A method to prevail false positive responses due to excess cations and viscous nature of Radiopharmaceuticals in Limulus Amebocyte Lysate Gel Clot test

Objective(s): Bacterial endotoxin test (BET) for detection and quantification of endotoxin in radiopharmaceuticals (RPs), used for therapy or diagnosis, is prerequisite to administration in patients. Out of the two established methods used for this purpose (Kinetic Chromogenic Assay: KCM and Gel Clot Bacterial Endotoxin Test: GC-BET), GC-BET is recommended by pharmacopeias to evaluate the interferences exhibited during the assay due to presence of various ingredients in samples. In the present study, the influence of excess of cations in [177Lu]Lu-DOTATATE, used for Peptide Receptor Radionuclide Therapy (PRRT), were studied and a protocol to negate the enhancement observed was developed. Additionally, a protocol for carrying out GC-BET for extremely viscous [131I]I-Lipiodol was standardized. Methods: GC-BET was performed for [177Lu]Lu-DOTATATE and [131I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [177Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl2) were added and evaluated for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [131I]I-Lipiodol various modification in the protocols like orbital vortexing at different rpm and time intervals were performed. KCM assays were also performed for studied RPs at MVD. Results: It was observed that at MVD, [177Lu]Lu-DOTATATE exhibited false positivity in GC-BET. However, all the individual reagents used in labeling of [177Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of 2mM CaCl2 (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [131I]I-Lipiodol due to its viscosity was resolved by orbital vortexing at 3000 rpm for 5 minutes. Conclusions: Our study proved that false positivity was observed in GC-BET for [177Lu]Lu-DOTATATE due to the presence excess M3+ ions. Further, our study is the first of its kind which demonstrated methods for negating these false positive results by using modified protocol and hypothesizing the reason behind the enhancement. Additionally, ours is the first study which proved that a simple step of vortexing the viscous RPs like [131I]I-Lipiodol can resolved the problems encountered during performing GC-BET due to viscosity of RPs.

Introduction
The use of radio-labeled peptides like ([ 177 Lu]Lu-DOTATATE) for PRRT has enhanced effectiveness in cancer treatment because of selective delivery of radioisotope to tumor sites which further reduces toxic side effects to healthy tissues. Additionally, target specific radiolabeled fatty acid (RFA) like [ 131 I]I-Lipiodol 110 Asia Ocean J Nucl Med Biol. 2022; 10(2): [109][110][111][112][113][114][115][116] and [ 177 Lu]Lu-DOTATATE, a radio-lanthanide labeled RP (RL-RP), has been extensively used for treatment of liver metastases and have obtained an approval from European Medicines Agency (EMA) and United States Food and Drug Administration (USFDA) in recent years (1,2). The success of RPs lies on the selection of suitable peptides, fatty acids, chelating cores and their ability to form stable conjugate and further to achieve efficient radio-labeling with various radioisotopes like Lutetium-177, Yttrium-90, Rhenium-188 and Technetium-99m. Most of these target specific therapeutic RPs are approved by USFDA and the EMA for treatment of various cancers in humans and these therapeutic RPs are considered to be in generic form. Hence, it is mandatory to test these radiolabeled formulations for apyrogenicity prior to administration in patients, to ensure that endotoxin levels are within permissible limits.
For RL-RPs, Endotoxin Limit (EL) is 175 EU/V (3,4) whereas for intra-arterial infused RFA, EL is 17.8 EU/mL (5,6). The standard method for assessing the bacterial endotoxin levels in any of these studied RPs and their cold kits is by rabbit pyrogen test as described in United State Pharmacopoeia (USP) chapter 151. However, common limitations with this test are; its labor intensive nature, potential radioactive dose to analyst and problems during administering radioactivity to animals.
GC-BET is the most economical and technically uncomplicated method available for evaluation of bacterial endotoxin in radiopharmaceuticals. However, it is very sensitive towards concentration of divalent calcium ions and the change in concentration during assay, contributed by the samples, lead to interference either in the form inhibition or enhancement. Radiometals like 177 Lu, 90 Y, 188 Re, 225 Ac etc are used for preparation of rdaiopharmaceuticals for therapeutic purpose and these radiometals undergo radiometallation or radiochelation reaction with suitable cyclic / acyclic / bifunctional chelators to which target specific peptides / enzyme inhibitors / monoclonal antibodies are attached. These final radiopharmaceutical preparations contain various monovalent / divalent / trivalent non-radioactive cations which do not interfere with physiological uptake. However, these cations, crucially get involved during enzymatic reaction of GC-BET and compete with Ca 2+ ions, thereby increasing (enhancement) or decreasing (inhibition) the sensitivity of assay. The enhancement observed in the assay results in false positivity, while inhibition leads to false negativity.

Methods
In BET assay, it is mandatory to calculate the Maximum Valid Dilution (MVD) which is the maximum allowable dilution of a sample at which the endotoxin limit can be determined and interfering test conditions can be avoided. MVD = EL/λ (Where EL is Endotoxin Limit of RPs and λ is Sensitivity in EU/mL of LAL reagent used). Considering the EL of studied RPs, MVD is calculated and sensitivity at which assay is to be performed is described in Table 1. Detailed protocol for GC-BET is shown in Table 2, and the assays were performed for all the RPs at MVD. Positive Product Control 2λ † … 50 50 100 Test 3 and 4 were also performed initially for stock solution of CaCl2 solution, saline (0.9% sodium chloride) and also for ultrapure water to ensure absence of endotoxin in stock solutions. The tests, Negative Product Controls (NPC) and Positive Product Controls (PPC) were incubated at 37 o C for 60 minutes in a dry heating block. To demonstrate reproducibility of the results 3 batches of each of RPs were tested. To demonstrate that the usage of CaCl2 does not interfere in the gel-clot formation and also does not reduce or enhance the sensitivity of the test, we have carried out assays with positive water control at different standard endotoxin concentrations with and without calcium chloride. The assay has also been done with negative water control with and without calcium chloride. The assays were repeated 10 times to study reproducibility. For all the studied RPs and active product ingredients (APIs), KCM assays using PTS were performed at the same MVD at which GC-BET were performed. Detailed protocol for the procedure is shown in Table 3.  Table 4. Even at MVD as high as 400, it is unfeasible to perform successful GC-BET for [ 131 I]I-Lipiodol without any modification, due to high viscosity of lipiodol and thick precipitation were observed in NPC test sample. However, this precipitation in NPC can be overcome by carrying out orbital vortexing at 3000 rpm for 5-8 minutes, at first dilution of the test samples. This short orbital vortexing step allows the dispersion of the viscous layer formed during dilution of samples.    Table 7 shows the result obtained for the GC-BET carried out at different concentrations of standard endotoxin from 4λ to λ/4 (λ: sensitivity of lysate used i.e 0.125 EU/mL and 0.03 EU/mL) and it can be seen that the presence of CaCl2 does not interfere in the assay.

GC-BET results for [ 131 I]I-Lipiodol at 400 MVD are depicted in
Further, the experiments gave consistent results over 10 replications indicating the validity and reproducibility of the procedure of reversing the enhancement of gel clot formation after addition of excess CaCl2.  T2  T3  T1  T2  T3  T1  T2  T3  T1  T2  T3  T1  T2

