Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade.

BACKGROUND: Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear. METHODS: A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed. RESULTS: Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade. CONCLUSIONS: These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.

Longitudinal gut microbiome changes in immune checkpoint blockade-treated advanced melanoma.

Multiple clinical trials targeting the gut microbiome are being conducted to optimize treatment outcomes for immune checkpoint blockade (ICB). To improve the success of these interventions, understanding gut microbiome changes during ICB is urgently needed. Here through longitudinal microbiome profiling of 175 patients treated with ICB for advanced melanoma, we show that several microbial species-level genome bins (SGBs) and pathways exhibit distinct patterns from baseline in patients achieving progression-free survival (PFS) of 12 months or longer (PFS ≥12) versus patients with PFS shorter than 12 months (PFS <12). Out of 99 SGBs that could discriminate between these two groups, 20 were differentially abundant only at baseline, while 42 were differentially abundant only after treatment initiation. We identify five and four SGBs that had consistently higher abundances in patients with PFS ≥12 and <12 months, respectively. Constructing a log ratio of these SGBs, we find an association with overall survival. Finally, we find different microbial dynamics in different clinical contexts including the type of ICB regimen, development of immune-related adverse events and concomitant medication use. Insights into the longitudinal dynamics of the gut microbiome in association with host factors and treatment regimens will be critical for guiding rational microbiome-targeted therapies aimed at enhancing ICB efficacy.

Factor structure of the modified Rivermead Post-concussion Symptoms Questionnaire (mRPQ): an exploratory analysis with healthy adult simulators.

OBJECTIVE: Persistent postconcussion symptoms (PPCS) are challenging to diagnose. An improved diagnostic process could consider typical and atypical postconcussion symptoms. This study examined the structure of a modified Rivermead Post-concussion Symptoms Questionnaire (mRPQ) with both symptom types. METHOD: 298 adult volunteers were randomized into groups: honest responders, mild traumatic brain injury (mTBI) simulators (MS), and biased mTBI simulators (BMS). Both mTBI simulating groups were coached about mTBI and primed about the simulation context (compensation evaluation). The BMS group was also encouraged to bias (exaggerate) symptoms. The participants completed an online battery of tests, including the mRPQ. RESULTS: An exploratory factor analysis of the mRPQ (full sample) revealed a three-factor solution, including a separate dimension for atypical symptoms (all item loadings >0.45, ~4% of explained variance). The overall and group analyses of the standard RPQ items (typical symptoms) found a one- or two-factor solution, as did the analyses of atypical symptoms. CONCLUSIONS: Consistent with prior RPQ research, a unidimensional or bifactor structure was measurable from standard RPQ symptoms. Whilst this study did not find support for domain-level symptom scores for either typical or atypical symptoms, the findings support the use of an overall atypical symptoms score.

Validating the modified Rivermead Post-concussion Symptoms Questionnaire (mRPQ).

OBJECTIVE: Response distortions in the reporting of postconcussion symptoms can occur for many reasons. The Rivermead Post-concussion Symptoms Questionnaire (RPQ) was recently modified to include an embedded symptom validity indicator to test for atypical symptoms. The present study used a simulation design to investigate the psychometric properties of the modified RPQ (mRPQ). METHOD: 298 adult volunteers were randomised into three groups: honest responding (Controls, C) who reported actual, current symptoms; mild traumatic brain injury (mTBI) simulators (MS) who role played being injured, and; biased mTBI simulators (BMS) who role played being injured and were asked to bias (exaggerate) their response. The MS and BMS participants received instructions to support the simulation. All participants completed the mRPQ and a modified Neurobehavioral Symptom Inventory (mNSI). RESULTS: A 2 × 3 mixed ANOVA with one within-group variable (Symptom type: Standard or Atypical) and one between-group variable (Instruction type: C, MS, BMS) found a significant two-way interaction (p < .05, ηp2 = .08). CONCLUSIONS: The BMS group had score elevations for both standard and atypical postconcussion symptoms; therefore, both symptom types should be considered when evaluating for biased responding. The mRPQ has promising psychometric properties and should be further developed.

Comparative Genomics Provides Etiologic and Biological Insight into Melanoma Subtypes.

Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes. SIGNIFICANCE: This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma. This article is highlighted in the In This Issue feature, p. 2711.

Choroidal melanoma with synchronous Fuchs' adenoma and novel ATRX mutation.

