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Twenty Friesian cows were randomly assigned to one of four prepartum diets in a 2 x 2 factorially designed experiment to determine the effect of anionic salts contained in a concentrate mixture and magnesium (Mg) intake on some blood and urine minerals in cows fed a grass silage based diet. Four diets provided either 16 g or 33 g total dietary Mg/day, and had either a low or high cation-anion difference. Dietary cation-anion balance (DCAB) of the diets, calculated as milliequivalents [(Na+ + K+) - (Cl- + S2-)], was +31 mEq/kg dry matter (DM) in the low DCAB group and +340 mEq/kg DM in the high DCAB group. DCAB was formulated using NH4Cl, (NH4)2SO4 and MgCl2 as anionic salts. Cows received grass silage (5.2 kg DM), hay (1.0 kg DM) and concentrate mixture (1.5 kg DM) until calving. Blood and urine samples were collected 4, 3, 2 and 1 week before the expected calving date, at calving, the day after calving and 1 week following calving. Cows fed the low DCAB diet had a lower urinary pH (P, Tutkimuksessa selvitettiin ummessaolevien lehmien kationi-anionitasapainon alentamisen vaikutuksia, kun magnesiumin saanti oli joko suomalaisten kivennäisnormien (16 g Mg/pv, 0.2% kuiva-aineesta) tai hollantilaistenkivennäisnormien mukaista (33 g/Mg, 0.4% kuiva-aineesta). Kationi-anionitasapaino laskettiin [(Na+ + K+) - (Cl- + S2-)] mEq/kg kuiva-ainetta (ka). Se oli joko +340 tai +31 mEq/kg ka. Magnesiumlisä annettiin magnesiumoksidina. Suoloina käytettiin magnesiumkloridia, ammoniumkloridia ja -sulfaattia. Lehmät saivat säilörehua (5.2 kg ka), heinää (1.0 kg ka) ja täysrehua (1.5 kg ka) neljä viikkoa ennen odotettua poikimista poikimispäivään saakka. Veri- ja virtsanäytteitä otettiin 4, 3, 2 ja 1 viikkoa ennen odotettua poikimista, poikimispäivänä sekä 1 vrk ja 1 viikko poikimisen jälkeen. Tulosten mukaan magnesiumoksidista ei ollut hyötyä ummessolevien lehmien magnesiumlisänä. Nykyistä suomalaista magnesiumsuositusta tulisi silti tarkistaa, koska kokeessa ilmeni muutamia hypomagnesiatapauksia poikimisvuorokautena. +31 mEq/kg ka ei aiheuttanut muutoksia verestä ja virtsasta mitattuihin parametreihin. Se ainoastaan lisäsi kalsiumin eritystä virtsaan ja alensi virtsan pH-arvoa verrattuna positiivisempaan kationi-anionitasapainoon (+340 mEq/kg/ka). Näin ollen kationi-anionitasapaino tulisi säätää pienemmäksi, jotta siitä olisi hyötyä poikimisen aikaiseen kalsiumaineenvaihduntaan.

Twelve Ayrshire and eight Friesian cows were randomly assigned to one of four prepartum diets in a 2 x 2 factorially designed experiment to determine the effect of anionic diet and calcium (Ca) intake on Ca metabolism, acid-base status and feed intake of grass silage based diets during the dry period. Four diets provided either 34 g or 74 g total dietary Ca/day, and were either anionic or cationic. Dietary cation-anion balance (DCAB), calculated as milliequivalents [(Na+ + K+) - (Cl- + S2-)], was -247 mEq/kg dry matter (DM) in the low DCAB group and +34 mEq/kg DM in the high DCAB group. DCAB was formulated using NH4Cl, (NH4)2SO4 and MgCl2 as anionic salts. Cows received grass silage (5.2 kg DM), hay (0.9 kg DM) and concentrate mixture (1.6 kg DM) until calving. Blood and urine samples were collected 4, 3, 2 and 1 week before the expected calving date, at calving, the day after calving and 1 week following calving. The results indicate that the reduction of cation-anion balance induced mild metabolic acidosis and increased the ability of the cow to maintain blood Ca concentration. However, DCAB should be higher since urinary pH decreased markedly (< 6) and so remarkable changes in some blood electrolyte concentrations were noticed., Kokeessa selvitettiin kationi-anionitasapainon ja kalsiumin saannin vaikutusta ummessaolevien lehmien tiettyihin verestä ja virtsasta mitattuihin kivennäisarvoihin sekä happo-emästasapainoon säilörehuvaltaisella ruokinnalla. Tarkoituksena oli testata, voidaanko anionisten suolojen lisäämisellä ennen poikimista vaikuttaa poikimisen aikaiseen kalsiumaineenvaihduntaan. Lisäksi tutkittiin, onko kalsiumin saannilla vaikutusta anionisten suolojen tehoon. Kationi-anionitasapaino laskettiin [(Na+ + K+) - (Cl- + S2-)] mEq/kg kuiva-ainetta (ka). Se oli joko -273 tai +34 mEq/kg ka.Kalsiumin saanti oli joko 34 g tai 74 g kalsiumia päivässä. Suoloina käytettiin magnesiumkloridia, ammoniumkloridia ja -sulfaattia. Lehmät saivat säilörehua (5.2 kg ka), heinää (0.9 kg ka) ja täysrehua (1.4 kg ka) neljä viikkoa ennen odotettua poikimista poikimispäivään saakka. Veri- ja virtsanäytteitä otettiin 4, 3, 2 ja 1 viikkoa ennen odotettua poikimista sekä poikimispäivänä, 1 vrk ja 1 viikko poikimisen jälkeen. Lehmien, joiden kationi-anionitasapaino oli säädetty negatiiviseksi, veren ionisoitunut kalsium säilyi poikimisen aikaan vakaana verrattuna lehmiin, joiden kationi-anionitasapaino oli positiivinen. Kationi-anionitasapaino -247 mEq/kg ka osoittautui kuitenkin liian negatiiviseksi, koska se aiheutti muutoksia lehmien veren happo-emäsarvoihin sekä alensi liikaa virtsan pH-arvoa (

Summary Ninety-seven dairy cows suffering from traumatic reticuloperitonitis, abomasal displacement, and other gastrointestinal disorders or dystocia were referred for abdominal surgery. Plasma protein concentrations, including plasma fibrinogen and haptoglobin, and conventional haematological variables were determined both pre- and postoperatively. The examined variables were not of value in predicting the outcome of the abdominal disorders in general, but fibrinogen concentrations may be helpful in postoperative monitoring of infectious complications, such as peritonitis. Fifty-five per cent of the cows had increased haptoglobin concentrations preoperatively and abdominal surgery led to an additional haptoglobin increase for a further 2–3 days. It is recommended that pre- and postoperative haptoglobin values should be compared when monitoring infectious complications. Non-parametric receiver operating characteristics (ROC) analysis suggested that plasma haptoglobin concentration was the most accurate parameter for differentiating traumatic reticuloperitonitis from other gastrointestinal disorders, but plasma fibrinogen and globulin concentrations were also of value in this respect.

