somatic cell counts (SCCs) in milk and determine whether isoflupredone acetate reduces mammary gland epithelial cell sloughing in cows with acute endotoxin-induced mastitis. 13 lactating Holstein cows. Determination of SCC and flow cytometric analysis of cytokeratin-positive (epithelial) cells in milk were performed before and 12 hours after induction of mastitis via intramammary administration of bacterial endotoxin in 8 cows and at the same time points in 5 cows without mastitis. Endotoxin-treated cows received isoflupredone acetate (20 mg) or saline (0.9% NaCl) solution (n = 4/group) IV after signs of mastitis developed. At the 12-hour time point, mean +/- SD percentage of epithelial cells in milk increased from 2.74 +/- 1.93% to 42.11 +/- 36.21% and decreased from 5.73 +/- 4.52% to 5.31 +/- 1.93% in milk from cows with and without mastitis, respectively. Median (range) SCC in milk increased from 195,000 cells/mL (17,000 to 442,000 cells/mL) to 5,437,500 cells/mL (69,000 to 11,036,000 cells/mL) and from 19,000 cells/mL (9,000 to 125,000 cells/mL) to 51,000 cells/mL (10,000 to 835,000 cells/mL) in cows with and without mastitis, respectively. Changes in these variables were significantly greater in mastitis-affected cows. Administration of isoflupredone acetate did not affect epithelial cell percentage or SCC in milk. During the early phase of endotoxin-induced mastitis in dairy cows, large numbers of epithelial cells were sloughed into the milk. Epithelial cell damage likely precedes an influx of immune cells into affected mammary glands and may contribute to breakdown of the blood-milk barrier.

Elimination kinetics of tilmicosin following intramammary administration in lactating dairy cattle

To determine elimination kinetics of tilmicosin in milk following intramammary administration in lactating dairy cattle. Prospective pharmacokinetic study. 6 lactating dairy cows. Following collection of baseline milk samples, 1,200 mg (4 mL) of tilmicosin was infused into the left front and right rear mammary glands of each cow. Approximately 12 hours later, an additional 1,200 mg of tilmicosin was infused into the left front and right rear glands after milking. Milk samples were then collected from each gland at milking time for 40 days. Concentration of tilmicosin was determined by means of ultraperformance liquid chromatography-mass spectrometry, and a milk withdrawal interval for tilmicosin was calculated on the basis of the tolerance limit method. Although there was considerable variation between glands, concentration of tilmicosin was high in milk from treated glands and had a long half-life in treated and untreated glands. Tilmicosin was detected in all treated glands for the entire 40-day study period and was detected in untreated glands for approximately 30 to 35 days. Findings indicated that tilmicosin should not be administered by the intramammary route in lactating dairy cows. Milk from all glands of any cows accidentally treated should be discarded for a minimum of 82 days following intramammary administration.

The Efficacy of a Cefoperazone - Danofloxacin Combination in the Treatment of Subclinical Mastitis in Dairy Cows Caused by Staphylococcus aureus

Summary We report on the effectiveness and profitability of a single-dose intramammary administration of cefoperazone and danofloxacin combination (CDC) against subclinical mastitis in lactating cows. The CDC proved to be effective against Staphylococcus aureus (S. aureus), in vitro checkerboard trials. The presence of mastitis examined on the 892 mammary quarters of 223 lactating cows by bacterial cultures and microscopic somatic cell counting (SCC). Selecting the subjects at random from the 223 animals, twenty-two cows (32 quarters) used for treatment and eleven cows (15 quarters) as controls. Milk samples received one week before the start of treatment and on days 0, 14, 21 and 28 of the experiment. Bacteriological cure examined to the treated quarters. Bacteriological cure defined as no signs of S. aureus after treatment of quarters previously confirmed as infected. The SCC remained unchanged 4 weeks after treatment started, but quarters that were cured (spontaneously or by CDC) showed significant differences on days 21 and 28 (P<0.05). There was no S. aureus in 53.1% of the treated quarters with intramammary CDC, while in the controls there was spontaneous cure of the infection in 13.3% of the untreated quarters with significant (P<0.05) difference. These preliminary results suggest the intramammary application of CDC is an effective and profitable therapy in treating S. aureus infection resulting in subclinical mastitis in lactating cows..

