First detection of kdr L1014F allele in Anopheles ziemanni and Anopheles pharoensis in Cameroon and distribution of the allele in members of the Anopheles gambiae complex.

BACKGROUND: Knockdown resistance (kdr) is one of the primary resistance mechanisms present in anopheline species. Although this mutation is largely spread across the Anopheles gambiae s.l. members, its prevalence in other species is still not well documented. METHODS: The present study investigated the distribution and allelic frequencies of kdr in An. gambiae s.l., An. pharoensis, and An. ziemanni samples collected in 2022 and 2023 in nine sites spread across five ecogeographical settings in Cameroon. Members of the An. gambiae complex were identified molecularly by polymerase chain reaction (PCR). kdr L1014F and L1014S alleles were screened by PCR and confirmed by sequencing. RESULTS: An. gambiae (49.9%), An. coluzzii (36.5%), and An. arabiensis (13%) were identified, and the frequency of the kdr L1014F was high in both An. gambiae and An. coluzzii in all sites. The kdr L1014F allele was detected for the first time in 8 out of 14 An. ziemanni samples examined and in 5 out of 22 An. pharoensis samples examined. The kdr L1014S allele was scarce and found only in the heterozygote "RS" state in An. arabiensis and An. gambiae in Yangah and Santchou. CONCLUSIONS: The present study sheds light on the rapid expansion of the kdr L1014F allele in malaria vectors in Cameroon and stresses the need for surveillance activities also targeting secondary malaria vectors to improve the control of malaria transmission.

Wolbachia and Asaia Distribution among Different Mosquito Vectors Is Affected by Tissue Localization and Host Species.

Microbial communities play an important role in the fitness of mosquito hosts. However, the factors shaping microbial communities in wild populations, with regard to interactions among microbial species, are still largely unknown. Previous research has demonstrated that two of the most studied mosquito symbionts, the bacteria Wolbachia and Asaia, seem to compete or not compete, depending on the genetic background of the reference mosquito host. The large diversity of Wolbachia-Asaia strain combinations that infect natural populations of mosquitoes may offer a relevant opportunity to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction. We surveyed Wolbachia and Asaia in 44 mosquito populations belonging to 11 different species of the genera Anopheles, Aedes, and Culex using qualitative PCR. Through quantitative PCR, the amounts of both bacteria were assessed in different mosquito organs, and through metagenomics, we determined the microbiota compositions in some selected mosquito populations. We show that variation in microbial community structure is likely associated with the species/strain of mosquito, its geographical position, and tissue localization. Together, our results shed light on the interactions among different bacterial species in the microbial communities of mosquito vectors, and this can aid the development and/or improvement of methods for symbiotic control of insect vectors.

De novo genome assembly of the invasive mosquito species Aedes japonicus and Aedes koreicus.

BACKGROUND: Recently, two invasive Aedes mosquito species, Ae. japonicus and Ae. koreicus, are circulating in several European countries posing potential health risks to humans and animals. Vector control is the main option to prevent mosquito-borne diseases, and an accurate genome sequence of these mosquitoes is essential to better understand their biology and to develop effective control strategies. METHODS: A de novo genome assembly of Ae. japonicus (Ajap1) and Ae. koreicus (Akor1) has been produced based on a hybrid approach that combines Oxford Nanopore long-read and Illumina short-read data. Their quality was ascertained using various metrics. Masking of repetitive elements, gene prediction and functional annotation was performed. RESULTS: Sequence analysis revealed a very high presence of repetitive DNA and, among others, thermal adaptation genes and insecticide-resistance genes. Through the RNA-seq analysis of larvae and adults of Ae. koreicus and Ae. japonicus exposed to different temperatures, we also identified genes showing a differential temperature-dependent activation. CONCLUSIONS: The assembly of Akor1 and Ajap1 genomes constitutes the first updated collective knowledge of the genomes of both mosquito species, providing the possibility of understanding key mechanisms of their biology such as the ability to adapt to harsh climates and to develop insecticide-resistance mechanisms.

Tripartite interactions comprising yeast-endobacteria systems in the gut of vector mosquitoes.

