Profiling Mannheimia haemolytica infection in dairy calves using near infrared spectroscopy (NIRS) and multivariate analysis (MVA)

Bovine respiratory disease (BRD) linked with Mannheimia haemolytica is the principal cause of pneumonia in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to inadequate treatment and further spread of disease. Near Infrared Spectroscopy (NIRS) is a rapid acquisition vibrational spectroscopy that can profile changes in biofluids, and when used in combination with multivariate analysis, has potential for disease diagnosis. This study characterizes the NIR spectral profile of blood plasma from dairy calves infected with M. haemolytica and validates the spectral biochemistry using standardized clinical and hematological reference parameters. Blood samples were collected for four days prior to (baseline), and 23 days after, a controlled intrabronchial challenge. NIR spectral profiles of blood plasma discriminated and predicted Baseline and Infected states of animal disease progression with accuracy, sensitivity, and specificity ≥ 90% using PCA–LDA models. These results show that physiological and biochemical changes occurring in the bloodstream of dairy calves during M. haemolytica infection are reflected in the NIR spectral profiles, demonstrating the potential of NIRS as a diagnostic and monitoring tool of BRD over time.

experiments were carried out with the approval of the MSU-Institutional Animal Care and Use Committee and all methods were performed in accordance with MSU-IACUC guidelines and regulations (IACUC-19-037). The bacterial challenge was given via bronchoalveolar lavage (BAL) catheter. Briefly, each calf was physically restrained in a chute, and a halter and lead were used to position the calf 's head straight up. Five mL of 2% lidocaine was used as a local anesthetic and was squirted into one nostril, and the BAL tube was introduced through this nostril and gently advanced into the trachea until the end of the catheter wedged into a bronchus. The appropriate volume of challenge inoculum, based on body weight, was administered through the BAL catheter, followed immediately by 60 mL of sterile 0.9% saline and 120 mL of air. Following the challenge procedure, a sub-sample of the challenge inoculum was used to prepare a quantitative culture assay on BHI agar and was checked at 24-48 h, and cfu were counted at 96-120 h. VCD was performed by an experienced veterinarian, and Complete Blood Counts (CBC) were assessed pre-and post-challenge before blood sample collection during four baseline days, 11 days immediately after challenge, and then every other day until 23 days post-infection.
Blood collection. A total of 18 blood samples were collected for each calf during the 23 days of the study (n = 90). Blood samples were collected through jugular venipuncture into two commercial blood collection tubes containing the anticoagulant Ethylenediaminetetraacetic acid (EDTA) and immediately placed on ice. One tube was centrifuged at 4000 rpm for 20 min for plasma separation and stored in duplicates of 1 mL at − 80 °C until NIRS analysis. The second tube was used for CBC analysis using a veterinary hematology analyzer for hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), red cell distribution width (RDW), white blood cell count (WBC), and platelets (PTLs). In addition, a microscopic differential count of nucleated cells was carried out to test the variability between neutrophils (polymorphonuclears, PMNs), eosinophils (polymorphonuclear eosinophils, PMEs), basophils (polymorphonuclear basophils, PMBs), monocytes (MOs), and lymphocytes (LYs).

Statistical analysis for clinical and hematological profiles.
