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A Gunnarsson

Publications and source records attributed to A Gunnarsson.

At least 19 recordsLinked to original sources

Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli.

Traditional culture and biochemical tests (CBT) were compared with PCR for sensitivity and detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in seeded faeces and clinical samples from diarrhoeic pigs. A duplex PCR system was developed based on primers detecting the tlyA-gene of B. hyodysenteriae and the 16S rRNA-gene of B. pilosicoli. Sensitivities for the PCR system were determined on seeded faeces, using DNA that had been recovered from primary cultures or extracted directly from faeces. Compared to CBT, PCR applied to DNA extracted directly from faeces lowered the sensitivity by a factor of 1000 to 10,000. B. hyodysenteriae and B. pilosicoli detection was compared for CBT and PCR using 200 clinical samples. CBT detected more B. hyodysenteriae isolates in the clinical samples than PCR, but fewer B. pilosicoli positive samples. An atypical strongly haemolytic isolate was detected only by CBT.

Animals↗

Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test.

There is no ring test for quality assessment available in Europe for diagnostics and antimicrobial susceptibility testing of the fastidious, anaerobic bacteria of the genus Brachyspira. Therefore, an international ring test for Brachyspira spp. was performed once a year during 2002-2004. Two sets of coded samples were prepared and distributed on each occasion. One set comprised six swabs dipped in pig faeces spiked with Brachyspira spp. intended for diagnostics. The other set comprised two pure strains intended only for susceptibility testing. All methods used were in-house methods. The species used were Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira innocens, Brachyspira murdochii and Brachyspira intermedia. In most cases, the correct Brachyspira spp. were detected. However, the results showed that Brachyspira spp. could be difficult to identify, especially if two Brachyspira spp. were mixed or if the concentration of Brachyspira in faeces was low. Additionally, some laboratories reported Brachyspira growth in control samples that were not seeded with any spirochaetes. The lowest detection level was 10(2) bacteria/ml faeces for both B. hyodysenteriae and B. pilosicoli. The susceptibility tests performed showed that disc diffusion was not recommendable for Brachyspira spp. Extended antimicrobial dilution series gave most congruent results. The diversity of the results highlights the importance of ring tests for a high quality of diagnostics and antimicrobial susceptibility tests for Brachyspira spp. This is the first ring test described for Brachyspira spp.

Animals↗

Prevalence of verotoxin-producing Escherichia coli (VTEC) 0157 in Swedish dairy herds.

A prevalence study of verotoxin-producing Escherichia coli O157 (VTEC O157) was performed in 371 randomly selected dairy herds distributed throughout Sweden. Faecal and manure samples were collected and analysed by immunomagnetic separation and culturing. Data were recorded for each herd regarding herd size, age of sampled animals and whether, in addition to cattle, the farm kept other animals. VTEC O157 was isolated from 33 (8.9%) of the 371 investigated herds. The prevalence was higher (23.3%) in Halland county than in the rest of Sweden (P > 0.01). Halland was also the county in Sweden that during the study period had the highest incidence of human VTEC O157 cases. VTEC O157 was not detected on any farm in northern Sweden. Identified risk factors, in the multivariate analyses, for herds being VTEC O157 positive were herd size, geographical localization, presence of pigs on the farm and median age of sampled animals.

Animals↗

Surveillance programme for Campylobacter species in Swedish broilers, July 2001 to June 2002.

A total of 20,599 samples from 4133 slaughter groups representing about 71 million broilers slaughtered between July 1,2001 and June 30, 2002 were tested for Campylobacter species. From each slaughter group, 40 cloacal samples and 10 samples of neck skin were taken from the carcases on the slaughter line and the samples were pooled, 10 to a pool. Campylobacter species were detected in cloacal samples from 17 per cent of the slaughter groups and in neck skin samples from 22 per cent of the groups, and the monthly prevalence figures ranged from 6 to 32 per cent. In most of the infected groups all four pooled cloacal samples were positive, but 18 per cent of the infected groups had only one or two positive samples. More than 90 per cent of the Campylobacter-positive slaughter groups were delivered by half the producers, and one in four of the producers delivered no Campylobacter-positive slaughter groups.

Abattoirs↗

Antimicrobial susceptibility of equine and environmental isolates of Clostridium difficile.

