https://doi.org/10.52973/rcfcv-e363981 Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI Recibido: 29/04/2026 Aceptado: 16/07/2026 Publicado: 12/08/2026 1 of 10 Aya BENSALEM * , Sana HIRECHE , Amina AMRAOUI , Samia AMEZIANE , Amir AGABOU Seroepidemiology and biochemical alterations associated with Neospora caninum infection in dairy cows from the northeastern region of Algeria Seroepidemiología y alteraciones bioquímicas asociadas con la infección por Neospora caninum en vacas lecheras de la región noreste de Argelia University of Constantine 1-Frères Mentouri, Institute of Veterinary Sciences El-Khroub, PADESCA Research Laboratory. Constantine, Algeria. ⃰Corresponding author: drayabensalem@gmail.com Bovine neosporosis is a major cause of abortion and stillbirth in cattle, causing significant economic losses worldwide. This study aimed to determine Neospora caninum seroprevalence, identify its potential risk factors and investigate alterations in selected biochemical traits in cattle from ﬁve Northeastern Algerian provinces. From March 2022 to April 2025, 368 cows belonging to 36 herds were randomly sampled and analyzed using an indirect enzyme-linked immunosorbent assay kit. Risk factors were determined using multivariable logistic regression model. For biochemical disturbances, sera from 30 seropositive and 30 seronegative cows were tested for glucose, total protein, albumin, calcium, phosphorus, urea, creatinine, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, lactate dehydrogenase and β- hydroxybutyrate, using standard laboratory methods. Biochemical values were compared statistically and the effects of N. caninum infection were evaluated using generalized linear models adjusting for confounding factors. According to results, herd and individual animal prevalence were 63.88 % (23/36; 95 % CI: 46.20 – 79.20) and 16.30 % (60/368; 95 % CI: 12.70 – 20.50) respectively. At individual level, the risk of being seropositive increased with age, history of reproductive problems, mastitis and types of vaccines. The use of artificial insemination, vaccination and birth of healthy calves were protective factors. At herd-level, quarantine and vaccination were protective factors. Infected cows showed significantly elevated serum levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, urea, total protein and globulin along with a significant decrease in calcium and albumin/globulin ratio. This survey highlights the widespread establishment of N. caninum within local cattle population and elucidates its associated risk factors as well as the metabolic and reproductive disorders it may generate. RESUMEN Palabras clave: Neosporosis; ganado; Argelia; seroprevalencia; factores de riesgo; cambios bioquímicos; trastornos metabólicos. La neosporosis bovina es una de las principales causas de abortos y mortinatalidad en el ganado vacuno, lo que provoca importantes pérdidas económicas en todo el mundo. El objetivo de este estudio fue determinar la seroprevalencia de Neospora caninum, identificar sus posibles factores de riesgo e investigar las alteraciones en determinados parámetros bioquímicos en el ganado vacuno de cinco provincias del noreste de Argelia. Entre marzo de 2022 y abril de 2025, se tomaron muestras aleatorias de 368 vacas pertenecientes a 36 rebaños y se analizaron utilizando un kit ensayo por inmunoabsorción ligado a enzimas indirecto. Los factores de riesgo se determinaron mediante un modelo de regresión logística multivariable. En cuanto a las alteraciones bioquímicas, se analizaron las muestras de suero de 30 vacas seropositivas y 30 seronegativas para determinar los niveles de glucosa, proteína total, albúmina, calcio, fósforo, urea, creatinina, alanina aminotransferasa, aspartato aminotransferasa, gamma- glutamil transferasa, fosfatasa alcalina, lactato deshidrogenasa y β-hidroxibutirato, utilizando métodos de laboratorio estándar. Los valores bioquímicos se compararon estadísticamente y los efectos de la infección por N. caninum se evaluaron mediante modelos lineales generalizados ajustados por factores de confusión. Según los resultados, la prevalencia en el rebaño y en los animales individuales fue del 63,88 % (23/36; IC del 95 %: 46,20 – 79,20) y del 16,30 % (60/368; IC del 95 %: 12,70 – 20,50), respectivamente. A nivel individual, el riesgo de ser seropositivo aumentaba con la edad, los antecedentes de problemas reproductivos, la mastitis y los tipos de vacunas. El uso de la inseminación artificial, la vacunación y el nacimiento de terneros sanos fueron factores protectores. A nivel de rebaño, la cuarentena y la vacunación fueron factores protectores. Las vacas infectadas mostraron niveles séricos signiﬁcativamente elevados de alanina aminotransferasa, aspartato aminotransferasa, gamma- glutamil transferasa, urea y proteína total y globulina, junto con una disminución signiﬁcativa del calcio y de la relación albúmina/globulina. Este estudio pone de relieve la amplia diseminación de N. caninum en nuestra población bovina local y aclara los factores de riesgo asociados, así como los trastornos metabólicos y reproductivos que puede provocar. ABSTRACT Key words: Neosporosis; cattle; Algeria; seroprevalence; risk factors; biochemical changes; metabolic disorders.

