Recurrent patent infections with Toxocara canis in household dogs older than six months : a prospective study

Background: To reduce environmental contamination with Toxocara canis eggs, the current general advice is to deworm all dogs older than six months on average four times a year. However, only a small proportion of non-juvenile household dogs actually shed T. canis eggs, and some dogs shed eggs more frequently than others. The identification of these frequent shedders and the associated risk factors is an important cornerstone for constructing evidence-based deworming regimens. The purpose of this study is to identify risk factors associated with recurrence of periods of shedding Toxocara eggs in a cohort of household dogs older than six months. Methods: We performed a prospective study (July 2011 to October 2014) on shedding Toxocara eggs in a cohort of 938 household dogs older than six months from all over the Netherlands. The median follow-up time was 14 months. Monthly, owners sent faecal samples of their dogs for Toxocara testing and completed a questionnaire. Dogs were dewormed only after diagnosis of a patent infection (PI). Survival analysis was used to assess factors influencing the time to first diagnosed PIs (FPI) and the time to recurrent PIs (RPI). Results: The overall prevalence of PIs was 4.5 %, resulting in an estimated average incidence of 0.54 PIs/dog/year. No PI was diagnosed in 67.9 % of the dogs, 17.5 % of the dogs went through only one PI and 14.6 % had > 1 PI. Prevalence of PIs always peaked during wintertime. Increased hazards for first diagnosed PIs were associated with coprophagy, geophagy, walking off-leash for = 80 % of walking time, reported worms in the faeces, feeding a commercial diet and suffering from urologic or respiratory conditions. Median time to reinfection was nine months. Factors associated with increased hazards for recurrent PIs were taking corticosteroids, changing dog's main purpose, and proxies for veterinary care-seeking behaviours. Conclusions: We concluded that targeted anthelmintic treatments in household dogs may be feasible as PIs tend to (re)occur in specific periods and in groups of dogs at high risk. Moreover, recurrent PIs appear to be influenced more by factors related to impaired immunity than environmental exposure to Toxocara eggs.</p

Environmental contamination with Toxocara eggs : A quantitative approach to estimate the relative contributions of dogs, cats and foxes, and to assess the efficacy of advised interventions in dogs

Background: Environmental contamination with Toxocara eggs is considered the main source of human toxocariasis. The contribution of different groups of hosts to this contamination is largely unknown. Current deworming advices focus mainly on dogs. However, controversy exists about blind deworming regimens for >6-month-old dogs, as most of them do not actually shed Toxocara eggs. We aim to estimate the contribution of different non-juvenile hosts to the environmental Toxocara egg contamination and to assess the effects of different Toxocara-reducing interventions for dogs. Methods: A stochastic model was developed to quantify the relative contribution to the environmental contamination with Toxocara eggs of household dogs, household cats, stray cats, and foxes, all older than 6 months in areas with varying urbanization degrees. The model was built upon an existing model developed by Morgan et al. (2013). We used both original and published data on host density, prevalence and intensity of infection, coprophagic behaviour, faeces disposal by owners, and cats' outdoor access. Scenario analyses were performed to assess the expected reduction in dogs' egg output according to different deworming regimens and faeces clean-up compliances. Estimates referred to the Netherlands, a country free of stray dogs. Results: Household dogs accounted for 39 % of the overall egg output of >6-month-old hosts in the Netherlands, followed by stray cats (27 %), household cats (19 %), and foxes (15 %). In urban areas, egg output was dominated by stray cats (81 %). Intervention scenarios revealed that only with a high compliance (90 %) to the four times a year deworming advice, dogs' contribution would drop from 39 to 28 %. Alternatively, when 50 % of owners would always remove their dogs' faeces, dogs' contribution would drop to 20 %. Conclusion: Among final hosts of Toxocara older than 6 months, dogs are the main contributors to the environmental egg contamination, though cats in total (i.e. both owned and stray) transcend this contribution. A higher than expected compliance to deworming advice is necessary to reduce dogs' egg output meaningfully. Actions focusing solely on household dogs and cats are unlikely to sufficiently reduce environmental contamination with eggs, as stray cats and foxes are also important contributors

Variation in haplotypes in single cysts of assemblages C and D, but not of assemblage E of Giardia duodenalis

