Seroprevalence of canine herpesvirus-1 and Brucella canis in Finnish breeding kennels with and without reproductive problems.

We compared the serological status of Brucella canis and canine herpesvirus-1 (CHV-1) in Finnish breeding kennels with and without reproductive problems. Dogs from kennels with reproductive problems had significantly higher CHV-1 titres than dogs from kennels having no reproductive problems (p < 0.001). In dogs from kennels with reproductive problems 100% (32/32) had positive titres, whereas in dogs from kennels without reproductive problems 65% (22/34) had positive titres. The median titre for dogs from kennels with reproductive problems was 1 : 160 and for dogs from kennels without reproductive problems 1 : 80. The high prevalence of positive CHV-1 titres in this study indicates that prevention of the disease is difficult and reinforces the need to minimize the reproductive problems caused by CHV-1. All 388 dogs from 94 kennels had negative B. canis titres.

Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

This report describes the development of in-house real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed as biological threats by the Centers for Disease Control and Prevention.

The effect of antimicrobial growth promoter withdrawal on the health of weaned pigs in Finland.

The use of the antimicrobial growth promoters (AGPs) carbadox and olaquindox has been banned in the European Union (EU) since September 1999. We studied the effects of the withdrawal on the health of weaned piglets on two types of piglet-producing farms (farrowing herds and farrow-to-finish herds) from the different regions of Finland. Farms with no major problems with post-weaning diarrhoea were selected for the study to better evaluate the effect of AGPs alone. Data on production, medication and incidence of diarrhoea were collected from 73 farms during 1 year after the withdrawal. On 29 of these farms, the data collection began 4 months before the withdrawal. The health management of the pigs is considered good in Finland, and special attention has been paid to improve the husbandry practices and management of the farms. Eighty-two percent of the farms in the study were free of both Mycoplasma hyopneumoniae and Sarcoptes scabiei infection. Brachyspira hyodysenteriae infection was not detected in any of the farms. The median number of sows in the herds was 56.0 (IQR=43.0; 72.5) in 2000. The level of antimicrobial use in each herd was classified as low, moderate and high when the percentage of weaned pigs treated for diarrhoea during a 4-month period was 0-5%, 6-19% and > or =20%, respectively. Only on four herds (14%), there was an increase in the level of antibiotic use after the AGP withdrawal, when seasonally corresponding 4-month periods were compared. Fourty-one percent of these 29 farms were categorized as low users of antimicrobials, 38% as moderate users and 21% as high users. The level of antimicrobial use for treatment of diarrhoea after weaning (and the incidence of diarrhoea in weaned piglets) did not increase significantly after the withdrawal of AGPs from weaner feeds according to farmers' evaluations. In this study, the Escherichia coli infection was the most-common cause of diarrhoea in weaned pigs. The age at weaning did not change after the withdrawal of AGPs.

Bovine mastitis in Finland 2001--prevalence, distribution of bacteria, and antimicrobial resistance.

A nationwide survey was conducted in Finland to estimate prevalence of bovine mastitis, distribution of mastitis pathogens, and in vitro antimicrobial susceptibility of different mastitis pathogens. In total, 12,661 quarter milk samples were collected from 3282 dairy cows at 216 farms. These were randomly selected from a database covering all Finnish dairy farms. Quarter milk samples collected by the dairy advisors were submitted for somatic cell counting, bacteriological examination, and testing for antimicrobial susceptibility. If the milk SCC of a cow or of a quarter exceeded 300,000/mL, the cow was defined as having mastitis. The results were compared with those of a previous survey done in 1995. The prevalence of mastitis continued to decrease from 38% in 1995 to 31% in 2001. Compared with the study from 1995, the number of quarters with bacterial growth in 2001 increased significantly from 21.0 to 33.5%. This mainly resulted from increased prevalence of Corynebacterium bovis. Coagulase-negative staphylococci remained the most common bacterial group, comprising almost one-half of the pathogens isolated, whereas the relative number of Staphylococcus aureus isolations decreased from the time of the previous study. According to in vitro antimicrobial susceptibility testing, the enterococci demonstrated the highest level of resistance. Compared with the other Nordic countries, penicillin resistance among the staphylococci was still at a relatively high level in Finland (52.1 and 32.0% for Staphylococcus aureus and coagulase-negative staphylococci, respectively). Streptococci isolated from mastitis were very susceptible to beta-lactam antibiotics, as also found in the previous survey in 1995.

