The 2 hemotropic Mycoplasma species (hemoplasmas) primarily associated with feline hemoplasmosis are Candidatus Mhm and Mhf.1–3 Clinical signs of infection vary by the organism and range from severe life-threatening hemolysis with Mhf to vague signs including lethargy and fever with Mhm.4 Dual infection with both organisms or other illness (eg, FeLV) usually exacerbates clinical signs of Mhm infection.5,6 The inability to culture these organisms in vitro complicates treatment and transmission studies. Although both hemoplasmas have been amplified from cat blood, fleas, and flea by-products by use of PCR assays, results do not definitively determine that the organisms are alive.7–10
In early experiments, hemotropic Mycoplasma spp were transferred between cats by administering uninfected cats infected blood via PO, IP, and IV injection.11 However, in that study, it was not known which of the hemotropic Mycoplasma spp infections were occurring. In more recent experiments that used genetically characterized isolates, IV inoculation of infected blood invariably resulted in infection of cats that previously had negative results of PCR assays.5,9,a Fighting behavior and arthropod vector-borne transmission have long been implicated as natural sources of transmission, although little experimental evidence exists to prove these theories.12,13
The DNA of both hemoplasmas has been amplified via PCR assay in Ctenocephalides felis collected from client-owned cats, potentially supporting the hypothesis that fleas are involved in the natural transmission of feline hemoplasmosis.10 In addition, C felis organisms that were allowed to feed on experimentally infected cats for 5 days yielded positive results of PCR assay; DNA of both hemoplasmas could be amplified from flea by-products (feces, eggs, and larvae).9 Although Mhm was not transmitted among cats by flea feeding, transfer of Mhf from an experimentally infected cat to a cat that previously had negative results for hemoplasma solely through hematophagous activity was detected.9 In those experiments, the fleas were contained in chambers; therefore, the cats were unable to ingest the fleas or flea by-products as they would while grooming during a natural flea infestation. Ingestion of C felis is known to be a mode of transmission for another infectious agent, Dipylidium caninum. Thus, we hypothesized that Mhf and Mhm are transmitted among cats by ingestion of infected C felis and their by-products. The purpose of the study reported here was to determine whether Mhf and Mhm can be transmitted by ingestion of Mycoplasma-infected C felis and their by-products.
Materials and Methods
Cats—Ten domestic shorthair cats (5 male and 5 female) from the ages of 1 to 3 years were used in this study. The cats were obtained from a commercial vendor, yielded negative results for FeLV antigen and FIV antibodies,b lacked physical examination abnormalities as determined by a licensed veterinarian, and were free of ticks and fleas. Of the 10 cats, 3 were chronic carriers of Mhf, 1 was a chronic carrier of Mhm, and 6 yielded negative results of a PCR assay for DNA of both hemoplasmas.7 The hemoplasma strains used in this study have been maintained in our laboratory by serial passage through multiple cats. The cats were provided with multiple litter boxes that were changed daily and fed a dry and canned commercial feline diet and fresh water ad libitum. The cats were housed together for the duration of the study and provided with environmental enrichments such as playthings and climbing structures. On completion of the study, all cats were adopted to private owners.
Fleas and flea chambers—The fleas used in these experiments were laboratory-reared, unfed adult fleas that were captured just after emergence from their pupae and raised completely in an indoor environment without exposure to animals.c The adult fleas used to produce the fleas used in that study had been fed only bovine blood, a species that neither feline hemoplasma is known to infect. Fleas were allowed to feed on subject cats by use of flea chambers and chamber attachment techniques.14 Fleas were placed into and then removed from the chambers in an area outside the study facility. After feeding, the fleas were removed from the chambers and sorted into viable males, viable females, nonviable males, nonviable females, and flea by-products (feces, eggs, and larvae). A random sample of fleas and flea by-products from each flea chamber was analyzed via PCR assay to confirm uptake of hemoplasma DNA. When fed to cats, the fleas were first transferred to a sterile test tube and immobilized by placement on ice for approximately 5 minutes. The immobilized fleas and flea by-products were then gently hand-mixed into 0.71 g of a commercially available human baby food that was onion powder–free to encourage ingestion by the cats.
Experimental design—The protocol used in this study was reviewed and approved by the Colorado State University Animal Care and Use Committee in accordance with federal regulations. Three sequential experiments were performed.
Experiment 1
Two flea chambers were loaded with 100 fleas of approximately equal male-to-female ratio and were attached to 2 of the 3 Mhf-infected carrier cats for 5 days. A random sample of fleas and flea by-products from each of the 2 flea chambers yielded positive results for Mhf DNA on day 5, confirming uptake of infected blood from the carrier cats. One cat with negative results for Mhf was then fed 39 viable female fleas, 54 viable male fleas, and 0.136 g of flea by-products, and another cat with negative results for Mhf was fed 37 viable female fleas, 54 viable male fleas, and 0.2 g of flea by-products. Blood samples were obtained for CBC and PCR assay from both of the cats on PI days 7, 14, 21, 28, 34, 51, and 65.
