Serological and molecular survey of Dirofilaria immitis infection in stray cats in Gyunggi province, South Korea.

The aim of this study was to carry out a serological and molecular survey for the presence of Dirofilaria immitis infection in stray cats using an ELISA kit and PCR assay. One hundred and fifty-five stray cats (77 females and 78 males) in Gyunggi province in South Korea, were used in this study. Four (2.6%) tested with the ELISA kit showed a positive reaction, and all positive samples by the ELISA kit showed a positive reaction by PCR analysis. No significant difference was observed between the male (2.6%) and female (2.6%) cat groups by ELISA kit. The positive rates for dirofilariosis were 2.8% in the 4-6-year-old group, and 18.2% in the > 6-year-old group by ELISA kit. With regard to the age element, older cats showed a higher prevalence of D. immitis infection in this study. A statistical analysis revealed that significant difference was observed in > 6-year-old group (p < 0.01). In conclusion, D. immitis infection in stray cats was present in Gyunggi province, although its incidence was low. Therefore, heartworm treatment and/or prophylaxis for stray cats captured are required in this area.

Intergenomic translocations of polyploid oats (genus Avena) revealed by genomic in situ hybridization.

Wild and cultivated hexaploid oats share the same genomes (AACCDD) and display a considerable level of interspecific variation in both plant and chromosome morphology. The GISH was utilized to detect the interspecific genomic compositions in four hexaploid and two tetraploid oats using total genomic DNA of Avena eriantha (a C-genome diploid) as probe. Intergenomic translocations between A/D and C-genome chromosomes were frequently observed in hexaploid and tetraploid species. In the hexaploid, two pairs of A/D genome segments on C-genome chromosome (A/D-C) translocation and four to six pairs of C-genome segments on A/D genome chromosome (C-A/D) translocation were clearly identified whilst the number of A/D-C translocations was constant among species. In the tetraploid A. maroccana (AACC), a pair of A-C and four pairs of C-A translocations were observed. Moreover, the A/D translocation segments on chromosome 5C was detected only in A. byzantina and A. maroccana, whilst A/D-C translocations were observed on the 1C and 7C of A. sativa, A. fatua and A. sterilis. A. byzantina did however also carry the 1C rearrangement. This result shows that A. byzantina has retained a similar genomic constitution to the tetraploid ancestor of hexaploid oats, A. maroccana. Three pairs of A-C translocations were detected only in A. murphyi (AACC), and two pairs of those were the 1C and 7C as well as the three hexaploid species except A. byzantina.

Intraspecific variation of 18S-5.8S-26S rDNA sites revealed by FISH and RFLP in wild oat, Avena agadiriana.

The tetraploid species Avena agadiriana that was first described in 1985 is distributed on the Atlantic coastal strip south of Casablanca in Morocco. Five accessions of this species (M55, M59, M60, M71 and M74) were compared by using FISH and RFLP analysis of 18S-5.8S-26S rDNA. The FISH data indicated that three pairs of major hybridization sites of the rDNA were located on satellite chromosomes in accessions M55, M59, M60 and M71. Accession M74, however, had only two pairs of major sites of hybridization. A pair of the major rDNA sites in M71 was very small and closely located at the terminal region of Nor-ST (Nucleolar organizing region of subtelocentrics) chromosomes. RFLP analysis of the rDNA sequence fragments identified differences among M55, M71 and M74, whilst M59 and M60 were the same with regard to the four restriction enzyme fragments utilized. M74 always lost single rDNA fragments in four restriction enzyme digests. The RFLP data made it possible to distinguish M55 from M59 and M60 in the northern Haut-Atlas Mountains group. A unique 20 kb EcoRI fragment characterized M71. Thus, a combination of FISH and RFLP analysis of rDNA was a good tool for inferring intraspecific evolutionary relationship of A. agadiriana.

Comparative study and cDNA cloning of the flavoprotein subunit of mitochondrial complex II (succinate-ubiquinone oxidoreductase: fumarate reductase) from the dog heartworm, Dirofilaria immitis.

