bacteriological and pathological studies on the … bacteriological examinations reflects the...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 22 No. 1 (January) 2009; 130 - 146

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Referred byReferred by Prof. Dr. Jakeen K. A. El-Jakee Professor of Microbiology & Immunity , Fac.

Vet. Med., Beni Sueif University Prof. Dr. Dawlat M. Amin Professor of Pathology, Animal Health Re-

saerch Institute, Dokki

Bacteriological and Pathological Studies on the Causes of Mortalities

among Sheep in Sharkia-Governorate Farms

By Hala Foad Habashy*; Fadel. N.G. ** and El Shorbagy. M.M. *

*Bacteriology Dept. Animal Health Res. Ins . Dokki Giza. ** Pathology Dept. Animal Health Res. Ins . Dokki Giza.

SUMMARY

S ome sheep farms in Sharkia Governorate showed mortality rate ranged from (12.2 to 16.9 %). Most of the affected cases showed

respiratory manifestation, while in other farms diarrhea with slight respi-ratory manifestation were the most observed symptoms. Samples were taken from dead, sick, emergency slaughtered and from contact apparently healthy cases. Samples include nasal swabs, serum and tissues for traditional bacterial examinations, ELISA and for histo-pathological examination.

The bacteriological examinations reflects the presence of mainly p. multocida associated with C. perfringens in some farms. The histopa-thological changes in the lung, liver, kidney, spleen, lymph node, intes-tine and heart were presented.

From this study it was concluded that P. multocida and C. perfrin-gens infection among sheep in Sharkia governorate farms constitute a major problem and need intensive precaution and preventive measures including; application of good hygiene and management in animal farms, application of vaccination programs allowed, feeding animals with balanced ration, and raising the immune status of the animals. Among methods used in diagnosis of pasteurella ELISA was more effi-cient. P. multocida and C. perfringens infection could be diagnosed histopathologically and more efficient in studying the severity of infec-tion.

INTRODUCTION

S mall ruminants play an impor-tant role in nutrition and in-

come of people around the world.

They serve primarily as source of meet also provide milk, skin, and wool (Mbilu, 2007). Among the causes of mortalities in sheep dis-


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eases of the respiratory system in-cluding pneumonia, debility, and unspecific toxemias were the ma-jor cause of deaths (Maru et al. 1993). Pneumonia is widespread among sheep and goat in sub-saharan Africa and it is considered to be one of the most important causes of losses in the small rumi-nant industry (Kusiluka and Kambarage, 1996).

The respiratory affection is complex involving stress factors, bacteria and virus infection. Bacte-rial pneumonia is regarded as the most frequent and serious causes of mortality and economic losses associated with respiratory dis-eases of sheep (Andrawis, 2001). However pasturelosis (Enzootic pneumonia) regarded as the most important respiratory disease af-fecting sheep caused mainly by Pasteurella multocida and Pas-teurella heamolytica. Pasteurella spp. Occur as apart of the normal flora of the nasopharynx but under initiation of complex environ-mental, genetic, stresses, and im-munological factors will cause dis-eases Gilmour and Angus (1983) and Andrawis (2001). On the other hand an aerobic bacteria especially clostridia are among the major bacteria which act as primary or secondary causes of many diseases threaten the health of the sheep (Quinn et al., 2002). In sheep as in many other

species of domestic animals clos-tridial disease still present chal-lenges to veterinary practioners, diagnosticians (Songer, 1998). En-terotoxaemia is a broad term used for a group of enteric intoxication associated with different types of C. perfringens. In sheep the dis-ease has been well characterized clinically (Blood et al., 1993).

