• Open Access

Efficacy of Pradofloxacin in Cats with Feline Upper Respiratory Tract Disease due to Chlamydophila felis or Mycoplasma Infections

Authors


  • An abstract of this work was presented at the 1st International Veraflox Symposium, Berlin, Germany, March 16–19, 2006 and at the ECVIM Congress, Amsterdam, the Netherlands, September 14–16, 2006.

Corresponding author: Katrin Hartmann, Prof., Dr. med. vet., Dr. habil., Dipl. ECVIM-CA, Medizinische Kleintierklinik, LMU Munich, Veterinaerstrasse 13, 80539 Muenchen, Germany; e-mail: Hartmann@uni-muenchen.de

Abstract

Background: Upper respiratory tract disease (URTD) of cats is caused by a number of pathogens, including Chlamydophila felis and Mycoplasma spp. For effective treatment of both infections, doxycycline and enrofloxacin are recommended, but adverse effects limit their use in cats.

Hypothesis: That the fluoroquinolone pradofloxacin is effective against C. felis and Mycoplasma infection in cats with URTD or conjunctivitis.

Animals: Thirty-nine cats with signs of URTD or conjunctivitis.

Methods: Placebo-controlled, double-blind clinical trial. Cats were randomly entered into 1 of 2 treatment groups: treated PO with either 5 mg/kg pradofloxacin q24h or 5 mg/kg doxycycline q12h for 42 consecutive days. Changes in health status and clinical scores were evaluated. The presence of C. felis and Mycoplasma spp. was determined by quantitative polymerase chain reaction (PCR) and nested PCR of conjunctival swabs, respectively.

Results: At the beginning of the study, C. felis and Mycoplasma spp. were detected in 23 and 20 cats, respectively. Cats of both groups responded rapidly with a marked improvement in clinical signs within the 1st week. During treatment with either drug, C. felis DNA copy number declined quickly. Complete elimination of Mycoplasma spp. was achieved in both groups; however, whereas all cats receiving doxycycline eliminated C. felis, 4 cats treated with pradofloxacin remained PCR-positive.

Conclusion and Clinical Importance: This study demonstrates that both pradofloxacin and doxycycline have good efficacy against C. felis and Mycoplasma spp., resulting in a marked improvement of clinical signs. However, C. felis DNA remained in some cats after treatment with pradofloxacin, suggesting that infection might not have been eliminated.

Upper respiratory tract disease (URTD) in cats is a common problem of predominantly young cats and kittens in the postweaning period. Many of the causes are infectious and so contact between cats facilitates transmission. Thus, cats grouped together in multiple cat households, breeding institutions, and animal shelters are infected frequently.1–3Chlamydophila felis is an obligate intracellular bacterium that replicates in conjunctival cells, causing conjunctivitis in cats. In a recent study in Germany, 8.2% of cats in multiple cat households with a history of URTD were infected with C. felis.4 Because C. felis can exist as an intracellular reticulate body, effective treatment is difficult, and complete elimination of the bacteria is not always achieved.5Mycoplasma spp. are wall-less bacteria detected on mucous membranes of the conjunctiva, respiratory tract, and genital tract of cats.6 They can be isolated frequently in cats with conjunctivitis, and in 1 study, Mycoplasma spp. DNA was amplified from conjunctival cells of more cats with conjunctivitis than of healthy controls.7,8

C. felis can be effectively treated with tetracyclines, including doxycycline, and the fluoroquinolone enrofloxacin.9,10 The drug of choice is currently doxycycline, which had been used effectively in a number of experimental studies.11–13 However, both drugs are associated with a variety of adverse effects. Tetracyclines can lead to gastrointestinal side effects, esophageal strictures, and administration to pregnant queens or kittens in the first few months of life should be avoided because of calcium chelate formation in bones and teeth resulting in growth retardation and tooth discoloration.14–16 Enrofloxacin has been associated with retinal degeneration in cats, especially if used at higher dosages.17 Clinical signs associated with Mycoplasma spp. infections also often resolve after administration of tetracyclines or fluoroquinolones; tylosin seems to be effective as well.18,19

The fluoroquinolone pradofloxacin is not toxic in cats and failed to induce fluoroquinolone-induced retinal changes even at high serum concentrations.a,b The drug exhibits a broad spectrum activity against Gram-negative and Gram-positive bacteria.c Therefore, it could be a good candidate for the treatment of cats with bacterial causes of URTD. The aim of this study was to investigate the efficacy of pradofloxacin in the treatment of cats with clinical signs of URTD and its ability to eliminate C. felis and Mycoplasma infection in these cats.

