Bovine Pathogens Research Articles

Comparison of bacteriological culture and PCR for detection of bacteria in ovine milk—Sheep are not small cows

Mastitis, inflammation of the mammary gland, is an important cause of disease, mortality, and production losses in dairy and meat sheep. Mastitis is commonly caused by intramammary infection with bacteria, which can be detected by bacterial culture or PCR. PathoProof (Thermo Fisher Scientific Ltd., Vantaa, Finland) is a commercially available real-time PCR system for the detection of bovine mastitis pathogens. Sheep differ from cattle in the bacterial species or bacterial strains that cause mastitis, as well as in the composition of their milk. The aim of this study was to evaluate whether the PathoProof system was suitable for detection of mastitis pathogens in sheep milk. Milk samples were collected aseptically from 219 udder halves of 113 clinically healthy ewes in a single flock. Aliquots were used for bacteriological culture and real-time PCR-based detection of bacteria. For species identified by culture, the diagnosis was confirmed by species-specific conventional PCR or by sequencing of a housekeeping gene. The majority of samples were negative by culture (74.4% of 219 samples) and real-time PCR (82.3% of 192 samples). Agreement was observed for 138 of 192 samples. Thirty-four samples were positive by culture only, mostly due to presence of species that are not covered by primers in the PCR system (e.g., Mannheimia spp.). Two samples were positive for Streptococcus uberis by culture but not by PCR directly from the milk samples. This was not due to inability of the PCR primers to amplify ovine Streptococcus uberis, as diluted DNA extracts from the same samples and DNA extracts from the bacterial isolates were positive by real-time PCR. For samples containing Staphylococcus spp., 11 samples were positive by culture and PCR, 9 by culture only, and 20 by PCR only. Samples that were negative by either method had lower bacterial load than samples that were positive for both methods, whereas no clear relation with species identity was observed. This study provides proof of principle that real-time PCR can be used for detection of mastitis pathogens in ovine milk. Routine use in sheep may require inclusion of primer sets for sheep-specific mastitis pathogens.

Hemostatic efficacy of latest-generation fibrin sealant after hepatic resection: a randomized controlled clinical study.

This randomized, controlled, single-blinded multicenter study evaluated the efficacy of latest-generation fibrin sealant containing synthetic aprotinin as fibrinolysis inhibitor as supportive treatment for hemostasis after elective partial hepatectomy. Adult subjects undergoing resection of at least one liver segment were assigned to treatment with fibrin sealant or manual compression with a surgical gauze swab if persistent oozing necessitated additional hemostatic measures after primary control of arterial and venous bleeding. The primary outcome measure was the proportion of subjects with intraoperative hemostasis at 4 min after start of randomized treatment application. Secondary efficacy outcome measures included intraoperative hemostasis at 6, 8, and 10 min, intra- and postoperative rebleedings, transfusion requirements, and drainage volume. Seventy subjects were randomized. Hemostasis at 4 min was achieved in 29/35 (82.9 %) fibrin sealant subjects compared with 13/35 (37.1 %) control subjects (p < 0.001). Significantly more fibrin sealant subjects achieved hemostasis at 6 (p < 0.001), 8 (p = 0.028), and 10 min (p = 0.017). The number of rebleedings was low in both study arms. Transfusion requirements and 48-h drainage volumes were similar between the study arms. No adverse events related to study treatment were reported. Fibrin sealant was shown to be safe and superior to manual compression in the control of parenchymal bleeding after hepatic resection. The use of synthetic aprotinin as fibrinolysis inhibitor further improves the safety margin of fibrin sealant by eliminating the risk of transmission of bovine spongiform encephalopathy and other bovine pathogens.

Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2).

