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Abstract

Blood transfusions are commonly administered to cats; associated risks include the transmission of various infectious diseases including Mycoplasma haemofelis (Mhf) and ‘Candidatus Mycoplasma haemominutum’ (Mhm). Blood transfusions in citrate-phosphate-dextrose-adenine (CPDA-1) solution are commonly administered immediately or stored for up to 1 month prior to administration. It is unknown whether Mhf or Mhm survive in this solution or temperature. The purpose of this study was to determine if Mhf or Mhm remain viable after storage in CPDA-1 for varying periods of time. The results provide evidence that transmission of hemoplasmas to naïve cats occurs after administration of infected feline blood that has been stored in CPDA-1 solution for 1 h (Mhf) and 1 week (Mhm). These findings support the recommendation that cats used as blood donors be screened for Mhf and Mhm infections by polymerase chain reaction (PCR) assay prior to use.

Whole blood transfusions are commonly administered to feline patients for treatment of anemia secondary to blood loss, hemolysis, or renal disease (Castellanos et al 2004, Weingart et al 2004). Important risks associated with transfusion of blood products include the transmission of various infectious diseases. Current recommendations to limit the risk of iatrogenic infection include screening of all blood donors for feline leukemia virus, feline immunodeficiency virus, Mycoplasma haemofelis (Mhf, formerly Haemobartonella felis Ohio strain (large form)), and ‘Candidatus Mycoplasma haemominutum’ (Mhm, formerly Haemobartonella felis California strain (small form)) (Haldane et al 2004, Lucas et al 2004, Reine 2004, Wardrop et al 2005).

Mhf and Mhm are two hemotropic bacteria that can result in feline infectious anemia (Berent et al 2000, Westfall et al 2001). Previously, the organisms were thought to be two forms of one species, Haemobartonella felis; however, recent publications have reclassified them as two distinct species that belong to the genus Mycoplasma (Foley and Pedersen 2001, Neimark et al 2001, 2002). Another genetically distinct hemoplasma infecting cats in Europe has also recently been described (Willi et al 2005). These hemotropic mycoplasmal species are collectively referred to as the hemoplasmas. The appearance of organisms on the surface of red blood cells during cytological examination of blood smears was once the only means of diagnosis. Currently, detection of hemoplasma DNA in blood by polymerase chain reaction (PCR) assay is the most sensitive diagnostic method. Although molecular methods are available to document infection, the inability to culture organisms has limited the ability to further investigate various aspects of the disease (Tasker and Lappin 2002).

The prevalence of hemoplasma infection in feline blood donors from several veterinary hospitals was evaluated in a recent study (Hackett et al in press). Of 118 active donors from the community, 15 (12.7%) were PCR positive for one or both hemoplasma species; nine (7.6%) donors were positive for Mhm alone, four (3.4%) were positive for Mhf alone, one (0.8%) was positive for Mhm and Mhf, and one (0.8%) was positive for Mhm and Bartonella henselae. The prevalence rates of hemoplasma infection in donors allowed outside or exposed to fleas were 19.7% and 22.7%, respectively, values comparable to recent PCR studies of feline populations in the US and Europe (Jensen et al 2001, Messick 2003, Tasker et al 2003a). Based on current evidence, it is possible that the feline blood supply in the United States is widely contaminated with hemoplasmas.

Feline whole blood transfusions in citrate-phosphate-dextrose-adenine (CPDA-1) solution are frequently given immediately or stored for up to 1 month prior to administration; however, the effect of storage and CPDA-1 on hemoplasmas is currently unknown. In experimental studies, infection with feline hemoplasmas is reliably transmitted by intravenous inoculation of as little as 1–2 ml of fresh EDTA-anticoagulated blood (Foley et al 1998, Westfall et al 2001). If hemoplasmas remain viable in CPDA-1 at 4°C, the widespread prevalence of the organisms in feline blood may place the recipient population at risk for infection. Because hemoplasmas are directly associated with feline red blood cells and cannot currently be cultured, organism viability studies must involve inoculation of cats. The purpose of the current study is to determine if Mhf and Mhm remain viable after storage in CPDA-1 for varying periods of time by inoculation of infected whole blood to hemoplasma-negative cats.

