Sheep with scrapie and mastitis transmit infectious prions through the milk

Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.Prion diseases have emerged globally as a significant threat to human and animal health. Recently, human-to-human spread of prions is believed to have occurred through blood transfusions (9, 12, 16), underscoring the importance of understanding possible transmission routes. PrP^Sc, a misfolded, aggregated form of the normal prion protein, PrP^C, commonly accumulates in the follicles of lymphoid tissues, prior to entering the central nervous system (2, 11, 14). Inflammation can cause lymphoid follicles to form in other organs, such as liver and kidney, which leads to prion invasion of organs that are not typically prion permissive (1). In mice, prion infection in the inflamed kidney has the untoward consequence of prion excretion in urine (13). This finding, together with our report of sheep with PrP^Sc in the inflamed mammary gland (8), has raised concerns of prion secretion into milk.Here, we investigated whether PrP^Sc in the inflamed mammary gland leads to prion secretion in milk and infection of naïve lambs through suckling. Prion infectivity has been detected in the milk of sheep expressing a prion gene (Prnp) coding for VRQ/VRQ or VRQ/ARQ at polymorphic codons 136, 154, and 171 (3, 4). However, whether (i) sheep-to-lamb transmission of prions in milk leads to clinical prion disease or (ii) sheep with the common ARQ/ARQ Prnp genotype can infect lambs through milk is unknown. We induced a chronic lentiviral mastitis and inoculated ARQ/ARQ Sarda breed sheep with infectious prions. After 14 months, we bred the sheep and collected the milk. To avoid cross-contamination of newborn lambs, we fed the milk to imported known-naïve lambs and then monitored the lambs for signs of prion infection (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.Sheep infected with prions and maedi-visna virus (MVV) develop lymphofollicular mastitis with PrP^Sc. (A) Experimental scheme. Sheep were inoculated with culture medium or MVV and were then orally exposed to scrapie prions and bred. Milk was collected near the time point that neurologic signs of scrapie developed and was fed to naïve lambs. The ratio of lambs with detectable PrP^Sc to lambs fed the indicated milk is shown for each experiment. (B) PrP immunohistochemistry assay of brain and tonsil from milk source sheep shows staining for PrP^Sc in the brainstem, particularly in the vagal nucleus (indicated by asterisks) and in the tonsillar follicles of scrapie-infected sheep (arrows). (C) Mammary gland (MG) of milk source sheep shows lymphoid follicles (arrowheads) with associated PrP^Sc (arrows) adjacent to milk ducts (md) in the MVV-inoculated sheep, whereas the medium-inoculated sheep had a histologically normal MG with no detectable PrP^Sc. Insets show a high magnification of follicles containing PrP^Sc. Scale bar = 100 μm; scale bar in inset = 25 μm. (D) Western blot analysis shows PrP^Sc detection in MG of sheep inoculated with MVV/scrapie agents but not in sheep inoculated with scrapie prions only. The sheep identification number is indicated for each lane. PK, proteinase K digested; pos. br, positive brain control; neg. br, negative brain control.To induce a chronic lymphofollicular mastitis, we exposed 7- to 10-day-old lambs (groups of 10) by intratracheal and intravenous routes to a common sheep lentivirus known as maedi-visna virus (MVV) or to cell culture medium only. To do this, lambs were inoculated with 3.5 ml intravenously and 0.5 ml intratracheally of MVV in culture supernatant containing 1.5 × 10⁶ tissue culture infectious doses/ml of the “rapid/high” MVV strain 85/34 (5, 15). Twenty days later, all lambs were orally