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1.
Artigo em Inglês | MEDLINE | ID: mdl-31778390

RESUMO

Public parks are leisure environments widely used by both, adults and children, often accompained by their pets. Soil contamination of these environments by enteric viruses and intestinal parasites occurs through these animals feces. The aim of this work was to detect Carnivore protoparvovirus 1 (CPV-1) and different species of Mastadenovirus in soils samples from a park located in a medium-sized city in Brazil and evaluate the presence of helminth eggs and larvae in 18 points of a public park soil samples, as well as feces found on this site during six months. Parasitological analyzes were conducted through flotation and sedimentation techniques, and polymerase chain reaction (PCR) was used for viral detection. Of the 216 soil and 16 feces samples, 49% (106/216) and 12% (2/16) were positivefor nematodes larvae, respectively, through sedimentation techniques. Toxocara spp eggs were found in one soil sample and one feces sample, Trichuris spp eggs were found in only one feces sample and Hookworms eggs were found in four soil samples. After reconstruction work in the streets near the park, 30% (64/216) of the samples were positive for Human Mastadenovirus C (HAdV-C), 1.4% (3/216) for HAdV-E and 0.4% (1/216) for Canine Mastadenovirus A (CAdV-A). The parasitic forms found in this study have demonstrated that the contamination of the park's soil pose a threat to human and animal health. This was the first study to report the presence of HAdVs and CAdVs in soil samples.


Assuntos
Ancylostomatoidea/isolamento & purificação , Mastadenovirus/isolamento & purificação , Microbiologia do Solo , Solo/parasitologia , Toxocara/isolamento & purificação , Ancylostomatoidea/classificação , Ancylostomatoidea/genética , Animais , Cães , Fezes/parasitologia , Humanos , Mastadenovirus/classificação , Mastadenovirus/genética , Parques Recreativos , Reação em Cadeia da Polimerase em Tempo Real , Toxocara/classificação , Toxocara/genética
2.
Physiol Biochem Zool ; 92(3): 326-338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30986114

RESUMO

Parasites can cause chronic stress in some animal species, and this type of stress response has been associated with adverse consequences for the host. In order to know whether parasitism elicited a stress response associated with decreased host fitness, hookworm (Uncinaria sp.) infection was studied in a colony of South American fur seals (Arctocephalus australis) in which hookworms infect nearly all pups born in a reproductive season. A parasite-free group was generated by treating a subset of pups with an antiparasitic drug before they developed patent hookworm infection. Stress and metabolic hormones, energy balance, and humoral and cellular immune parameters were measured in this group and hookworm-infected pups. Hookworms elicited a marked increase in plasma cortisol levels in fur seal pups. These hookworm-infected pups were able to maintain constant glucose levels, despite losing body mass over the course of infection potentially because of increased protein catabolism. Infected pups were able to mount an effective immune response against the parasite and eliminated hookworms from the intestine, recovering partial body mass lost as a result of hookworm infection at the end of the study period. As shown in previous studies, adequate glucose levels are critical for proper T lymphocyte reactivity, and it is possible that, through activation of a stress response, energy can be readily available for immune response against the parasite contributing to early recovery from infection. Although there are potential fitness costs to mounting a sustained stress response, these could also be adaptive and promote survival during critical life-history stages.


Assuntos
Otárias/parasitologia , Infecções por Uncinaria/veterinária , Estresse Fisiológico/imunologia , Ancylostomatoidea/genética , Ancylostomatoidea/isolamento & purificação , Animais , Antiparasitários/uso terapêutico , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Infecções por Uncinaria/tratamento farmacológico , Infecções por Uncinaria/parasitologia , Imunidade Celular , Imunidade Humoral , Ivermectina/uso terapêutico , Masculino
3.
J Helminthol ; 94: e43, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30813972

RESUMO

All canine hookworms are known to be zoonotic, causing infections ranging from transient skin irritations to prolonged 'creeping eruptions', eosinophilic enteritis and even patent intestinal infections. There is little information on canine hookworm species and their public health significance in sub-Saharan Africa. This study determined the prevalence and species of hookworms in dogs from different climatic zones of Kenya. Dog faecal samples were collected from the environment, and hookworm eggs were isolated by zinc chloride flotation and subjected to DNA extraction. Polymerase chain reaction (PCR) assays targeting the internal transcribed spacer (ITS) 1 and 2, 5.8S and 28S ribosomal RNA of Ancylostoma spp. and Uncinaria stenocephala were performed, and hookworm species were identified by PCR-restriction fragment length polymorphism (RFLP) or DNA sequencing. Hookworm eggs were detected by microscopy in 490/1621 (30.23%, 95% CI 28.01-32.54) faecal samples. Estimates of faecal prevalence were high in counties receiving higher rainfall (Narok 46.80%, Meru 44.88%) and low in those with a more arid climate (Isiolo 19.73%, Turkana 11.83%). In a subset of 70 faecal samples, Ancylostoma caninum (n = 59) was the most common species, followed by A. braziliense (n = 10) and A. cf. duodenale (n = 1). This study reports for the first time the detection of A. cf. duodenale in dog faeces and zoonotic hookworm species in Kenyan dogs. These findings emphasize the need for control measures such as enforcing laws for restraining stray dogs, regular deworming of dogs, and public health awareness programmes aimed at informing communities on outdoor use of footwear.


