Molecular identification of Ancylostoma species from dogs and an assessment of zoonotic risk in low-income households, São Paulo State, Brazil.

Hookworm infection stands out for its worldwide distribution and for its veterinary and public health relevance. Based on copromicroscopic examinations and polymerase chain reaction (PCR) amplification of the ITS1-5.8S-ITS2 region, we assessed, respectively, the prevalence of intestinal parasites and the identification of canine hookworm species in faeces recovered from 278 dogs living in households of an inland municipality of São Paulo State, Brazil. Intestinal parasites were found in 67.3% of dogs and hookworm infection was found at the highest prevalence rate (56.6%), followed by Toxocara canis (11.9%), Isospora spp. (11.9%), Giardia spp. (5.8%), Sarcocystis spp. (4.0%), 'Hammondia-like' (1.4%), Dipylidium caninum (1.1%) and Trichuris vulpis (0.7%). Of 158 samples positive for hookworm eggs, 106 (67.1%) were amplified by PCR and, of those, 88 (55.7%) were successfully sequenced for species identification. Single infections with Ancylostoma caninum and Ancylostoma braziliense were recorded in 61.4% and 12.5%, respectively, and mixed infections were found in 26.1%. The nucleotide sequences of both species showed high identity rates (98-100%) when compared with reference sequences. Although A. caninum was the most prevalent hookworm in the dogs assessed, the occurrence of both A. caninum and A. braziliense in single and/or mixed infections poses a potential risk for the local population in a low-income area, especially children, to acquire cutaneous larva migrans (CLM).

Genotyping of Giardia duodenalis isolates in asymptomatic children attending daycare centre: evidence of high risk for anthroponotic transmission.

Giardia duodenalis is a common intestinal parasite infecting children attending daycare centres. This study aimed to verify Giardia occurrence and the genotypes of isolates infecting children aged 0-6 years and workers at a daycare centre in the state of São Paulo, Brazil. The families of children who tested positive for Giardia, were asked to provide stool samples from household members and their dogs. Samples (123 children, 14 centre employees, 44 household members, 19 children after treatment, and 20 dogs) were examined for intestinal parasites using concentration methods. DNA extracted from all samples was submitted for polymerase chain reaction (PCR) testing and the amplicons generated were used for multilocus sequence typing of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Giardia was detected in 15·9% and 28·6% of the 220 samples by microscopy and PCR, respectively. Analysis of sequences retrieved from 29 isolates revealed both assemblages A (31%) and B (69%). Sub-assemblages AII, BIII and BIV were identified and the alignment of the bg, gdh and tpi sequences revealed the presence of some single nucleotide polymorphisms, especially in assemblage B sequences. The higher predominance of assemblage B and the identification of the AII type support the view that anthroponotic transmission appears to be an important route of transmission in environments that concentrate children at an age when poor hygiene practices make them more vulnerable to such infection.

Diagnosis of Giardia infections by PCR-based methods in children of an endemic area

The present study was designed to estimate the prevalence of Giardia infection in preschool- and school-aged children living in an endemic area. Fecal samples from 573 children were processed by zinc sulfate centrifugal flotation, centrifugal sedimentation (using a commercial device for fecal concentration - TF-Test kit®) and polymerase chain reaction (PCR)-based methods. Of the stool samples assessed, 277 (48.3 percent) were positive for intestinal parasites and/or commensal protozoa. Centrifugal flotation presented the highest diagnostic sensitivity for Giardia infections. The kappa index revealed that both coproparasitological techniques closely agreed on the Giardia diagnosis (86 percent) versus satisfactory (72 percent) and poor (35 percent) concordances for commensal protozoan and helminth infections, respectively. Concerning Giardia molecular diagnosis, from the 71 microscopy-positive samples, specific amplification of gdh and tpi fragments was noted in 68 (95.7 percent) and 64 (90 percent) samples, respectively. Amplification of gdh and tpi genes was observed, respectively, in 95.7 percent and 90 percent of microscopy-positive Giardia samples. For 144 microscopy-negative samples, gdh and tpi gene amplification products were obtained from 8.3 percent and 35.9 percent samples, respectively. The agreement between these genes was about 40 percent. The centrifuge-flotation based method was the most suitable means of Giardia diagnosis assessed in the present study by combining accuracy and low cost.