Materials and Methods

Cats

Forty-five cats with signs of URTD or conjunctivitis that were presented at the Veterinary Teaching Hospital of the LMU University of Munich between April 2004 and December 2005 were included in the study. Inclusion criteria were presence of URTD or conjunctivitis and negative serum test results for feline leukemia virus antigen and feline immunodeficiency virus antibodies.d Owners had to give their consent for participation. Exclusion criteria were an age of <6 months, pregnancy or weaning, evidence of renal or liver failure, CNS disease, and pretreatment with antibiotics during the past 6 weeks. Six cats did not complete the study: 4 were euthanized because of the development of feline infectious peritonitis (3 cats) or an oropharyngeal squamous cell carcinoma (1 cat), 1 cat was impossible to treat, and 1 cat developed severe diarrhea. Thirty-nine cats completed the study. Of these, 10 (26%) were client-owned, 11 (28%) were farm cats, and 18 (46%) came from animal shelters. All patients were domestic shorthair cats with an average weight of 2.6 ± 1.1 kg (0.6–5.0 kg). Of the cats, 21 were female (8 intact, 13 neutered) and 18 were male (6 intact, 12 neutered). The age of most cats could only be estimated. Thirteen cats were younger than 1 year, 16 cats were between 1 and 4 years of age, 8 cats were between 5 and 8 years of age, and 2 cats were between 8 and 12 years of age. The vaccination status was known only for the 10 client-owned cats, of which 4 were vaccinated regularly. The history about duration of clinical signs was unknown for the majority of cats.

Design of the Study

The study was performed as a controlled, double-blinded study. Cats were randomly assigned to one of 2 study groups. Cats in group P (n=17) were administered 5 mg/kg pradofloxacine PO every 24 hours and placebo every 12 hours. Cats in group D (n=22) were administered 5 mg/kg doxycyclinef every 12 hours and placebo every 24 hours. All compounds were given PO for 42 consecutive days. A treatment period of 6 weeks (42 days) is the currently recommended regime to eliminate C. felis infections.10 Substances used as placebos had the same color and consistency, but did not contain any antimicrobial agents. All investigators involved were blinded until the end of the study.

Examination Schedule and Sample Collection

Cats were examined daily during the 1st week, and thereafter on days 14, 28, and 42. Clinical signs evaluated included ocular and nasal discharge, sneezing, conjunctivitis, breathing pattern, and lung sounds (Table 1). Each sign was assessed separately and subsequently the total score was calculated summarizing the score of each sign. The maximum possible total clinical score was 18. General health status and quality of life were determined using the modified Karnofsky's score.20 This scoring system uses behavior patterns such as eating, playing, sleeping, grooming, and outdoor activities. The degree of quality of life is expressed in percentage ranging from 100% (cat with normal behavior) to 0% (death of the cat). CBC and chemistry profiles were investigated on days 0, 3, 7, 14, 28, and 42.

Table 1.   Scoring system for clinical signs evaluated in cats with URTD or conjunctivitis.
  1. URTD, upper respiratory tract disease.

Conjunctivitis0 = no clinical signs
1 = mild conjunctival hyperemia
2 = moderate conjunctival hyperemia and mild chemosis
3 = moderate to severe conjunctival hyperemia and moderate chemosis
4 = severe conjunctival hyperemia and severe chemosis
Ocular and nasal discharge0 = no clinical signs
1 = minor serous discharge
2 = moderate serous to mucoid discharge
3 = moderate mucopurulent discharge
4 = severe mucopurulent discharge
Sneezing0 = absent
1 = occasionally
2 = frequently
Breathing pattern0 = normal
1 = mild dyspnoe
2 = moderate to severe dyspnoe
Lung sounds0 = absent
1 = mild abnormal lung sounds
2 = moderate to severe lung sounds

On day 0, swabs for the detection of feline herpesvirus (FHV), feline calicivirus (FCV), C. felis, and Mycoplasma spp. were obtained. Follow-up swabs for C. felis and Mycoplasma spp. were taken on days 3, 7, 14, 28, and 42. Oropharyngeal swabs for the detection of FCV were obtained using sterile plain cotton swabs,g which were rolled over the mucous membrane of the oropharynx of each cat. The cotton tip of a swab was gently scraped over the conjunctival surface to collect ocular samples for the detection of FHV, C. felis, and Mycoplasma spp. All swabs were returned into their covers immediately after sampling and stored at − 70 °C until analysis. A multiplex real-time polymerase chain reaction (PCR) was run to detect FHV, FCV, and C. felis. For Mycoplasma spp., a nested PCR was performed.