BACKGROUND: Giardia duodenalis, a single-celled intestinal parasite, is divided into eight assemblages (A-H), with differences in host specificity. Giardia duodenalis reproduces asexually and cycles between the binucleated trophozoite (4 N) and the infectious cyst with four nuclei (16 N). Interaction between the nuclei is limited. Therefore, genetic drift causes differences in genetic make-up between the non-daughter nuclei; the allelic sequence heterozygosity (ASH). The ASH is low (0.01%-0.0023%) for the related assemblages A and E, higher (0.43-0.53) for assemblage B and much higher (0.74% -0.89%) for the assemblage C and D at the root of the phylogenetic tree. The heterozygosity in assemblage F, in the same clade as assemblage A and E, was unknown. The heterozygosity in the sequences of the gdh and dis3 genes was used as proxy for the ASH and whole genome amplification of single cysts followed by cloning and Sanger sequencing of dis3 fragment could reveal the genetic variation within the cyst. The aim of the study was to determine the level of heterozygosity within pooled and single cysts of different assemblages. RESULTS: The heterozygosity in gdh and dis3 was determined in pooled cysts of the assemblages A to F. Heterozygosity in the isolates of the assemblages C (n = 2) and D (n = 1) ranged from 0.41% to 0.82% for gdh and dis3 and no heterozygosity was found in the isolates of the assemblages A (n = 4), E (n = 3) and F (n = 3). The heterozygosity in assemblage B (n = 7) was intermediate (0% to 0.62%). Next, the number of haplotypes of dis3 was determined for single cysts of assemblages C, D and E. In the assemblages C and D, two to four haplotypes were found per cyst, while in assemblage E only one haplotype was identified. CONCLUSIONS: Having high heterozygosity is characteristic for the assemblages C and D, while having a low heterozygosity is characteristic for the clade with the assemblages A, E and F. Presence of more than 1 haplotype per cyst in assemblage C and D suggests differences between the non-daughter nuclei, in contrast to the one haplotype in assemblage E

Giardia duodenalis multi-locus genotypes in dogs with different levels of synanthropism and clinical signs

Background In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding. Methods One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR. Results Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool. Conclusion Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage

Environmental contamination with Toxocara spp. eggs in public parks and playground sandpits of Greater Lisbon, Portugal.

Toxocarosis is a zoonotic parasitic disease transmitted from companion animals to humans. Environmental contamination with Toxocara eggs is considered to be the main source of human infections. In Portugal, knowledge regarding the current situation, including density, distribution and environmental contamination by Toxocara spp., is largely unknown. The present study investigated environmental contamination with Toxocara spp. eggs, in soil and faecal samples collected from public parks and playground sandpits in Greater Lisbon, Portugal. A total of 151 soil samples and 135 canine faecal samples were collected from 7 public sandpits and 12 public parks, over a 4 month-period. Soil samples were tested by a modified centrifugation and sedimentation/flotation technique and faecal samples were tested by an adaptation of the Cornell-Wisconsin method. Molecular analysis and sequencing were performed to discriminate Toxocara species in the soil. Overall, 85.7% of the sandpits (6/7) and 50.0% of the parks (6/12) were contaminated with Toxocara spp. eggs. The molecular analysis of soil samples showed that, 85.5% of the sandpits and 34.4% of the parks were contaminated with Toxocara cati eggs. Faecal analysis showed that 12.5% of the sandpits and 3.9% of the parks contained Toxocara canis eggs. In total, 53.0% of soil and 5.9% of faecal samples were positive for Toxocara spp. Additionally, 56.0% of the eggs recovered from the samples were embryonated after 60 days of incubation, therefore considered viable and infective. The average density was 4.2 eggs per hundred grams of soil. Public parks and playground sandpits in the Lisbon area were found to be heavily contaminated with T. cati eggs, representing a serious menace to public health as the studied areas represent common places where people of all ages, particularly children, recreate. This study sounds an alarm bell regarding the necessity to undertake effective measures such as reduction of stray animals, active faecal collection by pet owners, awareness campaigns and control strategies to decrease the high risk to both animal and human health

Host factors associated with Giardia duodenalis infection in dogs across multiple diagnostic tests

