The Wistar Rat (Rattus Norvegicus) has a higher potential as an animal model of pneumonia than the others, which were used in previous studies
(Meyerholz et al., 2018), because of its larger body and lungs, so that chest radiographic examination is easier. Therefore, this study used Wistar rats as animal models (
Metersky and Waterer, 2020).
Bacterial inoculation techniques for animal models are primarily intratracheal/oropharyngeal
(Bergamini et al., 2021) or intranasal (
Mizgerd and Skerrett, 2008) or intravenous technique (
Widjiati, 2021) with anaesthesia. This study used intranasal techniques as the endotracheal tube (ET) and laryngoscope used for intratracheal inoculation were unavailable and this technique is more natural. The intranasal inoculation in the present study was carried out without anesthesia.
The result of the statistical analysis is shown in Table 1. The correlation between the dose of
Klebsiella pneumoniae solution and incubation period with the number of neutrophils, correlation between the dose of
Klebsiella pneumoniae solution and incubation period with histopathology of lung tissue and the correlation between the incubation period of pneumonia with the chest radiography were significant. Pneumonia with the highest neutrophil number parameters occurred on day 5 at a dose of
Klebsiella pneumoniae solution of 25 μL (neutrophils: 52.3%), on day 5 at a dose of
Klebsiella pneumoniae solution of 50 μL (neutrophils: 61.6%) and on day 1 at a dose of
Klebsiella pneumoniae solution of 100 μL (neutrophils: 46%). Normal values of neutrophils in Wistar Rats were in the range of 12-38% (
Giknis and Clifford, 2008). After inoculating
Klebsiella pneumoniae, neutrophil values increased on the first day (24 hours) and were commonly found in the air way or respiratory tract
(Liu et al., 2020; Madenspacher and Fessler, 2016). The dose of 100 μL, 0.5 McFarland standard on the first day (24 hours), neutrophil values in this group were high.
Previous studies reported leukocytes to increase at 24-48 hours post-inoculation and decrease after 72 hours post-inoculation
(Dong et al., 2012; Liu et al., 2020; Madenspacher and Fessler, 2016). Overall, the results of this study showed that the number of leukocytes was high (more than normal,
i.e. 2-10 × 10
3/mm
3) in post-inoculation because of the infection process. However, the results did not show a significant correlation between the dose of inoculated bacteria and post-inoculation time to the number of leukocytes
(Chakotiya et al., 2018). Statistical test results for the relationship between
Klebsiella pneumoniae dose and pneumonia incubation time based on the neutrophil count were significant (
p=0.030).
The relationship between
Klebsiella pneumoniae (
Kp) dose and incubation time was based on parameters of blood bacterial count (BBC) and pulmonal bacterial count (PBC) (Fig 2A) and based on chest radiography and histopathology (Fig 2B).
The highest degree of lesion on the thoracic radiography occurred on days 4 and 5 at the dose of
Klebsiella pneumoniae solution of 25 μL (score 3), on days 3, 4 and 5 at the dose of
Klebsiella pneumoniae solution of 50 μL (score 3) and on days 4 and 5 at the dose of
Klebsiella pneumoniae solution of 100 μL (score 3). The results of this study showed that chest radiography score had a significant correlation with the length of incubation time, which was about 3-5 days with a dose of
Klebsiella pneumoniae 25, 50 and 100 µL, 0.5 McFarland standard (Fig 2B). Pneumonia in the pulmonary parenchyma was caused by an inflammatory process due to
Klebsiella pneumoniae. The body’s defense factors can cause these bacteria to die, triggering macrophage alveolars, oedema fluid, polymorphonuclear cells (neutrophils) and erythrocytes filling the alveoli and interstitials. This inflammatory process appeared on thoracic radiography as opacity or consolidation in the pulmonary field. The consolidated picture could be seen on thoracic radiograph after the initial inflammatory/acute. The extent of consolidation on chest radiograph was influenced by the dose of bacteria entering the lungs, the length of occurrence time of infection and the location of the lesion
(Liu et al., 2020; Mizgerd and Skerrett, 2008). Chest radiograph is sometimes unable to show apparent lesions due to several reasons;, the location of the lesions that are in the posterior or parts of the lungs that are super positioned with other parts of the lungs, or large lesions that are still minimal. A more accurate radiological examination with a higher diagnostic value is the chest Computerized Tomography Scanning (CT-scan), but its application to animals is still very limited (
Fouriez-Lablée et al., 2017;
Ogur et al., 2019).
