# Use of commercial serological tests to detect schistosomiasis in sheep

Manon Blin, Félix Bornier, Nathalie Lazzarin, Justine Alonzi, Bruno Polack, Isabelle Vallée, Anne Rognon, Eglantine Mathieu-Bégné, Myriam Thomas, Denis Limonne, et al.

► To cite this version:

Manon Blin, Félix Bornier, Nathalie Lazzarin, Justine Alonzi, Bruno Polack, et al. Use of commercial serological tests to detect schistosomiasis in sheep. Veterinary Parasitology, 2025, 338, pp.110538. 10.1016/j.vetpar.2025.110538. hal-05338622

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# Use of commercial serological tests to detect schistosomiasis in sheep

Manon Blin<sup>a,b,*</sup>, Félix Bornier<sup>c</sup>, Nathalie Lazzarin<sup>c</sup>, Justine Alonzi<sup>c</sup>, Bruno Polack<sup>d,●</sup>,
Isabelle Vallée<sup>d</sup>, Anne Rognon<sup>a</sup>, Eglantine Mathieu-Bégné<sup>e,●</sup>, Myriam Thomas<sup>d</sup>,
Denis Limonne<sup>c</sup>, Jérôme Boissier<sup>a,●</sup>

## ARTICLE INFO

### Keywords:

Schistosomiasis

Zoonoses

Animal diagnostic

ELISA

Western-blot

S. haematobium

S. bovis

## ABSTRACT

Human schistosomiasis is a zoonotic disease, meaning that wild animals or livestock can serve as reservoirs for the disease. This sustains active parasite transmission in regions where extensive treatment initiatives are under way, potentially undermining efforts to combat schistosomiasis. In multi-host and multi-parasite systems, achieving disease elimination necessitates the incorporation of all reservoir hosts. For this reason, animal diagnosis is crucial to enable accurate targeting of treatment. However, methods for diagnosing animals remain underdeveloped. We have evaluated two commercially available serological diagnostics, the *Schistosoma* IgG ELISA produced by Bordier Affinity Products, and the Schisto II Western Blot IgG kit produced by LDBIO Diagnostics which are commonly employed for human diagnosis. The diagnostics were carried out on a collection of 175 sheep serum samples from 35 sheep experimentally infected with *S. haematobium*, *S. bovis* and various strains resulting from mate crossing between these two species. The diagnosis of these samples was possible using the two methods tested, with an adaptation of the protocol of both methods by replacing the conjugate. The kappa coefficient highlights substantial agreement between each newly tested method and the adult worm recovery during animal necropsy, suggesting robust consistency in the results obtained with the two serological diagnostics. In both instances, further studies are required for validation and standardization to facilitate field application and to specify the specificity of the Western-Blot.

## 1. Introduction

Human schistosomiasis is one of the main debilitating neglected tropical diseases (NTDs), caused by trematodes of the genus *Schistosoma*. Schistosomiasis is endemic in 78 countries located in intertropical regions, notably in sub-Saharan Africa. Worldwide, the number of people suffering from severe parasitic infections with measurable morbidity is estimated at 300 million, over 50 % of whom are school-age children (Tian-Bi et al., 2018). The schistosome is a heteroxenous parasite, i.e. it requires a complex life cycle with a freshwater gastropod invertebrate

host, in which the parasite asexually multiplies, and a definitive vertebrate host, in which the parasite sexually reproduces (Rollinson and Southgate, 1987). The diversity of definitive hosts that can be infected by schistosomes varies from Human only for *S. intercalatum* to 62 different species from 7 vertebrate orders for *S. japonicum* (Panzner and Boissier, 2021). Some Human schistosomiases such *S. mansoni* or *S. japonicum* are recognized as zoonotic disease, meaning that wild animals or livestock can act as a reservoir for the disease, maintaining active transmission of the parasite in areas where mass treatment campaigns are being carried out. In this way, animals can wipe out efforts to

**Abbreviations:** NTD, neglected tropical diseases; WHO, World Health Organization; POC-CCA, Point-of-Care Circulating Cathodic Antigen; ELISA, enzyme-linked immunosorbent assay; WB, Western-Blot; PCR, polymerase chain reaction; IgG, type G immunoglobulin; CI, Confidence interval.

* Corresponding author at: Hosts Pathogens Environment Interactions, UMR 5244, CNRS, IFREMER, UM, University of Perpignan Via Domitia, Perpignan F-66860, France

E-mail addresses: manon.blin@paradev.fr (M. Blin), fbornier@ldbiodiag.com (F. Bornier), nlazzarin@ldbiodiag.com (N. Lazzarin), jtritz@ldbiodiag.com (J. Alonzi), bruno.polack@vet-alfort.fr (B. Polack), isabelle.vallee@anses.fr (I. Vallée), rognon@univ-perp.fr (A. Rognon), eglantine.mathieu-begne@unibas.ch (E. Mathieu-Bégné), myriam.thomas@anses.fr (M. Thomas), dlimonne@ldbiodiag.com (D. Limonne), boissier@univ-perp.fr (J. Boissier).

<footnote><sup>a</sup> Hosts Pathogens Environment Interactions, UMR 5244, CNRS, IFREMER, UM, University of Perpignan Via Domitia, Perpignan F-66860, France</footnote>

<footnote><sup>b</sup> SAS ParaDev®, Perpignan 66860, France</footnote>

<footnote><sup>c</sup> LDBIO Diagnostics, Lyon 69009, France</footnote>

<footnote><sup>d</sup> Anses, INRAE, Ecole Nationale Vétérinaire d'Alfort, UMR BIPAR, Laboratoire de Santé Animale, Maisons-Alfort 94700, France</footnote>

<footnote><sup>e</sup> University of Basel, Department of Environmental Sciences, Zoology, Basel, Switzerland</footnote>
fight against schistosomiasis at a time when we are only just beginning to become aware of their contribution and the consequences this entails (Gower, Vince and Webster, 2017; World Health Organisation, 2020).

