71.,..12,222
GOJRATI, Hassan Ali Navvab, 1935 EPIZOOTIOLOGICAL SURVEY Of AVIAN I:iALARIA IN THE HAWAIIAN ISLANDS. '
University of Hawaii, Ph.D., 1970 Entomol,ogy
University Microfilms, A XEROX Company, Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED EPIZOOTIOLOGICAL SURVEY OF AVIAN MALARIA IN THE HAWAIIAN ISLANDS
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ENTOMOLOGY SEPTEMBER 1970
Hassan Ali Navvab Gojrati
Dissertation Committee
D. Elmo Hardy, Chairman Andrew J. Berger Mercedes D. Delfinado Wallace C. Mitchell Minoru Tamashiro ACh~OWLEDGMENT8
I would like to express my sincere appreciation to many individuals and organizations for their assistance and cooperation in various aspects of this study. Mr Jack Throp, Director of the Honolulu Zoo for allowing complete freedom in trapping and examination of birds at the zoo. Dr. Joseph E. Alicata of the
University of Hawaii for examination of some of the smears;
Dr. Allen Y. Miyahara of the University of Hawaii for very essential technical assistance; Miss Elaine M. L. Chang, technician of the Board of Agriculture; Mr. James K. Ikeda of the State
Department of Health for prOViding information on mosquito rearing;
Mr. Winston Banko of Hawaii National Park for great assistance in mist-netting the birds at higher elevations; Mr. John L. Sincock of the U. S. Fish and Wildlife Service, Koloa, Kauai, for providing large numbers of slides from birds trapped at high-elevations on
Kauai in addition to slides from Nihoa Finch, Nihoa Millerbird,
Laysan Finch, Laysan Duck, etc. The Pathology section, Queen1s
Medical Center for generously permitting me to use some of their laboratory facilities; Dr. Robert S. Gesowitz of the University of
Hawaii for examination of slides and technical assistance; and
Dr. Marshall Laird of the University of New Foundland, Canada, for indispensable assistance in diagnosis and identification of species of Plasmodium. TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS •.•.••••••••.•••.•.••••••.•.•••••• ii
ABSTRACT •.••••••.•••.•••••.••.••.•.••••••••...••• v
LIST OF TABLES ....•••.....•.•.•...... •...... •.•.. vii
INTRODUCTION ....••...... ••..•...... •..•...... 1
HISTORICAL BACKGROUND .....••..••...... 5
TAXONOMIC POSITION OF THE AVIAN MALARIA PARASITES •.....•...•..•••...•...... •.•....•...• 9 Class Sporozoa Leuckart 1879 ...•....•.•..... 9 Order Haemosporida Danilewsky 1886 •...•..... 10 Genus Plasmodium Marchiafava and Celli 1885 . 10
DEVELOPMENTAL CYCLE OF AVIAN PLASMODIA . 12
MATERIAL AND METHODS ...•.•.•.•••.•..•.•.•...•.•.. 16 Trapping and Handling the Birds •..• ...... 16 Collecting and Handling the Blood .....•••.•. 18 Collecting and Handling the Mosquitoes •..... 20 Description of the Collecting Area . 20 Rearing and Feeding the Mosquitoes . 21 Transmission Experiment.....••...... •... 22 Technical Obstacles...•.....•.....•.•... 24
HISTOLOGICAL METHODS .....•....•..•.....•.•...... 25 Preparation of Slides for Making Blood Smears . 26 Making Blood Smears ...... •.....••.....•.... 26 Staining Procedure....•.••...... ••...... 27 Buffered \\later....•••..••..•...... •...... 27 Technique for Dissecting the Salivary Glands of Mosquitoes .....•.••.....•...... 29 Technique for Preparation of Smear of the Salivary Glands for Sporozoites Study.•...... •...... •....•.. 30 Technique for Dissecting the Mid-gut of the Mosquitoes for Oocysts Study . 31 Technique for Preparation of Smear of the Mid-gut of Mosquitoes for Ookinetes Study...... •...•.....•...... 33 iv
Technique for Sectioning of Liver for Exoerythrocytic Study.•...•.•....•.•.•.•.•..•..... 34
RESULTS AND DISCUSSION ••.•••••••.•••.••..••••••.•••••...• 38 Information on Mosquitoes .••.•••.••••••••..•.••.••.• 48 Haemoproteus Infections in Birds . 51 Leucocytozoon Infections in Birds . 53
CONCLUSIONS ..•.•••..•••••.•.••.•••.•.•••.•••.••...•••.••. 55
SUMtv1A.RY •••••••••••••••.••••••••••••..•.•••••••••••••.•••• 58
LITERATURE CITED .....•.•.....••.••.•.•.••••••.••••••.•.•• 60 ABSTRACT
Avian malaria is believed to be one of the main factors in the extinction of the native Hawaiian avifauna. An epizoological investigation of the disease was conducted in the Hawaiian Islands primarily to determine whether avian malaria was present in Hawaii.
The birds were collected from the islands of Oahu, Maui, Kauai, and
Hawaii, at different elevations for a period of three years. Blood samples were taken either from the main wing vein, or from the toe nail. Blood smears were prepared and stained with Giemsa1s stain. Histological preparations for the study of the exoerythrocytic stage of the parasites were also made.
A total of 4,988 blood smears from 2,604 birds representing
38 species of native and introduced birds was examined. Plasmodium circumflexum Kikuth, f. gallinaceum Brumpt, f. cathemerium Hartman, and probably f. matutinum (Huff), were found in six species of native and introduced birds. Significantly, these records are the first
authenticated reports of these parasites from the Pacific Islands, and this is the first time that f. circumflexum has been reported from this part of the world. The presence of f. gallinaceum indicates that this parasite has accompanied its host to many
parts of the tropics.
Blood smears taken from pigeons and doves indicated that some
of these birds were infected with Haemoproteus and Leucocytozoon. vi It was found that 65 percent of the pigeons were infected with Haemoproteus. The doves, however, did not seem to be infected by this pathogen. Although both pigeons and doves were infected by the Leucocytozoon, the incidence was relatively low. Only four percent of the two species of birds were infected. This is the first record for Leucocytozoon from the Hawaiian Islands.
The presence of the larvae of the potential vector of avian malaria, Culex pipiens qUinguefasciatus Say, in a water container at 6511 feet elevation indicates that probably one of the limiting factors in the vertical distribution of Culex is the availability of suitable habitats. LIST OF TABLES
TABLE PAGE
I BUFFERED WATER FOR USE WITH GIEMSA r S STAIN...... 27
II OCCURRENCE OF PLASMODIUM IN NATIVE AND INTRODUCED BIRDS IN HAWAII . 39
III PREVALENCE OF PLASMODIUM IN NATIVE AND INTRODUCED BIRDS IN HAWAII . 42
IV DISTIRBUTION OF INFECTION RATES BY ISLAND AND ELEVATION ••••••••••••••••••.•• 44 INTRODUCTION
Research on avian malaria has offered to microbiologists, physiologists, biochemists, and many other scientists tools for solving many of their problems and this K~ll continue to be true regardless of whether human malaria is eradicated. Moreover, the growing feeling that eradication of malaria is just around the corner has given some people the idea that malarial research is no longer necessary. Because malaria has not as yet been eradicated, our tools for controlling it are rusting away leaving us unprepared for combatting an unexpected return of this great scourge. In this respect research on avian malaria provides us with a means for solving problems of basic importance to many fields of biology.
The discovery that birds are subject to malaria by Danielewsky in 1885 followed closely after the finding of malarial parasites in the blood of man by Laveran (1880). Since then, the course of experimental observation with both organisms has proceeded in a parallel course. Unfortunately, the study of bird malaria, and its possible important role in the extinction of the rich Hawaiian avifauna, has been almost completely neglected. It is believed that, the parasites of aaimals and their vectors have gained entrance with the importation of infected animals from various parts of the world. Because of the mild climate and other favorable factors, these parasites have become established. 2
It is characteristic of the parasitic diseases that they do not cause immediate high mortality, and more often their pathological symptoms develop gradually. This has been the case in the Hawaiian
Islands. It seems that parasites did not attract the attention of the
Hawaiians until about 1800 when the American and European ships began to call frequently. Since that time, parasites have been found in many species of birds and mammals (Alicata, 1969).
According to Hardy (1960), the night-biting mosquito (Culex pipiens quinquefasciatus Say) apparently was imported into the state of Hawaii in water casks on a ship (or ships) from Mexico between 1826 and 1830.
There are a number of accounts as to how they first arrived here. Van
Dine (1904) says, ITprevious to the year 1826 mosquitoes were unknown in Hawaii. During that year they were brought to the port of Lahaina, on the Island of Maui, in the ship TWp.llington T from San BIas, Mexico. 1T
Furthermore, up to the year 1826 there was no word in the Hawaiian language for mosquito. The native term is TMakika T, a corruption of the word mosquito (Hardy 1960). On the basis of available evidence,
Hardy (1960) also states that the two species of day-biting mosquitoes,
Aedes aegypti (Linnaeus) and ~. albopictus (Skuse), apparently did not reach the islands until a much later date.
Avian diseases aid greatly in controlling numbers of birds, but we know very little about these in Hawaii. With the exceptions of reports by Alicata (1939) on Haemoproteus columbae Kruse and of Fisher and Baldwin (1947) on Plasmodium vaughani Navy and Mac Neal, 3 practically no information is available. Warner (1968) reported on the susceptibility of drepaniid species to avian malaria and birdpox.
He concluded that avian malaria was probably one of the main factors in the extinction of native Hawaiian avifauna in the lowlands. The principle objective of this paper was to acquire more information concerning avian diseases. Therefore, an epizootiological investigation of avian malaria was undertaken in the Hawaiian Islands. This three year study confirmed the presence of Plasmodium, determined the species involved, the kind and proportion of the birds infected, the mosquito that probably serves as vector, and the relationships between Plasmodium, mosquito, and bird. It was demonstrated in the laboratory that Culex pipiens quinquefasciatus Say is capable of transmitting Plasmodium to canaries. It also has provided information concerning the prevalence of Haemoproteus and Leucocytozoon infection among Hawaiian birds.
In a broader sense, it was possible to study the ecology of avian malaria in the Hawaiian Islands in the hope of contributing to the epizoology of Plasmodium infection in native and wild birds. HISTORICAL BACKGROUND
The first mention of intracorpuscular parasites in the blood cells of birds was made by Danilewsky in 1885. Without knowledge of the description of malarial organisms in man by Laveran (1880), Danilewsky briefly but accurately described similar parasites from birds, although at the time he did not know what they were.
