The autotransporter protein from Bordetella avium,Baa1, is involved in host cell attachment

Bordetella avium is a Gram negative upper respiratory tract pathogen of birds. B. avium infection of commercially raised turkeys is an agriculturally significant problem. Here we describe the functional analysis of the first characterized B. avium autotransporter protein, Baa1. Autotransporters comprise a large family of proteins found in all groups of Gram negative bacteria. Although not unique to pathogenic bacteria, autotransporters have been shown to perform a variety of functions implicated in virulence. To test the hypothesis that Baa1 is a B. avium virulence factor, unmarked baa1 deletion mutants (Δbaa1) were created and tested phenotypically. It was found that baa1 mutants have wild-type levels of serum sensitivity and infectivity, yet significantly lower levels of turkey tracheal cell attachment in vitro. Likewise, semi-purified recombinant His-tagged Baa1, expressed in Escherichia coli, was shown to bind specifically to turkey tracheal cells via western blot analysis. Taken together, we conclude that Baa1 acts as a host cell attachment factor and thus plays a role B. avium virulence.     

The autotransporter protein from Bordetella avium, Baa1, is involved in host cell attachment

Bordetella avium is a Gram negative upper respiratory tract pathogen of birds. B. avium infection of commercially raised turkeys is an agriculturally significant problem. Here we describe the functional analysis of the first characterized B. avium autotransporter protein, Baa1. Autotransporters comprise a large family of proteins found in all groups of Gram negative bacteria. Although not unique to pathogenic bacteria, autotransporters have been shown to perform a variety of functions implicated in virulence. To test the hypothesis that Baa1 is a B. avium virulence factor, unmarked baa1 deletion mutants (Δbaa1) were created and tested phenotypically. It was found that baa1 mutants have wild-type levels of serum sensitivity and infectivity, yet significantly lower levels of turkey tracheal cell attachment in vitro. Likewise, semi-purified recombinant His-tagged Baa1, expressed in Escherichia coli, was shown to bind specifically to turkey tracheal cells via western blot analysis. Taken together, we conclude that Baa1 acts as a host cell attachment factor and thus plays a role B. avium virulence.     

Delivery of Aerosolized Liposomal Amikacin as a Novel Approach for the Treatment of Nontuberculous Mycobacteria in an Experimental Model of Pulmonary Infection

Pulmonary infections caused by nontuberculous mycobacteria (NTM) are an increasing problem in individuals with chronic lung conditions and current therapies are lacking. We investigated the activity of liposomal amikacin for inhalation (LAI) against NTM in vitro as well as in a murine model of respiratory infection. Macrophage monolayers were infected with three strains of Mycobacterium avium, two strains of Mycobacterium abscessus, and exposed to LAI or free amikacin for 4 days before enumerating bacterial survival. Respiratory infection was established in mice by intranasal inoculation with M. avium and allowing three weeks for the infection to progress. Three different regimens of inhaled LAI were compared to inhaled saline and parenterally administered free amikacin over a 28 day period. Bacteria recovered from the mice were analyzed for acquired resistance to amikacin. In vitro, liposomal amikacin for inhalation was more effective than free amikacin in eliminating both intracellular M. avium and M. abscessus. In vivo, inhaled LAI demonstrated similar effectiveness to a ∼25% higher total dose of parenterally administered amikacin at reducing M. avium in the lungs when compared to inhaled saline. Additionally, there was no acquired resistance to amikacin observed after the treatment regimen. The data suggest that LAI has the potential to be an effective therapy against NTM respiratory infections in humans.     

Mycobacterium avium Subspecies paratuberculosis Cultured from Locally and Commercially Pasteurized Cow's Milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

Expression of urease does not affect the ability of Bordetella bronchiseptica to colonise and persist in the murine respiratory tract

To investigate the role played by urease during the Bordetella bronchiseptica infection process, the ability to colonise and persist in the mouse respiratory tract of a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain. The results obtained showed that neither constitutive expression nor abolishment of urease activity had any significant effect on the course of B. bronchiseptica infection. Therefore, under our experimental conditions, urease is not essential for B. bronchiseptica to colonise and persist within the murine host.     

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.     

