sohair, i. badr* and eman, m. nasr** summary · sohair, i. badr* and eman, m. nasr** * pathology...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 22 No. 1 (January) 2009; 19 - 45

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Histopathological and bacteriological studies on livers affected with fascioliasis in cattle

By

Sohair, I. Badr* and Eman, M. Nasr**

* Pathology Dept., ** Bacteriology Dept. Anaerobic Unit, Animal Health Re-search Institute, Dokki, Giza.

SUMMARY

5 6 bovine liver samples were collected from El-Warak abattoir which appeared grossly infested with mature fasciola worms. Out of 56

cases, 17 (30.4%) were apparently infected with acute suppurative hepa-titis while 39 (69.6%) out of 56 cases appeared to be infected with chronic hepatitis.

A total of 35 bacterial isolates (18 anaerobic and 17 faculative an-aerobic) were recovered from acute suppurative hepatitis specimens. Fu-sobacterium necrophorum was the most predominant anaerobic isolates (7 isolates) and Arcanobacterium pyogenes was the most frequently iso-lated facultative anaerobic organisms (6 isolates).

While, polymicrobial isolation were detected in 13 (76.5%) in-stances. In case of chronic hepatitis Clostridium perfringens was the most predominant isolated anaerobe (26 isolates), Escherichia coli was the most frequently isolated facultative anaerobe (17 isolates) and Po-lymicrobial isolation were detected in 34 (87.2%) instances.

A total 31 C. perfringens isolates were tested for its toxin by using dermonecrotic reaction in guinea pig. C. perfringens type A represented 40% and 65.4% while, type D represented 60% and 23.1% for acute and chronic hepatitis, respectively.

The histopathological examination of acute hepatitis revealed he-patic necrosis and degeneration with presence of multiple variable sized abscesses in the hepatic parenchyma consisted of homogenous structur-less mass of necrotic cells surrounded by heavy aggregations of inflam-matory cells mainly neutrophils, histiocytes and lymphocytes, and the abscesses bounded by fibrous connective tissue capsule.

While the chronic hepatitis revealed increased fibrous connective tissue proliferation in the portal triads, around small and large bile ducts and in the Glisson;s capsule. The biliary epithelium were hyperplastic with formation of large numbers of newly formed bile ductules and pres-ence of mature fasciola worm within the lumen of the main bile ducts.


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INTRODUCTION

L iver is considered the most important organ for animal

health production and reproduc-tion. Many of the metabolic activi-ties of the body occurred in the liver. Liver infection is an impor-tant disease that affects all kinds of meat producing animals, this lead to great losses to live-stock pro-duction and national income due to condemnation of great numbers of livers in the slaughter houses (Foster and Woods, 1970 and Tomate, 1973).

Fascioliasis is an important helminth disease caused by two trematodes, Fasciola hepatica (the common liver fluke) and Fasciola gigantica. The disease belongs to the plant borne trematode zoono-ses. In Europe, the Amercas and Oceania only F. hepatica is a con-cern, but the distributions of both species overlap in many areas of Africa and Asia (Mas-Coma et al., 2005).

The definitive host range is very broad and includes many her-bivorous mammals including hu-mans.

Fascioliasis is now recognized as an emerging human disease (Spithill et al., 1999). In Egypt, human cases of fascioliasis is dis-

tributed in communities living in Nile Delta (Mas-Coma et al., 2005).

Fasciola plays an important role in the microbial invasion of the infected animals either by transportation or by depressing the vital resistance of the host.

Besides, infected liver consti-tute a good media for bacterial multiplication, transportation of microorganisms with the parasites occurs during the different stages of its life cycle either outside or inside the animal body. Anaerobic necrotic lesions of the liver pro-duced by immature flukes occa-sionally provides a suitable envi-ronment for the germination of spores of Clostridium novyi type B bacteria in the liver (Eguale and Abie, 2003). The bacteria will re-lease toxins into the blood stream resulting in what is known as black disease in sheep and sometimes cattle.

Importance of cattle fascio-liaosis in economic losses caused by condemnation of livers at slaughter and production losses es-pecially due to reduce weight gain.

