Immunohistochemistry as a diagnostic tool for ...

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VanKuppeveld et al., 1994. 5-GGG AGC AAA CAG GAT TAG ATA CCC T-3. 5-TGCACCATCTGTCACTCTGTTAACCTC-3. MarF. MarR. M.arginini. Kobayashi.
Egyptian society for Cattle Diseases C/O Faculty of Veterinary Medicine

12th Cong. Egyptian Society For Cattle Diseases 3 - 6 Dec. 2013, Hurgada, Egypt

Immunohistochemistry as a diagnostic tool for Mycopllasma bovis in buffalo calves Rawhya, Emran* ,Sarfinaz S. Abd El Ghany**, Dina Y.H. el-Shafey***and Dalia M. Mohsen** *Department of pathology, **Department of Buffalo diseases,*** Department of Mycoplasma, Animal Health Research Institute.

ABSTRACT Mycoplasma bovis (M.bovis) induced acute and chronic pulmonary infection in bovine animals ,many research works have been done to study M.bovis infection in cattle calves .In this study ,we studied its form in buffalo calves aging from three months to one year old suffering from respiratory manifestations, 37 pneumonic buffalo lungs were chosen from 200 animals examined at El- basatien abattoir , conventional mycoplasma culture method was done and positive samples confirmed to be Mycoplasma by PCR using 16S common gene for Mycoplasma. Pathological picture was described and Immunohistochemicalexamination usingM.bovis monoclonal antibodies were done. tionship between M. bovis infections and Bovine Respiratory Disease ( BRD) has been proven, the exact role of this organism in the pathogenesis of disease is still controversial. For many years M. bovis was considered an opportunistic bacterium present in the respiratory tract of healthy cattle (Rosendal and Martin, 1986). More recently, however, it has been shown that M. bovis is always associated with lung inflammatory lesions and rarely identified in healthy animals (Thomas et al., 2002). Several studies have confirmed that M.bovis is the most common bacterium identified in feedlot cattle affected by chronic unresponsive pneumonia (Haines et al., 2001) and in veal calves with fatal bronchopneumonia (Adegboye et al., 1995a; Shahriar et al., 2002; Gagea et al., 2006a). Diagnosis of M bovis infection can be performed by several methods including isolation of the agent (Sachse et al., 1993; Stipkovits et al., 2001), immunohistochemical staining (Adegboye et al., 1995b; Knudtson et al., 1996) and the use of specific PCR probe in the lung samples (Ghadersohi et al., 1997;and Hayman and Hirst, 2003) as well as detection of specific antibodies in the serum (Byrne et al., 2000; and Le Grand et al., 2001). Rodríguez et al, (1996) examined lung tissues from calves naturally and experimentally infected with Mycoplasma bovis which were subjected to histopathological and immunohistochemical ex-

INTRODUCTION Bovine respiratory disease (BRD) is the most significant and widespread cause of economic loss in the beef cattle industry (Ellis, 2001). Various factors, such as environmental conditions, stress, as well as concurrent viral and bacterial infections, play important roles in initiating and developing bovine pneumonic processes (Cusack et al., 2003 .)Buffalo is one of the most important animals in Egyptian animal welfare; it also considers the first of dairy production for Egyptians. Borghese ( 2010) reported that buffalo could be more and more important to develop job, economy and market with the richness of food coming from milk and meat of high quality for protein value, for fat with reduced cholesterol and higher unsaturated fatty acids and, finally, for luxurious taste of cheese and meat products. . Many researchers have been done for investigating Mycoplasma infection as a cause of mastitis in Egyptian buffaloes (El- Ebeedy et al.,1985; Gad et al., 1987; Ragab et al.,1987;Zaitoun et al.,1991;El- Shinnawy et al.,1993; ElShabiny,1994;El -Shater et al.,1999; Aliaa ,2002; Kamelia et al.,2008; and Marouf et al.,2011), while few researchers studied the role of Mycoplasma as a main cause of respiratory disease in buffalo calves. Mycoplasma bovis is considered one of the most important causes of calf pneumonia worldwide (Nicholas and Ayling, 2003). Although the rela-

