Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis

Abstract Background Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis. Methods To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. Results Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses. Conclusions Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis. Graphic Abstract

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Molecular Detection of Cattle Sarcocystis Spp. In North-West Italy Targeting Cox1 And 18S Genes Highlights Its Association With Bovine Eosinophilic Myositis.

10.21203/rs.3.rs-254835/v1 ◽

2021 ◽

Author(s):

Selene Rubiola ◽

Tiziana Civera ◽

Felice Panebianco ◽

Davide Vercellino ◽

Francesco Chiesa

Keyword(s):

Inflammatory Myopathy ◽

Molecular Techniques ◽

Economic Losses ◽

Intermediate Hosts ◽

Prevalence Studies ◽

North West ◽

Slaughter Cattle ◽

Significant Difference ◽

Pcr Targeting

Abstract Background: Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa due to the evidences supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based either on morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis.Methods: To reach our aim, individual cattle samples from BEM condemned carcasses (N=54) and randomly sampled carcasses (N=59) were obtained from Piedmont slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S and cox1 genes. PCR products amplified using the genus specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis, were sequenced to achieve species identification.Results: Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in 2 carcasses.Conclusions: Our study contributes to update the data on the prevalence of the different Sarcocystis spp. in cattle in Italy and emphasize the role of S. hominis and S. bovifelis as the major sarcosporidian species involved.

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Abstract 20433: Viral Myocarditis: Viral Genome Detection Yield on Endomyocardial Biopsy is Influenced by Methodological Factors

Circulation ◽

10.1161/circ.130.suppl_2.20433 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Elisa Carturan ◽

Stefania Rizzo ◽

Gaetano Thiene ◽

Cristina Basso

Keyword(s):

Endomyocardial Biopsy ◽

Viral Genome ◽

Viral Myocarditis ◽

Molecular Techniques ◽

Time Interval ◽

Tissue Fixation ◽

Viral Genomes ◽

Consensus Document ◽

Significant Difference

Introduction: Myocarditis is diagnosed on endomyocardial biopsy (EMB) by histological, immune-histochemical and molecular techniques to detect viral genomes. The role of EMB for the diagnosis and its contribution to patient management has been addressed by a Consensus document of American and European Cardiovascular Pathology Societies. Hypothesis: Different methods of tissue fixation and the number of myocardial samples could impact on viral genome yield on EMB. Methods: Consecutive EMBs referred at our Institution for histology/ immunohistochemistry evaluation in the time interval 1996-2012 were enrolled. Molecular investigation by RT-PCR and PCR technique was performed in all EMBs with inflammatory cardiomyopathy diagnosis at histology and immunohistochemistry. Results: A total of 467 EMBs have been diagnosed as myocarditis: 79 in pediatric (1 month to 18 years; 47M/ 32F) and 388 adult (19 to 75 years; 256M/132 F) patients (pts.). Viral etiology was identified in 28 (36%) pediatric and 101 (26%) adult pts, and the most prevalent type of viruses were enterovirus (8/28, 36%; 26/101, 24%). In a more recent subgroup of 137 EMBs (virus positive 30/137, 22%) seasonality, type of fixation and number of EMB samples were assessed. The seasonal distribution of myocarditis was higher in winter than in other seasons (33% vs. autumn 28%, spring 20% and summer 19%) without any significant difference in terms of virus positive EMBs. The number of EMB samples per pt. was ≤3 (either formalin or RNAlater) in 94/137 (69%) and >3 in 43/137 (31%), with a lower prevalence of virus positive in the former (17/94, 18% vs. 13/43, 30%). Eighty-one EMB samples were frozen while 56 were paraffin embedded, with a higher prevalence of viral genome in the former (26/81, 32% vs. 4/56, 7%; p=0.001). Conclusions: The diagnosis of myocarditis on EMB samples requires standardized protocols including molecular techniques. Viral genomes are identifiable in more than one third of pediatric and one fourth of adult cases. Methodological factors like the type of tissue fixation and the number of samples could impact on viral genome detection on EMB.

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Italian observational study on HPV infection, E6, and p16 expression in men with penile cancer

Virology Journal ◽

10.1186/s12985-020-01424-9 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Narcisa Muresu ◽

Giovanni Sotgiu ◽

Laura Saderi ◽

Illari Sechi ◽

Antonio Cossu ◽

...

