Molecular characterisation of a candidate gut sucrase in the pea aphid, Acyrthosiphon pisum.

The hydrolysis of sucrose, the principal dietary source of carbon for aphids, is catalysed by a gut alpha-glucosidase/transglucosidase activity. An alpha-glucosidase, referred to as APS1, was identified in both a gut-specific cDNA library and a sucrase-enriched membrane preparation from guts of the pea aphid Acyrthosiphon pisum by a combination of genomic and proteomic techniques. APS1 contains a predicted signal peptide, and has a predicted molecular mass of 68 kDa (unprocessed) or 66.4 kDa (mature protein). It has amino acid sequence similarity to alpha-glucosidases (EC 3.2.1.20) of glycoside hydrolase family 13 in other insects. The predicted APS1 protein contains two domains: an N-terminal catalytic domain, and a C-terminal hydrophobic domain. In situ localisation and RT-PCR studies revealed that APS1 mRNA was expressed in the gut distal to the stomach, the same localisation as sucrase activity. When expressed heterologously in Xenopus embryos, APS1 was membrane-bound and had sucrase activity. It is concluded that APS1 is a dominant, and possibly sole, protein mediating sucrase activity in the aphid gut.

Microbial forensics for natural and intentional incidents of infectious disease involving animals.

Microbial forensics is a relatively new scientific discipline dedicated to analysing microbiological evidence from a crime for attribution purposes. It builds on traditional microbiology and epidemiology but within a legal framework. Important motives for forensic investigations include interdiction of criminals, prosecution of justice, and ideally, deterrence of others from committing similar acts. Forensic capabilities in animal health should focus on building capacity for detection and reporting of increases in infectious disease morbidity and mortality among animals that might reflect a covert release of a pathogen. Suspicion should be raised when epidemiological patterns are different from those expected for the animal population and the pathogen in question. Existing capacities for the detection and reporting of epidemic and even endemic diseases should be an international priority for the prevention of catastrophic losses in animal and potentially in human life. The veterinary community needs to be more aware of the legal requirements related to forensic investigations so that veterinarians will be prepared to handle evidence properly within their own fields.

Leptospirosis among patients presenting with dengue-like illness in Puerto Rico.

Leptospirosis is difficult to distinguish from dengue fever without laboratory confirmation. Sporadic cases/clusters of leptospirosis occur in Puerto Rico, but surveillance is passive and laboratory confirmation is rare. We tested for leptospirosis using an IgM ELISA on sera testing negative for dengue virus IgM antibody and conducted a case-control study assessing risk factors for leptospirosis, comparing clinical/laboratory findings between leptospirosis (case-patients) and dengue patients (controls). Among 730 dengue-negative sera, 36 (5%) were positive for leptospirosis. We performed post mortem testing for leptospirosis on 12 available specimens from suspected dengue-related fatalities; 10 (83%) tested positive. Among these 10 fatal cases, pulmonary hemorrhage and renal failure were the most common causes of death. We enrolled 42 case-patients and 84 controls. Jaundice, elevated BUN, hyperbilirubinemia, anemia, and leukocytosis were associated with leptospirosis (p < .01 for all). Male sex, walking in puddles, rural habitation, and owning horses were independently associated with leptospirosis. Epidemiological, clinical, and laboratory criteria may help distinguish leptospirosis from dengue and identify patients who would benefit from early antibiotic treatment.

Carbohydrate analysis.

The analysis of the carbohydrate chains attached to proteins is becoming increasingly important as appreciation of the role of glycosylation in the structural and functional properties of biologically significant glycoproteins grows. Over the past year, a number of developments have been made that may improve and promote the analysis of the glycosylation of proteins.

The significance of gut sucrase activity for osmoregulation in the pea aphid, Acyrthosiphon pisum.