The VCD included rectal temperature (TEMP) recorded with a digital thermometer, heart rate (HR), respiratory rate (RR), and assessment of overall airway health (the type of cough, secretions) conducted by an experienced veterinarian using a stethoscope. Using VCD and the antibiotic therapy provided when the clinical signs aggravated as the reference parameters, blood plasma samples (n = 90) were categorized as Baseline (n = 20), Infected (n = 23), Infected and Treated with antibiotics (n = 21), and Recovered (n = 26); only the first two categories (Baseline and Infected) were selected for the application of the statistical analyses (Table 1) to avoid the interference of the antibiotic effect in the biochemical profile. VCD and CBC information was analyzed using univariate statistics to obtain the Mean and the standard deviation (SD). Significance in parameter response was tested between the two categories Baseline and Infected, using ANOVA and a pairwise mean comparison applying Student's t-test with alpha = 0.05 (JMP 14.0 SAS Institute Inc., NC. USA). In addition, Principal Component Analysis (PCA) was applied using full crossvalidation, and algorithm-SVD (Singular Value Decomposition) in order to determine the influence of clinical signs and CBC values in the baseline and infected periods (Unscrambler X v. 10.5 software CAMO Analytics, Oslo, Norway). NIR spectral signature collection. Transmittance NIRS spectra (n = 430) were collected using a portable ASD FieldSpec 3 + IndicoPro (Malvern Panalytical, ASD Analytical Spectral Devices Inc. Boulder, CO. USA). Samples were thawed over ice for 15 min and warmed between hands for approximately 1 min before NIR spectra collection. Plasma samples (300 µl) were analyzed in a 1.00 mm quartz cuvette mounted in an ASD-fibre optic cuvette adapter. Each NIR spectrum was collected across the range 350-2500 nm (interval = 1.4 nm for the region 350-1000 nm and 2.0 nm for the region 1000-2500 nm; 50 scans; 34 ms integration). Prior to plasma spectra collection, a reference spectrum was captured from an empty cuvette. Ten independent spectral signatures were collected per sample, repacking the cuvette with plasma between each replicate. Multivariate analysis (MVA). The chemometrics based MVA was carried out in Unscrambler X v. 10.5 and performed on the first overtone region of the near-infrared spectrum in the vibrational combination band between 1300 and 1600 nm. The mathematical pre-treatments of Linear Baseline Correction, Standard Normal Variate (SNV) with de-trending (polynomial order: 2), and a 2nd derivative (symmetric Savitzky-Golay smoothing, points = 12) were applied to all the databases described next. A balanced database (n = 300) was created by randomly selecting 30 spectral signatures per calf and per each category (Baseline or Infected), to ensure the homogeneity of variance and weight of the datasets by controlling for the imbalance and diversity of the total number of blood samples collected (Table 1). This database (DB0) included spectra from all five calves and was used to perform the PCA and the Aquaphotomic analyses. To test for mathematical pre-processing and modeling bias, and against the null hypothesis (no biological signature can be differentiated between samples from these two classes) in the discriminant analysis, five datasets were created by stratified random sampling and analyzed in a leave-one-animal-out approach, Table 2. Specifically, spectra obtained from samples from four calves were sorted into an 80/20% distribution to form the calibration and internal validation sets; plasma spectra from the remaining calf were used as the external validation set.
Principal component analysis. PCA was applied to the DB0 database and to the calibration sets created for the discriminant analysis, it was completed as a first step to observe spectral features from both baseline and infected plasma samples to determine the dataset factorizations and scores distributions, identify dominant peaks in the loadings and outliers using the Hotelling's T 2 influence plot. PCA on the mean-centered matrix Aquaphotomics. Aquaphotomics was applied as a complementary spectral analysis to distinguish the biochemical profile of blood plasma collected before and after the M. haemolytica challenge. Water microstructure is represented by 12 spectral bands in the first overtone of the OH stretching region between 1300 and 1600 nm; these Water Matrix Coordinates (WAMACS) were used to generate barcodes and aquagrams to distinguish NIR spectra of plasma from baseline and infected stages 29,30 . To generate the barcodes, which represent wavelength shifts, the mean spectral information from sterile distilled water was subtracted from the mean spectrum for each category (Baseline or Infected) obtained from the transformed balanced database DB0. Aquagrams emphasize changes in the magnitude of peaks and were created by comparing the mean normalized (SNV only) water absorbance spectral patterns (WASPs) of the blood plasma from baseline and infected stages of the M. haemolytica challenge.
Linear discriminant analysis. Linear Discriminant Analysis (LDA) was used in the transformed spectra containing the water information (1300-1600 nm) from the balanced databases (DB1-DB5). Before the application of LDA for spectra classification between Baseline and Infected categories, the dimensionality of each spectral database was reduced using PCA to overcome the constraint of requiring more objects (samples) than features (scores or PCs). PCs or factors capturing ≥ 99% of the variance in the calibration datasets were selected to build the PCA-LDA model for each balanced database 28,43 . The subsequent LDA based on Bayes' formula, identifies similar spectral features for intra-class groupings and differential spectral features to separate the classes of blood plasma collected before and after the M. haemolytica challenge 44 . The PCA-LDA for the five prediction models created from databases DB1-5 is reported from the confusion matrix as a percent (%) to describe the quality parameters of accuracy, sensitivity, and specificity to evaluate the performance of the classification method 45 . The sensitivity test quantifies the PCA-LDA model's ability to correctly identify the true positives of M. haemolytica infection 46 described by Eq. (2) where TP = True positive, and FN = False negative 45 . A sensitivity of 85% detects 85% of animals with the disease (true positives) but classifies 15% of infected animals as healthy (false negatives). A high sensitivity (≥ 90%) is essential where the prediction model is used to identify severe but treatable diseases 47 .
