ASCARIS SUUM EGG RECOVERY FROM SLUDGE SAMPLES AFTER PHASE EXTRACTION.

Some helminth test methods for sanitation samples include a phase extraction step to reduce lipid content and final pellet size before microscopy. Hydrophilic and lipophilic solutions are used to create 2 phases, with a plug of organic material or debris in between, whilst eggs are supposedly compacted at the bottom of the test tube. We tested 10% formalin, acetoacetic buffer, and acid alcohol as the hydrophilic solutions, and ethyl acetate and diethyl ether as the lipophilic solvents for egg recoverability from water, primary sludge, and fatty sludge. Normally, the supernatant and debris plug are discarded and the sedimented pellet of eggs is microscopically examined. We, however, also collected the entire supernatant plus debris plug to determine where eggs were possibly lost. We found that eggs were lost when samples were extracted with 10% formalin + ethyl acetate, 10% formalin + diethyl ether, acetoacetic buffer + ethyl acetate, and acetoacetic buffer + diethyl ether combinations (<50% egg recovery). Acid alcohol + ethyl acetate resulted in 93.2, 89.8, and 57.3% egg recovery in the pellet of water, primary sludge, and fatty sludge, respectively; however, the size of the final pellet was not reduced, defeating the purpose of the extraction step. We thus recommend that this step be excluded.

A case of asymptomatic infection of Ascaris suum identified by PCR-restriction fragment length polymorphism and DNA sequence analysis.

Human ascariasis is a soil-transmitted helminthiasis and remains a neglected tropical disease. Ascaris suum has the potential to cause cross-infections between humans and pigs. In this study, we present a rare case of a patient with asymptomatic infection by Ascaris suum. A 66-year-old male underwent colonoscopy, and a white linear worm body was found in the hepatic curvature. The worm was collected by aspiration and submitted to the laboratory for parasite identification. The patient had no symptoms related to parasitic infection. The worm was highly suspected to be of the genus Ascaris. Because of the difficulty of morphological classification, genetic analysis was performed. From PCR-restriction fragment length polymorphism results and sequence analysis of the internal transcribed spacer-1 region, it was determined to be A. suum. The experience with rapid differentiation of A. suum by performing genetic analysis will be useful for future examinations of parasitic infections.

Bayesian estimation of the sensitivity and specificity of coprological and serological diagnostic tests for the detection of Ascaris suum infection on pig farms.

Coprological and serological diagnostic tests were compared to define the status of a pig farm with regard to Ascaris suum. On each of the 100 farms in France visited for the study, 10 blood samples were taken from pigs at the end of fattening (at least 22 weeks old) and 20 to 30 faecal samples were taken, depending on the category of animals present on the farm (10 sows, 10 piglets aged 10 to 12 weeks and 10 pigs at the end of fattening, aged at least 22 weeks). A SERASCA® ELISA test (Laboratory of Parasitology, Ghent University) was performed on each blood sample (cut-off 0.5) and a coprological analysis on each faecal sample. A Bayesian approach was used to estimate the sensitivity and specificity of the coprological and serological tests. A farm was considered positive if at least one A. suum egg was observed in the faecal samples. With regard to the serological test, various hypotheses were tested in order to define the number of seropositive animals required to consider a farm positive for A. suum. The coprological test has very good specificity in the search for A. suum, whether 20 or 30 samples are taken per farm. However, even with an increase in the number of samples, the sensitivity of this diagnostic approach is very low (less than 30%). On the other hand, the serological diagnostic method, which consists of taking blood samples from 10 animals at the end of fattening, has good sensitivity and seems better suited to defining the status of a farm with regard to A. suum, provided that a farm is considered seropositive only if two out of 10 samples are positive.

Hepatobiliary Ascariasis in a Piglet.

