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In 2004, a new concept was introduced for simplifying identification of larvae of the common nematodes of cattle, sheep and goats that comprises estimates of the lengths of the sheath tail extensions of infective third-stage larvae (L3) of each genus and/or species to that of Trichostrongylus spp., instead of having to be dependent only on measurements in micrometre. For example, if the mean length of the sheath tail extension (the extension of the sheath caudad, beyond the caudal tip of the larva) of Trichostrongylus colubriformis and Trichostrongylus axei is assumed to be ‘X’, then that of Haemonchus contortus is 2.0–2.7 ‘X’ – a difference that is not difficult to estimate. An additional new approach suggested now, particularly for L3 of species and/or genera difficult to differentiate (such as Chabertia ovina and Oesophagostomum columbianum), is to estimate the proportion of the larval sheath tail extension comprising a terminal thin, whip-like filament. For the experienced person, it is seldom necessary to measure more than one or two sheath tail extensions of L3 in a mixed culture, because the identity of most of the remaining L3 can thereafter be estimated in relation to those measured, without having to take further measurements. The aim of this article was to present the novel approach in the form of a working guide for routine use in the laboratory. To facilitate identification, figures and a separate organogram for each of small ruminants and cattle have been added to illustrate the distinguishing features of the common L3.

Abstract Background Gastrointestinal nematodes (GIN) are ubiquitous in small ruminant farming, representing a major health and production concern. Given their differences in pathogenicity and the current problems regarding anthelmintic resistance, specific diagnosis of GIN is of significant importance. At present, the most widely applied method for this entails culture and microscopic analysis of third-stage larvae, allowing for identification at least to the genus level. Overall, a variety of keys for microscopic analysis have been published, showing substantial variation. Given this fact, this study aimed to produce a practical and updated guide for the identification of infective ovine GIN larvae. Methods Using existing keys and protocols, a total of 173larvae of the most common species/genera of ovine GIN from pooled faecal samples from Sardinia (Italy) were identified and extracted, and further individual molecular identification was performed. Morphometric and morphological data as well as high-quality photographs were collected and combined to produce the final guide. Results GIN microscopically and molecularly identified during this research include Trichostrongylus spp., Teladorsagia circumcincta , Haemonchus contortus , Cooperia curticei , and Chabertia ovina. Based on microscopic analysis, 73.5% of the larvae were correctly identified. Based on sheathed tail length, 91.8% were correctly classified into their respective preliminary groups. Conclusions It is crucial for the microscopic identification of infectious GIN larvae to examine each larva in its entirety and thus to take multiple characteristics into account to obtain an accurate diagnosis. However, a preliminary classification based on sheathed tail length (resulting in three groups: A, short; B, medium; C, long) was found to be effective. Further identification within group A can be achieved based on the presence of a cranial inflexion, caudal tubercles and full body measurements ( Trichostrongylus spp. < 720 µm, T. circumcincta ≥ 720 µm). Larvae within group B can be differentiated based on sheathed tail morphometry ( H. contortus > 65 µm, C. curticei ≤ 65 µm), the presence of cranial refractile bodies, total body length measurements ( H. contortus ≤ 790 µm, C. curticei > 790 µm) and shape of the cranial extremity. Finally, all characteristics proposed for the differentiation between Oesophagostomum spp. and C. ovina larvae (group C) were found to have considerable restrictions. Graphical abstract

The fruit fly Drosophila melanogaster is a vital model for studying the microbiome due to the availability of genetic resources and procedures. To understand better the importance of microbial composition in shaping immune modulation, we can investigate the role of the microbiota through parasitic infection. For this, we use entomopathogenic nematodes (EPN) of the genus Steinernema which exhibit remarkable ability to efficiently infect a diverse array of insect species, facilitated by the mutualistic bacteria Xenorhabdus found within their gut. To examine the microbiome changes in D. melanogaster larvae in response to Steinernema nematode infection, D. melanogaster late second to early third instar larvae were exposed separately to S. carpocapsae and S. hermaphroditum infective juveniles. We have found that S. carpocapsae infective juveniles are more pathogenic to D. melanogaster larvae compared to the closely related S. hermaphroditum . Our microbiome analysis also indicates substantial changes in the size and composition of the D. melanogaster larval microbiome during infection with either nematode species compared to the uninfected controls. Our results serve as a foundation for future studies to elucidate the entomopathogenic-specific effector molecules that alter the D. melanogaster microbiome and understand the role of the microbiome in regulating insect anti-nematode immune processes.

Resistance to fenbendazole, ivermectin, and moxidectin was explored by a fecal egg count reduction test in four meat sheep flocks in southwestern France where anthelmintic resistance was suspected. The FECR test results of the present study confirmed the presence of benzimidazole resistance in three out of the four farms and the presence of ivermectin resistance in one flock. In addition, a suspicion of moxidectin resistance was shown in this latter farm. Both conventional morphological and molecular identifications were performed on larval cultures before and after the treatment in the studied farms. A high positive correlation was found between the number of larvae counted under binocular microscope and the number of larvae estimated by the qPCR analysis (R2 = 0.88) and a high Cohen’s Kappa value (0.91) in the detection of strongylid larvae in larval cultures. According to qPCR results, Trichostrongylus species demonstrated high levels of BZ resistance and Teladorsagia circumcincta was involved in the IVM resistance in one farm. The molecular procedures used in this study have the potential to be beneficial for anthelmintic resistance surveillance in sheep industry.

