Fecal Polymerase Chain Reaction Research Articles

Environmental distribution of Echinococcus- and Taenia spp.-contaminated dog feces in Kyrgyzstan.

Recently, there have been epidemics of human cystic echinococcosis (CE) and alveolar echinococcosis (AE) in Kyrgyzstan. This study investigated 2 districts for the presence of Echinococcus granulosus s.l. and Echinococcus multilocularis eggs; species identity was confirmed by polymerase chain reaction in dog feces and the level of environmental contamination with parasite eggs in 2017–2018 was also investigated. In the Alay district 5 villages with a high reported annual incidence of AE of 162 cases per 100 000 and 5 villages in the Kochkor district which had a much lower incidence of 21 cases per 100 000 were investigated. However, the proportion of dog feces containing E. granulosus s.l. eggs was ~4.2 and ~3.5% in Alay and Kochkor respectively. For E. multilocularis, the corresponding proportions were 2.8 and 3.2%. Environmental contamination of Echinococcus spp. eggs was estimated using the McMaster technique for fecal egg counts, weight and density of canine feces. The level of environmental contamination with E. multilocularis eggs was similar at 4.4 and 5.0 eggs per m2 in Alay and Kochkor respectively. The corresponding values for E. granulosus s.l. were 8.3 and 7.5 eggs per m2. There was no association between village or district level incidence of human AE or CE and the proportion of dog feces containing eggs of Echinococcus spp. or the level of environmental contamination. Increased contamination of taeniid eggs occured in the autumn, after the return of farmers with dogs from summer mountain pastures.

Impact of customized electronic duplicate order alerts on microbiology test ordering: Financial and environmental cost savings.

To estimate cost savings after implementation of customized electronic duplicate order alerts. Alerts were implemented for microbiology tests at the largest public hospital in Victoria, Australia. These alerts were designed to pop up at the point of test ordering to inform the clinician that the test had previously been ordered and to suggest appropriate reordering time frames and indications. In a 6-month audit of urine culture (our most commonly ordered test) after alert implementation, 2,904 duplicate requesters proceeded with the request and 2,549 tests were cancelled, for a 47% reduction in test ordering. For fecal polymerase chain reaction (PCR), our second most common test, there was a 54% reduction in test ordering. For our most commonly ordered expensive test, hepatitis C PCR, there was a 42% reduction in test ordering: 25 tests were cancelled.Cancelled tests resulted in estimated savings of AU$52,382 (US$33,960) for urine culture, AU$34,914 (US$22,442) for fecal PCR, AU$4,506 (US$2,896) for hepatitis C PCR. For cancelled hepatitis B PCR and Epstein-Barr virus (EBV) and cytomegalovirus (CMV) serology, the cost savings was AU$8,472 (US$5445). The estimated financial cost saving in direct hospital costs for these 6 assays was AU$100,274 (US$67,925) over the 6-month period. Environmental waste cost saving by weight was estimated to be 280 kg. Greenhouse gas footprint, measured in carbon dioxide equivalent emissions for cancelled EBV and CMV serology tests, resulted in a saving of at least 17,711 g, equivalent to driving 115 km in a standard car. Customized alerts issued at the time of test ordering can have enormous impacts on reducing cost, waste, and unnecessary testing.