inoculated with 25 ml of 10% scrapie-infected brain homogenate from a pool of naturally infected Sarda sheep.We sequenced the entire Prnp gene and found that all lambs expressed the ARQ/ARQ Prnp genotype, indicating that the sheep should be susceptible to scrapie. As negative controls, 2 lambs of Prnp genotype ARR/ARR and ARQ/ARQ were mock inoculated with cell culture medium and healthy brain homogenate. All lambs originated from scrapie-free flocks that had been monitored for clinical scrapie cases for at least 3 years.All inoculated sheep were naturally bred to rams at 15 months postinoculation (p.i.) and produced lambs at 20 months p.i. Sheep developed early signs of scrapie just after the lambs were born. Milk from each sheep was manually collected and frozen daily.Eight of 10 MVV-and-scrapie (denoted MVV/scrapie)-inoculated sheep and 9 of 10 scrapie-inoculated sheep showed clinical signs of scrapie, with mean incubation periods of 22 ± 1.4 and 23 ± 1.5 months postinoculation, respectively, and were euthanized. There was no significant difference in incubation period between the groups (Student''s t test, P = 0.5), indicating that inflammation associated with the MVV infection does not accelerate prion disease. This finding is consistent with the results of previous studies that showed that chronic pancreatitis or nephritis did not affect the scrapie incubation period (1). Scrapie infection was confirmed postmortem by the detection of PrP^Sc in brain and lymphoid tissues by Western blot and immunohistochemistry assays (Fig. ​(Fig.1B).1B). Interestingly, scrapie did not develop in 3 sheep with a Prnp gene encoding a rare polymorphism at codon 176 (K), consistent with recent reports describing scrapie resistance for this genotype (10).Antibodies to MVV were detected in serum of all the MVV-inoculated sheep by indirect enzyme-linked immunosorbent assay (ELISA) (Elitest kit; Hyphen BioMed). Five of 8 MVV/scrapie-infected sheep (63%) showed a lymphofollicular mastitis (Fig. ​(Fig.1C),1C), and 3 had a diffuse interacinar lymphoid infiltrate. Of the 5 sheep with lymphofollicular mastitis, 4 had PrP^Sc deposits detectable by immunohistochemistry and Western blot assays (Fig. 1C and D), whereas no sheep with diffuse lymphoid infiltrates had detectable PrP^Sc. Surprisingly, 2 of 9 sheep inoculated only with scrapie also had lymphofollicular mastitis and anti-MVV antibodies, one of which had visible PrP^Sc deposits. MVV is a common pathogen in Europe, and it is possible that these sheep were infected from the dam. The remaining 7 scrapie-inoculated sheep had histologically normal mammary glands (Fig. ​(Fig.1C)1C) and no detectable PrP^Sc (Fig. ​(Fig.1D)1D) or anti-MVV antibodies.We selected the stored milk from the 4 MVV/scrapie-infected sheep with PrP^Sc in the mammary glands and from the 7 scrapie-infected sheep with histologically normal mammary glands. Milk samples from the early, middle, and late stages of lactation were pooled for each group. We imported naïve Cheviot lambs (n = 9) from flocks that originated from scrapie-free New Zealand and had been bred and housed under strict biosecurity containment in the United Kingdom to ensure that the lambs had not been exposed to scrapie. The Sarda lambs (n = 4) originated from a scrapie-free flock in Sardinia. We then fed pooled milk from MVV/scrapie-infected sheep to each of 8 naïve ARQ/ARQ lambs and from scrapie-infected sheep to 3 naïve ARQ/ARQ lambs ad libitum. Each lamb ingested a total volume of 1 to 2 liters over a total period of 3 days (Table ​(Table1).1). Two lambs were orally inoculated with brain homogenate pooled from the scrapie-infected milk donors as positive controls. Groups of lambs were housed in separate stalls and subjected to isolation conditions.

