Hepatic Profile of Gallus Gallus Linnaeus, 1758 Experimentally

Veterinary Parasitology 175 (2011) 207–211

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Hepatic proﬁle of Gallus gallus Linnaeus, 1758 experimentally infected by Plasmodium juxtanucleare Versiani & Gomes, 1941

Usha Vashist a, Aline Duarte Falqueto a, Danilo Lustrino b, Victor Menezes Tunholi a,b, Vinícius Menezes Tunholi-Alves a,b, Marcos Antônio José dos Santos c, Marta D’Agosto d, Carlos Luiz Massard a, Jairo Pinheiro b,∗

a Curso de Pós-Graduac¸ ão em Ciências Veterinárias, Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ 23.890-000, Brazil b Departamento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ 23.890-000, Brazil c Departamento de Biologia Animal, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ 23.890-000, Brazil d Departamento de Zoologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-900, Brazil

article info abstract

Article history: One of the species that causes avian malaria is Plasmodium juxtanucleare. It is commonly Received 24 June 2010 found in poultry, especially when the birds receive food free of coccidiostats. Since industrial Received in revised form 14 October 2010 and organic poultry breeding is increasing in the world and few studies have been conducted Accepted 14 October 2010 examining the clinical parameters of both healthy and infected birds, this work evaluated whether the infection caused by P. juxtanucleare in Gallus gallus provokes alterations in the Keywords: birds’ hepatic profile. We analyzed the activity of ALT and AST and carried out histological Avian malaria analyses of liver sections of infected fowls by intracelomic inoculation with infected blood Aminotransferases Plasmodium juxtanucleare from a donor fowl with a parasite load of around 7%. The infected birds’ parasite load was evaluated during 45 days by means of blood smears. There was a positive correlation between the increase in parasite load and higher ALT activity in the infected fowls, but there was no significant variation of the AST activity between the control and infected groups, possibly because of the non-specificity of this enzyme as an indicator of hepatic lesion. The results show that infection caused by P. juxtanucleare in G. gallus provokes hepatic alterations, indicated by the increase in the ALT enzyme activity and by the inflammatory infiltrates found in the liver sections of the infected fowls. © 2010 Elsevier B.V. Open access under the Elsevier OA license.

1. Introduction Besides domestic chickens, there are reports of other fowls being infected by P. juxtanucleare: Gallus lafayet- Plasmodium juxtanucleare Versiani & Gomes, 1941, the tei Linnaeus, 1758 (jungle fowl) in Sri Lanka (Dissanaike, agent that causes avian malaria in Gallus gallus Linnaeus, 1963); Bambusicola thoracica sonorivox Linnaeus, 1758 1758, was observed for the ﬁrst time in Brazil when (bamboo partridge) in Taiwan (Manwell, 1966); Fran- researchers studying avian spirochetosis at a street mar- colinus spp. (partridge) in Africa (Mohan and Manwell, ket, while examining blood samples drawn from live fowls, 1966); Crysolophus pictus Linnaeus, 1758 (golden pheas- observed a small parasite with rounded or irregular shape, ant), Lophura nyctemera Linnaeus, 1758 (silver pheasant), always found near the blood cell nuclei. For this reason, it Crysolophus amherstiae Linnaeus, 1758 (Lady Amherst’s was named P. juxtanucleare (Versiani and Gomes, 1941). pheasant) and Phasianus colchicus Linnaeus, 1758 (com- mon pheasant) in Brazil (Massard and Massard, 1981); and Meleagris galopavo Linnaeus, 1758 (Turkey), also in ∗ Corresponding author. Tel.: +55 21 26823222; fax: +55 21 26821763. Brazil (Serra-Freire and Massard, 1979). The invertebrate E-mail address: [email protected] (J. Pinheiro). hosts of P. juxtanucleare are mosquitoes of the Culicini