Diversity and stability of the Staphylococcus intermedius flora in three bitches and their puppies.

The study investigated the transfer and the stability of the S. intermedius flora in three bitches and their puppies. A total of 240 cutaneous and mucosal isolates of S. intermedius was collected from three healthy Cavalier King Charles spaniels and their puppies during the immediate prepartum period and after whelping, over a total of 15 weeks. The isolates were genotyped with random amplified polymeric DNA-polymerase chain reaction analysis (RAPD-PCR) using two primers. Seventeen different genotypes of S. intermedius were identified. One or two of the genotypes were dominant in each of the bitches and their puppies. The rest were isolated only once or twice from the bitches or their puppies. The study indicates that S. intermedius flora within each studied bitch mainly consisted of one or two dominating and persistent clones, which were transferred from the dam to her puppies immediately after birth.

Bovine mastitis in Finland in 1988 and 1995--changes in prevalence and antimicrobial resistance.

Two surveys were carried out (during 1988 and 1995) to estimate the prevalence of bovine mastitis in Finland. In 1988, 17,111 quarter milk samples were obtained from 4495 cows, and in 1995 the corresponding figures were 10,410 and 2648. Antimicrobial susceptibility of mastitis pathogens was studied. Prevalence of mastitis on cow basis decreased from 47.8% in 1988 to 37.8% in 1995. Staphylococci was the largest group of pathogens isolated. The proportion of Staphylococcus aureus decreased and that of coagulase-negative staphylococci (CNS) increased. The proportion of strains resistant to at least one antibacterial drug increased with regard to S. aureus from 36.9% in 1988, to 63.6% in 1995 and with CNS from 26.6% to 49.7%. Most of the increase in antibacterial resistance was due to a higher number of beta-lactamase producing strains. Multiresistance also increased, but it was proportional to the overall increase in resistance. All the predominant mastitis streptococci were susceptible to beta-lactams tested.

Persistence in bovine mastitis of Staphylococcus aureus clones as assessed by random amplified polymorphic DNA analysis, ribotyping and biotyping.

Staphylococcus aureus isolates (N = 40) from bovine mastitis were characterized by random amplified polymorphic DNA-PCR (RAPD-PCR), ribotyping and biotyping. The isolates were collected in the veterinary surveillance area of the Ambulatory Clinic, Faculty of Veterinary Medicine, University of Helsinki from 20 quarters during the acute phase of infection and from the same quarters 3 weeks after cessation of therapy. The aim of the study was to compare the S. aureus isolates taken from the same quarter at different times to verify persistence of virulent strains in infected quarters and to compare the discriminatory power of the diagnostic methods. Using all methods (except for a commercial diagnostic test), the paired isolates of S. aureus were identical. Results suggest that the chronic nature of S. aureus infections was due to the persistence of the original infective strain. More laborious ribotyping and the more convenient RAPD-PCR method produced identical results. The molecular methods differentiated the 40 isolates into 6 distinct genotypes. Biotyping produced partially identical results to RAPD and ribotyping. A commercial diagnostic test system identified only 3 S. aureus biotypes.

Resazurin reduction as a function of respiratory burst in bovine neutrophils.

OBJECTIVE: To determine whether the respiratory burst of neutrophils from bovine blood and milk can be analyzed by use of a fluorometric resazurin reduction assay. SAMPLE POPULATION: Neutrophils were obtained from EDTA-anticoagulated blood of 7 dairy cows. Neutrophils also were isolated from milk samples of a cow intramammarily challenge exposed with Escherichia coli lipopolysaccharide. PROCEDURE: The respiratory burst of neutrophils was analyzed in parallel, using the conventional luminol-enhanced luminometric procedure and a novel fluorometric procedure with resazurin as the fluorogenic substrate. Opsonized zymosan and phorbol myristate acetate were used as stimulants. The mechanism of the fluorescent response was analyzed, using metabolic inhibitors to various cell functions. Luminometry and fluorometry were carried out in parallel, using microtitration tray-reading instruments. RESULTS: Stimulation of neutrophils induced resazurin reduction to resorufin and a fluorescent response. The luminescent response was transient, but the fluorescent response (build-up of fluorescent resorufin) was cumulative. Therefore, a single end-point measurement can be used for the fluorometric assay. CONCLUSIONS: The proposed fluorometric microtitration tray technology is simple and has a high throughput capacity. The fluorometric and luminometric assays seem to have similar potential in the analysis of phagocyte functions.