TABLE 1.

Genotype, breed, and PrP^Sc detection in lambs fed milk from MVV/scrapie- or scrapie-infected sheep

Evidence for a New Avian Paramyxovirus Serotype 10 Detected in Rockhopper Penguins from the Falkland Islands

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table ​(Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).

TABLE 1.

Characteristics of prototype viruses APMV1 to APMV9 and the penguin virus

A Targeted Multilocus Genotyping Assay for Lineage,Serogroup, and Epidemic Clone Typing of Listeria monocytogenes

A 30-probe assay was developed for simultaneous classification of Listeria monocytogenes isolates by lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (EC) type (ECI, ECIa, ECII, and ECIII). The assay was designed to facilitate rapid strain characterization and the integration of subtype data into risk-based inspection programs.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious invasive illness (listeriosis) in humans and other animals. L. monocytogenes is responsible for over 25% of food-borne-disease-related deaths attributable to known pathogens and is a leading cause of food recalls due to microbial adulteration (12, 21). However, not all L. monocytogenes subtypes contribute equally to human illness, and substantial differences in the ecologies and virulence attributes of different L. monocytogenes subtypes have been identified (9, 13, 14, 23, 24, 33, 35, 36). Among the four major evolutionary lineages of L. monocytogenes, only lineages I and II are commonly isolated from contaminated food and human listeriosis patients (19, 27, 29, 33). Lineage I strains are overrepresented among human listeriosis isolates, particularly those associated with epidemic outbreaks, whereas lineage II strains are overrepresented in foods and the environment (13, 14, 24). Lineage III strains account for approximately 1% of human listeriosis cases but are common among animal listeriosis isolates and appear to be a host-adapted group that is poorly adapted to food-processing environments (6, 34-36). The ecological and virulence attributes of lineage IV are poorly understood, as this lineage is rare and was only recently described based on a small number of strains (19, 26, 29, 33).L. monocytogenes is differentiated into 13 serotypes; however, four major serogroups (4b, 1/2b, 1/2a, and 1/2c) from within lineages I and II account for more than 98% of human and food isolates (16, 31). Serogroups refer to evolutionary complexes typified by a predominant serotype but which include very rare serotypes that represent minor evolutionary variants (7, 9, 33). Phylogenetic analyses have indicated that rare serotypes may have evolved recently, or even multiple times, from one of the major serotypes (9), and numerous molecular methods fail to discriminate minor serotypes as independent groups (1, 4, 7, 9, 18, 22, 33, 38, 39). Serotyping is one of the most common methods for L. monocytogenes subtyping, and serogroup classifications are a useful component of strain characterization because ecotype divisions appear largely congruent with serogroup distinctions (16, 34). Serogroup 4b strains are of particular public health concern because contamination with these strains appears to increase the probability that a ready-to-eat (RTE) food will be implicated in listeriosis (16, 28). Serogroup 4b strains account for approximately 40% of sporadic listeriosis and also are responsible for the majority of listeriosis outbreaks despite being relatively rare contaminants of food products (9, 13, 17, 30, 34). In addition, serogroup 4b strains are associated with more severe clinical presentations and higher mortality rates than other serogroups (11, 16, 20, 31, 34). Serogroups 1/2a and 1/2b are overrepresented among food isolates but also contribute significantly to human listeriosis, whereas serogroup 1/2c rarely causes human illness and may pose a lower risk of listeriosis for humans (16). Serogroup-specific differences in association with human listeriosis are consistent with the prevalence of virulence-attenuating mutations in inlA within these serogroups (32, 34); however, a number of additional factors likely contribute to these differences.Four previously described epidemic clones (ECs; ECI, ECIa, ECII, and ECIII) of L. monocytogenes have been implicated in numerous listeriosis outbreaks and have contributed significantly to sporadic illness (15, 34). ECI, ECIa, and ECII are distinct groups within serogroup 4b that were each responsible for repeated outbreaks of listeriosis in the United States and Europe. ECIII is a lineage II clone of serotype 1/2a that persisted in the same processing facility for more than a decade prior to causing a multistate outbreak linked to contaminated turkey (15, 25). While there has been speculation that epidemic clones possess unique adaptations that explain their frequent involvement in listeriosis outbreaks (9, 34, 37), it is not clear that epidemic clones are more virulent than other strains with the same serotype. However, contamination of RTE food with EC strains would be cause for increased concern due to the previous involvement of these clones in major outbreaks of listeriosis (16).As a result of the L. monocytogenes subtype-specific differences in ecology, virulence, and association with human illness, molecular subtyping technologies have the potential to inform assessments of relative risk and to improve risk-based inspection programs. The objective of the present study was to develop a single assay for rapid and accurate classification of L. monocytogenes isolates by lineage, major serogroup, and epidemic clone in order to facilitate strain characterization and the integration of subtype data into inspection programs that are based on assessment of relative risk.A database of more than 5.3 Mb of comparative DNA sequences from 238 L. monocytogenes isolates (9, 33-35) was scanned for single nucleotide polymorphisms that could be used to differentiate lineages, major serogroups, and epidemic clones via a targeted multilocus genotyping (TMLGT) approach. The acronym TMLGT is used to distinguish this approach from previously published multilocus genotyping (MLGT) assays that were lineage specific and designed for haplotype discrimination (9, 33). To provide for simultaneous interrogation of the selected polymorphisms via TMLGT, six genomic regions (Table ​(Table1)1) were coamplified in a multiplex PCR. While the previous MLGT assays were based on three lineage-specific multiplexes and required prior identification of lineage identity, TMLGT was designed to target variation across all of the lineages simultaneously and is based on a unique set of amplicons. PCR was performed in 50-μl volumes with 1× High Fidelity PCR buffer (Invitrogen Life Technologies), 2 mM MgSO₄, 100 μM deoxynucleoside triphosphate (dNTP), 300 nM primer, 1.5 U Platinum Taq high-fidelity DNA polymerase (Invitrogen Life Technologies), and 100 ng of genomic DNA. PCR consisted of an initial denaturation of 90 s at 96°C, followed by 40 cycles of 30 s at 94°C, 30 s at 50°C, and 90 s at 68°C. Amplification products were purified using Montage PCR cleanup filter plates (Millipore) and served as a template for allele-specific primer extension (ASPE) reactions utilizing subtype-specific probes.

TABLE 1.