Mitochondrial complex II functions as a fumarate reductase (FRD), the reverse reaction of succinate dehydrogenase (SDH), and plays an important role in the anaerobic respiratory chain of parasitic helminths. In this study, complex II from the dog heartworm, Dirofilaria immitis adult, which is thought to act as a homolactatic fermenter, was examined in terms of its enzymatic features and primary structure in order to investigate the possible role of mitochondria in this filaria. Mitochondria from D. immitis adult showed high FRD activity when the enzymatic assay was performed using methylviologen as an artificial electron donor. The ratio of SDH to FRD in D. immitis was comparable to that in Ascaris suum adult, which is known to have an anaerobic mitochondrial respiratory chain with a high FRD activity of complex II. The FRD activity of D. immitis mitochondria was inhibited by the sulfhydryl reagent N-ethylmaleimide (NEM), while that of A. suum complex II was resistant to this inhibitor. The presence of the flavoprotein (Fp) subunit, which contains the substrate binding active site, was confirmed in D. immitis mitochondria by immunoblotting using a monoclonal antibody against the A. suum Fp subunit. By homology probing with the polymerase chain reaction, the entire cDNA for the D. immitis adult Fp was cloned and sequenced. The deduced amino acid sequence showed significant homology to that of A. suum and other mitochondrial Fps, in contrast to much less similarity to bacterial FRD, even though the D. immitis complex II showed high FRD activity. These results are the first indication of the presence of a functional complex II in D. immitis mitochondria.

Diurnal variation in microfilaremia in a cat experimentally infected with larvae of Dirofilaria immitis.

In five healthy mongrel female cats used, four cats (Cats 1-4) were experimentally inoculated with 100-123 larvae (L(3)) of Dirofilaria immitis. Cat 5 was an uninfected control. Only Cat 1 became microfilaremic on Day 201 after inoculation and the diurnal changes in the microfilaria population were monitored every 2 h for 24 h on Day 237 when a sufficient number of microfilaria were detected in the circulation. The maximum number of microfilaria in the blood (1,350/ml) occurred at 9:00 p.m. and then gradually decreased to the minimum of 300/ml at 7:00 a.m., indicating that the number of microfilaria shows a nocturnal sub-periodic pattern of diurnal rhythm even in peripheral blood of cats as an abnormal host. On postmortem examination, 10 live adult worms (three males and seven females) in Cat 1 and one live adult worm (one male) in Cat 2 were detected.

Seroprevalence of canine dirofilariosis in South Korea.

Eight-hundred and forty eight dogs (480 females and 368 males) not on chemoprophylaxis were examined for Dirofilaria immitis infection from 2001 to 2002. Three-hundred and thirty nine (40.0%) of 848 samples tested with antigen detecting ELISA kits showed positive reaction for D. immitis antigen. More male (43.5%) than female (37.3%) dogs were affected in this study, although there was no significant difference between both groups. Chi2 analysis showed that female and male dogs of shoreline areas had higher significant prevalence than other areas (P<0.001), respectively. The mean overall positive rates of heartworm infection were 10.4% in dogs <2-year-old group, 46.5% in 2-4-year-old group, 48.4% in 4-6-year-old group and 50.3% in >6-year-old group. The older the age, the higher the prevalence of D. immitis infection in this study. Chi2 analysis revealed a significant higher prevalence in 4-6-year-old group (100%) of shoreline areas and in 2-4-old-year group (51.2%) of urban areas (Chungnam province), respectively (P<0.001), and also revealed that the over 6-year-old group (50.3%) in mean overall prevalence had the significant higher prevalence than other age group (P<0.001). In climate comparison, our data showed that shoreline areas (69.5%) had the significant higher prevalence than urban areas and mountain areas (P<0.001). This survey confirms that the prevalence of canine heartworm infection increases, and also that the prevalence in shoreline areas is higher than in other areas in South Korea.

Re-migration of fifth-stage juvenile Dirofilaria immitis into pulmonary arteries after subcutaneous transplantation in dogs, cats, and rabbits.