Danilewsky described three types of parasites from the blood of birds. The first type occurred free in the plasma and appeared to him to be a type of gregarine or "little blood worm." The second blood parasite was a trypanosome, also noted free in the plasma. A third was found to be both intracorpuscular and, at certain times, free-swimming in the plasma; we now know this to be a species of bird malaria parasite. During the ten years which followed his discovery of malaria parasites in birds, Danilewsky published several papers describing further studies, chiefly in French and German journals. In 1889
Danilewsky published the monograph "La Parasitologie Comparee du Sang," in which he described his observations ffi<::.de on the malarial parasites of wild birds obtained in Southern Russia. Danilewsky continued his investigations with the malaria parasites of birds, and in 1890 published three papers in the Annals of the Pasteur Institute. The first of these deals chiefly with a description of Leucocytozoon.
In a second paper his observations on the phagocytosis of haemogregarines and malaria parasites by white blood cells were presented. A 5 distinction is made in the third paper between acute and chronic malaria infections in bi~ds, and a general comparison is given of malaria in birds and man.
Following Danilewsky, Grassi and Feletti were next in importance in the early history of bird malaria. In 1890 these authors reported two distinct types of intracorpuscular parasites in sparrows (Passer hispaniolensis) and in pigeons Columba livia. The genus Laverania was established to include the crescent-shaped parasites already described by Danilewsky in birds, as well as similar parasites in man described by Laveran. The parasite in birds was called Laverania danilewsky and in man, Laverania malariae. I believe the latter name is still used by some modern investigators, although others have placed it in synonomy as Plasmodium falciparum.
The second parasite described by Grassi and Feletti was closely related to the amoeboid forms of the human malaria parasite. In fact, it was their belief that this parasite was identical with the parasite of human malaria. This has given rise to an interesting situation regarding the correct scientific name for the parasite with which they were working. They suggest the genus Haemamoeba and proposed the name~. praecox for both the bird malaria parasite and the human parasite. A second species was described as ~' immaculata, because of the supposed absence of pigment. Later they changed the name ~. praecox to H. relicta retaining the name H. praecox for the parasite in human malaria.
During the Grassi and Feletti period, Kruse in 1890 proposed the 6 generic name Haemoproteus for the crescent-shaped bodies found by
Danilewsky in the blood cells of birds.
It is of interest that the discoverer of malaria parasites in man should play a part in the early researches on bird malaria.
Laveran began to publish a series of papers on this subject, being particularly interested in the use of birds as experimental laboratory hosts and in the relationships between human and bird malaria parasites.
In 1891 he published two papers on the general characteristics of malaria parasites in birds, and in the same year presented photo micrographs of both bird malaria and human malaria to the Academy of
Sciences in Paris.
Following Grassi and Feletti, the two most important Italian workers in the early studies on bird malaria were Celli and San Felice
(1891). They reclassified bird malaria parasites, but later work indicated that they were dealing with mixed infections of Haemoproteus and Plasmodium. A more concrete contribution by Celli and San Felice was the first successful transfer of malaria parasites from bird to bird by blood inoculation.
In 1894 Labbe's monograph appeared, in which blood-cell parasites were studied from a purely zoological point of view. He then outlined his own classification and suggested the generic name ProteosOffia for the parasites which Danilewsky described as Cytosporon and Grassi and
Feletti as Haemamoeba.
The endocorpuscular parasites of birds were not seriously considered in America until thirteen years after their discovery in
Europe. In 1898 Eugene Opie of the John Hopkins University in 7
Baltimore published a paper on the haemocytozoa of birds. Opie
examined a number of species of native American birds and found malaria parasites in several of them. One of the most important in the history
of malariology was the discovery of the process of fertilization in
Plasmodium falciparum (Welch) by MacCallum in 1898. Thus a sexual cycle in malaria was demonstrated. MacCallum also described the
pathology of bird malaria infections. In the same year (1898), the
role of mosquitoes as vectors of malaria was described by Ross, a discovery based in part on MacCallum1s description of the fertilization
process and Sir Patrick Manson1s encouragement. Koch (1899) verified the work of Ross by successfully transmitting avian plasmodia from bird to bird by mosquitoes. He was also the first worker to transmit
bird malaria to canaries.
The volume of literature on avian malaria in the 20th Century is
immense, many of the contributions having dealt with drug screening and other studies related to malaricidal properties of various drugs.
More than 30 species of Plasmodium have been described from birds of various kinds (see Hewitt, 1940), but fewer -than half are presently
regarded as valid (see Levine, 1961; Bray, 1957; Laird and Lari, 1958).
There are also a great many contributions on other aspects of experi mental research, such as studies on immunity, genetics, in vitro
cultivation, interrelationship with other infectio~s, and basic
physiology and biochemistry. A brief but excellent review of
representative contributions to these and other fields of experimental
research on avian malaria has been presented by Huff (1963). Besides, 8 several other workers such as Coatney and Roudabush (1937), Coatney and West, (1938), Herman (1938, 1944), Herman et al. (1954), Kikuth
(1931), Levine and Hanson (1953), Manwell (l935, 1938), and Wolfson
(1941) have made contributions to our knowledge of malariology.
In the period between the appearance of Hewitt1s (1940) monograph and this study, a very great increase in the amount of experimental work on avian malaria can be seen. The increase was largely due to the increased importance of human malaria during World War II. Avian malaria parasites were already known to be excellent screening agents for antimalarial compounds, and in fact had contributed significantly to the chemotherapy of malaria before World War II. Also, significant research was carried out on Leucocytozoon and Haemoproteus infections.
The history of malariology will record this period as one in which research on avian malaria played a most important part in gaining an understanding of the mode of transmission of malaria. From this great amount of interest and effort many new problems were uncovered and new techniques developed.
In the State of Hawaii the research which has been conducted on avian malaria is very limited. According to Alicata (1939), pigeons in Hawaii are commonly infected with Haemoproteus columbae Kruse.
Blood smears from 101 adult pigeons in Honolulu showed 83 to be infected; and of a total of 25 vectors, pigeon fly Pseudolynchia canariensis (Macquart) dissected, nine or 36 percent were infected with ~. columbae (Kartman, 1949). The true avian malarial organism,
Plasmodium vaughani Navy and Mac Neal, in the Pekin Nightingale, 9
Leiothrix lutea, has been reported by Fisher and Baldwin in 1947.
This parasite was noted in one of 11 birds examined from Hawaii
National Park, island of Hawaii.
TAXONOMIC POSITION OF THE AVIAN MALARIA PARASITES
Kudo (1966) proposed the new classification of the Protozoa in which the phylum is divided to two subphyla: Plasmodroma and
Ciliophora. The subphylum Plasmodroma is characterized by possession of one to many nuclei of one kind and flagellae or pseudopodia or no organelles of locomotion. This group is subdivided into four classes:
1) Mastigophora, 2) Sarcodina, 3) Sporozoa, and 4) Cnidosporidia.
In the subphylum Ciliophora are placed those protozoa which possess two kinds of nuclei (macronucleus and micronucleus) and have cilia or similar locomotor organelles in at least one stage of development.
It is subdivided into Ciliata and Suctoria. Each class is composed of several orders. However, we are primarily concerned here with the class Sporoaoz and specifically with the order Haemosporida. Therefore, a very brief discussion of this group is presented.
Class Sporozoa Leuckart 1879
Members of the Sporozoa are parasitic and produce spores.
They possess no organs of locomotion except in the gamete stage.
Reproduction is asexual by binary or multiple fission (schizogony) or sexual (gametogony). Gametogony leads to the formation of a zygote which in turn initiates the process of sporogony or spore formation. 10
The classification of the class Sporozoa has occasioned much discussion, but, for the purpose of this paper, that proposed by Kudo (1966) is being followed. Kudo divided the Sporozoa into four orders: 1) Gregarinida, 2) Coccidida, 3) Haplosporida, and
4) Haemosporida.
Order Haemosporida Danilewsky 1886
The Haemosporida require both vertebrate and invertebrate hosts to complete its life cycle. Schizogony occurs in vertebrates, and gametogony and sporogony occurs in blood-sucking invertebrates. The order is divided by Kudo into three families, namely:
Plasmodiidae, schizogony in the peripheral blood of vertebrates.
Pigment present. This family contains one genus of importance- Plasmodium, the malarial parasite of man and other animals.
Haemoproteidae, schizogony in the endothelial cells of inner organs; only gametocytes appear in the peripheral blood. Pigment present. Two genera of interest, Haemoproteus and Leucocytozoon, occur in this family. Babesiidae, small, non-pigmented parasites of erythrocytes.
Genus Plasmodium Marchiafava and Celli 1885
The true malarial organisms belong to the genus Plasmodium, which in turn is closely related to Haemoproteus and Leucocytozoon. The principal significant difference between Plasmodium and the other two genera is that the asexual stages (schizonts) of the 11
former occur in erythrocytes of the circulating blood, while those of the two latter genera occur in the internal organs (lung, liver, spleen, kidney, etc.). As a result, Plasmodium can be transmitted regularly from one susceptible host to another by injection of
infected blood from the vessels or heart, whereas in the case of the other two this procedure will result in infection only at certain times when merozoites are in the blood by chance, because the only
stages ordinarily in the blood are gametocytes which can o~~y ~o~t~~ue development inthe proper invertebrate host. Like Haemoproteus,
Plasmodium contains pigment. It is of special interest that mammalian malarias are carried by Anopheles mosquitoes, while those of birds
are generally carried by culicine (Culex, Aedes) mosquitoes, although some of the latter also have anopheline vectors.
Most of the avian species of Plasmodium are far less host specific than mammalian forms. Some occur naturally in a considerable
number of species of wild birds, and some have been adapted by experimental passage to the development in birds in which they are
not known to occur naturally. Only a few species have been reported as naturally infecting domestic birds, and it is not known whether all of these are of veterinary importance.
In the Haemoproteus, a part of the developmental cycle occurs in
Hippoboscid flies, commonly called Louse flies, and the only proven vector is Psuedolynchia canariensis (Macq.). However, it has been
shown by Fallis and Wood (1957) that a biting midge, Culicoides (possibly Eiliferus) Root and Hoffman, is an intermediate host and 12 transmitting agent of ~. nettionis Coatney in ducks. The vectors of Leucocytozoon are members of the genus Simulium, and Simulium venustum Say has beer recognized as an important vector species.
DEVELOPMENTAL CYCLE OF AVIAN PLASMODIA A major advance in the understanding of the life cycle of the malarial organism was made by the discovery that infective sporozoites did not enter erthrocytes directly, but rather developed as exoerythrocytic forms in cells of the reticuloendothelial system prior to invasion of the erythrocytes. Following the introduction of the sporozoites from infected culicine mosquitoes, numerous pre-erythrocytic schizonts are found in the macrophages and fibroblasts of the skin near the point of entry. These are referred to as cryptozoites. Merozoites from this first generation of pre-erythrocytic schizonts form a second generation of pre-erythrocytic schizonts, the metacryptozoites.
Merozoites from the metacryptozoites enter erythrocytes and other cells of the body and in the latter form exoerythrocytic schizonts.
In the case of P. gallinaceum, ~. relictum and ~. cathemerium, these other cells are endothelial cells, but in the case of P. elongatum and R. vaughani they are cells of the haemopoietic system.
