Use of pradofloxacin to treat experimentally induced Mycoplasma hemofelis infection in cats

Kristy L. Dowers Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80512

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Séverine Tasker School of Clinical Veterinary Science, University of Bristol, Bristol BS40 5DU, England

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Steven V. Radecki 150 N County Rd 3, Fort Collins, CO 80524

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Michael R. Lappin Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80512

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Abstract

Objective—To evaluate the efficacy of the fluoroquinolone pradofloxacin in the treatment of cats experimentally infected with Mycoplasma hemofelis.

Animals—23 young adult specific-pathogen–free cats.

Procedures—Cats were inoculated with M hemofelis from a chronically infected donor and assigned to 1 of 4 treatment groups: a doxycycline group, a low-dose–pradofloxacin group, a high-dose–pradofloxacin group, and an untreated control group. Treatment was initiated for 14 days when M hemofelis infection was detected via PCR assay and clinical signs of hemoplasmosis were present. Cats that had negative PCR assay results after treatment were administered a glucocorticoid and monitored via PCR assay for an additional 4 weeks.

Results—All cats yielded positive results for M hemofelis via conventional PCR and quantitative PCR assays and developed anemia. The low-dose–pradofloxacin group had significantly lower M hemofelis copy numbers than the doxycycline group. Six cats treated with pradofloxacin yielded negative results during treatment. Of those cats, 4 yielded negative conventional PCR assay results and all yielded negative quantitative PCR assay results for M hemofelis 1 month after administration of high-dose glucocorticoids.

Conclusions and Clinical Relevance—Pradofloxacin had anti–M hemofelis effects similar to those of doxycycline. In addition, pradofloxacin may be more effective at long-term M hemofelis organism clearance than doxycycline.

Abstract

Objective—To evaluate the efficacy of the fluoroquinolone pradofloxacin in the treatment of cats experimentally infected with Mycoplasma hemofelis.

Animals—23 young adult specific-pathogen–free cats.

Procedures—Cats were inoculated with M hemofelis from a chronically infected donor and assigned to 1 of 4 treatment groups: a doxycycline group, a low-dose–pradofloxacin group, a high-dose–pradofloxacin group, and an untreated control group. Treatment was initiated for 14 days when M hemofelis infection was detected via PCR assay and clinical signs of hemoplasmosis were present. Cats that had negative PCR assay results after treatment were administered a glucocorticoid and monitored via PCR assay for an additional 4 weeks.

Results—All cats yielded positive results for M hemofelis via conventional PCR and quantitative PCR assays and developed anemia. The low-dose–pradofloxacin group had significantly lower M hemofelis copy numbers than the doxycycline group. Six cats treated with pradofloxacin yielded negative results during treatment. Of those cats, 4 yielded negative conventional PCR assay results and all yielded negative quantitative PCR assay results for M hemofelis 1 month after administration of high-dose glucocorticoids.

Conclusions and Clinical Relevance—Pradofloxacin had anti–M hemofelis effects similar to those of doxycycline. In addition, pradofloxacin may be more effective at long-term M hemofelis organism clearance than doxycycline.

Mycoplasma hemofelis is one of the causative agents of feline infectious anemia, a hemolytic anemia of cats that can result in fever, lethargy, anorexia, splenomegaly, anemia, icterus, and death.1 Standard treatment of the disease has been tetracycline administration, specifically doxycycline. After recent reclassification within the Mycoplasmataceae family based on 16s rRNA sequencing,2, 3 several investigators have evaluated the efficacy of other anti-hemoplasma antimicrobials. Azithromycin was ineffective in 1 study,4 and use of imidocarb resulted in variable results in another.5 Fluoroquinolones, however, had promise in several studies6–8 of cats experimentally infected with M hemofelis. In one of those studies,6 cats treated with enrofloxacin at 5 mg/kg and 10 mg/kg daily improved clinically and hematologically, compared with nontreated cats. Two of the 8 cats in that study treated with enrofloxacin had negative results of PCR assay for M hemofelis for > 6 months despite administration of high doses of a glucocorticoid. That study established the fluoroquinolones as an additional class of drugs that may be useful against this disease. Subsequent studies with marbofloxacin revealed similar efficacy with regard to clinical and hematologic values. In 1 study 7 of cats experimentally infected with M hemofelis, cats treated with marbofloxacin had significant improvement of RBC variables, compared with untreated controls. In another experimental study,8 cats that were infected with M hemofelis and had a positive FIV status were treated with marbofloxacin and showed a significant decrease in M hemofelis DNA copy numbers, compared with untreated controls. These studies provide further evidence that the fluoroquinolones are useful in treating hemoplasmosis.

A separate issue is clearance of M hemofelis from affected cats. Earlier studies9, 10 revealed organism clearance via PCR assay during doxycycline administration at various dosages and durations of treatment, but M hemofelis DNA could be amplified from blood within days to weeks after treatment ended, suggesting that clearance had not occurred. The sensitivity of the PCR assays used in such studies is also a factor that could contribute to the temporary PCR assay negative status of the cats. Results of those studies and epidemiologic surveys documenting variable prevalence rates of M hemofelis in nonclinically affected cats11–14 suggested that most cats do not completely clear the organism, even with appropriate antimicrobial treatment. Clearance of experimentally induced M hemofelis infections for up to 6 months after infection and treatment with enrofloxacin was the first instance in which prolonged clearance, as evidenced by persistently negative PCR assay results, had been detected.6 Persistent PCR assay negative status also developed in 2 of 6 M hemofelis– infected cats treated with marbofloxacin for 28 days.7 However, in another study8 that used 28 days of marbofloxacin treatment, cats only intermittently yielded negative results of PCR assay following treatment, suggesting that clearance of infection was not consistently induced. Still, as newer fluoroquinolones with increased efficacy against Mycoplasma spp become available, it may be possible to not only treat the clinical disorder, but eradicate the organism from the blood.