0304-4017 © 2010 Elsevier B.V. Open access under the Elsevier OA license. doi:10.1016/j.vetpar.2010.10.031 208 U. Vashist et al. / Veterinary Parasitology 175 (2011) 207–211 tribe (Versiani and Gomes, 1941; Bennet et al., 1966; were fed Purina Natural® free of antibiotics and coccid- Garnham, 1966; Krettli, 1972; Lourenç o-de-Oliveira and iostats and given water ad libitum. At 60 days-old, the Castro, 1991). birds were separated into two groups of 12 animals each, The pathogenicity of P. juxtanucleare can be related to a control group and a group infected by P. juxtanucleare. the strains in which it produces exoerythrocytes stages, The control group was intracelomically inoculated with which can mainly be found in the spleen, along with 0.5 ml of physiological saline, while the infected group, the liver, lungs, bone marrow and brain (Paraense, 1947; by the same route, received 0.5 ml of blood from a donor Krettli, 1972). The number of parasited erythrocytes is gen- bird infected by P. juxtanucleare, with a parasite load of erally low in naturally infected fowls. For this reason, the around 7%. The parasite load of the birds in the two increase in blood parasite loads is usually slow due to the groups was then monitored by examining blood smears small number of merozoites produced by the schizonts in from a drop of blood drawn from the fine wing capil- relation to the plasmodia that afflict humans (Massard and laries. The blood smears were examined daily during the Massard, 1981). first 15 days after inoculation, the most critical period for Poultry breeding in recent decades has been growing experimental infection caused by P. juxtanucleare accord- in importance in the world, generating large capital move- ing to the literature (Vashist et al., 2008, 2009). Afterward, ments and increasing the number of rural jobs (Mota et al., they were examined every three days until the 42nd day 1998). Besides this, ecological problems related to cat- post-inoculation, the period in which according to the lit- tle raising, such as soil compactation and release of large erature the parasitemia tends to become chronic (Silveira amounts of methane by cattle, lead many experts to predict et al., 2009; Vashist et al., 2008, 2009). The smears were that the poultry industry will gain even more importance taken to the laboratory, fixed in methanol for 3 min and in the coming years. In addition, birds have shorter breed- stained with Giemsa stain, diluted in distilled water (1:4) ing cycles, require less space, a reduced amount of money for 45 min. A hundred fields per slide were observed using directed to its growth and they are an excellent source of a light microscope at 1000×. The total number of evo- protein for humans. But because industrial-scale breeding lutive forms of P. juxtanucleare found in each smear was operations make it easier for disease to spread among the recorded. Each week about 1 ml of blood was drawn from animals, there is a need for better methods to diagnose each fowl for hematocrit determination, by the microhe- avian diseases and to study the ways these diseases can matocrit technique, and for analysis of the activity of the affect the birds’ productivity. aspartate aminotransferase (AST) and alanine aminotrans- Biochemical variables have been used to diagnose dis- ferase (ALT) enzymes. For the aminotransferase analyses, eases in pets and producing animals (Borsa et al., 2006). 0.5 ml of ALT or AST substrate (a solution of 0.2 M l-alanine In the current literature there are no studies on the hep- or 0.2 M l-aspartate, respectively, 0.002 M ␣-ketoglutarate atic profile of birds infected by P. juxtanucleare, although and 0.1 M sodium phosphate buffer, pH 7.4) was incu- this is an important parameter that can be used to evaluate bated at 37 ◦C for 2 min. Then, 100 ␮lor200␮l of serum poultry health. (for ALT or AST, respectively) was added and the solution Aminotransferases (ALT and AST) are a group of was homogenized and incubated again at 37 ◦C for 30 min. enzymes that catalyze the interconversion of amino acids After that, 0.5 ml of 0.001 M 2.4-dinitrophenylhydrazine into ␣-ketoacids by transfer of amine groups (Moss and was added and maintained at 25 ◦C for 20 min. The reaction Henderson, 1998). Aminotransferases play an important was finalized by adding 5 ml of 0.4 M NaOH. The read- role in the link between the amino acids and carbohy- ings were taken in a spectrophotometer at 505 nm and drates metabolism. They are an essential group of enzymes the results were expressed as URF/ml (Kaplan and Pesce, for gluconeogenesis, besides being excellent indicators of 2003). hepatic lesions (Pinheiro et al., 2001). To study the possible types of hepatic lesions result- This article reports an experiment to verify changes ing from the infection by P. juxtanucleare, liver fragments in the hepatic profile of G. gallus in response to infection were taken from two fowls from each group at the end caused by P. juxtanucleare. of the 45-day experiment. For this, liver fragments of about 1 cm in diameter were taken during necropsy from 2. Materials and methods the left liver lobe of each fowl. The fragments were fixed in 4% formalin for 24 h at 4 ◦C and then main- This experiment was performed on 24 hens of the Cobb tained in 70% ethanol. These tissues were processed breed, purchased as day-old chicks. The chicks were vac- according to routine histological techniques, embedded cinated against fowl pox, gumboro disease and Marek’s in paraffin, sliced into 5-␮m sections with a microtome disease. The chicks were taken from the commercial estab- and mounted on glass slides. The sections were stained lishment and transferred to the W.O. Neitz Parasitology with hematoxylin–eosin and photographed with a Nikon Laboratory, Universidade Federal Rural do Rio de Janeiro, Coolpix 4300 digital camera coupled to a Hund Wetzlar in Seropédica, RJ, Brazil, where they were kept in asbestos H600 microscope. boxes (2 m × 1 m) with rounded edges and a layer of wood The parasite load values were expressed as the aver- chips spread at the bottom until they reached the age of age number of parasites. The values of the AST and ALT 15 days. Then they were transferred to a coop (5 m × 5m) activity measured in the birds during the experiment were previously cleaned and disinfected with a blowtorch. At expressed as the mean ± mean standard error (X ± SEM) 45 days of age the birds were transferred to suspended and were submitted to variance analysis (ANOVA) at 5% sig- cages (150 cm × 90 cm). Throughout the experiment, they nificance and the Tukey test for comparison of the means, as U. Vashist et al. / Veterinary Parasitology 175 (2011) 207–211 209