Binding of Staphylococcus aureus to milk fat globules increases resistance to penicillin-G.

The susceptibility to penicillin-G of Staphylococcus aureus strains that cause mastitis was tested in milk and in Iso-sensitest broth (ISB): The minimal inhibitory concentrations (MIC) of beta-lactamase-positive strains in milk were 10-100-fold those in ISB, whereas the MIC of beta-lactamase-negative strains in milk were some 10-fold those in ISB; beta-lactamase production was induced by milk in beta-lactamase-positive strains. Much of the increase in resistance to penicillin-G caused by milk can be attributed to milk fat globules; the increase in resistance was related to the binding capacity of the bacteria to milk fat globules as well as to capsule formation by the bacteria. It appears that the binding of the staphylococci to the fat globules and bacterial capsule formation resulted in a biofilm type of growth. In this case, the staphylococci behaved differently from the planktonic type of growth in artificial broth medium in which antibiotic susceptibility testing is usually carried out.

A study of phage- and ribotype patterns of Staphylococcus aureus isolated from bovine mastitis in the Nordic countries.

This study was conducted to investigate the geographical distribution of phage and ribotypes of Staphylococcus aureus causing bovine mastitis in the 5 Nordic countries. A total of 403 isolates of S. aureus was isolated from 403 different dairy herds. One hundred five strains were isolated in Denmark, 81 in Finland, 17 in Iceland, 96 in Norway and 104 in Sweden. The isolates were phage typed and characterized for their EcoRI restriction fragment length polymorphisms of the genes encoding ribosomal RNA (ribotyping). A total of 351 (87%) of the 403 isolates could be typed by phages assigning them to 25 different phage types. Two to 3 different phage types predominated within each country. One type (29/52) accounted for 36% of all the isolates and was found in 4 of the countries. A total of 87 different ribotypes was found among the isolates investigated. As for phage typing 2 to 3 different types predominated within countries. However, except for one type (ribotype 1), which was commonly found in Denmark, Sweden and Finland, different ribotypes predominated within each country. The combination of phage and ribotyping assigned the isolates to 178 different types. Ninety-six percent of the isolates of ribotype 1 belonged to phage type 29/52. This combined type accounted for 17% of all the 403 isolates. These findings show that a large number of different types of S. aureus can be isolated from cases of bovine mastitis. However, few types predominate within different countries. These predominating types seem to be specific in each country, however, a single type was common for both Denmark, Sweden and Finland. This could suggest differences in the virulence or in modes of transmission of predominating and rare types of S. aureus associated with bovine mastitis.

Comparisons of selenite and selenium yeast feed supplements on Se-incorporation, mastitis and leucocyte function in Se-deficient dairy cows.

The dairy cows at the Estonian Agricultural University appeared to have an extremely low selenium status. The selenium level was 5.6 micrograms/l in whole blood and 3.2 micrograms/l in milk, on average. The blood glutathione peroxidase was consequently extremely low. The effects of organic selenium (selenized yeast) and sodium selenite were compared in a feeding experiment on 100 dairy cows. Selenium incorporation, udder health and the in vitro function of blood neutrophils were monitored. Supplementation of the feed either with 0.2 ppm organic selenium or sodium selenite for 8 weeks, increased the blood selenium level (geometric mean) within this period from the back-ground level (about 5.6 micrograms/l) to 167 (Se-yeast) and to 91 micrograms/l (selenite). The respective change in whole blood glutathione peroxidase (GSH-PX) was from 0.22 to 3.0 (Se-yeast) and to 2.3 (selenite) microKat/g Hb. Blood GSH-PX continued to increase up to 10 weeks after the supplementation was stopped. The bioavailability of yeast selenium was superior to selenite: the relative bioavailability (selenite = 1) of yeast selenium was 1.4 if blood GSH-PX, 1.9 if blood selenium, and 2.7 if milk selenium was used as the response criterion. Selenium-supplementation showed a positive effect on udder health. The percentage of quarters harbouring mastitis pathogens dropped from 22.9 to 13.0 in the Se-yeast group and from 18.4 to 7.4 in the selenite group during the supplementation period. The effect of selenium on mastitis was also reflected as a decrease in the output of milk somatic cells and N-acetyl-beta-D-glucosaminidase (NAGase). The time-luminescence profile of zymosan-induced activity of blood neutrophils became skewed to the left in Se-supplemented cows.

