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FIELD EXPERIENCE WITH INTRAVENOUS MARBOFLOXACIN AGAINST ACUTE MASTITIS

537

Field experience with a single intravenous injection of marbofloxacin 16% in lactating dairy cows with acute clinical mastitis F. PILLET1, C. MONTREUIL1, O. ROY1, E. GRANDEMANGE2*, F. WOEHRLE2 Cebiphar, 1 rue de la Bodinière, 37230 Fondettes, FRANCE Vetoquinol Research Centre, BP189, 70204 Lure Cedex, FRANCE

1 2

* Corresponding author: [email protected]

SUMMARY

RESUME

In a non-controlled multicentre field trial conducted on commercial farms in France and Germany, local and general tolerance of marbofloxacin (Forcyl®, Vétoquinol SA) was evaluated after intravenous administration to 100 lactating dairy cows suffering from acute clinical mastitis. Clinical signs, concomitant therapy, disease evolution and bacteriological analysis of milk at inclusion were also monitored. All the 100 included cows presented local and general signs of acute clinical mastitis, involving one or two quarters. They were treated once with marbofloxacin (Forcyl®) at the dose rate of 10 mg/kg (i.e. 10 ml/160kg) by intravenous injection. Most of the included cows (83%) received a concomitant treatment. A clinical examination was performed before and immediately following treatment with the study product and 1, 2-3 and 7 days after this treatment, with some cases up to 14 days later (40%). Milk was sampled for microbiological examination before treatment in 81% of the cases. The time course of the target disease showed a rapid decrease of mean rectal temperature as well as mean score of all the local and general clinical parameters, whether the cows received concomitant therapy (83 cows) or not (17 cows). However, 8 cows were given a new treatment for mastitis during the study. The two main pathogens isolated before treatment were Escherichia coli and Streptococcus uberis. The most frequent side effects observed after injection were moderate and transient local reactions (swelling, slight pain at injection, hematoma). Diarrhoea was observed both before treatment and within the first days following treatment. Tremors, lameness and nervousness were also observed on rare occasions within the first days following injection. Intravenous administration of a high dose of marbofloxacin (Forcyl®) was efficacious and safe in cows suffering from acute clinical mastitis, in combination with various concomitant treatments (83% of the included animals) or alone (17%).

Utilisation en pratique terrain d’une administration intraveineuse unique de marbofloxacine 16% chez la vache laitière en lactation atteinte de mammite clinique aiguë

Keywords : Mastitis, dairy cows, lactation, marbofloxacin, fluoroquinolone, intravenous

Lors d’un essai de terrain non contrôlé et multicentrique, conduit dans des exploitations agricoles en France et en Allemagne, la tolérance locale et générale de la marbofloxacine (Forcyl®, Vétoquinol SA) a été évaluée après administration intraveineuse à 100 vaches laitières en lactation souffrant de mammite clinique aigüe. Les signes cliniques, les traitements concomitants, l’évolution de la maladie et l’analyse bactériologique du lait à l’inclusion ont également été enregistrés. Les 100 vaches incluses souffraient de signes locaux et généraux de mammite clinique aiguë de 1 ou 2 quartier(s). Elles ont été traitées une fois avec la marbofloxacine (Forcyl®) à la posologie de 10 mg/kg (soit 10 ml/160 kg) par injection intraveineuse. La plupart (83%) ont reçu également un traitement concomitant. Un examen clinique local et général a été réalisé avant et après le traitement avec le produit de l’étude, et 1, 2 ou 3, et 7 jours après ce traitement. 40% des vaches ont également été examinées après 14 jours. Un prélèvement de lait a été fait avant traitement pour 81% des cas. Le suivi de l’évolution de la maladie a montré une diminution rapide de la température rectale moyenne et du score moyen de tous les paramètres locaux et généraux, que les animaux aient reçu un traitement concomitant (83 vaches) ou non (17 vaches). Néanmoins, 8 vaches ont nécessité un nouveau traitement contre la mammite en cours d’étude. Les 2 pathogènes principaux isolés avant traitement étaient Escherichia coli et Streptococcus uberis. Les effets secondaires les plus fréquemment observés après injection étaient des réactions locales modérées et transitoires (gonflement, légère douleur à l’injection, hématome). De la diarrhée a été observée avant traitement et dans les tous 1ers jours qui suivaient. D’autres signes (tremblements, boiterie, nervosité) ont également été observés dans les premiers jours suivant le traitement de façon isolée. L’administration par voir intraveineuse d’une dose forte de marbofloxacine (Forcyl®) a été efficace et sûre pour le traitement de mammites cliniques aiguës en association avec divers traitements concomitants (83% des animaux inclus) ou seul (17%).

