Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing.

Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017-2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples were enriched for microbial DNA and subjected to shotgun next generation sequencing using Nextera DNA Flex library preparation kit and V2 chemistry sequencing kit on the Illumina MiSeq sequencing platform. Overall, 0.4-0.6 million reads per sample were analyzed using Kraken metagenomic sequence classification program. The taxonomic classification of the reads indicated that bacterial genomes were overrepresented (0.5 to 1%) among the total microbial reads. Most of the bacterial reads (~91%) belonged to phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Tenericutes in both groups. Importantly, 10-42.5% of Alphaproteobacterial reads in 5 of 10 animals with suspected tick-borne infection were identified as Anaplasma phagocytophilum. Of the 5 animals positive for A. phagocytophilum sequence reads, four animals tested A. phagocytophilum positive by PCR. Two animals with suspected tick-borne infection and A. phagocytophilum positive by PCR were found negative for any tick-borne microbial reads by metagenomic analysis. The present study demonstrates the usefulness of the NGS-based metagenomic analysis approach for the detection of blood-borne microbes.

Sialic Acid Receptors: The Key to Solving the Enigma of Zoonotic Virus Spillover.

Emerging viral diseases are a major threat to global health, and nearly two-thirds of emerging human infectious diseases are zoonotic. Most of the human epidemics and pandemics were caused by the spillover of viruses from wild mammals. Viruses that infect humans and a wide range of animals have historically caused devastating epidemics and pandemics. An in-depth understanding of the mechanisms of viral emergence and zoonotic spillover is still lacking. Receptors are major determinants of host susceptibility to viruses. Animal species sharing host cell receptors that support the binding of multiple viruses can play a key role in virus spillover and the emergence of novel viruses and their variants. Sialic acids (SAs), which are linked to glycoproteins and ganglioside serve as receptors for several human and animal viruses. In particular, influenza and coronaviruses, which represent two of the most important zoonotic threats, use SAs as cellular entry receptors. This is a comprehensive review of our current knowledge of SA receptor distribution among animal species and the range of viruses that use SAs as receptors. SA receptor tropism and the predicted natural susceptibility to viruses can inform targeted surveillance of domestic and wild animals to prevent the future emergence of zoonotic viruses.

Antimicrobial resistant enteric bacteria are widely distributed amongst people, animals and the environment in Tanzania.

Antibiotic use and bacterial transmission are responsible for the emergence, spread and persistence of antimicrobial-resistant (AR) bacteria, but their relative contribution likely differs across varying socio-economic, cultural, and ecological contexts. To better understand this interaction in a multi-cultural and resource-limited context, we examine the distribution of antimicrobial-resistant enteric bacteria from three ethnic groups in Tanzania. Household-level data (n = 425) was collected and bacteria isolated from people, livestock, dogs, wildlife and water sources (n = 62,376 isolates). The relative prevalence of different resistance phenotypes is similar across all sources. Multi-locus tandem repeat analysis (n = 719) and whole-genome sequencing (n = 816) of Escherichia coli demonstrate no evidence for host-population subdivision. Multivariate models show no evidence that veterinary antibiotic use increased the odds of detecting AR bacteria, whereas there is a strong association with livelihood factors related to bacterial transmission, demonstrating that to be effective, interventions need to accommodate different cultural practices and resource limitations.

Identification of risk factors associated with carriage of resistant Escherichia coli in three culturally diverse ethnic groups in Tanzania: a biological and socioeconomic analysis.

