Pathomorphological studies of respiratory infections in sheep with special reference to Mycoplasma ovipneumoniae in and around Hassan district

Introduction

Livestock production is essential for global food security and rural livelihoods, contributing significantly to national economies in low-and middle-income countries (Udhayavel et al., 2024) [1] . India possesses 74.26 million sheep and 148.88 million goats as per the 20th Indian Livestock Census, with Karnataka accounting for 14.88% of the national sheep population and ranking third after Telangana and Andhra Pradesh (Udhayavel et al., 2024; Behl et al., 2023) [1,2] . Respiratory diseases are major constraints in sheep farming, with pneumonia causing significant morbidity, mortality, and production losses, accounting for about 10% of annual deaths in India (Maru et al., 1990) [3] . Ovine pneumonia is multifactorial, involving pathogens such as Mycoplasma ovipneumoniae, Pasteurella haemolytica, Parainfluenza type-3 virus, adenovirus, and reovirus (Haziroglu et al., 1996) [4] . Among these, Mycoplasma ovipneumoniae is increasingly recognized as a primary cause of chronic, economically important respiratory disease. Mycoplasmas are cell wall-less bacteria causing diverse conditions in small ruminants, including mastitis, arthritis, respiratory and reproductive disorders (Yatoo et al., 2018) [5] . First isolated in 1963 and now reported worldwide, Mycoplasma ovipneumoniae affects both clinically ill and apparently healthy animals, facilitating silent transmission (Nicholas et al., 2015) [6] .

Clinical signs range from mild chronic cough to severe fatal pneumonia, influenced by immunity, age, and management factors (Ayling and Nicholas, 2007) [7] . Economic losses arise from mortality, poor growth, reduced milk yield, and reproductive inefficiency (Alley et al., 1999; Tigga et al., 2014) [8,9] , often triggered by stressors such as transport and overcrowding (Yatoo et al., 2016) [10] . Although culture remains the gold standard for diagnosis, it is time-consuming and technically demanding (Al-Momani et al., 2006) [11] . PCR offers rapid, sensitive, and specific detection, making it a valuable tool for confirming Mycoplasma ovipneumoniae infection (Madanat et al., 2001) [12] . Despite reports from various parts of India, detailed pathomorphological studies in Karnataka, particularly in Hassan district, are limited. Understanding the gross and microscopic lesions associated with Mycoplasma ovipneumoniae infection is crucial for early diagnosis and effective disease control strategies.

Therefore, the present study was undertaken with the following specific objectives a) To study the occurrence of respiratory infections of sheep in and around Hassan district, b) To diagnose Mycoplasma ovipneumoniae using polymerase chain reaction, and c) To record post-mortem findings and microscopic pathology of the respiratory system with special reference to Mycoplasma ovipneumoniae infection.

Materials and methods

Laboratory materials and reagents

Plastic wares such as tissue collection bottles, centrifuge tubes, PCR tubes, pipettes, and micro-tips were sterilized by autoclaving at 121 °C and 15 psi for 15 minutes. Glassware including slides, coverslips, petri dishes, and measuring cylinders was cleaned in detergent, rinsed with distilled water, kept overnight, and sterilized in a hot-air oven at 160 °C for 1.5 hours. Ten% neutral buffered formalin for tissue fixation was prepared using standard formulation (formalin 40% -100 ml, distilled water -900 ml, sodium phosphate monobasic -4 g, and sodium phosphate dibasic -6.5 g).

Gross pathological examination

Systematic necropsy was conducted for respiratory organs comprising the lungs, bronchus, bronchioles, trachea along with the mediastinal lymph nodes. Gross observations regarding colour, texture, consistency, areas of consolidation, congestion, oedema, fibrin accumulation, and exudation were recorded and photo-documented. Lesions of diagnostic relevance were selected for histopathology and PCR examination.

Histopathology

Tissue samples fixed in 10% neutral buffered formalin were washed in running tap water for 2 hours and dehydrated through graded ethanol (70, 80, 90, and 100%). Cleared tissues were embedded in paraffin wax and sectioned at 5 μm thickness using a rotary microtome. Sections were deparaffinized, rehydrated, and stained with Harris hematoxylin and eosin following the standard protocol (Suvarna et al., 2018) [13] . Stained slides were examined under a light microscope (10×, 40× and 100× objectives) and photomicrographs were captured.

