Are you PEPped and PrEPped for travel? Risk mitigation of HIV infection for travelers.

The HIV pandemic persists globally and travelers are at risk for infection by the Human Immunodeficiency Virus (HIV). While HIV-focused guidelines delineate risk stratification and mitigation strategies for people in their home communities, travel issues are not addressed. In this review, direct and indirect evidence on HIV risk among travelers is explored. The burgeoning practice of employing pre-exposure prophylaxis (PrEP) with anti-retroviral therapy in the non-travel setting is introduced, as well as the more established use of post-exposure prophylaxis (PEP). Challenges in applying these lessons to travelers are discussed, and a new guidelines process is scoped and recommended.

Herpes simplex virus type 2 and HIV infection among US military personnel: implications for health prevention programmes.

US military personnel are routinely screened for HIV infection. Herpes simplex virus type 2 (HSV-2) is a risk factor for HIV acquisition. To determine the association between HSV-2 and HIV, a matched case-control study was conducted among US Army and Air Force service members with incident HIV infections (cases) randomly matched with two HIV-uninfected service members (controls) between 2000 and 2004. HSV-2 prevalence was significantly higher among cases (30.3%, 138/456) than among controls (9.7%, 88/912, P < 0.001). HSV-2 was strongly associated with HIV in univariate (odds ratio [OR] = 4.2, 95% confidence interval [CI] = 3.1-5.8) and multiple analyses (adjusted [OR] = 3.9, 95% CI = 2.8-5.6). The population attributable risk percentage of HIV infection due to HSV-2 was 23%. Identifying HSV-2 infections may afford the opportunity to provide targeted behavioural interventions that could decrease the incidence of HIV infections in the US military population; further studies are needed.

Expression of FGF2 and TGFalpha and testis morphology during testicular hypertrophy subsequent to hemicastration in the neonatal boar.

The objective was to ascertain fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), and transforming growth factor-alpha (TGFalpha) mRNA expression and testis morphology during accelerated testicular growth after hemicastration in the neonatal boar. On Day 10 after birth (Day 0), boars were assigned to control (n = 28), no treatment; hemicastrated (n = 28), left testis removed. The right testis in both groups (n = 7) was removed on Days 5, 10, 15, and 20. Expression of mRNA for FGF2, EGF, and TGFalpha was determined by qRT-PCR using TaqMan. Testicular morphology was determined on Day 15. On Day 10, hemicastrated boars had a greater (P = 0.01) testis weight (6.2 +/- 0.8 g; mean +/- SEM) than controls (4.3 +/- 0.4 g) and on Day 15 testis weight in hemicastrated boars (8.8 +/- 0.8 g) was twice (P < 0.01) that of control boars (4.2 +/- 0.3 g). Seminiferous tubule volume was approximately doubled in hemicastrated boars (P < 0.01) and was associated with an increase (P < 0.01) in Sertoli cell number. Interstitial compartment volume was greater (P < 0.01) in hemicastrated boars. Leydig cell numbers were similar (P = 0.14) but volume was greater (P < 0.01) for hemicastrates. There were no differences (P > 0.05) between control and hemicastrated boars in TGFalpha or FGF2 expression on Day 5 or Day 10, and EGF was not detected. It was concluded that upregulation of TGFalpha or FGF2 expression is not a pre-requisite for enhanced testicular growth and increased Sertoli cell proliferation that occurs subsequent to hemicastration in the neonatal boar.

The characterization of lactic acid producing bacteria from the rumen of dairy cattle grazing on improved pasture supplemented with wheat and barley grain.

AIMS: To identify and characterize the major lactic acid bacteria in the rumen of dairy cattle grazing improved pasture of rye grass and white clover and receiving a maize silage and grain supplement with and without virginiamycin. METHODS AND RESULTS: Eighty-five bacterial isolates were obtained from the rumen of 16 Holstein-Friesian dairy cows. The isolates were initially grouped on the basis of their Gram morphology and by restriction fragment length polymorphism analysis of the PCR amplified 16S rDNA. A more definitive analysis was undertaken by comparing the 16S rDNA sequences. Many of the isolates were closely related to other previously characterized rumen bacteria, including Streptococcus bovis, Lactobacillus vitulinus, Butyrivibrio fibrisolvens, Prevotella bryantii and Selenomonas ruminantium. The in vitro production of L- and/or D-lactate was seen with all but five of the isolates examined, many of which were also resistant to virginiamycin. CONCLUSION: Supplementation of grain with virginiamycin may reduce the risk of acidosis but does not prevent its occurrence in dairy cattle grazing improved pasture. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that lactic acid production is caused, not only by various thoroughly researched types of bacteria, but also by others previously identified in the rumen but not further characterized.