The fluoroquinolone group is comprised of a number of chemical formulations. The fluoroquinolones approved for human use in the United States include ciprofloxacin, ofloxacin, norfloxacin, lomefloxacin, levofloxacin, enoxacin, and sparfloxacin. In veterinary medicine, the fluoroquinolones approved for use in cats are enrofloxacin, marbofloxacin, and orbifloxacin.15 As mentioned, enrofloxacin and marbofloxacin have anti-hemoplasma effects in experimentally inoculated cats.6–8 Although the other fluoroquinolones are assumed to have similar bacteriocidal activity because of their similar structure, results of a recent study16 suggest that there may be substantial differences in the in vitro activity of these individual drugs that may translate to differences in clinical efficacy. By use of quality control organisms as well as clinical isolates, MICs for ciprofloxacin were compared with the MICs for enrofloxacin, marbofloxacin, and orbifloxacin. The study revealed little correlation between ciprofloxacin MICs and the MICs for the other 3 veterinary fluoroquinolones. Likewise, a recent study17 revealed that pradofloxacin had lower MICs and higher in vitro activity against anaerobic bacterial isolates from cats and dogs than other veterinary fluoroquinolones, including enrofloxacin, marbofloxacin, difloxacin, and ibafloxacin. With pending introductions of other veterinary fluoroquinolones in the United States and Europe, the ciprofloxacin study emphasizes the importance of evaluating each new drug with regard to clinical efficacy against infections that have responded to older drugs in this class.

Pradofloxacin, a new fluoroquinolone under FDA review for veterinary use, is a novel 8-cyanofluoroquinolone that, compared with other fluoroquinolones, has increased in vitro efficacy against gram-negative and gram-positive organisms.18 Pradofloxacin has a great affinity for 2 targets within bacterial DNA, which may account for its decreased resistance profile.18,a Pharmacokinetic data for pradofloxacin have been evaluated in cats and dogs, and the drug has been used in trials in the European Union for clinical canine wound infections and canine pyoderma.b The purpose of the study reported here was to compare the clinical efficacy and organism clearance rates of pradofloxacin and doxycycline when administered to cats experimentally infected with M hemofelis.

Materials and Methods

Cats—The study protocol and animal treatment were reviewed and approved by the Colorado State University Animal Care and Use Committee in compliance with federal guidelines.

Twenty-six specific-pathogen–free cats (13 castrated males and 13 sexually intact females) between the ages of 1 and 2 years were used in the study. Two of the 26 cats, both males, served as donor cats for M hemofelis. The remaining 24 cats (11 males and 13 females) were randomly assigned to 1 of 4 treatment groups, for a total of 6 cats/group. Male cats were castrated prior to the start of the acclimation period. The cats, which were housed together for the duration of the study, were acclimated to group housing for 4 weeks prior to infection with M hemofelis. Food (dry feline maintenance diet) and fresh water were available ad libitum. One of the male cats developed a scrotal sac infection after castration and was removed from the study, resulting in 5 cats in the doxycycline group. A physical examination, CBC, serum biochemical analyses, FeLV antigen test,c FIV antibody test,d and cPCR assay for M hemofelis and Candidatus Mycoplasma hemominutum19 were performed to rule out concurrent disease and previous infection by a hemoplasma. In addition to conventional PCR assay, samples were frozen and later analyzed via quantitative real-time PCR assay. All surviving cats were adopted out to private homes at the end of the study.

Method of infection—Twenty-five days prior to the start of the study, the 2 donor cats were inoculated IV with 1 mL of heparinized blood from a cat that chronically carried M hemofelis. In addition, each donor was given methylprednisolone acetate (20 mg, IM) to maximize bacteremia. On PI day 24, results of the cPCR assay confirmed the presence of M hemofelis DNA in blood. The following day, 40 mL of blood was obtained in heparinized syringes from each cat and mixed aseptically. Each of the 23 study cats was inoculated IV within 15 to 30 minutes with 3 mL of the mixed donor blood.

Treatment groups—Each of the 4 treatment groups received one of the following treatment protocols: doxycyclinee (5 mg/kg, PO, q 12 h), low-dose pradofloxacinf (5 mg/kg, PO, q 24 h), high-dose pradofloxacin (10 mg/kg, PO, q 24 h), and no treatment (control group). All cats that received antimicrobials were treated for 14 consecutive days. Control group cats were to be treated with antimicrobials only if deemed necessary as a life-saving measure. Subcutaneous administration of fluids or blood transfusions was permitted for any of the cats as supportive care.

