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Infectious meningoencephalitides represent an important differential diagnosis for meningoencephalitis of unknown origin (MUO) in dogs. Treatment of the latter requires immunosuppression, but laboratory test results for infectious agents may take several days to return. This study investigated whether the presence of masticatory muscle changes on magnetic resonance imaging (MRI) of the head can be used to distinguish dogs with neosporosis from those with MUO at the time of diagnosis. Cases diagnosed with neosporosis or MUO at two referral centers in the United Kingdom (UK) were retrospectively collected. Clinical data were reviewed, and each MRI study was blindly assessed by a radiologist, a neurologist, and a neurology resident for the presence of masticatory muscle changes by consensus opinion. Statistical analysis was performed on obtained data. Twenty-two neosporosis cases and 23 MUO cases were enrolled. In the neosporosis group, six dogs (27%) had masticatory muscle changes, compared to one dog (4%) in the MUO group (p = 0.047). All six neosporosis cases had bilateral, multifocal, T2W and FLAIR hyperintense, contrast enhancing muscular changes, with three having concurrent masticatory muscle atrophy. The only MUO case with muscle changes had a mild, focal, unilateral temporal muscle lesion which was only visible in the T1W post-contrast images. Within the neosporosis group, dogs with masticatory muscle lesions had significantly higher cerebrospinal fluid WBC counts (p = 0.017) and protein concentrations (p = 0.025) compared to those without muscle changes. In conclusion, characteristic bilateral, multifocal masticatory muscle changes should raise the index of suspicion for neosporosis in dogs with an imaging diagnosis of meningoencephalitis and starting early antimicrobial treatment is recommended. However, the absence of masticatory muscle abnormalities does not exclude active Neospora caninum infection. In these cases, whether immunosuppressive or antimicrobial treatments are started prior to receiving further test results should still be based on the clinical status of the animal and index of suspicion using a combination of all available clinical information at that time. [ABSTRACT FROM AUTHOR]

OBJECTIVE This study aimed to report the impact of production practices and sanitary management on the prevalence of Neospora caninum (N caninum) and bluetongue virus (BTV) in Creole goats from the tropical dry forest of Utcubamba, Peru. METHODS 354 blood samples were obtained from male and female goats of diverse ages reared under an extensive productive system. The diagnosis of N caninum and BTV was conducted through a commercial ELISA kit (iD.vet) with readings taken on a Bio-Rad iMark microplate reader at 450 nm. Additionally, a survey was conducted with 18 goat producers to categorize them based on social, technical (production, health, reproduction, nutrition, and infrastructure), environmental, and economic factors. RESULTS The overall seroprevalence of N caninum and BTV in goats was 6.21% and 7.06%, respectively, Notably, group II farmers had the highest prevalence rates at 7.69% for N caninum and 23.08% for BTV. This group comprises producers with fewer than 21 goats, while group III includes 14.3% of producers with over 60 goats and better infrastructure. CONCLUSIONS This study confirms the presence of N caninum and BTV in goats of Utcubamba, Peru. Group II displayed the highest prevalence rates, suggesting that herd size and management infrastructure influence disease prevalence. CLINICAL RELEVANCE In the tropical dry forest of Utcubamba, inadequate management of N caninum carrier agents (eg, nondewormed dogs) heightens transmission risk, while the tropical climate supports BTV vectors. Effective diagnosis and health management strategies, including vector control and biosecurity practices, are crucial for ongoing herd health monitoring. [ABSTRACT FROM AUTHOR]