This study examined the capability of milk somatic cell count (SCC) and NAGase activity to discriminate between quarters that had been cured versus those that had not been cured at 4 wk after antimicrobial therapy for clinical mastitis. The distribution of microorganisms that were isolated before therapy from 630 quarters with mastitis was as follows: 225 strains of Staphylococcus aureus , 96 strains of coagulase-negative staphylococci, 152 strains of streptococci ( Streptococcus dysgalactiae and Streptococcus uberis ), and 157 strains of coliform bacteria. Bacteriological cure rates were 35% for mastitis caused by Staph. aureus , 75% for mastitis caused by coagulase-negative staphylococci, 66% for mastitis caused by streptococci, and 72% for mastitis caused by coliforms. Diagnostic accuracy of milk SCC and NAGase and their interquarter ratios for predicting bacteriological status of the control samples was assessed by calculating sensitivity, specificity, and accuracy and by means of receiver operating characteristic analysis. The efficiency of milk SCC and NAGase for predicting bacteriological cure was greatest for cows that had been infected with Staph. aureus . The main problem in detecting coagulase-negative staphylococci was low sensitivity, and the main problem in detecting streptococci and coliforms was low specificity. Receiver operating characteristic analysis is not completely suitable for the detection of mastitis because reference method bacteriology and indirect tests can never fully agree. To assess the recovery of cows from mastitis caused by Staph. aureus , bacteriology should be supplemented with an examination of milk SCC or NAGase activity at threshold values such as those presented here.

Two-hundred seventeen Staphylococcus aureus isolates from 116 dairy cows with intramammary infections were analyzed by pulsed-field gel electrophoresis to study the association between symptom severity, persistence of infection, and bacterial genotype. Among five main genotypes infecting 90% of the cows, one was associated with severe clinical symptoms but reduced persistence.

The present study reports a comparative proteome cataloging of a bovine mastitis and a human-associated Staphylococcus epidermidis strain with a specific focus on surfome (cell-wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC-MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house-keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy-metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein- and DNA-mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO2 conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 (http://proteomecentral.proteomexchange.org/dataset/PXD000404).

SummaryTen pregnant heifers were inoculated in both hind udder quarters with a mixture of Actinomyces pyogenes, Fusobacterium necrophorum and Peptostreptococcus indolicus. Development of the experimental mastitis was monitored by sequential clinical and bacteriological examinations, and by blood acute phase protein profiles. Sequential changes in plasma fibrinogen, serum haptoglobin, acid-soluble glycoproteins and α1-proteinase inhibitor activity were analysed and compared with both the clinical and bacteriological findings and the final outcome of the disease after calving. All ten heifers developed moderate to severe clinical mastitis. Four recovered completely, had a normal lactation after calving and exhibited only transient fever and moderate local signs. In six of the heifers the course of the disease was severe, and the inoculated quarters failed to produce milk after calving. The acute phase response of the four heifers that recovered was also significantly milder than that of the other six heifers. Haptoglobin and acid-soluble glycoproteins were most effective in indicating the severity of the infection and predicting the final outcome of the disease. Fibrinogen was a reliable indicator for detecting the presence of bacterial infection in all heifers. α1-Proteinase inhibitor activity was of low diagnostic value in this study.

A total of 169 Escherichia coli strains were isolated from cows with cases of clinical mastitis. β -Glucuronidase production, serum sensitivity, and susceptibility to selected antibacterials were analyzed using the fluorometric β -glucuronidase assay. About 89% (150 to 169) of the isolates tested positive for β -glucuronidase. Of these isolates producing β -glucuronidase, 102 (68%) were resistant or moderately resistant bovine serum. The antibacterial susceptibility of 96 isolates was tested in broth and milk. There was a significant shift from lower fluorometric minimum inhibitory concentration for tetracycline, sulfadoxin-trimethoprim, enrofloxacin, and gentamicin in broth to higher fluorometric minimum inhibitory concentration in milk. Serum sensitivity and susceptibility to tested antibacterials in broth or in milk were not related. Gentamicin and sulfadoxin-trimethoprim seemed to be more potent in mastitic milk than in normal milk, suggesting a possible synergistic effect between these exogenous antibacterials and the indigenous antibacterial agents in mastitic milk.

A high performance liquid chromatography (HPLC) method was developed, validated and used to analyse haptoglobin concentrations in serum samples taken from eight cows which had been challenged twice intramammarily with Escherichia coli. The results of the HPLC were compared with those from a photometric assay. The kinetics of the haptoglobin response were analysed with pharmacokinetic computer software. In contrast with the photometric assay, the HPLC was sufficiently sensitive to detect normal background levels of bovine serum haptoglobin. The serum haptoglobin concentrations of healthy cows ranged from 22 to 47 mg litre-1. As the concentration of haptoglobin increased, the results of the two methods correlated well (r = 0.96). A 52-fold increase in serum haptoglobin was detected after the first challenge with E coli. The mean pharmacokinetic parameters of the response after the first challenge were: lag phase 12 hours, t2/1increase 20 hours, tmax 72 hours, t1/2decrease 46 hours, and mean residence time 112 hours. The second challenge three weeks later resulted in a significantly lower haptoglobin response, the area under curve being 35 per cent of that after the first challenge. The clinical signs and inflammatory changes in the milk did not differ significantly between the challenges.

Five Ayrshire cows were given enrofloxacin (5 mg/kg body weight) intravenously (i.v.), intramuscularly (i.m.) and subcutaneously (s.c.). The antimicrobial activity was measured in milk and serum samples using the agar-diffusion technique. High-performance liquid chromatography (HPLC) assay was used to study the extent of metabolism of enrofloxacin to ciprofloxacin. Analysis of the serum concentration-time data was based on statistical moment theory. Mean t1/2 beta of antimicrobial activity in serum was 1.7, 5.9 and 5.6 h after i.v., i.m. and s.c. administration, respectively. Both i.m. and s.c. routes were associated with a marked flip-flop phenomenon. Based on HPLC analysis of serum samples, the half-lives of enrofloxacin and ciprofloxacin were approximately the same. A marked proportion of enrofloxacin was metabolized to ciprofloxacin. The enrofloxacin fraction bound in vitro to serum proteins was 36-45%. About 0.2% of the total enrofloxacin dose was found in milk during the first 24 h and the amount transferred did not depend on the route of administration. Based on the HPLC data, enrofloxacin concentration in milk was parallel to that in serum, while ciprofloxacin was concentrated in milk. After i.v. injection, the peak concentration of enrofloxacin in milk was reached between 0.7 and 1.3 h but occurred much later for ciprofloxacin (tmax 5-8 h). After i.m. and s.c. administration the concentration-time curves for both enrofloxacin and ciprofloxacin in milk were shallow and there were no obvious peaks.

Background The blaZ gene encoding penicillin resistance can be located either chromosomally or on plasmids. The aim of this study was to investigate the genetic relationships and to determine the location of the blaZ gene in S. aureus isolated in bovine mastitis in Finland and Sweden. Methods Seventy-eight β-lactamase positive S. aureus isolates from bovine mastitis (34 from Finland and 44 from Sweden) were included in the study. The localization of blaZ gene was determined by Southern blotting. The blaZ genes of the isolates were sequenced and the sequences were translated to beta-lactamase proteins and further grouped as different protein signatures. The isolates and, as control, 33 Swedish and 36 Finnish beta-lactamase negative isolates were typed with pulsed-field gel electrophoresis (PFGE). Results In 26 out of 34 Finnish isolates (76.5%) and in 25 out of 44 Swedish isolates (56.8%) the blaZ gene was localized on a plasmid. Six different protein signatures were found. One signature was found only in four Swedish isolates, but all other signatures were found both in Finnish and Swedish isolates. The PFGE results revealed a diversity of S. aureus clones. The protein signatures were not clearly associated with certain pulsotypes. Conclusions The plasmid location of the blaZ gene was not statistically significantly more common in Finland than in Sweden, and hence does not explain the higher proportion of penicillin-resistant isolates of S. aureus causing bovine mastitis in Finland compared to Sweden.

