Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358

Background Severe leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis. Methodology/Principal findings Male Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6–7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression. Conclusion/Significance This study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis.


Introduction
The severe manifestation of leptospirosis could be either Weil's disease, a triad of jaundice, renal impairment and haemorrhages; or severe pulmonary forms of leptospirosis (SPFL) without distinct renal and hepatic impairments [1]. The multi-organs involvement often appears as a sudden onset of clinical manifestations, rapidly progressive and associated with high mortality rates [2][3][4][5]. This severe manifestation of leptospirosis could be either due to infecting strain of Leptospira, the load of leptospiral inoculum and the age and immune status of the infected host.
Despite the existence of the disease for many years, the evolution and factors determining the development of severe leptospirosis in the infected host are still not well defined. Clinical features and pathological changes in severe leptospirosis are described and suggested its association with cytokine storm [6][7][8][9]. Several studies have been focused on identifying the factors associated with severe leptospirosis [10][11][12][13][14]. However, the majority of these studies were performed on samples collected at a single time point. For a detailed understanding of the parameters that could be monitored to prevent the illness from progressing to a severe form, a kinetics study of the pathogenesis is vital.
In our earlier study, we isolated and identified a new genotype of Leptospira interrogans strain HP358 (L. interrogans strain HP358) with Sequence Type (ST) 238 in rodents trapped from the hotspot of human leptospirosis in the forest area of Hulu Perdik, Selangor, Malaysia [15]. We performed an in vivo pathogenesis screening for the strain HP358 in the hamster model and found that this strain is highly pathogenic manifesting pulmonary haemorrhage, liver and kidneys damages and death as early as six days of post-infection (p.i.) [16]. The evidenced life-threatening clinical manifestations prompted us to investigate and understand the kinetics of the pathophysiology of severe leptospirosis. Therefore, this study was carried out to decipher the progression of the illness by monitoring the clinical manifestation of infected hamsters, histopathological changes in tissues (lungs, liver and kidneys), the leptospiral load, hemogram and serum biochemistry and the cytokines and chemokines expression profiles. We hypothesised that understanding the virulence severity and the time course progression of the disease development may identify factors that are expressed or altered during the early stage of infection which could be recruited for further evaluation and subsequently utilized as biomarkers in severe leptospirosis.

Ethics statement
All experiments were conducted following the guidelines of the Code of Practice for the Care and Use of Animals for Scientific Purposes, Universiti Putra Malaysia. Male golden Syrian hamsters aged between four and six weeks purchased from Monash Universiti Malaysia, Bandar Sunway, Selangor were housed (three per cage) with sterile sawdust bedding, fed with commercial feed and given water ad lib in sterile bottles during the study course. The hamsters were acclimatized for 14 days prior to the experiment. All animal procedures carried out in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Universiti Putra Malaysia with Animal Use Protocol (AUP) number: UPM/ IACUC/AUP-R044/2018. This study also is in compliance with the ARRIVE guidelines.

Infection, monitoring and euthanization of hamsters
Upon completion of two weeks of acclimatization, the hamsters (n = 21) were infected intraperitoneally (IP) with 2 x 10 8 of L. interrogans strain HP358 in 500μl Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. The bacterial load (to develop infection) to be inoculated were selected based on our earlier investigation [16] and in previous studies [17,18]. Control hamsters (n = 7) were injected intraperitoneally with 500μl sterile EMJH medium (without any Leptospira). The infectivity study was carried out for seven days. The hamsters were monitored throughout the study for clinical signs such as progressive loss of weight, loss of appetite, reduced physical activity and dyspnea. One control and three infected hamsters were euthanized from day 1 to 7 p.i. except for day 2 to study the pathological events. Due to unforeseen reasons, we were not able to sacrifice the hamsters on day 2. The hamsters were anaesthetized with 100 mg/kg ketamine and 5 mg/kg xylazine injected intraperitoneally and subsequently whole blood was collected by cardiac puncture. Blood was collected for (1) direct culture in EMJH medium, (2) detection of leptospiral DNA and haematological analysis in EDTA tube, (3) biochemistry analysis in plain tube and (4) detection of immune genes in RNAprotect animal blood tube. Hamsters were euthanized by atlanto-occipital dislocation and following dissection, lungs, liver and kidneys were harvested and examined macroscopically for any morphological changes. Twenty-five milligrams of each lung, liver and kidney tissues were collected and transferred into tubes containing absolute ethanol for leptospiral DNA detection and RNAlater for immune genes expression study. The remaining parts of the organs were fixed in 10% neutral buffered formalin for histopathological investigations.

