Elimination of Giardia duodenalis BIV in vivo using natural extracts in microbiome and dietary supplements.

In this study, a combination therapy of several natural products was evaluated in vivo in the Giardia duodenalis infection model. G. duodenalis infected mice were treated as follows: distilled water (infected control C+), BIOintestil® (BIO; natural products of Cymbopogon martinii and Zingiber officinale), MicrobiomeX® (MBX; extract of Citrus sinensis and Citrus paradisi), MBX + BIO, Camellia sinensis tea (CPR; black tea). These natural compounds were administered in a dose of 100 mg/day and were compared to G. duodenalis-infected mice treated with albendazole (ALB; 50 mg/Kg/day) and metronidazole (MET; 500 mg/Kg/day), the conventional therapies used to this day. One group remained un-infected and untreated as our control group (C-). Treatment started 8 days after infection, and after 5 days of treatment (7 days for MET), all animals were followed for 15 days. We continuously checked for the presence of G. duodenalis by Faust method, in association with detection of the parasite by PCR from feces, as well for the presence of trophozoites in the intestinal mucosa after sacrifice. Animals treated with MBX, BIO and MBX + BIO presented an undetectable parasitic load until the 15th day of monitoring, while animals treated with CPR, MET and ALB continued to release cysts. Animals in the MBX, MBX + BIO, ALB groups consumed lower feed, MBX, CPR, MET had greater weight and MBX, MBX + BIO, BIO, CPR, C- consumed more water when compared to infected-group control. MBX and BIO alone or associated eliminated G. duodenalis without apparent adverse effects and animals of these groups showed better clinical performance in relation to those with high parasitic load. MET, ALB and CPR only decreased the number of cysts, indicating limitations and therapeutic failure.

Comparative analysis of routine parasitological methods for recovery of cysts, molecular detection, and genotyping of Giardia duodenalis.

In order to improve the diagnosis of giardiasis, fecal samples (high/medium/low concentration of cysts) were processed by the parasitological methods used in the routine: Faust, Lutz e Ritchie modified (replacement of formaldehyde by distilled water). The cysts were quantified; the DNA was extracted and amplified by semi-nested PCR (GDH gene). Fifteen clinical samples were analyzed to validate the study by PCR-RFLP. The results showed that the parasite was only detected and genotyped correctly when samples from children with high, medium, and low parasitic load, belonging to genotype AII, were processed by the modified Ritchie method, different from what was observed for the other methods used in laboratory routine (Faust and Lutz). The modified Ritchie method proved to be more suitable, recovering a greater number of cysts from samples, regardless of parasitic load, which reduces the chance of false negative results and has epidemiological repercussions since individuals with low parasite load are usually asymptomatic and the main disseminators of this infection.

Improvement in cyst recovery and molecular detection of Giardia duodenalis from stool samples.

Molecular detection of Giardia duodenalis by polymerase chain reaction (PCR) is difficult in faecal samples due to inhibitors that contaminate DNA preparations, or due to low cyst concentrations. In order to eliminate inhibitors, improve cyst recovery and molecular detection of G. duodenalis, different types of water, distillates (MDs), deionized (MDz), injection (MI) or Milli-Q® (MM) were used instead of formaldehyde (F) in the laboratory routine method (Ritchie). Cysts were isolated from faecal samples with low cyst concentrations (< 1 cyst/field), medium (1-2 cysts/field) or high (> 2 cysts/field). Cyst recovery was improved using all water types (MDs, MDz, MI, MM) compared to formaldehyde. At all cyst concentrations, the use of MM consistently showed the greatest recovery of G. duodenalis cysts . DNA samples from recovered cysts were tested for the glutamate dehydrogenase (GDH) and ß-giardin (ßg) genes. The use of Milli-Q® water allowed to detect both genes in all cyst concentrations, including low. The method processed with the other types of water amplified these genes at high and medium cyst concentrations. GDH and ßg genes were not detected when the sample was processed with formaldehyde. These experimental results were confirmed in clinical samples. The results suggest that Milli-Q® water provides the highest cyst recovery from stool samples and, correspondingly, the highest sensitivity for detecting G. duodenalis by microscopy or PCR for GDH and ßg genes, even at low concentration of cysts.

Assemblages A and B of Giardia duodenalis reduce enteric glial cells in the small intestine in mice.

