In mammals, neural crest cells populate the gut and form the enteric nervous system (ENS) early in embryogenesis. Although the basic ENS structure is highly conserved across species, we show important differences between mice and humans relating to the prenatal and postnatal development of mucosal enteric glial cells (mEGC), which are essential ENS components. We confirm previous work showing that in the mouse mEGCs are absent at birth, and that their appearance and homeostasis depends on postnatal colonization by microbiota. In humans, by contrast, a network of glial cells is already present in the fetal gut. Moreover, in xenografts of human fetal gut maintained for months in immuno-compromised mice, mEGCs persist following treatment with antibiotics that lead to the disappearance of mEGCs from the gut of the murine host. Single cell RNAseq indicates that human and mouse mEGCs differ not only in their developmental dynamics, but also in their patterns of gene expression.

The global spread of the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the pandemic outbreak of coronavirus disease 2019 (COVID-19), an inflammatory disease that is primarily affecting the respiratory system. However, gastrointestinal symptoms in COVID-19 patients suggests that the gut may present another viral target organ. Disease development and severity is dependent on viral interaction with two cell surface human proteins, ACE2 and TMPRSS2, and on antiviral response which may lead to systemic hyperinflammatory syndrome and multiorgan dysfunction. Understanding the host response to SARS-CoV-2 infection and the pathology of the disease will be greatly enhanced by the development of appropriate animal models. Laboratory mice have been the mainstay of therapeutic and vaccine development, however, the virus does not grow in wild type mice and only induced mild disease in transgenic animals expressing human ACE2. As there are known differences between immune response in laboratory mice and humans we evaluated the response of human gut developed as xenografts and host mouse gut following systemic LPS injections as a hyperinflammation model system. The orthologous gene expression levels in the mouse and human gut were highly correlated (Spearman’s rank correlation coefficient: 0.28–0.76) and gene set enrichment analysis of significantly upregulated human and mouse genes revealed that a number of inflammatory and immune response pathways are commonly regulated in the two species. However, species differences were also observed, most importantly, in the inflamed human gut but not in the mouse gut, there was clear upregulation of mRNAs coding for TMPRSS2, ADAM17 and for RIG-I-like receptors, which are involved in the recognition of viruses and in antiviral innate immune response. Moreover, using species-specific immunofluorescence microscopy, we demonstrated the expression and localization of human ACE2 and TMPRSS2 proteins, which are essential elements of the molecular machinery that enables SARS-CoV-2 to infect and replicate in human gut cells. Our findings demonstrate that the intestinal immune response to inflammation in humans and mice are generally very similar. However, certain human-specific diseases, such as COVID-19, can only be successfully studied in an experimental model of human tissue, such as the gut xenograft.Competing Interest StatementThe authors have declared no competing interest.

The single-epithelial cell layer of the gut mucosa serves as an essential barrier between the host and luminal microflora and plays a major role in innate immunity against invading pathogens. Nuclear factor kB (NF-κB), a central component of the cellular signaling machinery, regulates immune response and inflammation. NF-κB proteins are activated by signaling pathways downstream to microbial recognition receptors and cytokines receptors. Highly regulated NF-κB activity in intestinal epithelial cells (IEC) is essential for normal gut homeostasis; dysregulated activity has been linked to a number of disease states, including inflammatory bowel diseases (IBD) such as Crohn's Disease (CD). Our aim was to visualize and quantify spatial and temporal dynamics of NF-κB activity in steady state and inflamed human gut. Lentivirus technology was used to transduce the IEC of human gut xenografts in SCID mice with a NF-κB luminescence reporter system. NF-κB signaling was visualized and quantified using low resolution, intravital imaging of the whole body and high resolution, immunofluorescence microscopic imaging of the tissues. We show that NF-κB is activated in select subset of IEC with low "leaky" NF-κB activity. These unique inflammatory epithelial cells are clustered in the gut into discrete hotspots of NF-κB activity that are visible in steady state and selectively activated by systemic LPS and human TNFα or luminal bacteria. The presence of inflammatory hotspots in the normal and inflamed gut might explain the patchy mucosal lesions characterizing CD and thus could have important implications for diagnosis and therapy.

