Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 89, Num. 2, 1994, pp. 145-151

Re-description of Trypanosoma corvi Stephens and Christophers, 1908 Emend. Baker, 1976 and Remarks on the Trypanosomes of the Avian Family Corvidae

NC Nandi, Gordon F Bennett (*)

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Zoological Survey of India, "M" Block, New Alipore, Calcutta 700 053, India (*) International Reference Centre for Avian Haematozoa and Department of Biology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B 3X9

The trypomastigote, epimastigote and amastigote stages of Trypanosoma corvi Stephens and Christophers, 1908 emend. Baker, 1976 from the peripheral blood, heart and bone marrow are described herein. Other trypanosomes described from the Corvidae are compared to T. corvi and their status is discussed.

Keywords: Trypanosoma corvi - redescription - trypanomastigote - amastigote - epimastigote

In 1885, Danilewsky described Trypanosoma avium from the European owl Athene noctua. Since that time, some 98 species and subspecies of avian trypanosomes have been described (Bishop & Bennett 1992). Most of these species are based on the presumption that avian trypanosomes show marked host specificity, a presumption shown to be false, at least in part, through the experimental studies of Bennett (1961, 1970a, b), Baker (1956a, b, c), and Woo and Bartlett (1982), who have all transmitted trypanosomes from one host to other avian hosts of different families. Recent studies on trypanosomes (Woo & Bartlett 1982, Kirkpatrick & Terway-Thompson 1986, Kirkpatrick et al. 1986) have stressed the value of using culture techniques and, in some cases, the identification of trypanosomes is approaching an analogous technology to that used by bacteriologists with differential growth on various culture media. However, these techniques have not as yet been universally applied and morphological description of the parasites in natural preparations is still of vital importance.

In 1908, Stephens and Christophers described T. corvi as follows: "T. corvi (S and C), in Corvus splendens, 40-56 by 3-5 um". This description, unaccompanied by illustrations, was a nomen nudum according to Article 12 of the International Code of Zoological Nomenclature. Therefore, the name is not an available name but may be made available later for the same or different concept. However, in 1976, Baker recognized that this was a nomen nudum and re-described the species from material he obtained from C. frugilegus in the United Kingdom. He designated the type host as C. splendens from India (the same host as specified by Stephens & Christophers) without having seen the material from this species, with additional material from the United Kingdom specimens of C. frugilegus. His rationale for this action was that this term would be used to apply to the large "Megatrypanum-like" trypanosomes found in corvids. One can question the wisdom and validity of this move as the only indication that Stephens and Christophers were looking at a "Megatrypanum" lies in the length measurements that they provided. However, this action has been taken and the species name is currently in the literature and it seems rather pointless at this time to change this designation.

In 1982, Woo and Bartlett described T. ontarioensis from the North American corvid, C. brachyrhynchos. This trypanosome was about half the size of T. corvi, resembling the small avian trypanosomes described by a number of authors from South American birds, and lacked the typical striated appearance of the "avium" group of trypanosomes. The species also differed from T. corvi in a number of cultural characters and was a clearly distinct species. Woo and Bartlett (1982) also found what they presumed to be T. paddae in the same corvid host, and this parasite was again considerably larger than T. ontarioensis. Chatterjee and Ray (1971) described T. avium bakeri from the bulbul, Pycnonotus jocosus in India. Baker (1976) emended the name of this striated trypanosome to T. brimonti bakeri nov. comb. and removed it from consideration as a trypanosome of the corvids. Bennett (1961, 1970b) studied the life cycle, morphology and transmission of a striated trypanosome originally isolated from a saw-whet owl (Aegolius acadicus) which he termed T. avium. He subsequently transmitted this trypanosome to a large number of species of North American birds of several families, including blue jays (Cyanocitta cristata) of the family Corvidae. He also described trypanosomes from naturally infected blue jays which had the same morphological appearance and developmental characteristics in invertebrate hosts as T. avium. Baker (1976) felt that this designation was in error, and in an extensive discussion, following Laveran's (1903) initial restriction of the term T. avium to strigids only, further restricted the term T. avium to those trypanosomes that were derived from Old World Strigiformes only, and restricted trypanosomes of similar morphology found in North American (New World) birds to the species T. confusum Luhe, 1906. Baker acknowledged that his own use of the name T. avium (1956a) for striated trypanosomes from corvids was in error and validated the Stephens and Christophers (1908) name T. corvi by presenting a valid description, although not one based on material from the type host which occurred several thousand kilometres away.

