Introduction
Sheep and goats play an important socioeconomic role within conventional animal husbandry systems, primarily providing high-quality animal protein and income (Al-Joboury et al., 1889; Ukwueze & Kalu, 2015; Rabana et al., 2022).
The small ruminant respiratory complex is one of the significant causes of morbidity and mortality in sheep flocks. The contributing factors comprise exposure to adverse weather conditions, animal movement, overcrowding, and stress, which increase the susceptibility of animals to viral and bacterial infections (Scott, 2011; Baghezza et al., 2024).
Respiratory diseases, particularly lamb pneumonia, stem from a multifaceted interplay of host factors (immunological and physiological), etiological agents (viruses, bacteria, mycoplasma), and environmental factors (Amit et al., 2012; Brogden et al., 1998; Kumar et al., 2011). Among various causative agents (Ali et al., 2005), Streptococcus pneumoniae, a commensal bacterium in the nasopharynx of animals, stands implicated in a majority of lamb pneumonia cases, precipitating reduced growth, increased mortality, and substantial economic burdens in terms of treatment costs and abattoir condemnations (Goodwin et al., 2004; Jones et al., 1982).
S. pneumoniae, a prominent gram-positive bacterium, is renowned for its involvement in respiratory tract infections, including pneumonia, in both human and animal populations (Proctor & Manning, 1990; Zivich et al., 2018). In veterinary medicine, S. pneumoniae poses a significant concern, particularly among livestock like lambs, where it causes notable morbidity and mortality (Kumar et al., 2013). While similarities exist between S. pneumoniae infections in lambs and humans (Alnajjar et al., 2021), the intratracheal route serves as a means of pathogen challenge, directly administering the pathogen into the animal’s respiratory tract (Borsa et al., 2019). Key aspects of S. pneumoniae pathogenesis warrant investigation, including bacterial colonization of the respiratory tract, adhesion to respiratory epithelial cells, evasion of host immune defenses, and induction of inflammatory responses leading to tissue damage (Sharma et al., 2020).
Upon bacterial invasion, the host mounts an immune response characterized by activating white blood cells (WBCs), notably neutrophils and lymphocytes (Korkmaz & Traber, 2023). Neutrophils, or polymorphonuclear leukocytes, play crucial roles in early infection stages through pathogen phagocytosis and cytokine-mediated immune responses such as tumor necrosis factor (TNF)-α release (Lee et al., 2022). Lymphocytes, encompassing B and T cells, are essential for specific immune responses against S. pneumoniae (He et al., 2021). Monocytes differentiate into macrophages, which engage in pathogen engulfment and digestion (Anandachar et al., 2023). TNF-α, a proinflammatory cytokine, facilitates immune cell recruitment and instigates inflammatory cascades (Silva et al., 2019).
Utilizing the lamb model of intratracheal route-induced pneumonia, this research endeavors to advance the understanding of S. pneumoniae infections in veterinary medicine and contribute to developing innovative strategies for preventing and treating S. pneumoniae in livestock populations.
Materials and Methods
Animals and housing
Six male lambs of the local Iraqi breed, aged 1-2 months and weighing between 5 and 7 kg, were procured from a local source in Al-Najaf Province, Iraq. The lambs were transported to the animal farm facility at the College of Veterinary Medicine, University of Baghdad, where they underwent a one-week acclimatization period to adapt to their new environment and diet.
Bacterial inoculum preparation
The S. pneumoniae strain ATCC 6303, serotype 3, was obtained from a local supplier. The bacteria were cultured on 5% sheep blood agar plates and incubated at 37 °C with 5% CO2 for 18 hours (Ashrafi et al., 2022). Subsequently, the bacterial suspension was prepared in sterile phosphate-buffered saline and incubated in brain heart infusion broth for 6 hours. The bacterial concentration was adjusted to 2×106 colony-forming units (CFU)/mL for experimental use (Alwash et al., 2017).
Experimental design
A single exposure to the prepared S. pneumoniae suspension was administered via injections precisely directed at the midsection of the trachea.
Bacterial suspension (3 mL) containing 2×106 CFU/mL was injected between two tracheal rings in the middle portion of the trachea. The procedure was conducted under controlled conditions to ensure consistent animal exposure.
Clinical evaluation
Following inoculation, lambs were monitored daily for clinical signs of pneumonia, including coughing, dyspnea, tachypnea, fever, and sputum production. Observations were recorded to track the progression and severity of the infection (Al-Khafagi et al., 2016).