2 of 10 Bovine neosporosis: seroepidemiology and metabolic disorders/BENSALEM et al. INTRODUCTION Neospora caninum is a cyst-forming coccidian protozoan parasite of the Toxoplasmatinae subfamily, the Sarcocystidae family and the Apicomplexa phylum. It is worldwide distributed and infects a large variety of endothermic species, especially ruminants and canids [1]. The global economic impact of N. caninum infection is estimated at over 1.3 billion USD per year in the dairy and beef industries, resulting from direct losses (related to abortions, stillbirths, fetal deaths and milk production drops) and indirect ones (due to Veterinary, diagnosis and control measures costs) [2]. The parasite is characterized by a complex biological cycle that includes sexual and asexual stages. Domestic dogs (Canis lupus familiaris), coyotes (Canis latrans) and dingoes (Canis lupus dingo) serve as deﬁnitive hosts that excrete sporulated oocysts in their feces and contaminate the environment [3, 4]. Cattle (Bos taurus), sheep (Ovis aries), goats (Capra hircus), horses (Equus caballus), chickens (Gallus gallus domesticus), dogs, cats (Felis catus) and rabbits (Oryctolagus cuniculus) act as intermediate hosts that get infected through a horizontal route by ingesting oocysts-contaminated food or water [1, 5]. Vertical transmission can also occur in cattle, when pregnant infected cows transfer the parasite transplacentally to their offspring [6, 7] . N. caninum’s life cycle includes three main infectious stages: sporozoites (contained within sporulated oocysts), bradyzoites (also termed cystozoites) and tachyzoites [8]. Neosporosis is characterized by the proliferation of tachyzoites in multiple cell types, inducing their lysis [9, 10]. Histopathological examination of N. caninum infected aborted calves reveals inflammatory lesions, such as non-suppurative hepatitis, myocarditis, pericarditis and encephalitis [11] . Polymerase chain reaction (PCR)-positive fetal tissues from aborted lambs show mononuclear cell infiltration, cerebral lesions, cardiac and hepatic necrosis (characterized by parenchymal destruction and hepatocytes atrophy) and ﬁbrosis [12]. Reproductive disorders are the principal outcomes of bovine neosporosis and have been related with late ﬁrst calving, reduced conception rates and prolonged calving intervals [13, 14] . The infection may manifest as sporadic, endemic or epidemic From march 2022 till april 2025, 368 dairy cows of more than two years old were randomly sampled from 36 herds located in Oum El Bouaghi (n = 33), Constantine (n = 78), Mila (n = 82), Guelma (n = 85) and Skikda (n = 90) provinces (Northeastern Algeria) (FIG.1) (TABLE I) [20, 21, 22]. MATERIALS AND METHODS Study design, population and sampling For significance representativeness, a two-stages sampling was performed. First, the minimum required sample size (355 serum samples) was calculated using formulas for simple random sampling as recommended by Thrusﬁeld [23], using a conﬁdence level of 95 %, a required precision of 5 % and an estimated prevalence of 36.2 % [19]. Then, at herd level, simple random sampling was performed using the records of the ministry of Agriculture. Random number tables have been employed to take random samples of cows from each herd. The minimal number of cows to be selected, so as to detect the presence of the disease, was calculated using the following formula (Eq.1): abortion, mostly at 5 - 6 months of gestation. Furthermore, the infection can lead to the birth of neurologically impaired calves [15, 16], or persistently-infected apparently-healthy calves [1,5, 17]. The occurrence of N. caninum in cattle has been recognized worldwide, showing regional fluctuations. Reported prevalence ranges from 4.1 to 43 % in Asia, 0.5 to 27.7 % in Europe, around 10.2 % in Oceania, 7.6 to 41 % in America and 10.7 to 19.6 % in Africa [18]. In Algeria, available studies indicate a prevalence of 12.4 to 36.2 % [19], with a very scarce data for the Northeastern region. Thus, we have undertaken the current study to deﬁne N. caninum seroprevalence and determine its potential risk factors. Recently, the epidemiology and diagnostic strategies of N. caninum have been widely documented, however, studies focusing on metabolic and biochemical alterations in dairy cows affected by neosporosis are rare. In this context, the present study also aimed to examine the effect of N. caninum on selected serum biochemical parameters in naturally infected cattle.