This study was carried out to enumerate the histopathological hepatic lesions induced by bacteria in liver infested with fasciola and

Referred byReferred by Prof. Dr. Mohamed O. El-Shazly Professor of Pathology, Fac. Vet. Med.,

Cairo University Prof. Dr. Mona El-Enbaewy Professor of Microbiology, Fac. Vet. Med.,

Cairo University


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to detect the relationship between these bacterial infections and liver fascioliasis.

MATERIALS AND METHODS Samples:

Specimens from 56 bovine livers were collected from El-Warak abattoir in the period be-tween 2007-2008.

Grossly, 39 samples were found to be infected with chronic hepatitis while, 17 samples showed liver abscesses in association with liver fluke infestation.

Parts of the affected livers were collected at the time of slaughter separately in plastic bags under sterile condition and trans-ferred directly to the laboratory for bacteriological examination. The other parts were kept in 10% neu-tral buffered formalin for patho-logical examination. I. Bacterial isolation and charac-

terization: The surface of the liver were

seared with hot spatula and then incised with a sterile scalpel.

a. Aerobic identification:

A loopful from each affected livers was streaked onto the nutri-ent agar, MacConkey agar and blood agar plates, then incubated aerobically at 37 °C for 24 hrs. Isolated colonies of various types (based on morphological appear-

ance) were picked from plates and subcultured on blood agar plates. Bacterial isolates were identified morphologically, culturally and biochemically according to Quinn et al. (2002).

b. Anaerobic identification:

A loopful from each affected deep tissue of livers was inocu-lated into two tubes of freshly pre-pared cooked meat broth, one of them was heated at 80 °C for 10 minutes, to eliminate non spore forming organisms while the other tube was left without heating, both tubes were incubated anaerobically at 37 °C for 48 hrs. A loopful from each heated tube were streaked onto blood agar plate for isolation of spore forming anaerobes while, loopfuls from non heated tubes were streaked onto neomycin blood agar and brain-heart infusion blood agar (BHIBA) plates for iso-lation of C. perfringens and non spore forming anaerobes, respec-tively. All plates were examined after anaerobic incubation for 2 to 3 days and each colony type was subcultured for identification ac-cording to Konoman et al. (1992). C. perfringens isolates were typed using the intradermal inoculation test in Albino Guinea pigs accord-ing to Sterne and Batty (1975).

II. Histopathological examina-

tion: Specimens from the liver and


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its associated lymph nodes were fixed in 10% neutral buffered for-malin for at least 24 hours and then routinely processed. The tissues were paraffin embedded and sec-tioned at 4-6 µ thickness, then the sections were stained with the fol-lowing stains according to Ban-croft et al. (1994), Haematoxylin and eosin stain, Masson's trichrom stain for connective tissue and Prussian blue stain for haemosidrin pigments.

RESULTS

I n the present work, by clinical examination, 17 cases (30.4%)

out of 56 liver samples were ap-peared to be infested with acute suppurative hepatitis, while, 39 cases (69.6%) were appeared to be infected with chronic hepatitis. Microbiological studies:

Microbiological examination of 17 liver specimens with acute suppurative hepatitis showed posi-tive results in all specimens. An-aerobic bacteria only were de-tected in 3 (17.6%) specimens, fac-ultative anaerobic bacteria were only in 5 (29.4%), while, mixed facultative anaerobic and anaero-bic organisms were in 9 (52.9%) (Table, 1).

As shown in Table (2), a total of 35 bacterial isolates (18 anaero-bic and 17 facultative) were ob-tained, from the above specimens.

The predominant anaerobes in

descending order of frequency were as follows: 7 F. necrophorum (41.2%), 5 C. perfringens (29.4%), 3 peptsreptococcus anaerobious (17.6%), 2 C. sordelli (11.8%) and 1 C. novyi ( 5.9%).

The most frequently isolated facultative anaerobic organisms were as follows. A pyogenes (6 isolates), S. aureus (4 isolates), E. coli (3 isolates) then Proteus and Staph. spp. 2 isolates for each. (Table, 3) showed that polymicro-bial isolation were detected in 13 (76.5%) instances and single bac-terial isolates in 4 specimens. The single bacterial isolates were A. Pyogenes (2) and S. aureus (2) in a percentage of 11.8 for each.

On the other hand, microbi-ological examination of 39 liver specimens with chronic hepatitis with fasciola infestation showed that anaerobic bacteria only were detected in 10 (25.6%) specimens, facultative bacteria only in 7 (17.9%) and mixed facultative an-aerobic and anaerobic organisms in 22 (56.4%) (Table, 1) .

