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Oxidative Stress and Immunosuppression Induced by Mycoplasma Infection in Cattle
Oxidative Stress and Immunosuppression Induced by Mycoplasma Infection in Cattle
Eissa, S. I.*, S.Z. Moussa **, Hanaa A. Ahmed* and Sahar A. El-Maedawy***
* Mycoplasma Dept., Animal Health Research Institute, Dokki
**Faculty of vet. Med., Biochemistry Dept, Cairo University
*** Biochemistry Dept., Animal Health Research Institute, Dokki.
SUMMARY
In a Private farm in Kalubia for Feedlot cattle, calves were suffering from severe respiratory manifestation with fever in 30 out of 110 calves; 3 cases of death were, also, recorded; with a history of no response to classical respiratory infection treatment,. Mycoplasma was suspected to be incriminated as a causative agent in that problem. Oropharyngeal swabs and blood samples were taken from the diseased and apparently healthy animals for biochemical and serological tests which revealed that the possible cause of that problem may be Mycoplasma and this was confirmed by Polymerase Chain Reaction (PCR) to be Mycoplasma bovis. The biochemical investigations declared that M. bovis induced oxidative stress through variable surface proteins (Vsps) on the cell surface protein as expressed by electrophoresis at 32 and 73 kDa causing a significant increase in malondialdahyde (MDA) concentration. The serum protein showed that there was hypoproteinaemia in all experimental groups manifested by decrease in α1, β2, and γ1 and γ2 globulin. These results augment the notion that mycoplasma has an immunosuppressive effect.
INTRODUCTION
Bovine respiratory diseases are the most significant cause of mortality and elimination of calves from feedlots, and have great economic impact on the cattle industry (Griffin, 1997). It is a good example of multifactorial disease in that its aetiology involves both infections by a variety of microorganisms and a number of environmental factors. (Nicholas and Ayling, 2003)
Mycoplasmas cause some of the most serious and economically most costly diseases of cattle (Nicholas and Ayling, 2003). Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum.(Gourlay et al., 1989). Recent evidence indicates that M. bovis may be an emerging pathogen in cattle around the world. In feedlot cattle, a history of respiratory disease unresponsive to treatment and followed by polyarthritis is suggestive of M. bovis infection (Dyer et al., 2008). The exact cause of the increased incidence and pathogenicity of M. bovis is not known (Shahriar and Edward, 2003). M. bovis is a proven cause of contagious mastitis in dairy cattle, pneumonia, septic arthritis (Pfützner and Sachse, 1996) and otitis media (Walz et al., 1997). It is, also, reported to cause keratoconjunctivitis (Kirby and Nicholas, 1996), meningitis (Stipkovits et al., 1993), abscesses (Kinde et al., 1993), genital disorders, abortion (Byrne et al., 1999) and reduction of fertility (Eaglesome and Garcia, 1990). M. bovis may be asymptomatically present as commensal organisms in the upper respiratory tracts of older animals, where the mycoplasmas form a constant source of infection for young animals that are more susceptible to developing clinical symptoms (Trevor, 1997).Other Mycoplasma species may be isolated from M. bovis infected lungs (Mycoplasma bovirhinis, M. arginini, M. bovigenitalium, M. californicum, and M. canis). Although these mycoplasmas are not considered primary bovine respiratory pathogens, it is reported that under certain conditions they may become pathogenic (Hewicker-Trautwein et al., 2002).
PCR is a powerful and valuable tool for ensuring the correct identification of M. bovis isolated from field specimens (Ghaderoshi et al., 1997). The technique is easy to perform, cost-effective and is highly discriminatory (Stakenborg, 2005). With the use of specific polymerase chain reaction (PCR) the chance of the detection of the organism is increased to both early and chronic infections, compared to culture and serological methods (Hirose et al., 2001).
Despite the fact that these organisms lack a cell wall and contain a remarkably small genome (Colman et al., 1990), the mycoplasmas are widespread in nature and many species are recognized as pathogens of humans, animals, and plants (Simecka et al., 1992). The persistence of this wall less agents in different environments as well as in various hosts indicates that mycoplasmas possess a capability to successfully adapt and respond to environmental fluctuations and to the defense mechanisms of the animal hosts. Studies have shown that populations of several pathogenic mycoplasmal species spontaneously and randomly generate distinct progenies with varied antigenic phenotypes (Lysnyansky et al., 1999). These antigenic variants may efficiently escape the host immune response and subsequently may play an important role in the chronic nature of mycoplasmal infections (Robertson and Meyer, (1992) and Wise, (1993).
