The outbreak of respiratory disease occurred at the peak of the laying period four months after the introduction of new partridges, i.e., the supposed source of infection in the game bird farm. The long incubation period is in agreement with reports on birds remaining asymptomatic until they are stressed . The disease broke out long after removing the birds from the one-month quarantine and the stress of the laying period was probably the factor triggering clinical manifestation. Pheasants and partridges are known to harbour many fast-growing mycoplasmas, making the isolation of the slower-growing M. gallisepticum difficult . In the present study, however, the isolation and identification of this mycoplasma species was straightforward. The high titres against M. gallisepticum together with its cultural and PCR identification witness for an ongoing systemic M. gallisepticum infection. While the high morbidity in the grey partridge flock corresponds with rates observed both in poultry and other avian species infected by M. gallisepticum as the sole pathogen, the high mortality is rather typical for mycoplasmosis complicated by some other infectious agent [11–15, 32]. Indeed, E. coli was cultured from infraorbital sinuses, lungs and air sacs of some mycoplasmal birds and there were bacterial colonies in the lungs upon investigation by histopathology. It has been experimentally shown that combined stressors exert enhanced effects in birds , and, apart from E. coli, other bacteria and viruses such as Pasteurella multocida and infectious bronchitis virus, respectively, may be implicated in synergistic respiratory infections with M. gallisepticum [13–15]. Serology, however, excluded other common respiratory infections in birds included in the control healthy and Mycoplasma-infected groups of partridges.
Mycoplasmosis affecting birds in the laying period results in reduced egg production and quality . The quantity of eggs laid by the grey partridge breeding flock decreased abruptly following the disease outbreak and eggs produced were not incubated to prevent contamination of the hatching device. It was, therefore, not possible to evaluate the biological quality of the clutch using such characteristics as viability and hatchability. Since an immune challenge decreases the reproductive allocation to the egg in the grey partridge , mycoplasmas are egg transmitted , and mycoplasmosis can also induce salpingitis in birds , the total effect of hatching (i.e., the percentage of chicks hatching from all eggs set) would certainly be very poor if the eggs were incubated.
Gross and microscopic pathology was specifically used to demonstrate the severity of the disease in grey partridges affected by M. gallisepticum in this biochemical study. Previous papers have reported sinusitis and bilateral swelling of the infraorbital sinuses as the most outstanding feature, airsacculitis in 46%, and tracheitis and lung lesions in 36 and 21% of cases, respectively [11, 14, 15]. Comparing the effects of experimental intranasal infection with M. gallisepticum and Mycoplasma imitans in red-legged partridges, nasal and sinus exudates were found in both groups, while tracheal exudates and airsacculitis were only seen in the M. gallisepticum infection . As the culture revealed M. gallisepticum in tracheal swabs and changes in the grey partridge were very similar to those mentioned above, pathological findings of the present study were in agreement with those observed by other authors [11, 14, 15].
As shown in Table 1, gender differences in healthy control birds do not interfere with the interpretation of significant responses in plasma chemical profiles of Mycoplasma-infected partridges. The results indicate that diagnosis of avian mycoplasmosis solely based on clinical biochemical parameters is not possible. They can, nevertheless, be used for the evaluation of the general health status in mycoplasmal birds . One would expect lower total protein and glucose due to starvation and weight loss in the Mycoplasma-infected group. Contrary to this, there was an increase in total plasma protein and glucose levels, probably as a consequence of inflammation or dehydration in the former parameter and stress in the latter. A response somewhat different from that in partridges was seen in caprine mycoplasmal pneumonia because total protein level was found to be lower, while the glucose level was increased . Amylase catalyses the hydrolysis of polysaccharides, it is associated with glycaemia and its increase corresponds with the observed higher levels of glucose . Both enzymes activities of which were significantly increased in mycoplasmal partridges, i.e., lactate dehydrogenase and creatine kinase, are closely associated. Lactate dehydrogenase is found in skeletal and cardiac muscle, liver, kidney, bone and erythrocytes and elevations can be observed with disruption of any of these. Distinguishing the source of lactate dehydrogenase elevation is based on measuring creatine kinase that originates mainly in skeletal and cardiac muscle. Elevated lactate dehydrogenase levels (three-fold) with concurrent elevation in creatine kinase (two-fold) in the present study are thus suggestive of skeletal or cardiac muscle damage .