The antimicrobial susceptibility of 50 Clostridium difficile isolates, 36 of them from horse feces and 14 from environmental sites, was determined by broth microdilution. The antimicrobial agents tested were avilamycin, cephalothin, chloramphenicol, clindamycin, erythromycin, gentamicin, neomycin, oxacillin, oxytetracycline, penicillin, spiramycin, streptomycin, trimethoprim/sulfamethoxazole, vancomycin, and virginiamycin. All isolates were susceptible to vancomycin (MIC </=1 microg/ml). The MICs of erythromycin, oxytetracycline, spiramycin, and virginiamycin showed a bimodal distribution. Compared with the majority of isolates, the MICs of erythromycin (MIC > 16 microg/ml), oxytetracycline (MIC >/=32 microg/ml), spiramycin (MIC > 16 microg/ml), and virginiamycin (MIC 8-16 microg/ml) were higher for 18 isolates. Those were mainly isolated from horses at animal hospitals and further from environmental sites at a stud farm. In contrast, all isolates, except one, from healthy foals had low MICs of erythromycin, spiramycin, virginiamycin, and oxytetracycline. The isolates from soil in public parks had also low MICs of these antimicrobial agents. Broth microdilution appeared both reliable and reproducible for susceptibility testing of C. difficile. The method was also readily performed and the MIC endpoints were easily read.

Animals↗

Molecular typing of isolates of Clostridium perfringens from healthy and diseased poultry.

The bacterium Clostridium perfringens can cause both clinical and subclinical disease in poultry. To study the pathogenesis and epidemiology of disease caused by C. perfringens, methods for typing its various strains need to be evaluated. C. perfringens isolates from healthy and diseased poultry from different parts of Sweden were analysed by polymerase chain reaction (PCR) in order to establish the presence of alpha-, beta-, beta2-, epsilon -, iota- and enterotoxin genes. In order to subtype C. perfringens isolates, the two methods amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) were compared on 21 C. perfringens isolates from 10 different farms. In a second study, 32 isolates of C. perfringens type A from three broilers from a healthy flock reared without ionophorous anticoccidials were subtyped by PFGE. All 53 isolates analysed with PCR belonged to the toxin type A of C. perfringens, with the gene coding for alpha-toxin production. Two isolates possessed the beta2-gene as well, but none had the other toxin genes. Both AFLP and PFGE differentiated 21 strains into 10 different subtypes. This differentiation correlated closely with the origins of the isolates. Unique subtypes were isolated from seven farms. Only isolates from birds of one farm demonstrated more than one subtype of C. perfringens. The subtyping of the isolates from a healthy flock showed that each bird carried two to three different subtypes and two different subtypes were found in the same kind of tissue sample in four cases. Three of the four different subtypes found in this study were new, compared with the first study. AFLP and PFGE were found to be equally suitable for subtyping of C. perfringens isolates. The wide variation in subtypes in the healthy broilers could be the result of the antibiotic-free rearing of these birds.

Animals↗

Leptospira in slaughtered fattening pigs in southern Vietnam: presence of the bacteria in the kidneys and association with morphological findings.

One kidney was collected from each of 32 fattening pigs at an abattoir in southern Vietnam in 2001 in order to demonstrate infecting Leptospira serovar and to associate renal macro- and microscopic findings with the presence of renal leptospires. Leptospires were demonstrated in 22 (69%) of the investigated kidneys by immunofluorescence. Multifocal interstitial nephritis (MFIN) and gross renal lesions (white spots) were each demonstrated in 24 (75%) kidneys. Leptospira interrogans serovar bratislava was isolated from one kidney. There was no association between presence of leptospires and MFIN (P=0.19), respectively and white spots (P=0.98), respectively. These data suggest that Leptospira infection is common among fattening pigs in the study area and that these animals may be considered as an occupational human health hazard. It is also suggested that the presence of white spots is an unreliable indicator of the presence of renal leptospires.

Agglutination Tests↗

Verocytotoxin-producing Escherichia coli O157:H7 in the Swedish pig population.