The single-epithelial cell layer of the gut mucosa serves as an essential barrier between the host and luminal microflora and plays a major role in innate immunity against invading pathogens. Nuclear factor kB (NF-kB), a central component of the cellular signaling machinery, regulates immune response and inflammation. NF-kB proteins are activated by signaling pathways downstream to microbial recognition receptors and cytokines receptors. Highly regulated NF-kB activity in intestinal epithelial cells (IEC) is essential for normal gut homeostasis; dysregulated activity has been linked to a number of disease states, including inflammatory bowel diseases (IBD) such as Crohns Disease (CD). Our aim was to visualize and quantify spatial and temporal dynamics of NF-kB activity in steady state and inflamed human gut. Lentivirus technology was used to transduce the IEC of human gut xenografts in SCID mice with a NF-kB luminescence reporter system. NF-kB signaling was visualized and quantified using low resolution, intravital imaging of the whole body and high resolution, immunofluorescence microscopic imaging of the tissues. We show that NF-kB is activated in select subset of IEC with low leaky NF-kB activity. These unique inflammatory epithelial cells are clustered in the gut into discrete hotspots of NF-kB activity that are visible in steady state and selectively activated by systemic LPS and human TNFa; or luminal bacteria. The presence of inflammatory hotspots in the normal and inflamed gut might explain the patchy mucosal lesions characterizing CD and thus could have important implications for diagnosis and therapy.Competing Interest StatementThe authors have declared no competing interest.

Mastitis, inflammation of the mammary gland, is a common disease of dairy animals. The disease is caused by bacterial infection ascending through the teat canal and mammary pathogenic Escherichia coli (MPEC) are common etiology. In the first phase of infection, virulence mechanisms, designated as niche factors, enable MPEC bacteria to resist innate antimicrobial mechanisms, replicate in milk, and to colonize the mammary gland. Next, massive replication of colonizing bacteria culminates in a large biomass of microbe-associated molecular patterns (MAMPs) recognized by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) mediating inflammatory signaling in mammary alveolar epithelial cells (MAEs) and macrophages. Bacterial lipopolysaccharides (LPSs), the prototypical class of MAMPs are sufficient to elicit mammary inflammation mediated by TLR4 signaling and activation of nuclear factor kB (NF-kB), the master regulator of inflammation. Using in vivo mastitis model, in low and high complements mice, and in vitro NF-kB luminescence reporter system in MAEs, we have found that the smooth configuration of LPS O-polysaccharides in MPEC enables the colonizing organisms to evade the host immune response by reducing inflammatory response and conferring resistance to complement. Screening a collection of MPEC field strains, we also found that all strains were complement resistant and 94% (45/48) were smooth. These results indicate that the structure of LPS O-polysaccharides chain is important for the pathogenesis of MPEC mastitis and provides protection against complement-mediated killing. Furthermore, we demonstrate a role for complement, a key component of innate immunity, in host-microbe interactions of the mammary gland.

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4β7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor alpha (RARA) and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. The pathway that regulates expression of integrin subunit alpha is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4β7, as well as CD62L and FOXP3, compared to cells incubated with rapamycin or rapamycin and ATRA. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labelled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4β7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. clinicaltrials.gov no: NCT03185000.

Neutrophil mobilization is a crucial response to protect the host against invading microorganisms. Neutrophil recruitment and removal have to be tightly regulated to prevent uncontrolled inflammation and excessive release of their toxic content causing tissue damage and subsequent organ dysfunctions. We show here the presence of live and apoptotic neutrophils in the cytoplasm of inflamed mammary, urinary and gall bladder epithelial cells following infection with E. coli and Salmonella bacteria. The entry process commenced with adherence of transmigrated neutrophils to the apical membrane of inflamed epithelial cells. Next, nuclear rearrangement and elongation associated with extensive actin polymerization enabled neutrophils to crawl and invaginate the apical membrane into cytoplasmic double membrane compartments. Scission of the invaginated cell membrane from the entry point and loss of these surrounding membranes released intracellular neutrophils into the cytoplasm where they undergone apoptotic death. The co-occurrence of this observation with bacterial invasion and formation of intracellular bacterial communities (IBCs) might link entry of infected neutrophils to the formation of IBCs and chronic carriage in E. coli mastitis and cystitis and Salmonella cholecystitis.

