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When Did They Forst Learn About Gardia 8n Dogs?

Introduction

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is the causative amanuensis of giardiosis and one of the virtually commonly institute parasite in dogs throughout the world (ane, 2). Molecular studies take shown that 1000. duodenalis comprises at to the lowest degree eight distinct genetic assemblages (A–H), of which assemblages C and D are found exclusively in dogs, while Giardia assemblages A and B have zoonotic potential (three–five).

Infection by G. duodenalis in dogs has a worldwide distribution with prevalence rates that vary according to the examination population, the diagnostic method used and the geographical area (6, 7). In Italy, prevalence values range between 11.i and 28.ix% in northern (8–x), between 6.four and 21.iv% in fundamental (11–14) and between 7.vii and 14.2% in southern regions (15, 16).

The diagnosis of Giardia infection in dogs may pose a claiming, due to a low infectious dose and marked persistence of cysts in the environment besides as a fluctuating excretion of the cysts in the feces that makes the monitoring of this parasite problematic (17, 18). Traditional tools for identification of Giardia cysts include fecal smear, zinc sulfate flotation technique (17, 19), immunofluorescence assay (IFA; the golden standard), immunochromatography, enzyme-linked immunosorbent assays (ELISA), and molecular analyses that let the different G. duodenalis assemblages to be distinguished (17, xx, 21). Amid the copromicroscopic techniques, a recent written report reported a high sensitivity and specificity of FLOTAC technique with zinc sulfate for diagnosis of Giardia cysts infection in dogs (19, 22).

Also the control of Giardia infection in dogs is decumbent to a number of issues. Several compounds have been tested against Giardia infections in dogs every bit benzimidazoles, in particular fenbendazole, and metronidazole that poses activity against the parasite (23–28). Other studies reported the efficacy of ronidazole, nitazoxanide, azithromycin, tinidazole, and ipronidazole and other drugs such as quinacrine, furazolidone, in reducing Giardia cysts shed by infected dogs (17, 29–32). Another option recommended by the European Scientific Counsel Companion Animal Parasites (ESCAAP) is the combination of febantel (a prodrug metabolized in vivo to fenbendazole)/pyrantel/praziquantel at the standard deworming dose (fifteen.0 mg/kg of febantel, xiv.4 mg/kg pyrantel, 5.0 mg/kg praziquantel) repeated once daily for 3 days (33). Recently, secnidazole, a molecule that is used for the treatment of giardiosis in humans, has been reported as an effective drug for the treatment of clinical canine giardiosis (34). However, fenbendazole and metronidazole are used routinely to treat giardiosis in dogs and are the only compounds registered in most European countries.

Resistance to antiparasitic drugs has been oftentimes suggested to explain treatment failure due to the incomplete parasite removal following treatment or other underlying diseases (due east.g., inflammatory bowel diseases, bacterial overgrowth, coinfection with other organisms). Shampooing of dogs (e.g., with a product containing chlorhexidine digluconate) at the beginning and at the cease of antiprotozoal treatment is too recommended to reduce re-infections through fecal fabric on the fur (xxx). Despite handling recommendation with fenbendazole for eliminating Giardia cysts in dogs, currently, unpublished data from veterinary practices are showing a low efficacy of this drug in eliminating the infection. Taking into business relationship that the ESCCAP Guidelines recommends fenbendazole (1x/d, 50 mg/kg for iii days) and metronidazole (2x/d, 25 mg/kg for 5-vii days) for the treatment of Giardia infections in dogs (33), the purpose of the present written report was to reassess the efficacy of these two specific drugs for the treatment of giardiosis in owned dogs, monitored for 50 days in home-conditions.