The specificity of the PCA-LDA model is the ability of the model to correctly identify uninfected subjects, or the true negatives, and is represented by Eq. (3), where TN = True negative, and FP = False positive 45 . For example, a specificity of 85% correctly reports 85% of true negatives, but 15% of uninfected animals will be incorrectly identified as false positives 47 .  B  I  B  I  B  I  B  I  B  I  B  I   1  30  30  CAL  External validation  24  24  24  24  24  24  24  24   VAL  6  6  6  6  6  6  6  6   2  30  30  CAL  24  24  External validation  24  24  24  24  24  24   VAL  6  6  6  6  6  6  6  6   3  30  30  CAL  24  24  24  24  External validation  24  24  24  www.nature.com/scientificreports/ The prediction equations from the five models were applied to the internal validation and external validation sets described in Table 2 and evaluated with the same quality parameters. PCA scores plots and PCA-LDA plots were visualized in JMP 14.0.

Clinical and hematological profile for M. haemolytica infection.
In terms of fever response and rectal temperature, all calves responded similarly before and after bacterial challenge on Day 0 (Fig. 1a). Due to severe disease on Day 3 post-challenge, four calves (ID 1, 3, 4, and 5) were treated over five days with a thirdgeneration cephalosporin antibiotic with broad-spectrum activity (Ceftiofur), which caused the recovery of the calves and the change in the trends for TEMP and WBC (Fig. 1b). In the case of calf 2, mild signs of the infection were present from Day 1 until Day 19 when his signs aggravated, and antibiotics were provided. Only two calves, 2 and 4, exhibited a variable increasing pattern in WBC starting on Day 8, while the other three calves 1, 3, and 5 maintained pre-challenge levels of WBC due to the antibiotic therapy (Fig. 1c). When the rectal temperature and the WBC are averaged across individuals, a noticeable increase can be observed 24 h after the M. haemolytica challenge, which is characteristic of this particular bacterial infection in cattle (Fig. 1b, d).
The mean and standard deviations from the current variables used to identify M. haemolytica infection in the VCD and CBC and the bovine reference values for blood parameters 48 , are listed in Table 3. When taking into account only the baseline and infected stages of the disease in the controlled challenge, there was a significant (p < 0.05) increase in the rectal temperature, heart rate, respiratory rate, WBC, and the percentage PMNs The PCA correlation plots from VCD and CBC for each category (Baseline or Infected) are shown in Fig. 2. The first two PCs explained 43.6% and 50.4% of the variance of the VCD and CBC databases for the baseline and infected periods, respectively. No outliers were found in the Hotelling's T 2 influence plot (not shown). Prior to bacterial challenge, the parameters that are positively correlated and have the most influence indicating baseline levels were RBC, HCT, HGB, MCH, MCHC, RDW, and MCV (Fig. 2a). During the infection period, the activation of the innate immunity showed a negative correlation in TEMP and the percentage of LYs and a positive correlation with the WBC and the percentage of PMNs (Fig. 2b). Table 3. Clinical and hematological values (Mean ± SD) compared to reference for dairy calves challenged with M. haemolytica. Data were analyzed using Student's t-test between the groups with an α < 0.05 delineating significant treatment effects (*). K/µl = thousands per cubic milliliter, M/µl = Millions per microliter, g/ dl = grams per deciliter.    49,50 . The mean raw and the transformed NIR spectral signatures from the bovine plasma collected before and after the M. haemolytica challenge displayed a characteristic and expected spectral water pattern in the wavelength range from 1300 to 1600 nm ( Supplementary Fig. S1 online). This reflects the need for the application of Aquaphotomics and chemometric analyses to unveil the biochemical profile of the infection in this complex biofluid. The complex chemical differences and similarities in the transformed spectra from bovine blood plasma between 1300 and 1600 nm where OH, CH, and NH bonds interact with NIR light 23 can be observed in the trends from the three dimensional PCA scores plot (Fig. 3a). Here the first three PCs explained 66.7% of the variation of the DB0 database. No outliers were found in the Hotelling's T 2 influence plot (not shown), eliminating the possibility of artificial bias. The PC loadings show the dominant peaks influencing the trends in the scores plot (Fig. 3b). The first PC loading, PC-1 explained 47.3% of the variance of the database; PC-2 and PC-3 explained another 11.4% and 8.0% of the variance, respectively.