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12th Sci. Cong., Egyptian Society For Cattle Diseases, 3-6 Dec. 2013, Hurgada, Egypt amination. The latter was carried out with a monoclonal antibody raised against M. bovis, and an avidin-biotin-peroxidase complex (ABC) detection substrate system. Pulmonary lesions in naturally infected calves included exudative bronchopneumonia and extensive foci of coagulative necrosis surrounded by inflammatory cells. Experimentally infected lungs showed suppurative bronchiolitis and varying degrees of peribronchiolar mononuclear cell cuffing. M. bovis antigen in field cases was mainly detected at the periphery of the areas of coagulative necrosis, in necrotic exudates, and in close association with infiltrating macrophages and neutrophils. In lung tissue from calves with induced M. bovis pneumonia, antigen was located in epithelial cells, within inflammatory cells in airway lumina, and in alveolar walls. Other microbiological observations suggested that the ability of M. bovis to invade and cause lung parenchymal damage could be influenced by the participation of other pathogens. Thus the aim of this work is to investigate and focus on the pathogenesis of Mycoplasma infection in buffaloes’ lung and to detect the perfect method of diagnosis, comparing the results with that found in previous researches on cattle.

falo aging from three months to one year at (ElBassatin abattoir ,Cairo Governorate) separately in sterile plastic bags for Mycoplasma culturing and PCR , other parallel samples from the same animals were fixed in 10% neutral buffered formalin for Immunohistochemistry (IHC) and histopathological examination( BioGenex). Mycoplasma Isolation Samples were cultivated for Mycoplasma isolation according to culture procedures (Razin and Tully 1983). Isolates obtained were differentiated from Achloplasma by digitonin test as described by Erno and Stipkovits (1973). Suspected purified Mycoplasma isolates were tested biochemically and serologically by growth inhibition and growth precipitation test (Sabry etal ;1971 and Krogsgaurd-Jensen 1972). Detection of Mycoplasma using polymerase chain reaction (PCR): Preparation of samples for DNA extraction (Yleana et al., 1995): 5ml of a 24 hour broth cultures of samples were centrifuged for 10 minutes at 12000 rpm. The pellet was washed. Twice in 1 ml of phosphate buffered saline pH 7.2 (PBS) and suspended in 50 ul PBS. The cell suspension was heated directly at 100oC for 10 minute. In a heat block to break the cell membrane and then cooled on ice for 5 min. Finally, the cell suspension was centrifuged for 5 min and the supernatant containing chromosomal

MATERIALAND METHODS Thirty seven samples collected from pneumonic lungs chosen from 200 of slaughtered buf-

Table (1): Primers used for detection of 16S ribosomal RNA for ruminant Mycoplasma, Mycoplasma bovis, Mycoplasma bovirhinis and Mycoplasma arginini. Species

Designation

Sequence

According to Alberto et al ., 2006

Sequence of 16S

MunivF

5- AGA CTC CTA CGG GAG GCA GCA -3

co mmo n gene

MunivR

5-ACT AGC GAT TCC GAC TTC ATG -3

for Mycoplasma M. bovis

MboF

5-CCT TTT AGA TTG GGA TAGCGGATG-3

Yleana et al ., 1995

M.arginini

MboR MarF

5-CCGTCAAGGTAG CGT CAT TTCCTAC-3 5-GGG AGC AAA CAG GAT TAG ATA CCC T-3

VanKuppeveld et

MarR

5-TGCACCATCTGTCACTCTGTTAACCTC-3

al., 1994

Mbr F

5’- GCT GAT AGA GAG GTC TAT CG-3’

Kobayashi

Mbr R

5’- ATTACT CGG GCA GTC TCC-3’

etal.,1998

M. bovirhinis

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12th Sci. Cong., Egyptian Society For Cattle Diseases, 3-6 Dec. 2013, Hurgada, Egypt Marker used for PCR: It was 100bp DNA ladder 100-3000bp (Sigma) PCR Master Mix ; ALLiance Bio: 1.1x Taq mix complet (1.5mM mg cl2 final) Materials used for agarose gel electrophoresis: Agarose 100g molecular biological Grade for electrophoresis separation of nucleic acids in range of 50bp 50kb