DNA Isolation

For isolation of the total nucleic acid (DNA and RNA), the cotton swabs were placed in a 96 deep-well block containing 300 μL phosphate-buffered saline, 300 μL buffer BQ1, and 30 μL proteinase Kh per well. The block was covered and the swabs were incubated in a shaker/incubatori for 15 minutes at 70 °C and 500 rpm. Three hundred microliters of 100% ethanol was added to the swab, mixed, and the liquid was transferred to a 96-well spin plate. Subsequent procedures were performed as outlined by the manufacturer. The total nucleic acid was eluted in 100 μL buffer BE and stored at −80 °C.

PCR for FCV, FHV, and C. felis

To reverse transcribe the FCV RNA present in the nucleic acid samples, the following reaction was set up on ice: 10 μL of 2X Platinum QRT-PCR Thermoscript,j 0.25 μL of 10 μM FCV reverse primer (Table 2), 0.5 μL Thermoscript/Platinum Taq mix, and 9.25 μL template nucleic acid. The reaction was incubated at 55 °C for 30 minutes to allow reverse transcription and at 85 °C for 5 minutes to inactivate the Thermoscript. A duplex real-time PCR assay to detect FCV and feline 28S rDNA was set up as follows: 12.5 μL 2 × Hotstartaq,k 1.5 μL of 50 mM magnesium chloride, 100 nM FCV primers and probe, 100 nM feline 28S primers and probe, 5 μL cDNA/gDNA from above, and water to bring the volume to 25 μL. Thermal cycling was performed in an iCycler iQl for 1 cycle of 95 °C for 15 minutes, followed by 45 cycles of 95 °C for 10 seconds and 60 °C for 30 seconds. Fluorescence was detected at 575 and 620 nm at each annealing step (60 °C).

Table 2.   Primer and probe sequences used in the PCR assays.
 Primer or Probe Sequence
  1. BHQ, black hole quencher; C. felis, Chlamydophila felis; CY-5, cyanine 5; FAM, carboxyfluorescein; FCV, feline calicivirus; FHV, feline herpesvirus; for, forward; Hex, hexachloro–fluorescein; Myc, Mycoplasma; PCR, polymerase chain reaction; rDNA, ribosomal deoxyribonucleic acid; rev, reverse.

C. felis for5′-GAACTGCAAGCAACACCACTG-3′
C. felis rev5′-CCATTCGGCATCTTGAAGATG-3′
C. felis Taqman5′-FAM-CGCTGCCGACAGATCAAATTTTGCC-BHQ1-3′
28S rDNA for5′-AGCAGGAGGTGTTGGAAGAG-3′
28S rDNA rev5′-AGGGAGAGCCTAAATCAAAGG-3′
28S rDNA Taqman5′-Texas Red-TGGCTTGTGGCAGCCAAGTGT-BHQ2-3′
FCV for5′-GTTGGATGAACTACCCGCCAATC-3′
FCV rev5′-CATATGCGGCTCTGATGGCTTGAAACTG-3′
FCV Taqman5′-Hex-TCGGTGTTTGATTTGGCCTGBHQ1-3′
FHV for5′-GGACAGCATAAAAGCGATTG-3′
FHV rev5′-AACGTGAACAACGACGCAG-3′
FHV Taqman5′-CY5-AATTCCAGCCCGGAGCCTCAATBHQ3-3′
Myc for 15′-ACACCATGGGAGCTGGTCAT-3′
Myc rev 15′-CTTC(AT)TCGACTT(CT)CAGACCAAGGCAT-3′
Myc for 25′-GTTCTTTGAAAACTGAAT-3′
Myc rev 25′-GCATCCACCA(AT)A(AT)ACTCT-3′

The 3-way multiplex real-time PCR assay to detect FHV, C. felis, and feline 28S rDNA was set up as follows: 12.5 μL 2 × Hotstartaq, 1.5 μL of 50 mM magnesium chloride, 100 nM FHV primers and probe, 100 nM C. felis primers and probe, 100 nM feline 28S primers and probe, 5 μL genomic DNA, and water to bring the volume to 25 μL. Thermal cycling was performed as described for FCV, except that the annealing step (60 °C) was increased to 60 seconds. Fluorescence was detected at 530, 620, and 680 nm at each annealing step (60 °C).

The duplex real-time PCR assay to detect C. felis and feline 28S rDNA on the swabs from all follow-up examinations was set up like the 3-way multiplex real-time PCR, but without the FHV primers and probe. Thermal cycling was performed as described for FCV, and fluorescence was collected at 530 and 620 nm at each annealing step (60 °C). All reactions were run in duplicate.