In some species of avian plasmodia, e.g. ~. gallinaceum, and P. elongatum, the exoerythrocytic developmental stages may be added to by forms which are derived from the erythrocytic cycle. These are 13 known as phanerozoites, being derived from the merozoites of the schizonts in the erythrocytic cycle.
The erythrocytic cycle is initiated seven to 10 days after infection by merozoites from metacryptozoites and at other times by merozoites ~rom exoerythrocytic schizonts located, according to species, in the endothelial or haemopoietic cells. On entering the red blood cell, the merozoite rounds up to form a trophozoite. This is a small rounded form containing a large vacuole which displaces the cytoplasm of the parasite to the periphe~ of the cell. The nucleus is situated at one of the poles, giving the young form a !signet ring! appearance when stained with Giemsa. The early trophozoites undergo schizogony to produce merozoites, the number produced depending on the species of parasite. During the process of schizogony, the parasite takes in host cell cytoplasm by invagination, haemoglobin is digested and the residual haematin pigment is deposited in granules within the food vacuoles. Apparently, schizogony may continue indefinitely, the length of each cycle of schizogony depending on the species of parasite. The release of merozoites from the schizonts occurs synchronously in the host, and in human malaria this is associated with a paroxysm of fever. Fever does not appear to be a significant part of the syndrome in avian hosts (Russell --et al., 1963). After a number of asexual generations has occurred, some merozoites undergo sexual development with the formation of micro- gametocytes and macrogametocytes. Levine (1961) claims that the '14 female forms should be referred to as macrogametes since they possess a haploid number of chromosomes. The haploid nature is maintained throughout the whole of the life cycle of the malarial parasite, except that a diploid state is found following fertilization and zygote formation. The female forms are generally more numerous than the male forms, and they stain more intensely blue with Giemsa than do the male forms. In addition, of course, the nucleus of the microgametocyte is more diffuse than in the female cell. Further development of the gametocytic stages can take place only when the blood is ingested by a suitable mosquito.
Development in the mosquito is rapid. Within 10 to 15 minute~ the nucleus of the microgametocyte divides, and through a process of exflagellation, six to eight long, thin, flagella-like microgametes are extruded from the parent cell. These remain attached to the parent cell for a few minutes, lashing actively; they then become detached and swim away to find, and fertilize, the macrogamete. The zygote resulting from fertilization is motile and is called an ookinete. This ookinete penetrates the mid-gut mucosa and comes to lie on the outer surface of the stomach, forming an early oocyst about 50 1)-60 u in diameter. The nucleus of the oocyst divides repeatedly to produce a very large number of sporozoites.
These are about 15 u in leng~h with a central nucleus. Maturation of the oocyst takes a variable period of time depending on the species of parasite, temperature, and the species of mosquito; but in general, it is 10-20 days. When mature, the oocyst ruptures, liberating the sporozoites into the body cavity of the mosquito, and these then 15 migrate allover the body of the mosquito but eventually reach the salivary glands. Here they may lie intracellularly, extracellularly, or in the ducts of the salivary glands. They are now infective to a new host, infection occurring when the mosquito takes a blood meal. A mosquito remains infected for its life span, transmitting malarial parasites every time it takes a blood meal. 16
MATERIAL AND METHODS
Trapping and Handling the Birds Birds were collected in a variety of ways. The majority of birds used in this work were captured by trapping. Numerous birds were sampled by mist netting, and collections were occasionally made by shooting. Two types of traps were utilized. One of these traps which was successfully used for capturing most of the common low-land birds was a square-mesh wire netting funnel trap 42 inches wide, 60 inches long, and 42 inches high. The trap had a funnel- or cone-shape entrance opening from both ends, ailU Cl small door on one side for releasing the birds. The trap was baited with natural bird food and placed in the Honolulu Zoo. Sometimes, for capturing a particular kind of bird, a few individuals were intentionally placed in the cage to attract other members of the same species. Another kind of trap utilized for capturing House Sparrows was a door trap. This trap is frequently called a lfpull-stringlf trap, and is a device which is merely an adaptation of the old and well known lfsievelf trap. It is easily made at little expense; and although not usually automatic in its operation, it is probably the best trap for a new operator to use until he has acquired proficiency in handling birds. The door trap used in this experiment was a square mesh wire netting 18 inches wide, 24 inches long, and 10 inches high. The trap was baited with natural bird foods and placed in the backyard of a residential area. 17
Several mist nets of different lengths and widths were also used for capturing the birds. The desired height of the net was adjusted with the help of telescopic poles. The mist nets were placed among the trees where the birds were actively moving from one place to another. The birds were collected at low elevations on the islands of
Oahu and Maui; at different elevations on Kauai; and at 4,000-6,500 feet elevations in the Volcano National Park on Hawaii. All the birds collected in the National Park and some of those collected for me from the island of Kauai were banded and rele~sed after they were sampled. Some of the birds were captured and blood smears made by John L. Sincock from the islands of Kauai, Nihoa, Necker, Laysan, and
Midway. These were: NewellTs Shearwater, Wedge-tailed Shearwater,
Bulwer's Petrel, Red-tailed Tropicbird, Laysan Duck, American Coot, Golden Plover, Black-necked Stilt, Gray-backed Tern, Barn Owl, Nihoa
Millerbird, Elepaio, Iiwi, Anianiau, Nihoa Finch, Laysan Finch.
If the bird was sacrificed (as in the case of Sparrows, Canaries, and few Mynahs), the liver was preserved either in 70 percent alcohol or ZenkerTs Fluid.* The former was used only when the latter was not available. The liver was fixed in Zenker's Fluid for approximately 24 harrs. It was then washed in several changes of 70 percent alcohol and finally preserved in 80 percent alcohol. Good results were obtained by using Zenker's Fluid.
* K2Cr207 - 2.5 grn.; HgC1 2 - 5 to 8 gm.; distilled water - 100 mI.; glacial acetic acid (added at time of use) 5% by volume. 18
Collecting and Handling the Blood
Blood was collected from the wing vein or by clipping the center toe nail of the birds. Some of the trapped birds were banded and released after being sampled, and some of them were released without banding. An attempt was made to minimize the handling period of the small birds prior to venipuncture. After removing caged birds from cages or wild birds from mist nets or traps, the birds were handled very gently for short periods before processing, because, even without venipuncture, small caged birds and small wild birds occasionally die if they are handled or manipulated excessively. Such handling or manipulation reduces the chance for survival following venipuncture. It had special importance in the case of this study because I was not allowed to kill the birds.
All preparations for venipuncture procedures were made before the blood was taken. These procedures included the banding and the parting of the feather in the blood-collecting site. Venipuncture was the last procedure before the bird was released. If blood collection from the wing vein was desired, one of the wings was held open so that the undersurface of the wing was facing the operator. The wing vein could be seen through the skin by parting the feathers and exposing the surface of the skin of the wing near the birdTs body. In most birds, especially the large ones, the wing
vein could be seen without much difficulty. In very small birds, application of slight pressure with the thumb against the wing bone facilitated the detection of the wing vein. 19
With the bird in the hand of the partner and the wing vein exposed, a 20-gauge needle attached to a 10 ml. syringe was inserted in the superficial blood vessel of the wing. About 5 mI. of venous blood was taken and poured into a clean blood-collection tube containing one drop of Sequester-Sol* as an anticoagulant agent. Care was taken to use the proper quantity of anticoagulant to prevent any distortion or change in the quality of the blood collected. The blood-collection tube containing the mixture of the blood and anti coagulant was inverted four to five times to insure the complete mixture of the blood with the anticoagulant. This was the procedure when blood was carried to the laboratory for the preparation of the blood smear. When the blood arrived at the laboratory, it was placed in the refrigerator for future use.
If the smear was made at the same time that the blood was taken, slightly different procedures were applied. A fingernail clipper was used to clip a toe nail about midway between the base and the end of the vessel visible in the nail. The nail was clipped in an anteroposterior direction, because this tends to dilate the vessel. The-blood was collected either from the wing vein or toe nail directly to a heparinized capillary tube 75 mm. long and 1.4 to 1.6 mm. in diameter. The blood collected in this way was put directly on a clean slide and the smear was immediately made. In general, bird blood coagulates rapidly. Because no anticoagulant was used in
* Active ingredient: Dipotassium Ethylenediamine Tetraacetone 20 this method, the smear had to be made very rapidly. In small birds, the blood flows very slowly, so that the blood had to be sucked out by attaching a rubber tube to the capillary tube.
Blood flow was stopped almost immediately by application of firm pressure to the toe nail or wing vein with the thumb and forefinger.
Collecting and Handling the Mosquitoes Description of the Collecting Area. The mosquitoes, Culex pipiens quinquefasciatus Say, were collected in two different ways. First, by collecting the eggs from the field, and second by collecting larvae in a small can containing water outside of the laboratory.
The former were collected for me in the field by the State Department of Health, Mosquito Control Division. The mosquito egg rafts were collected at the Hawaiian Maid farm in Ewa, Hawaii. These were found in a poultry effluent ditch which formed from the overflow of the chicken watering trough. The old feed and chicken feathers in the water provided a slightly acid media (pH=6.5) in the ditches. The fermenting feed attracted many rotifers and other pollution-hardy organisms. The highest concentration of mosquito eggs was amongst the weeds along the ditch and between patches of duckweed, Lemna spp. Heavy breeding is common along the edge of the ditch but the larvae in the deeper areas are eliminated by the guppies, Lebistes reticulatus 21
(Peters). Food sources available to the adult mosquitoes include the chickens and birds that frequent the poultry houses. Rearing and Feeding the Mosquitoes. The rearing procedure discussed below is commonly used by the Mosquito Control Division.
Egg rafts were collected and placed on damp Kimwipes in a baby-food jar. Initial field collections were made by transporting late instar larvae in plastic bags. This method proved to be detrimental to the larvae since more than 50 percent were dead upon arrival at the laboratory•
Five to six egg rafts (approximately 1000 eggs) were placed into a 6 x 8 x 12 inch plastic tub which was filled to a depth of two to three inches with tap water aged for one to two days. If the water was not aged, a pinch of Brewers! yeast was added to the fresh tap water to deoxygenate the media. Hatching occured after 24 hours and a small portion of a one-to-one mixture of Purina Dog Chow and Brewers! yeast (relatively inactive) were provided for larval food. Subsequently, daily portions of the same larval food was given, but care was taken to prevent overfeeding because this caused a scummy layer to appear on the water surface which made it necessary to transfer the larvae to a fresh container. By careful addition of the food, no scum developed.
After seven to eight days in the larval stage, the insec~ pupated. The pupae were removed from the rearing tubs by siphoning them through a modified pipitte. This pipitte was essentially a
11 syringe bulb fitted with a wide mouth glass tube (J.D. = 3/16 ). 22
The pupae were placed into small dixie cups and placed in 12 x 12 x 12 inch screened cages for emergence. A sleeve was attached to one side to facilitate the admission of birds and withdrawal of mosquitoes.