GLOBAL RETROSPECTIVE REVIEW OF SEVERE ACUTE RESPIRATORY SYNDROME SARS COV-2 INFECTIONS IN NONDOMESTIC FELIDS: MARCH 2020-FEBRUARY 2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in nondomestic felids have been documented in North America, South America, Africa, Europe, and Asia. Between March 2020 and February 2021, at nine institutions across three continents, infection was confirmed in 16 tigers (Panthera tigris), 14 lions (Panthera leo), three snow leopards (Panthera uncia), one cougar (Puma concolor), and one Amur leopard cat (Prionailurus bengalensis euptilurus) ranging from 2 to 21 yr old (average, 10 yr). Infection was suspected in an additional 12 tigers, 4 lions, and 9 cougars. Clinical signs (in order of most to least common) included coughing, ocular and/or nasal discharge, wheezing, sneezing, decreased appetite, lethargy, diarrhea, and vomiting. Most felids recovered uneventfully, but one geriatric tiger with comorbidities developed severe dyspnea and neurologic signs necessitating euthanasia. Clinical signs lasted 1-19 d (average, 8 d); one tiger was asymptomatic. Infection was confirmed by various methods, including antigen tests and/or polymerase chain reaction (PCR) of nasal or oral swabs, tracheal wash, and feces, or virus isolation from feces or tracheal wash. Infection status and resolution were determined by testing nasal swabs from awake animals, fecal PCR, and observation of clinical signs. Shedding of fecal viral RNA was significantly longer than duration of clinical signs. Postinfection seropositivity was confirmed by four institutions including 11 felids (5 lions, 6 tigers). In most instances, asymptomatic or presymptomatic keepers were the presumed or confirmed source of infection, although in some instances the infection source remains uncertain. Almost all infections occurred despite using cloth facemasks and disposable gloves when in proximity to the felids and during food preparation. Although transmission may have occurred during momentary lapses in personal protective equipment compliance, it seems probable that cloth masks are insufficient at preventing transmission of SARS-CoV-2 from humans to nondomestic felids. Surgical or higher grade masks may be warranted when working with nondomestic felids.

Effect of COVID-19 infection on the gastrointestinal tract considering preventive methods during endoscopic procedures.

This study aimed to prevent the infection risk of environmental contamination by feces during endoscopic procedures. We evaluated the effect of coronavirus disease 2019 (COVID-19) on the gastrointestinal tract using fecal polymerase chain reaction (PCR) and examined risk factors affecting endoscopic procedures, to develop infection prevention strategies. This single-center prospective observational study enrolled 32 patients diagnosed with COVID-19 at Tokyo Medical University Hospital between January and December 2022. We performed reverse transcriptase-PCR to detect severe acute respiratory syndrome coronavirus 2 in human stool specimens and evaluated the COVID-19 positivity rate in stool, the effect of vaccination on infection detection, and differences in positivity rates considering different patient backgrounds. Among the 32 nasal PCR-positive patients who underwent fecal PCR testing, the fecal PCR positivity rate was 21.8%. Compared to the negative cases, 71.4% vs. 32% were older than 65 years (p < 0.016), 71.4% vs. 0.8% (p < 0.001) had malignant tumors, the rate during BA.5 variant outbreaks was significantly higher (100% vs. 60% [p = 0.044]), and the rate of diarrheal symptoms was also higher (42.9% vs. 24%). The median collection period for fecal PCR-positive cases was 2 days after sampling. The severe acute respiratory syndrome coronavirus 2 affects not only the upper respiratory tract but also the gastrointestinal tract. These findings may indicate the risk of digestive fluid infection in older patients with gastrointestinal symptoms and immunocompromised patients with malignant tumor comorbidities, especially during the early stages of viral infection. Therefore, it is advisable to establish a system to prevent infection by using personal protective equipment, including eye guards, in future endoscopic procedures.

Molecular assay and in vitro culture for Blastocystis prevalence in Dakahlia governorate, Egypt

Blastocystis is a polymorphic enteric parasite with a worldwide distribution. It is one of the most common human intestinal protozoans in developing countries. The primary objective of this study was to determine the diagnostic value of microscopy, stool culture, and a polymerase chain reaction (PCR) technique for assessment of Blastocystis prevalence and risk factors. Human stool samples were collected from 110 individuals from Dakahlia governorate, Egypt as a part of a routine check-up or having gastrointestinal tract (GIT) symptoms. These samples were subjected to direct fecal smear microscopy, culture, and PCR for the detection of Blastocystis sp. Positive results for Blastocystis screening among the study population were 36 (32.7%), 41 (37.3%), and 43 (39.1%) by microscopy, PCR, and culture, respectively. Statistical analyses demonstrated that the agreement between the culture and PCR was perfect (Κ=0.925). Compared to culture, the sensitivity of PCR was 95% and the specificity was 97% while the sensitivity of microscopy was 84% and the specificity was 90.5%. We concluded that the in vitro culture and molecular assay have significant diagnostic value for the accurate detection and identification of Blastocystis in stool samples. The pathogenic potential of Blastocystis cannot be ruled out because our results found a link between Blastocystis carriage and gastrointestinal symptoms.