The osmotic pressure of the body fluids of aphids is lower than in their diet of plant phloem sap. It is hypothesised that aphids reduce the osmotic pressure of ingested food by sucrase-mediated hydrolysis of dietary sucrose to glucose and fructose, and the polymerisation of glucose into oligosaccharides of low osmotic pressure per hexose unit. To test this hypothesis, the impact of the alpha-glucosidase inhibitor acarbose on the sugar relations and osmoregulation of aphids was explored. Acarbose inhibited sucrase activity in gut homogenates and the production of monosaccharides and oligosaccharides in the honeydew of live aphids. Acarbose caused an increase in the haemolymph osmotic pressure for aphids reared on a diet (containing 0.75 M sucrose) hyperosmotic to the haemolymph and not on the isoosmotic diet containing 0.2 M sucrose. It did not affect aphid feeding rate over 2 days, except at high concentrations on 0.75 M sucrose diet, and this may have been a secondary consequence of osmotic dysfunction. Acarbose-treated aphids died prematurely. With 5 microM dietary acarbose, mean survivorship on 0.2 M sucrose diet was 4.2 days, not significantly different from starved aphids, indicating that, although these aphids fed, they were deprived of utilisable carbon; and on 0.75 M sucrose diet, mean survivorship was just 2.8 days, probably as a consequence of osmotic failure. It is concluded that the aphid gut sucrase activity is essential for osmoregulation of aphids ingesting food hyperosmotic to their body fluids.

Leptospirosis: an emerging health problem in Thailand.

Leptospirosis is an emerging health problem in Thailand, with dramatic increases in reported incidence since 1996. The annual number of reported leptospirosis cases increased from 398 cases in 1996 to 14,285 cases in 2000. In 2001, 2002, and 2003, the number of reported cases decreased, but still remained high at 10,217, 6,864, and 4,958 cases, respectively. The epidemiological characteristics of leptospirosis in Thailand include a peak incidence in September and October in association with the rainy season. A vast majority of the cases (90%) were reported in the Northeast region. The case fatality rate was as high as 4.4%, having a predominant association with male farmers aged 15 to 45 years. Outpatient cases were approximately 9 times more common than admitted cases, with an apparent recent shift in the pattern of infecting serovars among reservoir animals and humans.

Outbreak of histoplasmosis among cavers attending the National Speleological Society Annual Convention, Texas, 1994.

In June 1994, 18 people developed serologically confirmed histoplasmosis following cave exploration associated with the annual National Speleological Society Convention in Bracketville, Texas. Six others had an undiagnosed illness suspected to be histoplasmosis. Two persons were hospitalized. We conducted a survey of convention attendees and a nested case-control study of those entering caves. We also conducted a histoplasmin skin test survey of a subgroup of the society, the Texas Cavers Association, who were attending a reunion in October 1994. Among the national convention attendees, exposure to two caves was identified as responsible for 22 (92%) of the 24 cases; 12 (75%) of 16 people exploring one cave (Cave A) and 10 (77%) of 13 exploring a separate cave (Cave B) developed acute histoplasmosis. Additional risk-factors included fewer years of caving experience, longer time spent in the caves, and entering a confined crawl space in Cave A. Of 113 participants in the separate skin test survey, 68 (60%) were found to be skin test positive, indicating previous exposure to Histoplasma capsulatum. A positive skin test was significantly associated with male sex and more years of caving experience. Those less experienced in caving associations should be taught about histoplasmosis, and health care providers should pursue histories of cave exposure for patients with bronchitis or pneumonia that does not respond to initial antibiotic therapy.

Risk factors associated with leptospirosis in northeastern Thailand, 1998.

Leptospirosis is a zoonotic disease of worldwide distribution caused by spirochetes of the genus Leptospira. Humans are infected through direct contact with infected animals or through exposure to fresh water or soil contaminated by infected animal urine. Leptospirosis is characterized by acute fever that can be followed by a more severe, sometimes fatal illness that may include jaundice and renal failure (Weil's disease), meningitis, myocarditis, hemorrhagic pneumonitis, or hemodynamic collapse. To identify potential risk factors for leptospirosis in Thailand, we conducted a matched case-control study in Nakornratchasrima Province of the northeastern region. Fifty-nine cases and 118 controls were included in the study. Four activities in the two weeks prior to illness were independently associated with leptospirosis infection: walking through water (odds ratio [OR] = 4.9, 95% confidence interval [CI] = 1.7-14.1), applying fertilizer in wet fields for more than 6 hr a day (OR = 3.4, 95% CI = 1.5-7.8), plowing in wet fields for more than 6 hr a day (OR = 3.5, 95% CI = 1.1-11.6), and pulling out rice plant sprouts in wet fields for more than 6 hr a day (OR = 3.1, 95% CI = 1.02-9.3). Identification of these risk factors on admission might prove useful for early diagnosis and treatment of leptospirosis in Thailand.

Studies on the control of visceral leishmaniasis: validation of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) for field diagnosis of canine visceral leishmaniasis.

The Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA), the latest version of the enzyme-linked immunosorbent assay, uses antigen-coated beads. A 96-well plate can be run in 20 min without electricity or expensive equipment. We compared the FAST-ELISA, a standard ELISA, and an indirect immunofluorescent assay (IFA) for evaluation of canine leishmaniasis under field conditions using samples from 161 dogs from our longitudinal study in the endemic area of Jacobina, Bahia, Brazil. Organisms were isolated by culture (NN medium) or by inoculation of hamsters with samples from 59 of the dogs. When plasma were tested, we found a sensitivity of 88% and a specificity of 90% using the FAST-ELISA with a spectrophotometer. Using the same plasma samples, the IFA had a sensitivity of 75% and a specificity of 93%. The standard ELISA had a sensitivity of 90% and a specificity of 85%. When whole blood was tested with the FAST-ELISA, we found a sensitivity of 85%. There was no significant difference between visual and spectrophotometric results with plasma or whole blood. The FAST-ELISA system provides a sensitive, specific, and field-adaptable test for canine visceral leishmaniasis, which can be evaluated quickly without the use of a microscope or spectrophotometer.

Comparison of the polymerase chain reaction and serology for the detection of canine visceral leishmaniasis.

The polymerase chain reaction (PCR) and serology was evaluated for the diagnosis of canine visceral leishmaniasis in Bahia, Brazil in a study of 125 dogs. The PCR was 100% sensitive in 25 dogs that had Leishmania demonstrated by either culture or hamster inoculation. It was 100% specific for 35 dogs from the northeastern United States, all were PCR negative. However, 22 of 54 Brazilian dogs that were culture-hamster inoculation-negative were positive by PCR. The nature of the PCR product was identified by hybridization with specific Leishmania probes. Whereas the sensitivity of serology in relationship to infection, as determined by hamster or culture assay was more than 80%, sensitivity of serology was only 63% when compared with PCR. These results raise questions about the use of serology to detect Leishmania infection in dogs, and suggest that the PCR might serve as a better gold standard to define Leishmania infection than culture or hamster inoculation.

Studies on control of visceral leishmaniasis: impact of dog control on canine and human visceral leishmaniasis in Jacobina, Bahia, Brazil.

To assess the effect of removing leishmania-infected dogs on the incidence of visceral leishmaniasis, a controlled intervention study was performed in northeast Brazil. The attempted elimination of seropositive dogs resulted in an initial significant decrease in the annual incidence of seroconversion among dogs from 36% to 6% over the first two years. In the following two years, the incidence increased to 11% and 14%, respectively. In a control area in which dogs were surveyed but seropositive dogs were not removed, the cumulative incidence did not vary significantly from year to year, ranging from 16% to 27%. In the intervention area, the prevalence of dog seropositivity decreased from 36% before the intervention to 10% and remained stable. These findings suggest that attempting to remove seropositive dogs is insufficient as a measure for eradicating visceral leishmaniasis in dogs. However, the force of transmission of infection among dogs can be reduced by such programs. Also, when the number of human cases before and after the start of the intervention was calculated, a significant decrease in incidence of disease in the intervention area was observed among children less than 15 years of age (P < 0.01). The results of this intervention study suggest that the elimination of the majority of seropositive dogs may affect the cumulative incidence of seroconversion in dogs temporarily and may also diminish the incidence of human cases of visceral leishmaniasis.

Asymptomatic infection and risk factors for leptospirosis in Nicaragua.

As part of an investigation of a 1995 outbreak of leptospirosis in Nicaragua, a cross-sectional serologic survey was conducted in the town of El Sauce. Of 566 persons, 85 (15%) were positive for IgM anti-Leptospira antibodies, indicating recent leptospirosis infection. Asymptomatic leptospirosis infection was common, with only 25 (29.4%) of the 85 seropositive inhabitants reporting a febrile illness in the 2 months before the survey. Multivariable analysis revealed that having an indoor water source remained independently protective against leptospirosis. Gathering wood was independently associated with infection. These findings suggest that asymptomatic infection with Leptospira is common in endemic areas of Leptospira transmission. Improvement in water sanitation and behavioral modifications to reduce environmental exposure may reduce the risk of leptospirosis in endemic regions.

The glycosylation of glycoprotein lectins. Intra- and inter-genus variation in N-linked oligosaccharide expression.