A growing number of field studies have provided evidence of hybridization between sympatric *Schistosoma* species in Africa (Steinauer et al., 2008; Huyse et al., 2013; Léger et al., 2016; Boon et al., 2018; Rey et al., 2021; Onyekwere et al., 2022; Teukeng et al., 2022). Most *Schistosoma* hybrids have been identified in the *S. haematobium* clade, which comprises nine closely related species. Hybrids have been found between *S. haematobium* and *S. bovis*; *S. haematobium* and *S. curassoni*; *S. haematobium* and *S. guineensis*; *S. curassoni* and *S. bovis*; and *S. haematobium* and *S. mattheei* (Soentjens et al., 2016; Léger and Webster, 2017; Boon et al., 2018; Angora et al., 2022; Onyekwere et al., 2022; Teukeng et al., 2022; Agniwo et al., 2023). Several studies suggest the possibility that interspecific crosses generate lines with increased virulence or heterosis compared to parental species and lead to improved parasite selective value, with a broader definitive host spectrum (Rollinson et al., 1990; Huyse et al., 2013; King et al., 2015; Léger et al., 2016; Rey et al., 2021; Polack et al., 2024). This has been demonstrated in crosses of *S. bovis* x *S. haematobium* parasites infecting humans, cattle and rodents in Benin (Savassi et al., 2020). Due to climate change and other anthropogenic disturbances, interactions between humans and wild animals have intensified. This has led to increased contact between different *Schistosoma* species, which normally occur in separate hosts. This new transmission dynamic between *Schistosoma* spp., humans, rodents and cattle is helping to maintain the parasite in transmission areas (Miranda et al., 2022). In addition, it could potentially increase hybridization within the *Schistosoma* genus, as well as the emergence of new, more virulent strains (Léger and Webster, 2017; Colley and Loker, 2018; Catalano et al., 2020). During experimental infection of sheep, a previous study showed that parasite tropism within the host changes, and that certain hybrid lines are more virulent than the parental strains (Polack et al., 2024). Furthermore, animal schistosomiasis also holds paramount veterinary and socio-economic importance, given its substantial contribution to livestock mortality and morbidity, alongside a decline in productivity for their owners (Calvo-Urbano et al., 2023). Recent socio-economic surveys have unveiled significant yet often overlooked impacts of animal schistosomiasis on animal well-being, productivity, and financial aspects. These impacts further undermine the livelihoods, food security, and nutrition of neglected communities (Adeyemo et al., 2022). In fact, the financial burdens borne by subsistence farmers due to infected animals are projected to be considerably higher than those of a theoretical testing and treatment program (Adeyemo et al., 2022).

The World Health Organization (WHO)'s 2020–2030 roadmap establishes the goal to be achieved by 2030 in the fight against NTDs: their elimination as a public health problem (World Health Organisation, 2020). In the case of schistosomiasis, this means reducing the prevalence of infections with high parasite intensity and proven morbidity in school-age children (5–14 years) to 1 %. In other words, the aim is to reach 99 % of cases of low parasite intensity. In order to achieve the goal of the WHO schistosomiasis control roadmap 2021–2030, it is essential to consider an integrative approach and overcome the omission of animals from the schistosomiasis transmission equation. With this in mind, the question has already been raised as to whether mass livestock treatment campaigns would be profitable (Gower, Vince and Webster, 2017). Numerous cases of nematode resistance caused by the overuse of anthelmintics have already been reported (Kaplan, 2004; Kaplan and Vidyashankar, 2012). Considering livestock in mass treatment could help eradicate schistosomiasis. Currently, only 50 million out of 135 million pre-school aged children needing treatment receive it. Additionally, these campaigns target children, not adults. Treating animals thus remains a distant goal.

Diagnosis for animals is poorly documented, but more and more recent studies are emerging in this field. Diagnostic techniques tested on animals infected with *Schistosoma* spp. include (i) parasitological

techniques such as, for feces, Mini Flotac, Kato-Katz, Formalin-ethyl acetate sedimentation-digestion (FEA-SD), and, for urine, filtration (Xu et al., 2012; Catalano et al., 2019; Giovanoli Evack et al., 2020; Molla et al., 2022) (ii) antigen detection techniques such as Point-of-Care Circulating Cathodic Antigen (POC-CCA) cassette tests (Calvo-Urbano et al., 2023), (iii) serological tests such as Enzyme-linked immunosorbent assay (ELISA) (Ji et al., 2020), and (iv) molecular PCR tests (Pennance et al., 2021). Working with urine or feces presents challenges due to the large volumes excreted, resulting in highly unreliable outcomes (Giovanoli Evack et al., 2020). The review by Liang et al. listing all publications dealing with the diagnosis of schistosomiasis in non-human definitive hosts shows that the FEA-SD technique can allow to deal with large volume of excreta (Liang et al., 2022). Unsurprisingly, quantitative PCR is the molecular diagnostic that looks most promising for screening non-human hosts (Liang et al., 2022). The complexity of conducting quantitative PCR for animal diagnostics lies in its time-consuming nature, high cost, and the need for well-equipped facilities, which are often lacking in disease-endemic areas. The implementation of ELISA assays by Ji et al. enabled the detection of *S. turkestanicum* in goats using a laboratory-made protocol with a specific antigen for this species (Ji et al., 2020). Similarly, in Corsica, a laboratory protocol facilitated the detection of *S. bovis* through ELISA in 3519 domesticated animals, including 1147 cattle, 671 goats, and 1701 sheep (Oleaga et al., 2019). In this context, it would be intriguing to determine whether commercially available serological tests, comprising antigens from human pathogenic species such *S. mansoni* and *S. haematobium*, could be employed for animal diagnostics and thereby facilitate the detection of pathogenic animal schistosomes. The aim of this study is to test two commercially available serological tests on blood samples taken from sheep experimentally infected with *S. bovis*, *S. haematobium* and the 1st and 2nd generation hybrids resulting from their crossing. Cows are the preferred host for *S. bovis* (Léger et al., 2020). For technical reasons we have chosen another natural host, the sheep (Panzner and Boissier, 2021). The results of two selected serological tests, operating by ELISA and Western-Blot (WB), will be compared with the adult worm recovery during animal necropsy.