The aim of the present study is to determine the possible causes of mortalities in sheep among some farms in Sharkia Gover-norate and the pathological associ-ated lesions. MATERIAL AND METHODS

1- Animals: Five sheep farms in Sharkia Governorate showed mortality rate ranged from (12.2 to 16.9 %). Most of the affected cases showed respiratory manifestation as cough, nasal discharge, fever, while in other farms diarrhea with slight respiratory manifestation were the most observed symptoms. 2- Samples: Samples were taken from dead, sick, emergency slaughtered as well as contact apparently healthy cases. I. Bacteriological studies: Samples for bacteriological exami-nations include (124) nasal swabs, (104 were collected from diseased sheep and 20 from apparently


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healthy sheep) as presented in ta-ble (1).

A total of 90 tissue samples, 12 from dead, 6 from emergency slaughtered cases), were taken from lung, trachea, spleen, kidney and liver for aerobic examinations. Tissue samples (a total 72 sam-ples) for anaerobic examinations were taken from liver, intestine, kidney and spleen (table 2).

Blood samples were collected for ELISA and bacteriological ex-amination (104) were collected from diseased and 20 from appar-ently healthy. 1. Bacterial isolation and identi-

fications: The nasal swabs were cul-tured into peptone water 1% and inoculated over night and then cul-tured into DAS medium (1% crys-tal violet) , MacConky and Blood agar media. Samples which col-lected from slaughtered sheep were cultured anaerobically on cooked meat media (Willis, 1977); sheep blood agar and neomycin sulphate blood agar media (Carter and Cole, 1990). The same organs were inoculated directly onto DAS medium, MaConkey bile salt agar, and blood agar and incubated aero-bically at 37ºC for 24-48 hrs. The suspected growing colonies were studied for morphological and bio-chemical characters according to Kreig and Holt (1984).

2. Pathogenicity and virulence test for Pasteurella to mice; The bacterial suspension was made by plate washing technique (Stamp et al., 1955) from the original culture which was plated onto 10% sheep blood agar plate and incubated for 24 hrs. at 37ºC. Inoculated plate was flooded with 5ml saline solution and colonies were removed from the solid me-dium by gentle rubbing with glass rode. The resultant granular sus-pension contained an average 1.5 x 108 organism per ml by matching with McFarland tube no (2) (Wessman, 1964). Three mice were injected intraprotenial with each isolate.

٣. Potassium thiocyanate extract

(KSCN) antigen; Pasterulla multocida was grown on blood agar plates harvested with 0.5 m KSCN and 0.425 m NaCl PH 6.3 and incu-bated in water bath at 37ºC for 5 hrs. Cells were removed by cen-trifugation at 300 g for 30 min. the supernatant was dialyzed against 0.01 m Tris-HCl and 0.32 NaCl (Singh and Jayprakason, 2001). The protein content in the extract was adjusted to 5 g/ml (Lowery et al., 1951). 4. Serum ELISA. In micro titer plate the anti-gen was used performing the checker board titration to deter-mine the optimum antigen concen-


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tration and conjugate dilution. Plates were coated with KSCn ex-tract antigen (5 g/ ml) left over-night at 4°C. After washing with PBS, the 1:50 diluted serum sam-ples were added and incubated at 37°C for 1 hr and washed 3 times with the same washing buffer. Then 100 µ of 1:3000 rabbit anti-sheep radish peroxidase conjugate was added and left to react at 37°C for 30 minutes (The optimum con-centrations of the antigen and the conjugate were determined by checker board titration). The plates was washed again three times and finally 100 µl of the substrate O-phenylenediamine containing 0.001% H2O2 was added. After the color developed 25 µl of 8 NH2SO4 were added to stop the re-action and plates was read at 490 nm. Antigen, serum conjugate and substrate controls were set up as appropriate controls; end point was

calculated as the mean value of the negative sera plus two standard de-viation (Singh and Jayprakason, 2001). II. Histopathological studies: Tissue samples from sheep (lung and its associated lymph node ,liver, kidneys, spleen, heart, and intestine) were collected from twelve freshly dead cases as well as six emergency slaughtered cases. The collected tissue samples were preserved in 10% neutral buffered formalin washed in run-ning water and dehydrated in dif-ferent grades of concentrated alco-hol, cleared in xylene and embed-ded in paraffin. Paraffin sections of 5 µ thickness were obtained and stained by Haematoxylin and Eo-sin (Bancroft et al., 1996). Then covered and examined microscopi-cally.