A total of 73 bacterial isolates (40 anaerobic and 33 facultative) were obtained from these speci-mens.

As shown in table (2), the most frequently isolated facultative anaerobic organism were E. coli 17 (43.6%) followed by Staphylo-coccus spp. 7 (17.9%), Klebsiella 5 (12.8%), Proteus spp. 3 (7.7%) and S. aureus 1 (2.5%).


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The predominant anaerobes were C. perfringens (26 isolates) followed by C. sporogenes (6 ioslates), peptostreptococcus an-aerobius (5 isolates) and C. septi-cum (2 isolates) then C. novyi (one isolate). Table (3) clarified the po-lymicrobial isolation that were de-tected in 34 (87.2%) instances and single bacterial isolates in 2 speci-mens. The single isolates were C. perfringens 2 (5.1%), E. coli 2 (5.1%) and S. aureus 1(2.6%).

A total 31 C. perfringens iso-lates were typed for its toxin. Ta-ble (4) showed that C. perfringens type A represented 40% and 65.4% for acute and chronic hepa-titis respectively, while, type D represented 60% and 23.1% for acute and chronic hepatitis respec-tively. The pathological studies:

The histopathological exami-nation revealed 2 types of hepati-tis.

I. Acute suppurative hepatitis:

Grossly, the livers appeared hard, dark and brown in color with presence of multiple soft abscesses (ranged from 3-10 in diameter) on the liver surface. On cut section, a viscous yellow material oozed from the cut ends. The abscesses were surrounded by hyperemic zone.

Histopathologically, the he-patic blood vessels were dilated

and engorged with blood, the he-patic cords were disorganized and distorted while the hepatocytes of parenchymal cell revealed necrosis and degeneration (Fig. 1). The ne-crotic lesions emphasized by deeply eosinophilic cytoplasm with karyorhexis and karyolysis of their nuclei. Moreover, the degen-erative changes manifested by vacuolation of the hepatocytes par-ticularly around central vein (Fig. 2). The hepatic sinusoids showed presence of mononuclear inflam-matory cells in their lumina (Fig. 3) as well as the kupffer cells were swollen and increased in number (Fig. 4). Sometimes, multiple vari-able sized abscesses were detected in the hepatic parenchyma. The core of the abscess consisted of homogenous structureless mass of necrotic cells surrounded by heavy aggregations of inflammatory cells mainly neutrophils, histocytes, eosinophils and lymphocytes and bounded by fibrous connective tis-sue capsule (Fig. 5 & 6). Also, fo-cal infiltration of inflammatory cells were observed in the capsule (Fig. 7). Whereas, focal microab-scesses consisted of lymphocytes, histocytes, eosinophils and poly-morphnuclear leukocytes sur-rounded by thin layers of fibrous connective tissues were observed in the hepatic parenchyma.

II. Chronic hepatitis:

Grossly, the infested livers were hard, firm and tough in con-


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sistency with multiple, irregular pale brownish areas on the surface. Cut section of the liver revealed presence of large whitish areas of fibrosis. The affected ducts were enlarged, thickened, hard in con-sistency and in the form of cord-like structures and protruded above the surface of the liver. In most cases, mature fasciola worms were detected within the lumen of the affected bile ducts.

Histopathologically, the most

important alterations consisted of increased fibrous connective tis-sues proliferation within the portal triads with concentric arrangement (Portal cirrhosis), around hepatic blood vessels which revealed vac-uolation of its muscular layer and around small and large intrahepatic bile ducts (biliary cirrhosis) were seen (Figs. 8, 9 & 10). Sometimes, the proliferated fibrous tissues penetrated the hepatic lobules and subdivided them into lobule-like segments intersecting the classic lobules to produce pseudolobula-tion as well as the fibrous tissues infiltrated with lymphocytes, histo-cytes and eosinophils (multilobular cirrhosis) (Fig. 11) or insinuate in-be tween t he hepa t i c ce l l s (pericellular cirrhosis) (Fig. 12). In few instances, the proliferated fi-brous tissues replaced considerable part of the hepatic lobule leaving reminants of the hepatic cells and the proliferated fibrous connective

tissues showed diffuse infiltration with lymphocytes, histiocytes and eosinophils were seen. Meanwhile, the Glisson's capsule was variably thickened by fibrous connective tissue proliferation (Glisson's cir-rhosis) (Fig. 13).

