الفهرس 
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Abstract
Bovine Respiratory disease is the most infectious disease affecting calves and adults cattle and it remains a huge global economic burden for Both the dairy and beef industries causing severe economic losses as a major cause of deaths, loss of production, treatment costs and negatively impact growth, reproductive performance, longevity, carcass weight, marbling and other carcass value factors. BoHV-1 is a major pathogen in BRD. Clinical examinations of cattle with signs of BRD and necropsy examinations of recently succumbed cases and/or emergency slaughtered animals with signs of BRD inside and/or outside slaughtered house and laboratory detection of the most common respiratory pathogens in imported and native breed cattle in Abu-Simbel and different areas in Sohag, Assiut Upper Egypt, and Cairo governorates respectively were aimed in the present study.
During the period of investigation (29 consecutive months), a total number of 650 cattle were clinically inspected and were divided into two groups group I were native breed cattle (n= 300) and second group were the African breed cattle (imported breed) (n= 350). 195 (30%) cases were clinically suspected to have signs of BRD in various degrees. The severity of suspected cases with signs of BRD was categorized into mild, moderate and severe and the later cases were clinically examined in detail.
Comprehensively, the prevalence of Mycoplasma infection in the examined diseased cattle with severe signs of BRD was 68.5% (24/35) and 50% (14/28) in imported and native breed cattle respectively. Severely affected animals with BRD adopted a characteristic classic signs and posture, standing motionless with elbows abducted, neck extended, head lowered and extended with the mouth open (oral breathing) and the tongue protruding with frothing at the mouth this posture maximized the airway diameter and minimized the resistance to air flow. Other clinical signs included loss of weight with staring rough coat, dullness, lethargic, dehydrated and depressed. Milk yield were decreased. Carpal joint was enlarged.
Body temperature ranged from 40oc to 41oc, respiratory rate elevated more than 40 breaths per minute, rapid and shallow respiration especially rapid movement of flank regions both sides right and left. Heart rate increased more than 100 beats per minute, remarkable labored breathing (dyspnoeic) with increased thoracic and abdominal wall movements (abdominal respiratory line) but abdominal breathing was predominantly. Mucous membrane was congested then turned to cyanosed. Productive coughing was noticed. Nostrils were dilated, the cheeks were puffed, and there were bilateral tenacious, viscous, whitish creamy nasal discharge, epiphora and ears drooping. Prolonged antimicrobial therapy for nonresponsive or relapsing respiratory disease was characteristic.
Necropsy examination of recently succumbed animals (n= 20) and/or emergency slaughtered cattle (n= 43) inside abattoir and/or outside abattoir with severe signs of BRD revealed that sequestrum developed from the infarcted lung tissue and caused by M. haemolytica with M. bovis. Fibrin formed a lattice over the pleural surface of affected lung lobes, and Fibrinous pericarditis accompanied the pleuritis. Interlobular septa were distended with fibrinous exudate, and this lended a marbled appearance to the cut surface similar to beehive in structure. chronic suppurative bronchopneumonia were developed and the lesions were colonized by secondary pathogens, Trueperella pyogenes and the chronic infection resulted in pulmonary fibrosis, bronchiectasis, abscess formation, lung resembling bag containing pus and sequestration. Yellowish caseated purulent materials were found in bronchial and mediastinal lymph nodes and inside the kidney with systemic infection of Trueperella pyogenes. Cyanosis of lung lobes (bluish discoloration) due to hypoxic hypoxia with necrosis of lung alveoli, interlobular septa increased in thickness, edematous and fibrosed. Bluish discoloration of the heart due to hypoxaemic hypoxia with wide spread of petechial haemorrhages and atrophy of the coronary fat.
The histopathological findings showed that lung tissues infected with bovine herpesvirus showed hyperplasia in the bronchiolar epithelium, with massive neutrophil cell infiltration. Hyperplasia of pneumocytes type 2, intranuclear acidophilic inclusion bodies in macrophage cells associated with massive degeneration and necrosis of the alveolar tissues. Interstitial pneumonia with patchy dense fibrosis infiltrated with inflammatory cells was present.
The histopathological findings revealed that lung tissues infected with pus producing pathogen associated with bovine herpesvirus illustrated necrosis and sequestration of necrotic alveolar tissue. Those with a suppurative central core surrounded by a pyogenic membrane, in addition to the presence of abscesses, were found. Alveoli were replaced by a sac-like structure filled with necrotic tissue and containing edematous fluid. Fibrosis was infiltrated by massive inflammatory cells.
The most characteristic finding of lung emphysema was dilated air spaces throughout the lung parenchyma with no regional predilection. In Mycoplasma infection, marked eosinophilic cell infiltration was located. Severe vascular dilatation and congestion were associated with perivascular inflammatory cellular infiltration. Alveoli were destroyed and replaced by alveolar-like structures lined by cuboidal cells (honeycombing), which contained edematous fluid Fibrinopurulent exudate with necrotic epithelial lining obliterated the bronchioles. Bronchiolitis obliterans was present
Sixty-three pneumonic lungs samples of the examined diseased cattle with severe signs of BRD were subjected to PCR for molecular detection of BoHV-1. Fourty-eight (76.19%) of 63 pneumonic lung samples were molecularly BoHV-1 positive. The specific band of 173 bp after PCR amplification of gC gene of BoHV-1 could be detected with 66.7% (32/48). The specific band of 478 bp after PCR amplification of gB gene of BoHV-1 could be detected with 33.3% (16/48).
