Periodicity exhibited by Dirofilaria immitis microfilariae identified in dogs of Korea

Microfilarial periodicity of Dirofilaria immitis (the dog heartworm) was determined at two hr intervals for 72 consecutive hrs in 10 naturally infected war dogs, 3-9 years old, in Korea to facilitate harvest of the microfilariae for possible use in laboratory works and to elucidate further the periodicity of the microfilaria depending on geographic location. Although the periodicity had been observed as being low-grade nocturnal, maximal microfilarial counts were found at 21:00 hr and minimal at 11:00 hr, giving rise to an evident peak in fluctuation of the larval counts. This is the first record of the periodicity of the microfilariae identified as D. immitis in Korea.     

A serological survey of Dirofilaria immitis infection in pet dogs of Busan, Korea, and effects of chemoprophylaxis

The status of Dirofilaria immitis infection was assessed in pet dogs of Busan, Korea, and chemoprophylactic effects of microfilaricidal medication were evaluated. A total of 294 pet dogs older than 6 mo were examined, 217 of which had been maintained indoors, and 77 had been kept outdoors. The SnapR kit and direct microscopic examinations of the peripheral blood were used. The mean overall parasite positive rates were 10.2% and 6.5%, respectively. Outdoor dogs evidenced adult worm infection rate of 31.2% and microfilaria infection rate of 18.2%. The indoor dogs, however, evidenced adult worm infection rate of 2.8% and microfilaria infection rate of 2.3%. The prevalence in males was more than 2 times that of females. The changing pattern of infection rates by age evidenced a gradual increase, from 2- to 6-year-old dogs, after which, a decrease in infection rates was noted. With regard to chemoprophylaxis, the infection rates of complete and incomplete chemoprophylaxis groups were found to be 2-3 times lower than that of the non-chemoprophylaxis group. The results of the present study indicate that the risk of exposure to D. immitis in pet dogs is quite high, particularly in male outdoor dogs, and chemoprophylactic measures were quite effective.     

Prevalence of Toxoplasma gondii Infection in Stray and Household Cats in Regions of Seoul, Korea

The principal objective of this study was to investigate the prevalence of toxoplasmosis in household and stray cats in Seoul, Republic of Korea. We collected blood samples from 72 stray and 80 household cats, and all samples were examined by ELISA and nested PCR. The overall positive rates of Toxoplasma gondii in stray cats were 38.9% (28/72), with 15.3% (11/72) in ELISA and 30.6% (22/72) in PCR. The positive rate in male stray cats was slightly higher than that of female stray cats. The highest positive rate of T. gondii infection was noted in Gangnam and Songpa populations in ELISA and in Gwangjin population in PCR. In household cats, however, we could not detect any specific antibodies or DNA for T. gondii. In conclusion, we recognized that the infection rate of toxoplasmosis in stray cats in Seoul was considerably high but household cats were free from infection.     

Prevalence of Toxocara canis, Toxascaris leonina and Dirofilaria immitis in dogs in Chuncheon, Korea (2004)

The intestines and hearts of dogs were examined for Toxocara canis, Toxascaris leonina, and Dirofilaria immitis, after necropsy between June 26 and September 29, 2004 in Chuncheon, Korea. Of the 662 dogs examined, 6 were infected with T. canis (0.9%), 86 with T. leonina (13.0%). Fifty dogs were infected with D. immitis among 500 dogs examined (10.0%). Five were co-infected with T. canis and T. leonina, and three were co-infected with T. leonina and D. immitis. The cumulative positive infection rate for three species was 134/662 (20.2%). Considering previously reported seropositive rates of T. canis excretory-secretory antigen, i.e., 5% in the adult population in Korea, the possibility of toxocariasis caused by T. leonina should be reevaluated.     

Prevalence of Dirofilaria immitis in Dogs in Shenyang,Northeastern China

In the present study, we first report the seroprevalence of Dirofilaria immitis in dogs in Shenyang, northeastern China. Sera from 528 randomly selected dogs were examined for D. immitis antigen using SNAP®4Dx test kit; 12.7% tested showed seropositive. No significant difference of infection was observed in different genders and breeds (P>0.05), but the difference was significant in different age groups and rearing conditions (P<0.05). The result suggested that the risk of exposure to D. immitis in dogs is high in Shenyang, and should be given attention.     

