Escherichia coli causes a bacterial illness that frequently affects cats. Diseases caused by E. coli are treated using antibiotics. Because of their proximity to humans, cats possess an extremely high risk of contracting antibiotic resistance genes when their owners touch cat feces containing E. coli that harbor resistance genes. This study was conducted to identify multidrug-resistant E. coli and extended-spectrum β-lactamase (ESBL)-producing genes from cat rectal swabs collected at Surabaya City Veterinary Hospital to determine antibiotic sensitivity.TEM and CTX-M was performed using a polymerase chain reaction.Samples of cat rectal swabs were cultured in Brilliant Green Bile Lactose Broth medium and then streaked on eosin methylene blue agar medium for bacterial isolation, whereas Gram-staining and IMViC tests were conducted to confirm the identification results. The Kirby–Bauer diffusion test was used to determine antibiotic sensitivity, and the double-disk synergy test was used to determine ESBL-producing bacteria. Molecular detection of the genes E. coli. Furthermore, 23 E. coli isolates (32.39%) demonstrated the highest resistance to ampicillin. Four isolates were confirmed to be multidurg-resistant and ESBL-producing strains. Molecular examination revealed that three E. coli isolates harbored TEM and CTX-M.Based on morphological culture, Gram-staining, and biochemical testing, the results of sample inspection showed that of the 100 cat rectal swab samples isolated, 71 (71%) were positive for In conclusion, pet owners must be educated on the use of antibiotics to improve their knowledge about the risks of antibiotic resistance. Escherichia coli is a bacterium that frequently affects cats [E. coli bacteria are typical flora that are found in the digestive tracts of both humans and animals, but some of them are pathogenic and can cause diarrhea [E. coli [Cats are domesticated animals that frequently interact directly with humans . More thcts cats , which idiarrhea . Althoug[E. coli , their u[E. coli . Antibio[E. coli . Based o[E. coli . Animals[E. coli .Escherichia coli can be resistant to specific antibiotics even when it has never been in contact with them because other bacteria that have previously been resistant could have passed the resistance gene to it [E. coli that harbor resistant genes [et al. [E. coli isolates were multidrug-resistant to antibiotics from the classes of penicillin, cephalosporin, fluoroquinolones, aminoglycosides, and tetracycline (TE). Hence, MDR will exert a detrimental effect on the choice of therapies to be applied to manage the illness.ne to it . Becausent genes . Pet ownnt genes . Subsequnt genes . Multidrnt genes . Rzewusk [et al.  discoverE. coli resistance are still infrequently conducted. As cats are particularly susceptible to E. coli infections of the digestive system, it negatively affect the choice of medicine to be used for disease control [Resistance to third-generation cephalosporins generally occurs in Gram-negative bacteria due to extended-spectrum β-lactamase (ESBL) enzyme production . Extende control . A signi control .E. coli strains and ESBL-producing genes from cat rectal swabs collected at Surabaya City Veterinary Hospital to determine antibiotic sensitivity, which would be useful for reducing the development of antibiotic resistance and improving the effectiveness of treatment.This study aimed to identify multidrug-resistant Cat rectal swab samples were used in this study; hence, ethical approval was not necessary. Cat rectal swab samples were collected from Airlangga University Teaching Animal Hospital and Animal Husbandry Service Animal Hospital of East Java Province, Indonesia, during routine case handling.This study was conducted from March to April 2023. Cat rectal swab samples were collected at the Airlangga University Teaching Animal Hospital and the Animal Husbandry Service Animal Hospital of East Java Province, whereas bacterial isolation and sensitivity tests were conducted at the Veterinary Public Health Laboratory, Faculty of Veterinary Medicine.Approximately 100 samples of cat rectal swabs were collected. Swab results were labeled, and swab sampling was performed carefully. A tube containing Brilliant Green Bile Lactose Broth (BGBLB)  was placed over the cotton swab holding the sample. The samples were transported to the laboratory and placed in a cooler box.E. coli colonies that were metallic green in color [Each cat rectal swab sample was cultured in 10 mL of BGBLB enrichment media and incubated for 18–24 h at 37°C. Positive results are characterized by a change in media color from green to cloudy green and the presence of gas in the Durham tube . Next, 1in color . In addiEscherichia coli isolates were planted on agar plates using a sterile cotton swab with a standard McFarland turbidity of 0.5 and evenly rubbed on Mueller Hinton Agar (MHA) (Oxoid™ CM0337B) media surface [The Kirby–Bauer diffusion test was conducted to determine antibiotic sensitivity.  surface . The MHA surface .E. coli isolates were subjected to the double-disk synergy test (DDST) using antibiotic disks containing amoxicillin–clavulanate 20/10 μg, cefotaxime 30 μg, and ceftazidime 30 μg. After overnight incubation at 37°C, the diameter of the inhibitory zone was measured to interpret the data.Multidrug-resistant CTX-M and TEM  were positive for E. coli . The eme to TE, five isolates (7.04%) to C, four isolates (5.63%) to GM, and three isolates (4.22%) to ATM  of ) to ATM .E. coli resistance test to antibiotics demonstrated that of the 71 E. coli isolates, 16 (22.53%) were resistant to one class of antibiotics, whereas 5 isolates (7.04%) were resistant to two classes of antibiotics, and 4 isolates (5.63%) were confirmed to have MDR because they were resistant to three to five classes of antibiotics  were positive for ESBL  (CM0155 Oxoid™) media after a 24 h incubation period at 37°C is indicated in green. However, no color change was observed in the citrate test medium, indicating negative results for ironment .The Kirby–Bauer diffusion method was used to determine bacterial sensitivity on MHA medium using a standard McFarland 0.5 suspension of the bacterium. The inhibitory zone was measured using a caliper, which was then compared with the 2020 CLSI standard after incubating the media for 24 h at 37°C. Of the 71 isolates, 4 were labeled as multidrug-resistant because they were resistant to at least three to five different classes of antibiotics.et al. [E. coli isolates from healthy cats had acquired resistance to AMP. This is possible because AMP is a first-line antibiotic, meaning it is the first antibiotic used to treat E. coli infections [There were 23 isolates (32.39%) exhibited the maximum levels of antibiotic resistance to AMP. The β-lactam class of penicillin derivatives, which includes AMP, prevents the production of mucopeptides required for bacteria to produce their cell walls . These fet al. , who foufections . Moreovefections .E. coli, which can decrease antibiotic activity, is the mechanism underlying C resistance [In the antibiotic sensitivity test, the same results were obtained with the antibiotics C and TE, that is, five isolates (7.04%) were resistant. The production of the enzyme C acetyltransferase by sistance . Meanwhisistance .E. coli isolates (5.63%) demonstrated resistance to GM, an antibiotic of the aminoglycoside class. Zhou et al. [E. coli isolates exhibited resistance to GM. Kakoullis et al. [Four u et al.  reporteds et al.  reporteds et al. .In the present study, three isolates (4.22%) exhibited resistance to ATM, which is quite low because of the extremely low usage of the monobactam class of antibiotics in both veterinary and human medicine . The mecE. coli isolates in this study: ATM preparation for the β-lactam monobactam group, GM preparation for the aminoglycoside group, C preparation for the C group, TE preparation for the β-lactam penicillin group, and AMP preparation for the β-lactam penicillin group. Four E. coli isolates that were subjected to the antibiotic sensitivity test were found to be multidrug-resistant. Wibisono et al. [The following five different antibiotic preparations were used to test o et al.  recommeno et al. .E. coli, an ESBL is present when the diameter of the zone of inhibition for drugs containing β-lactamase inhibitors. This is because the β-lactamase inhibitor diffuses into the media and detects the presence of β-lactamase around cefotaxime or ceftazidime [Regarding the ESBL screening test results for tazidime . This isTEM and CTX-M were detected in the rectal swabs of cats in this study. According to a study in China, the epidemiological distribution of CTX-M is comparable between animals and humans [The genes d humans . Extended humans . Reportsd humans . Accordid humans .TEM and CTX-M. A similar study by Musa et al. [CTX-M, and 77.4% harbored both TEM and CTX-M. This is because the plasmid that encodes CTX-M is a form of large IncFII plasmid that also contains a gene for resistance to other antibiotic classes. Some CTX-M genes are associated with IncFII or IncI1 plasmids [CTX-M bla in Enterobacteriaceae isolates from other countries [CTX-M are highly transmissible, which enables the rapid and effective spread of resistance [CTX-M are multidrug-resistant. Because plasmids, which are mobile genetic components, include TEM-forming genes, they can spread rapidly. Li et al. [CTX-M is also associated with TEM, OXA, and aac-(6)lbr on the IncFII plasmid, which carries more than one replicon. A strong promoter exists in the insertion region of CTX-M, which is present in several clinical isolates [In samples A20, A25, and B6, this study also discovered a combination of a et al.  found thplasmids . The mosountries . Other isistance . Most bai et al.  mentioneisolates .E. coli is among the bacteria that are frequently discovered to be resistant to β-lactam antibiotics, aminoglycosides, TEs, and C, which are frequently used in veterinary hospitals [E. coli is caused by several processes, including the closure of cell wall pores, which reduces the quantity of antibiotics passing through the cell wall, and an increase in the synthesis of periplasmic β-lactamases, which damages the structure of β-lactams, increasing the pump output (efflux mechanism) on the transmembrane, which allows bacteria to excrete antibiotics before the perception of any effects [Antibiotics are administered to animals at veterinary hospitals alone, in combination with other antibiotics, or in conjunction with chemotherapeutics . Extendeospitals . The mec effects . Alterat effects .E. coli strains that are resistant to multiple antibiotics can be identified in urine samples, digestive tract, skin wounds, liquid waste, and wounds from surgery [E. coli strains, which facilitates the exchange of antibiotic resistance genes [In addition to their presence in feces,  surgery . The trace genes . Bacterice genes .Both public health and animal health welfare depend on the prudent use of antibiotics to prevent the development of antibiotic resistance . Animal E. coli isolates were discovered in the isolation and identification tests. Four multidrug-resistant and ESBL-producing E. coli isolates were detected in the sensitivity test and DDST. Based on molecular examination, three E. coli isolates harbored TEM and CTX-M. Therefore, pet owners must be educated on the use of antibiotics to increase their knowledge regarding the risks of antibiotic resistance.Of 100 cat rectal swab samples collected at Surabaya City Veterinary Hospital, 71 MTIF, SR, YKKW, GDSP, and SEK: Collection and/or assembly of data. ARK, SCK OSMS, and SKM: Analysis and interpretation of data and drafted the manuscript. MTIF and MHE: Performed the study. ISY, RTSA, and MHE: Concept anddesign of the study, critical revision of the manuscript. All authors have read, reviewed, and approved the final version of the manuscript.