Comparison of blood parameters according to fecal detection of Mycobacterium avium subspecies paratuberculosis in subclinically infected Holstein cattle.

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic and progressive granulomatous enteritis and economic losses in dairy cattle in subclinical stages. Subclinical infection in cattle can be detected using serum MAP antibody enzyme-linked immunosorbent assay (ELISA) and fecal polymerase chain reaction (PCR) tests. To investigate the differences in blood parameters, according to the detection of MAP using serum antibody ELISA and fecal PCR tests. We divided 33 subclinically infected adult cattle into three groups: seronegative and fecal-positive (SNFP, n = 5), seropositive and fecal-negative (SPFN, n = 10), and seropositive and fecal-positive (SPFP, n = 18). Hematological and serum biochemical analyses were performed. Although the cows were clinically healthy without any manifestations, the SNFP and SPFP groups had higher platelet counts, mean platelet volumes, plateletcrit, lactate dehydrogenase levels, lactate levels, and calcium levels but lower mean corpuscular volume concentration than the SPFN group (p < 0.017). The red blood cell count, hematocrit, monocyte count, glucose level, and calprotectin level were different according to the detection method (p < 0.05). The SNFP and SPFP groups had higher red blood cell counts, hematocrit and calprotectin levels, but lower monocyte counts and glucose levels than the SPFN group, although there were no significant differences (p > 0.017). The cows with fecal-positive MAP status had different blood parameters from those with fecal-negative MAP status, although they were subclinically infected. These findings provide new insights into understanding the mechanism of MAP infection in subclinically infected cattle.

YERSINIA PSEUDOTUBERCULOSIS INFECTION IN LIONS (PANTHERA LEO) AT A ZOOLOGICAL PARK.

Two co-housed 17-yr-old male lions (Panthera leo) and one 15-yr-old female lion in an adjacent enclosure developed acute lethargy, depression, anorexia, and ataxia at a zoological park in central North Carolina, United States. One of the male lions and the female lion were found dead 5 and 4 d after the onset of clinical signs, respectively. The other male lion recovered without any clinical treatment. A third male lion housed with the female lion never developed clinical signs. Postmortem examination, microscopic evaluation, and bacterial culture of the liver from both deceased lions confirmed systemic Yersinia pseudotuberculosis infection. Susceptibility testing revealed resistance to amoxicillin-clavulanic acid and cefazolin. To investigate the extent of the outbreak, qualitative real-time polymerase chain reaction (PCR) for Y. pseudotuberculosis was performed on feces and substrate from 15 enclosures housing nondomestic felids and canids, resulting in a positive sample from one enclosure housing four asymptomatic lions. This enclosure was adjacent to housing of the deceased female lion. Enrofloxacin was administered to all animals in the vicinity at doses of 5 mg/kg in nondomestic felids and 10 mg/kg in nondomestic canids, orally q24h for 14 d. Repeated fecal PCR performed 1 wk after completing the antibiotic treatment protocol found no positive samples. The source of the infection was not identified despite PCR testing of environmental samples from all enclosures in the vicinity, the remains of a prey item fed out prior to the outbreak, and a single dead rodent found on grounds. No further clinical cases have occurred within the following year.

Performance of diagnostic assays used to detect Cryptosporidium oocysts in faecal samples of cattle in Kuwait and genotyping of Cryptosporidium species