Genotyping of Brazilian Giardia duodenalis human axenic isolates

Giardia duodenalis is a complex species that comprises at least seven distinct genetic groups (A to G), but only genotypes A and B are known to infect humans and a wide variety of other mammals. Regardless of biological, biochemical and antigenic analysis, several isolates maintained in vitro were not genetically typed yet. So, in the present study, five Brazilian axenic isolates obtained from asymptomatic and symptomatic patients were typed in order to determine the major genetic groups to which the isolates belonged. DNA was extracted from axenic trophozoites, fragments of glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes were amplified by PCR and the isolate genotyping was carried out using restriction fragment length polymorphism (RFLP) and DNA sequencing for both genes. The results revealed that all isolates were assigned to genotype A at both analyzed loci. Indeed, DNA sequence analysis classified the four isolates obtained from asymptomatic individuals into subtype AII, while the isolate obtained from the symptomatic patient was typed as subtype AI. Despite of the limited number of isolates assessed, the findings presented herein provide interesting insights on the occurrence of Giardia genotypes in Brazil and hold the perspective for future molecular and epidemiological investigations.

Comparison of three concentration methods for the recovery of canine intestinal parasites from stool samples.

The aim of present study was to compare the efficiency of a commercial assay and two conventional methods for fecal concentration in detecting canine gastrointestinal parasites. Fecal samples from 254 dogs were processed by centrifugation-sedimentation (CS), centrifugation-flotation (CF) and a commercial assay for fecal concentration (TF-test). The following parasites were detected: Ancylostoma (37.8%), Giardia (16.9%), Toxocara canis (8.7%), Trichuris vulpis (7.1%), Isospora (3.5%), and Sarcocystis (2.7%). The calculated analytical sensitivity indicated that CF was more accurate (P<0.01) in detecting Ancylostoma, T. canis, T. vulpis and Giardia infections. However, CF showed significantly higher sensitivity only for Ancylostoma, compared to the other two methods. The kappa index value of diagnostic agreement between TF-test and CF was high for T. canis (83%) and moderate for Giardia (72%) and Ancylostoma (63%). The advantages and limitations of each method were assessed for individual diagnosis and epidemiological investigation.

Characterization of the excretory/secretory products of Dermatobia hominis larvae, the human bot fly.

Proteolytic activity in excretory/secretory products (ESP) of first- (L1), second- (L2) and third-instar (L3) larvae of Dermatobia hominis was analyzed through gelatin-gel and colorimetric enzyme assays with the chromogenic substrates azocasein and BApNA. The functional characterization of proteases was based on inhibition assays including synthetic inhibitors. ESP were obtained from new-hatched larvae reared in the laboratory and from second- and third-instar larvae removed from naturally infested cattle. Gelatin-gel analysis evidenced few bands of proteolysis, predominantly of high apparent molecular masses, in ESP of L1, whereas in the gel of L2 and L3 ESP there was a wide range of proteolytic activity most of them not resolved in a single species. Azocasein assays revealed a progressive increase of protease activity from first- to third-instar larvae. Protease inhibitor assays revealed a predominance of metalloproteases in L1 ESP that could be related to a skin penetration process and to a diversion of host immune response. The predominance of serine proteases in L2 and L3 and the great tryptic activity presented by L3 ESP were attributed to an increasing trophic activity by the growing larvae, since the viability of adult flies strictly depends on larval abilities to assimilate nutrients from the host. Taking together, these results suggest that Dematobia larvae secrete/excrete different proteases that may be related to diverse functions during host penetration and infestation, which reinforces the relevance of the study of such proteolytic enzymes.

Prevalence of dog intestinal parasites and risk perception of zoonotic infection by dog owners in São Paulo State, Brazil.

