Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
370
STUDY THE PATHOLOGICAL EFFECTS OF Klebsiella
pneumoniae ISOLATED FROM LUNGS OF PNEUMONIA'S
INFECTED SHEEP AND EXPERIMENTALLY INFECTED IN
MICE.
Muna Sachit Hashim*, Zahra Saleh Mahdi*, Taghreed Jabbar Humadi*
Eman Hashim Yousif* , Thikra Abdulla Mahmood**
*College of Veterinary Medicine. University of Baghdad,Baghdad,Iraq
**College of Medicine University of Kufa ,kufa,Iraq
Key word: bacterial; Pneumonia; Field Animals; mouse.
Corresponding Author;Sachitmuna@yahoo.com.
ABSTRACT
Currents study was designed to investigations about Klebsiella pneumoniae
infection in sheep then evaluation its effects in mouse by experimentally infection
Field study conducting on examination of one hundred100 sheep's lungs in Kerbala
Province, these lungs were send to laboratory of microbes for microbial examine.
Results showed 44 % of lungs samples were infected with Klebsiella pneumoniae;
18% of samples were negative for bacterial examination and the others 38 samples
were infected with different type of bacteria but not frequently occurs as Klebsiella e
Laboratory study, the isolated Klebsiella pneumoniae used to induce experimental
infection in mice. Thirty 30 mixed mice at age of (4-6) weeks were used and
subdivided in to three 3 group; 1st contains 10 ten males injected with 0.25ml.IP. (1.5
*10) suspension of bacteria; 2nd contains 10 ten females injected with 0.25ml.IP. (1.5
*10) suspension of bacteria; 3rd contains mixed ten mice injected with Distilled Water.
Scarified 4 four animals were done at the end of one week's until last 4 four week.
Samples of tissues (liver, Kidney, lung, spleen, testes, uterus) were taken from each
mouse. Maine pathological lesions in lung of sheep infected with Klebsiella
pneumoniae were suppurative broncho pneumonia, giant cell infiltration and
granulomatous foci. Tissues samples of experimentally infected mice showed general
abscesses at 1-2 weeks of infection, necrosis with granuloma and giant cell infiltration
at 3-4 weeks of infection. Conclusion: Klebsiella pneumoniae infects lung sheep
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
371
and cause chronic suppurative pneumonia due to presence of giant cell as well as
experimentally infects mice and causes general suppurative and granulomatous
lesions.
INTRODUCTION
Klebsiella spp., particularly Klebsiella pneumoniae, are important causes of
nosocomial infections due to the presence of capsular polysaccharide which is a major
surface-located virulence properties associated with the pathogenesis of Klebsiella
pneumoniae.(1). The capsule is an elaborate polysaccharide matrix that encases the
entire cell surface and provides resistance against many host defense mechanisms. Dgalacton
II has an important role in synthesising the lipopolysaccharide of Klebsiella
pneumoniae and many other gram negative bacteria.(1). Klebsiella species were found
to be the most frequently isolated gram negative bacteria in cases of primary
bacteremia (2). It is the second pathogen, next to E. coli that causes urinary tract
infection. It normally affects persons with low immune system such as hospital
patients, diabetes patients and people with chronic lung disease. Many a times,
alcoholics also suffer from K. pneumoniae infections. Thus, the infections are either
hospital-acquired or community-acquired (2). Characteristically, Klebsiella spp.
produce large mucoid colonies because of the synthesis of large amounts of capsular.
Klebsiella is one of the most important members of Klebsiella genus in
Enterobacteriacae family, which is responsible for pneumonia (3). Besides it is found
to cause infections in the urinary and lower biliary tract (4)(5).
Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactosefermenting,
facultative anaerobic, rod-shaped bacterium. It appears as a mucoid
lactose fermenter on MacConkey agar. Although found in the normal flora of the
mouth, skin, and intestines(2 )it can cause destructive changes to human and animal
lungs if aspirated (inhaled), specifically to the alveoli (in the lungs) resulting in
bloody sputum. In the clinical setting, it is the most significant member of
the Klebsiella genus of the Enterobacteriaceae. K. oxytoca and K.
rhinoscleromatis have also been demonstrated in human clinical specimens. In recent
years, Klebsiella species have become important pathogens in nosocomial infections.
It naturally occurs in the soil, and about 30% of strains can fix nitrogen in anaerobic
conditions.( 3)As a free-living diazotroph, its nitrogen-fixation system has been
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
372
much-studied, and is of agricultural interest, as K. pneumoniae has been demonstrated
to increase crop yields in agricultural conditions.(4)
Members of the genus Klebsiella typically express two types of antigens on their cell
surfaces. The first, O antigen is a component of the lipopolysaccharide (LPS), of
which 9 varieties exist. The second is K antigen, a capsular polysaccharide with more
than 80 varieties. (5)(6) Both contribute to pathogenicity and form the basis
for serogrouping. It is closely related to K. oxytoca from which it is distinguished by
being indole-negative and by its ability to grow on melezitose but not 3-
hydroxybutyrate.