Two udder hind quarters of ten pregnant heifers were inoculated experimentally with a combination of Actinomyces pyogenes, Peptostreptococcus indolicus and Fusobacterium necrophorum at a dose of 9×10 8 colony forming units (CFU) of each species of bacteria. The development of aerobic-anaerobic mastitis was followed-up clinically and through analysis of udder secretion samples. All heifers developed acute clinical mastitis. Thick, purulent secretions with foul odour were already present 24 h after infusion. At 32 h post inoculation, 19 of 20 quarters harboured all infused bacteria, the viable counts of bacteria varying from 10 4 to 10 5 CFU/ml. No bacteria were isolated from blood samples taken simultaneously. NAGase values of udder secretions, indicating tissue damage, were high. The heifers were treated systemically with penicillin G alone, or in combination with tinidazole. Treatment started 32 h after inoculation and was continued for three days. The anaerobic bacteria ( F. necrophorum and P. indolicus ) were usually eliminated within four days, while A. pyogenes infections often persisted in the quarters. Penicillin G with tinidazole was not more effective than penicillin G alone: of the four heifers which recovered from infection, two were treated with penicillin G alone and two with the drug combination. The most important factor affecting the recovery seemed to be the host response of individual heifers. The six heifers which did not recover from the infection were continuously infected after calving, harbouring A. pyogenes in ten quarters, and P. indolicus and F. necrophorum each in three quarters.

Antimicrobial agents are usually administered to cattle intramuscularly (im). After im injections, tissue damage at the injection sites may occur. This may be caused by the vehicle, the active ingredient, or both. Other factors affecting tissue reaction are pH, osmotic status, injection volume, concentration of the antimicrobial agent, and the injection technique used. Repeated injections and large injection volumes promote tissue damage (Kern 1987).

The predisposing effect of teat damage on mastitis caused by staphylococci and the pathogenicity of Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus epidermidis were investigated with an experimental model. The study included three experiments in which the teat canal orifice of 5 cows was slightly abraded. Experimental and control quarters were challenged with a staphylococcal suspension, and the status of the quarters was monitored. Virulence of the staphylococcal strains was studied using a protein-binding test with 125I-labeled proteins (fibronectin, fibrinogen, vitronectin, collagen type I and II, and IgG). Abrasion on the teat orifice epithelium was a predisposing factor for staphylococcal infections. Teat canal infection or colonization developed in 93% of experimental quarters and in 53% of control quarters; IMI developed in 73% of experimental quarters, but in none of the control quarters. Quarter IMI developed more consistently when the contaminating agent was S. aureus. Staphylococcus hyicus was very effective in causing teat canal infections, but S. epidermidis appeared to be less infectious. The S. aureus strain had strong binding sites for most of the proteins tested. The S. hyicus and S. epidermidis strains showed no binding, or only very weak binding, which correlated with lower infection rates.

Summary A total of 168 coagulase-negative staphylococci (CNS) strains were isolated from milk samples taken from cows with clinical mastitis. The samples were collected between January 1990 and August 1992 from cows in the veterinary surveillance area of the Ambulatory Clinic, College of Veterinary Medicine, Hautjarvi, Finland. In 100 cases the effect of antibiotic treatment was evaluated 3–4 weeks after initial sampling. Clinical symptoms of the animals were recorded, and the inflammatory status of their udders was evaluated using the CMT test and assessing milk NAGase activity. CNS mastitis was most common in young cows during early lactation. Staphylococcus hyicus, S. simulans and S. epidermidis were the most frequently isolated CNS. Clinical symptoms were most severe with S. hyicus. Cure rates for CNS induced mastitis were high.

Biofilm and slime formation assists bacteria in avoiding the host immune defence and antimicrobial therapy. It is suspected to affect the severity or persistence of mastitis caused by coagulase-negative staphylococci (CNS), which are a common cause of bovine mastitis. The phenotypic biofilm formation ability of 244 CNS isolates (199 isolates from bovine mastitis and 52 type and reference strains) was investigated with a tissue culture plate (TCP) assay and fluorescent in situ hybridization (FISH). Slime production of the strains was assessed using Congo red agar (CRA) plates. Additionally, genes encoding the adhesion proteins MSCRAMM (microbial surface components recognizing adhesive matrix molecules) and biofilm-associated proteins (bap) were detected. The severity of intramammary infection (IMI) in mastitis from which the isolates originated was measured with milk N-acetyl-β-d-glucosaminidase (NAGase) activity. One-third of isolates from mastitis produced biofilm when analysed with TCP or FISH. The kappa test value, measuring the agreement between two tests, differed between CNS species. Slime production was less frequent for isolates of the common mastitis species Staphylococcus chromogenes (0.2% of isolates produced slime) and Staphylococcus simulans (3.5%) compared to Staphylococcus epidermidis (40%). No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was rare in isolates originating from IMI. Only 12.7% of isolates from persistent IMI and 1.8% of isolates from spontaneously eliminated IMI produced slime. The eno gene encoding laminin-binding protein was most frequently detected among the isolates from mastitis, 75% of them having this gene. Only a few other MSCRAMM genes were detected.

Lactoferrin (Lf) is a molecule naturally present in bovine milk that affects the availability and transport systems of iron. Lf also binds endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria and modulates the immunological response. In the present study, concentrations of bovine Lf (bLf) and citrate in milk were determined in early (EL) and late (LL) lactating dairy cows, using an experimentally induced endotoxin mastitis model and a crossover design. Nine clinically healthy Finnish Ayrshire cows were challenged twice with 100 μg endotoxin infused into one udder quarter. Milk samples were collected from the challenged and control quarters of each cow before and after endotoxin infusion during 3 d, and bLf and citrate concentrations were measured. In all cows, clinical signs of mastitis were seen at both times of challenge, but the response was more severe in EL than in LL. Concentration of bLf in the milk started to rise approximately 8 h after endotoxin infusion and was still higher than normal on the third day, especially in the late-lactating cows. In milk of the LL group, concentrations of bLf were significantly higher than in the EL group. In contrast, concentrations of citrate were higher in milk of the EL cows compared with the LL cows. Concentration of bLf and citrate varied substantially among cows. The molar ratio of citrate to bLf before and after challenge was significantly higher during the EL period. The results of this study partly explain why cows in early lactation are more susceptible to intramammary infections and why mastitis is more severe in them.

Fast and reliable identification of the microorganisms causing mastitis is important for management of the disease and for targeting antimicrobial treatment. Methods based on PCR are being used increasingly in mastitis diagnostics. Comprehensive field comparisons of PCR and traditional milk bacteriology have not been available. The results of a PCR kit capable of detecting 11 important etiological agents of mastitis directly from milk in 4 h were compared with those of conventional bacterial culture (48 h). In total, 1,000 quarter milk samples were taken from cows with clinical or subclinical mastitis, or from clinically healthy quarters with low somatic cell count (SCC). Bacterial culture identified udder pathogens in 600/780 (77%) of the clinical samples, whereas PCR identified bacteria in 691/780 (89%) of the clinical samples. The PCR analysis detected major pathogens in a large number of clinical samples that were negative for the species in culture. These included 53 samples positive for Staphylococcus aureus by PCR, but negative by culture. A total of 137 samples from clinical mastitis, 5 samples from subclinical mastitis, and 1 sample from a healthy quarter were positive for 3 or more bacterial species in PCR, whereas culture identified 3 or more species in 60 samples from clinical mastitis. Culture identified a species not targeted by the PCR test in 44 samples from clinical mastitis and in 9 samples from subclinical mastitis. Low SCC samples provided a small number of positive results both in culture (4/93; 4.3%) and by PCR (7/93; 7.5%). In conclusion, the PCR kit provided several benefits over conventional culture, including speed, automated interpretation of results, and increased sensitivity. This kit holds much promise as a tool to complement traditional methods in identification of pathogens. In conventional mastitis bacteriology, a sample with 3 or more species is considered contaminated, and resampling of the cow is recommended. Further study is required to investigate how high sensitivity of PCR and its quantitative features can be applied to improve separation of relevant udder pathogens from likely contaminants in samples where multiple species are detected. Furthermore, increasing the number of species targeted by the PCR test would be advantageous.

