**4.3.3 Tylosin**

456 A Bird's-Eye View of Veterinary Medicine

administration. As AZT is so penetrating in tissues and cells, and mastitic milk is so rich in somatic and inflammatory cells, the drug becomes included into the more acidic cellular compartment, thus hindering its participation in the milk-serum diffusion process (despite its pKa), and this retains high amounts of AZT in milk by cell trapping. All this evidence supports the fact that AZT is a penetrating azalide with concentrations several times higher in tissues and milk than in plasma. If we observe the ratios AUC(milk)/AUC(serum) and we compare them with those obtained when the drug was administered intramuscularly we could conclude that the high availabilities in milk compared to those of serum are

But, in the study with cows at drying-off, we found some differences: AZT milk concentrations in mastitic and healthy animals were similar (Fig. 13 B and Table 17). One explanation for this is that physiology of the mammary gland during the dry period differs markedly from that during lactation. Very few cells (less than 2% are epithelial cells) and total leukocyte concentration increase rapidly in early involution and the milk fat and casein

Fig. 13. Mean serum and milk AZT concentrations after one IMM AZT syringe containing 125 mg in each mammary quarter for three consecutive milkings in healthy and mastitic lactating cows (A). Mean serum and milk AZT concentrations after IMM syringe containing

Finally, the excellent milk availability observed allows us to consider AZT as a potential antimastitic drug, although we have to fit the dosage through PK-PD modeling and corroborate with efficacy studies in the future. As was mentioned in previous paragraphs, the site where the pathogen is located represents one of the real challenges of ATM chemotherapy of the mammary gland. The pathogen can be in milk, or tissues. In the last case it can be in the interstitium or in cells. And in this case it can be in the cytoplasm or in fagolysosomes. As can be easily understood the deeper the location of the microorganism the more difficult will be to reach it by the ATM. Furthermore, if the ATM reaches the site of the microorganism, it has to exert its antibacterial effect and to do this it needs some special conditions, being the pH a critical one. And the pH becomes more acidic the deeper in tissues and cells it is measured. As illustrative figures we can mention the pH of plasma of 7.4, of interstitium 7.0, of cytoplasm 6.5 and of fagolysosome of 5.0. We evaluated the effect of the pH variation on the antibacterial activity of AZT against strains of *S. aureus* isolated of mastitic quarters. *S. aureus* strains isolated and *S. aureus* ATCC 25923 were tested at pH 7.4,

500 mg in each mammary quarter of healthy and mastitic cows at drying off (B).

independent of the route of administration.

may decrease the leukocytes phagocytic function.

Tylosin (TYL), other antibiotic of the macrolide group, is commonly used in food animal practice. Because it is an organic base (pKa = 7.1), moderately bound by serum proteins (40%), with a high degree of lipid solubility (Lucas et al., 2007), TYL would be expected to be widely distributed in body fluids and tissues. The MIC of TYL for *S. aureus* was <1 µg.ml-1 for most isolates studied. We determined the elimination milk profile of TYL after IM administration at multiple dose schemes.

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 459

review of clinical aspects and major PK characteristics of the preparations commercially available (Mestorino & Errecalde, 1997). The PK of OTC has been studied by several authors (Schifferli et al, 1982, Errecalde, 1992; Errecalde et al, 1997). The new long-acting formulations have allowed the maintenance of high concentrations for periods much longer than the classical preparations, which represented a significant change in the practicity and in the usage habits, but also in maintaining residual levels for long periods in different tissues. Moreover, these formulations result in a significantly longer persistence of milk concentrations. The popularity of these preparations is extended to virtually all types of exploitation. Although the use of these long acting formulations is justified especially in extensive cattle exploitations, in other kind of management (i.e. more intensive), where the animals can be daily treated and controlled, is not justified. The use of tetracyclines with long elimination half-lives in dairy cows has impacted on repeated violations of maximum permitted levels of these antibiotics in milk. Problems that may emerge from the misuse of antibiotics are especially related to the presence of sub-therapeutic concentrations and the resultant emergence and dissemination of bacterial resistant strains in both animals and humans. Indisputably, the possibility of transference of portions of DNA carrying genes encoding resistance between bacteria is one of the issues of major concern to people with

different responsibilities in the area. Direct toxicity must not be discarded.

Tetracyclines achieve milk concentrations that are approximately in the range of those of blood. They are second-choice parenteral antibiotics for serious infections of the udder caused by Gram positive organism and possibly by coliforms, although susceptibility among the later is controversial. In previous studies we evaluated the serum and milk PK behaviour of OTC after administration of therapeutic doses of three commercially available preparations at 5, 10 and 20 % by the IM route. In Figure 16 A and B, the plasma and milk OTC concentrations after IM administration of three different formulations is represented.

Fig. 16. Serum (A) and milk (B) OTC concentrations after IM administration of three

The 20% solution presented the higher area under the curve milk concentration versus time and the longest elimination half-life (Table 18). The withdrawal periods from milk for the

Addition of milk to Mueller-Hinton susceptibility test medium permitted measurement of milk effect on agar disc diffusion zone diameters obtained from *S. aureus* field isolates and

different formulations to milk cows in production.