Clinical response to infection
The earliest clinical sign observed in the infected hamsters was weight loss which occurred as early as day 3 p.i. Body weight continued to decrease over the days, with average weight loss of 0.3% (D3), 2.2% (D4), 4.6% (D5), 7.3% (D6) and 7.7% (D7). On day 5 p.i., all hamsters showed loss of appetite, reduced physical activity and developed dyspnea. On day 6 p.i., three hamsters died and one hamster died on day 7 p.i.. All control (non-infected) hamsters euthanized on days 1 and 3 to 7 p.i. showed normal behaviour and progressive weight increase (16.6% increase at the end of the study).

Macroscopically pulmonary haemorrhage occurred earliest
All three hamsters euthanized on day 1 p.i. showed normal morphology of the liver and kidneys, however, few focal haemorrhagic areas were observed in the lungs. Beginning from day 3 p.i., haemorrhage in the lung continued to spread (Fig 1). The kidneys appeared pale from day 6 p.i. while the liver did not show any marked changes. Another notable finding observed was yellowish discolouration of adipose tissues in some of the dead and euthanized hamsters on days 6 and 7 p.i. (Fig 2). The lungs, liver and kidneys harvested from the control hamsters showed no gross changes.

Microscopically all organs showed progressive damages
The earliest pathological changes observed were congestion of the lung and liver on day 1 p.i. and kidneys on day 3 p.i. (Figs 3-5; Table 2). Haemorrhage was observed in the lung as early    earlier before day 7 p.i. showed similar pathological changes which were haemorrhagic lungs and kidneys and congested liver. No lesions or any pathological changes were observed in the organs of control hamsters.

Leptospiral load in blood and organs
Blood and kidneys samples of infected hamsters from day 1 to day 7 p.i. cultured in the EMJH medium yielded positive growth for leptospires while no growth was observed in the control hamsters. Likewise, qPCR also showed positive amplification for all samples (blood, lungs, liver and kidneys) collected from the infected hamsters from day 1 to day 7 p.i. (Fig 6). No cultures were performed for the lungs and liver. The leptospiral load (qPCR) in blood and organs showed a progressive increase from day 1 to day 5 p.i. for blood, lungs and kidneys and until day 6 for the liver. On day 7 p.i., the leptospiral load was lower than day 1 p.i. in blood, lungs and liver while the load remained unchanged in the kidneys.

PLOS NEGLECTED TROPICAL DISEASES
Kinetic infection of L. interrogans strain HP358 in hamster model

Haematological and serum biochemical changes
Haematological changes. White blood cells (WBC), neutrophils and monocytes counts in infected hamsters showed an increase from as early as day 1 p.i. (p-value = <0.05) (Fig 7) while lymphocytes and platelets showed a decreasing trend (p-value = <0.05). Serum Biochemical changes. The level of total bilirubin (TB), direct bilirubin (DB) and creatinine kinase (CK) in infected hamsters showed a significant increase (p-value<0.05) compared to that of control hamsters from day 5 p.i. onwards (Fig 8). Similarly, alanine transaminase (ALT) and aspartate aminotransferase (AST) levels rose significantly (p-value = <0.05) beginning on day 5 p.i. The levels of creatinine and urea in infected hamsters began to increase beginning from day 3 p.i. and 5 p.i. respectively. DD: dead hamsters. The leptospiral copies number in blood and organs can be found in S1 Table. https://doi.org/10.1371/journal.pntd.0010409.g006

Expression of immune mediators in infected animals
Pro-inflammatory mediators. Among the pro-inflammatory cytokines and enzymes (interleukin-1beta: IL-1β, interleukin-6: IL-6, Tumor necrosis factor alpha: TNF-α, interferon gamma: IFN-γ, cyclooxygenase-2: COX-2 and inducible nitric oxide synthase: iNOS) tested, IL-1β was found to be significantly expressed in blood and all organs (Figs 9-12) from day 3 onwards. Expression of IL-6 and TNF-α was only observed in the lungs and kidneys. IL-6 was significantly higher in the lungs on days 1 and 7 p.i. while in kidneys, it demonstrated a progressive increase from day 3 p.i. TNF-α showed increased expression in the lungs from day 1 to day 4 p.i. while in kidneys, it showed a progressive increase from day 3 p.i. IFN-γ was higher until day 4 p.i. in blood and lungs. COX-2 was higher than the control in blood until day 5 p.i. while in kidneys, it increased progressively from day 3 p.i. In the lungs, COX-2 was found to be downregulated beginning on day 1 p.i. IL-1β, TNF-α and COX-2 were found to be highly expressed in the liver of dead hamsters. iNOS was downregulated in lungs and kidneys and not detected in blood and liver.