Characterization of clinical mastitis in primiparous heifers.

Using data from health records for cows in Finland from between 1983 and 1991, we investigated the general trend for frequency of mastitis in heifers, seasonal variation, breed differences, and etiology of the disease. The performance of the heifers was monitored during first lactation. Treatment of heifer mastitis, defined as mastitis treatment 1 wk before and 1 wk after calving, increased from 1.8 to 4.4% between 1983 and 1991. The increase can be partly attributed to increased milk production, subsequent reduced resistance to mastitis, and the general trend toward more efficient treatment of mastitis. Heifer mastitis was a characteristic disease of well-managed, productive herds with low SCC and a high frequency of mastitis treatments administered by a veterinarian. Of the feeding factors studied, only use of homemade concentrates was associated with higher mastitis frequency. Mastitic heifers had higher genetic potential for milk production than control heifers, but actual milk production was 70 to 80 kg lower than expected. Most mastitic heifers recovered well and were not more susceptible than control heifers to further incidence of mastitis or other diseases. However, heifer mastitis caused more heifers than usual to be culled.

Staphylococci in heifer mastitis before and after parturition.

Aseptic udder quarter secretion samples (n = 1112) from 200 mastitic and 65 non-mastitic control heifers from 160 different farms were collected for bacteriological examination and in vitro antimicrobial susceptibility testing. Coagulase-negative staphylococci (CNS) were the most frequently isolated bacteria (57.8%). The percentage of Staphylococcus aureus was 20.1, of streptococci 11.3 and of other pathogens 10.8. Staph. simulans, Staph. hyicus, Staph. xylosus and Staph. chromogenes were the most common CNS. Staph. simulans and Staph. hyicus were most frequently found in clinical samples after parturition, whereas other CNS were equally or more often found in control samples. Most CNS infections detected in the prepartum period were eliminated spontaneously or with antibiotic treatment during early lactation, but the infected quarters were more susceptible to new infection by other pathogens. Milk N-acetyl-beta-glucosaminidase activities were measured to monitor changes in inflammation. Among the CNS infections, N-acetyl-beta-glucosaminidase activities were highest for Staph. hyicus and lowest for Staph. xylosus in the lactating mammary gland of heifers. CNS were resistant to trimethoprim-sulphonamide, ampicillin and erythromycin. Staph. aureus was more resistant than CNS to beta-lactamase-sensitive penicillins.

Bovine clinical mastitis due to coagulase-negative staphylococci and their susceptibility to antimicrobials.

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, Hautjärvi, 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.

Antibacterial susceptibility of bovine-mastitis pathogens tested directly in milk from infected quarters.

Antibacterial susceptibilities of bovine-mastitis pathogens were analysed directly in 57 mastitic milk samples without inoculation with exogenous organisms. Aseptically collected milk was mixed with serial dilutions of antibacterials and the growth was observed using 2,3,5-triphenyltetrazolium chloride (TTC) reduction the following day. The results were compared with those obtained by using calibrated bacterial inocula in turbidimetric minimum-inhibitory-concentration (MIC) determination in broth cultures, and in TTC-broth culture-test and TTC-normal milk-test. The results of different methods all correlated positively when the entire data was used. However, taking the direct test in mastitic milk as the 'true' result, the total discrepancies varied from 34.7% to 48.8%. Antibacterial activities of the trimethoprim-sulphadoxine combination, and of spiramycin and ampicillin, decreased significantly when nutrient broth was replaced by milk as the test medium. The efficacy of trimethoprim-sulphadoxine as an antibacterial agent was also dependent on the source of milk.

Effect of abrasion of teat orifice epithelium on development of bovine staphylococcal mastitis.

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.

Association af changes in the bacterial ecology of bovine mastitis with changes in the use of milking machines and antibacterial drugs.