Blood sample collection
Blood samples were collected from the jugular vein at predetermined time points for hematological and biochemical analyses. Blood samples were evaluated in EDTA-anticoagulant tubes for total and differential WBC count using an automated hematology analyzer (Getein BHA-5000, China) (Karaşahi̇n et al., 2023). For TNF-α analysis, blood was collected in gel tubes, centrifuged at 3500 rpm for 5 minutes, and the serum was stored at -20 °C. Serum TNF-α levels were measured using a Sheep TNF-α ELISA Kit (Beijing Solarbio Science & Technology CO., China) following the manufacturer’s protocol (Shihab et al., 2022).
Bacterial isolation from lung tissues
To evaluate the persistence of S. pneumoniae post-intratracheal exposure, lung tissue samples were collected from the lambs at days 6 and 14 post-exposure. The tissues were processed under sterile conditions and cultured on blood agar plates. The presence of S. pneumoniae colonies was assessed qualitatively (AL Kutbi et al., 2001; Mohammed et al., 2020).
Histopathological examination
Tissue samples from the trachea and lungs were collected from euthanized lambs and subjected to histopathological analysis (Hashim, 2021). The samples were fixed, processed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined under a light microscope (Luna, 1968; Spitalnik & Witkin, 2017).
Statistical analysis
Data were analyzed using the statistical analysis system (SAS) software, version 2018. The least significant difference (LSD) test was employed for pairwise comparisons of means in the analysis of variance (ANOVA) (Omar & Ibrahim, 2023).
Results
Clinical signs
Following the onset of S. pneumoniae infection, clinical manifestations became evident, marked by symptoms such as coughing and nasal discharge accompanied by a mild elevation in body temperature (Figure 1).
Examination of S. pneumoniae post-infection
All lung tissue samples from lambs, collected 6 and 14 days after exposure, yielded positive results for bacterial colonies consistent with S. pneumoniae. Characteristic colony morphology observed on sheep blood agar included round, grayish-white, translucent colonies with alpha-hemolysis (Figure 2A). Microscopic examination further -confirmed the presence of gram-positive cocci arranged in pairs and chains (Figure 2B).
Subsequent analysis utilizing the VITEK 2 system provided a 98% probability of the isolates being S. pneumoniae, validating the initial observations and supporting the identification of the pathogen responsible for the infection in the lambs.
At day 6 post-exposure, S. pneumoniae was isolated from all three lambs (lamb 1: +, lamb 2: +, lamb 3: +). Similarly, at day 14 post-exposure, all three lambs exhibited the presence of S. pneumoniae in their lung tissues (lamb 4: +, lamb 5: +, lamb 6: +). This process was crucial in confirming the establishment and persistence of the S. pneumoniae infection in the lamb model, providing valuable insights into bacterial colonization dynamics following inhalation exposure.
The consistent presence of the bacteria in lung tissues at both time points served as compelling evidence of successful infection, shedding light on the progression and duration of the infection post-exposure.
Hematological parameters during the induction of pneumonia in lambs
The white blood cell (WBC) counts in lambs exposed to S. pneumoniae through the intratracheal route were measured pre-infection and post-infection. A significant increase in WBC counts was observed on day 3 post-infection (22.6±0.788×109/L), followed by slightly lower counts on day 6 (21.2±0.738×109/L) and day 14 (20.75±0.046×109/L), compared to day 0 (8.97±0.316×109/L), indicating an early and vigorous immune response. Differential WBC counts were also assessed, including neutrophils, lymphocytes, and monocytes. There was a significant increase in neutrophils on day 3 post-exposure (8.14±0.758×109/L) compared to day 0 (2.44±0.393×109/L), indicating an early neutrophilic response. Neutrophil counts gradually decreased by days 6 (6.83±0.701×109/L) and 14 (4.67±2.009×109/L) but remained significantly higher compared to day 0.
Intratracheal exposure to S. pneumoniae significantly increased the lymphocyte count on days 6 (10.5±1.706×109/L) and 14 (15.9±0.717×109/L) post-exposure compared to day 0 (6.43±0.951×109/L).
Lambs exposed to S. pneumoniae through the intratracheal route exhibited a significant increase in monocytes on day 3 post-exposure compared to day 0, indicating an early and robust monocyte response. Monocyte counts on days 6 and 14 post-exposure gradually decreased but were significantly higher than day 0 (Table 1).
The total WBC count and the breakdown of different types of white blood cells in blood samples from male lambs were examined following experimental intratracheal exposure to S. pneumoniae.
TNF-α level by intratracheal exposure
This study aimed to find changes in TNF-α levels after pneumococcal administration by intratracheal injection. The final results are shown below. On day 3 post-exposure, TNF-α levels increased significantly (P<0.001) compared to day 0, indicating an acute inflammatory response. TNF-α levels increased at intervals of 6 to 14 days but decreased steadily with subsequent testing. Although still greater than day 0, the decrease indicates continued resolution of inflammation and a return to homeostasis. Intratracheal exposure to S. pneumoniae in lambs significantly increases TNF-α levels, indicating an intense inflammatory response. TNF-α levels decrease over time, indicating that inflammation gradually resolves Figure 3.