Infection and treatment phase—The duration of the infection and treatment phase of study was 6 weeks (42 days). Blood samples were collected from cats weekly for weeks 1 to 3 and twice weekly for weeks 4 to 6 on PI days 7, 13, 21, 25, 28, 32, 35, 39, and 42. At each sampling, 1.5 mL was placed in an EDTA tube and submitted to the clinical pathology laboratory for a CBC. An additional 1 mL of whole blood was placed in a separate EDTA tube for DNA extraction and M hemofelis PCR assays. Daily scoring, which included evaluation of rectal temperature (30.2° to 39.7°C: 1 point; 39.8° to 40.2°C: 2 points; > 40.3°C: 3 points), heart rate (> 200 beats/min: 1 point), respiration rate (> 42 breaths/min: 1 point), mucous membranes (pale or icteric: 1 point), attitude (lethargy or depression: 1 point), appetite (decreased: 1 point), need for SC fluids (3 points), and need for a whole blood transfusion (10 points), was recorded by 2 clinical scorers who were unaware of treatment groups. In addition, cats were assessed daily by the same veterinarian (KLD) to ensure that cats did not need immediate medical attention; however, because this veterinarian was not unaware of treatment groups, those assessments were not included in the daily score. In an attempt to equate treatment responses to characteristic clinical abnormalities, treatment was started in individual cats on the first day that its PCV was < 30% or that a fever > 39.2°C was detected. A centrifuged Hct tube was used to measure the PCV of any cat that had clinical signs other than fever on those days on which a CBC was not performed. If anemia or fever was not present in the treatment group cats by day 28 of the study, treatment was initiated to assess the effect of antimicrobial treatment on clearance of M hemofelis in cats that became infected but remained clinically normal. Cats were evaluated for 6 weeks after inoculation because results of previous studies9, 10 indicated that although administration of antimicrobials may result in clinical improvement and temporary clearance of the organism during treatment, cats again have positive PCR assay results within days to weeks of discontinuation of administration of the antimicrobials. Cats that had positive cPCR assay results on day 42 PI were eliminated from the study.

Glucocorticoid administration—Cats that had negative M hemofelis cPCR results by PI day 42 were administered methylprednisolone sodium acetate (20 mg/kg, IM) in an attempt to induce immunosuppression and were evaluated daily for an additional 28 days by use of the described clinical scoring system. Cats that developed clinical hemoplasmosis during this phase were removed from the study and re-treated with the antimicrobial used during their infection and treatment phase.

Clinical pathology—After primary infection, CBCs were performed weekly for weeks 1 to 3 and twice weekly for weeks 4 to 6. Presence of M hemofelis detected cytologically was noted, but quantitative analysis was not attempted because the samples were also to be assessed by use of PCR assays.19, 20 A CBC and cPCR assay were performed on blood collected weekly for 4 weeks from the cats that were M hemofelis PCR negative on day 42 PI and administered glucocorticoids.

cPCR assay—Blood samples that could not be analyzed within the first 6 hours of collection were stored at 4°C. By use of a reported protocol,19 the cPCR assay was performed in batches of 23 (1 sample from each cat) on DNA extracted from blood collected in EDTA either within the first 6 hours after collection or within 5 to 7 days of the sample date from blood samples frozen at 4°C. Measures to prevent crosscontamination of DNA were maintained, and appropriate positive and negative controls were included in each cPCR assay. The DNA extracts were stored at −20°C for future assays.

Real-time qPCR assay—To determine potential effect of antimicrobial treatment on hemoplasma DNA copy numbers in blood, DNA extracts from samples collected during the 6-week PI and treatment periods were shipped overnight on ice to the laboratory of one of the investigators (ST) at the University of Bristol for qPCR analysis by use of a published protocol.20 Because of funding issues, these samples could not be shipped and processed for 12 months after the study ended, but were stored at −20°C during the interim. For the qPCR assay, a standardized log copy number of M hemofelis per ML of blood was calculated.

Statistical analysis—Data were analyzed with standard statistical software.g Clinical assessment data were transformed into a numeric score. Clinical signs with continuous outcomes (temperature, heart rate, and respiration rate) were analyzed as described for CBC results. Values of P < 0.05 were considered significant.

For CBC results, only variables with > 90% of the results presented were included in the statistical analysis. These included neutrophils, lymphocytes, monocytes, eosinophils, PCV, hemoglobin concentration, RBC count, mean corpuscular volume, mean cell hemoglobin concentration, nucleated RBCs, platelets, plasma protein, and mean platelet volume. All values were log transformed prior to statistical analysis on the basis of results of the Shapiro-Wilk test of normality. Given the repeated measures over time, the mixed procedure of the software programg was used to evaluate the effects of treatment group, time, and the interaction between group and time. If the group by time interaction was significant (P < 0.05), group effects within time were evaluated. Comparisons between the control and the other 3 groups were tested by use of a least significant difference test.

For cPCR results, given the repeated measures over time and the binary outcome, the generalized linear mixed model procedure of a statistical software programg was first used to evaluate the fixed effects of treatment group (control, doxycycline, low-dose pradofloxacin, and high-dose pradofloxacin), time, and the interaction of time and group during the postinfection treatment phase of the study. Because the algorithm would not converge, likely because of the high number of positive results, the Fisher exact test was used to evaluate the percentage of positive cats on day 42, the only day on which there were a large number of negative cPCR assay results.

qPCR assay results—For qPCR assay results, the statistical analysis was conducted as described for the CBC results. The M hemofelis copy number data were log transformed prior to statistical analysis (natural log). Comparisons of the cPCR and qPCR assay results are presented descriptively because the DNA extracts were stored before being shipped for qPCR assay, which may have affected the results.

Results

Clinical findings—All 23 cats developed at least 1 clinical sign (fever, icterus, pale mucous membranes, lethargy, signs of depression, and inappetence) consistent with hemoplasmosis. Fever (> 39.2°C) was documented in 22 of the 23 cats during the postinfection phase. Rectal temperatures ranged from 36.7° to 40.5°C. Pale mucous membranes were detected in 21 cats. Icterus was reported in 5 cats, all of which were in the control group. No time by group interactions were significant. The effect of group (regardless of time) was significant for temperature, respiration rate, and total clinical score. Comparisons with the controls indicated that temperature was significantly lower in the doxycycline and low-dose pradofloxacin groups. Respiration rates were significantly lower in the doxycycline group, compared with the control group. Total clinical scores were significantly lower in the doxycycline and low-dose– and high-dose–pradofloxacin groups, compared with the controls. None of the cats required blood transfusions or supportive care (SC administration of fluids or force-feeding). None of the control groups required antimicrobial. Drug toxicosis was not detected clinically in any cat. None of the cats were euthanatized.