TDP1 and TDP2 were discovered and named based on the fact they process 3'- and 5'-DNA ends by excising irreversible protein tyrosyl-DNA complexes involving topoisomerases I and II, respectively. Yet, both enzymes have an extended spectrum of activities. TDP1 not only excises trapped topoisomerases I (Top1 in the nucleus and Top1mt in mitochondria), but also repairs oxidative damage-induced 3'-phosphoglycolates and alkylation damage-induced DNA breaks, and excises chain terminating anticancer and antiviral nucleosides in the nucleus and mitochondria. The repair function of TDP2 is devoted to the excision of topoisomerase II- and potentially topoisomerases III-DNA adducts. TDP2 is also essential for the life cycle of picornaviruses (important human and bovine pathogens) as it unlinks VPg proteins from the 5'-end of the viral RNA genome. Moreover, TDP2 has been involved in signal transduction (under the former names of TTRAP or EAPII). The DNA repair partners of TDP1 include PARP1, XRCC1, ligase III and PNKP from the base excision repair (BER) pathway. By contrast, TDP2 repair functions are coordinated with Ku and ligase IV in the non-homologous end joining pathway (NHEJ). This article summarizes and compares the biochemistry, functions, and post-translational regulation of TDP1 and TDP2, as well as the relevance of TDP1 and TDP2 as determinants of response to anticancer agents. We discuss the rationale for developing TDP inhibitors for combinations with topoisomerase inhibitors (topotecan, irinotecan, doxorubicin, etoposide, mitoxantrone) and DNA damaging agents (temozolomide, bleomycin, cytarabine, and ionizing radiation), and as novel antiviral agents.

Potential applications for antiviral therapy and prophylaxis in bovine medicine.

Viral disease is one of the major causes of financial loss and animal suffering in today's cattle industry. Increases in global commerce and average herd size, urbanization, vertical integration within the industry and alterations in global climate patterns have allowed the spread of pathogenic viruses, or the introduction of new viral species, into regions previously free of such pathogens, creating the potential for widespread morbidity and mortality in naïve cattle populations. Despite this, no antiviral products are currently commercially licensed for use in bovine medicine, although significant progress has been made in the development of antivirals for use against bovine viral diarrhea virus (BVDV), foot and mouth disease virus (FMDV) and bovine herpesvirus (BHV). BVDV is extensively studied as a model virus for human antiviral studies. Consequently, many compounds with efficacy have been identified and a few have been successfully used to prevent infection in vivo although commercial development is still lacking. FMDV is also the subject of extensive antiviral testing due to the importance of outbreak containment for maintenance of export markets. Thirdly, BHV presents an attractive target for antiviral development due to its worldwide presence. Antiviral studies for other bovine viral pathogens are largely limited to preliminary studies. This review summarizes the current state of knowledge of antiviral compounds against several key bovine pathogens and the potential for commercial antiviral applications in the prevention and control of several selected bovine diseases.

The first study of bovine immunodeficiency virus (BIV) and bovine viral diarrhea virus (BVDV) co- infection in industrial herds of cattle in two provinces of Iran

BACKGROUND: BIV is a well-known bovine immunosup- pressive cause, but its pathogenesis has not been well characterized. It seems that it is possible that cofactors such as co- infection with other bovine viral pathogens may play a role in enhancing the pathogenesis of BIV infection; BVDV also has immunosuppressive effects. OBJECTIVE: The aim of this study was determination of possible correlation between BIV and BVDV infections. METHODS: Blood samples were randomly collected from a total of 1800 cattle in dairy industrial farms in Isfahan and Chaharmahal va Bakhtiari provinces of Iran. First BIV or BVDV positive sera were screened by ELISA, and then samples were analyzed to detect BIV proviral DNA or BVDV RNA, using PCR. RESULTS: Out of 1800 blood samples, 19 (1.06%) samples were BVDV positive, while BIV positive samples were 10 (0.55%). Nine (0.5%) samples contained both BIV and BVDV genomes and were positive in ELISA, while one of the samples (0.05%) was only BIV positive. CONCLUSIONS: In this study, there was a statistically significant relationship between BIV status and BVDV infection using Chi square and Pearson's correlation coefficient test (p=0, r=0.65).

Antimicrobial susceptibility and distribution of inhibition zone diameters of bovine mastitis pathogens in Flanders, Belgium

In dairy farms, antimicrobial drugs are frequently used for treatment of (sub)clinical mastitis. Determining the antimicrobial susceptibility of mastitis pathogens is needed to come to a correct use of antimicrobials. Strains of Staphylococcus aureus (n=768), Streptococcus uberis (n=939), Streptococcus dysgalactiae (n=444), Escherichia coli (n=563), and Klebsiella species (n=59) originating from routine milk samples from (sub)clinical mastitis were subjected to the disk diffusion method. Disks contained representatives of frequently used antibiotics in dairy. A limited number of clinical breakpoints were available through CLSI, and showed that susceptibility of Staph. aureus, E. coli, and Klebsiella was moderate to high. For streptococcal species however, a large variation between the tested species and the different antimicrobials was observed. In a next step, wild type populations were described based on epidemiological cut off values (EUCAST). Because of the limited number of official cut off values, the data were observed as a mastitis subpopulation and self-generated cut off values were created and a putative wild type population was suggested.The need for accurate clinical breakpoints for veterinary pathogens is high. Despite the lack of these breakpoints, however, a population study can be performed based on the distribution of inhibition zone diameters on the condition that a large number of strains is tested.