Primers used in multiplex amplification for the TMLGT assay

Environmental Sources of Scrapie Prions

Ovine scrapie and cervine chronic wasting disease show considerable horizontal transmission. Here we report that a scrapie-affected sheep farm has a widespread environmental contamination with prions. Prions were amplified by protein-misfolding cyclic amplification (sPMCA) from seven of nine environmental swab samples taken, including those from metal, plastic, and wooden surfaces. Sheep had been removed from the areas from which the swabs were taken up to 20 days prior to sampling, indicating that prions persist for at least that long. These data implicate inanimate objects as environmental reservoirs for prion infectivity that are likely to contribute to facile disease transmission.Prion diseases are fatal neurological disorders. The archetypal prion disease is scrapie in sheep, and in the last few decades novel prion diseases have emerged in a range of species, including bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and variant Creutzfeldt-Jakob disease (vCJD) in humans. The “protein-only” hypothesis dictates that a pathological isoform, PrP^Sc, of the cellular prion protein (PrP^C) constitutes the infectious agent, or prion (13). A wide range of tissues from CWD- and scrapie-affected animals contain PrP^Sc, and affected animals have been shown to excrete or secrete prions in milk, saliva, urine, and feces (2, 3, 6, 7, 8, 10, 16). This finding led to the hypothesis that infectivity resides in the environment, thus explaining the facile transmission of CWD and scrapie. In support of this hypothesis, CWD infectivity has been transmitted from a combination of exposed bedding, water, and food of captive animals (9), and CWD PrP^Sc has been detected within a single environmental water sample (11).Environmental prions are likely to be present at very low levels. The most sensitive method available for the detection of PrP^Sc is serial protein-misfolding cyclic amplification (sPMCA) (1). This technique was pioneered by Soto and colleagues (15), has been used for the amplification of scrapie (17), CWD (4), and vCJD (5) within their natural hosts, and has facilitated the detection of prions within ovine milk (6) and saliva (7) and within cervine urine (4). Here we investigated sources of environmental scrapie prions by applying sPMCA to samples taken from a range of surfaces that were accessible to animals on a farm where scrapie is endemic.Environmental samples were taken from the Veterinary Laboratories Agency, United Kingdom, farm where natural scrapie is endemic, with a high incidence since 1996. Sheep within the flock were exposed to the scrapie agent by natural routes of transmission. Control samples were taken from a farm (ADAS, United Kingdom) that houses a New Zealand-derived scrapie-free flock kept under strict biosecurity conditions. Environmental swabs were taken by wetting foam swabs (Edson Electronics, Northumberland, United Kingdom) in sterile water and then gently swabbing five times in both directions across a surface approximately 10 cm by 2 cm. Two swabs were taken from each area, and all samples were stored at −80^°C. A total of nine environmental samples from a scrapie-affected farm and a scrapie-free farm were analyzed by sPMCA.Two swabs taken from each area were thawed to room temperature and placed in a single container to which 6 ml of 150 mM PO₄ buffer plus 0.5% (vol/vol) Nonidet P-40 and 0.5% (wt/vol) sodium deoxycholate were added. The container was rotated for 2 h. Prions released into this buffer were precipitated on silicon dioxide and then eluted in 200 μl of 0.1% (wt/vol) sodium dodecyl sulfate. Ten microliters of the eluate was amplified within PCR tubes by sPMCA exactly as previously described (7). Samples from both a scrapie-exposed environment and a non-scrapie-exposed environment were analyzed concurrently within the same run on the same sonicator. Each extract was amplified at least in triplicate within a single run and then analyzed by Western blotting (14).Samples from four metal surfaces from an indoor pen occupied by sheep for a few days each week—a gate, a water trough, a feed trough, and penning—were analyzed. Samples from an outdoor environment that had contained sheep 20 days previously—a metal fence, a metal gate, a metal water trough, a plastic post where sheep frequently scratched, and a wooden fence post (Table ​(Table1)—were1)—were also analyzed. After 8 rounds of amplification, PrP^Sc was detected in all samples with the exceptions of the outdoor water trough and gate (Figure ​(Figure11 and Table ​Table1).1). Equivalent samples from a farm housing a scrapie-free flock were also analyzed, and PrP^Sc was not amplified even after 10 rounds of sPMCA. For indoor surfaces from the scrapie-affected farm, 83% of the sPMCA reactions were positive (n = 12), and 0% were positive for equivalent samples from a scrapie-free farm (n = 24). Similarly, 27% of analyses were positive for samples from outdoor surfaces (n = 15), and again no prion was amplified from equivalent samples taken from a scrapie-free farm (n = 30). For comparison of the percentages of positive sPMCA reactions for different cohorts of samples, data were set up as 2 by 2 contingency tables, and Fisher''s exact test (one-tailed) was applied to derive P values. Overall, prions were significantly more likely to be present in the scrapie-affected farm on indoor (P < 0.001) and outdoor (P = 0.009) surfaces. Analyses of all samples were carried out in two independent experiments that gave equivalent results.Open in a separate windowFIG. 1.Amplification of prions from environmental samples. Prions were extracted from swabs taken of surfaces from a scrapie-free farm or a farm where scrapie is endemic. Swabs were taken from a wooden post (1), a plastic scratching post (2), and the following metal surfaces: fencing (3), gate (4 and 6), pen (5), feed trough (7), and water trough (8 and 9). Samples 1, 2, 3, 4, and 8 were taken from outdoor surfaces and 5, 6, 7, and 9 from indoor surfaces. Extracts were used as seeds for 8 rounds of sPMCA. Products were digested with proteinase K and analyzed by Western blotting. PrP was detected with monoclonal antibodies SHA31 and P4. Molecular-weight markers are shown.

