MORPHOLOGY AND MORPHOMETRY OF THREE Plasmodium juxtanucleare ( APICOMPLEXA : PLASMODIIDAE ) ISOLATES *

ELISEI C.; FERNANDES, K.R.; FORLANO, M.D.; MADUREIRA, R.C.; SCOFIELD, A.; YOTOKO, K.C.; SOARES C.O.; ARAÚJO, F.R.; MASSARD C.L. Morphology and morphometry of three Plasmodium juxtanucleare (Apicomplexa: Plasmodiidae) isolates. [Morfologia e morfometria de três isolados de Plasmodium juxtanucleare (Apicomplexa: Plasmodiidae)]. Revista Brasileira de Parasitologia Veterinária, v. 16, n. 3, p. 139-144, 2007. Laboratório de Biologia Molecular Sanidade Animal, Embrapa Gado de Corte, Campo Grande, MS, Brasil. E-mail: carinaelisei@yahoo.com.br In this work, three isolates of Plasmodium juxtanucleare have been analyzed based on morphological, morphometric and parasitic parameters. Each isolate was sampled from naturally infected adult chicken (Gallus gallus) from rural areas of three Brazilian municipalities: Seropédica (22° 48’ S; 43° 41’ W), in the state of Rio de Janeiro; Cruzeiro (22° 33’ S; 44° 57’ W), in the state of São Paulo; and Santa Bárbara do Tugúrio (21° 15’ S; 43° 27’ W), in the state of Minas Gerais. The blood samples taken from each infected chicken were inoculated in three groups of ten young chicken (21 days old). Blood smears of the experimentally infected chicken were sampled every two days until the 69th day in order to evaluate the parasitemia. For the morphological-descriptive and morphometric analyses, we measured 30 individuals from each of the intraerythocytic states, measures of the major (MD) and minor diameters (md), the estimation of morphometric index (Mi=md/MD) and size (T=pab, a= md/2; b=MD/2). The results indicated low and homogeneous parasitemia rates in the three strains, which showed differences among shape and size of the parasitic stadia displayed.

The identification of the species of malaria parasites has been frequently based on morphological characteristics of the intraerythocytic stages (MANWELL, 1936).This probably led to precocious description of various new species of Plasmodium, which might be only plastic varieties of previously described species (MANWELL, 1936; VAN RIPER III et al., 1994).In this sense, Ishiguro (1957) considered Plasmodium japonicum as a new species of domesticated chicken parasite, but Akiba (1959), based on biological and morphological characteristics, found that P. japonicum and P. juxtanucleare are the same species.Manwell (1943), in studies based on morphological characteristics, raised the hypothesis that P. nucleophilum (parasites of Passeriformes) and P. juxtanucleare could be the same species.Nevertheless, this author had found that experimental infection of P. nucleophilum in chicken support the validity of distinct species.
The current studies concerning P. juxtanucleare do not clarify the biology of this parasite in a complete way.There are doubts about its biological cycle in vertebrate and invertebrate hosts, and little is known about its morphological and morphometric characteristics.In this work, we described the intraerythocytic parasites forms of three P. juxtanucleare isolates regarding their morphological and morphometric aspects and some biological parameters, such as the prepatent period and peak of parasitemia, under experimental infection.We measured these characteristics and compared their variance in order to verify the existence of significant differences within a group of organisms infected experimentally by a single isolate and also among groups infected by different isolates.