Six transgenic cows producing recombinant human lactoferrin (rhLf) in their milk and five normal cows at the same lactation stage were experimentally infected with Staphylococcus chromogenes to study the effect of a high concentration of lactoferrin in milk. Coagulase-negative staphylococci such as S. chromogenes have become very common as agents causing mild or subclinical mastitis. All transgenic cows became infected but showed no clinical signs, unlike the control cows, which developed mild clinical mastitis. Transgenic cows eliminated bacteria faster from the quarters than did the controls. Local clinical signs were milder, and the inflammatory reaction assessed by NAGase activity in the milk and by the concentration of milk amyloid A was lower in the transgenic cows. The mild response probably reflected the rapid elimination of bacteria. The milk concentration of rhLf remained constant throughout the study period, but the total concentration of bovine lactoferrin in the milk peaked in both groups at 46 h post-challenge. Three cows, all in the control group, exhibited systemic acute phase response as increased concentrations of serum amyloid A in the blood circulation. Transgenic cows with a high concentration of human lactoferrin in their milk seemed to be protected from clinical disease and from prolonged inflammatory reaction, but not from experimental intramammary infection induced by S. chromogenes .

The efficacy of the combination of systemic enrofloxacin (5mg/kg twice with a 24-h interval, first dose i.v., second dose s.c.) and the nonsteroidal antiinflammatory agent ketoprofen (3mg/kg i.m. or 4 mg/kg per os daily for 1 to 3 d) treatment was compared with antiinflammatory treatment only in dairy cows with naturally occurring acute clinical Escherichia coli mastitis. A total of 132 cows with acute clinical mastitis and with confirmed growth of E. coli in a pretreatment milk sample were randomly allocated to 1 of 2 treatment groups. Response to treatment was evaluated clinically and by bacteriological culturing and determination of N-acetyl-beta-d-glucosaminidase (NAGase) activity on d 2 and 21 posttreatment. Enrofloxacin treatment did not increase bacteriological (90.5% of treated vs. 86.8% of nontreated cured) or clinical cure (46.7% of treated vs. 57.1% of nontreated cured), cow survival (95.3% of treated vs. 92.7% of nontreated), or quarter milk production assessed 21 d posttreatment (21.8 vs. 29.3% return to preinfection level for nontreated cows), nor did it decrease mammary gland tissue damage estimated using determination of milk NAGase activity (24.0+/-0.3 vs. 18.3+/-1.3 pmol of 4-methylumbelliferone per min per microL for nontreated cows). Treatment did not influence the number of study cows remaining in the herd after 6 mo (71.9% of treated vs. 80.6% of nontreated). The only significant effects of enrofloxacin were enhancing the bacteriological cure (odds ratio=3.32 for treated cows) and decreasing the clinical cure (odds ratio=0.05 for treated cows) on d 2 posttreatment. Our results did not support the use of enrofloxacin to treat acute clinical E. coli mastitis.

In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The "no-growth" samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10(3) to 10(7) bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-beta-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such milk samples. Low bacterial concentration is commonly speculated to explain the no-growth milk samples. This hypothesis is not supported by the results of the current study.

Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal beta-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and beta-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.

Coagulase-negative staphylococci (CNS) have emerged as bovine mastitis pathogens in many countries. CNS mastitis is generally mild but can persist in the udder for long periods. Pathogenesis of CNS intramammary infection is not well understood. In the present study, adhesion, invasion and intracellular replication of twenty-two CNS strains isolated from bovine mastitis and the effect of bovine lactoferrin (bLf) on the internalization were studied in vitro in a bovine mammary epithelial (BME) cell model. The CNS strains were ofStaphylococcus chromogenes, Staph. simulans, Staph. epidermidis, Staph. haemolyticusandStaph. cohnii urealyticus; two strains ofStaph. aureuswere used as controls. Seven of the CNS strains originated from persistent and five from transient mastitis infections. The in-vitro susceptibility of the strains to bLf was also investigated. All CNS species examined had an adhesive ability equal to that ofStaph. aureus, but internalization varied among staphylococcal strains. The antagonistic effect of bLf on the adhesion and invasion of CNS strains was weak, but bLf significantly decreased intracellular replication and replication rates of CNS. No correlation between the in-vitro susceptibility of the strain to bLf or internalization among clinical signs of mastitis was established. No difference between the persistent and transient CNS strains in adhesion, invasion or replication rate was recorded. This in-vitro BME cell model can be used to study the virulence potential of mastitis pathogens, although the severity and persistence of eventual infections shall be further investigated in vivo. The role of bLf in intramammary infection caused by CNS may be limited.

Increasing dairy farm size and increase in automation in livestock production require that new methods are used to monitor animal health. In this study, a thermal camera was tested for its capacity to detect clinical mastitis. Mastitis was experimentally induced in 6 cows with 10 mu g of Escherichia coli lipopolysaccharide (LPS). The LPS was infused into the left forequarter of each cow, and the right forequarters served as controls. Clinical examination for systemic and local signs and sampling for indicators of inflammation in milk were carried out before morning and evening milking throughout the 5-d experimental period and more frequently on the challenge day. Thermal images of experimental and control quarters were taken at each sampling time from lateral and medial angles. The first signs of clinical mastitis were noted in all cows 2 h postchallenge and included changes in general appearance of the cows and local clinical signs in the affected udder quarter. Rectal temperature, milk somatic cell count, and electrical conductivity were increased 4 h postchallenge and milk N-acetyl-beta-D-glucosaminidase activity 8 h postchallenge. The thermal camera was successful in detecting the 1 to 1.5 degrees C temperature change on udder skin associated with clinical mastitis in all cows because temperature of the udder skin of the experimental and control quarters increased in line with the rectal temperature. Yet, local signs on the udder were seen before the rise in udder skin and body temperature. The udder represents a sensitive site for detection of any febrile disease using a noninvasive method. A thermal camera mounted in a milking or feeding parlor could detect temperature changes associated with clinical mastitis or other diseases in a dairy herd. Increasing dairy farm size and increase in automation in livestock production require that new methods are used to monitor animal health. In this study, a thermal camera was tested for its capacity to detect clinical mastitis. Mastitis was experimentally induced in 6 cows with 10 mu g of Escherichia coli lipopolysaccharide (LPS). The LPS was infused into the left forequarter of each cow, and the right forequarters served as controls. Clinical examination for systemic and local signs and sampling for indicators of inflammation in milk were carried out before morning and evening milking throughout the 5-d experimental period and more frequently on the challenge day. Thermal images of experimental and control quarters were taken at each sampling time from lateral and medial angles. The first signs of clinical mastitis were noted in all cows 2 h postchallenge and included changes in general appearance of the cows and local clinical signs in the affected udder quarter. Rectal temperature, milk somatic cell count, and electrical conductivity were increased 4 h postchallenge and milk N-acetyl-beta-D-glucosaminidase activity 8 h postchallenge. The thermal camera was successful in detecting the 1 to 1.5 degrees C temperature change on udder skin associated with clinical mastitis in all cows because temperature of the udder skin of the experimental and control quarters increased in line with the rectal temperature. Yet, local signs on the udder were seen before the rise in udder skin and body temperature. The udder represents a sensitive site for detection of any febrile disease using a noninvasive method. A thermal camera mounted in a milking or feeding parlor could detect temperature changes associated with clinical mastitis or other diseases in a dairy herd. Increasing dairy farm size and increase in automation in livestock production require that new methods are used to monitor animal health. In this study, a thermal camera was tested for its capacity to detect clinical mastitis. Mastitis was experimentally induced in 6 cows with 10 mu g of Escherichia coli lipopolysaccharide (LPS). The LPS was infused into the left forequarter of each cow, and the right forequarters served as controls. Clinical examination for systemic and local signs and sampling for indicators of inflammation in milk were carried out before morning and evening milking throughout the 5-d experimental period and more frequently on the challenge day. Thermal images of experimental and control quarters were taken at each sampling time from lateral and medial angles. The first signs of clinical mastitis were noted in all cows 2 h postchallenge and included changes in general appearance of the cows and local clinical signs in the affected udder quarter. Rectal temperature, milk somatic cell count, and electrical conductivity were increased 4 h postchallenge and milk N-acetyl-beta-D-glucosaminidase activity 8 h postchallenge. The thermal camera was successful in detecting the 1 to 1.5 degrees C temperature change on udder skin associated with clinical mastitis in all cows because temperature of the udder skin of the experimental and control quarters increased in line with the rectal temperature. Yet, local signs on the udder were seen before the rise in udder skin and body temperature. The udder represents a sensitive site for detection of any febrile disease using a noninvasive method. A thermal camera mounted in a milking or feeding parlor could detect temperature changes associated with clinical mastitis or other diseases in a dairy herd.