Assuntos
Ancylostomatoidea/isolamento & purificação , Doenças do Cão/parasitologia , Infecções por Uncinaria/veterinária , Ancylostomatoidea/classificação , Ancylostomatoidea/genética , Animais , Cães , Fezes/parasitologia , Feminino , Infecções por Uncinaria/parasitologia , Quênia , Masculino , Polimorfismo de Fragmento de Restrição
4.
J Helminthol ; 94: e39, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30789121

RESUMO

There is a paucity of information on hookworm species in humans, domestic animals and wildlife in southern Africa. Our study aimed to identify hookworm species from stray dogs, humans, and selected wildlife from South Africa. A total of 356 faecal samples were screened for the presence of hookworm-like eggs and subsequently coproculture from the positive samples was carried out to obtain larvae. Hookworm-like eggs were detected in 23.03% (82/356) of samples. Of these samples, 78/296 were from dogs, 3/50 from humans and 1/10 from wildlife. DNA was then isolated from the larvae of 55 positive samples, which were subjected to polymerase chain reaction (PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing of the nuclear ribosomal internal transcribed spacer (ITS1) and 5.8S rRNA region. Presence of Ancylostoma caninum, A. braziliense and A. ceylanicum-like species was recorded in stray dogs and A. caninum was recorded in wildlife and humans, using PCR-RFLP. Although PCR-RFLP results pointed to the presence of A. ceylanicum, we did not get a sequence that matched with A. ceylanicum from GenBank. This may have been due to the low proportion of A. ceylanicum larvae in our samples. Twenty-two of the 27 positive amplicons from stray dogs matched with A. caninum, three with A. braziliense and two had mixed infections of A. braziliense and A. caninum. Sequences from a lion and three humans matched with A. caninum. This is the first confirmation of a patent A. caninum infection in humans as evidenced by the presence of eggs in faeces, with the subsequent larvae from coproculture being identified as A. caninum.


Assuntos
Ancylostomatoidea/isolamento & purificação , Animais Selvagens/parasitologia , Doenças do Cão/parasitologia , Infecções por Uncinaria/parasitologia , Infecções por Uncinaria/veterinária , Ancylostomatoidea/classificação , Ancylostomatoidea/genética , Animais , DNA de Helmintos/genética , Cães , Fezes/parasitologia , Humanos , Larva/classificação , Larva/genética , Leões/parasitologia , Filogenia , Polimorfismo de Fragmento de Restrição , África do Sul , Zoonoses/parasitologia
5.
Parasitol Res ; 118(2): 701-706, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30610365

RESUMO

This study aimed to investigate the endoparasite fauna of wild European gray wolves, which are currently recolonizing Germany. In total, 69 fecal samples of wild wolves were collected in Lower Saxony, Germany, from 2013 to 2015, analyzed by the sedimentation-flotation and McMaster techniques and compared to previous results on captive European Gray wolves living in zoological gardens in Germany. In addition to coproscopy, taeniid-positive samples from wild as well as captive wolves were differentiated by amplification and sequencing of small subunit ribosomal RNA (SSU rRNA) and NADH dehydrogenase 1 (nad1) gene fragments. Missing Taenia krabbei SSU rRNA reference sequences were generated from two T. krabbei specimens. Overall, 60.87% (42/69) of wild wolve samples were microscopically positive for at least one of seven egg types. Capillaria/Eucoleus spp. showed the highest frequency (31.88% [22/69]), followed by Taeniidae (21.74% [15/69]), Ancylostomatidae (20.29% [14/69]), Alaria alata (15.94% [11/69]), Toxocara canis (13.04% [9/69]), and Toxascaris leonina and Trichuris vulpis (each 5.80% [4/69]). Amplification of SSU rRNA was successful for 7/15 Taeniidae-positive samples from wild and 20/39 samples from captive wolves, revealing T. hydatigena in two and 14 samples, respectively. Taenia krabbei was detected in two further samples of wild and three samples of captive wolves, while for the remaining samples, no differentiation between T. serialis/T. krabbei was possible. Echinococcus spp. were not detected. Sequence comparisons revealed that the SSU rRNA gene fragment was not suitable to differentiate between T. serialis and T. krabbei. Therefore, the use of this fragment alone cannot be recommended for species identification in future studies.


