Efficacy of marbofloxacin for naturally occurring contagious caprine pleuropneumonia

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Small Ruminant Research 77 (2008) 75–79

Short communication

Efficacy of marbofloxacin for naturally occurring contagious caprine pleuropneumonia Engin Balikci a,∗ , Omer Kizil a , Tolga Karapinar a , Murat Karahan b , Haydar Ozdemir a , Murat Dabak a a

University of Firat, Faculty of Veterinary Medicine, Department of Internal Diseases, 23119 Elazig, Turkey b University of Firat, Faculty of Veterinary Medicine, Department of Microbiology, 23119 Elazig, Turkey Received 28 May 2007; received in revised form 27 February 2008; accepted 27 February 2008

Abstract The aim of the present study is to determine the efficacy of marbofloxacin used in goats with naturally occurring contagious caprine pleuropneumonia (CCPP). The study was performed in two groups (consisting of 15 animals in each group) with two different doses of 10% aqueous solution of marbofloxacin injected intramuscularly into the semitendinous muscle. 2 mg/kg BW for 3 days (total dose administered: 6 mg/kg BW) was injected to the first group (group 1) and 3 mg/kg for 2 times every other day (total dose administered: 6 mg/kg BW) was injected to the second group (group 2). Microbiological analyses revealed that the causative agent of the disease was Mycoplasma capricolum subsp. capripneumoniae. Cure rates for groups 1 and 2 were determined as 100% (15/15 goats) and 93% (14/15 goats), respectively. The results of this field trial suggest that marbofloxacin could be an effective drug against CCPP in goats. © 2008 Elsevier B.V. All rights reserved. Keywords: Goat; Marbofloxacin; Contagious caprine pleuropneumonia

1. Introduction Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae (formerly Mycoplasma F38) has been known for many years and results in major losses in goat herds in many countries of Africa, the Middle East, Eastern Europe and other Asian countries, including Turkey (Thiaucourt et al., 1996; Nicholas, 2002; Ozdemir et al., 2005). CCPP is generally refractory to many commonly used antibiotics like penicilin, streptomycin, tetracyclines, and macrolides (Bergonier et al., 1997; Hernandez

∗

Corresponding author. Fax: +90 424 238 81 73. E-mail address: [email protected] (E. Balikci).

0921-4488/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2008.02.009

et al., 2006). Marbofloxacin, a fluoroquinolone, has been used for the treatment of a variety of microbial infections in animal and human medicine (Neu, 1988; Abadia et al., 1995; Petracca et al., 1993; Schneider et al., 1996; Brown, 1996). It shows a wide spectrum of activity against both gram-negative and gram-positive pathogens and against Mycoplasma spp. (Spreng et al., 1995; Dubreuil et al., 1996). Although marbofloxacin is used in different pathological conditions of ruminants like respiratory system infections and neonatal diarrhea (Thomas et al., 1997, 1998, 2001), an evaluation of marbofloxacin therapy in goats with naturally infected CCPP has not been reported. The objective of the field trial described in this paper is to determine the efficacy of marbofloxacin used in goats

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with naturally infected CCPP caused by M. capricolum subsp. capripneumoniae.

after the treatment. Tissue samples were obtained under aseptic conditions from the lungs for microbiological analyses.

2. Materials and methods

2.2. Bacterial culture and PCR

2.1. Field investigation

2.2.1. Isolation of the causative agents Lung and pleural fluid samples were processed according to the method described by Thiaucourt et al. (1992). Shortly, isolation of Mycoplasma spp. was done both by serial dilution in modified Hayflick medium (PPLO broth without crystal violet (21 g/l), 20% de-complemented horse serum, 10% fresh yeast extract, 0.2% glucose, 0.4% sodium pyruvate, 0.04% ampicillin) and by streaking onto solid agar (PPLO agar) of the same medium, simultaneously. Solidification of the broth media was done by adding 1% agar noble (Difco, Detroit, USA) to the medium. Samples were diluted by five subsequent 10-fold dilutions. The lung samples (1 cm3 ) were added to the first broth medium tube (5 ml) and were mixed properly. Then, 300 ␮l broth was taken from each tube and mixed respectively. The samples were incubated at 37 ◦ C in 5% CO2 for 7–10 days. The broths were checked daily and the samples with growth, indicated by turbidity in the broth cultures were sub-cultured onto agar plates from the last turbid tube. Turbidity was assessed by visual inspection. The plates were checked daily for the appearance of colonies. The isolated colonies were inoculated to the stock suspension (%50 broth/%50 horse serum) and were kept in deep-freeze. Inoculations from a loopful of pleural fluid samples were also carried out following the above procedure.