Toxoplasma gondii B1 Gene Detection in Feces of Stray Cats around Seoul,Korea and Genotype Analysis of Two Laboratory-Passaged Isolates

The increasing prevalence of Toxoplasma gondii infection in the human population in the Republic of Korea (= Korea) is due to various reasons such as an increase in meat consumption. However, the importance of cats in transmitting T. gondii infection through oocysts to humans has seldom been assessed. A total of 300 fecal samples of stray cats captured around Seoul from June to August 2013 were examined for T. gondii B1 gene (indicating the presence of oocysts) using nested-PCR. Fourteen (4.7%) of 300 cats examined were positive for B1 gene. Female cats (7.5%) showed a higher prevalence than male cats (1.4%). Cats younger than 3 months (5.5%) showed a higher prevalence than cats (1.5%) older than 3 months. For laboratory passage of the positive samples, the fecal suspension (0.2 ml) of B1 gene positive cats was orally inoculated into experimental mice. Brain tissues of the mice were obtained after 40 days and examined for the presence of tissue cysts. Two isolates were successfully passaged (designated KNIH-1 and KNIH-2) and were molecularly analyzed using the SAG5D and SAG5E gene sequences. The SAG5D and SAG5E gene sequences showed high homologies with the ME49 strain (less virulent strain). The results indicated the importance of stray cats in transmitting T. gondii to humans in Korea, as revealed by detection of B1 gene in fecal samples. T. gondii isolates from cats were successfully passaged in the laboratory for the first time in Korea.     

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.     

Prevalence of Toxoplasma gondii Infection in Household Cats in Korea and Risk Factors

Several epidemiological surveys have reported the prevalence of Toxoplasma gondii infection in stray cats in Korea, but little information is available on T. gondii infection in household cats. The aim of the present study was to assess the prevalence and risk factors of T. gondii infection among household cats reared in Seoul, Korea. A total of 474 blood samples were collected from clinically healthy household cats. All samples were tested using ELISA and PCR. The risk factor analysis was based on a questionnaire filled out by the owners. The overall positive rate for ELISA and PCR assays was 2.2% (10/437) and 2.1% (10/474), respectively. With regard to the origin of cats, the positive rates among cats adopted from the animal shelter and veterinary clinic for stray cats were significantly different (P<0.05). Our study demonstrated that the positive rate of T. gondii infection in household cats was low and that this low prevalence was assumed to be associated with keeping the cats indoors and restriction of eating raw food and uncooked meat. Therefore, we suggest that the owners check the origin of the cats prior to adoption to prevent infection of other animals, including humans.     

Trematodes Recovered in the Small Intestine of Stray Cats in the Republic of Korea

In 2005, we reported the infection status of 438 stray cats with various species of intestinal helminths, including nematodes (4 species), trematodes (23 species), and cestodes (5 species) in the Republic of Korea. However, morphologic details of each helminth species have not been provided. In the present study, we intended to describe morphologic details of 13 trematode species which were either new fauna of cats (10 species) or new fauna of not only cats but also all animal hosts (3 species). The worms were fixed in 10% neutral buffered formalin under a cover slip pressure, stained with Semichon''s acetocarmine, and then observed using a light microscope equipped with a micrometer. The 13 subjected species included members of the Heterophyidae (Stellantchasmus falcatus, Stictodora fuscata, Stictodora lari, Centrocestus armatus, Procerovum varium, and Cryptocotyle concava), Echinostomatidae (Echinostoma hortense, Echinostoma revolutum, Echinochasmus japonicus, and Stephanoprora sp.), Diplostomidae (Neodiplostomum seoulense), Plagiorchiidae (Plagiorchis muris), and Dicrocoeliidae (Eurytrema pancreaticum). By the present study, Cryptocotyle sp. and Neodiplostomum sp. recored in our previous study were identified as C. concava and N. seoulense, respectively. Three species, P. varium, C. concava, and Stephanoprora sp., are new trematode fauna in Korea.     

Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis

This study was performed to investigate whethere there is cross-reactivity between Dirofilaria immitis and three intestinal nematodes of dogs. In ELISA, D. immitis-infected dog sera obtained at the 4th molting stage (9-11 weeks) and microfilaremic stage (25-30 weeks) were shown to be highly reactive with crude extract of T. canis. In immunoblotting, some antigenic fractions, 44, 57, 88, 100 kDa of crude extract of T. canis, were found to be positive reaction with sera of dogs infected with D. immitis. However, little or no cross-reaction were observed between sera of D. immitis-infected dogs and crude extract antigen of T. vulpis or A. caninum. These result suggest that there are partial cross reaction between sera of D. immitis-infected dogs and the antigen of T. canis.     

Prevalence of Trichomoniasis by PCR in Women Attending Health Screening in Korea

Trichomoniasis is the most common curable sexually-transmitted infection (STI) worldwide. There are few reports on the prevalence of Trichomonas vaginalis in Korea. The purpose of this study was to examine the prevalence of trichomoniasis by PCR in Guri city, Korea. All adult women who visited Hanyang University Guri Hospital for health screening within the National Health Care Service were invited to participate in the study, and 424 women were enrolled between March and June 2011. PCR was used to detect Trichomonas vaginalis using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Fourteen women (3.3%) were found to have T. vaginalis. All were over 50, and they were significantly older on average than the 410 Trichomonas-negative women (mean ages 63.4 vs 55.3 years). It seems that T. vaginalis infection is not rare in women receiving health screening, especially among those over 50.     

Detection of Plasmodium vivax by Nested PCR and Real-Time PCR

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.     

An ivermectin-sensitive glutamate-gated chloride channel subunit from Dirofilaria immitis

Dirofilaria immitis is a filarial nematode that infects dogs and causes cardiopulmonary disease. The most effective way of controlling the infection is by chemoprophylaxis, using members of the avermectin/milbemycin (A/M) class of anthelmintics, which includes ivermectin; these drugs act at invertebrate glutamate-gated chloride channels (GluCl). We have cloned two cDNAs encoding D. immitis GluCl subunits and demonstrated that at least one may be an important molecular target for the A/Ms in vivo. The subunits are orthologues of the alternatively spliced GluClalpha3A and alpha3B subunits (encoded by the avr-14 gene) previously identified in Caenorhabditis elegans and in Haemonchus contortus. Although the alternative splicing of avr-14 is conserved across the species, the processing of the mature GluClalpha3A mRNA differs in D. immitis compared to C. elegans and H. contortus. Two-electrode voltage clamp recordings were made from Xenopus oocytes injected with subunit-specific cRNAs. The DiGluClalpha3B subunit formed channels that were gated by L-glutamate (1-100 mM) and ivermectin (1 microM). Oocytes injected with DiGluClalpha3A cRNA failed to respond to L-glutamate. The qualitative responses obtained were consistent with the pharmacology observed for the GluClalpha3 subunits from C. elegans and H. contortus.     

Rapid Detection and Identification of Wuchereria bancrofti,Brugia malayi,B. pahangi,and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.     

Prevalence of Toxoplasma gondii in stray cats of Gyeonggi-do, Korea

Toxoplasma gondii is an obligate intracellular zoonotic protozoan with a worldwide distribution. It infects humans as well as a broad spectrum of vertebrate hosts. Cats and wild felidae play crucial roles in the epidemiology of toxoplasmosis. This study was performed to survey the prevalence of T. gondii infection among stray cats in the Gyeonggi-do, Republic of Korea. A total of 174 stray cat blood samples were collected from Gwacheon-si (n=20), Bucheon-si (82), and Yangju-si (72). Positive sera for T. gondii were identified in 14 samples (8.1%) exclusively via the latex agglutination test, 28 (16.1%) via ELISA, and 23 (13.2%) via PCR analysis. The overall infection rate of female stray cats (29.2%) presented as higher than that of male cats (24.0%). This study suggests that T. gondii is widespread in the stray cat population of Gyeonggi-do, Korea. It is urgently needed to control urban stray cat population and to reduce the risk of zoonotic transmission of toxoplasmosis to other animal hosts and humans.     