Materials and Methods

Cats

Young adult (4 years old, n=4; 1 year old, n=2), mixed sex (female spayed, n=3; male castrated, n=1; intact male, n=2) laboratory reared cats were purchased from a commercial breeder (Liberty Research Inc, Waverly, NY, USA) and shipped to Colorado State University. A chronic carrier of Mhf and a chronic carrier of Mhm were used as organism donors. The blood types of the donors were type A. Because transfusion reactions resulting from incompatible blood types are acute and severe, the lack of a transfusion reaction suggests appropriate blood type compatibility. Thus, recipient cats were assumed to be type A although they were not tested. The cats were observed daily throughout the study including monitoring of temperature, respiratory rate, and heart rate. Food and water was available ad libitum. The protocol described was approved by a campus wide animal care and use committee.

PCR Assays

After collection, the samples were stored at −20°C prior to analysis by conventional PCR at Colorado State University (Jensen et al 2001). For the real-time PCR, the frozen samples were packaged on ice and shipped to the UK where they were stored at −20°C until assayed (Tasker et al 2003b). Each of the PCR assays amplifies the DNA of both hemoplasma species. Positive and negative controls were included on all PCR assays performed during the study. For the real-time PCR assay results, standardized log copy number/μl blood was calculated for each sample as previously described (Tasker et al 2003b). Percentage concordance between the results from all samples assayed by conventional (Conv) and real-time (Real) PCR assays was calculated by use of the following formula: [(Conv+, Real+)+(Conv−, Real−)]/[(Conv+, Real+)+(Conv−, Real−)+(Conv+, Real−)+(Conv−, Real+)]×100.

Sample Collection and Storage

The two hemoplasma carrier cats were sedated by administration of 20 mg of ketamine and 1 mg of diazepam IV. A sterile, closed-collection system (Teruflex; Terumo Co, Tokyo, Japan) with a total of three connected reservoirs was used; the primary collection bag contained 63 ml of CPDA-1. Prior to use, CPDA-1 was emptied from the primary collection reservoir into the first satellite bag (bag A). At the time of collection, the collection reservoir was free of anticoagulant while the blood collection line contained approximately 8 ml of CPDA-1. Fifty-two milliliters of blood were collected from each cat into the primary collection reservoir via jugular venepuncture and were immediately refrigerated in a temperature-monitored refrigerator at 4°C. Stored blood was mixed daily. At 1 h, one-third of the blood from the main unit was transferred to the second satellite bag (bag B) after thorough mixing of the blood; the sample for inoculation and PCR assay was collected from this reservoir. Subsequently, the excess CPDA-1 in bag A was transferred to bag B which was then detached and discarded. The blood transfer process was repeated at 1 week with the remaining reservoir, while the 1 month sample was obtained from the primary collection bag. Samples for inoculation were immediately administered without additional storage.

Experimental Design

Blood (1.5 ml) was collected by jugular venepuncture from the six study cats and the two hemoplasma carrier cats and placed in EDTA tubes for a complete blood cell count and hemoplasma PCR assay, twice prior to beginning of the study to confirm that the two carriers remained infected and the study cats remained negative. Whole blood was then collected from the two carrier cats for storage as previously described. At 1 h, 1 week, and 1 month after blood collection and storage, 2.2 ml of blood in CPDA-1 solution were aseptically collected from each of the two samples. Real-time and conventional hemoplasma PCR assays were performed on DNA extracted from 200 μl of blood, and 2 ml were inoculated IV into a hemoplasma-negative cat. After inoculation, 2 ml of blood were collected from each cat for complete blood count and PCR assay (performed following DNA extraction from 200 μl of blood) weekly for 4 weeks.

Data Analysis

The results of this study are reported descriptively. In previous studies (Foley et al 1998, Westfall et al 2001), inoculation of 2 ml of blood from cats carrying either hemoplasma into PCR assay negative cats has always resulted in development of persistently positive PCR assay results, a finding we believe documents infection. Thus, we believe that if hemoplasma DNA is detected persistently in the cats inoculated herein it will prove that the organism survived in CPDA-1 solution for the time period tested.

Results

Both chronic carrier cats were positive for Mhf or Mhm DNA throughout the study by both real-time and conventional PCRs. All six SPF cats were negative for Mhf and Mhm DNA before inoculation. DNA of Mhf or Mhm, respectively, was amplified from the CPDA-1 bags after 1 h, 1 week, and 1 month of storage with real-time and conventional PCRs. The standardized log copy number/μl blood results for Mhf in stored blood collected after 1 h, 1 week, and 1 month were 5.55, 5.61, and 5.40, respectively. The standardized log copy number/μl blood results for Mhm in stored blood collected after 1 h, 1 week, and 1 month were 4.20, 4.45, and 3.95, respectively.