5%, 10% and 20% solutions resulted 3.5, 5 and 8 days respectively.

Fig. 14. Inhibition of *S. aureus* exposed to different concentrations (expressed as Log10) of AZT (0.25 MIC; 0.5MIC; 1MIC; 2MIC; 4MIC and 8 x MIC) in function of the time and the medium pH (5; 6.5 and 7.4)

Fig. 15. Milk concentrations of TYL after its multiple (five doses every 24 hours) IM administration (10 mg.kg-1)

The objective was to calculate the withdrawal time in milk necessary for TYL to reach acceptable limits for human consumption. Healthy Holstein lactating cows received 5 intramuscular doses of TYL at 10mg.kg-1 every 24 hours (Figure 15). Milk samples were obtained from the four mammary quarters before the start of treatment, during and posttreatment every 12 hours until to 216 hours. The withdrawal time was calculated using the harmonized Time to Safe Concentration (TTSC) recommended by the European Union. The time required for milk to carry TYL concentration below the maximum residue limits (MRL 50 ppb) was 120 h post last TYL dose.

#### **4.4 Tetracyclines**

Oxytetracycline (OTC) is the most representative member of this class of ATMs. OTC is widely used in veterinary medicine worldwide. Its use is widespread due to its broad spectrum (not only against bacteria but also against some chlamydia, rickettsia and protozoa), and certain PK properties as its wide distribution throughout the body and prolonged therapeutic effects of some long-acting formulations. Our team has performed a

Fig. 14. Inhibition of *S. aureus* exposed to different concentrations (expressed as Log10) of AZT (0.25 MIC; 0.5MIC; 1MIC; 2MIC; 4MIC and 8 x MIC) in function of the time and the

Fig. 15. Milk concentrations of TYL after its multiple (five doses every 24 hours) IM

The objective was to calculate the withdrawal time in milk necessary for TYL to reach acceptable limits for human consumption. Healthy Holstein lactating cows received 5 intramuscular doses of TYL at 10mg.kg-1 every 24 hours (Figure 15). Milk samples were obtained from the four mammary quarters before the start of treatment, during and posttreatment every 12 hours until to 216 hours. The withdrawal time was calculated using the harmonized Time to Safe Concentration (TTSC) recommended by the European Union. The time required for milk to carry TYL concentration below the maximum residue limits (MRL

Oxytetracycline (OTC) is the most representative member of this class of ATMs. OTC is widely used in veterinary medicine worldwide. Its use is widespread due to its broad spectrum (not only against bacteria but also against some chlamydia, rickettsia and protozoa), and certain PK properties as its wide distribution throughout the body and prolonged therapeutic effects of some long-acting formulations. Our team has performed a

**Parameter Mean SD**  λ h-1 0.095 0.028 T½λ h 7.873 2.232 AUC0-t µg.h/mL 141.360 23.290 AUC0-∞ µg.h/mL 142.458 23.512 MRT h 78.395 3.380

medium pH (5; 6.5 and 7.4)

administration (10 mg.kg-1)

50 ppb) was 120 h post last TYL dose.

**4.4 Tetracyclines** 

review of clinical aspects and major PK characteristics of the preparations commercially available (Mestorino & Errecalde, 1997). The PK of OTC has been studied by several authors (Schifferli et al, 1982, Errecalde, 1992; Errecalde et al, 1997). The new long-acting formulations have allowed the maintenance of high concentrations for periods much longer than the classical preparations, which represented a significant change in the practicity and in the usage habits, but also in maintaining residual levels for long periods in different tissues. Moreover, these formulations result in a significantly longer persistence of milk concentrations. The popularity of these preparations is extended to virtually all types of exploitation. Although the use of these long acting formulations is justified especially in extensive cattle exploitations, in other kind of management (i.e. more intensive), where the animals can be daily treated and controlled, is not justified. The use of tetracyclines with long elimination half-lives in dairy cows has impacted on repeated violations of maximum permitted levels of these antibiotics in milk. Problems that may emerge from the misuse of antibiotics are especially related to the presence of sub-therapeutic concentrations and the resultant emergence and dissemination of bacterial resistant strains in both animals and humans. Indisputably, the possibility of transference of portions of DNA carrying genes encoding resistance between bacteria is one of the issues of major concern to people with different responsibilities in the area. Direct toxicity must not be discarded.

Tetracyclines achieve milk concentrations that are approximately in the range of those of blood. They are second-choice parenteral antibiotics for serious infections of the udder caused by Gram positive organism and possibly by coliforms, although susceptibility among the later is controversial. In previous studies we evaluated the serum and milk PK behaviour of OTC after administration of therapeutic doses of three commercially available preparations at 5, 10 and 20 % by the IM route. In Figure 16 A and B, the plasma and milk OTC concentrations after IM administration of three different formulations is represented.

Fig. 16. Serum (A) and milk (B) OTC concentrations after IM administration of three different formulations to milk cows in production.

The 20% solution presented the higher area under the curve milk concentration versus time and the longest elimination half-life (Table 18). The withdrawal periods from milk for the 5%, 10% and 20% solutions resulted 3.5, 5 and 8 days respectively.