Newly emerged adults were fed a five percent honey or sugar solution which was placed in a baby-food jar with a dental wick protruding th~ough a hole in the lid. The adults were able to feed on the material on the wick. They were also given raisins as additional food. Four days after emergence, the females were offered blood meals from an immobilized bird, as will be discussed later. Deposition of eggs by the engorged mated females took place three to five days after the blood meal. The oviposition media offered was aged water in a 1000 mI. beaker. Transmission Experiment. In connection with the experiment upon infectivity and transmission, two conditions had to be fulfilled-- first, the birds had to have large numbers of gametocytes in their blood, and second, there had to be a supply of adult mosquitoes in readiness for biting. This necessitated the taking and examination of smears frequently to determine whether or not the bird was in condition satisfactory for the feeding experiment. The birds were exposed to the mosquitoes four days after emergence of the latter. At this age; the mosquitoes theoretically should have shown a great tendency for a blood meal, but practically it was not always so, as will be discussed later. The birds used in the present work as a source of the pathogen were House Sparrows, (Passer domesticus), which were previously trapped and found to be infected with malaria by blood examination. 23
In order to allow the mosquitoes to feed on the diseased bird, the bird was immobilized by tying the feet, the wings, and the beak
snugly with a piece of masking tape, and the bird was placed on the top of the breeding chamber in such a way that its exposed breast would be accessible to the mosquitoes. The feathers of the bird were then parted in the pectoral region and wetted down. The bird could be left in this position for at least an hour without apparent dis comfort. When the female mosquitoes had engorged upon the blood of the infected bird, the bird was removed. Then, all engorged females in the cage were picked out individually and transferred to another breeding chamber which also was provided with a long sleeve. In so doing, it was necessary to wear a rubber glove to prevent the mosquitoes from engorging from my hand, for they might then be mistaken for mosquitoes which had had the infective meal.
The engorged mosquitoes were kept from eight to ten days and then
dissected. It was soon found, under the prevailing laboratory conditions (76 0 F. - 82 0 F temperature and 65 - 75% relative humidity), that the oocysts of the parasite reached their maximum size on the tenth day following the feeding. The mosquitoes were kept routinely thereafter for ten days. After a few had been anesthetized and dissected, and the presence of oocysts were observed, the rest of the mosquitoes in the chamber \'~ere kept long enough (i. e. usually about two weeks) to insure the finding of sporozoites in their salivary glands. I found that it was advisable to dissect a few mosquitoes
daily in order to observe the progress of infection and the day of 24 invasion of the salivary glands. No attempt at carefully counting the number of oocysts in the stomachs was made, because this count could in most cases have been only an estimate, due to the great number present and the difficulty of viewing all sides of the stomach without duplicating the count of certain oocysts.
When the presence of sporozoites in the salivary glands of the mosquitoes was observed, they were allowed to feed on healthy birds.
Five uninfected canaries, Serinus canaria were then exposed to the mosquitoes which had been fed on the infected birds fifteen days before, and they were allowed to be bitten by mosquitoes. In this way, the pathogen was transferred from the diseased House Sparrows to the healthy canaries. Canaries are usually used as an experimental animal for the study of malaria parasites because of their high susceptibility to the pathogen. A period of six to eight days elapsed until the parasites first appeared in the blood of the canaries bitten by infected mosquitoes.
An attempt was made to allow the mosquitoes to repeatedly feed on the infected birds because it is never possible to be quite certain that the gametocytes are matured. But, only partial success was obtained.
Technical Obstacles~ A great deal of difficulty was encountered at first in getting the adult females to feed upon birds. Three factors were essential for success in this attempt. It was found necessary to keep the mosquitoes away from water for at least 24 hours before attempting the feeding experiment. During extremely 25 hot weather, it was necessary to keep the breeding cages over moistened cotton so as to keep the air moist, but in such a way that the mosquitoes could not imbibe the liquid. The other important factor was darkness. Nearly all individuals of the species of mosquitoes dealt with in this experiment would bite much better in darkness than they would in the light. Hence all of the feedings
~ere made at night. Another problem in infecting the mosquitoes was that a certain percentage of the mosquitoes which were fed on parasitized birds failed to become infected even though other mosquitoes fed on the same bird exhibited full development of the parasite.
DifficUlty was frequently encountered in getting the mosquitoes to take a second blood meal. Some died immediately after depositing eggs; others drowned in the water tray; and some died shortly af~er the first feeding before the blood meal was digested. Many refused to feed a second time although they were given an opportunity to do so. It was necessary, therefore, in order to insure enough mosquitoes for successful transfers, that considerable allowance be made for the difficulties involved in carrying the transmission to completion.
Histological Methods In searching for blood protozoa in general, thick or thin smears of the blood are prepared and stained with one or another of the Romanowsky (methylene blue-eosin combination) stains. Thick smears are preferred to thin ones for mammalian blood because their use 26 permits one to examine a relatively large amount of blood in a relatively short time. However, they cannot be used for avian blood because of its nucleated erythrocytes. The protozoa may be distorted in thick smears enough so that much care and practice is needed to differentiate and interpret the species, especially of the malaria parasites. Therefore, I used only thin smears. Among many different kinds of stains available, Giemsa has been the most frequently used by different investigators of avian Plasmodia.
Rapid stains, such as Wright1s and Field1s stain, are used only if speed is necessary, because they stain unevenly; and they are not as precise as the slow stains.
Preparation of Slides for Making Blood Smears. The first step in making a good thin film and easily diagnosable smear is to have very clean slides. Therefore, the slides were rinsed in 95 percent alcohol and wiped with a cJ~an cloth
Making Blood Smears. To prepare a thin smear, a small drop of fresh blood was placed at one end of a slide, so that it was about
1 1/4 inch from the end. Quickly, the underside of a second spreader slide, the corner of which has been cut away, was touched to the drop of blood which would spread along the edge of the spreader slide in contact with the lower slide. Then, with the spreader slide held at a 30°_40° angle, rapidly drew (without pushing) the spread drop over the horizontal lower slide, and a uniform thin smear resulted. The well-spread smear appeared very pale with a feather-shaped end. The smears were allowed to dry in the air (a matter of a few seconds) 27 until they changed color. The host number or species was scratched on the slide with a diamond-point stylus. The smears were fixed and stained within four or five hours of smearing. Staining Procedure. After the thin blood smears had dried, they were immersed in absolute methyl alcohol (acetone-free) for three to five minutes for fixation. The slides were removed and allowed to dry in the air. Then, they were placed in GiemsaTs stain. The
Giemsa's stain was prepared fresh and diluted before staining.
Buffered Water. A phosphate buffered water having a pH of 6.5 to 6.8 was used for the dilution of the stock Giemsa and for rinsing the stained slides. The two salts were mixed thoroughly, as shows in
Table I, in a mortar and 1 gm. to 2000 c.c. of distilled water was used. It was necessary to vary the amounts of phosphates to obtain the desired results. Red is increased by lowering, and blue is increased by raising the pH.
TABLE I. BUFFERED WATER FOR USE WITH GIEMSATS STAIN
~ gm gm
6.5 2.723 8.316
6.8 4.539 5.940
Each drop of GiemsaTs stain was diluted with 1 c.c. of such a buffer. After dilution of Giemsa's stain, the smears were placed in the 28 stain for twenty to thirty minutes; then they were removed and washed with just enough buffered water to remove the excess stain.
The slides were then placed on end on a piece of blotting paper, and allowed to dry. The nuclear chromatin stains garnet or TIlby red and the cytoplasm a delicate sky blue, thus contrasting the reddish-purple nuclei of the leucocytes and thrombocytes (platelets). Many investigators do not cover blood smears because they think it is unnecessary to cover stained blood preparations unless they are to be observed with dry objectives. I covered the blood smears, and it seemed to me that this makes the color slightly more brilliant and the small details a little sharper. Moreover, if a given slide had to be checked several times, there was no danger of destroying the erythrocytes. If a smear was covered, a neutral medium such as diaphane was used, because fading may otherwise be very rapid. Diaphane-mounted Romanowsky-stained blood films will keep for years without much deterioration. All smears were examined under oil immersion objectives of a compound microscope for a sufficiently long period, usually at least ten minutes before the result was considered negative or positive.
Great care was exercised to avoid mistaking the blood platelets accidentally superimposed upon red cells for malarial parasites.
These platelets are frequently surrounded by an unstained halo. Precipitated stain, dirt, or bacteria may constitute other sources of error. It should be mentioned that a thorough working knowledge of the 29 thin blood film, i.e. the appearance of the normal constituents of blood, of the more common pathological changes in the blood cells, as well as of the different species of Plasmodia in their various stages, is necessary before attempting to learn to identify malaria parasite in a thin film. The thin film has the great disadvantage of failing to reveal a great number of positive cases, particularly of light infections. One also should be aware of the fact that the identification of species of naturally occurring malaria is not easily made from single blood smears even by specialists. Due to the lack of a protozoologist and expert on avian malaria in the Islands, I have spent a great deal of time familiarizing myself with the most commonly found species by consulting P. C. C. Garnham1s (1966) book. Finally, an attempt was made to assign each organism to the proper species. After preliminary identification of species, the slides were sent to Dr. M. Laird for final diagnosis. Technique for Dissecting the Salivary Glands of Mosquitoes. Several methods have been suggested by different investigators for extracting salivary glands from the mosquito. I have adopted the
Shute and Maryon (1966) method. These authors clearly describe and illustrate four stages of the process. This is one of the most usual methods for the removal of the salivary glands, and it is the modification and refinement of the method which is given by Barber and Rice (1936) and Giovannola (1934). Chloroform was used to anesthetize the mosquitoes. The wings and legs were then removed. The fly was laid on one side of a slide where a small drop of normal 30 saline was available. The head was severed by a sharp cut. Then, pressure was applied with a sharp dissecting needle to the side of the thorax near the base of the fore-legs whereupon the salivary glands would emerge, usually accompanied with a drop of a haemocoelic fluid. The salivary glands were separated quickly from adjacent structures and covered with a drop of saline. The glands were dissected in saline solution and were examined under the low power objective of the microscope without any coverslip in order to confirm the presence of sporozoites. They were then placed in a one percent aqueous solution of osmium tetroxide, kept at a pH of 7.3 by MichaelisTs buffer. Fixation proceeded at 32°_38° F. for half an hour, after which the glands were dehydrated rapidly through a series of graded alcohols. After a third change of ethyl alcohol, the salivary glands were stained for half an hour in a one percent solution of phosphotungstic acid in alcohol. Finally, a rinse in absolute alcohol was given, and they were embedded in Araldite. For more detailed information concerning this procedure, the reader can refer to Shute and Maryon (1966) and Barber and Rice (1936).