Assuntos
Ancylostomatoidea/isolamento & purificação , Helmintíase Animal/epidemiologia , Taenia/isolamento & purificação , Trematódeos/isolamento & purificação , Lobos/parasitologia , Ancylostomatoidea/classificação , Ancylostomatoidea/genética , Animais , Fezes/parasitologia , Alemanha/epidemiologia , Helmintíase Animal/parasitologia , NADH Desidrogenase/genética , Taenia/classificação , Taenia/genética , Trematódeos/classificação , Trematódeos/genética
6.
J Helminthol ; 93(3): 313-318, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-29606160

RESUMO

The presence and distribution of various species of canine hookworms in Africa are poorly known. The main objective of this study, therefore, was to identify the hookworm species present in canine faecal samples from Morogoro, Tanzania, using molecular techniques. Faecal samples from 160 local dogs were collected and hookworm positive samples processed to recover larvae for further molecular characterization. DNA was extracted from pools of larvae from individual samples (n = 66), which were analysed subsequently using two different molecular approaches, polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR coupled with Sanger sequencing. The PCR-RFLP technique detected only the presence of the ubiquitous Ancylostoma caninum in the 66 samples. However, by species-specific PCR coupled with Sanger sequencing we identified ten samples with A. braziliense, two with Uncinaria stenocephala and five with A. ceylanicum. Thus, all four known species of canine hookworms were identified in Morogoro, Tanzania. To our knowledge this is the first report of the detection of the presence of U. stenocephala and A. ceylanicum in Africa using molecular techniques. In addition to their veterinary importance, canine hookworms have zoonotic potential and are of public health concern.


Assuntos
Ancylostomatoidea/classificação , Ancylostomatoidea/isolamento & purificação , Fezes/parasitologia , Ancylostomatoidea/genética , Animais , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Doenças do Cão/parasitologia , Cães , Infecções por Uncinaria/parasitologia , Infecções por Uncinaria/veterinária , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Tanzânia
7.
J Helminthol ; 93(2): 159-165, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29400266

RESUMO

To investigate the prevalence of canine and feline hookworms in South China, and to assess the risk of zoonotic hookworms to humans, one pair of primers (HRM-F/HRM-R) was designed to establish a high-resolution melting (HRM) method based on internal transcribed spacer 1 (ITS-1) rDNA for the detection of Ancylostoma ceylanicum, A. caninum and A. tubaeforme infection. The results showed that the HRM for the three hookworms produced different melting-curve profiles, where melting temperature (Tm) values were 84.50°C for A. ceylanicum, 82.25°C for A. caninum and 81.73°C for A. tubaeforme, respectively. The reproducibility of intra- and inter-assay melting curves was almost perfect. The lowest concentration detected was about 5.69 ×10-4 g/µl. The HRM detection results from 18 canine and feline hookworm samples were in complete accordance with their sequencing results. The HRM method was more sensitive than the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique in the detection of 98 clinical samples. It is concluded that the HRM method can differentiate between A. ceylanicum, A. caninum, A. tubaeforme and their mixed infections, which may provide important technical support for the zoonotic risk assessment and molecular epidemiological survey of canine and feline hookworms.


Assuntos
Ancylostomatoidea/genética , Doenças do Gato/epidemiologia , DNA de Helmintos/genética , Doenças do Cão/epidemiologia , Infecções por Uncinaria/veterinária , Ancylostomatoidea/classificação , Animais , Doenças do Gato/parasitologia , Gatos , China/epidemiologia , Primers do DNA/genética , DNA Ribossômico/genética , Doenças do Cão/parasitologia , Cães , Fezes/parasitologia , Infecções por Uncinaria/epidemiologia , Infecções por Uncinaria/parasitologia , Limite de Detecção , Filogenia , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Prevalência , Reprodutibilidade dos Testes , Temperatura de Transição
8.
Infect Genet Evol ; 68: 105-112, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30508686

RESUMO

Hookworm infection persists focally in rural communities in Brazil. In this study, we analyze the mitochondrial nucleotide sequences obtained from hookworms infecting humans in order to characterize species composition and assess their genetic diversity and phylogenetic relationships. Field expeditions and cross-sectional surveys were carried out in three Brazilian municipalities from 2013 to 2017: Nossa Senhora de Nazaré (n = 605) and Teresina (n = 297), in the state of Piauí, and Russas (n = 213) in the State of Ceará. Parasitological methods were used to evaluate fecal samples. Hookworm-positive samples had a partial mtDNA cox1 amplified and sequenced. Maximum-likelihood and Bayesian analysis demonstrated two strongly-supported clades, including Group A, corresponding to Necator americanus, and Groups B and C, corresponding to Necator sp. Group A was divided into three main clusters: A1 grouped with Asian sequences, A2 grouped with African sequences, and A3 had only Asian sequences. Group B was closely related to Necator sp., showing a sequence similarity of 98%-99% with African samples circulating zoonotically among humans and non-human primates. Twenty three N. americanus haplotypes were identified. N. americanus Median-Joining network revealed three distinct groups, designated again as A1, A2, and A3. Group A1 presented a star-like shape, with one dominant haplotype. The molecular dating suggested that the two clades dividing N. americanus and Necator sp. began to diverge during the middle Pleistocene. The most recent common ancestor among N. americanus groups was dated to the late Pleistocene. Hookworms circulating in the studied communities are structured in well-defined subpopulations presenting both Asian and African genetic backgrounds. This reveals a double origin for hookworms in northeastern Brazil and opens up new possibilities in phylogeographic, evolutionary, and molecular epidemiological studies in regions where hookworms persists focally, despite control efforts. The presence of potentially zoonotic species and the specific identification of Necator sp. should be further investigated.