Overall decrease in the susceptibility of Mycoplasma bovis to antimicrobials over the past 30 years in France.

Mycoplasma (M.) bovis is frequently implicated in respiratory diseases of young cattle worldwide. Today, to combat M. bovis in Europe, only antimicrobial therapy is available, but often fails, leading to important economical losses. The antimicrobial susceptibility of M. bovis is not covered by antimicrobial resistance surveillance networks. The objectives of this study were to identify resistances that were acquired over the last 30 years in France and to determine their prevalence within comtemporary strains. The minimum inhibition concentration (MIC) values of 12 antimicrobials, considered active on M. bovis, were compared, using an agar dilution method, between 27 and 46 M. bovis isolates respectively obtained in 1978–1979 and in 2010–2012 from 73 distinct respiratory disease outbreaks in young cattle all over France. For eight antimicrobials, resistances were proven to be acquired over the period and expressed by all contemporary strains. The increase of the MIC value that inhibited 50% of the isolates (MIC50) was: i) substantial for tylosin, tilmicosin, tulathromycin and spectinomycin, from 2 to >64, 2 to >128, 16 to 128 and 4 to >64 µg/mL, respectively, ii) moderate for enrofloxacin, danofloxacin, marbofloxacin and oxytetracycline, from 0.25 to 0.5, 0.25 to 0.5, 0.5 to 1, 32 to >32 µg/mL, respectively. No differences were observed for gamithromycin, tildipirosin, florfenicol and valnemulin with MIC50 of 128, 128, 8, <0.03 µg/mL, respectively. If referring to breakpoint MIC values published for respiratory bovine pathogens, all contemporary isolates would be intermediate in vivo for fluoroquinolones and resistant to macrolides, oxytetracycline, spectinomycin and florfenicol.

An overview of calf diarrhea - infectious etiology, diagnosis, and intervention.

Calf diarrhea is a commonly reported disease in young animals, and still a major cause of productivity and economic loss to cattle producers worldwide. In the report of the 2007 National Animal Health Monitoring System for U.S. dairy, half of the deaths among unweaned calves was attributed to diarrhea. Multiple pathogens are known or postulated to cause or contribute to calf diarrhea development. Other factors including both the environment and management practices influence disease severity or outcomes. The multifactorial nature of calf diarrhea makes this disease hard to control effectively in modern cow-calf operations. The purpose of this review is to provide a better understanding of a) the ecology and pathogenesis of well-known and potential bovine enteric pathogens implicated in calf diarrhea, b) describe diagnostic tests used to detect various enteric pathogens along with their pros and cons, and c) propose improved intervention strategies for treating calf diarrhea.

Prevalence Survey of Selected Bovine Pathogens in Water Buffaloes in the North Region of Brazil

Although the largest buffalo herd in the occident is in the north region of Brazil, few studies have been conducted to assess the prevalence of selected parasitic diseases in buffalo herd. The present study was therefore conducted to investigate the epidemiological of Toxoplasma gondii, Neospora caninum, Anaplasma marginale, Babesia bigemina, and Babesia bovis in water buffaloes in the north region of Brazil. A total of 4796 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the IFAT and ELISA. The serological prevalence of T. gondii and N. caninum was 41.3% and 55.5% in ELISA and 35.7% and 48.8% in IFAT, respectively. The overall prevalence of A. marginale, B. bovis, and B. bigemina was 63%, 25%, and 21% by ELISA and 50.0%, 22.5%, and 18.8% by IFAT, respectively. This study shows valuable information regarding the serological survey of selected bovine pathogens in water buffaloes in the north region of Brazil which will likely be very beneficial for the management and control programs of this disease.

Bacteriological Quality and Detection of Bovine Mastitis Pathogens of Milk Sold in Jimma Town

Bacteriological Quality and Detection of Bovine Mastitis Pathogens of Milk Sold in Jimma Town

Short communication: Antimicrobial efficacy of intramammary treatment with a novel biphenomycin compound against Staphylococcus aureus, Streptococcus uberis, and Escherichia coli-induced mouse mastitis

Bovine mastitis undermines udder health, jeopardizes milk production, and entails prohibitive costs, estimated at $2 billion per year in the dairy industry of the United States. Despite intensive research, the dairy industry has not managed to eradicate the 3 major bovine mastitis-inducing pathogens: Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. In this study, the antimicrobial efficacy of a newly formulated biphenomycin compound (AIC102827) was assessed against intramammary Staph. aureus, Strep. uberis, and E. coli infections, using an experimental mouse mastitis model. Based on its effective and protective doses, AIC102827 applied into the mammary gland was most efficient to treat Staph. aureus, but also adequately reduced growth of Strep. uberis or E. coli, indicating its potential as a broad-spectrum candidate to treat staphylococcal, streptococcal, and coliform mastitis in dairy cattle.