Clinicopathologic findings—Differences in group mean PCV results over time were determined (Figure 1). Anemia (PCV < 30%) was documented in 21 of 23 cats; 16 cats developed a PCV < 25%. Although there were no differences between groups prior to inoculation, the group by time interaction was significant for PCV and total protein on days 21, 25, 28, 32, 35, 39, and 42; on days 21, 28, 32, 35, 39, and 42 for hemoglobin concentration; on days 28, 32, 35, 39, and 42 for RBC; on days 35, 39, and 42 for mean corpuscular volume; and on days 21, 32, 35, and 42 for mean cell hemoglobin concentration. On those days with significant group effects, all 3 treatment groups were significantly different from the control group. In addition to these group effects within time, a main effect of group (regardless of time) was detected for eosinophils (P = 0.001). All 3 treatment groups had significantly increased eosinophil counts, compared with the control group; however, this effect was detected prior to infection or treatment and so was not considered clinically important.

Figure 1—
Figure 1—

Changes in mean PCV values at various times after experimental inoculation with Mycoplasma hemofelis in cats. Doxy = Doxycycline. Prad – Low = Low-dose pradofloxacin. Prad – High = High-dose pradofloxacin.

Citation: American Journal of Veterinary Research 70, 1; 10.2460/ajvr.70.1.105

When the mean number of days of anemia (PCV < 30%) was compared among treatment groups, the doxycycline group (0.8 days), low-dose–pradofloxacin treatment group (2 days), and high-dose–pradofloxacin treatment group (1.6 days) had significantly fewer days of anemia, compared with the control group (6.6 days). Significant differences among the doxycycline and pradofloxacin treatment groups were not detected.

All cats yielded positive results for M hemofelis via cPCR assay within 7 days of inoculation. Treatment had no significant effect on the prevalence of M hemofelis as detected via cPCR assay for the doxycycline and the high-dose–pradofloxacin treatment groups, although 2 of the 6 high-dose–pradofloxacin cats had negative cPCR results on day 42, compared with none of the 5 doxycycline cats. The low-dose pradofloxacin group had significantly (P = 0.03) fewer cats with M hemofelis than the control group on day 42 (4/6 cats had negative cPCR results).

Via qPCR assay, all cats had detectable copy numbers for M hemofelis within 7 days (Figure 2). The group by time interaction was significant (P <0.001), suggesting that copy numbers were not the same across all groups at all time points. Further evaluation of the group by time interaction effect revealed that copy numbers were significantly lower in all treatment groups from days 21 through 42, compared with the control group. Additionally, the lowdose–pradofloxacin group copy numbers were significantly lower than the doxycycline group on days 28, 32, 35, and 42. The copy numbers in the highdose–pradofloxacin group were lower than those in the doxycycline group on days 25 and 28.

Figure 2—
Figure 2—

Changes in mean M hemofelis (Mhf) copy numbers at various times in the same cats as in Figure 1.

Citation: American Journal of Veterinary Research 70, 1; 10.2460/ajvr.70.1.105

Of the 232 postinfection samples (including the 16 samples collected after methylprednisolone acetate administration), 8 samples (3.5%) yielded positive results for M hemofelis DNA by use of cPCR but negative results for M hemofelis DNA by use of qPCR. Of the 232 samples, 6 (2.6%) yielded positive results for M hemofelis DNA by use of qPCR but negative results for M hemofelis DNA by use of cPCR. Of these 6 samples, 5 contained fewer than 8.2 M hemofelis copy numbers/ML, and the other sample contained 27.32 M hemofelis copy numbers/μL.

Immunosuppressive phase—Seventeen cats persistently yielded positive results via cPCR assay by day 42 and were retired from the study. Six cats, 4 from the low-dose–pradofloxacin group and 2 from the high-dose–pradofloxacin group, yielded negative results via cPCR by the final day (PI day 42) of the study. Retrospectively, it was shown that cats from the high-dose–pradofloxacin group and 1 cat from the lowdose–pradofloxacin group had small M hemofelis DNA copy numbers (27.32, 8.19, and 4.13 M hemofelis copy numbers/ML, respectively) as estimated via qPCR assay. There were no reports of fever, signs of depression, or anorexia in any of these cats after administration of methylprednisolone acetate. On several occasions, pale mucous membranes were noted by the clinical scorers, but PCV on sample days did not decrease to < 30% in any cat. One of the 6 cats had persistently negative results via cPCR. The other 5 cats had transient positive results via cPCR on days 7 (n =1 cat), 14 (5), 21 (2), and 28 (2). On day 28 after glucocorticoid administration, 4 of the 6 cats had negative results for M hemofelis DNA in blood via cPCR assay. Three of these cats were in the low-dose–pradofloxacin group, and 1 was in the high-dose–pradofloxacin group, and all 4 cats were also revealed retrospectively to have negative results via qPCR assay on this sample date.