Addition of milk to Mueller-Hinton susceptibility test medium permitted measurement of milk effect on agar disc diffusion zone diameters obtained from *S. aureus* field isolates and

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 461

Enrofloxacin (ENR) is a quinolone widely used for treatment of various infectious diseases in cattle caused both by Gram-positive and Gram-negative bacteria, but is not specifically recommended for bovine mastitis treatment; although high concentrations are reached and maintained in milk following parenteral administration. The MIC90 value found by Russi et al., (2008) was similar than the reported for isolates from Uruguay and (Gianneechini et al.,

Danofloxacin (DAF) is a fluoroquinolone (FQ) ATM drug developed for use in veterinary medicine. DAF shows a broad spectrum of activity against most Gram-negative, Grampositive bacteria and mycoplasma, but has poor activity against anaerobes (Shojaee Aliabadi & Lees, 2003). FQ share some characteristics such as a broad spectrum of bactericidal activity, a large volume of distribution, low plasma protein binding and relatively low minimal inhibitory concentrations (MICs) against target microorganisms (Otero et al., 2001a; 2001b; Mestorino et al., 2009). Danofloxacin 18% was demonstrated to be effective in the treatment of bacterial pneumonia caused by *P. multocida*, *M. hemolytica* and *H. somnus* or bacterial enteritis (Mestorino et al., 2009) given as a single injection at a dose rate of 6 mg.kg-1 of body weight, or two doses 48 hours apart, as needed. This formulation has the advantage of being safe and effective with a single dose or at maximum two doses, as handling animals many times for treatment is not practical. The concept of the high dosage in a single injection is that, after injection, the drug is available in high concentrations sufficient to kill all the sensitive bacteria during a relatively short period of time (Mestorino et al., 2009). This reduces the selection pressure for resistance. These characteristics suggest that it could be useful for the treatment of bovine mastitis caused by *S. aureus.* In order to investigate this possibility, the PK profile of DAF 18% was studied in plasma, milk and various tissues in dairy cows, when administered as a single subcutaneous injection at a dose of 6mg.kg-1 (Fig. 17 A, B, C and D). DAF was rapidly absorbed and reached peak plasma concentrations of 0.53 g.ml-1 within 2 hours after injection. Distribution of DAF from plasma to all sampled tissues and milk was extensive (Table 19). Peak milk concentrations of 1.37g.ml-1 were achieved 8 hours post-injection. Maximum concentrations (9.73 g.g-1) in udder tissue (average of 4 quarters) and uterus (2.53 g.g-1) were achieved 6 and 4 hours after injection, respectively. Maximum concentrations in intestinal tract tissue and lymph nodes ranged from 3.6 g.g-1 (duodenum) to 10.22 g.g-1 (lymph nodes), and were reached between 2 h and 12 hours after injection. Plasma area under the curve (AUCpl) was 9.69 g.h/ml. Plasma elimination half life (T½ß) was 12.53 h. DAF has a very high volume of distribution (Vd = 5.79 l.kg-1). AUC values for the various tissues and milk greatly exceeded AUCpl. Elimination half life from milk and tissues varied between 4.57 hours to 21.91 hours and the milk withdrawal time was 73.48 h (Table 19). The reported results support the potential use of DAF in the treatment of mastitis and

other infections in milk cows with three days of withdrawal.

Later we performed other study in cows with subclinical mastitis, who received 10 mg.kg-1 subcutaneous (SC) 18% DAF(Fig. 18). The MIC50 calculated for *S. aureus* isolated from

**4.5 Fluoroquinolones** 

2002; San Martín et al., 2002).

**4.5.1 Danofloxacin** 

stock strains. Milk reduced the activity of different ATM agents, as novobiocin, streptomycin, gentamycin, tetracycline, and vancomycin (Owens & Watts, 1987). As was previously said, MIC is used in conjunction with PK data to determine the more appropriate surrogate PK/PD parameter. Problems arise in vivo, however, due to the physiological status of the host, site of infection, and properties of the ATM. Often, the concentration of active ATM at the infection site is quite different from that in serum or milk. Antimicrobial concentrations are lower in ischemic areas, scar tissue, and abscess contents. Also, pH, protein binding, and also normal metabolism and renal excretion mechanism reduce concentration of available ATM (Thornsberry and Sherris, 1985). Standard susceptibility tests measure ATM effectiveness in conditions extremely different from those in the udder.


Table 18. Serum and milk PK parameters obtained after administration of three OTC formulations in Holstein healthy lactating cows

In bovine mastitis, the unique environment of the mammary gland presents a problem for determining usefulness of chemotherapeutic agents against a given organism. Milk proteins, lipids, pH, and ionic characteristics can reduce concentration of active ATM (Ziv, 1980a). A simple in vitro method for determining the effect of milk on ATM activity could be of use when examining ATMs for possible use in mastitis therapy (Owens & Watts, 1987).

The MIC90 of tetracycline (used as group representative) among *S. aureus* determined by different authors was between 0.5 - 1 µg.mL-1 (Pol and Ruegg, 2007). If we consider than the MIC of tetracycline in milk could increase 4 to 32 times that in MH, the *S. aureus* mastitis treatment with OTC has not real possibilities of success, because the OTC concentrations reached in milk are insufficient for achieving the appropriate PK/PD predictors of therapeutic efficacy against *S. aureus* (AUC24/MIC>100). An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles (Kuang, et al., 2009).