DNA extraction and polymerase chain reaction

Genomic DNA was extracted from frozen tissues by alkaline lysis method as described by Girish et al. (2013) [14] . Approximately 500 mg of homogenized tissue was treated with 0.2 N sodium hydroxide, incubated at 75 °C, and subsequently neutralized with Tris-HCl buffer. Extracted DNA was stored at -20 °C until use. Detection of the Mycoplasma genus was performed using primer pair GPO3 and MGSO, while species confirmation of Mycoplasma ovipneumoniae was carried out using primer pair LMF1 and LMR1 targeting the 16S rRNA gene (Yadav et al., 2020; Reddy et al., 2023) [15,16] . Polymerase chain reaction was performed in a 10 μl reaction mixture containing 5 μl of 2X PCR master mix (Hi-Media), 0.5 μl of each primer, 3 μl of template DNA, and 1 μl of nucleasefree water. Cycling conditions for GPO3 and MGSO included an initial denaturation at 94 °C for 4 minutes, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 50 °C for 30 seconds, and extension at 72 °C for 30 seconds, with a final extension of 72 °C for 10 minutes. Species-specific amplification using LMF1 and LMR1 followed identical conditions except for an annealing temperature of 55 °C.

Gel electrophoresis and visualization

Amplified PCR products were resolved on 1.5% agarose gel prepared in 1X Tris-borate-EDTA buffer containing ethidium bromide (0.5 μg/ml). A 1 kb DNA ladder was used for molecular size estimation. Electrophoresis was carried out at 80 V for approximately 2 hours and the resulting DNA bands were visualized under a UV transilluminator using a gel documentation system. The amplicon sizes of 280 bp (Mycoplasma genus) and 361 bp (Mycoplasma ovipneumoniae) were considered confirmatory.

Results

The present study was undertaken to investigate the pathomorphological alterations associated with respiratory infections in sheep, with special emphasis on Mycoplasma ovipneumoniae, in and around the Hassan district. A total of 100 sheep carcasses comprising 29 males and 71 females, collected between March 2025 and September 2025, were examined in detail. The results obtained from the molecular and pathological investigations are summarized below.

Occurrence of respiratory infection

Among the 100 carcasses examined, 62 lung samples originated from slaughterhouses in and around the Hassan region and the remaining 38 samples were obtained from animals submitted for post-mortem examination to the Department of Veterinary Pathology, Veterinary College, Hassan. Gross examination revealed that 71 animals exhibited respiratory-related lesions, whereas digestive system lesions were observed in 17 animals, circulatory system lesions in 6 animals, and urinary system lesions in 6 animals (Table 1). These observations indicated that respiratory disease constituted the most common pathology in the study population.

Occurrence of Mycoplasma ovipneumoniae infection

Out of the 71 sheep showing respiratory-related lesions, 23 animals tested positive for the Mycoplasma genus by PCR. Further screening of these 23 cases with species-specific primers confirmed Mycoplasma ovipneumoniae in 7 animals, representing a prevalence of 32.39% for Mycoplasma infection and 9.85% for Mycoplasma ovipneumoniae among the total samples (Table 2).

Age-wise occurrence:

The age-wise occurrence indicated that Mycoplasma infection was highest in sheep older than 12 months (43.47%; 10/23), followed by 3-6 months (30.43%; 7/23) and 6-12 months (26.08%; 6/23). Among Mycoplasma ovipneumoniae-positive cases, 42.85% (3/7) were between 6-12 months, while both 3-6 months and >12 months age groups accounted for 28.57% cases each (Table 3). These findings indicated a wider susceptibility across age groups with a slightly higher tendency among young and adult animals.

Breed-wise occurrence: Breed-wise analysis revealed the highest occurrence of Mycoplasma infection in non-descript animals (52.17%; 12/23), followed by the Hassan breed (34.78%; 8/23). Mandya (8.69%; 2/23) and Kenguri (4.34%; 1/23) recorded comparatively lower incidences. A similar pattern was observed for Mycoplasma ovipneumoniae, where non-descript and Hassan sheep exhibited the highest susceptibility (Table 4).

Taluka-wise occurrence

Geographical distribution of infection revealed variation across talukas of Hassan district. Mycoplasma infection was detected in Arasikere (20%; 4/20), Channarayapatna (20%; 2/10), Beluru (20%; 1/5), Arakalagudu (33.33%; 4/12), Hassan (25.92%; 14/54), and Holenarsipura (20%; 1/5). However, Aluru (0/2) and Sakaleshpura (0/1) recorded no infection. Mycoplasma ovipneumoniae infection was highest in Hassan taluka (7.4%; 4/54) and Channarayapatna (20%; 2/10), with a minor incidence in Arasikere (5%; 1/20), while other talukas recorded no positivity (Table 5).