Table (1): Type and Number of samples collected from sheep with differ-ent disease condition.

Animal status Nasal swabs Blood samples Organs

Diseased sheep 104 104 -

Dead & slaughtered sheep

- 18 90

Apparently healthy sheep

20 20 -

Total 124 142 90


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Table (2): Type and number of organs which collected from fresh dead and slaughtered sheep for bacteriological and pathological ex-aminations.

Type of organ examined

No. of examined organs

Aerobically Anaerobically Pathologically

Lungs 18 - 18

Trachea 18 - 18

Spleen 18 18 18

Kidneys 18 18 18

Liver 18 18 18

Intestine - 18 18

Lymph node - - 18

Total 90 72 126

RESULT Bacteriological results:

Isolation of Pasteurella multocida and clostridia from specimens were presented in tables (3, 4 and 5).

Table (3): Type and number of samples with number and percentage of isolates.

Animal status

Type of samples

No. of samples

P. multocida Clostridia

No. % No. %

Diseased sheep

Nasal swabs

104 57 54.8 - -

Blood samples

104 28 26.9 - -

Slaugh-tered sheep

Internal organs

18* 18 100 6 33.3

Appar-ently healthy sheep

Nasal swabs

20 6 30.0 - -

Blood samples

20 2 10 - -

* Number of slaughtered sheep examined for its internal organs.


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Table (4): Typing of isolated C. perfringens and prevalence of toxigenic and non-toxigenic strains in examined internal organs in sheep.

Site of isolate

No. of ex-amined samples

No. and % of isolated

strains

No. of type of toxigenic strains A B D

Non toxi-genic

No. % No. % No. % No. % No. %

Liver 18 5 27.7 3 60.0 - - 2 40.0 0 -

Intestine 18 6 33.3 3 50.0 - - 2 33.3 1 16.6

Spleen 18 3 16.6 3 100.0 - - - - 0 -

Kidneys 18 3 16.6 2 66.7 - - 1 33.3 0 -

Total 72 17 11 64.7 - - 5 29.4 1 5.9

Table (5): comparison between Pasteurella multocida isolation and ELISA technique in diagnosis.

Animal status No. of exam-ined Blood

samples

Traditional ELISA

No. % No. %

Diseased 104 28 26.9 35 33.6

Apparently healthy

20 2 10 10 50

As shown in table (3), it was notes that out of 104 nasal swabs revealed that isolation of P. multo-cida is 57 with an incidence of 54.8% while out of 104 blood sam-ples the number with positive iso-lation was 28 with an incidence of 26.9%. Pasteurella multocida were isolated from all internal organs of 18 cases dead and emergency slaughtered with an incidence of 100%. While the isolation of C. perfringens from internal organs were recorded in 6 cases out of 18

with an incidence of 33.3%. Mean-while the isolation of P. multocida from apparently healthy sheep out of 20 nasal swab revealed positive isolation in 6 cases with percent-age of 30%, while isolation from blood samples was 2 cases out of 20 with percentage of 10%. Typing of isolated C. perfrin-gens and prevalence of toxigenic and non-toxigenic strains in exam-ined internal organs of sheep was explained in table (4). It was noted that out of 18 internal organs ex-


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amined for C. perfringens isola-tion, the highest percentage of number of positive strains was re-corded in intestine (33.3%), while in liver (27.7%) in spleen and kid-ney (16.6%). Out of 5 C. perfrin-gens isolates from liver 3 was type A toxigenic while 2 was non toxi-genic type D with an incidence 60% and 40% respectively. Out of 6 C. perfringens isolates from in-testine, 3 was type A , 2 was type D and one of non toxigenic with an incidence of 50%, 33.3% and 16.6% respectively. All of 3 C. perfringens strains isolated from spleen was toxigenic type A with percentage of 100%. Finally out of 3 C. perfringens strains isolated from the kidney 2 was type A toxi-genic and 1 was type D with per-centage of 66.7% and 33.3 % re-spectively. The total incidence of C. perfringens isolated strains from internal organs was type A (64.7%), type D (29.4%) and non toxigenic (5.9%).