Background: The reproductive problem is an animal health-related bottleneck that constrains livestock genetic improvement efforts in tropical countries such as Ethiopia. The infectious causes of reproductive disorders are one cause of decreased reproductive efficiency. This study aimed to determine the seroprevalence to Bovine Herpesvirus-1 (BHV1), Bovine Viral Diarrhea Virus (BVDV), Neospora caninum (N. caninum) and C. burnetii (C. burnetii) exposures in dairy cows with reproductive disorders in selected areas of Ethiopia. Overall, 164 serum samples were collected from October 2018 to May 2019 from animals with a history of reproductive disorders. The collected sera were tested for antibody titers to Brucella species, N. caninum, BVDV, BHV1, C. burnetii and Chlamydophila abortus (C. abortus) using Rose Bengal and ELISA. Results: The apparent seroprevalence of BHV1, BVDV, N. caninum and C. burnetii were 61%, 33.5%, 4.9% and 0.6%, respectively. Among the selected study areas, the mean apparent seroprevalence was significantly greater in Bishoftu (35.9%), Holeta (34.2%) and Adaberga (28.6%) than in Mekelle (9.9%) and Ambo (16.2%). Among the specific seroprevalence in specific areas, BHV1 was the most common in Adaberga, with an apparent seroprevalence of 92.9%. Similarly, the seroprevalence of BVDV was the highest in Holeta, with an apparent seroprevalence of 73.3%. On the other hand, no seropositive animal to Brucella spp. or C. abortus was found in these study areas. Conclusion: BVDV and BHV1 seroprevalence was higher in dairy cattle with a history of reproductive disorder in Ethiopia as compared to the seroprevalence of N. caninum and C. burnetii. [ABSTRACT FROM AUTHOR]

Bovine brucellosis and neosporosis are reported as potential abortifacient infections in cattle worldwide. Brucellosis is additionally a zoonotic bacterial infection caused by numerous Brucella species. Meanwhile, neosporosis is a protozoan parasitic disease that is implicated in causing high economic losses in the cattle industry. Herein, we attempted to investigate the seroprevalence of specific antibodies to Brucella spp. and Neospora caninum using commercially available ELISAs. In addition, we conducted risk factor analysis and estimated the correlation of seropositivity of both pathogens with the recorded abortions in the tested herds. Serum samples from cattle (n = 460) collected from various governorates in the Delta region, northern Egypt, were targeted in this study. Overall, a seroprevalence of 5.4%, 33.3%, and 1.3% was revealed for Brucella spp., N. caninum, and mixed seropositivity, respectively. The location (Kafr El Sheikh vs. Dakahlia vs. Al-Qalyubiya vs. Damietta governorates) and a history of abortion (yes vs. no vs. unknown) were analyzed as risk factors of infection. Kafr El Sheikh governorate (57.7%, p = < 0.0001) and a history of abortion (54.1%, p = < 0.0001) were considered risk factors for Brucella spp. seropositivity compared to the reference factors Al-Qalyubiya (1.1%) and unknown abortion history (0.6%). In the case of N. caninum, the location was also considered a risk factor because the seropositive rates were significantly higher in Damietta (51%, p = 0.001) and Dakahlia (33.4%, p = 0.026) compared to Kafr El Sheikh (11.3%, set as a reference). Conversely to Brucella, animals without a history of abortion exhibited a higher seropositive rate for N. caninum (47.6%, p = 0.009) compared to those with a history of abortion (21.6%, set as reference). For further investigations into the association between abortion and the obtained seropositive rates, we also analyzed the reactivity by comparing samples of animals with, without, and unknown history of abortion. We detected high seroreactivity for Brucella spp. in samples collected from animals with a history of abortion, as demonstrated in the recorded antibody levels and correlation coefficient (Pearson r = 0.919). Based on our data, despite the higher seroprevalence of N. caninum compared to that of Brucella species, Brucella spp. might be the primary cause of abortion in our tested cattle population. [ABSTRACT FROM AUTHOR]

Dogs act as definitive hosts for several diseases caused by protozoa, some of which are zoonotic. Due to their close contact with humans and other animals, they play a crucial role in the transmission of these diseases. Although infection with Neospora caninum or Leishmania infantum is not a determining factor for another, co-infection with these protozoa can aggravate clinical signs and increase the mortality rate. Though, there are reports of success in the treatment of neosporosis, the prognosis is generally considered unfavorable, especially in young dogs. The objective of this study is to report a case of infection by both protozoa in a dog treated at a university veterinary hospital, highlighting the clinical remission of nervous signs of neosporosis after treatment with the combination of sulfadoxine and clindamycin, followed by reduction of clinical signs of canine leishmaniasis (CanL) with treatment with miltefosine and allopurinol. Despite the worse prognosis for co-infection with N. caninum and L. infantum, the dog presented remission of neurological signs and a reduction in parasite load and clinicopathological signs associated with CanL. [ABSTRACT FROM AUTHOR]