Keyword(s):

Penile Cancer ◽

Tertiary Care ◽

Hpv Infection ◽

Molecular Techniques ◽

Care Hospital ◽

Hpv 16 ◽

Hpv Dna ◽

Diagnosis And Prognosis ◽

Significant Difference

Abstract Background Human Papillomavirus (HPV) infection is one of the most important causes of cancer. It can play a role in cervical and extra-cervical cancers. Penile cancer is rare, even if an increasing trend was recently reported. Aim of the present study was to assess the prevalence and distribution of HPV genotypes in cases of penile cancer diagnosed in Sardinia, Italy. Surrogate markers of HPV infection (i.e., E6 and p16 genes) were also evaluated in all cases. Methods An observational, retrospective study which recruited all cases of penile cancer diagnosed between 2002 and 2019 at a tertiary care hospital in Sardinia, Italy, was carried out. HPV-DNA detection and genotyping were performed by Real-time PCR. Specimens were tested for oncogene E6 mRNA and for p16(INK4a) expression. Results HPV prevalence was 28.1% (9/32); HPV-16 was the most prevalent genotype (7/9, 77.8%). p16INK4a positivity was found in 66.7% of the samples with a statistically significant difference between HPV-positive and -negative groups. E6-transcript was detected in 71% of the HPV-16 positive samples. The overall survival was not statistically different between HPV-positives and -negatives. Discussion The present study confirms the etiologic role of HPV in penile cancer and supports the adoption of vaccination strategies in men and women. Further studies should clarify the diagnostic and prognostic role of E6 and p16 proteins. Conclusion HPV infection can favor the occurrence of penile cancer, whose diagnosis and prognosis could be improved with the implementation of validated molecular techniques.

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First molecular confirmation of Coenurus cerebralis in sheep and goats with neurological behaviors in Iraq

Veterinary World ◽

10.14202/vetworld.2021.1420-1425 ◽

2021 ◽

pp. 1420-1425

Author(s):

Eva Aisser Ajaj ◽

Hadeel Asim Mohammad ◽

Hasanain A. J. Gharban

Keyword(s):

Historical Data ◽

Intermediate Stage ◽

Molecular Techniques ◽

Economic Losses ◽

Sheep And Goats ◽

Neurological Signs ◽

Coenurus Cerebralis ◽

Polymerase Chain ◽

Female Sheep

Background and Aim: Coenurus cerebralis is the intermediate stage of the canine cestode, which infect sheep and goats, resulting mainly in neurological signs and causing direct and indirect economic losses. This study aimed to demonstrate the existence of C. cerebralis and to elucidate the role of this parasite in inducing neurological behaviors in sheep and goats. Materials and Methods: On the basis of historical data on neurological signs, we subjected 76 animals (49 sheep and 27 goats) of different ages, sexes, and geographical areas for molecular examination of their blood samples using the polymerase chain reaction assay. Results: Of the 76 animals, 23.68% tested positive for C. cerebralis infection. We found significant increases in infection (p<0.05) in sheep (26.53%) more than in goats (18.52%). Circling movement was prevalent significantly among both C. cerebralis-positive sheep and goats. The Nineveh region had a significant (p<0.05) increase in positive sheep and goats, and the sheep of all study regions were infected significantly (p<0.05) more than were the goats. We found no significant (p>0.05) variation between sheep ages ≥1-3 years and ≥3 years; however, both groups had a significantly (p≤0.043) higher positivity rate than did sheep ages <1 year. The findings of sheep ages <1 year and ≥1-3 years were significantly higher than those of the goats, but not for goats ages ≥3 years. Female sheep and goats showed a significant increase in positivity versus that for the males. Conclusion: To the best of our knowledge, this study is the first report in Iraq targeting detection of C. cerebralis in sheep and goats with neurological behaviors; therefore, additional studies involving different animals in other regions using molecular techniques are needed.

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Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya

10.21203/rs.2.21914/v1 ◽

2020 ◽

Author(s):

James Miser Akoko ◽

Roger Pelle ◽

Velma Kivali ◽

Esther Schelling ◽

Gabriel Shirima ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epidemiological Data ◽

Molecular Techniques ◽

Molecular Evidence ◽

Rt Pcr ◽

Growing Pig ◽

Pcr Targeting ◽

The Rose

Abstract BackgroundBrucellosis is an emerging, yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi, Kenya were screened in parallel, using the Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp.. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes.ResultsA prevalence of 0.57% (n=4/700) was estimated using RBT. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a multiple real-time PCR. ConclusionThe detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs indicate the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

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The Role of Birds of the Family Corvidae in Transmitting Sarcocystis Protozoan Parasites

Animals ◽

10.3390/ani11113258 ◽

2021 ◽

Vol 11 (11) ◽

pp. 3258

Author(s):

Evelina Juozaitytė-Ngugu ◽

Saulius Švažas ◽

Donatas Šneideris ◽

Eglė Rudaitytė-Lukošienė ◽

Dalius Butkauskas ◽

...