Materials and Methods

Animal Testing and Study Design

A field trial performed in-dwelling house conditions was conducted in 2018–2019 on 24 owned dogs naturally infected by Giardia spp. and living in the Campania region, southern Italy. The dogs included in the study were referred to the Laboratory of Parasitology and Parasitic Diseases at the Department of Veterinarian Medicine and Animal Production, University of Naples Federico Two, for coproparasitological analysis. Dog owners were informed about the study protocol and they gave their consent to inclusion of their pets. Animals were allocated in groups randomly in order to obtain two groups of 12 animals each with similar parasitic loads of Giardia cysts: group A-fenbendazole (hateful cyst per gram of feces-CPG = 20,995) and group B-metronidazole (CPG = xviii,580). The cysts were enumerated using a fecal flotation method (Flotation, Translation and Centrifugation) FLOTAC technique (encounter Laboratory Assay). In add-on, the dogs had the post-obit characteristics: xi males (five long-haired and half dozen curt-haired dogs) and 13 females (vi long-haired and seven short-haired dogs), aged between two months and 5 years, living indoor only with admission to outdoors. All the dogs included in the written report did not alive in the aforementioned household with other animals. Exclusion criteria were animals treated with any antiparasitic drug in the 2 weeks before, animals with any aggressive beliefs and animals showing clinical signs of any other diseases. For each brute included in the written report, data regarding age, fur length, clinical signs and consistency of fecal samples were recorded and analyzed. For ethical reasons, no untreated control-group of animals was available. Dogs in Group A were treated with fenbendazole tablets (Panacur®, Intervet Italia Srl) administered at the dose of 50 mg/kg orally once a solar day for 5 consecutive days. Dogs in Group B were treated with metronidazole tablets (Flagyl®, Zambon Italia Srl) administered orally at the dose of 25 mg/kg, orally, twice a day for 5 consecutive days. All the dogs started the start treatment at Day 0. All the dogs that were shedding Giardia cysts later the first treatment were retreated (either at Day 7 or at Day 14 or at Day 21) until a negative result was obtained with the same treatment. Additionally, all the dogs were re-examined at Day 50. Treatment efficacy was evaluated based on the hateful CPG found in the fecal samples on 24-hour interval−five (pre-handling) and on Days 7, xiv, 21, and 50 (post-treatment). Additionally, each dog was subjected to shampooing with chlorhexidine digluconate at the start and at the finish of each treatment practical. Furthermore, each owner was suggested to follow some bones rules to avoid the reinfection by cleaning and drying the environment, the use of clean utensils for feed and h2o and proper disposal of feces (33).

Laboratory Analysis

Dogs were initially identified and verified every bit infected with Giardia spp. and then subsequently analyzed at Twenty-four hour period−five (pre-treatment) and at Days 7, xiv, 21, and 50 after the first treatment practical (Day 0). The fecal samples analyzed at each sampling point were represented by pools collected on a daily basis for 2 days (prior to the treatment date). Analyses were performed within 24 h of sampling. At each sampling twenty-four hours the feces for all dogs were tested for the presence of Giardia cysts using the FLOTAC technique with an analytic sensitivity of ane cyst per gram (CPG) of feces (xix). Each sample was analyzed using zinc sulfate (specific gravity = ane.350).

Fifteen samples collected at Day−five were fixed in 2.5% potassium bichromate (dilution 1:4) and candy for molecular studies. Briefly, genomic Deoxyribonucleic acid was extracted using the FastDNA SPIN Kit for carrion (MP Biomedicals, Solon, Ohio, The states). A nested PCR assay was used to amplify a 511 bp fragment of the beta-giardin gene according to a published protocol (35). DNA from axenic cultures of Giardia duodenalis strains of assemblage A (WB, genotype AI) and assemblage B (GS, genotype BIV) was used every bit positive controls in PCR assays. PCR products were purified using spin columns (QiaQuick PCR Purification kit –Qiagen) and sequenced from both strands.

Handling Efficacy

The percent efficacy of the treatments (A and B) was calculated at each sampling betoken from the trial (Days 7, fourteen, 21, and 50) according to the following formula [adapted from (36)].

%Efficacy = Mean CPG 24-hour interval - 5 - Hateful CPG day 7 Mean CPG twenty-four hour period - 5 × 100 CPG = cysts per gram of feces .

Statistical Assay

Statistical assay was performed using Windows SPSS® (version 17.0). The non-parametric Isle of mann-Whitney U examination was used to determine the level of significant difference betwixt groups of handling (A, B).

Assessment Criteria and Clinical Consequence

An capricious scoring system (from 1 to 4) was used and a score assigned to each dog, based on fecal consistency and clinical symptoms (lethargic attitude, growth retardation, weight loss, vomiting and flatulence) as follows: (1) formed carrion and no clinical symptoms, (two) formed feces and clinical symptoms, (3) soft stools and clinical symptoms, and (iv) diarrhea and clinical symptoms.

Results

Table 1 shows dog'southward data (gender, historic period, fur length, clinical score), Giardia cysts per gram (CPG) of feces (hateful, standard deviation) and efficacy (%) of treatment with fenbendazole (Grouping A) and metronidazole (Group B) at the different report Days (Day−5, Day vii, Day 14, Day 21, and Day 50).

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Tabular array one. Groups A-Fenbendazole and B-Metronidazole: domestic dog's data (gender, age, fur length, clinical score), Giardia cysts per gram (CPG) of carrion (mean, standard departure), and efficacy (%) of the treatment at the different written report days.

Parasitological results at Twenty-four hours−5 showed hateful values of 20,995 Giardia CPG (minimum 2,780 CPG, maximum 48,000 CPG) in the Group A (fenbendazole) and 18,580 Giardia CPG (minimum 10,848 CPG, maximum 32,178 CPG) in the Grouping B (metronidazole). On Day 7, cysts of Giardia spp. were found in 33.3% (four/12; 95% Confidence Interval (CI) = 11.iii–64.5) of the dogs in the Group A and in 41.seven% (5/12; 95% CI = 16.5–71.4) of the dogs in the Group B.