Background Nippostrongylus brasiliensis —a nematode of rodents—is commonly used as a model to study the immunobiology of parasitic nematodes. It is a member of the Strongylida—a large order of socioeconomically important parasitic nematodes of animals. Lipids are known to play essential roles in nematode biology, influencing cellular membranes, energy storage and/or signalling. Methods The present investigation provides a comprehensive, untargeted lipidomic analysis of four developmental stages/sexes (i.e. egg, L3, adult female and adult male stages) of N. brasiliensis utilising liquid chromatography coupled to mass spectrometry. Results We identified 464 lipid species representing 18 lipid classes and revealed distinct stage-specific changes in lipid composition throughout nematode development. Triacylglycerols (TGs) dominated the lipid profile in the egg stage, suggesting a key role for them in energy storage at this early developmental stage. As N. brasiliensis develops, there was a conspicuous transition toward membrane-associated lipids, including glycerophospholipids (e.g. PE and PC) and ether-linked lipids, particularly in adult stages, indicating a shift toward host adaptation and membrane stabilisation. Conclusions We provide a comprehensive insight into the lipid composition and abundance of key free-living and parasitic stages of N. brasiliensis . This study provides lipidomic resources to underpin the detailed exploration of lipid biology in this model parasitic nematode. Graphical Abstract

Maintenance of laboratory colonies of insects and other arthropod pests offers significant research advantages. The availability, age, sex, housing conditions, nutrition, and relative uniformity over time of biological material for research facilitate comparison of results between experiments that would otherwise be difficult or impossible. A laboratory research colony of Phlebotomus papatasi (Scopoli), old world sand flies, was maintained with high-colony productivity for a number of years, but within a relatively short (4–6 mo) time period, colony productivity declined from over 10,000 flies per week to less than 100 per week. Mites and nematodes were both visible in the larval medium; however, the mites had been present throughout high productivity periods; therefore, it seemed reasonable to investigate the nematodes. PCR amplification of 18S rRNA yielded a clean cDNA sequence identified by BLAST search as Procephalobus sp. 1 WB-2008 (Rhabditida: Panagrolaimidae) small subunit ribosomal RNA gene, GenBank EU543179.1, with 475/477 nucleotide identities. Nematode samples were collected and identified as Tricephalobus steineri, (Andrássy, 1952) Rühm, 1956 (Rhabditida: Panagrolaimidae) based on morphological characteristics of the esophagus and the male copulatory apparatus. Mites (Tyrophagus putrescentiae [Acariformes: Acaridae]) may have played an additional predatory role in the loss of sand fly colony productivity. We hypothesized that the origin of the nematode infestation was rabbit dung from a local rabbitry used in preparation of the larval medium. Colony productivity was fully restored within 3 mo (two sand fly generational periods) by replacement of the rabbit dung from a clean source for use to prepare sand fly larval medium.

Background Gastrointestinal nematode (GIN) epidemiology is changing in many regions of the world due to factors such as global warming and emerging anthelmintic resistance. However, the dynamics of these changes in northern continental climate zones are poorly understood due to a lack of empirical data. Methods We studied the accumulation on pasture of free-living infective third-stage larvae (L3) of different GIN species from fecal pats deposited by naturally infected grazing cattle. The field study was conducted on three organic farms in Alberta, western Canada. Grass samples adjacent to 24 fecal pats were collected from each of three different pastures on each farm. Internal transcribed spacer-2 nemabiome metabarcoding was used to determine the GIN species composition of the harvested larvae. The rotational grazing patterns of the cattle ensured that each pasture was contaminated only once by fecal pat deposition. This design allowed us to monitor the accumulation of L3 of specific GIN species on pastures under natural climatic conditions without the confounding effects of pasture recontamination or anthelmintic treatments. Results In seven out of the nine pastures, grass L3 counts peaked approximately 9 weeks after fecal deposition and then gradually declined. However, a relatively large number of L3 remained in the fecal pats at the end of the grazing season. Nemabiome metabarcoding revealed that Cooperia oncophora and Ostertagia ostertagi were the two most abundant species on all of the pastures and that the dynamics of larval accumulation on grass were similar for both species. Daily precipitation and temperature across the whole sampling period were similar for most of the pastures, and multiple linear regression showed that accumulated rainfall 1 week prior to sample collection had a significant impact on the pasture L3 population, but accumulated rainfall 3 weeks prior to sample collection did not. Conclusions The results suggest that the pasture L3 population was altered by short-term microclimatic conditions conducive for horizontal migration onto grass. Overall, the results show the importance of the fecal pat as a refuge and reservoir for L3 of cattle GIN on western Canadian pastures, and provide an evidence base for the risk assessment of rotational grazing management in the region. Graphical Abstract