BACKGROUND: To report a case of Fuchs' adenoma occurring in an eye with a large choroidal melanoma. We have reviewed the literature to describe the clinical presentation, ultrasound characteristics and pathological features of these entities. CASE PRESENTATION: A 69-year-old Caucasian man presented with vision loss from a large choroidal melanoma. Enucleation showed an incidental Fuchs' adenoma in the same eye. Whole-exome sequence analysis was also performed on the patient's blood and melanoma, which showed a rarely-reported ATRX mutation. CONCLUSIONS: Fuchs' adenoma is an under-diagnosed benign age-related hyperplasia of the non-pigmented ciliary epithelium (NPCE). Given its location and characteristics, it can be mistaken for choroidal melanoma and clinicians are reminded how to differentiate between these pathologies and that they may co-exist.

Evaluation of the 4-point regional nerve block using 2% lidocaine in sheep.

PURPOSE: To determine whether a single 4-point regional nerve block using 2% lidocaine administered distal to the fetlock of sheep with a single distal limb lameness will result in analgesia of the digits. ANIMALS: Eighteen adult ewes with a single limb lameness originating from distal to the metacarpo/metatarsophalangeal joint were enrolled in the study. PROCEDURES: Digital lameness was confirmed and scored based on clinical examination. Pain associated with digital lesions was assessed in triplicate using a pressure algometer to quantify mechanical nociceptive threshold. The same procedure was repeated on the contralateral limb as a control, and maximum force and time to response recorded. A 4-point regional nerve block was performed using 8 mL of 2% lidocaine. Mechanical nociception was again applied in triplicate to both limbs as described above, by a blinded investigator. Following appropriate medical treatment, the ewe was released and lameness scoring repeated.Median values for pressure and time to withdrawal were determined for affected and control limbs, and differences between pre- and post-lidocaine block measures were compared using Friedman's ANOVA test. The Wilcoxon Signed-Rank test was used to compare lameness score pre- and post-block. Statistical significance was set at α = 0.05. MAIN FINDINGS: Application of the 4-point block resulted in a change in pressure required to elicit withdrawal (F-value 17.7; P < 0.0001) as well as time to withdrawal (F-value 20.4; P < 0.0001), for the affected limb as compared to the control limb. Lameness scores decreased following the block (Signed-rank statistic 85.5; P < 0.0001). PRINCIPAL CONCLUSION: The 4-point nerve block resulted in anesthesia of the distal limb in sheep in this clinical model.

Évaluation du bloc nerveux régional en quatre points avec de la lidocaïne à 2 % chez le mouton. OBJECTIF: Déterminer si un seul bloc nerveux régional en quatre points utilisant de la lidocaïne à 2 % administrée distalement du boulet d'un mouton présentant une boiterie d'un seul membre distal entraînera une analgésie des doigts. ANIMAUX: Dix-huit brebis adultes avec une boiterie d'un seul membre provenant de la partie distale de l'articulation métacarpo/métatarsophalangienne ont été incluses dans l'étude. PROCÉDURES: La boiterie digitale a été confirmée et notée sur la base d'un examen clinique. La douleur associée aux lésions digitales a été évaluée en triple à l'aide d'un algomètre à pression pour quantifier le seuil nociceptif mécanique. La même procédure a été répétée sur le membre controlatéral en tant que témoin, et la force maximale et le temps de réponse ont été enregistrés. Un bloc nerveux régional en quatre points a été réalisé avec 8 ml de lidocaïne à 2 %. La nociception mécanique a de nouveau été appliquée en triple exemplaire aux deux membres comme décrit ci-dessus, par un chercheur en aveugle. Suite à un traitement médical approprié, la brebis a été relâchée et le score de boiterie répété.Les valeurs médianes de la pression et du temps de retrait ont été déterminées pour les membres affectés et les membres témoins, et les différences entre les mesures du bloc avant et après le bloc de lidocaïne ont été comparées à l'aide du test ANOVA de Friedman. Le test de Wilcoxon (signed-rank) a été utilisé pour comparer le score de boiterie avant et après le bloc. Le seuil de signification statistique a été fixé à α = 0,05. PRINCIPAUX RÉSULTATS: L'utilisation du bloc à quatre points a entraîné une modification de la pression requise pour déclencher le retrait (valeur F 17,7; P < 0,0001) ainsi que du temps de retrait (valeur F 20,4; P < 0,0001), pour les membres affectées par rapport au membres témoins. Les scores de boiterie ont diminué après le bloc (statistique de Signed-rank 85,5; P < 0,0001). CONCLUSION PRINCIPALE: Le bloc nerveux en quatre points a entraîné une anesthésie du membre distal chez le mouton dans ce modèle clinique.(Traduit par Dr Serge Messier).