Discussion

In preclinical trials, pradofloxacin had lower MICs than most other veterinary fluoroquinolones when tested against Escherichia coli, Staphyloccocus aureus, Staphyloccocus intermedius, and select anaerobes in vitro.17, 18 In addition, pradofloxacin proved to be 2 to 3 times as active against Mycoplasma spp as were difloxacin, marbofloxacin, orbifloxacin, and enrofloxacin.h The greater efficacy is likely attributable to proprietary substitutions at key sites in the fluoroquinolone structure. Product information suggests that the novel structure provides additional killing mechanisms, compared with standard fluoroquinolones, by use of a dual-target strategy and thereby reduces the likelihood of the development of resistance. Pradofloxacin disrupts bacterial replication at 2 distinct sites. The first target is DNA gyrase, the bacterial topoisomerase II, and blocking this enzyme prevents DNA replication. The second target is topoisomerase IV, which is preferentially used for DNA replication as an additional bacterial strategy to resist antimicrobials.a Although older fluoroquinolones can disrupt both enzymes, the enzymes targeted depend on the bacterial species (gram positive vs gram negative), and usually, one enzyme is preferentially targeted over the other.21, 22 Pradofloxacin targets both enzymes and with increased affinity relative to earlier generations of fluoroquinolones.i This dual-target profile and the increased activity against other Mycoplasma spp made pradofloxacin an attractive alternative fluoroquinolone to consider for the treatment of hemoplasmosis.

All cats in the present study were successfully infected with M hemofelis as indicated by PCR assay results and cytologic results. Clinical findings were similar to those described following experimental inoculation with M hemofelis.6–10 Anemia was not detected in 2 cats; it is possible that anemia was present but missed because of the sampling schedule. Although those cats were administered the same volume of inoculum as the other cats, it is also possible the dose of viable organism varied among cats. However, under normal circumstances (using a pooled inoculum), we believe it was reasonable to assume that the organism was randomly distributed. The qPCR assay could have been used to attempt to determine whether the M hemofelis DNA load in each inoculum dose was the same, but this would not have proven that an equal infective dose was given to all cats because results of this assay do not prove the organism was alive. Host factors can influence whether an infectious agent results in manifestations of disease, and so the other major possibility, other than infective dose, was that those 2 cats were more resistant to development of anemia than others.

After M hemofelis infection, body temperatures were lower in the low-dose–pradofloxacin and doxycyclinetreated groups, compared with the untreated control group. In addition, all 3 antimicrobial-treated groups had lower total clinical scores, higher PCVs, quicker resolution of anemia, lower mean corpuscular volume, and lower copy numbers of M hemofelis, compared with the untreated control group. Lastly, after M hemofelis infection, the total protein concentrations were higher in the untreated control group, compared with the 3 treatment groups. Because the cats were not clinically dehydrated, the differences in total protein concentrations were most likely associated with greater production of antibodies or acute phase proteins in the untreated cats. All of these results indicated an antimicrobial effect for both doxycycline and pradofloxacin. However, differences among treatment groups were not detected via clinical and CBC findings, so none of the antimicrobial protocols could be considered superior to the others.

Use of the qPCR assay results provided an additional method for assessing efficacy of drug treatment of hemoplasmas.23 In the cats described here, detection of significantly lower M hemofelis copy numbers in the low-dose–pradofloxacin group, compared with the doxycycline group, for 4 of 5 consecutive weeks and detection of significantly lower M hemofelis copy numbers in the high dose-pradofloxacin group, compared with the doxycycline group for 2 consecutive weeks, suggested a greater effect. As with the previous fluoroquinolone studies, all groups treated with either doxycycline or a fluoroquinolone had more rapid resolution of anemia than the untreated control group. However, in the present study, none of the doxycycline-treated cats yielded negative results by use of cPCR or qPCR assays.

Although PCR assay results do not prove presence of viable organisms, it is generally believed that DNA of dead organisms will be cleared from the blood quickly. Thus, we believe that cats from which M hemofelis DNA can be amplified from blood are likely still infected. Prevalence studies from various regions of the world suggest that there is a subset of cats subclinically infected with 1 or more hemoplasmas.12,13,24 Carrier cats provide a reservoir of infection for other cats, and chronically infected cats can have a recrudescence of clinical disease in times of stress or immunosuppression.9 Other epidemiologic studies have detected a higher rate of infection with hemoplasmas when the FeLV or FIV status of the cat is also positive.12 Thus, optimal treatment of hemoplasmosis should include clearance of the organism from the body. In previous studies,6–8 administration of doxycycline, enrofloxacin, and marbofloxacin has all resulted in negative PCR assay results. In 1 study,6 all but one of the cats with negative results of M hemofelis PCR assay after doxycycline administration spontaneously yielded positive results for M hemofelis DNA in blood again, whereas administration of enrofloxacin at 10 mg/kg, PO, for 14 days resulted in persistently negative results in 2 cats. Two studies7, 8 have evaluated clearance of M hemofelis after administration of marbofloxacin; in 1 study,8 negative M hemofelis PCR assay results in cats assessed by use of qPCR for 87 days after inoculation were only transient. In the other study,7 2 of 6 M hemofelis–infected cats administered marbofloxacin and 1 untreated cat (of 5 controls) yielded persistently negative results of cPCR assay for 1 month (PI day 80) after administration of methylprednisolone acetate. The differences between these studies may be the results of the sensitivity of the 2 assays used and the duration of time the cats were monitored for M hemofelis DNA. In the study described here, 6 pradofloxacin-treated cats and none of the doxycycline-treated cats yielded negative results of M hemofelis cPCR assay. On day 28 after administration of methylprednisolone acetate, 4 of the cats had negative results by use of cPCR and qPCR assays. The outcome in all 3 studies suggests that clearance may be more likely achieved with the fluoroquinolones than with doxycycline. However, because different strains of M hemofelis and different PCR assays were used (conventional vs quantitative) and all cats were experimentally infected rather than naturally infected, the results of the studies cannot be compared directly. In addition, determination of organism clearance made on the basis of negative results of PCR assays on blood, regardless of technique, should not be considered completely reliable because the organism may be sequestered in the spleen, lung, or liver25 or the dose of glucocorticoid may not have been sufficient to induce bacteremia. For several samples, the results of cPCR and qPCR assays were discordant. However, because the samples were handled differently, the results should not be directly compared.