BackgroudCryptosporidium species are zoonotic protozoan parasites responsible for gastroenteritis in various animals and humans. The diagnosis of Cryptosporidium presents many challenges. This research attempted to match the diagnostic efficiency of the modified Ziehl–Neelsen technique (mZN), immunochromatographic assays (IC), and enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium in faecal samples of cattle in Kuwait. In addition, polymerase chain reaction (PCR) was utilised to determine the predominant species infecting cattle in Kuwait and correlating the detected species with the results of different diagnostic tests used, the presence or absence of clinical signs, and the age group of the infected cattle.ResultsOf 400 analysed faecal samples, Cryptosporidium positive samples were 23%, 15.25%, and 14% using IC, ELISA, and mZN. IC had the highest sensitivity (74.07%), and mZN had the highest specificity (98.29%) using a composite reference standard (CRS) as a gold standard. The rapid IC test results in high false-positive results of cryptosporidiosis, whereas using mZN alone is insufficient to declare a negative faecal sample. Only 74.5% (35/47) of Cryptosporidium-positive samples by the three assays could be amplified by PCR. This study was the first to genotype Cryptosporidium in Kuwait. Cryptosporidium parvum (n = 26) was the dominant species detected from cattle samples, followed by C. andersoni (n = 6), C. bovis (n = 2), and C. raynae (n = 1). The findings showed a statistically relevant relationship between diarrhoea and the detection of Cryptosporidium spp. in faecal samples of cattle (p-value = 0.0003). Pre-weaned calves were the most vulnerable age group to Cryptosporidium spp. infection (p-value = 0.0007).ConclusionFor screening of Cryptosporidium infection in faecal samples, antigen detection or PCR methods combined with one of the microscopy techniques should be used. Cryptosporidium parvum was the prepoderant Cryptosporidium spp. recovered from cattle samples in Kuwait followed by C. andersoni. Cryptosporidium parvum is a significant risk factor for diarrhoea in pre-weaned calves. However, further study is needed as many other causes of diarrhoea in calves must be ruled out before a diagnosis of Cryptosporidium diarrhoea can be made.

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Application of Bayesian modeling for diagnostic assays of Mycobacterium avium subsp. paratuberculosis in sheep and goats flocks

BackgroundThis study aimed to screen the sera of goats and sheep from flocks suspected of Mycobacterium avium subsp. paratuberculosis (MAP) infection by a newly standardized Mce-truncated ELISA (Mt-ELISA) kit for the detection of antibodies against MAP. Four diagnostic applied tests were evaluated including Indigenous plate-ELISA (IP-ELISA), Mt-ELISA, fecal Polymerase Chain Reaction (f-PCR) and fecal culture (FC).Materials and methodsAssuming the absence of a gold standard, latent-class models in a Bayesian framework were used to estimate the diagnostic accuracy of the four tests for MAP.ResultsMt-ELISA had higher Sensitivity (Se) in sheep (posterior median: 0.68 (95% Probability Interval (PI): 0.43–0.95), while IP-ELISA recorded the highest Se in goats as 0.83 (95% PI, 0.61–0.97). The f-PCR Se estimate slightly differed between species [sheep 0.36 (0.19–0.58), goats 0.19 (0.08–0.35)], while the Se of FC was similar between species [sheep 0.29 (0.15–0.51), goats 0.27 (0.13–0.45)]. The specificity estimates for all tests were high, close to unity, and similar between species.ConclusionOverall, the results showed that the Mt-ELISA method can be used for MAP detection in small ruminants’ flocks.

Comparative performance of different antigens on the lateral flow assay (LFA) platform for the rapid serodiagnosis of paratuberculosis

