Prevalence and genetic diversity of the lung nematode Eucoleus aerophilus in red foxes (Vulpes vulpes) in Central Europe (Poland) assessed by PCR and flotation.

Eucoleus aerophilus is the causative agent of respiratory capillariosis in wild and domestic carnivores and has sporadically affected humans. To date, this parasite has been detected in 38 countries, confirming its worldwide distribution. The aim of this study was to evaluate the prevalence of E. aerophilus in red fox populations from Central Europe and to describe the sequence variations in the partial cox1 gene of this parasite recovered from stool samples. An investigation was carried out using 342 samples of red foxes faeces investigated via seminested PCR and coproscopy. PCR results confirmed the presence of E. aerophilus DNA in 230 samples and E. boehmi DNA in 14 samples. Molecular analysis of the retrieved sequences revealed 22 haplotypes of E. aerophilus, EaPL1-EaPL22, and 4 haplotypes of E. boehmi, EbPL1-EbPL4. Coproscopic examination revealed that eggs of the Capillariidae family were most prevalent in all regions, with a mean prevalence of 73%. Furthermore, the following eggs were detected: Taeniidae (21.3%), Toxocara spp. (26.3%), Toxascaris leonina (5.3%), Trichuris vulpis (1.5%), trematodes (23.4%), hookworms (2.3%) and Mesocestoides spp. (1.8%). This study evaluated the prevalence of E. aerophilus in red fox populations in selected regions of Poland. To the best of our knowledge, this is the first molecular study on E. aerophilus in Poland.

Gelatin medium for preserving of Trichinella spp. for quality control in laboratories - estimation of larvae viability.

INTRODUCTION AND OBJECTIVE: Official food control laboratories ensure food safety using reliable, validated methods. Council Regulations (EC) No. 853/2004, 854/2004 and 882/2004 of the European Parliament established hygiene rules the production of food of animal origin, together with requirements for official controls. This leads to detailed requirements for Trichinella control set out in Commission Implementing Regulation (EU) 2015/1375 of 10 August 2015. These regulations require the laboratory to participate in proficiency testing (PT) to confirm their competence and improve the quality of testing, and require the PT Organizer to use methods for the preparation and preservation of parasite larvae in order to evaluate and improve detection. Traditional methods of preparing such larvae expose them to rapid degradation, making it necessary to simultaneously isolate the larvae and place them in meatballs to ensure quality. MATERIAL AND METHODS: We developed a technique for preserving of Trichinella spp. for quality control such as PT sample preparation. The procedure protects larvae against toxic oxygen activity and bacterial destruction via a gelatin barrier. To estimate the viability of larvae preserved by this method, gelatin capsules with 10 larvae of T. spiralis in each were stored (4-8 °C) during 45 days of an experiment. Samples were tested at 2 day intervals (3 samples each day of testing). RESULTS: In total, 75 samples were tested. Larvae remained alive up to 3 weeks. The number of living larvae diminished after 27 days through day 43, after which no living larvae were observed. CONCLUSIONS: The gelatin medium procedure facilitated easy, high-throughput sample preparation and supported 100% recovery for 3 weeks. The method allows fast, efficient and accurate PT sample preparation.

Molecular confirmation of the systemic toxoplasmosis in cat.

INTRODUCTION AND OBJECTIVE: Systemic toxoplasmosis with tissue-spread parasites occurring in intermediate hosts may also occur in immunocompromised cats (e.g., infected with FLV or FIV). To the best of our knowledge, no reports have been published on the detection and genotyping of T. gondii DNA in cats with extraintestinal toxoplasmosis in Poland. The article describes the case of the sudden death of 3 out of 4 cats in a cattery, and the detection and molecular characterization of T. gondii DNA detected in the tissues of one of the dead cats. MATERIAL AND METHODS: Samples of brain, lungs, heart, and liver of the cat that died suddenly were examined for the presence of T. gondii DNA (B1 gene) by nested PCR and real-time PCR. DNA positive samples were also genotyped at 12 genetic markers using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) and multilocus sequence typing (MLST). RESULTS: A total of 9 out of the 20 DNA samples were successfully amplified with nested and/or Real-time PCR. DNA from 3 out of 5 types of tested samples were genotyped (brain, heart and muscle). Mn-PCR-RFLP and MLST results revealed type II (and II/III at SAG1) alleles at almost all loci, except a clonal type I allele at the APICO locus. This profile corresponds to the ToxoDB#3 genotype, commonly identified amongst cats in Central Europe. CONCLUSIONS: To the best of our knowledge, this is the first study describing the genetic characteristics of T. gondii population determined in a cat in Poland. These data confirm the importance of this host as a reservoir for this pathogen, and demonstrate the genotypic variation of this parasite. Veterinarians should take into account that cats may develop disseminated toxoplasmosis, and that it is a systemic disease which may lead to the death of the cat, and to transmission of the pathogen to other domestic animals and to humans.