Introduction: Bovine neosporosis represents a significant threat to reproduction and production in livestock systems worldwide. This disease is caused by the protozoan Neospora caninum, resulting in abortions of cows and neurological signs in newborn calves. This leads to significant economic losses, decreasing meat and milk production, especially in tropical regions. The infection has an endogenous and exogenous cycle of transmission involving dogs that shed the oocysts, with the highest transmission successes in humid areas. Similarly, there is a lack of knowledge about the epidemiological risk factors and management practices involved in the transmission success in tropical humid regions. Methods: In this sense, a cross-sectional epidemiological survey was conducted on 150 farms from 24 municipalities of the Huila area. A total of 360 cattle were sampled, and information about the production system was collected using a structured poll with 128 questions. Results: In these cattle, 53% (191/360) were positive for antibodies against Neospora caninum using ELISA. The logistic regression analysis using the information collected from the poll identified the presence of flooring type, water access, production systems, and feed management as risk factors. Among the protective factors were identified the geographical area, molasses supplementation, and biosecurity practices such as animal separation and access control. Discussion: This study identified for the first time the epidemiological risk factors associated mainly with the exogenous cycle of neosporosis. The present study contributes to the design of intervention strategies oriented to minimize the impact of parasitism in Colombian herds. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii and Neospora caninum infections in dogs are predominantly manifest asymptomatic. However, these infections can also present highly varied and potentially severe clinical signs. This is due to the parasites' ability to replicate in a number of cell types within the host organism, with N. caninum exhibiting a particular tropism for the central and peripheral nervous systems, and T. gondii targeting the central nervous system and musculature. In clinical practice, toxoplasmosis and neosporosis are often considered to be closely related diseases, despite their distinct epidemiological, clinical, diagnostic, and therapeutic characteristics. The present review analyses the similarities and differences between these two protozoan infections, since an accurate and timely aetiological diagnosis is essential for establishing effective therapeutic protocols and control strategies. [ABSTRACT FROM AUTHOR]

Abstract Background Neospora caninum is a protozoan parasite in the Apicomplexa controlled by complex signaling pathways. Transcriptional control, an important way to regulate gene expression, has been almost absent in the N. caninum life process. However, to date, research on the transcriptional regulation of the AP2 family factors in N. caninum has been extremely limited. A prior study demonstrated that removing rhoptry protein 5 (ROP5), a significant virulence factor, resulted in abnormal expression levels of predicted NcAP2XII-4 in N. caninum, suggesting that the factor may regulate the function of ROP5. This study aimed to identify NcAP2XII-4 and its function in transcriptional regulation. Methods The NcAP2XII-4 gene was identified by analyzing the N. caninum genome. A polyclonal antibody against the protein was prepared and purified, and its expression and localization in the parasite were detected using western blot (WB) and immunofluorescence assay (IFA). The ΔNcAP2XII-4 strain was constructed from the Nc1 strain using CRISPR/Cas9 to study its effect on the growth and development of N. caninum, and DAP-Seq and electrophoretic mobility shift assay (EMSA) were used to verify the transcriptional regulatory functions of the gene. Results Bioinformatic analysis showed that NcAP2XII-4 consists of 11,976 bp and encodes 3991 amino acids, with a predicted molecular mass of 410 kDa. The protein has two AP2 domains, 1207aa-1251aa and 3453aa-3500aa, and is predicted to be located in the nucleus. The results of PCR, WB, and IFA were in accordance with the bioinformatics analysis. ΔNcAP2XII-4 was successfully constructed, but the strain could not be released and ultimately succumbed within parasitophorous vacuoles (PVs). Plaque assays demonstrated that parasites lacking this gene could not form plaques. One motif was successfully identified using DAP-Seq technique. Two prokaryotic expression vectors containing the AP2 domain of NcAP2XII-4 were successfully constructed, and two prokaryotic expression proteins, AP2-D1 and AP2-D2, and ROP5 biotinylated probes were prepared. Using EMSA, NcAP2XII-4 was shown to regulate ROP5 transcription by binding to its promoter. Conclusions NcAP2XII-4 is an essential gene in N. caninum. This study provides a foundation for further research on transcriptional regulation in N. caninum and identifies a new candidate factor for the development of vaccines against N. caninum. Graphical Abstract