Paratuberculosis is a globally prevalent disease, that adversely affects the economy of livestock farming. Control is largely based on early detection followed by ‘Test and Cull’ or ‘Test and Segregate’ Policy. Implementation of paratuberculosis control is a special challenge due to the non-availability of point of care diagnostics (PoCD). Therefore, the present study aimed to optimize and evaluate a lateral flow assay (LFA) for the rapid serodiagnosis of paratuberculosis in ruminant species, especially in the view of the resource-limited areas. Performance of three different antigenic preparations including native purified protoplasmic antigen (nPPA-LFA), commercial purified protoplasmic antigen (cPPA-LFA), and a cocktail of recombinant secretory proteins (RP-LFA) was evaluated as detection reagents for coating LFA strips. Comparative performance of the optimized LFA was also evaluated with gold standard tissue culture, fecal PCR (polymerase chain reaction), and plate ELISA. In addition, the onsite testing of animals belonging to different farms (endemic), species, and regions using optimized LFA was also done to highlight the on-farm testing approach. Findings revealed recombinant secretory proteins based LFA (RP-LFA) had a higher sensitivity of detection compared to other antigens. RP-LFA had a sensitivity of 77.7%, 75.44%, and 75.16% in comparison to gold standard tissue culture, fecal PCR, and plate ELISA, respectively. The specificity of RP-LFA was 100% with all reference tests. In comparison to plate ELISA, RP-LFA had a detection limit of 100% when the S/P ratio of the serum sample is ≥1.0 and 80% when the S/P ratio range of 0.8–1.0. Using RP-LFA, on-farm testing of 608 animals was done and 283 (46.5%) were found positive. Kappa analysis of present RP-LFA revealed ‘good strength of agreement’ with gold standard tissue culture, fecal PCR, and plate ELISA. Optimized RP-LFA had no cross-reactivity with bovine tuberculosis (bovine TB). The RP-LFA was found reproducible, user-friendly and test results can be interpreted within five minutes. In conclusion, the findings of the present study advocate the huge potential of LFA-based PoCD in the rapid diagnosis and control of paratuberculosis.

Comparison of polymerase chain reaction and microscopy for the detection of Fasciola spp. in the fecal matter of domestic bovines in Kalasin Province, Thailand.

Background and Aim:Fasciola spp. are important foodborne trematodes and waterborne zoonotic parasites that cause health problems and economic losses worldwide, including in Thailand. Fasciola spp. are usually detected by sedimentation or the formalin-ethyl acetate concentration technique (FECT) under microscopy, which is less specific and sensitive. Accurate detection is important to detect real incidence for protection against and elimination of fasciolosis in the area. This study aimed to determine the distribution of Fasciola spp. and compare the specificity and sensitivity of FECT under microscopy to that of polymerase chain reaction (PCR) in cattle feces.Materials and Methods:The study was conducted in Kalasin Province, Thailand. Feces of 46 cattle were investigated for infection with Fasciola spp. To detect infection, FECT under microscopy and PCR amplification of the 28S rRNA gene of Fasciola spp. were used to identify egg parasites.Results:Feces of 16 of 46 (34.78%) cattle were positive for Fasciola spp. using FECT under microscopy, whereas PCR showed that 67.39% (31 of 46) were positive for Fasciola spp. False-negative results were as high as 32.61% when diagnosed under microscopy.Conclusion:This study confirmed the infection of cattle with Fasciola spp. in Kalasin Province, indicating that PCR demonstrated higher sensitivity and specificity when diagnosing infection. FECT under microscopy can still be used as a primary and traditional method for diagnosis. However, relapse cases of Fasciola spp. and Paramphistomum spp. should be diagnosed by microscopy combined with PCR. This is the first report on the molecular distribution of fecal samples in cattle in Kalasin Province.

Comparison of interferon-gamma, neopterin, interleukin-10 and antibody levels in sheep with and without Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP), responsible for paratuberculosis, causes serious economic losses in the livestock industry worldwide. To develop accurate control strategies against MAP, it is necessary to understand the pathological basis of the disease and identify new diagnostic markers for the detection of infection. In this study, commonly used diagnostic tests for the detection of MAP infection were compared and the agreement between these tests was evaluated. The measurement of interleukin-10 (IL-10) and neopterin levels in the serum were also assessed for their potential to be used in the detection of MAP infection in sheep. The diagnostic tests revealed that 73 (48.66 %) of 150 sheep were MAP-positive. The highest proportion of positive sheep were determined by Ziehl-Neelsen (ZN) staining (27.33 %) and direct fecal polymerase chain reaction (PCR) (26.66 %), followed by interferon-gamma enzyme-linked immunosorbent assay (ELISA) (22 %), antibody ELISA (10.67 %) and fecal culture (3.33 %) methods. A significant, almost perfect, agreement was found between ZN staining and direct fecal PCR test (κ: 0.882, P < 0.0001). All isolated MAP strains were sheep-type MAP. Due to the significant difference in circulating serum IL-10 between the MAP-positive and MAP-negative sheep (P < 0.01) and its similar levels among the MAP-positive sheep, it was considered that IL-10 might be an important diagnostic marker to be used with other diagnostic tests in sheep. Due to the negative moderate correlation between serum neopterin and MAP-specific antibody levels, future studies should further investigate this to understand the role of neopterin.

