Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Natural pathology of the Baboon (Papio spp.).

BACKGROUND: Baboons are useful animal models for biomedical research, but the natural pathology of the baboon is not as well defined as other non-human primates. METHODS: A computer search for all morphologic diagnoses from baboon necropsies at the Southwest National Primate Research Center was performed and included all the natural deaths and animals euthanized for natural causes. RESULTS: A total of 10,883 macroscopic or microscopic morphologic diagnoses in 4297 baboons were documented and are presented by total incidence, relative incidence by sex and age-group, and mean age of occurrence. The most common diagnoses in descending order of occurrence were hemorrhage, stillborn, amyloidosis, colitis, spondylosis, and pneumonia. The systems with the most diagnoses were the digestive, urogenital, musculoskeletal, and respiratory. CONCLUSION: This extensive evaluation of the natural pathology of the baboon should be an invaluable biomedical research resource.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Fatal acute Chagas disease in a chimpanzee.

BACKGROUND: Chagas disease (CD) or American trypanosomiasis is caused by a hemoflagellate protozoan, Trypanosoma cruzi. This organism has been isolated from more than 100 mammalian species and several insect vectors demonstrating a wide host distribution and low host specificity. METHODS: A 23-year-old male chimpanzee died acutely and a complete necropsy was performed to evaluate gross and microscopic pathologic changes. After observation of trypanosomal amastigotes in the myocardium, PCR and immunohistochemistry was employed to confirm the diagnosis of T. cruzi. RESULTS: Gross findings were consistent with mild congestive heart failure. Microscopic findings included multifocal myocardial necrosis associated with severe lymphocytic to mixed inflammatory infiltrates, edema, and mild chronic interstitial fibrosis. Multifocal intracytoplasmic amastigotes morphologically consistent with T. cruzi were observed in cardiac myofibers. Trypanosoma cruzi was confirmed by PCR and immunohistochemistry. CONCLUSION: We report, to the best of our knowledge, the first fatal spontaneous case of T. cruzi infection in a chimpanzee.

Fowlicidin-3 is an alpha-helical cationic host defense peptide with potent antibacterial and lipopolysaccharide-neutralizing activities.

Cathelicidins are an important family of cationic host defense peptides in vertebrates with both antimicrobial and immunomodulatory activities. Fowlicidin-1 and fowlicidin-2 are two newly identified chicken cathelicidins with potent antibacterial activities. Here we report structural and functional characterization of the putatively mature form of the third chicken cathelicidin, fowlicidin-3, for exploration of its therapeutic potential. NMR spectroscopy revealed that fowlicidin-3 comprises 27 amino-acid residues and adopts a predominantly alpha-helical structure extending from residue 9 to 25 with a slight kink induced by a glycine at position 17. It is highly potent against a broad range of Gram-negative and Gram-positive bacteria in vitro, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range 1-2 microM. It kills bacteria quickly, permeabilizing cytoplasmic membranes immediately on coming into contact with them. Unlike many other host defense peptides with antimicrobial activities that are diminished by serum or salt, fowlicidin-3 retains bacteria-killing activities in the presence of 50% serum or physiological concentrations of salt. Furthermore, it is capable of suppressing lipopolysaccharide-induced expression of proinflammatory genes in mouse macrophage RAW264.7 cells, with nearly complete blockage at 10 microM. Fowlicidin-3 appears to be an excellent candidate for future development as a novel antimicrobial and antisepsis agent, particularly against antibiotic-resistant pathogens.

Structure-activity relationships of fowlicidin-1, a cathelicidin antimicrobial peptide in chicken.

Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti-infective agents. We recently identified three cathelicidin antimicrobial peptides from chicken, which have potent and broad-spectrum antibacterial activities in vitro (Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) J Biol Chem281, 2858-2867). Here we report that fowlicidin-1 mainly adopts an alpha-helical conformation with a slight kink induced by glycine close to the center, in addition to a short flexible unstructured region near the N terminus. To gain further insight into the structural requirements for function, a series of truncation and substitution mutants of fowlicidin-1 were synthesized and tested separately for their antibacterial, cytolytic and lipopolysaccharide (LPS)-binding activities. The short C-terminal helical segment after the kink, consisting of a stretch of eight amino acids (residues 16-23), was shown to be critically involved in all three functions, suggesting that this region may be required for the peptide to interact with LPS and lipid membranes and to permeabilize both prokaryotic and eukaryotic cells. We also identified a second segment, comprising three amino acids (residues 5-7) in the N-terminal flexible region, that participates in LPS binding and cytotoxicity but is less important in bacterial killing. The fowlicidin-1 analog, with deletion of the second N-terminal segment (residues 5-7), was found to retain substantial antibacterial potency with a significant reduction in cytotoxicity. Such a peptide analog may have considerable potential for development as an anti-infective agent.