## 2. Materials and methods

### 2.1. Animal infection procedure

#### 2.1.1. Hosts and parasite lines

The *S. haematobium* strain originating from Egypt, was supplied by Biomedical Research Institute, Rockville, Maryland (Lewis et al., 2008). The *S. bovis* strain originating from Spain was provided by Ana Oleaga from the Spanish laboratory of parasitology at the Institute of Natural Resources and Agrobiology in Salamanca (Silva et al., 1977). Female golden hamsters (*Mesocricetus auratus*) aged between eight to 10 weeks were obtained from Janvier-Labs. Female lambs of Romane breed, aged between 12 and 16 weeks, were supplied by an authorized provider.

#### 2.1.2. Experimental protocol

Methods for molluscs, hamsters and sheep infection were previously described (Boissier and Moné, 2000; Oleaga and Ramajo, 2004). The blood samples come from previously published experimental infection protocol (Polack et al., 2024). The general crossbreeding scheme (Fig. 1) consists of an initial cross between pure parasite strains to obtain F1 mirror hybrids; Each F1 hybrid is then backcrossed with either *S. bovis* or *S. haematobium*. Miracidia, derived from either *Schistosoma* species, were extracted from the liver of infected hamsters. Single miracidia were introduced to *B. truncatus* for infection. After 55 days, surviving positive snails emitting cercariae were chosen. PCR was utilized to determine the sex of the clonal population hosted by each positive snail (Kincaid-Smith et al., 2016). An equal number (150) of male and female cercariae of *S. bovis* and *S. haematobium* were employed for hamster infection to generate F1 hybrid crosses, resulting in two mirror hybrid lines: [♂Sb x
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Schematic diagram of the hybrid lines with which the sheep were infected. Parasitic strains are Sb: *S. bovis*; Sh: *S. haematobium*; two First generation hybrids: F1 [♀Sb x ♂Sh]; F1'(F1 mirror cross) [♂Sb x ♀Sh], and 8 back-crossed lines.</figcaption></figure>

♀Sh] and [♂Sh x ♀Sb]. Ninety days after infection, the hybrid miracidia were retrieved from the liver of the previously infected hamster. Two batches of snails (A1 and A2) were exposed to five miracidia from the F1 hybrid lines [♂Sb x ♀Sh] and [♂Sh x ♀Sb], respectively. Another two batches of snails (B1 and B2) were exposed to single miracidia from the F1 hybrid lines [♂Sb x ♀Sh] and [♂Sh x ♀Sb], respectively. After 55 days, the cercariae released by batches A1 and A2 were used to infect sheep with the two F1 hybrid lines (3 sheep per line). The sex of the clonal population hosted by each positive snail in batches B1 and B2 was identified through PCR analysis to produce backcrossed lines. An equal number (150) of male and female cercariae from F1 hybrid lines and pure strains were employed for hamster infection to generate four backcrossed lines: ♀ [♂Sb x ♀Sh] x ♂Sb, ♂ [♂Sb x ♀Sh] x ♀Sb, ♀ [♂Sh x ♀Sb] x ♂Sh, and ♂ [♂Sb x ♀Sh] x ♀Sh.

In the same way, ninety-days after the previous infection, the backcrossed miracidia were retrieved from the liver of the hamsters. Four batches of snails (C1 to C4) were exposed to five miracidia from each of the four backcrossed lines. After 55 days, the cercariae released by batches C1 to C4 were used to infect 3 sheep per line. Three sheep were also exposed to the pure strains: *S. bovis* and *S. haematobium*, and two sheep were kept uninfected as negative control. An equal number of cercariae (1500) was used for infecting each sheep. Sheep infections were carried out under anesthesia (ketamine and diazepam) on a forearm previously moistened with warm water. In each line, a minimum of 12 snails exposed to 5 miracidia was utilized to generate the cercariae for sheep infection.

Euthanasia was conducted by inducing deep anesthesia in the sheep through a combination of xylazine and ketamine, followed by blood collection through bleeding. Autopsies were executed under the guidance of a member of the ECVP board. For parasitemia assessment, a thorough examination of all internal organs was carried out, including inspection of the vessels in the small intestine, cecum, colon, rectum, and bladder using intense illumination.

### 2.1.3. Ethic statement

Experiments were conducted in accordance with national ethical standards outlined in the writ of February 1st, 2013 (NOR: AGRG1238753A), which defines the conditions for approval, planning, and operation of establishments, breeders, and suppliers involved in animal use for scientific purposes and controls.

The infections and euthanasia of hamsters took place at the

UMR5244 lab in Perpignan, France, operating under the approval A66040 granted by the French Ministry of Higher Education, Research, and Innovation. The investigator holds the official certificate for animal experimentation (Decree No. 87-848 of October 19, 1987; authorization number 007083), and the experiment adhered to protocols approved under APAFIS #39910-2022121915564694 v2.

For sheep infections and autopsies, procedures were conducted at the Ecole Nationale Vétérinaire d'Alfort in Maisons-Alfort, France. The animal facility there holds the agreement E940462 issued by the French Ministry of Higher Education, Research, and Innovation. Approval for the sheep experiment was granted by an independent ethics committee and registered with the French Ministry of Higher Education, Research, and Innovation under APAFIS#23786-2020012416159143 v2.

