A method to preserve low parasitaemia Plasmodium-infected avian blood for host and vector infectivity assays.

BACKGROUND: Avian malaria vector competence studies are needed to understand more succinctly complex avian parasite-vector-relations. The lack of vector competence trials may be attributed to the difficulty of obtaining gametocytes for the majority of Plasmodium species and lineages. To conduct avian malaria infectivity assays for those Plasmodium spp. and lineages that are refractory to in vitro cultivation, it is necessary to obtain and preserve for short periods sufficient viable merozoites to infect naïve donor birds to be used as gametocyte donors to infect mosquitoes. Currently, there is only one described method for long-term storage of Plasmodium spp.-infected wild avian blood and it is reliable at a parasitaemia of at least 1%. However, most naturally infected wild-caught birds have a parasitaemia of much less that 1%. To address this problem, a method for short-term storage of infected wild avian blood with low parasitaemia (even ≤ 0.0005%) has been explored and validated. METHODS: To obtain viable infective merozoites, blood was collected from wild birds using a syringe containing the anticoagulant and the red blood cell preservative citrate phosphate dextrose adenine solution (CPDA). Each blood sample was stored at 4 °C for up to 48 h providing sufficient time to determine the species and parasitaemia of Plasmodium spp. in the blood by morphological examination before injecting into donor canaries. Plasmodium spp.--infected blood was inoculated intravenously into canaries and once infection was established, Culex stigmatosoma, Cx. pipiens and Cx. quinquefasciatus mosquitoes were then allowed to feed on the infected canaries to validate the efficacy of this method for mosquito vector competence assays. RESULTS: Storage of Plasmodium spp.--infected donor blood at 4 °C yielded viable parasites for 48 h. All five experimentally-infected canaries developed clinical signs and were infectious. Pathologic examination of three canaries that later died revealed splenic lesions typical of avian malaria infection. Mosquito infectivity assays demonstrated that Cx. stigmatosoma and Cx. pipiens were competent vectors for Plasmodium cathemerium. CONCLUSIONS: A simple method of collecting and preserving avian whole blood with malaria parasites of low parasitaemia (≤ 0.0005%) was developed that remained viable for further experimental bird and mosquito infectivity assays. This method allows researchers interested in conducting infectivity assays on target Plasmodium spp. to collect these parasites directly from nature with minimal impact on wild birds.

Comparison of bovine hematology reference intervals from 1957 to 2006.

BACKGROUND: The most commonly used bovine hematology reference intervals were published in 1965. We found the results from healthy cattle in 2001 differed from those in many ways. Discovery of the original laboratory book used to calculate the 1965 values gave us the opportunity to evaluate whether hematology values of healthy cattle have changed over time. OBJECTIVE: The purpose of this study was to establish hematology reference intervals for Holstein cows, compare selected hematologic results with similar population data from 1957, and compare these reference intervals with those of other North American veterinary schools and published values. METHODS: Reference intervals were developed in 2001 using clinically healthy, bovine leukemia virus-negative, mid-lactation Holstein cows. Selected parts of the hemograms and neutrophil:lymphocyte (N:L) ratio were compared with those from healthy, age-matched Holstein cows evaluated in 1957. Bovine reference intervals were solicited from clinical pathology laboratories in North American veterinary colleges and analyzed for population characteristics and method of analysis. RESULTS: Between 1957 and 2001, mean neutrophil counts increased significantly, whereas lymphocyte, monocyte, and eosinophil counts and hemoglobin concentration decreased significantly. Mean N:L ratio increased significantly to 1.17. Most surveyed laboratories were using the 1965 reference intervals. Two other institutions that had developed reference intervals after 2000 had results similar to ours. CONCLUSIONS: Continued use of older bovine hematology reference intervals could lead to misinterpretation of within-reference neutrophil counts as neutrophilia and under-recognition of neutropenia, eosinophilia, monocytosis, or lymphocytosis. Use of N:L>1 as evidence of inflammation should be discontinued or used with great caution.

Evaluation of a rapid agglutination method for detection of equine red cell surface antigens (Ca and Aa) as part of pretransfusion testing.

BACKGROUND: Blood typing before transfusion minimizes the risk of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. Standard blood-typing protocols are time-consuming (2.5-3.0 hours) and impractical in emergency settings. OBJECTIVES: The purpose of this study was to determine whether equine RBCs could be typed for Ca and Aa antigens using sera from horses with RBC antibodies in a modified rapid (15 minute) blood-typing protocol. METHODS: Serum was obtained from a horse with anti-Ca antibodies and from another horse with anti-Aa antibodies. The presence of agglutinating antibodies was confirmed with antibody screening. Venous blood samples, collected in citrate-phosphate-dextrose, were obtained from 21 horses of various breeds. Samples were blood typed in the Veterinary Medical Teaching Hospital Hematology Laboratory using standard methodology. Washed RBCs from each of the 21 horses were incubated individually with anti-Ca and anti-Aa sera at dilutions of 1:4, 1:8, and 1:16 for 15 and 30 minutes at room temperature and 37 degrees C. RESULTS: Of the 21 horses, 13 were identified as Aa+/Ca+, four were Aa+/Ca-, two were Aa-/Ca+, and two were Aa-/Ca-. All 17 Aa-positive horses had a positive agglutination reaction at all dilutions of anti-Aa serum, incubation times, and temperatures, while all Aa-negative horses were negative. Each Ca-positive horse had a positive agglutination reaction at all incubation time points and temperatures up to the 1:16 dilution of the anti-Ca serum. All Ca-negative horses were negative at all times, temperatures, and dilutions of anti-Ca serum. Use of the modified protocol on 26 hospitalized horses resulted in accurate typing, based on complete antibody screens. CONCLUSIONS: These results support the hypothesis that equine RBCs can be blood typed using a rapid (15 minute) protocol, at room temperature, for the presence of Ca and Aa antigens using equine-derived antisera. This technique may be beneficial for pretransfusion testing of equine patients in an emergency setting.