COMPARATIVE STUDY OF PATHOGENICITY BETWEEN ASPERGILLUS FUMIGATUS AND PENICILLIUM CHRYSOGENUM IN MICE

168 COMPARATIVE STUDY OF PATHOGENICITY BETWEEN ASPERGILLUS FUMIGATUS AND PENICILLIUM CHRYSOGENUM IN MICE S. N. Yassein Assist. Prof. Dept. of Microb., Coll. of Vet. Med. / University of BaghdadIraq. Shaimaa_nabhan@yahoo.com ABSTRACT This study was conducted to compare between two types of opportunistic fungi (Aspergillus fumigatus and Penicillium chrysogenum) in concerning their pathogenicity after intraperitoneal inoculation of mice. A total of twenty four male albino mice were used in this study which divided equally into 3 groups, The first and second groups were inoculated with 0.2ml of 1x 10 7 spores/ml of A. fumigatus and P. chrysogenum intraperitonially respectively, while the third group was inoculated with normal saline which served as control group. All animals were monitored for 2 weeks after infection. The blood samples were collected by heart puncture after 18 days post infection to isolate of serum that used for biochemical analysis of liver and kidney functions. After that, all animals were sacrificed. Some internal organs of infected groups (liver, kidney, intestine, heart, spleen and lung) were taken to study the histopathological changes. It was found that there was severe histopathological changes in studied organs of infected mice particularly liver, kidney, spleen and intestine which corresponding with significant variation (p<0.01) in enzyme activities of liver and kidney like (Alanine Aminotransferase (ALT), Urea and Creatinine). Also, It was found that P. chrysogenum had more impact on these enzymes (15.65 ± 0.78, 135.23 ± 8.75 and 0.928 ± 0.02 respectively) than A. fumigatus (21.70 ± 1.04, 57.91 ± 5.99 and 0.587 ± 0.03 respectively). Therefore, the present study indicated that fungi present in the environment can induce severe inflammation reach to tissue damage in most vital internal organs So, further studies should be performed to determine the specific virulence factors and active components, which are responsible for pathogenesis of A. fumigatus and P. chrysogenum in spite of the fact that P. chrysogenum can produce antibiotic.


INTODUCTION
Aspergillosis is an umbrella term that contain wide range of diseases from localized to fatal disseminated infections in human and various types of animals. (24). Aspergillus fumigatus is the most common and life-threatening ubiquitous airborne opportunistic fungal pathogen, especially significant among immunocompromised hosts (15,26).It is present in the environment, particularly in air, water, plants and soil (1,20) When its conidia are inhaled, it can be responsible for wide range of distinct clinical entities (8). The ability of this species to cause different types of diseases is due to possess various virulence factors which help with fungal survival and persistence in the host resulting in tissue damage and disease (7). Otherwise, Penicillium is one of the largest and most important genera of fungi, more than 400 described species distributed worldwide (2,32).These fungi are broadly present in nature including air, soil, cereal grains, hay and other plant materials or foodstuff. Exposure to these fungi has been associated with a variety of adverse health outcomes including respiratory, heamatological, immunological, and neurological system disorders and/or diseases (21). Penicillium chrysogenum is a filament tous fungus of major medicaland historical importance (5). Many studies reported high percentage of respiratory problems, mammary gland infection and other diseases caused by Penicillium spp. (3, 6, 10) but there is no or rare information about the effect Penicillium chrysogenum on the internal organs. So, the main objective of this study was to determine the similarity or different MATERIALS AND METHODS Source of fungal isolates A. fumigatus and P. chrysogenum were isolated from nasal swabs of sheep suffering from recurrent pulmonary infections in the winter of 2017. These isolates were cultured on Sabouraud dextrose Agar (Himedia -India) and incubated at 28±2ºC for 4-7 days then diagnosed macroscopically and microscopically according to Washinton et al., (29). Spore suspension was prepared for both fungi according to Van der Velden et al., (28) and adjusted to 1x 107spores/ml according to Mirkov et al., (17).