High prevalence of HIV infection among rural tea plantation residents in Kericho, Kenya.

Human immunodeficiency virus type 1 (HIV-1) epidemiology among residents of a rural agricultural plantation in Kericho, Kenya was studied. HIV-1 prevalence was 14.3%, and was higher among women (19.1%) than men (11.3%). Risk factors associated with HIV-1 for men were age (>or=25 years), marital history (one or more marriages), age difference from current spouse (>or=5 years), Luo ethnicity, sexually transmitted infection (STI) symptoms in the past 6 months, circumcision (protective), and sexual activity (>or=7 years). Among women, risk factors associated with HIV-1 were age (25-29 years, >or=35 years), marital history (one or more marriages), age difference from current spouse (>or=10 years), Luo ethnicity, STI symptoms in the past 6 months, and a STI history in the past 5 years. Most participants (96%) expressed a willingness to participate in a future HIV vaccine study. These findings will facilitate targeted intervention and prevention measures for HIV-1 infection in Kericho.

A nascent HIV type 1 epidemic among injecting drug users in Kabul, Afghanistan is dominated by complex AD recombinant strain, CRF35_AD.

Injecting drug use (IDU), common in global centers of heroin production, confers significant risk for HIV-1 infection. Once introduced into IDU networks, an explosive rise in HIV-1 infection typically occurs, fueled principally by needle sharing. New HIV-1 epidemics in IDUs have occurred in Russia, China, Thailand, Spain, Iran, and in other countries, and some have spread into other risk groups in their respective countries. In Afghanistan, the introduction of HIV-1 into IDU networks has begun, but a recent report of 3% HIV-1 prevalence suggests that the epidemic is still at an early stage. Here we establish, by complete genome sequencing and phylogenetic analysis of four viral strains from Afghan IDUs, that all are the same complex recombinant strain, combining HIV-1 subtypes A and D and herein termed CRF35_AD. Published partial HIV-1 sequences from an HIV-1 epidemic among IDUs in Iran, already at 23.2% HIV-1 prevalence, are either CRF35_AD or a related recombinant. Voluntary HIV-1 screening and harm reduction programs in Afghanistan, applied now, could limit the spread of HIV-1, both in IDUs and in other social networks.

Characterization of Streptococcus bovis from the rumen of the dromedary camel and Rusa deer.

AIMS: Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. METHODS AND RESULTS: Bacteria were isolated from the rumen contents of four camels and two deer fed lucerne hay by culturing on the semi-selective medium MRS agar. Based on Gram morphology and RFLP analysis seven isolates, MPR1, MPR2, MPR3, MPR4, MPR5, RD09 and RD11 were selected and putatively identified as Streptococcus. The identity of these isolates was later confirmed by comparative DNA sequence analysis of the 16S rRNA gene with the homologous sequence from S. bovis strains, JB1, C14b1, NCFB2476, SbR1, SbR7 and Sb5, from cattle and sheep, and the Streptococcus equinus strain NCD01037T. The percentage similarity amongst all strains was >99%, confirming the identification of the camel isolates as S. bovis. The strains were further characterized by their ability to utilize a range of carbohydrates, the production of volatile fatty acids (VFA) and lactate and the determination of the doubling time in basal medium 10 supplemented with glucose. All the isolates produced l-lactate as a major fermentation end product, while four of five camel isolates produced VFA. The range of carbohydrates utilized by all the strains tested, including those from cattle and sheep were identical, except that all camel isolates and the deer isolate RD11 were additionally able to utilize arabinose. CONCLUSIONS: Streptococcus bovis was successfully isolated from the rumen of camels and deer, and shown by molecular and biochemical characterization to be almost identical to S. bovis isolates from cattle and sheep. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus bovis is considered a key lactic acid producing bacterium from the gastrointestinal tract of ruminants, and has been implicated as a causative agent of lactic acidosis. This study is the first report of the isolation and characterization of S. bovis from the dromedary camel and Rusa deer, and suggests a major contributive role of this bacterium to fermentative acidosis.