BACKGOUND: Improved antimicrobial stewardship, sanitation, and hygiene are WHO-inspired priorities for restriction of the spread of antimicrobial resistance. Prioritisation among these objectives is essential, particularly in low-income and middle-income countries, but the factors contributing most to antimicrobial resistance are typically unknown and could vary substantially between and within countries. We aimed to identify the biological and socioeconomic risk factors associated with carriage of resistant Escherichia coli in three culturally diverse ethnic groups in northern Tanzania. METHODS: We developed a survey containing more than 200 items and administered it in randomly selected households in 13 Chagga, Arusha, or Maasai villages chosen on the basis of ethnic composition and distance to urban centres. Human stool samples were collected from a subset of households, as were liquid milk samples and swabs of milk containers. Samples were processed and plated onto MacConkey agar plates, then presumptive E coli isolates were identified on the basis of colony morphology. Susceptibility of isolates was then tested against a panel of nine antimicrobials (ampicillin, ceftazidime, chloramphenicol, ciprofloxacin, kanamycin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim) via a breakpoint assay. Susceptibility findings were matched with data across a wide range of household characteristics, including education, hygiene practices, wealth, livestock husbandry, and antibiotic use. FINDINGS: Between March 23, 2012, and July 30, 2015, we interviewed 391 households (118 Arusha, 100 Chagga, and 173 Maasai). Human stool samples were collected at 226 (58%) households across the 13 villages. 181 milk samples and 191 milk-container swabs were collected from 117 households across seven villages. 11 470 putative E coli samples were isolated from stool samples. Antimicrobial use in people and livestock was not associated with prevalence of resistance at the household level. Instead, the factors with the greatest predictive value involved exposure to bacteria, and were intimately connected with fundamental cultural differences across study groups. These factors included how different subsistence types (pastoralists vs farmers) access water sources and consumption of unboiled milk, reflecting increased exposure to resistant bacteria in milk. INTERPRETATION: When cultural and ecological conditions favour bacterial transmission, there is a high likelihood that people will harbour antimicrobial-resistant bacteria irrespective of antimicrobial use practices. Public health interventions to limit antimicrobial resistance need to be tailored to local practices that affect bacterial transmission. FUNDING: US National Science Foundation; Biotechnology and Biological Sciences Research Council, UK Medical Research Council; and the Allen School.

A two-month follow-up evaluation testing interventions to limit the emergence and spread of antimicrobial resistant bacteria among Maasai of northern Tanzania.

BACKGROUND: In sub-Saharan Africa, efforts to control antimicrobial resistance (AMR) are aggravated by unregulated drug sales and use, and high connectivity between human, livestock, and wildlife populations. Our previous research indicates that Maasai agropastoralists-who have high exposure to livestock and livestock products and self-administer veterinary antibiotics-harbor antibiotic resistant Escherichia coli (E. coli). Here, we report the results of a public health intervention project among Maasai aimed at reducing selection and transmission of E. coli bacteria. METHODS: Research was conducted in two Maasai communities in Northern Tanzania. Participants were provided with health knowledge and technological innovations to facilitate: 1) the prudent use of veterinary antibiotics (tape measures and dosage charts to calculate livestock weight for more accurate dosage), and, 2) the pasteurization of milk (thermometers), the latter of which was motivated by findings of high levels of resistant E. coli in Maasai milk. To determine knowledge retention and intervention adoption, we conducted a two-month follow-up evaluation in the largest of the two communities. RESULTS: Retention of antimicrobial knowledge was positively associated with retention of bacterial knowledge and, among men, retention of bacterial knowledge was associated with greater wealth. Bacterial and AMR knowledge were not, however, associated with self-reported use of the innovations. Among women, self-reported use of the thermometers was associated with having more children and greater retention of knowledge about the health benefits of the innovations. Whereas 70% of women used their innovations correctly, men performed only 18% of the weight-estimation steps correctly. Men's correct use was associated with schooling, such that high illiteracy rates remain an important obstacle to the dissemination and diffusion of weight-estimation materials. CONCLUSION: Our results indicate that dietary preferences for unboiled milk, concerns over child health, and a desire to improve the health of livestock are important cultural values that need to be incorporated in future AMR-prevention interventions that target Maasai populations. More generally, these findings inform future community-health interventions to limit AMR.

Antimicrobial Use and Veterinary Care among Agro-Pastoralists in Northern Tanzania.

Frequent and unregulated use of antimicrobials (AM) in livestock requires public health attention as a likely selection pressure for resistant bacteria. Studies among small-holders, who own a large percentage of the world's livestock, are vital for understanding how practices involving AM use might influence resistance. We present a cultural-ecological mixed-methods analysis to explore sectors of veterinary care, loosely regulated AM use, and human exposure to AMs through meat and milk consumption across three rural to peri-urban Tanzanian ethnic groups (N = 415 households). Reported use of self-administered AMs varied by ethnic group (Maasai: 74%, Arusha: 21%, Chagga: 1%) as did consultation with professional veterinarians (Maasai: 36%, Arusha: 45%, Chagga: 96%) and observation of withdrawal of meat and milk from consumption during and following AM treatment (Maasai: 7%, Arusha: 72%, Chagga: 96%). The antibiotic oxytetracycline was by far the most common AM in this sample. Within ethnic groups, herd composition differences, particularly size of small-stock and cattle herds, were most strongly associated with differences in lay AM use. Among the Arusha, proxies for urbanization, including owning transportation and reliance on "zero-grazing" herds had the strongest positive associations with veterinarian consultation, while distance to urban centers was negatively associated. For Maasai, consultation was negatively associated with use of traditional healers or veterinary drug-shops. Observation of withdrawal was most strongly associated with owning technology among Maasai while Arusha observance displayed seasonal differences. This "One-Health" analysis suggests that livelihood and cultural niche factors, through their association with practices in smallholder populations, provide insight into the selection pressures that may contribute to the evolution and dissemination of antimicrobial resistance.