### **4.5 Fluoroquinolones**

460 A Bird's-Eye View of Veterinary Medicine

stock strains. Milk reduced the activity of different ATM agents, as novobiocin, streptomycin, gentamycin, tetracycline, and vancomycin (Owens & Watts, 1987). As was previously said, MIC is used in conjunction with PK data to determine the more appropriate surrogate PK/PD parameter. Problems arise in vivo, however, due to the physiological status of the host, site of infection, and properties of the ATM. Often, the concentration of active ATM at the infection site is quite different from that in serum or milk. Antimicrobial concentrations are lower in ischemic areas, scar tissue, and abscess contents. Also, pH, protein binding, and also normal metabolism and renal excretion mechanism reduce concentration of available ATM (Thornsberry and Sherris, 1985). Standard susceptibility tests measure ATM effectiveness in conditions extremely different from those in the udder.

**Cmax (µg.ml-1)** 2.94±0.74 2.46±0.49 3.37±0.54 2.35±0.27 3.39±0.43 2.30±0.33 **Tmax (h)** 3±0 9.50±1.91 7.50±1.91 9.00±1.15 6.25±3.30 17.50±7.55 **ß (h-1)** 0.035 ±0.02 0.0490.018 0.037±0.018 0.0240.006 0.022±0.007 0.0250.017 **T½ ß (h)** 23.06±8.53 16.167.31 22.94±13.04 31.1610.16 34.07±11.00 36.7417.61 **MRT (h)** 33.68±12.01 29.97±11.96 33.35±16.07 51.88±16.19 52.21±11.85 64.94±19.57 **AUC (µg.ml/h)** 92.72 ±26.77 62.6713.60 114.64 ±32.81 125.21 21.18 196.05 ±32.50 166.43 28.10

**(µg.ml/h)** 32.51±7.59 40.49±6.03 43.14 ± 6.88 **CmaxS/CmaxM** 1.19 1.43 1.47 **AUCM/AUCS** 0.68 1.09 0.85

In bovine mastitis, the unique environment of the mammary gland presents a problem for determining usefulness of chemotherapeutic agents against a given organism. Milk proteins, lipids, pH, and ionic characteristics can reduce concentration of active ATM (Ziv, 1980a). A simple in vitro method for determining the effect of milk on ATM activity could be of use

The MIC90 of tetracycline (used as group representative) among *S. aureus* determined by different authors was between 0.5 - 1 µg.mL-1 (Pol and Ruegg, 2007). If we consider than the MIC of tetracycline in milk could increase 4 to 32 times that in MH, the *S. aureus* mastitis treatment with OTC has not real possibilities of success, because the OTC concentrations reached in milk are insufficient for achieving the appropriate PK/PD predictors of therapeutic efficacy against *S. aureus* (AUC24/MIC>100). An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles (Kuang, et al., 2009).

Table 18. Serum and milk PK parameters obtained after administration of three OTC

when examining ATMs for possible use in mastitis therapy (Owens & Watts, 1987).

formulations in Holstein healthy lactating cows

**OXT 5% OXT 10% OXT 20% Serum Milk Serum Milk Serum Milk Mean±SD Mean ±SD Mean ± SD Mean ±SD Mean ±SD Mean ±SD** 

**Parameters** 

**AUC0-24** 

Enrofloxacin (ENR) is a quinolone widely used for treatment of various infectious diseases in cattle caused both by Gram-positive and Gram-negative bacteria, but is not specifically recommended for bovine mastitis treatment; although high concentrations are reached and maintained in milk following parenteral administration. The MIC90 value found by Russi et al., (2008) was similar than the reported for isolates from Uruguay and (Gianneechini et al., 2002; San Martín et al., 2002).