The efficacy of fenbendazole was lxxx.nine% at Day 7, 100% at day 14, 97.0% at day 21, and 95.0% at day l. The efficacy of metronizazole was 70.8% at Solar day vii, 99.0% at Day xiv, 100% at Day 21, and 97.one% at Day 50. Overall, the efficacies of Fenbendazole and Metronidazole confronting the infection past G. duodenalis were not significantly different (P = 0.686).

No other co-infections with intestinal parasites were found at copromicroscopic examinations. Briefly, six dogs from Group A and five dogs from Group B remained negative until Mean solar day 50 afterward they received the start treatment at Day 0. 8 dogs (iv from each group), received the second handling at Day 7 (subsequently the kickoff therapy at 24-hour interval 0), and two other dogs (one from each grouping) were retreated for the second time at Solar day 21 from the Group A and Day 14 from the Group B. Moreover, in the Group B, there was one dog that received 3 treatments during the study at Days 0, seven and 14. Finally, two dogs (i from each grouping), that had received the kickoff treatment at Mean solar day 0, were nonetheless shedding Giardia cysts at the concluding written report Day (Day 50).

Cess Criteria and Clinical Upshot

At Day−5, feces were unformed to diarrhoeic in dogs belonging to both handling groups simply no adverse responses due to the medications were observed. 33.3% of the dogs had score i, 25% score 2, 25% score 3, and xvi.7% score four. Regarding the dogs that tested positive for Giardia after the treatment (13/24), five belonged to the starting time category (38.6%), four to the second category (30.8%), one to the tertiary category (7.7%), and three to the 4th (23%).

Molecular Characterization

Giardia DNA could be amplified only from six of the fifteen fecal samples tested, 3 from the Group A (dog'southward ID numbers = F1, F6, F8) and three from the Grouping B (domestic dog's ID numbers = M4, M7, M11). The beta-giardin distension products were sequenced and compared, using Nail, with all available sequences in the GenBank database. This revealed full homology (due east.1000., 100% with JN416550) with the canine specific aggregation D in the six positive dogs. The lack of distension in the remaining samples is likely due to cyst impairment resulting from storage weather.

Discussion

During the final 60 years, a number of chemotherapeutic agents have been introduced and are still in use in the therapy for giardiosis (37). Unfortunately, most drugs used have considerable agin effects such every bit vomiting, bloody diarrhea, abortion, neurological dysfunctions and so they are contraindicated (38). In this context, studies on chemotherapeutic agents play a central role in the rationale for the treatment of giardiosis.

Several benzimidazoles (39, twoscore), and in particular fenbendazole (23), or the combinations of febantel/fenbendazole with other compounds proved to be effective (24, 25, 41). Metronidazole, a nitroimidazole, and fenbendazole are used routinely to treat giardiosis in dogs (30). Ronidazole showed a good antiprotozoal effect against Giardia in dogs (xxx) and oxfendazole showed a meaning decrease in the number of cysts (40).

The findings of the present study did not reveal a meaning departure of efficacy between the drugs used (fenbendazole 80.9% vs. metronidazole 70.viii% at Day 7). Moreover, after applying the first handling, both drugs failed to eliminate the Giardia cysts in all the dogs. Re-appearance of the cysts in the carrion could exist attributed to a re-infection, treatment failure, the correct medication given by the owners, the diet, as well as biological problems related to the intermittent excretion of Giardia cysts. Notwithstanding, in that location are no specific information of the dog'southward surroundings at home if any try was made by the owner to make clean up contaminated areas. Faure et al. (42), reported a pregnant departure of efficacy betwixt fenbendazole and metronidazole (xxx.3% vs. 91.nine%). In our study, fenbendazole showed lower efficacy than that reported by Zajac et al. (26), who obtained 90% efficacy (26). Even so, information technology is important to note that in our present study both drugs showed 100% efficacy afterward two sequent cycles of treatments, i.eastward., at Days 14 in the Group A and at Day 21 in the Group B.

Fifteen (62.five%) out of 24 dogs treated from both groups (eight dogs from Grouping A and 7 dogs from Grouping B) were tested negative for Giardia cysts after the first therapy at Solar day vii. Moreover, there were dogs from both groups A and B, thus showing a persistent infection that has been cleared either at Day 14 or at Day 21 after two or three treatments of v days each. It could be possible, that some Giardia remained at low abundance in the intestine of these dogs that were even so positive after the first handling applied, or for the dogs that needed to be treated three times until a negative outcome was obtained, or perhaps we were facing with a treatment failure due to drug resistance. However, future investigations are needed, in gild to found the availability of the drugs in the blood and the capacity of these to eliminate the Giardia cysts by in vitro methods.