Focal areas of haemorrhages

were detected in the hepatic paren-chyma with presence of golden brown haemosidrin pigments ei-ther free or within the cytoplasm of phagocytic cells which con-firmed by using prussion blue stain (stained blue) (Figs. 14 & 15). Ne-crosis and desquamation of the epithelial lining of the bile ducts were evident in many instances (Fig. 16). Hyperplasia of the bil-iary epithelium where the epithe-lial lining of the bile ducts thrown as papillomatous projections into the lumen were also seen (Fig. 17). Moreover, there were large num-ber of newly formed bile ductules which assumed an adenoid pattern (adenomatoid hyperplasia) were also seen (Fig. 18). Focal and dif-fuse inflammatory cells infiltration were observed in the proliferated fibrous tissue between the hyper-plastic newly formed bile ductules (Fig. 19). Sometimes, the epithelial lining of the newly formed bile ductules showed metablastic changes into mucous secreting cells (Fig. 20). In most cases, ma-ture fasciola worms with promi-nent spines were detected within


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the lumen of the main bile ducts (Fig. 21).

The hepatic lymph nodes in all

cases revealed depletion of lym-phoid cells of the lymphatic follicles (Fig. 22), follicular hyalinosis (Fig. 23) and presence of R. B. Cs in the dilated lymphatic sinuses mixed with histiocytes and lymphocytes were also seen (Fig. 24).

Table (1): Frequency of isolation of facultative anaerobic and anaerobic bacteria from the examined liver samples.

Types of liver samples

No. of ex-amined samples

Facultative an-aerobic bacte-

ria

Obligatory an-aerobic bacte-

ria

Mixed bacteria

No. % No. % No. %

Acute suppurative hepatitis with fas-ciola

17 5 29.4 3 17.6 9 52.9

Chronic hepatitis with fasciola 39 7 17.9 10 25.6 22 56.4


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Table (2): Incidence of the aerobic and anaerobic microorganisms isolated from examined livers samples.

Type of liver samples Bacterial isolates

Acute suppurative hepatitis with fasciola

(No. = 17)

Chronic hepatitis with fasciola

(No.=39)

Aerobic bacteria A. Pyogenes

No. %* No. %**

6 35.3 - -

S. aureus 4 23.5 1 2.5

E. coli 3 17.6 17 43.6

Staphylococcus Spp. 2 11.8 7 17.9

Proteus spp. 2 11.8 3 7.7

Klebsiella - - 5 12.8

Subtotal 17 100 33 84.6

Anaerobic bacteria F. necrophorum 7 41.2 - -

C. perfringens 5 29.4 26 66.7

Peptstreptococcus anaerobious

3 17.6 5 12.8

C. sordelli 2 11.8 - -

C. novyi 1 5.9 1 2.6

C. septicum - - 2 5.1

C. sporogenes - - 6 15.4

Subtotal 18 105.9 40 102.6

Total 35 205.9 73 187.2

The percentage was calculated inn relation to the total number of * livers with acute suppurative hepatitis and ** chronic hepatitis with fasciola.


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Table (3): Pattern of isolation of anaerobic and facultative microorganisms from livers specimens infected with fasciola.

Single and polymicrobial culture isolated from liver with fasciola Acute suppurative hepa-

titis (No. 17) No. %* Chronic suppurative hepatitis (No. 39) No. %**

Single cultures: Single cultures:

A. pyogenes 2 11.8 C. perfringens 2 5.1

S. aureus 2 11.8 E. coli 2 5.1

Polymicrobial cul-tures: S. aureus 1 2.6

F. necrophorum + C. perfringens 1 5.9 Polymicrobial cul-

tures:

C. perfringens + C. novyi 1 5.9 C. perfringens + C.

sporogenes 3 7.7

C. perfringens + C. sor-delli + peptostreptococ-cus

1 5.9 C. perfringens + C. novyi 1 2.6

F. necrophorum + A. pyogenes 2 11.8 C. perfringens + C.

septicum 2 5.1

F. necrophorum + A pyogenes + E. coli 2 11.8 C. perfringens + pepto-

streptococcus 2 5.1

F. necrophorum + S. aureus 1 5.9 E. coli + Staphylococ-

cus spp. 1 2.6

Peptostreptococcus + C. sordelli + Staphylo-coccus spp.