Hydrogen peroxide (H202), the end product of respiration in mycoplasmas, has been implicated as a major pathogenic factor ever since it was shown to be responsible for the lysis of erythrocytes by mycoplasmas in vitro (Chochola et al., 1995); however, the production of H2O2 alone does not determine pathogenicity. For the H2O2 to exert its toxic effect, the mycoplasmas must adhere closely enough to the host cell surface to maintain a toxic, steady-state concentration of H2O2 sufficient to cause direct damage, such as lipid peroxidation, to the cell membrane. The accumulation of malonyldialdehyde, an oxidation product of membrane lipids, supports this notion (Almagor et al., 1984).
The current work was performed to identify the cause of that outbreak and to follow the biochemical changes concerning the pathogenesis of the disease especially for lipid peroxidation and its relation to the whole mycoplasma protein profile. Also, serum protein electrophoresis was performed for tracking the immune response to the disease.
Material and Methods
The present study was conducted in a private farm in Kalubia governorate for feedlot cattle, which suffered from severe respiratory manifestations with fever, inappetence, conjunctivitis, swelling of joints and loss of weight (morbidity rate 27.3%, mortality rate 2.8%). The animals did not respond to treatment with classical treatment. These animals were vaccinated against viral diseases and under strict program of internal and external parasite control. There were about 30 out of 110 calves exhibiting the signs, with 3 cases of death. Oropharyngeal swabs were taken from 19 of both diseased and apparently healthy animals. For identification of the causative microorganism, blood samples were, also, taken similarly before and after treatment by tylosin for two weeks at a dose of one ml/20kg body weight IM (200mg/ml) for biochemical investigation.
Isolation and propagation of mycoplasma using liquid and solid media was prepared as described by Sabry and Ahmed (1975). Digitonin sensitivity and biochemical characterization (glucose fermentation and arginine deamination) were done for the obtained isolates according to Erno and Stipkovits (1973). Serological identification was conducted by growth inhibition test as described by Clyde (1964). Electrophoresis was done as 10% separating gel and 4% stacking gel in denatured dissociating buffer system (SDS-PAGE) for mycoplasma antigen (Avakian et al., 1992) following the method described by Laemmli (1970). SDS-PAGE Molecular Weight standards, Low range (cataloge number 161-0304, (Pharmacia) was used as marker. DNA extraction and primer selection was done according to Yleana et al. (1995) and the PCR products were electrophoresed on 1.5% agarose gel with DNA Ladders: 100 bp (Pharmacia). Malondialdehyde was determined according to the method described by Albro et al. (1986), total serum protein and protein polyacrylamide gel electrophoresis was made according to Hames and Rickwood (1990) in 7% separating gel using non-denatured non-dissociating buffer system. Image analysis was made by ImageQuantTL-V2005 software of Amersham Bioscience.
RESULTS and DISCUSSION
M. bovis is not ubiquitous but widely spread within the bovine population in enzootically infected areas. The infection is usually introduced to M. bovis-free herds by clinically healthy calves or young cattle shedding the mycoplasmas and once established on multi-age sites becomes very difficult to eradicate. Its appearance on some farms suffering low grade respiratory disease can lead to increased morbidity and mortality (Gourlay et al., 1989). Infected cattle can shed the mycoplasmas via the respiratory tract for many months even years and may act as a reservoir of infection (Pfützner, 1990). Contact animals become infected via the respiratory tract, the teat canal, genital tract or by artificial insemination with infected semen (Pfützner, 1990).
In the present study, 30 out of 110 calves were infected (the morbidity rate was 27.27%); 3 died calves (the mortality rate was 2.73%). Six out of 19 samples were found to be mycoplasma (the recovery rate was 31.58%). All isolates were digitonin positive and by biochemical identification; 5 isolates proved to be M. bovis (glucose fermentation, arginine hydrolysis negative and positive to film and spot formation) while only one isolate suspected to be M. bovirhinis (glucose fermentation positive, arginine hydrolysis negative and doubt formation of film and spot) but unfortunately their antisera were not available (untyped mycoplasma) as shown in Table 1.