2.1.1. Study area and animals One goat (2-year-old) from a flock of 80 goats was presented to the Veterinary Teaching Hospital of Firat University with loss of appetite, dyspnoea, nasal discharge and cough. Two dead goats had also been brought for necropsy. After a tentative diagnosis of CCPP based on the results of clinical and pathological examination of the goats, the remainder of the flock was enrolled in the study. The study was conducted in the province of Elazig, Turkey, during October 2006. The area is characterised by a climate with erratic rainfall. Thirty goats of the herd showing signs of CCPP were admitted to the study. These animals were judged to have CCPP when it presented with a rectal temperature of 40.5 ◦ C or higher, coughing, hyperaemia of conjunctiva, abnormal lung sounds, loss of appetite and tachypnoea. Ages of the animals varied between 2 and 3 years with mean body weights of approximately 40–50 kg. The herd was on pasture in mornings and was housed in a barn at nights. The goats were fed straw, barley and concentrated feed in addition to pasture grass and had access to water ad libitum. None of the animals had been vaccinated against CCPP. 2.1.2. Experimental design The study was performed in two groups (consisting of 15 animals in each group) with two different doses of 10% aqueous solution of marbofloxacin (Marbocyl® , Vetoquinol, Lure, France) injected intramuscularly into the semitendinous muscle. 2 mg/kg estimated BW for 3 days (total dose administered: 6 mg/kg BW) was injected to first group (group 1) and 3 mg/kg BW for 2 times every other day (total dose administered: 6 mg/kg BW) was injected to second group (group 2). 2.1.3. Clinical and postmortem examination A detailed physical examination was performed on each selected animal before the treatment. The goats were closely monitored on day 1, 3, 5 (between 8 and 10 a.m.) after the treatment. Rectal temperatures, pulse and respiratory rates and rumen contractions were recorded. Clinical assessment (coughing, nasal discharge, hyperaemia of conjunctiva, injection of scleral vessels, abnormal sounds of lung auscultation, loss of appetite and dehydration) was performed. Clinical assessment of the goats was also carried out on day 1, 3, 5, 15 and 30 after the treatment. The effect of the treatment was evaluated in both groups based on the alterations in the following parameters: rectal temperature (T), pulse (P) and respiratory (R) rates, rumen contractions (Rc), loss of appetite, nasal discharge, hyperaemia of conjunctiva, injection of scleral vessels, abnormal lung sounds, cough and dehydration. A detailed necropsy was performed on three goats, two which died before treatment and one in group 2 which died

2.2.2. Polymerase chain reaction (PCR) After DNA samples were extracted from the specimens, PCR was performed in a TC 512 Temperature Cycling System (Techne, Staffordshire, UK). A primer pair specific to M. capricolum subsp. capripneumoniae (Mccp-spe-F 5 -ATCATTTTTAATCCCTTCAAG-3 and Mccp-spe-R 5 TACTATGAGTAATTATAATATATGCAA-3 ) was used in the PCR (Woubit et al., 2004). PCR products with a molecular size of 316 bp were considered indicative for M. capricolum subsp. capripneumoniae. 2.3. Statistical analysis Data were analyzed according to the different days of the study, by using one-way analysis of variance (ANOVA). T-test was used to compare P, R, T and Rc parameters between groups 1 and 2.

3. Results The predominant clinical findings observed in both groups were coughing, hyperaemia of conjunctiva, wheezes and crackles at lung auscultation, loss of appetite, nasal discharge, injection of scleral vessels and dehydration. Few of them displayed signs of mouth breathing and lying down behind rest of the flock.

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Table 1 Some clinical parameters before treatment and 1, 3, 5 days after treatment in group 1 (n = 15) and group 2 (n = 15) Before treatment

After treatment

0

1 days

3 days

5 days

Pulse rate (breath/min)

Group 1 Group 2 Group 1 vs. 2

96.93 ± 2.58 a 104.13 ± 4.52 a NS

89.07 ± 2.68 ab 79.47 ± 3.04 b NS

88.40 ± 3.06 ab 77.33 ± 1.77 b P < 0.05

84.00 ± 4.13 b 76.80 ± 2.06 b NS

Respiratory rate (breath/min)

Group 1 Group 2 Group 1 vs. 2

51.20 ± 2.18 a 54.93 ± 3.69 a NS

41.60 ± 3.55 b 40.53 ± 3.09 b NS

34.67 ± 2.16 b 32.80 ± 1.66 bc NS

33.60 ± 4.13 b 30.93 ± 2.26 c NS

Rectal temperature (◦ C)

Group 1 Group 2 Group 1 vs. 2

40.85 ± 0.14 a 40.71 ± 0.16 a NS

39.31 ± 0.19 b 39.21 ± 0.09 b NS

39.14 ± 0.11 b 39.16 ± 0.06 b NS

39.07 ± 0.45 b 39.11 ± 0.11 b NS

Rumen contractions (number/5 min)

Group 1 Group 2 Group 1 vs. 2

3.33 ± 0.39 a 3.33 ± 0.29 a NS

4.93 ± 0.38 b 5.53 ± 0.32 b NS

6.27 ± 0.41 c 7.27 ± 0.37 c P < 0.05

7.40 ± 0.50 c 8.00 ± 0.35 c NS

a, b, c: means in the same row and within the same variable with different superscripts differ (P < 0.05), NS: non significant.

isolates (three lung isolates and three pleural fluid isolates) were identified as Mycoplasma species when the plates viewed under a stereoscopic microscope (Olympus 209376, Tokyo, Japan) for ‘fried egg’ colonies. In the PCR analysis of DNA extracted from the agar media, amplification was obtained in all the samples with M. capricolum subsp. capripneumoniae specific primers.