Mucormycosis is an uncommon, yet deadly invasive fungal infection caused by the Mucorales moulds. These pathogens are a WHO-assigned high-priority pathogen group, as mucormycosis incidence is increasing, and there is unacceptably high mortality with current antifungal therapies. Current diagnostic methods have inadequate sensitivity and specificity and may have issues with accessibility or turnaround time. Patients with diabetes mellitus and immune compromise are predisposed to infection with these environmental fungi, but COVID-19 has established itself as a new risk factor. Mucorales also cause healthcare-associated outbreaks, and clusters associated with natural disasters have also been identified. Robust epidemiological surveillance into burden of disease, at-risk populations, and emerging pathogens is required. Emerging serological and molecular techniques may offer a faster route to diagnosis, while newly developed antifungal agents show promise in preliminary studies. Equitable access to these emerging diagnostic techniques and antifungal therapies will be key in identifying and treating mucormycosis, as delayed initiation of therapy is associated with higher mortality. Rhizopus, Mucor, and Lichtheimia . A. A87]. AAlthough Mucorales cause mostly sporadic infections in the community setting, nosocomial outbreaks are increasingly recognised ,90,91. OMucor spp. (44%) . M. Mp = 0.olletiae ,204,207.L. corymbifera, R. arrhizus, and R. microsporus is 1 mg/L, while the ECV for M. circinelloides is 4 mg/L , its efficacy is poor in animal models [Posaconazole and isavuconazole have good in vitro activity against many Mucorales. The 95% ECV of posaconazole for s 4 mg/L  200]. W. WL. corole MICs ,205,209.ole MICs ,210,211.l models ,203.R. arrhizus isolates [There have been a small number of studies looking at the synergistic activity of antifungal combinations using chequerboard microdilution methods . Certainisolates . Schwarzisolates .A limited number of studies have compared the reference broth microdilution method against alternative methods such as disc diffusion or gradient strip testing. Disc diffusion appears feasible, with a low rate of major and very major errors demonstrated for posaconazole and amphotericin B when using cutoffs of 1 and 4 mg/L to pragmatically define sensitivity and resistance, respectively . There hR. arrhizus has demonstrated that CYP51A is also responsible for its intrinsic voriconazole and fluconazole resistance [Rhizopus and Mucor spp. may be explained by a key F129 residue of the CYP51 F5 gene in Mucorales spp. which confers decreased binding of short-side-chain  but not long-side-chain azoles (posaconazole) [Molecular determinants of antifungal resistance are not well studied or described. Research into sistance ,221. Modonazole) .R. arrhizus), as well as the presence of whole-genome duplication events [R. microsporus var. rhizopodiformis strains were ≅25 Mbp, but some isolates had larger genomes (43–51 Mbp), which are indicative of genome expansion [R. pusillis (≅25 Mbp), Apophysomyces (≅32 Mbp), and Lichtheimia spp. (≅58 Mbp) are larger still [Earlier investigations of mucormycosis case clusters have detailed epidemiological linkages without data on the genetic relatedness between strains, partly as there are no established genotyping makers for Mucorales ,132. Then events . Most asxpansion . The gener still ,224. GenM. circinelloides 1006 PhL [M. indicus B7402 [R. microsporus (GenBank: GCA_006680115.1) [R. pusillus (GenBank: GCA_900175165.2) [Secondly, reference genomes for Mucorales are less rigorously standardized in comparison with reference bacterial genomes. Nonetheless, there are publicly available reference genomes that have been assembled by research groups, such as 1006 PhL , M. indius B7402 ,226, R. 80115.1) , and R. 75165.2) . Variabi75165.2) .Mucor circinelloides sensu stricto wound infection [M. circinelloides 1006PhL. This study set the premise that the genome of M. circinelloides was stable, established parameters of the sequencing process, and assigned a genetic threshold (by estimating evolutionary distance and number of single nucleotide polymorphisms (SNPs) between pairs of isolates) to determine the genetic proximity between genomes from identical or clonal strains. Unexpectedly, approximately 5296 SNPs were identified as a cutoff below which two isolates could be designated as being of the same strain. Four clades were identified, where each clade was separated by >290,000 SNPs, while isolates within each clade varied by ≅20,000 SNPs [A. trapeziformis and exposure [Garcia-Hermoso et al. were amongst the first to use WGS analysis to investigate a possible nosocomial cluster of six patients with nfection . Twenty-000 SNPs ,227. Con000 SNPs ,224 wherR. pusillus strains yielded > 5900 core SNPs between them; the third isolate was Lichtheimia ramosa [R. microsporus var. rhizopodiformis, however, 21 of 24 isolates associated with a cluster of surgical infections were of a single clade, with 17 of the 21 forming an inner clade consisting of only 1235 SNPs . Although this is indicative of a likely common geographic origin, the genomic diversity was reported to be inconsistent with a point source outbreak [In a WGS-directed investigation of three cases of mucormycosis in heart and lung transplant patients over 6 months, genome assembly of two a ramosa . This reoutbreak .Rhizopus spp. and one Lichtheimia sp.) over a 10-month period and performed WGS on 72 clinical and environmental Mucorales including the outbreak isolates [Similarly, Nguyen et al. investigated a cluster of four transplant-associated cases of mucormycosis , while the deoxycholate formulation is recommended only in cases where other endorsed amphotericin B formulations are unavailable. Isavuconazole and posaconazole (delayed release tablets and intravenous formulations only) are both reasonable first-line alternatives when amphotericin is contraindicated, or they can alternatively be used as salvage therapy. Both are preferred over posaconazole oral suspension, as the latter is associated with higher rates of treatment failure ,229. WheCombination antifungal therapy has been studied. For treatment refractory cases, the evidence suggests improved outcomes with combined amphotericin and echinocandin, or amphotericin and posaconazole; however, other retrospective evaluations report treatment equivalence, particularly in first line therapy . Thus, tThe treatment arsenal for Mucorales infection remains limited, and, even with current best-practice therapy, mortality rates over 60% are frequently described. Furthermore, current antifungal agents have limitations in terms of substantive toxicity and/or variable in vitro activity . Thus, aR. arrhizus var. arrhizus; however, the potency against R. arrhizus var. delemar was poor. In vivo R. arrhizus var. arrhizus infection studies in immunocompromised mice demonstrated a ≅1 log decrease in lung and brain fungal burdens in treated mice compared to placebo controls [Oteseconazole , a long side-chain azole, is a novel metalloenzyme inhibitor which prevents ergosterol synthesis through selective inhibition of fungal CYP51 (lanosterol 14α-demethylase) . Early scontrols ,234. WhiR. arrhzius var arrhizus and R. arrhizus var. delemar isolates, and efficacy has been demonstrated when used as a single agent to treat R. arrhizus pulmonary and disseminated infection in immunosuppressed mice [Fosmanogepix (prodrug of manogepix) prevents the maturation of glycerolphosphatidyl inositol (GPI)-anchored proteins by inhibiting the enzymatic action of inositol acyltransferases (Gwt1), compromising fungal growth. In a murine model, combined fosmanogepix with liposomal amphotericin B was superior to amphotericin B alone in the treatment of invasive mucormycosis . Potent sed mice . ClinicaR. arrhizus, L. corymbifera, and M. circinelloides. Additionally, enhanced survival and a decreased fungal burden were demonstrated in murine models with pulmonary infection due to R. arrzhius var. delemar [Jawsamycin (FR-900848) inhibits the Spt14/Gpi3-mediated biosynthesis of glycosylphosphatidylinositol, which is essential to maintain the integrity of the fungal cell wall. Low MICs were recorded when ATCC isolates of six common species of Mucorales were exposed to jawsamycin in vitro, including  delemar .Calcineurin and mTOR inhibitors are more novel treatment options which have both demonstrated synergistic in vitro activity against Mucorales isolates when combined with posaconazole, isavuconazole, or amphotericin B ,239,240.Rhizopus spp. These agents are detailed in There are several other novel antifungal agents in early stages of evaluation against Mucorales, with some promising in vitro and in vivo activity, particularly for In this paper, we described significant advances in diagnostic methods and promising signals for new antifungal therapy options, but significant challenges still exist in regard to controlling the burden of disease caused by this WHO high-priority pathogen group. First, an accurate estimate of the burden is required, along with a better understanding of the epidemiology of disease—especially as at-risk populations continue to emerge and expand. This requires systematic surveillance across countries, using common data points and ensuring that laboratory data are married to clinical meta-data.Research and development efforts must focus on developing accurate, cheap, rapid diagnostic tests—ideally available in a point-of-care format. Advanced molecular epidemiology is complicated and challenging but holds significant promise; however, robust pathology stewardship will be needed.Research must continue to focus on the development of new antifungal agents and adjunctive therapies. Mortality is unacceptably high with the current armamentarium, and newer agents, while promising, still require further investigation before being introduced to routine clinical care. Finally, there must be equitable access to available treatments and diagnostics. Improved supply chains, fair pricing, and capacity building will contribute to this goal.