Coprological examination was used to estimate the prevalence of gastrointestinal parasites in stray and domiciled dogs from Botucatu, São Paulo State, Brazil. Risk factors for dog infection were assessed in relation to demographic, husbandry and management data. The dog owners completed a questionnaire survey on some aspects of dog parasitism such as parasite species, mechanisms of infection, awareness of zoonotic diseases and history of anthelmintic usage. Parasites were found in the faeces of 138 dogs, with an overall prevalence of 54.3%. Dogs harbouring one parasite were more common (31.4%) than those harbouring two (18.5%), three (3.2%) or four (1.2%). The following parasites and their respective frequencies were detected: Ancylostoma (37.8%), Giardia (16.9%), Toxocara canis (8.7%), Trichuris vulpis (7.1%), Dipylidium caninum (2.4%), Isospora (3.5%), Cryptosporidium (3.1%) and Sarcocystis (2.7%). Stray dogs were found more likely to be poliparasitized (P<0.01) and presented higher prevalence of Ancylostoma, T. canis and Giardia (P<0.01) than domiciled ones. Toxocara canis was detected more frequently in dogs with <6 months of age (P<0.05) and no effect of sex or breed could be observed (P>0.05). Except for Ancylostoma, that showed a significantly higher prevalence in dogs living in a multi-dog household (P<0.01), parasite prevalences were similar in single- and multi-dog household. The answers of dog owners to the questionnaire showed that the majority does not know the species of dog intestinal parasites, the mechanisms of transmission, the risk factors for zoonotic infections, and specific prophylactic measures. The predominance of zoonotic species in dogs in the studied region, associated with the elevated degree of misinformation of the owners, indicates that the risk of zoonotic infection by canine intestinal parasite may be high, even in one of the most developed regions of Brazil.

Detection of Babesia bigemina in cattle of different genetic groups and in Rhipicephalus (Boophilus) microplus tick.

Babesia bigemina infections were investigated in four genetic groups of beef cattle and in Rhipicephalus (Boophilus) microplus engorged female ticks. Blood samples and engorged female ticks were collected from 15 cows and 15 calves from each of the following genetic groups: Nelore, Angus x Nelore, Canchim x Nelore, and Simmental x Nelore. Microscopic examination of blood smears and tick hemolymph revealed that merozoites of B. bigemina (6/60) as well as kinetes of Babesia spp. (9/549) were only detected in samples (blood and ticks, respectively) originated from calves. PCR-based methods using primers for specific detection of B. bigemina revealed 100% infection in both calves and cows, regardless the genetic group. Tick infection was detected by nested-PCR amplifications showing that the frequency of B. bigemina was higher (P<0.01) in female ticks collected from calves (134/549) than in those collected from cows (52/553). The frequency of B. bigemina was similar in ticks collected from animals, either cows or calves, of the four genetic groups (P>0.05).

Occurrence of Tetrameres confusa (Nematoda, Tetrameridae) in Ara ararauna (Psittacidae)

Relata-se a ocorrência de Tetrameres confusa Travassos 1917 (= T. americana (Cram, 1927) Baylis, 1929) (Nematoda, Tetrameridae) em um novo hospedeiro, Ara ararauna Linnaeus, 1758 (Aves, Psittacidae). Este é o primeiro registro da ocorrência do nematódeo em psitacídeos no Brasil.

Babesia spp. infection in Boophilus microplus engorged females and eggs in Sao Paulo State, Brazil.

Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01).

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle.

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.

Response of Corriedale and Crioula Lanada sheep to artificial primary infection with Haemonchus contortus.

Clinical, parasitological and biochemical parameters were evaluated in Corriedale and Crioula Lanada sheep after a single experimental infection with Haemonchus contortus. Ten 4-month-old worm-free lambs, of each breed, were infected with 200 L3 H. contortus per kg live weight and four uninfected animals of each breed were used as controls. Every week, the animals were weighed and blood and faecal samples were collected for measurement of packed cell volume (PCV), total serum protein (TSP) and albumin (ALB), and the number of eggs per gram of faeces (EPG), respectively. Twelve weeks after infection, the animals were slaughtered. The worm burden was determined and samples of the abomasal mucosa were processed for determination of the number of eosinophils, mast cells and globule leukocytes. No significant differences in PCV, TSP, ALB, parasite burden or the cell populations of the abomasal mucosa were observed between breeds, but Crioula lambs had a lower EPG count. The comparison of the infected groups with their respective controls revealed significant alterations in PCV, TSP and ALB in the Corriedale lambs and in PCV, TSP, ALB and the density of eosinophils and mast cells in the Crioula lambs.