Infection of Giardia duodenalis is one of the most common human parasitic disease worldwide. This infection may be related to important changes in the enteric nervous system. The objective of this study was to evaluate the myenteric and submucosal plexuses, the intestinal muscle layer, and gastrointestinal transit in mice infected with assemblages A and B of G. duodenalis. Swiss albino mice (Mus musculus) were infected with assemblages A and B of G. duodenalis for 15 days. Gastrointestinal transit time was evaluated before euthanasia. Duodenum and jejunum were removed for histological and immunohistochemical analyses. It was observed a reduction in the enteric glial cell count and a decrease in the ratio of enteric glial cells to neurons. The number of neurons did not change, but morphological changes were observed in the duodenum and jejunum in both plexuses, including an increase in the nuclear area and a reduction of cell bodies in the myenteric plexus and a decrease in the nuclear area in the submucosal plexus. A reduction of the thickness of the muscle layer was observed in the duodenum, with no significant differences in the gastrointestinal transit times. Assemblages A and B of G. duodenalis decrease the number of enteric glial cells in the myenteric and submucosal plexuses, decrease the thickness of the muscle layer, and change the morphology of neurons. Graphical abstract ᅟ.

Synergistic effects of fenbendazole and metronidazole against Giardia muris in Swiss mice naturally infected.

In this study were proposed different protocols for the treatment of mice naturally infected with Giardia muris. Male Swiss mice were divided into seven groups, with five animals each, in a blind, controlled, randomized by drawing lots and once-repeated experiment. Parasite detection and cure control were performed using the Faust method and search by trophozoites in the intestinal mucosa. Clinical parameters (weight, water and feed consumption, elimination of excreta, aspect of the fur and feces) were also evaluated. All animals were treated with metronidazole (M), fenbendazole (F), and probiotics (P), administered intragastrically, during 7 days. M1, FM1, and F1 groups were treated 1×/day; M3, FM3, and PM3 groups 3×/day; and ST (control group) received only water. After the 5th and 7th days of treatment, the animals in FM1/FM3 and PM3/M3 groups presented, respectively, negative results and remained negative in the following 10 days. Animals in F1 group consumed less water (p = 0.00010) compared with FM1/FM3/PM3. The animals in M1 group compared with FM3/M3, F1 compared with M3, and ST compared with FM1/FM3/M3/PM3 consumed a larger amount of feed (p = 0.00001). The animals in F1 group compared with FM3/M1/M3/PM3, FM1 compared with FM3, and ST compared with FM3/M1/M3/PM3 eliminated lower volume of excreta (p = 0.00001). The results show that the association between F and M potentiates the effects, indicating a synergistic action of these two drugs, and FM1 is the best protocol due to early negativity in the animals, lower concentrations of the drugs, lower risk of toxicity and stress, and less alterations in clinical parameters.

Food handlers as a link in the chain of transmission of Giardia duodenalis and other protozoa in public schools in southern Brazil.

BACKGROUND: Food handlers (FHs) may facilitate transmission and dissemination of pathogens. The importance of FHs as a link in the epidemiological chain of transmission of Giardia duodenalis and other intestinal protozoa was assessed. METHODS: Fecal and subungual material from 27 FHs were analyzed using parasitological methods. G. duodenalis was identified by direct immunofluorescence and genotyped by PCR-RFLP for the bg and gdh genes, and gdh was sequenced. RESULTS: At least one protozoan was detected in 30% (8/27) of the FHs and G. duodenalis (19%; 5/27) was the most common species. The AII and BIV genotypes were found in 20% (1/5) and 60% (3/5) of FHs infected with G. duodenalis, respectively. CONCLUSIONS: FHs can be involved in the chain of transmission of G. duodenalis and other protozoa. GENBANK ACCESSION NUMBERS: KJ741310 - KJ741313.

Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them.

BACKGROUND: Giardia duodenalis infects humans and other mammals by ingestion of cysts in contaminated water or food, or directly in environments with poor hygiene. Eight assemblages, designated A-H, are described for this species. METHODOLOGY/PRINCIPAL FINDINGS: We investigated by microscopy or by direct immunofluorescence technique the occurrence of G. duodenalis in 380 humans, 34 animals, 44 samples of water and 11 of vegetables. G. duodenalis cysts present in samples were genotyped through PCR-RFLP of ß giardin and glutamate dehydrogenase (gdh) genes and sequencing of gdh. The gdh gene was amplified in 76.5% (26/34) of the human faeces samples with positive microscopy and in 2.9% (1/34) of negative samples. In 70.4% (19/27) of the positive samples were found BIV assemblage. In two samples from dogs with positive microscopy and one negative sample, assemblages BIV, C, and D were found. Cysts of Giardia were not detected in water samples, but three samples used for vegetable irrigation showed total coliforms above the allowed limit, and Escherichia coli was observed in one sample. G. duodenalis BIV was detected in two samples of Lactuca sativa irrigated with this sample of water. BIV was a common genotype, with 100% similarity, between different sources or hosts (humans, animals and vegetables), and the one most often found in humans. CONCLUSIONS/SIGNIFICANCE: This is the first study in Brazil that reports the connection among humans, dogs and vegetables in the transmission dynamics of G. duodenalis in the same geographic area finding identical assemblage. BIV assemblage was the most frequently observed among these different links in the epidemiological chain.