The AgI/II Family Adhesin AspA Is Required for Respiratory Infection by Streptococcus pyogenes

Streptococcus pyogenes (GAS) is a human pathogen that causes pharyngitis and invasive diseases such as toxic shock syndrome and sepsis. The upper respiratory tract is the primary reservoir from which GAS can infect new hosts and cause disease. The factors involved in colonisation are incompletely known however. Previous evidence in oral streptococci has shown that the AgI/II family proteins are involved. We hypothesized that the AspA member of this family might be involved in GAS colonization. We describe a novel mouse model of GAS colonization of the nasopharynx and lower respiratory tract to elucidate these interactions. We used two clinical M serotypes expressing AspA, and their aspA gene deletant isogenic mutants in experiments using adherence assays to respiratory epithelium, macrophage phagocytosis and neutrophil killing assays and in vivo models of respiratory tract colonisation and infection. We demonstrated the requirement for AspA in colonization of the respiratory tract. AspA mutants were cleared from the respiratory tract and were deficient in adherence to epithelial cells, and susceptible to phagocytosis. Expression of AspA in the surrogate host Lactococcus lactis protected bacteria from phagocytosis. Our results suggest that AspA has an essential role in respiratory infection, and may function as a novel anti-phagocytic factor.     

Epidemic Enteropathogenic Escherichia Coli,Newfoundland, 1963: Autopsy Study of 16 Cases

The autopsy findings and certain other features are described in 16 fatal cases of infantile diarrhea associated with enteropathogenic Escherichia coli infection during the 1963 epidemic in Newfoundland which resulted in 100 deaths. Age is an important factor in the severity of the infection and in the outcome. Almost one-half the patients were under 4 months and a third under 2 months. Pathological changes in the gastrointestinal tract were meagre and were not pathognomonic. Other pathological findings and their possible role in the severity and fatal outcome in enteropathogenic Escherichia coli gastroenteritis are discussed. Such conditions as prematurity, congenital anomaly, trauma, neoplasm and metabolic disorders were, no doubt, also important factors in the outcome. Severe respiratory tract infection was the leading terminal cause of death. No sex, seasonal or geographic variation was found. Enteropathogenic Escherichia coli serotype 0111:B4 was the prevalent infecting organism.     

Clustering of Mycobacterium avium subsp. paratuberculosis in Rabbits and the Environment: How Hot Is a Hot Spot?

Clustering of pathogens in the environment leads to hot spots of diseases at local, regional, national, and international levels. Scotland contains regional hot spots of Johne's disease (caused by Mycobacterium avium subsp. paratuberculosis) in rabbits, and there is increasing evidence of a link between paratuberculosis infections in rabbits and cattle. The spatial and temporal dynamics of paratuberculosis in rabbits within a hot spot region were studied with the overall aim of determining environmental patterns of infection and thus the risk of interspecies transmission to livestock. The specific aims were to determine if prevalence of paratuberculosis in rabbits varies temporally between seasons and whether the heterogeneous spatial environmental distribution of M. avium subsp. paratuberculosis on a large scale (i.e., regional hot spots) is replicated at finer resolutions within a hot spot. The overall prevalence of M. avium subsp. paratuberculosis in rabbits was 39.7%; the temporal distribution of infection in rabbits followed a cyclical pattern, with a peak in spring of 55.4% and a low in summer of 19.4%. Spatially, M. avium subsp. paratuberculosis-infected rabbits and, thus, the risk of interspecies transmission were highly clustered in the environment. However, this is mostly due to the clustered distribution of rabbits. The patterns of M. avium subsp. paratuberculosis infection in rabbits are discussed in relation to the host's socioecology and risk to livestock.     

Animal model of Mycoplasma fermentans respiratory infection

Background

Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.

Results

Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.

Conclusions

Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.     

Nucleotide-binding oligomerization domain-2 (NOD2) regulates type-1 cytokine responses to Mycobacterium avium but is not required for host control of infection

Nucleotide-binding oligomerization domain-2 (NOD2) is an innate immune receptor that recognizes peptidoglycan-derived muramyl dipeptide from intracellular bacteria and triggers proinflammatory signals. In this study, we sought to evaluate the role played by this receptor during early and late stages of infection with Mycobacterium avium in mice. We demonstrated that NOD2 knockout (KO) animals were able to control M. avium infection similarly to wild-type mice at all time points studied, even though IL-12 and TNF-α production was impaired in NOD2-deficient macrophages. At 100 days following infection with this bacterium, but not at 30 days post-infection, NOD2-deficient mice showed significantly diminished production of IFN-γ, as confirmed by reduced accumulation of IFN-γ and IL-12 mRNA in the spleens of KO mice. Additionally, a reduction in the size and in the number of lymphocytes/granulocytes of hepatic granulomas from NOD2 KO animals was observed only during late time points of M. avium infection. Taken together, these data demonstrate that NOD2 regulates type-1 cytokine responses to M. avium but is not required for the control of infection with this bacterium in vivo.     