Discussion
The use of peptides, enzyme inhibitor and other molecules, radiolabeled with different radioisotopes of different half-lives and energies, is ever increasing in treatment as well as diagnosis. In view of the spot preparations of RPs, in house endotoxin evaluation before administration to patients, has become mandatory in hospital radiopharmacy unit of the Nuclear Medicine Departments of hospitals. As mentioned earlier the endotoxin in samples can be estimated using GC-BET or KCM. Coagulation cascade reaction in GC-BET is based on presence of natural substrates i.e. proline rich proteins and 8.6 kDa proteins of limulus transglutaminase present in non-granular hemocytes (L granules) of horse shoe crab8 and also on optimum proteolytic activity of transglutaminase enzyme. Optimum proteolytic activity of transglutaminase is dependent on the balanced free divalent cations concentration9, 10,11,12 and overall this concentration in ionic form (Ca 2+ ) has to be maintained for optimized transglutaminase activity (13).
Transglutaminase binds to three Ca 2+ ions at three different sites, which means optimum concentration should be 1.0 Ca 2+ /mol in absence of any other metals. The Ca 2+ ion in site one is constitutively bound with binding affinity of Kd~0.3 µM (ΔH ~ -6.70 kcal/mol) and is required for stabilization of the enzyme (14). Upon proteolysis transglutaminase exothermally acquires two more Ca 2+ ions at site 2 and 3, which activates the transglutaminase by opening a channel which exposes tryptophan-236 (Trp-236) and tryptophan-327 (Trp-327) residues that controls substrate access to the active site (15).
[ 177 Lu]Lu-DOTATATE is used for PRRT for patients with neuroendocrine tumors (NET) and in present study, it was prepared at Centralized Radiopharmacy Centre (CPC) with RCP of ≥95 to 99%, using carrier added [ 177 Lu]LuCl3 as per the standard procedures (7). At CPC when GC-BET was performed at 30 MVD (total production volume is 7-9 ml) along with other QC analysis, the preparation did not show any false positive results .
However  (12), we reported that some of the 99m Tc-labeled radiopharmaceuticals ( 99m Tc-RPs) and their cold kits (TCKs) exhibited inhibition during GC-BET. The PPC test samples ( 99m Tc-DMSA-III, 99m Tc-DMSA-V, 99m Tc-EC, 99m Tc-DTPA and their respect-tive cold kits) did not form any firm clots and false negative results were observed. These false negative results were reversed by adding excess 300 µM Ca 2+ ions (CaCl2 form) in test samples at MVD. We reported that, the free carboxyl groups (-COOH) present in some of the 99m Tc-RPs and their respective TCKs inhibit GC-BET by depleting Ca 2+ ions concentration, thereby inhibiting transglutaminase activity. However, though DOTATATE acetate, was with presence of free -COOH groups, no false negative results in PPC test samples were observed. This may be because the molar concentration for DOTATATE acetate was 0.565 µmoles at final GC-BET, which is very low as compared to TCKs. Thus the molar concentrations of DMSA-III and DMSA-V were 2.74 µmoles at final GC-BET assay which was almost 5 times the molar concentration of DOATATATE acetate. While molar concentrations for EC and DTPA were as high as 3.73 and 29.51 µmoles at final GC-BET .
Highly viscous [ 131 I]I-Lipiodol (EL <17 EU/mL) (6,18) forms two different phases (viscous lipophilic and aqueous hydrophilic phase) on diluting with LRW (MVD -400), thereby making this product unfeasible to perform GC-BET. We used orbital vortexing at 3000 rpm for 5-8 minutes for [ 131 I]I-Lipiodol at first dilution (200) which resulted in uniform dispersion of [ 131 I]I-Lipiodol in LRW allowing successful GC-BET of this product. Use of heating prior to performing GC-BET for RFA is not recommended as heating of diluted [ 131 I]I-Lipiodol at 125 o C though may solubilize highly viscous lipiodol but will volatilize highly radioactive Iodine-131 which is very hazardous. Centrifugation of 131 I-Lipiodol is strictly not recommended for dispersion of RFA as it will lead to separation of hydrophilic and lipophilic phase. BET by KCM using PTS cannot be performed for [ 131 I]I-Lipiodol at 400 MVD, since these highly viscous RFA will not mix thoroughly with lyophilized LAL, synthetic CS and CSE present in channel of PTS test cartridges (PTS-TC) and lead to blockage of channel which has diameter of 2 mm.

Conclusion
The present study is the first one to report that simple 3000 rpm orbital vortexing can solve the problem of performing GC-BET for [ 131 I]I-Lipiodol. Further, this is also the first study to report addition of 2 mM CaCl2 solution for [ 177 Lu]Lu-DOTATATE to overcome the enhanced false positive result due to increased transglutaminase enzymatic activity and formation of 4 to 6 fold stiffer gel. The adaptation of these modified LAL test methods provides a reliable BET quality control testing option for both the RPs at Hospital Radiopharmacy Settings. Thus an expensive assay like KCM-BET can be replaced by these modified GC-BET for the RPs mentioned in our study.


Intestine-Specific NHE3 Deletion in Adulthood Causes Microbial Dysbiosis

In the intestine, the Na+/H+ exchanger 3 (NHE3) plays a critical role for Na+ and fluid absorption. NHE3 deficiency predisposes patients to inflammatory bowel disease (IBD). In mice, selective deletion of intestinal NHE3 causes various local and systemic pathologies due to dramatic changes in the intestinal environment, which can influence microbiota colonization. By using metagenome shotgun sequencing, we determined the effect of inducible intestinal epithelial cell-specific deletion of NHE3 (NHE3IEC-KO) in adulthood on the gut microbiome in mice. Compared with control mice, NHE3IEC-KO mice show a significantly different gut microbiome signature, with an unexpected greater diversity. At the phylum level, NHE3IEC-KO mice showed a significant expansion in Proteobacteria and a tendency for lower Firmicutes/Bacteroidetes (F/B) ratio, an indicator of dysbiosis. At the family level, NHE3IEC-KO mice showed significant expansions in Bacteroidaceae, Rikenellaceae, Tannerellaceae, Flavobacteriaceae and Erysipelotrichaceae, but had contractions in Lachnospiraceae, Prevotellaceae and Eubacteriaceae. At the species level, after removing those with lowest occurrence and abundance, we identified 23 species that were significantly expanded (several of which are established pro-inflammatory pathobionts); whereas another 23 species were found to be contracted (some of which are potential anti-inflammatory probiotics) in NHE3IEC-KO mice. These results reveal that intestinal NHE3 deletion creates an intestinal environment favoring the competitive advantage of inflammophilic over anti-inflammatory species, which is commonly featured in conventional NHE3 knockout mice and patients with IBD. In conclusion, our study emphasizes the importance of intestinal NHE3 for gut microbiota homeostasis, and provides a deeper understanding regarding interactions between NHE3, dysbiosis, and IBD.

INTRODUCTION
The Na + /H + exchanger 3 (NHE3) mediates Na + and fluid absorption in the intestine and reabsorption in the kidney. Knockout of NHE3 selectively in the small intestine and colon of mice results in disruption of intestinal structural integrity, persistent alkaline diarrhea, metabolic acidosis, hyponatremia and hyperkalemia associated with drastically elevated plasma aldosterone levels, and increased mortality rate (Xue et al., 2020;Xue et al., 2022). Together, these symptoms are consistent with patients experiencing congenital sodium diarrhea (CSD).
In patients, germline mutations in guanylyl cyclase C, a regulator of NHE3, and in NHE3 itself result in CSD (Janecke et al., 2015;Janecke et al., 2016). Of note, a number of patients with these mutations develop inflammatory bowel disease (IBD), implying that NHE3 dysfunction directly or indirectly predisposes patients to IBD (Anderson et al., 2011;Jostins et al., 2012). Several studies have demonstrated an inverse relationship between NHE3 activity and pathogenesis of IBD. Conventional NHE3 knockout (NHE3 -/-) mice develop spontaneous distal colitis (Laubitz et al., 2008) and are highly susceptible to dextran sodium sulfate (DSS)-induced mucosal injury (Kiela et al., 2009). Similarly, intestinal NHE3 dysfunction has been observed in other murine colitis models such as interleukin (IL) 2-deficient mice (Barmeyer et al., 2004), IL10deficient mice (Lenzen et al., 2012), and mice treated with DSS or trinitrobenzene sulfate acid (Sullivan et al., 2009). Development of IBD is thought to occur as a result of pathologic interactions between the intestinal environment, immune system, and microbial factors in a genetically susceptible host. Several studies have shown that NHE3 deficiency predispose patients and animals to microbial dysbiosis and IBD (Engevik et al., 2013;Larmonier et al., 2013;Janecke et al., 2016;Laubitz et al., 2016;Harrison et al., 2018). NHE3 -/mice show reduced microbial diversity with expansion of the inflammation-associated Bacteroidetes phylum and contraction of the Firmicutes phylum (Engevik et al., 2013;Harrison et al., 2018), which is consistent with what has been found in IBD patients (Ott et al., 2004;Sokol and Seksik, 2010;Fava and Danese, 2011). When NHE3 -/mice were re-derived into a germ-free facility, they exhibited no inflammatory phenotype and show a delayed mortality in response to DSS (Larmonier et al., 2013). On the other hand, reintroduction of conventional microflora in germfree NHE3 -/mice restored spontaneous distal colitis, highlighting the role of microbiota in NHE3 dysfunctioninduced colitis (Larmonier et al., 2013). It should be noted that colonization of microbiota begins at birth; thus, studies utilizing NHE3 -/mice are evaluating the effect of NHE3 deletion on the gut microbiome across the entire development span. However, it is not known if lack of NHE3 in adulthood exerts similar effects on gut microbiome.
The aim of the current study was to determine how inducible intestinal-specific knockout of NHE3 in adulthood impacts the gut microbiome. Our data demonstrate that functional NHE3 is required for a healthy microbiome, and lack of intestinal NHE3 results in microbial changes that favor inflammation and downstream pathological consequences.