PCR data were analyzed using the iCycler software version 3 as described previously.3,11 Threshold cycle values (Ct) were calculated using a threshold of 100 relative fluorescence units (RFU) for the FAM channel, 20 RFU for the Hex channel, 50 RFU for the Cy5 channel, and 25 RFU for the Texas Red channel. To facilitate comparisons between swabs and calculate relative copy numbers, the C. felis Ct values were normalized to the 28S Ct values. For each swab, the 28S Ct was adjusted to 30 through the application of a correction factor (CF), where CF=28S Ct− 30. The CF value was then subtracted from the C. felis Ct for each swab to create a normalized C. felis Ct value: normalized C. felis Ct=C. felis Ct− CF. Relative copy number (RCN) values were generated based on the notion that a C. felis Ct value of 45 equates to a template copy number of 1. Each swab was then given a delta CtCt) value, where ΔCt=45 – corrected C. felis Ct. An RCN was assigned to each swab by calculating 1.9 ΔCt, as the reaction has been shown to be 92% efficient.21 To allow the results to be plotted on a log scale, 1 was added to each relative copy number result.

PCR for Mycoplasma Species

The PCR was performed using previously described primers for both the flanking (Myc for 1, Myc rev 1) and nested reactions (Myc for 2, Myc rev 2) (Table 1).22 The 50 μL reactions (both flanking and nested) were carried out with 1 μL of sample DNA and a final concentration of the following: 1 μM each primer, 800 μM dNTP mix, 1.5 mM MgCl2, 1 × PCR Bufferm (16 mM (NH4)2SO4, 67 mM Tris-HCl [pH 8.8 at 25 °C], 0.01% Tween-20), and 3.75 U Taq polymerase.j The flanking and nested reactions were carried out using the previously described protocols.23

Statistics

Statistical analysis was performed using a commercial software package.n The Mann-Whitney U-test was used to detect (1) differences in the distribution of pathogens among both treatment groups on day 0; (2) differences in Karnofsky's score, mean clinical scores, and mean relative copy numbers at each time point between treatment groups; and (3) differences in the clinical scores between groups infected with either Mycoplasma spp. or C. felis at the beginning and end of the study. To detect differences in laboratory variables, Student's t-test was used. The Fisher's exact test was used to investigate differences in the number of cats in which C. felis and Mycoplasma infections were eliminated. The mean time necessary to clear infections was estimated by Kaplan-Meier survival analysis. A P value of ≤0.05 was considered statistically significant.

Results

Pathogens Present at the Beginning of the Study

Nucleic acids of FCV, FHV, C. felis, or Mycoplasma spp. were present in 38 of 39 (97%) cats at the beginning of the study (Table 3). There was no statistically significant difference regarding the distribution of pathogens between both treatment groups.

Table 3.   Infectious pathogens present in cats with URTD or conjunctivitis at the beginning of the study and their distribution among treatment groups.
 Both
Groups
Group
P
Group
D
P
  1. Results are based on pharyngeal swabs (FCV, Mycoplasma spp.) and conjunctival swabs (FHV, C. felis, Mycoplasma spp.). Prevalence rates between groups P and D were compared by the Mann-Whitney U-test with significance defined as P <.05.

  2. C. felis, Chlamydophila felis; FCV, feline calicivirus; FHV, feline herpesvirus; spp., URTD, upper respiratory tract disease.

Number of cats included39 (100%)1722 
Total number of pathogens
 FHV11 (28%)74.114
 FCV28 (72%)1216.883
 C. felis23 (59%)1013.987
 Mycoplasma spp. (conjunctival swab)18 (46%)810.921
 Mycoplasma spp. (pharyngeal swab)25 (64%)1114.945
Coinfections
 FHV + FCV5 (13%)41.079
 C. felis+Mycoplasma spp.11 (28%)65.387
 All 4 pathogens4 (10%)22.785

Change in Clinical Signs and Laboratory Parameters

On day 0, the mean body temperatures were 38.9 ± 0.5 and 38.8 ± 0.3 °C in group P and group D, respectively. Treatment in both groups resulted in rapid improvement of the general health status. According to Karnofsky's score, the mean general health status of 88 ± 11% and 89 ± 14% on day 0 improved to 99 ± 2% and 100 ± 0% on day 7 in cats treated with pradofloxacin and doxycycline, respectively (Fig 1). There was no statistically significant difference between both groups on any examination day.

Figure 1.

 Improvement of the Karnofsky's score (0–100%) in cats with upper respiratory tract disease treated with pradofloxacin or doxycycline during the first week of treatment. Solid line = pradofloxacin; dashed line = doxycycline.