PCR Comparison of Mycobacterium avium Isolates Obtained from Patients and Foods

Mycobacterium avium is a cause of disseminated disease in AIDS patients. A need for a better understanding of possible sources and routes of transmission of this organism has arisen. This study utilized a PCR typing method designed to amplify DNA segments located between the insertion sequences IS1245 and IS1311 to compare levels of relatedness of M. avium isolates found in patients and foods. Twenty-five of 121 food samples yielded 29 mycobacterial isolates, of which 12 were M. avium. Twelve food and 103 clinical M. avium isolates were tested. A clinical isolate was found to be identical to a food isolate, and close relationships were found between two patient isolates and two food isolates. Relatedness between food isolates and patient isolates suggests the possibility that food is a potential source of M. avium infection. This study demonstrates a rapid, inexpensive method for typing M. avium, possibly replacing pulsed-field gel electrophoresis.     

Mouse Model of Respiratory Tract Infection Induced by Waddlia chondrophila

Waddlia chondrophila, an obligate intracellular bacterium belonging to the Chlamydiales order, is considered as an emerging pathogen. Some clinical studies highlighted a possible role of W. chondrophila in bronchiolitis, pneumonia and miscarriage. This pathogenic potential is further supported by the ability of W. chondrophila to infect and replicate within human pneumocytes, macrophages and endometrial cells. Considering that W. chondrophila might be a causative agent of respiratory tract infection, we developed a mouse model of respiratory tract infection to get insight into the pathogenesis of W. chondrophila. Following intranasal inoculation of 2 x 10⁸ W. chondrophila, mice lost up to 40% of their body weight, and succumbed rapidly from infection with a death rate reaching 50% at day 4 post-inoculation. Bacterial loads, estimated by qPCR, increased from day 0 to day 3 post-infection and decreased thereafter in surviving mice. Bacterial growth was confirmed by detecting dividing bacteria using electron microscopy, and living bacteria were isolated from lungs 14 days post-infection. Immunohistochemistry and histopathology of infected lungs revealed the presence of bacteria associated with pneumonia characterized by an important multifocal inflammation. The high inflammatory score in the lungs was associated with the presence of pro-inflammatory cytokines in both serum and lungs at day 3 post-infection. This animal model supports the role of W. chondrophila as an agent of respiratory tract infection, and will help understanding the pathogenesis of this strict intracellular bacterium.     

Roles for Cell Wall Glycopeptidolipid in Surface Adherence and Planktonic Dispersal of Mycobacterium avium

The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. avium and by other Mycobacterium species. In order to test this hypothesis in a directed fashion, three model systems were used to examine biofilm formation by mutants of M. avium with transposon insertions into pstAB (also known as nrp and mps). pstAB encodes the nonribosomal peptide synthetase that catalyzes the synthesis of the core GPL. The mutants did not adhere to polyvinyl chloride plates; however, they adhered well to plastic and glass chamber slide surfaces, albeit with different morphologies from the parent strain. In a model that quantified surface adherence under recirculating water, wild-type and pstAB mutant cells accumulated on stainless steel surfaces in equal numbers. Unexpectedly, pstAB mutant cells were >10-fold less abundant in the recirculating-water phase than parent strain cells. These observations show that GPLs are directly or indirectly required for colonization of some, but by no means all, surfaces. Under some conditions, GPLs may play an entirely different role by facilitating the survival or dispersal of nonadherent M. avium cells in circulating water. Such a function could contribute to waterborne M. avium infection.     

Isolation of Mycobacterium avium subsp. paratuberculosis from Free-Ranging Birds and Mammals on Livestock Premises

Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants (“7,5”) appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock.     

Replication and Long-Term Persistence of Bovine and Human Strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.     