color ,firm in texture and on cut surface showed small spaces representing dilated lymphatic or minute pulmonary abscesses . lung samples were cultured for mycoplasma isolation only, 7 ( 18.9%) were positive giving typical mycoplasma colonies Photo (1) ,this result matches with(Gabinaitiene et al., 2011) and about (12.1%) that has been found by (El-Shinnawy et al.,1993) and (30%) found by El-Shabiny,1994) in case of buffaloes mastitic milk samples and much more than the incidence found by ( Marouf et al.,2011) which was17.1% . Several studies have shown that Mycoplasma bovis (M. bovis ) is the most common bacterium in feedlot cattle affected by chronic unresponsive pneumonia (Haines et al.,2001)and in veal calves with fatal broncho pneumonia (Shahriar et al., 2002) . To confirm that the isolates belonged to the class Mollicutes isolated microorganisms were tested using a common 16S rRNA mycoplasma primer. It was determined that all isolated strains from lung samples belonged to the class Mollicutes but neither of our isolates could be identified as M.bovis ,M.bovirhinis nor M.arginini using PCR. Positive samples gave DNA band at 1000 bp (Alberto et al ., 2006) Photo (2) , while the same samples were negative using specific primers to M.bovis, M.bovirhinis and M.arginini otherwise 4(10.8%) lung samples showed positive IHC reaction to M.bovis by specific monoclonal antibodies Table (2) ,this result may suggest that IHC examination is effective in detecting quite fastidious organism like M. bovis than conventional culture method specially when there are mixed infection with other faster growing mycoplasma strain like in our study which is

Pathological Examination Collected samples were examined grossly for any gross lesions and then fixed in 10% buffered neutral formalin solution and processed routinely, paraffin sections were prepared, stained by H&E and Trichromo stain then examined microscopically for histopathology (Bancroft andGamble 2008). Immunohistochemical Stainin (Adegboye et al; 1995b): Immunohistochemistry applied on paraffin section using Murine monoclonal antibody anti M.bovis (Biox- Diagnostics) and supersensitive polymer-HRPIHC detection system according to the associated manufacture manual on coated slides, Sections were counterstained with Myer’s hematoxylin and examined microscopically. Results and Discussion Buffaloes are obviously more resistant to microbial infection, during our investigation 200 animals were examined by inspection, 37 (18.5%) chosen lung samples showed severe signs of pneumonia. Macroscopically lungs showed congestion, firm in texture and emphysema. Our results are nearly similar to that reported by (Rawhia et al, 2002) who described gross lesions of pneumonic bovine lungs as dark in 3

12th Sci. Cong., Egyptian Society For Cattle Diseases, 3-6 Dec. 2013, Hurgada, Egypt matches with (Dina el shafey 2005). These morphometric and histochemical broncoepithelial changes may be able to be used as markers of the severity of bovine respiratory mycoplasmosis (Nadeeka et al; 2012). Table (2): Results of mycoplasma culture, PCR, and IHC No of ex-

No of pneumonic

No of positive

No of positive

No of positive

amined

lung samples

samples to my-

samples to my-

lung tissues to

coplasma isola-

coplasma by

mycoplasma bovis

animals 200

37

7

7

4

Incidence

(37\200*100)18.5%

(7\37*100)18.9%

(7\7*100)100%

(4\37*100)10.8%

Photo (1): Mycoplasma colonies on agar 24 hours old culture (x 10) isolated from pneumonic buffalo calf lungs.

Photo (2): Agarose gel electrophoresis showing amplification of the 1000 bp fragment of 16S ribosomal RNA for ruminant. Lane1:DNA ladder, Lane 2: M.bovis Reference strain PG45 (control positive), Lane 3 and 5: positive Mycoplasma isolates, Lane 4: negative control ,Lanes from 6 to 12 : negative samples.

pluritis was obvious among most of the cases (photo 4A and 4B) . Some cases showed lung fibrosis especially around bronchiole and pulmonary vasculature associated with thrombosis (photo 5,6). Rosendal, (1994) reported that most of the mycoplasma produces peroxide and super oxides which impair host cell integrity. Simultaneously inactivating host cell catalase and super oxide dismutase may help the mycoplasma to survive in the host cells. Our results revealed that the histopa-

Immunohistochemical and histopathological results The histopathological examination of the lungs of mycoplasma positive cases showed variable degrees of bronchopneumonia characterized by hyperplasia and /or necrosis of the bronchial epithelia associated by necrotic exudates filling the bronchial lumen (photo 3 B,C)accompanied with round cells in and around the bronchioles. Lung alveoli showed different grades of hepatization, edema and /or emphysema(photo 3A and 4A) . Also

