Genetic heterogeneity of Pneumocystis carinii from rats of several regions and strains

Pneumocystis carinii is a major opportunistic pathogen which has been found in the lungs of a wide variety of mammalian host species, and the fact suggests the possibility of intraspecific variation. Until now, P. carinii from different mammalian species are differentiated as subspecies, and the rats are known to be infected by two subspecies. The present study investigated genetic heterogeneity of P. carinii isolates from two strains of rats in Korea and China by molecular karyotyping, RFLP and sequencing analysis. Karyotypes of P. carinii were grouped into three, two from two strains of rats in Korea and one from rats in China. However RFLP of PCR product of ribosomal and MSG gene of the P. carinii isolates showed same pattern. The sequence homology rates of alpha-tubulin DNA of the P. carinii isolates were 96% in Seoul Wistar rats, 93% in Seoul Sprague-Dawley rats, and 85% in Chinese Sprague-Dawley rats. The present finding confirmed that P. carinii from rats in Korea are grouped into two karyotype strains which are different from that of P. carinii from rats in China. The Chinese isolate shows a little different sequences of alpha-tubulin DNA.     

Purification of a 68-kDa cysteine proteinase from crude extract of Pneumocystis carinii

The present study intended to verify activities of cysteine proteinase of Pneumocystis carinii from rats and to purify the enzyme. In order to exclude the contamination of host-derived enzymes, concentrates of P. carinii was primarily treated with a mixture of proteinase inhibitors before lysis of P. carinii. A 68-kDa cysteine proteinase was finally purified from the crude extract of P. carinii by 4 sequential chromatographic methods. The enzyme showed an optimal activity at pH 5.5 in 0.1 M sodium acetate, and its activity was specifically inhibited by L-trans-epoxy-succinylleucylamido (4-guanidino) butane (E-64) and iodoacetic acid, suggesting that the enzyme is a cysteine proteinase. The 68-kDa proteinase weakly digested macromolecules such as collagen, hemoglobin and fibronectin. The present study demonstrated the activity of cysteine proteinase at the 68-kDa band of P. carinii, and purified and characterized the molecule.     

Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.     

Variation of antigenicity and serological reaction to Pneumocystis carinii in Korea.

The present study observed the variation of antigenicity of Pneumocystis carinii and serum IgG antibody reaction to the antigens from different localities in Korea. Antigens of rat P. carinii and sera of inhabitants were collected at Chunchon. Chungju, Kwangju, and Seoul during 1995-1996. Enzyme-linked Immunosorbent Assay and immunoblot were used for immune reaction. Absorbance of 1,294 human sera ranged between 0.01 and 0.93. Sera from Chunchon showed higher absorbances than those from other areas. Immunoblotting revealed IgG antibody reactions to 116, 100, and 45-55 kDa antigenic bands of rat P. carinii, but the frequencies of positive reaction to individual bands were variable by localities. Total 62.6% of the sera showed the reaction to 116 kDa band while 37.7% reacted to 100 kDa band and 32.0% to 45-55 kDa bands. For the reaction to 116 kDa, the reaction rate was 60.0% to 82.6% by localities. It is found that the reaction rates of the human sera to rat P. carinii antigen are variable according to the localities. Also, the high molecular antigen of 116 kDa of rat P. carinii is the most frequent antigenic band reacting to human sera.     

Adjunctive steroid in HIV-negative patients with severe Pneumocystis pneumonia

Background

High-dose steroid therapy has been proven effective in AIDS-related Pneumocystis pneumonia (PCP) but not in non-AIDS-related cases. We evaluated the effects on survival of steroids in HIV-negative patients with PCP.

Methods

Retrospective study patients admitted to the ICU with hypoxemic PCP. We compared patients receiving HDS (≥1 mg/Kg/day prednisone equivalent), low-dose steroids (LDS group, <1 mg/Kg/day prednisone equivalent), and no steroids (NS group). Variables independently associated with ICU mortality were identified.

