Mixed infection of Mycoplasma and bacteria in the respiratory tract of sheep with reference to the histopathological picture in Sharkia Governorate

Rania, H. Abd-Algawad*; Rehab, E.  Mowafy**

Amira, E. Lamey***; Noha, M.A. Atia; *** and Heba, A.M. Ewes**

*Mycoplasma Department, AHRI, Dokki - ARC

**Pathology Department, Zagazig Province Lab. AHRI- ARC

***Bacteriology Department, Zagazig Province Lab. AHRI- ARC

INTRODUCTION:

Sheep production is encouraged in Egypt as it can improve the daily protein intake of humans; it is constituted as an important component of Egypt food security plan (Elshazly and Youngs2019). In 2017, 2.34 million head was produced representing approximately 7.4% of all red meat production in Egypt (FAO STAT, 2018).

Sheep has occupied an advanced position within the Egyptian livestock sector because of their suitability to different agricultural conditions in the country(Elshazly and Youngs 2019), However sheep diseases represent major limiting factor for such huge industry where pneumonia and neonatal diarrhea represents the more leading health problems (Tarabees et al., 2016).

The respiratory problems particularly different types ofpneumonia are common in all species of domestic animals.The causative agents are multifactorial and the diseaseappears due to the interaction of the infectious micro-organisms (bacteria; Pasteurella and Mycoplasma, viruses; PI3, reovirus, and adenovirusand fungi), hostdefense, environmental factors and stress (Lacasta et al., 2008 and Azizi et al., 2011).

Different species of Mycoplasma are associated with many pathological problems in small ruminants including respiratory manifestation, this problem results in significant losses, especially in African countries (Pálma et al., 2018). It is highly fastidious microorganisms that required very precise media to develop in vitro (Abdel-Halium et al., 2019).

Mycoplasma species commonly associated with pneumonia in small ruminants are M. ovipneumoniae, M. arginini, M. capri, M. capripneumoniae, and M. capricolum (Chinedu et al., 2016). M. arginini is frequently isolated with M. ovipneumoniae from cases of atypical pneumonia in sheep and goats (Kumar et al., 2013) and other cases with lung consolidation (Fernández et al., 2016). In Egypt, different Mycoplasma species have been isolated including M. arginini, M. ovipneumoniae, and M. agalactiae (Ammar et al., 2008).

Although the role of M. ovipneumoniae in sheep respiratory infection is often overlooked, it may be the primary agent with various manifestations (chronic soft persistent cough with ocular and nasal discharges) but the real problem lies in that the disease condition become exaggerated by other bacterial infections (Ozturkler and Otlu  2020).

E. coli is the most common member of family Enterobacteriacea frequently isolated from respiratory affection in sheep either from nasal swabs or from pneumonic lungs (Zaghawa et al., 2010). Also Klebsiella pneumonia and Klebsiella oxytoca ofthe same family are mostly associated with ovine pneumonic cases (Patel et al., 2017 and Franco et al., 2019).

Pseudomonas aeruginosa induces various disease conditions in sheep and goats with respiratory illness considered as one of the major issues mainly represented by pneumonia leading to significant mortalities and increased economic losses (Dapgh et al., 2019).

Mixed infection with different bacterial causes in the same sheep was common andthis was attributed to the respiratory problems which were considered as multifactorial diseases where interaction between different microbial agents as bacteria leads to increase the incidence of those problems (Thrusfield and Robert, 2018).

There has been some degree of resistance of pathogens isolated from pneumonic sheep to some of the commercially used antibiotics (Goodwin-Ray, 2006). However, there are some effective antimicrobial agents as ciprofloxacin, ceftriaxone and oxytetracycline for the treatment of sheep pneumonia (Kumar et al., 2018).

Severe congestion of the nasal sinus with catarrhal mucous, bilateral pneumonia with pulmonary congestion of cranial lobes with progression to the caudal lobes, fibrinous pleurisy, adhesions to the chest. Histological sections of the lung revealed suppurative bronchopneumonia with alveoli and bronchioles filled with variable proportions of neutrophils, macrophages, serofibrinous exudation, degenerated leukocytes and necrotic debris, and multifocal presence of aggregates of bacteria. Fibrinous pneumonia and severe distension of the interlobular septa by fibrin and edema was also observed and occasional peribronchiolar lymphocytic accumulation (Rosárioetal., 2010).