Animals
All animal experimentation was conducted in accordance with the Guide for Care and Use of Laboratory Animals (National Institute of Health, Bethesda, MD) and was approved by the Institutional Animal Care and Use Committee (3338R). The generation of tamoxifen-inducible intestinal specific NHE3 knockout mice (NHE3 IEC-KO ) has been described previously (Xue et al., 2020). Mice were genotyped by polymerase chain reaction from genomic DNA isolated from ear punch. At 11 weeks of age, NHE3 deletion was induced in mice by application of tamoxifen (67 mg/kg), initially dissolved in 5% (v/v) of ethanol followed by adding 95% (v/v) of corn oil. Tamoxifen was administered via oral gavage (volume 1% of body weight) for 5 consecutive days to control and NHE3 IEC-KO mice. Only male mice were used for microbiome analysis.

Sample Collection, and Shotgun Metagenomic Sequencing and Quality Control of Reads
At 12 weeks of age, mice (n = 9/genotype) were single housed for 2 weeks and colon content was collected into collection tubes containing DNA stabilization buffer. Whole metagenome shallow shotgun sequencing (at least 2 million paired-end reads/sample) was performed using the Illumina miSeq or Illumina NextSeq instrument (the instrument used is dependent on the number of samples in a batch, Illumina, San Diego, CA). For the analysis, samples were extracted using the Qiagen PowerMag Microbiome DNA Isolation kit (Hilden, Germany) on the King Fisher automated platform (Thermo Scientific, Waltham, MA). Isolated DNA was quantitated using a fluorescent concentration assay and normalized to prepare for library prep using the Illumina Nextera XT DNA Library prep recommendations. Runs were spiked with 1% PhiX. Standard processing used 2 x 150 base pair paired-end sequencing with dual 8 base pair indexes. The instrument run takes~29 hours. Criteria for acceptable results: final run must have a cluster density of 180-230K/mm 2 with greater than 80% of clusters passing filter, and at least 75% of bases must call at a minimum Phred score of Q30 (99.5%).

Data Analysis and Statistics
The One Codex Database consists of~114,000 complete microbial genomes, including 62,000 distinct bacterial genomes, 48,000 viral genomes, and approximately 4,000 fungal, archaeal, and eukaryotic genomes. The human genome was included to screen out host reads, and a complete list of references is available in the One Codex application at https:// app.onecodex.com/references. The database is assembled from both public and private sources, with a combination of automated and manual curation steps to remove low quality or mislabeled records. Comparing a microbial sample against the One Codex Database consists of three sequential steps. First, every individual NGS read was compared against the One Codex Database by exact alignment using k-mers where k=31 [(Ames et al., 2013;Wood and Salzberg, 2014) for details on kmer based classification]. The k-mer classification results were filtered based on the relative frequency of unique k-mers in the sample, sequencing artifacts were filtered out of the sample. This filtering only removes probable sequencing or reference genomebased artifacts and does not filter out low abundance or low confidence hits. Finally, the relative abundance of each microbial species was estimated based on the depth and coverage of sequencing across every available reference genome. Microbial profiles were generated using the OneCodex analysis platform using the targeted loci module with summarization at the phylum through species levels. Results were normalized to an even level of coverage through subsampling without replacement (11,000 observations per sample). Statistical analyses were performed using the R Statistical Environment (v.3.5.3). Normalized species level profiles were utilized to calculate aand b-diversity measures with the vegan R package. Principal coordinates analysis (PCoA) was performed using the ape R package. PERMANOVA calculations were executed using the adonis function in vegan. Differential abundance analysis included calculation of the  (Segata et al., 2011) was applied to normalized taxonomic data to compare taxonomic membership between genotypes, followed by visualization of identified taxa by cladogram and individual LDA scores. Additional visualizations of stacked histograms and boxplots utilized the ggplot2 package in R. Aggregation of abundance estimates for aerobic, facultative anaerobe, and obligate anaerobe categories applied a taxonomic classification strategy based on literature review in which (1) aerobic included members of Pseudomonas, Mycobacterium, Nocardia, Bacillus or Streptomyces, (2) those not assigned to aerobic that were members of the phyla Actinobacteria, Firmicutes, Proteobacteria were assigned as facultative anaerobes, and otherwise (3) obligate anaerobes were members matching the taxa Tenericutes, Bacteroidetes, Actinomyces, Bacteroides, Clostridium, Fusobacterium, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, and Veillonella.

Intestinal NHE3 Deletion in Adulthood Distinctly Impacts Microbiome Diversity
The analysis of gut microbiome b-diversity, a measure of diversity differences between genotypes, identified that NHE3 IEC-KO mice have a distinct gut microbiome signature that clusters differently when compared with their age-and sex-matched control counterparts. Pairwise compositional dissimilarity analysis using Bray-Curtis (P = 0.0002), Jaccard (P = 0.0002), and Gower (P = 0.0002) indices each showed significant differences between genotypes ( Figure 1). Further analysis of microbial a-diversity, a measure of variance within a specific genotype, showed that NHE3 IEC-KO and control mice harbor distinct populations of gut microbes. Operational taxonomic units (OTU richness, taxa level, P = 0.009), Chao1 (richness estimator, phylum level, P = 0.007), and Shannon index (reflects species numbers and evenness of species abundance, P = 0.046) all showed a significantly greater microbial diversity in NHE3 IEC-KO compared with control mice (Figure 2).

Differential Abundance of Microbiota Between NHE3 IEC-KO and Control Mice
We systematically analyzed the microbial composition distribution and differential abundance from phyla down to species levels between NHE3 IEC-KO and control mice. Compared with control mice, NHE3 IEC-KO mice showed significant differential distribution across taxonomic levels ( Figure 3). At the phyla level, the most abundant phylum was Bacteroidetes, which showed no significant difference between the genotypes (65 ± 2% vs 59 ± 6%, NS) ( Table 1). The second most abundant phylum, Firmicutes, showed a tendency to be lower in NHE3 IEC-KO mice compared with control mice (21 ± 1% vs 33 ± 6%, P = 0.062). The Firmicutes/Bacteroidetes (F/B) ratio is widely regarded as an indicator of dysbiosis, with a decreased F/B ratio observed in IBD. We found that the F/B ratio in NHE3 IEC-KO mice trended to be lower than in control mice (0.33 ± 0.03 vs 0.74 ± 0.20, P = 0.079). Abundance of the phylum Proteobacteria, commonly considered to be inflammophilic pathobionts, was nearly 2-fold higher in NHE3 IEC-KO mice compared with control mice (11 ± 2% vs 6 ± 1%, P < 0.01) ( Table 1).
Linear discriminant analysis effect size (LEfSe) identified microbiota with the greatest differences in abundance between NHE3 IEC-KO and control mice. The family Bacteroidaceae, genus Bacteroides, and phylum Proteobacteria (LDA >0.5) are the representative taxa with significant expansions; whereas the family Lachnospiraceae, family Prevotellaceae and genus Prevotella (LDA >0.5) are the representative taxa with significant contraction in NHE3 IEC-KO compared with control mice (Figure 4). However, in our LEfSe analysis the LDA scores were low (LDA score <1) because all significant differences were highly correlated. In addition, NHE3 IEC-KO mice showed no significant differences in the distributions of aerobes, facultative anaerobes, or obligate anaerobes compared to control mice ( Figure 5).