It is shown that bacteria use yeast as a niche for survival in stressful conditions, therefore yeasts may act as temporary or permanent bacterial reservoirs. Endobacteria colonise the fungal vacuole of various osmotolerant yeasts which survive and multiply in sugar-rich sources such as plant nectars. Nectar-associated yeasts are present even in the digestive system of insects and often establish mutualistic symbioses with both hosts. Research on insect microbial symbioses is increasing but bacterial-fungal interactions are yet unexplored. Here, we have focused on the endobacteria of Wickerhamomyces anomalus (formerly Pichia anomala and Candida pelliculosa), an osmotolerant yeast associated with sugar sources and the insect gut. Symbiotic strains of W. anomalus influence larval development and contribute digestive processes in adults, in addition to exerting wide antimicrobial properties for host defence in diverse insects including mosquitoes. Antiplasmodial effects of W. anomalus have been shown in the gut of the female malaria vector mosquito Anopheles stephensi. This discovery highlights the potential of utilizing yeast as a promising tool for symbiotic control of mosquito-borne diseases. In the present study, we have carried out a large Next Generation Sequencing (NGS) metagenomics analysis including W. anomalus strains associated with vector mosquitoes Anopheles, Aedes and Culex, which has highlighted wide and heterogeneous EB communities in yeast. Furthermore, we have disclosed a Matryoshka-like association in the gut of A stephensi that comprises different EB in the strain of W. anomalus WaF17.12. Our investigations started with the localization of fast-moving bacteria-like bodies within the yeast vacuole of WaF17.12. Additional microscopy analyses have validated the presence of alive intravacuolar bacteria and 16S rDNA libraries from WaF17.12 have identified a few bacterial targets. Some of these EB have been isolated and tested for lytic properties and capability to re-infect the yeast cell. Moreover, a selective competence to enter yeast cell has been shown comparing different bacteria. We suggested possible tripartite interactions among EB, W. anomalus and the host, opening new knowledge on the vector biology.

N400 differences between physical and mental metaphors: The role of Theories of Mind.

Whether Theory of Mind (ToM) contributes to metaphor understanding has been largely investigated in language acquisition and decay. Yet we know very little about the role of ToM in real-time processing of metaphors in neurotypical adults. Here, we tested the relationship between ToM and metaphor through Event Related Potentials (ERPs) by capitalizing on the difference between metaphors inviting inferences on physical (Boxers are pandas) vs. mental aspects (Teachers are books). Physical metaphors involved a larger and sustained negativity compared to mental ones. This pattern resembled concreteness effects and suggests that physical metaphors may benefit from both verbal and perceptual information. Moreover, higher scores in the Reading the Mind in the Eyes Test (RMET), but not in the Animation task, were associated with a reduction of the N400 amplitude for both physical and mental metaphors. When exploring the ERP temporal trajectory with Generalized Additive Mixed Modeling, earlier differences between metaphors characterized individuals with higher RMET scores. Among the various ToM components, thus, emotion recognition seems to be involved in the processing of metaphors in general, with an earlier impact on the mental type. These findings highlight the multifaceted nature of metaphor, at the crossroad of language, social and perceptual experience.

Bacterial Symbionts in Ceratitis capitata.

Ceratitis capitata (Diptera: Tephritidae) is responsible for extensive damage in agriculture with important economic losses. Several strategies have been proposed to control this insect pest including insecticides and the Sterile Insect Technique. Traditional control methods should be implemented by innovative tools, among which those based on insect symbionts seem very promising. Our study aimed to investigate, through the 16S Miseq analysis, the microbial communities associated with selected organs in three different medfly populations to identify possible candidates to develop symbiont-based control approaches. Our results confirm that Klebsiella and Providencia are the dominant bacteria in guts, while a more diversified microbial community has been detected in reproductive organs. Concertedly, we revealed for the first time the presence of Chroococcidiopsis and Propionibacterium as stable components of the medfly's microbiota. Additionally, in the reproductive organs, we detected Asaia, a bacterium already proposed as a tool in the Symbiotic Control of Vector-Borne Diseases. A strain of Asaia, genetically modified to produce a green fluorescent protein, was used to ascertain the ability of Asaia to colonize specific organs of C. capitata. Our study lays the foundation for the development of control methods for C. capitata based on the use of symbiont bacteria.