Figure (1): K. pneumoniae on a MacConkey agar plate
MATERIAL AND METHODS
Field study:
Samples: one hundred 100 lung were collected from slaughtered sheep in Kerbala
Province and put in cleaned container and sent to microbial laboratory . Examination
of bacteria Klebsiella pneumonia was doing according to authors (7).
Bacterial suspension: After examination of bacteria Bacterial Challenge Preparation:
K.peumonia isolated from lung sample of sheep with pulmonary infection was
employed in this study. Challenge dose was adjusted in mice by preparation of
bacterial suspension and bacterial turbidity according to McFarland test to (1.5x10
CFU/ml).Experimental animals were infected intarperitoneally with (0.25 ml) of
suitable concentration with (1.5x10 CFU)virulent viable K.peumoniae according to
author(8)surror, 2017).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
373
Laboratory study: White mice 30 male and female were injected with bacterial
suspension at different time (72hours; 2weeks; 3weeks; 4weeks).
RESULTS AND DISCUSSION
Results of field study: microbiological examination detects that 44% of lung samples
were infected with Klebsiella pneumoniae diagnosis performed according to authors
(7)(9).
Results of Laboratory study:
A: Group 1 at 72 hours of infection: Liver showed inflammatory cells aggregation
around congested central veins and in the interstitial tissue. Figure(2 ).Increase
thickness of intra alveolar septa of lung and inflammatory cell infiltration Figure(3).
Figure(2) :Histopathological section of liver mice showing infiltration of
inflammatory cell around congested blood vessels and central vein.
Figure(3 ) :histopathological section of lung in mice in 72 h. showing increase
thickness ofInter alveolar septa and infiltration of phagocytic cell with
presence of fibrinous exudation.
Spleen showed hyperplasia of endothelial cell of the central artery with hyperplasia
per arterial area .Figure(4) . Kidney showed congestion of blood vessels , hemorrhage
,infiltration of inflammatory cells in the interstitial tissue, desquamation of
epithelial lining cells of renal tubules ,dilatation of glomerular space .Figure(5 ).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
374
Figure(18 ) :Histopathological section of spleen showing inflammation around
spleen
characterized by acute fibrinopurelent perisplanitis.H&E.100X.
Figure( 19 ) :Histopathological section of kidney showing lymphocytic focal
infiltration lead to atrophy of renal tubules.H&E.100X.
Group2 at 2 weeks of infection: At the period of two week of infection with
K.pneumoniae liver showed multiple variable size micro abscess lesions around
congestion of blood vessels .Figure(6). Thickening of intraalveolar septa of lung and
infiltration of inflammatory cell .Figure (7).
Figure( 6 ) :Histopathological section of liver showing micro-abscesses lesion
around the central vein and scatter the liver.H&E.100X.
Figure (7) : Histopathological section of lung showing increase thickness of inter
alveolar septa, congestion of blood vessels and fibroblasts
proliferation.H&E.100X.
Kidney showed infiltration of inflammatory cell in the renal parenchyma, congestion
of blood vessles and stenosis of renal tubules. Figure (8).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
375
Figure( 8 ) :Histopathological section of kidney showing infiltration of
inflammatory Cell congestion of blood vessels, stenosis of renal
tubules.H&E.400X.
Group 3 at 3 weeks of infection: liver showed foci granulomatous lesion. Figure
(9). Lung showed increase thickness of inter alveolar septa due to excessive
inflammatory reaction(10) .
Figure ( 9 ) :Histopathological section of liver showing granulomatous lesion
.H&E.400.X
Figure (10) : Histopathological section of lung showing increase thickness of
inter alveolar septa from heavy inflammatory response .H&E.400.X.
Spleen showed amyloid deposition and reactive hyperplasia (11).Kidney showed
necrosis, hemorrhage and atrophy of glomerular tuft .figure (12).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
376
Figure (11 ) :Histopathological section of kidney showing necrosis, hemorrhage
amyloid deposition and atrophy of glomerular tuft.H&E.400X.
Figure (12 ) :Histopathological section of kidney showing necrosis, hemorrhage
amyloid deposition and atrophy of glomerular tuft.H&E.400X
Group4 at 4 week of infection: liver showed necrosis, hepatic cord disorganization.
Figure (13). Lung showed infiltration of inflammatory cell, pervasculitis. Figure (14)
Spleen showed hypoplasia and proliferation of reticuloendothelial cell. Figure (15) .