Cross-cohort gut microbiome associations with immune checkpoint inhibitor response in advanced melanoma.

The composition of the gut microbiome has been associated with clinical responses to immune checkpoint inhibitor (ICI) treatment, but there is limited consensus on the specific microbiome characteristics linked to the clinical benefits of ICIs. We performed shotgun metagenomic sequencing of stool samples collected before ICI initiation from five observational cohorts recruiting ICI-naive patients with advanced cutaneous melanoma (n = 165). Integrating the dataset with 147 metagenomic samples from previously published studies, we found that the gut microbiome has a relevant, but cohort-dependent, association with the response to ICIs. A machine learning analysis confirmed the link between the microbiome and overall response rates (ORRs) and progression-free survival (PFS) with ICIs but also revealed limited reproducibility of microbiome-based signatures across cohorts. Accordingly, a panel of species, including Bifidobacterium pseudocatenulatum, Roseburia spp. and Akkermansia muciniphila, associated with responders was identified, but no single species could be regarded as a fully consistent biomarker across studies. Overall, the role of the human gut microbiome in ICI response appears more complex than previously thought, extending beyond differing microbial species simply present or absent in responders and nonresponders. Future studies should adopt larger sample sizes and take into account the complex interplay of clinical factors with the gut microbiome over the treatment course.

Definition of Biologically Distinct Groups of Conjunctival Melanomas According to Etiological Factors and Implications for Precision Medicine.

Conjunctival melanoma (ConjMel) is a potentially deadly ocular melanoma, originating from partially sunlight-exposed mucosa. We explored the mutational landscape of ConjMel and studied the correlation with etiological factors. We collected 47 primary ConjMel samples and performed next-generation sequencing of 400 genes. Hotspot mutations in BRAF, NRAS, HRAS, and KIT were observed in 16 (34%), 5 (11%), 2, and 2 cases, respectively. Patients with BRAF and CDKN2A-mutated ConjMel tended to be younger while the NF1-mutated one tended to be older. The eight tumors arising from nevi were enriched in CTNNB1 mutations (63% vs. 8%; Fisher's exact p-test = 0.001) compared to non-nevi ConjMel and five were devoid of BRAF, RAS, NF1, or KIT mutations, suggesting a specific oncogenic process in these tumors. The two KIT-mutated cases carried SF3B1 mutations and were located on sun-protected mucosa, a genotype shared with genital and anorectal mucosal melanomas. Targetable mutations were observed in ERBB2, IDH1, MET, and MAP2K1 (one occurrence each). Mutational landscape of ConjMel characterizes distinct molecular subtypes with oncogenic drivers common with mucosal and skin melanomas. CTNNB1 mutations were associated with nevus-derived ConjMel. Concomitant KIT/SF3B1 mutations in sun-protected cases suggest a common tumorigenic process with genital and anorectal mucosal melanomas.

Characterization of Efficacy and Animal Safety across Four Caprine Disbudding Methodologies.

There is a strong industry demand for technically simple and highly efficacious alternatives to heat cautery disbudding in goat kids that can be performed as a stand-alone procedure without adjunct anesthesia, and that result in improved overall welfare through reduced acute pain, reduced tissues healing interval, and a consistent safety record. The objective of this study was to consider the net effect of disbudding techniques on goat welfare by examining vocalization frequency, long-term efficacy and animal safety associated with four alternative caprine disbudding methods against sham-disbudded and heat-cautery controls. Sixty-five commercial male dairy kids were disbudded at 3-10 days of age with one of six disbudding treatments (clove oil injection, caustic paste, two cryosurgical methods, heat-cautery, and sham procedure). Heat cautery was 91% effective, caustic paste was 55% effective, and the other treatments were ineffective. Heat cautery and sham procedures resulted in similar vocalization efforts; freezing with a liquid-nitrogen cooled iron resulted in significantly greater vocalization numbers. No unintended paste transfer injuries were observed with short-term application of the caustic paste. Heat cautery resulted in numerous superficial infections but no permanent injury. Clove oil injection was associated with several unexpected and severe complications including unintended tissue necrosis, temporary paresis, skull defects, meningitis, and death. Collectively, we did not find that any of the alternative methods of disbudding provided a feasible option over heat cautery to improve welfare.

Ultraviolet radiation drives mutations in a subset of mucosal melanomas.