An assessment was made of the capacity for re-migration of the 5th-stage juvenile canine heartworm, Dirofilaria immitis. Live, 5th-stage juvenile worms recovered from the pulmonary arteries of infected dogs were transplanted into the subcutaneous tissue of uninfected dogs, cats, and rabbits. A mean of 45%, 61%, and 18% of the transplanted worms were recovered from the pulmonary arteries of dogs, cats, and rabbits, respectively, 1 and 3 mo later. The 5th-stage juvenile worms thus have the ability to re-migrate through tissue into the pulmonary arteries of the host.

Immunological analysis of agglutination in Dirofilaria immitis microfllariae.

The mechanism of agglutination phenomenon of Dirofilaria immitis microfilariae was analyzed. Circulating microfilariae were collected from a D. immitis-infected microfilaremic dog and cultured in the several kinds of sera from dogs and animals. The agglutination of D. immitis microfilariae is a specific phenomenon due to some immune complexes formed with the anti-microfilarial antibody, heat-instable factor(s) and excretory-secretory products of microfilariae. Only live microfilariae were agglutinated and the agglutinated microfilariae remained alive as long as 27 days in culture in vitro.

Immunoblotting analysis of somatic components of Dirofilaria immitis.

SDS-PAGE analysis of Dirofilaria immitis extracts demonstrated the complexicity of protein components of microfilariae, similar to that of adult male and female worms. Immunoblotting analysis using sera from microfilaremic and amicrofilaremic dogs with D. immitis infection suggest that antigenic components in the low molecular weight region may be related to the anti-microfilarial mechanism of the host.

Immunological treatment of cough occurring in dogs with dirofilariosis.

A persistent, spasmic and productive cough known as filarial cough often occurs in dogs with dirofilariosis, and has been considered to be the consequence of an allergic response to Dirofilaria immitis. Twenty-one dogs with filarial cough were subcutaneously injected with worm antigen (200 micrograms of protein concentration) extracted from adult D. immitis once a day for 5 days. These injections were effective for 17 (81%) of the dogs, resulting in a complete cure for 7 dogs and marked improvement for 10 dogs.

[Fundamental studies of cefoxitin in the field of obstetrics and gynecology].

The concentration of cefoxitin (CFX, Merxin) in dead space exudate was studied in 14 patients following total extirpation of diffuse uterine cervical cancer. A two-compartment model was used for the analysis. The results obtained were as follows: Calculated maximum concentrations of CFX in the pelvic dead space exudate were 26.55 micrograms/ml at 2.11 hours, 31.07 micrograms/mg at 2.01 hours and 51.51 micrograms/ml at 2.10 hours after 1 hour intravenous drip infusions of CFX 2, 3 and 4 g, respectively. These concentrations were higher than the MIC80 of 12.5 micrograms/ml against E. coli and B. fragilis and were maintained for a sufficient period of time. Based on the results of this study, CFX is considered to be an important and valuable drug in the field of obstetrics and gynecology.

[Fundamental and clinical studies on aztreonam in the field of obstetrics and gynecology].

Aztreonam (AZT) was studied for the transfer into the pelvic dead space exudate in 5 patients who received radical hysterectomy due to uterocervical cancer, and for the clinical efficacy in the treatment of 5 cases of obstetrical and gynecological infections. Transfer into the pelvic dead space exudate Data analysis was performed by the simulation curves prepared from pharmacokinetic parameters using the two-compartment model. On examination, the serum concentration of cubital venous serum was found to be 53.20 micrograms/ml at 1 hour after the start of 1-hour intravenous drip infusion of 1 g of AZT. Also, the shifting concentration of the pelvic dead space exudate was found to have reached its peak value of 12.79 micrograms/ml at 3.22 hours after the start of the infusion, and still showed the value of more than 7 micrograms/ml even at 8 hours after the start of the infusion. The AUC was 112.81 micrograms X hr/ml. Clinical evaluation AZT was administered at a daily dose of 2-4 g in 2 divided doses by intravenous injection and intravenous drip infusion for 60 minutes. Clinical efficacy of the 5 patients with acute and moderate degree obstetrical and gynecological infections was; excellent in 1 case, good in 3 cases and poor in 1 case, with the overall efficacy rate of 80.0%.