### 2.2. Sheep blood sampling and treatment

The experimental infection of the 38 sheep was performed in three batches for convenience. For the first batch, six sheep underwent five blood samples according to the following kinetics: the first time just before infection, then 29, 65, 133 after infection and finally the last sample was taken on the day the animal was euthanized, 142 days after infection. For the second batch, the kinetic of sampling for 10 additional sheep was the day of infection, then 18, 47, 83, and 125 days after infection. For the third batch, the kinetic of sampling for the 20 remaining sheep was the day of infection, then 50, 100, 156, and 164 days after infection. *Schistosoma* adults in renal venous system were isolated after portal perfusion performed on the day of euthanasia. The number of adult worms recovered serves as a point of comparison with the serological reactivity of the WB and ELISA tests in terms of infection intensity. Sera of these blood samples were collected and immediately stored at -20°C until analysis. The blood bank is composed of a total of 190 samples. Samples from 3 of these sheep were excluded from all following analyses because of a concern noted at the time of infection (one sheep infected with *S. bovis*, another infected with ♀[♂Sh x ♀Sb] x ♂Sh and one with ♀[♂Sb x ♀Sh] x ♂Sh).

### 2.3. Enzyme-linked immunosorbent assay (ELISA)

Sera were screened for anti-*Schistosoma* antibodies using the *Schistosoma* IgG ELISA (Bordier Affinity Products, Crissier, Switzerland) according to manufacturers' instructions except for incubation step with
the conjugate which was replaced by a Rabbit Anti-Sheep IgG H&L conjugated with Alkaline Phosphatase (ab6748, Abcam, Cambridge, UK) which was incubated during 1 h at 37°C.

This secondary IgG was 1/2000 diluted with dilution buffer from the ELISA kit. All sera, including positive and negative controls, were diluted as the same way than the secondary IgG (1/2000). A serum was considered negative if the absorbance value was below the highest value measured for the negative controls (i.e. <1). Negative controls were sheep n°22013 and 21960 (unexposed sheep). Positive control was n°21937 (sheep with the highest worm recovery rate). Note that this commercial test includes exclusively *S. mansoni* antigens.

Results were visualised on a heatmap created using the ggplot 2 and pheatmap packages (versions 3.4.3 and 1.0.12 respectively) in Rstudio 2023.06.1 software.

## 2.4. Western Blot (WB) assay

Sera were screened for anti-*Schistosoma* antibodies using the WB assay Schisto II Western Blot IgG kit (LDBIO Diagnostics, Lyon France), according to the manufacturer's instructions except for incubation step with the conjugate which was replaced by a Rabbit Anti-Sheep (Alkaline Phosphatase AffiniPure™ Rabbit Anti-Sheep IgG (H+L), ref 313-055-045, Jackson ImmunoResearch Laboratories, USA))(Houze et al., 2003; LDBio-Diagnostics, 2024). This commercial test includes a mix of *S. mansoni* and *S. haematobium* antigens. As indicated in the Schisto II kit leaflet, the bands of interest for the specific diagnosis of schistosomiasis by WB in human serum are located between 8 and 34 kDa. Of these bands, P22-24 and P30-34 are the most easily identifiable and are indicative of schistosomiasis. The presence of one of these bands is sufficient to confirm the positivity of a serum analysed for infection with *Schistosoma* spp. The presence of a more intense P22-24 band could be indicative of *S. haematobium* infection, while a pronounced P30-34 band could be indicative of *S. mansoni* infection. Note that these bands can take the form of a single broad band, a doublet of thinner bands or one of the two bands making up the doublet. Sheep sera were analyzed according to the same criteria, assuming that sheep antibodies would react in the same way during the WB. As a result, the serology was considered positive if one or both *Schistosoma*-specific band were observed (i.e. P22-24, P30-34 bands) and negative if not.

## 2.5. Statistical analysis

Sensitivities and specificities obtained with both ELISA and WB assays were calculated with a 95 % confidence interval using stats package (version 3.6.2) in Rstudio 2023.06.1 software. The agreement between results obtained with ELISA, WB and worm recovery rate, in the detection *Ig-Schistosoma* in sera sampling were statistically analysed calculating a Kappa coefficient analysis using psych package (version 2.2.9). In addition, the car (version 3.1-2), carDara (version 3.0-5) and corrplot (version 0.92) packages were used to analyze the correlation between antibody production by ELISA with worm recovery rate in sheep.

# 3. Results

## 3.1. Worm recovery

All parasites were found in the veins surrounding the digestive tract, from the small intestine to the rectum. No parasites were found in the veins surrounding the bladder (see Polack et al., 2024 for more details). The pure *Sh* strain does not infest sheep, unlike the pure *Sb* strain. All hybrids created in the laboratory are infective. *Sb*-backcrossed strains were also more infective than *Sh*-backcrossed strains.

## 3.2. Enzyme-linked immunosorbent assay (ELISA)

The adjustments made to the *Schistosoma* IgG ELISA kit protocol produced the results shown in Fig. 2. The raw absorbance values at 405 nm for the assay are provided in Supplementary File 1. All unexposed sheep sera or exposed with *S. haematobium* (incompatible with sheep) and therefore supposed to be negative were indeed negative with ELISA assay. All sheep exposed with pure *S. bovis*, or one hybrid lines are ELISA-positive for at least one of their samples. If we consider only the late sample to establish sensitivity and specificity compared with the worm recovery rate used as a reference, the sensitivity of the ELISA test is 90.0 % (95 % CI 73.4–97.9) and its specificity is 83.3 % (95 % CI 35.8–99.6). If we consider only the 4th sample, sensitivity increases to 100 % and specificity stays at 83.3 % (95 % CI 35.8–99.6).

Sheep exposed with *S. haematobium* back-crossed lines seem to give lower optical densities compared to sheep exposed with *S. bovis* backcrossed lineages. Indeed, at the last sampling, most of sheep exposed with *S. haematobium* backcrossed lines showed a weaker signal than those exposed with F1, F1' or *S. bovis* backcrossed lines. All sheep in the first and second batches were negative at the second sampling, carried out at 18- and 29-days post-infection respectively. Some sheep in the third batch were already positive on the second sample taken at 50 days post-infection. This shows that antibodies start to be produced between 29 and 50 days after infection. Statistical analysis showed no significant correlation between worm recovery rate, and antibody production measured by ELISA (p-value=0.2148). The gender of the F1 or F1' hybrid involved in backcrossing does not seem to have any real impact on the nature and intensity of the bands.