Keyword(s):

Light Microscope ◽

Sarcocystis Species ◽

Protozoan Parasites ◽

Intermediate Hosts ◽

Specific Pcr ◽

Sarcocystis Spp ◽

The Family ◽

Species Specific ◽

Pcr Targeting

Members of the family Corvidae are ecologically flexible omnivorous birds, particularly adaptive to urban habitats, and living in proximity to humans; these birds may serve as definitive hosts (DH) for Sarcocystis spp., but research about this is lacking. In the present study, intestinal samples from 91 corvids collected in Lithuania were molecularly tested by species-specific PCR targeting the ITS1 and cox1 genes and subsequently sequenced for the presence of Sarcocystis spp. Under a light microscope, oocysts of Sarcocystis spp. were observed in 43 samples (47.3%), while molecular methods, detected Sarcocystis spp. in 77 birds (84.6%). Eleven Sarcocystis spp. (S. columbae, S. cornixi, potentially pathogenic S. halieti, S. kutkienae, S. lari, S. turdusi, S. wobeseri, S. arctica, S. lutrae, S. ovalis, and S. oviformis) were identified in the intestinal samples from six corvid species from Lithuania. Infections with multiple Sarcocystis spp. were detected in 79.2% of the infected corvid birds. Three of the identified Sarcocystis spp. use corvids as intermediate hosts (IH); therefore, corvids may serve as IH and DH of the same Sarcocystis species. Based on molecular results and on corvid diet, omnivorous corvids may play an important role in transmitting Sarcocystis spp.

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The Role of Mustelids in the Transmission of Sarcocystis spp. Using Cattle as Intermediate Hosts

Animals ◽

10.3390/ani11030822 ◽

2021 ◽

Vol 11 (3) ◽

pp. 822

Author(s):

Petras Prakas ◽

Linas Balčiauskas ◽

Evelina Juozaitytė-Ngugu ◽

Dalius Butkauskas

Keyword(s):

American Mink ◽

Single Species ◽

Meles Meles ◽

Cox1 Gene ◽

Intermediate Hosts ◽

Specific Pcr ◽

Sarcocystis Spp ◽

Species Specific ◽

Pcr Targeting

There is a lack of research on the role of mustelids in the transmission of various Sarcocystis spp. In the present study we tested the hypothesis that widespread mustelids in Lithuania could be involved in the transmission of Sarcocystis spp. using cattle as intermediate hosts. In 2016–2020, intestinal samples of 84 mustelids were examined. Sarcocystis spp. were identified by species-specific PCR targeting the cox1 gene and subsequent sequencing. Under a light microscope, oocysts/sporocysts of Sarcocystis spp. were observed in 40 samples (47.6%), while using molecular methods, they were detected in 75 animals (89.3%). Four Sarcocystis spp. were identified in the intestinal samples of American mink (Neovisonvison), Beech marten (Martes foina), European pine marten (Martes martes), European badger (Meles meles) and European polecat (Mustela putorius). The prevalence of predominant Sarcocystis spp., S. bovifelis (89.3%) and S. cruzi (73.8%) was significantly higher than that of S. hirsuta (3.6%) and S. hominis (1.2%). In an individual sample, most frequently two Sarcocystis spp. were identified (69.0%), then a single species (15.5%) and three species (4.8%). The present study provides strong evidence that mustelids serve as definitive hosts for Sarcocystis spp. using cattle as intermediate hosts.

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THE INFLUENCE OF LIQUID CALCIUM SOURCE ON EGGSHELL QUALITY

Archives of Veterinary Medicine ◽

10.46784/e-avm.v12i1.40 ◽

2019 ◽

Vol 12 (1) ◽

pp. 87-96

Author(s):

Ksenija Nešić ◽

Nikola Pavlović ◽

Vladimir Radosavljević ◽

Miloš Gavrilović

Keyword(s):

Egg Production ◽

Laying Hens ◽

Egg Quality ◽

Economic Losses ◽

Significant Difference ◽

Beneficial Impact ◽

Calcium Source ◽

Liquid Calcium ◽

Production Increase

It is estimated that the world’s population will enlarge by 25% by the middle of this century, resulting in the food production increase by at least 60%. Intensifying egg production is one of the most affordable solutions to provide animal protein. Among many other efforts to improve the quality of eggs, special attention is paid to attempts to provide a better endurance and strength of eggshell, due to the fact that the production of eggs with broken, cracked or soft shells incurs significant economic losses. One of the most important factors to achieve this goal is careful adjustment of calcium in the diet of laying hens, but the nutritional role of calcium is closely linked to that of phosphorus and the effect of vitamin D. The aim of the experiment was to determine whether two different nutritional supplements of calcium, applied in drinking water of laying hens for two weeks have any significant influence on the shell egg quality. Statistically very significant difference in egg production was observed in the treatment with the product containing calcium and phosphorus. On the other hand, the product consisting of calcium and vitamin D3 had no beneficial impact on the egg parameters. Based on the obtained results and literature data, it can be concluded that the amount and source of calcium in the diet of laying hens is a very complex and not fully solved issue. Therefore, especially keeping in mind the duration of the experiment, additional research is needed on this subject.