Pathology gross and microscopic
Gross changes
The experimental intratracheal infection with S. pneumoniae in lambs revealed affected lungs at day 6 post-infection, represented by moderate to mild pulmonary congestion, hemorrhagic spots, and linear congestion of the mucosal internal layer along the trachea (Figures 4A, and 4B). At day 14, post-infection shows mild enlargement, congestion of trachea with dorsal hemorrhagic spots, apical lobular congestion and edematous fluid, ecchymotic hemorrhage in ventral lobes and hemorrhage, emphysema in lower lobes (Figures 4C and 4D).
Microscopic examination
The microscopic examination of lambs’ post-infection intratracheally with S. pneumoniae revealed marked disruption of tracheal and bronchial mucosal epithelia represented by vacuolar degeneration of mucosal epithelia (Figure 5a), intraepithelial and subepithelial infiltration of mononuclear cells (MNCs) mainly lymphocytes and polymorph neutrophils (PMNs), also in submucosa to serosa (Figure 5b) at day 6. The microscopic examination At day 14 post-infection (Figure 5c) shows massive infiltration of inflammatory exudate replaced the normal mucosa and submucosa of pulmonary passages (trachea and bronchi), focal proliferation of lymphoid cells in submucosa was the prominent lesion (Figure 5d). On day 6, the lung tissues (Figures 5e, and 5f) revealed mild to moderate thickening of interalveolar septa due to hyperemia of alveolar capillaries and infiltration of inflammatory cells, mainly lymphocytes, macrophages, and few neutrophils in interalveolar septa with bronchitis and bronchiolitis as necrotic damage of mucosal epithelia and infiltration of inflammatory cells. On day 14, post-infection, focal MNCs around the bronchioles were found (Figure 5g). Figure 5h shows lobular interstitial thickening of the lungs.
Discussion
The pathogenesis of pneumococcal infection is complex and involves the host, infectious agent, and environmental factors (Narayan et al., 2023). Serotype 3 is the most common serotype isolated from severe infections in animals and humans (Wong et al., 2023). Different isolates of S. pneumoniae can cause various diseases, from fatal systemic diseases to asymptomatic ones. This difference in disease severity is not related to genome size, serotype, sequence type, or virulence-associated phenotypes in vitro (Norman, 2015; Chaguza et al., 2022; Jacques et al., 2023; Wong et al., 2023). The importance of understanding the pathogenesis and immune response induced by pneumococci is underscored by the appearance of clinical symptoms after infection (Morimura et al., 2021), as observed in this study by signs of respiratory disease such as rhinorrhea, mild cough, and mild temperature depending on the time of infection. After 6 and 14 days, Arends et al. (2022) have reported typical clinical signs, such as cough, nasal discharge, and a slight increase in body temperature, which are often associated with pneumococcal respiratory infections and indicate the body’s effort to confront invading pathogens (Arends et al., 2022). These clinical observations were consistent with the study of (Small et al., 1986), who observed that a moderate increase in body temperature corresponded with activation of the host immune response against pneumococci.
Fever, which is often mediated by the release of pyrogenic cytokines, such as interleukin 1 beta and TNF-α, is vital to prevent bacterial growth and enhance immune cell function, which explains the significant rise in serum TNF-α levels and increased number of immune cells. On days 6 and 14 after infection with S. pneumoniae, neutrophils emerge to reduce the number of pathogens and eliminate their harmful effects in target tissues by stimulating immune responses through the action of proinflammatory cytokines of interleukins and tumor necrosis factors. They cause an increase in temperature in infected lambs due to infectious tissue injury and pyrogen production combined with the innate immune response, increased reconstitution of polymorphonuclear neutrophils in the pulmonary tissue of the trachea and lungs, and finally, increased levels of lymphocytes and macrophages (MNCs), which represent the adaptive immune response. This finding is consistent with Jimistan’s results, who recorded a peak body temperature on the third day after infection. The initial stages of the immune response, characterized by the recruitment of neutrophils and macrophages to the site of infection, are followed by the initiation of adaptive immune responses (Periselneris et al., 2014).
The subsequent decrease in body temperature on days 6 and 14 marks the end of the acute phase of infection, indicating successful containment and elimination of the pathogen. This observation is consistent with the idea that pneumococci usually elicit local protective immune responses, as evidenced by mild clinical signs in the present investigation. These local responses may include activating mucosal immunity and producing specific antibodies targeting pneumococcal antigens, contributing to the resolution of infection without major systemic complications. This finding is consistent with Wong et al. (2023) results, who reported a significant increase in total WBC counts, neutrophils, and monocytes, agreeing with the present findings of higher total WBCs and differential neutrophil counts on day 3 in both groups and increased numbers of lymphocytes on days 6, 14 along with high percentages of monocytes reached on day 14 after infection. This rise in leukocyte count can be attributed to bacterial invasion, where colonization causes infection of epithelial tissue and endothelial cells, leading to the release of growth factors, cytokines, and other inflammatory mediators, thus stimulating the proliferation and maturation of white blood cells.