Comparison of antibiotic resistant Escherichia coli obtained from drinking water sources in northern Tanzania: a cross-sectional study.

BACKGROUND: Antimicrobial resistance (AMR) is a growing and significant threat to public health on a global scale. Escherichia coli comprises Gram-negative, fecal-borne pathogenic and commensal bacteria that are frequently associated with antibiotic resistance. AMR E. coli can be ingested via food, water and direct contact with fecal contamination. METHODS: We estimated the prevalence of AMR Escherichia coli from select drinking water sources in northern Tanzania. Water samples (n = 155) were collected and plated onto Hi-Crome E. coli and MacConkey agar. Presumptive E. coli were confirmed by using a uidA PCR assay. Antibiotic susceptibility breakpoint assays were used to determine the resistance patterns of each isolate for 10 antibiotics. Isolates were also characterized by select PCR genotyping and macro-restriction digest assays. RESULTS: E. coli was isolated from 71 % of the water samples, and of the 1819 E. coli tested, 46.9 % were resistant to one or more antibiotics. Resistance to ampicillin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim was significantly higher (15-30 %) compared to other tested antibiotics (0-6 %; P < 0.05). Of the ß-lactam-resistant isolates, bla TEM-1 was predominant (67 %) followed by bla CTX-M (17.7 %) and bla SHV-1 (6.0 %). Among the tetracycline-resistant isolates, tet(A) was predominant (57.4 %) followed by tet(B) (24.0 %). E. coli isolates obtained from these water sources were genetically diverse with few matching macro-restriction digest patterns. CONCLUSION: Water supplies in northern Tanzania may be a source of AMR E. coli for people and animals. Further studies are needed to identify the source of these contaminants and devise effective intervention strategies.

Prevalence of Antibiotic-Resistant Fecal Escherichia coli Isolates from Penned Broiler and Scavenging Local Chickens in Arusha, Tanzania.

We compared the prevalence of antibiotic-resistant Escherichia coli isolates from household-level producers of broiler (commercial source breeds) and local chickens in the Arusha District of Tanzania. Households were composed of a single dwelling or residence with independent, penned broiler flocks. Free-range, scavenging chickens were mixed breed and loosely associated with individual households. A total of 1,800 E. coli isolates (1,200 from broiler and 600 from scavenging local chickens) from 75 chickens were tested for their susceptibility against 11 antibiotics by using breakpoint assays. Isolates from broiler chickens harbored a higher prevalence of antibiotic-resistant E. coli relative to scavenging local chickens, including sulfamethoxazole (80.3 versus 34%), followed by trimethoprim (69.3 versus 27.7%), tetracycline (56.8 versus 20%), streptomycin (52.7 versus 24.7%), amoxicillin (49.6 versus 17%), ampicillin (49.1 versus 16.8%), ciprofloxacin (21.9 versus 1.7%), and chloramphenicol (1.5 versus 1.2%). Except for resistance to chloramphenicol, scavenging local chickens harbored fewer resistant E. coli isolates (P < 0.05). Broiler chickens harbored more isolates that were resistant to ≥7 antibiotics (P < 0.05). The higher prevalence of antibiotic-resistant E. coli from broiler chickens correlated with the reported therapeutic and prophylactic use of antibiotics in this poultry population. We suggest that improved biosecurity measures and increased vaccination efforts would reduce reliance on antibiotics by these households.

Not All Antibiotic Use Practices in Food-Animal Agriculture Afford the Same Risk.