Bordetella pertussis Infection or Vaccination Substantially Protects Mice against B. bronchiseptica Infection

Although B. bronchiseptica efficiently infects a wide range of mammalian hosts and efficiently spreads among them, it is rarely observed in humans. In contrast to the many other hosts of B. bronchiseptica, humans are host to the apparently specialized pathogen B. pertussis, the great majority having immunity due to vaccination, infection or both. Here we explore whether immunity to B. pertussis protects against B. bronchiseptica infection. In a murine model, either infection or vaccination with B. pertussis induced antibodies that recognized antigens of B. bronchiseptica and protected the lower respiratory tract of mice against three phylogenetically disparate strains of B. bronchiseptica that efficiently infect naïve animals. Furthermore, vaccination with purified B. pertussis-derived pertactin, filamentous hemagglutinin or the human acellular vaccine, Adacel, conferred similar protection against B. bronchiseptica challenge. These data indicate that individual immunity to B. pertussis affects B. bronchiseptica infection, and suggest that the high levels of herd immunity against B. pertussis in humans could explain the lack of observed B. bronchiseptica transmission. This could also explain the apparent association of B. bronchiseptica infections with an immunocompromised state.     

Characterization of a Novel Plasmid,pMAH135, from Mycobacterium avium Subsp. hominissuis

Mycobacterium avium complex (MAC) causes mainly two types of disease. The first is disseminated disease in immunocompromised hosts, such as individuals infected by human immunodeficiency virus (HIV). The second is pulmonary disease in individuals without systemic immunosuppression, and the incidence of this type is increasing worldwide. M. avium subsp. hominissuis, a component of MAC, causes infection in pigs as well as in humans. Many aspects of the different modes of M. avium infection and its host specificity remain unclear. Here, we report the characteristics and complete sequence of a novel plasmid, designated pMAH135, derived from M. avium strain TH135 in an HIV-negative patient with pulmonary MAC disease. The pMAH135 plasmid consists of 194,711 nucleotides with an average G + C content of 66.5% and encodes 164 coding sequences (CDSs). This plasmid was unique in terms of its homology to other mycobacterial plasmids. Interestingly, it contains CDSs with sequence homology to mycobactin biosynthesis proteins and type VII secretion system-related proteins, which are involved in the pathogenicity of mycobacteria. It also contains putative conserved domains of the multidrug efflux transporter. Screening of isolates from humans and pigs for genes located on pMAH135 revealed that the detection rate of these genes was higher in clinical isolates from pulmonary MAC disease patients than in those from HIV-positive patients, whereas the genes were almost entirely absent in isolates from pigs. Moreover, variable number tandem repeats typing analysis showed that isolates carrying pMAH135 genes are grouped in a specific cluster. Collectively, the pMAH135 plasmid contains genes associated with M. avium’s pathogenicity and resistance to antimicrobial agents. The results of this study suggest that pMAH135 influence not only the pathological manifestations of MAC disease, but also the host specificity of MAC infection.     

Inefficient Toll-Like Receptor-4 Stimulation Enables Bordetella parapertussis to Avoid Host Immunity

The recognition of bacterial lipopolysaccharide (LPS) by host Toll-like receptor (TLR)4 is a crucial step in developing protective immunity against several gram negative bacterial pathogens. Bordetella bronchiseptica and B. pertussis stimulate robust TLR4 responses that are required to control the infection, but a close relative, B. parapertussis, poorly stimulates this receptor, and TLR4 deficiency does not affect its course of infection. This led us to hypothesize that inefficient TLR4 stimulation enables B. parapertussis to evade host immunity. In a mouse model of infection, B. parapertussis grew rapidly in the lungs, but no measurable increase in TLR4-mediated cytokine, chemokine, or leukocyte responses were observed over the first few days of infection. Delivery of a TLR4 stimulant in the inoculum resulted in a robust inflammatory response and a 10- to 100-fold reduction of B. parapertussis numbers. As we have previously shown, B. parapertussis grows efficiently during the first week of infection even in animals passively immunized with antibodies. We show that this evasion of antibody-mediated clearance is dependent on the lack of TLR4 stimulation by B. parapertussis as co-inoculation with a TLR4 agonist resulted in 10,000-fold lower B. parapertussis numbers on day 3 in antibody-treated wild type, but not TLR4-deficient, mice. Together, these results indicate that inefficient TLR4 stimulation by B. parapertussis enables it to avoid host immunity and grow to high numbers in the respiratory tract of naïve and immunized hosts.