Respiratory illness-induced stress triggers the release of endogenous corticosteroids, affecting the regulation of circulating leukocyte concentrations in moderate to severe cases of pneumonia (Sayad et al., 2002; El-Naser and Khamis, 2009; Saleh & Allam, 2014; Donia et al., 2018). These virulent pathogens have polysaccharide capsules, M protein, and group A streptococci. Hyaluronidase is one of the enzymes of pathogenic bacteria Streptococcus spp., Staphylococcus spp., and Clostridium spp., which hydrolyzes hyaluronic acid from connective tissue and facilitates the invasion of bacteria into the depths of layers of affected tissue (Borriello, 1998; Paton et al., 1993; Wong et al., 2023).
S. pneumoniae infection triggers an inflammatory response in exclusively infected respiratory organs. S. pneumoniae is characterized by a severe inflammatory reaction, as occurred in the present study, due to direct stimulation by cell wall components of pneumococci and pneumolysin, which has been shown to stimulate TNF-α production. by human monocytes in vitro (Heumann et al., 1994; Houldsworth et al., 1994). Increased levels of TNF-α have been observed in serum and cerebrospinal fluid in patients with acute lower respiratory tract infection and meningitis, respectively, caused by S. pneumoniae (Glimåker et al., 1993; Nohynek et al., 1991). In addition, TNF-α is an important mediator of inflammation and tissue damage in pneumococcal meningitis (Xu et al., 2017). However, the role of TNF-α in the pathogenesis of pneumococcal pneumonia is unclear. Takashima et al. created a model of pneumonia in mice with pneumocystis pneumonia that closely mimics the situation in humans (Takashima et al., 1996). Using this model, endogenous TNF-α production and its role in pneumonia were studied by administration of anti-TNF-α antibody, demonstrating the protective role of the endogenous product (TNF-α) in pneumococcal pneumonia, which may explain the persistent respiratory infection during the trial period.
Post-mortem examination of affected lambs revealed gross signs of inflammation, such as redness, swelling, congestion of the affected lungs, and bleeding (petechiae and ecchymosis). Gross changes in the trachea showed linear congestion of the internal mucosa. The histopathological changes caused by intratracheal pneumococcal infection on days 6 and 14 after infection revealed focal interstitial bronchitis represented by focal infiltration of mainly MNCs. A few lymphocytes and macrophages were also seen around the bronchi, bronchioles, and perivascular cuffs, reflecting the host immune response against the invading microorganisms from the action of cytokines and chemokines to the site of pulmonary infection with bacterial toxins, the same results suggested by Lucas et al., (2020). The cytokines of interest in the present study, TNF-α, were elevated in their levels a few days after pneumonia as proinflammatory cytokines at the time of pathogen invasion and petechial and ecchymotic hemorrhages in the affected lungs.
Thickening of the barrier between the alveoli and the pleura was observed 14 days after the injury due to congestion of alveolar capillaries and the accumulation of edema in the interstitium, as well as infiltration of lymphocytes, and the same pathological changes occurred in an experiment by Alnajjar et al. (2021), who demonstrated pneumonia as a secondary bacterial infection after viral pneumonia or viral-bacterial coinfection (Madhi & Klugman, 2004; Smith et al., 2013). They used the lamb rather than the mouse model to understand that viral-bacterial coinfections are crucial for studying treatments. Sheep are also permissive to S. pneumoniae infection and have served as a model for sepsis because they show clinical signs similar to human infection (Legesse Garedew et al., 2010; Zaghawa et al., 2010). In conclusion, pneumonia causes pathological lesions of epithelial cell degeneration and necrosis that represent acute lung injury in the trachea and bronchi that may facilitate coinfection with other pathogens and increase morbidity and even mortality.
Conclusion
S. pneumoniae, as a pathogenic agent, has a significant role in inducing inflammatory reactions in air passages when introduced directly into the trachea.
Ethical Considerations
Compliance with ethical guidelines
Ethical approval was granted by the Local Committee of Animal Care and Use at the College of Veterinary Medicine within the University of Baghdad, Baghdad, Iraq (Code: 2152/PG).
Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.
Authors' contributions
All authors contributed equally to the conception and design of the study, data collection and analysis, interception of the results and drafting of the manuscript. Each author approved the final version of the mnuscript for submission.
Conflict of interest
The authors declared no conflict of interest.
Acknowledgments
The authors appreciate and thank everyone who helped with laboratory and theoretical devices.
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