[Basic and clinical studies on cefminox in the field of obstetrics and gynecology].

In order to determine transference of cefminox (CMNX, MT-141) into the female genital organ tissues, the drug concentration in the pelvic dead space exudate was measured in cases undergone radical hysterectomy due to uterocervical cancer. For analysis of data the three-compartment model was used. When CMNX was given at a dose of 1 g as an intravenous drip infusion taking 1 hour, the serum concentration of the cubital vein reached its peak of 89.53 microgram/ml at 1 hour after start of administration. In the pelvic dead space exudate the peak concentration of 39.84 microgram/ml was reached at 2.55 hours after administration and a concentration higher than 7 micrograms/ml was still detected even at 12 hours. The area under the curve (AUC) for CMNX concentration in the pelvic dead space exudate was 295.63 microgram X hr/ml. These results suggest that CMNX achieves high concentration in the pelvic dead space exudate and that it is an antibiotic with clinical utility. CMNX was used in the treatment of 6 cases of obstetrical and gynecological infections. The clinical results were excellent in 1 case, good in 2 cases and poor in 2 cases. In the remaining 1 case the results was unknown.

[Clinical studies of cefotaxime (CTX, HR 756) for infectious diseases in the field of obstetrics and gynecology (author's transl)].

Ten cases of gynecological infection were treated successfully by a new cephalosporin derivative, cefotaxime (HR 756, CTX), which proved excellent in 4 cases and good in 6 cases. No adverse reaction was found to be induced by administration of this drug. Post-administration results were all normal for both renal and hepatic function tests. It is extremely difficult to accurately assess bacteriological efficacy of a drug in treatment of female genital infections. Nevertheless, we determined its MICs for a few number of strains we could isolate from clinical cases. Its MIC against 4 strains of B. fragilis inoculated at 10(8) cells/ml was 25 approximately 50 mcg/ml and was 1.56 approximately 3.13 mcg/ml at 10(6) cells/ml inoculation. Its MIC against 2 strains of Ps. cepacia was 12.5 mcg/ml at 10(8) cells/ml inoculation and 6.25 mcg/ml at 10(6) cells/ml inoculation. These MICs suggest that this drug is a very promising antimicrobial agent for treatment of gynecological infections, as been already indicated by clinical results of this drug.

[Pharmacokinetic studies on ceftazidime in the obstetrical and gynecological field].

Concentrations of ceftazidime ( CAZ ) were examined in the pelvic dead space exudate in 6 patients who received radical hysterectomy due to uterocervical cancer. Data analysis was performed by the simulation curves prepared from pharmacokinetics parameters using the three-compartment model. Following 1-hour intravenous drip infusion of 1 g of CAZ , the mean drug concentration in venous blood was 75.54 micrograms/ml at 1 hour after start of the infusion. The mean CAZ level in the pelvic dead space exudate was as high as 23.87 micrograms/ml at 2.09 hours after start of the infusion, and the AUC was 149.11 micrograms . hr/ml. The above results suggest that CAZ is a useful drug clinically with good penetration into the pelvic dead space.

[Fundamental and clinical studies on sulbactam/cefoperazone in the field of obstetrics and gynecology].

Concentrations of sulbactam/cefoperazone (SBT/CPZ) in the pelvic dead space exudate were examined in 5 patients who received radical hysterectomy due to uterocervical cancer. Data analysis was performed by the simulation curves prepared from pharmacokinetics parameters using a three-compartment model. Following 1 hour intravenous drip infusion of 2 g of SBT/CPZ, the mean drug concentration in venous blood was 50.62 micrograms/ml for SBT and 120.16 micrograms/ml for CPZ at 1 hour after start of the infusion. The peak SBT level in the pelvic dead space exudate was 21.24 micrograms/ml at 1.58 hours after start of the infusion. The peak CPZ level was 19.13 micrograms/ml at 3.46 hours after start of the infusion. In the treatment of 4 cases with gynecological infections, the clinical efficacy of SBT/CPZ was assessed as effective in 3 cases and poor in 1 case.