The results of mastitis bacteriology made by the National Veterinary and Food Research Institute in Finland during the past 50 years (1.15 million samples) are viewed in relation to simultaneous changes in dairy cow management. Although intensive preventive measures have been applied for decades, the prevalence of bovine mastitis has not decreased. Instead, pathogenic bacteria are becoming progressively less susceptible to the available therapy. In part this must be due to the emergence of antibiotic resistant strains of bacteria, but it seems that the bacterial spectrum has also changed. The incidence of Streptococcus agalactiae (group B streptococci) has decreased, while in contrast, the incidence of staphylococci, initially Staphylococcus aureus and later coagulase-negative staphylococci, has increased. Results suggest that external pressure, like changes in animal husbandry, including antimicrobial treatments and introduction of modern milking machines, act as selective forces on the bacterial species which cause bovine mastitis.

Effect of treatment on the jejunal and colonic bacterial flora of dogs with exocrine pancreatic insufficiency.

The bacterial flora from the jejunum and colon of 19 dogs with naturally occurring exocrine pancreatic insufficiency (EPI) was examined. In six dogs with EPI that did not receive any treatment the bacterial count in the small intestine was > 10(6) colony-forming units (CFU)/g. This was consistent with bacterial overgrowth (BOG) (control dogs: < 10(5) CFU/g). Six dogs with EPI that received pancreatic enzyme supplementation and were in good condition had bacterial counts (> 10(6) CFU/g) that were nonsignificantly different from the untreated group. The remaining seven dogs with EPI that received both pancreatic enzyme supplementation and oral antibiotic therapy (tylosin), had bacterial flora counts that were significantly lower than the values in the other two groups of EPI dogs, and did not differ significantly from those of the control group. There were no statistical differences between the number of bacteria in the colon among the different groups of dogs with EPI and the control dogs. We conclude that the prevalence of BOG in dogs with EPI is high. Pancreatic enzyme supplementation did not affect the microflora of dogs with naturally occurring EPI. Oral antibiotic therapy with tylosin, however, effectively decreased BOG in these dogs.

Comparison of penicillin-G susceptibility testing methods of staphylococci isolated from bovine mastitis.

Penicillin-G susceptibility was analyzed on forty staphylococcal strains isolated from mastitic bovine quarters (29 coagulase positive and 11 coagulase negative) by the standard Kirby-Bauer agar diffusion method, the epsilon-Test, the Vetmic, turbidimetric MIC analysis in Iso-Sensitest Broth (ISB) and whey. Penicillinase production was tested. Parallel susceptibility tests were carried out in whole milk, whey and ISB using resazurin and triphenyltetrazolium as the indicators of bacterial activity. The traditional susceptibility testing methods (radial agar diffusion, MIC in broth culture) showed good agreement with each other and confirmed that the tests can be used interchangeably with the current breakpoint values (0.25 micrograms/ml and phi 26 mm). The tests carried out in whey showed good correlation with the traditional tests. However, the susceptibility testings in milk resulted in additional variation. Therefore, traditional susceptibility tests of Penicillin-G in artificial media are limited in estimation of bacterial susceptibility when they grow in whole milk. The relevance of this observation regarding mastitis therapy is discussed.

Relevance of sensitivity testings (MIC) of S. aureus to predict the antibacterial action in milk.

Bacterial susceptibility testings were carried out in parallel Iso-sensitest broth (ISB) and bovine milk cultures using 16 antibacterials and 4 sensitive strains of mastitic isolates of Staphylococcus aureus. Bacterial activities were analyzed by continuous turbidity monitoring (broth cultures), continuous fluorometric monitoring of the resazurin-reducing redox activity, and by analyzing the triphenyltetrazolium (TTC)-reducing capacity at the end of the incubation period. To obtain an equipotent bacteria-suppressing activity, milk cultures required in general several times more antibiotic than the respective ISB cultures. Antibacterial activities of sulfadoxine-trimethoprim, vancomycin, novobiocin, macrolides, aminoglycosides and oxytetracycline were most effectively suppressed by milk. Aminoglycosides suffered additionally from reduction of oxygen in the incubation environment. The beta-lactams (penicillin G, oxacillin, cephalothin, ceftiofur, ampicillin, ampicillin-clavulanic acid), gentamicin and enrofloxacin showed extremely variable sensitivity results depending on the S. aureus/milk combination.