Echinococcus multilocularis genetic diversity based on isolates from pigs confirmed the characteristic haplotype distribution and the presence of the Asian-like haplotype in Central Europe.

Introduction: The aim of the study was to determine the genetic diversity of Echinococcus multilocularis in pigs in highly endemic areas in Poland, as well as to attempt to confirm the occurrence and geographical distribution of haplotypes characteristic for these areas, which were previously described on the basis of examination of adult tapeworms isolated from foxes. Material and Methods: Twenty samples of E. multilocularis larval forms were obtained from pigs' livers in four provinces of Poland. Genetic analyses were conducted on sequences of two mitochondrial genes: cox1 and nad2. Results: Seven haplotypes were found for the cox1 gene (OQ874673-OQ874679) and four haplotypes for nad2 (OQ884981-OQ884984). They corresponded to the haplotypes described earlier in foxes in Poland (some of them differing only in one nucleotide). The analysis showed the presence of the Asian-like haplotype in both the cox1 and nad2 genes. The remaining haplotypes were grouped in the European clade. The geographical distribution of haplotypes identified in the pig samples was noticed to bear a similarity to the distribution of haplotypes previously isolated from foxes in the same regions. Conclusion: The characteristic geographical distribution of E. multilocularis haplotypes in Central Europe (including the presence of the Asian-like haplotype) previously described in the population of definitive hosts (foxes) has now been confirmed by the analysis of samples from non-specific intermediate hosts (pigs).

Occurrence of Eucoleus aerophilus in wild and domestic animals: a systematic review and meta-analysis.

BACKGROUND: Eucoleus aerophilus (syn. Capillaria aerophila) is a nematode with a worldwide geographical distribution. It causes a disease called lung capillariosis by affecting the respiratory tract of wild and domestic animals, and has also occasionally been described in humans. Despite steady increases in knowledge of the morphology of this neglected parasite, many aspects are still poorly understood. Epidemiological data regarding, for example, geographic distribution, range of hosts, clinical relevance and the actual zoonotic potential of this nematode are scarce and incomplete. METHODS: This article is a systematic review based on the screening of three databases (PubMed, Web of Science and Science Direct) to identify eligible studies published from 1973 to the end of 2022. RESULTS: From a total of 606 studies describing the occurrence of E. aerophilus, 141 articles from 38 countries worldwide were included in this meta-analysis, all of which presented results obtained mainly with flotation and necropsy. Due to the occurrence of E. aerophilus in many different species and different matrices (lungs and faeces), we decided to conduct the meta-analysis separately for each species with a given matrix. This systematic review confirmed the status of the Red fox as the main reservoir and main transmitter of E. aerophilus (average prevalence of 43% in faeces and 49% in lungs) and provided evidence of a higher prevalence of E. aerophilus in wild animals in comparison to domestic animals, such as dogs (3% in faeces) and cats (2% in faeces and 8% in lungs). Previous studies have investigated many host-related factors (age, sex, environmental/living conditions) in relation to the prevalence of E. aerophilus, but they show wide variations and no simple relationship has been demonstrates. Furthermore, mixed infections with other pulmonary nematodes, such as Crenosoma vulpis and/or Angiostrongylus vasorum, are reported very frequently, which greatly complicates the diagnosis. CONCLUSIONS: This systematic review focused on identifying data gaps and promoting future research directions in this area. To the best of our knowledge, this is the first systematic review that evaluates and summarizes existing knowledge on the occurrence and prevalence of E. aerophilus in wild and domestic animals originating from different geographical locations worldwide.

