Effect of Soil Temperature on Canine Soil-Transmitted Nematodes in Kandy District with the First Record of Hookworm, Ancylostoma tubaeforme from Sri Lanka.

PURPOSE: Soil-transmitted nematodes (STNs) are widespread in tropical and subtropical regions, particularly where the communities are socio-economically challenged. We investigated the effect of soil temperature on the prevalence and intensity of STN infection in free-roaming dogs. METHODS: Fresh faecal samples collected from free-roaming dogs in Digana and Pussellawa town areas in the Kandy District, Sri Lanka, were microscopically analysed for canine STNs. Soil temperature was measured at each sampling site. Highly prevalent canine hookworm Ancylostoma, was further studied using PCR and sequencing, followed by phylogenetic analysis. RESULTS: The soil temperature ranged between 28 and 31 °C (mean = 29.79 °C) and 18-21 °C (mean = 19.52 °C) in Digana and Pussellawa, respectively, showing a significant difference in the two sites (Students t-test t = 1.68, p < 0.0001). Of the total 44 dogs sampled, 41 (93.2%) were positive for STNs. During microscopic analysis, five nematodes: Ancylostoma spp., Capillaria sp., Strongyloides sp., Toxocara canis, and Trichuris sp., were identified. Ancylostoma species (93.2%) were the most prevalent, followed by Strongyloides sp. (22.7%) and Toxocara canis (15.9%). Infection prevalence of Strongyloides sp. was higher in Digana (40.9%) compared to that in Pussellawa (4.5%; Chi-square test, χ2 = 8.28, p = 0.004) and also the infection intensity from Digana (EPG = 8.02 ± 20.2) compared to that from Pussellawa (0.45 ± 2.1; Mann Whitney U test, p = 0.006). Amplicons (ITS1-5.8S-ITS2) of the expected size for A. caninum, and A. tubaeforme were produced. An A. caninum sequence reported here (OQ101719) illustrated the highest similarity of 99.2% to one of the local sequences (MZ707153) upon pairwise comparison. CONCLUSION: Digana, with a higher soil temperature than Pussellawa, had a significantly higher prevalence and infection intensity, particularly Strongyloides sp. This study also signifies the first molecular identification of hookworm species A. tubaeforme in Sri Lanka.

Association of helminth infestation with childhood asthma: a nested case-control study.

OBJECTIVES: The association between helminthiasis and asthma remains inconclusive but can only be investigated in counties where helminthiasis is transitioning from a high to low burden. We investigated this association using data from a childhood respiratory cohort in Sri Lanka. METHODS: A case-control study was nested within a population-based cohort of children aged 6-14 years in Sri Lanka. The stool samples of 190 children with asthma and 190 children without asthma were analyzed to assess the burden of helminth infestation. Logistic regression models were fitted to investigate the association of gastrointestinal helminth species with asthma. RESULTS: Helminthiasis in children with and without asthma was 23.3% (n = 44) and 15.3% (n = 23), respectively. Those with asthma were more likely to have helminthiasis (odds ratio 3.7; 95% confidence interval 1.7, 7.7; P = 0.001), particularly with Trichiuris trichura (odds ratio 4.5; 95% confidence interval 1.6, 12.3; P = 0.004). Helminth eggs per gram of feces were not associated with asthma (P >0.05). CONCLUSION: Our findings demonstrate a positive association between T. trichura infestation and asthma and point to the need to fully characterize this association to understand the likely immunological mechanism that drives it. This association highlights an important public health intervention in countries where these infestations are still prevalent, affecting 24% of the population worldwide.

Filariasis in dogs brought to the Veterinary Teaching Hospital, University of Peradeniya, Sri Lanka.