DISCUSSION
There is emerging evidence that the ionic milieu in the intestine plays a critical role in inducing gut inflammation and colitis (Prasad and Visweswariah, 2021). Impaired Na + and water transport, which occurs as a result of NHE3 deficiency, may mediate pathogenesis in gastrointestinal disorders such as CSD and IBD. It is becoming increasingly clear that the ionic milieu, alongside changes in luminal pH, can directly affect the composition of the gut microbiome. Several studies have shown that NHE3 deficiency predispose patients and animals to microbial dysbiosis and IBD (Engevik et al., 2013;Larmonier et al., 2013;Janecke et al., 2016;Laubitz et al., 2016;Harrison et al., 2018). We previously developed a novel inducible intestine-specific NHE3 knockout mouse that mimics symptoms of CSD, including alkaline diarrhea, increased luminal Na + concentration, metabolic acidosis, hyponatremia and hyperkalemia (Xue et al., 2020). The aim of the current study was to determine how deletion of intestinal NHE3 in adulthood impacts the gut microbiome. We utilized metagenomic shotgun sequencing to study how the gut microbiome is altered from A B FIGURE 4 | LEfSe analysis of gut microbiota from NHE3 IEC-KO and control mice. Cladograms (A) shows the microbial clades with the greatest differences in abundance in microbiota from NHE3 IEC-KO and control mice. LDA scores (B) of microbial clades differing in abundance between NHE3 IEC-KO and control mice (with LDA score >0.1 and significance of P < 0.05, determined using Kruskal-Wallis test). N = 9/genotype. phyla to species levels with high resolution following inducible intestine-specific NHE3 deletion. Our data demonstrate that functional NHE3 is required for a healthy microbiome, and lack of intestinal NHE3 results in microbial changes that favor inflammation and downstream pathological consequences. Changes to the intestinal microenvironment can lead to selective pressure on microbiota, ultimately leading to certain microbes gaining a growth advantage while others become restricted. This dysbiosis can make the host more susceptible to inflammatory or infectious diseases processes, which can further reshape the gut microbiome. Thus, it has become an area of interest to better understand how normal physiological functions, such as Na + /H + exchange, can alter the microenvironment and therefore change the composition of the gut microbiome. Dysbiosis is typically characterized by a reduced overall microbial diversity (so called a-diversity) (Pickard et al., 2017). Indeed, patients with IBD have been shown to have less complex profiles of microbiota compared to healthy individuals (Ott et al., 2004). Similarly, conventional NHE3 deletion also resulted in reduced microbial diversity (Larmonier et al., 2013;Laubitz et al., 2016). Surprisingly, our study showed that inducible intestine-specific deletion of NHE3 results in higher microbial a-diversity compared with control mice. We speculate that increased diversity doesn't necessarily reflect a healthy microbiome, but rather that the microenvironment has changed in such a way that there is expansion of less dominant pathogenic bacterial species. Importantly, differences in microbial diversity could also relate to animals being housed at difference facilities and/or fed different diets (Parker et al., 2018). In addition, we used an inducible NHE3 IEC-KO mouse model, in which NHE3 was deleted in the intestine during adulthood rather than NHE3 being absent throughout the entire lifespan. Given that colonization of microbiota begins at birth and can be derived from direct maternal transmission (including breast feeding), conventional NHE3 deletion could have a different effect on the microbiome compared with inducible NHE3 deletion in adult animals. Moreover, metagenomic shotgun sequencing provides high resolution at the species level which, when compared with previous studies using 16S rRNA sequencing, could specifically lead to differences in microbial a-diversity. Of note, we found that microbial b-diversity was significantly different in NHE3 IEC-KO compared with control mice, underscoring that the microbiome is distinctly different when NHE3 is deficient compared to healthy control mice.
The gut microbiome is predominantly composed of two bacterial phyla, Bacteroidetes and Firmicutes. NHE3 -/mice have been shown to have higher Bacteroidetes abundance and reduced Firmicutes abundance in both the luminal and mucosaassociated microbiomes compared to wild-type littermates (Engevik et al., 2013;Larmonier et al., 2013;Harrison et al., 2018). Despite not seeing significant differences at the phylum level for the abundance of Bacteroidetes and Firmicutes in our study with inducible intestinal epithelial cell-specific NHE3 deletion, we did find that the Firmicutes : Bacteroidetes (F:B) ratio tended to be lower in NHE3 IEC-KO compared with control mice, which is consistent with previous studies demonstrating that a reduced F:B ratio favors IBD progression (Stojanov et al., 2020).
Even though we did not see differences in Bacteroidetes between genotypes, we did see significant expansion at the family (e.g., Bacteroidaceae, Tannerellaceae, Rikenellaceae), genus (e.g., Bacteroides, Parabacteroides, Alistipes), and species (e.g., B. thetaiotamicron, P. distasonis) levels in NHE3 IEC-KO compared with control mice. Given that NHE3 -/- (Schultheis et al., 1998) and NHE3 IEC-KO (Xue et al., 2020) mice have alkaline diarrhea, these findings are consistent with other studies that indicate Bacteroidetes thrive in conditions with a slightly higher pH (Ilhan et al., 2017). Of note, the Bacteroides member B. thetaiotamicron was also found to be increased in NHE3 -/mice and was also found to be Na + sensitive (Engevik et al., 2013). This suggests that the increased luminal salinity that occurs with NHE3 deficiency may give this microbe a competitive edge. Indeed, B. thetaiotaomicron has been identified as a pathobiont in models of IBD (Bloom et al., 2011;Hansen et al., 2012). The Tannerellaceae family has a relatively new genus Parabacteroides, which now has approximately 10 valid species, including P. distasonis (Ezeji et al., 2021). Recent studies have shown that P. distasonis has ambivalent effects in models of IBD, with reports of both proinflammatory and anti-inflammatory effects (Kverka et al., 2011). We found that P. distasonis was significantly higher in abundance in NHE3 IEC-KO compared with control mice; however, it remains unclear if this is beneficial or harmful. The genus Alistipes has recently emerged as having major implications in diseases such as IBD, cardiovascular disease, and cancer (Parker et al., 2020). In our study, we found that NHE3 IEC-KO mice had enrichments in 5 Alistipes species: A. finegoldii, A. shahii, A. putredinis, A. obesi and A. senegalensis. Interestingly, it has been shown that A. finegoldii attenuates colitis in mice (Dziarski et al., 2016). Although Firmicutes have previously been shown to be contracted in NHE3 -/mice and patients with IBD (Engevik et al., 2013;Larmonier et al., 2013), we did not see significant changes between genotypes at the phylum level. However, we did see a trend for contraction at the class (Clostridia) and order (Clostridiales) levels, and significant contraction at the family (Lachnospiraceae), genus (Roseburia), and species (e.g., C. scindens, R. hominis) levels in NHE3 IEC-KO compared with control mice. The Lachnospiraceae and Ruminococcaceae families are known to be fairly abundant members of the normal gut microbiome; however, have been shown to be decreased in NHE3 -/mice (Laubitz et al., 2016;Harrison et al., 2018). We found that Lachnospiraceae was contracted in NHE3 IEC-KO compared with control mice, but we did not find any differences in Ruminococcaceae. Reduced abundance of Lachnospiraceae correlates with decreased microbial production of short chain fatty acids (particularly butyrate), which serve as the primary fuel source for colonocytes and also exert immunomodulatory roles in the gut mucosa. Of note, Lachnospiraceae and Ruminococcaceae have been shown to be pH sensitive; a shift in pH from 5.5 to 6.5 dramatically stunted both microbial abundance and butyrate production (Walker et al., 2005). One of the most notable family members, Roseburia, resides in the mucus layer of the intestine (Van den Abbeele et al., 2013), making this microbe particularly vulnerable to changes in luminal pH as a result of dysfunctional Na + /H + exchange. Our study and others have shown that NHE3 deficiency results in a dramatic reduction in the abundance of Roseburia (Harrison et al., 2018), which has also been seen in patients with IBD (Sellon et al., 1998;Chassaing et al., 2012). We observed more than a 3-fold reduction in Roseburia in NHE3 IEC-KO compared with control mice, which was predominantly attributed to the reduction of the species R. hominis (which accounts for >95% abundance of Roseburia). Of note, a significant reduction in R. hominis in the intestine of ulcerative colitis patients was recently described (Patterson et al., 2017). Our data also revealed that other members within the family Lachnospiraceae are contracted in NHE3 IEC-KO mice, e.g., at the genus level, Robinsoniella was nearly depleted, 3-fold reduction in Kineothrix, nearly 2-fold reduction in Lachnoclostridium and Butyrivibrio (although did not reach statistical significance); and at the species level, e.g. C. scindens (protective against Clostridium difficile infection (Mills et al., 2018) and gut inflammation (Burgess et al., 2020)), L. phytofermentans, K. alysoides, R. peoriensis, E. massiliensis, and C. polysaccharolyticum.
The subdominant phylum Proteobacteria includes a wide variety of pathogens, and its expansion is a common risk factor for many diseases including IBD (Berry and Reinisch, 2013;Rizzatti et al., 2017). Our study and those in NHE3 -/mice found a significant expansion in Proteobacteria (Engevik et al., 2013;Larmonier et al., 2013), which is consistent with our previous report that~50% of NHE3 IEC-KO mice develop mild to moderate colitis three weeks after induction of intestinal NHE3 deletion (Xue et al., 2020). Helicobacter species have been shown to be endemic in mouse colonies and colonization with H. ganmani has been associated with increased release of proinflammatory cytokines in IL-10 deficient mice (Alvarado et al., 2015). We found that this species was absent in control mice, but present in NHE3 IEC-KO mice, suggesting it may play a role in the pathogenesis of intestinal disease.
Our study has several limitations, which warrant further studies. First, the microbiome composition can be drastically influenced by animal facility (Parker et al., 2018), so we cannot exclude facility-specific effects in our mouse colony. Replicating these microbiome experiments in animal facilities at other institutions would allow for confirmation that changes in the microbiome were specific to intestinal NHE3 deletion. Second, previous studies using microbial b-diversity analysis showed distinct signatures between mucosa-associated and fecal microbiota (Lo Presti et al., 2019). However, our study only evaluated changes in fecal microbiota. Future studies will further delineate the effect of loss of intestinal NHE3 on mucosaassociated microbiota. Third, only male mice were used in this study; whether or not there are sex differences in NHE3 deficiency-reshaped gut microbiota needs to be determined. Lastly, in mice, NHE3 is differentially expressed in distinct segments of small intestine (jejunum > duodenum > ileum) and colon (Fenton et al., 2017;Wang et al., 2019). Given that distinct segments of intestine harbor different microbial compositions, it remains to be determined if NHE3 modulates microbiota in a segment-specific way.
Taken together, our data provide high-resolution microbial composition analysis to dissect the differential abundance of microbes between NHE3 IEC-KO and control mice, elucidating the effects of intestinal NHE3 on gut microbiota. Overall, intestinespecific NHE3 deletion in adulthood creates an intestinal microenvironment in which certain inflammophilic species gain a competitive advantage over other anti-inflammatory species. Interestingly, microbiome analysis in patients with IBD demonstrated a reduction of obligate anaerobes and a sharp expansion of facultative anaerobes (Berry and Reinisch, 2013;Henson and Phalak, 2017). However, our data showed no change in aerobic/anaerobic microbial composition between NHE3 IEC-KO and control mice. The reason for this is not fully understood and warrants further analysis. Even though the causative relationship between NHE3 deficiency, dysbiosis, and IBD has not been completely understood to date, we have provided additional evidence that changes in the ionic milieu (Na + and pH) as a result of intestine-specific deletion of NHE3 provides a competitive advantage for certain microbiota (e.g., Bacteriodetes and Proteobacteria) and a disadvantage for others (e.g., Firmicutes). This may predispose and/or promote pathogenesis of intestinal diseases such as IBD. In turn, the pathological state of IBD can further reshape the gut microbiota by influencing expansion of certain pathobionts that consequently inhibit NHE3 activity (Hayashi et al., 2004;Subramanya et al., 2007;Hodges et al., 2008), which reciprocally aggravates dysbiosis and IBD.