Glycosylated lectins represent a series of glycoproteins with related activities and, in the case of the Leguminosae, related amino acid sequences. Therefore, they offer a model system in which to study the diversity of N-linked oligosaccharide structures of plant glycoproteins. The influence of the polypeptide on the type of oligosaccharide substitution and the problem of inter- and intra-genus variation in glycosylation can also be addressed. Analysis of the glycosylation of 18 lectins has shown that they can be classified into four qualitatively similar groups on the basis of the Bio-Gel P-4 elution profiles of the oligosaccharides released by hydrazinolysis: (a) The Erythrina cristagalli profile, with a major component at 8.8 glucose units (gu) and minor components at 8.0, 7.2, and 5.8 gu. The major component is the heptasaccharide, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-[beta-D-Xyl p-(1----2)]- beta-D-Manp-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-(1----3)]- D-GlcNAc. (b) The Phaseolus vulgaris profile, which was characterized by peaks at 12.5, 11.7, 10.8, and 9.9 gu, in addition to the peaks at 8.8, 8.0, 7.2, and 5.8 gu mentioned above. These higher-mol.-wt. components were oligo-D-mannose oligosaccharides containing 9, 8, 7, and 6 D-mannose residues, respectively. (c) The Lonchocarpas capassa profile, which had a major peak at approximately 8 gu. (d) The soybean agglutinin profile, which has a single peak at 12.5 gu. This peak consisted solely of an oligomannose undecasaccharide containing 9 D-mannose residues. This lectin is unique in that it shows no microheterogeneity.

Infectivity of low numbers of Toxoplasma gondii oocysts to pigs.

To define the infectiousness of the VEG strain of Toxoplasma gondii, 42 pigs were fed doses estimated at 10, 1, or < 1 mouse infective oocysts. They were killed 38-99 days after inoculation and 50 g of tissues from their tongue, heart, and brain were individually homogenized in acidic pepsin solution and bioassayed in mice. Pools of brain, heart, tongue, and skeletal muscle (total 500 g) were bioassayed in cats. Toxoplasma gondii was isolated by bioassays in mice and in cats from 13 of 14 pigs fed 10 oocysts, 13 of 14 pigs fed 1 oocyst, and 4 of 14 pigs fed "less than" 1 oocyst, indicating high infectivity of VEG strain of T. gondii to pigs. All infected pigs developed modified agglutination test antibodies (> 1:50). Control pigs (n = 6) remained seronegative (< 1:20) and T. gondii was not isolated from their tissues. Toxoplasma gondii was isolated from tongues of 27 (93%), brains of 21 (72%), and hearts of 13 (45%) of 29 experimentally infected pigs by bioassay in mice. The number of T. gondii-positive mice after inoculation of tongue, brain, and heart from infected pigs was 240 (80%), 84 (28%), and 36 (12%) of 300 mice inoculated with each organ, respectively. Thus, the VEG strain of T. gondii was localized more often and in higher numbers in the tongue than in the brain and the heart of pigs. The apparent muscle localization after infection with the low dose of the VEG strain of T. gondii agrees with other studies in livestock that suggest T. gondii is more neurotropic in mice than in livestock.

Epidemiology of selected mycobacteria that infect humans and other animals.

This paper provides a summary of salient clinical and epidemiological features of selected mycobacterial diseases that are common to humans and other animals. Clinical and diagnostic issues are discussed and related to estimates of the incidence and prevalence of these diseases among humans. Source of infection, route of transmission and control measures are also presented. The mycobacteria discussed in this paper are Mycobacterium bovis, M. ulcerans, M. leprae and M. avium complex, although this is by no means a complete list of the mycobacteria common to humans and other animals. Certain generalities can be made regarding these species of mycobacteria and their occurrence in humans and other animals; firstly, current understanding of the epidemiology and control of many of the resultant diseases is incomplete; secondly, environmental sources other than animal reservoirs may play a role in transmission (with M. leprae perhaps being the exception); and thirdly, the incidence and prevalence of these diseases in many countries of the world are unclear, principally because of the complexity of diagnosis and lack of reporting systems.

Analysis of the 1998 outbreak of leptospirosis in Missouri in humans exposed to infected swine.