Although identified as the key environmental driver of common cutaneous melanoma, the role of ultraviolet radiation (UVR)-induced DNA damage in mucosal melanoma is poorly defined. We analyze 10 mucosal melanomas of conjunctival origin by whole genome sequencing and our data shows a predominance of UVR-associated single base substitution signature 7 (SBS7) in the majority of the samples. Our data shows mucosal melanomas with SBS7 dominance have similar genomic patterns to cutaneous melanomas and therefore this subset should not be excluded from treatments currently used for common cutaneous melanoma.

Genomic analysis of adult case of ocular surface giant congenital melanocytic nevus and associated clinicopathological findings.

INTRODUCTION: Conjunctival nevi are the most common tumor of the ocular surface. There are some rare reports of so-called 'giant' conjunctival nevi. We report a case of a 47-year-old female with a cutaneous and ocular surface giant congenital melanocytic nevus and describe her clinical course. CASE DESCRIPTION: This is a retrospective case report of a single patient. A 47-year-old female with a history of biopsy-proven periorbital congenital melanocytic nevus, with an associated giant conjunctival nevus presented for structural and functional rehabilitation. Serial surgeries were performed and excised tissue was sent for histopathological and genetic examination. The conjunctival nevus had a low tumor mutation burden, and of the 647 somatic mutations, only one occurred within a protein coding region, namely NRAS p.Gln61Arg. CONCLUSION: This is the first reported adult case including genomic analysis of an ocular surface giant congenital melanocytic nevus. The case shows a possible association between periorbital congenital melanocytic nevi and giant conjunctival nevi, and underscores the possible role that targeted drug therapies may have in malignant transformation of these conditions.

Non-specific protection from respiratory tract infections in cattle generated by intranasal administration of an innate immune stimulant.

Alternatives to antibiotics for prevention of respiratory tract infections in cattle are urgently needed given the increasing public and regulatory pressure to reduce overall antibiotic usage. Activation of local innate immune defenses in the upper respiratory tract is one strategy to induce non-specific protection against infection with the diverse array of viral and bacterial pathogens associated with bovine respiratory disease complex (BRDC), while avoiding the use of antibiotics. Our prior studies in rodent models demonstrated that intranasal administration of liposome-TLR complexes (LTC) as a non-specific immune stimulant generated high levels of protection against lethal bacterial and viral pathogens. Therefore, we conducted studies to assess LTC induction of local immune responses and protective immunity to BRDC in cattle. In vitro, LTC were shown to activate peripheral blood mononuclear cells in cattle, which was associated with secretion of INFγ and IL-6. Macrophage activation with LTC triggered intracellular killing of Mannheimia hemolytica and several other bacterial pathogens. In studies in cattle, intranasal administration of LTC demonstrated dose-dependent activation of local innate immune responses in the nasopharynx, including recruitment of monocytes and prolonged upregulation (at least 2 weeks) of innate immune cytokine gene expression by nasopharyngeal mucosal cells. In a BRDC challenge study, intranasal administration of LTC prior to pathogen exposure resulted in significant reduction in both clinical signs of infection and disease-associated euthanasia rates. These findings indicate that intranasal administration of a non-specific innate immune stimulant can be an effective method of rapidly generating generalized protection from mixed viral and bacterial respiratory tract infections in cattle.

Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE).

Germline variants in oculocutaneous albinism genes and predisposition to familial cutaneous melanoma.

Approximately 1%-2% of cutaneous melanoma (CM) is classified as strongly familial. We sought to investigate unexplained CM predisposition in families negative for the known susceptibility genes using next-generation sequencing of affected individuals. Segregation of germline variants of interest within families was assessed by Sanger sequencing. Several heterozygous variants in oculocutaneous albinism (OCA) genes: TYR, OCA2, TYRP1 and SLC45A2, were present in our CM cohort. OCA is a group of autosomal recessive genetic disorders, resulting in pigmentation defects of the eyes, hair and skin. Missense variants classified as pathogenic for OCA were present in multiple families and some fully segregated with CM. The functionally compromised TYR p.T373K variant was present in three unrelated families. In OCA2, known pathogenic variants: p.V443I and p.N489D, were present in three families and one family, respectively. We identified a likely pathogenic SLC45A2 frameshift variant that fully segregated with CM in a family of four cases. Another four-case family harboured cosegregating variants (p.A24T and p.R153C) of uncertain functional significance in TYRP1. We conclude that rare, heterozygous variants in OCA genes confer moderate risk for CM.