As shown in table (5), out of 124 blood samples P. multocida was identified in 28(26.9%) and 35(33.6%) from diseased sheep ex-amined by traditional bacterial iso-lation and by ELISA technique re-spectively, while out of 20 blood samples from apparently healthy sheep P. multocida was identified in 2 (10%), and 10 (50%) of sam-ples examined by traditional bacte-rial isolation and by ELISA tech-nique respectively.

Pathological results: 1- Pathological feature in freshly

dead animals as well as scari-fied sheep:

The lungs showed mainly hepatization of the apical lobe mostly with involvement of other lobes occasionally. The associated lymph node showed swelling. The liver showed mainly congestion while in few cases friable liver with grayish red pinpoint foci were observed. Kidney appear normaly in most of the cases , while in few cases kidney and urinary bladder appeared severely increased in size and congested . The intestine ap-peared congested. No apparent pathological alterations in spleen and heart.

2- Histopathological findings; Lungs showed mainly edema and dilatation of interlobular area, congestion of blood vessels and capillaries associated with perivas-cular and peribronchial infiltration of mononuclear inflammatory cells. Diffuse infiltration of neutro-philes filling the alveolar lumen with few alveolar macrophages were observed in some cases (Figs., 1-4). The associated lymph nods showed hyperplasia of lym-phoid follicles with sever infiltra-tion of neutrophiles in few cases (Figs., 5 & 6).

The liver mostly showed di-lated engorged blood vessels and sinusoids sever diffuse necrobiotic changes in hepatocytes with peri-


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Fig. (1): Lung of sheep showing areas of atlectasis with engorged capil-laries and mononuclear inflammatory cells infiltration (H&E X 200).

Fig. (2): Lung of sheep showing peribronchial, diffuse and impacted al-veolar lumen with inflammatory cells infiltration (H&E X 100).

Fig. (3 & 4): Lung of sheep showing thickened interalveolar septa, con-gestion, impacted alveolar lumen by polymorphneuclear inflammatory cells mainly , with few alveolar macrophages. (H&E X 400).

vascular mononuclear inflamma-tory cell infiltration. In few cases focal area of necrosis with loss of cellular details and architecture, sever neutrophilic infiltration and hemorrhage were observed (Figs., 7 & 8).

The kidney mostly showed dilated engorged blood vessels, hemorrhages, granular and vascu-lar degenerative changes in renal tubular epithelium, and inter tubu-lar mononuclear inflammatory cells infiltration. Hypercellularity of glomeruli and cystic tubules

were seen in few cases (Figs., 9 & 10).

The spleen showed, depletion of lymphocytes was the most com-mon lesion, while hemorrhage was observed in few cases (Fig. 11).

The intestine in most of the cases examined showed degenera-tion and detachment of intestinal epithelium with congestion, hem-orrhage and mononuclear inflam-matory cell infiltration in propria sub mucosa (Fig 12). The heart showed no characteristic changes except congestion of blood vessels.


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Fig. (5): Lymph node of sheep showing hyperplasia of lymphoied folli-cles with polymorphneuclear inflammatory cells infiltration (H&E X 200).

Fig. (6): Lymph node of sheep showing hyperplasia of lymphoied folli-

cles with sever polymorphneuclear inflammatory cells infiltration (H&E X 400).

Fig. (7): Liver of sheep showing focal area of necrosis , degenerative

changes in hepatocytes mainly vaccular degeneration with diffuse mononuclear inflammatory cells infiltration ( H&E X 100).

Fig. (8): Liver of sheep showing focal area of necrosis, degenerative

changes in hepatocytes mainly vaccular degeneration with diffuse polymorphneuclear inflammatory cells ( H&E X 400).