The World Health Organization has identified quinolones, third- and fourth-generation cephalosporins, and macrolides as the most important antibiotics in human medicine. In the context of agricultural use of antibiotics, the principle zoonotic agents of concern are , spp., , and spp. Antibiotic exposure provides a selective advantage to resistant strains of these bacteria relative to their susceptible conspecifics. This is a dose-dependent process, and consequently antibiotic use practices that involve higher doses will exert greater and longer-lasting selective pressure in favor of resistant bacterial populations and will therefore increase the probability of transmission to people and other animals. Oral administration has a greater impact on enteric flora with the exception of fluoroquinolone treatments, which appear to affect the enteric flora equally if administered orally or parenterally. The use of quinolones in agriculture deserves heightened scrutiny because of the ease with which these broad-spectrum antibiotics favor spontaneously resistant bacteria in exposed populations. When present at sufficient concentrations, excreted antibiotics have the potential to selectively favor resistant bacteria in the environment and increase the probability of transmission to people and animals. The bioavailability of antibiotics varies greatly: some antibiotics remain active in soils (florfenicol, ß-lactams), whereas others may be rapidly sorbed and thus not bioavailable (tetracycline, macrolides, quinolones). When considering the risks of different antibiotic use practices in agriculture, it would be prudent to focus attention on practices that involve high doses, oral delivery, and residues of antibiotics that remain active in soils.

IncF Plasmids Are Commonly Carried by Antibiotic Resistant Escherichia coli Isolated from Drinking Water Sources in Northern Tanzania.

The aim of this study was to identify the replicon types of plasmids, conjugation efficiencies, and the complement of antibiotic resistance genes for a panel of multidrug resistant E. coli isolates from surface waters in northern Tanzania. Standard membrane filtration was used to isolate and uidA PCR was used to confirm the identity of strains as E. coli. Antibiotic susceptibility was determined by breakpoint assay and plasmid conjugation was determined by filter-mating experiments. PCR and sequencing were used to identify resistance genes and PCR-based replicon typing was used to determine plasmid types. Filter mating experiments indicated conjugation efficiencies ranged from 10(-1) to 10(-7). Over 80% of the donor cells successfully passed their resistance traits and eleven different replicon types were detected (IncI1, FIC, P, FIIA, A/C, FIB, FIA, H12, K/B B/O, and N). IncF plasmids were most commonly detected (49% of isolates), followed by types IncI1 and IncA/C. Detection of these public health-relevant conjugative plasmids and antibiotic resistant traits in Tanzanian water suggests the possible pollution of these water sources from human, livestock, and wild animal wastes and also shows the potential of these water sources in the maintenance and transmission of these resistance traits between environments, animals, and people.

Soil-borne reservoirs of antibiotic-resistant bacteria are established following therapeutic treatment of dairy calves.

We determined if antibiotics residues that are excreted from treated animals can contribute to persistence of resistant bacteria in agricultural environments. Administration of ceftiofur, a third-generation cephalosporin, resulted in a ∼ 3 log increase in ceftiofur-resistant Escherichia coli found in the faeces and pen soils by day 10 (P = 0.005). This resistant population quickly subsided in faeces, but was sustained in the pen soil (∼ 4.5 log bacteria g(-1)) throughout the trial (1 month). Florfenicol treatment resulted in a similar pattern although the loss of florfenicol-resistant E. coli was slower for faeces and remained stable at ∼ 6 log bacteria g(-1) in the soil. Calves were treated in pens where eGFP-labelled E. coli were present in the bedding (∼ 2 log g(-1)) resulting in amplification of the eGFP E. coli population ∼ 2.1 log more than eGFP E. coli populations in pens with untreated calves (day 4; P < 0.005). Excreted residues accounted for > 10-fold greater contribution to the bedding reservoir compared with shedding of resistant bacteria in faeces. Treatment with therapeutic doses of ceftiofur or florfenicol resulted in 2-3 log g(-1) more bacteria than the estimated ID50 (2.83 CFU g(-1)), consistent with a soil-borne reservoir emerging after antibiotic treatment that can contribute to the long-term persistence of antibiotic resistance in animal agriculture.

Load and Prevalence of Antimicrobial-Resistant Escherichia coli from Fresh Goat Meat in Arusha, Tanzania.