Our study aimed to throw a spot of light on the mixed infection of Mycoplasma with other bacterial agents isolated from respiratory manifested sheep and studying their antibiotic susceptibility patterns for selection of the appropriate ones. In addition,the current study elucidated the macroscopic and histopathological lesions in the affected organs.

MATERIAL AND METHODS

Samples collection:

One hundred and ten samples were collected from respiratory manifested sheep (fever, nasal discharge, râles, cough, accelerated respiration, dyspnea), freshly dead and slaughtered sheep. Thirty nasal swabs and eighty tissue specimens including pneumonic lungs and tracheas from 60 freshly dead and 20 slaughtered sheep were aseptically collected. These samples were collected from different farms at Sharkia Governorate and immediately transported in icebox to the laboratory.

Bacteriological isolation:

All samples were submitted for bacteriological and mycoplasmal examinations. Bacteriological isolation was done by inoculation into brain heart infusion broth then streaked into and MacConkey, Ethylene Methelyne Blue (EMB) (Oxoid- UK) and Cetramide agars (HIMEDIA) followed by further identification for suspected isolates.The various members of family Enterobacteriacae were subjected to biochemical identification according to Krieg and Holt (1984).Pseudomonas was identified biochemically as described by Quinn et al. (1994).Mycoplasma isolation was done byinoculation into PPLO broth media, then plated onto PPLOagar media (Sabry and Ahmed, 1975) and maintained at 37ºC for 3-7 days with 24-48h observation interval for “fried egg” colonies under dissecting microscope (Reichert Wien – Germany).

Identification of thebacterial isolates:

Biochemical identification of the purified Mycoplasma isolates was applied according to Watson etal. (1988). Digitonin sensitivity, glucose fermentation, arginine deamination and film and spot formation tests were applied as mentioned by (Erno and Stipkovits, 1973 &Razinetal., 1998). Serological identificationof the isolated E. coli andKlebsiellaisolates was done according to Koketal. (1996) and Carter (1984), respectively in “Food Analysis Center - Faculty of Veterinary Medicine - Benha University.

Molecular confirmation of the bacterial isolates:

The bacterial genomic DNA was obtained using the extraction kit (GeneJET Genomic DNA purification KitThermo-scientific) following the manufacturer’s instructions. DNA concentration was determined using nanodrop. The PCR primers were synthesized by metabion international AG, (Germany), (Table1). The PCR reaction was performed in agradient thermal cycler (1000S Thermal cycler Bio-RAD, USA).The total volume of thereaction mixture (50µl) contains: 25µl PCR master mix (Thermo Scientific™ PCR Master Mix (2X) Cat. no: K0171, USA.), 3µl target DNA, 1µl of each primers (10p mole/µl) and the mixture was completed to 50µl by PCR grade water.

Table (1): Primers sequences used of target genes

After amplification as shown in Table(2),the PCR products for the target geneswere separated by 1.5% agarose gel electrophoresis (Sigma,USA) using Tris-boric EDTA buffer and stained with ethidium bromide using GeneRuler 100base pair DNA ladder (Thermoscientific Company, Cat.No.SM0243,USA).

Table (2): PCR amplification conditions for thebacterial PCR products

Pathological investigations:

          All investigated eighty tissue samples including freshly 60 dead and 20 slaughtered sheep were carefully examined for gross abnormalities. The gross tissue lesions were observed and recorded carefully, and representative parts of the tissue samples were fixed in 10% neutral formalin buffer for further histopathological studies. Afterward, the preserved samples were dehydrated in alcohol, cleared in xylene, impregnated and embedded in paraffin wax, sectioned at 4μm and finally stained with hematoxylin and eosin (H&E) for histopathological examination as described by Survarna et al. (2013) and examined microscopically.

Antibiotic susceptibility test:

It was performed forthe obtained bacterial isolates by disc diffusion method for testing their susceptibly to ampicillin, ceftriaxone, chloramphenicol, ciprofloxacin,colistin, doxycycline, erythromycin, gentamycin, lincomycin, oxytetracycline, spectinomycin and sulfamethoxazole+trimethoprimrepresenting different antibiotic classes according to CLSI (2017).

RESULTS

Prevalence and identification:

M. ovipneumoniae, E.coli, Klebsiella sp. and P. aeruginosa were detected with recovery rates (31.8, 21.8, 11.8 and 9.1%, respectively) (Table 3). Biochemical identification declared that all Mycoplasma isolates were positive for digitonin inhibition and glucosefermentation but negative for arginine hydrolysis and film and spot formation. Different typesof pneumonia including; alveolar pneumonia, bronchopneumonia, fibrinous bronchopneumonia, hemorrhagic pneumonia and interstitial pneumonia were correlated with their causitive agents and thus, multiple types were observed in the mixed infections. Alveolar pneumonia was the most prominent type associated with M. ovipneumoniae, E. coli and P. aeruginosa (Table 3).