PURPOSE: Ascariasis caused by the helminth Ascaris suum is the most common parasitosis of swine worldwide and it may involve all age categories of pigs. The present study reports an unusual localization of A. suum worms in the biliary system of a piglet slaughtered for human consumption. METHODS: The liver was subjected to ultrasound scan and pathological examination. The isolated worms were morphologically examined and the DNA was extracted for the molecular identification of the species involved. RESULTS: A total of 43 preadult nematodes were found within the gallbladder and the bile ducts. Parasites were morphologically identified as belonging to the genus Ascaris and molecularly as A. suum. At gross examination, the liver was moderately enlarged, with the bile ducts severely dilated. A chronic inflammatory infiltrate was noted, often centered around ectatic bile ducts (up to 5 mm in diameter), lined by hyperplastic epithelium and filled with sections of nematodes. The worm sections showed smooth cuticle, coelomyarian musculature, and an intestinal tract lined by columnar, uninucleated cells within a pseudocoelom. The ex vivo ultrasonographic examination of the liver allowed the visualization of several nematodes in the bile duct lumen and could be suggested for in vivo diagnosis. Unfortunately, the absence of the intestine did not allow to define the pathogenesis of the infection. CONCLUSION: Although, given the unusual nature of this finding, it is difficult to identify predisposing factors for this A. suum localization, it suggests that ascariasis should be considered in the differential diagnosis of pigs with hepatobiliary disease.

ASCVac-1, a Multi-Peptide Chimeric Vaccine, Protects Mice Against Ascaris suum Infection.

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

A two-step morphology-PCR strategy for the identification of nematode larvae recovered from muscles after artificial digestion at meat inspection.

To ensure that meat from livestock and game is safe for human consumption, European legislation lays down rules for mandatory testing. Helminth larvae are a category of zoonotic foodborne pathogens that can contaminate meat. Among helminths, the only zoonotic nematode regulated in Europe regarding meat inspection is Trichinella spp.. It is precisely during Trichinella testing that other potentially zoonotic larvae can be found. Due to current lack of tools, their identification is often very complicated. Nematode larvae other than Trichinella, recovered from artificial digestions of pig and wild boar muscles from France and Germany, were subjected to a newly developed two-step identification scheme, which includes both morphological examination and molecular assays. The first step is a general orientation towards a broad taxonomic group; the second step consists of targeted identification based on the results of first step. Different parasites were identified, some of which were not zoonotic such as Metastrongylus spp. and Angiostrongylus vasorum, but others are known to be zoonotic such as Toxocara cati, Ascaris suum, and Uncinaria stenocephala. The strategy is efficient for the identification of nematode larvae recovered from muscles but could also be applied for larvae from other sources.

Evaluation of copromicroscopy and serology to measure the exposure to Ascaris infections across age groups and to assess the impact of 3 years of biannual mass drug administration in Jimma Town, Ethiopia.

BACKGROUND: The scientific community has recently summarized the desired characteristics for diagnostic tools across the different phases of a soil-transmitted helminth (STH) mass drug administration (MDA) program. Although serology meets some of the desired criteria, there is a scarcity of data on baseline serological profiles in human populations, both prior to and during MDA programs. METHODS: In this study, we compared the copromicroscopic and the serological infection profiles in 600 school-aged children (SAC) and 600 adults at the advent of the MDA program in Jimma Town, Ethiopia. The serological profiles were examined by two ELISAs that measure IgG4 responses to the Ascaris suum haemoglobin antigen (AsHb) and a somatic extract of lung stage larvae (AsLungL3). Three years into the MDA program, we sampled another group of 600 SAC from the same schools to assess the reduction in prevalence and intensity of Ascaris infections measured by copromicroscopy and serology. PRINCIPAL FINDINGS: Prior to the start of MDA, copromicroscopy revealed an Ascaris prevalence of 31.0% and a mean fecal egg count of 2,919 eggs per gram (EPG) in SAC. Following three years of biannual treatment, the prevalence reduced to 13.2% (57.8% reduction) and the mean fecal egg count to 1,513 EPG (48.1% reduction). This reduction was also reflected in the serological results. The seroprevalence reduced with 40.9% and 27.4% and the mean optical density ratio reduced with 44.2% and 38.2% as measured by the AsHb or AsLungL3 ELISA respectively. We also showed that, despite a decreasing coproprevalence, seroprevalence to Ascaris increased with age. CONCLUSIONS: This study is the first to provide IgG4 response profiles of an endemic population to two different A. suum antigens. The results suggest that exposure to the infectious stages of Ascaris reaches beyond SAC alone. Furthermore, it highlights the possible use of serological assays to monitor changes in STH exposure during MDA programs.

High anti-Ascaris seroprevalence in fattening pigs in Sichuan, China, calls for improved management strategies.