Verocytotoxin-producing Escherichia coli O157:H7 (VTEC O157:H7) was detected in two of 2446 individual faecal samples collected from pigs slaughtered at five Swedish slaughterhouses, indicating a prevalence of 0.08 per cent, with a 95 per cent confidence interval from 0 to 0.16 per cent Four Swedish VTEC O157:H7-positive farms which kept ruminants and pigs were studied by repeated faecal sampling; VTEC O157:H7 was isolated from the ruminants and pigs on all the farms and the same strains were present in the pigs and the ruminants. On one of the farms, the organism persisted in the pig population for 11 months. On all four farms, management practices which might have influenced the isolation rate in pigs were identified. A group of young VTEC O157:H7-positive pigs was moved from one of the VTEC O157:H7-positive farms to a fattening herd where there were no ruminants. The number of VTEC O157:H7-positive faecal samples decreased gradually and after nine weeks the pigs were all negative; at slaughter none of the pigs was VTEC O157:H7-positive.

Abattoirs↗

Diarrhoea in the growing pig - a comparison of clinical, morphological and microbial findings between animals from good and poor performance herds.

Diarrhoea among growing pigs (8-13 weeks old) is a significant problem in many herds. Nine herds with poor performance and diarrhoea among growing pigs were selected on the basis of their piglet mean age at a body weight of 25 kg, compared to the overall mean age in Swedish herds. In addition, four herds with good average performance and no problems with diarrhoea were selected. Pigs were necropsied and samples for histology and microbiology were collected. Based on the necropsy findings, the pigs from the good performing herds were all judged to be healthy. The presence of Brachyspira pilosicoli and Lawsonia intracellularis was significantly correlated to poor performing herds and the results indicate that these microbes are main pathogens involved in enteric diseases among Swedish grower pigs. In addition, concomitant infections with other presumptive pathogens were commonly found.

Animals↗

Antimicrobial susceptibility testing of porcine Brachyspira (Serpulina) species isolates.

No standardized method for susceptibility testing of Brachyspira spp. is currently available. A broth dilution procedure was evaluated and used to test the activities of six antimicrobial agents for 108 isolates of Swedish porcine Brachyspira spp. representing biochemical groups I, II, and III. Group I corresponds to Brachyspira hyodysenteriae, group II corresponds to B. intermedia, and group III corresponds to B. murdochii and B. innocens. A panel was designed with the antimicrobial agents dried in tissue culture trays with wells that allowed a liquid volume of 0.5 ml in each and agitation of the broth when incubated on a shaker. The MICs were determined by using brain heart infusion broth with 10% fetal calf serum. For 10 isolates, the results obtained in broth were compared to the MICs obtained on two different types of agar. Different inoculum densities and incubation times were also compared. The concentrations at which 90% of the B. hyodysenteriae isolates (n = 72) were inhibited in the broth dilution test by tiamulin (0.25 micro g/ml), tylosin (>256 micro g/ml), erythromycin (>256 micro g/ml), clindamycin (>4 micro g/ml), virginiamycin (4 micro g/ml), and carbadox (0.06 micro g/ml) were determined. The MICs tended to be lower in broth than on agar. Differences in inoculum densities and incubation times had little influence on the MICs. The evaluated broth dilution test was simple to perform, the end points were easily read, and the results were reproducible and reliable. No isolates with decreased susceptibility to tiamulin were found among the Swedish isolates tested.

Animals↗

Clostridium difficile: prevalence in horses and environment, and antimicrobial susceptibility.