Mots-clés : Mammite, vaches laitières, lactation, marbofloxacine, fluoroquinolone, intraveineux

INTRODUCTION

dairy cows [2]. It can be classified into severe, moderate or mild clinical mastitis, and subclinical mastitis.

Mastitis is by far the most common and most costly disease in milk production in industrialised countries. Costs are related to loss in milk quality, decreased milk production related to subclinical mastitis, discarded milk, replacement cows, cost of treatment and labour costs [1]. This disease is the most frequent reason for the use of antimicrobials in

Clinical mastitis is a condition characterised by the presence of functional symptoms (macroscopic modifications of the quantity and quality of milk composition), local inflammatory signs (painful, hard, swollen udder quarter, for example) and general clinical signs (hyperthermia, anorexia,

Revue Méd. Vét., 2013, 164, 12, 537-545

538 depression, for example). Practically, mastitis is considered clinical as soon as the milk composition or the mammary secretion is modified [3]. In case of severe acute mastitis, supportive therapy such as fluid replacement and anti-inflammatory drugs is usually indicated. Fluid replacement is necessary for the animals showing signs of dehydration and is essential to control the toxic shock syndrome that can occur in acute mastitis. Treatment with hypertonic saline solution, by parenteral or oral route, provides immediate extracellular fluid volume and temporarily rehydrates animals [4, 5, 6]. Compensation of possible hypocalcemia, hypokalaemia or hypoglycaemia may also be part of the supportive therapy [5, 7]. As with the glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs) may provide symptomatic relief and promote well-being [6, 8]. Administration early in the course of the infection is likely to increase clinical benefit [7, 8]. Severe and moderate clinical mastitis usually needs antibiotic therapy and should be treated in the interests of animal welfare [1, 8]. The goals of therapy include the return of the cow to normal milk production and composition, prevention of mortality in peracute cases, elimination of infectious microorganisms, diminution of somatic cell counts and reduction of contamination of other cows [4]. The administration of intramammary antibiotic treatment is more widespread than the administration of systemic antibiotic treatment. This is explained by practical aspects of the administration by the farmer and by the necessity to provide a sufficient dose of antibiotic rapidly at the infection site [3]. The antibiotic treatment by parenteral route is indicated when some general signs are present, life threatening situations [2, 9] or in the case of chronic infections for which diffusion of the antibiotic into the parenchyma is necessary. Moreover, the early bacteraemia reported during toxic mastitis also leads to advocate the systemic route [3, 2]. When administered by a parenteral route, an active molecule must be able to penetrate the mammary tissue by diffusing from the blood to tissues of the udder and to the milk cistern, as well as to stay in the blood at a bactericidal concentration. Broadspectrum antimicrobial drugs such as fluoroquinolones have such properties and have been recommended and used to treat coliform mastitis [10]. A few systemic antibiotic treatments indicated for mastitis are currently available in France. Among them, three fluoroquinolones have a specific indication for the treatment of acute mastitis due to Escherichia coli [11]. In case of severe, acute or peracute, E. coli clinical mastitis, some authors considered fluoroquinolones as the most appropriate treatment [12, 7]. Fluoroquinolones are used in both human and veterinary medicine. Marbofloxacin [a third generation

GRANDEMANGE (E.) AND COLLABORATORS fluoroquinolone] has been specifically developed for veterinary use. This fluoroquinolone was first approved in Europe at a dose of 2 mg/kg daily for 3 to 5 days for the treatment of Bovine Respiratory Disease (BRD) [13, 14, 15], then for acute mastitis. The efficacy of daily dosage of 2 mg/ kg with Marbocyl® ([marbofloxacin])10% for the treatment of acute clinical mastitis was already demonstrated in vitro [16] and in vivo after experimental [17] or natural infection [8, 18]. Following the introduction of new concepts for the use of fluoroquinolones at higher doses for reduced treatment duration and reduction of the risk of resistance selection among pathogenic bacteria [19], Single Injection Short Acting AntiBiotic [SISAAB] protocols have been developed for the treatment of acute BRD and mastitis with marbofloxacin in cattle [20, 21, 22]. The objective of the study presented hereafter was to confirm the field safety of a single intravenous injection of marbofloxacin (Forcyl®, Vétoquinol SA) in lactating dairy cows suffering from acute clinical mastitis, to complement a controlled trial that has demonstrated the efficacy and safety after single intramuscular administration [9]. The disease evolution, the bacteriological analysis of milk, as well as the concomitant therapy administered during the study, are also presented hereafter.