Figure ( 13 ) :Histopathological section of liver showing coagulative necrosis ;
kuffer cell infiltration. H&E. 400X.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
377
Figure ( 14 ) :Histopathological section of lung showing hyperplasia of
lymphoid tissue .H&E.100 X
Figure (15 ) :Histopathological section of spleen showed amyloid deposition
appear as pink area.H&E.400X.
Isolation of Klebsilla pneumonia from Iraqi sheep was agreed with author (10) who
isolates the bacteria from ticks infects sheep living in Basrah province. But there is highly
incidence of infection in lungs of sheep living in Kerbala province. Infected lungs of
slaughtered sheep as well as experimentally infected mice showed interstitial pneumonia
with suppurative bronchopneumonia and fibrinous pneumonia according to immune
status of animals.(11). Many differences in severity of pathological finding of
Pneumonia's characters in examined lungs depend on stages of infection and immunity of
body against infection and virulence of microbes (12)(13)(14). Author(15) found similar
finding in detection of Klebsilla pneumonia in Mousl city .Virulence factor of Klebsilla
pneumonia play important role in continuously or persistence of infection(11). K.
pneumoniae utilizes a variety of virulence factors, especially capsule polysaccharide,
lipopolysaccharide, fimbriae, outer membrane proteins and determinants for iron
acquisition and nitrogen source utilization, for survival and immune evasion during
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
378
infection(11) which acts as antimicrobial activity (16).and may cause fatal
pneumonia(18).other authors revealed the same pathogen in other city in iraq(19)(20) In
experimental infection in mice ,the histopathalogical change of liver after 72 hours
showed inflammatory cell aggregation around the interstitial tissue and central veins, this
as reported by author (21) liver parenchyma was infiltrate by large number of
neutrophils associated with degeneration ,lung showed increase thickness of interalveolar
septa due to infiltration of inflammatory cell this agrees with (22) who characteristics
lobar pneumonia of Klebsiella infection had developed, also ,area of apparent cellular
destruction could be visualized, as revealed by author( 23) who showed histopathalogical
changes like extension of poly nuclear cells through pleural membrane. At 2 weeks of
infection, the histopathalogical changes of liver showed hydropic degeneration in the
cytoplasm of of hepatocytes with infiltration of inflammatory cell in the parenchyma of
liver and congestion of blood vessels, as said by (24) who explained occurrence mild
inflammatory changes in spleen almost. The histopathalogical changes of lung showed
infiltration of inflammatory cell and increase thickness of interalveolar septa, this agreed
with (25) who reported sever necrosis of parenchymal tissue and infiltration of
inflammatory cell. Histopathlogical changes of spleen showed hyperplasia of endothelial
cells of central artery, this agreed with (26) who explained inflammatory aspects of
infection in spleen.
REFERENSECSE
1-R F Berendt, G G Long, F B Abeles, P G Canonico, M R Elwell, and M C
Powanda(1977). Pathogenesis of respiratory Klebsiella pneumoniae infection in rats:
bacteriological and histological findings and metabolic alterations. Infect Immun.
1977 Feb; 15(2): 586–593.
2-Klebsiella pneumoniae (Schroeter, 1886) Trevisan, 1887 in GBIF Secretariat
(2017) GBIF Backbone Taxonomy. Checklist dataset
https://doi.org/10.15468/39omeri accessed via GBIF.org on 2018-09-28.
3-Quinn,P.J; Cartet,M,E; Markey,B,K; Donelt.W.J; Leonnard,F.C.and
Maghire,D.(2002).Veterinary Microbiology and Microbial disease .Blackwell
Publishing Company.
4-Bruere,A; West,D and Ridler,A.(2000).Enzootic pneumonia, in sheep :health,
disease and production :written for veterinarians and farmers. Veterinary containing
Education massey university ,palmerston north,N.Z.,PP 100-108.
5-Saleh,N and Allam,T.(2014).Pneumonia in sheep: Bacteriological and clinic
pathological studies .American Journal of Research Communication. 2(11):73-88.
6-Jones, T.C.; Hunt, R.D. and King, N.W.(1997). Veterinary pathology .6th ed.
Williams and Wikins. Philadephia .U.S.A.657-658.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
379
7--Abeer Ghassan Munther Al-Agha1*, Nazar Jabar Muslih Al-Khafaji2 and
Amer Khazal Salih Al-Azawi(2017). Isolation and Identification of Klebsiella
pneumoniae using API-20E analytical system and conventional PCR assay.
International Journal of Current Microbiology and Applied Sciences ISSN: 2319-
7706 Volume 6 Number 8 (2017) pp. 203-210.
8- Surror, A.T.(2017). Immunopathological study of Klebsiella pneumonia in mice
immunized with two types of antigen .M.Sc. Thesis in Vet. Med. College of Vet
Med./ Zoonosis Unit.