Experimental design
A total of twenty four male albino mice with 10-12 weeks in age and weight 20-28gm were used in this study which divided equally into 3 groups (8 animal for each group) and kept in large plastic cages for one week before beginning the experiment. The first group was inoculatedwith 0.2ml of 1x10 7 spores/ml of A. fumigatus intraperitonially, the second group was inoculated with the same dose as well as the same rout with P. chrysogenum according to Odebode and Adekunle (21), while the third group was inoculated with normal saline which served as control group. All animals were monitored for 2 weeks after infection. The blood samples were collected by heart puncture after 18 days post infection to obtain of serum that used for biochemical analysis.

Histopathological examination
All animals were sacrificed and one cm3 of some internal organs of infected groups like (live, kidney, intestine, heart, spleen and lung were taken and fixed in 10% natural formalin buffer solution which replaced after 24 hrs. till the preparation of histological section. Tissue were embedded in paraffin and several tissue sections were prepared for histopathological sections were stai-ned with Hematoxylin -Eosin stain (H&E) according to Bancroft and Stevens (4).

Statistical analysis
The statistical analysis system-SAS (22) program was used to detect the effect of difference groups in study parameters. One way analysis of variance (ANOVA) was used to compare between means using Least Significant Difference-LSD in this study.

RESULTS AND DISCUSSION
A. fumigatus is considered one of the most common saprophytic fungi in the environment, and in the same time, It is opportunistic pathogen can cause different types of mycosis ranging from allergy to invasive and fatal disseminated Aspergillosis through it's presence as ubiquitous airborne conidia (7,15,16), So, there are many studies were performed to determine effect of A. fumigatus when inoculated intranasally into mice and conducted to that the normal host can be suffering from systemic Aspergillosis ( 13,14,25). But the present study focused on the efficacy this type of fungi on the some internal organs when adminis-tered intraperitonially into mice as a sequel to disseminated Aspergillosis. Therefore, the histopathological lesions of the animals infected intraperitonially with A. fumigatus for two weeks showed increase thickness of alveolar septa due to infiltration of mononuclear cell (MNC) and presence of hemosiderin pigment in the interstitial tissue of lung (Fig.1), and infiltration of mononuclear cell (MNC) in lamina propria of intestine with hyperplasia of epithelial cells as in (Fig.2).Kidney sections showed infiltration of inflammatory cells like mononuclear cell (MNC) in interstitial tissue and in glomeruli with dilation of bowman's space (Fig.3) Liver sections revealed aggregation of mononuclear cell (MNC) as granuloma in hepatic interstitial and necrosis of hepatocytes (Fig.4). While other section showed apoptotic cell and infiltration of inflammatory cells in dilated sinusoids as in Fig. 5.
Also, the spleen sections revealed infiltration of inflammatory cells like mononuclear cell (MNC) and presence of hemosiderin pigment (Fig. 6). Heart sections showed infiltration of mononuclear cell (MNC) between dilated myocardial fibers (Fig.7).