Conventional and real-time PCR results of SPF cats inoculated with stored blood are shown in Table 1. The percentage concordance between the real-time PCR assay results and the conventional PCR assay results were 100% and 88.9% for Mhf and Mhm, respectively. In both samples from Mhm inoculated cats with discordant results, the conventional PCR assay result was positive and the real-time PCR assay result was negative. For the samples that were positive in the real-time PCR assay, the Mhm and Mhf standardized copy numbers/μl of blood were maximal on week 1 and then decreased numerically over time (Table 1).

Table 1

PCR assay results from cats inoculated with Mhf or Mhm stored in CPDA-1 solution for varying time periods

	Storage time in CPDA-1 solution
	1 h	1 week	1 month
Mhf results*
Week 0†	neg/neg	neg/neg	neg/neg
Week 1	pos/pos (7.97)	neg/neg	neg/neg
Week 2	pos/pos (4.86)	neg/neg	neg/neg
Week 3	pos/pos (4.38)	neg/neg	neg/neg
Week 4	pos/pos (4.41)	neg/neg	neg/neg
Mhm results*
Week 0†	neg/neg	neg/neg	neg/neg
Week 1	pos/pos (2.76)	pos/neg	neg/neg
Week 2	pos/pos (2.49)	neg/neg	neg/neg
Week 3	pos/neg	neg/neg	neg/neg
Week 4	neg/neg	neg/neg	neg/neg

Conventional PCR result/real-time PCR result (standardized log copy number/μl blood if real-time PCR positive); neg=negative; pos=positive.

†

Week 0 performed twice with identical results.

Throughout the study, few clinical signs were noted as a result of hemoplasma infection. Thirteen days after inoculation, the Mhf-infected cat was lethargic with a fever of 102.8°F for 1 day; clinical signs rapidly resolved. Neither anemia nor a decrease in hematocrit was noted at any time throughout the study. Lymphocytosis was occasionally noted on complete blood counts, but the results were otherwise unremarkable. Acute onset of conjunctivitis was noted in one cat; clinical signs rapidly resolved during the topical administration of a generic triple antibiotic ointment for ophthalmic use.

Discussion

DNA of Mhf and Mhm was amplified from the blood stored in CPDA-1 solution throughout the study and the standardized log copy number/μl blood of the samples was similar throughout the study. These results document the presence of hemoplasma DNA in the CPDA-1 solution, but do not prove the organisms were alive. However, because standardized log copy number/μl blood did not increase over time, it is unlikely the hemoplasmas replicated in the CPDA-1 solution.

Amplification of hemoplasma DNA in samples directly from infected cats also does not directly prove the organisms were alive. However, we believe it is likely that DNA of organisms that have died is cleared from the blood quickly. This hypothesis is supported by some of the results of this study. While DNA of both hemoplasmas was amplified from the stored blood throughout the study, none of the cats inoculated with Mhf-infected blood stored for 1 week or blood infected with Mhf or Mhm stored for 1 month became PCR positive. These results suggest that under those conditions, neither Mhf nor Mhm survived and that the previously negative cats rapidly cleared the DNA of the dead organisms given by IV inoculation. However, it is also possible that living organisms were present, but the healthy recipient cats were capable of eliminating the infections or minimizing the infections to levels below the sensitivity limits of the PCR assays.

In contrast, we believe the amplification of Mhf or Mhm DNA from blood of previously negative recipient cats for weeks after inoculation of blood stored for 1 h is most likely evidence of transfer of live organisms. Additionally, replication of Mhf in vivo is supported by the finding that more DNA copies of Mhf were present in the blood of the recipient cat given Mhf after 1 h of storage (7.97 standardized log copy number/μl blood) than in the stored blood (5.55 standardized log copy number/μl blood). Our results also suggest that Mhm remains viable for at least 1 week under the same conditions.

Previously negative cats inoculated with Mhm containing blood stored in CPDA-1 for 1 h and 1 week were only PCR positive for 3 weeks and 1 week after inoculation, respectively. In contrast, cats experimentally infected with Mhm immediately after collection generally remained PCR positive for months after inoculation (Foley et al 1998, Westfall et al 2001). In the majority of cats given Mhf immediately after collection, severe clinicopathologic evidence of infection (anemia and fever) occurs within 3 weeks of inoculation (Harvey and Gaskin 1977, Berent et al 1998, Foley et al 1998, Westfall et al 2001). In this study, the previously negative recipient cat administered Mhf containing blood stored in CPDA-1 for 1 h only developed transient fever. These results suggest that the viability or virulence of Mhf and Mhm was decreasing over time allowing the recipient cats to eliminate the infections or minimize the infections to levels below the sensitivity limits of the PCR assays.