The complementary Aquaphotomic analysis displayed a different spectral pattern for the Baseline and Infected blood plasma in the wavelength range 1300-1600 nm (Fig. 4a). Barcodes highlight a shift in the WAMACS 31 for the baseline and infected blood plasma samples, reflecting changes in the plasma constituents and the water matrix. In the coordinate C8 (1448-1454 nm), a peak shift to higher frequency can be observed in the infected blood plasma spectra to 1448 nm in comparison with the baseline peak at 1454 nm, indicating a shift from bulk water dynamics to an increase in the formation of complex three-dimensional molecular spheres or hydration shells around solute molecules 31,51 . Additionally, in the spectra collected from blood plasma collected during the infected stage, NIR spectral peaks are right-shifted in C9 (1458-1468 nm), C11 (1482-1495 nm), and C12 (1506-1516 nm), more specifically to1466, 1492 and 1516 nm in comparison with spectral peaks at 1465, 1489, and 1510 nm from the blood plasma collected during the baseline stage; suggesting a shift towards more strongly bound water and longer-lived water complexes 52 . More highly organized water complexes and limited water exchange from solvation shells to bulk water occurs as solubility decreases, either in the context of concentration or reduced polarity of solutes (Fig. 4b).
The aquagram was created with the highest absorbances from the WAMACS in the baseline plasma at 1336 ,  1366, 1370, 1380, 1398, 1421, 1432, 1454, 1465, 1482, 1489, and 1510 nm (Fig. 4c). Overall, different WASPs can be observed for the blood plasma collected before and after the M. haemolytica challenge. Higher absorbances at 1366 and 1510 nm for the Infected and Baseline plasma are observed. These spectral absorbance points belong to the coordinates C2 (1360-1366 nm) and C12 (1370-1376 nm), and represent a higher number of strongly bound water molecules to non-water constituent molecules in the baseline period, and molecules organized in water symmetrical and asymmetrical stretching vibrations associated with changes in the solute composition of the blood plasma during the infection period 31 .

Classification of the biochemical NIRS profile during M. haemolytica infection. The PCA-LDA
was conducted on the spectra of plasma from both baseline and infected states simultaneously. A total of 18 PCs explaining 99 ± 0.2% of the variance in the PCA of the calibration sets were selected for the creation of the discriminant models using databases DB1 to DB5 (Supplementary Table S1 online). The derived prediction equations www.nature.com/scientificreports/ were then applied to the internal and external validation sets of spectra (Table 4). On average, the calibration of this supervised pattern recognition approach gave accuracy, sensitivity, and specificity of 98.5 ± 0.9, 97.5 ± 1.2, and 99.6 ± 0.9%, respectively. These values indicate that only 2.5 ± 1.2% of the infected blood plasma spectra were classified as false negatives, and 0.4 ± 0.9% of the baseline spectra corresponded to false positives to M. haemolytica infection. All the calibration databases (DB1-DB5) displayed similar trends in the PCA-LDA plot where two defined groups are observed; in Fig. 5, database DB1, excluding calf 1, is shown as a representation of the five databases analyzed. In addition, the internal validation set exhibited average values of 94.2 ± 1.7, 91.7 ± 2. 9, and 96.7 ± 1.9% for the accuracy, sensitivity, and specificity, respectively. In this process, 8.3 ± 2. 9% and 3.3 ± 1.9% of the spectra were classified as false negatives and false positives to the bacteria infection, correspondingly. Furthermore, the external validation set containing the spectra from the calf excluded during the calibration process was accurately classified with an average percentage of 83.3 ± 7.5%, a sensitivity of 79.2 ± 21.3%, and a specificity of 87.5 ± 10.9% when applying the prediction model. This indicates 20.8 ± 21.3% and 12.5 ± 10.9% of the spectra from the excluded calf to be classified as false negatives and false positives to M. haemolytica infection, respectively, when VCD is used as the reference method. These results suggest that the biochemical changes in the bloodstream as a result of the calves' response to the bacterial infection have the potential to be accurately detected and classified using NIR spectroscopy.