Macroscopic sarcocystosis in a pig carcass from an Italian abattoir.

Different food-safety institutions, including the European Food Safety Authority, encourage monitoring and characterising Sarcocystis spp. in animals and foodstuffs; among meat-producing animals, domestic pigs (Sus scrofa domesticus) can host two different Sarcocystis spp., that is Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis. Herein, we report for the first time the presence of macrocysts of Sarcocystis miescheriana in a domestic pig resulting in carcass condemnation. In North-West Italy, in June 2022 the carcass of a clinically healthy sow was condemned due to the detection of multifocal macroscopic whitish fusiform lesions. Affected muscle samples were submitted to histological and molecular analyses targeting the mtDNA cox1 and 18S rRNA genes. At gross examination and histology, well demarcated, oval or elongated macrocysts up to 8 mm in length characterized by a calcified central core surrounded by fibrosis were detected. The molecular amplification and sequencing of the cox1 mtDNA and 18S rRNA genes revealed the presence of Sarcocystis miescheriana DNA in all sampled macrocysts. Our study provides the first molecularly confirmed case of Sarcocystis miescheriana infection in a domestic pig in Italy. The present report highlights the need to increase data related to the occurrence and the prevalence of Sarcocystis spp. in meat-producing animals, and in wild and domestic pigs in particular, taking into account the zoonotic potential of Sarcocystis suihominis and the possible financial losses related to carcass discard due to macroscopic Sarcocystis spp. cysts.

Scheme of Effective Epidemiological Investigations in Trichinella Outbreaks on Pig Farms.

Trichinellosis is a parasitic, zoonotic disease caused by larvae of the genus Trichinella. Infection occurs via the consumption of raw or undercooked meat containing this parasite. Symptoms of the disease manifest as intestinal disorders, followed by facial swelling, fever, muscle pain and other symptoms, eventually leading to neurological and cardiac complications and even death. In Europe, trichinellosis is most often associated with the consumption of meat from wild boars, pigs and horses. In recent years, wild boars that are hunted illegally and not tested for Trichinella spp. have been the most common cause of trichinellosis in humans; however, there have also been cases where infected pigs have been the source of infection. When trichinellosis is suspected in humans, epidemiological measures are taken to identify the source. Similarly, an epidemiological investigation should be initiated whenever Trichinella spp. has been detected in pigs. However, commonly used actions do not provide sufficient data to determine the source of infection for pigs and to prevent further transmission. Therefore, in this article, we propose a scheme for effective epidemiological investigations into Trichinella outbreaks on pig farms that can help trace the transmission mechanisms of the parasite and that takes into account currently available testing tools. The proposed pathway can be easily adopted for epidemiological investigations in routine veterinary inspection work.

Validation Parameters of the Magnetic Stirrer Method for Pooled Sample Digestion for Trichinella spp. in Horse Meat Based on Proficiency Tests Results.

Meat of horses may be infested with nematodes of the genus Trichinella spp. and can cause serious disease in humans. Rules for the carcasses sampling of species susceptible to Trichinella spp. infection and examination are laid down in Commission Regulation 1375/2015, where the magnetic stirrer method for pooled-sample digestion is recommended (Commission Regulation 1478/2020). All personnel involved in the examination should be properly trained and participate in quality control programs. Proficiency tests (PTs) play a key role in the quality verification process. This paper presents the results of PTs organized for 68 Polish laboratories in 2014-2019. Results were assessed qualitatively at three levels of sample contamination (0, 3, 5 larvae) and quantitatively at one level (5 larvae). The laboratories have achieved the average correct qualitative results 100%, 96.2% and 96.8% for the samples contaminated with 0, 3 and 5 larvae, respectively. In the quantitative evaluation, an average 94.1% of the reported results were correct. The data from PTs enabled us to define, for the first time, validation parameters of the digestion method for the horse meat matrix in a large-scale experiment including: specificity (100%), sensitivity (95.6%), accuracy (97.1%), the limit of detection (LOD) (1.14 ≈ 1) and the limit of quantification (LOQ) (3.42 ≈ 3).