Abstract Background Early diagnosis of neosporosis in dogs is challenging. Objectives To evaluate the feasibility of a compound multimodal testing approach for diagnosing in dogs neuromuscular and combined forms of neosporosis. Animals A total of 16 dogs diagnosed with solely neuromuscular neosporosis or with a combination of neuromuscular and central nervous system neosporosis. Methods Retrospective review of clinical signs, laboratory findings, treatment, and outcome with focus on the diagnostic utility of different tests. Development of a chromogenic in situ hybridization (ISH) assay for the identification of Neospora caninum in paraffin‐embedded muscle samples. Results 13/16 dogs had only neuromuscular signs of neosporosis, 3/16 had disease signs with concomitant central nervous system (CNS) involvement. Serology was performed in 15/16, with 10/15 showing titers >1 : 160 at admission. PCR on muscle samples detected N. caninum DNA in 11/16. Immunohistochemistry (IHC) detected N. caninum in 9/16 and ISH in 9/16. Histopathology revealed inflammatory myopathy in 10/16, necrotizing myopathy in 5/16, borderline changes in 1/16 and tachyzoites in 9/16. In 4 cases, N. caninum infection was confirmed with all 5 diagnostic methods, 3 cases with 4, 2 with 3, 6 with 2, and 1 animal with 1. Conclusions and Clinical Importance Diagnosis of N. caninum infection should rely on a multimodal diagnostic approach and negativity of 1 single test should not allow for exclusion. Serology in combination with direct parasite identification via histopathology, DNA via PCR, or both modalities, appears a reliable diagnostic approach.

Egress represents a crucial process employed by Neospora caninum in the establishment of infection. Dense granule proteins (GRAs), secreted by the dense granule, play significant roles in modifying the parasitophorous vacuole, maintenance of morphology, and regulating host-cell interactions. However, their precise involvement in tachyzoite egress remains inadequately characterized. In this study, we identified a homologous gene, Ncgra41, corresponding to the dense granule protein 41 (GRA41) of Toxoplasma gondii, which is associated with egress, utilizing NCBI and ToxoDB databases. NcGRA41 is localized extracellularly within dense granules and intracellularly within parasitic vacuoles. Deletion of NcGRA41 did not affect tachyzoites invasion or proliferation but significantly reduced egress capacity and pathogenicity in mice. The phenotypic characteristics were restored in a complementary strain. Further investigation revealed that the absence of NcGRA41 reduced gliding motility and the transcription level of the subtilisin-like protein (SUB1). A microneme secretion assay demonstrated a significant decrease in NcMIC1 secretion, along with reduced expression levels of NcMIC1, NcMIC4, and NcMIC8. These findings demonstrate that NcGRA41, a novel dense granule protein in N. caninum, modulates tachyzoites egress and influences pathogenicity by regulating microneme secretion. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii and Neospora caninum infect a wide range of domestic and wild animals, including humans, in the case of T. gondii, and cause economic losses in livestock due to abortion and neonatal mortality. In Somalia, zoonotic diseases are concerning due to cultural practices and livestock’s economic importance, but surveillance is limited. This study aimed to determine the seroprevalence and molecular prevalence of T. gondii and N. caninum in Somali sheep, goats, and cattle. A cross-sectional study was conducted between December 2018 and January 2020 in Benadir and Lower Shabelle regions of Somalia. Blood samples were collected from 128 cattle, 184 goats, and 46 sheep. Serum samples were tested for anti-T. gondii and anti-N. caninum antibodies using IFAT, and PCR was performed on extracted DNA to detect T. gondii and N. caninum DNA. Overall, 106/358 (29.6%) animals tested positive for anti-T. gondii antibodies, with the highest prevalence in sheep (62.5%), followed by goats (30.4%) and cattle (15.6%) (P < 0.001). For anti-N. caninum antibodies, 13/358 (3.6%) animals tested positive, with cattle showing the highest prevalence (6.2%), followed by goats and sheep (both 2.2%). Co-seropositivity for both antibodies was found in cattle and sheep. Molecular detection of T. gondii DNA revealed a prevalence of 9/358 (2.5%), primarily in sheep (15.2%) and cattle at 1.6% while all goat samples tested negative. No samples were positive for the N. caninum Nc5 gene. This study reveals T. gondii and N. caninum prevalence in Somali ruminants, highlighting the need for better surveillance and control. [ABSTRACT FROM AUTHOR]