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Fig. (9): Kidney of sheep showing focal area of necrosis, heamorrhage, degenerative changes in renal tubular epithelial cells, and destruction of Bowmans capsule (H&E X 400).

Fig. (10): Kidney of sheep showing degenerative changes in renal tubular

epithelial cells formation of cystic tubules (H&E X 400). Fig. (11): Spleen of sheep showing depletion of lymphocytes and few

heamorrhage (H&E X 200). Fig. (12): Intestine of sheep showing congestion of blood vessels mono-

nuclear inflammatory cells infiltration in propria sub mucosa (H&E X 400).


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DISCUSSION

T he clinical symptoms in farms under observation in our

study were respiratory manifesta-tion associated with diarrhea in few cases and mortality ranged from 12.2-16.9%. This symptoms were referred to the isolation of Pasteurella multocida associated with C. perfringens in few cases, similar clinical symptoms were re-ported by (Soher and Ali, 1993; Ali and Mahmoud, 1993 and Mi-yashiro et al., 2007) associated with Pasteurella multocida and C. perfringens in sheep. Pneumonic pasteurellosis was first described in Iceland and sub-sequently has been reported in many countries such as Australia, Britain, Ethiopia, Norway, South Africa, Somalia and USA (Gilmo-ur and Angus, 1983). Isolation of Pasteurella mul-tocida from nasal swabs and blood samples from diseased cases were recorded in the present study (table 3), the incidence were 54.8% and 26.9% respectively. A higher inci-dence was reported by Selim et al. (2003) who revealed an incidence of 89.1 %, 67.2 % from nasal swabs and blood samples respec-tively. Previous investigator re-ported isolation of Pasteurella multocida from diseased and ap-parently healthy sheep as (Ores et al., 1997; Rusavai and Fodder,

1998 and Andrawis, 2001). In the present investigation sheep harbor Pasteurella multocida (85 isolates out of 104) more or less close to results reported by Ugochukwu (1985) who found the incidence among diseased sheep was 75%. On the other hand higher inci-dences were recorded by El Sherif and Abd El-Ghani (1974), Baul-jihad and Leipold (1995) who re-ported it as 85.9% and 90.2%, re-spectively. Mean while apparently healthy cases in the present study revealed an incidence of 40% which agreed with Andrawis (2001), who reported an incidence of 39.6% other investigators re-ported higher incidence 68.5% and 78.5% (Elsherif and Abd el Ghani, 1974 and Foder et al., 1990). The difference in incidence among apparently healthy cases may be explained due to carrier cases of sheep (Al Tarazi and Dognall, 1997). Our study re-vealed that Pasteurella multocida was isolated in an incidence of 30% - 10% from nasal swabs and blood samples of the apparently healthy sheep, these results were almost agreed with Selim et al. (2003) who reported 33.3% and 8.1% incidence from nasal and blood samples of apparently health cases, respectively. The incidence of Pasteurella multocida which were isolated from the internal organs from


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sheep (18 cases) were 100%, these result agreed with the obtained re-sults recorded by Sunder and Kumar (2001) and Selim et al. (2003) of Pasteurella multocida isolation from internal organs of freshly dead sheep. Our study showed that apply-ing the serum ELISA for diagnosis of pasteurella in living sheep was accurate and rapid when compared to the traditional isolation and identification of pasteurella organ-isms. As presented in table 5 the percentage of positive diseased cases were 33% while the tradi-tional bacteriological studies re-vealed 26.9%. Also, among the ap-parently healthy cases ELISA showed 50% positive comparing to 10% by the traditional isolation and identification. These findings agreed with Singh and Jaypr-akason (2001) and Smith and Holdman (1968).