Experiment 2
Two months after completion of experiment 1, both cats fedMhf-infected fleas and flea by-products had repeatedly yielded negative results for DNA of both hemoplasmas; these 2 cats were considered to be uninfected and were used again in experiment 2. A flea chamber was loaded with 200 fleas of approximately equal male-to-female ratio and was attached to the cat with chronic Mhm infection for 5 days. A random sample of fleas and flea by-products from the flea chambers yielded positive results for Mhm DNA on day 5, confirming uptake of infected blood from the carrier cat. One previously uninfected cat was then fed 43 viable female fleas, 50 viable male fleas, and 0.274 g of flea by-products, and the other previously uninfected cat was fed 41 viable female fleas, 49 viable male fleas, and 0.354 g of flea by-products. Blood samples were obtained for a CBC and PCR assay from both cats on PI days 7, 14, 21, 28, 35, 42, 49, and 56.
Experiment 3
Two flea chambers were loaded with 200 fleas of approximately equal male-to-female ratio; 1 was attached to the third Mhf carrier cat, and 1 was attached to the same Mhm carrier cat used in experiment 2. After 3 days, the chambers were removed and a random sampling of the fleas and flea by-products from the flea chambers yielded positive results for Mhf or Mhm DNA, respectively, confirming uptake of infected blood from the carrier cats. The Mhf-infected fleas and flea by-products were split and fed to 2 previously uninfected cats; 1 cat was fed 44 viable female fleas, 51 viable male fleas, and 0.412 g of flea by-products, and the other cat was fed 44 viable female fleas, 50 viable male fleas, and 0.35 g of flea by-products. The Mhm-infected fleas and flea by-products were split and fed to 2 previously uninfected cats; 1 cat was fed 41 viable female fleas, 50 viable male fleas, and 0.397 g of flea by-products, and the other cat was fed 40 viable female fleas, 51 viable male fleas, and 0.293 g of flea by-products. Blood samples were obtained for a CBC and PCR assay from the previously uninfected cats that were fed Mhf-infected fleas and flea by-products on PI days 7, 14, 21, 28, 36, 43, and 50 and from uninfected cats that were fed Mhm-infected fleas and flea by-products on PI days 10, 17, 24, 35, 42, and 49.
Mycoplasma spp PCR assay—All cat blood and flea samples assessed in this study were assayed by use of the same PCR assay that amplifies both Mhf and Mhm.7 Blood in EDTA was digested for DNA extraction and assayed as described after a maximum of 4 days' storage at 4°C. Random, but not quantified, amounts of flea feces or flea feces and eggs or flea feces and larvae were assayed together. No attempt was made to remove residue of feces from the fleas, eggs, or larvae. These samples were individually placed in a 1.7-mL microcentrifuge tube and frozen at −70°C for 5 minutes. On removal from −70°C storage, the tube was placed on dry ice and the contents were thoroughly pulverized with a pestle, to which a genomic DNA isolation reagentd (30 μL/flea) was added. The resultant mixture was heated for 3 minutes at 95°C and centrifuged at 16,000 × g for 3 minutes to pellet the flea components. The supernatant was placed in a separate 1.7-mL microcentrifuge tube, to which 100% ethanol (15 μL/flea) was added, and the combination was vortexed briefly. The mixture was centrifuged at 20°C for 5 minutes at 16,000 × g. The supernatant was discarded, 150 μL of cold 70% ethanol was added to the DNA pellet, and the resultant mixture was centrifuged for 5 minutes at 16,000 × g. The supernatant was removed and discarded, and the resultant pellet was air-dried for 30 minutes. The DNA was suspended in 30 to 100 μL of water. Five microliters of the resultant mixture was assayed by use of the PCR assay.7 A negative control consisting of water and a positive control consisting of blood from known laboratory-maintained carriers of both Mycoplasma spp being studied were assayed with all experimental samples.
Results
For each of the experiments, the random sample of fleas or flea by-products obtained from the flea chambers after the fleas were allowed to feed on Mycoplasma-infected cats yielded positive results for either Mhf or Mhm DNA, respectively, which indicated that the fleas or flea by-products fed to previously uninfected cats contained hemoplasma DNA. However, DNA of Mhm or Mhf was not detected in any previously uninfected cat after being fed fleas or flea by-products. Additionally, neither anemia nor clinical findings consistent with hemoplasmosis were detected for any cat.
Discussion
Details of several important aspects of feline hemoplasmosis remain elusive despite a half century of clinical and experimental trials. One of the most obvious gaps in knowledge is the natural route of transmission of the organisms. Although hematophagous arthropods have long been incriminated as the primary suspects in the transmission of the disease, a paucity of data exists in the literature to strongly support this contention. Recently, C felis was able to transmit Mhf DNA from an infected cat to a naïve cat, although the efficiency of transmission by C felis in that study9 was poor. In addition, transmission of Mhm was not detected by use of similar methods. In those experiments, the cats were unable to ingest infected fleas during grooming, thereby eliminating a potentially large source of exposure to Mycoplasma organisms. Thus, in the study described here, we hypothesized that feline hemoplasmas could be transmitted among cats by ingestion of infected fleas or flea by-products. However, the study reported here failed to detect transmission of either Mhf or Mhm by ingestion of hemoplasma-infected C felis.
