Cloning and characterization of the gene (rfc) encoding O-antigen polymerase of Pseudomonas aeruginosa PAO1.

The lipopolysaccharide (LPS) O-antigen polymerase is the product of the rfc gene. Loss of O-antigen polymerase activity due to mutation in rfc gives rise to a characteristic LPS phenotype known as core-plus-one or semi-rough, wherein the LPS core is capped with a single oligosaccharide unit. Pseudomonas aeruginosa (Pa) AK1401, a derivative of strain PAO1 (serogroup O5), expresses a semi-rough LPS; this mutant phenotype was complemented by a 2.2-kb NsiI-SacI fragment of Pa PAO1 DNA. Sequence analysis of this fragment revealed a 1317-bp open reading frame (ORF) potentially encoding a 438-amino-acid (aa) protein of 48,849 Da. This DNA sequence and the inferred aa sequence contain many of the features of other O-antigen polymerases, including an aberrantly low G + C content (particularly apparent in the high-G + C background of Pa), an unusual codon usage pattern, and a hydrophobicity profile indicative of a membrane protein. A 345-bp fragment internal to the ORF hybridized to genomic DNA from two of ten Pa serogroup strains examined by Southern blot; these two strains express O antigens structurally related to that of strain PAO1.

The regulation of mortality and fecundity in Schistosoma mattheei following a single experimental infection in sheep.

The regulation of mortality and fecundity of Schistosoma mattheei in sheep was examined using a series of mathematical models applied to data culled from the literature. Parasite mortality (mu) was found to be an increasing linear function of the magnitude of the initial infection over the ranges of doses examined (200-91,000 cercariae) where mu = 9.78 x 10(-3) + 3.476 x 10(-7) infection dose. Parasite fecundity (lambda) was found to be inversely related to the duration of the infection. The best fit model for parasite fecundity was one in which fecundity decreased exponentially with time since initial infection, lambda = lambda 0e-delta(t-tau). There was no evidence for density-dependent regulation of fecundity.

The development and mortality of the free-living stages of Haemonchus contortus in laboratory culture.

Haemonchus contortus eggs were cultured in intact fecal pellets at various temperatures (5-35 degrees C) for 22 days. Temperature and relative humidity were kept constant throughout the incubation period. Nl larval development occurred at 5 degrees C; peak third-stage larval recovery occurred at 20 degrees C. Egg mortality was an age-dependent phenomenon, whereas larval mortality remained constant irrespective of larval age. Development was characterized by a minimum development time followed by a transition to the next stage which occurred at a constant rate. All rates were temperature dependent. The minimum development times reported here are much less than those previously reported. Based on these results a mathematical model was used to describe the demography of the free-living stages of H. contortus at various temperatures.

The mortality and fecundity of Haemonchus contortus in parasite-naive and parasite-exposed sheep following single experimental infections.

Parasite-exposed lambs and their parasite-naive controls were experimentally infected once only with 30,000 H. contortus larvae at 3, 9, 12, and 20 weeks following termination of a moderate immunizing infection of 30,000 H. contortus larvae. Previously exposed lambs, challenged at 3 weeks, had a significant reduction in the total H. contortus worm burden as compared to parasite-naive controls. No difference in the total H. contortus worm burden was found between parasite-exposed or parasite-naive lambs challenged at 9 weeks or thereafter. Female worms were found to be significantly smaller in lambs previously exposed to the parasite as compared to those found in parasite-naive lambs. The average parasite fecundity was 4700 eggs per female worm per day. Previous exposure of the lambs to the parasite had no effect on parasite fecundity. Various mathematical models were used to examine parasite fecundity. Parasite fecundity was found to increase in the initial post-challenge period reaching a constant value approximately 58 days after challenge infection. No density-dependent constraints on fecundity were observed.

A study of the mortality and fecundity of Haemonchus contortus in sheep following experimental infections.

The regulation of the fecundity and mortality of H. contortus in sheep was examined using a series of mathematical models. Six-month-old Dorset crossbred lambs were infected once only with various doses of infective H. contortus larvae (500-20,000 larvae). Parasite mortality was found to be an increasing linear function of the magnitude of the initial infection over the range of doses examined. Parasite fecundity was found to remain constant over the intensity and duration of the infection. The average fecundity for H. contortus at the time of slaughter was found to be 7037 eggs per female worm per day. There was no evidence of time-dependent changes in fecundity or density-dependent regulation of fecundity.