Phylogenetic positions of phytoplasmas associated with dieback, yellow crinkle and mosaic diseases of papaya, and their proposed inclusion in 'Candidatus Phytoplasma australiense' and a new taxon, 'Candidatus Phytoplasma australasia'.

DNA extracted from three papaya (Carica papaya L.) plants, individually affected by dieback, yellow crinkle or mosaic diseases, was subjected to PCR using phytoplasma-specific primers to amplify the 16S rRNA gene plus 16S-23S rRNA intergenic spacer region. Near-complete DNA sequences obtained for the three PCR amplimers were subjected to phylogenetic analyses and direct sequence comparison with other phytoplasma 16S rDNA and 16S-23S spacer region DNA sequences. The papaya yellow crinkle (PpYC) and papaya mosaic (PpM) sequences were identical to each other, but distinctly different from the papaya dieback (PpDB) sequence, showing 90.3% identity in the he 16S rDNA and 87.8% identity in the 16S-23S spacer region DNA sequences. A phylogenetic tree based on 16S rDNA sequences was calculated, in which PpYC and PpM are most closely related to the tomato big bud phytoplasma (TBB; 99.7% 16S rDNA sequence identity) from Australia, within subclade iii. This subclade consists of strains only reported occurring in the Southern Asian region and Australia, which indicates an Asian/Australasian origin. PpDB is most closely related to the Phormium yellow leaf phytoplasma from new Zealand (PYL; 99.9% identity) and the Australian grapevine yellows phytoplasma (AGY; 99.7% identity). These three phytoplasma strains form a distinct clade within subclade xii, which also includes the European strains STOL and VK as another distinct clade. The origin of the closely related but geographically separated AGY-like strains and STOL-like strains of subclade xii is unclear. It is proposed that phytoplasma strains PpDB, PYL and AGY be included in the previously described taxon 'Candidatus Phytoplasma australiense', and that PbYC, PpM and TBB be assigned to a new taxon, "Candidatus Phytoplasma australasia'.

Changes in testicular steroidogenic acute regulatory (STAR) protein, steroidogenic enzymes and testicular morphology associated with increased testosterone secretion in bulls receiving the luteinizing hormone releasing hormone agonist deslorelin.

Testosterone secretion and the expression and relative contents of steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450SCC), 3beta-hydroxysteroid dehydrogenase/delta(5)-->delta(4)-isomerase (3 beta-HSD), and (17)alpha-hydroxylase cytochrome P450/C17-20 lyase (P450(17)alpha) were determined in testicular tissues of bulls treated with a LHRH agonist. Testis morphology and spermatogenesis were also examined. In Experiment 1, bulls (30-mo-old) received no treatment (control, n = 7) or were implanted for 10 days with the LHRH agonist deslorelin (n = 7). Bulls were castrated on Day 10 and testis tissues prepared for Western and Northern blotting. At castration, bulls implanted with deslorelin had greater plasma testosterone (5-fold) and testis content of testosterone (10-fold) compared with control bulls. Relative content (per micrograms total testis protein or RNA) of StAR protein, 3beta-HSD, P450SCC, and mRNA for P450(17)alpha in bulls treated with deslorelin ranged from 3- to 6-fold that of control bulls. In Experiment 2, bulls (20-mo-old) were left untreated (control, n = 6) or implanted with deslorelin (n = 12) for 120 days. On Day 120, bulls were castrated and right testis tissues prepared for morphology. Testis volume and weight were increased (P < 0.01) in bulls treated with deslorelin compared with control bulls. Stereological analysis revealed that this increase occurred in all compartments (seminiferous epithelium, lumen and interstitium) studied, but was significant (P < 0.01) only for the seminiferous epithelium. Absolute numbers of round spermatids per testis were increased (P < 0.05) in bulls treated with deslorelin compared with control bulls. Increased testosterone secretion in bulls treated with deslorelin was associated with increased testicular StAR protein and steroidogenic enzymes. Bulls treated long-term with deslorelin had a faster rate of testis growth and increased daily sperm production at the end of the experiment.

Epidemiology of Phytoplasma-Associated Papaya Diseases in Queensland, Australia.