Each of the 6 pradofloxacin-treated cats had negative results for M hemofelis DNA in blood after completion of the treatment period. This contrasts with results of most studies6,7,9,10 of doxycycline, enrofloxacin, and marbofloxacin, in which negative PCR assay results were detected primarily during treatment. In a marbofloxacin study,8 negative qPCR results were first obtained toward the end of a 28-day course of marbofloxacin or after administration had ceased. Pradofloxacin is known to have a postantimicrobial effect, which may explain a potentially delayed response.i However, because this effect was only detected in vitro as a shortterm effect (ie, hours, rather than days or weeks), extrapolations from the laboratory to the clinical setting are difficult to make.

Pradofloxacin was tolerated by the cats described here with no evidence of hypersalivation, vomiting, diarrhea, or clinical evidence of visual impairment or blindness. Some fluoroquinolones have been associated with retinal degeneration in several species,26 including both enrofloxacin and orbifloxacin in cats.27, 28 The ocular safety margin of pradofloxacin in cats appears high. In 1 studyj that used optical coherence tomography for retinal evaluation of cats treated with pradofloxacin at 10 times the recommended dosage, no changes in retinal thickness were detected, compared with controls. In the same report, cats that received enrofloxacin at 6 times the recommended dosage had significant retinal thinning relative to the pradofloxacin-treated group. A recent experimental study of healthy cats administered pradofloxacin at 6 and 10 times the recommended dose (30 mg/kg/d and 50 mg/kg/d, respectively) revealed, via electroretinogram and histologic analyses, no toxic retinal effects on either rod or cone function.29 In the present study, the low-dose–pradofloxacin protocol was as effective as, or more effective than, the high-dose protocol. Thus, it is likely pradofloxacin could be safely used at the dosage of 5 mg/kg/d to treat cats naturally infected with M hemofelis.

Pradofloxacin is presently unavailable commercially in the United States or Europe, but is pending approval in several countries. Results of the present study indicated equivalent efficacy of pradofloxacin, compared with standard doxycycline treatment, in terms of clinical recovery, significantly decreased M hemofelis organisms for some cats treated with pradofloxacin, and possible elimination of infection in some cats; therefore, we believe that pradofloxacin should be considered an effective anti-hemoplasma drug. In addition, alternatives to doxycycline are also needed, given the concern regarding esophageal stricture associated with the use of doxycycline in cats.30, 31 More studies of spontaneous cases of M hemofelis infections are necessary to determine whether the same efficacy and clearance potential exists for naturally occurring hemoplasmosis.

ABBREVIATIONS

PI

Postinoculation

cPCR

Conventional PCR

qPCR

Quantitative PCR

a.

Heisig P. Bacterial resistance to antimicrobials—the exceptional case of the fluoroquinolones (abstr), in Proceedings. 1st Int Veraflox Symp 2006;10–11.

b.

Stephan B. Summary of clinical efficacy and palatability of Veraflox® (abstr), in Proceedings. 1st Int Veraflox Symp 2006;32–33.

c.

FeLV antigen test, Snap Combo, IDEXX Laboratories, Portland, Me.

d.

FIV antibody test, Snap Combo, IDEXX Laboratories, Portland, Me.

e.

Doxycycline hyclate powder, PCCA, Houston, Tex.

f.

Veraflox, Bayer Animal Health, Merriam, Kan.

g.

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References

  • 1.

    VanSteenhouse JL, Taboada J, Millard JR. Feline hemobartonellosis. Compend Contin Educ Pract Vet 1993;15:535545.

  • 2.

    Neimark H, Johansson KE, Rikihisa Y, et al. Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of ‘Candidatus Mycoplasma haemofelis’, ‘Candidatus Mycoplasma haemomuris’, ‘Candidatus Mycoplasma haemosuis’ and ‘Candidatus Mycoplasma wenyoni.’ Int J Syst Evol Microbiol 2001;51:891899.

    • Search Google Scholar
    • Export Citation
  • 3.

    Neimark H, Kocan KM. The cell wall-less rickettsia Eperythrozoon wenyonii is a Mycoplasma. FEMS Microbiol Lett 1997;156:287291.

  • 4.

    Westfall DS, Jensen WA, Reagan WJ, et al. Inoculation of two genotypes of Hemobartonella felis (California and Ohio variants) to induce infection in cats and the response to treatment with azithromycin. Am J Vet Res 2001;62:687691.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Lappin MR, Radecki S. Effects of imidocarb diproprionate in cats with chronic haemobartonellosis. Vet Ther 2002;3:144149.

  • 6.

    Dowers KL, Olver C, Radecki SV, et al. Use of enrofloxacin for treatment of large-form Haemobartonella felis in experimentally infected cats. J Am Vet Med Assoc 2002;221:250253.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Ishak AM, Dowers KL, Cavanaugh MT, et al. Marbofloxacin for the treatment of experimentally-induced Mycoplasma haemofelis infection in cats. J Vet Intern Med 2008;22:288292.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Tasker S, Carey SMA, Day MJ, et al. Effect of chronic FIV infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection. Vet Microbiol 2006;117:169170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Foley JE, Harrus S, Poland A, et al. Molecular, clinical, and pathologic comparison of two distinct strains of Haemobartonella felis in domestic cats. Am J Vet Res 1998;59:15811588.

    • Search Google Scholar
    • Export Citation
  • 10.

    Berent LM, Messick JB, Cooper SK. Detection of Haemobartonella felis in cats with experimentally induced acute and chronic infections, using a polymerase chain reaction assay. Am J Vet Res 1998;59:12151220.