BACKGROUND: Ascariasis, caused by Ascaris suum, is an important soil-transmitted parasitic disease of pigs worldwide. It leads to significant economic losses in the pork industry, as a consequence of low feed conversion efficiency in pigs and liver condemnation at slaughter. Despite ascariasis still being widespread on pig farms in many developing and the industrialised countries, there are surprisingly limited data on porcine ascariasis in China, where nearly half of the world's total pork is produced. METHODS: In the present study, using the recently developed A. suum-haemoglobin (As-Hb) antigen-based serological test, we screened 512 individual serum samples from fattening pigs from 13 farms across seven distinct locations of Sichuan Province in China for anti-Ascaris antibody. RESULTS: The prevalence of anti-Ascaris antibody ranged from 0% to 100% on the distinct farms, with the mean (overall) seroprevalence being > 60%. There was no significant difference in seroprevalence between the intensive and extensive farms. CONCLUSIONS: To our knowledge, this is the first study to measure anti-Ascaris seroprevalence in China. The results of this 'snapshot' investigation indicate that Ascaris infection in commercial pig farms in Sichuan Province is seriously underestimated, encouraging future, large-scale serological studies to assess the distribution and extent of Ascaris exposure and infection in various regions of China and the world.

Establishment of a serodiagnosis system for the detection of Toxocara spp. and Ascaris suum infection in chickens.

Chickens are considered to act as paratenic hosts for agents, Toxocara canis, T. cati and Ascaris suum; which cause ascarid larva migrans syndrome (ascarid LMS) in humans. In addition, they are the definitive host for Ascaridia galli, considered not to be infective for humans. All ascarid parasites can have a high homology of antigenicity, leading to cross-reactivity in serodiagnostic assays. This study was conducted to establish a procedure for the serological detection of those roundworm infections in chickens. Twenty-five male Julia chickens were divided into five groups (n = 5); T. canis-, T. cati-, Ascaris suum- and Ascaridia galli-infected, and an uninfected control group. In Ascaris suum-soluble worm antigen preparation (As-SWAP) ELISA, all infected groups showed an elevation of anti-ascarid antibodies, indicating the usefulness of As-SWAP as a screening antigen for the detection of ascarid infections. For infecting species identification, T. canis-excretory/secretory (Tc-ES) and Ascaris suum-ES (As-ES) antigen ELISA were conducted by serial dilution sera. Toxocara spp.-infected sera showed stronger binding to Tc-ES than As-ES, while Ascaris suum and Ascaridia galli-infected sera bound to As-ES more strongly than Tc-ES. To discriminate between Ascaris suum and Ascaridia galli infection, sera were pre-incubated with Ascaridia galli-SWAP antigen and applied to Tc-ES and As-ES ELISAs. In this pre-adsorbed ES antigen ELISAs, only the Ascaris suum infected group showed positive binding to As-ES, resulting from the adsorption of cross-reactive antibodies in Ascaridia galli-infected sera. Finally, anti-Toxocara specific antibodies were confirmed by Tc-ES western blot (WB). Toxocara spp.-infected sera showed toxocariasis-specific band pattern in Tc-ES WB, while no specific band appeared on any strip incubated with Ascaris suum, Ascaridia galli-infected and uninfected sera. In conclusion, the serodiagnostic assays evaluated in this study are useful for the detection of ascarid infections in chickens.

Effect of endoparasites occurrence in sows from intensive production system.

The goal of this study was to assess the effect of farm size (FS) and farrowing order (FO) on the occurrence of endoparasites eggs in commercial sows housed in maternity and gestation areas during the period from May to July 2014. Forty-three piglet production units were classified by FS: small (100 to 250 sows), medium (251 to 510 sows), large (511 to 1,000 sows) and very large (more than 1,000 sows). Sows were classified by FO: up to two, three to five or more than five parturitions. Faecal samples were processed using the simple flotation technique in a hypersaturated salt solution (30-35% NaCl). The results revealed that the overall prevalence of gastrointestinal endoparasites obtained in this study was 12.47%, in that 4.64% were positive for Ascaris suum, 0.56% for Trichuris suis and 8.27% for coccidia oocysts. The prevalence of endoparasites obtained for small and medium size farm, and for large and very large farm was 34.58% and 15.52%, respectively. In conclusion, the study shows that more than half of the farms were positive for A. suum and coccidia oocysts, but mainly for younger females. In general, sows with up to two parturitions and small farms showed a higher endoparasites percentage.