TABLE 1.

sPMCA of prions found in the environment^a

Comparative Analysis of Myxococcus Predation on Soil Bacteria

Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.Predation plays a major role in shaping both the ecology and evolution of biological communities. The population and evolutionary dynamics of predators and their prey are often tightly coupled and can greatly influence the dynamics of other organisms as well (1). Predation has been invoked as a major cause of diversity in ecosystems (11, 12). For example, predators may mediate coexistence between superior and inferior competitors (2, 13), and differential trajectories of predator-prey coevolution can lead to divergence between separate populations (70).Predation has been investigated extensively in higher organisms but relatively little among prokaryotes. Predation between prokaryotes is one of the most ancient forms of predation (27), and it has been proposed that this process may have been the origin of eukaryotic cells (16). Prokaryotes are key players in primary biomass production (44) and global nutrient cycling (22), and predation of some prokaryotes by others is likely to significantly affect these processes. Most studies of predatory prokaryotes have focused on Bdellovibrionaceae species (e.g., see references 51, 55, and 67). These small deltaproteobacteria prey on other Gram-negative cells, using flagella to swim rapidly until they collide with a prey cell. After collision, the predator cells then enter the periplasmic space of the prey cell, consume the host cell from within, elongate, and divide into new cells that are released upon host cell lysis (41). Although often described as predatory, the Bdellovibrionaceae may also be considered to be parasitic, as they typically depend (apart from host-independent strains that have been observed [60]) on the infection and death of their host for their reproduction (47).In this study, we examined predation among the myxobacteria, which are also deltaproteobacteria but constitute a monophyletic clade divergent from the Bdellovibrionaceae (17). Myxobacteria are found in most terrestrial soils and in many aquatic environments as well (17, 53, 74). Many myxobacteria, including the model species Myxococcus xanthus, exhibit several complex social traits, including fruiting body formation and spore formation (14, 18, 34, 62, 71), cooperative swarming with two motility systems (64, 87), and group (or “wolf pack”) predation on both bacteria and fungi (4, 5, 8, 9, 15, 50). Using representatives of the genus Myxococcus, we tested for both intra- and interspecific variation in myxobacterial predatory performance across a broad range of prey types. Moreover, we examined whether prey vary substantially in the degree to which they support predatory growth by the myxobacteria and whether patterns of variation in predator performance are constant or variable across prey environments. The latter outcome may reflect adaptive specialization and help to maintain diversity in natural populations (57, 59).Although closely related to the Bdellovibrionaceae (both are deltaproteobacteria), myxobacteria employ a highly divergent mode of predation. Myxobacteria use gliding motility (64) to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth (66). Myxobacterial predation is cooperative both in its “searching” component (6, 31, 82; for details on cooperative swarming, see reference 64) and in its “handling” component (10, 29, 31, 32), in which secreted enzymes turn prey cells into consumable growth substrates (56, 83). There is evidence that M. xanthus employs chemotaxis-like genes in its attack on prey cells (5) and that predation is stimulated by close contact with prey cells (48).Recent studies have revealed great genetic and phenotypic diversity within natural populations of M. xanthus, on both global (79) and local (down to centimeter) scales (78). Phenotypic diversity includes variation in social compatibility (24, 81), the density and nutrient thresholds triggering development (33, 38), developmental timing (38), motility rates and patterns (80), and secondary metabolite production (40). Although natural populations are spatially structured and both genetic diversity and population differentiation decrease with spatial scale (79), substantial genetic diversity is present even among centimeter-scale isolates (78). No study has yet systematically investigated quantitative natural variation in myxobacterial predation phenotypes across a large number of predator genotypes.Given the previous discovery of large variation in all examined phenotypes, even among genetically extremely similar strains, we anticipated extensive predatory variation as well. Using a phylogenetically broad range of prey, we compared and contrasted the predatory performance of 16 natural M. xanthus isolates, sampled from global to local scales, as well as the commonly studied laboratory reference strain DK1622 and representatives of three additional Myxococcus species: M. flavescens (86), M. macrosporus (42), and M. virescens (63) (Table ​(Table1).1). In particular, we measured myxobacterial swarm expansion rates on prey lawns spread on buffered agar (31, 50) and on control plates with no nutrients or with prehydrolyzed growth substrate.