Fig. 3. Percentual variation in the hematocrit of birds uninfected (control) and Plasmodium juxtanucleare-infected through the period post inoculation analyzed: (0) week before inoculation, (1) ﬁrst week after inoculation, (2) second week after inoculation, (3) third week after inoculation, (4) fourth week after inoculation, (5) ﬁfth week after inoculation. Values are Fig. 1. Erythrocytic stage of Plasmodium juxtanucleare (arrow) found in expressed by X ± SEM, ˛ = 5%. the Gallus gallus of the infected group. well as Spearman’s test at 5% to check the relation between parasitemia and the AST and ALT activity.

3. Results

All the erythrocytic stages of P. juxtanucleare (trophozoites, schizonts and gametocytes) were observed in the infected group (Fig. 1). The pre-patent period and peak parasite load occurred between the seventh and eighth day, after which it declined (Fig. 2). The fowls of both groups Fig. 4. ALT activity in the uninfected (control) and Plasmodium had hematocrits above 26.9%, considered normal for fowls juxtanucleare-infected through the period post inoculation analyzed: (0) (Fig. 3). week before inoculation, (1) first week after inoculation, (2) second week With respect to ALT activity, there was a significant after inoculation, (3) third week after inoculation, (4) fourth week after increase in the first week of infection (27.99 ± 1.58) in inoculation, (5) fifth week after inoculation. Values are expressed by ± ˛ a,b ± X SEM, = 5%. Different letter indicate means with significant differ- relation to the baseline value (week 0; 13.83 1.57), but ence among them (˛ = 5%). this increase was also observed in the control group (25.3 ± 1.21). However, the second week of infection, the ALT activity of the infected group (24.14 ± 2.41) There were significant differences in the AST activity in remained significantly higher than that of the control group the first week of the experiment in both the infected group (19.99 ± 1.74). There was also a correlation between the (144.67 ± 3.65) and control group (149.68 ± 2.73) in rela- peak parasite load and highest ALT activity in the infected tion to the baseline value (109.86 ± 2.23), but starting in group in the second week of the experiment. Starting in the third week the values leveled off (Fig. 5). the third week, there was no longer a significant difference between the control and infected groups in relation to the baseline value (group 0) (Fig. 4).

Fig. 5. AST activity in the uninfected (control) and Plasmodium juxtanucleare-infected through the period post inoculation analyzed: (0) week before inoculation, (1) first week after inoculation, (2) second week after inoculation, (3) third week after inoculation, (4) fourth week after inoculation, (5) fifth week after inoculation. Values are expressed by Fig. 2. Average number of parasites (Plasmodium juxtanucleare) found in X ± SEM, ˛ = 5%. a,bDifferent letter indicate means with significant differ- the birds (Gallus gallus) infected during 42 days of the experiment. ence among them (˛ = 5%). 210 U. Vashist et al. / Veterinary Parasitology 175 (2011) 207–211