In some recently published papers in the Journal of Dairy Science, new terminology was introduced. The terms were new at least to me as a nonnative speaker of English. I would like to know the definition for the term “generic mastitis” (Bar et al., 2008) and how it differs from the simple term “mastitis.” I understand that the aim is to present information on mastitis caused by different pathogens. Another term used was “nonambulatory cows” and its counterpart “no nonambulatory cows” (Green et al., 2008). Eventually I understood that the term referred to recumbent cows and normal, walking cows. I would suggest that only defined, wellestablished terms understandable to all readers should be used in this scientific journal.

In this review of the literature, mastitis-causing coagulase-negative staphylococci (CNS) and Staphylococcus aureus are compared. Staphylococci are the bacteria most commonly isolated from bovine mastitis, and CNS are now predominant over S. aureus in most countries. CNS include various species, but only a few prevail in bovine mastitis. S. aureus can cause clinical mastitis, but often causes subclinical mastitis, which remains persistent and increases milk somatic cell count. CNS, traditionally regarded as minor pathogens, seem to lack the ability to cause severe mastitis. CNS can, however, persist in the mammary gland and moderately increase milk somatic cell count. Resistance to various antimicrobials is more common in CNS than in S. aureus, but CNS mastitis responds much better to antimicrobial treatment than S. aureus mastitis.

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein-Friesian cows were challenged with S. chromogenes 4 weeks after calving. One udder quarter of each cow was inoculated with 2.1 x 10(6)cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-beta-D-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on-off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.

Coagulase-negative staphylococci (CNS) have become the most common bovine mastitis isolate in many countries and could therefore be described as emerging mastitis pathogens. The prevalence of CNS mastitis is higher in primiparous cows than in older cows. CNS are not as pathogenic as the other principal mastitis pathogens and infection mostly remains subclinical. However, CNS can cause persistent infections, which result in increased milk somatic cell count (SCC) and decreased milk quality. CNS infection can damage udder tissue and lead to decreased milk production. Staphylococcus simulans and Staphylococcus chromogenes are currently the predominant CNS species in bovine mastitis. S. chromogenes is the major CNS species affecting nulliparous and primiparous cows whereas S. simulans has been isolated more frequently from older cows. Multiparous cows generally become infected with CNS during later lactation whereas primiparous cows develop infection before or shortly after calving. CNS mastitis is not a therapeutic problem as cure rates after antimicrobial treatment are usually high. Based on current knowledge, it is difficult to determine whether CNS species behave as contagious or environmental pathogens. Control measures against contagious mastitis pathogens, such as post-milking teat disinfection, reduce CNS infections in the herd. Phenotypic methods for identification of CNS are not sufficiently reliable, and molecular methods may soon replace them. Knowledge of the CNS species involved in bovine mastitis is limited. The dairy industry would benefit from more research on the epidemiology of CNS mastitis and more reliable methods for species identification.

Isolates of various species of coagulase-negative staphylococci (CNS) from extramammary swab samples were compared with isolates of bovine mastitis CNS species. Swab samples were taken from perineum skin and udder skin, teat apices and teat canals of lactating dairy cows of the research dairy herd of the University of Helsinki in 1999 and 2002. In addition, hands of herd staff and liners of teat cups were sampled for CNS. CNS isolates from milk samples of subclinical or clinical mastitis in the same herd were collected during 1998–2002. Species identification was performed using phenotyping (API Staph ID 32 test) and by constructing a 16 and 23S rRNA RFLP library (ribotyping). Based on phenotype, 84% of mastitis isolates and 57% of extramammary isolates were identified at species level with >90% probability. Ribotype patterns formed 24 clusters, and 15 of them included a CNS type strain. If the ribotype clusters contained isolates of both extramammary and mastitis origin, they were further typed using pulsed-field gel electrophoresis (PFGE). The predominant CNS species in mastitis, based both on phenotyping and genotyping, wereStaph. chromogenesandStaph. simulans. Phenotyping failed to identify half of the extramammary isolates. Based on phenotyping,Staph. equorumandStaph. sciuri, and based on ribotyping,Staph. succinusandStaph. xylosus, were the predominant CNS species in extramammary samples. The most common species in milk samples,Staph. chromogenes, was also isolated from several extramammary samples, and five out of ten pulsotypes were shared between mastitis and extramammary isolates, indicating that strains from udder skin are highly similar. The second commonest mastitis species,Staph. simulans, was isolated only from three extramammary samples, indicating thatStaph. simulansmay be more specifically associated with mastitis. Consequently, the origin of CNS mastitis may vary depending on the causing CNS species.

Transition from the dry period to lactation is a high risk period for the modern dairy cow. The biggest challenge at that time is mastitis. Environmental bacteria are the most problematic pathogens around parturition. Coliforms are able to cause severe infections in multiparous cows, and heifers are likely to be infected with coagulase-negative staphylococci. During the periparturient period, hormonal and other factors make the dairy cows more or less immunocompromised. A successful mastitis control programme is focused on the management of dry and calving cows and heifers. Clean and comfortable environment, proper feeding and adequate supplementation of the diet with vitamins and trace elements are essential for maintaining good udder health. Strategies which would enhance closure of the teat canal in the beginning of the dry period and would protect teat end from bacteria until the keratin plug has formed decrease the risk for mastitis after calving. Dry cow therapy has been used with considerable success. Yet, a selective approach could be recommended rather than blanket therapy. Non-antibiotic approaches can be useful tools to prevent new infections during the dry period, in herds where the risk for environmental mastitis is high. Vaccination has been suggested as a means to support the immune defence of the dairy cow around parturition. In some countries, implementation of Escherichia coli core antigen vaccine has reduced the incidence of severe coliform mastitis after calving.

Serum tinidazole concentrations were monitored in five clinically healthy adult horses after intravenous (i.v.) and oral administration of the drug (15 mg/kg and 25 mg/kg, respectively). After i.v. administration, the mean residence time was 7.0 h, the elimination half-life 5.2 h and the body clearance rate 1.6 ml/min/kg. The distribution volume was found to be 660 ml/kg. After oral administration, the mean residence time was 8.5 h, the absorption half-life 1.1 h and the bioavailability essentially 100%. In view of the in-vitro sensitivities of various anaerobic bacteria, a dosage of 10-15 mg/kg of tinidazole, orally, at 12-h intervals, can be recommended for the treatment of anaerobic infections in horses.