TABLE 1.

List of myxobacteria used, with geographical origin

Impact of Viral Dose and Major Histocompatibility Complex Class IB Haplotype on Viral Outcome in Mauritian Cynomolgus Monkeys Vaccinated with Tat upon Challenge with Simian/Human Immunodeficiency Virus SHIV89.6P

The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6P_cy243. In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID₅₀]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load (P = 0.0280) and CD4 loss (P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID₅₀ (P < 0.0001), and contained acute CD4 loss at 15 MID₅₀ (P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility (P = 0.0199) and resistance (P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion (P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.Advances in typing of the major histocompatibility complex (MHC) of Mauritian cynomolgus macaques (14, 20, 26) have provided the opportunity to address the influence of host factors on vaccine studies (13). Retrospective analysis of 22 macaques vaccinated with Tat or a Tat-expressing adenoviral vector revealed that monkeys with the H6 or H3 MHC class IB haplotype were overrepresented among aviremic or controller animals, whereas macaques with the H2 or H5 haplotype clustered in the noncontrollers (12). More recently, the H6 haplotype was reported to correlate with control of chronic infection with simian immunodeficiency virus (SIV) mac251, regardless of vaccination (18).Here, we performed a retrospective analysis of 112 Mauritian cynomolgus macaques, which included the 22 animals studied previously (12), to evaluate the impact of the challenge dose and class IB haplotype on the acquisition and severity of simian/human immunodeficiency virus (SHIV) 89.6P_cy243 infection in 45 control monkeys and 67 monkeys vaccinated with Tat from different protocols (Table ​(Table11).

TABLE 1.

Summary of treatment, challenge dose, and outcome of infection in cynomolgus monkeys

Hydroxylation and Further Oxidation of Δ9-Tetrahydrocannabinol by Alkane-Degrading Bacteria

The microbial biotransformation of Δ⁹-tetrahydrocannabinol was investigated using a collection of 206 alkane-degrading strains. Fifteen percent of these strains, mainly gram-positive strains from the genera Rhodococcus, Mycobacterium, Gordonia, and Dietzia, yielded more-polar derivatives. Eight derivatives were produced on a mg scale, isolated, and purified, and their chemical structures were elucidated with the use of liquid chromatography-mass spectrometry, ¹H-nuclear magnetic resonance (¹H-NMR), and two-dimensional NMR (¹H-¹H correlation spectroscopy and heteronuclear multiple bond coherence). All eight biotransformation products possessed modified alkyl chains, with hydroxy, carboxy, and ester functionalities. In a number of strains, β-oxidation of the initially formed C₅ carboxylic acid led to the formation of a carboxylic acid lacking two methylene groups.Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) is the decarboxylated product of the corresponding Δ⁹-THC acid, the major cannabinoid present in the cannabis plant (Cannabis sativa L., Cannabaceae). This compound is officially registered as a drug for the stimulation of appetite and antiemesis in patients under chemotherapy and human immunodeficiency virus therapy regimens. Other biological activities ascribed to this compound include lowering intraocular pressure in glaucoma, acting as an analgesic for muscle relaxation, immunosuppression, sedation, bronchodilation, and neuroprotection (11).Δ⁹-THC and many of its derivatives are highly lipophilic and poorly water soluble. Calculations of the n-octanol/water partition coefficient (K_o/w) of Δ⁹-THC at neutral pH vary between 6,000, using the shake flask method (15), and 9.44 × 10⁶, by reverse-phase high-performance liquid chromatography estimation (19). The poor water solubility and high lipophilicity of cannabinoids cause their absorption across the lipid bilayer membranes and fast elimination from blood circulation. In terms of the “Lipinsky rule of 5” (14), the high lipophilicity of cannabinoids hinders the further development of these compounds into large-scale pharmaceutical products.To generate more water-soluble analogues, one can either apply de novo chemical synthesis (as, e.g., in reference 16) or modify naturally occurring cannabinoids, e.g., by introducing hydroxy, carbonyl, or carboxy groups. Chemical hydroxylation of compounds such as cannabinoids is difficult (Δ⁹-THC is easily converted into Δ⁸-THC under mild conditions), and therefore microbial biotransformation of cannabinoids is potentially a more fruitful option to achieve this goal.So far, studies on biotransformation of Δ⁹-THC were mainly focused on fungi, which led to the formation of a number of mono- and dihydroxylated derivatives. Previous reports on the biotransformation of cannabinoids by various microorganisms are summarized in Table ​Table1.1. The aim of the present study was to test whether bacterial strains are capable of transforming Δ⁹-THC into new products (with potentially better pharmaceutical characteristics) at a higher yield and specificity than previously found for fungal strains. For this purpose, we have chosen to use a collection of alkane-degrading strains, since it was shown in previous studies (8, 18, 20) that alkane oxygenases often display a broad substrate range. Production of novel cannabinoid derivatives that might have interesting pharmacological activities was another objective of this project.