PLOS NEGLECTED TROPICAL DISEASES
Anti-inflammatory mediators. The expression of transforming growth factor-beta 1 (TGF-β1) showed an increasing pattern in the liver and kidneys (Fig 14) beginning on day 4 p. i. and significantly high on days 6 and 7 p.i. TGF-β1 was found to be downregulated in blood while in the lungs it was slightly higher on days 3 and 4 p.i. Two hamsters showed amplification of IL-10 in lungs, liver and kidneys with a ct value of > 35 on days 5 and 6 p.i. One dead hamster showed amplification of IL-10 in the lung with a ct value of 33.63.
Association between clinical manifestations and pathophysiology in infected hamsters: Identification of biomarkers for severe leptospirosis. As summarized in Table 3, the pathophysiological presentations observed for severe leptospirosis in the hamster model included: (1) occurrence of pulmonary haemorrhage earlier than liver and kidney damages (before any clinical manifestations), (2) increased WBC, monocytes and neutrophils and decreased lymphocytes and platelets (before severe signs and symptoms), (3) serum biochemistry parameters changes were concurrent with the apparent clinical manifestations and (4) earlier expression of pro-inflammatory mediators IL-1β, CXCL10/IP-10 and CCL3/MIP-α in all organs (blood, lungs, liver and kidneys) prior to observable damages. The early expression of IL-1β, CXCL10/ IP-10 and CCL3/MIP-α, increase of neutrophils and decrease of lymphocytes and platelets suggesting that these parameters could be used as a cumulative panel for potential biomarkers in severe leptospirosis.  Table. https://doi.org/10.1371/journal.pntd.0010409.g012

Discussion
Leptospirosis presents a protean clinical manifestation and most cases (90%) are mild. Severe cases account for 5 to 15% and usually occur in the immune phase of illness [12,24,25]. Severe leptospirosis also presents with a fulminant monophasic illness [26][27][28]. In both conditions, the evolution of the disease is rapid and potentially fatal if not treated. Hence, early management of the disease is vital. Given the fact that the prompt diagnosis of this illness is challenging and the sudden progression to severe leptospirosis is life-threatening; understanding the sequence of disease progression and determination of early prognosis markers are of utmost importance for a favourable outcome.
In the present study, the first investigation was focused on the clinical manifestation developed in the hamsters when infected with the L. interrogans strain HP358. Hamsters reproduce the severe form of human leptospirosis [29] thereby suitable to be used as a model for studying the progression of severe leptospirosis. As seen from the results, loss of body weight started from day 3 p.i. and from day 5 p.i., all hamsters showed loss of appetite, reduced physical activities and difficulty in breathing (dyspnea). Similar clinical signs were also reported in several previous studies [19,30,31].