In improver, 2 dogs from each group (A and B) remained negative after the first therapy (Day 0), until Day 50, when turned positive. In these cases, it was suggested that reinfections from the environs are the most common cause for treatment failure (25, 30) considering that the prepatent menstruation can be as curt equally iv days (33). Also, the presence of cysts on the creature's fur, associated with the resistance of the parasite to disinfectants (chlorhexidine gluconate in our report) and environmental conditions and with stress, may explicate reinfections (30). Since this is a field trial performed in-home weather condition we were non sure about the failure of cleaning and bathing due to the lack of compliance past the possessor, thus, the use of hygiene measures such as chlorhexidine digluconate shampoo earlier and after the handling might accept a considerable influence on our results representing a major event of this study. Moron-Soto et al. also reported that dogs re-shed cysts following a cursory menses after antiparasitic treatment when no disinfection or cleaning of their enclosures was performed (31).

Some experimental studies have evaluated as well the efficacy of the oral administration of probiotics together with albendazole (38), therefore during the treatment the veterinarian tin recommend the assistants of probiotics that play a synergistic role with the drug regenerating the intestinal flora. However, in the present study, none of the dogs received a special nutrition, or probiotics supplements during and subsequently the therapy. Moreover, in that location are few recent studies that showed that the microbial diversity was not altered by fenbendazole assistants, which is in contrast to metronidazole which significantly altered microbial structure and multifariousness (43–47). Administration of metronidazole to healthy dogs caused meaning changes in the microbiome, some of which persist following discontinuation of the drug with unknown clinical effects. Furthermore, some dogs on metronidazole can go neurotoxicity (48, 49). However, in the present study, no adverse responses due to the medications were observed including transient neurological signs due to metronidazole.

There take been ii reports examining the effects of secnidazole on Giardia in dogs. One, reported 100% efficacy of cyst reduction in two groups of six hospitalized dogs (50). The other ane, showed that at 43 days afterwards the outset treatment, all 9 dogs were considered to accept normal stools, but 4 of the 9 puppies were positive for Giardia antigen (34). Bearing these facts in listen, maybe secnidazole practical twice, 2–3 weeks autonomously, could be an culling and easier treatment, with a better compliance as well for treating Giardia infection. Further data are needed to prove its efficacy both in field and experimental studies.

Finally, genotyping of Giardia demonstrated the host-specific assemblage D in all positive dogs, in agreement with other studies in Italia (12, 35). However, our report presented limitations due to the sample size and due to the poor and inappropriate storage atmospheric condition to ensure the effective isolation of DNA from fecal samples. Therefore, further enquiry on distinguish G. duodenalis strains, are needed, in order to perform a realistic interpretation of zoonotic risk (51).

Based on the findings, equally expected in a trial with a small number of individuals in-abode atmospheric condition, and especially for a parasite with the characteristics of Giardia (i.e., having a direct infection and short prepatent menstruation), the authors could only speculate that treatment lone is not sufficient for controlling Giardia infection.

A chief limitation of our study is based on the small-scale sample size (no. = 12 dogs), the absence of a control group and the allotment of the animals in the two treatment Groups based only on individual Giardia CPG assessed at Day−5. Even so, the limited number of dogs in our report was mainly due to type of study (field trial in-home conditions) that requires a full compliance of both vet practitioners and dog owners. Upstanding problems were the main reasons for not including a control group in such a kind of field trial performed in-dwelling house conditions. In a similar study, Mirò et al. (52) assessed the efficacy of fenbendazole and mebendazole using 10 per group. As a upshot, in the allocation phase we could not consider other primal epidemiological parameters as age, fur length and style of living. Different hypotheses might explain the re-appearance of the Giardia cysts in some dogs after treatment, eastward.grand., re-infection from the home environment, the correct medication given past the owners, the diet, equally well every bit treatment failure, but too biological issues related to the intermittent excretion of Giardia cysts. Therefore, due to the uncontrolled parameters in this field trial performed in-home conditions, future studies are warranted to produce conclusive show for the evaluation of integrated approaches needed for the treatment of dogs naturally infected past Giardia.

Data Availability Statement

The raw information supporting the conclusions of this article will exist made available by the authors, without undue reservation.

Ethics Statement

The animals used in the nowadays report were sampled following approval past the animal ethics and welfare committee of the University of Naples Federico 2 (in Italian, Comitato Etico-scientifico per la Sperimentazione Animale dell' Università di Napoli Federico II; protocol number PG/20170055343). Written informed consent was obtained from the owners for the participation of their animals in this report.

Author Contributions

MG, LR, LC, LK, SMC, ARS, and GC contributed to the conception and design of the study. PP, AB, AV, and MPM organized the database and performed the statistical analysis. MG, LR, and LC wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

Conflict of Involvement

The authors declare that the inquiry was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Source: https://www.frontiersin.org/articles/626424

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