All tested nasal swabs were molecularly negative for BoHV-1 to both gB and gC gene.
Partial nucleotide sequencing of the glycoprotein C (gc) and glycoprotein B (gB) genes for Bovine alphherpesvirus1 isolated during 2022 from pneumonic lung of cattle from Sohag Governorate was carried out. Sequence was submitted to GenBank under accession number of (OP777904)for gC gene and (OQ161992 and OQ161993) for gB gene.
Comparative sequence analyses of glycoprotein C gene reveal that field Bovine alphherpesvirus 1 obtained in the current study shared 99% to 100% identity on both nucleotide (nt) and amino acid (AA) sequences with BoHV-1 isolates for Bovine alphherpesvirus1 from USA, India, China, Serbia, Cooper strain and commercial vaccines TSV-2, Nasalgen IP and Bovishield gold FP 5 MLV. Comparative sequence analyses of glycoprotein B gene reveal that field Bovine alphherpesvirus 1 obtained in the current study (OQ161992 and OQ161993) shared 99% to 100% identity on both nucleotide and amino acid sequences in between and 99 to 100% with BoHV-1 isolates from Egypt, USA, and commercial available vaccines vaccinal strains.
In imported breed cattle Mycoplasma was isolated with 68.5% (24/35) of pneumonic lungs of the examined diseased cases with severe signs of BRD by culturing technique. In native breed cattle Mycoplasma was isolated with 50% (14/28) of pneumonic lungs of the examined diseased cases with severe signs of BRD and showed characteristic fried egg colonies under dissecting microscope. All isolates were digitonin sensitive and classified as belonging to members of genus mycoplasma and not belonging to the genus Acholeplasma. Biochemically isolated Mycoplasma was glucose-and arginine-negative and film and spots positive.
After culturing and biochemical identification for the major bacterial respiratory pathogens associated with BoHV-1 infection in diseased cases with severe signs of BRD of imported breed cattle emergency slaughtered and/or recently succumbed (n= 24) we found that 12 (50%) of 24 diseased cases were mixed infection of Mycoplasma, Trueperella pyogenes (T. pyogenes) and Mannheimia haemolytica (M. haemolytica). Six (25%) of 24 diseased cases were mixed infection of Mycoplasma, M. haemolytica, staph aureus and T. pyogenes. Four (16.71%) of 24 diseased cases were mixed infection of Mycoplasma, Pasteurella multocida spp multocida (P. multocida spp multocida), β haemolytic streptococci (β.H.S) and E. coli. Two (8.3%) of 24 diseased cases were single infection of Mycoplasma.
In diseased cases with severe signs of BRD of native breed emergency slaughtered and/or recently succumbed (n= 14) we found that 7 (50%) of 14 diseased cases were mixed infection of Mycoplasma, M. haemolytica and Staph aureus. Six (42.86%) of 14 diseased cases were mixed infection of Mycoplasma, P. multocida spp multocida, β haemolytic streptococci (β.H.S.) and M. haemolytica. One (7.14%) of 14 diseased cases was mixed infection of Mycoplasma, T. pyogenes and E. coli.
The obtained results indicated that the frequent distribution of the isolated respiratory pathogens from the examined imported breed cattle with severe signs of BRD (total isolates; n= 86) were as the following, Mycoplasma 27.90% (24/86), T. pyogenes 16.27% (14/86), M. haemolytica 16.27% (24/86), Staph aureus 11.62% (10/86), P. multocida spp multocida 9.3% (8/86), βHS 9.3% (8/86) and E. coli 9.3% (8/86). Moreover, the frequent distribution of the isolated respiratory pathogens from the examined native breed cattle with severe signs of BRD (total isolates; n= 50) were as the following, Mycoplasma 28% (14/50), M. haemolytica 26% (13/50), Staph aureus 18% (9/50), P. multocida spp multocida 12% (6/50), βHS 12% (6/50), E.coli 2% (1/50) and T. pyogenes 2% (1/50).
The most common isolated gram positive pneumonic bacterial pathogens in diseased cases of imported breed cattle were Trueperella pyogenes (16.27%) as purulent producing pathogens and the most common isolated gram negative pneumonic bacterial pathogens were Mannheimia haemolytica (16.27%). More than 50% of the tested samples were Mycoplasma, Trueperella pyogenes and Mannheimia haemolytica.
The most common isolated gram positive pneumonic bacterial pathogens in diseased cases of native breed cattle were Staph aureus (18%). The most common isolated gram negative pneumonic bacterial pathogens were Mannheimia haemolytica (26%). More than 50% of the tested samples were Mycoplasma and Mannheimia haemolytica. Gold standard diagnostic method is still bacterial culture.
In conclusion, BoHV-1 is one of the major pneumogenic pathogens and its occurrence frequently and early in respiratory disease suggested that it is an initiating factor which increases the severity of Bovine respiratory disease by unemployment of lung clearence mechanism facilitating the way for the entrance of other pathogens. BoHV-1 was detected molecularly from deep pulmonary tissues and was not detected from nasal swabs. There is a synergistic action between BoHV-1, Mycoplasma spp especially M. bovis and Mannheimia haemolytica. Unemployment of lung clearence mechanism facilitates the way for the entrance of Trueperella pyogenes which is responsible for lung abscesses and production of yellowish caseated purulent materials. Synergistic action between BoHV-1 and Mycoplasma bovis and has been demonstrated. BRD is non sex linked disease. BRD is a multifactorial disease including environmental and managmental factor, host immune status and pathogens.