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Neosporosis primarily affects cattle and dogs and is not currently considered a zoonotic disease. Toxoplasmosis is a zoonosis with a worldwide distribution that is asymptomatic in most cases, but when acquired during pregnancy, it can have serious consequences. The seropositivity rates determined by the indirect fluorescent antibody test for Neospora caninum ( N. caninum ) and Toxoplasma gondii ( T. gondii ) were 24.3% (49 samples) and 26.8% (54 samples), respectively. PCR positivity for N. caninum was observed in two samples of cord blood (1%) using the Nc5 and ITS1 gene, positivity for T. gondii was observed in 16 samples using the primer for the B1 gene (5.5% positivity in cord blood and 2.5% positivity in placental tissue). None of the samples showed structures characteristic of tissue cysts or inflammatory infiltrate on histopathology. Significant associations were observed only between N. caninum seropositivity and the presence of domestic animals (p = 0.039) and presence of dogs (p = 0.038) and between T. gondii seropositivity and basic sanitation (p = 0.04). This study obtained important findings regarding the seroprevalence and molecular detection of N. caninum and T. gondii in pregnant women; however, more studies are necessary to establish a correlation between risk factors and infection.

Canine leishmaniasis (CanL) is a zoonotic parasitic disease caused by Leishmania infantum in the Mediterranean area and transmitted by phlebotomine sand fly vectors. The domestic dog is the main reservoir host. The aim of this study was to assess the influence of different individual, environmental and spatial risk factors on the dog exposure to L . infantum and to estimate the seroprevalence among owned and kennel dogs, in the Lazio region (central Italy), where canine leishmaniasis is endemic. In the period 2010–2014, 13,292 sera from kennel and owned dogs were collected by official and private veterinarians. The presence of anti- Leishmania IgG was analysed by indirect fluorescent antibody test (IFAT), using a 1:80 titre cut-off. At the univariable analysis, CanL seropositivity was associated with sex, size, breed, coat length, living with other dogs and forest/semi-natural land cover. At the multivariable analysis, age, ownership and attitude were confirmed as risk factors, being more than 2 years old, owned, and hunting dogs at higher risk. Being a Maremma sheepdog was a protective factor. A true overall seroprevalence of 6.7% (95% CI: 6.2–7.2) was estimated in the whole population while 7.3% (95% CI: 6.8–7.8) was estimated in kennel dogs and 74.3% (95% CI: 70.8–77.6) in owned dogs. The role of kennels as a key component for CanL active and passive surveillance was also highlighted. This study confirmed the endemicity of CanL in the Lazio region and focused some factors that can influence the seropositivity of dogs in a Mediterranean region.

Neosporosis is a proven disease of farm animals and dogs caused by Neospora caninum. This cross-sectional study investigates N. caninum prevalence and seroprevalence among 268 dogs. Nc5 gene PCR was carried out on dog faeces and confirmed by sequencing. Seroprevalence was detected using an indirect fluorescent antibody test (IFAT). Three age groups, gender, locality (Amman, Irbid, and Zarqa Governorates), dog type (stray, pet, and breeding), place of living (indoor/outdoor), food type (raw/cooked), having diarrhoea, having abortion in the area, and having animals nearby were tested as independent variables for associations with positivity to N. caninum using univariate and multivariable logistic regression analyses. The true prevalence of N. caninum was 34.3% (95% CI 28.4, 40.5) using the Nc5 -PCR test. The true seroprevalence rate of N. caninum among dogs in Jordan was 47.9% (95% CI 41.4, 54.5) using IFAT. The sequenced isolates of Nc5 -PCR products ( n  = 85) matched three N. caninum strains, namely, NcHareGre ( n  = 70, 82.4%, 95% CI 72.6–89), NC MS2 ( n  = 14, 16.5%, 95% CI 9.3–26.1), and L218 ( n  = 1, 1.2%, 95% CI 0.03–6.4). The three strains were isolated previously from three different countries and continents. N. caninum shedding is associated with abortion among dogs and animals in the area (odds ratio = 3.6). In Amman and Zarqa, living indoors reduced seroprevalence at 0.45, 0.24, and 0.02 odds ratios, respectively. Jordan shares three molecular N. caninum strains with three different countries and continents.