Assuntos
Ancylostomatoidea/classificação , Ancylostomatoidea/genética , DNA Mitocondrial , Filogenia , Animais , Brasil/epidemiologia , Biologia Computacional/métodos , Genes Mitocondriais , Variação Genética , Geografia Médica , Infecções por Uncinaria/epidemiologia , Infecções por Uncinaria/parasitologia , Análise de Sequência de DNA
9.
J Helminthol ; 94: e8, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30428941

RESUMO

Hookworms of the genus Uncinaria parasitize pinniped pups in various locations worldwide. Four species have been described, two of which parasitize pinniped pups in the southern hemisphere: Uncinaria hamiltoni parasitizes Otaria flavescens and Arctocephalus australis from the South American coast, and Uncinaria sanguinis parasitizes Neophoca cinerea from the Australian coast. However, their geographical ranges and host specificity are unknown. Uncinaria spp. are morphologically similar, but molecular analyses have allowed the recognition of new species in the genus Uncinaria. We used nuclear genetic markers (internal transcribed spacer (ITS) and large subunit (LSU) rDNA) and a mitochondrial genetic marker (cytochrome c oxidase subunit I (COI)) to evaluate the phylogenetic relationships of Uncinaria spp. parasitizing A. australis and O. flavescens from South American coasts (Atlantic and Pacific coasts). We compared our sequences with published Uncinaria sequences. A Generalized Mixed Yule Coalescent (GMYC) analysis was also used to delimit species, and principal component analysis was used to compare morphometry among Uncinaria specimens. Parasites were sampled from A. australis from Peru (12°S), southern Chile (42°S), and the Uruguayan coast, and from O. flavescens from northern Chile (24°S) and the Uruguayan coast. Morphometric differences were observed between Uncinaria specimens from both South American coasts and between Uncinaria specimens from A. australis in Peru and southern Chile. Phylogenetic and GMYC analyses suggest that south-eastern Pacific otariid species harbour U. hamiltoni and an undescribed putative species of Uncinaria. However, more samples from A. australis and O. flavescens are necessary to understand the phylogenetic patterns of Uncinaria spp. across the South Pacific.


Assuntos
Ancylostomatoidea/crescimento & desenvolvimento , Ancylostomatoidea/isolamento & purificação , Caniformia/parasitologia , Infecções por Uncinaria/veterinária , Ancylostomatoidea/classificação , Ancylostomatoidea/genética , Animais , Chile , DNA de Helmintos/genética , DNA Espaçador Ribossômico/genética , Otárias/parasitologia , Infecções por Uncinaria/parasitologia , Peru , Filogenia
10.
Am J Trop Med Hyg ; 99(4): 1033-1040, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30062984

RESUMO

Appropriate diagnostic techniques are crucial to global soil-transmitted helminth (STH) control efforts. The recommended Kato-Katz method has low sensitivity in low-transmission settings. Quantitative polymerase chain reaction (qPCR) is a highly sensitive alternative diagnostic option. However, little is known about the variability in qPCR results, and there are few published comparisons between qPCR and other microscopy-based techniques such as sodium nitrate flotation (SNF). Using 865 stool samples collected from 571 individuals, we compared SNF and qPCR in terms of diagnostic sensitivity and infection intensity measurements. In addition, we conducted repeated examinations on a single Necator americanus-positive stool sample over a 6-month period. Results showed good diagnostic agreement between SNF and qPCR for Ascaris spp. (κ = 0.69, P < 0.001), and moderate agreement for hookworm (κ = 0.55, P < 0.001) and Trichuris spp. (κ = 0.50, P < 0.001). Quantitative polymerase chain reaction demonstrated higher sensitivity than SNF for Ascaris spp. (94.1% versus 68.1%) and hookworm (75.7% versus 66.9%) but not for Trichuris spp. (53.1% versus 81.3%), which had very low prevalence. Sodium nitrate flotation and qPCR infection intensity measurements were strongly correlated for Ascaris spp. (ρ = 0.82, P < 0.001) and moderately correlated for hookworm (ρ = 0.58, P < 0.001). Repeated examinations using qPCR showed that N. americanus cycle threshold values decreased significantly at 1 month and remained stable thereafter. Results confirm the high diagnostic sensitivity of qPCR for Ascaris spp. and hookworm, particularly for light-intensity infections, which is ideal for settings approaching transmission elimination. Results support the potential for qPCR to be used as a quantitative assay for STH. Further research is needed in settings where Trichuris trichiura is endemic.