Discussion
The trends in the clinical and hematological parameters observed in our controlled challenge are related to the physiological response to M. haemolytica virulence factors associated with pathogenesis and needed to overcome the innate and adaptive immune response from the host. Our results are in line with similar profiles documented in previous studies when calves were challenged with either M. haemolytica alone or after they were exposed to Bovine Viral Diarrhea Virus Type 1B 8,53,54 . After the bacterial challenge, adherence and invasion of the respiratory tract cells occurs in response to the bacterial capsule, adhesins, and neuraminidase 5 . Once the host's cells are infected, the LPS complex and other pyrogenic features of M. haemolytica are released, activating the innate immunity or nonspecific defense mechanisms, triggering the observed fever and an increase in phagocytic cells (macrophages and neutrophils) after 24 h of the infection 8,53 . Subsequently, adaptive immunity is affected, which is reflected by the decrease in lymphocytes due to LKT release by the pathogen. The interaction of LKT with the CD18 subunit of the β2 integrin receptor of leukocytes forms transmembrane pores causing leakage of oxygen radicals and other products, such as nitric oxide, lysosomal enzymes, and inflammatory mediators such as cytokines IL-1β and TNFα, into the surrounding pulmonary parenchyma 7 . This contributes to cell apoptosis and pulmonary necrosis associated with the irregular breathing pattern, grunting on expiration, increased bronchial sounds, crackles, and wheezes 5,7 . The differences found in the univariate and multivariate results here demonstrate physiological and hematological changes occurred in response to the M. haemolytica challenge. More importantly, these parameters reflect a characteristic physiological response of disease state and provide the basis of the NIR spectral profiles used to discriminate healthy from infected calves, which will be useful for future diagnosis and monitoring of infection over time.
The NIR spectral profiles obtained in the chemometrics based MVA and the complementary Aquaphotomics, indicate changes in the biochemical make-up of the bovine blood plasma between the baseline and infected stages of the M. haemolytica challenge reflecting a calf 's immune response to the infection through changes in inflammatory cellular energy metabolism and antimicrobial pathways 55 . The recruitment of inflammatory cells results in a shift in their energy demand such that the supply almost exclusively originates from glycolysis, in order to accomplish the processes of phagocytosis to promote microbial death 55,56 . At the same time, increased flux through the pentose phosphate pathway (PPP) occurs, generating the reducing equivalent NADPH 55 . When glucose becomes limiting, T-cells utilize alternative energy sources, such as glutamine (critical for nitrogen metabolism and protein transamination), or fatty acids which feed the tricarboxylic acid cycle (TCA) generating intermediates such as citrate and the reducing agent NADH for the electron transport chain and aerobic metabolism 56 . The NADPH and NADH resulting from the PPP and TCA cycle, respectively, are required to produce reactive oxygen species (ROS) by NADPH oxidase in the membrane of activated macrophages, which are the crucial mechanisms for killing phagocytosed bacteria 55 . Additionally, nitric oxide (NO) and downstream reactive nitrogen species (RNS) produced by NO synthase in activated macrophages exert microbicidal or microbiostatic activity against bacteria by using L-arginine imported into the cell for the synthesis of NO as an antimicrobial product 55 . The increase of energy substrates, products, and key metabolites from the metabolic shifts occurring in the host as a response to M. haemolytica infection provide a changing milieu of components contributing to the biochemical NIR spectral profile. In this way, NIR spectra obtained from the blood plasma collected from the dairy calves before and after the M. haemolytica challenge differ, thus providing the information needed for spectra classification representing disease state.