Fecundity of gastrointestinal trichostrongylid nematodes of sheep in the field.

Twenty-two Dorset Rambouillet lambs were moved to contaminated pasture on Apr 1, 1987. At regular intervals thereafter, pairs of lambs were withdrawn and euthanatized. Gastrointestinal parasites in the abomasum, small intestine, cecum, and large intestine were removed and counted. The last pair of lambs was euthanatized 8 months after original placement on the contaminated pasture. Fecal samples were taken at 3- to 4-week intervals throughout the grazing season and the fecal egg counts were used to estimate parasite fecundity (output of eggs per female parasite per day). The principal parasite genera found included Haemonchus spp, Trichostrongylus spp, and Nematodirus spp. In each of the genera examined, parasite fecundity remained the same irrespective of the intensity or duration of infection. Estimated average fecundities (eggs/female/day) were as follows: Haemonchus contortus, 6,582; Trichostrongylus spp, 262; Nematodirus spp, 40; and Oesophagostomum venulosum, 11,098.

Mathematic model for the population biology of rabies in raccoons in the mid-Atlantic states.

A series of coupled differential equations was used to model the temporal dynamics of rabies in raccoons in the mid-Atlantic region of the United States. The model takes explicit account of the development of natural immunity to rabies and was used to evaluate culling and vaccination elimination strategies. For habitats typical of the mid-Atlantic states, and given the assumptions of the model, it was estimated that elimination of rabies in raccoons by culling may involve the annual removal of over 32% of the raccoon population or the yearly vaccination of up to 99% of the susceptible fraction. Assuming a constant marginal cost for both culling and vaccination, the model suggests that, whatever the actual cost of each method, the cheapest strategy will always involve either culling or vaccination alone. A combined strategy of culling and vaccination will be cheaper than culling alone only when the per capita cost of vaccination is around one-fifth or less the per capita cost of culling.

Estimated prevalence of injection-site sarcomas in cats during 1992.

OBJECTIVE: To obtain an estimate of the yearly prevalence of injection-site sarcomas in cats. DESIGN: Mail survey of members of the American Association of Feline Practitioners. PROCEDURE: A questionnaire was sent to 1,112 veterinarians. RESULTS: 235 responses were sufficiently complete for inclusion in the study. Overall, responding veterinarians reported 744,993 cat visits in 1992, representing 434,638 individual cats (1.7 visits/cat). The estimated overall prevalence of injection-site sarcomas during 1992 was 0.00021 cases/cat visit (2.1 cases/10,000 cat visits) or 0.00036 cases/cat (3.6 cases/10,000 cats). CLINICAL IMPLICATIONS: Results suggest that injection-site sarcomas were rare during 1992.

Canine parvovirus.

Canine parvovirus is a truly new pathogen of dogs that emerged in the late 1970s. Initially seen as epidemic disease in all dogs, parvoviral enteritis is now primarily a disease of 1- to 6-month-old dogs. Maternal antibody interference with immunization accounts for the vast majority of vaccine "breaks." Molecular virologic methods have revealed continued evolution of the virus, but this appears to be of greater academic than practical interest. Clinical diagnosis can be definitive in fulminant cases but requires laboratory support--usually demonstration of virus in the feces--in less clear-cut cases. Treatment remains symptomatic, based simply on principles of good supportive care. As the virus is firmly entrenched in both the wild and domestic canine population, elimination of the virus is impossible, and CPV-2 will remain a concern for the small animal practitioner indefinitely.

Duration of immunity in cats after vaccination or naturally acquired infection.

The necessity for cats to be vaccinated annually against common pathogens has been questioned because sarcomas have infrequently been reported at the injection site. However, with few exceptions, the duration of immunity induced by vaccination or infection is uncertain, and there may therefore be a risk associated with a decision not to revaccinate. This article reviews the information available about the duration of immunity induced by vaccination or infection in cats, and reveals many shortcomings that make blanket recommendations impossible. Each vaccine must be considered individually.

Duration of immunity in dogs after vaccination or naturally acquired infection.