Assuntos
Bioensaio/normas , DNA de Helmintos/genética , Testes Diagnósticos de Rotina/normas , Helmintíase/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/normas , Adolescente , Ancylostomatoidea/classificação , Ancylostomatoidea/genética , Ancylostomatoidea/isolamento & purificação , Animais , Ascaris/classificação , Ascaris/genética , Ascaris/isolamento & purificação , Criança , Pré-Escolar , Fezes/parasitologia , Feminino , Helmintíase/parasitologia , Humanos , Masculino , Necator americanus/classificação , Necator americanus/genética , Necator americanus/isolamento & purificação , Nitratos/química , Projetos Piloto , Sensibilidade e Especificidade , Solo/parasitologia , Trichuris/classificação , Trichuris/genética , Trichuris/isolamento & purificação
11.
Parasitol Res ; 117(6): 1891-1898, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29696393

RESUMO

As the Scandinavian wolf population is limited in size, it is only rarely subject to systematic studies on its disease biology, especially gastrointestinal parasites. Therefore, this study aims to describe the prevalence of gastrointestinal helminths of gray wolves hunted on a limited license as a part of a wildlife management program. Helminths of 20 wolves were examined post mortem by macroscopy and coprology. Intestinal worms of five species were recovered from 18 wolves (90%): Uncinaria stenocephala (90%), Taenia spp. (45%), Alaria alata (25%), and Mesocestoides spp. (5%). Of the taeniid specimens typed by multiplex PCR and sequencing of the cox1 gene, 25% belonged to Taenia hydatigena and 25% to Taenia krabbei. The overall species diversity was low compared to findings from wolves of the northern hemisphere. Fecal eggs of Eucoleus boehmi were detected in 12 wolves (60%). Fecal metastrongylid larvae were found in seven individuals (39%), but PCR analyses specific for Angiostrongylus vasorum were negative. The wolves were in good body condition suggesting that the parasite infestation had no negative impact on the general health of the examined wolves. Although some of the recovered parasite species have zoonotic or veterinary impact, it is not likely that the spare wolf population pose substantial threat to human or veterinary health.


Assuntos
Ancylostomatoidea/isolamento & purificação , Angiostrongylus/isolamento & purificação , Helmintíase/parasitologia , Enteropatias Parasitárias/parasitologia , Enteropatias Parasitárias/veterinária , Mesocestoides/isolamento & purificação , Taenia/isolamento & purificação , Lobos/parasitologia , Ancylostomatoidea/genética , Angiostrongylus/genética , Animais , Animais Selvagens/parasitologia , Fezes/parasitologia , Helmintíase/epidemiologia , Enteropatias Parasitárias/epidemiologia , Mesocestoides/genética , Prevalência , Suécia/epidemiologia , Taenia/genética
12.
PLoS Negl Trop Dis ; 12(1): e0006130, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29346412

RESUMO

BACKGROUND: Proper collection and storage of fecal samples is necessary to guarantee the subsequent reliability of DNA-based soil-transmitted helminth diagnostic procedures. Previous research has examined various methods to preserve fecal samples for subsequent microscopic analysis or for subsequent determination of overall DNA yields obtained following DNA extraction. However, only limited research has focused on the preservation of soil-transmitted helminth DNA in stool samples stored at ambient temperature or maintained in a cold chain for extended periods of time. METHODOLOGY: Quantitative real-time PCR was used in this study as a measure of the effectiveness of seven commercially available products to preserve hookworm DNA over time and at different temperatures. Results were compared against "no preservative" controls and the "gold standard" of rapidly freezing samples at -20°C. The preservation methods were compared at both 4°C and at simulated tropical ambient temperature (32°C) over a period of 60 days. Evaluation of the effectiveness of each preservative was based on quantitative real-time PCR detection of target hookworm DNA. CONCLUSIONS: At 4°C there were no significant differences in DNA amplification efficiency (as measured by Cq values) regardless of the preservation method utilized over the 60-day period. At 32°C, preservation with FTA cards, potassium dichromate, and a silica bead two-step desiccation process proved most advantageous for minimizing Cq value increases, while RNA later, 95% ethanol and Paxgene also demonstrate some protective effect. These results suggest that fecal samples spiked with known concentrations of hookworm-derived egg material can remain at 4°C for 60 days in the absence of preservative, without significant degradation of the DNA target. Likewise, a variety of preservation methods can provide a measure of protection in the absence of a cold chain. As a result, other factors, such as preservative toxicity, inhibitor resistance, preservative cost, shipping requirements, sample infectivity, and labor costs should be considered when deciding upon an appropriate method for the storage of fecal specimens for subsequent PCR analysis. Balancing logistical factors and the need to preserve the target DNA, we believe that under most circumstances 95% ethanol provides the most pragmatic choice for preserving stool samples in the field.