Post-partum metritis is among the most prevalent disease in dairy cows affecting animal welfare and inflicting considerable economic loses. While post-partum contamination of the uterus is rife in dairy cows, only a fraction of these animals will develop metritis. Our main objective was to compare the bacterial communities and the inflammatory response in the endometrium of healthy and metritic dairy cows. Holstein-Friesian cows (n = 35) were sampled immediately following clinical classification as healthy (n = 21), suffering from metritis (n = 13) or septic metritis (n = 1), based on veterinary examination at 5-10 days post-partum. Polymorphonuclear cells (PMN) percentage in endometrial cytology was significantly higher in cows with metritis. Full-thickness uterine biopsy analysis revealed that the luminal epithelium in inter-caruncle areas was preserved in healthy cows, but in metritis it was compromised, with marked PMN infiltration particularly in the apical endometrium. Gram staining revealed that bacterial load and spatial distribution was associated with disease severity. 16S-rDNA bacterial community analysis revealed unique endometrial bacterial community composition in metritic cows, as compared to more diverse communities among healthy cows. The most abundant phyla in healthy cows were Proteobacteria (31.8 +/- 9.3%), Firmicutes (27.9 +/- 8.4%) and Bacteroidetes (19.7 +/- 7.2%), while Bacteroidetes (60.3 +/- 10.3%), Fusobacteria (13.4 +/- 5.9%) and Firmicutes (10.5 +/- 3.3%) were most abundant in the endometrial mucosa of metritic cows. Relative abundance of Bacteroidetes (19.7 +/- 7.2% vs. 60.3 +/- 10.3%), Fusobacteria (7.5 +/- 5.2% vs. 13.4 +/- 5.9%) and Proteobacteria (31.8 +/- 9.3% vs. 7.3 +/- 5.6%) phyla differed significantly between healthy and metritic cows. In summary, endometrial PMN abundance, spatial distribution and bacterial communities differed between healthy and metritic dairy cows at early post-partum.

Background and Aims: Enteric fistulas represent a severe and medically challenging co-morbidity commonly affecting Crohn's disease [CD] patients. Gut fistulas do not develop in animal models of the disease. We have used transplantation of the human fetal gut into mice as a novel platform for studying inflammatory enterocutaneous fistulas. Methods: Human fetal gut segments were transplanted subcutaneously into mature SCID mice, where they grew and fully developed over the course of several months. We first analyzed the resident immune cells and inflammatory response elicited by systemic LPS in normal, fully developed human gut xenografts. Thereafter, we used immunostaining to analyze fully-developed xenografts that spontaneously developed enterocutaneous fistulas. Results: Resident human innate and adaptive immune cells were demonstrated in gut xenografts during steady state and inflammation. The expression of human IL-8, IL-1β, IL-6, TNF-α, A20 and IkBα was significantly elevated in response to LPS with no change in IL-10 gene expression. Approximately 17% [19/110] of fully developed subcutaneous human gut xenografts spontaneously developed enterocutaneous fistulas revealing striking histopathological similarities with CD fistula specimens. Immunohistochemical analyses of fistulating xenografts revealed transmural lymphocytic enteritis associated with massive expansion of resident human CD4 + lymphocytes and their migration into the intraepithelial compartment. Regionally, mucosal epithelial cells assumed spindle-shaped mesenchymal morphology and formed fistulous tracts towards chronic non-healing wounds in the host mouse skin overlying the transplants. Conclusions: Inflammation and fistulas developed in human gut xenografts lacking IL-10 gene response. This novel model system will enable systematic studies of the inflamed and fistulating human gut in live animals.

BACKGROUND: Current efforts to develop stem cell therapy as a novel treatment for neurointestinal diseases are limited by the unavailability of a model system to study cell transplantation in the human intestine. We propose that xenograft models support enteric nervous system (ENS) development in the fetal human intestine when transplanted into mice subcutaneously or intra-abdominally. METHODS: Fetal human small and large intestine were grafted onto the small intestinal mesentery and into the subcutaneous tissue of immunodeficient mice for up to 4 months. Intestinal cytoarchitecture and ENS development were studied using immunohistochemistry. KEY RESULTS: In both abdominal and subcutaneous grafts, the intestine developed normally with formation of mature epithelial and mesenchymal layers. The ENS was patterned in two ganglionated plexuses containing enteric neurons and glia, including cholinergic and nitrergic neuronal subtypes. c-Kit-immunoreactive interstitial cells of Cajal were present in the gut wall. CONCLUSIONS & INFERENCES: Abdominal xenografts represent a novel model that supports the growth and development of fetal human intestine. This in vivo approach will be a useful method to study maturation of the ENS, the pathophysiology of neurointestinal diseases, and the long-term survival and functional differentiation of neuronal stem cells for the treatment of enteric neuropathies.