Potential Zoonotic Trematodes Recovered in Stray Cats from Kuwait Municipality,Kuwait

Stray cats are a common feature roaming the streets and alleys of Kuwait; they could be a source of parasites, including trematodes, that affect humans. A survey was conducted to identify feline trematodes and throw the light on their public health significance in Kuwait. Out of 240 stray cats trapped from different localities of Kuwait from June 2011 to May 2012, 59 (24.6%) were found to be infected with 14 species of trematodes. The most common were trematodes of the genus Heterophyes, particularly H. heterophyes and H. dispar that were found in respectively 15.8% and 10.8% of the cats examined. Other trematodes recorded, with lower prevalences, were Heterophyes nocens (2.9%), Haplorchis taichui (3.8%), Stictodora sawakinensis (2.1%), Stellantchasmus falcatus (1.6%), Echinochasmus japonicus (1.6%), and Mesostephanus dottrensi (1.3%). Centrocestus cuspidatus, Galactosomum fregatae, Ascocotyle sp., Mesostephanus appendiculatus, Haplorchis yokogawai, and Pygidiopsis genata showed the lowest prevalence (0.4%) and intensity. The majority of the trematodes are recorded for the first time in Kuwait and even in the Gulf region. The study reveals that stray cats are good indicators of fish-borne trematodes in the environment. As all trematodes recovered are zoonotic, their significance to public health should be considred.     

Zoonotic Intestinal Trematodes in Stray Cats (Felis catus) from Riverside Areas of the Republic of Korea

The present study was performed to survey the infection status of zoonotic intestinal trematode (ZIT) in stray cats from 5 major riverside areas in the Republic of Korea. Total 400 stray cats were captured with live-traps in riverside areas of Seomjingang (‘gang’ means river) (203 cats) from June to October 2010, and of Yeongsangang (41), Nakdonggang (57), Geumgang (38), and Hangang (61 cats) from June to October 2011, respectively. Small intestines resected from cats were opened with a pair of scissors in a beaker with 0.85% saline and examined with naked eyes and under a stereomicroscope. More than 16 ZIT species were detected in 188 (92.6%) cats from Seomjingang areas, and the number of worms recovered was 111 per cat infected. In cats from riverside areas of Yeongsangang, Nakdonggang, Geumgang, and Hangang, more than 9, 8, 3, and 5 ZIT species were recovered, and the worm burdens were 13, 42, 11, and 56 specimens per infected cat, respectively. As the members of family Heterophyidae, more than 10 species, i.e., Metagonimus spp., Pygidiopsis summa, Heterophyes nocens, Stellantchasmus falcatus, Heterophyopsis continua, Acanthotrema felis, Centrocestus armatus, Procerovum varium, Cryptocotyle concava, and Stictodora lari, were recovered. More than 5 species of echinostomes, i.e., Echinostoma hortense, Echinochasmus japonicus, Echinochasmus sp., Echinoparyphium sp., and unidentified larval echinostomes, were collected. Plagiorchis spp. were detected in cats from areas of Seomjin-gang and Yeongsangang. From the above results, it has been confirmed that stray cats in 5 major riverside areas of Korea are highly infected with various species of ZITs.     

Vascular endothelial cell activation by adult Dirofilaria immitis antigens

Dirofilaria immitis is the causal agent of cardiopulmonary dirofilariosis (heartworm disease). Adult worms lodge in the pulmonary arteries and right ventricle, thus vascular endothelium is exposed to high concentrations of Dirofilaria antigenic products. Heartworm disease habitually develops as a chronic foreseeable pathology. Moreover, the simultaneous death of many adult worms, naturally or induced by a filaricide treatment, can cause acute thromboembolisms and endarteritis. To better understand the effects of the massive death of D. immitis adult worms on the blood vessel endothelium, we cultured vascular endothelial cells in the presence or absence of an antigenic extract of D. immitis adult worms (DiSA). The parasite products increased the expression of enzymes and the synthesis of eicosanoids related to inflammation, such as COX-2, 5-LO, PGE₂ and LTB₄. The expression of ICAM-1 and PECAM-1 adhesion molecules and endothelial and inducible Nitric Oxide Synthases (eNOS and iNOS) was also increased in cultures treated with DiSA. Nevertheless, DiSA decreased endothelial permeability and does not alter both proliferation and apoptosis. These results suggest that the somatic extract of D. immitis adult worms stimulate inflammatory mechanisms in endothelial cells, without altering their basic physiologic processes.     

Xenomonitoring of Different Filarial Nematodes Using Single and Multiplex PCR in Mosquitoes from Assiut Governorate,Egypt

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.