#### **4.5.1 Danofloxacin**

Danofloxacin (DAF) is a fluoroquinolone (FQ) ATM drug developed for use in veterinary medicine. DAF shows a broad spectrum of activity against most Gram-negative, Grampositive bacteria and mycoplasma, but has poor activity against anaerobes (Shojaee Aliabadi & Lees, 2003). FQ share some characteristics such as a broad spectrum of bactericidal activity, a large volume of distribution, low plasma protein binding and relatively low minimal inhibitory concentrations (MICs) against target microorganisms (Otero et al., 2001a; 2001b; Mestorino et al., 2009). Danofloxacin 18% was demonstrated to be effective in the treatment of bacterial pneumonia caused by *P. multocida*, *M. hemolytica* and *H. somnus* or bacterial enteritis (Mestorino et al., 2009) given as a single injection at a dose rate of 6 mg.kg-1 of body weight, or two doses 48 hours apart, as needed. This formulation has the advantage of being safe and effective with a single dose or at maximum two doses, as handling animals many times for treatment is not practical. The concept of the high dosage in a single injection is that, after injection, the drug is available in high concentrations sufficient to kill all the sensitive bacteria during a relatively short period of time (Mestorino et al., 2009). This reduces the selection pressure for resistance. These characteristics suggest that it could be useful for the treatment of bovine mastitis caused by *S. aureus.* In order to investigate this possibility, the PK profile of DAF 18% was studied in plasma, milk and various tissues in dairy cows, when administered as a single subcutaneous injection at a dose of 6mg.kg-1 (Fig. 17 A, B, C and D). DAF was rapidly absorbed and reached peak plasma concentrations of 0.53 g.ml-1 within 2 hours after injection. Distribution of DAF from plasma to all sampled tissues and milk was extensive (Table 19). Peak milk concentrations of 1.37g.ml-1 were achieved 8 hours post-injection. Maximum concentrations (9.73 g.g-1) in udder tissue (average of 4 quarters) and uterus (2.53 g.g-1) were achieved 6 and 4 hours after injection, respectively. Maximum concentrations in intestinal tract tissue and lymph nodes ranged from 3.6 g.g-1 (duodenum) to 10.22 g.g-1 (lymph nodes), and were reached between 2 h and 12 hours after injection. Plasma area under the curve (AUCpl) was 9.69 g.h/ml. Plasma elimination half life (T½ß) was 12.53 h. DAF has a very high volume of distribution (Vd = 5.79 l.kg-1). AUC values for the various tissues and milk greatly exceeded AUCpl. Elimination half life from milk and tissues varied between 4.57 hours to 21.91 hours and the milk withdrawal time was 73.48 h (Table 19). The reported results support the potential use of DAF in the treatment of mastitis and other infections in milk cows with three days of withdrawal.

Later we performed other study in cows with subclinical mastitis, who received 10 mg.kg-1 subcutaneous (SC) 18% DAF(Fig. 18). The MIC50 calculated for *S. aureus* isolated from

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 463

(A) (C)

(B) (D) Fig. 17. Mean DAF plasma and milk concentrations (A). Mean DAF mammary, uterus and plasma concentrations (B), mean DAF duodenum, jejunum, ileum, large intestine vs. plasma concentrations (C) and mean DAF mesenteric lymph nodes vs. plasma concentrations (D),

As described, *S.aureus* represents a major problem in bovine mastitis because of the poor cure rate despite the in vitro susceptibility of the acting bacteria. One possible reason for this is its intracellular location within the udder phagocytes, a place difficult to access for the majority of ATMss, wich combines with reduced activity at the acidic pH of lysosomes. Other possible obstacle for antibacterial efficacy is the non-diffusion of acidic antibiotics through the lysosomal membrane due to their ionic presentation at extracellular (7.4) or cytoplasmic (6.5) pH and the very poor retention in cells of antibiotic which penetrate freely such lincomycins. Consequently, there is a clear need for more specialized dosage forms to be developed for use in the treatment of *S. aureus* bovine mastitis. Indeed, in view of the magnitude of the economic losses, every effort to develop new dosage forms should be undertaken. Such new formulations should be designed to counteract the causes of failure of antibiotic therapy for *S.aureus* and should have the following features (Gruet et al., 2001):

Ability to penetrate phagocytes and to be retained in cells for adequate time;

each after its SC administration in lactating cows

No substrate or low metabolism in the cells;

**5. Perspectives for new therapeutic formulations** 

bovines with subclinical mastitic was 0.25 *µ*g.mL-1 and the MIC90 was 1 *µ*g.mL-1. Gramnegative bacteria have a MIC range of 0.05-2.5 *µ*g.mL-1, while for Gram-positive bacteria it was 0.25-5 *µ*g.mL-1 (Shem-Tov et al, 1998). Although, there are no references for DAF MIC against *S. aureus*, it has been proved that enrofloxacin MIC is ≥1 *µ*g.mL-1 (Cester et al, 1992). The MIC90 was determined in order to calculate PK/PD parameters. Milk maximum concentration (Cmax) was 2.99 ± 0.88 µg.mL-1 (6.13 h post-administration) whereas plasma Cmax was 1.45 ± 0.26 µg.mL-1 (1.17 h post-administration). DAF was eliminated with mean half-lives of 7.56 ± 2.53, and 4.43 ± 1,36 h from milk and plasma respectively. The mean area under the concentration versus time curve from 0 to 24 hours (AUC0-24h) was 34.84 ± 10.97 µg.h/mL in milk and 8.71 ± 1.14 µg.h/mL in plasma.


Table 19. DAF PK parameters (mean ± SD) obtained in plasma and after its subcutaneous administration (6 mg.kg-1)

When comparing PK parameters by grouping quarters by level of production, it was observed that DAF was eliminated more slowly from quarters of low-producing cows (Fig 18 B). Concomitantly, the MRT in milk from quarters of low-producing cows was higher than the MRT determined in milk from quarters of high-producing cows.