1 5.9 Klebseilla + Staphylo-coccus spp. 3 7.7

C. perfringens + E. coli 2 11.8 C. perfringens + E. coli 11 28.9

F. necrophorum + S. aureus + Proteus spp. 1 5.9 C. perfringens +

Staphylococcus spp. 3 7.7

Staphylococcus spp. + Proteus spp. 1 5.9 Peptostreptococcus +

E. coli 3 7.7

C. perfringens + Kleb-seilla spp. 2 5.1

C. perfringens +proteus spp. 3 7.7

The percentage was calculated in relation to the total number of liver with * acute suppurative hepatitis or ** with chronic hepatitis with fasciola.


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Fig. (1): Liver of cattle showing distortion and individualiza-tion of the hepatic cells (H & E X 400)

Fig. (2): Liver of cattle revealed dilated central vein and para-central fatty change of the ad-jacent hepatocytes (H & E X 400).

Fig. (3): Liver of cattle showing dilated hepatic sinusoids with mononuclear inflammatory cells in their lumina (H & E X 400).

Fig. (4): Liver of cattle showing swollen and increased num-bers of Kupffer cells (H & E X 200).

٢ ١

٤ ٣


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Fig. (5): Liver of cattle showing large abscess in the hepatic pa-renchyma (H & E X 200)

Fig. (6): High magnification power of Fig. (5) showing heavy aggrega-tion of inflammatory cells com-posed of neutrophils, histiocytes and lymphocytes and surrounded by fibrous connective tissue cap-sule (H & E X 400).

Fig. (7): Liver of cattle showing fi-brous capsule of abscess depi-cating focal infiltration of in-flammatory cells (H & E X 100).

Fig. (8): Liver of cattle showing exten-sive fibrous connective tissue pro-liferation in the portal triade (portal cirrhosis) (Trichrom stain X 200)

٦ ٥

٨ ٧


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Fig. (9): Liver of cattle showing vacuolations of the hepatic blood vessel with perivascular fibrosis (H & E X 400).

Fig. (10): Liver of cattle showing bil-iary cirrhosis, extensive fibrous connective tissue proliferation around the intrahepatic bile duc-tules (H & E X 200).

Fig. (11): Liver of cattle showing multilobular cirrhosis. The proliferated fibrous connective tissue around the hepatic lob-ules was infiltrated with in-flammatory cells (H & E X 200).

Fig. (12): Liver of cattle showing de-picating fibrous connective tissue proliferation in between the he-patic cells (pericellular cirrhosis) which brings about pressure atro-phy on the adjacent hepatic cells (H & E X 200).

١٠ ٩

١٢ ١١


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Fig. (13): Liver of cattle showing biliary cirrhosis and Glisson's cirrhosis [Extensive fibrous connective tissue proliferation in the hepatic capsule] (H & E X 200).

Fig. (14): Liver of cattle showing golden brown haemosidrin pig-ment in the proliferated fibrous connective tissue of the portal area either free or in the cyto-plasm of phagocytic cells (H & E X 200)

Fig. (15): Liver of cattle showing haemosidrin pigment stained blue (Prussion blue stain X 200).

Fig. (16): Liver of cattle showing ne-crosis and desquamation of the epithelial lining the bile ducts (H & E X 200).

١٤ ١٣

١٦ ١٥


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Fig. (17): Liver of cattle depicating hyperplastic proliferation of the epithelial lining of the main bile ducts with the for-mation of papillary like pro-jections toward the lumen (H & E X 400).

Fig. (18): Liver of cattle showing hy-perplastic proliferation of biliary epithelium with the formation of newly formed bile ductules (Adenomatoid hyperplasia) (H & E X 100)

Fig. (19): Liver of cattle showing multifocal inflammatory cell infiltrations in the proliferated fibrous connective in between the newly formed bile ductules (H & E X 100).

Fig. (20): Liver of cattle depicating metaplastic lesions of the epithe-lial lining of the newly formed bile ductules into mucous secre-tory cells (H & E X 400).