Rhipicephalus (Boophilus) annulatus, has been recognized as a prime vector of tick-borne diseases in South Indian regions. Over time, the use of chemical acaricides for tick control has promoted the evolution of resistance to these widely used compounds through metabolic detoxification. Identifying the genes related to this detoxification is extremely important, as it could help detect valid insecticide targets and develop novel strategies for effective insect control. We performed an RNA-sequencing analysis of acaricide-treated and untreated R. (B.) annulatus and mapped the detoxification genes expressed due to acaricide exposure. Our results provided high-quality RNA-sequenced data of untreated and amitraz-treated R. (B.) annulatus, and then the data were assembled into contigs and clustered into 50,591 and 71,711 uni-gene sequences, respectively. The expression levels of the detoxification genes across different developmental stages of R. (B.) annulatu identified 16,635 transcripts as upregulated and 15,539 transcripts as downregulated. The annotations of the differentially expressed genes (DEGs) revealed the significant expression of 70 detoxification genes in response to the amitraz treatment. The qRT-PCR revealed significant differences in the gene expression levels across different life stages of R. (B.) annulatus.Ticks are hematophagous ectoparasites of economic consequence by virtue of being carriers of infectious diseases that affect livestock and other sectors of the agricultural industry. A widely prevalent tick species,  Ticks are vectors that have a worldwide distribution and are of huge economic relevance due to the direct harm they inflict on their hosts. Ticks acquire pathogens from infected vertebrate hosts and transmit diseases to other animals through blood meal . As a reDue to the increasing cases of cattle tick infestations in tropical and subtropical regions, tick control is a priority for developing countries . Tick inRhipicephalus (Boophilus) annulatus (R. (B.) annulatus) is a widespread exophilic tick species that live and feed on the host for several days; they ingest blood meal by attaching their hypostome to the skin of the cattle. Once fed, ticks commence engorgement, molting, reproduction, and finally, they die [R. (B.) annulatus infestation has a negative impact on milk productivity due to the induced stress, immune dysfunction, blood loss, and the transmission of pathogens such as Babesia bovis and Babesia bigemina [R. (B.) annulatus is a member of the family Ixodidae and pose a threat to livestock in South India due to their capacity to spread many variants of tick-borne diseases, including anaplasmosis and babesiosis in cattle [they die . R. (B.)bigemina ,8. R.  microplus and R. (B.) annulatus [Ixodidae [In the field, commercially available vaccines have been effective at enhancing the immunity of cattle against ticks ,20. Howennulatus ,22 that Ixodidae ,24,25,26In this context, chemical acaricides continue to be a control method for cattle producers to mitigate tick infestations. The availability and ease of handling has made acaricide treatment the dominant method for tick control. However due to their frequent use these acaricide residues have been shown to contaminate the environment . The lonR. (B.) annulatus can easily develop resistance properties to many classes of acaricides. R. (B.) annulatus have been constantly challenged by the repeated usage of chemical acaricides, and as a result, a vast array of defenses has been employed, including biochemical defenses such as AchE insensitivity, sodium channel insensitivity, and GABA receptor (Cl—channel) insensitivity.Being one host ticks, Ixodes scapularis [Boophilus microplus [Dermacentor variabilis [Rhipicephalus sanguineus [R. (B.) annulatus. Exploring the interaction between the detoxification genes and insecticide resistance could help with the development of integrated pest management programs, thereby defining the precise role of these genes in the development of acaricide resistance in R. (B.) annulatus.In many cases, ticks express a different set of genes at high levels to counter acaricide treatments that could be used as a target to further develop efficient tick control approaches ,34,35. Tapularis , Boophilicroplus , Dermaceriabilis , and Rhinguineus  through R. (B.) microplus [R. decoloratus against amitraz [R. (B.) annulatus populations.A formamidine acaricide, amitraz, was initially introduced alongside synthetic pyrethroids in 1986 to restrain organophosphate-resistant ticks, but its usage was limited due to the high cost. The resistance mechanism of icroplus ,40,41,42 amitraz  has been amitraz  and the  amitraz . HoweverR. (B.) annulatus larvae following amitraz treatments and compared the results with a non-treated control group. The most likely candidates for the differentially expressed detoxification genes were considered for validation using qRT-PCR. We demonstrated the differences in the expressed genes/transcripts across different developmental stages of R. (B.) annulatus. In addition, a comparison of the gene expression profiles associated with treatments of amitraz and flumethrin (a commonly used pyrethroid) was performed. The information revealed through this study could enhance our understanding and help in the development of potential strategies for controlling this menacing pest.In this study, we leveraged a transcriptomic approach to identify and quantify the transcripts of The adult engorged female ticks were obtained from the Instructional Farm, Kerala Veterinary and Animal Sciences University, Wayanad, Kerala. The ticks were collected from infested cattle and maintained in a biochemical oxygen demand (BOD) cabinet with 80 ± 5% relative humidity. Under this incubation condition, ticks were allowed to oviposit, with subsequent hatching into larvae. It took roughly 20 days for egg laying, 10–15 days for the eggs to hatch into larvae, and another 20 days for the larvae to mature into an adult. Both larvae (8–10 days old) and newly engorged adult ticks were used for further experiments.50, which is the median lethal concentrations of both acaricides, was assessed in R. (B.) annulatus using the Abbott formula.The larval packet test (LPT) was performed based on the Food and Agricultural Organization (FAO’s) standardized protocol (1971)  with sliR. (B.) annulatus were doused in distilled water and drained on absorbent paper before being used for AIT. The different concentrations of amitraz (200 ppm–350 ppm) and flumethrin (20 ppm–100 ppm) were prepared using methanol, as described above. Four replicates, containing six ticks each, were immersed in the respective concentration for two minutes. After two minutes, ticks were transferred to plastic tubes and kept in a BOD incubator with 80% relative humidity at 28 °C. The specimen tubes were observed for oviposition, and mortality was assessed up to 15 days. The egg mass was continuously monitored in the BOD incubator, under the same incubation state, for 30 days.The stage specific expression analysis was performed by comparing the acaricide treated larval and adult tick populations. The optimum acaricide concentration for the treatment of adult ticks was obtained through the Adult Immersion Test (AIT). The AIT was based on the procedure detailed in . Fully eR. (B.) annulatus larvae untreated or treated with amitraz at 0.56 ppm using the trizol RNA isolation protocol . The purity and concentration of the total RNA of R. (B.) annulatus was determined using a Nanodrop spectrophotometer 2000 . To obtain transcriptome data, a cDNA library was constructed using the procedure from the Illumina TruSeq stranded mRNA library prep kit. The cDNA library sequencing was conducted on the Illumina HiSeq 500 platform at the Genotypic technology’s Genomics facility . The RNA quantity was measured using a Qubit fluorometer and validated using a high-sensitivity Bioanalyzer chip . Then, cDNA libraries were prepared for paired-end sequencing on the Illumina HiSeq 500 platform. The raw RNA-seq data for R. (B.) annulatus were deposited into the SRA database (NCBI) and assigned the accession number SRP235650.Total RNA extraction was performed from the Quality checks for the raw data  was achieved using FASTQC software. Low-quality reads were filtered out using NGS QC Toolkit  with a sR. (B.) annulatus transcript and functionally classified into different categories, such as cellular component, biological process, and molecular function. In addition, the R. (B.) annulatus transcriptome data were screened to identify simple sequence repeats (SSRs) using MIcroSAtellite Identification Tool (MISA) [For annotation, BLASTX was performed against arthropod protein sequences from UniProt, followed by NCBI non-redundant databases. Standalone Gene ontology (GO) was performed using the BLAST2GO program  and pathl (MISA) .R. (B.) annulatus larvae. The gene expression level of untreated ticks was used to normalize the gene expression of treated ticks. To gauge the expression level of each unigene (amitraz treated and untreated), the CD-hit program was used to cluster quality reads from each set of samples and to compare gene expression. Differentially expressed genes were identified in treated versus untreated samples; these were further analyzed using DESeq software and Bioconductor (http://bioconductor.org/ (accessed on 25 August 2015) R-packages [Expression profiling was performed to offer insight into the genes driving acaricide resistance by classifying the Differentially Expressed Genes between amitraz treated and untreated packages . Cluster50 to normalize the drug exposure to body surface area/volume.To identify resistant genes in adult ticks and larvae, we used Real-Time PCR analysis. Due to the large size differences between adult ticks and larvae, the drug dosage for the gene expression studies was determined by their respective LC−ΔΔCt method [t-tests.Some of the candidate genes were selected for expression validation using real time PCR. The RT-qPCR was performed with the Roche-Light Cycler 480 System, which is a high-performance, high-throughput PCR platform. The versatile Light cycler 480 instrument provides fast, highly sensitive, and reproducible gene expression results. We selected 7 CYP450 genes and 2 GST genes, CYP (30980), CYP (27576), CYP (51361), CYP (38633), CYP (41222), CYP (48268), CYP (36267), GST (34065), and GST 55171), based on the fold differences pattern from the transcriptome data for the qRT-PCR assay. Expression analysis of the nine identified detoxifying genes in the two developmental stages (larvae and adult) in response to the two separate groups of acaricides at its median lethal concentration (amitraz and flumethrin) was also tested using quantitative real time PCR. The expression of each sample was normalized by the reference gene Beta actin. Three biological replicates were developed for each gene, and all samples were assayed in technical triplicates. The abundance of expressed genes in the acaricide treated and untreated ticks was measured using the 2171, base50) value of amitraz and flumethrin against R. (B.) annulatus was calculated using the larval packet test (LPT). Concentrations ranging from 300 ppm to 0.146 ppm were used to find the lethal concentration of amitraz to R. (B.) annulatus larvae. Bioassays on R. (B.) annulatus with amitraz revealed 100% mortality at 300 ppm and 150 ppm, whereas insignificant mortality was observed in the untreated sample  annulatus when treated with amitraz. The ticks that survived the treatment at the LC50 concentration were taken for RNA isolation and transcriptome analysis.The median lethal concentrations (LCd sample . The morstimated . At 0.56R. (B.) annulatus responded highly even at the lowest concentration. The toxic interaction of flumethrin with R. (B.) annulatus showed 100% mortality when the dose concentration was between 25 ppm and 1.5625 