MATERIAL AND METHODS
Three adult chicken (G.gallus) naturally infected with P. juxtanucleare were sampled from different rural areas: Seropédica, in the state of Rio de Janeiro (22° 48' S; 43° 41' W), Cruzeiro, in São Paulo (22° 33' S; 44° 57' W), and Santa Bárbara do Tugúrio, in Minas Gerais (21° 15' S; 43° 27' W).In order to raise the number of parasites in each individual, they received an intramuscular injection of 26 mg/Kg of methylprednisolone acetate (SOUZA, 1998) in the breast region (single dose) to suppress the immunological system.After that, the birds were accompanied and peripheral blood smears were prepared daily, until the 28th day, when 15 mL of the blood of each individual were extracted through radial puncture.
To measure the parasitemia, infection dynamics and parasite size and form, 40 one-day-old chicks of commercial laying lineage were acquired and kept in a covered enclosure protected against mosquitoes.At 21 days of age, the chicken were examined through blood smears and divided into four groups of ten chicks each.In three of these groups, each chick was inoculated, by intraperitoneal injection, with 1mL of the infected blood of the infected chicken of Seropédica (Irj), Cruzeiro (Isp) and Santa Bárbara do Tugúrio (Img).The control group received 1mL of physiological solution.
The parasitemia of the chicken in each group were accompanied by peripheral blood smears every two days, from the first to the 69th day after inoculation (DAI).The blood smears were fixed in methanol (methyl alcohol P.A. -Merck®), air dried, stained with Giemsa (methyl blue eosin, Giemsa -Merck®) and observed under an optical microscope (Leitz Wetzalar -Dialux 20 EB) with a 10X eyepiece and 100X immersion objective lens.
Since most of the time each erythrocyte is infected by only one parasite, the number of parasites were counted by the number of infected erythrocytes in 100 homogeneous microscopic fields (SOUZA, 1998) of each blood smear (parasite density), and the parasitemia was calculated by multiplying this number by 100 and dividing it by 10,000 (x*100/10.000).During the count process, we also classified the intraerythocytic stage of each parasite.
For the morphological-descriptive and morphometric analyses, we measured 30 individuals from each of the intraerythocytic stadia (trophozoites, schizonts, macrogametocytes and microgametocytes).The measurements were taken from the blood smears with the highest parasite peak per isolate.During the morphometric analysis, we measured the major and minor diameters (MD and md) of each parasite.For that, an ocular micrometer (Wild-pZo -15x) was attached to the microscope.These measures were used to calculate the parasites morphometric index (MI=md/MD) and size index (T=pab, with a=md/2 and b=MD/2, based on the formula for the area of an ellipse).
The statistical treatment of the data was done with variance analysis (ANOVA), to detect heterogeneity within and between samples.Since the data obtained were not normally distributed, a condition for use of parametric tests, Log (x+1) correction was applied.

RESULTS
The naturally infected chickens of the states of Rio de Janeiro, São Paulo and Minas Gerais presented parasitemia of 0.03%, 0.01% and 0.04% respectively, before the administration of methylprednisolone acetate.After 28 days, the parasitemia has increased to 13.4%, 8.6% and 14.2%, respectively, and the blood was extracted and inoculated into the three experimental groups studied here.The following results refer to the three experimental groups of chicken inoculated with three isolates of P. juxtanucleare: Irj, Isp and Img.The control group remained parasite free during the entire experiment, showing that the protection against infection by natural vectors was effective.Figure 1 shows the different stadia of P. juxtanucleare (Trophozoite, Schizont, Microgametocyte and Macrogametocyte).
The pre-patent period (PPP) of the experimental inoculation of the three P. juxtanucleare isolates was, on average, 14.2, 14 and 16.8 days in the Irj, Isp and Img isolates, respectively.The maximum parasitism (MP) occurred, on average, 45 days after inoculation (DAI) in Irj, 55 DAI in Isp and 45 DAI in Img (Figure 2).The parasitism for the three inoculates remained low, with some fluctuations within the groups inoculated with the different isolates (Figure 2).Nevertheless, we found no significant differences among the parasite densities within (P > 0.05) and among (P > 0.05) the isolates, which averaged 4.8% (Table 1).
The mean trophozoite parasitemia of the Irj, Isp and Img isolates were, respectively, 3.41, 2.52 and 3.76; and we found no significant differences in the parasitemia of trophozoites within (P> 0.05) and among (P> 0.05) the isolates (Table 2).Within the parasite stadia, the trophozoites presented the largest number; a result previously obtained both in observations of natural (SOUZA, 1998;SANTOS-PREZOTO, 2004) and experimental infections (SOUZA, 1998;ELISEI, 2001).The mean parasitemia of the schizonts was 0.54 (Irj),   0.44 (Isp) and 0.50 (Img), and there were no significant differences among the parasitemia of schizonts within (P > 0.05) and among (P > 0.05) isolates (Table 2).From two to four merozoites per schizont were observed in the three isolates.
The trophozoites presented significant differences in form (MI) and size (S) among the isolates.The schizonts did not have any significantly different forms in comparing the isolates, but there were size differences, which were on average greater in the group inoculated with Irj.The variance in the sizes was greatest in Img and smallest in Isp (Table 3).
Regarding the macrogametocytes and microgametocytes, only in the former we have detected significant differences in form among the groups studied.There were significant size differences among the groups in both parasite stadia (Table 3).