١٨ ١٧

٢٠ ١٩


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Fig. (21): Liver of cattle showing mature fasciola worm in the lu-men of the intra-hepatic bile duct. Heavy infiltration with in-flammatory cells in the fibrous connective tissue between newly formed bile ductules (H & E X 400).

Fig. (22): Lymph node of cattle show-ing marked depletion of lymphoid cells in the lymphoid follicles (H & E X 100).

Fig. (23): Lymph node of cattle showing follicular hyalinosis (H & E X 400).

Fig. (24): Lymph node of cattle show-ing R.B.Cs in the dilated lymphatic sinuses admixed with histiocytes and lymphocytes. (H & E X 400).

٢٢ ٢١

٢٤ ٢٣


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DISCUSSION

L iver is considered the most important organ for animal

health production and reproduc-tion. It is important to evaluate the state of health of liver since this organ is involved in many disease processes either primarily or sec-ondarily and also because any liver damage disturb metabolic proc-esses that are vital for normal health and optimum productivity (Sayed et al., 2008).

In the present study the bacte-rial infections associated with cases of bovine fascioliasis were investigated as the intercurrent in-fections could undoubtelly compli-cate the disease process. It is wor-thy to mention that, 17 out of 56 specimens infested with fasciola were accompanied by liver ab-scesses and this may be attributed to that bacteria were acquired by the flukes in the small intestine of ruminants and during migration, that may suggest that the flukes can spread highly pathogenic bac-teria (Al-Khafaji and Rhaymah, 1993).

Bacteriological examination of the samples with acute suppura-tive hepatitis revealed that the inci-dence of facultative anaerobic and anaerobic organisms were 100% and 105.9%; respectively. Also, Zaki et al. (2000) detected aerobic and anaerobic organisms from liver samples in an incidence of 63.9% and 77.8%; respectively.

While, Lechtenberg et al. (1988) isolated anaerobic bacteria from liver abscesses in an incidence of 100%, whereas facultative bacteria were isolated in an incidence of 44.9%.

A. pyogenes was the predomi-nant facultative anaerobic bacte-rium which isolated in an inci-dence of 35.3%, while, F. necro-phorum and C. perfringens were the most obligatory anaerobic bac-teria which isolated from livers with acute suppurative hepatitis and chronic hepatitis in incidences of 41.2% and 29.4%, respectively. These results nearly similar to that mentioned by Scanlan and Ed-wards (1990) and Nagaraja and Chengappa (1998).

Fusobacterium necrophorum and A. pyogenes isolated either as a single or mixed together or with other facultative anaerobic and/or anaerobic bacteria (Table, 3). As evidence exists for pathogenic syn-ergy between A. pyogenes and F. necrophorum (Takeuchi et al., 1983), and F. necrophorum and other bacteria (Jang and Hirsh, 1994), F. necrophorum produces a potent leukotoxin, a fact that may explain some of these observations (Hofstad, 1989). Blood and Ra-dostitis (1989) also stated that F. necrophorum was considered to be one of the most common causes of hepatic abscesses. Similarly, Lot-follahzadeh et al. (2005) isolated F. necrophorum as a unique bacte-


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rial cause of hepatic abscesses in an incidence of 63.63% and they isolated A. pyogenes in an inci-dence of 27.27%.

Other bacteria isolated from acute suppurative hepatitis, speci-mens have included A. pyogenes, S. aureus, E. coli, Proteus spp., C. novyi and Peptostreptococcus an-aerobious (Table, 2). Similar find-ings were described by Rosa et al. (1989), El-Sayed et al. (1991) and Zaki et al. (2000). Also, Dore et al. (2007) mentioned that the most common bacterium isolated from the liver abscesses were A. pyo-genes and anaerobic bacterium in incidences of 22.2% and 38.9%, respectively, and they recorded po-lymicrobial isolates with both aerobic and anaerobic bacteria in an incidence of 75% of positive samples. Bacteriological examina-tion of samples with chronic fas-cioliasis revealed that the inci-dence of facultative anaerobic and anaerobic organisms were 8436% and 102.6%, respectively. Table (2) showed the most frequently isolated aerobic or facultative or-ganisms were E. coli (43.6%) and the predominant anaerobes were C. perfringens, mixed infection oc-curred in 56.4% instances. No con-sistent patterns of bacterial combi-nation were noted except between C. perfringens and E. coli in 28.2% instances. These results agree with the previous findings of Srokina (1987); Samad and

Haque (1987) and Zaki et al. (2000) who found that fasciola in-festation plays an important role in stimulation of clostridium infec-tion especially C. perfringens due to the damages which were attrib-uted to toxic environment created by the organisms in liver tissue.