FQ are considered to have a concentration-dependent effect, although a time dependent bactericidal effect against some Gram-positive bacteria has also been described (Cester et al. 1996). DAF exhibited the same behavior against *S.aureus*, because it was strongly bactericidal at higher concentrations and when increases contact time. Importantly, the potency of DAF, evaluated by the MIC, is not affected by pH changes (Fig.19). The WT resulted longer in milk of low production than in milk of high production cows. Separating the groups according to the health status (Fig 18 A), it was observed that there were no statistically significant differences between the PK behaviour in both groups. The level of production had a significant effect (*P* < 0.05) on the milk elimination half-life (T½) and on the mean residence time (MRT) of DAF in milk. The infection presence in the mammary quarters had a significant effect (*P* < 0,05) on Tmáx and MRT in milk. The incidence of infection and level of production on the PK of danofloxacin in milk cows have to be considered when designing dosage regimens for this drug.

bovines with subclinical mastitic was 0.25 *µ*g.mL-1 and the MIC90 was 1 *µ*g.mL-1. Gramnegative bacteria have a MIC range of 0.05-2.5 *µ*g.mL-1, while for Gram-positive bacteria it was 0.25-5 *µ*g.mL-1 (Shem-Tov et al, 1998). Although, there are no references for DAF MIC against *S. aureus*, it has been proved that enrofloxacin MIC is ≥1 *µ*g.mL-1 (Cester et al, 1992). The MIC90 was determined in order to calculate PK/PD parameters. Milk maximum concentration (Cmax) was 2.99 ± 0.88 µg.mL-1 (6.13 h post-administration) whereas plasma Cmax was 1.45 ± 0.26 µg.mL-1 (1.17 h post-administration). DAF was eliminated with mean half-lives of 7.56 ± 2.53, and 4.43 ± 1,36 h from milk and plasma respectively. The mean area under the concentration versus time curve from 0 to 24 hours (AUC0-24h) was 34.84 ± 10.97

> Parameter Plasma X SD Milk X SD ß (h-1) 0.06 0.01 0.15 0.02 T½ ß (h) 12.53 1.47 4.57 0.46 Kabs (h-1) 1.46 0.91 0.32 0.06 T½ abs (h) 0.64 0.39 2.27 0.48 MRT (h) 18.38 2.52 8.34 1.31 Cmax (µg.mL-1) 0.53 0.13 1.37 0.73 CmaxM/CmaxPL --- --- 2.68 1.35 T½ßM/T½ßPL --- --- 0.37 0.08 Tmax (h) 2.17 0.98 8.67 2.07 AUC (µg.mL/h) 9.69 1.41 15.46 5.42

WT (h) 73.48

than the MRT determined in milk from quarters of high-producing cows.

Table 19. DAF PK parameters (mean ± SD) obtained in plasma and after its subcutaneous

When comparing PK parameters by grouping quarters by level of production, it was observed that DAF was eliminated more slowly from quarters of low-producing cows (Fig 18 B). Concomitantly, the MRT in milk from quarters of low-producing cows was higher

FQ are considered to have a concentration-dependent effect, although a time dependent bactericidal effect against some Gram-positive bacteria has also been described (Cester et al. 1996). DAF exhibited the same behavior against *S.aureus*, because it was strongly bactericidal at higher concentrations and when increases contact time. Importantly, the potency of DAF, evaluated by the MIC, is not affected by pH changes (Fig.19). The WT resulted longer in milk of low production than in milk of high production cows. Separating the groups according to the health status (Fig 18 A), it was observed that there were no statistically significant differences between the PK behaviour in both groups. The level of

the mean residence time (MRT) of DAF in milk. The infection presence in the mammary quarters had a significant effect (*P* < 0,05) on Tmáx and MRT in milk. The incidence of infection and level of production on the PK of danofloxacin in milk cows have to be

) and on

production had a significant effect (*P* < 0.05) on the milk elimination half-life (T½

considered when designing dosage regimens for this drug.

µg.h/mL in milk and 8.71 ± 1.14 µg.h/mL in plasma.

administration (6 mg.kg-1)

Fig. 17. Mean DAF plasma and milk concentrations (A). Mean DAF mammary, uterus and plasma concentrations (B), mean DAF duodenum, jejunum, ileum, large intestine vs. plasma concentrations (C) and mean DAF mesenteric lymph nodes vs. plasma concentrations (D), each after its SC administration in lactating cows

#### **5. Perspectives for new therapeutic formulations**

As described, *S.aureus* represents a major problem in bovine mastitis because of the poor cure rate despite the in vitro susceptibility of the acting bacteria. One possible reason for this is its intracellular location within the udder phagocytes, a place difficult to access for the majority of ATMss, wich combines with reduced activity at the acidic pH of lysosomes. Other possible obstacle for antibacterial efficacy is the non-diffusion of acidic antibiotics through the lysosomal membrane due to their ionic presentation at extracellular (7.4) or cytoplasmic (6.5) pH and the very poor retention in cells of antibiotic which penetrate freely such lincomycins. Consequently, there is a clear need for more specialized dosage forms to be developed for use in the treatment of *S. aureus* bovine mastitis. Indeed, in view of the magnitude of the economic losses, every effort to develop new dosage forms should be undertaken. Such new formulations should be designed to counteract the causes of failure of antibiotic therapy for *S.aureus* and should have the following features (Gruet et al., 2001):


Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 465

administration to cows at drying off. The microparticles exhibited a good encapsulation ratio and were shown in vitro to release the drug slowly over a long period of time. This dosage form could be considered an example of possible paths to follow looking for suitable