DATA AVAILABILITY STATEMENT
The data presented in the study are deposited in the Figshare repository (https://figshare.com/articles/dataset/Effect_of_ intestinal_NHE3_deletion_on_microbiome/19358456).

ETHICS STATEMENT
The animal study was reviewed and approved by University of South Florida Institutional Animal Care and Use Committee.

AUTHOR CONTRIBUTIONS
TR, JDR and JX conceived and designed the work. TR, JDR, JX, LT and JW contributed to the acquisition, analysis, or interpretation of data for the work. TR, JDR and JX drafted the work; TR, JDR, JX, LT and JW revised it critically for important intellectual content. TR, JDR, JX, LT and JW approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases grant 1R01DK110621 (to Dr. Rieg), VA Merit Review Award IBX004968A (to Dr. Rieg) and an American Heart Association Transformational Research Award 19TPA34850116 (to Dr. Rieg). Financial support for this work was also provided by the NIDDK Diabetic Complications Consortium (RRID : SCR_001415, www.diacomp.org), grants DK076169 and DK115255 (to Dr. Rieg). Dr. Thomas by an American Heart Association postdoctoral fellowship (828731).


Molecular Detection of Parvovirus in Captive Siberian Tigers and Lions in Northeastern China From 2019 to 2021

The fact that wild felines are carriers of pernicious infectious viruses should be a major concern due to the potential cross-species transmission between the felines and human or domestic animals. However, studies on the virus in the captive wild felines, especially in tigers, are thin on the ground. In this study, we screened four infectious viruses, namely, feline parvovirus (FPV), feline coronavirus (FCoV), canine distemper virus (CDV), and influenza A virus (IAV), in the blood samples of 285 captive Siberian tigers (Panthera tigris altaica) and in the spleen samples of two deceased lions (Panthera leo), which were collected from 2019 to 2021 in three Siberian Tiger Parks from the northeast of China. Nucleic acids isolated from the blood samples collected from tigers and the spleen samples collected from two deceased lions were positive for FPV by PCR, and the positive rate was 4.6% (13/285) in tigers. Furthermore, the VP2 gene of FPV was amplified by nested PCR, and the sequences of the VP2 gene from these six FPV positive strains shared 98.3–99.9% homology with the reference. The key amino acid sites of VP2 protein were consistent with that of FPV reference strains. Phylogenetic analysis based on the VP2 gene showed that in this study, FPV-positive strains were grouped within the FPV clade and closely related to the Asian strains clade. The results of this study showed that FPV circulated in the captive Siberian tigers and lions in northeastern China and provided valuable information for the study of FPV epidemiology in wild felines. Therefore, we suggest that regular antibody monitoring and booster immunization for tigers should be performed.

INTRODUCTION
The captive breeding mode of feline species increases the possibility of cross-species transmission between the species and humans (Foley et al., 2013;Carver et al., 2016), which can potentially impact the survival of wildlife populations. For example, feline parvovirus (FPV), feline coronavirus (FCoV), canine distemper virus (CDV), and influenza A virus (IAV) are widespread in wild felines (Duarte et al., 2012;Candela et al., 2019;McCauley et al., 2021). FPV is a pathogen that causes panleukopenia in felines, the symptoms of which include high fever, vomiting, severe leukopenia, and enteritis (Stuetzer and Hartmann, 2014). It is closely related to blue fox parvovirus (BFPV), mink enteritis virus (MEV), raccoon dog parvovirus (RDPV), and canine parvovirus (CPV) in gene, structure, and antigen. FPV affects cats of all ages and has a wide range of hosts, affecting all members of the family felines (Allison et al., 2013). Goodrich et al. (2012) found that the antibodypositive rate of FPV among Siberian tigers in eastern Russia was 68% (n = 44). In addition, FPV exposure or infection has been reported in captive Siberian tigers in Guangzhou and Jilin, China (Wang et al., 2017(Wang et al., , 2019. CDV is a multi-host pathogen with various clinical outcomes of interspecies and cross-species transmission. PCR and antibody detection of CDV infection have been reported in wolves, tigers, lions, ferrets, minks, red foxes, giant pandas, and genets (Feng et al., 2016;Loots et al., 2017;Weckworth et al., 2020). FCoV infects domestic cats and wild felines; although FCoV tends to cause mild or inconspicuous illness, a small percentage of felines die from the fatal systemic disease feline infectious peritonitis (FIP) . IAV infects a variety of vertebrates, including birds, humans, and other mammals (Driskell et al., 2013). Although felines were previously considered to be resistant to IAV infection, the H5N1 subtype virus is wreaking havoc in many countries and has caused fatal infection in domestic cats, leopards, and tigers (Driskell et al., 2013;He et al., 2015).
To investigate the prevalence of FPV, FCoV, CDV, and IAV in Siberian tigers and lions in northeastern China, and to enrich the epidemiological data of tigers, FPV, FCoV, CDV, and IAV were detected by PCR, and the key amino acid site mutation and phylogenetic analysis of VP2 gene were performed.

Samples Collection
A total of 285 blood samples of captive Siberian tigers were randomly collected from the Siberian Tiger Parks in three cities: Harbin (n = 190, 200-300 tiger/km 2 ), Hailin (n = 55, 1,300-1,400 tiger/km 2 ), and Shenyang (n = 40, 600-700 tiger/km 2 ) in northeastern China (41-46 • N, 122-130 • E), from January 2019 to March 2021. The samples used in this study are the same as our previous studies, but after the detection of four viruses [i.e., feline herpesvirus 1 (FHV-1), feline calicivirus (FCV), feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV)] and persistent organic pollutants, some samples have been run out, so the sample size in this study is 285, smaller than the sample size of 324 last time (Liu et al., 2021a,b;Huang et al., 2022). All tigers fasted for 1 day before the blood sample collection, and the tigers were anesthetized with a dart containing 10 mg/kg of ketamine (Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd., China). In addition, spleen samples were collected from two dead lions in the Siberian Tiger Park of Harbin, and all samples were stored at −80 • C for later use. All tigers were dewormed once a year (Hailechong, Zhejiang Hai Zheng Animal Health Products Co., Ltd., China) and were vaccinated twice. The first immunization was taken after birth and the second was 1 month later (Fel-O-Vax PCT, Zoetis, United States). No animals were killed for investigative purposes, and all samples were collected by the veterinarian of Siberian Tiger Park, which was approved by the Northeast Forestry University Institutional Review Board of Ethics and Administration of Experimental Animals (NEFU-IRBEA) (Liu et al., 2022).

Virus Detection by PCR
The viral nucleotide extraction from the blood of tigers and the spleen of deceased lions was performed by the Baypure TM Universal Magnetic Bead Viral DNA/RNA Rapid Extraction Kit (BayBio, Guangzhou, China), and reverse transcription was performed to get the cDNA of FCoV. The extracted DNA, RNA, and cDNA were stored at −80 • C.
Feline parvovirus, CDV, and IAV were detected by real-time PCR, and FCoV was detected by nested PCR, as previously described (Herrewegh et al., 1995;Meli et al., 2009;Streck et al., 2013); the PCR primers are shown in Supplementary Table 1. VP2 gene of FPV was amplified by nested PCR (Mochizuki et al., 1996;Steinel et al., 2000;Battilani et al., 2001;Sacristán et al., 2021). FPV and FCoV positive controls were cat samples with the corresponding disease, which were collected in pet hospitals. CDV positive control is the CDV live attenuated vaccine. Birds infected with IAV are used as positive controls for testing IAV. To avoid contamination of samples, RNase-free water was used as a negative control for each assay.