Technique for Preparation of Smear of the Salivary Glands of
Mosquitoes for Sporozoites Study. To prepare the smear of the salivary glands, the slide containing the salivary glands was transferred to the microscope and the specimen was brought into focus with low power, using a reduced light. The glands were placed in the center of the slide, and a large square coverslip was dropped onto 31 them at an angle, so that one corner of the coverslip was over the glands and the opposite corner was protruding beyond the slide. The coverslip was dropped sufficiently heavily onto the glands to rupture the cells without displacing them. The object of applying the cover
slip at an angle is to prevent the saline from becoming too widely distributed and leaving the glands and their contents concentrated in
a very small area. Before staining, a circle surrounding the specimen was drawn with
a blue grease pencil on the reverse side of the slide. This made it
easy to locate the specimen when the time came to examine it. The technique for the staining was the same as for the blood films, and the GiemsaTs stain was used. It was found by experiment that the pH of the distilled water had to be adjusted to neutral or pH 7.1 to 7.2;
otherwise, the nucleus and the cytoplasm would not be prominently
displayed. If the specimen was worth keeping, it was mounted in
Euparal.
Technique for Dissecting the Mid-gut of the Mosquitoes for Oocysts Study. When the salivary glands were dissected with care,
the remainder of the insect was undamaged. It was then placed on the
center of a glass slide. The insect was placed on a drop of saline with the thorax pointing to the left. The thorax was steadied on the slide by transfixing it with the point of the left-hand needle. With
the right-hand needle, the integument above and below on either side of the sixth and seventh abdominal segment was nicked. The detached 32 segments were pulled with the right-hand needle until the mid-gut, and the attached malpighian tubes came into view. The alimentary canal was severed sufficiently far forward to bring away a portion of the fore-gut. If the correct quantity of saline had been used, the malpighian tubules floated backwards, and the whole mid-gut was left clear. The gut was held by placing the point of the left-hand needle on the fore-gut, and the right-hand needle was used to cut through the alimentary canal at the junction of the mid- and hind-gut. The bases of the malpighian tubules were severed at the same time. The hind-gut and the malpighian tubules were removed so that only the mid-gut with the attached oesophagus remained on the slide. One edge of the coverslip was rested on the slide, but not in contact with the stomach, and the opposite edge rested on the point of the dissecting needle which was then gradually lowered until the cover slip came to rest on the stomach. The amount of saline on the slide at this time plays an important role because if the stomach is lying
on too small a drop of saline, the weight of the coverslip will
rupture the stomach cells; and this will spoil the preparation and make examination for oocysts difficult. On the other hand, if too
much saline is used, the stomach will not flat~en sufficiently.
Therefore, an attempt was made to use more saline on the slide; and
the excess saline was removed by holding a strip of filter paper against the edge of the coverslip. With practice, the right quantity of saline required to flatten the tissue was learned.
To examine for oocysts, the slide containing the specimen was 33 examined with the low power of microscope and reduced light.
Technique for Preparation of Smear of the Mid-gut of Mosquitoes for Ookinetes Study. The mosquitoes were anesthetized about 24 hours after the blood meal, and the legs and wings were removed, as was discussed before. The mid-gut was dissected in saline and the mid-gut containing the blood clot was excised without tearing the wall of the stomach. The malpighian tubules were cut away, and the clot was lifted on to the point of a dissecting needle and was transferred to a drop of saline which was previously placed about one inch from the end of another clean slide. With the aid of a dissecting microscope against a white background, the clot was torn so that the blood became mixed with the saline fluid, trying to confine the released blood to as small a space as possible to prevent any part of it from drying. When the blood and the fluid were thoroughly mixed, the thin films on clean slides were made in the same way as if the material was a drop of normal blood from the wing or the toe nail. After the films were dried, they were stained in the same way as a blood film.
The amount of the saline used was about the same volume as the blood clot. Some dilution, but not too much, was necessary because the blood was semi-digested, coagulated and contained much debris; if the blood was not undiluted, the examination would have been difficult.
Search for ookinetes was made under the oil immersion. 34
Technique for Sectioning of Liver for Exoerythrocytic Study.
At the present time two methods are in use for the staining of sections by Romanowsky stains: one is that described by Hewitt (1939), and the other is that of Shortt and Cooper (1948). The majority of investigators have used the Giemsa-colophonium method of Shortt and
Cooper because they believe that the Giemsa-colophonium method is the better TIlethod and is the only staining method which give good and evenly brilliant staining. Therefore, for this work this stain was also used. The Giemsa-colophonium technique of Shortt and Cooper was originally introduced by Wolbach in 1911 and is more fully described by Bray and Garnham (1962).
Upon autopsy the liver was exposed, and a small piece about 1/2 or 3/4 of an inch in length was excised from the lower border. Care was taken in order to avoid squeezing and distorting the schizonts in the liver. It \vas suggested by Garnham (1966) that it is better not to fix the piece of liver immediately, but to allow it to regain its shape for five or ten minutes first.
The piece of liver was fixed in Carnoy1s fluid (60 mI. absolute ethyl alcohol, 30 IDl. chloroform, 10 mI. glacial acetic acid) for three to four hours depending on the size of the piece. The fixative was replaced by fresh fixative about haJf way through the three or four hours.
The tissue was then placed in 90 percent alcohol for two changes of an hour each. The tissue remained in a third change of 90 percent alcohol for approximately an hour. If a longer period of storage 35 was required, the tissue was put into 70 percent alcohol after all acetic acid had been removed by further washings in 90 percent alcohol. The tissue was dehydrated in 95 percent alcohol and two or three changes of 100 percent alcohol or one hour each. The tissue was cleared in xylene for four to six hours. If for some reason the time was not available, the tissue was cleared in clove oil. If clove oil was used, the xylene was then used for one hour to remove the oil. The tissue was placed in 50 percent wax in xylene, then in graded waxes and embedded. Sections were cut four to six microns in thickness. Four micron sections were especially useful in studying the detail of the parasites. The sections were brought to water by treatment with xylene and alcohol. The sections were washed in water and stained for one hour or longer with Giemsa1s stain. The composition of the staining fluid was as follows:
10 ml. concentrated Giemsa1s stain
10 ml. methyl alcohol
10 mI. acetone
100 ml. distilled water buffered by phosphates to pH 7.2. The sections were washed indiVidually with tap water. All the water was then drained except one or two drops which were always left covering the sections. The slides were held in a level position and about 1/2 ml. of colophonium resin in acetone (15 percent resin) 36
was added to the water covering the slide. Then, the slide was shaken and agitated to ensure mixture and even flow of the mixture over the sections. Agitation continued for three to ten seconds. The
mixture was then poured off the slide, and the section was quickly
washed with colophonium in acetone two or three times until no green color remained in the washings. The slides were washed very quickly in a mixture of 70 percent xylene and 30 percent acetone. In order to remove all traces of
acetone, the slides were repeatedly washed in pure xylene. The xylene was drained off without allowing the sections to dry.
The sections were mounted in green Euparal. As has been
mentioned by Bray and Garnham (1962), the critical part of this procedure is the differentiation of the Giemsa stain by the colo phonium in acetone. In this step the blue color is leached out, the underlying red color is brought out and a balance of blue
cytoplasm and red nuclei in the specimen results. The process of
differentiation is speeded up by the drops of water left covering the section. In the absence of water, colophonium in acetone
differentiates the Giemsa1s stain only weakly and slowly. Experience plays a very important role in this step because the amount of water
to be left on the slide and mixed with the colophonium in acetone is learned only by experience. Consequently, I sectioned and stained the liver gradually in order to avoid the danger of wasting the tissues. 37
Some of the sections were stained in half-strength Giemsa (i.e. 5 ml. of concentrated GiemsaTs stain, 10 ml. methyl alcohol, 10 mI. acetone, 100 ml. distilled water buffered by phosphate to
pH 7.2) overnight. Differentiation was made by adding acetic acid. The acetic acid was washed off, and very little water was left on the slide. Further differentiation with colophonium in acetone was made for 15 to 30 seconds, adding more colophonium in acetone until it was pure for dehydration. Very good results were obtained in this way. RESULTS AND DISCUSSION
A total of 4,988 blood smears from 2,604 birds, representing
38 species was examined. Of these, 20 smears from six species contained Plasmodium.
Table II lists the species found to be infected with Plasmodium during the three-year study. Due to the nature of the avian malaria, th.i.s L not to be interpreted as its incidence or prevalence in the birds examined. In most cases the figure represents the result of examination of two blood smears; in some cases, three or four smears procured at varying intervals. The number and species of birds in which no Plasmodia were found were: 56 Newell ls Shearwater (Puffinus puffinus newelli) 22 Wedge-tailed Shearwater (Puffinus pacificus) 2 Bulwerls petrel (Bulweria bulwerii)
5 Red-tailed Tropicbird (Phaethon rubricauda)
1 Brown Booby (Sula leucogaster) 20 Laysan Duck (Anas laysanensis) 14 Japanese Quail (Coturnix coturnix japonica)
5 Blue (Indian) Peacock (Pavo cristatus) 4 Common Gallinule (Gallinula chloropus) 1 American Coot (Fulica americana)
1 Golden Plover (Pluvialis dominica)
26 Black-necked Stilt (Himantopus himantopus) TABLE II
OCCURRENCE OF PLASMODIUM IN NATIVE AND INTRODUC.w BIRDS IN HA\oJAII
HOST No. of No. of No. of Positive COMMON NAME SCIENTIFIC NAME Birds Smears Smears
Domestic hen Gallus gallus 10 20 1
Rock Dove Columba --1ivia 924 1,845 6
Acridotheres tristis Indian Mynah ---- 73 146 4
House Sparrow Passer domesticus 9 27 3
White-eye Zosterops pa1pebrosus japonicus 85 170 5
Apapane Himatione sanquinea 27 50 1
C;:) \0 40 1 Common or Brown Noddy (Anous stolidus)
2 Gray-backed Tern (Sterna lunata) 482 Spotted Dove (Streptopelia chinensis chinensis) 468 Barred Dove (Geopelia striata striata)
9 Galapagos Dove (Nesopelia galapogoensis) 10 Barn Owl (Tyto alba)
10 Hawaiian Owl (Asio flammeus)
5 Nihoa Millerbird (Acrocephalus familiaris kingi)
8 Elepaio (Chasiempis sandwichensis sclateri)
14 Amakihi (Loxops virens)
14 Anianiau (Loxops parva)
19 Laysan Finch (Psittirostra cantans cantans) 33 Nihoa Finch (Psittirostra cantans ultima) 5 Iiwi (Vestiaria coccinea) 10 Ricebird (Lonchura punctulata)
30 Gray Singing Finch (Serinus leucopygius) 82 Kentucky Cardinal (Richmondena cardinalis) 112 Brazilian Cardinal (Paroaria cucul1ata)
Several species of Plasmodium were observed in this study. These species were as follows:
Plasmodium circumflexum Kikuth 1931
Plasmodium gal1inaceum Brumpt 1935 Plasmodium cathemerium Hartman 1927
and the confirmation of either Plasmodium relictum (Grassi and 41
Feletti, 1891) or Plasmodium matutinum (Huff, 1937) (probably
the latter according to Dr. Marshall Laird, 1970, in litt) need
more investigation or experimental evidence. Plasmodium circumflexum, by far the most frequently observed was found in nine of the 20 infected birds(Table III). This is the first record of this species in the Pacific region (Laird, 1970, in litt.). The species of hosts and numbers of individuals infected are given in Table II. Plasmodium circumflexum is a cosmopolitan avian parasite, and was probably seen on different occasions by the early workers on bird malaria. According to Garnham (1966), the parasite which Labbe
(1899) recorded, under the name of Halteridium danilewskyi, from a lark caught near Paris was possibly this species. Amongst the corpuscular parasites of native American birds described by Opie
(1898), ~. circumflexum may be tentatively identified. The formal description of the organism was made in 1931 by Kikuth when he observed it in a mixed infection with P. relictum in a German thrush
(Turdus pilaris) in Germany. The passerine birds are the favored hosts, but the parasite has been found in the Ruffed Grouse in Canada by Fallis (1945, 1946), and in Canada Geese in Illinois by Levine and
Hanson (1953).