In 1976, 17 tree pies (Dendrocitta vagabunda, Corvidae) were examined for blood parasites over a period of one month; two of the birds were found to harbour trypanosomes in the blood. The birds were sacrificed and various stages of the parasite were found in the blood, liver and bone marrow. This trypanosome from the tree pie is presented herein as a re- description of T. corvi Stephens and Christophers, 1908 emend. Baker, 1976, as it comes from a corvid host from the same geographical area as the original region, in contrast to the description by Baker (1956a) based on material from corvids in the United Kingdom. The description also presents additional material obtained from bone marrow preparations and thus extends Baker's (1956a) description, which was based only on circulating blood forms as well as some forms seen in NNN culture.

MATERIALS AND METHODS

Seventeen tree pies, D. vagabunda (Latham), were captured around Barasat and Basirhat, 24-Parganas District of West Bengal, and maintained in the laboratory for about one month. Two of the birds had trypanosomes in the peripheral blood. The birds were sacrificed and preparations of peripheral blood, heart blood and bone marrow mixed with citrated saline were made. Some of the citrated preparations were examined under cover glass preparations to study the living trypanosomes, while blood smears and tissue imprints were stained with Leishman's, Wright's or Giemsa's stain for more critical morphological study.

Cross-infection studies were also carried out by intra- peritoneal inoculations of citrated heart blood and bone marrow into two pigeons (Columba livia intermedia), two white-throated munias (Lonchura malabarica), two white- breasted water hens (Amaurornis phoenicurus), four house crows (Corvus splendens) and two tree pies (D. vagabunda). One bird of each species was retained as an untreated control and the inoculated birds were examined at irregular intervals for the presence of trypanosomes. The birds were sacrificed after 7-15 days and heart blood and bone marrow (tibia-tarsus) examined for the presence of the parasites.

Re-description of Trypanosoma corvi Stephens and Christophers, 1908 emend. Baker, 1976.

Type host: the house crow, Corvus splendens

  Trypomastigote (Fig. 1; A-D; Table) - 
Trypomastigotes were rare in the peripheral blood (0-3
parasites per slide), slightly more numerous in the heart
blood and numerous in citrated preparations of bone marrow and
occurred as slender, intermediate and broad morphs which
varied in width, length and the shape of the caudal end. The
cytoplasm was increasingly coarser in the broad form and the
number of cytoplasmic vacuoles increased. Longitudinal
striations (myonemes) were most pronounced in the intermediate
forms. An oval vacuole (2-3 um in diameter) adjacent to the
kinetoplast, and larger than the other cytoplasmic vacuoles,
was a constant feature. The kinetoplast was round to oval,
stained deep red and was 0.9-1.8 um in diameter. The nucleus
was granulated, staining pink with the granules arranged
around the periphery on some occasions. A large vacuole, 3.0-
6.0 um was occasionally lodged within the nucleus. The
anterior end gradually tapered to a point in all three morphs.
The slender and intermediate forms were most frequently
encountered and may be considered as most typical of this
species of trypanosome.



     Epimastigote (Figs 3; E, F) - Epimastigotes were
spear-shaped with both ends tapering. They measured 15.0-21.0
um in length (excluding the free flagellum), 2.5-3.8 um in
width at the level of the nucleus; nucleus round, 2.4-3.0 um
in diameter, staining pink and located centrally, 5.5-11.5 um
from the posterior end. Cytoplasm somewhat coarse and staining
blue. Undulating mem-brane narrow but distinct, free flagellum
7.0-9.0 um in length. Material from bone marrow.



     Amastigote (Figs 3, 4; G-I) - Amastigote typically
round, 5.0-7.5 um in diameter, with centrally or eccentrically
placed spherical nuclei. Cyto-plasm somewhat coarse and
vacuolated. All spe-cimens from bone marrow preparation.



  Basis of description - All material from the tree
pie, Dendrocitta vagabunda (Latham), collected by NC
Nandi at Barasat, North 24-Parganas district, West Bengal,
India, 15 June 1976.  All material deposited in the National
Zoological Collections at the Zoological Survey of India,
Calcutta.


                           
                           TABLE


Morphological parameters (in um) of three forms of Trypanosoma 
corvi from Dendrocitta vagabunda compared with measurements 
of the same species by Baker (1956a), Subramanian and Singh (1962) 
and of Trypanosoma ontarioensis by Woo and Bartlett (1982)

=================================================================
Distance                       From tree pie (this paper)^a   
                               -------------------------------
                                slender  intermediate  broad
-----------------------------------------------------------------
Posterior end to kinetoplast 
                (PK)           6.5-10.0   7.0- 8.5   3.5- 6.0 
Kinetoplast to centre of 
        nucleus (KN)           8.0-10.0  11.5-13.5   7.5- 9.0 
Posterior end to centre of 
        nucleus (PN)          15.0-19.5  17.5-21.5  12.5-14.5 
Centre of nucleus to anterior 
            end (NA)          18.0-19.5  30.5-32.5  22.0-23.5 
Length of body without free 
      flagellum (PA)          34.8-38.0  48.5-54.0  35.5-37.5 
Free flagellum  (FF)           5.0-7.0    7.5- 9.0   9.0-11.5 
Width excluding undulating 
       membrane (BW)           3.5-4.5    5.0- 6.0   7.0-8.0  
               PK/PA             0.21       0.15       0.14   
               PN/PA             0.48       0.36       0.38   
Nuclear index (PN/NA)            0.94       0.64       0.58   
Kinetoplast index (PN/KN)        2.00       1.66       1.64    