Assuntos
Ancylostomatoidea/isolamento & purificação , DNA de Helmintos/análise , Fezes/parasitologia , Infecções por Uncinaria/diagnóstico , Manejo de Espécimes/métodos , Ancylostomatoidea/genética , Animais , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Temperatura Ambiente , Fatores de Tempo
13.
Mol Biochem Parasitol ; 219: 24-32, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29157661

RESUMO

The phenomenon of unequal usage of synonymous codons encoding an amino acid in which some codons are more preferred to others is the codon usage bias (CUB) and it is species specific. Analysis of CUB helps in understanding evolution at molecular level and acquires significance in mRNA translation, design of transgenes and new gene discovery. In our current study, we analyzed synonymous codon usage pattern and the factors influencing it on mitochondrial protein coding genes of 6 different hookworms i.e. Ancylostoma ceylanicum, Ancylostoma duodenale, Necator americanus, Ancylostoma tubaeforme, Ancylostoma caninum and Uncinaria sanguinis as no work was reported yet. The effective number of codons for mitochondrial genes suggested that codon usage bias was high in most species. The GC content was lower than AT content i.e. genes were AT rich as indicated by nucleotide composition analysis. The overall nucleotide composition along with its composition at 3rd codon position and correspondence analysis suggested that both natural selection and mutation pressure might have affected the codon usage bias in mitochondrial genes. However, neutrality plot revealed that mutation pressure might have played a major role in A. ceylanicum while natural selection might have played the dominant role in Ancylostoma duodenale, Necator americanus, Ancylostoma tubaeforme, Ancylostoma caninum and Uncinaria sanguinis.


Assuntos
Ancylostomatoidea/genética , Códon , Proteínas Mitocondriais/genética , Animais , Composição de Bases , Evolução Molecular , Genes Mitocondriais , Seleção Genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-28504099

RESUMO

This study investigated the presence of zoonotic parasites and vector-borne pathogens in dogs housed in kennels and shelters from four sites of Italy. A total of 150 adoptable dogs was examined with different microscopic, serological and molecular methods. Overall 129 dogs (86%) were positive for one or more parasites and/or pathogens transmitted by ectoparasites. Forty-eight (32%) were positive for one infection, while 81 (54%) for more than one pathogen. The most common zoonotic helminths recorded were hookworms, roundworms and Capillaria aerophila, followed by mosquito-borne Dirofilaria spp. and Dipylidium caninum. One hundred and thirteen (77.9%), 6 (4.1%) and 2 (1.4%) dogs were positive for Rickettsia spp., Leishmania infantum and Anaplasma spp., respectively. The results show that dogs living in rescue facilities from the studied areas may be infected by many zoonotic internal parasites and vector-borne pathogens, and that control measures should be implemented.


Assuntos
Vetores de Doenças , Doenças do Cão/epidemiologia , Enteropatias Parasitárias/veterinária , Intestinos/parasitologia , Zoonoses , Ancylostomatoidea/genética , Ancylostomatoidea/isolamento & purificação , Animais , Capillaria/genética , Capillaria/isolamento & purificação , Dirofilaria/genética , Dirofilaria/isolamento & purificação , Doenças do Cão/parasitologia , Doenças do Cão/transmissão , Cães , Humanos , Enteropatias Parasitárias/epidemiologia , Enteropatias Parasitárias/parasitologia , Enteropatias Parasitárias/transmissão , Itália , Leishmania infantum/genética , Leishmania infantum/isolamento & purificação
15.
Korean J Parasitol ; 54(4): 471-6, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27658599

RESUMO

To know the infection status of helminths in primary schoolchildren of southern parts of Vietnam, we performed an epidemiological study in Krong Pac district, Dak Lak Province, Vietnam. A total of 1,206 stool specimens were collected from ethnic Ede schoolchildren in 4 primary schools in 2015 and examined by the Kato-Katz technique. In addition, stool cultures were done by the Harada-Mori method to obtain hookworm larvae and then to clarify the species of hookworms infected. The results showed that the helminth infection rate was 25.0%, including 2.0% Ascaris lumbricoides, 0.33% Trichuris trichiura, and 22.8% hookworm infections. The average intensity of infection was 102.0 eggs per gram of feces (EPG) for Ascaris, 36.0 EPG for Trichuris, and 218.0 EPG for hookworms. ITS1 gene sequences of the hookworm larvae were identical with those of Necator americanus (100% homology) reported in GenBank. It has been confirmed in this study that the hookworm, N. americanus, is a dominant helminth species infected in primary schoolchildren of a southern part of Vietnam. Public health attention is needed for control of hookworm infections among schoolchildren in surveyed areas of Vietnam.


Assuntos
Ancylostomatoidea/classificação , Ancylostomatoidea/genética , Helmintíase/epidemiologia , Helmintíase/parasitologia , Helmintos/isolamento & purificação , Enteropatias Parasitárias/epidemiologia , Enteropatias Parasitárias/parasitologia , Adolescente , Ancylostomatoidea/anatomia & histologia , Animais , Criança , Análise por Conglomerados , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Fezes/parasitologia , Feminino , Humanos , Masculino , Microscopia , Carga Parasitária , Filogenia , Prevalência , Instituições Acadêmicas , Análise de Sequência de DNA , Estudantes , Vietnã/epidemiologia
16.
Afr Health Sci ; 16(1): 83-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27358617

RESUMO

BACKGROUND: Hookworm eggs identification and quantification is usually carried out by Kato-Katz method. However various structures present in the smear may be confused with eggs of such parasites. OBJECTIVE: To document the presence of structures in Kato-Katz slides that could initially be misinterpreted as hookworm eggs. METHOD: 497 faecal samples were analysed by Kato-Katz technique, diphasic concentration technique, agar-plate coprocultive and larvae obtained were analysed by PCR and characterized by sequencing. RESULT: Hookworm-like eggs were found in 159 (32%) of the samples by Kato-Katz, finally identified as Caenorhabditis elegans by PCR technique. CONCLUSION: The diagnosis of human hookworm eggs, only by the use of Kato-Katz technique can lead to false positives because of similarities with eggs of other free-living worms, from wet soils like those of Rwanda that could contaminate stool samples.