TABLE 1.

Previous biotransformation experiments conducted using various microorganisms to transform cannabinoids

Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of Thermobifida fusca Cel9A

Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.Cellulases catalyze the breakdown of cellulose into simple sugars that can be fermented to ethanol. The large amount of natural cellulose available is an exciting potential source of fuels and chemicals. However, the detailed molecular mechanisms of crystalline cellulose degradation by glycoside hydrolases are still not well understood and their low efficiency is a major barrier to cellulosic ethanol production.Thermobifida fusca is a filamentous soil bacterium that grows at 50°C in defined medium and can utilize cellulose as its sole carbon source. It is a major degrader of plant cell walls in heated organic materials such as compost piles and rotting hay and produces a set of enzymes that includes six different cellulases, three xylanases, a xyloglucanase, and two CBM33 binding proteins (12). Among them are three endocellulases, Cel9B, Cel6A, and Cel5A (7, 8), two exocellulases, Cel48A and Cel6B (6, 19), and a processive endocellulase, Cel9A (5, 7).T. fusca Cel9A-90 (Uniprot {"type":"entrez-protein","attrs":{"text":"P26221","term_id":"2506384","term_text":"P26221"}}P26221 and {"type":"entrez-protein","attrs":{"text":"YP_290232","term_id":"72162575","term_text":"YP_290232"}}YP_290232) is a multidomain enzyme consisting of a family 9 catalytic domain (CD) rigidly attached by a short linker to a family 3c cellulose binding module (CBM3c), followed by a fibronectin III-like domain and a family 2 CBM (CBM2). Cel9A-68 consists of the family 9 CD and CBM3c. The crystal structure of this species (Fig. ​(Fig.1)1) was determined by X-ray crystallography at 1.9 Å resolution (Protein Data Bank [PDB] code 4tf4) (15). Previous work has shown that E424 is the catalytic acid and D58 is the catalytic base (11, 20). H125 and Y206 were shown to play an important role in activity by forming a hydrogen bonding network with D58, an important supporting residue, D55, and Glc(−1)O1. Several enzymes with amino acid changes in subsites Glc(−1) to Glc(−4) had less than 20% activity on bacterial cellulose (BC) and markedly reduced processivity. It was proposed that these modifications disturb the coordination between product release and the subsequent binding of a cellulose chain into subsites Glc(−1) to Glc(−4) (11). Another variant enzyme with a deletion of a group of amino acids forming a block at the end of the catalytic cleft, Cel9A-68 Δ(T245-L251)R252K (DEL), showed slightly improved filter paper (FP) activity and binding to BC (20).Open in a separate windowFIG. 1.Crystal structure of Cel9A-68 (PDB code 4tf4) showing the locations of the variant residues, catalytic acid E424, catalytic base D58, hydrogen bonding network residues D55, H125, and Y206, and six glucose residues, Glc(−4) to Glc(+2). Part of the linker is visible in dark blue.The CBM3c domain is critical for hydrolysis and processivity. Cel9A-51, an enzyme with the family 9 CD and the linker but without CBM3c, had low activity on carboxymethyl cellulose (CMC), BC, and swollen cellulose (SWC) and showed no processivity (4). The role of CBM3c was investigated by mutagenesis, and one modified enzyme, R557A/E559A, had impaired activity on all of these substrates but normal binding and processivity (11). Variants with changes at five other CBM3c residues were found to slightly lower the activity of the modified enzymes, while Cel9A-68 enzymes containing either F476A, D513A, or I514H were found to have slightly increased binding and processivity (11) (see Table ​Table1).1). In the present work, CBM3c has been investigated more extensively to identify residues involved in substrate binding and processivity, understand the role of CBM3c more clearly, and study the coordination between the CD and CBM3c. An additional goal was to combine amino acid variants showing increased crystalline cellulose activity to see if this further increased activity. Finally, we have investigated whether the changes that improved the activity of Cel9A-68 also enhanced the activity of intact Cel9A-90.