## 3.3. Western Blot (WB) assay

The Schisto II Western Blot IgG kit produced the results shown in Fig. 3 where are represented the sheep's serological profile on transfer strips. No specific bands were observed in the sera of sheep that had not been experimentally exposed (sheep 22013 and 21960). Low-intensity P22-24 bands are visible in animals exposed with *S. haematobium* (22014, 21957 and 21969). These were observed in sera collected between 50- and 164-days post-infection but seem to disappear rapidly since sheep 21969 lost the signal during the last sampling and the band intensity of sheep 957 decreases (Table 1). Sheep exposed with *S. bovis* (21054, 21062 and 21092) showed a variable serological profile, with appearance of an intense P22-24 band (21054), a medium-intensity P30-34 band (21062). A low-molecular-weight band (possibly P14-15) of late onset was observed in all three animals. The sera from sheep exposed with this species show bands of high intensity indicating a consistent immune response. Animals exposed with first generation hybrids F1 (21068, 21069 and 21991) and F1' (21051 and 21922) showed serological profiles varying between individuals and conditions, but overall close to a *S. haematobium* profile, with a more intense P22-24 band than P30-34. Similar results were observed when analyzing the serological profiles of animals exposed by second-generation hybrids. In fact, these vary according to the hybrid strains inoculated and between animals in the same condition. The P22-24 specific band appears rapidly, for all sera and seems to testify once again to the antigenic inheritance brought by *S. haematobium* different crosses. The gender of the F1 or F1' hybrid involved in backcrossing does not seem to have any real impact on the nature and intensity of the bands. However, the P12-13 and P14-15 bands, hardly visible in the sera of sheep exposed with F1 or F1', reappeared in several serological profiles of animals exposed with hybrids resulting from backcrossing (at least 5 out of 8 conditions).

Considering only the results of the 5th sampling to establish sensitivity and specificity compared with the worm recovery rate used as a reference, the sensitivity of the WB test is 100 % (95 % CI 88.4–100) and its specificity is 50.0 % (95 % CI 11.8–88.2).
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Heatmap of optical density at 405 nm obtained after ELISA diagnosis detecting anti-Schistosoma antibodies present in sheep sera. The values shown in the heatmap are those read by the spectrophotometer after subtraction of the serum-free blank. A serum was considered negative if the absorbance value was below the highest value measured for the negative controls (i.e. <1). A line refers to an experimentally infected sheep. Parasitic strains are indicated on the right of the heatmap. Parasitaemia is the number of male and female worms present in the bloodstream of each sheep the day each animal was euthanized. The batch number indicates the experimental isolate for infection of each sheep. The experimental infection of 38 sheep was divided into three batches for convenience, with variable kinetics for each batch. This explains why the x-axis “days post infection” indicate the sampling range in days for the three batches.</figcaption></figure>

**3.4. Correlation between ELISA, WB and worm recovery rate**

Kappa agreement calculated by crossing the results obtained with the ELISA and WB methods for the 5th sampling of each sheep and the worm recovery rate quantified the same day is equal to 0.64 and 0.62 respectively. The correlation analysis of the results obtained between the ELISA and WB methods, again for the 5th sample from each sheep, gave a kappa approval of 0.48. The cross-tables for each comparison are shown in Table 2.

**4. Discussion**

Schistosomiasis is a prevalent zoonotic disease among numerous animals (De Bont and Vercruysse, 1997; Panzner and Boissier, 2021). Achieving the WHO's objective of reducing cases of high parasite intensity to less than 1 % by 2030 requires an integrated approach to schistosomiasis control (Rollinson et al., 2013; World Health Organisation, 2020). As part of this approach, the mass screening of livestock herds living near humans is one of the tasks to be carried out. Furthermore, accurate detection of *Schistosoma* spp. infection in animals would provide not only crucial data to guide surveillance and influence control measures (World Health Organisation, 2020; Lo et al., 2022) but it would also play a role in improving livestock-keeping communities

wellbeing, their financial situation, and the welfare of the animals (Gower, Vince and Webster, 2017; Adeyemo et al., 2022). Therefore, the development of new diagnostic tests for animals or testing of existing commercial tests for human diagnosis is necessary. In the present study, we have evaluated the clinical performance of two commercially available serological diagnostic, ELISA and WB, which are used to human diagnostic. The diagnosis of a bank of 175 sera samples from 35 sheep experimentally exposed with *S. haematobium*, *S. bovis* and various strains resulting from mating between these two species was possible using the two methods tested. The kappa coefficient highlights substantial agreement between each newly tested method and the worm recovery rate, but also between the two methods themselves, suggesting consistency in the results obtained with the two serological diagnostics. The *Schistosoma* IgG ELISA kit from Bordier Affinity products can be used simply by replacing the kit's protein conjugate with an antibody directed against the animal species to be tested, in our case an anti-sheep antibody. The protein conjugate A in the kit did not have sufficient affinity for sheep antibodies. By respecting the positivity threshold set at OD= 1, there was no signal misinterpretation; results are easy to interpret. ELISA produced a sensitivity of 90,0 % (95 % CI 73.4–97.9) or 100 % (95 % CI 88.4–100), depending on the kinetics of serological sampling, and a specificity of 83.3 % (95 % CI 35.8–99.6). The similar study by Ji et al. showed a sensitivity and specificity of both 100 % on
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Kinetic analysis of the serological profile on Schisto II transfer strips. In this figure, each transfer strip represents a WB performed on one of the 5 sheep samples, positioned in chronological order of infection kinetics. The WB transfer strips have been grouped by condition, regardless of transfer production number, in order to validate the identity of the bands present by comparison. The identifier of each animal is indicated by a bold number. The strain of *Schistosoma* spp. with which each animal was experimentally exposed is indicated above the corresponding transfers. Bands P22–24 and P30–34 are indicated by a green band. Bands P12–13 and P14–15 are indicated by a blue band.</figcaption></figure>

goats sera analyzed by ELISA (Ji et al., 2020). It should be noted that they utilized antigens directly from the targeted species; perhaps the lack of sheep's antibody affinity for *S. mansoni* antigens could explained the observed lower sensitivity in the tested ELISA during the fifth sampling.