Bovine necrotic vulvovaginitis (BNVV) is a severe and potentially fatal disease of post-partum cows that emerged in Israel after large dairy herds were merged. While post-partum cows are commonly affected by mild vulvovaginitis (BVV), in BNVV these benign mucosal abrasions develop into progressive deep necrotic lesions leading to sepsis and death if untreated. The etiology of BNVV is still unknown and a single pathogenic agent has not been found. We hypothesized that BNVV is a polymicrobial disease where the normally benign vaginal microbiome is remodeled and affects the local immune response. To this end, we compared the histopathological changes and the microbial communities using 16S rDNA metagenetic technique in biopsies taken from vaginal lesions in post-partum cows affected by BVV and BNVV. The hallmark of BNVV was the formation of complex polymicrobial communities in the submucosal fascia and abrogation of neutrophil recruitment in these lesions. Additionally, there was a marked difference in the composition of bacterial communities in the BNVV lesions in comparison to the benign BVV lesions. This difference was characterized by the abundance of Bacteroidetes and lower total community membership in BNVV. Indicator taxa for BNVV were Parvimonas, Porphyromonas, unclassified Veillonellaceae, Mycoplasma and Bacteroidetes, whereas unclassified Clostridiales was an indicator for BVV. The results support a polymicrobial etiology for BNVV.

Background: We have recently shown that Tregs isolated from the peripheral blood of patients with Crohn's Disease (CD) play a critical role in controlling both phenotype and expansion of auto-reactive T cells (1). This is a critical step to developing cell based therapy for Crohn's disease, where where primary and secondary non response rates to biologics remain unacceptably high. The immune system used retinoic acid (ATRA) to induce a4b7 and thus prime Tregs to home to the gut. We sought use ATRA in-vitro in order to engineer gut specific Tregs for our Phase 1 trial in Crohn's Disease. We then validated our findings in-vitro and in-vivo.Methods: Tregs were isolated from peripheral blood of CD patients. ATRA supplementation was tested in standard culture conditions. The expression of a4b7 was assessed by flow cytometry. Suppression assays were performed using autologous effector T cells (Teff). An ibidi flow chamber system coated with recombinant human MAdCAM-1 was used for in-vitro trafficking experiments. SCID mice xenografted with foetal intestinal small bowel were used for in-vivo experiments. Parametric and non-parametric data were calculated as the mean ± s.d. and median (IQR). For comparison of parametric and non-parametric data, t-test, or ANOVA were used.Results:Ex-vivo expansion of Tregs in the presence of ATRA significantly induced the expression of a4b7 compared to Rapamycin alone (5.57%±3.12 vs 82.8%±9.5, p=0.0057) Cells treated with Rapa+ATRA maintained their superior suppressive ability compared to Rapamycin treated Tregs (95.8%±3.5% vs 91.15%±10.1% p=ns; at Treg:Teff 1:1 ratio). RAPA+ATRA Tregs did not produce IFNy or IL17 under pro-inflammatory cytokine challenge. When flowed through a MAdCAm-1 coated chamber, significantly higher numbers of Rapa+ATRA treated cells were observed to roll (Rapa 0.83±0.40 vs Rapa+ATRA 10.17±2.54 p=0.005), crawl (Rapa 0 vs Rapa+ATRA 4±0.89 p=0.001) and firmly adhere (Rapa 0.33±0.21 vs Rapa+ATRA 36.8±1.78, p<0.001) than those treated with Rapa alone. When Tregs were transferred into mice, a higher proportion of Tregs were found in xenografts of animals treated with Rapa+ATRA Tregs compared Rapa Tregs (12.10 (7.54–22.83) vs 4.97 (1.72–7.63), p=0.0056). Importantly there was a higher proportion of Tregs in inflamed xenografts of animals treated with Rapa+ATRA Tregs compared to those treated with Rapa Tregs (18.35 (12.95–28.63) vs 6.78 (2.65–9.61), p=0.0095).Conclusions: The addition of ATRA to Treg culture in-vitro confers a gut homing phenotype. This is functionally relevant in-vitro and in-vivo. The treatment maintains the highly suppressive and phenotypically stable phenotype of these cells. These gut specific Tregs will be implemented in our first in man trial Treg therapy for Crohn's disease.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe intestinal disease and infant mortality in developing countries. Virulence is mediated by a type three secretion system (T3SS) causing the hallmark lesions of attaching and effacing (AE) and actin-rich pedestal formation beneath the infecting bacteria on the apical surface of enterocytes. EPEC is a human-specific pathogen whose pathogenesis cannot be studied in animal models. We therefore established an EPEC infection model in human gut xenografts in SCID mice and used it to study the role of T3SS in the pathogenesis of the disease. Following EPEC O127:H6 strain E2348/69 infection, T3SS-dependent AE lesions and pedestals were demonstrated in all infected xenografts. We report here the development of T3SS-dependent intestinal thrombotic microangiopathy (iTMA) and ischemic enteritis in approximately 50% of infected human gut xenografts. Using species-specific CD31 immunostaining, we showed that iTMA was limited to the larger human-mouse chimeric blood vessels which are located between the muscularis mucosa and circular muscular layer of the human gut. These blood vessels were massively invaded by bacteria which adhered to and formed pedestals on endothelial cells and aggregated with mouse neutrophils in the lumen. We conclude that endothelial infection, iTMA and ischemic enteritis might be central mechanisms underlying severe EPEC-mediated disease.