    • Search Google Scholar
    • Export Citation
  • 11.

    Yamaguchi N, Macdonald DW, Passanisi WC, et al. Parasite prevalence in free-ranging farm cats, Felis silvestris catus. Epidemiol Infect 1996;116:217223.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Luria BJ, Levy JK, Lappin MR, et al. Prevalence of infectious diseases in feral cats in Northern Florida. J Feline Med Surg 2004;6:287296.

  • 13.

    Ishak AM, Radecki S, Lappin MR. Prevalence of Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, Bartonella species, Ehrlichia species, and Anaplasma phagocytophilum DNA in the blood of cats with anemia. J Feline Med Surg 2007;9:17.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Criado-Fornelio A, Martinez-Marcos A, Buling-Saraña A, et al. Presence of Mycoplasma haemofelis, Mycoplasma haemominutum and piroplasmids in cats from southern Europe: a molecular study. Vet Microbiol 2003;93:307317.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Bahri LE, Blouin A. Fluoroquinolones: a new family of antimicrobials. Compend Contin Educ Pract Vet 1991;13:14291433.

  • 16.

    Riddle C, Lemons CL, Papich MG, et al. Evaluation of ciprofloxacin as a representative of veterinary fluoroquinolones in susceptibility testing. J Clin Microbiol 2000;38:16361637.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Silley P, Stephan B, Greife HA, et al. Comparative activity or pradofloxacin against anaerobic bacteria isolated from dogs and cats. J Antimicrob Chemother 2007;60:9991003.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Wetzstein H-G. Comparative mutant prevention concentrations of pradofloxacin and other veterinary fluoroquinolones indicate differing potentials in preventing selection of resistance. Antimicrob Agents Chemother 2005;49:41664173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Jensen WA, Lappin MR, Kamkar S, et al. Use of a polymerase chain reaction assay to detect and differentiate two strains of Haemobartonella felis in naturally infected cats. Am J Vet Res 2001;62:604608.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Tasker S, Helps CR, Day MJ, et al. Use of real-time PCR to detect and quantify Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ DNA. J Clin Microbiol 2003;41:439441.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Peterson LR. Quinolone molecular structure-activity relationships: what we have learned about improving antimicrobial activity. Clin Infect Dis 2001;33(suppl 3):S180S186.

    • Search Google Scholar
    • Export Citation
  • 22.

    Drlica K, Malik M. Fluoroquinolones: action and resistance. Curr Top Med Chem 2003;3:249282.

  • 23.

    Tasker S, Helps CR, Day MJ, et al. Use of a Taqman PCR to determine the response of Mycoplasma haemofelis to antimicrobial treatment. J Microbiol Methods 2004;56:6371.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Lappin MR, Griffin B, Brunt J, et al. Prevalence of Bartonella species, haemoplasma species, Ehrlichia species, Anaplasma phagocytophilum, and Neorickettsia risticii DNA in the blood of cats and their fleas in the United States. J Feline Med Surg 2006;8:8590.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Maede Y. Sequestration and phagocytosis of Haemobartonella felis in the spleen. Am J Vet Res 1979;40:691695.

  • 26.

    Dietz BL. The fluoroquinolones. Clin Toxicol Rev [serial online]. 1997;20:12. Available at: www.maripoisoncenter.com/ctr/9712Fluoroquinolones.html. Jul 13, 2006.

    • Search Google Scholar
    • Export Citation
  • 27.

    Gelatt KN, van der Woerdt A, Ketring KL, et al. Enrofloxacin-associated retinal degeneration in cats. Vet Ophthalmol 2001;4:99106.

  • 28.

    Wiebe V, Hamilton P. Fluoroquinolone-induced retinal degeneration in cats. J Am Vet Med Assoc 2002;221:15681571.

  • 29.

    Messias A, Gekeler F, Wegener A, et al. Retinal safety of a new fluoroquinolone, pradofloxacin, in cats: assessment with electroretinography. Doc Ophthalmol 2008;116:177191.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Melendez LD, Twedt DC, Wright M. Suspected doxycycline induced esophagitis with esophageal stricture formation in three cats. Feline Pract 2000;28(2):1012.

    • Search Google Scholar
    • Export Citation
  • 31.

    German AJ, Cannon MJ, Dye C, et al. Oesophageal strictures in cats associated with doxycycline therapy. J Fel Med Surg 2000;7:3341.

  • Figure 1—

    Changes in mean PCV values at various times after experimental inoculation with Mycoplasma hemofelis in cats. Doxy = Doxycycline. Prad – Low = Low-dose pradofloxacin. Prad – High = High-dose pradofloxacin.

  • Figure 2—

    Changes in mean M hemofelis (Mhf) copy numbers at various times in the same cats as in Figure 1.

  • 1.

    VanSteenhouse JL, Taboada J, Millard JR. Feline hemobartonellosis. Compend Contin Educ Pract Vet 1993;15:535545.

  • 2.

    Neimark H, Johansson KE, Rikihisa Y, et al. Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of ‘Candidatus Mycoplasma haemofelis’, ‘Candidatus Mycoplasma haemomuris’, ‘Candidatus Mycoplasma haemosuis’ and ‘Candidatus Mycoplasma wenyoni.’ Int J Syst Evol Microbiol 2001;51:891899.

    • Search Google Scholar
    • Export Citation
  • 3.

    Neimark H, Kocan KM. The cell wall-less rickettsia Eperythrozoon wenyonii is a Mycoplasma. FEMS Microbiol Lett 1997;156:287291.

  • 4.