Signs of URTD improved within 7 days after initiating treatment. Mean total clinical score of cats treated with pradofloxacin improved from an average of 4.9 ± 1.7 on day 0 to 0.9 ± 0.9 on day 7. Cats treated with doxycycline had a mean total clinical score of 4.5 ± 2.4 on day 0, which decreased to 1.4 ± 1.5 on day 7 (Fig 2). Differences in clinical signs were not statistically significant between the treatment groups on each examination day. At the end of the treatment period, however, 12 of 17 cats in group P and 8 of 22 cats in group D still had minor signs of URTD. Coinfections with both viruses were present in 9 and 8 cats of group P and group D, respectively. This was not statistically significantly different between both groups.

Figure 2.

 Improvement of mean total clinical scores (maximum 18) in cats treated with pradofloxacin or doxycycline during the first 7 days of treatment. Solid line = pradofloxacin; dashed line = doxycycline.

Occasionally, cats in both groups developed vomiting and diarrhea. Vomiting was observed on single days and occurred only once per day (5 cats in group P, 4 cats in group D). Diarrhea was present in 5 cats in total (2 in group P, 3 in group D) and persisted up to 2 days in 4 cats. The 5th cat treated with doxycycline had to be withdrawn from the study on day 7 because of severe diarrhea for 5 days. Data obtained from this cat were not included into analysis. Clinical signs resolved in all other cats without any specific treatment. On day 42, pododermatitis on all 4 paws was observed in 1 cat treated with doxycycline.

On day 0, no laboratory values of any cat included in the study were above the reference range. Mean serum alanine transferase (ALT) activity was within the reference range (0–114 U/L) in both treatment groups on each day of investigation. However, mean values in cats treated with doxycycline increased from 64 ± 29 U/L on day 0 to 92 ± 72 U/L and 93 ± 61 U/L on day 28 and 42, respectively. Elevations above the reference range were present in 3 cats on day 28 and in 4 cats on day 42. Values on days 28 and 42 were statistically significantly (day 28: P=.022; day 42: P=.042) higher than values on day 0. No changes in any other serum parameters developed during treatment with doxycycline and no changes were observed during pradofloxacin treatment.

The mean white blood cell count was 20.92 ± 8.99 × 103/μL (range: 6.03–36.3 × 103/μL) on day 0. Seventeen of the 39 patients (44%) had leukocytosis (>20.00 × 103/μL). A mild left shift with 0.83 ± 1.65 × 103/μL banded neutrophils was present in 20 of 39 cats (53%). In both groups, a normalization of the leukocyte counts was achieved within 1 week of initiating therapy.

Presence of C. felis

C. felis PCR was positive in 23 of 39 cats (59%) on day 0 (10 cats in group P, 13 cats in group D). Two cats treated with pradofloxacin remained positive throughout the treatment period. Two cats displayed positive results at the end of treatment despite negative test results in between. One cat was PCR negative on day 28, but was positive on the previous (day 14) and the following sampling day (day 42). The other cat showed a positive test result on day 42, but tested negative on days 7, 14, and 28 (Figs 3 and 4, Table 4). On day 42, the number of cats with positive C. felis PCR assay results was significantly different (χ2=5.77, P=.016) between the treatment groups. The estimated mean duration of treatment for C. felis infections in cats was 28.8 ± 2.2 days for pradofloxacin and 23.4 ± 1.7 days for doxycycline (Fig 5).

Figure 3.

 Effect of treatment with pradofloxacin for 42 days on relative copy numbers of Chlamydophila felis (10 cats).

Figure 4.

 Effect of treatment with doxycycline for 42 days on relative copy numbers of Chlamydophila felis (13 cats).

Table 4.   Mean clinical scores, individual conjunctivitis score, and status of infection at each time point in cats treated with pradofloxacin (FCV and FHV only tested on day 0) and with doxycycline (FCV and FHV only tested on day 0).
Cat #Day of Study
037142842
  1. Cf, positive for Chlamydophila felis; FCV, positive for feline calicivirus; FHV, positive for feline herpesvirus; M, positive for Mycoplasma spp.