Effect of endoparasites occurrence in sows from intensive production system / Efeito da ocorrência de endoparasitas em matrizes suínas de sistema de produção intensivo

Abstract The goal of this study was to assess the effect of farm size (FS) and farrowing order (FO) on the occurrence of endoparasites eggs in commercial sows housed in maternity and gestation areas during the period from May to July 2014. Forty-three piglet production units were classified by FS: small (100 to 250 sows), medium (251 to 510 sows), large (511 to 1,000 sows) and very large (more than 1,000 sows). Sows were classified by FO: up to two, three to five or more than five parturitions. Faecal samples were processed using the simple flotation technique in a hypersaturated salt solution (30-35% NaCl). The results revealed that the overall prevalence of gastrointestinal endoparasites obtained in this study was 12.47%, in that 4.64% were positive for Ascaris suum, 0.56% for Trichuris suis and 8.27% for coccidia oocysts. The prevalence of endoparasites obtained for small and medium size farm, and for large and very large farm was 34.58% and 15.52%, respectively. In conclusion, the study shows that more than half of the farms were positive for A. suum and coccidia oocysts, but mainly for younger females. In general, sows with up to two parturitions and small farms showed a higher endoparasites percentage.

Resumo O objetivo deste estudo foi o de avaliar o efeito de tamanho de granja (TG) e a ordem de parição (OP) sobre a ocorrência de ovos de endoparasitas em matrizes suínas comerciais alojadas na maternidade e gestação durante o período de maio a julho de 2014. Quarenta e três unidades produtoras de leitões foram classificadas por TG: pequena (100 a 250 porcas), média (251 a 510 porcas), grande (511 a 1.000 porcas) e muito grande (mais de 1.000 porcas). As porcas foram classificadas por OP: até dois, três a cinco e mais que cinco partos. As amostras fecais foram processadas usando a técnica de flotação em solução salina hipersaturada a 30-35%. Os resultados revelaram que a prevalência global de endoparasitas gastrointestinais obtidos neste estudo foi de 13,59%, em que 4,64% foram positivas para Ascaris suum, 0,56% para Trichuris suis e 8,27% para oocistos de coccídeos. A prevalência de endoparasitas obtidos para fazendas de pequeno e médio porte, e para fazendas grandes e muito grandes foi de 34,58% e 15,52%, respectivamente. Em conclusão, o estudo mostra que mais da metade das fazendas foram positivas para A. suum e oocistos de coccídeos, mas principalmente para as fêmeas mais jovens. Em geral, as porcas com até dois partos e pequenas propriedades mostraram uma porcentagem maior de endoparasitas.

Comorbidity associated to Ascaris suum infection during pulmonary fibrosis exacerbates chronic lung and liver inflammation and dysfunction but not affect the parasite cycle in mice.

Ascariasis is considered the most neglected tropical disease, and is a major problem for the public health system. However, idiopathic pulmonary fibrosis (IPF) is a result of chronic extracellular deposition of matrix in the pulmonary parenchyma, and thickening of the alveolar septa, which reduces alveolar gas exchange. Considering the high rates of ascariasis and pulmonary fibrosis, we believe that these two diseases may co-exist and possibly lead to comorbidities. We therefore investigated the mechanisms involved in comorbidity of Ascaris suum (A. suum) infection, which could interfere with the progression of pulmonary fibrosis. In addition, we evaluated whether a previous lung fibrosis could interfere with the pulmonary cycle of A. suum in mice. The most important findings related to comorbidity in which A. suum infection exacerbated pulmonary and liver injury, inflammation and dysfunction, but did not promote excessive fibrosis in mice during the investigated comorbidity period. Interestingly, we found that pulmonary fibrosis did not alter the parasite cycle that transmigrated preferentially through preserved but not fibrotic areas of the lungs. Collectively, our results demonstrate that A. suum infection leads to comorbidity, and contributes to the aggravation of pulmonary dysfunction during pulmonary fibrosis, which also leads to significant liver injury and inflammation, without changing the A. suum cycle in the lungs.