BACKGROUND AND AIM: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express alpha4beta7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

The bovine gastrointestinal tract is a natural reservoir for commensal and pathogenic Escherichia coli strains with the ability to cause mastitis. Here, we report the whole-genome sequences of six E. coli isolates from acute mastitis cases and six E. coli isolates from the feces of udder-healthy cows.

Lumpy skin disease (LSD) is a viral disease of cattle and buffalo, caused by a Capripox virus. A field study was performed during an LSD epidemic which occurred in 2012-2013 in Israel, in order to assess the efficacy of two commercial vaccines for protection against LSD. Fifteen dairy herds, vaccinated 2-5 months prior to study onset with a single dose of 10(2.5) TCID50 of RM65 attenuated sheep-pox vaccine, and not affected previously, were enrolled in the study. 4694 cows were randomized to be either vaccinated with a 10(3.5) TCID50/dose of RM65 vaccine (x10RM65) or with a same dose of an attenuated Neethling LSD virus vaccine. A case of LSD was defined as the appearance of at least 5 lesions typical to LSD and a severe case was defined if this sign was accompanied by either fever (>39.5 degrees C) or/and a 20% reduction in milk production. Deep lesion biopsies and blood samples were collected from 64.5% of the cases in an attempt to detect DNA of LSD virus by PCR and to differentiate between the wild strain and the vaccine Neethling strain. Seventy-six cows were affected by LSD in 8 herds with an incidence of 0.3-5.7%. Mantel-Haenszel relative risk (RRMH) for LSD morbidity at least 15 days after vaccination in x10RM65 vs. Neethling was 2.635 (CI95%=1.44-4.82) and 11.2 (2.3-54.7) for severe morbidity. RRMH for laboratory confirmed cases was 4.28 (1.59-11.53). An incidence of 0.38% (9/2356) of Neethling associated disease was observed among Neethling vaccinated cows while no such disease occurred in x10RM65 vaccinated cows. We conclude that the Neethling vaccine is significantly more effective than x10RM65 in preventing LSD morbidity, though it might cause a low incidence of Neethling associated disease. No transmission of the Neethling strain to non-Neethling vaccinated cows was observed in this study.

On dairy farms in hot climates worldwide, cows suffer from heat stress, which is alleviated by the use of water cooling systems. Sprinklers and showerheads are known to support the development of microbial biofilms, which can be a source of infection by pathogenic microorganisms. The aim of this study was to investigate the presence of microbial biofilms in dairy cooling systems, and to analyze their population compositions using culture-independent technique, 16S rRNA gene sequencing. Biofilm samples were collected on eight dairy farms from 40 sprinklers and the microbial constituents were identified by deep sequencing of the 16S rRNA gene. A total of 9,374 operational taxonomic units (OTUs) was obtained from all samples. The mean richness of the samples was 465 +/- 268 OTUs which were classified into 26 different phyla; 76% of the reads belonged to only three phyla: Proteobacteria, Actinobacteria and Firmicutes. Although the most prevalent OTUs (Paracoccus, Methyloversatilis, Brevundimonas, Porphyrobacter, Gp4, Mycobacterium, Hyphomicrobium, Corynebacterium and Clostridium) were shared by all farms, each farm formed a unique microbial pattern. Some known potential human and livestock pathogens were found to be closely related to the OTUs found in this study. This work demonstrates the presence of biofilm in dairy cooling systems which may potentially serve as a live source for microbial pathogens.