Wolbachia in Aedes koreicus: Rare Detections and Possible Implications.

The emerging distribution of new alien mosquito species was recently described in Europe. In addition to the invasion of Aedes albopictus, several studies have focused on monitoring and controlling other invasive Aedes species, as Aedes koreicus and Aedes japonicus. Considering the increasing development of insecticide resistance in Aedes mosquitoes, new control strategies, including the use of bacterial host symbionts, are proposed. However, little is known about the bacterial communities associated with these species, thus the identification of possible candidates for Symbiotic Control is currently limited. The characterization of the natural microbiota of field-collected Ae. koreicus mosquitoes from North-East Italy through PCR screening, identified native infections of Wolbachia in this species that is also largely colonized by Asaia bacteria. Since Asaia and Wolbachia are proposed as novel tools for Symbiotic Control, our study supports their use for innovative control strategies against new invasive species. Although the presence of Asaia was previously characterized in Ae. koreicus, our study characterized this Wolbachia strain, also inferring its phylogenetic position. The co-presence of Wolbachia and Asaia may provide additional information about microbial competition in mosquito, and to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction in Ae. koreicus.

Formulation and Safety Tests of a Wickerhamomyces anomalus-Based Product: Potential Use of Killer Toxins of a Mosquito Symbiotic Yeast to Limit Malaria Transmission.

Wickerhamomyces anomalus strain WaF17.12 is a yeast with an antiplasmodial property based on the production of a killer toxin. For its symbiotic association with Anopheles mosquitoes, it has been proposed for the control of malaria. In an applied view, we evaluated the yeast formulation by freeze-drying WaF17.12. The study was carried out by comparing yeast preparations stored at room temperature for different periods, demonstrating that lyophilization is a useful method to obtain a stable product in terms of cell growth reactivation and maintenance of the killer toxin antimicrobial activity. Moreover, cytotoxic assays on human cells were performed, showing no effects on the cell viability and the proinflammatory response. The post-formulation effectiveness of the killer toxin and the safety tests indicate that WaF17.12 is a promising bioreagent able to impair the malaria parasite in vector mosquitoes.

Phylogenomics Reveals that Asaia Symbionts from Insects Underwent Convergent Genome Reduction, Preserving an Insecticide-Degrading Gene.

The mosquito microbiota is composed of several lineages of microorganisms whose ecological roles and evolutionary histories have yet to be investigated in depth. Among these microorganisms, Asaia bacteria play a prominent role, given their abundance in the gut, reproductive organs, and salivary glands of different mosquito species, while their presence has also been reported in several other insects. Notably, Asaia has great potential as a tool for the control of mosquito-borne diseases. Here, we present a wide phylogenomic analysis of Asaia strains isolated from different species of mosquito vectors and from different populations of the Mediterranean fruit fly (medfly), Ceratitis capitata, an insect pest of worldwide economic importance. We show that phylogenetically distant lineages of Asaia experienced independent genome reductions, despite following a common pattern, characterized by the early loss of genes involved in genome stability. This result highlights the role of specific metabolic pathways in the symbiotic relationship between Asaia and the insect host. Finally, we discovered that all but one of the Asaia strains included in the study possess the pyrethroid hydrolase gene. Phylogenetic analysis revealed that this gene is ancestral in Asaia, strongly suggesting that it played a role in the establishment of the symbiotic association between these bacteria and the mosquito hosts. We propose that this gene from the symbiont contributed to initial pyrethroid resistance in insects harboring Asaia, also considering the widespread production of pyrethrins by several plants.IMPORTANCE We have studied genome reduction within several strains of the insect symbiont Asaia isolated from different species/strains of mosquito and medfly. Phylogenetically distant strains of Asaia, despite following a common pattern involving the loss of genes related to genome stability, have undergone independent genome reductions, highlighting the peculiar role of specific metabolic pathways in the symbiotic relationship between Asaia and its host. We also show that the pyrethroid hydrolase gene is present in all the Asaia strains isolated except for the South American malaria vector Anopheles darlingi, for which resistance to pyrethroids has never been reported, suggesting a possible involvement of Asaia in determining resistance to insecticides.