Table (3): Prevalence of bacterial isolates associated with type of pneumonia

 n= number

Mixed infections between different bacterial isolates were illustrated in Table (4). M. ovipneumoniae was observed in mixed infection with E.coli and P. aeruginosa while K. pneumoniae. was detected with E.coli and P. aeruginosa (Table 4).

Table (4): Mixed infection between the bacterial isolates

n= number of samples

Members of family Enterobactariaceae were serologicaly identified and results revealed the detection of E. coli serogroups (O153, O44, O91, O84 and O8) where O153 was the most prominent serogroup. All Klebsiella sp. were identified as K. pneumoniae except for only one isolate was identified as K. oxytoca in a single infection (Table 5).

Table (5): Serological identification of E. coli and Klebsiella sp.:

Molecularidentification:

Conventional PCR test was carried out on the culturally positive bacterial isolates after being biochemically and serologicallyidentified for confirmation of these isolates. The obtained results revealed the detection of 16SrRNA genes specific for Mycoplasma sp. and M. ovipneumoniae in seven and eight examined isolates which gave their characteristic bands at 280bp and 361bp, respectively as shown in Fig (1 and 2).

Fig (1):Agarose gel electrophoresisof the group-specific primer set of Mycoplasma16SrRNAgene. Lane (1): 100bp DNA ladder. Lane (2): control negative. Lane (3): control positive. Lanes(4-10): positive samples for the target gene at 280bp.

Fig(2):Agarose gel electrophoresis of M. ovipneumoniae16SrRNA gene.Lane (1): 100bp DNA ladder. Lane (2): control positive. Lane (3): control negative. Lanes (4-11): positive samples for the target gene at 361bp.

16SrRNA specific gene of P. aeruginosa was amplified in five isolates showing its characteristic band at 965bp as illustrated in Fig (3).

Fig (3):Agarose gel electrophoresis of P. aeruginosa 16SrRNA gene.Lane (1):100bp DNA ladder.Lane (2): control positive. Lane (3): control negative. Lanes (4-8): positive samples for the target gene at 956bp.

E. coli 16SrRNA gene was successfully amplified in six tested E. coli isolates representing different serogroups at 662bp (Fig 4).

Fig (4):Agarose gel electrophoresis of E.coli16SrRNA gene. Lane (1):100bp DNA ladder. Lane (2): control positive. Lane (3): control negative. Lanes (4-9): positive samples for the target gene at 662bp.

Klebsiella gyrA gene was amplified in six isolates (5 K. pneumonaie and 1 K. oxytoca) and gave a specific band at 441bp (Fig 5).

Fig (5):Agarose gel electrophoresis of KlebsiellagyrA gene. Lane (1): 100bp DNA Ladder. Lane (2): control positive.Lane (3): control negative.Lanes(4-9): positive samples for the target geneat 441bp.

Gross Findings:

Grossly,tracheas showed limited gross lesions restricted in mild to moderate congestion in some cases and petechial hemorrhage in other few cases. Lung lesions were observed and described then categorized into the following types: (1) Congestion (15%), (2) hemorrhage (20%), (3) mixed congestion and hemorrhage (35%), (4) hepatization of lungs (20%) and (5) emphysema (10%) (Fig 6).

Microscopic findings:

          Various pictures of tracheal lesions in infected sheep were observed. Tracheal lesions were not specific to certain pathogen. Most cases showed submucosal and periglandular leukocytic cells infiltration (Fig7a& 7b). Only heavy infected cases revealed necrosis of some chondrocytes of tracheal rings cartilage (Fig7c). Metaplasia of some tracheal epithelium into goblet cells common lesion (Fig7d). Five types of pneumonia were identified, microscopically, categorized and summarized in Table (3) with its related pathogens. Peribronchial hyalinization of connective tissues with hyperplasia of bronchial epithelium was observed (Fig 8a). Alternative red and grey hepatization of pulmonary tissue (Fig 8b) was the most observed lesion as common stages of pneumonia. Alveolar pneumonia was characterized by dilated and congested pulmonary blood vessels with the presence of serous alveolar exudate with leukocytic cells infiltrations. Bronchopneumonia was characterized by the pneumonia presence of inflammatory cells consisted of mainly neutrophils within the lumen of bronchiole. Fibrinous bronchopneumonia (Fig 8c) characterized by peribronchial inflammatory zone with intrabronchial fibrinous exudates with visible fibrin strand, thickened interlobular septa, congestion of pulmonary blood vessels. Hemorrhagic pneumonia was also detected and characterized by excessive extravasated erythrocytes within the bronchi, alveoli and interalveolar septa related to leukocytic cells infiltration. Leukocytic cells infiltrated pulmonary tissues suffering from extravasated erythrocytes (Fig 8d) were observed in some cases. Lung showed emphysema (Fig 8e).Interstitial pneumonia characterized by congestion with reactive cells in and around the bronchial wall, and sometimes intrabronchial exudate contained few fibrin threads with peribronchial inflammatory zone with fibroblast and thickened interlobar septa.