Out of 146 blood smears made from 73 Indian Mynah (Acridotheres tristis), the Plasmodium infection could be detected in only four (5.8%) birds. Three individuals of the mynahs were collected at the Honolulu Zoo and one was from the Island of Maui. All collections were TABLE III
PREVALENCE OF PLASMODIUM IN NATIVE AND INTRODUCED BIRDS IN HA\oJAII
, SPECIES OF PLASMODIUM
Number s Bird Number of birds ;:l s s x ;:l ;:l Examined infected OJ '0-1 OJ S ~ C) ;:l s (j:j OJ res c: ;:l s s c: '0-1 +.J ;:l OJ '0-1 +.J C) C) ..c: rl ;::l '0-1 ~ +.J rl +.J rl '0-1 res res OJ C) C)...b'b s ~ P-II P-II P-II P-Il P-Il
Domestic hen 10 1 - - + --
Rock Dove 924 6 - - - -.1: ~'<
Indian Mynah 73 4 + - -- - -
House Sparro\'l 99 3 - + - --
\\lhite-eye 85 5 + -- - -
Apapane 27 1 - ** - - o;':"'l:
The species is not completely confirmed but a~cording to Laird, 1970, it is probably P. matutinum. ,j:>. * !'J ..::~': Experimental evidences are needed for clear distinction in this case. - 43 made at sea level(Table IV). Among 170 smears made from 85 White-eyes which were collected from the Islands of Oahu, Maui, Kauai, and Hawaii, only five (5.8%) birds from Oahu and Hawaii were infected by Plasmodium circumflexum.
A high incidence of disease was observed among the White-eyes collected from the Volcanoes National Park at an elevation of 4,000 feet; three
out of nine birds collected there were found to be infected. The
other two White-eyes were trapped at sea level on Oahu. The rest of the White-eyes which were not infected by the R. circumflexum, were from two different sources. Some were captured by me at the sea level on Oahu and Maui, and at 4,000 feet elevation on Hawaii; some were trapped by Sincock in the Alakai Swamp and at different elevations on Kauai. The presence of typical Plasmodium cathemerium was observed in the blood smears prepared from House Sparrows. This record is the first certain one from an oceanic Pacific island, although there are reports (some doubtful) from Southeast Asia and adjacent major islands (Laird, in litt. 1970). A total of nine House Sparrows was captured
on Oahu, of which three (33.3%) proved to be infected with ~.
cathemerium,(Table III). In order to facilitate the diagnosis of these
birds T blood smears, the pathogen was transferred from the diseased
House Sparrows to healthy canaries, as described before. The canaries are highly susceptible to certain species of Plasmodium (i.e., cathermerium), and therefore, they are a very good laboratory
animal for malaria studies. All of the canaries finally died after TABLE IV
DISTRIBUTION OF INFECTION RATES BY ISLAND AND ELEVATION
, Locality of :(:nfection rate Elevation Bird collection %
Domestic hen Oahu-Honolulu Zoo 10
Rock Dove Oahu-Honolulu Zoo 0.63 Indian Mynah Oahu-Honolulu Zoo 4.35
Indian Mynah Maui-Hana Section 1.45 Sea-level House Sparrow Oahu-Moiliili area 33.3
White-eyes Oahu-Honolulu Zoo 2.34
\~hite-eyes Hawaii-Volcanoes 3.46 National Park
2,000 feet ana above Apapane Hawaii 3.7
,j::>. ,j::. 45 a period of high infection of J. cathemerium. Some of the slides prepared from House Sparrows showed very distinctive rod-shaped pigment granules more clearly than in most preparations of this c:)mparatively seldom-encountered parasite. Of 50 smears made from 27 Apapane captured on the islands of
Kauai and Hawaii, only one (3.7%) of the Apapane at Volcanoes
National Park at 4,000 feet elevation proved to be infected with Plasmodium. According to my preliminary diagnosis, the parasite seemed to be P. cathemerium, but a more precise diagnosis by Laird (1970) did not confirm my identification, but suggested the possibility of confusion with P. relictum. It should be mentioned in this connection that P. cathemerium has often been confused with Po relictum "which is something of a blanket repository species and subspeciesfT (Laird, in litt.). In many cases, either because there were few parasites in the blood smears or because the staining was unsatisfactory (such as the smear made from Apapane), it was difficult to determine to which species the gametocyte belonged. Further study of the Apapane is required in order to make a clearcut diagnosis of the species Plasmodium involved. In this connection, I might add that
Dr. Miyahara (1968) has been able to detect Plasmodium (species not determined) in the blood smear of another Apapane which was captured by Dr. Berger on Kauai and hand-raised in Honolulu, where the infection undoubtedly was contracted. The remainder of Apapane which showed no sign of Plasmodium infection, were collected at 4,000 feet elevation at Volcanoes National Park on Hawaii, and along 46
the Alakai Swamp trail or other high elevations on Kauai. The Rock Dove was by far the most frequently captured species at
the Honolulu Zoo. A total cf 924 Rock Doves was captured, and 1,845
smears were prepared(Table II). From such a large number of smears
only six cases (.63%) of Plasmodium could be found(Table II~; although a large percentage (65%) of the Rock Doves were infected with
Haemoproteus. None of these smears seemed to have a mixed infection
of both Haemoproteus and Plasmodium. According to my preliminary diagnosis, the possibility of the
presence of Plasmodium relictum was suggested, but careful examination
of slides by Laird (1970) revealed that I was possibly dealing with R. matutinum (Huff, 1937) rather than P. relictum. It should be
mentioned, that, without any experimental evidence, it is som~vhat hazardous to be confident on this point, but, because of a number of
factors, Laird concluded that it was probably~. matutinum. For example, multiple invasion of erythrocytes as seen in the slides is
characteristic of]? matutinum. Also, the appearance of a high degree
of synchronicity, merozoite-invaded RBCs, is strikingly dominant in these slides. Moreover, the vacuolation and pigmentation are in
agreement with!. matutinum, as are the secondary exoerythrocytic
forms. A further point to be considered here is that made by Levine (1962) and Garnham (1966) that R. matutinum is the relictum-like
species characteristic of columbiform birds. Therefore, more bl~od
preparation, and specifically more experimental evidence, is needed in order to draw a clear line of distinction between the two species 47 of Plasmodium mentioned. In spite of close coexistance between the pigeons and the doves, no Plasmodium could be detected in the dove smears. It was interesting to find that 964 smears of Spotted Doves and 936 smears of Barred
Doves were all Plasmodium free. This was, however, not a new discovery, because an attempt was made by Kartman (1949) to learn whether or not the introduced doves are naturally infected with the pigeon parasites. He was not able to find any blood parasite in the blood smears of 43 doves that he examined.
Another species of malaria found during the course of this study was Plasmodium gallinaceum, commonly known as chicken malaria. A limited number (only 10) of domestic hen Gallus gallus was examined, and 20 blood smears were prepared. The rate of infection was 10 percent because only one hen proved to harbor the Plasmodium~able III). Crawford (1945) thought that Jungle Fowls are the natural hosts of P. gallinaceum. These are Gallus lafayetti in Ceylon, Q. sonnerati in Sumatra, and G. bankiva in India. Brumpt (1935), hrn~ever, thought that the natural, wild host is still unknown. He also believed that the Jungle Fowls are relatively resistant, although outbreaks of disease do occur in domestic chickens introduced into areas where the parasite is endemic in wild birds. Brumpt (1935) was able to infect partridges, peacocks, pheasants, and geese experimentally, but according to his studies pigeons, ducks, doves, canaries, House Sparrows, and some other birds are resistant.
According to Garnham (1966), the domestic hen Gallus gallus is 48 the secondary host. I was not able to find any report in the literature regarding the presence of ~. gallinaceum in the Hawaiian Islands, but this organism seems to have accompanied its host to many parts of the tropics.
INFORMATION ON MOSQUITOES
At the present time there are four biting species of mosquitoes present on the Hawaiian Islands. The first species of mosquito
introduced to the Islands was Culex pipiens quinguefasciatus Say, commonly known as the house mosquito. It was brought to the port of Lahaina on the Island of Maui between 1826 and 1830, and, according to Hardy (1960), it is very abundant throughout the Islands wherever suitable breeding habitats occur. The second species of mosquito to be reported in Hawaii was Aedes aegypti (Linnaeus), commonly known as the yellow fever mosquito. The exact date of introduction is not clear, but, according to
Perkins (1913), it was widespread in Hawaii when he first started his work in 1892. Now it is present on the Islands of Molokai, Hawaii, and Lanai.
The third species of introduced mosquito which was Aedes (Stegomyia) albopictus (Skuse), commonly known as the forest mosqUito. According to Perkins (1913), this species did not attract his attention when he first came to Hawaii in 1892, but became abundant sometime later
on. The species was abundant in 1896-1897, when he was continuing his collecting. Now it is present on all Hawaiian Islands. 49
The last mosquito species which was accidentally introduced and established is Aedes vexans nocturnus (Theobald), commonly known as the flood water mosquito. This species was first recorded in
1962 at the Public Health Quarantine Station at Fort Armstrong in Honolulu (Joyce and Nakagawa, 1962). Now it is present on the
Islands of Oahu, Molokai, and Kauai. Among these four species, the night-biting mosquito, Culex pipiens quinquefasciatus Say, has been incriminated as the potential vector of avian malaria. There is experimental evidence which supports this idea. Tempelis et ale (1965-1968) have studied the feeding preferences of the four species of mosquitoes in the Hawaiian Islands. The results of their study indicates that Culex pipiens quinquefasciatus seemed to feed principally on birds (69%). More than half (52%) of the feedings were on chicken, 8.8% on Passeriformes, 2.7% on Red- footed Boobies, and 1% on Columbiformes.
Among other species of mosquitoes, Tempelis et ale found that only 5.8% of~. albopictus had fed on birds, while 94.2% of them had fed on a wide range of mammals. ~. aegypti had a feeding rate on birds of 1.6%; no A. vexans nocturnus had fed on birds.