3 of 10 Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI RESULTS AND DISCUSSION To investigate the relationship between certain biochemical parameters and N. caninum serostatus, sera from 30 seropositive and 30 seronegative animals were selected. In order to minimize confounding variables, each seropositive cow was matched with a seronegative one of similar age, breed, lactation phase and pregnancy status. Serum biochemical parameters including calcium, phosphorus, glucose, total protein, albumin, creatinine, urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), were measured colorimetrically using biochemistry reagent kits (Biosystems S.A., Spain) on a BA-88A analyzer (Mindray, China). Globulin concentrations were obtained by calculating the difference between serum total protein and albumin concentration. Albumin/globulin ratio (A/G) was also calculated. Serum β- hydroxybutyrate (BHB) was determined using a rapid test kit (On Call GK Dual, Acon Laboratories, USA). Data from questionnaires, serological and biochemical analyses were analyzed using SPSS (Version 26, IBM Corp, USA). Chi-square tests were performed to identify factors associated with N. caninum seropositivity at individual and herd levels. Variables with P ≤ 0.25 were entered to multivariate logistic regression analysis. After normality testing (Shapiro-Wilk test), biochemical parameters (mean ± SD) were compared between seronegative and seropositive cattle using Student’s t test or Mann- Whitney U test. Generalized linear models (GLMs) evaluated the independent effect of N. caninum seropositivity on each biochemical parameter. N. caninum serostatus (seropositive/ seronegative) was entered as the main predictor factor. The following variables were also included in each model as independent variables: age (< 3, [3 – 6], and > 6 years), breed (Holstein, Atlas Brown, Montbeliarde), lactation phase (early, mid, late, dry, gravid heifer) and parity (0, 1, 2, 3, 4, 5, and > 5). For all analysis, P < 0.05 indicated statistical signiﬁcance. A pre-structured questionnaire based on previously reported associated factors to N. caninum infection was administered to farmers. Farm-level variables included farming system (extensive/intensive/semi-intensive), farm type (dairy/mixed), presence of other animal species (dogs, rodents, cats, sheep, poultry, and wild animals), forest access, water source, feeding practices, and disinfection methods. Individual animal data covered age, breed, vaccination and deworming status, lactation phase, parity, pregnancy status, and reproductive disorders (abortion, stillbirths, mastitis, and infertility) during the last 24 months. Serum antibodies (IgG) against N. caninum were detected by an indirect enzyme-linked immunosorbent assay (ELISA) kit (ID Screen®, Innovative Diagnostics, France) as recommended by the manufacturer. Both specificity and sensitivity of the test attain 100 % (95 % CI: 99.41 – 100 % and 95 % CI: 98.8 – 100 %, respectively). Optical density (OD) values were measured at 450 nm using an ELx800 microplate reader (BioTek Instruments Inc., USA). Results were calculated as sample to positive (S/P) ratios, with values ≥ 0.5 considered as positive. Out of the 368 dairy cattle tested for N. caninum, 60 samples (16.30 %) were found seropositive. The prevalence varied across provinces: 15.15 % (5/33) in Oum El Bouaghi, 15.29 % (13/85) in Guelma, 15.38 % (12/78) in Constantine, 17.07 % (14/82) in Mila and 17.78 % (16/90) in Skikda. The over-all herd-level prevalence was 63.88 % (23/36). Published data on bovine N. caninum epidemiology in Algeria are limited, with only five studies documented between 2012 and 2025, yielding prevalence rates ranging from 12.22 to 36.2 % across different regions [19, 24, 25, 26, 27]. The present study recorded a prevalence of 