Fig (6): Lung of infected sheep with E. coli ₊ M. ovipneumoniae showing multiple focal areas of congestion and hepatization of lung tissue (arrows).

Fig (7): Photomicrograph of trachea of infected sheep. (a): trachea infected with Klebsiellasp. showing submucosal leukocytic cells infiltration (arrow)(H&E x200).(b):trachea infected with M. ovipneumoniae showing periglandular leukocytic cells infiltration (arrow)(H&E x100).(c):trachea infected with P. aeruginosa showing necrosis of some chondrocytes (arrows)(H&E x400). (d): trachea infected with E.colishowing metaplasia of some tracheal epithelium into goblet cells(arrows)(H&E x200).

Fig (8): Photomicrograph of lung of infected sheep. (a): lung infected with M. ovipneumoniae + P. aeruginosa showing peribronchial hyalinization (arrows) with hyperplasia of bronchial epithelium (arrow head) (H&E x400). (b): lung infected with Klebsiella sp. + M. ovipneumoniae showing alternative red and grey hepatization of pulmonary tissue (H&E x100). (c): lung infected with P. aeruginosa + E. coli showing intrabronchial exudate and peribronchial inflammatory zone with fibrin threads and mild thickened interlobar septa (H&E x200). (d): lung infected with P. aeruginosa showing leukocytic cells infiltrated pulmonary tissue (arrow) suffering from extravasated erythrocytes (H&E x100). (e): lung infected with M. ovipneumoniae showing emphysema (arrow) (H&E x400).

Anti-bogramsusceptibility:

Results revealed absolute resistance to some antibiotics andvariable resistance levels to other ones that vary from one bacterium to another.M.ovipneumoniae were sensitive to doxycycline lincomycin, oxytetracycline and spectinomycin. E. coli isolates showed highest susceptibility tocolistin and ciprofloxacin with absolute resistance to oxytetracycline and ceftriaxoneand variable resistance levels to rest of the used antibiotics. Klebsiellaisolates represented susceptibility to ciprofloxacin,gentamycin and chloramphenicoland variable susceptibility to other antibiotics P. aeruginosawas only sensitive to ciprofloxacin and gentamycin and highly resistant to other tested antibiotics.Highest susceptibility levels were recorded for ciprofloxacin on all examined bacteria(Table 6).

Table (6): Anti-biogram susceptibility results of the isolated bacteria

S= sensitive                                         R= resistant                

DISCUSSION

Sheep respiratory diseases are sometimes acute and fatal. Early rapid and specific diagnosis allowed designing appropriate prevention and control strategies to alleviate the economic losses (Chakrabortyet al. 2014),consequently the present study aimed to correlate the histopathological findings in respiratory diseased sheep with their bacterial etiologies.

Prevention and control of fatal infectious respiratory diseases of small ruminants require many diagnostic strategies to be adopted and routine antibiotic sensitivity tests to be done. The diagnostic tests include combination of conventional and advanced diagnostic investigations. However, the initial suggestive diagnosis involves clinical signs observation accompanied by postmortem examination followed by isolation, serological and molecular detection of etiological agents (Chakrabortyet al., 2014).

The obtained data of the bacteriological examination indicated the role of M. ovipneumoniae, E. coli, Klebsiella sp. and Pseudomonas sp. in occurrence of respiratory diseases.The total bacterial recovery rate was 82/110 (74.54%) which is in accordance with Zagwa et al. (2010) (78.3%).Furthermore, results revealed the presence of these bacterial species as single or mixed infection as reported in the previous studies (Kumar etal., 2018, Mona, 2019 & Nahed and Allam, 2019). Mixed infections with different bacterial etiologies in respiratory affection are common and that can be explained by the fact that infection with one bacterial agent can increase the chance of infection with another (Mona, 2019).