 Status of infection conjunctivitis score     
P1FHV, FCV     
100000
P2FHV     
100000
P3FHV, FCV     
200000
P4FCV     
111101
P5FHV, FCV     
100000
P6FCV, Cf, MCf, MCf, MCf, MM 
200000
P7FCV     
110000
P8FHV, FCV, CfCf, MCf M 
321001
P9FHV, FCV, Cf, MCf   Cf
311100
P10FCV     
100000
P11Cf, MCfCfCfCfCf
311111
P12Cf     
100000
P13Cf, MCfCfCfCfCf
321101
P14FCV, Cf, MCf    
211111
P15Cf, MCfCfCf Cf
320011
P16FCV, Cf, MCf    
110000
P17FHV, FCV, Cf, MCf    
210000
Mean clinical scores
Breathing pattern0.2 ± 0.60.1 ± 0.20.1 ± 0.20.1 ± 0.30.1 ± 0.20
Lung sounds0.3 ± 0.60.1 ± 0.30.1 ± 0.20.3 ± 0.50.1 ± 0.30.1 ± 0.3
Sneezing0.5 ± 0.70.2 ± 0.400.3 ± 0.60.1 ± 0.30.1 ± 0.3
Nasal discharge0.8 ± 0.80.2 ± 0.40.1 ± 0.20.2 ± 0.60.1 ± 0.20.2 ± 0.4
Ocular discharge1.4 ± 0.80.8 ± 0.60.4 ± 0.50.3 ± 0.50.4 ± 0.50.5 ± 0.5
Conjunctivitis1.8 ± 1.00.8 ± 0.80.4 ± 0.50.3 ± 0.50.1 ± 0.30.4 ± 0.5
Figure 5.

 Efficacy of treatment in cats with Chlamydophila felis infections using pradofloxacin and doxycycline (percentage of positive cats at each time point). Solid line = pradofloxacin; dashed line = doxycycline.

On day 0, C. felis mean relative copy numbers in cats were 376,296 ± 653,108 and 27,614 ± 53,960 in group P and group D, respectively (P=.23). Two C. felis genomic DNA samples with Ct values of 19 and 27 were run in triplicate on 4 occasions. The maximum difference was 1.2 Ct, which equates to 2-fold. Treatment resulted in a decrease in C. felis relative copy numbers in both groups. At the end of the treatment period, there was no statistically significant difference in mean relative copy numbers between both groups, although all cats in group D became negative, whereas 4 cats in group P were still positive for C. felis by PCR on day 42 (P=.12).

Detection of Mycoplasma Spp.

Mycoplasma spp. DNA was amplified in conjunctival swabs from 18 of 39 cats (46%) (8 cats in group P, 10 cats in group D) and in oral swabs from 25 of 39 cats (64%) (11 cats in group P, 14 cats in group D). Two cats negative for Mycoplasma spp. on day 0 were positive on day 3 and 1 cat on day 28. All cats positive for Mycoplasma spp. on day 0 or on day 3 or day 28 were negative on day 42 (Table 5). Mean elimination of Mycoplasma spp. was estimated to be 19.7 ± 2.1 and 18.9 ± 1.7 days for treatment with pradofloxacin and doxycycline, respectively (Fig 6).

Table 5.   Mean clinical scores, individual conjunctivitis score, and status of infection at each time point in cats treated with doxycycline.
Cat #Day of Study
037142842
  1. Cf, positive for Chlamydophila felis; FCV, positive for feline calicivirus; FHV, positive for feline herpesvirus; M, positive for Mycoplasma spp.

 Status of infection conjunctivitis score     
D1Cf     
310000
D2      
121111
D3FCV     
100000
D4FCV, CfCf    
100000
D5FCV, CfCf    
000000
D6FCV, CfCf    
211100
D7FCV     
100100
D8FHV, FCV     
000000
D9FCV, CfCfCf   
211000
D10FCV, M MM  
222100
D11M M   
110002
D12FCVM    
100000
D13FCV     
321100
D14M     
210000
D15FCV, Cf, MCf    
201000
D16FHV, FCV, Cf, MCf Cf  
211000
D17FCV, Cf, MCf, MCf, MCfM 
321000
D18FCV, Cf, MCfCf   
421000
D19FCV, Cf     
111111
D20FHV, Cf, MCf    
110010
D21Cf, MCf  Cf 
120000
D22FHV, FCV, Cf, MCfCfCfCf 
122000
Mean clinical scores
Breathing pattern0.2 ± 0.40.1 ± 0.40.1 ± 0.20.1 ± 0.30.1 ± 0.30.1 ± 0.2
Lung sounds0.3 ± 0.60.3 ± 0.50.2 ± 0.40.2 ± 0.40.2 ± 0.40.1 ± 0.4
Sneezing0.4 ± 0.70.2 ± 0.40.1 ± 0.30.1 ± 0.40.2 ± 0.40.1 ± 0.3
Nasal discharge0.9 ± 0.80.5 ± 0.60.1 ± 0.40.1 ± 0.30.1 ± 0.40.1 ± 0.3
Ocular discharge1.1 ± 0.80. 7 ± 0.70.3 ± 0.50.2 ± 0.50.2 ± 0.40.2 ± 0.4
Conjunctivitis1.6 ± 1.01.0 ± 0.80.6 ± 0.70.3 ± 0.50.1 ± 0.30.2 ± 0.5
Figure 6.