Assuntos
Ancylostomatoidea/isolamento & purificação , Fezes/parasitologia , Infecções por Uncinaria/diagnóstico , Infecções por Uncinaria/parasitologia , Contagem de Ovos de Parasitas/métodos , Solo/parasitologia , Adolescente , Ancylostomatoidea/genética , Animais , Criança , Estudos Transversais , Humanos , Reação em Cadeia da Polimerase , População Rural , Ruanda , Sensibilidade e Especificidade , Análise de Sequência
17.
Vet Parasitol ; 224: 65-67, 2016 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-27270392

RESUMO

The tetraprimer ARMS-PCR technique is efficient for SNP detection and can be used to search for polymorphisms associated with drug resistance. However, the establishment of this methodology is not always straightforward because of the constraints on primer design due to the restrictions of the polymorphic regions. Here, we describe the standardization of the tetraprimer ARMS-PCR methodology for the detection of a SNP at codon 198 of the Ancylostoma caninum ß-tubulin gene. This SNP is associated with resistance to albendazole in various nematodes. The methodology was used to screen 327 individuals from 6 different locations. No mutation was found in any of the samples. This methodology will be useful for screening for the E198A SNP in the ß-tubulin gene of canine hookworms in a broader population to determine whether this SNP is associated with benzimidazole resistance in this species. The method could also be adapted for the analysis of other SNPs in other nematode species.


Assuntos
Doenças do Cão/parasitologia , Resistência a Medicamentos/genética , Infecções por Uncinaria/veterinária , Parasitologia/métodos , Reação em Cadeia da Polimerase/veterinária , Tubulina (Proteína)/genética , Albendazol/farmacologia , Ancylostoma/genética , Ancylostomatoidea/efeitos dos fármacos , Ancylostomatoidea/genética , Animais , Anti-Helmínticos/farmacologia , Brasil , Primers do DNA/genética , Primers do DNA/normas , Cães , Infecções por Uncinaria/parasitologia , Reação em Cadeia da Polimerase/normas , Polimorfismo de Nucleotídeo Único/genética
18.
Parasit Vectors ; 8: 401, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26216353

RESUMO

BACKGROUND: Differentiation of canine hookworm species is crucial from both a veterinary and public health standpoint. In Vietnam, three hookworm species, namely Ancylostoma caninum, Ancylostoma braziliense and Uncinaria stenocephala are reported to infect dogs. In light of the emerging distribution of A. ceylanicum in Asia, this study aims to re-evaluate the status of Ancylostoma in dogs in Vietnam. METHODS: Faecal samples collected from 200 community dogs in Dak Lak province were subjected to faecal floatation for the detection of hookworm eggs. Hookworm-positive samples were subjected to a PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay targeting the internal transcribed spacer (ITS) region of rDNA for hookworm species identification. A subset of hookworm-positive samples was also subject to haplotype characterisation at the cytochrome oxidase-1 (COX-1) gene. Detailed morphological criteria were utilised in addition to molecular markers, to identify adult hookworms recovered from necropsied dogs. RESULTS: Of 200 canine faecal samples, 111 (55.5 %) were positive for hookworm eggs on faecal flotation. Of these, 94/111 (84.7 %) were successfully amplified and assigned species status by PCR-RFLP targeting the ITS region. In total, 54.3 % (51/94) dogs harboured single infections with A. ceylanicum, 33.0 % (31/94) with A. caninum, and 12.7 % (12/94) harboured mixed infections with both A. ceylanicum and A. caninum. Adult worms recovered from necropsied dogs matched morphological description provided for A. ceylanicum, Looss (1911) for which the mediolateral and posteriolateral rays are parallel. Characterisation of the COX-1 gene placed all Vietnamese canine isolates of A. ceylanicum within the 'zoonotic' haplotype. CONCLUSION: Based on this information, it is apparent that the hookworms present in dogs in Vietnam are those of A. ceylanicum and not A. braziliense. Owing to the endemic nature of this significant zoonosis in dogs, the study strongly advocates for specific identification of this hookworm in human hookworm surveys.