Human toxoplasmosis, a protozoonosis caused by Toxoplasma gondii, has been described as a worldwide foodborne disease with important public health impact. Despite infection has reportedly varied due to differences in alimentary, cultural and hygienic habits and geographic region, social vulnerability influence on toxoplasmosis distribution remains to be fully established. Accordingly, the present study has aimed to assess T. gondii seroprevalence and factors associated to social vulnerability for infection in households of Ivaiporã, southern Brazil, with 33.6% population making half minimum wage or less, ranked 1,055th in population (31,816 habitants), 1,406th in per capita income (U$ 211.80 per month) and 1,021st in HDI (0.764) out of 5,570 Brazilian cities. Serum samples and epidemiological questionnaires were obtained from citizen volunteers with official City Secretary of Health assistance in 2015 and 2016. In overall, serosurvey has revealed 526/715 (73.57%) positive samples for anti-T. gondii antibodies by Indirect Fluorescent Antibody Test. Logistic regression has shown a significant increase associated to adults (p = 0.021) and elderly (p = 0.014) people, illiterates (p = 0.025), unemployment (p <0.001) and lack of household water tank (p = 0.039). On the other hand, sex (male or female), living area (urban or rural), yard hygiene, meat ingestion, sand or land contact, owning pets (dog, cat or both) were not significant variables of positivity for anti-T. gondii antibodies in the surveyed population. Although no significant spatial cluster was found, high intensity areas of seropositive individuals were located in the Kernel map where the suburban neighborhoods are located. In conclusion, socioeconomic vulnerability determinants may be associated to Toxoplasma gondii exposure. The increased risk due to illiteracy, adult or elderly age, unemployment and lack of household water tank were confirmed by multivariate analysis and the influence of low family income for seropositivity by the spatial analysis.

Protozoa of the genus Sarcocystis have an obligate two-host-prey-predator life cycle, in which South American Camelids (SAC) are among the intermediate hosts for certain species. Sarcocystis spp. infection has been well documented in SAC in endemic areas of the South America, while data from Europe are absent. This study assessed the seroprevalence of Sarcocystis spp. in SAC in Italy investigating related risk factors. A total of 506 SAC sera (486 alpacas and 20 llamas) from 38 sampling sites were analysed to detect antibodies to Sarcocystis spp. using the Indirect Fluorescent Antibody Test (IFAT). A seropositivity of 15.2% (77/506) was found, corresponding to 14.4% (70/486) in alpacas and 35.0% in llamas (7/20). Risk factor analysis showed that Sarcocystis spp. seropositivity increased with age, while no statistically significant association was found with regard to sex. Access to stables and/or pasture by dogs was identified as a putative risk factor, particularly as the number of dogs increased. Further association was found between seropositivity to Sarcocystis spp. and N. caninum . Although the life cycles of Sarcocystis spp. in SAC are not fully elucidated, these findings highlight the need for further studies to clarify the potential role of dogs as definitive hosts for Sarcocystis species infecting SAC in Europe. This study provides for the first time data on specific antibodies for Sarcocystis spp. in SAC in Europe, highlighting the need of improving the surveillance of this protozoan in camelids, particularly given the growing interest in camelid meat production.