Three phytoplasma-related diseases of papaya (Carica papaya), dieback, yellow crinkle, and mosaic, are recognized within Australia. Immature leaf material was sampled every week for 8 months from a cohort of 60 female plants, located within a commercial papaya plantation, to determine the minimum time between infection and symptom expression. Phytoplasma DNA was detected using the polymerase chain reaction (PCR) with primers specific for phytoplasmas in general, and for the stolbur group of phytoplasmas. The dieback-associated phytoplasma was detected 1 week prior to (four cases) or the same week (nine cases) as symptom expression, while phytoplasma DNA was detected between 3 and 11 weeks prior to expression of mosaic symptom (six cases). Lateral shoot regrowth on the lower stem of plants which had suffered dieback disease failed to generate stolbur-specific PCR products in 15 cases. A dual infection with dieback and yellow crinkle or mosaic was diagnosed in a further two cases, using restriction fragment length polymorphism digests, and both cases were interpreted as secondary infections by the dieback-associated phytoplasma. Regrowth in three of seven cases of yellow crinkle- and three of nine cases of mosaic-affected plants tested positive for phytoplasma-specific DNA. Ratooning of dieback-affected plants and removal of yellow crinkle- or mosaic-affected plants is suggested for the management of these diseases.

Histopathology and Within-Plant Distribution of the Phytoplasma Associated with Australian Papaya Dieback.

Dieback-affected papaya plants were characterized by a discoloration of the contents of laticifers, while the anatomy of sieve elements was healthy in appearance until the necrotic stages of the disorder were reached. Laticifer discoloration was not always associated with the presence of phytoplasma in affected tissue, as judged by polymerase chain reaction (PCR) using primers based on the 16S rRNA gene and 16S-23S intergenic spacer region. Phytoplasma DNA was detected in a range of plant tissues, including roots, but not in mature leaves which would act as photoassimilate sources. As plants recovered from a dieback period, the extent of the distribution of both laticifer discoloration and phytoplasma DNA decreased. Phytoplasma cells were not observed in transmission electron microscopy studies of mature sieve elements of dieback-affected leaf, stem, or fruit tissue from plants at various stages of symptom expression, although PCR tests indicated the presence of phytoplasma DNA. Membrane-bound structures, similar in shape and size to phytoplasma cells but interpreted as autophagic vesicles or latex vesicles in immature laticifers, were observed within vacuoles of cells in phloem tissue in leaves displaying tissue breakdown in the form of a water-soaked appearance to veins ("X-Y" patterning). In contrast, phytoplasmas were readily observed in papaya leaves displaying symptoms of yellow crinkle. We conclude that phytoplasma cells are present in very low titer in dieback-affected tissues and that, while the plant appears to limit proliferation of the dieback-associated pathogen, this defense strategy is ultimately unsuccessful because it is associated with a rapid decline of the papaya plant.

Pertussis: an update on primary prevention and outbreak control.

Pertussis is a highly contagious respiratory disease. Infected adolescents and adults with mild illness are the source of potentially life-threatening illness in infants and young children. The incidence of pertussis has been rising steadily in recent years. Primary vaccination is 80 percent effective, but protection is transient. Pertussis can be difficult to diagnose because classic whooping cough is uncommon, disease manifestations are often atypical, and laboratory and radiologic aids are frequently nonspecific. Diagnosis is usually based on the clinical picture, but culture, direct fluorescent antibody tests and serology can be helpful. Antibiotic therapy can decrease the duration and severity of illness, and prevents secondary spread if started early. Effective management of pertussis outbreaks requires early diagnosis and treatment of cases, antibiotic prophylaxis of contacts and accelerated vaccination of susceptible infants and children. Acellular pertussis vaccine preparations have recently been recommended for the entire primary vaccination series.

Increased testosterone secretion in bulls treated with a luteinizing hormone releasing hormone (LHRH) agonist requires endogenous LH but not LHRH.

The requirement for endogenous LHRH and LH action in the maintenance of elevated plasma concentrations of testosterone in bulls receiving the LHRH agonist deslorelin was examined. In Experiment 1, bulls were either (i) left untreated (control); (ii) implanted with deslorelin; (iii) actively immunized against LHRH; or (iv) implanted with deslorelin and immunized against LHRH. Experiment 2 was of similar design to Experiment 1, except that bulls were immunized against LH in place of LHRH. In Experiment 1, plasma LH declined in bulls immunized against LHRH, but not in the bulls immunized against LHRH and implanted with deslorelin. Also in Experiment 1, plasma testosterone declined in bulls immunized against LHRH but was elevated in bulls treated with deslorelin and bulls treated with deslorelin and immunized against LHRH. In Experiment 2, bulls immunized against LH and treated with deslorelin had plasma concentrations of testosterone similar to controls, whereas bulls treated only with deslorelin had elevated plasma testosterone. It was concluded from these experiments that endogenous LHRH action was not required for increased steroidogenic activity in bulls treated with a LHRH agonist. However, circulating LH was necessary for increased plasma testosterone in bulls implanted with deslorelin. LH is therefore involved in mediating the response of bulls to treatment with deslorelin, either by acting directly at the testes or through a permissive role that allows a direct action of deslorelin at the testes.