Correction to: Prolonged stable disease in a uveal melanoma patient with germline MBD4 nonsense mutation treated with pembrolizumab and ipilimumab.

The authors regret that the online version of this article contains an error. The MBD4 mutation in sample MM138 was given an incorrect dbSNP ID. The correct ID is rs769076971.

A phase 2 trial of sunitinib in patients with progressive paraganglioma or pheochromocytoma: the SNIPP trial.

BACKGROUND: Pheochromocytoma (PCC) and paraganglioma (PGL) are uncommon neoplasms with high morbidity in advanced stages. Effective systemic treatments are limited. METHODS: A multisite phase 2 trial evaluated sunitinib in patients with progressive PCC/PGL. Patients received 50 mg orally for 4-6 weeks. RESULTS: Between May 2009 and May 2016, 25 patients were enroled. The median age was 50 years and 56% were male. Three patients (12%) received prior chemotherapy and 16 (64%) prior surgery. The DCR was 83% (95% CI: 61-95%) and median PFS 13.4 (95% CI: 5.3-24.6) months. Of 23 evaluable patients, 3 (13%) with germline mutations (SDHA, SDHB, RET) achieved a PR. The patient with mutated RET and MEN2A remains on treatment after 64 cycles. The median time on treatment was 12.4 (1-88.0) months. Grade 3 or 4 toxicities were as expected and manageable; fatigue (16%) and thrombocytopenia (16%) were most common. One patient with grade 3 hypertension and 2 with grade 3 cardiac events discontinued treatment. CONCLUSION: Although the primary endpoint of disease control was met, the overall response rate of sunitinib was low in unselected patients with progressive PCC/PGL. Patients with germline variants in RET or in the subunits of SDH may derive greatest benefit.

High Prevalence of Fluoroquinolone-Resistant Campylobacter Bacteria in Sheep and Increased Campylobacter Counts in the Bile and Gallbladders of Sheep Medicated with Tetracycline in Feed.

Campylobacter is a major foodborne pathogen in humans and a significant cause of abortion in sheep. Although ruminants are increasingly recognized as important reservoirs for Campylobacter species, limited information is available about the molecular epidemiology and antimicrobial resistance (AMR) profiles of sheep Campylobacter Here, we describe a two-trial study that examined Campylobacter profiles in sheep and determined whether in-feed tetracycline (TET) influenced the distribution and AMR profiles of Campylobacter Each trial involved 80 commercial sheep naturally infected with Campylobacter: 40 of these sheep were medicated with tetracycline in feed, while the other 40 received feed without antibiotics. Fecal and bile samples were collected for the isolation of Campylobacter The bacterial isolates were analyzed for antimicrobial susceptibility and genotypes. The results revealed that 87.0% and 61.3% of the fecal and bile samples were positive for Campylobacter (Campylobacter jejuni and Campylobacter coli), with no significant differences between the medicated and nonmedicated groups. All but one of the tested Campylobacter isolates were resistant to tetracycline. Although fluoroquinolone (FQ) resistance remained low in C. jejuni (1.7%), 95.0% of the C. coli isolates were resistant to FQ. Genotyping revealed that C. jejuni sequence type 2862 (ST2862) and C. coli ST902 were the predominant genotypes in the sheep. Feed medication with tetracycline did not affect the overall prevalence, species distribution, and AMR profiles of Campylobacter, but it did increase the total Campylobacter counts in bile and gallbladder. These findings identify predominant Campylobacter clones, reveal the high prevalence of FQ-resistant C. coli, and provide new insights into the epidemiology of Campylobacter in sheep.IMPORTANCECampylobacter is a major cause of foodborne illness in humans, and antibiotic-resistant Campylobacter is considered a serious threat to public health in the United States and worldwide. As a foodborne pathogen, Campylobacter commonly exists in the intestinal tract of ruminant animals, such as sheep and cattle. Results from this study reveal the predominant genotypes and high prevalence of tetracycline (TET) and fluoroquinolone (FQ) resistance in sheep Campylobacter The finding on fluoroquinolone resistance in sheep Campylobacter is unexpected, as this class of antibiotics is not used for sheep in the United States, and it may suggest the transmission of fluoroquinolone-resistant Campylobacter from cattle to sheep. Additionally, the results demonstrate that in-feed medication with tetracycline increases Campylobacter counts in gallbladders, suggesting that the antibiotic promotes Campylobacter colonization of the gallbladder. These findings provide new information on Campylobacter epidemiology in sheep, which may be useful for curbing the spread of antibiotic-resistant Campylobacter in animal reservoirs.