Table (1): Recovery rate of mycoplasma from the examined samples and the Biochemical characterization and serological identification of the obtained isolates
| Samples | Recovery rate | Biochemical behavior | Serological typing | |||||
| No. Exam. | No. +ve recovered | %+ve | No. Examined | G | A | F & S | | |
| Oropharyngeal swabs | 19 | 6 (4 diseased + 2 apparently healthy) | 31.58 | 6 | - 1+ve | - - | 5+ve ±ve | 5 M. bovis 1 untyped mycoplasma |
G = glucose fermentation test
A= arginine hydrolysis test
F&S= film and spot formation test
In a survey conducted by the Office Internationale de Epizooties (OIE) in over 48 countries, M. bovis was a major component of the calf pneumonia complex, with isolation rates of 23–35% (Nicholas et al., 2000). Similar results were obtained by Tenk et al. (2004) who screened 34 large cattle herds for the presence of Mycoplasma bovis infection by examining slaughtered cattle for macroscopic lung lesions, by culturing M. bovis from lung lesions and found in 25.2% of the cases; M. bovis was isolated from lungs with no macroscopic lesions.
DNA-based techniques, especially PCR can yield rapid and specific diagnosis of infections caused by M. bovis (Hirose et al., 2001). PCR result had confirmed that the isolates were M. bovis by the presence of the specific band at 360 bp. Similar results were obtained by Yleana et al. (1995) who used the M. bovis PCR system to detect the microbein nasal samples of calves from a herd suffered from an outbreak of pneumonia. Also, Ghaderoshi et al. (1997)concluded that the PCR assay was 10 times more sensitive than dot blot hybridization (Fig. 1).
Fig. (1): Electrophoretic analysis of PCR products of M. bovis (PG45 reference strain) and field isolates.
| 1- 100 bp DNA ladder |
| 2- M. bovis (PG45 reference strain) |
| 3- Field isolate 1 |
| 4- Field isolate 2 |
| 5- Field isolate 3 |
| 6- Field isolate 4 |
| 7- Field isolate 5 |
| 8- M. bovis (PG45 reference strain) |
The mycoplasma membrane classically consists of two bilipid sheets and membrane proteins (You et al., 2006). Mycoplasma species variably express structurally heterogeneous cell surface antigens. Variations in the genes encoding cell surface adherence molecules reveal distinct patterns of mutations capable of generating changes in mycoplasma cell surface molecular size and antigenic diversity. Variable surface antigenic structures and rapid changes in their expression are thought to play important roles in the pathogenesis of mycoplasmal infections by providing altered structures for escape from immune responses and protein structures that enhance cell and tissue colonization and penetration of the mucosal barrier (Nicolson et al., 1998).
The antigenic repertoire of the M. bovis cell surface was found to be subjected to rapid changes due to the presence of a set of antigenically and structurally related variable membrane surface lipoproteins designated Vsps (Behrens et al., 1994). These Vsps undergo highly dynamic and spontaneous changes in size and expression and are key immunogenic components. They may play a critical role as mediators of adherence to host cells and in escaping immune destruction(Behrens et al., 1996).
The electrophoretic pattern of M. bovis reference strain revealed 21 bands extended from 9.9 – 147.4 kDa, while M. bovis field isolates gave 21-24 bands varied from 9.9 – 141.9 kDa resembling the reference strain. While, the untyped mycoplasma gave 29 bands varied from 9.9– 141.9 kDa. It was obviously clear that M. bovis field isolates resembled the reference strain in most protein bands especially at 67.6 -9.9 kDa but field isolates did not have protein bands at 81.1, 24.3 and 18.4 kDa. While field isolates of M. bovis have extra protein bands at 137.5 and 90.5 kDa and share with the untyped mycoplasma in bands at 112.1, 83.1, 75.2, 52.3, 33.7, 28.2, 20.5, 15.7, 11.8 and 10.4 kDa this may indicate that they came from one source or their accommodation to their environment.. The untyped mycoplasma resembled the field isolates in the rest of protein bands with the reference strain. Similar results were obtained by Sachse et al., (1992) and Rosengarten et al., (1994) revealing a high degree of similarity between most of the strains. While Sachse et al. (1992) established a high degree of similarity between most of the strains with strain-to-strain differences mainly confined to quantitative variations of certain protein bands, particularly in the molecular weight regions of 64-68, 55 and 26 kDa with two of the isolates provided more deviating patterns from the examined 34 isolates.