Phylogenetic Analysis
Multiple sequence alignments were conducted using the MegAlign program of DNAStar 11 software and were translated into putative amino acid sequences using the MEGA 7.0 software. 1 Reference sequences were selected based on the VP2 gene of panleukopenia viruses from the GenBank database. 2 The phylogenetic tree was established in MEGA 7.0 software. The neighbor-joining method with 1,000 bootstrap replications was used with the Kimura 2-parameter model to analyze the relationship between the partial VP2 sequences in this study and other reference sequences.

RESULTS
In this study, only parvovirus infection was detected in the tigers (13/285; 4.6%) and two deceased lions (2/2; 100%) from Harbin, but no FCoV-, CDV-, and IAV-positive samples were found. FPVpositive samples were detected only in the Harbin area, and there is no much difference in the positive rate between male (4.9%) and female (4.3%) animals. However, the positive rate of young tigers (5.5%) was higher than that of adult tigers (3.8%) ( Table 1).
strains shared 99.3-99.9% homology with FPV isolates and 98.3-98.9% homology with CPV isolates. The amino acids at 80, 93, 103, 323, 564, and 568 of the VP2 gene were the key sites to distinguish FPV and CPV antigen types (Yi et al., 2021). To further analyze the mutation of positive strains, the amino acid sequences of VP2 are compared in Table 2. The result showed that the amino acid mutation sites of six VP2 proteins were identical with FPV reference strains. However, due to the quality of samples or primers, the full-length sequence of the VP2 gene was not successfully amplified.
A phylogenetic tree about the VP2 nucleotide sequences was constructed based on the positive strain sequences in this study and 37 reference sequences from Europe, America, and Asia (Supplementary Table 2). The phylogenetic analysis indicated that all the parvoviruses can be grouped into two large branches: the FPV branch and the CPV branch (Figure 1). FPV branch comprised TPV (The feline parvovirus isolated from tigers was named TPV), FPV, MEV, and BFPV. In the FPV branch, 26 strains from China, Korea, Japan, India, United States, and Italy formed a cluster, and four sequences from Portugal (EF418568, EF418569, EU221279, JF422105) formed a different clade. All positive strains in this study belong to the FPV strains branch and were closely related to the Asia-isolated strains, but they were distant from the Portugal-isolated strains. The Asian isolates were mainly divided into two differentiated clades, and HB1811 strain was closely related to a tiger strain isolated in China (MN908257), but was not in the same clade as other strains in this study.

DISCUSSION
Wild felines are susceptible to virus infection due to crossspecies transmission potentially by domestic cats or dogs (Canuti et al., 2020). Early detection of viral shedding in asymptomatic or symptomatic felines may help inspect the health status of wild felines and are critical for early treatment and prevention plans for animal diseases. In this study, the positive rate of FPV in captive Siberian tigers was 4.6%, higher than previous results of FPV in Malaysian tigers was 2.7% (Nur-Farahiyah et al., 2021), and the positive rate of the virus in Italian red foxes was 2.8% (Ndiana et al., 2021). The tigers in this study are captive, and such a large sample size may be the main reason for this result. Furthermore, there are no coinfection among FPV and the other four viruses (i.e., FHV-1, FCV, FIV, and FeLV) . FIGURE 1 | Phylogenetic tree of six FPV partial VP2 genes compared with other references of parvovirus strains obtained from GenBank. The tree was generated using the neighbor-joining method in MEGA 7.0 using the Kimura 2-parameter model with 1,000 bootstrap values. Bootstrap values ≥ 50 at the nodes of the tree. " " represents the strains in this study; "L" represents vaccine strains; EU498680 and EU498681 are the FPV vaccine strain; MW650832 is the CPV vaccine strain. The length of the six VP2 genes is as follows: HB1807: 973 bp; HB1752: 790 bp; HB1765: 984 bp; HB1819 bp: 973; HB1811: 1683 bp; LWL: 825 bp.
Real-time PCR detection of FPV in the two dead deceased lions was also positive. The two deceased lions showed depressed spirit, lost appetite with shame and tears, dry nose, dark red vomit, bloody stool, dehydration, a body temperature of 39.5 • C, and leukocyte elevation during the paroxysm of symptoms. The pathological changes after autopsy mainly included gastric bleeding, bile reflux, severe intestinal bleeding, hemorrhagic enteritis, mesenteric lymphadenitis enlargement, and hemorrhage, so we believe that FPV infection may be the main reason for the death of lions. These results suggest that FPV has a certain prevalence in captive Siberian tigers and lions in northeastern China. However, we have not found FCoV, CDV, and IAV in any of these samples, which showed that there was no epidemic of FCoV, CDV, and IAV in the three Siberian Tiger parks.
Feline parvovirus was first discovered in 1928, and canine parvovirus type 2 (CPV-2) was discovered in 1978. CPV-2 originated from FPV or an FPV-like parvovirus in carnivores. In 1979, CPV-2a mutated away from the original type 2 in five amino acids (aa) in VP2 that alter relevant biological properties between the strains. Another antigenic variant, CPV-2b, with a single additional substitution (Asn426Asp) in the VP2 protein appeared in 1984 and spread globally (Parrish et al., 1985;Parrish, 1991). Since then, many new CPV variants throughout the world have been detected, such as the "new CPV-2a" and "new CPV-2b" with an additional 297-Ala mutation, CPV-2c with an additional 300-Asp mutation, and 426-Glu mutation (Buonavoglia et al., 2001;Decaro and Buonavoglia, 2012). In this study, due to the sample's quality and DNA concentration reasons, such as samples stored for a long time, repeated freezing, and thawing, we only got six positive sample sequences, and we failed to amplify the entire VP2 gene. The main amino acid mutation sites obtained in this study were identical to FPV ( Table 2).
The viruses are named after the host from which they are isolated, such as FPV, TPV, MEV, BFPV, and CPV. In this study, five TPV strains from tigers and one FPV (LWL) of lion strain are identified. We selected 37 parvovirus VP2 genes from GenBank for phylogenetic analysis, and all six positive strains in this study belong to the Asia-isolated FPV strains but had a distant relationship to the Portugal-isolated FPV strains. These results suggest that the spreading and prevalence of FPV are related to the geographical location of the animals. The relationship between positive strains and commercial FPV vaccine strains (i.e., EU498680 and EU498681) is also distant. Inactivated vaccines (Fel-O-Vax PCT, Zoetis, United States), which combine FCV, FHV, and FPV strains, are used to prevent disease in tigers. However, tigers and lions are still infected with FPV; the reason for this result may be the low antibody level and short antibody duration of the FPV-inactivated vaccine (Bergmann et al., 2019). In the FPV clade, both the tigers and lion strains in this study belong to the same evolutionary clade, and in this clade, the tiger and lion strains are closely related to the reference strains from tigers and cats. The Siberian tigers in these three regions were all fenced with wire mesh that prevents the tigers from escaping but does not restrict domestic cat movement, and the staffs often see the stray cats pass in and out of the Siberian Tiger Park freely, which allows for the possibility of contact, and therefore transmission, between the stray cats and tigers or lions (Liu et al., 2022). Considering the widespread infection of FPV (Franzo et al., 2017) and the captive environment, FPV might be transmitted from the stray cats to tigers and lions. Previous studies have confirmed the possibility of cross-species transmission of FPV in domestic cats and other felines (Wang et al., 2017;Shetty et al., 2019). Furthermore, human participation in the landscape is an important factor affecting the spread of pathogens across species (Carver et al., 2016).
Combined with our previous study , FHV-1, FCV, FIV, and FPV have certain prevalence rates in the Siberian Tiger Park of northeastern China, and interspecies and cross-species transmission may happen. This study not only provides new data on the prevalence of FPV among tigers and lions but also contributes to improving the management of captive felines.

DATA AVAILABILITY STATEMENT
The data presented in the study are deposited in the NCBI repository, accession numbers OM810192, OM810193, OM810194, OM810195, and OM810196.


Genetic Variation in Rhipicephalus sanguineus s.l. Ticks across Arizona

Rhipicephalus sanguineus s.l. (Latreille, 1806), the brown dog tick, is the most widely distributed tick species in the world. The two dominant lineages, a temperate group and a tropical group, are recognized as important disease vectors for both dogs and humans. The temperate and tropical lineages overlap in range in some regions of the world, including the southwestern United States, where recent outbreaks of Rocky Mountain spotted fever are linked to R. sanguineus s.l. While it is unclear to what extent they may differ in their capacity to transmit pathogens, finer-scale resolution of temperate and tropical lineage distribution may provide insight into the ecology of these two tick groups and the epidemiology of R. sanguineus s.l.-vectored diseases. Using diagnostic polymerase chain reaction assays, we examined the geospatial trends in R. sanguineus s.l. lineages throughout Arizona. We found the temperate and tropical lineages were well delineated, with some overlap in the eastern part of the state. In one county, tropical and temperate ticks were collected on the same dog host, demonstrating that the two lineages are living in sympatry in some instances and may co-feed on the same host.