Differential responses in anterior pituitary luteinizing hormone (LH) content and LH beta- and alpha-subunit mRNA, and plasma concentrations of LH and testosterone, in bulls treated with the LH-releasing hormone agonist deslorelin.

Anterior pituitary gland contents of LH and LH beta- and alpha-subunit mRNAs, and circulating concentrations of LH and testosterone, were determined in bulls treated with the LH-releasing hormone (LHRH) agonist deslorelin. Brahman (Bos indicus) bulls (14-month-old) were allocated to two groups and received the following: Control (n = 5), no treatment; Deslorelin (n = 4), four deslorelin implants (approximately 200 micrograms total deslorelin/day) for 36 d. Plasma concentrations of LH were higher in bulls treated with deslorelin on Day 1, had returned to typical levels by Day 8, and did not differ for control bulls and bulls treated with deslorelin from Day 8 to Day 29. Pituitary content of LH on Day 36 was reduced (P < 0.001) in bulls treated with deslorelin (33 +/- 4 ng/mg) compared with control bulls (553 +/- 142 ng/mg). Relative pituitary content of LH beta-subunit mRNA was also reduced on Day 36 in bulls treated with deslorelin (Control, 0.65 +/- 0.10; Deslorelin, 0.22 +/- 0.04; P = 0.003). However, alpha-subunit mRNA relative content did not differ (Control, 0.73 +/- 0.15; Deslorelin, 1.06 +/- 0.12; P > 0.05). Plasma concentrations of testosterone were increased over the period of the experiment in the bulls treated with deslorelin compared with control bulls. This is the first demonstration of reduced pituitary content of LH beta-subunit mRNA and LH, and unaltered content of alpha-subunit mRNA, in bulls treated with LHRH agonist. This was associated with apparently typical plasma concentrations of LH and elevated plasma testosterone. The anterior pituitary in bulls treated with LHRH agonist, therefore, undergoes classical desensitization and downregulation, but plasma LH and testosterone are not suppressed.

Direct actions of the luteinizing hormone-releasing hormone agonist, deslorelin, on anterior pituitary contents of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), LH and FSH subunit messenger ribonucleic acid, and plasma concentrations of LH and FSH in castrated male cattle.

The objective in this study was to characterize direct effects of the LHRH agonist, deslorelin, on anterior pituitary gland function in male cattle in the absence of gonadal feedback. Castrated bulls (steers), 30 mo old, were allocated to four groups: group 1, control, no treatment (n = 8); group 2, five deslorelin implants (approximately 250 micrograms total deslorelin/day) for 42 days (n = 8); group 3, control+ LHRH (50 micrograms i.m.) at weekly intervals (n = 3); group 4, five deslorelin implants+LHRH as for group 3 (n = 3). Plasma LH was similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 4, and continued to decrease to Day 41 (group 1, 1.71 +/- 0.20 ng/ml [mean +/- SEM]; group 2, 0.38 +/- 0.03 ng/ml [p < 0.001]). Mean plasma concentrations of FSH were similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 7, and declined to Day 41 (group 1, 43.5 +/- 3.9 ng/ml; group 2, 17.5 +/- 1.5 ng/ml [p < 0.001]). Steers in group 3 showed increases in plasma LH after injection of LHRH on all occasions, while steers in group 4 did not show increases in plasma LH from Day 14 onward. Mean relative pituitary contents (arbitrary units) of LH beta- and FSH beta-subunit mRNAs were reduced on Day 42 in steers treated with deslorelin (LH beta: groups 1 and 3, 1.56 +/- 0.27; groups 2 and 4, 0.08 +/- 0.01 [p < 0.001]; FSH beta: groups 1 and 3, 1.01 +/- 0.08; groups 2 and 4, 0.34 +/- 0.07 [p < 0.001]). However, alpha-subunit mRNA was similar for control steers and steers treated with deslorelin (groups 1 and 3, 1.00 +/- 0.11; groups 2 and 4, 0.86 +/- 0.12 [p > 0.1]). Pituitary content of LH, but not FSH, was reduced in steers treated with deslorelin. In summary, steers treated with deslorelin showed desensitization to natural LHRH, and this was associated with reduced pituitary contents of LH and FSH beta-subunit mRNAs, a reduction in pituitary content of LH, and decreases in plasma concentrations of LH and FSH. This demonstrated, for the first time, a direct action of LHRH agonist on LH and FSH beta-subunit gene expression in cattle, independent of gonadal feedback. Also, there was a differential effect of treatment with deslorelin on gonadotropin alpha- and beta-subunit mRNA contents in the anterior pituitary.

