Ecological Archives M075-022-A2

Robert E. Ricklefs, Bethany L. Swanson, Sylvia M. Fallon, Alejandro Martínez-Abraín, Alexander Scheuerlein, Julia Gray, and Steven C. Latta. 2005. Community relationships of avian malaria parasites in southern Missouri. Ecological Monographs 75:543–559.

Appendix B. Comparison of infection prevalence estimated by visual examination of blood smears and PCR analysis of DNA extracted from host blood samples.

Of 188 hosts from 1999 for which DNA samples were available, 32 (17.0%) were positive by inspection of smears whereas 101 (53.7%) produced amplification products using PCR screening primers. Cytochrome b primers produced PCR products that could be sequenced in one case in which neither smears nor screening primers were positive, and in four cases in which the smear was positive but the screening primers were not. Of 32 positive smears, the screening primers were positive for all but four cases. Sequences were obtained for 58 (30.9%) of the samples including 53 of the 101 samples that were positive with the screening primers. Failure to obtain sequences was partly a result of mixed infections yielding uninterpretable data, but primarily because the cytochrome b primers did not amplify the sequence. This may have been due to small variations in nucleotides within the primer sites or to the low concentration of parasite DNA in the whole blood samples. We presume that the concentration of parasite DNA was higher in samples that were visibly infected on blood smears, and the proportion of sequences obtained from samples with positive smears and positive screens (25/28 = 89.3%) was higher than that from samples with negative smears and positive screens (28/73 = 38.4%). Fallon et al. (2003) estimated that the screening primers could detect infection intensities as low as 10-5 to 10-6 erythrocytes whereas the lower limit of detection on blood smears in our study was ca. 10-4 erythrocytes (see also Hellgren et al. 2004) .

Table B1. Host blood samples obtained in 1999 (n = 188) and scored positive or negative with respect to visual inspection of smears, PCR screening primers, and cytochrome b sequences obtained. For smears, screens and sequences, 0 = negative and 1 = positive; the number of cases of each of the eight combinations is shown in the right-hand column.

Smears

Screens

Sequences obtained

Number of cases

0

0

0

82

0

0

1

1

0

1

0

45

0

1

1

28

1

0

0

0

1

0

1

4

1

1

0

3

1

1

1

25

   Notes: The single sequence obtained when neither the smear nor the screen was positive was obtained from a Yellow-breasted Chat (Icteria virens, OZ99-072) infected with Plasmodium lineage OZ04 having an infection intensity of 3.7 cells 10-4. This infection should have been picked up on the initial screening of 1999 smears, but was not.

LITERATURE CITED

Fallon, S. M., R. E. Ricklefs, B. L. Swanson, and E. Bermingham. 2003. Detecting avian malaria: An improved polymerase chain reaction diagnostic. Journal of Parasitology 89:1044–1047.

Hellgren, O., J. Waldenstrom, and S. Bensch. 2004. A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. Journal of Parasitology 90:797–802.



[Back to M075-022]