ppm  annulatus. The higher concentrations of 200 ppm to 350 ppm were used for amitraz and 20 ppm to 100 ppm for flumethrin. At a concentration used in field conditions, amitraz (300 ppm) showed 16.6625 ± 6.8% mortality and 75.80% inhibition of fecundity, while flumethrin (80 ppm) showed 12.49 ± 4.165% mortality and 100% inhibition of fecundity against R. (B.) annulatus  with an average length of 151 bp were clustered using Trinity, a short-read assembling program. The assembled contigs produced 50,591 (untreated) and 71,711 (treated) unigenes with a 662 and 542 bp average length, respectively. The entire identified transcripts were 76,491 for the untreated sample and 52,471 for the treated sample. The maximum contig lengths obtained in the untreated and treated samples were 15,075 and 15,881, respectively, and the minimum contig length obtained was 201 for both samples. The N50 values were 1169 for untreated samples and 731 for treated samples. The summary of the De novo Assembly Statistics and the Clusters of Orthologous Groups (COGs) Statistics are in To understand the putative protein functions, the COGs database was used to forecast and categorize the potential functional involvement of the identified unigenes. Since a reference genome was not available, a sequence similarity search was performed with all available arthropod protein sequences (UniProt Database) to identify homologous genes in other species putatively. Annotation of transcripts was conducted using the BLASTALL program and only transcripts with more than 30% identity were considered for analysis. A total of 92,341 transcripts were generated; of these, there were 35,585 annotated transcripts and 56,756 unannotated transcripts. BLAST searching of non-redundant databases provided important matches, allowing the gene families most likely encoding gene products engaged in xenobiotic sequestration and detoxification to be curated and annotated.R. (B.) annulatus unigenes based on sequence homology. The Gene Ontology database was used to classify the functions of R. (B.) annulatus unigenes into three main categories associated with molecular function, cellular component formation, and the biological process. Those were further categorized into nine biological processes, seven cellular components, and ten molecular functions, respectively (R. (B.) annulatus showed that a high percentage of genes are in the molecular function category, of which about 19.26 % are involved in nucleic acid binding.Annotation with BLAST2GO generates a functional classification of the identified ectively . Among tR. (B.) annulatus tick species, although a total of 38,472 pathways were identified. These unigenes were highly represented in metabolic pathways, signal transduction pathways, cellular community, and transport and catabolism pathways (R. (B.) annulatus.The Kyoto Encyclopedia of Genes and Genomes (KEGG) database gathers manually drawn pathway maps, allowing pathway-based analysis to understand gene interactions and biological functions. Using the KASS server, the identified unigenes were functionally assigned to several different pathways specific to pathways . These aR. (B.) annulatus transcriptome data. The identified SSRs were predominantly mononucleotide and tetranucleotide repeats, representing about 63%  and 18% (3457) of the SSRs, respectively. A small fraction of dinucleotide repeats (12%) and pentanucleotide repeats (7%) were also identified in the R. (B.) annulatus transcriptome. Features of SSRs identified in the R. (B.) annulatus transcriptome are shown in Due to the high degree of polymorphism, abundance, and ease of development , Simple Anopheles gambiae, T. castaneum, and Drosophila melanogaster [R. (B.) annulatus. In our study, a total of 70 detoxifying genes were identified from the R. (B.) annulatus transcriptome, including 44 CYP450 genes, 23 GST genes, and 3 EST genes encoding detoxifying enzymes. These detoxifying genes were differentially expressed when triggered by amitraz , and Glutathione S-transferases (GSTs) genes. Previously published insect genome data of nogaster ,60,61 we amitraz . The genR. (B.) annulatus tick larvae were determined using the DESeq and Bioconductor (http://bioconductor.org/ (accessed on 25 August 2015) R-packages (R. (B.) annulatus larvae resulted in statistically significant changes in gene expression, the quantum of gene expression was analyzed after normalizing the gene abundance from each library to reads per kb per million reads (RPKM). Comprehensive DESeq computational analysis facilitated the recognition of differentially expressed transcripts vis-à-vis amitraz. Overall, 16,635 transcripts exhibited upregulation, 15,539 exhibited downregulation, and 56,864 transcripts were neutral. Notably, 1898 transcripts showed expression only in untreated conditions, whereas 1403 showed expression only in treated conditions. The complete transcriptome data revealed substantial differences in the expression patterns among amitraz-treated and untreated R. (B.) annulatus larvae. We also compared the differentially expressed detoxifying genes upon amitraz treatment, which revealed 29 upregulated genes, 12 downregulated genes, and 27 genes that were neutral  annulatus and in response to different insecticides. Genes such as CYP (30980) and CYP (41222)  A and GST (41222) B were si (41222) C showed  (41222) D. In ourRhipicephalus (Boophilus) annulatus ticks are adept at transmitting a large variety of infectious diseases, such as anaplasmosis and babesiosis, and at causing huge impediments in the cattle industry. R. (B.) annulatus reside in large areas of Southern India [R. (B.) annulatus voraciously sucks blood meal, injects toxins, transmits pathogens, and finally modifies immune responses in cattle, which negatively impacts cattle productivity and results in significant economic losses [R. (B.) annulatus with high resistance levels are insensitive to certain amitraz formulations, rendering this acaricide method ineffective for tick control. Thus, it is necessary to understand the mechanism through which R. (B.) annulatus counteracts the effect of insecticide. Ongoing research and development initiatives are geared toward finding a successful management strategy for this pest.rn India ,45. R.  annulatus at sublethal doses of flumethrin and amitraz.Previous studies on the underlying resistance mechanism in ticks led to the understanding that behavioral, biochemical, and genetic mechanisms are involved in neutralizing acaricide ,64,65,66R. (B.) annulatus. Larval toxicity experiments revealed that R. (B.) annulatus showed a slight susceptibility difference against amitraz and flumethrin, indicating that flumethrin is comparatively more effective at larval mortality than amitraz. The LC50 value observed was 100 ppm for amitraz and 80 ppm for flumethrin against R. (B.) annulatus adult ticks. Based on this result, flumethrin seems to be comparatively effective to amitraz. This result is similar to the acaricidal efficacy of flumethrin in R. (B.) microplus collected from Northern India, which showed high efficacy compared to other compounds [R. (B.) annulatus [R. (B.) annulatus. We used the larval stage of R. (B.) annulatus for de novo sequencing, assembly, annotation, and downstream analysis. According to the annotated results, 70 genes were involved in the detoxification mechanism in R. (B.) annulatus. Among these 70 genes, 44 were annotated as CYP450, 23 as GSTs, and 3 as ESTs. Considering the abundance of detoxifying genes, it was intriguing that more than half of the genes belonged to the Cytochrome P450 monooxygenase family. To further investigate gene expression changes associated with the amitraz treatment in R. (B.) annulatus, differentially expressed contigs were compared using DESeq. A total of 16,635 transcripts were upregulated and 15,539 were downregulated. Among the results obtained from the DESeq analysis, we compared differentially expressed detoxifying genes upon amitraz treatment. Of these, 29 upregulated (11 CYP450 and 18 GST) and 12 downregulated (11 CYP450 and 1 GST) detoxifying genes were identified. Further, a total of nine differentially expressed genes obtained from the Illumina analysis were selected to examine expression changes during various developmental stages of R. (B.) annulatus mediated by amitraz and flumethrin using qRT-PCR. Among the 9 genes, 3 CYP genes CYP (41222), CYP (27576), and CYP (30980) and 1 GST gene, GST (34065) were highly expressed in both amitraz-treated R. (B.) annulatus adults and larvae. Similarly, for the flumethrin-treated samples, 3 CYP genes CYP (38633), CYP (51361) and CYP (30980) were significantly upregulated in adults and larvae.In vitro bioassays, such as the larval packet test and adult immersion test, were conducted to compare the acaricidal effects of amitraz and flumethrin against ompounds . The resompounds . Severalompounds ,71,72. Tompounds ,74. Consnnulatus ,45,70, oR. (B.)microplus [R. (B.)microplus, have developed tolerance to nearly all synthetic and chemical acaricides over the past few decades due to their intensive use [R. (B.) microplus -associated pyrethroid acaricide resistance with the level of CYP expression [R. (B.) annulatus during the acaricide-stressed condition might implicate their role in resistance development and the increased survival rate of R. (B.) annulatus. These genes could be used as targets for pest control, such as by inhibiting their action through sequence-specific gene silencing via RNA interference [Cytochrome P450 Monooxygenases are well known for oxidizing widely diverse substrates and are capable of producing an array of molecular reactions . The exiicroplus . Differeicroplus . As a resive use . Comprehpression . Studiespression ,69,79,80pression ,81. In orference . AdditioThe larval insensitivity to flumethrin may indirectly highlight its efficiency as a drug used to target the larval stage compared to amitraz. This is consistent with the larval packet and adult immersion tests, where flumethrin caused more lethality than amitraz. This could be attributed to the unaltered gene expression pattern of various metabolic resistance genes in the larval stages of the ticks. This provides evidence that overexpression could confer acaricide resistance, possibly in the absence of target site insensitivity.R. (B.) annulatus transcriptome. The identified gene targets from this study may be used as valid targets for the currently evolving gene-based tick management strategies. These outcomes are geared to evaluate the possible molecular mechanisms involved in tick-borne diseases, thus positively benefiting medical and veterinary professionals, farmers, and the common public. The knowledge gained from this study has the potential to aid practical applications, especially when generating new strategies to control menacing ticks.Overall, our research exploited a combination of transcriptome analysis, differential gene expression, and gene expression validations to study the acaricide resistance mechanism. These results showed that our approach relying on the Illumina Hi-Seq platform enabled a comprehensive representation of the R. (B.) annulatus is indispensable to perform a functional analysis to understand the complex processes involved in acaricide resistance, thus providing valuable insights into the various pathways and processes that are crucial for the survival of ticks. This study quantified the relative expression of detoxifying genes in insecticide-treated and untreated R. (B.) annulatus and correlated their expression using qRT-PCR and Illumina sequencing to discern the genetic and molecular basis of insecticide resistance. Having realized the overriding significance of these genes in R. (B.) annulatus, we have analyzed their expressional variations at two stages: the larval and adult stages, in response to two different acaricides. Increasing access to Next-generation Sequencing technology, as well as transcriptome technology, can accelerate research on acaricide resistance and could play a significant role in developing novel tools to control the tick population. Taken together, this will propel further research in the field of toxicology and insecticide resistance, as well as advance our understanding of the role of environmental adaptation and microevolution as vital processes underlying resistance development.An improved understanding of the detoxifying genes in