Ascaris suum infection was associated with a worm-independent reduction in microbial diversity and altered metabolic potential in the porcine gut microbiome.

The effect of infection of pigs with Ascaris suum on the microbial composition in the proximal colon and fecal matter was investigated using 16S rRNA gene sequencing. The infection significantly decreased various microbial diversity indices including Chao1 richness, but the effect on Chao1 in the colon luminal contents was worm burden-independent. The abundance of 49 genera present in colon contents, such as Prevotella and Faecalibacterium, and 179 operational taxonomic units was significantly changed as a result of infection. Notably, infection was also associated with a significant shift in the metabolic potential of the proximal colon microbiome, where the relative abundance of at least 30 metabolic pathways including carbohydrate metabolism and amino acid metabolism was reduced, while the abundance of 28 pathways was increased by infection. Furthermore, the microbial co-occurrence network in infected pigs was highly modular. Two of 52 modules or subnetworks were negatively correlated with fecal butyrate concentrations (r < -0.7; P < 0.05) while one module with 18 members was negatively correlated with fecal acetate, propionate and total short-chain fatty acids. A partial Mantel test identified a strong positive correlation between node connectivity of the operational taxonomic units assigned to ß-Proteobacteria (especially the family Alcaligenaceae) and fecal acetate and propionate levels (r = 0.82 and 0.74, respectively), while that of the family Porphyromonadaceae was positively correlated with fecal egg counts. Overall, Ascaris infection was associated with a profound change in the gut microbiome, especially in the proximity of the initial site of larval infection, and should facilitate our understanding of the pathophysiological consequence of gastrointestinal nematode infections.

Molecular analysis of human- and pig-derived Ascaris in Honduras.

Ascaris sp. is a soil-transmitted helminth (STH) significantly affecting the health of human and swine populations. Health inequities and poverty, with resulting deficiencies in water, sanitation and hygiene, are directly associated with Ascaris lumbricoides prevalence in humans. Resource constraints also lead to small-scale livestock production under unsanitary conditions. Free-ranging pigs, for instance, are exposed to a number of infectious agents, among which Ascaris suum is one of the most common. Under these conditions, close proximity between people and pigs can result in cross-contamination; that is, pigs harbouring human Ascaris and vice versa. Moreover, the potential interbreeding between these two Ascaris species has been demonstrated. The present study analysed Ascaris worms obtained from children and pigs in Honduras. Adult worms were collected from stool samples of children after pharmacological treatment, and from pigs' intestines after slaughter for commercial purposes at a local abattoir. A nuclear ribosomal internal transcribed spacer (ITS) region was amplified by polymerase chain reaction (PCR) and digested with a restriction enzyme in order to separate putative human- and pig-derived Ascaris isolates. PCR products were also sequenced, and cladograms were constructed. All parasites isolated from children showed the typical human-derived genotype of Ascaris, whereas 91% of parasites from pigs showed the expected pig-derived genotype. Cross-infections between hosts were not demonstrated in this study. Nine per cent of pig-derived worms showed a restriction band pattern highly suggestive of a hybrid human-pig Ascaris genotype. These results contribute to the understanding of ascariasis epidemiology and its zoonotic potential in a highly endemic region.

Molecular Confirmation of Ascaris suum: Further Investigation into the Zoonotic Origin of Infection in an 8-Year-Old Boy with Loeffler Syndrome.

An 8-year-old male from south Louisiana was diagnosed with Loeffler syndrome of suspected Ascaris origin. Further investigation of the farm recovered larvated, infective Ascaris eggs from the soil in drains surrounding pens on the family's small hog farm. Molecular analysis of the recovered eggs, in conjunction with Ascaris-specific IgE, inadequate farm management and sanitation, and behavioral risk factors indicate the patient had an Ascaris suum soil-transmitted infection.

Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats.

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100 pg to 100 ag and found to be 10 fg, 1 fg and 100 fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.

Molecular identification of Ascaris lumbricoides and Ascaris suum recovered from humans and pigs in Thailand, Lao PDR, and Myanmar.