The presence of some mem-bers of family Enterobacteriaceae in combination with other Clos-tridial bacteria such as C. perfrin-gens may be explained by Wern-ery (1992) that infection with pathogenic E. coli may cause alter-ing the mucous membrane of the intestine, enable the clostridial mi-croorganisms to vegetate and re-lease its toxins which absorbed through the damaged gut and reached to the blood circulation and then to the liver Wernery (1992). On the other hand, Yassein et al. (1989) and Morshdy et al. (1991) considered E. coli as an in-cidental pathogen which contami-nated the animal tissues during preparation of the carcasses from faecal material, skin and hides.

C. sporogenes, C. speticum and C. novyi were isolated in an inci-dence of 15.4%, 5.1%, 2.6%, re-spectively. These results nearly co-incide with that observed by Dar-wish (1996) and Mohamed et al. (1997) and Zaki et al. (2000). Sim-ilarly Leloglu (1972) isolated C. movyi and C. septicum in incid-ences of 6% and 2%; respectively.

C. perfringens was isolated in


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mixed culture with C. sporogenes, C. sordelli and Peptostreptococcus anaerobious. Concerning Pepto-streptococcus anaerobious and C. sporogenes. It is believed that they may have a secondary role in initi-ating hepatic lesions in animals (Taydon et al., 1980 and Itman et al., 1989). C. perfringens type A was the most prevalent isolates re-covered from chronic hepatitis samples in an incidence of 65.4% while C. perfringens type D was recovered in an incidence of 23.1 %. Similar findings were recorded by Darwish (1996). While, in acute hepatitis C. perfringens type D was the most prevalent isolates in an incidence of 60%. This type accord with that of Zaki et al. (2000).

In current study, the macro-scopic examination of acute cases revealed multiple soft abscess on the liver surfaces surrounded by hyperemic zone. This result was agreed with Jubb et al. (1985); Lechtenberg et al. (1988); Hun-gerford (1989); Gutierrez (1990); Woods and Gutierrez (1993) and Jones et al. (1997).

Histopathologically, the acute cases revealed dilated blood ves-sels with disorganization and dis-tortion of the hepatic cords while, the hepatic cells showed variable degrees of necrosis and degenera-tion. Similar results were also re-corded by Hungerford (1989); Darwish (1996); Jones et al.

(1997); Mohamed et al. (1997); Andrews (1998); MacGavin et al. (2001) and Sayed et al. (2008).

Invasion of the liver by mi-grating immature liver fluke dam-ages the tissue and results in re-duction of the oxygen tension (anaerobic condition), that allowed the germination and proliferation of closteridial spores with release of its toxins and induce hepatocel-lular necrosis (Jubb et al., 1985; Jones et al., 1997; Topley and Wilson, 1998 and Sayed et al., 2008). Hepatic abscesses were also appeared in the current study in cases infected with Fusebacterium, Staph. aureus and C. pyogenes which consisted of homogenous structurless eosinophilic core sur-rounded by inflammatory cells mainly lymphocytes, histiocytes, o e s i n o p h i l e s a n d p o l y -morphnucealr cells and bounded by fibrous connective tissues cap-sule. Similar results were observed by Lechtenberg et al. (1988); It-man et al. (1989); Scanlan and Edwards (1990) and Darwish (1996). Jubb et al. (1985); Woods and Gutierrez (1993) and Carl-ton and MacGavin (1995) men-tioned that there was a synergistic relationship between F. necropho-rum and S. aureus in which S. aureus stimulates the growth of F. necrophorum and its leukotoxin substance were antigenic to the he-patic tissue.

Regarding to the chronic liver


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hepatitis, the gross examination of fasciola infested livers revealed that the liver were hard, firm and tough in consistency and the cut section showed large whitish areas of fibrosis while the affected ducts were thickened, enlarged and cord-like in structure. Similar findings were observed by Motwally-and Sami (1982); Jubb et al. (1985); Hungerford (1989); Jones et al. (1997) and Sayed et al. (2008).