Yaks are tough animals living in Tibet’s hypoxic stress environment. However, the metabolite composition of yak milk and its role in hypoxic stress tolerance remains largely unexplored. The similarities and differences between yak and human milk in hypoxic stress tolerance are also unclear. This study explored yak colostrum (YC) and yak mature milk (YMM) using GC–MS, and 354 metabolites were identified in yak milk. A comparative metabolomic analysis of yak and human milk metabolites showed that over 70% of metabolites were species-specific. Yak milk relies mainly on essential amino acids- arginine and essential branched-chain amino acids (BCAAs): L-isoleucine, L-leucine, and L-valine tolerate hypoxic stress. To slow hypoxic stress, human breast milk relies primarily on the neuroprotective effects of non-essential amino acids or derivates, such as citrulline, sarcosine, and creatine. In addition, metabolites related to hypoxic stress were significantly enriched in YC than in YMM. These results reveal the unique metabolite composition of yak and human milk and provide practical information for applying yak and human milk to hypoxic stress tolerance. ThiIn this study, the metabolites of yak colostrum (YC) and yak mature milk (YMM) were investigated by gas chromatography–mass spectrometry (GC–MS). Comparative metabolomic analysis identified distinct metabolites and explored the mechanisms underlying compositional changes between YC and YMM. The comparative metabolomics between yak and human breast milk was also analyzed. Common metabolites between yak and human breast milk were also analyzed in detail. This study provides practical information on the nutritional components of yak milk and lays a foundation for the development and research of yak milk products.2.2.1.The yak milk samples were collected from the “Yushu Yak” at the Zangbala Farm  in Yushu, Qinghai Province, China. Ninety cows with similar nutritional levels were selected as the experimental group. Six yak YC or YMM samples were randomly chosen for metabolomics analysis, respectively. These yaks were all around six years old and under similar physical conditions. The milk sample was frozen to liquid nitrogen and stored at −80°C until analysis.The human breast milk data was from a previous publication and the human breast milk sampling method is described again . Briefly2.2.2O, Pyridine, N-hexane, O-methyl hydroxylamine hydrochloride (97%), and BSTFA+1%TMCS were all from CNW technologies. The acetonitrile and 2-chloro-l-phenylalanine were separately provided by Fisher and Shanghai HC Biotech Co. The eleven fatty acid methyl esters were supported by Larodan, Nc-chek, DR. The quality control (QC) samples were prepared by equally mixing the metabolite extracts from 12 samples, including six Yak colostrum and six mature milk samples. The metabolites were identified based on three identical QC samples to increase the reliability of experiments.The chemical reagents and the catalog numbers were identical to the previous study . The met2.3.The sample extracts were prepared following the standard protocol for GC–MS metabolomics analysis . Briefly2.4.The derivatized samples were analyzed on an Agilent 7890B gas chromatography system coupled to an Agilent 5977A MSD system  with standard protocol . A DB-5M2.5.The GC/MS raw data were processed by AnalysisBaseFileConverter software and then imported into MS-DIAL software for further processing to get the original data matrix . The Unt2.6.https://www.kegg.jp/kegg/) was used for qualitative and metabolic pathway analysis.The raw data clean matrix was then imported into the R ropls package for Principal component analysis (PCA),  partial least-squares-discriminant analysis (O)PLS-DA . Correla2.7.(species 1) – FC(species 2)| > 1.The human breast milk metabolomics data were obtained from the previous publication . Common 3.3.1.According to the GC–MS data, a total of 354 metabolites were identified in Yak milk , and the3.2.t-test: p < 0.05) were identified, and 109 of them were selected as significantly different metabolites (SDM) between the YC and YMM groups with an additional criterion: the variable importance in the projection (VIP) values exceeding 1.0 , amino acids (12.99%), fatty acids, and conjugates (6.21%), which accounted for 38.89% of all 354 identified metabolites . To undeding 1.0 . Interes%, amino ding 1.0 . Howeverding 1.0 . Metabolding 1.0 . In addiding 1.0 . 74 SDMsding 1.0 . These r3.3.To identify the relationship between different SDMs, the correlation between metabolites was calculated using the Pearson correlation coefficient and was then grouped according to the type of metabolite . Visualihttps://www.metaboanalyst.ca)   for furtlyst.ca) . Amino alyst.ca) . These r3.4.p value and the number of metabolites, 24 key pathways were screened out, which might correlate with the metabolism composition dynamics between YC and YMM  in yak milk  are specific to yak and not found in human milk, according to PubChem CID . SimilarKEGG enrichment analysis was performed for yak-specific metabolites and human breast-specific metabolites using HMDB IDs to explore the functions of yak-specific metabolites further. Galactose metabolism, starch and sucrose metabolism, glycine, serine, and threonine metabolism, neomycin, kanamycin, and gentamicin biosynthesis, and Linoleic acid metabolism are the most significantly enriched pathways in the human breast-specific metabolites . Especia4.Metabolites are essential in milk, whereas yak milk metabolites are mainly unexplored. The only report on yak milk was the identification of 21 volatile compounds using GC–MS and GC-O-MS methods . In thisYaks live in the harsh environment of the Qinghai-Tibet Plateau, and hypoxia is an unavoidable major environmental factor in the life of animals on the plateau . ArgininAlthough yak milk has diverse metabolites to increase cellular adaptation to hypoxia, some human breast milk metabolites also have their specificity in response to hypoxia . Citrull5.Yak milk is essential for the survival of Tibetans, and the composition of yak milk during lactation remains unexplored. In this study, 354 metabolites were identified in YC and YMM during lactation. Incredibly, 109 of them were significantly differentially expressed between YC and YMM. Therefore, this study could provide the composition of metabolites of yak milk during lactation, providing a preliminary understanding of yak milk lactation. In addition, comparative metabolomics analysis unveils that yak milk-specific metabolites were enrichment in hypoxic stress-tolerant pathways. Thus, these data also provided helpful information to decipher how the metabolites composition of yak milk contributed to the hypoxic stress tolerance and provide practical information for Yak milk production in the future.The original contributions presented in the study are included in the article/The studies involving animals were reviewed and approved by the Experimental Animal Welfare and Ethical Review Certificate of Qinghai Academy of Animal Husbandry and Veterinary Sciences . Informed consent was obtained from the owners for the participation of their animals in this study.WL and LM: conceptualization. WL, WZ, and XJ: methodology. YZ and XF: software. YZ and ZM: validation. XF: formal analysis. ZM and WL: investigation. YZ: resources. WZ: data curation. WL, WZ, and LM: writing—original draft preparation. WL, LM, WZ, and XJ: writing—review and editing. YZ: supervision. LM: project administration. WL and YZ: funding acquisition. YH and YS: visualization. All authors contributed to the article and approved the submitted version.


Cryptosporidium spp. and Giardia duodenalis, enteric protozoan pathogens affecting humans and animals, elicit substantial global public concern. This study conducted in Taiwan sought to determine the prevalence and co-infection rates of Cryptosporidium spp. and G. duodenalis in dogs and cats. The investigation encompassed an analysis of infection rates and associated risk factors within the surveyed population. Predominantly identified species were C. canis and C. felis, aligning with canine-specific assemblages C and D. In contrast, the infrequent presence of human-specific assemblage A was noted in Giardia-positive samples. Phylogenetic analysis alluded to the potential for zoonotic transmission originating from domesticated animals. This underscores the role of pets as possible reservoirs for the transmission of cryptosporidiosis and giardiasis to humans in Taiwan.Cryptosporidium spp. and Giardia duodenalis are enteric protozoan pathogens in humans. and animals. Companion animals infected with zoonotic species/assemblages are a matter of major public concern around the world. The objectives of the present study were to determine the prevalences of Cryptosporidium spp. and G. duodenalis infections and their co-infection statuses in dogs and cats living in Taiwan and to identify the species and assemblages. Fecal samples were collected from local animal shelters (n = 285) and a veterinary hospital (n = 108). Nested polymerase chain reaction (PCR) was performed using the SSU-rRNA, β-giardin, and glutamate dehydrogenase genes for Cryptosporidium spp. and G. duodenalis, respectively. Results showed that the overall prevalences of Cryptosporidium and G. duodenalis were 7.38% (29/393) and 10.69% (42/393). In addition, co-infection was detected in 1.02% (4/393) of all samples. Sample source, clinical sign, and breed may be risk factors that influence the infection rate. In Cryptosporidium-positive samples, C. canis and C. felis were detected most frequently. Although the canine-specific assemblages C and D (37/42) were dominant, the zoonotic human-specific assemblage A (1/42) was also found in Giardia-positive samples. Phylogenetic analysis revealed that most positive samples belonged to host-specific subtypes/assemblages, while some Cryptosporidium or Giardia-positive samples could be zoonotic. The findings suggested that pet animals could be a cause of zoonotic transmission, causing human cryptosporidiosis and giardiasis in Taiwan. Cryptosporidium spp. and Giardia intestinalis , protozoan intestinal parasites which are of significant public health concern worldwide because infections are transmitted primarily through zoonotic, water-borne, and foodborne routes [Cryptosporidium or Giardia spp. to the environment [Cryptosporidium infections in immunocompetent people are mild and self-limiting. However, the lives of young children, the elderly and the immunocompromised can be threatened by infections [Cryptosporidium and Giardia infections are only occasionally found, such as in immunocompromised patients, they still represent a significant health concern.Cryptosporidiosis and giardiasis are emerging infectious diseases caused by e routes ,2. Humanironment . The maiironment . Generalfections ,5. In huCryptosporidium species, C. hominis, C. parvum, C. meleagridis, C. canis, and C. felis are the most prevalent in humans [G. duodenalis, only assemblages A and B pose significant human health risks [Cryptosporidium spp. and G. duodenalis are frequently found in dogs and cats worldwide [Cryptosporidium canis and C. felis are the primary Cryptosporidium species in dogs and cats, respectively. However, occasional detections of C. hominis, C. parvum, C. muris, and C. ubiquitum have been reported in these animals [G. duodenalis genotypes in these