    Westfall DS, Jensen WA, Reagan WJ, et al. Inoculation of two genotypes of Hemobartonella felis (California and Ohio variants) to induce infection in cats and the response to treatment with azithromycin. Am J Vet Res 2001;62:687691.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Lappin MR, Radecki S. Effects of imidocarb diproprionate in cats with chronic haemobartonellosis. Vet Ther 2002;3:144149.

  • 6.

    Dowers KL, Olver C, Radecki SV, et al. Use of enrofloxacin for treatment of large-form Haemobartonella felis in experimentally infected cats. J Am Vet Med Assoc 2002;221:250253.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Ishak AM, Dowers KL, Cavanaugh MT, et al. Marbofloxacin for the treatment of experimentally-induced Mycoplasma haemofelis infection in cats. J Vet Intern Med 2008;22:288292.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Tasker S, Carey SMA, Day MJ, et al. Effect of chronic FIV infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection. Vet Microbiol 2006;117:169170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Foley JE, Harrus S, Poland A, et al. Molecular, clinical, and pathologic comparison of two distinct strains of Haemobartonella felis in domestic cats. Am J Vet Res 1998;59:15811588.

    • Search Google Scholar
    • Export Citation
  • 10.

    Berent LM, Messick JB, Cooper SK. Detection of Haemobartonella felis in cats with experimentally induced acute and chronic infections, using a polymerase chain reaction assay. Am J Vet Res 1998;59:12151220.

    • Search Google Scholar
    • Export Citation
  • 11.

    Yamaguchi N, Macdonald DW, Passanisi WC, et al. Parasite prevalence in free-ranging farm cats, Felis silvestris catus. Epidemiol Infect 1996;116:217223.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Luria BJ, Levy JK, Lappin MR, et al. Prevalence of infectious diseases in feral cats in Northern Florida. J Feline Med Surg 2004;6:287296.

  • 13.

    Ishak AM, Radecki S, Lappin MR. Prevalence of Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, Bartonella species, Ehrlichia species, and Anaplasma phagocytophilum DNA in the blood of cats with anemia. J Feline Med Surg 2007;9:17.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Criado-Fornelio A, Martinez-Marcos A, Buling-Saraña A, et al. Presence of Mycoplasma haemofelis, Mycoplasma haemominutum and piroplasmids in cats from southern Europe: a molecular study. Vet Microbiol 2003;93:307317.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Bahri LE, Blouin A. Fluoroquinolones: a new family of antimicrobials. Compend Contin Educ Pract Vet 1991;13:14291433.

  • 16.

    Riddle C, Lemons CL, Papich MG, et al. Evaluation of ciprofloxacin as a representative of veterinary fluoroquinolones in susceptibility testing. J Clin Microbiol 2000;38:16361637.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Silley P, Stephan B, Greife HA, et al. Comparative activity or pradofloxacin against anaerobic bacteria isolated from dogs and cats. J Antimicrob Chemother 2007;60:9991003.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Wetzstein H-G. Comparative mutant prevention concentrations of pradofloxacin and other veterinary fluoroquinolones indicate differing potentials in preventing selection of resistance. Antimicrob Agents Chemother 2005;49:41664173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Jensen WA, Lappin MR, Kamkar S, et al. Use of a polymerase chain reaction assay to detect and differentiate two strains of Haemobartonella felis in naturally infected cats. Am J Vet Res 2001;62:604608.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Tasker S, Helps CR, Day MJ, et al. Use of real-time PCR to detect and quantify Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ DNA. J Clin Microbiol 2003;41:439441.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Peterson LR. Quinolone molecular structure-activity relationships: what we have learned about improving antimicrobial activity. Clin Infect Dis 2001;33(suppl 3):S180S186.

    • Search Google Scholar
    • Export Citation
  • 22.

    Drlica K, Malik M. Fluoroquinolones: action and resistance. Curr Top Med Chem 2003;3:249282.

  • 23.

    Tasker S, Helps CR, Day MJ, et al. Use of a Taqman PCR to determine the response of Mycoplasma haemofelis to antimicrobial treatment. J Microbiol Methods 2004;56:6371.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Lappin MR, Griffin B, Brunt J, et al. Prevalence of Bartonella species, haemoplasma species, Ehrlichia species, Anaplasma phagocytophilum, and Neorickettsia risticii DNA in the blood of cats and their fleas in the United States. J Feline Med Surg 2006;8:8590.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Maede Y. Sequestration and phagocytosis of Haemobartonella felis in the spleen. Am J Vet Res 1979;40:691695.

  • 26.

    Dietz BL. The fluoroquinolones. Clin Toxicol Rev [serial online]. 1997;20:12. Available at: www.maripoisoncenter.com/ctr/9712Fluoroquinolones.html. Jul 13, 2006.

    • Search Google Scholar
    • Export Citation
  • 27.

    Gelatt KN, van der Woerdt A, Ketring KL, et al. Enrofloxacin-associated retinal degeneration in cats. Vet Ophthalmol 2001;4:99106.

  • 28.

    Wiebe V, Hamilton P. Fluoroquinolone-induced retinal degeneration in cats. J Am Vet Med Assoc 2002;221:15681571.

  • 29.

    Messias A, Gekeler F, Wegener A, et al. Retinal safety of a new fluoroquinolone, pradofloxacin, in cats: assessment with electroretinography. Doc Ophthalmol 2008;116:177191.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Melendez LD, Twedt DC, Wright M. Suspected doxycycline induced esophagitis with esophageal stricture formation in three cats. Feline Pract 2000;28(2):1012.

    • Search Google Scholar
    • Export Citation
  • 31.

    German AJ, Cannon MJ, Dye C, et al. Oesophageal strictures in cats associated with doxycycline therapy. J Fel Med Surg 2000;7:3341.

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