Vowel variability and contrast in Childhood Apraxia of Speech: acoustics and articulation.

Phoneme production may be affected by limited speech motor control in Childhood Apraxia of Speech (CAS), with a general instability of acoustic targets across multiple repetitions of speech stimuli. This acoustic and Ultrasound Tongue Imaging (UTI) study shows that increased variability and reduction of contrast in vowel production is found in native Italian speakers with CAS, particularly as far as the height dimension is concerned. The data suggest that vowel production should play a major role in CAS diagnosis and treatment. Moreover, this study shows that a combined acoustic and articulatory approach allows direct observation of lingual dynamics together with an estimation of changes in the acoustic dimension. The two dimensions are shown not to correspond in a straightforward way in the speech of children with CAS, and encourage consideration of articulatory compensation strategies aimed at saving the acoustic identity of vowels.

Mosquitocidal and Anti-Inflammatory Properties of The Essential Oils Obtained from Monoecious, Male, and Female Inflorescences of Hemp (Cannabis sativa L.) and Their Encapsulation in Nanoemulsions.

Among the various innovative products obtainable from hemp (Cannabis sativa L.) waste biomass originating from different industrial processes, the essential oil (EO) deserves special attention in order to understand its possible application in different fields, such as cosmetics, pharmaceuticals, and botanical insecticides. For the purpose, in the present work, we studied the chemical composition of EOs obtained from different hemp varieties, namely Felina 32 and Carmagnola Selezionata (CS) using monoecious, male, and female inflorescences, and we evaluated their mosquitocidal activities on larvae and pupae of two main malaria vectors, Anopheles gambiae and An. stephensi. Then, in order to evaluate the safe use of hemp EOs for operators, the potential pro- or anti-inflammatory effect of hemp EOs together with their toxicological profile were determined on dermal fibroblasts and keratinocytes. Given the promising results obtained by insecticidal and anti-inflammatory studies, a preliminary evaluation of EOs encapsulation into nanoemulsions (NEs) has been performed with the aim to develop a formulation able to improve their poor physicochemical stability. Felina 32 and CS inflorescences provided EOs with an interesting chemical profile, with monoterpene and sesquiterpene hydrocarbons as the major components. This study highlighted the potential application of male inflorescences, which are usually discharged during hemp product processing. These EOs could be exploited as potential sustainable and eco-friendly insecticides, given their capability to be toxic against mosquitoes and the possibility to use them to prepare stable and safe formulations. The LC50 values found in this study (<80 ppm) are lower, on average, than those of many plant EOs, with the advantage of using an industrial waste product. From MTT assay and gene and protein expression analysis, EOs showed no cytotoxicity at the appropriate doses and exerted an anti-inflammatory effect on the human cell lines tested. These findings encourage further applied research on hemp EOs in order support their industrial exploitation.

Inhibition of Asaia in Adult Mosquitoes Causes Male-Specific Mortality and Diverse Transcriptome Changes.

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females.

Wickerhamomyces anomalus in Mosquitoes: A Promising Yeast-Based Tool for the "Symbiotic Control" of Mosquito-Borne Diseases.

The ascomycete yeast Wickerhamomyces anomalus is a mutualistic symbiont of different insects, including diptera vectors of diseases. Although fungal symbioses have been so far poorly characterized, the topic is gaining attention as yeast-insect interactions can provide pivotal information on insect biology, such as their environmental adaptation or vectorial capability. We review the symbiosis between W. anomalus and mosquitoes, which implies nutritional and protective functions. Furthermore, we focus on antiplasmodial effects of W. anomalus in malaria vectors and discuss the yeast potential for the "symbiotic control" (SC) of mosquito-borne diseases (MBDs).

Asaia Activates Immune Genes in Mosquito Eliciting an Anti-Plasmodium Response: Implications in Malaria Control.