Ascaris lumbricoides is the largest roundworm known from the human intestine while Ascaris suum is an internal parasite of pigs. Ascariasis, caused by Ascaris lumbricoides, has a worldwide distribution. Here, we have provided the first molecular identification of Ascaris eggs and adults recovered from humans and pigs in Thailand, Lao PDR, and Myanmar. We amplified and sequenced nuclear ribosomal DNA (ITS1 and ITS2 regions) and mitochondrial DNA (cox1 gene). Sequence chromatograms of PCR-amplified ITS1 region revealed a probable hybrid genotype from two human ascariasis cases from Chiang Mai Province, northern Thailand. All complete ITS2 sequences were identical and did not differ between the species. Phylogenetic trees and haplotype analysis of cox1 sequences showed three clusters with 99 haplotypes. Forty-seven samples from the present study represented 14 haplotypes, including 7 new haplotypes. To our knowledge, this is the first molecular confirmation of Ascaris species in Thailand, Lao PDR, and Myanmar. Zoonotic cross-transmission of Ascaris roundworm between pigs and humans probably occurs in these countries.

Devitalisation of pathogens in stored pig slurry and potential risk related to its application to agricultural soil.

The study investigated the risks arising from application of pig slurry to soil in relation to viability of Salmonella typhimurium, Escherichia coli, total coliforms, faecal enterococci and eggs of Ascaris suum at different temperatures. Potential effect of changes in physico-chemical parameters, particularly dry matter (DM), pH and ammonia, were also investigated. Examination showed that S. typhimurium was devitalised after storage in the slurry for 115 days at 4 °C and after 90 days at 20 and 42 °C. Devitalization of E. coli and faecal entrerococci required more than 115 at temperature of 4 °C and faecal enterococci were recovered from slurry after 115 days of storage even at temperature of 20 °C. Total coliforms survived for 115 days at all investigated temperatures. Complete devitalization of A. suum eggs was not achieved even after 115 days at 42 °C. Our investigations indicated potential microbial and parasitic risk related to application of pig slurry to soil even after 115 days of storage.

Analysis of the internal transcribed spacer region of Ascaris suum and Ascaris lumbricoides derived from free range Tibetan pigs.

Ascaris suum (A. suum) is the most commonly occurring worldwide internal parasite of pigs; however, little is known about this organism in Tibetan pigs in China. A study was carried to isolate and identify the characteristics of internal transcribed spacer region (ITS) gene of A. suum derived from Tibetan pigs. Adult nematodes were collected from Tibetan pigs in 2015-2016. Total genomic DNA of the extracted parasites was performed and a fragment of the ITS of mitochondrial (mt) gene was amplified. The amplicons were cloned into PGEM®-T Easy Vector (Promega, WI) and the positive clones were sequenced by ABI 3730 × 1 sequencer. The sequence and phylogenetic analysis were performed by ClustalWVer. 1.4 and MEGA 6.0 software, respectively. Results indicated that the identity of A. suum isolates was 98.4%-99.9% with previously reported pig isolates, and 99.4%-99.7% with A. lumbricoides isolates. To the best of our knowledge, this is the first report describing the characteristics of ITS gene of A. suum derived from the Tibetan pigs from high and remote areas depicting high identity with the isolates of both A. suum and A. lumbricoides.

Effect of reagents used during detection and quantification of Ascaris suum in environmental samples on egg viability.

Soil-transmitted helminths (STHs) are a major health concern globally. Infection is mostly through contact with contaminated water, food or soil. Therefore to break the cycle of viable transmission STH eggs must be quantitatively detected in the environment. The effect of different reagents on the viability of Ascaris suum eggs during laboratory detection and quantification was assessed and different incubation solutions compared. Sulphuric acid gave a slightly higher recovery percentage of viable eggs (91.2%) than distilled water (90.0%) and 0.5% formalin (87.6%), although the difference was not statistically significant (p > 0.05). Acetoacetic acid, ethyl acetate, ammonium bicarbonate, zinc sulphate, magnesium sulphate and Tween 80, are reagents widely used in test protocols for the detection and quantification of STH eggs. Eggs were exposed to these reagents for different time durations. Acetoacetic acid resulted in the highest loss of viability (3.4 ± 0.7% viable), while magnesium sulphate resulted in the least effect (88.5 ± 1.2% viable). In conclusion the use of the selected reagents in the detection of these eggs was found to affect the viability of exposed eggs, especially during prolonged exposures. Therefore we recommended that eggs be exposed for ≤5 minutes, to reduce the risk of viability loss.