Felippe Danyel Cardoso Martins

Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil

The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.

ISOLATION AND GENOTYPING OF Toxoplasma gondii IN SERONEGATIVE URBAN RATS AND PRESENCE OF ANTIBODIES IN COMMUNICATING DOGS IN BRAZIL

The role of rodents in the epidemiology of toxoplasmosis was investigated in Londrina, Paraná State, Brazil. One hundred and eighty-one Rattus rattus and one Mus musculus were caught in 37 places. Blood and tissues were collected and submitted to the indirect fluorescence antibody test (IFAT) and the bioassay. Serum samples from 61 contacting dogs were also collected. Sixteen rats (8.8%) were positive for Toxoplasma gondii, but just two of them were positive by serology and bioassay test. Antibodies were found in nine (4.9%) rats. Tissues of nine rats bioassayed were positive and four isolates were obtained. Restriction fragment length polymorphism (RFLP) analysis was performed using 12 markers (SAG1, SAG2, SAG2-alt, C22-8, C29-2, L358, PK1, BTUB, GRA6, SAG3, Apico, CS3). Genotyping revealed that the four strains isolated from this study have been isolated before in cats and chickens from Brazil. None of the isolates was identified like clonal archetypal T-types I, II, and III. The rats presented lower serologic Toxoplasma gondii prevalence (8.8%) compared to contacting dogs (70.5%).

Cryptosporidium spp. and Giardia spp. in feces and water and the associated exposure factors on dairy farms

The aims of this study were to verify the prevalence of Cryptosporidium spp. and Giardia spp. in animal feces and drinking water on dairy farms and to identify a possible relation between the exposure factors and the presence of these parasites. Fecal samples from cattle and humans and water samples were collected on dairy farms in Paraná, Brazil. Analysis of (oo)cysts in the feces was performed by the modified Ziehl-Neelsen staining and centrifugal flotation in zinc sulfate. Test-positive samples were subjected to nested PCR amplification of the 18SSU ribosomal RNA gene for identification of Cryptosporidium and Giardia and of the gp60 gene for subtyping of Cryptosporidium. Microbiological analysis of water was carried out by the multiple-tube method and by means of a chromogenic substrate, and parasitological analysis was performed on 31 samples by direct immunofluorescence and nested PCR of the genes mentioned above. Identification of the species of Cryptosporidium was performed by sequencing and PCR with analysis of restriction fragment length polymorphisms. The prevalence of Giardia and Cryptosporidium was higher in calves than in adults. Among the samples of cattle feces, Cryptosporidium parvum was identified in 41 (64%), C. ryanae in eight (12.5%), C. bovis in four (6.3%), C. andersoni in five (7.8%), and a mixed infection in 20 samples (31.3%). These parasites were not identified in the samples of human feces. Thermotolerant coliform bacteria were identified in 25 samples of water (45.5%). Giardia duodenalis and C. parvum were identified in three water samples. The gp60 gene analysis of C. parvum isolates revealed the presence of two strains (IIaA20G1R1 and IIaA17G2R2) in the fecal samples and one (IIaA17G2R1) in the water samples. The presence of coliforms was associated with the water source, structure and degradation of springs, rain, and turbidity. The prevalence of protozoa was higher in calves up to six months of age. C. parvum and G. duodenalis were identified in the water of dairy farms, as were thermotolerant coliforms; these findings point to the need for guidance on handling of animals, preservation of water sources, and water treatment.

Spatial and simultaneous representative seroprevalence of anti- Toxoplasma gondii antibodies in owners and their domiciled dogs in a major city of southern Brazil

Toxoplasmosis, caused by Toxoplasma gondii, has traditionally been considered an important water and foodborne protozoonosis with important public health considerations. Although felids play a well-established role as definitive hosts, canine epidemiological involvement in the parasite’s life cycle remains questionable and controversial. The increasing closeness of the human-dog bond, particularly seen in urban settings, has been recognized as a historically unprecedented worldwide movement. Sharing daily lives in the same households, dogs may be exposed to similar associated risks of T. gondii infection as their owners. Thus, epidemiological assessment of the intra-domiciled environment, especially among socio-economically different human populations, may provide novel information regarding the actual role of dogs in animal and human toxoplasmosis. Despite spatial approaches being recently used for other water and foodborne diseases, no study has been conducted on the simultaneous spatial seroprevalence of both human and animal IgG anti-T. gondii antibodies in urban areas of major cities. Accordingly, the aim of the present study was to assess the seroprevalence and associated variables of Toxoplasma infection in owners and their domiciled dogs in Londrina, southern Brazil. Human and canine seroprevalence rates and variables associated with seroprevalence were investigated through representative random sampling among 564 households, which included 597 owners and 729 dogs. Overall, statistically significant differences between the seroprevalence of human and dog anti-T. gondii antibodies were found by Immunofluorescence Antibody Testing in 248/597 (41.54%) owners and 119/729 (16.32%) dogs. Through multiple analysis, significant concomitant variables for seropositivity of household individuals (people and dogs) were determined, including public sewer service, yard cleaning frequency, and having a dirty yard. Although no statistically significant multiple logistic model was observed among owners, univariate analysis detected associations with monthly income, soil contact, and occupation. Among dogs, the absence of other dogs and the absence of a dirty yard were concomitant significantly protective associated factors. Age differences between seropositive and seronegative individuals was significant only for human beings, with the median age of negative individuals significantly higher than positive individuals. Although no spatial clusters were identified for humans or residences, a significant cluster was identified for dogs. In conclusion, characteristics of urban toxoplasmosis may include significantly higher owner seroprevalence than their owned dogs, with canine seroprevalence directly associated with having more dogs and a dirty backyard, and spatial differences in both human and dog exposures. Although not a good indicator for human foodborne diseases, dogs may be a reliable sentinel for environmental infection. Moreover, such a holistic approach may provide crucial information for more focused prevention and monitoring programs, particularly in households with multiple pets and trash-filled backyards.