DISCUSSION
In this work, we measured three isolates of P. juxtanucleare for their morphometric characteristics and parasitemia indices.To avoid deviations due to the particularities of the original hosts, each isolate was inoculated in 10 experimental birds, and all the measurements were homogeneous in each host group studied, showing that the differences found are due to real differences among the isolates.Since we did not found any infection signal in the control group, we can conclude that the protection system used during the experiments was efficient against infection by natural vectors.
The morphometric analysis revealed significant differences of form (measured by the MI) among the isolates in the trophozoite and macrogametocyte stadia, while size differences were found in all parasite stadia (Table 3).These differences emphasize the need of the study of a larger number of samples, in order to reach a more complete scenario of the variation spectrum of this parasite, and avoid the miss description of new species.As an example, this kind of data, the description of P. japonicum (ISHIGURO, 1957) could be avoided.On the other hand, the morphometric measurements of P. nucleophilum appear to be within the variation of P. juxtanucleare, but the maintenance of its status as a species was possible due to the characteristics of association with the host.Hence, other techniques of characterization, such as using molecular markers, could be useful in species identification.Besides this, since experimental hosts are theoretically homogeneous, one can infer that this variation is largely due to the intrinsic genetic variation of this type of characteristic.
Most of the parasitemia measures, particularly total parasitism (which after all can be used as a measure of the parasite's pathogenicity), showed that the three isolates are homogeneous.Nevertheless when they were separated into their components, only the parasite densities of the trophozoites and schizonts did not differ significantly among the isolates, while there were differences in the parasite densities of macro and microgametocytes.It is possible, however, that these differences rose due to problems of visual distinguing between macro and microgametocytes.Since the main distinction between these forms is basically revealed with coloration differences, with macrogametocytes generally being more colored (AIKAWA; JORDAM, 1968), it is possible that gametocytes with coloring problems or in intermediate stadia of formation were not adequately identified ( VAN RIPER III et al., 1994;AIKAWA;JORDAM, 1968;GARNHAM, 1966).With this, there are two contrasting results: the isolates of P. juxtanucleare differ in form and size, as well as in some aspects of parasitism, but not differ in those characteristics related with the pathogenicity.The parasitism in all isolates was considered as moderate to low.Besides this, none of the birds involved in the experiment showed clinical symptoms of malaria.Souza (1998) and Elisei (2001) found the same results under experimental conditions.Santos-Prezoto et al. ( 2004) also found low parasitism in animals naturally infected with P. juxtanucleare, showing that low parasitism is not a exclusive consequence of experimental parasite inoculation.Together, these results corroborate the hypothesis that infection by P. juxtanucleare is an illness of secondary character due to an intrinsic limitation to the development of this parasite, which may be associated with its low virulence or an efficient host immune response (SANTOS-PREZOTO et al., 2004).
In this way it is interesting to note that P. juxtanucleare is part of the Novyella group, composed exclusively of species of Plasmodium known for low pathogenicity (BENNETT, 1970).Two alternative hypotheses can be invoked to explain the low variability in parasitemia and the low pathogenicity of P. juxtanucleare in G. gallus.
Supposing that the parasitemia is directly related to pathogenicity, the first hypothesis is exclusively adaptive, in the sense that highly virulent strains (with high parasitemia) tend to kill their hosts, with consequent elimination of the strain.On the other hand, strains with very low parasitism are not transmitted by vectors.Hence, natural selection would favor intermediate parasitemies.The second hypothesis is that the low parasitemia variability is an inherited characteristic of P. juxtanucleare ancestors (theoretically ancestors of the genus Novyella).A test of the second hypotheses would be to construct a phylogenetic species tree of Plasmodium using independent characteristics of the parasitemia measures, permitting measurement of the phylogenetic signal of this characteristic through comparative methods (which has been performed elsewhere).If an evident phylogenetic signal is detected, the first hypothesis is weakened, dispensing the need for new tests.If the phylogenetic signal is weak, the first hypothesis will be strengthened, which should then be tested more intensely.

Table 1 .
Analysis of the parasite density among isolates of Plasmodium juxtanucleare, experimentally inoculated in chicks (Gallus gallus).

Table 3 .
Analyses of variance of the form and size of the parasite stadia of different isolates of Plasmodium juxtanucleare

Table 2 .
Analysis of variance of the parasite density of the intraerythocytic stadia of the different isolates of Plasmodium juxtanucleare.