PLOS NEGLECTED TROPICAL DISEASES
Kinetic infection of L. interrogans strain HP358 in hamster model haemorrhage and blood vessel congestion in the lungs, liver and kidneys. The earliest sign of haemorrhage was observed in the lungs from day 1 p.i. while in kidneys on day 4 p.i. Marked organ damages (Table 3) were detected beginning from day 4 p.i concurrent with the clinical manifestations. These were followed by the death of four hamsters on days 6 and 7 p.i. while progressive moribund conditions were observed in the remaining hamsters. A similar observation was reported in a recent study where pulmonary haemorrhage appeared much earlier followed by liver and renal damages prior to the animal's state of moribund and death [31]. In human leptospirosis, pulmonary haemorrhage is the severe form of the illness, though it occurs only in a small number of cases, mortality is seen higher among these patients (more than 70%) [32,33]. The ability of the leptospires to invade multiple organs also depends on the Leptospira species or strain [16]. It could be postulated that in patients with severe leptospirosis, the patients might be infected with a highly virulent Leptospira strain invading multiple organs. As observed in the present study, the lungs were the first organ showing damage, hence it is important to monitor the respiratory problems or bleeding in leptospirosis patients as a prognostic factor for severe leptospirosis. As reported in previous studies, pulmonary haemorrhage could occur prior to jaundice and renal failure and led to severe disease and fatality in human leptospirosis [34,35]. Leptospiral DNA was detected in the blood and all organs on day 1 p.i. indicating rapid dissemination and successful colonization of L. interrogans strain HP358 in the hamster as reported in other studies [17,36]. The bacterial load in blood and all organs continued to increase until day 5 p.i. denoting the replication of leptospires. We observed a decrease in the leptospiral load from day 6 p.i. onwards in blood, lungs and liver while it was maintained in kidneys. Hamsters that died before the completion of the study had a high load of leptospires in all organs (lungs, liver and kidneys) compared to those euthanized on day 7 p.i.
Changes in haematological parameters were observed to occur as early as on day 1 p.i. indicating the response of the innate immunity of hamsters against the invading leptospires. Monocytes and neutrophils continued to increase while lymphocytes and platelets showed decreasing trends. Although neutrophils and monocytes could recognize leptospires, both have limited capacity to control the pathogen [37-42]. It was reported that pathogenic Leptospira spp. could bind to platelets and induce cytotoxic effects resulting in dysfunction and clearance of platelets [43][44][45][46][47]. A low level of platelets noticed in the present study could also play important role in the haemorrhagic presentation as observed in both animal [46] and human leptospirosis [48-50]. Significant neutrophilia and lymphocytopenia had also been reported in severe and fatal cases of human leptospirosis [6,[51][52][53][54][55]. The significant changes in total and direct bilirubin, AST, ALT, creatinine, urea and CK appeared much later than the haematological parameters which were on days 4 and 5 p.i. henceforth concurrent with the appearance of damage in the liver and kidneys. These changes in the liver and kidney function tests were in agreement with changes in human leptospirosis [51,[55][56][57][58][59].
Both pro-inflammatory cytokines and chemokines were expressed in the infected hamsters. Pro-inflammatory cytokines and enzymes which were IL-1β, IL-6, TNF-α, IFN-γ and COX-2 and chemokines CXCL10/IP-10 and CCL3/MIP-α showed upregulation as reported in both human and animal leptospirosis [6,17,18,23,[60][61][62]. Pro-inflammatory enzyme iNOS was downregulated in lungs and kidneys and not expressed in blood and liver which was similarly reported in a previous study [63]. Anti-inflammatory cytokine TGF-β1 showed expression from day 3 p.i. while IL-10 was slightly induced in some of the dead hamsters on days 5 and 6 p.i. as similarly reported in the previous study [64].
Overall, two main manifestations of pathophysiology in severe leptospirosis were observed; haemorrhage and organ damage where pulmonary haemorrhage appeared as the earliest pathological event. The mechanism of pulmonary haemorrhage is still poorly understood and could result from multiple factors [65]. Direct injury by leptospires or their circulating products (leptospiral outer membrane proteins, glycoproteins, hemolysins and lipopolysaccharides) and indirectly by the host's immune dysregulation have been proposed to contribute to the haemorrhagic manifestation in leptospirosis [66][67][68][69]. Pathogenic leptospires could bind to the endothelial lining of the blood vessels [50,67,[70][71][72] and potentially disrupts the endothelial cell layer [69,73]. IL-6 has been associated with severe pulmonary haemorrhage [6]. In the present study, IL-6 was significantly high in the lung on day 1 p.i. and while in the kidney, it only appears on day 4 p.i. concurrent with the haemorrhagic presentation, thereby could support the role of this cytokine in the haemorrhagic presentation in leptospirosis.
Haemorrhagic manifestation of leptospirosis could also be due to vascular cell damage by reactive oxygen species (ROS) and arterial hypertension [74]. Neutrophils could produce ROS [75], thus the high production of neutrophils in the infected animals may indirectly contribute to the haemorrhagic manifestation observed in this study. Nitric oxide (NO) production catalyzed by the enzyme iNOS [76,77] functions as a vasodilator [78] and is also able to control the production and activity of ROS [79,80] in inhibiting the replication of the pathogen [81][82][83]. The down-regulation of iNOS influences the release and activities of NO [76,77]. Low or specific inhibition of iNOS is associated with pulmonary haemorrhage [63], increased mortality, bacterial load in the kidney and reduced specific humoral response [84] in the hamster model and patients with severe disease [85].