In mosquitoes, the discovery of the numerous interactions between components of the microbiota and the host immune response opens up the attractive possibility of the development of novel control strategies against mosquito borne diseases. We have focused our attention to Asaia, a symbiont of several mosquito vectors who has been proposed as one of the most potential tool for paratransgenic applications; although being extensively characterized, its interactions with the mosquito immune system has never been investigated. Here we report a study aimed at describing the interactions between Asaia and the immune system of two vectors of malaria, Anophelesstephensi and An. gambiae. The introduction of Asaia isolates induced the activation of the basal level of mosquito immunity and lower the development of malaria parasite in An. stephensi. These findings confirm and expand the potential of Asaia in mosquito borne diseases control, not only through paratransgenesis, but also as a natural effector for mosquito immune priming.

Identification of a Killer Toxin from Wickerhamomyces anomalus with ß-Glucanase Activity.

The yeast Wickerhamomyces anomalus has several applications in the food industry due to its antimicrobial potential and wide range of biotechnological properties. In particular, a specific strain of Wickerhamomyces anomalus isolated from the malaria mosquito Anopheles stephensi, namely WaF17.12, was reported to secrete a killer toxin with strong anti-plasmodial effect on different developmental stages of Plasmodium berghei; therefore, we propose its use in the symbiotic control of malaria. In this study, we focused on the identification/characterization of the protein toxin responsible for the observed antimicrobial activity of the yeast. For this purpose, the culture medium of the killer yeast strain WaF17.12 was processed by means of lateral flow filtration, anion exchange and gel filtration chromatography, immunometric methods, and eventually analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on this concerted approach, we identified a protein with a molecular weight of approximately 140 kDa and limited electrophoretic mobility, corresponding to a high molecular weight ß-glucosidase, as confirmed by activity tests in the presence of specific inhibitors.

Killer yeasts exert anti-plasmodial activities against the malaria parasite Plasmodium berghei in the vector mosquito Anopheles stephensi and in mice.

BACKGROUND: Wickerhamomyces anomalus is a yeast associated with different insects including mosquitoes, where it is proposed to be involved in symbiotic relationships with hosts. Different symbiotic strains of W. anomalus display a killer phenotype mediated by protein toxins with broad-spectrum antimicrobial activities. In particular, a killer toxin purified from a W. anomalus strain (WaF17.12), previously isolated from the malaria vector mosquito Anopheles stephensi, has shown strong in vitro anti-plasmodial activity against early sporogonic stages of the murine malaria parasite Plasmodium berghei. RESULTS: Here, we provide evidence that WaF17.12 cultures, properly stimulated to induce the expression of the killer toxin, can directly affect in vitro P. berghei early sporogonic stages, causing membrane damage and parasite death. Moreover, we demonstrated by in vivo studies that mosquito dietary supplementation with activated WaF17.12 cells interfere with ookinete development in the midgut of An. stephensi. Besides the anti-sporogonic action of WaF17.12, an inhibitory effect of purified WaF17.12-killer toxin was observed on erythrocytic stages of P. berghei, with a consequent reduction of parasitaemia in mice. The preliminary safety tests on murine cell lines showed no side effects. CONCLUSIONS: Our findings demonstrate the anti-plasmodial activity of WaF17.12 against different developmental stages of P. berghei. New studies on P. falciparum are needed to evaluate the use of killer yeasts as innovative tools in the symbiotic control of malaria.

'Honey, shall I change the baby? - Well done, choose another one': ERP and time-frequency correlates of humor processing.

We studied the electrophysiological correlates of verbal humor comprehension by comparing Event Related Potentials (ERPs) and time-frequency representations recorded while 50 participants read humorous and non-humorous passages. Using linear mixed models on single trials we showed that humorous target words elicited a larger Left Anterior Negativity (LAN), sustained in time and followed by a positive shift involving P600 and Late Positive Complex (LPC) components. In the time-frequency domain, humor was associated with a power decrease in the beta-band of the EEG. Furthermore, participants' Autism-spectrum Quotient correlated with the size of the LAN, suggesting that social skills may affect humor comprehension during the early processing phase. Our results describe a sequence of events where incongruity detection (associated with the LAN) precedes a composite set of mechanisms serving resolution and acting in parallel: the sustained LAN might reflect the search for an alternative script, while the P600 might index inferential processes arriving at the resolution and the updating of the discourse model. The processing differences associated with the LPC and the changes in beta power may reflect a later stage of more elaborative and reflective processing (where the receiver reflects upon the joke's solution) and the abandonment of the current discourse model.