16.30 %, consistent with this range and comparable to ﬁndings of Derdour et al. [27] of 15 % and Ghalmi et al. [25] of 19.64 %, but it is higher than those (12.37 and Where n is the sample size, P is the probability of detection of at least one seropositive cow, N is the herd size, and d is the number of seropositive cows in the herd. The probability to detect at least one seropositive cow in a herd was determined at 95 % (P = 0.95), while the number of seropositive cows in each herd (d) was estimated assuming that within herd prevalence equals 10 %. Blood samples (10 mL) were obtained from the jugular vein, using disposable needles and plain tubes. They were transported on ice to the laboratory and centrifuged at 0.805 G for 10min (Sigma, model 1–6P, maximum speed: 2.837 G, Germany). Serum was divided into two separated aliquots (for serological and biochemical analyses) that were kept at – 20 °C until testing (ENIEM, model CF 1301, 350 L, Algeria). Epidemiological data collection Serological analysis Seroprevalence Biochemical analyses Statistical analysis FIGURE 1. Geographic map of Algeria showing the locations of the sampled dairy herds for Neospora caninum infection

4 of 10 Bovine neosporosis: seroepidemiology and metabolic disorders/BENSALEM et al. At individual level, multivariable logistic regression analysis (TABLE II) identified age, type of vaccines used, mastitis and reproductive disorders as significant risk factors. However, vaccination, artificial insemination and birth of healthy calves were protective factors. At herd level, there were no significant risk factors, vaccination and quarantine were identiﬁed as protective (TABLE III). In this study, older animals were more likely to be seropositive to N. caninum, in agreement with the findings of Selim et al. in Egypt [36] and Llano et al. in Colombia [37], who observed a positive association between age and N. caninum seroprevalence. This could be attributed to prolonged exposure time, that increases the probability of acquiring the infection via horizontal transmission and contact with definitive hosts [1]. Conversely, in Iran, Noori et al. [38] observed a seropositivity decrease with age, in relation to gradual antibody titers decline (in absence of re-infection), reduced longevity or selective culling of seropositive cattle [1, 39]. Cattle with reproductive disorders history had significantly increased odds of seropositivity, speciﬁcally, those with metritis, fertility disorders and multiple reproductive issues. These ﬁndings support earlier studies that identified various reproductive problems, like abortion, stillbirth and uterine infections, to be significantly associated to N. caninum seropositivity [8, 14, 17, 36]. In addition, mastitis was significantly associated with increased risk of N. caninum seropositivity, in accordance with Thurmond and Hietala [40], who reported seropositive cows to Associated risk factors 12.22 %) described by Achour et al. and Miroud et al. [24, 26], respectively, and lower as compared to prevalence of 36.2 % documented by Abdeltif et al. [19]. At the international level, the observed prevalence is comparable to those observed in Portugal (17.2 %) [28] and Egypt (18.6 %) [29] . However, this rate is lower than those reported in Brazil (47.36 %) [30] and Greece (21.03 %) [17] and higher than those found in Ethiopia (13.3 %) [31] and China (13.6 %) [32]. In neighboring countries, there are also fewer surveys of N. caninum in domestic animals in general. In Morocco (on the western borders of Algeria), Zouagui et al. found 25 % of dairy cattle to be seropositive for this protozoan [33]; while in Tunisia (on the eastern borders), this parasite had been molecularly identiﬁed in genital apparatus of 21.1 % naturally infected cows [34] which confirms the establishment of this pathogen. For the other surrounding countries, no data could be found in the published scientiﬁc literature. The observed inter-regional and international variations in prevalence may be ascribed to differences in environmental conditions, herd management, diagnostic methods, study design, and sampling strategies [35].