animals. However, zoonotic assemblages A and B have been identified in some studies [Among the approximately 40 recognized n humans . Similarth risks . Cryptosorldwide ,9. Crypt animals ,12,13,14 studies ,15.Cryptosporidiosis and Giardiasis [vulnerable to exposure to zoonotic diseases [Cryptosporidiosis and Giardiasis are transmitted to humans through the fecal–oral route or contaminated food and water, infections can be more prevalent in crowded conditions if the shelter settings are unsanitary. In this research, we focused on sources in local animal shelters and household pets and analyzed the associations with risk factors. Hence, in this study, we collected stool specimens from one veterinary teaching hospital and three local animal shelters in Taiwan.Companion animals may play an essential role in the transmission routes of ardiasis . Animal diseases . Since bCryptosporidium and Giardia relies on detecting specific oocyst/cyst characteristics in stool samples, typically using acid-fast staining [Traditionally, the diagnosis of staining  or immunCryptosporidium and Giardia with the traditional visual microscopic method is difficult due to the inability to differentiate between different species using morphology and/or host occurrence. Instead, a molecular technique such as polymerase chain reaction (PCR) should be used to characterize Cryptosporidium and Giardia in feces or environmental samples [Diagnosing  samples . Compare samples .Cryptosporidium oocysts and Giardia cysts have been conducted in livestock drinking water in Taiwan over the past decade, references or reports pertaining to Cryptosporidium and Giardia infection in small animal shelters are relatively limited. Thus, in this study, we performed the first cross-sectional investigation from different sources to offer a molecular assessment for future shelter animal medicine research. The primary aim of this research was to determine and elucidate the prevalences of Cryptosporidium and Giardia infection and their co-infection status in companion animals in Taiwan. The resulting information could serve as baseline data here. Although some surveys of In 2020, from February 1 to December 31, fresh canine and feline stool specimens were randomly collected from three local animal shelters  and one veterinary teaching hospital in Taiwan. The majority of the stool specimens were specifically recruited for the study, while the rest were obtained from routine diagnostics at the Section of Clinical Pathology, National Taiwan University Veterinary Hospital (NTUVH), Taipei. Among the 393 canine and feline specimens, 285 were from 3 local animal shelters, and 108 were from NTUVH. All the specimens were collected from live animals, and the collection processes were approved and followed local government regulations on animal protection. The study was approved by the Institutional Animal Care and Use Committee of National Taiwan University, Taipei, Taiwan (protocol code: IACUC No. NTU107-EL-00200).Each specimen (0.5–1 g) was collected from the ground immediately after animal defecation, placed into a 2 mL microcentrifuge tube, and then mixed with an appropriate amount of distilled deionized water (DDW). The properties of each specimen and the breed, age, gender, and past history of the animal were recorded in detail if possible. After several rounds of mechanical agitation, aliquots of the stool specimen were exposed to 5 freeze–thaw cycles, as described previously . The proAfter the freeze–thaw cycles, total genomic DNA (gDNA) was isolated from the feces sample using the NautiaZ Stool DNA Extraction Mini Kit  and eluted according to the manufacturer’s instructions. DNA concentration and purity were measured with a spectrophotometer . The isolated gDNA samples were used immediately or stored at −20 °C for up to 1 month prior to screening.Cryptosporidium spp. was detected via nested-PCR amplification of the small subunit (SSU) rRNA gene. The DNA of G. intestinalis was amplified, targeting β-giardin (BG) and glutamate dehydrogenase (GDH) genes. As seen in Cryptosporidium or Giardia DNA) and a negative control (distilled water) were run with every PCR batch. After nested-PCR amplification, 10 µL of each PCR product was electrophoresed by 2% agarose gel at 100 V for 30 min to identify the size of the products. PCR-positive products were purified using the PCR Clean-Up and Gel Extraction Kit , following the manufacturer’s instructions.To calculate the estimated prevalence rate, we count the number of positive samples and divide it by the total number of samples tested using nested PCR. The overall prevalence rate is determined by dividing the number of positive samples by the total number of companion animal fecal samples that are being tested, irrespective of whether they are dogs or cats. We also calculated stratum-specific prevalence rates for dogs and cats separately.Cryptosporidium spp. and Giardia duodenalis assemblages in canines and felines, all positive secondary PCR amplicons  were sent for nucleotide sequencing at Mission Biotech . The results were compared with corresponding sequences on the National Center for Biotechnology Information (NCBI) website using the BLAST® program . A phylogenetic tree was constructed using the MEGA-X program with neighbor-joining (NJ) analysis of the SSU rRNA and BG gene sequences.To examine the genetic relationships among 2) test and Fisher’s exact tests were performed to test for any significant differences in location, gender, age, breed, and clinical signs between the two groups of canine and feline populations. Results were considered statistically significant at p < 0.05.Geological maps were illustrated in the software QGIS 3.10.14. Statistical analyses were performed in Python 3.7 scipy.stats modules. Pearson’s chi-squared  and 10.69% (42/393) (Cryptosporidium infection in dogs and cats were 8.4% (21/250) and 5.6% (8/143), while those of G. duodenalis infection in dogs and cats were 12.4% (31/250) and 7.69% (11/143), respectively  of the specimens were collected from the veterinary hospital (TW-NTU) and 72.5% (285/393) from local shelters  A. Of the(42/393) B. In thiectively . Moreoveectively C,D. For mblages. .p values < 0.05 were regarded as associated with infection. Animal gender, fecal source, breed, and clinical signs were evaluated as possible variables. Only gender was found to be not significantly related to both Cryptosporidium and G. duodenalis infections. The rates of G. duodenalis infection in canines and felines were higher in those from the local animal shelters than in those from the veterinary hospital. The probability of being infected by G. duodenalis was more than five times higher  for the animal shelter source than for the veterinary hospital source. Dogs presenting diarrhea had a 2.9 times higher  probability of Cryptosporidium infection. In contrast, cats, whether displaying noticeable clinical signs or not, were not linked to the prevalence of either disease. Interestingly, compared to mixed-breed felines, purebred cats were 9.5 times  more likely to be Cryptosporidium-positive. However, mixed-breed dogs were more likely to be Giardia-positive than purebred dogs (p = 0.0406).The univariable analyses of risk factors associated with positive detection results are shown in Cryptosporidium infection was detected in 7.38% of all fecal samples, whereas Giardia infection was detected in 10.69%. After DNA sequencing, the results showed that the species of Cryptosporidium were mainly C. canis, C. felis, and C. meleagridis  retrieved from the GenBank database. Within the nucleotide sequences of Giardia-positive samples isolated from companion animals, 61.9% (26/42) were successfully characterized as some specific assemblages, with most being assemblage C (17/26), followed by assemblage D (8/26) and assemblage A (1/26). Other parts of isolated Giardia-positive samples still presented as β-giardin-positive but matched as partial codons  and Giardia canine-specific assemblage C .Nested PCR and DNA sequencing results revealed that eagridis A. Sequenl codons B. The moCryptosporidium infection rate was 7.38%. The prevalence of Giardia infection in companion animals identified at different genetic loci (bg and gdh) were 9.16% (36/393) and 3.56% (14/393), respectively. After the integration of positive samples of Giardia at the two loci, an overall 10.69% (42/393) prevalence was detected. A systemic review reported prevalence rates of Giardia of around 15.2% in dogs and 12% in cats worldwide [Giardia infection by ELISA antigen test or PCR have reported rates of 18.7% in Japan [Our data showed that the overall in Japan , 11.2% iin Japan , and 11%in Japan . Our finCryptosporidium spp. was 2.9-fold higher in the diarrhea canine group than in the no-obvious-clinical-sign canine group. Meanwhile, our study also indicated that Cryptosporidium in felines and Giardia in both companion animals were not associated with diarrhea clinical signs. That is, although Cryptosporidium was confirmed in diarrhea cases, it was also commonly found in normal feces from patients without clinical signs. Both pathogens were detected regardless of companion animal diarrhea status. The detection of both endoparasites in normal feces suggests that the possibility of contamination with (oo)cysts in an entire population cannot be ruled out. This finding is important and has public health significance because pet owners and clinical veterinarians may neglect the possibility of infection of Cryptosporidium or Giardia due to their clinical presentation. These results should be considered by small-animal veterinarians in differential diagnosis.The most noteworthy symptom in companion animals infected with protozoan parasites is diarrhea. According to our risk factor analysis, the risk of being positive for Cryptosporidium species, human cryptosporidiosis is mostly caused by five species: C. parvum, C. hominis, C. meleagridis, C. felis, and C. canis [C. meleagridis may be as common as C. parvum [C. meleagridis (n = 1) and C. canis (n = 18) in dogs, while C. felis (n = 5) and C. canis (n = 3) were identified in cats. For Giardia, 88.1% were classified as assemblage C or D. Moreover, one feline sample was successfully classified as Giardia assemblage A . The three Cryptosporidium species and Giardia spp. genotype A recognized by DNA sequencing is known as zoonotic endoparasites.Among the known C. canis ,28,29. I. parvum ,31. In oC. parvum and C. hominis, which are waterborne, C. canis and C. felis may infect humans through intrafamilial transmission by companion animals [Cryptosporidiosis and Giardiasis in local animal shelters in Taiwan may present a public health threat to humans. As noted in our previous discussion, there is no significant difference between clinical signs in Giardia-positive pets. Therefore, we may neglect the potential risk when contacting clinically healthy companion animals.Although human cryptosporidiosis infection through anthroponotic transmission is mainly caused by  animals . Thus, tCryptosporidium are virtually all vertebrate animal species. In Taiwan, the shelter facilities vary greatly by size, capacity, budget, and the number of governmental staff. These shelters offer temporary housing for not only stray dogs and feral cats but also a wide range of animals brought in by animal rescue services in local regions. Thus, wild birds and wildlife such as ferret badgers may be housed simultaneously in a high-density facility. The increased probability of exposure to contaminated water and food in animal shelters may explain the higher percentage of parasitic opportunistic infections. For example, birds are the natural host of C. meleagridis [The hosts of eagridis . It is nGiardia cysts and Cryptosporidium oocysts can survive for months in environments with high humidity, low temperature, and low exposure to sunlight [Cryptosporidium oocysts and Giardia cysts have been documented