This paper reviews current scientific information about the duration of immunity induced in dogs by infection or vaccination. It describes the shortcomings of the methods used to measure the immune responses of dogs, and explains the need for basic studies on the nature of protective humoral and cellular responses, and standardised assays for the long-term duration of immunity to pathogens other than rabies. The information is inadequate to warrant uniform recommendations on the ideal intervals for vaccination; each vaccine must be evaluated on the basis of its own merits and the characteristics of the disease it is intended to guard against.

Gallstone disease.

Most gallstones are composed largely or entirely of cholesterol. The larger calculi are more often associated with acute cholecystitis than are smaller stones. Factors predisposing to gallstone formation include sex, age, race, child-bearing, and possibly diet and obesity. About half of all persons with cholelithiasis have symptoms referable to the biliary tract. The most important symptom in the diagnosis of gallstone disease is biliary colic. Biliary pain lasting longer than five or six hours is indicative of acute cholecystitis, with obstruction of the cystic duct by a calculus as the primary event in most instances. The reliability of cholecystography in detecting gallstones is at least 95 percent. In patients over age 60, cholecystectomy is indicated only in those with specific symptoms referable to the biliary tract. The effectiveness of chenodeoxycholic acid in dissolving radiolucent gallstones in asymptomatic patients has been confirmed in several clinical trials. Early operation in patients with acute cholecystitis is advocated.

Efficacy of two canine parvovirus vaccines for inducing seroconversion in Rottweiler and Doberman pinscher pups with various levels of maternally derived antibodies.

The study reported here investigated the efficacy of two commonly used modified-live virus vaccines to induce seroconversion against canine parvovirus (CPV) in 213 Rottweiler and Doberman pinscher pups with various titers of maternally derived CPV antibody. Beginning at 6 to 8 weeks of age, pups were given a subcutaneous vaccination every 21 days (range, 18-24 days) in the dorsal region of the neck or shoulder area. Pups vaccinated with vaccine A(a) received three vaccinations and completed the vaccination series by 12 to 14 weeks of age. Pups vaccinated with vaccine Bb received four vaccinations and completed the vaccination series by 15 to 17 weeks of age. Antibody titers against CPV in both vaccine groups were similar before vaccination. Pups in the vaccine-A group seroconverted significantly earlier than those in the vaccine-B group. After the first vaccination, more pups with a CPV-2b hemagglutination inhibition (HI) titer of < or = 1:80 responded to vaccine A than to vaccine B. In addition, CPV-2b HI titers after vaccination were also significantly (P < or = 0.05) higher for the pups in the vaccine-A group after first, second, and third vaccinations, compared with those of pups in the vaccine-B group.

Seroconversion of puppies to canine parvovirus and canine distemper virus: a comparison of two combination vaccines.

Sixty puppies were randomly assigned to receive one of two commercially available combination vaccines, and responses to the canine parvovirus and canine distemper virus components of the vaccines were determined by measuring serum antibody titers. The percentage of puppies that seroconverted to canine parvovirus was significantly higher and the mean time for seroconversion was significantly shorter for puppies that received one of the vaccines than for puppies that received the other vaccine. Percentages of puppies that seroconverted to canine distemper virus were not significantly different.

Carbohydrate malabsorption in black and Hispanic dialysis patients.

Patients on chronic hemodialysis have decreased food intake and decreased fat stores. Malabsorption of carbohydrates such as lactose, sorbitol, or fructose cause functional bowel symptoms. The aim of this study was to assess the role of carbohydrate malabsorption in the nutritional abnormalities of chronic hemodialysis (CHD). Eleven patients on dialysis (six Hispanic, five black Americans) were studied, compared to 11 healthy volunteers age-, race-, and sex-matched. Lactulose 10 g (transit time), lactose 12.5 g, sorbitol 5 g, and fructose 37.5 g were tested fasting. Breath [H2] was measured 4 h postprandially by gas chromatograph analysis. Positive test was defined as 20 ppm [H2] above baseline. Weight, height, and triceps skinfold were measured. One hundred percent of CHD patients were below the 50th percentile for triceps skinfold measurement and 55% were below the 10th percentile. No biochemical abnormalities were noted. Breath [H2] tests: lactulose: all patients in both groups responded with positive tests. No difference in transit time was noted. Lactose: 73% of CHD had positive test compared to 36% control. Sorbitol: 73% of CHD had positive test compared to 27% control (p less than 0.05). Fructose: 27% CHD compared to 0% control. This study confirmed that CHD patients have decreased fat stores. It demonstrates for the first time that CHD patients have increased incidence of malabsorption of sorbitol. This carbohydrate malabsorption may contribute to the nutritional abnormalities of CHD.