Background Indication-based data on the use of antimicrobials in animals were collected using a prospective cross-sectional survey, similarly as for surveys carried out in human medicine, but adapting the questionnaire to include veterinary-specific issues. The participating veterinarians were randomly selected from a sample population of practising veterinarians. The sampling was stratified to take into account the proportions of different types of veterinary practice in the country. All patients consulting the veterinary practice during a 1-week period were included in the study and veterinarians returned a completed questionnaire for each patient receiving antimicrobial treatment. As cattle received most of the treatments, results from the survey are given using cattle as an example species. Results The survey was sent to 681 veterinarians, of whom 262 (39%) responded. In total 2850 questionnaires were completed. The largest quantities of antimicrobials, measured in kilograms, were used for cattle, followed by pigs, dogs and horses. The species that were treated most were cattle (n = 1308), dogs (n = 989) and cats (n = 311). For cattle, the most common reason for treatment was acute mastitis (52%), followed by dry-cow therapy (21%), subclinical mastitis (6%) and treatment for acute enteritis (4%). The remaining treatments covered 17% of cattle patients and 15 different indications. For acute mastitis, parenteral or intramammary treatment was used in 36% and 34% of the cases, respectively. The remaining 30% received both treatments simultaneously. Of the parenteral treatments (n = 459), benzyl penicillin was used in 83% of the treated animals (n = 379), while fluoroquinolones were used in 49 cases (11%). Of the 433 cows receiving intramammary treatment, ampicillin combined with cloxacillin was most commonly used (n = 157; 36%), followed by cephalexin+streptomycin (n = 113; 26%). Conclusion This cross-sectional prospective survey provided a useful method for the collection of information on the indication-based use of antimicrobials in different animal species. Cattle were the most commonly treated animal species during the study period. The most common indication for antimicrobial use in cows was mastitis. Benzyl penicillin was the drug most frequently used for the treatment of mastitis, which seems appropriate according to the national guidelines on the use of antimicrobials in cattle in Finland.

Persistence of coagulase-negative staphylococci (CNS) in intramammary infections during lactation was studied in a research dairy herd of University of Helsinki. Milk samples from 328 udder quarters of 82 dairy cows (30 primiparous, 52 multiparous) were collected 2 wk before calving, at calving, and every 4 wk thereafter until the end of lactation or until the cow left the herd. The CNS isolated from the milk samples were analyzed with the API Staph ID 32 (bioMerieux, Marcy l'Etoile, France) test (API) and genotyped using amplified fragment length polymorphism (AFLP) analysis. The AFLP patterns were used for similarity analysis between CNS isolates and for species identification. For the latter, AFLP patterns of CNS isolates and staphylococcal type strains were used as operational taxonomic units in numerical analysis. In addition, the somatic cell count (SCC) of the milk samples was measured during lactation. A CNS infection was considered persistent when isolates originating from the same quarter had identical AFLP patterns on at least 3 consecutive samplings. In total, 63 CNS infections were detected during lactation in 30 and 33 quarters in the first and later lactations, respectively. Twenty-nine of these infections persisted and 34 were transient. Most of the persistent infections lasted until the end of lactation. In 57 quarters, CNS infection was detected before calving, at calving, or both, but only half of these quarters were infected by CNS during subsequent lactation. The geometric mean of SCC in quarters during persistent CNS infection was 657,600 cells/mL, and the mean of SCC in quarters with transient CNS infection was 619,100 cells/mL. The median of SCC in quarters during persistent CNS infection was 355,400 cells/mL, and the median of SCC in quarters with transient CNS infection was 133,500 cells/mL. According to both the API test and AFLP results, Staphylococcus chromogenes and Staphylococcus simulans were the CNS species isolated most often. Identification results for API and AFLP corresponded in 71.9% of the isolates.

This is the first study describing an experimental mastitis model using transgenic cows expressing recombinant human lactoferrin (rhLf) in their milk. The aim of the study was to investigate the concentrations in milk and protective effects of bovine and recombinant human lactoferrin in experimental Escherichia coli mastitis. Experimental intramammary infection was induced in one udder quarter of seven first-lactating rhLf-transgenic cows and six normal cows, using an E. coli strain isolated from cows with clinical mastitis and known to be susceptible to Lf in vitro. Clinical signs were recorded during the experimental period, concentrations of human and bovine Lf and indicators of inflammation and bacterial counts were determined for milk, and concentrations of acute-phase proteins and tumor necrosis factor alpha were determined for sera and milk. Serum cortisol and blood hematological and biochemical parameters were also determined. Expression levels of rhLf in the milk of transgenic cows remained constant throughout the experiment (mean, 2.9 mg/ml). The high Lf concentrations in the milk of transgenic cows did not protect them from intramammary infection. All cows became infected and developed clinical mastitis. The rhLf-transgenic cows showed milder systemic signs and lower serum cortisol and haptoglobin concentrations than did controls. This may be explained by lipopolysaccharide-neutralizing and immunomodulatory effects of the high Lf concentrations in their milk. However, Lf does not seem to be a very efficient protein for genetic engineering to enhance the mastitis resistance of dairy cows.

Resistance of Staphylococcus aureus to penicillin G is common among isolates from bovine mastitis. We determined phenotypic resistance to penicillin G for 151 S. aureus isolates derived from dairy cows with intramammary infection by two methods. The methods were determination of minimum inhibitory concentration (MIC) by a standard agar dilution technique and direct testing of beta-lactamase production using a chromogenic cephalosporin, nitrocefin. The results from these tests were compared with the presence of the beta-lactamase (blaZ) gene in the isolates, which was detected by polymerase chain reaction (PCR). Testing beta-lactamase production with nitrocefin was more predictive for the presence of the blaZ gene than the agar dilution method and the results of the former agreed highly with the presence of the blaZ gene in the isolates. In contrast, the resistance breakpoint generally used in the agar dilution method may be too high for prediction of penicillin resistance in S. aureus isolates with borderline MICs. Using this method, 40% of the isolates possessing the blaZ gene were classified as susceptible; however, majority of these isolates produced beta-lactamase when tested with nitrocefin.

International audience; A cross-over study was conducted to investigate the effect of intramammarily infused lipopolysaccharide (LPS) on the acute phase reaction in early (EL) and in late (LL) lactation. Nine cows received intramammary injections of 100 µg of Escherichia coli 0111:B4 LPS during EL and LL. The severity of each cows systemic and local signs and change in milk appearance were recorded and scored throughout the experiment. Systemic and local signs were found to be more serious in EL cows. Tumor necrosis factor $\alpha$ (TNF$\alpha$) was detected in milk but not in serum. Serum amyloid A (SAA) concentrations increased both in serum and in milk. The milk TNF$\alpha$ concentrations peaked at 8 h post-challenge (PC). SAA concentrations started to increase at 8 h PC, and peak concentrations were seen at 32 and 48 h PC in milk and serum, respectively. The milk TNF$\alpha$ and SAA seemed to be correlated, being on average higher in EL. Serum SAA concentration was not correlated with milk TNF$\alpha$ or SAA, nor with the severity of local or systemic signs, but was correlated with changes in milk appearance.

Materials and methods The study was conducted in 1998-1999. It included 18 cattle herds which had problems with bovine respiratory tract disease complex. Four to five diseased calves from each farm were chosen for closer examination. A thorough clinical examination and tracheobronchial lavage were done. Blood samples were taken from the calves at the acute phase and 3-4 weeks later. In addition, 10 blood samples from cattle of different age were taken from the same herds. Because bovine herpesvirus-1 (BHV-1) does not exist in Finland and also bovine virus diarrhoea virus (BVDV) is very rare, serum samples were tested for antibodies to bovine parainfluenza virus3 (PIV-3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCV), bovine adenovirus-3 (BAV-3) and bovine adenovirus-7 (BAV-7). Some samples were also tested for antibodies to BVDV. ELISA-test was used for antibodies against PIV-3, BRSV, BCV and BVDV. Virus neutralization test was used for BAV-3 and BAV-7. Bacteria were cultured from lavage fluid and in vitro susceptibility for selected antimicrobials (ampicillin, penicillin, trimethoprim-sulfamethoxazole, gentamicin, tetracycline and ciprofloxacin) tested. One calf was autopsied.