Background Leishmania infantum is a sand fly-transmitted zoonotic protozoan, endemic in the Mediterranean basin and responsible for human, canine (CanL), and feline (FeL) leishmaniosis. While dogs are the primary reservoir host, a growing number of FeL cases have been reported in this region despite the absence of pathognomonic clinical signs and limited diagnostic tools. Herein, we evaluate the performance of seven serological tools for CanL in detecting antibodies to Leishmania in cats, aiming to improve FeL diagnosis. Methods Five ELISAs based on Leishmania -specific antigens (soluble promastigote Leishmania antigens, SPLA; recombinant Leishmania proteins K39 [rK39], K28, and KDDR, and L. infantum cytosolic peroxiredoxin, LicTXNPx), indirect fluorescent antibody test (IFAT), and direct agglutination test (DAT) were compared for detecting anti- Leishmania antibodies in 274 cats. Blood samples from the same cats were molecularly tested. Statistical analysis was performed based on clustering of multivariate serological data. Reference serological profiles were first defined in a control group. Study group data were subsequently classified according to these profiles, with principal component analysis used for dimensionality reduction and graphical representation. Associations between seropositivity and clinicopathological alterations were determined using seropositivity thresholds. Results Cats exhibited attenuated and heterogeneous antibody responses to L. infantum serological tests. Agreement between individual tests was variable, with poor concordance when single markers were considered. Multivariate analysis, based on clustering of serological responses, showed that positivity to multiple antigens was associated with clinically affected cats. Positivity to multiple Leishmania -specific ELISA antigens was associated with diverse clinical presentations and prognostic laboratory alterations, including anaemia, thrombocytopenia, and hypergammaglobulinaemia. Conclusions Integrating multi-antigen ELISA, particularly rK39, SPLA, and LicTXNPx, into FeL diagnostic workflows, alongside molecular and clinical assessment, improves epidemiological surveillance, early detection, and disease management. These findings support the development of serological strategies tailored to feline hosts for enhanced surveillance and management. Graphical Abstract

Canine leishmaniosis (CanL) is a neglected zoonotic disease caused by Leishmania spp. Leishmania infantum is the species responsible for the zoonotic form of the disease where dogs are reservoir hosts. This study aimed to determine the seroprevalence of CanL in asymptomatic dogs in Kosovo. Blood samples were collected from 285 dogs in all seven regions in Kosovo (35–50 samples per region) from summer 2021 to spring 2022. Sera were tested using enzyme-linked immunosorbent assay (ELISA), and the presence of anti- Leishmania IgG was confirmed by an indirect fluorescent antibody test (IFAT). The true overall seroprevalence of CanL of asymptomatic dogs in Kosovo with ELISA was 4.21% (95% CI: 2.42–7.21) while with IFAT was 3.51% (95% CI: 1.92–6.34). The highest rates were found in the Prishtina region to be 8.0% (4/50) by ELISA and 6.0% (3/50) by IFAT, and in the Mitrovica region, the prevalence was 0% (0/40). There were no significant differences among the different regions, gender, age, health status, and breed. These findings highlight the presence of CanL in most regions of Kosovo and underline the veterinary relevance of clinically asymptomatic dogs infected with Leishmania .

Diagnosis of equine Lyme borreliosis (LB), an infection caused by members of the Borrelia burgdorferi sensu lato complex ( Bb sl), is challenging due to the nonspecific clinical signs of the disease and due to the variety of non-standardized serological tests. Specific vaccine-induced antibodies against LB, providing an effective protection against the infection, complicate the issue further. The standard for the detection of specific antibodies against Bb sl is a two-tier test system based on an enzyme-linked immunosorbent assay (ELISA) or indirect fluorescent antibody test (IFA) for antibody screening combined with a qualitative, highly specific immunoassay (e. g. line immunoassay (LIA)) for confirmation. In this study, three LIAs available for detection of antibodies in equine serum samples were evaluated and compared. A total of 393 serum samples of 131 horses with known serostatus were used. It included groups of non-vaccinated horses, immunized horses (vaccinations against LB on days 0 and 14), and horses that had received an initial immunization plus an additional booster on day 180. Sera were collected on days 0, 135 and 210 of the study. Results were compared considering the tests’ sensitivity, specificity, diagnostic outcome, and the operability of each test. Agreements of the diagnostic results among the LIAs were calculated for overall test results and single antigen-antibody-complex signal results. They are presented as inter-rater agreement and statistic reliability, represented by the Fleiss’ kappa coefficient. Agreement scores ranged from poor to moderate depending on group and time-point of blood sample collection. Depending on LIA used, deficiencies were observed in the form of non-sufficient sensitivity of antigen signals on the LIA strips (especially for outer surface protein A (OspA) or variable major protein like sequence expressed (VlsE)) or as an inappropriate test interpretation of the OspA signal. Operability of the three LIAs was equally user-friendly with minor variations. In two LIAs, test-evaluation was simplified by a supplied scanner and evaluation software. To improve functionality of available LIAs for equine serum samples it is advisable to adjust sensitivity and specificity of single test antigen signals and establish appropriate evaluation protocols.