REASONS FOR PERFORMING STUDY: Clostridium difficile has been associated with acute colitis in mature horses. OBJECTIVES: To survey C. difficile colonisation of the alimentary tract with age, occurrence of diarrhoea and history of antibiotic therapy; and to study the occurrence and survival of C. difficile in the environment and antimicrobial susceptibility of isolated strains. METHODS: A total of 777 horses of different breeds, age and sex were studied. Further, 598 soil samples and 434 indoor surface samples were examined. Antimicrobial susceptibility of 52 strains was investigated by Etest for 10 antibiotics. RESULTS: In horses that developed acute colitis during antibiotic treatment, 18 of 43 (42%) were positive to C. difficile culture and 12 of these (28%) were positive in the cytotoxin B test. Furthermore, C. difficile was isolated from a small number of diarrhoeic mature horses (4 of 72 [6%]) with no history of antibiotic treatment, but not from 273 healthy mature horses examined or 65 horses with colic. An interesting new finding was that, in normal healthy foals age < 14 days, C. difficile was isolated from 1/3 of foals (16 of 56 [29%]). All older foals (170) except one were negative. Seven of 16 (44%) nondiarrhoeic foals treated with erythromycin or gentamicin in combination with rifampicin were also excretors of C. difficile. On studfarms, 14 of 132 (11%) outdoor soil samples were positive for C. difficile in culture, whereas only 2 of 220 (1%) soil samples from farms with mature horses were positive for C. difficile (P = < 0.001). By PCR, it was demonstrated that strains from the environment and healthy foals can serve as a potential reservoir of toxigenic C. difficile. The experimental study conducted here found that C. difficile survived in nature and indoors for at least 4 years in inoculated equine faeces. The susceptibility of 52 strains was investigated for 10 antibiotics and all were susceptible to metronidazole (MIC < or = 4 mg/l) and vancomycin (MIC < or = 2 mg/l). CONCLUSIONS: C. difficile is associated with acute colitis in mature horses, following antibiotic treatment. Furthermore, C. difficile was isolated from 1 in 3 normal healthy foals age < 14 days. POTENTIAL RELEVANCE: Strains from healthy foals and the environment can serve as a potential reservoir of toxigenic C. difficile.

Acute Disease↗

Animal- and herd-level risk factors for leptospiral seropositivity among sows in the Mekong delta, Vietnam.

In 1998, a total of 424 sows had sera collected in the Mekong delta in Vietnam. Of these, 283 sows were from 151 small-scale family farms in 19 villages, and 141 from seven large-scale state farms. The sera were subjected to the microscopic agglutination test (MAT) for antibodies to 13 Leptospira serovars. The overall leptospiral seroprevalence for titres > or =1:100 and > or =1:400, was 73 and 29%, respectively, and was higher (P=0.001) at small- than at large-scale farms. The highest seroprevalence was recorded for Leptospira interrogans serovar (sv) bratislava (52%). At small-scale farms, higher prevalences were found to serovars L. interrogans sv icterohaemorrhagiae (P=0.04) and L. interrogans sv pomona (P=0.02). Epidemiological information (at the individual-animal and herd-levels) was collected with a questionnaire. The data were analysed using logistic multiple regression. At the animal-level, sows seropositive for L. interrogans sv australis and sv autumnalis had less direct contact with sows in neighbouring pens (odds ratio (OR)=0.3 and 0.4, respectively) and sows seronegative for L. interrogans sv bratislava were of lower age (OR=0.1 for seropositivity). Also, sows seropositive for L. interrogans sv icterohaemorrhagiae had higher odds (OR=5.8) if they had not been born on the farm (had been introduced to it as gilts). Herds seropositive for sv javanica showed association with farms not taking measures to control the local rodent population (OR=7.8). Serovar pomona was also linked to the use of artificial insemination (AI), as opposed to natural-breeding services (OR=11.2). These results indicate that housing and management could affect the seroprevalence of Leptospira infection in pigs.

Age Factors↗

Validation of a polymerase chain reaction/restriction enzyme analysis method for species identification of thermophilic campylobacters isolated from domestic and wild animals.

AIMS: To compare and evaluate a polymerase chain reaction/restriction enzyme analysis (PCR/REA) method with standard phenotypic tests for the identification and differentiation of the thermophilic campylobacters Campylobacter jejuni, C. coli, C. lari and C. upsaliensis. METHODS AND RESULTS: One hundred and eighty-two presumptive thermophilic campylobacters from 12 different animal species were tested by a recently published PCR/REA and standard phenotypic tests. By PCR/REA, 95% of the isolates were clearly identified as either one of the four thermophilic Campylobacter species or as not belonging to this group of organisms at all. By standard phenotyping, 174 of the 182 isolates were initially identified as either C. jejuni, C. coli, C. lari or C. upsaliensis. Additional genotypic tests and phenotyping showed that 52 of these identifications were either incorrect or unreliable. Of the C. jejuni isolates, 19% were identified as C. coli by initial phenotyping and 27 sheep isolates phenotyped as C. coli or C. lari were, in fact, arcobacters. CONCLUSIONS: The PCR/REA was more reliable than standard phenotyping for the identification of thermophilic campylobacters from different animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Routinely used phenotypic tests often resulted in unreliable identifications, requiring additional testing. The PCR/REA, however, gave unequivocal results and was considered useful for the routine identification of thermophilic campylobacters from different animals.