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12th Sci. Cong., Egyptian Society For Cattle Diseases, 3-6 Dec. 2013, Hurgada, Egypt thological pictures of buffalo infected lung were milled as compared with that observed with cattle by (Rawhia et al., 2002) . (Aliaa 2002,Khodakaram-Tafti and Lopez 2004 and Caswel and Archambault 2007) reported that the lesions of pneumonia associated with M. bovis infection in cattle and calves were characterized by subacute or chronic suppurative bronchopneumonia with multiple foci of caseous or coagulative necrosis or abscesses. Also (Mondal et al., 2004) described caprine mycoplasmal pneumonia as interstitial pneumonia with serofibrinous exudates and necrosis of lining cells, thrombosis in lung's blood vessels, thickening of interlobular septa and infiltration with neutrophils and macrophages. While Radaelli,et al., ( 2008) found that M.bovis antigen which was detected by immunohistochemistry in veal calves and adult cattle was associated with bronchogenic necrosuppurative or fibrinonecrotizing lesions.(Amit Kumar et al., 2012) observed M. bovis in Sheep settles down predominantly in broncho-alveolar region. The histopathological examination revealed lesions comprised of interstitial pneumonia accompanied by perivascular and peribronchial lymphoid cell infiltration .Accumulation of desquamated cells and serous exudate in bronchioles and alveoli with catarrhal pneumonia and the presence of inflammatory foci encapsulated by connective fibrous tissues. That differentiation of pulmonary lesions of buffalo calves comparative to other species could be attributed to the immune status of buffaloes. By application of immunohistochemistry on different lung tissues M.bovis antigen was detected

within the epithelial lining the bronchi and bronchioles (photo 7 A,B); within pnemocyte and round cells infiltrating around pulmonary vasculature, bronchioles and lung alveoli (photo 7 C,D). Also M.bovis antigen was detected on the round cell filling the lung alveoli ,the reaction appeared as specific dark-brown granular staining. Our result are going hand in hand with (Dina,2005) and that described by (Khodakaram and Lopez,2004) who said M.bovis can adhere to ,invade, and survive in epithelial and inflammatory cells. These characteristics can facilitate the persistence of M.bovis and the development of chronic pneumonia in the face of an immune response and prolonged antibiotic therapy. But our results are disagree with (Gagea,2006 and Dina and Rawhia ,2007) who recorded that M.bovis antigen in feedlot beef cattle was often distributed at the interface between coagulative and caseous lesions and at the periphery of the caseous foci. In bronchioles containing caseous debris, antigen was present within the depris and adjacent to but not within bronchiolar epithelial cells. In the cases of fibrinosuppurative bronchopneumonia, M.bovis antigen was detected in the cytoplasm of necrotic neutrophils and macrophages (oat cell) that filled the alveoli. Conclusion: 1. We can concluded that M. bovis can affect buffalo causing pneumonia but in mild form as compared with that of cattle. 2. IHC is an effective tool for diagnosis of Mycoplasma bovis in buffalo.

fig (4): Lung A,B showing variable grades of pleuritis associated with lung edema and congestion, the invading inflammatory cells are round cells H&E A×100 ,B×200

fig (3): Lung A, showing edema, emphysema, associated with focal hepatization B,C showing variable degrees of bronchitis H&E A×200, B,C×400

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12th Sci. Cong., Egyptian Society For Cattle Diseases, 3-6 Dec. 2013, Hurgada, Egypt

Fig (5): lung A, showing marked bronchial hyperplasia, B showing marked thickening of pulmonary blood vessels accompanied with thrombosis (arrow), H&E A&B×200.

Fig (6): lung A, showing fibrous thickness of pleura associated with round cells aggregation (arrow) B, showing fibrous tissue proliferation through lung alveoli Trichromo stain A×400 and B, ×200.