Genome Reduction in the Mosquito Symbiont Asaia.

Symbiosis is now recognized as a driving force in evolution, a role that finds its ultimate expression in the variety of associations bonding insects with microbial symbionts. These associations have contributed to the evolutionary success of insects, with the hosts acquiring the capacity to exploit novel ecological niches, and the symbionts passing from facultative associations to obligate, mutualistic symbioses. In bacterial symbiont of insects, the transition from the free-living life style to mutualistic symbiosis often resulted in a reduction in the genome size, with the generation of the smallest bacterial genomes thus far described. Here, we show that the process of genome reduction is still occurring in Asaia, a group of bacterial symbionts associated with a variety of insects. Indeed, comparative genomics of Asaia isolated from different mosquito species revealed a substantial genome size and gene content reduction in Asaia from Anopheles darlingi, a South-American malaria vector. We thus propose Asaia as a novel model to study genome reduction dynamics, within a single bacterial taxon, evolving in a common biological niche.

Denaturing Gradient Gel Electrophoresis Analysis of Bacteria in Italian Ticks and First Detection of Streptococcus equi in Rhipicephalus bursa from the Lazio Region.

Tick-borne diseases are an increasing problem for the community. Ticks harbor a complex microbial population acquired while feeding on a variety of animals. Profiling the bacterial population by 16S rDNA amplification and denaturing gradient gel electrophoresis enables detection of the broad spectrum of bacteria that settles in the ticks. This study identified known and unknown tick-infecting bacteria in samples from Italy. Seven adult ticks from different hosts and origins were analyzed: two Rhipicephalus sanguineus ticks from dogs (Lombardia), two Rhipicephalus bursa ticks from bovines (Lazio), and three Ixodes ricinus ticks from humans (Marche). The major result was the first report of the zoonotic agent Streptococcus equi in ticks. S. equi is a species complex of highly contagious pathogens. Subsequent to S. equi detection in a R. bursa tick removed from a bovine of Lazio in 2012, we studied 95 R. bursa samples collected from 3 bovines, 3 ponies, and 1 sheep grazing in the same area in 2012 and from 6 ponies grazing there in 2017. The results of a specific PCR assay indicated a not sporadic occurrence of S. equi in ticks. This finding provides a basis for assessing the potential of ticks to harbor and disperse S. equi.

Qualitative and quantitative methods for estimating Spirorchiidiasis burden in sea turtles.

Infection by blood flukes Hapalotrema mistroides and Neospirorchis sp. (Digenea: Spirorchiidae) has been recently reported in Caretta caretta (Linnaeus, 1758) in the Mediterranean Sea. Observations of post mortem lesions are generally used to assess disease severity, and few attempts have been made to standardize the evaluation of the parasitic burden from tissue egg counts. Faeces and spleen homogenates of 105 loggerheads from the northwestern Adriatic Sea were submitted to a sedimentation-flotation technique for the research of spirorchiid eggs; molecular techniques were used for unequivocal identification. Egg quantification for positive faeces and spleen samples was achieved using a modified McMaster method. Spleen samples were also submitted to quantification through the only method cited in the literature for similar purposes, which involves preventive chemical digestion. Correlations between splenic counts obtained from the two different methods and between faecal and splenic egg burdens were calculated using Spearman's rho test. Concordance between studies on eggs in faeces and spleen tissue was also calculated. Eggs of H. mistroides and Neogen-11 were found in spleen and faecal samples. Strong correlations were found between splenic egg burdens calculated from the two methods for H. mistroides, demonstrating that the modified McMaster method can be used for quantification. A multiplying factor must be used before drawing comparisons, as egg burdens are higher in value when measured after chemical digestion. High concordance was obtained from a qualitative examination of faeces and spleen tissue of H. mistroides, showing that copromicroscopic examination can be used for in vivo diagnosis. As weak correlations were found between faecal and splenic egg counts, faecal burden cannot be regarded as indicative of disease severity. For Neogen-11, low concordance was found between faeces and spleen tissue, likely reflecting lower levels of egg embolization in organs.