Electrophoretic conditions for high resolution citrus isozymes in polyacrylamide gel electrophoresis.

Electrophoretic conditions including electrode and gel buffers, acrylamide concentration, use of stacking gels, voltage, current, and run time were investigated in order to produce isozyme bands of high resolution which would facilitate densitometric quantification of enzyme activity following polyacrylamide gel electrophoresis (PAGE). Electrode buffers which provided optimal conditions for gels stained for the isozymes of malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGD), phosphoglucose isomerase (PGI), and shikimate dehydrogenase (SkDH) were 0.02 M Tris-glycine, pH 8.5, 0.1 M sodium borate, pH 6.0, 0.1 M sodium borate, pH 8.7, and 0.07 M sodium borate, pH 7.0, respectively. A 0.5 M Tris-HCl, pH 7.5, gel buffer was optimal for gels stained for the isozymes of 6-PGD, PGI and SkDH. A 0.5 M Tris-HCl, pH 8.5, gel buffer was best for gels stained for MDH. Stacking gels were found to be detrimental to enzyme activity and showed no improvement in resolution for any of the enzymes. Acrylamide concentration for gels stained for MDH were 8.7%, gels stained for 6-PGD and PGI were 7.5%, while gels stained for SkDH had an acrylamide concentration of 5.0%. Higher concentrations above these levels caused a reduction and in some cases loss of band activity, while below this concentration there was a decrease in band resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of extracts for electrophoresis from citrus leaves.

Extracting proteins from vegetative tissues while maintaining good enzyme activity and electrophoretic resolution presents numerous problems due to the presence of phenols, quinones, proteases and other components released during cell disruption. To overcome this problem in Citrus leaves, an extraction buffer was developed which contained EDTA, potassium chloride, magnesium chloride hexahydrate, PVP-40, 2-mercaptoethanol and bovine serum albumin in a Tris-HCl buffer, pH 7.5. This extraction buffer was used in association with liquid nitrogen for sample preparation. Buffers used in previous studies for Citrus isozyme extraction for PAGE were found to provide unsatisfactory resolution and activity for the three enzyme systems investigated (malate dehydrogenase, 6-phosphogluconate dehydrogenase and shikimate dehydrogenase). This extraction buffer maintains high enzyme activity and provides good resolution in PAGE gels suitable for densitometric analysis.

Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid.

A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.

Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens.

A reliable and efficient method has been developed for the electroporation-mediated transformation of Clostridium perfringens with plasmid DNA. Transformation of vegetative cells of C. perfringens strain 13 with the 7.9-kb Escherichia coli-C. perfringens shuttle plasmid pHR 106 required pretreatment with lysostaphin (2 to 20 micrograms/ml) for 1 h at 37 degrees C. Cells harvested early in the logarithmic stage of growth were transformed more efficiently than cells at other growth phases. The transformation frequency increased with the DNA concentration, to a saturating level at 5 to 10 micrograms DNA/ml. The transformation frequency was proportional to the field strength and time constant of the electroporation pulse; however, the field strength was a far more important parameter. A cell density between 1 x 10(8) and 5 x 10(8) cells/ml proved to be optimal for transformation. The procedure was capable of generating up to 3.0 x 10(5) transformants per micrograms DNA. The potential value of the method for the cloning of C. perfringens genes was demonstrated by the cloning of the clostridial tetracycline-resistance determinant, tetP, from the E. coli recombinant plasmid pJIR71, into C. perfringens strain 13.