MATERIAL AND METHODS STUDY DESIGN The field trial was conducted in compliance with VICH GCP at farms in France and Germany. Approval was obtained from the appropriate regulatory authorities and conformed to local animal welfare standards. The efficacy and safety of Forcyl® were already demonstrated in previous experimental and field studies in comparison with active and negative control products [9, 21]. As the objective of this study was to confirm the tolerance of the product in a greater number of animals of the target species under field conditions, no control product was used. Lactating dairy cows with clinical mastitis were included in the study and received a single injection of marbofloxacin (Forcyl®, 10 mg/kg) in the jugular vein on Day 0. Each animal was subjected to a clinical examination from Day 0 before treatment to Day 7 or Day 14. Moreover, milk samples were performed for bacteriological analysis on Day 0 before treatment for 81% of the cases.

ANIMAL INCLUSION Lactating dairy cows showing clinical signs of mastitis were enrolled by 14 investigators in France and Germany on 61 different commercial farms. Inclusion in the study required that lactating dairy cows presented with at least one local clinical sign of mastitis [quarter inflammation or modification of milk composition Revue Méd. Vét., 2013, 164, 12, 537-545

FIELD EXPERIENCE WITH INTRAVENOUS MARBOFLOXACIN AGAINST ACUTE MASTITIS of one or several udder quarters], accompanied by at least one general clinical sign [rectal temperature ≥39.5°C, depression or inappetence]. The animals did not receive any systemic antibiotic treatment in the 7 days preceding their inclusion. The administration of intramammary treatment before inclusion was authorised. The concomitant use of antibiotic products by intramammary route, anti-inflammatory drugs and hypertonic solutions by infusion or oral route, products intended for kidney or liver support, demulcents and rumination activators was authorised.

ETHICAL CONSIDERATION At any time the investigator could decide to administer another treatment of his choice due to worsening of the mastitis.

RESULTS STUDY POPULATION A total of 100 lactating dairy cows presenting with clinical signs of acute mastitis were included in the study and received marbofloxacin once at Day 0 (tables II and III).

SAFETY ASSESSMENT AND DISEASE OUTCOME The following signs were monitored for the assessment of the general tolerance of the treatment: salivation, tremors, diarrhoea, behaviour change, constipation, nervous signs, lameness and any other general reaction. Pain at injection (Day 0) or at palpation (next visits), swelling (size and consistency), phlebitis, and any other observations were considered for the evaluation of the local tolerance. Also, to assess disease outcome, rectal temperature, local and general clinical signs were recorded by the investigator on Day 0 before and after treatment, Day 1, 2 or 3, 7 and if necessary Day 14 (table I).

Score Appetite General behaviour Hydration status Milk yield Quarter inflammation Quarter milk composition

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The safety of marbofloxacin was assessed throughout the study for the 100 included cows. 114 affected quarters were included, as 14 cows had 2 udder quarters presenting signs of acute mastitis. Evaluation of the disease outcome was assessed at Day 7 for 93 out of the 100 included animals, as 7 were withdrawn due to rescue therapy administration (antibiotic treatment by systemic route for 6 cows) or mortality (1 cow). Other clinical signs observed at inclusion (before marbofloxacin injection) included diarrhoea (9 cases), recumbent cow (2 cases) and panting (1 case).

0

1

2

3

Normal Normal Normal Normal No Normal

Slightly reduced Mild depression Dehydration ≤ 8% Impaired Mild Small flakes

Severely reduced Marked depression Dehydration > 8% Nil or hardly nil Moderate Clots

Anorexia Severe depression Acute Serous

Table I: Scoring parameters of general and local clinical signs

Breed 85 Holstein, 8 Normande, 6 Montbeliarde, 1 Brown cattle.