Molecular confirmation by pcr

PCR amplification using Mycoplasma genus-specific primers produced a 280 bp amplicon confirming the presence of Mycoplasma in 23 samples (Fig. 1). Subsequent species-specific PCR using LMF1 and LMR1 primers yielded a 361 bp product confirming Mycoplasma ovipneumoniae in 7 samples (Figs 2a &2b). These findings validated the role of molecular assays in accurate detection of respiratory mycoplasmosis in sheep.

Pathomorphological changes

Most affected sheep displayed reduced body weight, coarse hair coat, and nasal discharge (Fig 3). Systematic necropsy examination documented organ-wise lesions.

Trachea

Grossly, congestion of the tracheal mucosa with frothy to reddish-mucoid exudate was recorded in 78.26% (18/23) of Mycoplasma-positive and 85.71% (6/7) of Mycoplasma ovipneumoniae-positive cases (Fig 4). Histologically, epithelial sloughing, loss of cilia, goblet cell hyperplasia, lymphocytic infiltration, haemorrhages around tracheal glands and apoptotic bodies were evident (Figs 5678).

Lungs: Multiple pneumonic patterns and accompanying lesions were observed.

Pulmonary lesion

M. ovipneumoniaepositive

PCRnegative

Discussion

The present study investigated gross and histopathological alterations of the respiratory system in sheep from Hassan district, with molecular confirmation of Mycoplasma ovipneumoniae infection using PCR. A high incidence (71%) of respiratory lesions was observed among the examined carcasses, reaffirming that respiratory disease remains a major health constraint in small ruminants. Similar observations have been reported by Kumar et al.

(2014) [17] and Mekibib et al. (2019) [18] , who identified respiratory disorders as leading causes of morbidity and mortality in sheep. The relatively high occurrence recorded in this study may be attributed to environmental stress, flock density, immune status, and managemental practices prevalent in the region.

Occurrence of Mycoplasma and M. ovipneumoniae Among pneumonic lungs, 32.39% were positive for the Mycoplasma genus and 9.85% were confirmed as M. ovipneumoniae. These findings are comparable to previous reports, although inter-study variation exists. Reddy et al.

(2023) [16] and El-Mashad et al. (2020) [19] reported higher species-level prevalence, possibly reflecting regional differences, host factors, or diagnostic sensitivity. Comparable genus-level prevalence has also been documented in goats (Erra, 2024) [20] , suggesting that Mycoplasma infections are consistently present across small ruminant populations, though the proportion attributable specifically to M. ovipneumoniae varies.

The variability in species-specific detection may reflect differences in sample type, stage of infection, antimicrobial exposure, co-infections, or managemental conditions.

Additionally, the multifactorial nature of respiratory disease may mask the primary role of M. ovipneumoniae when secondary pathogens are involved.

Age-wise occurrence: Age significantly influenced infection dynamics. General Mycoplasma infections were more frequent in animals above 12 months, whereas M. ovipneumoniae showed peak prevalence in the 6-12-month age group. This pattern supports earlier findings by Besser et al. (2013) [21] , who reported higher susceptibility among post-weaning lambs, likely due to stress, immune transition, and increased exposure. The higher detection of Mycoplasma in adults may indicate chronic carriage and subclinical persistence, contributing to flock-level transmission. Thus, younger sheep appear more vulnerable to active M. ovipneumoniae-associated pathology, while older animals may serve as reservoirs.

Breed-wise occurrence: Breed variation was evident. Nondescript sheep exhibited the highest infection rates, followed by Hassan breed, while Mandya and Kenguri showed lower prevalence. This variation likely reflects management practices, flock density, and possible genetic resilience. The higher susceptibility in non-descript animals may be associated with uncontrolled breeding and semi-intensive rearing systems. Although breed-specific studies in Karnataka are limited, these findings suggest that managemental and environmental factors may outweigh inherent breed resistance. Close-contact transmission, as demonstrated experimentally by Dassanayake et al. (2013) [22] , likely contributes to higher prevalence in densely managed flocks.

Taluka-wise distribution: Geographical variation was observed across agro-climatic zones. Higher prevalence was recorded in dry and densely populated talukas such as Hassan and Arakalagudu, while no cases were detected in Aluru and Sakaleshpura, though sample size limitations must be considered. Environmental stressors, stocking density, and animal movement likely influence pathogen circulation. Chen et al. (2024) [23] emphasized that high density and ventilation deficiencies enhance M. ovipneumoniae transmission. The observed variability highlights the combined influence of ecological and managemental determinants in disease epidemiology.