Human larva migrans is a zoonotic disease caused by larvae of various nematode species, with all previously confirmed pathogens originating from mammalian reservoirs. In 2020, a case series in Vietnam reported instances of cutaneous larva migrans caused by avian eyeworm larvae of the Oxyspirura genus. This nematode genus, belonging to the Thelaziidae family, includes 84 species that mainly parasitize the eyes of various bird species. They utilize different arthropod intermediate hosts, such as cockroaches, grasshoppers, and crickets, in their life cycle. Although the 18S sequences of the larvae were analyzed, the precise identification of the pathogen at the species level remains inconclusive. This study aims to identify the exact causative agent at the species level by analyzing larvae from a patient and adult eyeworms collected from chickens raised in his family using morphological and molecular methods. Molecular analysis of 18S rDNA, ITS2, and cox 1 sequences revealed genetic identity between the larva from the patient and adult eyeworms from chickens, as well as with Oxyspirura mansoni from Thailand and Bangladesh. Additionally, morphological examinations further confirmed the adult chicken eyeworms as Oxyspirura mansoni . These findings confirm that O. mansoni larvae are responsible for human larva migrans, marking the first confirmation of nematode larvae from birds (chickens) as a pathogen causing an emerging neglected tropical disease in humans. Given the widespread distribution of this nematode, further research is crucial to investigate this neglected disease, not only in Vietnam but also in other regions. Understanding appropriate treatments and transmission routes is essential to prevent infections in both chickens and humans.

Crenosoma vulpis is a metastrongyloid nematode primarily associated with respiratory tract infections of red foxes in North America and Europe. Sporadic cases have also been reported in domestic dogs. The present study aimed to provide morphological, molecular, and epidemiological data on the geographical distribution of this nematode throughout Italy. From 2012 to 2014, 12 of the 138 foxes examined, three dogs and one badger scored positive for C. vulpis. Forty adults were isolated from foxes and the badger, whereas first-stage larvae were detected in the three dogs. All specimens were morphologically identified as C. vulpis, and 28 nematodes were also molecularly characterized by sequencing mitochondrial (12S ribosomal DNA (rDNA)) and nuclear (18S rDNA) ribosomal genes. Four haplotypes were identified based on the 12S rDNA target gene, with the most representative (78.5 %) designated as haplotype I. No genetic variability was detected for the 18S rDNA gene. The molecular identification was consistent with the distinct separation of species-specific clades inferred by the phylogenetic analyses of both mitochondrial and ribosomal genes. Data herein reported indicates that C. vulpis has a wide distribution in foxes from southern Italy, and it also occurs in dogs from southern and northern regions of the country. Practitioners should consider the occurrence of this nematode in the differential diagnosis of canine respiratory disease, particularly in dogs living close to rural areas where foxes are present.

Anisakis simplex, Pseudoterranova decipiens, and Contracaecum osculatum third-stage larvae (L3) are fish-borne nematodes that can cause human anisakidosis. Although A. simplex is a known source of allergens, knowledge about the allergic potential of P. decipiens and C. osculatum is limited. Therefore, we performed comparative proteomic profiling of A. simplex, P. decipiens, and C. osculatum L3 larvae using liquid chromatography–tandem mass spectrometry. In total, 645, 397, and 261 proteins were detected in A. simplex, P. decipiens, and C. osculatum L3 larvae, respectively. Western blot analysis confirmed the cross-reactivity of anti-A. simplex immunoglobulin (Ig)G antibodies with protein extracts from P. decipiens and C. osculatum L3 larvae. The identified proteins of the Anisakidae proteomes were characterized by label-free quantification and functional analysis, and proteins involved in many essential biological mechanisms, such as parasite survival, were identified. In the proteome of A. simplex 14, the following allergens were identified: Ani s 1, Ani s 2 (2 isomers), Ani s 3 (2 isomers), Ani s 4, Ani s 8, Ani s 9, Ani s 10, Ani s 11-like, Ani s 13, Ani s fructose 1,6-bisphosphatase, Ani s phosphatidylethanolamine-binding protein (PEPB), and Thu a 3.0101. The following 8 allergens were detected in P. decipiens: Ani s 2, Ani s 3 (2 isomers), Ani s 5, Ani s 8, Ani s 9, Ani s PEPB, and Ani s troponin. In C. osculatum 4, the following allergens were identified: Ani s 2, Ani s 5, Ani s 13, and Asc l 3. Furthermore, 28 probable allergens were predicted in A. simplex and P. decipiens, whereas in C. osculatum, 25 possible allergens were identified. Among the putative allergens, heat shock proteins were most frequently detected, followed by paramyosin, peptidyl-prolyl cis-trans isomerase, enolase, and tropomyosin. We provide a new proteomic data set that could be beneficial for the discovery of biomarkers or drug target candidates. Furthermore, our findings showed that in addition to A. simplex, P. decipiens and C. osculatum should also be considered as potential sources of allergens that could lead to IgE-mediated hypersensitivity.