and Massard, 1981; Oliveira et al., 2001). This can also be related to the isolate’s pathogenicity. The infected group had lower hematocrit values than those in the control group in the last weeks of the experiment, but all the values observed for both groups were within the normal range according to other authors (Lucas and Jamroz, 1961; Campbell and Dein, 1984). Some authors mention anemia in poultry infected by P. juxtanucleare with high parasitemia, but this did not occur in this study, prob- ably due to the low parasite load in the infected birds. With respect to the activity of the hepatic ALT and AST enzymes, reports in the literature mention that factors related to climate, type of food and management can influ- Fig. 6. Dark-colored liver (asterisk) from one of the birds (Gallus gallus) ence the results of these analyses (Borsa et al., 2006). In of the Plasmodium juxtanucleare-infected group. the first week of the experiment, the control and infected The livers of the infected birds had darker colored areas groups showed higher activity of AST and ALT than in the than normally observed (Fig. 6). Microscopic examination other weeks. A possible explanation is that the fowls, still of the hepatic tissue fragments revealed the presence of adapting to being handled to collect blood samples, could vacuolized and tumefied hepatocytes, extensive hemor- have been stressed, but as the experiment proceeded the rhage, proliferation of fibrous conjunctive tissue in the birds were better acclimated, reducing the stress, and the portal space, multifocal and diffuse subcapsular (lym- parameters were only affected by the infection. In the sec- phoplasmocytic) inflammatory infiltrate in the portal and ond week, both enzymes presented a profile similar to the periportal areas and the parenchyma, sinusoidal conges- baseline value, except for the infected group in relation to tion, dilatation and intrahepatic cholestasis (Fig. 7). the ALT activity, demonstrating that this enzyme is altered by infection by P. juxtanucleare, since its respective con- 4. Discussion trol did not differ significantly from the baseline value. The peak parasite load occurred at the start of the sec- The most abundant blood stages in this study, the ond week after inoculation and the highest ALT activity in trophozoites, were the same as those found in other stud- the infected group occurred at the end of the first week ies (Santos-Prezoto et al., 2004; Silveira et al., 2009; Vashist and start of the second week. There was a positive correla- et al., 2008, 2009). tion between the peak parasitemia and highest ALT activity. The pre-patent period of the isolate studied, about ALT is predominantly found in the hepatocytes, located in two days, was shorter than the majority of the peri- the cytoplasm. Aggression to the hepatocytes triggers the ods mentioned in the literature for infection caused by release of ALT. The activity of this enzyme in small ani- P. juxtanucleare, which vary from four to eighteen days mals such as chickens is widely used to determine hepatics (Versiani and Gomes, 1941; Dhanaphala, 1962; Massard pathologies (Kramer, 1989). Studies revealed that ducks and Massard, 1981; Oliveira et al., 2001). This varia- inoculated with the hepatitis virus showed higher activ- tion can be explained by the strain’s pathogenicity: more ity of ALT (Ahmed et al., 1961). The increase in the serum pathogenic strains have shorter pre-patent periods. activity of these enzymes attributed to hepatic dysfunction In this study the peak parasite load of the infection can be due to the rupture of hepatocytes, resulting in necro- occurred earlier than reported elsewhere in the literature sis or alterations of the permeability of the cell membrane (Versiani and Gomes, 1941; Dhanaphala, 1962; Massard or a process of cholestasis (Kaneco, 1989). The activity of AST did not vary significantly in the infected group in relation to the control group during the experiment. Some authors have mentioned cases of chronic hepatic damage that produced subtle ruptures in the hepatics cells, but without altering the normal serum activity of AST (Fudge, 2000). This enzyme is considered a non- specific market because it is found in various tissues, but it is also considered a highly sensitive indicator of tissue lesion, more closely related to recent tissue injury and reduced organ function (Lumeij and Westerhof, 1987). The baseline level of the enzyme in the plasma is generally a reflection of its accumulation and the change undergone by the tissue containing the enzyme. Analysis of the activity of AST is often utilized to complement another diagnostic method rather than as a principal parameter in itself. The fact that the AST activity in this study did not vary significantly between the two groups was possibly due to the Fig. 7. Hepatic tissue from a bird (Gallus gallus)ofthePlasmodium juxtanucleare-infected group, with inflammatory infiltrates (arrow head) unspecific nature of this enzyme as an indicator of hepatic (scale bar = 50 ␮m). lesion. U. Vashist et al. / Veterinary Parasitology 175 (2011) 207–211 211

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