-----------------------------------------------------------------
                                Baker (1956a)^b    Subramanian   
                                                       and
                                                 Singh (1962)^c
-----------------------------------------------------------------                              
Posterior end to kinetoplast  
                (PK)           10.9 ( 4.7-20.0)   7.5 ( 5.5-13.2)
Kinetoplast to centre of      
        nucleus (KN)                 -                 -         
Posterior end to centre of    
        nucleus (PN)           22.3 (15.7-27.7)        -         
Centre of nucleus to anterior 
            end (NA)                             16.2 (12.5-19.5)
Length of body without free   
      flagellum (PA)           49.2 (35.0-57.5)  34.7 (29.0-44.0)
Free flagellum  (FF)            7.4 ( 4.5-10.2)        -         
Width excluding undulating    
       membrane (BW)            4.6 ( 3.5- 6.0)   5.5 ( 3.5- 7.0)
               PK/PA                 0.22              -         
               PN/PA                 0.45              -         
Nuclear index (PN/NA)                 -                -         
Kinetoplast index (PN/KN)             -                -         

-----------------------------------------------------------------
                                  T. ontarioensis ^d
-----------------------------------------------------------------                                  
Posterior end to kinetoplast      
                (PK)                 1.4 ( 0.8- 2.0)
Kinetoplast to centre of          
        nucleus (KN)                 6.8 ( 5.9- 9.2)
Posterior end to centre of        
        nucleus (PN)                 8.3 ( 7.0-11.6)
Centre of nucleus to anterior     
            end (NA)                 9.6 ( 7.5-16.7)
Length of body without free       
      flagellum (PA)                18.0 (15.3-24.5)
Free flagellum  (FF)                 8.4 ( 5.7-11.6)
Width excluding undulating        
       membrane (BW)                 2.6 ( 2.0- 3.9)
               PK/PA                      0.17
               PN/PA                      0.46
Nuclear index (PN/NA)                     0.86
Kinetoplast index (PN/KN)                 1.22        
=================================================================

a = from Dendrocitta vagabunda. b = from Corvus frugillegus. 

c = from Garrulus lanceolatus.  d = from Corvus brachyrhynchos.



      Camera lucida drawings of Trypanosoma corvi from the tree pie,
      Dendrocitta vagabunda



COMMENTS


The frequency of occurrence of the different forms in the bone
marrow was as follows: amastigotes (9%), epimastigotes (15%),
trypomastigotes (76%). Only 18% of the forms were dividing,
82% were non-dividing parasites.


Dividing forms of the trypanosome (Figs 5-10) were encountered
only in the bone marrow of the tree pies. In addition to the
typical longitudinal binary fission, the trypomastigotes were
also found to divide by a form of plasmotomy in which
elongated tri- or tetra-nucleated trypomastigotes probably
gave rise to epimastigotes (Figs J-L). Most of the
multinucleated trypomastigotes possessed a single kinetoplast,
indicating that the kinetoplast did not divide synchronously
with the nucleus. Normally, the multiplication in the
trypomastigote occurs in a definite sequence, involving
successively division of the basal body, flagellum,
kinetoplast and nucleus, culminating in the cleavage of the
cytoplasm (Hoare 1972). However, Hoare's work was conducted on
mammalian trypanosomes and the same sequence may not always be
followed by avian species of the group. However, Bennett
(1961, 1970b) demons-trated this sequence in the division of
T. avium re-producing in the mid-gut of Aedes
aegypti, so this sequence is not totally foreign to the
avian species. A few dividing amastigotes (Fig. L) with not
more than four nuclei were seen in the smears but a dividing
epimastigote was not detected. Baker (1956a) also described
multi-nucleated crithidial forms in culture that were similar
to those seen in the bone marrow preparations described
here.


The dimensions of the trypanosomes from the tree pie presented
here are similar to those given by Baker for T. corvi
(Table). Although the measurements are slightly smaller, the
range of the measurements embraces those presented by Baker
(1956a) and they are clearly the same species. The sample size
here is considerably larger than that presented by Baker, who
worked with a total of only four specimens (three from the
rook C. frugilegus and one from the jackdaw C.
monedula). Thus the mean values presented for T.
corvi by Baker are not really statistically of importance.
Although three morphs could be distinguished within the tree
pies (including bone marrow), the range of measurements for
each morph broadly overlap the next morph and the measurements
really present a continuum of what is a highly pleomorphic
species.