Background The aim of this study was to assess potential associations between Giardia duodenalis infection in dogs, as determined by three diagnostic tests, and dog’s group of origin, fecal consistency, age, sex, neuter status, and co-infections with other gastrointestinal parasites. Methods Fecal samples from 1291 dogs from four groups (household, shelter, hunting and clinical dogs) were tested with qPCR, rapid enzyme immunochromatographic assay (IDEXX SNAP® Giardia), and direct immunofluorescence (DFA, Merifluor) for presence of G. duodenalis. Moreover, fecal samples were tested with centrifugation sedimentation flotation (CSF) coproscopical analysis for presence of gastrointestinal parasites. Associations were expressed as odds ratios (ORs). Results Several significant associations were found, of which a few were consistent for all three tests and Giardia positivity in general (positive with at least one of these tests). Dogs older than one year were significantly less likely to test positive for Giardia than younger dogs. Group-housed dogs, especially hunting dogs, were significantly more likely to test positive for Giardia compared to household and clinical dogs. A consistently significant association with Trichuris appeared to be driven by the high prevalence in hunting dogs. Although there was no significant association between loose stool and Giardia infection in the overall population, household dogs were significantly more likely to test Giardia-positive when having loose stool. Overall, Giardia-positive dogs with loose stool shed significantly more cysts, both determined semi-quantitatively with CSF and quantitatively by qPCR, than positive dogs with no loose stool. When other gastrointestinal parasites were present, significantly fewer cysts were detected with CSF, but this was not confirmed with qPCR. Conclusion Giardia is the most common gastrointestinal parasite in Dutch dogs, except for hunting dogs, in which Trichuris and strongyle-type eggs (hookworms) prevailed. Giardia infection was not significantly associated with loose stool, except for household dogs. Young dogs and group-housed dogs were significantly more often Giardia-positive. These associations were consistent across diagnostic tests. Young dogs, clinical dogs and dogs with loose stool shed Giardia cysts in the highest numbers. If another gastrointestinal parasite was present lower numbers of cysts were observed by microscope (CSF), but not with a molecular method (qPCR)

Giardia duodenalis multi-locus genotypes in dogs with different levels of synanthropism and clinical signs.

Background: In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding. Methods: One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR. Results: Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool. Conclusion: Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage

Longitudinal study of extended-spectrum-β-lactamase- and AmpC-Producing Enterobacteriaceae in household dogs

A longitudinal study was performed to (i) investigate the continuity of shedding of extended-spectrum-beta-lactamase (ESBL)-producing Enterobacteriaceae in dogs without clinical signs, (ii) identify dominant plasmid-mediated ESBL genes, and (iii) quantify ESBL-producing Enterobacteriaceae in feces. Fecal samples from 38 dogs were collected monthly for 6 months. Additional samples were collected from 7 included dogs on a weekly basis for 6 weeks. Numbers of CFU per gram of feces for non-wild-type Enterobacteriaceae were determined by using MacConkey agar supplemented with 1 mg/liter cefotaxime (MCC), and those for total Enterobacteriaceae were determined by using MacConkey agar. Cefotaxime-resistant isolates were screened by PCR and sequence analysis for the presence of blaCTX-M, blaCMY, blaSHV, blaOXA, and blaTEM gene families. Bacterial species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. PCR-negative isolates were tested by a double-disk synergy test for enhanced AmpC expression. A total of 259 samples were screened, and 126 samples were culture positive on MCC, resulting in 352 isolates, 327 of which were Escherichia coli. Nine dogs were continuously positive during this study, and 6 dogs were continuously negative. Monthly or weekly shifts in fecal shedding were observed for 23 dogs. Genotyping showed a large variety of ESBL genes and gene combinations at single and multiple consecutive sampling moments. The ESBL genes blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaSHV-12, and blaCMY-2 were most frequently found. The mean number of CFU of non-wild-type Enterobacteriaceae was 6.11 × 108 CFU/g feces. This study showed an abundance of ESBL-producing Enterobacteriaceae in dogs in the Netherlands, mostly in high concentrations. Fecal shedding was shown to be highly dynamic over time, which is important to consider when studying ESBL epidemiology

Host factors associated with Giardia duodenalis infection in dogs across multiple diagnostic tests.

Fecal samples from 1291 dogs from four groups (household, shelter, hunting and clinical dogs) were tested with qPCR, rapid enzyme immunochromatographic assay (IDEXX SNAP® Giardia), and direct immunofluorescence (DFA, Merifluor) for presence of G. duodenalis. Moreover, fecal samples were tested with centrifugation sedimentation flotation (CSF) coproscopical analysis for presence of gastrointestinal parasites. Associations were expressed as odds ratios (ORs)