Dirofilariasis is the predominant emerging zoonotic filariasis in the world. The two most frequent filarial worms that infect dogs are Dirofilaria repens and Dirofilaria immitis. This study reports filariasis among dogs brought to the Veterinary Teaching Hospital (VTH) at the University of Peradeniya and signifies the first molecular characterization of D. repens, responsible for an emerging zoonotic filarial disease in Sri Lanka. Blood samples were collected and were morphologically analyzed using Modified Knott's Technique, followed by molecular analyses. The difference in filariasis prevalence among gender, breed, and age categories was analyzed using a chi-square test. Infection intensities were analyzed using the Mann-Whitney U test and the Kruskal Wallis test. The dogs were brought to the clinic for either vaccination and/or for a regular checkup, and most were sick having non-specific clinical signs. Among the 87 dogs tested, 27.6% were positive for Dirofilaria. Conventional PCR and bi-directional sequencing of genomic DNA of microscopically tested positive samples revealed that the species in Sri Lanka was D. repens. The infection was significantly higher in males (39.1%) than in females (14.6%; χ2 = 0.447, p = 0.011), though it is not significant between puppies (age < 1 year) and adult dogs. More crossbred dogs were infected compared to older and purebred dogs. There was no difference in intensity of infection based on their gender, age, or breed. Sequences obtained from the current study were unique and were only 63% identical to those of D. repens reported from South India. The high number of Dirofilaria infections in domestic dogs indicates a potential reservoir for emerging human dirofilariasis cases in Sri Lanka. Thus, morphological and molecular diagnosis, along with epidemiological assessment of these zoonoses, is critical for the formulation of effective public health programs and control mechanisms.

First Molecular Identification of Ancylostoma Species in Dogs in a Rural Tea Estate Community in Sri Lanka and the Detection of Other Zoonotic Gastro-intestinal Parasites.

PURPOSE: Ancylostoma spp. causing canine hookworm infections are distributed worldwide with a high prevalence in the South Asian region. Here, we report the first molecular identification of the canine hookworm infecting the dogs in Sri Lanka. METHOD: Fresh faecal samples were collected from free-roaming dogs in tea estate communities in Talawakelle, processed and microscopically analysed for gastro-intestinal parasites. The common dog hookworm Ancylostoma sp. was further investigated using molecular diagnostic tools including PCR, sequencing, and phylogenetic analyses. RESULTS: Of the individual dogs sampled, 97.0% (97/100) were positive for GI parasites, identifying 14 species, all of which were zoonotic. Entamoeba sp. (58.0% (58/100)) was the most prevalent infection and Ancylostoma sp. (45.0% (45/100)) was the most prevalent helminth infection. Overall, helminth prevalence was higher in female dogs (92.5% (37/40)) than male dogs (73.3% (44/60)), potentially due to immune suppression during pregnancy and lactation. The prevalence and burden of Toxocara canis was higher in puppies (≤ 1 year) (68.8%(11/16); Chi-square test; χ2 = 20.912, p < 0.001 and FEC = 5.91; Mann-Whitney U test; p < 0.001), possibly due to vertical trans-mammary or trans-placental transmission of infective larvae. This study also presents the first record of Hymenolepis sp. from dogs in Sri Lanka. Ancylostoma sp. was molecularly confirmed as A. caninum. CONCLUSION: The local variant of A. caninum was phylogenetically unique and showed 99.2% similarity to that from India. The high prevalence of zoonotic GI parasites with public health significance highlights the importance of improving community well-being, veterinary care for dogs, and public awareness on zoonoses.

Gastro-intestinal parasites in two subspecies of toque macaque (Macaca sinica) in Sri Lanka and their zoonotic potential.

Gastro-intestinal (GI) parasites of primates have a greater potential of becoming zoonotic. This potential may vary in different primates based on multiple factors such as proximity to human settlements and the climate of their habitat. We examined the GI parasites in two subspecies of toque macaque: Macaca sinica sinica (confined to the dry zone) and Macaca sinica aurifrons (confined to the wet zone) of Sri Lanka. Fresh faecal samples were collected and analysed following a modified Sheather's sucrose floatation method. A total of 90.8% (89/98) macaques were infected with one or more parasite species. There was no difference in the overall prevalence of GI parasites between the two subspecies, M. s. aurifrons (95.9%) and M. s. sinica (85.7%; χ2;χ = 3.059, p = 0.080). Sixteen parasite species were recorded including, 15 species in the M. s. sinica and 12 species in the M. s. aurifrons. Among the helminths identified, Anatrichosoma sp., Ancylostoma spp., Capillaria spp., Oesophagostomum /Bunostomum spp. and Physaloptera spp. are known to be zoonotic while Ascaris spp., Enterobius sp., Strongyloides spp. and Trichuris spp. have both zoonotic and anthroponotic potential. Among the protozoans, Balantidium coli and Buxtonella sp. are known to be zoonotic, while Entamoeba spp. and Cryptosporidium spp. have both anthroponotic and zoonotic potential. This study provides the first record of Anatrichosoma sp. and Buxtonella sp. in Sri Lanka and the first record of Cryptosporidium spp. in M. s. aurifrons. The molecular data allowed further identification and differentiation of Entamoeba nuttalli and E. coli that are known to be zoonotic and anthroponotic, respectively. The two subspecies of macaques have close interactions with humans; hence, in-depth epidemiological studies are required to understand the potential public-health risks to humans and conservation implications for macaque populations.