The research presented here showed modulations of antioxidant parameters, the total antioxidant capacity and oxidative damage in the form of lipid peroxidation associated with the respiratory disease caused by a natural infection with M. gallisepticum of grey partridges. Oxidative stress is an unspecific biochemical process involved in the adverse action of many stressors. There is a clear association between oxidative stress and immune responses of birds to infectious agents . Endogenous antioxidant defences of an enzymatic and non-enzymatic nature are essential for the control of reactive-molecular-species-mediated oxidative damage of biomolecules . Interestingly, changes in oxidative stress parameters were not restricted to the respiratory apparatus of mycoplasmal birds, but also occurred in non-respiratory organs and plasma. Similarly, extrapulmonary complications were recognised in humans with M. pneumoniae infection . Statistical analysis also revealed significant correlations among responses of the oxidative stress parameters in the heart, lungs, spleen, liver and plasma, and numerous inter-tissue correlations of all the studied oxidative stress parameters. Correlations among the oxidative stress parameters illustrate the complex character of the response and interdependence of parameters. Significant correlations among the studied parameters observed in the liver confirm the major metabolic role of this organ in birds. It would be an interesting issue for future studies to evaluate the relationship between the tissue mycoplasmal burden and oxidative stress parameters, as has been done in other bacterial infections .
Hydrogen peroxide and superoxide radicals produced by mycoplasmas are coupled with endogenous toxic oxygen molecules generated by the host cells to induce oxidative stress that then results in host cell damage [24, 41]. It has been suggested that the pathogenesis of mycoplasmosis comprises the following sequence of events: (a) adherence of mycoplasmas to host cells; (b) generation of superoxide and hydrogen peroxide by the microorganisms and their introduction into host cells; (c) irreversible inhibition of host cell catalase by intracellular reactive-oxygen-species accumulation; and (d) oxidative damage to vital cell constituents [18, 19, 22]. In agreement with the above mentioned in vitro findings, catalase showed significantly lower activity in the heart, lungs, liver and gonads of Mycoplasma-infected partridges.
Significant changes were found for the glutathione-related parameters. Glutathione-S-transferase was elevated in the eye and the associated infraorbital sinus and kidney, and decreased in the liver. Decreased levels of reduced glutathione were in the heart, kidney, liver and gonads and the activity of glutathione reductase was lower only in the lungs of birds affected by mycoplasmosis. These results correspond basically with data on the protective role of the glutathione redox cycle and its adaptive responses observed in cultured fibroblasts and mice infected with M. pneumoniae, respectively [20, 23]. Similarly, decreases in plasma glutathione concentrations and glutathione peroxidase activity were reported in goats naturally infected with M. agalactiae .
It is possible to evaluate the total antioxidant capacity of biological fluids using the ferric reducing antioxidant power assay as a clinical marker of oxidative stress. Non-enzymatic antioxidants such as ascorbic acid, uric acid, bilirubin, vitamin E, α-tocopherol and albumin contribute to the ferric reducing antioxidant power, the reaction is linearly related to their molar concentrations, and uric acid is estimated to make around 60% of the contribution to the plasma value . Importantly, the primary route of excretion of nitrogenous waste in birds is via the formation of uric acid in the liver and its elimination by renal tubular secretion . As shown in Table 1, uric acid levels were increased in the blood of mycoplasmal birds, but not significantly owing to the greater variability of data. Despite it, the total antioxidant capacity of plasma was nearly the same in both groups of birds and agreed with normal plasma values already published for the grey partridge . The ferric reducing antioxidant power values were significantly lower only in the heart and kidneys of Mycoplasma-infected birds.
While ingested carotenoids are both ornamental pigments and antioxidants in birds, M. gallisepticum infection can disrupt their utilisation and result in a trade-off between immune system activation, oxidative stress and the health or sexual quality traits [44, 45]. These and other diet-derived antioxidants can therefore be supplemented to feeds for Mycoplasma-infected captive birds as a means of supportive therapy.
Compared to healthy birds, mycoplasmosis in the grey partridge caused significant differences in the level of lipid peroxidation, i.e., a parameter of damage to membrane lipids, measured as the total thiobarbituric acid reactive species in avian lungs and plasma. Contrary to the situation in goats naturally infected with M. agalactiae , lipid peroxidation was decreased in plasma samples collected from mycoplasmal partridges. This is, however, understandable in light of the birds from the group infected by M. gallisepticum being able to maintain normal blood antioxidant capacity and induction of higher levels of the non-enzymatic antioxidant glutathione. As expected, avian mycoplasmosis was associated with increased lipid peroxidation in the lungs.
Mycoplasmas are considered to be extracellular pathogens. It has, however, been demonstrated recently that M. gallisepticum has the capability of entering nonphagocytic host cells, where it resists host defences and antibiotic therapy. This is also a mechanism of establishing chronic infections, while passage through the respiratory mucosal barrier is responsible for the ability to cause systemic infections . As eradication is very difficult or even impossible once M. gallisepticum has been introduced, the breeding flock of grey partridges should be depopulated rather than used for repopulation .