Susceptibility to antimicrobial agents among bovine mastitis pathogens isolated from North American dairy cattle, 2002–2010

Approximately 8,000 isolates of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Escherichia coli, isolated by 25 veterinary laboratories across North America between 2002 and 2010, were tested for in vitro susceptibility to beta-lactam, macrolide, and lincosamide drugs. The minimal inhibitory concentrations (MICs) of the beta-lactam drugs remained low against most of the Gram-positive strains tested, and no substantial changes in the MIC distributions were seen over time. Of the beta-lactam antimicrobial agents tested, only ceftiofur showed good in vitro activity against E. coli. The MICs of the macrolides and lincosamides also remained low against Gram-positive mastitis pathogens. While the MIC values given by 50% of isolates (MIC50) for erythromycin and pirlimycin and the streptococci were all low (≤0.5 µg/ml), the MIC values given by 90% of isolates (MIC90) were higher and more variable, but with no apparent increase over time. Staphylococcus aureus showed little change in erythromycin susceptibility over time, but there may be a small, numerical increase in pirlimycin MIC50 and MIC90 values. Overall, the results suggest that mastitis pathogens in the United States and Canada have not shown any substantial changes in the in vitro susceptibility to beta-lactam, macrolide, and lincosamide drugs tested over the 9 years of the study.

Herd-level relationship between antimicrobial use and presence or absence of antimicrobial resistance in gram-negative bovine mastitis pathogens on Canadian dairy farms

Concurrent data on antimicrobial use (AMU) and resistance are needed to contain antimicrobial resistance (AMR) in bacteria. The present study examined a herd-level association between AMU and AMR in Escherichia coli (n=394) and Klebsiella species (n=139) isolated from bovine intramammary infections and mastitis cases on 89 dairy farms in 4 regions of Canada [Alberta, Ontario, Québec, and Maritime Provinces (Prince Edward Island, Nova Scotia, and New Brunswick)]. Antimicrobial use data were collected using inventory of empty antimicrobial containers and antimicrobial drug use rate was calculated to quantify herd-level AMU. Minimum inhibitory concentrations (MIC) were determined using Sensititre National Antimicrobial Resistance Monitoring System (NARMS) gram-negative MIC plate (Trek Diagnostic Systems Inc., Cleveland, OH). Isolates were classified as susceptible, intermediate, or resistant. Intermediate and resistant category isolates were combined to form an AMR category, and multivariable logistic regression models were built to determine herd-level odds of AMR to tetracycline, ampicillin, cefoxitin, chloramphenicol, trimethoprim-sulfamethoxazole combination, sulfisoxazole, streptomycin and kanamycin in E. coli isolates. In the case of Klebsiella species isolates, logistic regression models were built for tetracycline and sulfisoxazole; however, no associations between AMU and AMR in Klebsiella species were observed. Ampicillin-intermediate or -resistant E. coli isolates were associated with herds that used intramammarily administered cloxacillin, penicillin-novobiocin combination, and cephapirin used for dry cow therapy [odds ratios (OR)=26, 32, and 189, respectively], and intramammary ceftiofur administered for lactating cow therapy and systemically administered penicillin (OR=162 and 2.7, respectively). Use of systemically administered penicillin on a dairy farm was associated with tetracycline and streptomycin-intermediate or -resistant E. coli isolates (OR=5.6 and 2.8, respectively). Use of cephapirin and cloxacillin administered intramammarily for dry cow therapy was associated with increasing odds of having at least 1 kanamycin-intermediate or -resistant E. coli isolate at a farm (OR=8.7 and 9.3, respectively). Use of systemically administered tetracycline and ceftiofur was associated with cefoxitin-intermediate or -resistant E. coli (OR=0.13 and 0.16, respectively); however, the odds of a dairy herd having at least 1 cefoxitin-intermediate or -resistant E. coli isolate due to systemically administered ceftiofur increased with increasing average herd parity (OR=3.1). Association between herd-level AMU and AMR in bovine mastitis coliforms was observed for certain antimicrobials. Differences in AMR between different barn types and geographical regions were not observed.