Fig (7): lung A,B,C and D showing intense dark-brown staining M.bovis antigen within bronchial epithelium (A,B) and showing specific dark-brown granular immunoperoxidase staining within macrophages and pneumocytes (alveolar epithelial cells (C) and within macrophages (arrow) around pulmonary vasculature and lung alveoli (D) Avidin-biotin immunoperoxidase complex with counterstaining mayer,shaematoxylinA×100&B×200. C×400 and D×1000.

niaAsian Journal of Animal and Veterinary AdvancesV. 7: 149-157 Bancroft JD and Gamble M (2008): Theory and Practice of Histological Techniques. 5thed., Churchill Livingstone. New York, London, Philadelphia. Borghese A., (2010): Development and perspective of Buffalo and Buffalo market in Europe and Near East. Via Salaria 31, 00015 Monterotondo, Roma (Italy) [email protected] Byrne, W.J., R. Mc Cormack, N. Brice, B. Markey and H.J. Ball, (2000): Isolation of Mycoplasma bovis from bovine clinical sample in the Republic of Irel and. Vet. Res., 148: 331-333. Caswell JL, Archambault M. (2007): .Mycoplasma bovis pneumonia in cattle. Anim Health Res Rev. 8 (2):161-86. Cusack, P.M., McMeniman, N., Lean, I.J., (2003): The medicine and epidemiology of bovine respiratory disease in feedlots. Australian Veterinary Journal 81, 480–487 Dina Y.El-Shafey (2005): Advanced studies on My-

References Adegboye, D.S., Hallbur, P.G., Cavanaugh, D.L., Werdin, R.E., Chase, C.C., Miskimins, D.W., Rosenbusch, R.F., (1995a): Immunohistochemical and pathological study of Mycoplasma bovis – associated lung abscesses in calves. Journal of Veterinary Diagnostic Investigation 7,333–337. Adegboye, D.S., U. Rasberry, P.G. Halbur, J.J. Andrews and R.F. Rosenbusch, (1995b): Monoclonal antibody based Immunohistochemical technique for the detection of Mycoplasma bovis in formalin-fixed paraffin embedded calf lung tissues. J. Vet. Diagn. Invest., 7: 261-265. Alberto, A;M.F. Addis;B. Chessa; T.Cubaddu; M.B. profiti; S. Rossati; Aliaa A.E. Mohamed(2002): Some bacteriological and mycoplasmalogical studies on respiratory tract infection in buffaloes and caws. M.V. Sc. Thesis Fac. Vet. Med. Zagazig University. Amit Kumar, Verma A.K., Gangwar N.K. and Rahal A. (2012): Isolation, Characterization and Antibiogram of Mycoplasma bovis in Sheep Pneumo6

12th Sci. Cong., Egyptian Society For Cattle Diseases, 3-6 Dec. 2013, Hurgada, Egypt coplasma bovis in cattle. Thesis presented to Cairo University Faculty of Vet. Med. Dina, Y.H. el shafey and Rawhia, M.A. Emran (2007): Detection of Mycoplasma bovis antigen in cattle tissues using Monoclonal antibodies by avidin biotin coplex technique and indirect immunoflorecent antibodies test.9th sci. Cong., Egyptian Socity for cattle Diseases, 2-4 dec.2007, Assiut, Egypt. El-Ebeedy, A.A.; Gad, A.S.; Rashwan, A.; Moustapha, A.; El-Ahli, S.S.; Ismail, S.and Allam, N.M. (1985): Isolation of bovine mastitis in Egypt. Egypt. Vet. Med. Ass., 45 (1): 247-253. Ellis JA. (2001): The immunology of the bovine respiratory disease complex. Vet Clin North Am Food Anim Pract. Nov; 17(3):535-50, vivii. Review. El-Shabiny, L.M. (1994): Enzyme linked immunosorbent assay for the diagnosis of Mycoplasma mastitis in cows and buffaloes. Vet. Med. J., 42 (2): 51-53. El-Shater, S.A.; Eissa, S.I.; Dardeer, M.A. and ElShabiny, L.M. (1999): Antigenic variability among Mycoplasmas isolated from cattle, buffalo and camels by SDS-PAGE and immunoblot.Proceeding of the 5th Sci. Cong., Egypt. Society for cattle diseases, 180-195. El-Shinnawy M.M.;S. Abd- El- Karim; A. Zaghawa;Kh.M. Akl; A.Metwalli and G.E.M. abou-El-Enean (1993): Immuno-fluorescent studies of clinical and subclinical mycoplasma mastitis in buffaloes.2nd Sci.Cong., Egyptian society for cattle Diseases, 5-7Dec.1993, Assiut, Egypt. Erno, H. and Stipkovits, L. (1973): Bovine Mycoplasma cultural and biochemical studies. Acta. Vet. Scand. 14, 450-463. Gabinaitiene1 A., Siugzdaite1 J., Zilinskas1 H., Siugzda 2 R. and Petkevicius1 S., (2011): Mycoplasma bovis and bacterial pathogens in the bovine respiratory tract. Veterinarni Medicina, 56 (1): 28–3428 Gad, A.S.; El-Balkimi, F.A.; El-Shennawy, M.M.; Eissa, S.I.; Rashwan, A. and El-Shabiny, L.M. (1987): Serological studies on Mycoplasma mastitis. Alex. J. Vet. Sci., 3 (1): 335-344. Gagea, M.I., Bateman, K.G., van Dreumel, T., McEwen, B.J., Carman, S., Archambault, M., Shanahan, R.A., Caswell, J.L.,(2006a): Diseases and pathogens associated with mortality in Ontario beef feedlots. Journal of Veterinary Diagnostic Investigation 18, 18–28. Gagea M. I., Bateman K.G., Shanahan R A., van Dreumel T., McEwen B. J., Carman S., Marie Archambault, Caswell J.L. (2006b): Naturally occurring Mycoplasma bovis–associated pneumonia and polyarthritis in feedlot beef calves J Vet Diagn Invest 18:29–40.