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Development of a Whole-Virus ELISA for Serological Evaluation of Domestic Livestock as Possible Hosts of Human Coronavirus NL63

Viruses ◽

10.3390/v11010043 ◽

2019 ◽

Vol 11 (1) ◽

pp. 43 ◽

Cited By ~ 3

Author(s):

Philip El-Duah ◽

Benjamin Meyer ◽

Augustina Sylverken ◽

Michael Owusu ◽

Lina Theresa Gottula ◽

...

Keyword(s):

High Throughput Screening ◽

Immunofluorescence Assay ◽

Intermediate Hosts ◽

Human Samples ◽

Significant Difference ◽

Domestic Livestock ◽

Density Values ◽

Human Coronaviruses ◽

Testing Algorithm

Known human coronaviruses are believed to have originated in animals and made use of intermediate hosts for transmission to humans. The intermediate hosts of most of the human coronaviruses are known, but not for HCoV-NL63. This study aims to assess the possible role of some major domestic livestock species as intermediate hosts of HCoV-NL63. We developed a testing algorithm for high throughput screening of livestock sera with ELISA and confirmation with recombinant immunofluorescence assay testing for antibodies against HCoV-NL63 in livestock. Optimization of the ELISA showed a capability of the assay to significantly distinguish HCoV-NL63 from HCoV-229E (U = 27.50, p < 0.001) and HCoV-OC43 (U = 55.50, p < 0.001) in coronavirus-characterized sera. Evaluation of the assay with collected human samples showed no significant difference in mean optical density values of immunofluorescence-classified HCoV-NL63-positive and HCoV-NL63-negative samples (F (1, 215) = 0.437, p = 0.509). All the top 5% (n = 8) most reactive human samples tested by ELISA were HCoV-NL63 positive by immunofluorescence testing. In comparison, only a proportion (84%, n = 42) of the top 25% were positive by immunofluorescence testing, indicating an increased probability of the highly ELISA reactive samples testing positive by the immunofluorescence assay. None of the top 5% most ELISA reactive livestock samples were positive for HCoV-NL63-related viruses by immunofluorescence confirmation. Ghanaian domestic livestock are not likely intermediate hosts of HCoV-NL63-related coronaviruses.

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MRI Study of Paraspinal Muscles in Patients with Amyotrophic Lateral Sclerosis (ALS)

Journal of Clinical Medicine ◽

10.3390/jcm9040934 ◽

2020 ◽

Vol 9 (4) ◽

pp. 934

Author(s):

Luca Diamanti ◽

Matteo Paoletti ◽

Umberto Di Vita ◽

Shaun Ivan Muzic ◽

Cristina Cereda ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Inflammatory Myopathy ◽

Psoas Muscle ◽

Longissimus Dorsi ◽

Paraspinal Muscles ◽

Psoas Muscles ◽

Significant Difference ◽

Als Patients ◽

Lateral Sclerosis

Background: the study of paraspinal muscles is pivotal for the diagnosis and staging of Amyotrophic Lateral Sclerosis (ALS), and is usually performed by electromyography. Objective: to evaluate the role of paraspinal muscle MRI as a diagnostic biomarker in ALS. Methods: we evaluated T1-w images of newly diagnosed ALS patients (n = 14), age-matched healthy controls (n = 11), patients affected by inflammatory myopathy (n = 10), and lumbar radiculopathy (n = 19), and compared them semiquantitatively by using the Mercuri Scale. Results: a significant difference in the appearance of the psoas muscle was observed between ALS patients and patients with radiculopathy (p = 0.003); after stratifying ALS patients into spinal and bulbar onsets, we found a significant difference in the appearance of the longissimus dorsi muscle between the spinal onset ALS subgroup and bulbar onset ALS subgroup (p = 0.0245), while no difference was found for multifidus (p = 0.1441), iliocostal (p = 0.0655), and psoas muscles (p = 0.0813) between the cohort subgroups. Conclusions: paraspinal T1-w MRI could help to distinguish spinal ALS patients from healthy and pathological controls. Specifically, the study of longissimus dorsi could play the role of a diagnostic ALS biomarker.

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