Mycoplasma is considered a part of the respiratory tract micro biota in sheep that can turn pathogenic under certain conditions. Primary infection with M. ovipneumoniae can lead to secondary infection by other micro-organism (Franco et al., 2019). Isolation rates showed that M. ovipneumoniae was the highly detected bacterium (31.8%). Our findings are in accordance with Ibrahim et al. (2018) who isolated Mycoplasma from 29% of pneumonic sheep where M. ovipneumoniae was the main detected species but higher than that reported by Ozturkler and Otlu (2020) who isolated Mycoplasma form 10.4% of sheep pneumonic lungs showing that M. ovipneumoniae was the most predominant specie and the isolation rate variation may be attributed to the different circumstances in the geographies studied.

Our study clarified the potential role of E. coli in the occurrence of respiratory disease in sheep, its incidence rate was 21.81% which was nearly similar to that reported by (Ertan, 2006) (24.56%) and Kumar et al., (2018) (25%), but higher than reports of Garedew et al. (2010) (11.41%) and Nehra and Jakhar (2018)(12.5%). However, Azizi et al. (2013) failed to isolate E. coli from trachea and lung of pneumonic goats.

Klebsiellasp. isolation emphasized its importance as an etiological pathogenin respiratory disorders in sheep with total recovery rate (11.8%).Isolation rate of Klebsiellasp. varied greatly among studies (Azizietal., 2013(15.09%); Obaid and Khudair, 2016 (13.6%)and El-Mashadetal., 2020(3.84%). Herein, we isolate K. pneumonia and K. oxytoca, both species were detected in former studies (Patel et al.,2017 and Franco et al. 2019) in the contrary many previous studies did not isolate Klebsiellasp.from ovine pneumonic lungs (Garedewetal., 2010).

P. aeruginosa involvement was obvious in the present study as it was isolated with recovery rate (9.09%). This was somewhat similar to that reported by Nahed and Allam (2019) who isolated P. aeruginosa from sheep with 10%and Mona (2019) where its isolation rate was 11.4% fromthe examined lung tissues. In the contrary Dapgh et al. (2019) found that P. aeruginosa was isolated from 5.8% of the examined sheep and  Zagwa et al. (2010) isolated P. aeruginosa with low rates 5.85% from both nasal swabs and pneumonic lung tissues.

Molecular techniques have proved beneficial impact in bacterial diagnosis as it overcomes some disadvantages of conventional methods in addition to its sensitivity and rapidity (Kaber et al., 2004) and in spite of that cultured based methods remain to the forefront of clinical microbial detection (Srinivasan et al., 2015). Bacteria identification by PCR is founded mainly on target gene amplification that should be highly conserved (Gangwal and Kashyap, 2017).16SrRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain (Srinivasanet al., 2015).

Therefore this gene gave positive amplifications at 280 and 361bp for Mycoplasma sp. and M. ovipneumoniae, respectively (Besser et al., 2008) which was in accordance to (Mona, 2019) who proved that 16SrRNA PCR based assay for Mycoplasma species identification insheep is more accurate than other methods.

The results of conventional method was confirmed by PCR applied on different E. coli isolates representing different serotypes indicating that the primer set used herein is highly specific to E. coli species and could be used in the future in direct E. coli detection from various clinical cases without need to time consuming culturing methods as mentioned before (Saei et al., 2012).

In most laboratories, the detection of P. aeruginosa is still accomplished by culturing and biochemical tests. Although this result is reliable, they are time consuming (Deschaght et al., 2009). Moreover, in some cases in which the bacterial count is low it gave false-negative results. Thus, access to rapid and specific methods that have a high sensitivity as PCR is of a great importance. Suspected P. aeruginosa isolates gave positive amplification for 16SrRNAat 956bp as mentioned by (Spilker et al., 2004).

DNA-based method has been developed for the detection of pathogenic Klebsiella sp. (Jonas et al. 2004). In the present study tested isolates for gyrA gene amplified a region of 441bp. This is in accordance to Brisse and Verhoef (2001) who mentioned that subunit A of DNA gyrase is encoded by gyrA gene that is considered the main target of fluroquinololnes in Klebsiella and also used for confirmation of genus Klebsiella.