5 of 10 Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI be more susceptible to mastitis, with lower milk production and a duple risk of being culled due to udder disorders. This suggests that chronic N. caninum infection may alter mammary immunity and increase vulnerability to intramammary pathogens. On the other hand, Peregrine et al . [41] found better udder health markers in seropositive cows. Although artificial insemination may control bovine neosporosis [42] , Algerian family farms (especially small holders) still share a same bull to breed all their cows, even between herds, facilitating horizontal transmission of the parasite. In the present study, artificially inseminated cows had signiﬁcantly lower risk compared to naturally bred ones. Farms practicing quarantine showed significantly lower odds to be seropositive to N. caninum. However, this protective effect is not necessarily attributed to quarantine itself. In the visited farms, quarantine consisted of isolating newly introduced animals up to 15 days to evaluate their overall health. Since N. caninum infection is asymptomatic in cattle, and serological diagnosis of the parasite is not systematically performed in Algeria, this measure cannot identify infected animals or directly reduce seropositivity. The lower odds observed may reflect better farm management, including good hygiene practices and rigorous biosecurity measures that control horizontal transmission [35]. To date, no specific commercial vaccines for N. caninum are available [43]. Although, cattle vaccinated against other pathogens had significantly lower N. caninum seropositivity, at both individual and herd level. Regular vaccination reflects good herd management. Farms that invest in routine vaccination demonstrate greater hygiene vigilance and apply good biosecurity measures, both of which are known to reduce the risk of horizontal transmission of N. caninum [1]. On the contrary, cattle receiving multiple vaccines (Aphtovirus, Rhabdovirus, Pasteurella, Clostridium) had higher seropositivity Serum biochemical concentrations in N. caninum-seropositive and seronegative cattle are presented in TABLE IV. Results of generalized linear models for signiﬁcant biochemical parameters associated with N. caninum serostatus and predictor factors (age, breed, parity and lactation phase) are detailed in TABLE V. Serum biochemical alterations than those vaccinated only against Aphtovirus and Rhabdovirus. Similar associations between specific vaccination protocols and increased N. caninum seropositivity have been recorded by Cavirani et al. [44]. This increase may result from vaccine- induced immune stress, that can reactivate latent encysted bradyzoites. Furthermore, as cattle are vulnerable to endotoxins, the administration of vaccines against Gram-negative bacteria such as Pasteurella, which contain lipopolysaccharide endotoxins, may alters host immunity and increase the likelihood of acquiring secondary infections [45]. Cows that gave birth to healthy calves had less chance of being seropositive for N. caninum, as confirmed by the pathophysiology of vertical transmission. In fact, infected cows can transmit the parasite transplacentally to their fetuses, leading to fetal losses, stillbirths or birth of weak or non-viable calves [1, 6].