Equally, Schisto II WB from LDBIO Diagnostics kit could be used to diagnose cases of infection with different *Schistosoma* spp. species (*S. haematobium* and *S. bovis*) or hybrids in sheep, after the modification of the protein conjugate. Although some intra- and inter-condition variability is observed within the serological profiles of sheep exposed with different strains of *Schistosoma* spp. it is relatively consistent between conditions. This is marked by the appearance, in most cases, of a P22-24 band. Thus, the various hybrids (1st and 2nd generations) seem

to generate serological profiles close to those of *S. haematobium*, due to their close antigenic composition of this species as is also the case for their genomic composition (Rey et al., 2021). However, we found an unexpected *S. mansoni*-type immune profile type immune profile (presence of P30–34 bands) on transfers produced from *S. bovis* and hybrid F1 and F1' hybrid antigens, suggesting the presence in these antigens of epitopes that are not, or only weakly, expressed by the *S. haematobium*. This phenomenon was also observed in WB tests performed on sera from hamsters infected in the same experiment. These observations suggest a certain common antigenic community between *S. bovis* and *S. mansoni* (Losada et al., 2005). This cross-reactivity is known even between phylogenetically more distant species such as *S. mansoni* and *S. japonicum* (Kitani, Miura and Iuchi, 1979). With the
<caption>**Table 1**</caption>

WB diagnostic bands for each time step. Interpretation of bands read on WB Schisto II transfer strips. Results are sorted by parasite strain and separated into batches with different reaction kinetics. “-” means no band has been read.

<table><caption>**Table 2**</caption><thead><tr><th rowspan="2">Batch</th><th rowspan="2">Sheep-ID</th><th rowspan="2">Schistosome strain</th><th colspan="4">WB diagnostic bands for each time sampling (in days post infection)</th><th rowspan="2">Parasitaemia</th></tr><tr><th>0</th><th>18-50</th><th>47-100</th><th>83-156</th><th>125-164</th></tr></thead><tbody><tr><td>3</td><td>22013</td><td>Unexposed</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td></tr><tr><td>3</td><td>21960</td><td>Unexposed</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td></tr><tr><td>3</td><td>22014</td><td>Sh</td><td>-</td><td>-</td><td>22-24</td><td>-</td><td>22-24</td><td>0</td></tr><tr><td>3</td><td>21957</td><td>Sh</td><td>-</td><td>22-24</td><td>22-24</td><td>22-24</td><td>-</td><td>0</td></tr><tr><td>3</td><td>21969</td><td>Sh</td><td>-</td><td>-</td><td>-</td><td>22-24</td><td>-</td><td>0</td></tr><tr><td>2</td><td>21054</td><td>Sb</td><td>-</td><td>-</td><td>-</td><td>22-24</td><td>14-15/22-24/30-34</td><td>244</td></tr><tr><td>2</td><td>21062</td><td>Sb</td><td>-</td><td>-</td><td>30-34</td><td>30-34</td><td>14-15/30-34</td><td>342</td></tr><tr><td>2</td><td>21068</td><td>F1</td><td>-</td><td>-</td><td>-</td><td>22-24/30-34</td><td>14-15/22-24/30-34</td><td>190</td></tr><tr><td>2</td><td>21069</td><td>F1</td><td>-</td><td>-</td><td>30-34</td><td>30-34</td><td>22-24/30-34</td><td>221</td></tr><tr><td>3</td><td>21991</td><td>F1</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>22-24/30-34</td><td>22-24/30-34</td><td>11</td></tr><tr><td>2</td><td>21051</td><td>F1'</td><td>-</td><td>-</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>268</td></tr><tr><td>3</td><td>21966</td><td>F1'</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>181</td></tr><tr><td>3</td><td>21922</td><td>F1'</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>191</td></tr><tr><td>1</td><td>21048</td><td>δF1 x ♀Sh</td><td>-</td><td>-</td><td>22-24</td><td>22-24</td><td>22-24</td><td>34</td></tr><tr><td>2</td><td>21070</td><td>δF1 x ♀Sh</td><td>-</td><td>-</td><td>22-24</td><td>22-24</td><td>22-24</td><td>8</td></tr><tr><td>2</td><td>21090</td><td>δF1 x ♀Sh</td><td>-</td><td>-</td><td>22-24</td><td>22-24</td><td>22-24</td><td>2</td></tr><tr><td>1</td><td>21058</td><td>♀F1 x δSh</td><td>-</td><td>-</td><td>22-24</td><td>14-15/22-24/30-34</td><td>14-15/22-24/30-34</td><td>49</td></tr><tr><td>2</td><td>21085</td><td>♀F1 x δSh</td><td>-</td><td>-</td><td>22-24</td><td>22-24</td><td>22-24</td><td>29</td></tr><tr><td>2</td><td>21065</td><td>δF1' x ♀Sh</td><td>-</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>22-24/30-34</td><td>11</td></tr><tr><td>2</td><td>21087</td><td>δF1' x ♀Sh</td><td>-</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>22-24/30-34</td><td>32</td></tr><tr><td>2</td><td>21093</td><td>δF1' x ♀Sh</td><td>-</td><td>-</td><td>22-24</td><td>22-24</td><td>22-24</td><td>19</td></tr><tr><td>1</td><td>21049</td><td>♀F1' x δSh</td><td>-</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>22-24/30-34</td><td>0</td></tr><tr><td>1</td><td>21061</td><td>♀F1' x δSh</td><td>-</td><td>-</td><td>22-24</td><td>22-24</td><td>22-24</td><td>4</td></tr><tr><td>3</td><td>21915</td><td>δF1 x ♀Sb</td><td>-</td><td>-</td><td>22-24/30-34</td><td>14-15/22-24/30-34</td><td>14-15/22-24/30-34</td><td>638</td></tr><tr><td>3</td><td>21937</td><td>δF1 x ♀Sb</td><td>-</td><td>22-24</td><td>22-24</td><td>14-15/22-24/30-34</td><td>14-15/22-24/30-34</td><td>832</td></tr><tr><td>3</td><td>21940</td><td>δF1 x ♀Sb</td><td>-</td><td>-</td><td>-</td><td>14-15/22-24</td><td>14-15/22-24</td><td>644</td></tr><tr><td>3</td><td>22000</td><td>♀F1 x δSb</td><td>-</td><td>-</td><td>22-24</td><td>12-13/22-24/30-34</td><td>12-13/22-24/30-34</td><td>206</td></tr><tr><td>3</td><td>22020</td><td>♀F1 x δSb</td><td>-</td><td>-</td><td>22-24</td><td>12-13/22-24/30-34</td><td>12-13/22-24/30-34</td><td>42</td></tr><tr><td>3</td><td>21954</td><td>♀F1 x δSb</td><td>-</td><td>-</td><td>22-24</td><td>12-13/14-15/22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>3</td></tr><tr><td>3</td><td>22023</td><td>δF1' x ♀Sb</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>14-15/22-24/30-34</td><td>14-15/22-24/30-34</td><td>150</td></tr><tr><td>3</td><td>22024</td><td>δF1' x ♀Sb</td><td>-</td><td>22-24/30-34</td><td>14-15/22-24/30-34</td><td>14-15/22-24/30-34</td><td>14-15/22-24/30-34</td><td>79</td></tr><tr><td>3</td><td>21972</td><td>δF1' x ♀Sb</td><td>-</td><td>30-34</td><td>22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>204</td></tr><tr><td>3</td><td>22017</td><td>♀F1' x δSb</td><td>-</td><td>30-34</td><td>14-15/22-24/30-34</td><td>14-15/22-24</td><td>14-15/22-24</td><td>323</td></tr><tr><td>3</td><td>21943</td><td>♀F1' x δSb</td><td>-</td><td>-</td><td>22-24/30-34</td><td>22-24/30-34</td><td>22-24/30-34</td><td>562</td></tr><tr><td>3</td><td>21973</td><td>♀F1' x δSb</td><td>-</td><td>-</td><td>14-15/22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>12-13/14-15/22-24/30-34</td><td>260</td></tr></tbody></table>