Mean P, R, T and Rc and differences between groups 1 and 2 are presented in Table 1. No significant differences were observed in relative to T, P, R and Rc between the groups out of P and Rc values on day 3 after the treatment (P < 0.05). T, P, R and Rc improved after the treatment compared with those of before treatment in both groups (P < 0.05). Results of clinical assessment and clinical cure rates for groups 1 and 2 are presented in Table 2. Cure rates for groups 1 and 2 were determined as 100% (15/15 goats) and 93% (14/15 goats), respectively. After the treatment, no general or local adverse reactions were observed in any goats. Growth, as indicated by turbidity in the broths, was observed in all lung and pleural fluid samples of three goats. Also growth was obtained in all samples when the broths were inoculated onto agar medium. These six

4. Discussion The treatment of mycoplasmoses is currently based on antibiotics, such as tetracyclines, macrolides, florfenicol and fluoroquinolones (Bergonier et al., 1997; Hernandez et al., 2006). However, if the therapeutic dose is not well defined and the antibiotic is not administered for a sufficiently long period, results of treatment may be poor or ineffective against mycoplasmas (Madanat et

Table 2 Results of general clinical assessment rates of goats throughout the trial Clinical parameters

Coughing Nasal discharge Hyperaemia of conjunctiva Injection of scleral vessel Abnormal sounds at lung auscultation Loss of appetite Dehydration Cure rate

Group 1 (n = 15)

Group 2 (n = 15)

Before treatment

After treatment (days)

Before treatment

After treatment (days)

0

1

3

5

15

30

0

1

3

5

15

30

100 80 100

27 53 7

13 7 7

7 0 7

0 0 0

0 0 0

100 87 100

33 53 33

13 13 13

7 13 13

0 0 0

0 0 0

93 100

33 33

7 7

7 7

0 0

0 0

93 100

40 60

13 13

7 13

0 0

0 0

100 100 0

33 47 30

7 20 67

0 13 93

0 0 100

0 0 100

100 100 0

40 40 27

13 20 60

13 20 87

0 0 93

0 0 93

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al., 2001). Moreover, it has been reported that tilmicosin and oxytetracycline-resistant strains of Mycoplasma spp. were present (Ayling et al., 2000a,b and Ayling et al., 2005). Marbofloxacin, a fluoroquinolone, has been used against some respiratory infections of calves (Thomas et al., 1998, 2001) and sheep (Skoufos et al., 2007) but it has not been used in goats with CCPP. In a comparative study, marbofloxacin has induced a faster improvement than tilmicosin based on the time course of the overall clinical score and the time to cure in treatment of bovine respiratory disease (Thomas et al., 2001). The rapid bactericidal activity of marbofloxacin may be an advantage for this superiority as tilmicosin action is bacteriostatic. In the present study, the dose of marbofloxacin given 2 mg/kg for 3 days was found to be more effective than the dose of 3 mg/kg for 2 times, every other day. This may probably be caused by maintaining of effective antimycoplasmal concentrations in the lungs of the goats by every day administration of the drug. Fluoquinolones are known to accidentally cause adverse cutaneous reactions in treated animals (Ihrke, 1996; Doukas et al., 2005). However, no local reaction has also been reported in any lamb injected with marbofloxacin (Skoufos et al., 2007). Marbofloxacin has been found to be better tolerated at the i.v. injection than s.c. injection site (Thomas et al., 2001). In the present study the solution rate of 10% marbofloxacin was found to be well tolerated at the site of i.m. injection and there were no systemic and local adverse reaction. The results of this field trial suggest that marbofloxacin could be an effective drug against CCPP in goats. Moreover, among two doses of marbofloxacin, the dose of 2 mg/kg for 3 days was found to be more effective than the dose of 3 mg/kg for 2 times every other day. References Abadia, A.R., Aramayona, J.J., Munoz, M.J., Pla Delfina, J.M., Bregante, M.A., 1995. Ciprofloxacin pharmacokinetics in dogs following oral administration. Zbl. Vet. A 42, 505– 511. Ayling, R.D., Baker, S.E., Peek, M.L., Simon, A.J., Nicholas, R.A., 2000a. Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against recent field isolates of Mycoplasma bovis. Vet. Rec. 146, 745– 747. Ayling, R.D., Baker, S.E., Nicholas, R.A., Peek, M.L., Simon, A.J., 2000b. Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against Mycoplasma mycoides subspecies mycoides small colony type. Vet. Rec. 146, 243–246. Ayling, R.D., McAuliffe, L., Bisgaard-Frantzen, S., Bashiruddin, S., Miles, K., Nicholas, R.A.J., 2005. Mycoplasma ovipneumoniae:

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