Milk producing cows are a very special kind of animal. An average good level of milk production is in the order of 5% of body weight daily, but everyday more "top" producers are rounding 10%. These are values that years ago were considered impossible to reach. The huge milk production increase was due to a very efficient process of selection. But these processes of increase of milk production pushed these animals to the border between physiology and pathology. In these conditions slight modifications in the diet, environment and management can result in defensive disequilibrium and disease. It has to be noted that the defensive aspects of the mammary gland did not receive the same attention than production itself, and, as a consequence, the higher the level of production, the higher the risks of mammary pathology. The mammary gland is a rather immunologically weak organ, the phagocitic activity in the mammary gland is poor. During the first few hours after milking, neutrophils coming into the gland are active, however, in a short time, they become to "engorge" with lipid molecules and their activity slows. At the end of the intermilking interval, phagocytes are almost inactive. Dilution of defensive elements in great producers is another factor. A special consideration has to be made when *S. aureus* is present. This bacterium has the ability of penetrate cells and to reproduce very slowly there. The majority of ATMs have serious difficulties to penetrate cells, and, if they reach the site where bacteria are, they fail acting against slowly growing organisms or low pH. The combination of these factors constitutes a complex problem. The knowledge of PK and PD of the different ATMs in consideration of the physiological characteristics of the mammary gland can contribute to a more rational use of ATMs. In the present paper we presented some data on the behaviour of some ATMs in milk producing cows. The consideration of the available data in a frame of prudent use surely will increase the number of therapeutically positive results with less possibilities of

Antibiotic therapy is an essential component in programs of mastitis control, but does not replace preventive hygienic measures. A rational approach to ATM treatment involves knowing the different variables that influence the outcome of therapy. Accordingly, it is of fundamental importance to determine what type of etiological agents are predominant in each farm, which are their susceptibilities and what drugs can be used to combat them, having a special consideration on the penetration and tissue distribution in the mammary

The increase in the knowledge of the microbiology and pathophysiology of mastitis by *S. aureus*, together with the new tools to model PK and PD of ATMs will contribute to the design of new administration systems and therapeutic plans with improvement of clinical and microbiological efficacy without the emergence and dissemination of resistant

compartment, penetration into cells, and ATM activity once in the target place.

emergence and dissemination of resistant bacterial strains.

new therapeutic systems.

**6. Conclusion** 

microorganisms.


Fig. 18. **(A)** Mean concentrations of DAF in mastitic quarters and healthy quarters after 10 mg.kg-1 IM dose **(B)** Mean plasma and milk concentrations of DAFin high-producing and low-producing cows after 10 mg.kg-1 IM dose.

Fig. 19. Inhibition of *S. aureus* exposed to different concentrations (expressed as Log10) of DAF (0.25 MIC; 0.5MIC; 1MIC; 2MIC; 4MIC and 8 x MIC) in function of the time and the medium pH (5; 6.5 and 7.4)

Delivery systems such as injectable microparticles or colloidal suspensions could fulfill some of the expectations for a new dosage form for IMM infusion. Microparticles are small, spherical particles with a diameter larger than 5µm, and can be made out of natural (gelatin or alginate) or synthetic material. Only the smallest could potentially be taken up by the phagocytes and therefore offer therapeutic value against intracellular bacteria. Colloidal suspensions include liposomes and nanoparticles. Both types are spherical particles with an average diameter of between 0.05 µm and <5µm. Liposomes are phospholipidic particles with an aqueous core. Due to their amphiphilic structure, they can incorporate either lipophilic or hydrophilic compounds. Nanoparticles are polymeric particles.

Thus liposomes, microparticles and nanoparticles may be considered potential delivery systems in the treatment of bovine mastitis by *S. aureus* since they may be taken up by the phagocytes liberating the active once inside. From the available literature, microparticles appear to be the most appropriate candidates for clinical trials in cows. Bodmeier et al. (1997) have reported the preparation of ceftiofur microparticles for the purpose of administration to cows at drying off. The microparticles exhibited a good encapsulation ratio and were shown in vitro to release the drug slowly over a long period of time. This dosage form could be considered an example of possible paths to follow looking for suitable new therapeutic systems.

#### **6. Conclusion**

464 A Bird's-Eye View of Veterinary Medicine

Fig. 18. **(A)** Mean concentrations of DAF in mastitic quarters and healthy quarters after 10 mg.kg-1 IM dose **(B)** Mean plasma and milk concentrations of DAFin high-producing and

Fig. 19. Inhibition of *S. aureus* exposed to different concentrations (expressed as Log10) of DAF (0.25 MIC; 0.5MIC; 1MIC; 2MIC; 4MIC and 8 x MIC) in function of the time and the

Delivery systems such as injectable microparticles or colloidal suspensions could fulfill some of the expectations for a new dosage form for IMM infusion. Microparticles are small, spherical particles with a diameter larger than 5µm, and can be made out of natural (gelatin or alginate) or synthetic material. Only the smallest could potentially be taken up by the phagocytes and therefore offer therapeutic value against intracellular bacteria. Colloidal suspensions include liposomes and nanoparticles. Both types are spherical particles with an average diameter of between 0.05 µm and <5µm. Liposomes are phospholipidic particles with an aqueous core. Due to their amphiphilic structure, they can incorporate either lipophilic or hydrophilic compounds. Nanoparticles are polymeric

Thus liposomes, microparticles and nanoparticles may be considered potential delivery systems in the treatment of bovine mastitis by *S. aureus* since they may be taken up by the phagocytes liberating the active once inside. From the available literature, microparticles appear to be the most appropriate candidates for clinical trials in cows. Bodmeier et al. (1997) have reported the preparation of ceftiofur microparticles for the purpose of

Effective at low pH against S. aureus;

low-producing cows after 10 mg.kg-1 IM dose.

medium pH (5; 6.5 and 7.4)

particles.