Neutrophils as one of the major haptoglobin sources in mastitis affected milk

The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following intramammary administration of endotoxin or bacteria. However, the sources of milk Hp responsible for such increases are not fully understood. The purpose of this study was to define the source of milk Hp from dairy cows with naturally occurring mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups according to SCC as group A: < 100 (n = 19); B: 100–200 (n = 10); C: 201–500 (n = 10); and D: > 500 × 103 (n = 11) cells/mL. Our results reveal that milk Hp concentrations were correlated with SCC (r = 0.742; P < 0.01), and concentrations in group D were ~10-fold higher than in group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that the milk somatic cells from group D were not only capable of synthesizing Hp but could also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double confocal immunofluorescence, and Hp releasing experiments demonstrate that neutrophils were associated with the biosynthesis and release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium of mammary gland tissue with mastitis and was also expressed in the cultured mammary epithelial cells. We propose that neutrophils and epithelial cells may play an essential role in elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary tissues and circulation.

Elimination Kinetics of Tilmicosin Following Intramammary Administration in Lactating Dairy Cattle

Tilmicosin is a macrolide antibiotic approved for both the treatment and control of respiratory disease in beef cattle and dairy cattle under 20 months of age. The drug is not approved for use in lactating dairy cattle; however, tilmicosin is sometimes used in an extralabel manner by bovine practitioners for the intramammary treatment of mastitis. This practice results in detectible residues and has resulted in several instances of milk residue violation referred to the Food Animal Residue Avoidance Databank (FARAD). The purpose of this study was to determine the elimination kinetics of tilmicosin in milk following an intramammary infusion so an appropriate proper milk withdrawal interval could be determined.

Tissue distribution of benzylpenicillin after intramammary administration in the isolated perfused bovine udder

Udders from previously healthy lactating cows were perfused with warmed and gassed Tyrode solution in vitro. Benzylpenicillin was administered in three formulations: an oily suspension with micronized particles of <10 μm diameter, an oily suspension with average particle size of 40 μm and an aqueous solution (3 million IU benzylpenicillin–potassium, volume 15 mL). The antibiotics were administered intracisternally to six front and six rear quarters each. Moreover, a dry-off-ointment (100 000 IU benzylpenicillin–potassium and 100 000 IU benzylpenicillin–benzathine, volume 7.5 mL) was tested in four udder halves. Perfusate samples were collected over 3 h. Furthermore, glandular tissue at different vertical distances from the teat base and the regional lymph node were sampled after 3 h. The determination of benzylpenicillin was performed by high pressure liquid chromatography with UV detection. With increasing distance from the teat base, the concentration of benzylpenicillin in tissue exponentially decreased. Using the aqueous solution or oily suspension that contained micronized active principle, higher concentrations were reached compared to the formulation with particle sizes of 40 μm. In udder lymph nodes, the concentration was highest after treatment with the coarse suspension. The transfer from the dry-off-ointment with benzathine-salt into perfusate was very low. These results suggest that it is possible to study tissue distribution of antibiotics in the isolated perfused bovine udder.

Exposure to endotoxin during estrus alters the timing of ovulation and hormonal concentrations in cows

The effect of intramammary (IMM) or intravenous (IV) administration of E. coli endotoxin (LPS), at the onset of estrus, at the time of ovulation was examined. Steroid and gonadotropin concentrations around ovulation were also determined. Lactating Holstein cows ( n = 33) were assigned to saline-controls ( n = 12) and treated with LPS-IV (0.5 μg/kg; n = 13) or LPS-IMM (10 μg; n = 8). Synchronized cows were observed continuously for estrus. LPS (or saline) was injected within 30 min from the onset of standing estrus, at peak estradiol concentrations. The typical rise of body temperature, somatic cell count, cortisol, and NAGase activity was noted. One-third of both LPS-IV- and LPS-IMM-treated cows were manifested by an extended estrus to ovulation (E–O) interval of around 75 h or did not ovulate, compared with about 30 h in the other 2/3 of LPS cows and all controls. Estradiol concentrations 24 h before and after LPS did not differ between groups. However, LPS-IV cows with extended intervals exhibited another estrus and an additional rise of estradiol followed by delayed ovulation. LPS-treated cows with a delayed E–O interval had low or delayed LH surge; two LPS-treated cows did not exhibit LH surge and did not ovulate. All control cows exhibited normal hormone levels. Delayed ovulation was associated with a delayed rise of luteal progesterone. The results indicated that exposing cows to endotoxin during estrus induced a decreased and delayed LH surge in one-third of the cows. This was associated with delayed ovulation, which reduces the chances of successful fertilization.