OBJECTIVE: To determine the extent of leptospirosis in persons exposed to infected swine, confirm the source of disease, define risk factors for infection, and identify means for preventing additional infections during an outbreak in Missouri in 1998. DESIGN: Cross-sectional study. SAMPLE POPULATION: 240 people and 1,700 pigs. PROCEDURE: An epidemiologic investigation was conducted of people exposed to infected pigs from the University of Missouri-Columbia swine herd. The investigation included review of health of the pigs, a cross-sectional study of the people handling the pigs, serologic testing of human and porcine sera, and risk-factor analysis for leptospirosis within the human population. RESULTS: Serologic testing of samples collected at the time of the investigation indicated that 59% of the pigs had titers to leptospires, denoting exposure. Of the 240 people in the exposed study population, 163 (68%) were interviewed, and of these, 110 (67%) submitted a blood sample. Nine (8%) cases of leptospirosis were confirmed by serologic testing. Risk factors associated with leptospirosis included smoking (odds ratio [OR], 14.4; 95% confidence interval [CI], 1.39 to 137.74) and drinking beverages (OR, 5.1; 95% CI, 1.04 to 24.30) while working with infected pigs. Washing hands after work was protective (OR, 0.2; 95% CI, 0.03 to 0.81). CONCLUSIONS AND CLINICAL RELEVANCE: Leptospirosis is a risk for swine producers and slaughterhouse workers, and may be prevented through appropriate hygiene, sanitation, and animal husbandry. It is essential to educate people working with animals or animal tissues about measures for reducing the risk of exposure to zoonotic pathogens.

Injury among cavers: results of a preliminary national survey.

BACKGROUND: To estimate the frequency of and risk factors for caving-associated injuries. METHODS: A standardized questionnaire covering demographics, caving exposure, and injury history was distributed to all members of the National Speleological Society by inclusion in the monthly newsletter. RESULTS: Of 9,532 members sent a questionnaire, 301 responded (3.2%). Respondents had an average of 18 years of caving experience, and 37% had sustained one or more injuries while caving. Hypothermia was the most frequent injury, followed by fractures, animal bites, and concussions. The rate of injury was about 1 per 1,990 hours in a cave. Injury rates for females were about twice those of males; older persons and those with more than 5 years of caving experience seemed to have lower injury rates. CONCLUSIONS: Many caving injuries appear potentially preventable. Proper technique for safe climbing should be a part of exploration training. There is a need for proper belaying or rappelling for even short ascents or descents. Helmet use should be stressed, as should adequate protection from hypothermia.

Susceptibility to Buruli ulcer is associated with the SLC11A1 (NRAMP1) D543N polymorphism.

Similar to other mycobacterial diseases, susceptibility to Buruli ulcer (Mycobacterium ulcerans infection) may be determined by host genetic factors. We investigated the role of SLC11A1 (NRAMP1) in Buruli ulcer because of its associations with both tuberculosis and leprosy. We enrolled 182 Buruli ulcer patients (102 with positive laboratory confirmation) and 191 healthy neighbourhood-matched controls in Ghana, and studied three polymorphisms in the SLC11A1 gene: 3' UTR TGTG ins/del, D543N G/A, and INT4 G/C. Finger prick blood samples from study subjects were dried on filter papers (FTA) and processed. D543N was significantly associated with Buruli ulcer: the odds ratio (adjusted for gender, age, and region of the participant) of the GA genotype versus the GG genotype was 2.89 (95% confidence intervals (CI): 1.41-5.91). We conclude that a genetic polymorphism in the SLC11A1 gene plays a role in susceptibility to develop Buruli ulcer, with an estimated 13% population attributable risk.

Living on a high sugar diet: the fate of sucrose ingested by a phloem-feeding insect, the pea aphid Acyrthosiphon pisum.

The natural diet of aphids, plant phloem sap, generally contains high concentrations of sucrose. When pea aphids (Acyrthosiphon pisum) were fed on chemically defined diets containing sucrose radiolabelled in the glucose or fructose moiety, 2 to 12-fold and 87 to 110-fold more radioactivity was recovered from the tissues and honeydew, respectively, of aphids that ingested [U-(14)C-glucose]-sucrose than from those ingesting [U-(14)C-fructose]-sucrose. The total radioactivity recovered was 70% of the ingested [U-(14)C-glucose]-sucrose and <5% of ingested [U-(14)C-fructose]-sucrose. The dominant honeydew sugars produced by aphids feeding on 0.75 M sucrose diets were oligosaccharides comprising glucose. In vitro the guts of pea aphids had high sucrase activity, 1-5 U mg(-1) protein, generating equimolar glucose and fructose except at high sucrose concentrations where glucose production was inhibited (K(si)=0.1 M). These data suggest that the fructose moiety of ingested sucrose is assimilated very efficiently and may be preferentially respired by the aphid, and that the glucose moiety of sucrose is incorporated into oligosaccharides by the transglucosidase activity of the gut sucrase at high sucrose concentrations. These differences in the fate of sucrose-derived glucose and fructose are important elements in both the carbon nutrition and osmoregulation of aphids.