Diagnostic and Therapeutic Considerations Towards Dientamoeba fragilis in Children: A Survey Amongst General Practitioners and Pediatricians in the Netherlands.

This survey was undertaken to obtain insight in the attitude of Dutch physicians towards pathogenicity, diagnostic- and therapeutic approach towards Dientamoeba fragilis in children. Physicians were invited by e-mail for a questionnaire. A total of 211 of 450 physicians (46.9%) completed the questionnaire, including 67 general practitioners (GPs) and 144 pediatricians. Of all respondents, 175 of 211 (82.9%) considered D fragilis a "potential pathogen", when other causes of gastro-intestinal complaints are ruled out. Only 16 of 211 (7.6%) performed diagnostic tests regularly. Diagnostic tests were performed by 162 of 211 (77%) of respondents in children with diarrhea and abdominal pain in consideration of duration of symptoms. Fecal polymerase chain reaction (PCR) was diagnostic modality of preference. Eighty-nine of 142 (62.7%) prescribed metronidazole as antibiotic of first choice. This study shows heterogeneity in clinical practice amongst Dutch physicians regarding diagnostic- and therapeutic approach of D fragilis in children. Different attitude towards pathogenicity and inconsistent guidelines could be causative factors.

Management of Entamoeba histolytica in the non-human primates at the Singapore Zoo.

Amebic dysentery caused by Entamoeba histolytica accounts for significant morbidity in the non-human primates (NHP) at the Singapore Zoo. This includes the animals in the collection as well as a sizeable free-roaming wild crab-eating macaque (Macaca fascicularis) population. The disease is of great concern because of its zoonotic potential. Passive surveillance, both ante and post-mortem, of NHP displaying clinical symptoms and active surveillance of NHP assessed to be at a higher risk of infection were carried out via fecal real-time polymerase chain reaction (PCR) testing for 4years. Treatment of the disease with 25mg/kg metronidazole BID for 10days followed by 15mg/kg paromomycin BID for 7days achieved good clinical resolution in most cases that tested positive. Three diseased NHP with severe clinical signs of weight loss, lethargy, and diarrhea were anesthetized for veterinary diagnostic investigation. Mesenteric lymphadenopathy was consistently seen on ultrasound examination in these severe cases of entamoebiasis. Two animals eventually died of severe chronic enteritis due to the disease. The eradication of entamoebiasis in the NHP at the Singapore Zoo may be complicated by the maintenance of a disease reservoir in wildlife, but a combination of timely treatment and efforts at maintaining biosecurity can help manage the disease in the collection.

Screening for Helicobacter pylori infection and Clarithromycin resistance using Real-Time Polymerase Chain Reaction.

Helicobacter pylori (H. pylori) infection is spread worldwide and affects at least half of the world's population. Infected people are at increased risk of several diseases' development, including gastric adenocarcinoma. The aim of this study was to screen patients with dyspeptic symptoms for H. pylori infection and assess Clarithromycin resistance prevalence among the infected patients. Screening for H. pylori infection was performed in all patients using molecular test based on Real-Time Polymerase Chain Reaction (RT-PCR) in feces after RNA-DNA extraction. Stool samples from all participants were collected 1 to 3 days after patients' hospitalization. The positive results were furthermore assessed for confirmation by breath test and stool antigen test. By point mutations detection in 23S rRNA gene was possible to detect Clarithromycin resistance. Statistical analysis was performed via SPSS 22.0 (IBM Corp., Armonk, NY, USA). This study enrolled 50 patients (18 males) at mean age 46.46±15.10 years. Using molecular test based on RT-PCR in feces we identified H. pylori infection in 24 patients (48.00%). Clarithromycin resistance was observed in 7 of them (29.17%). None of those patients was eradicated before. There was no significant difference by age and gender between infected and non-infected patients. Gastrointestinal symptoms were more often reported in infected patients (p<0.05). The molecular test showed 85.71% sensitivity and 100% specificity, with a diagnostic accuracy of 92.00%. H. pylori screening by molecular test based on RT-PCR in feces might be beneficial as the test's accuracy is high and include Clarithromycin resistance assessment, which could improve the outcome of eradication therapy.