Dietary cholesterol affects chenodeoxycholic acid action on biliary lipids.

Chenodeoxycholic acid (CDC) decreases biliary saturation and dissolves gallstones in one-half of the treated patients. Dietary cholesterol also affects biliary lipids and is a possible factor explaining unsuccessful CDC therapy. The aim of this investigation was to study the effect of high and low dietary cholesterol on the CDC-induced decrease of biliary saturation and activity of hepatic hydroxymethylglutaryl coenzyme A reductase (HMG-CoAR). Seventy two hamsters in six groups were fed for 1 month one of three diets: 0.8 mg of cholesterol per g of food, 2.4 mg of cholesterol per g, or cholesterol-free. On each diet hamsters received no CDC or CDC 30 mg per kg per day. When animals were killed, biliary lipids were determined and the activity of hepatic HMG-CoAR was assayed. CDC administration decreased the saturation index (SI)(P less than 0.01) in hamsters on the high cholesterol and standard diets but not on the cholesterol-free diet. The SI in CDC-treated hamsters on the high cholesterol (0.78 +/- 0.03) and cholesterol-free (0.68 +/- 0.02) diets were greater (P less than 0.02) than in CDC-treated hamsters on the standard diet (0.48 +/- 0.03). CDC decreased (P less than 0.01) HMG-CoA reductase activity on each diet. In comparison to HMG-CoAR activity (190 +/- 7.6 pmoles per mg per min) in CDC-treated hamsters on the standard diet, the activity in CDC-treated hamsters on the high cholesterol diet (176 +/- 5.8 pmoles per mg per min) was decreased ( less than 0.05), whereas the activity on the cholesterol-free diet (495 +/- 11.5 pmoles per mg per min) was greater (P less than 0.01). It is concluded that: (1) dietary cholesterol is necessary for optimum CDC inhibition of HMG-CoAR; (2) high cholesterol and cholesterol-free diets prevent maximum CDC decrease of the biliary saturation index; (3) dietary cholesterol alterations may therefore be one cause of the failure of CDC dissolution of gallstones.

Interstrain variation of the polysaccharide B biosynthesis locus of Bacteroides fragilis: characterization of the region from strain 638R.

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.

The Pseudomonas aeruginosa algC gene encodes phosphoglucomutase, required for the synthesis of a complete lipopolysaccharide core.

We have constructed strains of Pseudomonas aeruginosa with mutations in the algC gene, previously shown to encode the enzyme phosphomannomutase. The algC mutants of a serotype O5 strain (PAO1) and a serotype O3 strain (PAC1R) did not express lipopolysaccharide (LPS) O side chains or the A-band (common antigen) polysaccharide. The migration of LPS from the algC mutant strains in Tricine-sodium dodecyl sulfate-polyacrylamide gels was similar to that of LPS from a PAO1 LPS-rough mutant, strain AK1012, and from a PAC1R LPS-rough mutant, PAC605, each previously shown to be deficient in the incorporation of glucose onto the LPS core (K. F. Jarrell and A. M. Kropinski, J. Virol. 40:411-420, 1981, and P. S. N. Rowe and P. M. Meadow, Eur. J. Biochem. 132:329-337, 1983). We show that, as expected, the algC mutant strains had no detectable phosphomannomutase activity and that neither algC strain had detectable phosphoglucomutase (PGM) activity. To confirm that the PGM activity was encoded by the algC gene, we transferred the cloned, intact P. aeruginosa algC gene to a pgm mutant of Escherichia coli and observed complementation of the pgm phenotype. Our finding that the algC gene product has PGM activity and that strains with mutations in this gene produce a truncated LPS core suggests that the synthesis of glucose 1-phosphate is necessary in the biosynthesis of the P. aeruginosa LPS core. The data presented here thus demonstrate that the algC gene is required for the synthesis of a complete LPS core in two strains with different LPS core and O side chain structures.