The incidence of pneumonia with the parameter of the bacterial count in pulmonary preparations indicated the growth of gram-negative bacteria on days 1, 2, 3 and 5. Whereas in the blood specimen, the bacterial count showed growth on the first and third days. The results of statistical tests showed no significant correlation between the dose of bacterial solution and the incubation time with the bacterial count from lung tissue and blood specimens. Microorganisms (
Klebsiella pneumoniae) can enter through the respiratory tract and get to the alveoli after 8 hours. This study showed positive culture results in both samples, namely from lung tissue and blood on day 1 at a dose of 25 µL, 0.5 McFarland standard (Fig 2A,3). The bacteria can persist for a long time in the lung tissue and can be found earlier in the first 24 hours in the lung tissue of the Wistar Rat intranasally. Intravenous administered bacterial solution inoculation caused bacteria to be found in the blood after the first 24 hours of incubation. In the lung tissue, the bacteria were found after 48 hours and will be systemically circulated into blood vessels (bacteriemia) (
Madenspacher and Fessler, 2016). Fig 3A-E shows the culture results of blood and lung specimens on McConkey’s selective media showing the growth of
Klebsiella pneumoniae colonies.
Fig 4 (N, A-C) shows the results of chest radiography from Wistar Rats with a pneumonia model with an appearance of the lungs without lesions (N) and an appearance of opacity lesions indicating an inflammatory reaction (Pneumonia).
Histopathological examination; Inflammatory degree on the first day (day 1) already indicated the presence of mild and moderate degree inflammations. Severe inflammatory degree occurred on day 1 (dose of
Klebsiella pneumoniae solution of 100 μL), day 2 (dose of
Klebsiella pneumoniae solution of 50 μL) and days 4-5 (dose of
Klebsiella pneumoniae solution of 50-100 μL). Some histopathological parameters indicated an inflammatory degree based on the number of polymorphonuclear cells (leukocytes, neutrophils, erythrocytes/bleeding), vascular thickening, bronchus wall and the alveoli areas thickened (epithelial layer of the alveoli)
(Ogur et al., 2019).
Data from the study showed that the number of leukocytes and neutrophils in blood samples, which were parameters of pneumonia in Wistar Rats, showed high results from day 1 at all doses of bacterial solution. An increase in leukocytes and neutrophils number are reported
(Dong et al., 2012) at 24 hours and 48 hours after inoculation of
Klebsiella pneumoniae in animal models (Table 1). Systemically, the leukocytes and neutrophils numbers in the inflammatory process of acute pneumonia can be judged from peripheral blood preparations. Pro-inflammatory cytokines and Chemocin (IL-1β, IL-6, IL-12, TNF-α) showed improvement on hours 24 and 48 (
Madenspacher and Fessler, 2016).
Histopathological examination showed inflammation with an average score of 2 on day 1 post-inoculation. It occurred at all doses of the bacterial solution of
Klebsiella pneumoniae. Histopathology of the pulmonary parenchyma indicated alveolar inflammation and an increase in the number of intra-alveolar neutrophils on days 1,2,3 (
Di Bonaventura et al., 2010). The bacterial count in the blood and lung tissue cannot be used as a parameter because it gives invalid or inconsistent results. Based on these data, researchers concluded that pneumonia in model animals occurred on day 3 at doses of 25, 50 and 100 μL, 0.5 McFarland standard. Pneumonia diagnosis based on the number of leukocytes, neutrophils and histopathology of the lung tissue occurred since the first day of post-inoculation, but visually the density of the lung tissue increased in chest radiography only seen on day 3. This study showed that lung density increased on day 3 post-bacterial inoculation. Chest radiography showed abnormal results with an increase in pulmonary density occurring from day 3 at all doses of
Klebsiella pneumoniae solution. Inflammatory parameters in the radiographic picture (Fig 4 A-C) indicated improvement in the density of the pulmonary parenchyma, indicating inflammation of the alveoli. The fluid filled the lumen alveoli, transudate, or exudate
(Meyerholz et al., 2018).
The parameters used for the successful development of pneumonia animal models can be inferred from the explanation in Table 1. The data presented showed a significant correlation between: doses of 25, 50 and 100 μL, 0.5 McFarland standard on day 1 post-inoculation with an increase in number of neutrophils (
p: 0.030); doses of 25, 50, 100 μL, 0.5 Mc Farland standard on days 1 to 4 post-inoculation with pulmonary histopathological parameters (HE) (
p: 0.00) and (
p: 0.013); doses of 25, 50 and 100 μL, 0.5 McFarland standard on days 3 to 5 post-inoculation with thoracic radiography screening parameters (
p: 0.000) (Table 1).
Some factors need to be considered in the development of animal models. They are the dose of inoculated bacteria, the length of incubation time, the host’s immune response factor, the virulence level (hypervirulent) of microorganisms (
Klebsiella pneumoniae), rat homogenization (strain uniformity, weight, sex, feeding, rat rearing site), microorganism inoculation techniques (
Klebsiella pneumoniae and others) (
Bengoechea and Sa Pessoa, 2019;
Joseph et al., 2021; López Hernández et al., 2015).