Attenuation of Mhm has been previously noted after passage through multiple cats (George et al 2002). The transmission of Mhm but not Mhf after 1 week of storage in CPDA-1 may relate to differences between the organisms. Genetically, the two species are distinct with only 83% homology between 16S rRNA sequences (Sykes 2003). In other studies, we showed Mhf but not Mhm to be transmitted by flea feeding under one set of experimental conditions (Woods et al 2005). Further research is needed to further elucidate other differences between the two species.

Possible reasons for the decreased viability of hemoplasmas after storage include effects of temperature, effects of CPDA-1, and alterations in RBC function. Because red blood cells and hemoplasmas are closely associated, it is possible that organism viability is affected by changes in feline red blood cells that occur with storage (Schneider 2000). While there are no studies evaluating the shelf-life of feline red blood cells in CPDA-1, packed red blood cells in dogs retain 75% viability for up to 20 days in solution (Price et al 1988). Thus, while the mechanisms of host–parasite interaction are not entirely known, it may be reasonable to assume that changes in red blood cell function and viability may adversely affect hemoplasmas.

The percent concordance between conventional and real-time PCR assays was 100% for Mhf. For Mhm, two of 15 samples were discordant, with the conventional PCR assay result being positive and the real-time PCR assay result being negative. For these samples in the conventional PCR assay, the negative controls performed concurrently were negative. Thus, it seems unlikely the results of the conventional PCR assay are falsely positive. Based on our other studies, we believe the real-time PCR assay is as sensitive as or more sensitive than the conventional PCR assay. Thus, it is possible that by the time the samples were assayed, in the real-time PCR assay, the Mhm copy numbers were below the threshold limit of 3.63 copies/μl blood (Tasker et al 2003b). The samples were shipped to the United Kingdom for assay which may have affected the results because we cannot completely account for quality of storage during shipping. Larger sample numbers, assayed in parallel, will be needed to directly compare the sensitivities of the two PCR assays.

The consequence of infection with Mhm and Mhf may range from mild or absent clinical signs to severe anemia and death (Harvey and Gaskin 1977, Berent et al 1998, Foley et al 1998, Westfall et al 2001). Although it appears that the risk of transmitting hemoplasmas by blood is decreased with prolonged storage, short-term storage appears unlikely to decrease the transmissibility of hemoplasmas in infected blood. However, our experimental design varied distinctly with what occurs in clinical practice and so even hemoplasma-infected blood stored for longer periods of time may be dangerous. Anemic cats are generally administered 45–60 ml of blood. Thus, even blood stored for 30 days may contain enough viable hemoplasma to cause infection when this volume is used. In addition, the recipient cats described herein were healthy adults. In general, cats needing blood transfusions are extremely ill and frequently would be expected to be immune compromised which might increase the likelihood that transfusion of low numbers of hemoplasmas would result in infection with or without clinical disease. Because these organisms are common in cats, and because cytology can be falsely negative, we support the recommendation that all cats used as blood donors are screened for hemoplasmas by PCR assay and excluded from blood donor programs if positive, even if the blood is to be stored before use. These recommendations are emphasized by the fact that there is no known therapy that can consistently eliminate hemoplasma infection once established. In addition, donors should be housed indoors with proper ectoparasite control to decrease the risk of newly acquired infection (Schneider 2000, Lucas et al 2004, Reine 2004).

Footnotes

Acknowledgements

The authors would like to thank Melissa Brewer, Jennifer Hawley, and Arianne Morris for aid in sample handling and performance of all PCR assays, Maura Green for her assistance and expertise in collection and storage of the whole blood used in this study, and Jason Eberhart, Erin Kennedy, and Caryn Reynolds for their help with monitoring of the cats throughout the project. The project was funded in part by the Kenneth W. Smith Professorship at Colorado State University and an unrestricted gift from Heska Corporation, Fort Collins, Colorado.

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Survival of Mycoplasma haemofelis and ‘ Candidatus Mycoplasma haemominutum’ in blood of cats used for transfusions

Abstract

Materials and Methods

Cats

PCR Assays

Sample Collection and Storage

Experimental Design

Data Analysis

Results

Discussion

Footnotes

Acknowledgements

References