Efficacy and pharmacokinetics of enrofloxacin and flunixin meglumine for treatment of cows with experimentally induced Escherichia coli mastitis. J. vet. Pharmacol. Therap.25, 251–258. The efficacy of flunixin alone and together with enrofloxacin in treatment of experimental Escherichia coli mastitis was compared using six cows. The cross-over study design was used. Pharmacokinetics of flunixin and enrofloxacin were also studied in these diseased cows. The response of each cow was similar after the first and second challenge and the individual reaction seemed to explain the severity of clinical signs. The most important predictive factor for outcome of E. coli mastitis was a heavy drop in milk yield. Treatment with enrofloxacin and flunixin enhanced elimination of bacteria, but the difference from those receiving flunixin alone was not significant. Two cows, which had received no antimicrobial treatment (Group 1), were killed on day 4 postchallenge. One cow was killed after the first and the other after the second challenge. Cows receiving combination therapy produced 0.9 L more milk per day during the study period than cows which had only received flunixin (P

Bovine mastitis remains as the disease causing the biggest economic losses to the dairy industry, despite the intensive research and prevention measures at herd level carried out for decades. Antibiotics are widely used to combat mastitis, but focus should be shifted from treatment to more economical and efficient prevention. The bacteriological aetiology of mastitis has changed from contagious to environmental pathogens, which has reduced the efficacy of the traditional mastitis control strategies. Considerable progress in the understanding of epidemiology, immunology, diagnostics and pathogenesis of mastitis has been made. The modern molecular biological methods offer good possibilities for the research of the epidemiological and virulence aspects of bacteria, which may help in building-up specific mastitis control strategies for dairy herds. Studies on the host response and relationship between somatic cell count and susceptibility to mastitis offer tools for genetic improvement of dairy cows. Biotechnological approaches for mastitis prevention are in the developmental stage, but many problems are associated, e.g. with vaccination of dairy cows against mastitis. Different methods of immunomodulation for the prevention of mastitis have shown promise in experimental trials, but the evidence is not yet enough to support commercial applications. Improving nutrition, housing and environment of dairy cattle are still crucial in the prevention of mastitis, especially during the most susceptible period after parturition. New milking techniques including robotic milking may provide better possibility for proper milking and improved udder health. Mastitis control should be part of the herd health programme in the dairy herds. In this paper, results from recent research and proposals for new prevention strategies in the field of mastitis are reviewed.

International audience; The pharmacokinetics of sulphadoxine-trimethoprim was studied in 6 pre-ruminant calves using two different products. Product A, which contained 200 mg sulphadoxine and 40 mg trimethoprim per mL, was administered intravenously or subcutaneously at a dosage of 25 mg sulphadoxine and 5 mg trimethoprim$\cdot$kg$^{-1}$ bodyweight. Product B, containing 62.5 mg sulphadoxine and 12.5 mg trimethoprim per mL plus lidocaine (1 mg$\cdot$mL$^{-1}$), was given subcutaneously at the same dosage. After intravenous administration of product A the mean time of half-life of elimination phase (t$_{1/2}$) for sulphadoxine was 12.9 h, steady-state volume of distribution (V$_{{\rm d(ss)}}$) was 0.44 L$\cdot$kg$^{-1}$ and clearance was 0.024 L$\cdot$kg$^{-1}\cdot$h$^{-1}$. Respective values for trimethoprim were 1.9 h, 2.0 L$\cdot$kg$^{-1}$ and 0.9 L$\cdot$kg$^{-1}\cdot$h$^{-1}$. After subcutaneous administration, the bioavailability of sulphadoxine was 96% and 98% and the time to reach a maximum concentration was 6.3 and 8.0 h for products A and B, respectively. The C$_{{\rm max}}$ for trimethoprim was higher for product A (0.49 $\mu$g$\cdot$mL$^{-1}$) than for product B (0.32 $\mu$g$\cdot$mL$^{-1}$) ($p = 0.014$). Slow absorption from the injection site appeared to delay the elimination of trimethoprim after subcutaneous administration when compared to that after intravenous administration: apparent elimination t$_{1/2}$ for trimethoprim after intravenous administration of product A was 1.9 h compared to 3.9 h and 3.6 h after subcutaneous administration of products A and B, respectively. The difference between intravenous and subcutaneous administrations was statistically significant ($p < 0.05$). Also the mean residence time was significantly shorter ($p < 0.05$) after intravenous administration (2.4 h) than that after subcutaneous administration of product A (6.9 h) and B (7.1 h). The bioavailability of trimethoprim was lower than that of sulphadoxine: 76% and 74% for products A and B, respectively. All 6 calves showed pain after subcutaneous administration of product A and the injection sites were warm and showed soft oedematous reactions 5-8 cm in diameter. Three of the calves also showed some pain after subcutaneous administration of product B; the local reactions were less severe. A marked increase was seen in creatine kinase activity after subcutaneous administration of both products. Product A caused a more pronounced increase but the difference was not statistically significant. We suggest 30 mg$\cdot$kg$^{-1}$ at 24-h intervals or alternatively 15 mg$\cdot$kg$^{-1}$ at 12-h intervals as the minimum dosage of sulphadoxine-trimethoprim combination for pre-ruminant calves. Extravascular routes of administration should be avoided due to marked tissue irritation at the injection site.; Pharmacocinétique de la sulphadoxine et de la triméthoprime et irritation des tissus causée par deux produits contenant de la sulphadoxine et de la triméthoprime après une administration sous-cutanée chez des veaux préruminants. La pharmacocinétique de la sulphadoxine-triméthoprime a été étudiée chez six veaux préruminants en utilisant deux produits différents. Le produit A qui contenait 200 mg$\cdot$mL$^{-1}$ de sulphadoxine et 40 mg$\cdot$mL$^{-1}$ de triméthoprime a été administré par voie intraveineuse (i.v.) ou sous-cutanée (s.c.) à un dosage de 25 mg de sulphadoxine et 5 mg de triméthoprime$\cdot$kg$^{-1}$ de poids vif. Le produit B contenant 62,5 mg$\cdot$mL$^{-1}$ de sulphadoxine et 12,5 mg$\cdot$mL$^{-1}$ de triméthoprime et 1 mg$\cdot$mL$^{-1}$ de lidocaïne, a été donné au même dosage par voie sous-cutanée. Après administration i.v. du produit A, le temps de demi-vie d'élimination (t$_{1/2}$) pour la sulphadoxine était de 12,9 h, le volume de distribution à l'équilibre était de 0,44 L$\cdot$kg$^{-1}$ et la clairance de 0,024 L$\cdot$kg$^{-1}\cdot$h$^{-1}$. Les valeurs respectives pour la triméthoprime étaient de 1,9 h, 2,0 L$\cdot$kg$^{-1}$ et 0,9 L$\cdot$kg$^{-1}\cdot$h$^{-1}$. Après administration s.c., la biodisponibilité de la sulphadoxine était respectivement de 96 % et de 98 % et le temps nécessaire pour atteindre une concentration maximale était respectivement 6,3 et 8,0 h pour les produits A et B. La C$_{{\rm max}}$ de la triméthoprime était plus élevée pour le produit A (0,49 $\mu$g$\cdot$mL$^{-1}$) que pour le produit B (0,32 $\mu$g$\cdot$mL$^{-1}$). A cause de la lente absorption au niveau de la zone d'injection, l'élimination de la triméthoprime a été plus lente après administration s.c. qu'après l'administration i.v.: le t$_{1/2}$ pour la triméthoprime après l'administration i.v. du produit A était de 1,9 h tandis qu'il était de 3,9 et de 3,6 h après l'administration s.c. des produits A et B. La différence entre l'administration i.v. et l'administration s.c. était statistiquement significative ($p < 0,05$). Aussi le temps moyen de résidence après l'administration i.v. (2,4 h) était significativement plus court ($p < 0,05$) que celui constaté après l'administration s.c des produits A (6,9 h) et B (7,1 h). La biodisponibilité de la triméthoprime était plus basse que celle de la sulphadoxine: respectivement 76% et 74% pour les produits A et B. Les six veaux ont montré de la douleur après l'administration s.c. du produit A et les zones d'injection étaient chauds et présentaient des réactions oedémateuses souples de 5-8 cm de diamètre. Trois veaux ont également montré une légère douleur après l'administration du produit B; les réactions locales ont été moins sévères. Une augmentation notable de l'activité de la créatine kinase a été notée après l'administration s.c. des deux produits. Le produit A a causé une augmentation plus prononcée mais la différence n'était pas statistiquement significative. Nous proposons 30 mg$\cdot$kg$^{-1}$ toutes les 24 h ou alternativement 15 mg$\cdot$kg$^{-1}$ toutes les 12 h comme dosage minimum de la combinaison sulphadoxine-triméthoprime pour les veaux préruminants. Le produit devrait être injecté par voie intraveineuse pour éviter l'irritation du tissu dans la zone d'injection.