Body weight [kg] 659.8 ± 89.86 [min.: 490; max.: 960]

Age [years] 5.7 ± 1.92 [min.: 2.2; max.: 10.0]

Lactation number

Pregnancy

3.3 ± 1.52 [min : 1; max : 7]

79 not pregnant 21 pregnant [2.7 months of pregnancy ± 1.89]

Table II: Main general characteristics of animals at inclusion

Rectal temperature [°C] *

Appetite *

General behaviour *

Hydration status *

Milk yield *

Quarter inflammation**

Quarter milk composition **

39.8 ± 0.86 [min.: 37.0; max.: 41.3]

1.6 ± 0.87 [min.: 0; max.: 3]

1.2 ± 0.81 [min.: 0; max.: 3]

0.3 ± 0.57 [min.: 0; max.: 2]

1.2 ± 0.41 [min.: 0; max.: 2]

1.9 ± 0.78 [min.: 0; max.: 3]

2.0 ± 0.91 [min.: 0; max.: 3]

Table III: Mean rectal temperature and mean score of clinical signs at inclusion [* n=100; ** n=114]

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GRANDEMANGE (E.) AND COLLABORATORS

CONCOMITANT THERAPY -Before inclusion: Twenty cows (20%) received an intramammary antibiotic therapy before inclusion. Eight different products were administered (see table IV). One cow received an antiinflammatory treatment by systemic route on Day 0 prior to inclusion (non-inclusion criterion) rather than concomitantly with Forcyl® at D0.

-Throughout the study (between Day 1 and 14): Eight cows (8%) were administered a new treatment for mastitis during the study (7 failures, 1 relapse), from Days 2, 4, 7 or 14 onwards. Antibiotic products were administered by intravenous, intramuscular or intramammary routes (respectively 1, 5 and 5 cows). Anti-inflammatory products were also administered to 3 out of the 8 failed cows, by intravenous or intramuscular route (1 and 2 cows respectively).

-From D0 onwards: Most of the cows (83%) received a concomitant treatment on Day 0 or from Day 0 onwards (figure 1). Among these 83 cows, 3 were given an unauthorised treatment on Day 0 according to the protocol definition (antibiotic product by systemic route). In summary, 57 cows received a concomitant antiinflammatory treatment, 39 cows intravenous fluid therapy, 5 cows oral fluids and 6 cows a liver support or rumination activator. Moreover, 69 cows out of the 100 cows received a concomitant intramammary treatment (including those for which the treatment started before inclusion and continued after). 13 different products were used (table IV).

Number of antibiotics in the speciality

1

1 [+ 1 anti-inflammatory]

2

3 [+ 1 anti-inflammatory]

Figure 1: Summary of treatments administered concomitantly with the study product

Active ingredient[s]

Number of cows concerned

cefquinome

3 [before inclusion] 2 [concomitant]

cefapirin

2 [before inclusion] 3 [concomitant]

cloxacillin

2

oxacillin

3 [concomitant]

cefoperazone

1 [concomitant]

pirlimycin

1 [before inclusion] 1 [concomitant]

cefalexin

3 [concomitant]

procain penicillin G

5 [concomitant]

amoxicillin - clavulanic acid + prednisolone

4 [before inclusion] 20 [concomitant]

colistine + cloxacillin

2 [concomitant]

gentamycin + cloxacillin

2 [concomitant]

benzylpenicillin + dihydrostreptomycin

1

[before inclusion]

[before inclusion]

colistine + cloxacillin

1 [concomitant]

cefalexin + kanamycin

2 [before inclusion] 18 [concomitant]

tetracyclin + neomycin + bacitracin + prednisolone

5 [before inclusion] 8 [concomitant]

Table IV: Details of antibiotic intramammary treatments administered before marbofloxacin injection or concomitantly Revue Méd. Vét., 2013, 164, 12, 537-545

FIELD EXPERIENCE WITH INTRAVENOUS MARBOFLOXACIN AGAINST ACUTE MASTITIS

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OUTCOME OF THE DISEASE

BACTERIOLOGICAL ANALYSIS

The time course of the different parameters from Day 0 to 14 (overall population) showed a rapid decrease of mean rectal temperature as well as of the mean score of each clinical parameter observed (appetite, general behaviour, hydration status, milk yield, inflammation of udder quarter, and milk composition of quarter) (figures 2 and 3). The 17 animals that did not receive any concomitant therapy showed, as the overall population described above, a rapid decrease of temperature and clinical parameters.