The present work showed that numbers of positive samples of C. perfringens isolated from liver was 5 isolates by percentage 27.7%, while in intestine 6 isolates 33.3% and in spleen and kidney 3 isolates 16.7% as present in table (4), these findings agreed with Ghoniem et al. (1972). From the obtained re-sults it could be noticed that the incidence of type A (64.7%) while type D (29.9%) and non toxigenic strain (5.9%), so the C. perfringens type A, D, as a normal flora may

indicate the incidence of entero-toxaemia in sheep (Sterne and Warrack, 1964). The pathological alterations observed in the different internal organs were similar to those previ-ously described by several authors associated with infection by Pas-teurella multocida and C. perfrin-gens infection (Niilo, 1980; Han-cock et al., 1991; Soher and Ali, 1993; Ali and Mahmoud, 1993; Center for Food Security and Public Health, 2004, Center for Food Security and Public Health, 2005 and Miyashiro et al., 2007). In farms with mixed in-fection by both organisms the pathological and histopathological lesions were more sever than farms with P. multocida infection only. The lung showed lesions var-ied from congestion of capillaries with thickened interlobular septa and atlectasis, to sever lesions of perivascular and perbronchial mononuclear inflammatory cell in-filtration and diffuse neutrophilic infiltration filling the alveolar space with few alveolar macro-phages. Similar lesions were de-scribed by Hancock et al. (1991); Ali and Mahmoud (1993): Kusiluka and Kambarage, (1996) and Nasser (2000) associ-ated with P. multocida infection, while, Center for Food Security and Public Health (2004 and


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2005) described only pulmonary edema and congestion in sheep in-fected by C. perfringens. The mixed P. multocida with C. per-fringens infection in our study re-sulted in sever histopathological changes in tissues of the lung. The associated pulmonary lymph node shows lymphoid hyperplasia with sever neutrophilic infiltration. Ab-scesses in lymph node was de-scribed by Kusiluka and Kam-barage (1996) and Nasser (2000). The histopathological chang-es in the liver were necrobiotic changes in hepatocytes, focal ne-crosis with neutrophillic as well as mononuclear inflammatory cells infiltration. These changes were more pathgnomonic in sheep from farms with mixed P. multocida and C. perfringens infection. Similar lesions were described by (Soher and Ali, 1993, Ali and Mah-moud, 1993 and Center for Food Security and Public Health, 2004). The histopathological chang-es in the kidney of sheep from farms with P. multocida infections only were mostly congestion, haemorrhages, and few necrobiotic changes in renal tubular epithe-lium. While in sheep from farms with mixed P. multocida and C. perfringens the kidney showed hy-percellularity of glomeruli, throm-bosis and mononuclear inflamma-

tory cells infiltration, in addition to the above mentioned changes. Similar lesion were described by (Niilo, 1980; Ali and Mahmoud, 1993 and Miyashiro et al., 2007).

The spleen showed depletion of lymphocytes and hemorrhage and such lesion may represent an immune reaction to infection (Ali and Mahmoud, 1993).

The microscopical changes in the intestine were demonstrated as detached epithelium, hemorrhage, and mononuclear inflammatory cells infiltration and mostly seen in cases of mixed P. multocida and C. perfringens infection, similar lesions were described by (Niilo, 1980 and Miyashiro et al., 2007) Associated with C. perfringens in-fection.

The microscopic changes in the heart were only congestion of blood vessels; similar lesion was described by Miyashiro et al. (2007), and Ali and Mahmoud (1993), associated with P. multo-cida and C. perfringens infection.

The different lesions found in the examined organs in our opin-ion may caused by the toxin of the P. multocida and the sever lesions found in mixed infected cases may resulted from double action of P. multocida and C. perfringens toxin.

From this study it is con-cluded that P. multocida and C.


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perfringens infection among sheep in Sharkia Governorate farms con-stitute a major problem and need intensive precaution and preven-tive measures including; applica-tion of good hygiene and manage-ment in animal farms, application of allowed vaccination programs, feeding animals with balanced ra-tion, and raising the immune status of the animals. Among methods used in diagnosis of pasteurella ELISA was more efficient. P. mul-tocida and C. perfringens infection could be diagnosed histopathologi-cally and the histopathological changes were valuable in studying the severity of the infection.

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