9- Brown , A. and Smith, K. (2015). Microbiological Application , Laboratory
Manual in General Microbiology. 13th Ed. Pg.227.
10-Mohanad .F.A.Al-Amura and Moaed .H.Almyahii (2012). ISOLATION
AND IDENTIFICATION Escherichia Coli AND Klebsiella Pneumonia FROM
TICKS Hyalomma SPP. KOCH, 1844FROM SHEEP IN BASRAH CITY.
Bas.J.Vet.Res.Vol.11,No.1,2012 229.
11-Bei Li; Zhenhong Chen; Changting Liu(2014). Molecular pathogenesis of
Klebsiella pneumoniae Article in Future MicrobiologyVOL. 9, NO. 9 REVIEW .
12- Ryan, KJ; Ray, CG, eds. (2004). Sherris Medical Microbiology (4th
ed.). McGraw Hill. ISBN 0-8385-8529-9.
13-Lee, C.-R.; Lee, J. H.; Park, K. S.; Jeon, J. H.; Kim, Y. B.; Cha, C.-J; Jeong,
B. C.; Lee, S. H. (2017). "Antimicrobial resistance of hypervirulent Klebsiella
pneumoniae: epidemiology, hypervirulence-associated determinants, and resistance
mechanisms". Front. Cell. Infect. Microbiol. 7: 483. doi:10.3389/fcimb.2017.00483.
14-Chanishvili, N, ed. (2012). A Literature Review of the Practical Application of
Bacteriophage Research. Hauppauge, NY: Nova Science. ISBN 978-1-62100-851-4.
15- Hadeel, B.(2017). Pulmonary and Hepatic lesions in slaughtered sheep in Mousl
city. Tikrit Journal of Pure Science 22(6).
16.Hussein NH..(2017). Emergence of NDM-1 among carbapenem-resistant
Klebsiella pneumoniae in Iraqi hospitals. Acta Microbiol Immunol Hung. 2018 Jun
1;65(2):211-227. doi: 10.1556/030.64.2017.026. Epub 2017.
17- Bhagwan,P and Singh, N.(1972) .Pneumonia in sheep and goat in tarai; A
Pathological study.Indian Journal Animal Science .42: 938-942.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
380
18- Abubaker, M; Abdala ,S; Elfaki, M and Kamal, S.(1980). Pathological studies
on sheep and goat pneumonia in Sudan. Africa. Journal volum. (28):288-293.
19-. Abadmahmoud, M.(2017). Etiopathological aspects on some Pneumonic lesions
in Slaughtered sheep in Diyala Province. M.Sc. Thesis in Vet. Med. College of Vet
.Med./ Department of Pathology.
20- Abbas, H.O. and Zainab, W.Kh.(2016). The role of Klebsiella pneumonia for
effect on pneumonia in the sheep. Kufa Journal for Veterinary Medical Sciences
Vol.(7).No.(2).
21.Fungn, C ,P.; Chang, F.Y; Lin, J.C.; Ho ,D. M.; Chen, C .T. ;Chen, J. H., Yeh,
K. M. ;Chen ,T .L .,Lin ,Y .t; Siu ,L.K.(2011): Immune response and
pathophysiological features of K.pneumoniae liver abscesses in an animal model .J.
Lab Invest .91(7):1029-39.
22-Lawlor, M.S.; Hsu, J. ;Rick, P.D.; Miller, V.L.(2005). Identification of
Klebsiella pneumoniae virulence determinants using an intranasal infection model. J.
Mol .Microbiol.58(4):1054-73.
23-Nishi, T. and Tsuchiya, K.(1980).Therapeutic effect of Cefotiam and Cefazolin
on Experimental pneumonia caused by Klebsiella pneumonia DT-S in Mice. J.
Antimicrobial agent and chemotherapy p.549-556 vol.18, No. 4 00664804.
24-Tokyo, V.; Sharma, S.; Arora ,B. B.; Chhibber, S.(2003). Establishment of
asepsis model following implantation of Klebsiella pneumonia-infected fibrin clot
into the peritoneal cavity of mice .J. Folia Microbial (Praha). 48(5):665-9.
25-Raheema, R.H.(2016). Experimental study for the effect of
Trigonellafoenumgraeum (fenugreek) seeds extract on some biochemical and
histopathological study in induced diarrhea in mice by Klebsiella pneumonia. J.
Journal of pharmacy Volume 6. Issue (2016), PP.04-13.
26-Tokyo, V.; Sharma, S.; Arora ,B. B.; Chhibber, S.(2003). Establishment of
asepsis model following implantation of Klebsiella pneumonia-infected fibrin clot
into the peritoneal cavity of mice .J. Folia Microbial (Praha). 48(5):665-9.