The main pathogens isolated on Day 0 before treatment were Escherichia coli, Streptococcus uberis (30.5 and 29.3% of the samples, respectively), Staphylococcus aureus and coagulase-negative Staphylococcus (11.0 and 4.9% of the samples, respectively) (table V). Single bacterial infections were the most common: 77.8% of the cases (63 out of 81 sampled cows). For 8 cases (9.9%), 2 pathogens were isolated on Day 0. Three out of the 75 bacterial isolates (4%) were resistant (as assessed by antibiogram) to marbofloxacin, whereas 5 (6.7%) were intermediate. Two cows with intermediate isolates (out of 5, Streptococcus uberis and Enterococcus sp.) required rescue therapy (failure on D4 and D2, respectively). One out of the 3 resistant isolates concerned a cow that received a new treatment for mastitis by systemic and intramammary route on D14 (relapse).

Failure on D7 was considered for 7% of the cases, as a new treatment for mastitis was prescribed between D1 and D7. Moreover, only one cow received a new treatment from D14 due to relapse of the mastitis. None of the 17 animals that did not receive any concomitant therapy were concerned by failure or relapse.

SAFETY ASSESSMENT -General adverse signs: No nervous signs, salivation or constipation were observed throughout the study. Diarrhoea, tremors, behaviour change and lameness were observed on Day 0 after injection, Day 1, Day 2 and/or Day 3 on rare occasions. They were not observed on Day 7 and Day 14 (see table VI).

Figure 2: Time course of mean rectal temperature [°C] and of mean score of general clinical parameters

The following observations were also recorded: Cow found recumbent on Day 1, involving a concomitant therapy on Day 1 (NaCl 10% infusion and tolfenamic acid intravenous injection), worsening of the general condition on Day 4 requiring euthanasia. Toxic shock due to acute mastitis was suspected to be the cause of the death. Escherichia coli was isolated from the milk sample performed before treatment. Cardiac disorder and metallic foreign body finding on Day 1 requiring a treatment; suspicion of endocarditis on Day 7; and abnormal cardiac noises still present on Day 14. Foot abscess on Day 5, requiring hoof trimming. It is unlikely that those adverse events were related to the treatment with marbofloxacin. Fourteen cows exhibited one or more of the general clinical signs mentioned above. They all received at least one additional product as well as marbofloxacin: 12 cows received at least one product by general route on Day 0, and the 2 last cows received only an intramammary treatment.

Figure 3: Time course of mean score of local clinical parameters

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Six cows received an overdose of marbofloxacin (from 10.8% to 14.3% more than 10  mg/kg). None of these cows showed particular adverse signs after treatment (considering that the diarrhoea observed in 2 cows was pre-existing to treatment hence unrelated).

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GRANDEMANGE (E.) AND COLLABORATORS Susceptibility to marbofloxacin [disk method]

Number of isolates

Bacterial species

Resistant

Intermediate

Sensitive

Escherichia coli

25

1

0

24

Streptococcus uberis

24

0

4

20

Staphylococcus aureus

9

0

0

9

Streptococcus dysgalactiae Coagulase-negative Staphylococcus [including 1 S. epidermidis and 1 S. xylosus] Enterococcus sp. [including 1 E. faecium]

4

0

0

4

4

-

-

-

4

1

1

2

Arcanobacterium pyogenes

2

1

0

1

Streptococcus equisimilis

1

-

-

-

Pasteurella multocida

1

0

0

1

Enterobacter sp.

1

0

0

1

Citrobacter freundii

1

-

-

-

Pantoea sp.

1

-

-

-

Candida sp. [including 1 C. kefyr]

2

-

-

-

Unusable sample [multiple contamination]

3

-

-

-

Negative

7

-

-

Table V: Bacteriological results [Day 0 before treatment individual milk samples]

Sign

Total [Day 0 after injection to Day 14]

Day 0 after injection

Day 1

Day 2 or 3

Diarrhoea

8%*

7

5

1

Tremors

3%

1

3

0

Behaviour change: nervous cow

1%

1

1

0

Lameness

1%

0

0

1

* Sign also observed on D0 before injection for all the concerned cows

Table VI: Frequency of general adverse signs observed [percentage of cows concerned]

Sign

Total [Day 0 after injection to Day 14]

Day 0 after injection

Day 1

Day 2 or 3

Day 7

Day 14

Pain at injection

7%

7 [slight]