Molecular Confirmation of Taenia crassiceps Cysticercosis in a Captive Ring-Tailed Lemur (Lemur catta) in Poland.

(1) Background: Taenia crassiceps is a cosmopolitan tapeworm endemic to the northern hemisphere with an indirect lifecycle. Its definitive hosts are carnivores, and its intermediate hosts are rodents and rabbits. Nonhuman primates in zoos appear to be highly susceptible to T. crassiceps cysticercosis. The aim of this study was to confirm the presence and the molecular characterization of T. crassiceps cysts isolated from a captive ring-tailed lemur. (2) Methods: Surgery revealed multifocal, transparent saccules containing several thin-walled tapeworm cysticerci. In some of the metacestodes, single or multiple exogenous buds from daughter cysticerci were spotted. A molecular analysis was performed to confirm our morphological examinations, using two protocols to obtain the partial nad1 and cox1 genes of the Taenia sp. (3) Results: On the basis of morphological features and molecular analysis, the cysticerci were identified as T. crassiceps metacestodes, and products taken from the PCRs were sequenced. With respect to interpreting the sequencing results of the obtained amplicons, we compared them with data in the GenBank database, proving that, in this case, the causative agent was indeed T. crassiceps. (4) Conclusions: The received data can be used to supplement descriptions of this species. To the best of our knowledge, this is the first case of cysticercosis caused by T. crassiceps in a nonhuman primate in Poland.

Validation of the Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples Based on Proficiency Tests Results.

Trichinellosis is a zoonotic disease caused by the nematodes of the genus Trichinella. Infection takes place through the consumption of infected meat containing live larvae. The only way to prevent the disease is to break its epizootic chain. To ensure effective control of Trichinella spp., a range of preventive and control measures have been undertaken. These efforts have been focused on controlling Trichinella in domestic pigs, the main source of the disease. Artificial digestion is also the reference point for other methods for Trichinella risk control. Descriptive data validation of the digestion assay was presented in 1998 based on results published by scientific laboratories. Herein, we supplement those data by characterizing the method's performance in inter-laboratory comparisons. The source of data was the results of Proficiency Testing conducted in 2015-2019. Samples were contaminated by 0, 1, 3, and 5 larvae. In total, 7580 samples were examined by the laboratories. Based on Proficiency Testing results, the main parameters characterizing the method performance in field conditions were established as follows: specificity, 97.3%; sensitivity, 86.5%; accuracy, 89.2%; uncertainty, 0.3; limit of detection (LOD), 1 larva; and limit of quantification (LOQ), 3 larvae.

Grass Snakes (Natrix natrix) as a Reservoir of Alaria alata and Other Parasites.

The aim of the study was to investigate the occurrence of Alaria alata (Goeze, 1782) in fifty-one grass snakes (Natrix natrix) collected in Gostyninsko-Wloclawski Landscape Park. Each snake was tested for the presence of A. alata mesocercariae using the AMT and MSM methods. 18S ribosomal RNA (18S rRNA), cytochrome C oxidase subunit I (COI) and 28S ribosomal RNA (28S rRNA) genes were amplified by PCR and sequenced for the purpose of species identification. Fifty grass snakes were infected with helminths. The molecular characterization of trematodes allowed us to identify A. alata in 30 snakes (58.8%), Conodiplostomum spathula (Dubois, 1937) in 16 snakes (31.3%), Strigea falconis (Szidat, 1928) in 12 snakes (23.5%), and Neodiplostomum attenuatum (Linstow, 1906) in 2 snakes (3.9%), while, in 4 snakes (7.8%), the trematodes species could not be identified. Based on the analysis of 18S and COI sequences, Crenosoma vulpis (Dujardin, 1845) was identified in four snakes (7.8%), while nematodes collected from three snakes remained unidentified. The tapeworm sample was identified as Ophiotaenia. The obtained results indicate that grass snakes are an excellent vector of A. alata and may be a potential source of infection for mammals, e.g., wild boars and foxes, which results in an increased risk of alariosis for consumers of raw or undercooked game meat.

Proteomic Profiling and In Silico Characterization of the Secretome of Anisakis simplex Sensu Stricto L3 Larvae.