Tissue irritation after intramuscular injections of 4 nonsteroidal anti-inflammatory agents was studied in 5 lactating cows. Preparations containing phenylbutazone, flunixin, metamizole (dipyrone) and ketoprofen were investigated; physiological saline was used as a control substance. Tissue reactions at the injection sites were examined by palpation and by determining serum creatine kinase. A kinetic method based on creatine kinase released from the injured muscle tissue was used, which allowed estimation of the amount of damaged muscle. The metamizole preparation clearly provoked signs of pain in all the cows. After flunixin and phenylbutazone injections slight reactions were observed, and ketoprofen and saline did not cause any clinical signs. Some palpatory findings after injections were found for all the preparations except saline. Based on serum creatine kinase, the 2 most irritating preparations were the ones containing flunixin and phenylbutazone. After injections of these 2 substances, the estimated amount of damaged muscle was about 80 grams. The statistical difference between flunixin and phenylbutazone and the other 2 preparations was significant. Physiological saline had no effect on serum creatine kinase. For preparations containing phenylbutazone and flunixin, intravenous administration is recommended.

Pharmacokinetic behaviour of enrofloxacin was studied in 6 horses after intravenous (i.v.) or intramuscular (i.m.) administration of enrofloxacin (5 mg/kg bwt). Concentration of enrofloxacin and ciproflaxin were measured by high performance liquid chromatography in serum. Antimicrobial activity of the samples was determined with an agar-diffusion technique. Reactions at the site of i.m. injection were monitored clinically and by determination of serum creatine kinase (CK) activity. After i.v. administration, elimination half-life of enrofloxacin was 4.4 h and volume of distribution was 2.3 1/kg bwt. Enrofloxacin was rapidly metabolised to ciprofloxacin. The half-life of ciprofloxacin parallelled that of the parent drug, its concentration in serum reached 20-35% of that of the parent drug. After i.m. administration, elimination half-life of enrofloxacin was longer (9.9 h) than after i.v. administration. Mean absorption time of enrofloxacin was also long (9.9 h). No statistically significant differences were found when half-life and mean residence time of antimicrobial activity were compared with those of enrofloxacin and ciprofloxacin from chemically analysed data. Intramuscular injection of enrofloxacin was found to be very irritating. After i.m. administration, CK activity in serum, compared with pre-injection levels, increased over 10-fold. CK activity also stayed high during the 32 h follow-up period. Clinical reactions, such as swelling or tenderness at the i.m. injection sites, were observed in 2 horses.

Methicillin-resistant staphylococci (MRS) are increasingly being isolated in bovine mastitis. The aim of our study was to evaluate the occurrence of MRS in Finnish mastitis milk samples and characterize the MRS isolates using molecular methods. Methicillin-resistant S. aureus (MRSA) was a rare finding in bovine mastitis in Finland. Only two out of 135 (1.5%) S. aureus isolates were positive for mec genes. One of these carried mec A and was of spa type t172, SCCmec type IV and ST375, and the other harboured mec C, being spa type t3256, and ST130. MRSA ST375 is common among human MRSA isolates in Finland, but this is the first report in the country of bovine mec C MRSA. In coagulase-negative staphylococci (CoNS) originating from bovine mastitis, methicillin resistance was more common. In the two CoNS collections studied, 5.2% (17/324) and 1.8% (2/110) of the isolates were mecA positive. Eighteen of these were methicillin-resistant S. epidermidis (MRSE), which were divided into 6 separate PFGE clusters. One pulsotype was detected in different parts of the country, indicating clonal spread. Most MRSE (13/18) were of SCCmec type IV, one was of type V and four were non-typeable. Comparison with a human staphylococcal database indicated that bovine MRSE strains were not closely related to human MRSE isolates. The occurrence of MRS, especially MRSA, in bovine mastitis in Finland was low. Most methicillin-resistant bovine CoNS are MRSE, and we found evidence of a bovine MRSE strain that may spread clonally. This is the first report of a Finnish bovine isolate of MRSA mecC ST130. The study provides a baseline for further MRS monitoring.

The objective of the study was to monitor the effect of two therapy regimens on experimental Escherichia coli mastitis. Single udder quarters of 12 cows that were at least 30 d postpartum were inoculated with 1500 cfu of E. coli. The inoculation was repeated in the contralateral quarter after a 3- to 4-wk interval. Initially, half of the cows were treated with antimicrobials, and the remaining half were left untreated. At the second inoculation, the cows that were originally treated were not treated, and vice versa. Therapy began 12h after inoculation and consisted of parenteral trimethoprimsulfadiazine (6 cows) or intramammary colistin sulfate (6 cows). Clinical signs, daily milk yield, bacterial count, and endotoxin content of the milk were recorded. Milk SCC, NAGase activity, and trypsin inhibitor capacity were also monitored. The response to bacterial challenge varied greatly among cows. Bacteria were eliminated from the quarters within 7 d in all but 1 cow. Treatment did not significantly affect the elimination rate of bacteria or any of the measured parameters. Significant positive correlations existed among milk bacterial counts, endotoxin concentrations, and clinical signs at the acute stage of the infection. Based on these findings, antimicrobial therapy of E. coli mastitis during lactation apparently is no more beneficial than no treatment.

The concentrations of haptoglobin (Hp) and serum amyloid A (SAA) and the activity of N-acetyl-β-D-glucosaminidase (NAGase) in milk from 234 cows with spontaneous mastitis caused by different pathogens were measured to assess whether they corresponded with the clinical signs of mastitis and whether there were any differences between pathogens. Ninety-eight of the cows had clinical mastitis and 136 had subclinical mastitis. There were statistically significant positive correlations between the concentrations of SAA and Hp and the activity of NAGase. Significant differences in the concentrations of acute phase proteins and NAGase activity were found in milk from cows with mastitis caused by different pathogens. The highest concentrations of Hp and NAGase were found in cases of mastitis caused by Escherichia coli and Arcanobacterium pyogenes, and the lowest concentrations were from cases of mastitis caused by coagulase-negative staphylococci. Very low SAA concentrations were found in milk from the cases caused by A pyogenes, in contrast to cases caused by other major mastitis pathogens. The median concentration of SAA was over 10 times higher in cases of mastitis caused by E coli than in mastitis caused by other pathogens. There were significant differences in the mean Hp concentration and NAGase activity between clinical and subclinical mastitis. In approximately one-third of the samples, the Hp concentration was below the detection limit, potentially compromising the use of Hp as a mastitis marker.