Immune regulatory activities of fowlicidin-1, a cathelicidin host defense peptide.

Appropriate modulation of immunity is beneficial in antimicrobial therapy and vaccine development. Host defense peptides (HDPs) constitute critically important components of innate immunity with both antimicrobial and immune regulatory activities. We previously showed that a chicken HDP, namely fowlicidin-1(6-26), has potent antibacterial activities in vitro and in vivo. Here we further revealed that fowl-1(6-26) possesses strong immunomodulatory properties. The peptide is chemotactic specifically to neutrophils, but not monocytes or lymphocytes, after injected into the mouse peritoneum. Fowl-1(6-26) also has the capacity to activate macrophages by inducing the expression of inflammatory mediators including IL-1ß, CCL2, and CCL3. However, unlike bacterial lipopolysaccharide that triggers massive production of inflammatory cytokines and chemokines, fowl-1(6-26) only marginally increased their expression in mouse RAW264.7 macrophages. Additionally, fowl-1(6-26) enhanced the surface expression of MHC II and CD86 on RAW264.7 cells, suggesting that it may facilitate development of adaptive immune response. Indeed, co-immunization of mice with chicken ovalbumin (OVA) and fowl-1(6-26) augmented both OVA-specific IgG1 and IgG2a titers, relative to OVA alone. We further showed that fowl-1(6-26) is capable of preventing a methicillin-resistant Staphylococcus aureus (MRSA) infection due to its enhancement of host defense. All mice survived from an otherwise lethal infection when the peptide was administered 1-2 days prior to MRSA infection, and 50% mice were protected if receiving the peptide 4 days before infection. Taken together, with a strong capacity to stimulate innate and adaptive immunity, fowl-1(6-26) may have potential to be developed as a novel antimicrobial and a vaccine adjuvant.

Tissue expression and developmental regulation of chicken cathelicidin antimicrobial peptides.

Cathelicidins are a major family of antimicrobial peptides present in vertebrate animals with potent microbicidal and immunomodulatory activities. Four cathelicidins, namely fowlicidins 1 to 3 and cathelicidin B1, have been identified in chickens. As a first step to understand their role in early innate host defense of chickens, we examined the tissue and developmental expression patterns of all four cathelicidins. Real-time PCR revealed an abundant expression of four cathelicidins throughout the gastrointestinal, respiratory, and urogenital tracts as well as in all primary and secondary immune organs of chickens. Fowlicidins 1 to 3 exhibited a similar tissue expression pattern with the highest expression in the bone marrow and lung, while cathelicidin B1 was synthesized most abundantly in the bursa of Fabricius. Additionally, a tissue-specific regulatory pattern was evident for all four cathelicidins during the first 28 days after hatching. The expression of fowlicidins 1 to 3 showed an age-dependent increase both in the cecal tonsil and lung, whereas all four cathelicidins were peaked in the bursa on day 4 after hatching, with a gradual decline by day 28. An abrupt augmentation in the expression of fowlicidins 1 to 3 was also observed in the cecum on day 28, while the highest expression of cathelicidin B1 was seen in both the lung and cecal tonsil on day 14. Collectively, the presence of cathelicidins in a broad range of tissues and their largely enhanced expression during development are suggestive of their potential important role in early host defense and disease resistance of chickens.

Butyrate enhances disease resistance of chickens by inducing antimicrobial host defense peptide gene expression.

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. In this study, we tested the hypothesis that exogenous administration of butyrate, a major type of short-chain fatty acids derived from bacterial fermentation of undigested dietary fiber, is capable of inducing HDPs and enhancing disease resistance in chickens. We have found that butyrate is a potent inducer of several, but not all, chicken HDPs in HD11 macrophages as well as in primary monocytes, bone marrow cells, and jejuna and cecal explants. In addition, butyrate treatment enhanced the antibacterial activity of chicken monocytes against Salmonella enteritidis, with a minimum impact on inflammatory cytokine production, phagocytosis, and oxidative burst capacities of the cells. Furthermore, feed supplementation with 0.1% butyrate led to a significant increase in HDP gene expression in the intestinal tract of chickens. More importantly, such a feeding strategy resulted in a nearly 10-fold reduction in the bacterial titer in the cecum following experimental infections with S. enteritidis. Collectively, the results indicated that butyrate-induced synthesis of endogenous HDPs is a phylogenetically conserved mechanism of innate host defense shared by mammals and aves, and that dietary supplementation of butyrate has potential for further development as a convenient antibiotic-alternative strategy to enhance host innate immunity and disease resistance.

A fowlicidin-1 analog protects mice from lethal infections induced by methicillin-resistant Staphylococcus aureus.