Mild alveoli dilation observed on day 5 p.i. is a contributing factor for dyspnea which is in agreement with a report in a recent study [86]. The dilated alveolus is a characteristic of chronic obstructive pulmonary disease (COPD) with dyspnea as the cardinal symptom. Enlargement of the alveolus destructs the alveoli walls through inflammation [87]. Cytokine IL-1β has been shown to exert airway inflammation and emphysema in the COPD mice model [88][89][90][91][92]. The raised level of serum IL-1β in patients with mild alveolar dilation is in agreement with the present investigation where expression of IL-1β was significantly high from day 4 p.i. onwards [93].
The progression of damage in the liver and kidneys is associated with the changes in the serum biochemistry and increased expression of inflammatory cytokines and chemokines. The liver and kidney functions test markers (total bilirubin, direct bilirubin, ALT, AST, creatinine and urea, CK) showed a significant increase from day 4 p.i. The progressive upregulation of inflammatory cytokines and chemokines in the kidney (IL-1β, IL-6, TNF-α, COX-2, TGF-β1, CXCL10/IP-10 and CCL3/MIP-α) and liver (IL-1β, TGF-β1, CXCL10/IP-10 and CCL3/ MIP-α) beginning from day 3 p.i. support the possibility that damage in these organs is associated with the increased inflammatory response. CXCL10/IP-10 and CCL3/MIP-α are known to mediate the migration of T cells, monocytes, neutrophils and natural killer (NK) cells from the bloodstream to tissues in response to inflammation [94][95][96][97].
A severe manifestation of leptospirosis is comparable to sepsis that occurs due to an imbalance in the inflammatory responses in the host infected with pathogens. The infected hosts release inflammatory mediators in an attempt to neutralize the pathogenic effect. The occurrence of a sustained and increased expression of pro-inflammatory cytokines characteristic of a "cytokine storm" will lead to persistent inflammation [9] and this is followed by a massive and systemic production of anti-inflammatory mediators resulting in a state of "immunoparalysis" and tissue oedema [98,99]. Tissues oedema could impair the local organ perfusion leading to loss of organ function and endothelial permeabilization [98,100]. In asymptomatic or mild leptospirosis and mice animal models, homeostasis between pro-inflammatory and antiinflammatory is maintained where both are produced early and strictly regulated [9,17]. In severe leptospirosis in humans, two scenarios have been reported; either high IL-10 and low TNF-α [6,61] or low IL-10 and high TNF-α [17,101,102]. In this present study, we saw a sudden surge of the pro-inflammatory mediators (cytokines and chemokines) beginning from day 3 p.i.without the prominent expression of anti-inflammatory IL-10. A low expression of IL-10 (ct value of >33 cycles) and early (day 3) expression of TNF-α, TGF-β and IP-10 in hamsters infected with L. interrogans serovar Pyrogenes has been reported earlier [64]. In conclusion, severe leptospirosis due to the L. interrogans strain HP358 could be characterized as a sudden and increased pro-inflammatory response with delayed and significantly low expression of anti-inflammatory IL-10. The severe leptospirosis characterized in the hamster model in the present study is in accordance with the severe form of leptospirosis in humans where patients showed mild symptoms during the early course of the disease and developed a rapidly worsening condition leading to fatality within 72 hours [70,103]. As observed in this study, the rapid evolution to severe illness and fatality in hamsters occurred when most inflammatory mediators were expressed and all organs (lungs, liver and kidneys) were affected. Likewise, the severe form of human leptospirosis involves haemorrhage and multiple organ damages.
Identification of biomarkers in leptospirosis is important not only for diagnosis but also to predict the progression to severity. The main characteristic of ideal biomarkers is their early detection [104] for timely intervention in patients management. A panel of biomarkers will increase the specificity and sensitivity of the diagnosis compared to a single biomarker [101,105]. Serum biochemistry may not be a good predictor for severe leptospirosis as these markers are detected only after the occurrence of serious damage to the liver and kidneys. Cytokines play a major role in host-pathogen interaction and prognosis [18]. Several earlier studies have identified significant expression of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-α in severe leptospirosis [6,102,106]. However, these studies were conducted in one-time sampling. Progressive monitoring is important to elucidate the progression of cytokines levels and to determine the most appropriate biomarker for disease severity. As the damage in leptospirosis surge rapidly, we recommend performing blood and cytokines profiling at 24 hours interval to monitor the biomarkers for the severe illness that could prevent substantial damage to the organs. From the present investigation, we found the expression of IL-1β, CXCL10/IP-10 and CCL3/MIP-α increased in the blood and most organs day by day as the infection progressed. On the other hand, neutrophils increased progressively from day 1 p.i. while lymphocytes and platelets showed a declining trend. Taken all these data together, we suggest that high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets could serve as a cumulative panel of potential biomarkers in the disease progression from mild to severe in leptospirosis. As this study was conducted in an animal model, a progressive validation study in human leptospirosis is recommended.

Conclusion
Severe leptospirosis is characterized by a sudden over-expression of pro-inflammatory cytokines after infection of L. interrogans strain HP358 and without prominent expression of regulatory cytokines. The massive expression of cytokines and chemokines led to sudden and rapid damage to the liver and kidneys. Damages in the lungs, liver and kidneys were preceded by the early occurrence of haemorrhage in the lungs. High levels of IL-1β, CXCL10/IP-10, CCL3/ MIP-α, neutrophils and low levels of lymphocytes and platelets might serve as a cumulative panel of biomarkers in severe leptospirosis.
Supporting information S1