Toxoplasma gondii is a widespread zoonotic protozoan parasite, with domestic cats as definitive hosts. To improve serodiagnosis surveillance and control, this study aimed to develop and evaluate an indirect enzyme-linked immunosorbent assay (iELISA) using recombinant Tg GRA14 protein for the serological detection of T. gondii in domestic cats. The Tg GRA14 gene (nucleotides 114–858 encoding amino acids 38–286) was synthesized and cloned into the pET-28a vector containing an N-terminal FLAG tag. The recombinant plasmid was transformed into E. coli BL21, and protein expression was purified via anti-FLAG affinity chromatography. Protein integrity, antigenicity and identity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blotting, while mass spectrometry verified protein identity. The purified Tg GRA14 protein was used as a coating antigen in iELISA, which was applied to 149 feline serum samples and compared with results from the indirect fluorescent antibody test (IFAT). The recombinant Tg GRA14 protein (approximately 29 kDa) was successfully expressed and purified, with confirmed antigenicity and 99.6–100% identity by mass spectrometry. The iELISA detected  38.3% seroprevalence in feline samples, compared to 32.9% by IFAT, and showed high diagnostic performance with 95.9% sensitivity, 90.0% specificity, and a kappa value of 0.824. These results indicate that the iELISA based on recombinant Tg GRA14 is a highly sensitive and specific diagnostic tool for detecting T. gondii infection in domestic cats, which has strong potential for application in epidemiological surveillance and serological screening programs.

Background Lysine lactylation (Kla) is a novelposttranslational modification (PTM) identified in histone and nonhistone proteins of several eukaryotic cells that directly activates gene expression and DNA replication. However, very little is known about the scope and cellular distribution of Kla in apicomplexan parasites despite its significance in public and animal health care. Methods Toxoplasma gondii , the causative agent of toxoplasmosis, is an obligate intracellular apicomplexan parasite that can infect different nucleated cell types of animals and humans. We used this parasite as a model organism and extracted the total protein of tachyzoites to produce the first global lysine lactylome profile of T. gondii through liquid chromatography–tandem mass spectrometry. We also investigated the level and localization of the Kla protein in T. gondii using western blotting and the indirect fluorescent antibody test (IFA), respectively. Results A total of 983 Kla sites occurring on 523 lactylated proteins were identified in the total protein extracted from Toxoplasma tachyzoites, the acute toxoplasmosis-causing stage. Bioinformatics analysis revealed that the lactylated proteins were evolutionarily conserved and involved in a wide variety of cellular functions, such as energy metabolism, gene regulation and protein biosynthesis. Subcellular localization analysis and IFA results further revealed that most of the lactylated T. gondii proteins were localized in the nucleus, indicating the potential impact of Kla on gene regulation in the T. gondii model. Notably, an extensive batch of parasite-specific proteins unique to phylum Apicomplexa is lactylated in T. gondii . Conclusions This study revealed that Kla is widespread in early dividing eukaryotic cells. Lactylated proteins, including a batch of unique parasite proteins, are involved in a remarkably diverse array of cellular functions. These valuable data will improve our understanding of the evolution of Kla and potentially provide the basis for developing novel therapeutic avenues. Graphical Abstract