6 of 10 Bovine neosporosis: seroepidemiology and metabolic disorders/BENSALEM et al. The biochemical profile of dairy cattle can be influenced by various factors including physiological status, nutrition, breed predispositions and pathologies [46, 47]. Based on the literature on N. caninum pathogenesis, we targeted 15 biochemical indicators of hepatic, renal and metabolic functions. As shown in TABLE V, certain predictor factors, including parity, breed, and lactation phase, significantly influenced some biochemical parameters. It should be noted, however, that this study was designed to investigate the impact of N. caninum serostatus on biochemical parameters, and not to assess the effects of physiological variables that the study will not discuss. Infected cattle showed significantly increased hepatic enzymes: AST, ALT and GGT compared with healthy ones. Alike previous studies [48, 49] , this could result from the hepatocellular damage caused by N. caninum. As well, elevated AST may also reflect necrosis in other organs [1, 12]. Univariate comparative analysis revealed significant differences in serum levels of total protein, globulin, A/G ratio, AST, ALT, ALP, urea, creatinine and calcium between N. caninum seropositive and their seronegative counterparts (TABLE IV). After adjusting by generalized linear models (TABLE V), seropositive cattle showed significantly higher levels of total protein (P < 0.0001), globulin (P < 0.0001), AST (P < 0.0001), ALT (P = 0.046), GGT (P = 0.020) and urea (P = 0.000), and significantly lower levels of calcium (P < 0.0001) and A/G ratio (P = 0.009). No significant associations were noticed for BHB, glucose, albumin, LDH, ALP, creatinine or phosphorus (P > 0.05). After adjusting for confounding factors, GGT became statistically signiﬁcant (P = 0.054 vs P = 0.020) while, the ALP (P = 0.033 vs P = 0.231) and creatinine (P = 0.007 vs P = 0.520) turned into non- signiﬁcant (TABLES IV and VI).

7 of 10 Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI Regarding mineral metabolism, no significant differences in phosphorus concentrations were observed between seropositive and seronegative cows. But, seropositive cows had signiﬁcantly lower calcium levels. Under physiological conditions, the coordinated action of 1.25-dihydroxyvitamin D3, parathyroid hormone (PTH) and calcitonin, controls calcium levels [50] . The pathological effects of neosporosis on hepatic and renal functions may disturb the hydroxylation steps of vitamin D to 25-hydroxyvitamin D3 in the liver and to 1.25-dihydroxyvitamin D3 in the kidneys [50], that lower the calcium levels. Moreover, proinflammatory interleukins produced during the host immune response activated during neosporosis can reduce PTH secretion, causing hypocalcemia [51]. A previous study reported significantly elevated BHB in N. caninum infected cows [49]. Differently to these findings, no significant differences were detected in serum levels of BHB and glucose between seropositive and seronegative cows in the present work. Raised urea and creatinine levels have been reported in sheep with renal damage and dysfunction following infection with Toxoplasma gondii [48] , another protozoan coccidian parasite that shares many similarities with N. caninum [1]. In the current survey, comparable results were observed, but, elevated creatinine in seropositive cows was not significant after GLM adjustment, suggesting confounding factors. In contrast, elevated urea remained signiﬁcantly associated with seropositivity. Although, this dissociation between urea and creatinine indicates that the observed hyperuremia may reflect increased proteins catabolism due to inflammation process or may be related to other factors rather than renal dysfunction, such as nutrition, that were not explored in this study. As opposed to Alekich et al . [49] , who observed a non- significant decrease in total protein in N. caninum seropositive cows, the findings of the current study showed a significant increase, even after adjustment. This may be due to the non- suppurative inflammation induced by N. caninum in multiple organs [1, 11, 47]. The elevated total protein amount was associated with a significant increase in globulin levels and a decrease in A/G ratio in seropositive cattle, which reflects the immunoglobulin production during the immune response [1, 47, 52] . These ﬁndings are reinforced by those of Appelt et al. [53]. CONCLUSION The present study provides evidence of subclinical hypocalcemia in dairy cows in relation to neosporosis. Altered hepatic enzymes reflect an overall metabolic disorder beyond the well-known reproductive issues. Further researches must be conducted to better understand pathological mechanisms of this infection on dairy cows’ metabolism. 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