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.
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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