Using NIRS for determining infection from M. haemolytica in dairy calves has a threshold of 90% sensitivity and specificity within the calibration and validation spectra, which is comparable to other techniques currently considered for BRD diagnosis. For example, ELISA determination of serum haptoglobin has a sensitivity of 93% and a specificity of 86% using a cutoff of ≥ 0.81 mg/mL 8 . Likewise, triplex real-time PCR based on the V3/V4 region of the 16S rRNA gene of three Mycoplasma species (M. bovis, M. bovirhinis, and M. dispar), is reported to have a diagnostic specificity of 98.2, 99.1, and 100%, respectively, when using bronchoalveolar lavage fluid (BALF) from infected calves 19 . Moreover, blood plasma from cattle with or without signs of BRD was evaluated with NMR for metabolite identification and diagnosis using visual clinical diagnosis as the reference method. In this case, animals with signs of BRD demonstrated increases in plasma α-glucose chains, hydroxybutyrate, and phenylalanine, and decreases with tyrosine, glutamine, citrate, and glutamate compared to healthy control animals. The accuracy, sensitivity, and specificity in this NMR study were 93, 99, and 88% for the calibration and 81, 84, and 74% for the validation process, respectively 20 .
Blood plasma spectra containing water information was used for the discriminant analysis (PCA-LDA) of baseline and infected samples to include the biochemical information of a fluid imbalance that may occur during the bacterial infection, affecting the blood volume and the blood pressure and consequently, the blood plasma composition 57 . Our results for M. haemolytica infection in dairy calves are comparable with previous studies where human blood plasma was evaluated using NIRS to profile, detect, and classify diseases. In one study, PCA and soft independent modeling of class analogy (SIMCA) were applied to identify features of NIR spectra of plasma from patients infected with HIV-1 in comparison with healthy individuals 35 . Both the PCA scores plot and the SIMCA Cooman's plot suggested that HIV-1 infections caused specific blood plasma changes and affect its Vis-NIR spectra from 600 to 1100 nm 35 . In other studies, infrared spectroscopy analysis of human blood plasma has been performed to discriminate mild, moderate, and severe cases of Alzheimer's disease (AD), in comparison with normal elderly persons used as a control [36][37][38] . In one study, NIR spectra in combination with regression analysis based on an oxidative stress index for group classification of AD patients, reported an 80% sensitivity and 77% specificity to discriminate patients with this disease 38 . Similarly, NIR spectra in combination with PCA and quadratic discriminant analysis (PCA-QDA) displayed 92.8% accuracy, 87.5% sensitivity and 96.1% specificity to classify blood plasma from patients at various stages of AD 36  www.nature.com/scientificreports/ transform infrared (FT-IR) spectra were discriminated with a sensitivity of 89% and specificity of 92% for the presence of Alzheimer's disease in blood plasma 37 . In ruminants, blood plasma has been evaluated using NIRS for early diagnosis of pregnancy in sheep, discerning spectra from pregnant and non-pregnant females by partial least squares discriminant analysis (PLS-DA), and using ELISA for pregnancy-associated glycoprotein (PAG), progesterone (P 4 ), and abdominal ultrasonography at 45 days after artificial insemination (AI) as reference methods. At Day 18 after AI, the sensitivity and specificity of NIRS and P 4 for pregnancy detection were 98.9% and 100%, respectively. Likewise, on Day 25, these parameters were 100% for NIRS and PAG 40 . All these studies suggest that NIRS can be an accurate method of diagnosis with similar sensitivities and specificities as the reference methods used to analyze blood plasma.

Conclusion
Clinical and hematological profiles for M. haemolytica infection were assessed using MVA, identifying the most reliable indicators of BRD in cattle among the numerous data parameters usually collected when disease is suspected. Based on these reference parameters and the associated biochemical changes occurring with the immune response, NIR spectral profiles were established for blood plasma collected before and after M. haemolytica infection. The NIR spectral profiles were used to develop a PCA-LDA model for discrimination of blood samples that can identify cattle infected with M. haemolytica with an accuracy, sensitivity, and specificity of > 90% within the model. Model prediction had an accuracy of 83.3 ± 7.5%, a sensitivity of 79.2 ± 21.3%, and a specificity of 87.5 ± 10.9% for NIR spectra of blood plasma collected from different animals than used in the model structure.
Our data indicate NIRS is a promising approach to rapid in-line diagnosis of BRD caused by M. haemolytica.

Data availability
Data is available upon request from Carrie K. Vance (ckv7@msstate.edu).