in both drinking water for livestock and urban tap water in Taiwan [G. duodenalis, while Cryptosporidium variably requires from 9 to 1024 oocysts [sunlight . In Taiwsunlight . Addition Taiwan ,37. One  oocysts ,38. The Cryptosporidium or Giardia (oo)cytes are transmitted in animal shelters, the horizontal transmission may spread dramatically because of oral–fecal infection within companion animals, owing to the narrow space and high-contact environment. Thus, to reduce the risk of zoonotic disease occurrence, fecal samples of pet animals should be routinely submitted for parasitic diagnostic tests, and owners should be informed about the public health issues related to pet fecal pollution. In local animal shelters, expansion of the disinfection checkpoints to maintain the biosecurity of the animal shelters is warranted.On the other hand, dogs and cats in animal shelters commonly come into contact with adopters and visitors. Although veterinarians regularly impose the disinfection process and exo-parasite chemical deworming program before the animals are admitted, the infection risk of endoparasites still exists. Moreover, once Cryptosporidium and Giardia, molecular examinations should not only be based on fecal samples so as to prevent underestimation of the prevalence of each parasite. Secondly, increases in the levels of Cryptosporidium spp. and Giardia infection in young companion animals have been documented previously [Cryptosporidium spp., and Giardia spp. has been reported with double or triple infections [Cryptosporidium spp., and Giardia spp. has also been noted in Brazil [There are some limitations to this research. Firstly, given the intermittent shedding pattern of (oo)cytes of eviously ,40. The fections . The conn Brazil . HoweverCryptosporidium and Giardia species/assemblage characteristics in companion animals were C. canis, C. felis, and Giardia assemblages C and D, respectively. This is the first cross-sectional molecular evaluation of animals derived from local animal shelters and household pets in Taiwan. Notably, C. canis, C. felis, C. meleagridis, and Giardia assemblage A have the potential for zoonotic transmission. These molecular results can serve as a baseline to assess the zoonotic potential of companion animals in Taiwan. Further epidemiological studies are necessary to understand the transmission better.The present study demonstrated that the most dominant


Clostridioides difficile infection (CDI) presents a major public health threat by causing frequently recurrent, life-threatening cases of diarrhea and intestinal inflammation. The ability of C. difficile to express antibiotic resistance and to form long-lasting spores makes the pathogen particularly challenging to eradicate from healthcare settings, raising the need for preventative measures to curb the spread of CDI. Since C. difficile utilizes the fecal–oral route of transmission, a mucosal vaccine could be a particularly promising strategy by generating strong IgA and IgG responses that prevent colonization and disease. This mini-review summarizes the progress toward mucosal vaccines against C. difficile toxins, cell–surface components, and spore proteins. By assessing the strengths and weaknesses of particular antigens, as well as methods for delivering these antigens to mucosal sites, we hope to guide future research toward an effective mucosal vaccine against CDI. Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacterium [C. difficile infection (CDI) usually arises following antibiotic usage that drives gut microbiota dysbiosis, allowing C. difficile to overgrow [C. difficile, a C. difficile vaccine could offer a significant economic advantage by avoiding the costs of treating CDI in the first place [C. difficile vaccine could be a cost-effective disease management tool for both primary and recurrent CDI [C. difficile toxin A (TcdA) and toxin B (TcdB) are undergoing clinical trials [C. difficile vaccination strategies. Current C. difficile vaccine candidates in clinical trials are administered via the parenteral route , which samples and processes antigens to induce adaptive immune responses . GALT isOne of the most important effector cell types in the mucosal immune response is the plasma cell, a terminally differentiated B cell that produces immunoglobulins (Ig) against invading pathogens . Tissue-C. difficile invasion is highly crucial, as the pathogenesis of this bacteria starts at the gut mucosal interface  secretion, causing the loss of epithelial barrier integrity. Toxin binding through respective receptors on intestinal epithelial cells leads to the disruption of the skeletal structure and the tight junctions, forming a leaky gut [C. difficile bacterium also prime the antigen-presenting cells, such as macrophages and dendritic cells (APCs). The APCs further interact with B cells and T cells to trigger humoral and cell-mediated immunity . The detailed immune response against the C. difficile invasion has been extensively reviewed elsewhere [Mucosal immunity against nterface . C. diffeaky gut . In respeaky gut . This inlsewhere ,56,62,66C. difficile strains induce Th1/Th2/Th17 and T-reg cell responses. Specifically, hypervirulent C. difficile R20291 induces a strong Th1 and Th17 response in terms of IFN-γ+ and IL-17A CD4 T cells compared to the non-virulent C. difficile 630 strain in co-cultured murine bone-marrow-derived dendritic cells (BMDCs) and splenocytes [C. difficile-infected patients showed a shift from Th1 to Th17 response or Th2 response with the increasing severity of the disease [Briefly, it has been shown that enocytes . Clinica disease . Further disease . Elevate disease . C. difficile-mediated pseudomembranous colitis is an inflammatory disease associated with the dysregulation of immune homeostasis. Mouse models of inflammatory diseases suggest the important role of regulatory T-cells (Treg) in ensuring proper immune system function [C. difficile infection.function . Upon enfunction . A detaifunction . TherefoC. difficile pathogenicity locus (PaLoc) codes for toxin A (TcdA) and toxin B (TcdB), the primary drivers of CDI symptoms [The symptoms . TcdB issymptoms , but botsymptoms . TcdA ansymptoms . This glsymptoms .C. difficile toxins is passive vaccination, whereby neutralizing antibodies are delivered directly into the body. The advantages of passive vaccination include the high specificity and low toxicity of purified antibodies [C. difficile, multiple studies have utilized animal-derived antibody preparations as passive mucosal vaccines [One method of mitigating the effects of tibodies . Regardivaccines ,78,79,80vaccines . Althougvaccines ,82.C. difficile toxins yielded promising results in animal models. A 1991 study demonstrated that intragastric vaccination of hamsters with bovine anti-TcdB and anti-TcdA polyclonal IgG antibodies provided complete protection from death [Initial studies of passive mucosal vaccination against om death . Buildinom death , and theom death . Besidesom death . Betweenom death  reportedOne of the disadvantages of passive mucosal vaccination, however, is that orally delivered antibodies can be degraded in the GI tract before they reach the site of infection ,84. HighC. difficile IgG cocktail in whey protein (CDIW) found that the vaccine was only equally as effective as metronidazole at preventing rCDI [Two human trials of passive anti-TcdA and anti-TcdB vaccines have been conducted to date, suggesting this vaccination strategy may be able to prevent recurrent CDI (rCDI). A human trial of the WPC-40 bovine antibody preparation provided full protection from CDI relapse for sixteen patients throughout the 333-day monitoring period with no major adverse effects . A latering rCDI . The stuC. difficile cells and a toxoid mixture, whereas the CDIW study only inoculated cows with inactivated C. difficile cells. Despite the authors’ hypotheses that CDIW might neutralize toxins, the poorer performance of CDIW relative to WPC-40 could have been due to lower toxin-neutralizing capabilities. Future studies into WPC-40, CDIW, or novel passive vaccines should thoroughly characterize the mechanism of protection in vitro to better understand their performance in human trials.One possible advantage of WPC-40 over CDIW is that WPC-40 contains a significant amount of secretory IgA (SIgA), which is known to play critical roles in mucosal immune responses . MoreoveC. difficile toxins, future studies could consider alternative strategies to overcome a number of limitations. As previously mentioned, vaccine antibodies must be protected from degradation in the GI tract. Fortunately, there are many existing technologies for encapsulation that could be evaluated in CDI models [Although the above studies show that animal-derived polyclonal antibodies could be harnessed for a passive mucosal vaccine against I models . SecondlAdditionally, the frequent inclusion of milk proteins (due to the use of cows to generate the antibodies in most studies) could present allergy restrictions. Monoclonal antibodies (mAbs) would seem ideal for tackling these problems, as manufacturing is animal-free and would not contain milk allergens. While anti-TcdB mAb (bezlotoxumab) has been approved for clinical use to prevent rCDI , both beLactobacilli, which naturally inhabit the human microbiome, can be safely consumed, resist degradation in the GI tract, and can be engineered to deliver drug payloads. Recently, Lactobacillus paracasei strains were developed that produced anti-TcdB variable domain heavy chain-only (VHH) antibodies either on the cell surface or as a surface-immobilized fragment [C. difficile infection (likely due to degradation), oral administration of Lactobacilli bearing surface-immobilized VHH fragments greatly improved hamster survival (50%) relative to negative controls (0%). Interestingly, the authors chose not to test VHH-secreting strains because the oral administration of VHH fragments failed at doses greater than what the engineered strains would produce in the gut. Future studies would be warranted to test if VHH-secreting strains could be effective, perhaps using more stable VHH fragments. It would also be interesting to examine how long these engineered bacteria persist in the gut, as this raises the possibility of establishing long-lasting protection against rCDI.Besides mAbs, other passive vaccination technologies could be explored. fragment . While oC. difficile toxins have been directed at intramuscularly-delivered toxoids, which previous reviews have examined [In contrast to providing a patient with anti-TcdA and anti-TcdB antibodies through passive vaccination, active vaccination stimulates the host immune system to produce antibodies in response to attenuated toxins (toxoids) or toxin fragments presented by the vaccine. Most efforts towards an active vaccine against examined ,95. To sexamined ,20 and texamined  have beeexamined  due to pexamined .To our knowledge, very few studies have attempted to administer toxoid or recombinant toxin fragments directly to mucosal surfaces. One study administered formalin-inactivated TcdA and TcdB to hamsters through a variety of mucosal and non-mucosal routes . The autC. difficile vectors, Ryan and colleagues orogastrically vaccinated rabbits with an engineered Vibrio cholerae strain that expressed a TcdA fragment [V. cholerae successfully colonized the rabbits’ guts, triggering a significant anti-TcdA IgG response. Vaccination offered some protection from TcdA damage in an ileal loop challenge assay, but increasing the toxin levels overwhelmed the limited protective effect of the vaccine [Salmonella typhimurium strain, expressing the C-terminal of TcdA, triggered significant IgG and IgA responses in mice upon intranasal or intragastric vaccination, with intranasal delivery outperforming intragastric delivery [S. typhimurium strain capable of expressing the receptor binding domains (RBDs) of TcdA and TcdB [As an alternative to administering toxoids at mucosal surfaces, multiple studies have used various bacterial vectors as toxoid delivery systems. In a 1997 publication on CDI vaccination using non-fragment . The eng vaccine . A few yand TcdB . The autand TcdB .Lactococcus lactis strain was engineered to express recombinant fragments of TcdA and TcdB [In a 2015 study, a and TcdB . Oral vaL. lactis mucosal vaccine shows great promise, and this can also be said for two more recent studies on bacterially-delivered TcdA/TcdB vaccines. In the first of these studies, Hong and colleagues modified Bacillus subtilis spores to express the TcdA C-terminal [C. difficile colonization, while an intramuscular toxoid vaccine did not [C. difficile [B. subtilis vaccine delivery platform, but they also showed that a TcdA antigen could induce antibody responses capable of neutralizing both TcdA and TcdB [Nonetheless, the 75% protection offered by terminal ,99. A co did not . Oral vaand TcdB ,99. ThisRegardless of whether toxin-based vaccines are administered via mucosal routes or through more traditional methods, there are a number of challenges that could hamper their ultimate effectiveness. Both animal models  and clinC. difficile toxins alone may not provide comprehensive protection against diverse strains, colonization, and multiple exposures. However, vaccinating against toxins and C. difficile surface components may provide the benefits of toxin neutralization along with greater colonization protection.Additionally, the long-run effectiveness of toxoid vaccines is uncertain. Anti-TcdB antibodies from recovered patients displayed moderate affinity and limited neutralization capabilities , so it iC. difficile membrane preparations. For example, rectal vaccination with a membrane preparation containing the adhesin Cwp66 and S-layer proteins reduced C. difficile colonization in mice [C. difficile membrane fraction (ntCDMF) as a rectal mucosal vaccine that was also protective in a mouse model [C. difficile membrane protein, it is unclear what specific antigens were protective in the study [C. difficile bacterium or spore in CDI patients generate IgG responses to cell–surface antigens , present in mice . Anotherse model . While nhe study ,108. OthC. difficile bacterium, and they are implicated in bacterial adhesion and immune activation [Surface-layer (S-layer) proteins (SLPs) cover the outside of the tivation . The S-ltivation ,112. Vactivation . Due to tivation . Both sttivation ,113. Sintivation , findingC. difficile encodes a number of SlpA paralogues classified as cell wall proteins (CWPs) [C. difficile [C. difficile to colonize animals despite mucosal vaccination with Cwp84 [s (CWPs) , which cs (CWPs) . The cels (CWPs) , is immus (CWPs) , and recs (CWPs) . A folloifficile . While rifficile . A laterifficile . Althougifficile . Moreoveth Cwp84  suggeststh Cwp84  C-terminth Cwp84 .C. difficile colonization [C. difficile adherence [C. difficile adherence by vaccine-induced antibodies [C. difficile surface components.Another more promising mucosal vaccine candidate is CD0873. This surface-localized lipoprotein is immunogenic and is thought to support nization ,119,120.dherence . This stdherence . A subsedherence . Not onltibodies . The autC. difficile colonization [C. difficile adhesion, is an understudied vaccine candidate [A few other surface protein candidates could be considered for further mucosal vaccine development. GroEL is a heat-shock protein that can be secreted or surface-expressed, and it is suspected to be involved with nization . Intrananization . Intrarenization . Lastly,andidate . Most CDandidate . Future C. difficile during infection [The flagellin FliC and flagellar cap FliD are involved with bacterial attachment . Comparenfection .C. difficile FliC in mice was immunogenic and offered complete protection from CDI [So far, only a few studies have tested FliC and FliD mucosal vaccines. Rectal administration of FliD generated the highest level of IgA and only slightly lower IgG levels compared with parenteral delivery in mice . Mice thfrom CDI , but mucC. difficile FliD with the B. subtilis spore coat proteins CotB, CotC, CotG, and CotZ [B. subtilis spores, paving the way for potential oral spore-based vaccines against FliD.It should be noted that FliC/FliD vaccine performance may vary widely depending on the delivery route. Despite the aforementioned successes with intrarectal vaccination, intranasal and intragastric FliD vaccination was not strongly immunogenic (even when the latter method was combined with antigen encapsulation) . Furtherand CotZ . These fC. difficile spores were shown to be reactive against strain R20291 spore components [C. difficile colonization.Numerous proteins are responsible for the spore coat structure, such as CotA, CotE, and CotCB, among others  Several mponents ,130. Altmponents , developC. difficile [Two potential spore antigens for mucosal vaccination include CdeC and CdeM . Both arifficile ,133. Oneifficile . Moreoveifficile . Future B. subtilis spores expressing the TcdA C-terminal [C. difficile  [C. difficile adherence to intestinal cells [Recent studies demonstrated, perhaps unintentionally, the promise of mucosal vaccines targeting spore proteins, such as CdeC. We previously discussed a study demonstrating the use of modified terminal ,99. Aftend LdhA) . Vaccineal cells . This woCTD) into mice. A second vaccine design was tested by adsorbing BclA2CTD to Bacillus subtilis (B. subtilis) spores [CTD triggered similar and significant IgG responses in mice after two immunizations, but neither vaccine blocked colonization or mitigated CDI symptoms in vivo.Unfortunately, other spore coat targets have proven more difficult to use for vaccine development. One study intranasally delivered the C-terminal domain of the spore surface protein BclA2 (BclA2) spores . Both frCTD) produced IgG responses in mice. This time, however, a B. subtilis spore-displayed BclA3CTD was less immunogenic than the protein alone. Administration of the free BclA3CTD vaccine reduced spore levels in murine feces slightly faster than controls and prevented weight loss, a noticeable improvement on the BclA2CTD study. However, the BclA3CTD vaccine was unable to reduce diarrhea prevalence, diarrhea severity, spore load in the gut, or toxin levels in feces. Another group observed similar results—that their intranasal, recombinant BclA3 vaccine could not mitigate symptoms, prevent colonization, or reduce the spore load [B. subtilis spores did not improve results [Similar strategies were applied to a recombinant BclA3 vaccine with only slightly better performance . Intranaore load . Express results . Since t results , this maC. difficile used in the test because RT027 strains express truncated BclA1 [C. difficile to bypass the effects of SleC vaccination [C. difficile mucosal vaccine. However, future research should consider the intranasal delivery methods presented in the BclA2 and BclA3 vaccination studies. Even though intranasal vaccination with spore coat proteins is not strongly protective based on current findings, additional refinement or the use of other antigens could yield an effective mucosal vaccine that could be easily administered in a clinical setting.Other antigens that have proven challenging so far include the exosporium protein BclA1 , the spoed BclA1 . Meanwhied BclA1 . Lastly,cination . Taken tC. difficile (NTCD) strain as a mucosal vaccine against CDI [C. difficile [Recently, our research group was the first to report an engineered non-toxigenic inst CDI . NTCD hainst CDI . In bothifficile . A cliniifficile . In our ifficile . In a subsequent publication, our group again proposed a new design for a modified NTCD vaccine expressing toxin subunits . For thiC. difficile to Caco-2 cells [Building off of our work on NTCD-based vaccines, another group sought to develop a modified NTCD vaccine that placed special emphasis on neutralizing colonization factors to block early pathogenesis . The NTC-2 cells . Since o-2 cells , this ra-2 cells ,146.C. difficile vaccination and IBD, there are no specific guidelines for how to apply preventative therapies against CDI specifically [C. difficile vaccines in IBD patients specifically. The incidence of primary and recurrent CDI is generally higher in immunocompromised populations  . Moreoveifically . More geifically . HoweverC. difficile vaccines in immunocompromised patients [C. difficile vaccines, then component vaccine technology would likely be easiest to use in both immunocompetent and immunocompromised patients. Fortunately, immunocompromised patients could rely on other treatments until a safe, effective vaccine is validated in vulnerable patient subgroups. For example, a recent systematic review of fecal microbiota transfer (FMT) found that 87% of immunocompromised patients recovered after only one round of FMT, with 93% of immunocompromised patients recovering after several rounds of therapy [Regarding immunocompromised patients, a systematic review of vaccine responses in diverse immunocompromised populations found that the effectiveness of vaccines varied by the exact nature of the immune deficiency. For example, patients with solid tumors or immune-mediated inflammatory diseases responded similarly to vaccines when compared with non-immunocompromised patients, whereas B-cell deficiencies were associated with poor responses . Regardipatients , and we  therapy . The fre therapy .C. difficile, effective vaccines are needed to prevent outbreaks. Several C. difficile antigens have been evaluated, thus far, as potential vaccine candidates, including toxins, surface proteins, spore proteins, and engineered C. difficile. Although current data suggest certain strengths and weaknesses in relation to these antigens , it does not appear that toxin-based vaccines can prevent colonization. Future designs need to address this weakness to halt the spread of CDI in healthcare facilities through asymptomatic carriage. Combining toxin antigens with surface antigens of vegetative cells and/or spores may be the most promising strategy to facilitate the clearance of C. difficile. The effectiveness of surface–antigen vaccine candidates against hypervirulent strains should also be given increased attention to determine if binary toxin antigens should be included in a CDI vaccine.While considerable groundwork has already been performed testing both passive and active vaccination against C. difficile, each method available has a number of advantages and downsides. While many animal models successfully made use of intrarectal delivery, patient compliance could be an issue in a clinical setting. Intranasal vaccination would be far easier to administer, but more work is needed to determine which C. difficile antigens will be most protective using this strategy. Oral delivery of antigens would also be simpler than intrarectal delivery, and multiple oral vaccines have shown promise in testing so far. Perhaps the greatest challenge of oral vaccination is preventing the degradation of antigens in the GI tract. Fortunately, either encapsulation or delivery by orally administered recombinant bacteria appears to mitigate the problem of degradation. Recombinant bacteria, especially non-toxigenic C. difficile, could combine the advantages of stimulating strong mucosal immunity with the added bonus of occupying the metabolic niche that toxigenic C. difficile would attempt to use upon colonization of the gut. Moreover, engineered NTCD strains can present multiple antigens simultaneously, simplifying the challenge of producing and delivering multiple recombinant proteins to the mucosa. Future efforts could explore the mechanisms of protection offered by engineered bacteria, as well as alternative delivery methods for expressing C. difficile antigens at mucosal surfaces.Regarding how to deliver a mucosal vaccine against