An appraisal of oriental theileriosis and the Theileria orientalis complex, with an emphasis on diagnosis and genetic characterisation.

Oriental theileriosis, a tick-borne disease of bovids caused by members of the Theileria orientalis complex, has a worldwide distribution. Globally, at least 11 distinct genotypes of T. orientalis complex, including type 1 (chitose), type 2 (ikeda), type 3 (buffeli), types 4 to 8, and N1-N3, have been described based on the sequence of the major piroplasm surface protein (MPSP) gene. Of these 11 genotypes, mainly ikeda and chitose are known to be pathogenic and cause considerable morbidity (including high fever, anaemia, jaundice and abortion), production losses and/or mortality in cattle. Mixed infections with two or more genotypes of T. orientalis is common, but do not always lead to a clinical disease, posing challenges in the diagnosis of asymptomatic or subclinical forms of oriental theileriosis. The diagnosis of oriental theileriosis is usually based on clinical signs, the detection of piroplasms of T. orientalis in blood smears, and/or the use of serological or molecular techniques. This paper reviews current methods used for the diagnosis of T. orientalis infections and the genetic characterisation of members of the T. orientalis complex, and proposes that advanced genomic tools should be established for investigations of these and related haemoparasites.

Infections by human gastrointestinal helminths are associated with changes in faecal microbiota diversity and composition.

Investigations of the impact that patent infections by soil-transmitted gastrointestinal nematode parasites exert on the composition of the host gut commensal flora are attracting growing interest by the scientific community. However, information collected to date varies across experiments, and further studies are needed to identify consistent relationships between parasites and commensal microbial species. Here, we explore the qualitative and quantitative differences between the microbial community profiles of cohorts of human volunteers from Sri Lanka with patent infection by one or more parasitic nematode species (H+), as well as that of uninfected subjects (H-) and of volunteers who had been subjected to regular prophylactic anthelmintic treatment (Ht). High-throughput sequencing of the bacterial 16S rRNA gene, followed by bioinformatics and biostatistical analyses of sequence data revealed no significant differences in alpha diversity (Shannon) and richness between groups (P = 0.65, P = 0.13 respectively); however, beta diversity was significantly increased in H+ and Ht when individually compared to H-volunteers (P = 0.04). Among others, bacteria of the families Verrucomicrobiaceae and Enterobacteriaceae showed a trend towards increased abundance in H+, whereas the Leuconostocaceae and Bacteroidaceae showed a relative increase in H- and Ht respectively. Our findings add valuable knowledge to the vast, and yet little explored, research field of parasite-microbiota interactions and will provide a basis for the elucidation of the role such interactions play in pathogenic and immune-modulatory properties of parasitic nematodes in both human and animal hosts.

Investigating the first outbreak of oriental theileriosis in cattle in South Australia using multiplexed tandem PCR (MT-PCR).

This study investigated the first outbreak of oriental theileriosis in a herd of beef cattle in South Australia using a newly established multiplexed tandem PCR (MT-PCR) to identify, differentiate and quantitate the four genotypes (buffeli, chitose, ikeda and type 5) of Theileria orientalis recognised to occur in Australasia. Following clinical diagnosis of oriental theileriosis (based on clinical signs, laboratory findings and post mortem examination), 155 blood samples were collected from individual cows (n = 85) and calves (n = 70), and tested by MT-PCR. In total, 117 (75.48%) cattle were shown to be test-positive for T. orientalis. All four genotypes were detected, and ikeda had the highest prevalence (90.6%; 106/117), followed by buffeli (83.8%; 98/117), chitose (18.8%; 22/117) and type 5 (5.1%; 6/117). Mixed infections with genotypes buffeli and ikeda had a higher prevalence (55.5%; 65/117) than any other combination of genotypes. The prevalences of buffeli and ikeda were significantly higher (P<0.005) than those of chitose and type 5. The average intensity of infection with genotype ikeda (329,775 DNA copies) was significantly higher (P<0.0001) than buffeli (212,843) and chitose (125,462). This study reinforces the utility of MT-PCR as a diagnostic tool for rapidly investigating oriental theileriosis outbreaks in cattle herds and as a pre-movement screening test for preventing the introduction of this disease into non-endemic regions.