Results

139 HIV-negative patients with PCP were included. Median age was 48 [40–60] years. The main underlying conditions were hematological malignancies (n=55, 39.6%), cancer (n=11, 7.9%), and solid organ transplantation (n=73, 52.2%). ICU mortality was 26% (36 deaths). The HDS group had 72 (51.8%) patients, the LDS group 35 (25%) patients, and the NS group 32 (23%) patients. Independent predictors of ICU mortality were SAPS II at ICU admission (odds ratio [OR], 1.04/point; [95%CI], 1.01-1.08, P=0.01), non-hematological disease (OR, 4.06; [95%CI], 1.19-13.09, P=0.03), vasopressor use (OR, 20.31; 95%CI, 6.45-63.9, P<0.001), and HDS (OR, 9.33; 95%CI, 1.97-44.3, P=0.02). HDS was not associated with the rate of ICU-acquired infections.

Conclusions

HDS were associated with increased mortality in HIV-negative patients with PCP via a mechanism independent from an increased risk of infection.     

A Case of Pneumonia Caused by Pneumocystis jirovecii Resistant to Trimethoprim-Sulfamethoxazole

A 50-year-old male visited the outpatient clinic and complained of fever, poor oral intake, and weight loss. A chest X-ray demonstrated streaky and fibrotic lesions in both lungs, and chest CT revealed multifocal peribronchial patchy ground-glass opacities with septated cystic lesions in both lungs. Cell counts in the bronchoalveolar lavage fluid revealed lymphocyte-dominant leukocytosis, and further analysis of lymphocyte subsets showed a predominance of cytotoxic T cells and few T helper cells. Video-assisted wedge resection of the left upper lobe was performed, and the histologic examination was indicative of a Pneumocystis jirovecii infection. Trimethoprim-sulfamethoxazole (TMP-SMX) was orally administered for 3 weeks; however, the patient complained of cough, and the pneumonia was aggravated in the follow-up chest X-ray and chest CT. Molecular studies demonstrated mutations at codons 55 and 57 of the dihydropteroate synthase (DHPS) gene, which is associated with the resistance to TMP-SMX. Clindamycin-primaquine was subsequently administered for 3 weeks replacing the TMP-SMX. A follow-up chest X-ray showed that the pneumonia was resolving, and the cough was also alleviated. A positive result of HIV immunoassay and elevated titer of HCV RNA indicated HIV infection as an underlying condition. This case highlights the importance of careful monitoring of patients with P. jirovecii pneumonia (PCP) during the course of treatment, and the molecular study of DHPS mutations. Additionally, altering the anti-PCP drug utilized as treatment must be considered when infection with drug-resistant P. jirovecii is suspected. To the best of our knowledge, this is the first case of TMP-SMX-resistant PCP described in Korea.     

Separation of Pneumocystis carinii from the lung of the steriod-suppressed rat

Pneumocystis carinii pneumonia was induced in rats by chronic corticosteroid immunosuppression. The parasite was separated from virtually all contaminating host cells using the technique of unit gravity sedimentation. Cellular contamination was routinely below 0.02%. The same technique allowed partial separation of the cyst from the trophozoite stage.     

Parasitic infections in HIV-infected patients who visited Seoul National University Hospital during the period 1995-2003

The prevalence of parasitic infections was investigated in human immunodeficiency virus (HIV)-infected patients (n = 105) who visited Seoul National University Hospital, Seoul, Korea, during the period from 1995 to 2003. Fecal samples were collected from 67 patients for intestinal parasite examinations, and sputum or bronchoalveolar lavage samples from 60 patients for examination of Pneumocystis carinii. Both samples were obtained from 22 patients. Thirty-three (31.4%) of the 105 were found to have parasitic infections; Cryptosporidium parvum (10.5%; 7/67), Isospora belli (7.5%; 5/67), Clonorchis sinensis (3.0%; 2/67), Giardia lamblia (1.5%; 1/67), Gymnophalloides seoi (1.5%; 1/67), and Pneumocystis carinii (28.3%; 17/60). The hospital records of the 11 intestinal parasite-infected patients showed that all suffered from diarrhea. This study shows that parasitic infections are important clinical complications in HIV-infected patients in the Republic of Korea.     