Results of this study suggest that ingestion of hemoplasma-infected C felis is not an important means of transmission for either of these organisms. Although we believe that the uninfected cats in these experiments were fed infected fleas on the basis of detection of hemoplasma DNA, it is truly unknown whether the positive PCR assay results represented viable organisms or fragmented, nonviable DNA. Thus, it is possible that C felis organisms mechanically uptake hemoplasma DNA while feeding, but they do not survive in the arthropod. This possibility will be difficult to assess further until feline hemoplasmas are successfully grown in vitro.
Other limitations of this study that might explain the findings include the differences between C felis field strains and those used here, differences between biological behavior of Mhf and Mhm field strains and those used here, feeding of suboptimal quantities of infective material, inappropriate handling of fleas and flea by-products, and use of inappropriate flea feeding times.
Each of these experiments was conducted with laboratory-maintained C felis and feline hemoplasmas, which may vary in biological behavior, compared with field strains. The hemoplasmas have been maintained via serial passage through experimental cats for several years, which may have altered the virulence or transmissibility of these strains. Similarly, the strain of C felis used in these experiments has been maintained in the laboratory by continuous inbreeding through hundreds of generations. These possibilities could be explored by performing similar experiments with fleas collected from cats with naturally occurring hemoplasmosis.
Each cat in these experiments was fed approximately 100 fleas and a varying quantity of flea by-products consisting of feces, larvae, and eggs. The number of fleas and weight of by-products fed were arbitrarily determined. We wanted the cats to rapidly ingest the entire amount in 1 feeding to ensure that the fleas were ingested while alive and approximate what occurs during grooming during natural flea infestation. Results of recent studies indicate that feral cats in flea-endemic areas in the summer months maintain a mean flea population of 13.6 fleas/cat.15 Therefore, ingestion of 100 fleas as performed in this experiment should represent an adequate exposure, especially compared with cats in areas of low flea prevalence, cats with suboptimal flea control, or cats that primarily live inside the home. However, it is plausible that strictly outdoor cats in flea endemic areas ingest vastly higher quantities of fleas during continuous and repeated natural exposure; therefore, it is possible that the negative results stemmed from ingestion of an inadequate number of infected fleas, flea by-products, or both.
To facilitate voluntary ingestion of the flea and flea by-products, these items were mixed into a food that the cats were not normally offered. Mixture of the fleas and flea by-products into human baby food resulted in almost complete voluntary ingestion by most of the cats. Care was taken to select baby food that did not contain onion powder because of concerns over inducing Heinz-body anemia. Potentially, mixture of the fleas and flea by-products into this substance may have reduced the viability of both the fleas and hemoplasmas and contributed to transmission failure. Subsequent experiments should avoid this form of contamination.
To facilitate mixing of the fleas and baby food, the fleas were first immobilized to avoid having fleas escape and cause a flea infestation within the study facility. Hypothermia by use of ice for several minutes was chosen, which adequately immobilized the fleas and allowed their mixture into the baby food and sub-sequent ingestion by the cats. This severe, albeit only brief, change in temperature for the organisms (both fleas and hemoplasmas) could have contributed to loss of viability of fleas and hemoplasmas, resulting in the lack of transmission seen in this experiment. However, in a previous study,a both hemoplasmas have been successfully transmitted by IV inoculation of blood stored for 1 hour at 4°C. Thus, it is unlikely that slowing the fleas by temporary immersion in the ice bath altered results of the study.
It is possible that Mycoplasma spp survive outside cats only in fleas for a short period and that successful transmission of these organisms is dependent on an infected flea being ingested by a naïve cat sooner than our study allowed. In this study, fleas were allowed to feed on Mycoplasma spp–infected cats for a period of 3 and 5 days and then these fleas were fed to naïve cats. It is known that C felis begin feeding within minutes of finding a host; therefore, it is feasible that uptake of hemoplasmas into the flea occurs in a short time. The period between initiation of C felis feeding and subsequent ingestion of C felis by naïve cats may have decreased the viability of any Mycoplasma organisms in the fleas. This hypothesis could be evaluated by feeding fleas to naïve cats more quickly after placing them on infected cats.
| Mhm | Mycoplasma haemominutum |
| Mhf | Mycoplasma haemofelis |
| PI | Postingestion |
Gary AT, Richmond HL, Hackett TB, et al. Survival of Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ in blood of cats used for transfusions (abstr). J Vet Intern Med 2005;19:435.
SNAP FIV Antibody/FeLV Antigen Combo Test, IDEXX Laboratories Inc, Westbrook, Me.
Fleas (Ctenocephalides felis) and flea chambers, Heska Corp, Fort Collins, Colo.
DNAzol, Molecular Research Center, Cincinnati, Ohio.
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