Improving the detection of integrative conjugative elements in bovine nasopharyngeal swabs using multiplex recombinase polymerase amplification.

Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively. DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.

Multidrug Resistance in Pasteurellaceae Associated With Bovine Respiratory Disease Mortalities in North America From 2011 to 2016.

Multidrug-resistant (MDR; resistance to ≥3 antimicrobial classes) members of the Pasteurellaceae family may compromise the efficacy of therapies used to prevent and treat bovine respiratory disease (BRD) in feedlot cattle. This study examined the prevalence of multidrug resistance in strains of Mannheimia haemolytica and Pasteurella multocida collected from BRD cattle mortalities in North America. Isolates of M. haemolytica (n = 147) and P. multocida (n = 70) spanning 69 Alberta feedlots from 2011 to 2016 and two United States feedlots from 2011 to 2012 were examined for antimicrobial resistance (AMR) in association with integrative and conjugative elements (ICEs). Overall, resistance was high in both bacterial species with an increase in the prevalence of MDR isolates between 2011 and 2016. Resistance to >7 antimicrobial drugs occurred in 31% of M. haemolytica and 83% of P. multocida isolates. Resistance to sulfadimethoxine, trimethoprim/sulfamethoxazole, neomycin, clindamycin oxytetracycline, spectinomycin, tylosin, tilmicosin, and tulathromycin was most common. Although >80% of strains harbored three or more ICE-associated genes, only 12% of M. haemolytica and 77% of P. multocida contained all six, reflecting the diversity of ICEs. There was evidence of clonal spread as P. multocida and M. haemolytica isolates with the same pulsed-field gel electrophoresis profile from the United States in 2011 were isolated in Alberta in 2015-2016. This work highlights that MDR strains of Pasteurellaceae containing ICEs are widespread and may be contributing to BRD therapy failure in feedlot cattle. Given the antimicrobial resistance gene profiles identified, these MDR isolates may be selected for by the use of macrolides, tetracyclines, and/or in-feed supplements containing heavy metals.

A Sensitive and Accurate Recombinase Polymerase Amplification Assay for Detection of the Primary Bacterial Pathogens Causing Bovine Respiratory Disease.

Rapid and accurate diagnosis of bovine respiratory disease (BRD) presents a substantial challenge to the North American cattle industry. Here we utilize recombinase polymerase amplification (RPA), a fast and sensitive isothermal DNA-based technology for the detection of four BRD pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis), genes coding antimicrobial resistance (AMR) and integrative conjugative elements (ICE) which can harbor AMR genes. Eleven RPA assays were designed and validated including: a) one conventional species-specific multiplex assay targeting the 4 BRD pathogens, b) two species-specific real-time multiplex RPA assays targeting M. haemolytica/M. bovis and P. multocida/H. somni, respectively with a novel competitive internal amplification control, c) seven conventional assays targeting AMR genes (tetH, tetR, msrE, mphE, sul2, floR, erm42), and d) one real-time assay targeting ICE. Each real-time RPA assay was tested on 100 deep nasopharyngeal swabs (DNPS) collected from feedlot cattle previously assessed for targets using either culture methods and/or polymerase chain reaction (PCR) verification (TC-PCR). The developed RPA assays enabled sensitive and accurate identification of BRD agents and AMR/ICE genes directly from DNPS, in a shorter period than TC-PCR, showing considerable promise as a tool for point-of-care identification of BRD pathogens and antimicrobial resistance genes.

Pretreatment of crop residues by ammonia fiber expansion (AFEX) alters the temporal colonization of feed in the rumen by rumen microbes.