Assuntos
Ancylostomatoidea/classificação , Doenças do Cão/parasitologia , Infecções por Uncinaria/veterinária , Ancylostomatoidea/genética , Animais , DNA de Helmintos/genética , Doenças do Cão/epidemiologia , Cães , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica , Haplótipos , Infecções por Uncinaria/epidemiologia , Infecções por Uncinaria/parasitologia , Filogenia , Vietnã/epidemiologia
19.
Parasit Vectors ; 8: 380, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26183074

RESUMO

BACKGROUND: In resource-limited countries, stool microscopy is the diagnostic test of choice for intestinal parasites (soil-transmitted helminths and/or intestinal protozoa). However, sensitivity and specificity is low. Improved diagnosis of intestinal parasites is especially important for accurate measurements of prevalence and intensity of infections in endemic areas. METHODS: The study was carried out in Orán, Argentina. A total of 99 stool samples from a local surveillance campaign were analyzed by concentration microscopy and McMaster egg counting technique compared to the analysis by multi-parallel quantitative real-time polymerase chain reaction (qPCR). This study compared the performance of qPCR assay and stool microscopy for 8 common intestinal parasites that infect humans including the helminths Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, and the protozoa Giardia lamblia, Cryptosporidium parvum/hominis, and Entamoeba histolytica, and investigated the prevalence of polyparasitism in an endemic area. RESULTS: qPCR showed higher detection rates for all parasites as compared to stool microscopy except T. trichiura. Species-specific primers and probes were able to distinguish between A. duodenale (19.1%) and N. americanus (36.4%) infections. There were 48.6% of subjects co-infected with both hookworms, and a significant increase in hookworm DNA for A. duodenale versus N. americanus (119.6 fg/µL: 0.63 fg/µL, P < 0.001) respectively. qPCR outperformed microscopy by the largest margin in G. lamblia infections (63.6% versus 8.1%, P < 0.05). Polyparasitism was detected more often by qPCR compared to microscopy (64.7% versus 24.2%, P < 0.05). CONCLUSIONS: Multi-parallel qPCR is a quantitative molecular diagnostic method for common intestinal parasites in an endemic area that has improved diagnostic accuracy compared to stool microscopy. This first time use of multi-parallel qPCR in Argentina has demonstrated the high prevalence of intestinal parasites in a peri-urban area. These results will contribute to more accurate epidemiological survey, refined treatment strategies on a public scale, and better health outcomes in endemic settings.


Assuntos
Ancylostomatoidea/isolamento & purificação , Infecções por Uncinaria/epidemiologia , Enteropatias Parasitárias/epidemiologia , Parasitos/isolamento & purificação , Ancylostomatoidea/genética , Animais , Argentina/epidemiologia , Fezes/parasitologia , Feminino , Infecções por Uncinaria/parasitologia , Humanos , Enteropatias Parasitárias/parasitologia , Contagem de Ovos de Parasitas , Parasitos/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , População Urbana
20.
Acta Trop ; 150: 210-7, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26215130

RESUMO

Human infections with the helminth species Strongyloides stercoralis encompass a wide clinical spectrum, ranging from asymptomatic carriage to life-threatening disease. The diagnosis of S. stercoralis is cumbersome and the sensitivity of conventional stool microscopy is low. New molecular tools have been developed to increase sensitivity. We compared the diagnostic accuracy of real-time PCR with microscopy for the detection of S. stercoralis and hookworm in human stool samples, and investigated the inter-laboratory agreement of S. stercoralis-specific real-time PCR in two European laboratories. Stool specimens from 256 randomly selected individuals in rural Côte d'Ivoire were examined using three microscopic techniques (i.e. Kato-Katz, Koga agar plate (KAP) and Baermann (BM)). Additionally, ethanol-fixed stool aliquots were subjected to molecular diagnosis. The prevalence of S. stercoralis and hookworm infection was 21.9% and 52.0%, respectively, whilst co-infections were detected in 35 (13.7%) participants. The diagnostic agreement between real-time PCR and microscopy was excellent when both KAP and BM tested positive for S. stercoralis, but was considerably lower when only one microscopic technique was positive. The sensitivity of KAP, BM and real-time PCR for detection of S. stercoralis as compared to a combination of all diagnostic techniques was 21.4%, 37.5% and 76.8%, respectively. The inter-laboratory agreement of S. stercoralis-specific PCR was substantial (κ=0.63, p<0.001). We conclude that a combination of real-time PCR and stool microscopy shows high accuracy for S. stercoralis diagnosis. Besides high sensitivity, PCR may also enhance specificity by reducing microscopic misdiagnosis of morphologically similar helminth larvae (i.e. hookworm and S. stercoralis) in settings where both helminth species co-exist.


Assuntos
Ancylostomatoidea/isolamento & purificação , Infecções por Uncinaria/diagnóstico , Strongyloides stercoralis/isolamento & purificação , Estrongiloidíase/diagnóstico , Adolescente , Adulto , Idoso , Ancylostomatoidea/genética , Animais , Criança , Pré-Escolar , Coinfecção , Costa do Marfim/epidemiologia , Estudos Transversais , Fezes/parasitologia , Feminino , Infecções por Uncinaria/complicações , Infecções por Uncinaria/epidemiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , População Rural , Sensibilidade e Especificidade , Strongyloides stercoralis/genética , Estrongiloidíase/complicações , Estrongiloidíase/epidemiologia , Adulto Jovem
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