Anisakis simplex sensu stricto (s.s.) L3 larvae are one of the major etiological factors of human anisakiasis, which is one of the most important foodborne parasitic diseases. Nevertheless, to date, Anisakis secretome proteins, with important functions in nematode pathogenicity and host-parasite interactions, have not been extensively explored. Therefore, the aim of this study was to identify and characterize the excretory-secretory (ES) proteins of A. simplex L3 larvae. ES proteins of A. simplex were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the identified proteins were then analyzed using bioinformatics tools. A total of 158 proteins were detected. Detailed bioinformatic characterization of ES proteins was performed, including Gene Ontology (GO) analysis, identification of enzymes, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, protein family classification, secretory pathway prediction, and detection of essential proteins. Furthermore, of all detected ES proteins, 1 was identified as an allergen, which was Ani s 4, and 18 were potential allergens, most of which were homologs of nematode and arthropod allergens. Nine potential pathogenicity-related proteins were predicted, which were predominantly homologs of chaperones. In addition, predicted host-parasite interactions between the Anisakis ES proteins and both human and fish proteins were identified. In conclusion, this study represents the first global analysis of Anisakis ES proteins. The findings provide a better understanding of survival and invasion strategies of A. simplex L3 larvae.

Analysis of a Trichinellosis Outbreak in Poland after Consumption of Sausage Made of Wild Boar Meat.

An outbreak of trichinellosis due to the consumption of sausage made from wild boar meat unexamined for the presence of Trichinella spp. was reported in Poland in December 2020. The outbreak affected eight people. Examination of the sausages made of wild boar meat collected during epidemiological investigation indicated a high level of Trichinella spp. Larvae per gram (>30 lpg) and therefore the threat of an infection in humans after consumption of such product was significant. Over the years, the main source of trichinellosis in Poland has been wild boar meat, and the majority of trichinellosis cases were related to the consumption of traditional raw meat products such as Polish sausage. Taking this into account, there is the need for better education of consumers in the Trichinella spp. endemic regions and among cultures consuming traditional raw meat products.

Results of Proficiency Testing for Trichinella in Poland, 2015-2019.

Trichinellosis is a zoonotic meat-borne disease caused by the nematodes of the genus Trichinella. Meat containing live Trichinella larvae is a source of infection. The examination of meat for Trichinella was introduced in 1869, but the digestion method for this did not appear in Poland until the late 1970s. Nowadays, the meat of all food animals susceptible to Trichinella spp. is examined in the frame of official post mortem control with the digestion method. The majority of laboratories in Poland meet the requirements of the ISO/IEC 17025 Standard (352 field laboratories). Laboratory personnel participate in quality control programs. This paper presents the results of proficiency tests (PTs) organized within 2015-2019 in Poland. Over this period, the laboratories examined 7580 samples (contamination levels: zero, one, three, and five larvae). Each laboratory was provided with a set of samples (one negative and three positive). Over 95% of the samples were considered correct in qualitative assessments, though the results of the quantitative evaluations were slightly lower, with 89% of samples being considered correct. Based on a sample evaluation, 88% of laboratories passed the PT comparison. A slight decrease was observed in the examination of samples spiked with five larvae, and great progress was achieved in samples containing three larvae. Low levels of sample contamination are sought after in laboratories but may make evaluations difficult. For this reason, we must consider increasing the number of larvae added to the samples in the next PTs.

Trichinella Outbreaks on Pig Farms in Poland in 2012-2020.

Trichinella nematodes continue to circulate in various hosts both in the domestic and sylvatic cycles. In the majority of countries in Europe, wild boars have been noticed as a primary source of Trichinella spp. infections in humans. However, in some regions, the meat of pigs containing Trichinella spp. larvae can still be a cause of trichinellosis. Therefore, in the present study, we aimed to determine and present actual data on the occurrence of Trichinella spp. on pig farms (Sus scrofa f. domestica) in Poland. In this study, over 194 million pigs, slaughtered for commercial and personal purposes between 2012 and 2020, were tested with a digestion method according to the official rules for Trichinella control. Positive results were noticed in 172 pigs which gives an overall prevalence of 0.000088%. On seven farms, rats (Rattus norvegicus) infected with Trichinella spp. were also discovered. The species identification showed pigs were infected with Trichinella spiralis on 26 farms, and on four farms pigs with Trichinella britovi infections were found. Therefore, it is important to constantly monitor pigs for the presence of these parasites, especially in view of the growing interest in organic meat originated from ecological farms.