 Efficacy of treatment in cats with Mycoplasma species infections using pradofloxacin and doxycycline (percentage of cats at each time). Solid line = pradofloxacin; dashed line = doxycycline.

Discussion

The aim of this study was to evaluate the efficacy of pradofloxacin and doxycycline in the treatment of URTD and infections with C. felis and Mycoplasma in cats. The prevalence and variety of pathogens detected by PCR in the current study are similar to those reported by other authors. In this study, 72% of the cats were infected with FCV and 28% of the cats with FHV. In other publications, FCV and FHV had been detected in cats with URTD with a prevalence of 33–80% and 11–54%, respectively.1,3,24–26 The prevalence of C. felis (59%) and of Mycoplasma (46%) in this study was higher than reported in most other investigations, which can be explained by the selected inclusion of cats with conjunctivitis.8 Additionally, the majority of cats included in the study originated from multiple-cat environments (shelters, farms) in which a higher percentage of infections is expected compared with cats from single-cat households.

C. felis was detected in 23 cats in the current study. Mean relative copy numbers declined over time and clinical scores declined over time regardless of the drug administered. In 3 cats (1 cat in group P, 2 cats in group D), however, C. felis DNA was intermittently detectable without clinical signs of conjunctivitis at these time points. In a study of Dean et al11 similar observations were made in 3 experimentally infected cats receiving a 14-day course of doxycycline. C. felis has also been detected in organs, for example, the gastrointestinal tract, lungs, and genital tract.12,27 Hence, persistent infections in other organs, without presence in the conjunctiva, may contribute to intermittingly negative PCR results when the infection has not been eliminated.11

Clinical signs of URTD improved within a week of initiating treatment with no statistically significant difference between the treatment groups. Treatment response might be caused by the elimination of C. felis and Mycoplasma spp., but might also be caused by treatment of secondary infections such as Staphylococcus spp. and Streptococcus spp. This seems to be especially likely in virally infected cats without Mycoplasma and C. felis infections. Field studies in cats with URTD using other fluoroquinolones such as marbofloxacin and enrofloxacin also demonstrated good efficacy.9,28 However, in the study a clinical cure was not achieved in all cats. Remaining signs mainly consisted of conjunctivitis and ocular discharge. It can be assumed that in some cats signs persisted because of coinfection with FHV. In 4 other cats, C. felis was not eliminated because of treatment failure of pradofloxacin. Three of these cats showed clinical signs on day 42. Alternatively, chronic changes such as occlusion of the nasolacrimal duct, symblepharon, and adhesions of the conjunctiva to itself or to the cornea are known to develop during chronic URTD and are unlikely to fully resolve.29

Treatment resulted in a rapid decline in mean relative C. felis copy numbers in both groups. Six of 10 cats treated with pradofloxacin became negative at day 42 of the study. Two cats, however, remained positive throughout the study. In these cats, copy numbers did not decrease. In the 2 other cats, C. felis was not detected on day 28, but the cats were positive again on day 42. An increase in relative copy numbers on day 42 was correlated with clinical signs in one of these 2 cats. Treatment failure may be caused by insufficient absorption or distribution of the drug, poor compliance, or antimicrobial resistance. Pradofloxacin has excellent pharmacokinetic properties, including quick absorption, development of high serum concentration, and good bioavailability.30,31 One study demonstrated a high concentration of pradofloxacin in neutrophils.o However, data are missing if pradofloxacin penetrates into tissue cells. It may be possible that it is taken up into cells to a lesser extent than doxycycline, which may explain the treatment failure in 2 cats of group P. Vomiting or diarrhea, which might have interfered with the absorption of the antimicrobial agent in the gastrointestinal tract, were observed only occasionally and occurred in the doxycycline group as well. Hence, gastrointestinal problems as a reason for treatment failure is unlikely. Poor compliance of the owners and failure to administer the drugs are the major reasons for reoccurrence of disease.12,32 Both of the cats in which C. felis persisted until the end of the treatment period stayed in the hospital throughout the whole study period, and treatment was part of the daily routine of hospitalized cats. Staff members were specifically instructed about handling of the drugs and application. Thus, it is very unlikely that the drugs were administered incorrectly in these cats. Resistance of Chlamydophila spp. to antimicrobials is difficult to test and does not seem to be very common. Susceptibility testing in culture has been shown not to be reliable and test results did not correlate well with the clinical outcome of the patients.33 A few case reports about treatment failure caused by resistant strains of Chlamydia trachomatis in humans have been published.34–36 Acquired resistance of Chlamydia trachomatis to fluoroquinolones can be caused by mutation in the DNA gyrase gene and can be produced experimentally in vitro during serial cell passages.37,38 This phenomenon did not develop in Chlamydophila pneumoniae, suggesting that the efficacy of antimicrobial drugs depends on the degree of metabolic activity in the target bacteria.38 No data exist on the development of antibiotic resistance in C. felis, and thus resistance remains a potential cause for the treatment failure.