Cross-tables of serological methods (WB and ELISA) and between serological methods and parasitaemia deduced from the worm recovery during animal necropsy. These results are based on the serological tests carried out on the last blood sample taken from the sheep at the time of euthanasia (between 125 and 164 days post-infestation).

<table><caption>ELISA x Parasitaemia</caption><thead><tr><th>ELISA Results</th><th>Parasitaemia positive</th><th>Parasitaemia negative</th><th>Total</th><th>Kappa value</th></tr></thead><tbody><tr><td>Positive</td><td>26</td><td>1</td><td>27</td><td rowspan="3">0.64</td></tr><tr><td>Negative</td><td>3</td><td>5</td><td>8</td></tr><tr><td>Total</td><td>29</td><td>6</td><td>35</td></tr><tr><td colspan="5">WB x Parasitaemia</td></tr><tr><td>WB Results</td><td>Parasitaemia positive</td><td>Parasitaemia negative</td><td>Total</td><td>Kappa value</td></tr><tr><td>Positive</td><td>29</td><td>3</td><td>32</td><td rowspan="3">0.62</td></tr><tr><td>Negative</td><td>0</td><td>3</td><td>3</td></tr><tr><td>Total</td><td>29</td><td>6</td><td>35</td></tr><tr><td colspan="5">ELISA x WB</td></tr><tr><td>ELISA Results</td><td>WB positive</td><td>WB negative</td><td>Total</td><td>Kappa value</td></tr><tr><td>Positive</td><td>27</td><td>0</td><td>27</td><td rowspan="3">0.48</td></tr><tr><td>Negative</td><td>5</td><td>3</td><td>8</td></tr><tr><td>Total</td><td>32</td><td>3</td><td>35</td></tr></tbody></table>

exception of the Western blot, all commercial tests use *S. mansoni* antigens and are capable of detecting all human schistosomiasis (Luzón-García et al., 2023). Conversely, ELISA tests using *S. bovis* antigens have also demonstrated their ability to detect *S. mansoni* infestation in humans (Pardo et al., 2007). WB produced a sensitivity of 100% (95% CI 88.4–100) and a specificity of 50.0% (95% CI 11.8–88.2).

Western blots are known to be a highly sensitive and specific technique, and are used in human diagnostics to screen for mild infections as confirmation of a first-line test (Van Lieshout et al., 2000). On our study, the obtained specificity is quite poor, though this figure needs to be taken with caution due to (i) the limited number of negative samples within the tested sheep cohort, and (ii) the comparison of WB results with the worm recovery rate used as a reference technique, which is known to lack sensitivity. Furthermore, it should be borne in mind that the SCHISTO II WB kit have not been developed for use in animals, and we don't know whether a single strip can be used to confirm the positivity of animals. Uncertainties persist regarding the specificity arising from the not always straightforward interpretation of bands on transfer strips, which is subject to the reader's judgment. An illustration of this is the presence of very weak P30-34 bands that are visible on the sample taken on the day of infection from sheep 21065, which is supposed to be negative regarding to worm recovery rate.