The high frequency of feeding of Culex pipiens quinquefasciatus
on birds and its wide range of distribution allover the Hawaiian Islands indicate that it should be ecologically capable of serving as an enzootic vector of certain diseases. Although both ~. albopictus and~. aegypti occasionally take blood meals from birds, the low rate of feeding on birds and the restricted distribution (in 50
the case of~. aegypti) in the Islands make it unlikely that they would be important in the transfer of 'arthropod-borne diseases from one susceptible host to another.
So far, the vertical distribution of Culex pipiens guinguefasciatus
has not been thoroughly studied. Winston Banko and others have
recorded Culex pipiens quinquefasciatus breeding at various elevations up to 6,500 feet. These have all been in temporary pools, or in
stagnant water collected in water tanks and watering basins, and it
seems evident that this mosquito is not capable of establishing permanent breeding sites above possibly 2,000 feet elevation. It
seems probable that a major limiting factor in the vertical distribution of this species in Hawaii is the availability of suitable habitats. The aquatic habitats at higher elevations in the Hawaiian Islands are not suitable for this species. Small localized populations sometimes occur in puddles left after a stream has dried up or in stagnant water pools. It seems reasonable to assume that the native birds of high elevations are susceptible to malaria infection. There is some
evidence which supports this idea. One Amakiki brought to Paradise
Park in Manoa Valley on Oahu, died after six weeks. Blood examinations by Dr. Miyahara (1968) revealed the presence of Plasmodium. However,
several other Amakiki survived six months or longer at Paradise Park without contracting malaria. It appears possible that Culex
mosquitoes could have played an important part in the extinction of
native low land birds; however, the experimental evidence is not 51 enough to draw a clear conclusion. Some other ecological factors may have possibly played as important a role as malaria in this matter.
HAEMOPROTEUS INFECTIONS IN BIRDS
During the course of this study, I frequently encountered halter-shaped gametocytes while checking the erythrocytes of the pigeon smears. Haemoproteus infections can be diagnosed by finding and identifYing the protozoa in the stained blood smears. However, not all infections in which gametocytes alone are found are necessarily Haemoproteus infections. Some of them may be Plasmodium.
This fact brought about some confusion for the writer in the beginning of this study, espec~ally when the slide was not properly stained.
A factor that was of great help in diagnosing the Haemoproteus was that schizogony does not take place in the erythrocytes (as it does in Plasmodium) but in the endothelial cells of the blood vessels, especially in the lungs.
As was indicated earlier in this paper, a total of 1,845 blood smears was prepared from 924 Rock Doves at the Honolulu Zoo. I found that 65% of the birds had different degrees of infection with Haemoproteus. I have not had the opportunity so far to determine the exact species involved, but, according to previous investigators, ~. columbae is the species in Rock Doves in Hawaii.
This finding substantiates the work of Alicata (1939) who indicated 52 that Rock Doves in Hawaii are commonly infected with H. columbae, and of Kartman (1949) who claimed that 83% of the Rock Doves were positive for this parasite. The only difference was the rate of the infection: 65% in this work.
It is interesting that the introduced doves are not infected with Haemoproteus even though they live in very close association with the pigeons. In addition to the domestic and wild pigeons,
Mourning Doves, Turtle Doves, and a number of other columbiform birds are the normal hosts of Haemoproteus in other areas.
Of 950 Spotted and Barred Doves examined during the period of
2.5 years, none seemed to harbor the Haemoproteus infection. This work substantiates the findings of Kartman (1949) who failed to transmit the pigeon Haemoproteus to three species of doves and to a young chicken, either by means of the fly vector or by inoculation of macerated flies. This high degree of natural immunity toward the pigeon Haemoproteus can be ascribed to the known high degree of host specificity among many of the Haemosporida.
According to Levine (1961), Haemoproteus species are not an important cause of disease, because they are only slightly, if at all, pathogenic. Infected birds usually show no signs of disease.
In relatively heavy infections, the birds may appear restless, and go off feed, and anemia may result from destruction or erythrocytes, but this is unusual. The pathogenecity of some of the species of
Haemoproteus is still unknown. If the species we are dealing with in Hawaii is columbae; it is only slightly pathogenic, and with the 53 exception of very heavy infection, the host is not harmed.
LEUCOCYTOZOON INFECTIONS IN BIRDS
In addition to Plasmodium and Haemoproteus, Leucocytozoon was another blood parasite I encountered in this study. Leucocytozoon is one of the genera of the family Haemoproteidae in which schizogony takes place in the parenchyma of the liver, kidney, heart, or other organs; but as in Haemoproteus, only sexual stages occur in the peripheral blood. This limitation is somewhat a handicap in studying the biology of the pathogen. Leucocytozoon is common in many wild birds and also causes disease in ducks, turkeys, geese, and chickens.
During this study I was able to detect the Leucocytozoon infection in doves and pigeons. Of 1,900 smears prepared from 950 Barred and
Spotted Doves from the islands of Oahu and Maui, and 1,845 smears made from 924 Rock Doves in Oahu, 38 doves (4%) and 36 Rock Doves
(3.8%) were found to be infected. I am not aware of any previous report on Leucocytozoon infections among Hawaiian birds. Therefore, this report stands as the first one from these islands. The species of Leucocytozoon is not identified yet; therefore, the pathogenecity of the species involved is unknown at the present time. No sign of illness in the infected birds was seen.
Some of the species of Leucocytozoon are markedly pathogenic, and the heaviest losses occur among young birds, but in some sI,ecies the pathogenecity is uncertain or unknown. O'Roke (1934) reported mortalities of 35% to 85% among young ducks in three different years 54 in Michigan, and Savage and Isa (1945) described an outbreak in
Manitoba in which more than 3,000 out of 8,000 birds died in two months. Death in birds is mostly due to obstruction of the circulatory system by large numbers of parasites (Johnson et al., 1938).
According to Levine (1961), the outstanding feature of an outbreak of Leucocytozoonosis is the suddenness of its onset. A group of birds may appear normal in the morning, may become ill in the afternoon, and may be dead by the next morning. Actually affected birds are usually listless and do not feed. Some of the birds which become ill, may recover, but such young recovered birds fail to grow normally, and remain carriers.
The present knowl'~dge of Leucocytozoon infections in the
Hawaiian birds is very limited. Further investigation is needen to determine the prevalence of disease among other birds, the species of the disease, its pathogenecity, and the species of the vector involved. CONCLUSIONS
The presence of several species of Plasmodium among the introduced and native birds at different elevations indicates that the epizootiological significance of the disease and its possible role in the extinction of native Hawaiian avifauna in the lowlands requires further investigations. Circumstantial evidence would seem to indicate that mosquito-borne diseases may have played an important part in the extinction of native birds from lowland areas but are obviously of no epizootiological significance in the highlands, although, the presence of the mosquito vector at 6,511 feet elevation is an indication of the possibility of the disease occasionally occuring at higher elevations. Nevertheless, I feel that no one can say definately that the native birds have been adversely affected by malaria.
It is surprising that in spite of the possible importance of blood parasites among the native and introduced birds, very little care or thought has been given to the complexity of the problem, or how these diseases originally came to Hawaii. I am not aware that any quarantine existed for the birds imported to the territory in the olden days. The birds were imported in the past on the simple basis that someone wanted them. Even today there is no quarantine for pet store birds, except for psittacine birds and poultry from foreign countries.
Importations were made largely as a result of the activities of 56 a few organizations (Fisher, 1948), and the main idea was to introduce and establish songbirds in the Hawaiian Islands. No doubt these groups and individuals believed they were improving the natural attractiveness of the island. However, the benefits derived from these activities can be questioned. It is quite possible that as a result of importations, the native birds and already established exotic birds, were subjected to the danger of unknown diseases. It is also possible that some other factors such as competition for food, etc., or other complicated ecological factors, which were brought about as the by-products of importations, were responsible for the extinction of native Hawaiian avifauna.
The various organisms in any particular environment establish themselves by natural processes over a long period of time. This ecological balance has been so greatly altered in the Hawaiian
Islands that years will be necessary for any sort of equilibrium to be reached. It is impossible to attain the original equilibrium.
In general, a great deal remains to be done before one can claim to have really good information on the blood parasite situation in the Hawaiian birds. The casual observations are all very well; but extensive, careful, thorough surveys would be much more valuable. Furthermore, surveys made at one time of the year or on one age group of hosts may not represent the situation at another time of year or on another age group of the same host. The information on all this is lackin~. 57
The incidence of blood parasites of birds in the Hawaiian
Islands is not the only area on which information is scattered and superficial; but our knowledge of the life cycles, vectors, and pathogenesis is also very poor. We do not know the role of these parasites in the interplay of favorable and unfavorable factors on which their hosts! survival in nature depends. More information is also needed to learn to what extent Plasmodium, Haemoproteus, and Leucocytozoon are pathogenic or nonpathogenic. The writer is hopeful that future investigation will throw further light on our present kno\vledge of avian malaria in the Hawaiian Islands. SUMMARY
1. Avian diseases aid greatly in controlling number of birds,
but we know very little about these in Hawaii. Therefore, an
epizootiological survey of avian malaria in the Hawaiian
Islands was undertaken to acquire some information on the
matter. 2. A total of 4,988 blood smears from 2,604 birds representing
38 species of native and lowland were examined. Of these,
20 smears from 6 species contained Plasmodium. 3. The presence of Plasmodium circumflexum Kikuth, R. gallinaceum Brumpt, R. cathemerium Hartman were proved, and the possibility of confusion between R. matutinum (Huff) and P. relictum (Grassi and Feletti) was revealed. 4. Significantly, these records are the first from the Hawaiian
Islands. P. cathemerium and P. circumflexum are new records
from an oceanic Pacific island.
5. The presence of the potential vector of avian malaria, Culex
pipiens quinguefasciatus Say in artificial receptacle at
6,511 feet elevation indicates that probably one of the
limiting factors in the vertical distribution of Culex is the
availability of suitable habitats.
6. Of a total of 1,845 blood smears from 924 Rock Doves, 65
percent demonstrated different degrees of infection with 59 Haemoproteus. None of the Spotted or Barred Doves examined
seemed to harbor the infection.
7. Of 1,900 blood smears prepared from 950 Spotted and Barred
Doves and 1,845 smears from 924 Rock Doves, only four oercent
were found to be infected with Leucocytozoon. LITERATURE CITED
Aikawa, M., P. K. Hepler, C. G. Huff, and H. Sprinz. 1966. The feeding mechanism of avian malarial paLasites. J. Cell BioI. 28(2):355-373. Alicata, J. E. 1939. Poultry parasites. Hawaii Agr. Expt. Sta. Rept. 1938: 79-82.
1969. Parasites of man and animals in Hawaii. S. Karger, Basel, Switzerland. 190 p.
Banko, W. 1969. Personal communication with D. E. Hardy.
Barber, M. A., and J. B. Rice. 1936. Methods of dissecting and making permanent preparations of the salivary glands and stomachs of Anopheles. Amer. J. Hyg. 24:32-40.