Administration via local infusion through the teat canal

Milk producing cows are a very special kind of animal. An average good level of milk production is in the order of 5% of body weight daily, but everyday more "top" producers are rounding 10%. These are values that years ago were considered impossible to reach. The huge milk production increase was due to a very efficient process of selection. But these processes of increase of milk production pushed these animals to the border between physiology and pathology. In these conditions slight modifications in the diet, environment and management can result in defensive disequilibrium and disease. It has to be noted that the defensive aspects of the mammary gland did not receive the same attention than production itself, and, as a consequence, the higher the level of production, the higher the risks of mammary pathology. The mammary gland is a rather immunologically weak organ, the phagocitic activity in the mammary gland is poor. During the first few hours after milking, neutrophils coming into the gland are active, however, in a short time, they become to "engorge" with lipid molecules and their activity slows. At the end of the intermilking interval, phagocytes are almost inactive. Dilution of defensive elements in great producers is another factor. A special consideration has to be made when *S. aureus* is present. This bacterium has the ability of penetrate cells and to reproduce very slowly there. The majority of ATMs have serious difficulties to penetrate cells, and, if they reach the site where bacteria are, they fail acting against slowly growing organisms or low pH. The combination of these factors constitutes a complex problem. The knowledge of PK and PD of the different ATMs in consideration of the physiological characteristics of the mammary gland can contribute to a more rational use of ATMs. In the present paper we presented some data on the behaviour of some ATMs in milk producing cows. The consideration of the available data in a frame of prudent use surely will increase the number of therapeutically positive results with less possibilities of emergence and dissemination of resistant bacterial strains.

Antibiotic therapy is an essential component in programs of mastitis control, but does not replace preventive hygienic measures. A rational approach to ATM treatment involves knowing the different variables that influence the outcome of therapy. Accordingly, it is of fundamental importance to determine what type of etiological agents are predominant in each farm, which are their susceptibilities and what drugs can be used to combat them, having a special consideration on the penetration and tissue distribution in the mammary compartment, penetration into cells, and ATM activity once in the target place.

The increase in the knowledge of the microbiology and pathophysiology of mastitis by *S. aureus*, together with the new tools to model PK and PD of ATMs will contribute to the design of new administration systems and therapeutic plans with improvement of clinical and microbiological efficacy without the emergence and dissemination of resistant microorganisms.

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 467

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**23** 

*Turkey* 

Sima Sahinduran

*University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine, Department of Internal Medicine,* 

**Protozoan Diseases in Farm Ruminants** 

Protozoa are ubiquitous throughout aqueous environments and the soil, and play an important role in their ecology. Protozoa occupy a range of trophic levels.They also play a vital role in controlling bacteria population and biomass. Farm animals are usually infected with several species of parasites and they are also confined to pasture or pens. Parasite eggs, larvae, and cysts are intense in soil. One of the most important aspect of animal protozoology is transmission to humans. These are called zoonosis. Some of zoonotic diseases are common and important to public health. This chapter is about of the etiology, epidemiology, pathogenesis, clinical findings, diagnosis, treatment and control of the

Babesiosis is an infectious tick- borne disease of livestock that characterised by fever, anemia, haemoglobinuria and weakness. The disease also is Known by such names as bovine babesiosis, piroplasmosis, Texas fever, redwater, tick fever, and tristeza (Zaugg, 2009). The disease also is a hemoparasitic disease caused by protozoa of the genus Babesia (Phylum: Apicomplexa), which infects mainly ruminants (Melendez, 2000). Infection of a vertebrate host is initiated by inoculation of sporozoite form of parasites into the blood stream during the taking of a blood meal (Radostits et al. 2008). The list of babesia species is

Bovine babesiosis disease is caused by at least six Babesia species (Table 1). Bovine babesiosis associated with B. bigemina and B. bovis is the most important disease of tropical and subtropical regions between 40N and 32S. Both species are transmitted transovarially by Boophilus ticks, but only tick larvae transmit B. bovis, whereas nymphs and adults transmit B. bigemina. (Radostits et al. 2008). In Europe, babesiosis is caused by Babesia divergens, an intraerythrocytic parasite that can persist for >13 months in the organs of

**1. Introduction** 

**2. Babesiosis 2.1 Etiology** 

shown in table 1.

**2.2 Epidemiology** 

**2.2.1 Bovine babesiosis** 

pathogenic protozoa in farm ruminants.

susceptible or penicillin-resistant Staphylococcus aureus. *Acta veterinaria Scandinavica*; 44(1-2):p.53-62. ISSN 1751-0147