TABLE 1.

Activities of Cel9A-68 CBM3c variant enzymes and CD variant enzymes used to create the double variants

Mutant PrPSc conformers induced by a synthetic peptide and several prion strains

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP^Sc(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4°C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP^Sc(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a β-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP^Sc(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP^Sc in the brains of Tg196 mice. Our results argue that MoPrP^Sc(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP^C(P101L) as well as in Tg196 mice expressing low levels of MoPrP^C(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a β-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.The discovery that brain fractions enriched for prion infectivity contain a protein (rPrP^Sc) that is resistant to limited proteolytic digestion advanced prion research (8, 37). N-terminal truncation of rPrP^Sc produced a protease-resistant fragment, denoted PrP 27-30, that is readily measured by Western blotting, enzyme-linked immunosorbent assay, or immunohistochemistry. The measurement of PrP^Sc was dramatically changed with the development of the conformation-dependent immunoassay (CDI), which permitted detection of full-length rPrP^Sc as well as previously unrecognized protease-sensitive forms of PrP^Sc (39).The CDI depends on using anti-PrP antibodies that react with an epitope exposed in native PrP^C but that do not bind to native PrP^Sc. Upon denaturation, the buried epitope in PrP^Sc becomes exposed and readily reacts with anti-PrP antibodies. Using the CDI, we discovered that most PrP^Sc is protease sensitive, which we designate sPrP^Sc. Whether sPrP^Sc is an intermediate in the formation of rPrP^Sc remains to be determined. In Syrian hamsters inoculated with eight different strains of prions, the ratio of rPrP^Sc to sPrP^Sc was different for each strain and the concentration of sPrP^Sc was proportional to the length of the incubation time (39).In earlier studies, transgenic (Tg) mice, denoted Tg2866, expressing high levels of PrP(P101L) were used to model Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L point mutation. In the brains of several lines of mice expressing high levels of PrP(P101L), no rPrP^Sc(P101L) was detectable (26, 27, 47). This was particularly perplexing since these Tg mice expressing high levels of PrP(P101L) developed all facets of prion-induced neurodegeneration, including multicentric PrP amyloid plaques. Moreover, brain extracts from ill Tg2866 mice transmitted disease to Tg196 mice expressing low levels of PrP(P101L) that infrequently developed spontaneous neurodegeneration (29).In humans with GSS, several different mutations of the PrP gene (PRNP) resulting in nonconservative amino acid substitutions have been identified (23). In these patients, the clinical presentation, disease course, and amounts of rPrP^Sc in the brain are variable. Brain extracts from humans who died of GSS were inoculated into apes and monkeys, but the transmission rates were not correlated with the levels of PrP^Sc in the inoculum (1, 2, 9, 32). In a limited study, GSS(P102L) was transmitted to Tg mice expressing a chimeric mouse-human (MHu2 M) PrP transgene carrying the P102L mutation but not to Tg mice expressing MHu2M PrP without the mutation (47). In another study, GSS(P102L) human prions were transmitted to Tg mice expressing MoPrP(P101L) in which the transgene was incorporated through gene replacement (31). The use of gene replacement permits all of the regulatory elements that control the wild-type (wt) MoPrP gene to modulate the expression of MoPrP(P101L). In these mice, the expression level of MoPrP(P101L) in brain is likely to be similar to that in Tg196 mice.When we synthesized a 55-mer MoPrP peptide composed of residues 89 to 143 containing the P101L mutation and folded it under conditions favoring a β-structure, it induced neurodegeneration in Tg196 mice (29). When the peptide was not folded into a β-structure, it did not produce disease in Tg196 mice. We report here that the peptide-initiated disease in Tg196 mice could be serially transmitted to other Tg196 mice using brain extracts from the peptide-inoculated Tg196 mice. Using procedures derived from the CDI, brain extracts from inoculated Tg196 mice were found to contain sPrP^Sc(P101L), from which a 22- to 24-kDa PrP fragment was generated by limited digestion with proteinase K (PK) at 4°C and selective precipitation with phosphotungstate (PTA) (25, 39). In the interest of clarity, we have designated digestion at 4°C as “cold PK” and simply refer to standard digestion at 37°C as “PK.” To aid in distinguishing rPrP^Sc(P101L) from sPrP^Sc(P101L), their properties based on the work reported here and in other previously published papers are listed in Table ​Table11 (39, 40).

TABLE 1.

Characteristics of PrP(P101L) isoforms