Molecular confirmation by PCR

PCR proved to be a rapid and reliable diagnostic tool. Genus-specific amplification of the 16S rRNA gene (280 bp) identified 32.39% positivity, while species-specific amplification (361 bp) confirmed M. ovipneumoniae in 30.43% of genus-positive cases. These findings align with earlier molecular studies (Reddy et al., 2023; Udhayavel et al., 2024) [1,16] and reaffirm PCR as superior to conventional culture methods, which are limited by the fastidious nature of Mycoplasma. The detection of M. ovipneumoniae in sheep from Hassan district confirms its endemic presence and underscores the importance of molecular diagnostics in routine surveillance.

Pathomorphological changes:

Trachea: Grossly, congestion and mucosal exudation were observed. Histologically, epithelial sloughing, ciliary loss, lymphocytic infiltration, and goblet cell hyperplasia were evident. These lesions correspond to the organism's adherence to ciliated epithelium, leading to ciliostasis and impaired mucociliary clearance (Jones et al., 1985; Lin et al., 2008) [24,25] . Apoptotic epithelial cells suggest oxidative stress-mediated injury, consistent with Li et al. (2016), who demonstrated ROS-induced apoptosis in infected airway epithelium.

Lungs: Pneumonia was the predominant lesion and was categorized as interstitial (33.8%), broncho-interstitial (29.6%), and bronchopneumonia (36.6%). These patterns are consistent with reports by Kumar et al. (2014) [17] and Daee et al. (2020) [27] . Although bronchopneumonia was common in Mycoplasma-positive cases, its presence in PCR-negative lungs confirms the multifactorial etiology of ovine pneumonia. Pathogenesis involves adherence of M. ovipneumoniae to ciliated epithelium, production of hydrogen peroxide and reactive oxygen species, oxidative stress, and epithelial apoptosis (Lin et al., 2008; Li et al., 2016) [25,26] . Impaired mucociliary clearance facilitates secondary bacterial colonization, particularly by Mannheimia haemolytica (Alley et al., 1999) [8] . Immune modulation further contributes, as the organism can suppress lymphocyte responses and resist phagocytosis (Shahzad et al., 2010; Al-Kaissi and Alley, 1983) [28,29] . Strain variability and recurrent colonization prolong inflammatory responses and contribute to chronic pneumonia (Bottinelli et al., 2017) [30] . The occurrence of similar lesions in PCRnegative cases indicates involvement of other pathogens such as viral agents (PI-3, respiratory syncytial virus) and bacteria including Pasteurella multocida and Mannheimia haemolytica, emphasizing the polymicrobial nature of small ruminant respiratory disease.

Mediastinal lymph nodes

Mediastinal lymph nodes showed swelling, congestion, lymphoid depletion, and follicular atrophy, particularly in M. ovipneumoniae-positive cases. These findings suggest systemic immunological involvement and possible lymphoid exhaustion. Similar observations were reported by Mondal et al. (2004) [31] and Mohamed and Bader (2011) [32] , indicating that infection extends beyond pulmonary tissues and influences regional lymphoid architecture. The study confirms that Mycoplasma ovipneumoniae is a significant respiratory pathogen in sheep of Hassan district, associated with multiple pneumonic patterns. Disease occurrence is influenced by age, breed, agro-climatic factors, and management practices, with pathogenesis involving epithelial damage, oxidative stress, immune modulation, and secondary bacterial infections. However, lesions in PCR-negative cases indicate a multifactorial etiology of ovine respiratory disease. These findings emphasize the need for improved management, routine molecular diagnostics, and comprehensive disease surveillance for effective control.

Conclusion

The present study confirmed that Mycoplasma ovipneumoniae is one of the predominant respiratory pathogens of sheep in and around Hassan district of Karnataka. PCR targeting the 16S rRNA gene proved to be a rapid, specific, sensitive, and reliable tool for confirmatory diagnosis. Infections at both genus and species levels were associated with significant pathomorphological changes, including interstitial and broncho-interstitial pneumonia, bronchopneumonia, pulmonary congestion, edema, atelectasis, emphysema, lymphoid depletion, periportal lymphocytic infiltration, and fibrinous pericarditis. Concomitant and opportunistic infections likely aggravated the lesions, highlighting the need for further investigation. Effective control measures, regular diagnostic surveillance, and improved management practices are essential to reduce disease severity and economic losses in sheep farming.

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