Berger, A. J. 1961. Bird study. Wiley, New York. 389 p. Boyd, G. H. 1925. The influence of certain factors upon the course of infections with Plasmodium praecox. Amer. J. Hyg. 5:818-837.
Bray, R. S. 1957. Studies on the exoerythrocytic cycle in the genus Plasmodium. London Sch. Hyg. Trop. Ned. Mem. 12. H. K. Lewis, London.
Brumpt, E. 1935. Paludisme aviarie: Plasmodium gallinaceum n. Spa de la poule domestique. C. r. hebd. Seance Acad. Sci. Paris. 200:783-786. Celli, A., and F. San Felice. 1891a. Sui parasiti del globulo rosso nell ' uomo c negli animali. Ann. d'lgiene Sperim. Roma. 1:33-65.
1891b. Uber die parasiten der rothen Blutkopperchens in Menschen und in Thieren. Fortso der Med. 9:499-511, 541-552, 581-586.
Coatney, G. R., and R. I. Roudabush. 1937. Some blood parasites from Nebraska birds. Amer. MidI. Nat. 18:1005.
____~.---.-., and E. West. 1938. Some blood parasites from Nebraska birds. II. Amer. MidI. Nat. 19:6010
Conn, H. J., M. A. Darrow, and V. M. Emmel. 19650 Staining procedure. Williams and Wilkins, Baltimore. 289 p. 61
Corradetti, A., P. C. C. Garnham, and M. Laird. 1963. New classification of the avian malaria parasites. Parasitologia. 5(1):1-4.
Crawford, M. 1945. Plasmodiu~ gallinaceum, a malaria parasite of the domestic fowl. Vet. Rec. 57:395-396.
Danilewsky, B. 1885. Zur parasitologie des Blutes. BioI. Centralb. 5:529-537.
1889. Parasitologie comparee du sang. Vol. I and II. Karl Richer, St. Petersberg. 179 p.
1890a. Development des parasites malariques dans les leucocytes des oiseaux. Ann. Inst. Past. 4:427-431.
1890b. Contribution a ITetude des phagocytes. Ann. Inst. Past. 4:432-439. 1890c. Sur les microbes de ITinfection malarique aigue et chronique ches les oiseaux et chez IThomme. Ann. Inst. Past. 4:753-760.
Fallis, A. M., and D. M. Wood. 1957. Bitiug midges (Diptera: Ceratopogonidae) as intermediate host for Haemoproteus of ducks. Canad. J. Zool. 35:425-435.
Fisher, H. I. 1948. The question of avian introductions in Hawaii. Pac. Sci. 2(1):59-64• •, and P. H. Baldwin. 1947., Notes on the red-billed ----~L~e~i~o~t~h-rix in Hawaii. Pac. Sci. 1:54-51.
Garnham, P. C. C. 1966. Malaria parasites and other haemosporidia. Blackwell Sci, Publ., Osford, England. 1114 p.
Giovannola, A. 1934. La colorazione vitale degli sporozoiti ed il suo impiego nella diagnosi dellTinfettivita degli anofeli. Riv. Malar. 13:3-7.
Grassi, B., and R. Feletti. 1890a. Parasites malariques chez les oiseaux. Arch. Ital. de BioI. Turin. 13:297-300.
1890b. Ancora sui parassiti malarici degli uccelli. Boll. mens dellTAcc. Gioenia di Sci. Nat. Catania. Pasco 14:2-7. Hardy, D. E. 1960. Insects of Hawaii. Vol. 10. Diptera: Nematocera-Brachycera. Univ. Hawaii Press, Honolulu. 368 p. 62
Hepler, P. K., C. G. Huff, and H. Sprinz. 1966. The fine structure of exoerythrocytic stages of Plasmodium fallax. J. Cell BioI. 30(2):333-358. Herman, C. M. 1938. The relative incidence of blood Protozoa in some birds from Cape Cod. Trans. Amer. Micr. Soc. 57:132.
1944. The blood Protozoa of North American birds. Bird banding. 15:89.
1954. Haemoproteus infections in waterfowl. Proc. Helminth. Soc. Wash. 21(1):37-42 •
•, W. C. Reeves, H. E. McClure, E. M. French, and ----~W~.~M~c~D-. Hammon. 1954. Studies on avian malaria in vectors and hosts of encephalitis in Kern County, California. I. Infections in avian hosts. Amer. J. Trop. Med. Hyg. 3(4):676-695.
Hewitt, R. 1940. Bird malaria. Amer. J. Hyg., Monograph Sere No. 15. John Hopkins Press, Baltimore.
Huff, C. G. 1927. Studies on the infectivity of Plasmodia of birds for mosquitoes, with special reference to the problem of immunity in the mosquito. Amer. J. Hyg. 7:706-734.
1948. Natural immunity and susceptibility of doves and pigeons to exoerythrocytic stages of Plasmodium relictum. Proc. Fourth IntI. Congr. Trop. Med. Malaria. 602-606.
1954. Merozoite size in exoerythrocytic infections of Plasmodium gallinaceum, P. fallax, P. lophurap., and P. cathemerium. Expt. Parasitol. 3(5):433-444.
1957. Organ and tissue distribution of the exoerythro cytic stages of various avian malarial parasites. Expt. Parasitol. 6(2):143-162.
1963. Experimental research on avian malaria. Adv. Parasitol. 1:1-65.
1965. Susceptibility of mosquitoes to avian malaria. Expt. Parasitol. 16:107-132• ., and F. Coulston. 1944. The development of Plasmodium ----:;-::--:-gallinaceum from sporozoite to erythrocytic trophozoite. J. Inf. Dis. 75:231-249. 63
Johnson, E. P., G. W. Underhill, J. A. Cox, and W. L. Threlkeld. 1938. A blood protozoon of turkeys transmitted by Simulium nigroparvum (Twinn). Amer. J. Hyg. 27:649-665.
Joyce, C. R., and P. Y. Nakagawa. 1962. Aedes vexans nocturnus (Theobald) in Hawaii. Proc. Haw. Ent. Soc. 18(2):273-280.
Kartman, L. 1949. Observations on the Haemoproteus of pigeons in Honolulu, Hawaii. Pacif. Sci. 3:127-132.
Kerlin, R. E. 1964. Venipuncture of small birds. J. Amer. Vet. Med. Assoc. 144(8):870-874.
Kikuth, W. 1931. Immunbiologische und chemotherapeutische studien an verschiedenen stammen von voge1malaria. Centralb. f. Bakt. I. Orig. 121:401.
Koch, R. 1899. Uber die Entwickelung der Malariaparasiten. Ztschr. f. Hyg. U. Infekt. 32:1-24.
Kruse, W. 1890. Ueber Blutparasiten. VirchowTs Arch. 120: 541-560, 121:359-372.
Kudo, R. R. 1966. Protozoology. 5th ed. Thomas Publ., Springfield. 1174 p.
Labbe, A. 1894. Recherches zoologiques et biologiques sur les parasites endoglobulaires du sang des vertebres. A. Hennuyer, Paris. 208 p.
Laird, M. 1962. Malayan protozoa. 5. Two avian malaria parasites. J. Protozool. 9:21-26.
Laird, R. L. 1941. Observations on m0squito transmission of Plasmodium lophurae. Amer. J. Hyg. 34:163-167 .
., and F. A. Lari. 1958. Observations on Plasmodium ----:---circumflexum Kikuth and Plasmodium vaughani Novy and MacNeal from East Pakistan. J. Parasitol. 44:136.
Laveran, A. 1891a. Des Hematozoaires des Oiseaux voisins de IT Hematozoaire du paludisme. Mem. de Ie Soc. BioI. 21 nov.
1891b. Sur des Hematozoaires des Oiseaux voisins de ceux du paludisme. C. R. Soc. BioI. 3:383-386.
Levine, N. D. 1961. Protozoan parasites of domestic animals and of man. Burgess Publ. Co., Minneapolis. 412 p. 64 1962. Geographic host distribution of blood parasites in columborid birds. Trans. Ill. St. Acad. Sci. 55(2):92-111.
., and H. C. Hanson. 1953. Blood parasites of the -----=----.----Canada goose. J. Wildlife Mgt. 17:185.
Lucas, A. M., and C. Jamrox. 1961. Atlas of avian hematology. Agr. Monograph. No. 25. U.S.D.A. 271 p.
Lund, E. L., and M. M. Farr. 1965. Diseases of poultry. H. E. Biester, and L. H. Schwarte (Eds.). Iowa State Univ. Press, Ames, Iowa. 1382 p.
MacCallum, W. 1898a. Notes on the pathological changes in the organs of birds infected with haematozoa. J. Expt. Med. 3:103-116.
1898b. On the haematozoan infections of birds. J. Expt. Med. 3:117-136.
Manwell, R. D. 1933. The behaviour of the avian malaria in the common fowl, an abnormal host. Amer. J. Trop. Med. 13: 97-112.
1935. How many species of avian malaria parasites are there? Amer. J. Trop. Med. 15:265.
1938. The identification of the avian malarias. Amer. J. Trop. Med. 18:565.
•, and C. Herman. 1935. The occurrence of the avian ----::----:-malaria in nature. Amer. J. Trop. Med. 15:661-673.
Miyahara, A. Y. 1968. Personal communication.
Opie, E. 1898. On the hematozoa of birds. J. Expt. Med. 3: 79-101.
O'Roke, E. C. 1934. A malaria-like disease of ducks caused by Leucocytozoon anatis. Wickware. Univ. Mich. School of Forestry and Conservation Bull. 4:1-44.
Ross, R. 1898. Report on the cultivation of Proteosoma, Labbe, in grey mosquitoes. Indian Med. Gaz. 33:401-448.
Russel, P. F., L. S. West, R. D. Manwell, and G. MacDonald. 1963. Practical malariology. 2nd ed. Osford Univ. Press, London. 65
Savage, A. and J. M. Isa. 1945. An outbreak of Leucocytozoon disease in turkeys. Cornell. Vet. 35:270-272.
Shute, P. G., and M. E. Maryon. 1966. Laboratory technique for the study of malaria. 2nd ed. J. & A. Churchill Ltd. 112 p.
Tempelis, C. H., R. O. Hayes, A. D. Hess, and W. C. Reeves. 1965 1968. The blood feeding habits of four species of mosquitoes found in Hawaii. Unpublished data. 16 p.
Van Dine, D. L. 1904. Mosquitoes in Hawaii. Hawaii Agr. Expt. Sta. Bul. 6:1-30.
Van Tyne, J., and A. J. Berger. 1966. Fundamentals of ornithology. Wiley, New York. 624 p.
Warner, R. E. 1968. The role of introduced diseases in the extinction of the endemic Hawaiian avifauna. Condor 70(2): 101-120.
Weathersby, A. B. 1954. The ectopic development of malarial oocysts. Expt. Parasitol. 3(6):538-543.
Wolfson, F. 1941. Avian hosts for malaria research. Quart. Rev. BioI. 16:462.