Ghadersohi, A., R.J. Coelen and R.J. Hirst, (1997): Development of specific DNA probe and PCR for the detection of Mycoplasma bovis. Vet. Microbiol., 56:87-98 Haines, D.M., Martin, K.M., Clark, E.G., Jim, G.K., Janzen, E.D., (2001): The immunohistochemical detection of Mycoplasma bovis and bovine viral diarrhea virus in tissues of feedlot cattle with chronic, unresponsive respiratory disease and/or arthritis. The Canadian Veterinary Journal 42, 857– 860. Hayman, B. and Hirst, R. (2003): Development of Seminested PCR for the detection of Mycoplasma bovis from bovine milk and mucosal samples. Vet. Microbiol., 91: 91-100. 1Kamelia M. Osman, 2K.A. Abd El-Razik, 1E.E. Barbar, 3Dina Y.H. ELShafey and 2Amany A. Arafa 2008: Molecular Typing of Mycoplasma Species Recovered from Bovine Mastitis. Global Veterinaria 2 (6): 360-368. Khodakaram-Tafti A, Lopez A(2004): Immunohistopathological findings in the lungs of calves naturally infected with Mycoplasma bovis. J Vet Med A Physiol Pathol Clin Med 51:10–14. Knudtson, W.U., D.E. Reed and G. Daniels, 1996: Identification of mycoplasmatales in pneumonic calf lungs. Vet. Microbiol., 11: 79-91. Kobayashi H, Hirose K, Worarach A, Paugtes P, Ito N, Morozumi T, Yamamoto K, (1998): In vitro amplification of the 16S rRNA genes from Mycoplasma bovirhinis, Mycoplasma alkalescens and Mycoplasma bovigenitalium by PCR. J Vet Med Sci. Dec;60(12):1299-303. Krogsgaurd-Jensen, A. (1972): Mycoplasma: Growth precipitation as a serodiagnostic method.App. Microbiol., 23,: 553-558. Le Grand, D., D. Calavas, M. Brank, C. Citti, R. Rosengarten, P. Bezzile and F. Poumarat, (2001): Serological prevalence of Mycoplasma bovis infection in suckling beef cattle in France. Vet. Rec., 150: 268-273. Marouf SA ,EL-Jakee J, Mohamed Kh F (2011): Preparation of autogenous bivalent vaccine for M. bovis and M. bovigenitalium in Egypt. Life Science Journal, 2011;8(4). Mondal D., Pramanik A.K. and Basak D.K.(2004): Clinico-haematology and pathology of caprinemycoplasmalpneumonia in rain fed tropics of West Bengal. Small ruminant research 51: 285-2995. Nadeeka K. Wawegamaa, Anna Kancia, Marc S. Marendaa, Peter D. Mansellb, Glenn F. Browninga, , Philip F. Markhama (2012): Histochemical and morphometric characterization of bronchopneumonia in calves caused by infection 7