Production and characterization of egg yolk antibodies against bovine alimentary tract pathogens

Aim of the study was to evaluate the effect of immunization of hens with bovine vaccines (C, R, T) on the course of IgY antibodies production against selected bovine E. coli strains, rota- and coronaviruses in egg yolk in farm conditions. The hens (40 individuals per group) were vaccinated twice, subcutaneously in four week interval and eggs were harvested once a week. Control group consisted of eggs sampled from non-vaccinated hens located in neighbouring cages. The antibody activity was measured by ELISA. All used vaccines induced the rise of IgY antibody in egg yolks. Based on the duration and the highest level of IgY antibody against bovine alimentary tract pathogens C vaccine was further used in next two trials for vaccination of 1000 hens each time. Double immunization seems to be enough in mounting response against examined pathogens for several weeks. Immunization with C vaccine allowed to harvest eggs with satisfactory levels of E. coli, rotavirus and coronavirus IgY antibodies which may be used to evaluate their protective effect by oral administration in calves.

Detection and discrimination of common bovine mastitis-causing streptococci

Detection and typing of bovine mastitis pathogens are currently limited by time-consuming and culture-based techniques. In this work, a novel genus-specific DNA marker for Streptococcus and species-specific DNA markers for the prevalent mastitis pathogens Streptococcus agalactiae and Streptococcus uberis were designed and assessed. In order to enable further discrimination of these mastitis-causing streptococci, metabolic and pathogenicity-related genes were used to infer additional functional markers. A total of 12 DNA markers were validated with a set of 50 reference strains and isolates, representative of the Streptococcus genus, of closely related species and of microorganisms with matching habitats. The experimental validation, using dot blot hybridization under high stringency conditions, confirmed the specificity of the selected markers. The broad-spectrum taxonomic marker (ST1) was specific to the Streptococcus genus and the markers selected for S. agalactiae (A1 and A2) and S. uberis (U1 and U2) were shown to be species-specific. The functional markers revealed strain-specific patterns of S. agalactiae and S. uberis. Markers derived from the fructose operon (FO1 and FO3) were specific to bovine isolates of S. agalactiae, and the nisin operon markers (NU1 and NU3) were able to discriminate isolates belonging to S. agalactiae and S. uberis. The virulence-associated markers (V1, V2 and V3) allowed the detection of S. uberis and of closely related species. This work suggests that the combined use of these novel taxa-specific markers coupled with discriminatory functional markers presents a promising approach for the rapid and cost-effective detection and discrimination of common bovine mastitis-causing pathogens, which will contribute to an improved treatment and control of this disease.

Mutations induced in the NS5B gene of bovine viral diarrhea virus by antiviral treatment convey resistance to the compound

Bovine viral diarrhea virus (BVDV) is a widespread bovine pathogen for which there is no specific therapeutic agent. A previous study using 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) to treat calves persistently infected with BVDV resulted in a decrease in the viral load of infected calves but treatment resulted in the rapid selection of drug-resistant mutant isolates. In this article we describe three mutations found in the mutant isolates associated with in vivo and in vitro resistance to DB772. All three mutations are found in the NS5B which functions as the RNA-dependent-RNA-polymerase during viral replication. Growth curves for the mutant isolates were not largely different from those of wild-type isolates when cultured in the absence of DB772. Thus, DB772 appears to act by binding to the specified domain but binding is disrupted or inhibited by the described mutation.

Pharmacodynamics of florfenicol for calf pneumonia pathogens

The antimicrobial properties of florfenicol were investigated for the bovine respiratory tract pathogens, Mannheimia haemolytica and Pasteurella multocida. Three in vitro indices of efficacy and potency were determined; minimum inhibitory...