-

-

-

-

Swelling at the injection site

8%

3

5

5

2

1

Hematoma at the injection site

1%

1

0

0

0

0

Table VII: Frequency of local adverse signs [percentage of cows concerned]

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FIELD EXPERIENCE WITH INTRAVENOUS MARBOFLOXACIN AGAINST ACUTE MASTITIS - Local adverse signs: A total of 14 cows presented with local signs throughout the study. Pain at injection, swelling, hematoma, and swelling of jugular vein were observed on Day 0 after injection, Day 1, Day 2 and/or Day 3, Day 7 and/or Day 14 (table VII). No phlebitis was observed.

DISCUSSION The field trial presented here followed 100 cows on 63 different farms in France and Germany for up to 2 weeks following treatment. The evaluation of the field safety of a single intravenous injection of marbofloxacin 16% solution [Forcyl®] was performed in lactating dairy cows suffering from acute clinical mastitis, characterised by local and general clinical signs, and confirmed by the isolation of pathogen bacteria in milk sampled before treatment. No control product was included in the study design as the purpose of the study was to assess the safety of the test product: it has been already studied in experimental and field conditions, including in comparison with existing reference products. No placebo could be used for ethical reasons due to the possible severity of the disease on welfare grounds. The purpose of such a study is also to assess efficacy and safety in conditions closer to general practice than a comparative registration study: inclusion of a wider range of animals [e.g. several, rather than just one, affected quarters, teat or sphincter with lesions] and with a wider range of concomitant treatments.

TOLERANCE In the presented study, diarrhoea was the most frequent general sign observed [8% of the included cows], only within the first days after treatment [Day 0, 1, and 2]. However, diarrhoea was already present for all of the cows concerned before the administration of marbofloxacin. Tremors were observed once or twice in 3% of the cows, on Day 0 or Day 1 after injection. One cow required euthanasia on Day 4 due to a worsening of condition [suspicion of toxic shock due to acute mastitis], despite other treatments prescribed by the investigator from D1 onwards. Another cow presented with a concurrent disease, which required other treatments. The local tolerance evaluation showed that swelling at the injection site was the most frequent local sign observed [8% of the included cows] and occurred at different times [from Day 0 after injection to Day 7]. Duration ranged from 1 to 7 days. It was still present 15 days after injection in a single cow [1%]. Size was always less than 2 cm for 6 of the 8 cows concerned and between 2 and 5 cm for 2% of the cows. Consistency was soft [4%], soft then hard [1%] or hard [3%]. Slight pain at injection was observed in 7% of the cows on Day 0 after injection. No pain on palpation was observed thereafter. Hematoma was observed in 1% of the cows on Day 0 after injection and was likely caused by movement of the animal during the injection. Revue Méd. Vét., 2013, 164, 12, 537-545

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These moderate and transient local reactions, commonly observed after injection, resolved spontaneously within 1 to 7 days, except for 1 cow [local reaction observed on D7 and D14, no further follow-up]. General adverse signs such as diarrhoea and fatality are frequently described in cases of coliform mastitis and are considered to be due to the effects of endotoxin [25]. In the study, Escherichia coli was isolated in the milk sample performed for 5 out the 9 cows exhibiting diarrhoea or fatality. Similarly to the presented study, marbofloxacin administered at different dosages (2, 4, 8 or 10 mg/kg) by systemic route (intramuscular or intravenous) appeared to be well tolerated in bovines in other studies, whether in calves with BRD [20, 23], in healthy pregnant cows [24] or in lactating dairy cows [9, 18, 21]. The results of safety evaluated after a single administration of marbofloxacin, in comparison with those of a single administration of florfenicol, were comparable with those obtained in the presented trial. A total of 231 cattle with BRD were treated either with marbofloxacin (118 animals, intramuscular route, 10 mg/kg) or with florfenicol (113 animals, subcutaneous route, 40 mg/kg). A total of 16 animals were reported to experience an adverse event; including diarrhoea (for 5 and 3 animals, in the marbofloxacin and florfenicol group, respectively), complication of the BRD leading to death (3 animals in the florfenicol group) or concurrent disease (1 and 4 animals, in the marbofloxacin and florfenicol group, respectively). Pain at injection was recorded for 24.6% of the animals in the marbofloxacin group [vs. 45.1% for the florfenicol group] and no swelling at D7 was reported in animals of the marbofloxacin group [vs. 9.8% for florfenicol]. Both treatments were considered well tolerated [20].