Animals↗

Cross-sectional study of the seroprevalence to Borrelia burgdorferi sensu lato and granulocytic Ehrlichia spp. and demographic, clinical and tick-exposure factors in Swedish horses.

A cross-sectional study of the seroprevalence to Borrelia burgdorferi sensu lato and granulocytic Ehrlichia spp. in Swedish horses was conducted to evaluate associations with demographic, clinical and tick-exposure factors. From September 1997-1998, blood samples from 2018 horses were collected from the animals presented to veterinary clinics affiliated with the Swedish Horserace Totalizator Board (regardless of the primary cause for consultation). Standardized questionnaires with information both from owners and attending veterinarians accompanied each blood sample. The apparent seroprevalences to B. burgdorferi s. l. and granulocytic Ehrlichia spp. were 16.8 and 16.7%, respectively. The northern region had the lowest seroprevalences. Four logistic models were developed (controlling for demographic variables). In the disease model of seropositivity to B. burgdorferi s. l., age, breed, geographic region, the serologic titer to granulocytic Ehrlichia spp., season and the diagnosis coffin-joint arthritis were significant. In the tick-exposure model of B. burgdorferi s. l., pasture access the previous year and gender were significant. Age, racing activity, geographic region, season and the serologic titer to B. burgdorferi s. l. were associated with positivity to granulocytic Ehrlichia spp. In the tick-exposure model of granulocytic Ehrlichia spp., pasture access was a risk factor. An interaction between racing activity and geographic region showed that the risk of positive serologic reactions to Ehrlichia spp. was increased in the horse population in the south and middle of Sweden, but only among horses not used for racing. Except for the positive association between coffin-joint arthritis and serologic reactions to B. burgdorferi s. l., there were no significant associations in the multivariable models between non-specific or specific clinical sign or disease with seropositivity to either of these agents.

Animals↗

Clinical and bacteriological aspects on the use of oxytetracycline and flunixin in primiparous cows with induced retained placenta and post-partal endometritis.

Retention of the fetal membranes and post-partal endometritis (RFM) are common problems in dairy cows. Treatment often includes manual removal of the placenta in combination with antibiotic treatment. Earlier studies have shown that cows with endometritis post-partum have a strong tendency to recover spontaneously. The present study focused on treatments of post-partal endometritis with the prostaglandin synthesis inhibitor, flunixin (F) either alone or combined with oxytetracycline (T). The study was conducted in two experiments, using 12 primiparous cows in each. As a model for RFM, premature parturition was induced in late pregnant heifers by injecting PGF2alpha (25 mg i.m.) twice with a 24 h interval. In each experiment the cows were set into four groups and treated with either T (10 mg/kg BW i.m. once daily), F (2.2 mg/kg BW p.o. twice daily), a combination of T and F (dosage, as above) or conservatively (group 0, no drugs). The treatment periods lasted from days 11-14 post-partum in experiment I (groups T1, F1, TF1 and 0) and from days 3-6 post-partum in experiment 2 (groups T2, F2, TF2 and 0). Jugular vein blood samples were collected for analyses of flunixin and total white blood cells. Uterine biopsies were collected twice weekly for investigation of endometrial microbiology. Rectal palpation and ultrasonographic examinations were performed three times weekly for investigations of uterine involution and ovarian activity. No attempts were made to remove the placentas manually. The experiment lasted until day 56 post-partum. The induction of parturition was successful in all heifers and 22 of 24 animals had RFM. All RFM cows had bacterial endometritis. The predominant bacteria were Escherichia coli alpha-haemolytic streptococci, Fusobacterium necrophorum, Arcanobacterium (Actinomyces) pyogenes, Bacteroides spp., Pasteurella spp. and Proteus spp. Fusobacterium necrophorum and A. pyogenes could be isolated for 3-5 weeks post-partum and E. coli Pasteurella and Proteus could be isolated for 2-3 weeks post-partum. Animals treated with tetracycline after placental shedding (T1 and TF1) had a more rapid recovery from infections with A. pyogenes and F. necrophorum than animals that were not treated with tetracycline. No other genera were affected. Antibiotic treatment before placental shedding (T2 and TF2) did not shorten the uterine infection but altered the bacterial flora, seen as an overgrowth of Proteus spp. (p < 0.05) and increased frequency of Pasteurella (p < 0.05). The alpha-haemolytic streptococci were less common in T2 and TF2 than in other groups (NS). Antibiotic treatment of cows before placental shedding (T2 or TF2, n = 6) postponed detachment of placenta compared to cows were no antibiotics were administered before placental shedding (T1, TF1, F1, F2 and 0, n = 16. 9.8 days pp (median) versus p = 0.004). Neither treatment shortened uterine involution. Flunixin treatments did not seem to influence recovery from infection or uterine involution. It was concluded that early oxytetracycline treatment of retained fetal membranes in the cow did not shorten the uterine involution or uterine infection but it did slow down the detachment process of the retained placenta. Oxytetracycline treatment after placental shedding might shorten the uterine infection but otherwise did not affect the clinical results. Flunixin treatment had no influence on the clinical outcome of the disease.