The histopathological changes revealed cirrhosis which appeared as portal, multilobular, biliary, pericellular and Glissonian's ac-cording to the fibrous connective tissues distribution. Theses find-ings also reported by Anthony et al. (1977); Blamire et al. (1990); Bojkind and Greenwel (1993); Darwish (1996); Jones et al. (1997) and Sayed et al. (2008). In spite of the role played by the bac-terial microorganisms as a causa-tive agents for hepatic fibrosis, Jones et al. (1983) believed that the most cases of cirrhosis in ani-mals originated from the ingestion of toxic substances and the infec-tious agents may be also causative in animals. Also, Conn and Fessel (1971) mentioned that E. coli oc-curs in patients with liver cirrhosis. The biliary cirrhosis associated with papillomatous protrusions of the epithelial lining of the intra-hepatic bile ducts with hyperplas-tic proliferation of the ductul epi-thelium and newly formed bile

ductules in adenomatous arrange-ment were observed in our results. These findings were agreed with Motwally and Sami (1982); Swa-rup and Pachaur (1987); Blamire et al. (1990); Jones et al. (1997); Sayed et al. (2008) and Darwish (1996) in liver of camel and El-Mahdy (1975) in liver of sheep. The hyperplasia of the bile ducts is an attempt to regenerate hepatic parenchyma when the par-enchymal cells have lost their ca-pacity to regenerate themselves (Kelly, 1985). Also, Popper and Hutterer (1970) mentioned that the hyperplasia of the ductul epi-thelium occurs as a result of fluke toxic products which cause chang-ing in the structural integrity of the ductul cells in non specific and po-tentially destructive manner. The ductul epithelium revealed necro-sis and desquamation with pres-ence of mature worms within its lumina were appeared in the cur-rent study. These findings were at-tributed to the effect of toxic prod-ucts elaborated by fasciola worms (Mahmoud et al., 1989) and Sayed et al. (2008). The presence of mature worms within the lumen of intra hepatic bile ducts brings about a continous irritation and lead to hyperplastic proliferations which emphasized by papilloma-tous projections and formation of newly formed bile ductules (El-Mahdy, 1975). The ductul epithe-lium of the newly formed bile duc-


39

tules undergoes metaplastic changes to mucous secreting cells in our study. These findings were coincided with El-Mahdy (1975) who reported catarrhal cholangitis in fasciola infested sheep liver; Fahmy and El-Attar (1990) and Darwish (1996) in camel's liver. In our study, presence of areas of heamorrhages in the liver. This re-sults may resulted from the injury to the wall of the blood vessels by toxins elaborated by the bacterial agents, so the presence of Prussian blue positive haemosidrin pig-ments within the proliferated fi-brous connective tissues either free or engulfed by the phagocytic cells was considered incidental findings of old haemorrhages Jones et al. (1997). Also, he added that haem-orrhages resulted from migrating larvae as well as blood –sucking by adult worms cause iron defi-ciency anaemia. The vacuolar de-generation within the muscular wall of blood vessel in our results may be attributed to the irritating effect of the toxins elaborated by the bacterial agents.

The hepatic lymph nodes in the current study revealed deple-tion of lymphoid cells, follicular hyalinosis and presence of RBCs with histiocytes and lymphocytes in the dilated lymphatic sinuses. Similar results were also observed by Darwish (1996). Vallei (1993) attributed that the occurrence of haemorrhage in lymph nodes of

cattle mostly related to bacterial infection. Also he attributed the follicular hyalinosis to stressful condition that cause lymphoid at-rophy, where the antigen focusing dendritic cells located in the center of the follicle became apparent and forming matrix of fibrinoid deposi-tion defined as follicular hyalino-sis. Meanwhile, the presence of histiocytes in the sinuses is consid-ered as a sign of reactive lympha-denitis (Kumar et al., 1997) and also indication of antigenic stimu-lation of the node (Jones et al., 1997).

F rom the achieved results, it concluded that livers of cattle

showed a very high proportion of gross lesions rather than different histopathological disorders. Also fasciola worms may incriminated in aiding bacterial infections spe-cially C. perfringenes and E. coli which lowering the hepatic viabil-ity. Generally, livers of slaugh-tered cattle are considered as haz-ardous source of different mixed bacterial species. Moreover, their low value referred to high inci-dence of pathological lesions.

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