Given the potential public health risks associated with a burgeoning goat meat industry in Tanzania, we estimated the load of Escherichia coli and the prevalence of antibiotic-resistant strains for goat meat by using a cross-sectional study design (June to July 2015). Five large (n = 60 samples) and five small (n = 64 samples) slaughterhouses were sampled over a period of four to six visits each. Meat rinsate was prepared and plated onto MacConkey agar, and presumptive E. coli colonies were enumerated and reported as CFU per milliliter of rinsate. In total, 2,736 presumptive E. coli isolates were tested for antibiotic drug sensitivity by using breakpoint assays against 11 medically important antibiotics. E. coli was recovered from almost all the samples (96.8%), with counts ranging from 2 to 4 log CFU ml-1, and there was no significant difference (P = 0.43) in recovery according to facility size (average, 3.37 versus 3.13 log CFU ml-1, large and small, respectively). Samples from large facilities had relatively higher prevalence (P = 0.026) of antibiotic-resistant E. coli compared with small facilities. This was mostly explained by more ampicillin (30.1 versus 12.8%) and amoxicillin (17.6 versus 4.5%) resistance for large versus small facilities, respectively, and more tetracycline resistance for small facilities (5.6 versus 10.6%, respectively). Large slaughter operations may serve as foci for dissemination of antibiotic-resistant bacteria via food products. More effective hygiene practices during slaughter and meat handling would limit the probability of transmitting antibiotic-resistant E. coli in goat meat.

Do antibiotic residues in soils play a role in amplification and transmission of antibiotic resistant bacteria in cattle populations?

When we consider factors that contribute to the emergence, amplification, and persistence of antibiotic resistant bacteria, the conventional assumption is that antibiotic use is the primary driver in these processes and that selection occurs primarily in the patient or animal. Evidence suggests that this may not always be the case. Experimental trials show that parenteral administration of a third-generation cephalosporin (ceftiofur) in cattle has limited or short-term effects on the prevalence of ceftiofur-resistant bacteria in the gastrointestinal tract. While this response may be sufficient to explain a pattern of widespread resistance to cephalosporins, approximately two-thirds of ceftiofur metabolites are excreted in the urine raising the possibility that environmental selection plays an important additive role in the amplification and maintenance of antibiotic resistant E. coli on farms. Consequently, we present a rationale for an environmental selection hypothesis whereby excreted antibiotic residues such as ceftiofur are a significant contributor to the proliferation of antibiotic resistant bacteria in food animal systems. We also present a mathematical model of our hypothesized system as a guide for designing experiments to test this hypothesis. If supported for antibiotics such as ceftiofur, then there may be new approaches to combat the proliferation of antibiotic resistance beyond the prudent use mantra.

Urine from treated cattle drives selection for cephalosporin resistant Escherichia coli in soil.

The U.S. Food and Drug Administration recently issued new rules for using ceftiofur in food animals in part because of an increasing prevalence of enteric bacteria that are resistant to 3(rd)-generation cephalosporins. Parenteral ceftiofur treatment, however, has limited effects on enteric bacteria so we tested the hypothesis that excreted ceftiofur metabolites exert significant selection pressure for ceftiofur-resistant Escherichia coli in soil. Test matrices were prepared by mixing soil with bovine feces and adding urine containing ceftiofur metabolites (CFM) (0 ppm, ∼50 ppm and ∼100 ppm). Matrices were incubated at 23°C or 4°C for variable periods of time after which residual CFM was quantified using a bioassay. Bla(CMY-2) plasmid-bearing ceftiofur resistant (cef(R)) E. coli and one-month old calves were used to study the selection effects of CFM and transmission of cef(R) bacteria from the environment back to animals. Our studies showed that urinary CFM (∼13 ppm final concentration) is biologically degraded in soil within 2.7 days at 23°C, but persists up to 23.3 days at 4°C. Even short-term persistence in soil provides a >1 log(10) advantage to resistant E. coli populations, resulting in significantly prolonged persistence of these bacteria in the soil (∼two months). We further show that resistant strains readily colonize calves by contact with contaminated bedding and without antibiotic selection pressure. Ceftiofur metabolites in urine amplify resistant E. coli populations and, if applicable to field conditions, this effect is far more compelling than reported selection in vivo after parenteral administration of ceftiofur. Because ceftiofur degradation is temperature dependent, these compounds may accumulate during colder months and this could further enhance selection as seasonal temperatures increase. If cost-effective engineered solutions can be developed to limit ex vivo selection, this may limit proliferation for ceftiofur resistant enteric bacteria while preserving the ability to use this important antibiotic in food animal production.

ß-lactams and florfenicol antibiotics remain bioactive in soils while ciprofloxacin, neomycin, and tetracycline are neutralized.