Treatment with doxycycline resulted in a clearance of C. felis DNA by day 42 in all patients. Thus, it can be confirmed that the efficacy of doxycycline in the treatment of C. felis infections in cats is similar to previously published work.11–13,32 A 21-day course of doxycycline is recommended to treat C. felis infection, but clinical signs reoccurred after a treatment period of 21 days, suggesting that a treatment period of 21 days is not enough.11,12 A 28-day course of doxycycline therefore has been recommended.11 In the present study, 2 cats still showed shedding of C. felis on day 28. Thus, a treatment period of 42 days was necessary to eliminate infection in all cats.

Mycoplasma spp. have been shown to be susceptible to several fluoroquinolones and tetracyclines in vitro.39,40 In our study, 9 cats treated with pradofloxacin and 11 cats treated with doxycycline became Mycoplasma spp. DNA negative after a mean treatment period of 20 and 19 days, respectively. This clinical trial, therefore, demonstrated a good efficacy of both substances in the elimination of Mycoplasma spp. in vivo. However, to treat these organisms effectively, a treatment period of 42 days is recommended because of the positive PCR results on day 28 in 3 of 20 cats.

Vomiting and diarrhea occurred in cats of both treatment groups. Many antimicrobial agents may cause gastrointestinal side effects. With doxycycline, gastrointestinal upset is believed to develop because of changes in the natural enteric flora as well as esophageal, gastric, and intestinal irritation.16 ALT activity increased continuously during treatment with doxycycline. Increase in ALT is a result of liver cell damage or membrane destabilization rather than enzyme activation. Tetracycline-induced hepatotoxicity had been shown to cause an increase in liver enzyme activity such as ALT and alkaline phosphatase.41 Tetracyclines are also known to have effects on the skin, such as inducing photosensitivity with erythema and edema after UV light exposure.42 Whether the development of the pododermatitis in 1 cat was connected to the treatment, however, remains uncertain. No cases of pododermatitis after doxycycline therapy have been published so far.

In this study, treatment of C. felis infections was significantly more effective when doxycycline was used. Whereas administration of pradofloxacin also effectively eliminated Mycoplasma and led to similar improvement of clinical signs and well-being, C. felis infection was not eliminated in 4 of 10 cats. Pradofloxacin, however, can be used as an alternative in URTD and conjunctivitis caused by C. felis or Mycoplasma in young cats and in cats with liver disease in which doxycycline should be avoided.

Footnotes

aWegener A, Laser H. Evaluation of the retinal drug safety profile of pradofloxacin with Optical Coherence Tomography. 1st International Veraflox® Symposium, Berlin, 2006, 28 (abstract)

bZrenner E, Messias A, Gekeler F. Pradofloxacin: an electroretinographic study on retinal toxicity in cats. 1st International Veraflox® Symposium, Berlin, 2006, 30–31 (abstract)

cDe Jong A, Bleckmann I. Comparative activity of pradofloxacin against clinical canine and feline strains of Germany. 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, 2003, 223 (abstract)

dSNAP FIV Antibody/FeLV Antigen Combo Test, IDEXX Laboratories, Woerrstadt, Germany

eVeraflox Oral Suspension 2.5%, Bayer HealthCare AG, Leverkusen, Germany

fVibravet, Pfizer, West Ryde, Australia

gPlain dry swab in peel pouches 155C, Copan Italia S.P.A., Brescia, Italy

hNucleospin 96 blood kit, Macherey-Nagel GmbH & Co KG, Dueren, Germany

iVortemp 56, Appleton Woods, Birmingham, UK

jInvitrogen, DH Breda, the Netherlands

k Qiagen, Crawley, UK

l Bio-Rad Laboratories Ltd, Hempstead, UK

m Biolase DNA polymerase with 10 × PCR buffer, Bioline Inc, Boston, MA

n SPSS 13.0.0., SPSS Inc, Chicago, IL

oBoothe DM. The accumulation of pradofloxacin in phagocytes. Abstract: 1st International Veraflox® Symposium, Berlin 2006; 18

Acknowledgments

This study was funded by Bayer HealthCare AG, Division Animal Health, Leverkusen, Germany. The authors thank Jennifer R. Hawley for performing the Mycoplasma PCR.

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