Introduction
Ticks (Acari: Ixodida: Ixodidae) are important vectors of many human and animal diseases, especially within North America. Among the most significant of these diseases is Rocky Mountain spotted fever (RMSF), a bacterial infection caused by Rickettsia rickettsii, which kills more people within North America than any other tickborne disease [1]. In 2018, the Centers for Disease Control and Prevention (CDC) reported 5544 cases of spotted fever rickettsioses, including RMSF reported in the United States, a twelve-fold increase since the year 2000 [2,3]. Most of the reported cases occurred in southeastern states, including North Carolina, South Carolina, Tennessee, Oklahoma, and Arkansas, where the principal vector is Dermacentor variabilis, and Montana, Idaho, and Wyoming, where the principal vector is D. andersoni.
Rhipicephalus sanguineus sensu lato-vectored RMSF has been recognized in Mexico since the 1930s, though data on estimated incidence is limited [4]. Since the first case of locally transmitted RMSF was identified in Arizona in 2003, there have been nearly 500 cases and 28 deaths on six tribal reservations, with a case fatality rate almost 15 times higher than the national rate [5][6][7]. While the disease is easily treated with antibiotics, it can be fatal if left untreated. Delays in seeking health care and the resemblance of symptoms to many other diseases further exacerbate the mortality rate of RMSF in these communities [6].
Dogs are the preferred hosts of R. sanguineus s.l., but the ticks will feed on humans as incidental hosts and can transmit R. rickettsii at all life stages. The close relationship between domestic dogs and humans enables infestations of R. sanguineus s.l. in and around human homes and allows the tick to complete its entire life cycle indoors [8]. Throughout Arizona and northern Mexico, the warm climate and peridomestic habitat provide suitable conditions for the ticks to remain active year-round [9]. Affected communities report a high incidence of free-roaming dogs, which can spread infected ticks among households [5,6,8]. The association of R. sanguineus s.l. with RMSF may have important implications for the epidemiology and expansion of this disease to other parts of the world and should be explored in greater detail.
R. sanguineus s.l. refers to a complex of 11-17 related species or subspecies with a worldwide distribution [9][10][11]. The existence of more than one species within this taxon was first recognized by Szabo et al. in 2005 [12]. Historically, a non-specific morphological description and loss of the holotype have led systematists to disagree on how to classify the species complex [13,14]. Morphological distinctions among the two main recognized R. sanguineus s.l. lineages (a tropical group and a temperate group) and other Rhipicephalus species (specifically R. turanicus) are minute, further complicating the taxonomic status [15]. In 2018, Nava et al. [13] designated a neotype and renewed morphological and molecular descriptions of the species and identified the previously designated temperate lineage as R. sanguineus sensu stricto. The aforementioned tropical lineage within the Americas is currently under revision, with specimens from Australia and southern Asia declared a separate species, R. linnaei, by Šlapeta et al. (2021) [16]. To date, however, there has been no revision that includes the tropical lineage as described in North America. While the redesignations are acknowledged as valid, given the current transitionary state of the taxonomical status of the R. sanguineus species complex, ticks will be referred to in terms of temperate and tropical lineage throughout this text.
While it has been suggested that the two main R. sanguineus s.l. lineages occupy distinct ecological niches [17][18][19][20][21][22], various studies have found both the temperate and tropical groups living in sympatry in central eastern Arizona [17,22,23]. A recent study by Sanchez-Montes et al. (2021) found both lineages present in northern Mexico, with the tropical lineage widespread throughout the country and the temperate lineage present in Chihuahua and Sonora states [21]. Both lineages may harbor Rickettsia spp. bacteria, but to date, only the tropical lineage has been associated with the distribution of RMSF in Mexico [18,19,24]. It is unclear whether R. sanguineus s.l. lineages differ in their capacity to transmit R. rickettsii. However, various experimental transmission studies suggest that the temperate and tropical lineages do differ in their ability to transmit a variety of other pathogens, including Ehrlichia canis, Hepatozooan canis, and Anaplasma platys [25][26][27][28][29][30]. Understanding the distribution of the two R. sanguineus s.l. species complex ticks within Arizona may, therefore, provide insight into the epidemiological distribution of R. sanguineus s.l.-vectored diseases across the state in the future.
Much work has been done to investigate the molecular dissimilarities between divergent populations within the R. sanguineus species complex. Given the higher rate of base substitution relative to most nuclear genes, mitochondrial DNA sequences are useful phylogenetic markers for clades with relatively recent divergence [31]. A systematic review of molecular markers for acarological phylogenetics by Cruickshank (2002) references the 12S rRNA gene as being historically useful to investigate intraspecific relationships within tick species [32]. The mitochondrial 12S rRNA gene is frequently targeted for molecular identification of R. sanguineus s.l. taxa [12,17,23,33], often in conjunction with 16S rRNA [14,16,20,22,23,[34][35][36][37][38]. Attempts to verify mitochondrial findings with nuclear genes have thus far proven unsatisfactory. Abdullah et al. (2016) compared 12S, 16S, CO1, ITS2, and 18S sequences of R. sanguineus s.l. ticks with those of Hyalomma dromedarii [39]. The authors determined that while the mitochondrial genes were suitable for phylogenetic analyses, neither 18S nor ITS2 were suitable for such purpose in tick taxonomy. Indeed, Nava et al. (2018) and Šlapeta et al. (2021) relied upon mitochondrial sequences to delineate species within the R. sanguineus complex after finding that nuclear markers, specifically ITS2, were insufficiently variable and therefore not informative or robust enough to provide resolution on the necessary scale [13,16].
The aim of this work was to determine the spatial distribution of R. sanguineus s.l. lineages within Arizona. A diagnostic polymerase chain reaction (PCR) protocol was developed using lineage-specific primers to differentiate between the temperate and tropical lineages of R. sanguineus s.l. based on the mitochondrial 12S rRNA gene with Sanger sequencing confirmation on a subset of samples. Given the current understanding of the global distribution of R. sanguineus s.l. ticks, it was hypothesized that the temperate lineage will be more abundant, particularly in northern latitudes and higher elevations across Arizona. However, the presence of the tropical lineage in the six regions of the state where historic and ongoing transmission of RMSF is occurring was predicted.

Sample Collection
In 2018 and 2019, R. sanguineus s.l. ticks of all life stages and engorgement levels were solicited throughout the year as a convenience sample from animal shelters, tribal vector control agencies, and University of Arizona Cooperative Extension offices across Arizona. Most ticks included in this study were collected from dogs at animal shelters (Maricopa, Pima, and Pinal Counties) or in collaboration with local stakeholders during vector control activities, such as RMSF prevention campaigns and mobile rabies clinics. The majority of ticks were collected during peak tick activity, during the months of March through August. Ticks were identified to species complex using standard taxonomic keys [10], and the life stage and sex determined.

DNA Isolation and Sequencing
Ticks were placed in a 1.5 mL tube, submerged in liquid nitrogen, homogenized with a sterile pestle, and treated with 20 mg/mL lysozyme solution (Thermo Fisher Scientific, Waltham, MA, USA). DNA was isolated using the Qiagen QiaAmp DNA Mini Kit, with an AE buffer elution volume of 50 µL.

Primer Design, PCR, and Sequencing
Primers were designed using the NCBI Primer Design Tool and sequences published by René-Martellet et al. [23] (Accession numbers KU255848-56; Supplementary Table S1). Lineage-specific primers for R. sanguineus s.l. were based on differences of two bp at the binding sites for both the forward and reverse primers, and amplified a sequence of the mitochondrial 12S rRNA approximately 350-360 base pairs in length ( Table 1). The "standard 12S" primer was designed to be inclusive of the lineage-specific primer binding sites and amplified a 400 bp region of the 12S rRNA gene that contains significant nucleotide differences between the lineages. Positive controls for each lineage were provided by the Centers for Disease Control and Prevention Rickettsial Zoonoses Branch. Each sample was prepared using both temperate and tropical primer pairs within the same thermocycler session (primer sequences and parameters described in Table 1). A 4 µL volume of each reaction was visualized by 1% agarose-gel electrophoresis stained with SYBR Safe (Thermo Fisher Scientific, Waltham, MA, USA). PCR product from 5 samples was cleaned and sequencing confirmed that the lineage-specific assay was correctly amplifying the targeted fragment of 12S rRNA for both lineages.
Results were further verified on a subset of specimens from each county via Sanger sequencing using the "standard 12S" primer set. Additional sequencing was performed on a~425 bp fragment of the 16S rRNA gene to verify results for a subset of specimens [13,36]. 12S rRNA sequencing was performed at the University of Arizona Genetics Core. 16S rRNA sequencing was performed at the University of Texas Arlington. Sequences were manually corrected by visual analysis of the electropherogram using Geneious Prime.