Novel formulation with essential oils as a potential agent to minimize African swine fever virus transmission in an in vivo trial in swine.

Background and Aim:African swine fever (ASF) is currently the most prevalent disease in swine. The disease is spreading throughout primary swine-producing countries with heavy losses in population and revenue. To date, no successful vaccines or medications have been reported. This study aimed to design and develop a blend of natural essential oils and test its efficacy against the ASF virus (ASFV) in swine.Materials and Methods:We attempted to develop a natural oil blend formulation (NOBF) and determine its efficacy against the ASFV. This study follows on from a previously published in vitro study that reported that the NOBF has anti-ASFV properties. A study was designed using 21 healthy piglets of triple-cross (Landrace + Yorkshire + Durok) crossbred pathogen-free pigs with an average weight of 15 kg. The study consisted of NOBF-incubated, NOBF, positive control, and negative control groups. The NOBF groups were administered NOBF (80 mL/ton mixed in drinking water) beginning 10 days before the challenge and continuing throughout the experiment. The positive and negative control pigs consumed regular drinking water. The pigs were challenged by a sublethal dose of pure isolate ASFV strain Vietnam National University of Agriculture-ASFV-L01/HN/04/19 inoculation with 103.5 HAD50/dose through the intramuscular route. There were sic pigs in each group, three pigs directly IM challenged, and three pigs were considered cohoused pigs.Results:Both challenged (three) and cohoused (three) pigs in the positive control showed clinical signs of ASFV infection, as detected by real-time polymerase chain reaction (RT-PCR) in blood samples, oral swabs, and feces. There was 100% cumulative mortality, that is, both challenged and contact pigs died in the positive control group on day 20 of infection. No signs of infection or mortality were observed in the NOBF-incubated group. The challenged pigs in the NOBF-direct challenge group showed clinical signs and mortality, whereas no clinical signs or symptoms occurred in the cohoused pigs. The immunoglobulin G (IgG) level of the contact pigs was the highest in the treatment group and the lowest in the positive control group. The IgM level of the contact pigs in the treatment groups was the lowest, whereas that of the positive control was the highest. The RT-PCR test showed that the ASFV was deactivated in the NOBF-incubated group. The challenged and contact pigs of the positive control group had high Ct values. The challenged pigs of the NOBF group had high Ct values, whereas the contact pigs from the same group and those of the negative control were negative for the ASFV, determined by PCR, in all samples. The comparison of the challenged groups showed that the appearance of the virus was delayed by at least 2 days in the NOBF group compared to the positive control group.Conclusion:The results showed that NOBF can prevent the spread of the ASFV in a population. Moreover, NOBF can enhance the pig humoral immune system by enhancing IgG levels and reducing IgM levels. This study successfully demonstrated that NOBF is an anti-ASFV agent, which prevents horizontal transmission and enhances pig humoral immunity.

Comparison of diagnostic methods (wet mount, trichrome staining, formol-ether, PCR, and xenic in vitro culture) for the detection of Blastocystis in stool samples in Urmia educational hospitals, the Northwest of Iran.