The results obtained seem to show that sheep exposed with *S. haematobium* back-crossed lines have fewer antibodies and therefore a lower immune response observed with ELISA. This weaker response is corroborated by the results observed with the WB where the intensity of the bands on the transfer strips was much higher for sheep exposed with first generation hybrids backcrossed with *S. bovis* than for those exposed with first generation hybrids backcrossed with *S. haematobium*. Even though there is no statistical correlation between parasitemia and OD in ELISA, the dissimilarity in signal intensity observed between first generation hybrids backcrossed with *S. haematobium* and first generation hybrids backcrossed with *S. bovis* in ELISA and WB likely stems from a lower parasitic intensity as indicated by parasitemia. Conducting a larger-scale study could have provided us with a sufficient dataset to potentially establish the significance of this relationship between
immune response and parasitic intensity. Indeed, there is a strong lik-
lihood that this stems from the incompatibility of S. haematobium to
infect sheep as observed, in sheep exposed with the pure strain of
S. haematobium, no parasitemia is reported, and no positives are detected
with ELISA assay. It has been previously demonstrated that only sera
from compatible hosts recognize the schistosome antigens used in In-
direct Immunofluorescence tests (Kourilová and Kolárová, 2002).
However, some bands of low intensity appeared on the western blot of
sheep exposed with S. haematobium, suggesting a slight persistence of
the humoral response in the presence of an incompatible strain. Alter-
natively, antigens from S. bovis might be produced in larger quantities
than those from S. haematobium, thereby eliciting a stronger host im-
mune response. In fact, Kittur et al. suggested the possibility that
S. haematobium and S. mansoni worms might generate varying antigen
quantities or that S. haematobium could metabolize them more effi-
ciently (Kittur et al., 2016). Interestingly, these bands emerged around
time point three of the kinetics, specifically between 50- and 100-days
post-infection, but appear to decrease, as two out of three sheep tested
negative at the fifth sampling. This contrasts with all other conditions,
where the time needed for the immune system to initiate a humoral
response to the parasitic infection appears to be between 29 and 50 days,
and this response is maintained until the final sampling. An earlier study
revealed a timeframe of less than a week for the immune response of
chickens to infection by Syngamus trachea (Petavy, Vernes and Biguet,
1980). Thus, this delay appears to be highly variable, depending on host
metabolism and parasite species. Serological reactivity data on humans
confirms this, since Ig detection times are extremely longer in humans
than in animals, which goes hand in hand with the parasite's develop-
ment time within its host (Hinz et al., 2017; Hoermann et al., 2022).

5. Conclusions

The *Schistosoma* IgG ELISA produced by Bordier Affinity Products, and the Schisto II Western Blot IgG kit produced by LDBIO Diagnostics WB which are commonly employed for human diagnosis enable the efficient detection of infections caused by various strains of *Schistosoma* spp. in sheep, with the adjustments implemented. In both instances, further studies are required for validation and standardization to facilitate field application. Particularly, the specificity of the Western-Blot assays needs to be verified. It seems relevant to concurrently develop molecular detection tests that are feasible for mass livestock diagnostics in endemic areas using isothermal amplification, which could offer the potential to be realized in such contexts.

Ethics approval and consent to participate

Experiments were carried out according to national ethical standards established in the writ of February 1st, 2013 (NOR: AGRG1238753A), setting the conditions for approval, planning and operation of establishments, breeders and suppliers of animals used for scientific purposes and controls. Hamster infections and euthanasia were performed at UMR5244 lab (Perpignan, France). The animal facility possesses the agreement A66040 delivered by the French ministry of Higher Education, Research and Innovation. The investigator possesses the official certificate for animal experimentation (Decret n 87-848 du 19 octobre 1987; number of the authorization 007083). The experiment followed APAFIS #39910-2022121915564694 v2 protocols approval. Sheep infections and autopsies were performed at Ecole Nationale Vétérinaire d'Alfort (Maisons-Alfort, France). The animal facility possesses the agreement E940462 delivered by the French ministry of Higher Education, Research and Innovation. The sheep experiment was registered by the French ministry of Higher Education, Research and Innovation and approved by an independent ethics committee: APAFIS#23786-2020012416159143 v2.

Funding

This study was supported by the programme HySWARM (ANR18-
CE35-0001) from the French Research National Agency, awarded to J.
Boissier. This study is set within the framework of the "Laboratoires
d'Excellence (LABEX)" TULIP (ANR-10-LABX-41).

CRediT authorship contribution statement

**Myriam Thomas:** Resources, Methodology. **Denis Limonne:** Writing – review & editing, Supervision, Formal analysis, Conceptualization. **Jérôme Boissier:** Writing – review & editing, Supervision, Funding acquisition, Formal analysis, Conceptualization. **BLIN Manon:** Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. **Bruno Polack:** Writing – original draft, Supervision, Resources, Methodology, Conceptualization. **Isabelle Vallée:** Resources, Methodology. **Anne Rognon:** Resources, Methodology. **Eglantine Mathieu-Bégné:** Writing – review & editing, Resources, Methodology. **Félix Bornier:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Nathalie Lazzarin:** Resources, Methodology. **Justine Alonzi:** Resources, Methodology.

Declaration of Competing Interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests,
Jerome Boissier reports financial support was provided by French Na-
tional Research Agency. Denis Limonne reports a relationship with
LDBIO Diagnostics that includes: board membership. Felix Bornier re-
ports a relationship with LDBIO Diagnostics that includes: employment.
Nathalie Lazzarin reports a relationship with LDBIO Diagnostics that
includes: employment. Justine Alonzi reports a relationship with LDBIO
Diagnostics that includes: employment. If there are other authors, they
declare that they have no known competing financial interests or per-
sonal relationships that could have appeared to influence the work re-
ported in this paper.

Acknowledgments

The authors are thankful to all members who were involved in
monitoring the sheep within the present study: personnel of the
Biomedical Research Center of Maisons-Alfort, ENVA, and personnel of
the BioPôle Alfort, ENVA. We would like to thank Pr. Jean-Jacques
Fontaine, European Specialist in Veterinary Pathology and now
retired, for his great help with the animal experimentation part of the
project. We are grateful to the Biomedical Research Institute (BRI),
Maryland, USA, for provision of S. haematobium strain and to Dr Ana
Oleaga, for provision of S. bovis strain.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.vetpar.2025.110538.

Availability of data and materials

All data generated or analysed during this study are included in this
published article.

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