Phylogenetic and Geospatial Analyses
Phylogenies were constructed for both the 12S rRNA and 16S rRNA sequences. Sequences used in this analysis are available on the NCBI Sequence Read Archive. Sequences were aligned using MAFFT [40] in Geneious Prime with the default settings. Maximumlikelihood phylogenetic trees were inferred using RAxML version 8.2.10 [41] employing the GTR + Gamma model of nucleotide substitution. Node support values were calculated using rapid bootstrapping halted at 500 replications. The tree was rooted with the outgroup R. turanicus.
Data on lineage was mapped to county level using ArcGIS Pro (ESRI). Presence/absence data were recorded for the two lineages at each sampling site, and elevation and latitude data were inferred based on nearest city/town utilizing the ArcGIS Data and Maps global databases.

Results
A total of 306 R. sanguineus s.l. ticks were included in this study from eleven counties in Arizona (Apache, Cochise, Gila, Graham, La Paz, Maricopa, Navajo, Pima, Pinal, Yavapai, and Yuma, Figure 1). The majority (84.3%) of samples were adults. The bias towards adults likely reflects the difficulty in finding nymphal and larval ticks on hosts, given their size. While most Arizona counties were sampled, many more ticks were sampled in the most populous county, Maricopa ( Table 2). Month of collection was recorded for 77.5% of ticks collected. While ticks were collected in every month, with the exceptions of February, October, and December, temperate ticks were only collected from March through July, whereas tropical ticks were collected between August and July. This disparity may be an artifact of the convenience sampling method, as many collaborating stakeholders only hold tick prevention events during peak tick season.

PCR and Sequencing
As expected, positive controls for the temperate and tropical lineages amplify with the appropriate lineage-specific primer but not the other (Figure 2). Twenty-eight percent (n = 86) of the samples amplified using the temperate primers, whereas 72% (n = 220) amplified using the tropical primers. Occasionally, there was low amplification with the second lineage-specific primer set, resulting in a faint band in the second lane for a sample (n = 39, 14%). This may have been due to non-specific binding, contamination, or an indication of hybridization and paternal leakage of mitochondrial DNA [42]. However, we do not have the data to resolve this. The samples amplified similarly in subsequent repetitions of the assay and we assigned all samples in question to their dominant PCR product. Sanger sequencing was performed on the 12S PCR amplicon of 117 (38.2%) samples from eleven counties in Arizona and compared against lineage-specific diagnostic PCR results. Four samples (two larvae and two nymphs) were removed from the analysis due to low DNA concentrations and subsequent low-quality Sanger sequencing reads. These samples amplified using the lineage-specific primers but could not be verified with sequencing. For the remaining samples, there was 100% consensus between amplification with lineage-specific primers and sequencing-based identification.

Geospatial Analysis
The distributions of the two R. sanguineus s.l. lineages were surprisingly well-delineated across the state. However, both lineages were found in two counties-Gila and Cochise-indicating some overlap in their ranges. Figure 1 depicts the distribution of temperate and tropical ticks and number of dogs sampled across the eleven counties sampled. On two occasions in Gila County, both tropical and temperate ticks were collected on the same dog host, demonstrating that the two lineages are living in sympatry and co-feed on hosts.
In addition to the sites where both lineages were collected, the temperate and tropical ranges overlapped in latitude. Specifically, both temperate and tropical R. sanguineus s.l. ticks were collected between 31.5 • and 34 • latitude ( Figure 3). Interestingly, the temperate lineage was present in higher latitudes at higher elevations, leading the combination of these two variables to near perfectly predict the presence of that lineage (Table 3, Figure 3). However, because latitude and elevation were highly correlated in this dataset, it was not possible to tease apart the effect of the variables within levels of each other without more thorough sampling. Table 3. Distribution of R. sanguineus s.l. lineages across elevation (in feet) in Arizona.

Phylogenetic Analysis
Samples identified as temperate and tropical using diagnostic PCR clustered separately on the 12S rRNA with high bootstrap value (100%) (Figure 4a). On the 16S rRNA phylogeny, all sequences from the tropical lineage formed a distinct clade (Figure 4b). Temperate sequences were basal to the tropical clade but did not form their own distinct clade.

Discussion
The spatial analysis of R. sanguineus s.l. lineage demonstrates that the two lineages overlap in range in Arizona. While this study utilized a convenience sampling method and therefore the lack of a lineage in a county may not be representative of an actual absence, the results support the published literature on the distribution of the temperate lineage [22,34]. We found temperate ticks in the present study in lower latitudes at higher elevations. This matches findings from Sanchez-Montes et al. [21], where the presence of the temperate lineage in northern Mexico was at or above 4000 feet elevation. This also corroborates the interpretation of Zemtsova et al. [22] that the two lineages are climatically restricted within certain temperature ranges. Labruna et al. [43] further elucidated this geospatial relationship by identifying that the temperate lineage has a pronounced diapause in winter months, suggesting adaptation to colder regions, whereas the tropical lineage, which inhabits warmer regions, lacks a distinct diapause.
It is well established that vector-borne diseases, including those transmitted by ticks, increase in incidence as the global climate warms [44]. The results presented here contrasted expectations that the temperate lineage would be dominant in this region based on global patterns of distribution [22,43]. The distribution of the tropical lineage across Arizona may represent an ecological range expansion event northward [19], and suggests potential implications of changing global conditions on a local disease vector. A better understanding of the specific range-limiting factors of the two R. sanguineus s.l. lineages is necessary to assess future risks of tickborne diseases vectored by this tick species complex in a warming world.
The diagnostic PCR protocol described here quickly and reliably distinguishes between temperate and tropical lineages of R. sanguineus s.l. ticks without the need for additional sequencing. While not sufficient for phylogenetic analyses, it may prove useful for researchers seeking to identify R. sanguineus s.l. ticks to lineage without additional investments in time and resources. This protocol may be especially useful in regions where both lineages have been found to screen large numbers of ticks and define the distribution of R. sanguineus s.l. with high resolution.
Due to lingering taxonomic questions, several hybridization experiments have been conducted using geographically isolated, allopatric R. sanguineus s.l. populations [33,45]. A cross-breeding experiment of two temperate R. sanguineus s.l. populations showed reproductive success, and also indicated the occurrence of paternal inheritance of mitochondria and mitochondrial heteroplasmy [45]. Hybridization between tropical and temperate R. sanguineus s.l. lineages has also been observed experimentally, although most of the hybrid progeny did not produce viable offspring [33,45]. However, these experiments involved geographically distant tick populations rather than the sympatric tropical and temperate ticks observed in this study. While unresolved in this study, the faint double bands using the lineage-specific primer sets may be an indication of hybridization between the two lineages with paternal mitochondrial DNA leakage. Future researchers may consider investigating the hybridization success of sympatric populations of the temperate and tropical R. sanguineus s.l. lineages, and whether an R. sanguineus s.l. tick may behaviorally present as one lineage while mitochondrial DNA determines it to be the other. Identification of a nuclear genetic marker with sufficient resolution at this taxonomic level would aid in this line of research.
While R. sanguineus s.l. is the primary vector of RMSF in Arizona, much remains to be investigated. The temperate and tropical R. sanguineus s.l. lineages are present in Arizona and living in sympatry in some areas. A systematic assessment of the presence/absence of each lineage across the state using non-convenience sampling strategies may highlight additional areas of sympatry. Future research on this topic should focus on whether the two lineages differ in their vectorial capacity for RMSF. Additionally, a more thorough assessment of climatic variables relevant to range limitation and overlap may be helpful to better understand the distribution of the temperate and tropical R. sanguineus s.l. ticks in this region of the United States. Finally, researchers should seek evidence of hybridization among sympatric temperate and tropical R. sanguineus s.l. lineages to further understand the implications of this on the epidemiology of RMSF and other tickborne diseases.

Conclusions
This research explores the distribution of the two recognized lineages of R. sanguineus s.l., temperate and tropical, at a fine scale in Arizona. We were able to sample ticks from eleven of fifteen counties in the state, including regions of the state affected by R. sanguineus s.l.-transmitted RMSF. The data presented here demonstrates a clear pattern of R. sanguineus s.l. lineage distribution across both latitude and elevation within Arizona. We also verify the presence of both lineages in two counties in the eastern part of the state. Our findings are somewhat limited by convenience sampling and limited access to dog hosts in some counties, as it is possible that our dog sample sizes in some areas were insufficient to obtain a representative sample of lineage.