Alaria alata in Terms of Risks to Consumers' Health.

Alaria alata flukes are cosmopolitan parasites. In Europe, the definitive hosts are red foxes (Vulpes vulpes), wolves (Canis lupus), and raccoon dogs (Nyctereutes procyonoides), as well as animals that belong to the Felidae family. Intermediate hosts, such as snails and frogs, are the sources of infection for definitive hosts. The developmental stages of A. alata mesocercariae may occur in paratenic hosts, including many species of mammals, birds, and reptiles, as well as in wild boars (Sus scrofa), which are important from the zoonotic point of view. Because there are no regulations concerning the detection of A. alata in meat, this fluke is usually detected during official obligatory Trichinella spp. inspections. However, a method dedicated to A. alata detection was developed. The growing popularity of game and organic meat has led to an increased risk of food-associated parasitic infections, including alariosis, which is caused by the mesocercarial stage of A. alata. The aim of this article is to highlight the problem of A. alata as an emerging parasite, especially in the terms of the increasing market for game and organic meats that have been processed with traditional methods, often without proper heat treatment.

Molecular Characterization of Enterotoxigenic Staphylococcus aureus Isolated from Raw Cow Milk in Poland.

The aim of this study was to characterize enterotoxigenic Staphylococcus aureus recovered from raw cow milk from two geographical regions of Poland using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Among 610 samples tested, 229 (37.5%) were positive for S. aureus and 30 (13.1%) of them possessed at least one gene encoding enterotoxins. The sec marker was the most commonly identified (12; 40.0% isolates), followed by the sed (9; 30.0%), sea (6; 20.0%), and seb (1; 3.3%) genes. Some S. aureus possessed a combination of the sea and sec or sea and seb toxin markers. Only two (6.7%) of the enterotoxin gene-positive isolates were not able to produce enterotoxins in vitro. Genotypic analysis with the PFGE method of a total of 50 toxigenic S. aureus isolates from the present and previous studies identified 16 clonal groups. Furthermore, MLST revealed the presence of 15 sequence types with the most common being ST45 and ST1. The results of this study indicate that raw cow milk may be a source of S. aureus with classical enterotoxin genes, which may pose a potential threat for the consumers' safety.

Occurrence of Staphylococcus aureus on Farms with Small Scale Production of Raw Milk Cheeses in Poland.

This paper describes the results of a 3-year study on the prevalence, enterotoxinogenicity and resistance to antimicrobials of S. aureus isolated on dairy farms with small scale production of raw cow milk cheeses. The samples of raw milk, semi-finished products and the final products as well as swabs were collected between 2011 and 2013 from nine dairy farms in Poland. A total of 244 samples were examined, of which 122 (50.0%) were contaminated with S. aureus including 18 of 26 (69.2%) mature cheese samples with log10 CFU g(-1) between <1- and 7.41. In swabs collected from the staff and production environment the highest contamination rate with coagulase positive staphylococci (CPS) was detected on hands of cheese makers (4.34 log10 CFU/swab). None of the cheese samples contaminated with CPS contained staphylococcal enterotoxins (SEs). However, 55 of 122 (45.1%) S. aureus isolates possessed SEs genes, mainly (26 of 55; 47.3%) a combination of the sed, sej and ser genes. Furthermore, the sep (15 of 55; 27.3%) as well as seg and sei (9 of 55; 16.4%) genes were also identified. The remaining S. aureus isolates possessed the sea gene (one isolate), the combination of sec, seg and sei (three isolates) as well as the sed, sej, sep and ser markers together (one CPS). Resistance to penicillin (62 of 122 isolates; 50.8%) was the most common among the tested isolates. Some CPS were also resistant to chloramphenicol (7; 5.7%) and tetracycline (5; 4.1%). The obtained results indicated that the analyzed cheeses were safe for consumers. To improve the microbiological quality of traditional cheese products more attention should be paid to animal welfare and hygiene practices during the process of cheese manufacturing in some dairy farms.