Comparison of the performance of three PCR assays for the detection and differentiation of Theileria orientalis genotypes.

BACKGROUND: Oriental theileriosis is a tick-borne disease of bovines caused by the members of the Theileria orientalis complex. Recently, we developed a multiplexed tandem (MT) PCR to detect, differentiate and quantitate four genotypes (i.e., buffeli, chitose, ikeda and type 5) of T. orientalis. In this study, we used MT PCR to assess the prevalence and infection intensity of four T. orientalis genotypes in selected cattle herds that experienced oriental theileriosis outbreaks in New Zealand, and compared the sensitivities and specificities of MT PCR, PCR-high resolution melting (PCR-HRM) and a TaqMan qPCR. METHODS: MT PCR, PCR-HRM analysis for T. orientalis and a TaqMan qPCR assay for ikeda genotype were employed to test 154 and 88 cattle blood samples from North (where oriental theileriosis outbreaks had occurred; designated as Group 1) and South (where no outbreaks had been reported; Group 2) Islands of New Zealand, respectively. Quantitative data from MT PCR assay were analyzed using generalized linear model and paired-sample t-test. The diagnostic specificity and sensitivity of the assays were estimated using a Bayesian latent class modeling approach. RESULTS: In Group 1, 99.4% (153/154) of cattle were test-positive for T. orientalis in both the MT PCR and PCR-HRM assays. The apparent prevalences of genotype ikeda in Group 1 were 87.6% (134/153) and 87.7% (135/154) using the MT PCR and Ikeda TaqMan qPCR assays, respectively. Using the MT PCR test, all four genotypes of T. orientalis were detected. The infection intensity estimated for genotype ikeda was significantly higher (P = 0.009) in severely anaemic cattle than in those without anaemia, and this intensity was significantly higher than that of buffeli (P < 0.001) in the former cattle. Bayesian latent class analysis showed that the diagnostic sensitivities (97.1-98.9%) and specificities (96.5-98.9%) of the three PCR assays were very comparable. CONCLUSION: The present findings show the advantages of using the MT PCR assay as a useful tool for in-depth epidemiological and transmission studies of T. orientalis worldwide.

Use of multiplexed tandem PCR to estimate the prevalence and intensity of Theileria orientalis infections in cattle.

This study employed a semi-quantitative, multiplexed tandem PCR (MT-PCR) to assess the prevalence and infection intensity of four genotypes (buffeli, chitose, ikeda and type 5) of Theileria orientalis in cattle in Australia. Genomic DNA samples from blood samples (n=448) collected from 27 to 32 dairy cows from each of 15 dairy herds with a history of recent theileriosis outbreaks (Group 1), and from blood samples available from 24 cows with or without oriental theileriosis (Group 2) were tested using MT-PCR. Results revealed that all four genotypes were present in Group 1 cattle; genotype buffeli had the highest prevalence (80.5%), followed by genotypes ikeda (71.4%), chitose (38.6%) and type 5 (20.3%). Genotype ikeda had the highest average infection intensity in the cattle (relating to 55,277 DNA copies), followed by buffeli, chitose and type 5 (6354-51,648 copies). For Group 2, results indicated that genotype ikeda had a significantly higher average intensity of infection than buffeli in symptomatic cattle (P<0.001), and symptomatic cattle had a higher intensity of ikeda than asymptomatic cattle (P=0.004). Future studies should assess the utility of the present MT-PCR assay as a diagnostic and epidemiological tool in other parts of Australasia and the world.

Semiquantitative multiplexed tandem PCR for detection and differentiation of four Theileria orientalis genotypes in cattle.

Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region.

Assessment of sequence variability in a p23 gene region within and among three genotypes of the Theileria orientalis complex from south-eastern Australia.