[Fundamental study of ceftriaxone in the field of obstetrics and gynecology].

Excretion of ceftriaxone (Ro 13-9904, CTRX) into female genital organs was studied by determining its concentration in the dead space exudate of the patients who received radical hysterectomy due to uterocervical cancer. Data analysis was performed by the three-compartment model. The serum peak level of CTRX in the cubital vein was 177.74 micrograms/ml 1 hour after the initiation of intravenous drip infusion for 1 hour with 1 g. The concentration of CTRX in the exudate of pelvic dead space reached a peak of 62.75 micrograms/ml 4.61 hours after the start of administration and still showed a very high level of 25 micrograms/ml at 24 hours. The AUC of the excretion into the exudate of pelvic dead space was 1,432.43 micrograms X hr/ml. These results indicate that CTRX is a clinically useful drug, being excreted into the exudate of pelvic dead space favorably.

[Fundamental and clinical evaluation of imipenem/cilastatin sodium in the perinatal period].

To evaluate the efficacy of imipenem/cilastatin sodium (IPM/CS) in the field of obstetrics and gynecology, fundamental (measurement of IPM/CS concentrations in mothers' milk) and clinical studies were performed. Concentrations of IPM/CS in mothers' milk were measured every 1 hour up to 6 hours after a 30-minute drip infusion of 500 mg/500 mg of IPM/CS. IPM/CS was distributed similarly to other cephalosporins. In the clinical study, a 500 mg/500 mg dose of IPM/CS was administered to 5 patients with puerperal intrauterine infections and to 3 patients with urinary tract infections by a 30-minute drip infusion b.i.d. or t.i.d. Good responses were observed in all 8 patients, though the infections were mild or moderate. From these results, IPM/CS appeared to be a useful drug for the treatment of perinatal infections.

[Fundamental and clinical studies on cefpiramide in the field of obstetrics and gynecology].

Transfer of cefpiramide (SM-1652, CPM) to female genital organs was studied. CPM concentration was determined in the uterine artery, portio vaginalis, myometrium, ovary and oviduct of patients undergoing hysterectomy, and in the pelvic dead space exudate of patients undergoing radical hysterectomy due to uterocervical cancer. Data obtained were analyzed by three-compartment model. The maximum concentration at 1 hour after intravenous drip infusion of CPM in a dose of 1 g were 244.31 micrograms/ml in both the uterine arterial serum and uterine venous serum, 31.41 micrograms/g in the portio vaginalis, 32.99 micrograms/g in the myometrium, 31.67 micrograms/g in the ovary and 31.99 micrograms/g in the oviduct. The concentration in the pelvic dead space exudate reached to the maximum level of 5.32 micrograms/ml at 5.84 hours and thereafter decreased slowly. The clinical effect of CPM was examined in 6 patients with various female genital infections and found to be effective in all cases. Side effects and abnormal laboratory finding values were not observed at all.

[Pharmacokinetic and clinical studies on flomoxef in perinatal period].

Pharmacokinetic and clinical studies on flomoxef (FMOX) in the perinatal period were carried out. The results are summarized as follows. 1. The concentration of FMOX in umbilical cord serum was about 10 micrograms/ml in about 30 minutes after 1 g one shot intravenous injection. Amniotic fluid concentration was 7 micrograms/ml in 41 minutes after administration. By 1 hour intravenous drip infusion, FMOX concentration in umbilical cord serum was about 5 micrograms/ml in 2-3 hours after administration. Amniotic fluid concentration of about 20 micrograms/ml was found in 1 case. 2. FMOX 1-2 g x 2-3/day was given by intravenous drip infusion to 7 cases of perinatal infection for 4-26 days. Clinical efficacies were evaluated a good for all cases. Neither side effect nor abnormal laboratory test value was observed. Consequently, FMOX was considered to be highly effective and safe for its clinical use in perinatal period infections.