CONCOMITANT THERAPY The majority of the included cows (83%) received a concomitant treatment by intramammary route only (24 cows), by systemic route only (14 cows), or both (45 cows). These results could be compared to other published data, in particular a French prospective cohort study published in 2003. 97% of the clinical mastitis cases were treated with one or more intramammary treatments, in association with a systemic product in 37.5% of cases. More than 55% of the cases were given an intramammary product with both antibiotic and anti-inflammatory indications. More than 30% of the cases were given an antibiotic treatment by systemic route, alone or in combination with anti-inflammatory. Only 0.9% of the cases received a fluid therapy. Veterinary intervention was mostly observed for mastitis with severe clinical signs (general and local), which probably explained the higher frequency of systemic treatment in this study [26].

544 The differences with this field trial could be explained by the selection of clinical cases that required an antibiotic treatment by systemic route, which could then influence the choice of concomitant treatment by the veterinarian investigator. The choice of the intramammary antibiotic given in association with the systemic fluoroquinolone in this study was most commonly a beta-lactam antibiotic containing therapy. This completed the antibiotic spectrum towards gram positive pathogens with a longer duration of treatment for those bacteria. Recent in-vitro synergy studies have confirmed that the association of marbofloxacin with beta-lactam antibiotics is not antagonistic [27], and might enable mutant prevention concentrations to be reached for gram negative and positive pathogens in the udder. The main systemic anti-inflammatory products used were non-steroidal anti-inflammatory drugs: flunixin, meloxicam, carprofen and tolfenamic acid, were administered to 21, 20, 9 and 7 cows respectively. Steroidal anti-inflammatory drugs [dexamethasone or prednisolone] were administered to 10 cows. In this field clinical study, despite the wide variety of treatments administered concomitantly with Forcyl®, no adverse effect that can be imputed to drug interactions was observed.

DISEASE EVOLUTION AND MILK BACTERIOLOGY The time course of the target disease from Day 0 to Day 14 showed a rapid decrease of mean rectal temperature as well as mean score of each of the clinical parameters observed (appetite, general behaviour, hydration status, milk yield, inflammation of udder quarter, and milk composition of udder quarter). These results are similar to those reported in a European field study evaluating the efficacy of marbofloxacin (10 mg/kg, single intramuscular injection) in the treatment of bovine acute E. coli mastitis in comparison to danofloxacin (6 mg/kg, single subcutaneous injection). A high success rate was obtained for both treatments, for both clinical and bacteriological cure rates [8]. The two main pathogens isolated on Day 0 were Escherichia coli and Streptococcus uberis [30.5 and 29.3% of the samples, respectively]. Staphylococcus aureus, coagulase-negative Staphylococcus, Streptococcus dysgalactiae, Enterococcus sp. and Arcanobacterium pyogenes were also isolated in the study, but less frequently. One isolate of Escherichia coli, 1 of Enterococcus sp. and 1 of Arcanobacterium pyogenes were resistant to marbofloxacin. These results can be compared to those already published. In particular, the high sensitivity to marbofloxacin of the main pathogenic bacterial species isolated from bovine mastitis cases was confirmed in a European survey conducted between 2002 and 2008 [28]. Furthermore, in a study performed in France, a total of 1770 milk samples were

GRANDEMANGE (E.) AND COLLABORATORS taken between January 2007 and March 2008 from cases of subclinical or clinical mastitis, leading to the identification of 1631 bacterial isolates. The same bacterial species were identified as the major causative agents of clinical mastitis but in different proportions: Streptococcus uberis (22.1% of samples), Escherichia coli (16%), and coagulase-positive staphylococci (15.8%) [29].

Conclusion

In this field study, marbofloxacin 10  mg/kg single intravenous injection (Forcyl®) was safe and efficacious for the treatment of acute mastitis, confirming the previous studies.

ACKNOWLEDGMENT The authors would like to thank the investigators, farmers, and laboratory staffs who took part in this clinical study.

CONFLICT OF INTEREST Florence PILLET, Cécile MONTREUIL and Olivier ROY are employees of CEBIPHAR. Erik GRANDEMANGE and Frédérique WOEHRLE are employees of Vétoquinol S.A.

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