Fowlicidin-1 is a newly identified alpha-helical cathelicidin host defense peptide. We have shown that fowlicidin-1 possesses potent antibacterial activity, but also displays considerable toxicity toward mammalian cells. To further identify fowlicidin-1 analog(s) with enhanced therapeutic potential, a series of amino-terminal truncation analogs were synthesized and functionally evaluated. Relative to the full-length peptide, fowl-1(6-26), an analog with omission of five amino-terminal amino acid residues, maintained the antibacterial potency against a range of Gram-negative and Gram-positive bacteria including antibiotic-resistant strains. Fowl-1(6-26)-NH(2), a carboxyl-terminal amidated form of fowl-1(6-26), retained the antibacterial activity for a minimum of 2h in the presence of 100% serum. In addition, an intraperitoneal administration of 10mg/kg of fowl-1(6-26)-NH(2) led to a 50% increase in the survival of neutropenic mice over a 7-day period from a lethal dose of methicillin-resistant Staphylococcus aureus (MRSA), concomitant with a reduction in the bacterial titer in both peritoneal fluids and spleens of mice 24h post-infection. Fowl-1(6-26)-NH(2) at 20 microM was further found to suppress lipopolysaccharide-mediated production of TNF-alpha and nitric oxide in macrophages by 77% and 96%, respectively. Therefore, with potent endotoxin-neutralizing and bactericidal activities, fowlicidin-1(6-26)-NH(2), may have strong therapeutic potential for drug-resistant infections and sepsis.

The central kink region of fowlicidin-2, an alpha-helical host defense peptide, is critically involved in bacterial killing and endotoxin neutralization.

Fowlicidins are a group of newly identified chicken cathelicidin host defense peptides. We have shown that the putatively mature fowlicidin-2 of 31 amino acid residues possesses potent antibacterial and lipopolysaccharide (LPS)- neutralizing activities, but with a noticeable toxicity to mammalian cells. As a first step in exploring the structure-activity relationships of fowlicidin-2, in this study we determined its tertiary structure by nuclear magnetic resonance spectroscopy. Unlike the majority of cathelicidins, which are composed of a predominant alpha-helix with a short hinge sequence near the center, fowlicidin-2 consists of 2 well-defined alpha-helical segments (residues 6-12 and 23-27) connected by a long extensive kink (residues 13-20) induced by proline. To further investigate the functional significance of each of these structural components, several N- and C-terminal deletion analogs of fowlicidin-2 were synthesized and analyzed for their antibacterial, cytotoxic and LPS-neutralizing activities. Our results indicated that neither the N- nor C-terminal alpha-helix alone is sufficient to confer any function. Rather, fowlicidin-2(1-18) and fowlicidin-2(15-31), 2 alpha-helical segments with inclusion of the central cationic kink region, retained substantial capacities to kill bacteria and neutralize the LPS-induced proinflammatory response, relative to the parent peptide. More desirably, these 2 peptide analogs showed substantially reduced toxicity to human erythrocytes and epithelial cells, indicative of improved potential as antibacterial and antisepsis agents. To our knowledge, fowlicidin-2 is the first alpha-helical cathelicidin, with the central kink region shown to be critically important in killing bacteria and neutralizing LPS.

Identification and functional characterization of three chicken cathelicidins with potent antimicrobial activity.

Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain. Here we report that the entire chicken genome encodes three cathelicidins, namely fowlicidin-1 to -3, which are densely clustered within a 7.5-kb distance at the proximal end of chromosome 2p. Each fowlicidin gene adopts a fourexon, three-intron structure, typical for a mammalian cathelicidin. Phylogenetic analysis revealed that fowlicidins and a group of distantly related mammalian cathelicidins known as neutrophilic granule proteins are likely to originate from a common ancestral gene prior to the separation of birds from mammals, whereas other classic mammalian cathelicidins may have been duplicated from the primordial gene for neutrophilic granule proteins after mammals and birds are diverged. Similar to ovine cathelicidin SMAP-29, putatively mature fowlicidins displayed potent and salt-independent activities against a range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range of 0.4-2.0 microm for most strains. Fowlicidin-1 and -2 also showed cytotoxicity, with 50% killing of mammalian erythrocytes or epithelial cells in the range of 6-40 microm. In addition, two fowlicidins demonstrated a strong positive cooperativity in binding lipopolysaccharide (LPS), resulting in nearly complete blockage of LPS-mediated proinflammatory gene expression in RAW264.7 cells. Taken together, fowlicidin-1 and -2 are clearly among the most potent cathelicidins that have been reported. Their broad spectrum and salt-insensitive antibacterial activities, coupled with their potent LPS-neutralizing activity, make fowlicidins excellent candidates for novel antimicrobial and anti-sepsis agents.