Subramanian and Singh (1962) described T. garruli
from the black-throated jay, Garrulus lanceolatus
from northern India. The description of the species, including
both measurements and illustrations, are clearly those of
T. corvi (Table).  The two authors were unaware of
Baker's (1956a, b, c) work as they failed to cite any of his
publications and compare their material with his.  They also
describe their trypanosome as lacking a free flagellum, a
condition (see below) that is frequently associated with
moribund trypanosomes, either through faulty preparation or
because they were obtained from dead birds. We consider
T. garruli Subramanian and Singh, 1962 to be a
synonym of T. corvi. Zeiniev (1975) named T.
dschunkowskii from the Eurasian jay G.
glandarius. Unfortunately, as with all Zeiniev's names
presented in this paper (Peirce & Bennett 1979), they were
listed only in a table without illustration or description and
are all nomina nuda. T. dschunkowskii is hereby
declared a nomen nudum. 


The measurements of T. ontarioensis (Table) given by
Woo and Bartlett (1982) indicate a trypanosome which is
considerably smaller in all dimensions and is clearly a
distinct species that is readily separable from T.
corvi. Thus the two trypanosome species described from the
Corvidae are easily separated. Baker (1976, p. 160) concludes
that there are probably no more than 12 valid species of avian
trypanosomes. On the basis of descriptions presented by Baker
(1976) and Bennett (1961) these can be roughly grouped
morphologically into the "avium-like" species with
marked striations (myonemes), the "paddae-like" group
in which the kinetoplast is close to the nucleus, the
"calmettei-like" series in which the kinetoplast is
nearly at the posterior end, and T. everetti Molyneux,
1973 "which does not resemble any previously described avian
trypanosome" (Molyneux 1973a).


Baker considers that T. johnstoni Dutton and Todd, 1903
and T. delhiense Grewal, 1963 may be distinct on the
basis that they lack a free flagellum. However, this is
difficult to accept as most trypanosomes have a free
flagellum, even though such flagella may not be long or very
distinct as in the stumpy forms of T. brucei. It is
more likely that blood smears containing trypanosomes lacking
free flagella were made from dead or moribund birds and that
the trypanosomes involved were either dead or moribund. Such
trypanosomes, in our experience, rapidly change their
morphology, including the loss of the free flagellum. T.
ontarioensis is typical of New World (especially South
American) avian trypanosomes which are extremely small,
usually less than half the length of those seen in Old World
birds. In the over 130,000 records of the International
Reference Centre for Avian Haematozoa, there is only a single
record of such "ontarioensis-like" trypanosomes outside
of the New World (in the collared flycatcher Ficedula
albicollis from Gotland, Sweden). Baker (1976) also
considers that T. macfiei Cotton, 1970 is a distinct
species on the basis of its life cycle and vector. However,
morphologically it falls within the "everetti" group
and, as the life cycles and natural vectors of most avian
trypanosomes are unknown, this becomes a difficult character
to use for speciation of avian trypanosomes at this time. As
virtually all of the named avian trypanosomes fall within the
above species complexes, it is difficult and probably of
little value to make specific comparisons of the morphological
forms seen in a variety of species of birds. It is probably
better to assume at least a familial specificity at this time,
and then synonymize as opportunity and life cycle and cross-
transmission experiments confirm or disprove the species
status of any given form. It is on this basis that we are
assuming that T. corvi is a distinct species, as both
culture and cross-transmission experiments using natural
vectors have been carried out. The re-description of the
morphological forms is given to further substantiate the
specific status of this trypanosome and to provide a broader
base for interpretation of the morphometric measurements than
was previously available.


While a degree of plasmotomy and asynchrony was demonstrated
in bone marrow preparations, the significance of these
observations is as yet unclear. This type of observation has
been so rarely made on avian trypanosomes it is probably
unwise to generalize further until many more such observations
have been made on other species of avian trypanosomes.
However, the appearance of amastigote and epimastigote forms
in the avian host was also reported by both Molyneux (1973b)
for epimastigotes of T. bouffardi and by Chatterjee and
Ray (1970) for both amastigote and epimastigote forms of T.
avium bakeri (= T. brimonti bakeri).


The results of the cross-transmission studies in which
macerated infected bone marrow or heart blood was inoculated
into a variety of bird species proved to be negative. The fact
that neither the type host house crow nor the tree pie became
infected suggested that the transfer technique left something
to be desired and little can be concluded from these
trials.



ACKNOWLEDGEMENTS


To the Director of the Zoological Survey of India, Calcutta,
for providing the facilities used in this study. 



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