It is generally assumed that antibiotic residues in soils select for antibiotic-resistant bacteria. This assumption was tested by separately adding 10 different antibiotics (≥200 ppm) to three soil-water slurries (silt-loam, sand-loam, and sand; 20% soil [wt/vol]) and incubating mixtures for 24 h at room temperature. The antibiotic activity of the resultant supernatant was assessed by culturing a sensitive Escherichia coli strain in the filter-sterilized supernatant augmented with Luria-Bertani broth. We found striking differences in the abilities of supernatants to suppress growth of the indicator E. coli. Ampicillin, cephalothin, cefoxitin, ceftiofur, and florfenicol supernatants completely inhibited growth while bacterial growth was uninhibited in the presence of neomycin, tetracycline, and ciprofloxacin supernatants. High-performance liquid chromatography (HPLC) analysis demonstrated that cefoxitin and florfenicol were almost completely retained in the supernatants, whereas tetracycline and ciprofloxacin were mostly removed. Antibiotic dissipation in soil, presumably dominated by adsorption mechanisms, was sufficient to neutralize 200 ppm of tetracycline; this concentration is considerably higher than reported contamination levels. Soil pellets from the tetracycline slurries were resuspended in a minimal volume of medium to maximize the interaction between bacteria and soil particles, but sensitive bacteria were still unaffected by tetracycline (P = 0.6). Thus, residual antibiotics in soil do not necessarily exert a selective pressure, and the degree to which the pharmaceutical remains bioactive depends on the antibiotic. Efforts to control antibiotic contamination would be better directed toward compounds that retain biological activity in soils (e.g., cephalosporins and florfenicol) because these are the antibiotics that could exert a selective pressure in the environment.

Selection pressure required for long-term persistence of blaCMY-2-positive IncA/C plasmids.

Multidrug resistance blaCMY-2 plasmids that confer resistance to expanded-spectrum cephalosporins have been found in multiple bacterial species collected from different hosts worldwide. The widespread distribution of blaCMY-2 plasmids may be driven by antibiotic use that selects for the dissemination and persistence of these plasmids. Alternatively, these plasmids may persist and spread in bacterial populations in the absence of selection pressure if a balance exists among conjugative transfer, segregation loss during cell division, and fitness cost to the host. We conducted a series of experiments (both in vivo and in vitro) to study these mechanisms for three blaCMY-2 plasmids, peH4H, pAR060302, and pAM04528. Results of filter mating experiments showed that the conjugation efficiency of blaCMY-2 plasmids is variable, from <10(-7) for pAM04528 and peH4H to ∼10(-3) for pAR060302. Neither peH4H nor pAM04528 was transferred from Escherichia coli strain DH10B, but peH4H was apparently mobilized by the coresident trimethoprim resistance-encoding plasmid pTmpR. Competition studies showed that carriage of blaCMY-2 plasmids imposed a measurable fitness cost on the host bacteria both in vitro (0.095 to 0.25) and in vivo (dairy calf model). Long-term passage experiments in the absence of antibiotics demonstrated that plasmids with limited antibiotic resistance phenotypes arose, but eventually drug-sensitive, plasmid-free clones dominated the populations. Given that plasmid decay or loss is inevitable, we infer that some level of selection is required for the long-term persistence of blaCMY-2 plasmids in bacterial populations.

Bilateral malacia associated with sodium poisoning in turkey poults.

Improper cleaning of the water storage tank resulted in a toxic concentration of sodium in drinking water in a commercial turkey flock. Within the first week after placement 40% of the birds in the flock died. Clinically, poults were depressed and weak, huddled together, and reluctant to walk. At necropsy the birds had crops and gizzards filled with rice hulls, moderately swollen livers, distended gall bladders, and congested lungs. Neither ascites nor round heart was observed. The major microscopic lesion was multifocal symmetrical malacia of brain and spinal cord. Laboratory results revealed a high concentration of sodium in water (2340 mg/liter). The concentration of sodium in brain and liver ranged from 1870 to 2680 (mean = 2185; SD = 321.5) mg/liter wet weight and from 1810 to 2360 (mean = 2191.67; SD = 193.2) mg/liter wet weight, respectively, whereas the normal expected sodium concentration in the brain and liver tissues from young turkeys (< 7 days old) that were submitted for other causes averaged 1233 and 983 mg/liter wet weight, respectively. Based on the histological and toxicological results, a diagnosis of salt poisoning was made. This case investigation demonstrated that sodium analysis of brain and liver are diagnostically useful when confirming sodium poisoning in young turkeys.