Administration, Oral↗

The use of culture, pooled samples and PCR for identification of herds infected with Brachyspira hyodysenteriae.

The sensitivity of culturing Brachyspira hyodysenteriae was determined after sampling with swabs from porcine fecal specimens inoculated with tenfold dilutions of a field strain of these microbes. After storage of swabs, Brachyspira hyodysenteriae was recovered throughout the first 3 weeks after inoculation from feces with more than 140 cells/g. Viable spirochetes could still be recovered after up to 83 days of storage from feces, with 1.4 x 10(6) cells or more per gram. Culture for Brachyspira spp. was performed on 285 rectal swabs, which were pooled in batches of five. The number of pooled samples positive for B. hyodysenteriae corresponded with the sum results of individual analysis of the corresponding collections of five samples. A PCR system based on the tlyA gene of B. hyodysenteriae was developed and tested on primary cultures of pooled samples. The results of the PCR assay showed a 97% correlation with the culture results. The prevalence of Brachyspira spp. was determined in five swine herds and found to be highest among breeding gilts and boars aged 13-16 weeks and among 6-12-week-old weaned pigs. In contrast, Brachyspira spp. were only rarely found in sows, which may reflect the development of immunity by adult pigs to all species of the genus.

Age Factors↗

Susceptibility to pleuromutilins in Brachyspira (Serpulina) hyodysenteriae.

The pleuromutilins are the only antimicrobial agents with sufficient minimum inhibitory concentration (MIC) values left to treat swine dysentery in Sweden. Other antimicrobials are either not approved for use against swine dysentery or only partly active against Brachyspira hyodysenteriae. To date, in Sweden two pleuromutilins, tiamulin and valnemulin, are authorized for use in pigs. This study includes a comparison between MICs of tiamulin and valnemulin for Swedish field isolates of B. hyodysenteriae, as determined by broth dilution. For different isolates the MIC of tiamulin was between 0 and 8 times higher than that of valnemulin. No resistance to pleuromutilins was recorded (tiamulin MIC range 0.031-2 microg/ml, valnemulin MIC range < or =0.016-1 microg/ml). In vitro development of tiamulin resistance was also studied. Two B. hyodysenteriae and two B. pilosicoli strains became resistant to tiamulin following reiterated passages on agar containing tiamulin in increasing concentrations. The resistance emerged slowly and three of the strains that went through more than 60 passages increased their tiamulin MICs from 0.031-0.25 to more than 128 microg/ml. The tiamulin MIC for one B. hyodysenteriae strain that went through 29 passages increased from 0.0125 to 4 microg/ml. One B. pilosicoli strain developed cross-resistance to valnemulin; the MIC increased from 0.25 to more than 64 microg/ml. The valnemulin MIC for one B. hyodysenteriae strain increased from 0.031 microg/ml to 32 microg/ml. Valnemulin MIC was not determined for the B. hyodysenteriae strain that only went through 29 passages. The valnemulin MIC of the other B. pilosicoli strain increased from 0.031 to 4 microg/ml.

Animals↗