Karyotypes of two rare rodents, Hapalomys delacouri and Typhlomys cinereus (Mammalia, Rodentia), from Vietnam

Karyotypes of Hapalomys delacouri (Rodentia, Muridae) and Typhlomys cinereus (Rodentia, Platacanthomyidae) from Vietnam are described for the first time. The diploid karyotype of Hapalomys delacouri is 38 (NFa=48), consisting of six pairs of bi-armed and 12 pairs of acrocentric autosomes decreasing in size; plus a large metacentric X chromosome and Y chromosome, also metacentric, that is equal in size to the largest pair of acrocentric autosomes. The newly described karyotype differs significantly from that reported for Hapalomys delacouri from northern Thailand. The latter record very likely represents a different species of Hapalomys, possibly the taxon Hapalomys pasquieri described from north-central Laos.The diploid karyotype of Typhlomys cinereus is 38 (NF=48), consisting of five pairs of meta- to submetacentric and 14 pairs of acrocentric chromosomes varying in size from large to small; sex chromosomes were not defined.     

Echinostoma macrorchis in Lao PDR: Metacercariae in Cipangopaludina Snails and Adults from Experimentally Infected Animals

The echinostome metacercariae encysted in Cipangopaludina sp. snails that were purchased from a market in Vientiane Municipality, Lao PDR, were identified as Echinostoma macrorchis (Digenea: Echinostomatidae) through recovery of adult flukes after experimental infection to rats and a cat. The metacercariae were round, 113-128 (121)×113-125 (120) µm, having a thick cyst wall, a head collar armed with collar spines, and excretory granules. The adult flukes recovered from the rats and cat at day 14 and 30 post-infection, respectively, were elongated, ventrally curved, and 3.9-6.3×0.7-1.1 mm in size. The head collar was distinct, bearing 43-45 collar spines with 5 angle spines on each side. Two testes were large (as the name implies), tandem, and slightly constricted at the middle, with irregular margins. Eggs were operculated, ovoid to elliptical, and 88-95×56-60 µm. In scanning electron microscopy, the head collar was prominent, with 43-45 collar spines. Scale-like tegumental spines were densely distributed on the ventral surface between the oral and ventral suckers. Sensory papillae were distributed mainly on the tegument around the 2 suckers. It is confirmed that E. macrorchis is distributed in Lao PDR using Cipangopaludina sp. snails as the second intermediate host.     

Species distribution of staphylococci from small wild mammals

A total of 197 isolates of Staphylococcus from small wild animals (insectivores and rodents) were identified by partial sequencing of the rpoB and dnaJ genes. Among the identified isolates the predominant species was S. succinus (28%), followed by S. xylosus (20.8%) and S. stepanovicii (18.3%). The other 14 Staphylococcus species were occasionally isolated. PCR-RFLP of the rpoB gene digested by Hpy8I was a fast and simple method to distinguish the two subspecies of S. succinus. More than 90% of the 55 S. succinus strains isolated belonged to S. succinus subsp. casei and only 9% to S. succinus subsp. succinus. Moreover, the present study describes the first ever isolation of S. fleurettii from healthy animals.     

Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5''and 3''ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.     

Late Holocene extinction of Puerto Rican native land mammals

West Indian land mammals have suffered the most severe extinctions of any Holocene mammal faunas. However, 'last-occurrence' dates based on radiometric or robust stratigraphic data remain unavailable for most West Indian species, making it impossible to identify factors responsible for these extinctions. Here, we present new radiometric dates from archaeological and palaeontological sites on Puerto Rico, the only Greater Antillean island to have lost all native land mammals. Although it has been suggested that these species died out earlier than other West Indian mammals, we demonstrate that Puerto Rican mammal last-occurrence dates are in close agreement with those from other Antillean islands, as several species in fact persisted for millennia following Amerindian arrival. Echimyid rodents and nesophontid 'island-shrews' were still present on Puerto Rico approximately 1000 years BP, and probably became extinct following European arrival. The large (13kg) heptaxodontid rodent Elasmodontomys obliquus also appears to have survived for over 2000 years after Amerindian colonization, suggesting that at least some large West Indian mammals became extinct in protracted pre-European 'sitzkrieg'-style events rather than 'blitzkrieg'-style overkill.     