Figure 7. Histopathological changes in heart of infected animal with A. fumigatus for two weeks shows infiltration of inflammatory cells between dilated myocardial fibers (red arrow) (H&E stain, X400)
The liver of animals that infected with P. chrysogenum showed granulomatous aggregation of inflammatory cells around blood vessels and necrosis of hepatocytes as in (Fig. 8) and presence of apoptotic cells with arrangement of hepatocytes like cords (Fig. 9), The kidney revealed degeneration of epithelial cells of renal tubules, infiltration of inflammatory cells in glomerulous and renal parenchyma and atrophy of some glomeruli (Fig. 10). While the Intestine showed infiltation of inflammatory cells in mucosa and submucosa and hyperatrophy of goblet cells as in (Fig. 11). Heart sections of infected animal with P. chrysogenum for two weeks revealed infiltration of inflammatory cells between myocardial fibers which appeared like cords (12), while spleen sections showed necrosis and infiltration of MNC (Fig. 13).    kidney showed highly significant differences (P<0.01) among the groups. The results revealed very highly significant differences (P<0.01) in the activity of ALT in animals of the1 st and 2 nd groups as compared with control groups, while the biochemical tests of the kidney showed highly significant differences (P<0.01) in creatinin and urea in the animals treated with P. chrysogenum when compared with the 1 st and control groups (Table 1). This study was performed to compare of pathogenicity between two types of opportunistic fungi (A. fumigatus and P. chrysogenum) not only in the liver or Kidney but also in other internal organs of mice. This pathogenicity which responsible for alterations and tissue damage is associated with many types of virulence factors. As mentioned by numerous studies about the virulence factors of A. fumigatus which help this fungus in survival and persistence in the host resulting in tissue damage and disease (30). These factors include an ability to adhere to hosts' tissues through conidia of A. fumigatus are covered with hydrophobic proteins known as rodlets. These proteins are mediate adhesion of the conidia to albumin and collagen., Production of enzymes like proteases and phospholipases which degrade elastin present in lung tissue, Thermotolerance that can the fungi grow at temperatures of up to 55-77 ºC through HSP 70, as well as Melanin production (14,30). Moreover, Mycotoxins like Gliotoxin which is considered potent immunosuppressive and cytocidal action and this may be discuss the reason of presence of apoptotic body in some samples of liver and this finding agree with the results of (19) who studied the impact of Gliotoxin in the liver of rats and found presence of apoptotic hepatic cell in the section. Moreover, Penicillium is large genus that more than 400 spp. have been described (32). Some of them can produce mycotoxins while another parts of them have chemical compounds used as enzymes and antibiotic (27). P. chrysogenum which is known previously P.notatum that have medical importance due to Penicillin production, also have the ability to produce some extrolites and mycotoxins mainly PR-toxin as mentioned by (11). There are very few or rare reports about the pathogenicity of P. chrysogenum on the internal organs. Odebodea and Adekunlea (21) investigated the effect of this fungus in lung depending on doses and periods of time and found there was acute inflammation through a strong and rapid monocytes cells production in the lung. The results of current study indicated that there was severe pathological alterations in most organs taken in this experiment. This may be return to induction of oxidative damage and free radicals generation that reacted with cellular components leading to pathological changes. Also, if this isolate of Penicillium can produce mycotoxin particularly PR toxin, this will lead to tissue damage. This evidence is in consistence with Dubey et al., (9) who recorded damage in the vital internal organs, GIT perturbations, carcinogenicity, immunotoxicity, necro-sis and enzyme inhibition due to action of PR toxin in case of ingestion food products enriched with this toxin. Morever, the cytochrome P450 which is secreted from liver cells, renal tubular cells and lung cells can cause detoxification of toxin complex compound and this will lead to degeneration of organ cells as mentioned by Yassein and Zghair (31).In addition to histopathological study that carried out in this research, abnormalities on chemistry panel were also identified represented by significant differences (P<0.01) in ALT, Creatinin and Urea in all groups for both fungi. This result was corresponding with histopathological findings due to the relationship with degree of organ damage, when the hepatocytes loss their functions and suffering from apoptosis this will lead to decline in ALT activity in serum. this evidence is in line with work of Shafiq and Al-Joofy (25) on nasal Aspergi-llosis. But the result of kidney function markers in the group that treated with P. chrysogenum revealed high significant values when compared with other groups. This may be return to increase lipid peroxidation in the kidney, then lead to renal insufficiant because these indices are considered a characteristic feature of renal injury. Based on the current findings, It can be concluded that although A.fumigatus and P. chrysogenum are saprophytic fungi, but they can be caused systemic mycosis with severe pathogenesis when inoculated intraperitoneally to mice in spite of the fact of P. chrysogenum produce antibiotic, So, further studies should be performed to determine the specific virulence factors in these fungi.