Intramammary Application of Non-Methylated-CpG Oligodeoxynucleotides (CpG) Inhibits both Local and Systemic Mammary Carcinogenesis in Female BALB/c Her-2/neu Transgenic Mice

CpG are powerful drugs activating the innate immune system. In this study, the ability of their intramammary administration in impeding the devastating progression of carcinogenesis in all the mammary glands of female BALB/c mice transgenic for the rat neu transforming oncogene was assessed. Starting when in situ carcinomas were scattered over all their mammary glands (week 10), mice received CpG injections in the stroma of the fourth left gland. Local neoplastic progression was inhibited by six monthly administrations. CpG not only delayed the onset of carcinomas in the injected gland, but also hampered their progression. Extended latency was observed for tumors in glands both close to and far from the injection site. When the experiment ended (week 45), no tumors were palpable in 67% of the injected glands and a markedly impaired tumor growth was evident in the others. An impressive local infiltrate of CD11b(+) cells with the morphologic features of macrophages, plasma cells, B220(+) B cells, and CD4(+) and CD8(+) T cells was quickly recruited to the CpG-treated glands. High quantities of IFN-gamma producing cells were only present in the ipsilateral axillary draining lymph nodes of the treated glands. Enhanced natural killer (NK) lytic activity was also detected in the spleens. Inhibition of progression was weaker when only four injections were given, and abolished by in vivo depletion of NK cells. CpG monotherapy is thus effective in an aggressive model of autochthonous cancer. The results strongly support the administration of CpG as a local monotherapy of multiple invasive microscopic lesions.

Dose-dependent immune response in milk cells and mammary tissue after intramammary administration of lipopolysaccharide in dairy cows

The immune response in milk cells and the status of mammary tight junctions (TJ) in response to intramammary (IM) infusion of different doses of &lt;i&gt;Escherichia col&lt;/i&gt;i lipopolysaccharide (LPS) was investigated. &lt;i&gt;Experiment I&lt;/i&gt;: Seven German Braunvieh cows were IM infused into one quarter with 1 &amp;mu;g (LPS-1) and 3 &amp;mu;g (LPS-3) of LPS, respectively, and the contralateral control quarter with saline (9 g/l; C). Milk samples were taken immediately before and 12, 24, 36, 48, 60, 84 and 108 h after infusion and analysed for somatic cell counts (SCC), lactose, sodium (Na) and chloride (Cl) ions, and electrical conductivity (EC). Milk cell mRNA expression of various inflammatory factors was quantified by real-time RT-PCR. Blood samples were taken immediately after milking for the analysis of leukocytes (WBC), polymorphonuclear neutrophils (PMN), Na and Cl. Milk SCC, lactose, Na, Cl and EC did not differ significantly between LPS-1 and C quarters after the challenge. In LPS-3 quarters SCC levels increased within the first 12 h, reached peak levels between 12 and 36 h (&lt;i&gt;P&lt;/i&gt; &amp;le; 0.001) and decreased (&lt;i&gt;P&lt;/i&gt; &amp;le; 0.05) thereafter to reach baseline at 108 hours. Lactose in LPS-3 quarters decreased (&lt;i&gt;P&lt;/i&gt; &amp;le; 0.05) to a minimum at 24 h and increased slightly thereafter while EC, Na, and Cl increased transiently in response to LPS-3. WBC and PMN levels in both groups decreased numerically within 24 h after LPS administration. In LPS-1, WBC at 24, 48 and 108 h were significantly lower whereas in LPS-3 they were significantly higher than at time 0. TNF&amp;alpha;-mRNA expression in both groups did not change in response to IM LPS-challenge. IL-1&amp;beta;-mRNA expression at 12, 24 and 36 h in LPS-1 quarters increased significantly as compared to time 0. In LPS-3 quarters the mRNA expression values of all tested ILs increased significantly as compared to time 0 within 12 h after LPS-challenge. IL-1&amp;beta;-mRNA expression decreased (&lt;i&gt;P&lt;/i&gt; &amp;le; 0.05) at 48 and 84 h in LPS quarters. IL-8 mRNA was significantly decreased at 84 h after challenge in LPS-3 quarters. COX-2-mRNA expression in LPS-1 quarters decreased significantly as compared to time 0 at 48, 84 and 108 h, with a minimum at 84 h (&lt;i&gt;P&lt;/i&gt; &amp;le; 0.05). In LPS-3 quarters COX-2-mRNA levels increased (&lt;i&gt;P&lt;/i&gt; &amp;le; 0.05) within 48 h after the LPS-challenge. &lt;i&gt;Experiment II&lt;/i&gt;: Six cows (5 German Braunvieh, 1 Brown Swiss) were injected in one quarter with 100 &amp;mu;g LPS and in the contralateral quarter with saline (9 g/l; C). Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of TJ proteins occludin (OCLN) and zonula occludens (ZO-) 1, 2 and 3 were quantified by real-time RT-PCR. OCLN-mRNA expression did not change in response to the IM infusion while that of ZO-1, ZO-2 and ZO-3 decreased significantly within six hours. In conclusion, a dose of 1 &amp;mu;g LPS did not initiate a immune response in the mammary gland. Furthermore the dose of 100 &amp;mu;g of LPS enhanced TJ permeability by reducing TJ plaque proteins density.