First identification of Cryptosporidium parvum subtype IIaA20G1R1 in water buffalos ( Bubalus bubalis )

Cryptosporidium can infect a wide variety of vertebrate animals, including mammals, birds, amphibians, reptiles, and fish. There are few molecular characterizations of Cryptosporidium isolated from water buffalo. Thus, the present study investigated the occurrence and molecular characterization of Cryptosporidium spp. in water buffalos by nested-PCR. Non-diarrheic feces were obtained from 122 water buffalo calves. All samples were tested by nested-PCR based on the 18S rRNA gene, after which positive samples were analyzed by RFLP and genetic sequencing. Sixteen fecal (13.1%) samples were positive, and RFLP showed that fifteen presented patterns consistent with C. ryanae and one with C. parvum. Sequencing of the gp60 gene from the C. parvum positive sample indicated the subtype IIaA20G1R1. This is the first identification of the IIaA20G1R1 subtype in water buffalos.

Spatial and simultaneous seroepidemiology of anti-Leishmania spp. antibodies in dog owners and their dogs from randomly selected households in a major city of southern Brazil

Although leishmaniasis has been described as a classic example of a zoonosis requiring a comprehensive approach for control, to date, no study has been conducted on the spatial distribution of simultaneous Leishmania spp. seroprevalence in dog owners and dogs from randomly selected households in urban settings. Accordingly, the present study aimed to simultaneously identify the seroprevalence, spatial distribution and associated factors of infection with Leishmania spp. in dog owners and their dogs in the city of Londrina, a county seat in southern Brazil with a population of half a million people and ranked 18th in population and 145th in the human development index (HDI) out of 5570 Brazilian cities. Overall, 564 households were surveyed and included 597 homeowners and their 729 dogs. Anti-Leishmania spp. antibodies were detected by ELISA in 9/597 (1.50%) dog owners and in 32/729 (4.38%) dogs, with significantly higher prevalence (p = 0.0042) in dogs. Spatial analysis revealed associations between seropositive dogs and households located up to 500 m from the local railway. No clusters were found for either owner or dog case distributions. In summary, the seroepidemiological and spatial results collectively show a lack of association of the factors for infection, and the results demonstrated higher exposure for dogs than their owners. However, railway areas may provide favorable conditions for the maintenance of infected phlebotomines, thereby causing infection in nearby domiciled dogs. In such an urban scenario, local sanitary barriers should be focused on the terrestrial routes of people and surrounding areas, particularly railways, via continuous vector surveillance and identification of phlebotomines infected by Leishmania spp.

Molecular detection of Cryptosporidium spp. and the occurrence of intestinal parasites in fecal samples of naturally infected dogs and cats

Cats and dogs are hosts of a large number of gastrointestinal parasites and can shed helminth eggs and protozoan oocysts in their feces. The close relationship between companion animals and humans intensifies human exposure to zoonosis caused by parasites. In this study, 177 fecal samples were collected: 128 from dogs and 49 from cats of both sexes and varied ages. One or more intestinal parasites were observed in 56.2% (72/128) of the dog fecal samples and in 53.0% (26/49) of the cat fecal samples. Parasitic monoinfection was present in 70.8% (51/72) of dog samples and in 46.1% (12/26) of cat samples, whereas multi-infection was observed in 29.2% (21/72) and 53.8% (14/26) of dog and cat samples, respectively. The detection frequency of Cryptosporidium spp. was 22.6% (40/177) using Ziehl-Neelsen staining. DNA was extracted from all samples and the Cryptosporidium small subunit ribosomal ribonucleic acid (SSU rRNA) gene was amplified from 5.6% (10/177) of the fecal samples using nested polymerase chain reaction (PCR). Amplification was achieved in 4.6% (6/128) of the dog samples and in 8.2% (4/49) of the cat samples. DNA sequencing of the nested PCR positive samples identified Cryptosporidium canis in 66.6% (4/6) and Cryptosporidium parvum in 33.3% (2/6) of the dog samples and Cryptosporidium felis in 75% (3/4) and Cryptosporidium parvum in 25% (1/4) in the cat samples. The present study thus demonstrated significant levels of gastrointestinal parasite infection in companion animals and highlighted the presence of zoonosis agents.

Molecular surveillance of Cryptosporidium and Giardia duodenalis in sludge and spent filter backwash water of a water treatment plant

The purpose of this study was to monitor the presence of Cryptosporidium spp. and Giardia duodenalis in a water treatment plant (WTP) using settling sludge and backwash water (BW) samples in previous and post the deflocculation of polyaluminium chloride (PAC) flacks. Eleven collections were performed. BW and settling sludge (SSF) were concentrated by calcium carbonate flocculation, and another aliquot of settling sludge (SSC) by centrifugation. The samples were divided as follows: Group A, containing 33 samples without degradation of PAC flakes, and Group B, with degradation by alkalinization with 10 M NaOH. Sample DNA was extracted with a commercial kit, and nested polymerase chain reaction (PCR) was used to detect Cryptosporidium and G. duodenalis. All samples from Group A were negative for Cryptosporidium spp., and 6.1% (2/33) were positive for G. duodenalis in SSC samples. While the absence of Cryptosporidium may be due to a low contamination level of the water resource, the presence of G. duodenalis indicates contamination of the raw water. The detection of G. duodenalis in SSC samples indicates that this detection method was the most effective. The 33 samples from Group B were negative for both protozoa, probably due to the presence of aluminium and humic substances.