This study examines the colonization of barley straw (BS) and corn stover (CS) by rumen bacteria and how this is impacted by ammonia fiber expansion (AFEX) pre-treatment. A total of four ruminally cannulated beef heifers were used to investigate in situ microbial colonization in a factorial design with two crop residues, pre-treated with or without AFEX. Crop residues were incubated in the rumen for 0, 2, 4, 8 and 48 h and the colonizing profile was determined using 16 s rRNA gene sequencing. The surface colonizing community clustered based on incubation time and pre-treatment. Fibrobacter, unclassified Bacteroidales, and unclassified Ruminococcaceae were enriched during late stages of colonization. Prevotella and unclassified Lachnospiraceae were enriched in the early stages of colonization. The microbial community colonizing BS-AFEX and CS was less diverse than the community colonizing BS and CS-AFEX. Prevotella, Coprococcus and Clostridium were enriched in both AFEX crop residues, while untreated crop residues were enriched with Methanobrevibacter. Several pathways associated with simple carbohydrate metabolism were enriched in the primary colonizing community of AFEX crop residues. This study suggests that AFEX improves the degradability of crop residues by increasing the accessibility of polysaccharides that can be metabolized by the dominant taxa responsible for primary colonization.

Corrigendum: Multidrug Resistance in Pasteurellaceae Associated With Bovine Respiratory Disease Mortalities in North America From 2011 to 2016.

[This corrects the article DOI: 10.3389/fmicb.2020.606438.].

Zoonotic Fecal Pathogens and Antimicrobial Resistance in Canadian Petting Zoos.

This study aimed to better understand the potential public health risk associated with zoonotic pathogens in agricultural fairs and petting zoos in Canada. Prevalence of Salmonella, Shiga toxin-producing Escherichia coli (STEC) O157:H7, and top six non-O157 STEC serogroups in feces (n = 88), hide/feather (n = 36), and hand rail samples (n = 46) was assessed, as well as distributions of antimicrobial resistant (AMR) broad and extended-spectrum ß-lactamase (ESBL)-producing E. coli. Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in pig nasal swabs (n = 4), and Campylobacter, Cryptosporidium, and Giardia in feces was also assessed. Neither Salmonella nor MRSA were detected. Campylobacter spp. were isolated from 32% of fecal samples. Cryptosporidium and Giardia were detected in 2% and 15% of fecal samples, respectively. Only one fecal sample was positive for STEC O157, whereas 22% were positive for non-O157 STEC. Multi-drug resistance (MDR) to antibiotics classified as critically and highly important in human medicine was proportionally greatest in E. coli from cattle feces. The ß-lactamase-producing E. coli from pig, horse/donkey feces, and hand rail samples, as well as the STEC E. coli from handrail swabs were MDR. The diversity and prevalence of zoonotic pathogens and AMR bacteria detected within agricultural fairs and petting zoos emphasize the importance of hygienic practices and sanitization with respect to reducing associated zoonotic risks.

Effect of severe weather events on the shedding of Shiga toxigenic Escherichia coli in slaughter cattle and phenotype of serogroup O157 isolates.

High-event periods (HEPs) occur sporadically when beef carcasses and meat have episodes of acute contamination with Shiga toxin-producing Escherichia coli (STEC). In this study, severe weather events were investigated as catalysts for HEPs based on PCR and isolate prevalence of seven E. coli serogroups in slaughter cattle feces. Winter ambient temperatures with daily means 10.5oC warmer or 12.3°C colder than seasonal norms (-10.4°C) most altered STEC shedding. Fecal samples yielded increased proportions (P < 0.05) of O26 and O157 isolates during winter warm periods, and reduced (P < 0.05) O45 isolates during cold periods compared to samplings during seasonal norms. Based on changing PCR prevalence and isolates collected, O157 was the serogroup most responsive to severe weather events. Consequently, O157 isolates (n = 219) were evaluated for heat resistance, biofilm-forming potential and virulence gene subtypes. Two isolates had heat-resistant phenotypes with thermal death time at 60°C (D60) > 10 min and one also had strong biofilm-forming potential. However, this isolate lacked eae and stx genes. Severe weather can influence STEC shedding, particularly of O157, and could possibly trigger HEPs. However, our data suggest that it is unlikely for isolates to carry virulence genes and possess phenotypes capable of evading post-harvest microbiological interventions.

Farm Fairs and Petting Zoos: A Review of Animal Contact as a Source of Zoonotic Enteric Disease.