Oriental theileriosis is a tick-borne, protozoan disease of cattle caused by one or more genotypes of Theileria orientalis complex. In this study, we assessed sequence variability in a region of the 23kDa piroplasm membrane protein (p23) gene within and among three T. orientalis genotypes (designated buffeli, chitose and ikeda) in south-eastern Australia. Genomic DNA (n=100) was extracted from blood of infected cattle from various locations endemic for oriental theileriosis and tested by polymerase chain reaction (PCR)-coupled mutation scanning (single-strand conformation polymorphism (SSCP)) and targeted sequencing analysis. Eight distinct sequences represented all DNA samples, and three genotypes were found: buffeli (n=3), chitose (3) and ikeda (2). Nucleotide pairwise comparisons among these eight sequences revealed considerably higher variability among the genotypes (6.6-11.7%) than within them (0-1.9%), indicating that the p23 gene region allows the accurate identification of T. orientalis genotypes. In the future, we will combine this gene with other molecular markers to study the genetic structure of T. orientalis populations in Australasia, which will pave the way to establish a highly sensitive and specific PCR-based assay for genotypic diagnosis of infection and for assessing levels of parasitaemia in cattle.

Oriental theileriosis in dairy cows causes a significant milk production loss.

BACKGROUND: Oriental theileriosis is a tick-borne, protozoan disease of cattle caused by members of the Theileria orientalis-complex. Recent outbreaks of this disease in eastern Australia have caused major concerns to the dairy and beef farming communities, but there are no published studies of the economic impact of this disease. On a farm in Victoria, Australia, we assessed whether oriental theileriosis has an impact on milk production and reproductive performance in dairy cows. METHODS: Blood samples collected from all 662 cows on the farm were tested using an established molecular test. For individual cows, milk production and reproductive performance data were collected. A clinical assessment of individual cows was performed. Based on clinical findings and molecular test results, the following groups of cows were classified: group 1, with cardinal clinical signs of oriental theileriosis and molecular test-positive for T. orientalis; group 2, with mild or suspected signs of theileriosis and test-positive; group 3, with no clinical signs and test-positive; and group 4, with no clinical signs and test-negative. Milk production and reproductive performance data for groups 1, 2 and 3 were each compared with those for group 4 using linear and logistic regression analyses, respectively. RESULTS: At 100 days of lactation, group 1 cows produced significantly less milk (288 l; P = 0.001), milk fat (16.8 kg; P < 0.001) and milk protein (12.6 kg; P < 0.001) compared with group 4. At this lactation point, group 2 also produced significantly less milk fat (13.6 kg; P = 0.002) and milk protein (8.6 kg; P = 0.005) than group 4. At 305 days of lactation, group 1 cows produced significantly less milk (624 l; P = 0.004), milk fat (42.9 kg; P < 0.001) and milk protein (26.0 kg; P < 0.001) compared with group 4 cows. Group 2 cows also produced significantly less milk fat (21.2 kg; P = 0.033) at this lactation point. No statistically significant difference in reproductive performance was found upon pairwise comparisons of groups 1-3 with group 4 cows. CONCLUSIONS: The present findings demonstrate that clinical oriental theileriosis can cause significant milk production losses in dairy cattle.

Epidemiological survey following oriental theileriosis outbreaks in Victoria, Australia, on selected cattle farms.

This study investigated Theileria orientalis following outbreaks of oriental theileriosis in cattle in the state of Victoria, Australia, from September 2010 to January 2012, using traditional and molecular methods of diagnosis. A questionnaire was used to collect epidemiological information from cattle farms. Blood samples (n=301), collected from individual symptomatic and asymptomatic cattle from 19 cattle farms, were examined for the presence of Theileria on stained blood smears and tested using a PCR-based approach, employing a region within the major piroplasm surface protein (MPSP) gene as a marker. The microscopic examination of stained blood smears detected stages consistent with Theileria piroplasms in 28.1% (79/281) of the samples. PCR products were amplified from 70.8% (213/301) of the samples. Mutation scanning analysis of all amplicons displayed seven distinct profiles. Following the direct sequencing of representative amplicons, the genotypes ikeda, chitose, buffeli and type 5 were detected in 91.1%, 32.9%, 2.4% and 1.4% of 213 blood samples, respectively. The distribution of these four genotypes varied among the 19 farms; genotype ikeda was detected on all farms, whereas genotypes chitose, buffeli and type 5 were detected on 14, 3 and 2 farms, respectively. Mix infections with genotypes ikeda and chitose were common (21.6%). Survey results revealed that oriental theileriosis affected mainly beef cows of more than two years of age, prior to calving, and disease was associated with abortion and cow deaths. Future investigations should focus on developing improved tools for investigating and managing oriental theileriosis.