Blastocystis spp. is known as a common intestinal protozoan parasite in human and animals. The parasite has a worldwide distribution and is frequently detected in faecal samples in clinical parasitology laboratories. The goal of the study was to compare the sensitivity and specificity of formol-ether technique (FECT), trichrome staining, xenic in vitro culture (XIVC), microscopy of faecal smears, and polymerase chain reaction (PCR) methods for detecting Blastocystis spp. in human stool samples. The prevalence of the parasite in the stool samples referred to educational hospitals was also determined. A total of 575 cases were assessed to detect the parasite. After collecting from patients referring to Urmia educational hospitals, the samples were examined by microscopy of faecal smears, trichrome staining, FECT, XIVC using Jones' medium, and PCR, to evaluate the presence of Blastocystis spp. Microscopy of faecal smears, trichrome staining, FECT, and PCR technique detected 94, 100, 96, and 44 positive cases, with the sensitivity of 71.3%, 74.4%, 74.4%, and 80.4% and the specificity of 99.6%, 99.1%, 100%, and 93.1%, respectively. XIVC method identified the highest number of positive cases (129 cases) among the other methods. Our findings indicates that XIVC technique is more sensitive method for the detection of Blastocystis spp. in human stool, as compared to direct smear, trichrome staining, FECT, and PCR methods.

Bowel Wall Thickening on Computed Tomography in Children: A Novel Method of Measurement and Its Clinical Significance.

PurposeThe clinical implications of bowel wall thickening (BWT) on abdominal computed tomography (CT) among children are unknown. We aimed to suggest a new method for measuring BWT and determining its clinical significance in children.MethodsWe retrospectively analyzed 423 patients with acute abdomen who underwent abdominal CT; 262 were classified into the BWT group. For this group, the pediatric radiologist described the maximal bowel wall thickness (MT), normal bowel wall thickness (mm) (NT), and their ratios for each segment of the bowel wall.ResultsIn the thickened bowel walls, the thickness differed significantly between the small bowel (6.83±2.14 mm; mean±standard deviation) and the colon (8.56±3.46 mm; p<0.001). The ratios of MT to NT in the small bowel (6.09±3.17) and the colon (7.58±3.70) were also significantly different (p<0.001). In the BWT group, 35 of 53 patients had positive fecal polymerase chain reaction results; 6 patients infected with viruses predominantly had BWT in the small intestine, while the terminal ileum and the colon were predominantly affected in 29 patients with bacterial infections. In the initially undiagnosed 158 patients with BWT, the symptoms improved spontaneously without progression to chronic gastrointestinal disease.ConclusionThis study provides a clinical reference value for BWT in the small intestine and colon using a new method in children. The BWT on abdominal CT in children might indicate nonspecific findings that can be observed and followed up without additional evaluation, unlike in adults.

Detection of Mycobacterium avium sub sp. paratuberculosis (MAP) by PCR in the faeces of dairy cattle of Chitwan, Nepal

Johne's disease or Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic granulomatous enteritis with decrease in production resulting huge economic losses with high negative impact on the livestock industry. Diagnosis of MAP is difficult due to lack of characteristics clinical signs, prolong incubation period in cultivation of MAP, and non-specific results in diagnostic tests. To the best of our knowledge there is no report on faecal culture and molecular detection of MAP in dairy cattle of Nepal. The main objective of this research was to access the herd level prevalence of Johne’s disease in the representative dairy farms of Chitwan district with the use of modern techniques as faecal polymerase chain reaction (fPCR) to know the MAP distribution in dairy cattle. A total of 265 individual dairy cattle faeces sample were collected during February 2017 to January 2018 from dairy farms of three different geographical location of Chitwan district, Nepal. Faeces were decontaminated and subjected for faecal culture as well as fPCR to have molecular detection of MAP. Findings revealed that bio-load of MAP in dairy cattle were 13.57% by faecal culture, and 16.59% by fPCR detection method. The overall prevalence of MAP in dairy cattle was detected as 16.59 % by fPCR. Likewise, IS900 PCR assay proved to be a more sensitive and reliable test than faecal culture for the detection of MAP in faecal sample of clinically suspected dairy cattle as the PCR assay was able to detect significantly (p &lt; 0.01) more positive cases than faecal culture. Findings of this study suggests that IS900-PCR-based detection of MAP could be used as a potential diagnostic tool for rapid and effective Johne’s disease (JD) surveillance as compared with faecal culture detection method due to its advantage for JD control programs by reducing the time of definitive diagnosis from several months to a few days. This is the first molecular level of diagnostic research performed and reporting of MAP in dairy cattle of Nepal. These results will be useful in designing suitable disease control strategy for livestock industry.