The buffy coat method: a tool for detection of blood parasites without staining procedures

Abstract

Background: Blood parasites belonging to the Apicomplexa, Trypanosomatidae and Filarioidea are widespread in birds and have been studied extensively. Microscopical examination (ME) of stained blood films remains the gold standard method for the detection of these infections in birds, particularly because co-infections predominate in wildlife. None of the available molecular tools can detect all co-infections at the same time, but ME provides opportunities for this to be achieved. However, fixation, drying and staining of blood films as well as their ME are relatively time-consuming. This limits the detection of infected hosts during fieldwork when captured animals should be released soon after sampling. It is an obstacle for quick selection of donor hosts for parasite experimental, histological and other investigations in the field. This study modified, tested and described the buffy coat method (BCM) for quick diagnostics (~ 20 min/sample) of avian blood parasites. Methods: Blood of 345 birds belonging to 42 species was collected, and each sample was examined using ME of stained blood films and the buffy coat, which was examined after centrifugation in capillary tubes and after being transferred to objective glass slides. Parasite detection using these methods was compared using sensitivity, specificity, positive and negative predictive values and Cohen's kappa index. Results: Haemoproteus, Leucocytozoon, Plasmodium, microfilariae, Trypanosoma and Lankesterella parasites were detected. BCM had a high sensitivity (> 90%) and specificity (> 90%) for detection of Haemoproteus and microfilariae infections. It was of moderate sensitivity (57%) and high specificity (> 90%) for Lankesterella infections, but of low sensitivity (20%) and high specificity (> 90%) for Leucocytozoon infections. Trypanosoma and Plasmodium parasites were detected only by BCM and ME, respectively. According to Cohen's kappa index, the agreement between two diagnostic tools was substantial for Haemoproteus (0.80), moderate for Lankesterella (0.46) and fair for microfilariae and Leucocytozoon (0.28) infections. Conclusions: BCM is sensitive and recommended as a quick and reliable tool to detect Haemoproteus, Trypanosoma and microfilariae parasites during fieldwork. However, it is not suitable for detection of species of Leucocytozoon and Plasmodium. BCM is a useful tool for diagnostics of blood parasite co-infections. Its application might be extended to studies of blood parasites in other vertebrates during field studies.

Challenges in the Diagnostic Performance of Parasitological and Molecular Tests in the Surveillance of African Trypanosomiasis in Eastern Zambia

“…An on-site microscopic examination was conducted on cattle blood stored in heparinized capillary tubes. The sealed capillary tubes were spun on-site in a microhematocrit centrifuge for five minutes at 10,000 rpm [ 19 ], after which packed cell volumes (PCVs) were determined using a PCV reader. The buffy coat from each sample was then placed on a microscopic slip with a cover slip and examined on site at a ×400 magnification for the presence of motile trypanosomes.…”

Molecular and morphological characterization of three new species of avian Onchocercidae (Nematoda) with emphasis on circulating microfilariae

“…These are the prominent obstacles for the diversity research. It is easier to detect circulating microfilariae, particularly because euthanasia of the host is unnecessary [ 20 ]. This study combined traditional morphological characters with molecular markers, and the results support previous speculations that identification of circulating microfilariae is possible at both the genus and species levels in wild birds [ 4 ].…”

“…30 µl) were taken from the vena ulnaris cutanea (wing vein). A few drops of fresh blood were used to prepare three thin blood films for microscopic examination; the remaining blood (approximately 25 µl) was used in the buffy coat method to detect individuals infected with microfilariae [ 20 ]. More specifically, the heparinized capillary tubes with blood were centrifugated in a microhematocrit centrifuge for 5 min at 10,000 rpm, following each tube was placed above a glass slide and the buffy coat area was examined under low magnification.…”

Lankesterella (Apicomplexa, Lankesterellidae) Blood Parasites of Passeriform Birds: Prevalence, Molecular and Morphological Characterization, with Notes on Sporozoite Persistence In Vivo and Development In Vitro

“…In vitro development experiments were conducted during fieldwork at Ventės Ragas Ornithological Station in May 2018. An intensively naturally Lankesterella infected sedge warbler Acrocephalus schoenobaenus (AS1) (donor of parasite blood stages, sporozoites) was selected using the buffy coat method as described by Chagas et al [ 22 ] ( Table 1 ). The intensity of parasitemia was 53% or 53 sporozoites per 100 thrombocytes.…”

“…To check if the S. canaria were susceptible, blood was collected from the experimental birds every 3–4 DPE for almost 6 months (160 DPE). Blood collection was performed as described above, and each sample was examined using the buffy-coat method [ 22 ], microscopic examination of Giemsa-stained blood films [ 21 ], and PCR-based methods, as described below. Control birds remain non-infected throughout entire observation.…”