The presence of Mycoplasma bovis cytadhesion protein at 32 kDa (p26 antigen) was diagnostic to M. bovis which occur at both reference and field isolates. The p26 antigen is one of the Vsp families of M. bovis which played a major part as adhesion factors (Sachse et al., 1996). In addition, all isolates have a 73-kDa band which was identified by Tenk (2005) as thermo-stable glycoprotein, localized in the cytoplasm membrane protein which has toxic properties.
Adhesion of the mycoplasmas to the host cells is the primary and key factor of pathogenesis. According to the experiments by Thomas et al. (1991). M. bovis adheres to the neutrophils and the macrophage in a dose dependent way and there is a lack of phagocytotic activity to M. bovis by these cells. It had been previously proved that M. bovis is able to persist and multiply on the surface of these cells (Howard, 1984).
| Fig. (2): Comparing the protein profile of M. bovis reference strain and field isolate |
| Fig. (3): Comparing the protein profile of M. bovis reference strain and the untyped mycoplasma field isolate. |
Malondialdhyde (MDA) constitutes both a biomarker of lipid membrane oxidation and a potential concurrent cause of mutation and cancer initiation (Del Rio et al., 1998). It is evidenced from the results recorded in (Table 2) that mycoplasma infected calves (Group 2) had significantly higher level of MDA than other groups followed by the apparently healthy calves. While treated calves (Group 4) showed a non significant difference in comparison with control value. Khan et al. (2005)recorded similar results and proved the production of H2O2 during the oxidation of NADH and L-alpha-glycerophosphate by lysed cells of M. bovis.
Mycoplasmal lipoproteins (MLP) induced sustained phosphorylation of p38 mitogen-activated protein kinase (MAPK), accompanied by generation of reactive oxygen species (ROS) in human embryonic kidney 293 cells (Into andShibata,2005).These ROS include superoxide anion (O2), hydrogen peroxide (H2o2), hydroxyl radical(OH), peroxy radical (Roo), alkoxyl radical (RO) and peroxy nitrite (HOONO) (Regoli et al., 2000).
ROS may be involved in lipid peroxidation directly or indirectly in various combination in vivo and auto-oxidation in vitro (Kanner and Kinsella., 1983).The generation of free radicals in biological systems containing poly- unsaturated fatty-acids is a potentially harmful phenomena because the damage induced by these radicals can be greatly magnified by a chain reaction involving peroxyl radicals (Vuillaume, 1987). These results indicated that the simultaneous infection by mycoplasma leads to the release of H2O2, a toxic and potentially lethal metabolite (Chochola et al., 1995) which may lead to host cell catalase activity inhibition as stated by Almagor et al. (1986) who mentioned that oxidative damage induced in M pneumonia infected cells due to the increase in intracellular levels of H2O2 and O2- is involved in the inhibition of host cell catalase activity.These findings, also, suggest that the inactivation of this antioxidative defense mechanism results in progressive oxidative damage in infected cell by M. bovis (Almagor et al., 1984).
Plasma proteins had the shortest half-life of all the proteins in the body and were therefore particularly sensitive to acute or chronic changes in amino acid and protein metabolism (Silverman and christensone, 1996). It has been recognized that mycoplasma infection affects the host's immune system in different ways. The current work was performed to characterize the influence of M. bovis infection on the immunological status of infected host in acute phase of the disease.
Table (2) showed results of serum protein electrophoresis, it declared that the mean significant value of albumin was significantly decreased in infected and apparently healthy calves. Hypoalbuminemia occurs due to increased catabolism as a result of tissue damage or inflammation (Limidi and Hyde, 2003).These results are in agreement with that of Wise and Evans (1975), who mentioned that serum albumin concentrations, were markedly reduced in poults with M meleagridis. It is argued that M meleagridis induced ascites may be an acute manifestation of a pathological process. It is, also, suggested that low serum albumin concentrations may play a primary role in the pathogenesis of mycoplasma.
Table (2): Serum MDA(nmol/ml), total protein and its electrophoretic patterns concentrations (gm/dl) in different experimental groups
| Parameter | Control | Apparent Healthy | Diseased | Treated | LSD |
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