Strongyloides venezuelensis infection susceptibility of seven inbred strains of mice

A trial was carried out to investigate the susceptibility of seven strains of mice to Strongyloides venezuelensis primary and secondary experimental infections, in order to provide the basis for genetic studies about resistance. Twelve six-week-old male inbred mice of the A/J, BALB/c, CBA/J, C3H/Hepos, C57BL/6, DBA/2 and NIH strains were infected s.c. with 2000 infective larvae. The mean worm counts (± SD) in the small intestine six days after infection were, in increasing order: 28 (± 19) in NIH; 647 (± 228) in BALB/c; 709 (± 425) in DBA/2; 731 (± 151) in C3H/Hepos, 801 (± 174) in CBA/J; 1024 (± 267) in C57BL/6 and 1313 (± 483) in A/J. C57BL/6 mice showed the highest fecal egg counts and NIH, the lowest. No eggs in fecal exams or nematodes in small intestines were recovered from animals reinfected 14 days after primary infection. NIH strain was highly resistant to primary infection by S. venezuelensis. The most susceptible of the other six strains appeared to be the C57BL/6 strain which presented a high nematode counting in intestine and the highest egg output

Prevalence of intestinal parasites in dogs from São Paulo State, Brazil.

The prevalence of gastrointestinal parasites in stray dogs, and dogs with owners was investigated by fecal examinations from 271 dogs employing sedimentation, simple flotation and centrifugation-flotation methods. The centrifugation-flotation method, when compared to simple flotation or sedimentation methods was generally more accurate in the diagnosis of all intestinal parasites, but statistical differences were detected only in relation to Giardia spp. and Cystoisospora spp. (synonym Isospora spp.). The following parasites, with their respective prevalence, were diagnosed in the fecal samples: Ancylostoma spp. (23.6%); Toxocara canis (5.5%); Trichuris vulpis (4.8%); Spirocerca lupi (1.9%); Dipylidium caninum (0.7%); Giardia spp. (12.2%); Hammondia heydorni (2.6%); Cystoisospora spp. (8.5%); and Sarcocystis spp. (2.2%). The prevalence of most parasites was similar for dogs of mixed-breed and for dogs of a defined-breed, except for Cystoisospora spp. and T. canis which showed a significantly higher prevalence in mixed-breed dogs. The prevalence of Ancylostoma spp. (17.1%) was significantly lower in stray dogs than in those with an owner (31.9%) and the prevalence of Giardia spp. and Cystoisospora spp. was higher in stray dogs (P < 0.05). No effect of season on the occurrence of the different parasite genera could be observed, except for Ancylostoma spp., for which an increase in the percentage of dogs shedding eggs was observed at the beginning of Summer with a peak occurrence during April and May (Autumn). The prevalence of Ancylostoma spp., T. canis, T. vulpis, Giardia spp. and Cystoisospora spp. was higher in adult males than in adult females, but significant differences between the two groups occurred only with Giardia spp. Young animals were found to more frequently shed Nematode eggs in feces than adult animals.

Parasitological characteristics and tissue response in the abomasum of sheep infected with Haemonchus spp

A histopathological study was conducted on the abomasal mucosa of 40 Corriedale sheep exposed to natural infection with Haemonchus spp. The sheep were allowed to graze on contaminated pastures for 14 days and, after being housed for 28 days, they were slaughtered. Fecal samples were collected for fecal egg counts (FEC) and abomasum samples were obtained for histopathological examination and eosinophil, mast cell and globule leucocyte counts. The number of Haemonchus spp. present in the abomasum was estimated from a 10 per cent aliquot of its content. The number of eggs per female was estimated for 10 Haemonchus spp. females collected from each animal. The following significant correlation coefficients were obtained between the characteristics analyzed: number of Haemonchus and FEC (r = 0.86), female length and number of eggs per female (r = 0.60), female length and FEC (r = 0.53), number of eosinophils and number of Haemonchus (r = 0.48), number of eosinophils and number of globule leucocytes (r = 0.54), number of mast cells and female length (r = -0.39), and number of mast cells and number of globule leucocyte (r = 0.34). The characteristics that showed the highest correlation with animal load was FEC