Molecular Detection of Giardia intestinalis from Stray Dogs in Animal Shelters of Gyeongsangbuk-do (Province) and Daejeon,Korea

Giardia is a major public health concern and considered as reemerging in industrialized countries. The present study investigated the prevalence of giardiosis in 202 sheltered dogs using PCR. The infection rate was 33.2% (67/202); Gyeongsangbuk-do and Daejeon showed 25.7% (39/152, P<0.0001) and 56% (28/50), respectively. The prevalence of infected female dogs (46.7%, P<0.001) was higher than in male dogs (21.8%). A higher prevalence (43.5%, P<0.0001) was observed in mixed breed dogs than purebred (14.1%). Although most of the fecal samples collected were from dogs of ≥1 year of age which showed only 27.4% positive rate, 61.8% (P<0.001) of the total samples collected from young animals (<1 year of age) were positive for G. intestinalis. A significantly higher prevalence in symptomatic dogs (60.8%, P<0.0001) was observed than in asymptomatic dogs (23.8%). Furthermore, the analysis of nucleotide sequences of the samples revealed that G. intestinalis Assemblages A and C were found in the feces of dogs from Gyeongsangbuk-do and Daejeon. Since G. intestinalis Assemblage A has been known to infect humans, our results suggest that dogs can act as an important reservoir of giardiosis in Korea. Hence, hygienic management should be given to prevent possible transmission to humans.     

Intestinal Helminth Infections in Feral Cats and a Raccoon Dog on Aphaedo Island, Shinan-gun, with a Special Note on Gymnophalloides seoi Infection in Cats

Four feral cats and a raccoon dog purchased from a local collector on Aphaedo Island, Shinan-gun, where human Gymnophalloides seoi infections are known to be prevalent, were examined for their intestinal helminth parasites. From 2 of 4 cats, a total of 310 adult G. seoi specimens were recovered. Other helminths detected in cats included Heterophyes nocens (1,527 specimens), Pygidiopsis summa (131), Stictodora fuscata (4), Acanthotrema felis (2), Spirometra erinacei (15), toxocarids (4), and a hookworm (1). A raccoon dog was found to be infected with a species of echinostome (55), hookworms (7), toxocarids (3), P. summa (3), and S. erinacei (1). No G. seoi was found in the raccoon dog. The results indicate that feral cats and raccoon dogs on Aphaedo are natural definitive hosts for intestinal trematodes and cestodes, including G. seoi, H. nocens, and S. erinacei. It has been first confirmed that cats, a mammalian species other than humans, play the role of a natural definitive host for G. seoi on Aphaedo Island.     

Genetic Diversity of Pneumocystis carinii Derived from Infected Rats, Mice, Ferrets, and Cell Cultures

ABSTRACT. The degree of strain and/or species diversity among Pneumocystis carinii isolates is unknown. As a first approach to the study of P. carinii genetic relatedness, we compared the pulsed field gel electrophoretic karyotypes of P. carinii derived from lung homogenates of three immunosuppressed host animals: rats transtracheally inoculated with P. carinii -infected rat lung; mice transtracheally inoculated with P. carinii -infected mouse lung; and ferrets which developed reactivated latent P. carinii pneumonia. Rat P. carinii propagated on HEL299 cells was also examined. Karyotypes of P. carinii DNA from both rat lung homogenate and cell culture were identical (14 bands, 315–680 kb). In contrast, mouse and ferret P. carinii DNA karyotypes were each distinctly different from the rat P. carinii samples (mouse P. carinii 15 bands, 315–610 kb; ferret P. carinii nine bands, 410–760 kb). Three distinct rat P. carinii gene probes reacted with both Southern-transferred rat and mouse P. carinii DNA but not with ferret P. carinii DNA. Thus, P. carinii from rat, mouse, and ferret are genetically diverse. The results are consistent with recently reported antigenic and nucleic acid sequence differences among P. carinii isolates recovered from different hosts.     

Karyological Studies of Biomphalaria tenagophila (d'Orbigny, 1835) (Gastropoda: Planobidae) from Rio de Janeiro,Brazil

The karyotypes of Biomphalaria tenagophila collected from Rio de Janeiro, Brazil were studied using the air-drying method. Somatic cells of this species had 2n=36. The 18 chromosome pairs were identified and classified into 3 groups. The diploid cell has 7 pairs of metacentric, 8 pairs of submetacentric, and 3 pairs of subtelocentric chromosomes. Observed chromosomes ranged from 2.4 to 6.4 µm, and the total length was 122.3 µm. This is the first report on the chromosome of B. tenagophila.