Concerning the antibiotic sensitivity test results, M. ovipneumoniae declared susceptibility to both oxytetracycline and ciprofloxacin. Maksimović  et al. (2020) mentioned that oxytertacycline had the highest values of minimal inhibitory concentration (MIC) but ciprofloxacin showed the lowest ones.

E. coli exhibited high susceptibility to colistin and ciprofloxacin, moderate sensitivity to gentamycin and high resistance level to others. These results are similar to some extent with Nehra and Jakhar (2018) who statedthat E. coli showedhigh sensitivity to gentamycin and ciprofloxacin and high resistance to tetracycline. Seai et al. (2014) mentioned that E. coli isolated from sheep showed absolute resistance to penicillin and erythromycin, low resistance level to tetracycline and gentamicin. Our results also detected multiple drug resistance in E. coliisolates and that highlighted that ovine host can act as reservoir of resistant isolates which may transfer to human.

Klebsiella isolates showed susceptibility to ciprofloxacian, gentamycin and doxycycline and chloramphenicol which was in agreement with Wassif and El-Kattan (2015) as they provedthesusceptibility of Klebsiella isolated from sheep to cephalosporin, gentamycin, ciprofloxacin and chloramphenicol.

Infection caused by P. aeruginosa is one of the major issues in sheep and associated with significant mortality rates, consequently early diagnosis and correct medical treatments are the best strategies for saving animal life (Bangar et al., 2016). Our results showed that P. aeruginosa displayed elevated resistance level to all tested antibiotics except for ciprofloxacin and gentamycin as mentioned by Dapgh et al. (2019) who detected their susceptibility to both ciprofloxacin and norfloxacin and complete resistance to ampicillin, erythromycin, tetracycline, sulfamethoxazole +trimethoprim and chloramphenicol. P. aeruginosa is commonly resistant to antibiotics are consequently difficult to eliminate when they infect a compromised site (Kadhim, 2020).Antibiotics resistance property among P. aeruginosa isolates could be attributed to the existence of the unusually restricted outer membrane permeability which acts as a safeguard barrier for antibiotics also there is other second­ary intrinsic factors as multidrug efflux pumps (Xu et al., 2014).

Grossly, our study showed limited gross lesions restricted in mild to moderate congestion and petechial hemorrhage in the trachea which were in partial accordance with those described by Radostits et al. (2002) who described tracheal congestion, hemorrhage, edema, obstruction and pustular. In the present study, the lung exhibited several gross forms as congestion, hemorrhages, emphysema and pulmonary tissue hepatization similar to those described by many authors (Akbor et al., 2007 and Sukanta et al., 2018).On the other hand, Jubb et al. (1993) observed pus and cyst in the pulmonary tissue which could be explained on the base of differences in pathogen nature and severity of the disease. Our current study could not find out any pathognomonic lesions related to any of the previously isolated pathogen in both lungs and tracheas and this was in accordance with that recorded by Damassa et al. (1992) who mentioned that clinical mycoplasmosis often lacks pathognomonic characteristics and symptoms can be shared by or can mimic other clinically significant infections. Pneumonic lesions in our study were summarized in five types including; alveolar pneumonia, bronchopneumonia, fibrinous bronchopneumonia, hemorrhagic pneumonia and interstitial pneumonia; with their related frequencies in each pathogen. Similar pneumonic lung tissues were described by many authors (Collie et al., 2013, Lindstrom et al., 2018 and Sukanta et al., 2018).The most pneumonic lesion recorded was; alveolar pneumonia followed by fibrinous bronchopneumonia and hemorrhagic pneumonia then interstitial pneumonia.No purulent pneumonianor purulent bronchopneumonia were detected,which was opposite to that recorded by Akbor et al. (2007) and Sukanta et al. (2018) in spite of the isolation of E .coli which is one of the pyogenic pathogens having the ability to progress the purulent lesion.This could be attributed to the virulence of the isolated strains and the response of thesheep immune system. Mixed infectionshave obvious pneumonic lesions than those declared in single ones similar to that mentioned by Novert (2002). Histopathology of bronchopneumonia and hemorrhagic pneumonia described in this investigation corresponded to the lesions of other investigators(Ashok et al., 2004).

Conclusion:

Mixed bacterial infection including M. ovipneumoniae, E. coli, K. pneumonaie, K. oxytoca and P. aeruginosa causing different degrees of respiratory disease in sheep is observed. They are accompanied with general respiratory manifestationsand five characteristic types of pneumonia are related to these pathogens. Molecular investigations are beneficial in confirming the isolated bacterial agents. High level of antibiotics resistance especially inP. aeruginosa is alarming.