Many public venues such as farms, fairs, and petting zoos encourage animal contact for both educational and entertainment purposes. However, healthy farm animals, including cattle, small ruminants, and poultry, can be reservoirs for enteric zoonotic pathogens, with human infections resulting in nausea, vomiting, diarrhea, and, in some cases, severe complications that can lead to death. As animals shed these organisms in their feces, contamination of themselves and their surroundings is unavoidable. The majority of North Americans reside in urban and suburban settings, and the general public often possess limited knowledge of agricultural practices and minimal contact with farm animals. Furthermore, there is a lack of understanding of zoonotic pathogens, particularly how these pathogens are spread and the human behaviors that may increase the risk of infection. Human risk behaviors include hand-to-mouth contact immediately after physical contact with animals and their environments, a practice that facilitates the ingestion of pathogens. It is often young children who become ill due to their under-developed immune systems and poorer hygienic practices compared with adults, such as more frequent hand-to-mouth behaviors, and infrequent or improper hand washing. These illnesses are often preventable, simply through adequate hygiene and hand washing. Our objective was to use a structured approach to review the main causal organisms responsible for human illnesses acquired in petting zoo and open farm environments, Shiga toxin-producing Escherichia coli, nontyphoidal Salmonella, Campylobacter, and Cryptosporidium. Notable outbreaks involving direct contact with farm animals and farm, fair, or petting zoo environments are discussed and recommendations for how public venues can increase safety and hand hygiene compliance among visitors are proposed. The most effective protective measures against enteric illnesses include education of the public, increasing overall awareness of the risks and the importance of hand hygiene, as well as access to hand-washing facilities.

Further development of sample preparation and detection methods for O157 and the top 6 non-O157 STEC serogroups in cattle feces.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens responsible for outbreaks of human infections worldwide. Ruminant livestock harbor STEC in their intestinal tract, and through fecal contamination possess the potential to compromise the safety of food and water. As a human health safety risk, STEC detection methods on beef carcasses and trim are needed as mandated by the USDA-FSIS. In order to monitor STEC prior to harvest and human consumption, our goal was to evaluate and/or improve detection of seven STEC serogroups in cattle feces. In comparison to traditional approaches, sample processing methods in bovine feces were evaluated using a multi-factorial Latin square design which involved freezing or freeze drying feces. Autoclaved versus non-autoclaved feces were spiked with O26:H11 or O157:H7 serotypes in various dilutions and enriched for up to 6h. Each hour, enriched aliquots were compared using traditional culture methods and quantitative polymerase chain reaction (qPCR). Furthermore, a 7-serogroup multiplex PCR (mPCR) was developed to detect O26, O45, O103, O111, O121, O145 and O157 serogroups simultaneously. The diagnostic sensitivity of our mPCR assay following 6h enrichment was superior (10CFU/g across all serogroups) compared to a previously established PCR assay (10CFU/g for O26, and O103; ≥10(4)CFU/g for all other serogroups). Obtaining viable isolates appeared to be limited by the efficiency of current immunomagnetic separation (IMS) methods, which ranged from 20 to 100% effectiveness at retrieving colonies depending on serogroup. After IMS, 70 putative STEC isolates were screened for Shiga toxin and attachment genes by mPCR. Sixty-five isolates contained one or both Shiga toxin genes.

Synthesis of O-serogroup specific positive controls and real-time PCR standards for nine clinically relevant non-O157 STECs.

Non-O157 Shiga toxin producing Escherichia coli (STEC) are gaining recognition as human pathogens, but no standardized method exists to identify them. Sequence analysis revealed that STEC can be classified on the base of variable O antigen regions into different O serotypes. Polymerase chain reaction is a powerful technique for thorough screening and complex diagnosis for these pathogens, but requires a positive control to verify qualitative and/or quantitative DNA-fragment amplification. Due to the pathogenic nature of STEC, controls are not readily available and cell culturing of STEC reference strains requires biosafety conditions of level 2 or higher. In order to bypass this limitation, controls of stacked O-type specific DNA-fragments coding for primer recognition sites were designed to screen for nine STEC serotypes frequently associated with human infection. The synthetic controls were amplified by PCR, cloned into a plasmid vector and transferred into bacteria host cells. Plasmids amplified by bacterial expression were purified, serially diluted and tested as standards for real-time PCR using SYBR Green and TaqMan assays. Utility of synthetic DNA controls was demonstrated in conventional and real-time PCR assays and validated with DNA from natural STEC strains.