Molecular survey of bovine vector-borne pathogens in Cebu, Philippines

Vector-borne diseases (VBDs) continue to threaten the worldwide livestock industry, but comprehensive epidemiological surveys on such diseases have not been conducted in the Philippines. In the present study, we screened 408 bovine blood samples from 9 areas in Cebu, Philippines, for various VBD pathogens using specific PCR assays. The results revealed prevalences of 54.7, 15.4, 10.0, and 12.0% for Anaplasma spp., Babesia bigemina, Babesia bovis, and Trypanosoma (Tr.) theileri, respectively. In contrast, none of the samples were positive for Trypanosoma (Tr.) evansi, Theileria (Th.) orientalis, and Theileria (Th.) annulata. Mixed infections were observed in 24.2% of the samples tested. Phylogenetic analysis based on the 16S rRNA gene revealed that the Anaplasma spp. sequences from the present study were genetically close either to Anaplasma marginale or Anaplasma phagocytophilum. In addition, B. bovis RAP-1 and Babesia bigemina AMA-1 gene sequences were identical and monophyletic to other known B. bovis and B. bigemina sequences. On the other hand, Tr. theileri cathepsin-L like protein gene sequences shared 97.1–100% identities with those from the USA and Brazil and clustered within a single genotype in the phylogenetic tree. The molecular identification of several VBD pathogens in Cebu cattle calls for the implementation of control measures to prevent the spread of these pathogens to nearby localities or islands, and ultimately, economic losses to the Philippine economy.

Novel Aspects of the Z and R3 Antigens of Streptococcus agalactiae Revealed by Immunological Testing

Group B streptococci (GBS) are important human and bovine pathogens which can be classified by a variety of phenotype- and gene-based techniques. The capsular polysaccharide and strain-variable, surface-anchored proteins are particularly important phenotypic markers. In an earlier study, a previously unrecognized protein antigen called Z was described. It was expressed by 27.2% of GBS strains from Zimbabwe, usually in combination with R3 protein expression. In this study, a putative Z-specific antiserum actually contained antibodies against two different antigens named Z1 and Z2; Z1 was >250 kDa in molecular mass. Z1, Z2, and R3 generated multiple stained bands on Western blots and showed similar chromatographic characteristics with respect to molecular mass, aggregate formation, and charge. Of 28 reference and prototype GBS strains examined, 8/28 (28.5%) isolates expressed one, two, or all three of the Z1, Z2, and R3 antigens; 4/28 expressed all three antigens; 2/28 expressed Z2 and R3; 1/28 expressed Z1 only; and 1/28 expressed R3 only. Twenty (71.5%) of the 28 isolates expressed none of the three antigens. Expression of one or more of these antigens was shown by isolates of the capsular polysaccharide types Ia, Ib, V, and IX and NT strains and occurred in combination with expression of various other strain-variable and surface-localized protein antigens. When used as serosubtype markers, Z1, Z2, and R3 affected existing GBS serotype designations for some of the isolates. For instance, the R3 reference strain Prague 10/84 (ATCC 49447) changed serotype markers from V/R3 to V/R3, Z1, and Z2. Other isolates may change correspondingly, implying consequences for GBS serotyping and research.

Pharmacodynamics of marbofloxacin for calf pneumonia pathogens

The pharmacodynamic (PD) properties of the fluoroquinolone, marbofloxacin, were determined for the bovine respiratory tract pathogens Mannheima haemolytica and Pasteurella multocida. For six pathogenic isolates of each organism, three in vitro indices of efficacy and potency were determined, namely, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves. Each parameter was determined in two matrices, Mueller Hinton Broth (MHB) and calf serum. For serum, MBC:MIC ratios were 2.7:1 (M. haemolytica) and 2.4:1 (P. multocida). The killing action of marbofloxacin had the characteristics of concentration dependency against M. haemolytica and co-dependency (on time and concentration) against P. multocida. To confirm the characteristics of the time-kill profiles, growth inhibition produced by marbofloxacin was also established ex vivo in three biological fluids, calf serum, exudate and transudate, harvested from a tissue cage model. The in vitro time-kill data were modelled with pharmacokinetic properties of marbofloxacin, established by intramuscular administration in calves at a dose of 2mg/kg; three levels of activity, namely bacteriostatic, 3 log10 reduction and 4 log10 reduction in bacterial counts were determined. Mean AUC24h/MIC values (with percentage coefficients of variation indicating inter-isolate variability) for M. haemolytica, based on serum MICs, were 31.3 (41.6), 57.7 (42.4) and 79.2 (44.6)h, respectively. Corresponding values for MHB were 20.5 (58.0), 40.5 (51.8) and 51.2 (24.30)h, respectively. When allowance was made for binding of marbofloxacin to serum protein, the AUC24h/MIC values for serum were similar to those for MHB. Numerical AUC24h/MIC values for P. multocida were slightly lower than those obtained for M. haemolytica. These data establish for the first time inter-isolate variability in AUC24h/MIC values required for three levels of bacterial kill for two pathogenic species and thereby provide an indication of variability in serum concentration that might be required to achieve efficacy in clinical subjects.