12th Sci. Cong., Egyptian Society For Cattle Diseases, 3-6 Dec. 2013, Hurgada, Egypt with Mycoplasma bovis. Volume 158, Issues 1–2, 6 July, Pages 220–224. Nicholas, R.A.J., Ayling, R.D., (2003): Mycoplasma bovis: disease, diagnosis, and control. Research in Veterinary Science 74, 105–112. Radaelli E., Luini M., Loria G.R., Nicholas R,A.J. and Scanziani E. (2008): Bacteriological, serological, pathological and immunohistochemical studies of Mycoplasma bovis respiratory infection in veal calves and adult cattle at slaughter. Research in Vet.Scie.85 : 282-290. Ragab A.M. ;Laila M. El-Shabini; A.S. Mostafa ,and Amal Rashwan (1987): Clinico-pathological Aspect of Mastitis due to Mycoplasma bovigenitalium with special reference to some milk lipids .Vet. Med. J.35,no,1,61-71(1987). Rawhia M.A.Omran, Dina Y.H.El-Shafey and Amal M.R ashwan (2002): Histopathological examination and immunohistochemical technique for detection of Mycoplasma bovis in formalin fixed paraffin embedded calf lung tissues using monoclonal antibodies. Egypt J.Comp.& Clinic.Path. 15 (2) 140-154. Razin, S. and Tully J.G. (1983): Textbook. Methods in mycoplasmology. Vol. II.Acad. Press. I.N.C., USA. Rodríguez F, Bryson DG, Ball HJ, Forster F.(1996): Pathological and immunohistochemical studies of natural and experimental Mycoplasma bovis pneumonia in calves.J Comp Pathol. 115(2):151-62. Rosendal, S. (1994): Toxicity and toxins of mycoplasmas. In: Gyles, C. L., Theon, C.O. (Eds.) pathogenesis of bacterial infections in animals 2 nd ed. International book distribution Co. Lucknow, India Rosendal, S., Martin, S.W., (1986): The association between serological evidence of mycoplasma infection and respiratory disease in feedlot calves. Canadian Journal of Veterinary Research 50, 179–183. Sabry, M.Z.; Erno, H. and Freundt, E.A. (1971): Manual of technical methods for the characterization and serotyping of Mycoplasma.Ed. by mycoplasma

division, Ani. Helth. Res. Inst. of Agric, Dokki,Giza Egypt. Sachse, K., H. Pfutzner, H. H otzel, B. Demuth, M. Heller and E. Berthold, (1993): Comparison of various diagnostic methods for the detection of Mycoplasma bovis. Rev. Sci. Tech. Off. Int. Epiz., 12: 571-580 Shahriar, F.M., Clark, E.G., Janzen, E., West, K., Wobeser, G., (2002): Coinfection with bovine viral diarrhea virus and Mycoplasma bovis in feedlot cattle with chronic pneumonia. The Canadian Veterinary Journal 43, 863–868 Stipkovits, L., P.H . Ripley, J. Varga and V. Palfi, (2001): Use of valnemulin in the control Mycoplasma bovis infection under field conditions. Vet. Rec., 148:399-402. Thomas, A., Ball, H., Dizier, I., Trolin, A., Bell, C., Mainil, J., Linden, A., (2002): Isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium. The Veterinary Record 151, 472–476. Yleana, R. Chaves Gonzalez, Carlos Ros Bascunana, Goran Bolske, Jens G. Mattsson, crmen Fernandez Molina Karl-Erik Johansson(1995): In Vitro amplification of the 16S RNA gene from Mycoplasma bovis and Mycoplasma agalactae byPCR. Vet Microbial.,47:183-190. van Kuppeveld FJ, Johansson KE, Galama JM, Kissing J, Bölske G, Hjelm E, van der Logt JT, Melchers WJ.1994: 16S rRNA based polymerase chain reaction compared with culture and serological methods for diagnosis of Mycoplasma pneumoniae infection. Eur J Clin Microbiol Infect Dis.May;13(5):401-5. Zaitoun, A.M.; El-Allowy, T.A.; Abdallah, I.S.; ElEbeedy, A.A.; Eissa, S.I. and El-Shabiny, L.M. (1991): Incidence of Mycoplasma infection in mastitic cows and buffaloes in upper Egypt. Assiut Vet. J., 25 (50): 108-114.