Avaliação da dosagem de própolis pulverizada nas folhas de amoreira (morus sp.) sobre o desenvolvimento biológico e produtivo do bicho-da-seda (bombyx mori l.) / Evaluación de la dosis de propóleos esparcidas en las hojas de morera (morus sp.) en el desarrollo biológico y productivo del gusano de seda (bombyx mori l.) / Evaluation of the doses of propolis pulverization in mulberry leaves (morus sp.) on the biological and productive development of silkworm (bombyx mori l.)

Este experimento foi realizado no Laboratório de Sericicultura, no Campus Sede da Universidade Paranaense (UNIPAR) de Umuarama, no período de 17/09/2009 a 17/10/2009, com o objetivo de verificar o efeito da própolis em diferentes dosagens na alimentação durante o desenvolvimento biológico do bicho-da-seda (Bombxy mori L). O método empregado na parte experimental foi a pulverização do extrato alcoólico de própolis, diluído em 500 mL de água destilada nas folhas de amoreira, nas seguintes dosagens, água-controle, 5mL, 10mL, 15mL e 20mL compondo os tratamentos T1, T2, T3, T4, T5, respectivamente. As folhas de amoreira foram fornecidas cinco vezes ao dia, durante o manejo alimentar. Verificou-se pelos resultados obtidos, que as diferentes dosagens de própolis utilizadas interferem no ganho de peso das lagartas, no peso dos casulos verdes e crisálidas quando comparado ao tratamento controle, influenciando também no número de casulos formados e, para os teores de seda bruto e líquido não apresentaram resultados significativos, quando comparados com o tratamento controle. Portanto, verificou-se que a própolis, nas dosagens utilizadas, não trouxe efeitos depressivos à biologia e produção do bicho-da-seda.

This experiment was conducted at the Sericulture Laboratory, in the Campus Sede of the Universidade Paranaense (UNIPAR) in Umuarama, from 17/09/2009 to 17/10/2009, in order to verify the effects of propolis in different dosages during the development of silkworm (Bombyx mori L). The method used during the experiment was the dillution of an alcohoolic solution of propolis, in 500 mL of destilled water with its further pulverization on mullberry leaves, with the following doses: water control; 5 mL; 10 mL; 15 mL; 20 mL, composing the following treatments: T1, T2, T3, T4, T5, respectively. The mullberry leaves were sprinkled five times a day, during the feed management. It was verified by the results that the different doses of propolis used interfere with gain-weight of the larvae, weight of cocoons and chrysalis, when compared to control. The treatment also influenced the number of cocoons formed. The contento fraw and liquid silk were not significant when compared with the control. Therefore, it was concluded that the propolis, used in those dosages, brought no depressive effects to the development and the production of silkworm.

Este experimento se realizó en el Laboratorio de Sericicultura del Campus Sede de la Universidad Paranaense (UNIPAR) de Umuarama, en el período de 17/09/2009 a 17/10/2009, con el objetivo de verificar el efecto de propóleos en diferentes dosis en la alimentación durante el desarrollo biológico del gusano de seda (Bombyx mori L.). El método usado en la parte experimental fue la pulverización del extracto alcohólico de propóleos, diluido en 500 ml de agua destilada en las hojas de morera, en las siguientes dosis: agua control, 5 ml, 10 ml, 15 ml y 20 ml componiendo los tratamientos T1, T2, T3, T4, T5, respectivamente. Las hojas de morera fueron suministradas cinco veces al día, durante el manejo alimentar. Por los resultados obtenidos se verificó que las dosis diferentes de propóleos utilizadas interfieren en el gano de peso de los gusanos, en el peso de los capullos verdes y crisálidas cuando comparado al tratamiento control, influenciando también en el número de capullos formados y, para contenidos de seda bruta y líquida no presentan resultados significativos, mientras comparado con el tratamiento control. Sin embargo, se puede verificar que propóleos, en dosis utilizadas, no trajo efectos depresivos a la biología y producción del gusano.