‫اﻟﻣﻠﺧص اﻟﻌرﺑﻲ‬ ‫إﺧﺗﺑﺎر اﻟﻧﺳﯾﺞ اﻟﻣﻧﺎﻋﻲ اﻟﻛﯾﻣﺎوي ﻛﺄداة ﺗﺷﺧﯾﺻﯾﺔ ﻟﻠﻣﯾﻛوﺑﻼزﻣﺎ ﺑوﻓس ﻓﻲ ﻋﺟول اﻟﺟﺎﻣوس‬ ‫*روﺣﯾﺔ ﻋﻣران و**ﺳﺎرﻓﯾﻧﺎز ﺳﺎﻣﻰ ﻋﺑد اﻟﻐﻧﻲ و**داﻟﯾﺎ ﻣﺣﻣد ﻣﺣﺳن و ***دﯾﻧﺎ ﯾﺣﻰ اﻟﺷﺎﻓﻌﻰ‬ ‫*ﻗﺳم اﻟﺑﺎﺛوﻟوﺟﯾﺎ و**ﻗﺳم أﻣراض اﻟﺟﺎﻣوس و***ﻗﺳم اﻟﻣﯾﻛوﺑﻼزﻣﺎ‬ ‫ﻣﻌﮭد ﺑﺣوث ﺻﺣﺔ اﻟﺣﯾوان‬ ‫ وﻗد أﺟرﯾت اﻟﻌدﯾد ﻣن اﻷﻋﻣﺎل اﻟﺑﺣﺛﯾﺔ ﻟدراﺳﺗﮭﺎ ﻓﻲ اﻟﻌﺟول‬، ‫ﺗﺳﺑب اﻟﻣﯾﻛوﺑﻼزﻣﺎ ﺑوﻓز اﻟﺗﮭﺎب رﺋوي ﺣﺎد أو ﻣزﻣن ﻓﻲ اﻷﺑﻘﺎر واﻟﺟﺎﻣوس‬ ‫ أﺷﮭر اﻟﻰ ﻋﺎم و ﯾﻌﺎﻧون ﻣن أﻋراض‬3 ‫ درﺳﻧﺎ ﺻورﺗﮭﺎ ﻓﻲ ﻋﺟول اﻟﺟﺎﻣوس اﻟذﯾن ﺗﺗراوح اﻋﻣﺎرھم ﻣن‬،‫اﻟﺑﻘرﯾﺔ و ﻓﻲ ھذه اﻟدراﺳﺔ‬ ‫ ﻋﯾﻧﺔ رﺋﺔ ﻣﺻﺎﺑﺔ اﺧﺿﻌت ﻟﻠﻔﺣص اﻟﻣﻌﻣﻠﻲ ﻟﻌزل ﻣﯾﻛروب‬37 ‫ ﺣﯾوان ﻓﻲ ﻣﺟزر اﻟﺑﺳﺎﺗﯾن وﺗم اﺧﺗﯾﺎر‬200 ‫ ﻓﻘد ﺗم ﻓﺣص‬.‫ﺗﻧﻔﺳﯾﺔ‬ ‫اﻟﻣﯾﻛوﺑﻼزﻣﺎ وﺗم اﻟﺗﺄﻛد ﻣن ﻧﺗﺎﺋﺞ اﻟﻔﺣص ﻟﻠﻌﯾﻧﺎت اﻹﯾﺟﺎﺑﯾﮫ ﺑواﺳطﺔ اﺧﺗﺑﺎر اﻧزﯾم اﻟﺑﻠﻣرة اﻟﻣﺗﺳﻠﺳل ﺑﺎﺳﺗﺧدام اﻟﺟﯾن اﻟﻣﺷﺗرك ﻟﻠﻣﯾﻛوﺑﻼزﻣﺎ‬ ‫ أس ﻛﻣﺎ وﺻﻔت اﻟﺻورة اﻟﺑﺎﺛوﻟوﺟﯾﺔ ﻟﻼﻧﺳﺟﺔ اﻟرﺋوﯾﺔ اﻟﻣﺧﺗﺎرة واﺟري ﻋﻠﯾﮭﺎ اﺧﺗﺑﺎر اﻟﻧﺳﯾﺞ اﻟﻣﻧﺎﻋﻲ ﺑﺎﺳﺗﺧدام اﻷﺟﺳﺎم اﻟﻣﻧﺎﻋﯾﺔ‬16 . ‫وﺣﯾدة اﻟﻧﺳﻠﺔ ﻟﻠﻣﯾﻛوﺑﻼزﻣﺎ ﺑوﻓز‬ 8