Simple Summary: Disseminated neosporosis is an uncommon but important differential for progressive lameness in young dogs. Neospora caninum is a protozoan parasite with a two-host life cycle with canids as definitive hosts. Disease in immunocompetent adult dogs is rare; however, puppies may serve as aberrant intermediate hosts if infected congenitally. Affected puppies appear healthy at birth, and clinical disease usually develops from 3 to 9 weeks of age. Puppies show hindlimb weakness and paresis, which ascends and is generally fatal without treatment. A PCR of cerebrospinal fluid or affected tissues allows for antemortem diagnosis, though high IgG antibody titers are presumptively diagnostic in puppies with consistent clinical signs. A 3-month-old Cane Corso puppy presented to the Purdue Veterinary Hospital emergency service with a 1-week history of progressive left hindlimb lameness. The puppy was non-ambulatory paraparetic in both pelvic limbs at presentation. The puppy became tetraparetic, developed a dull mentation, cranial nerve deficits, and eventually suffered a cardiac arrest over the next four days. At necropsy, lymphohistiocytic inflammation was seen in the brain, spinal cord, myocardium, and skeletal muscle, with a few protozoal cysts seen in the brain and skeletal muscle. Antemortem titers returned positive for IgG (dilution titer of 1:4096, baseline 1:32), and Neospora caninum PCR of the skeletal muscle and brain was positive, with a Ct value of 22.09, confirming infection with Neospora caninum. We present a case of fatal disseminated neosporosis in a 3-month-old Cane Corso puppy. The puppy presented to the Purdue Veterinary Hospital emergency service with a 1-week history of progressive left hindlimb lameness. On presentation, the puppy was nonambulatory paraparetic. Signs progressed over two days to nonambulatory tetraparesis, and decreased to absent limb reflexes, cranial nerve deficits, and dull mentation. Blood work showed eosinophilia, elevated ALT, and extremely elevated creatine kinase at 36,000 IU/L (reference interval 22–491 IU/L). Two days after diagnostics were performed, the puppy experienced acute cardiac arrest, and the body was submitted for necropsy. At necropsy, skeletal muscle in all four limbs was diffusely pale tan. Tan streaks were disseminated through the diaphragm, abdominal wall, and myocardium. Histologically, Skeletal myocytes and cardiomyocytes were frequently degenerative, with abundant lymphohistiocytic inflammation and fibrosis. White matter within the brain and spinal cord was inflamed and contained frequent dilated myelin sheaths and spheroids. A few protozoal cysts were within the brain and skeletal myocytes. Antemortem antibody titers were positive for IgG against Neospora caninum (dilution titer of 1:4096, baseline 1:32), confirming protozoal cysts as Neospora caninum. Disseminated neosporosis is an uncommon but important clinical differential for ascending paresis in young dogs. [ABSTRACT FROM AUTHOR]

Environmental changes associated with global warming create new opportunities for pathogen and parasite transmission in Arctic wildlife. As an apex predator ranging over large, remote areas, changes in pathogens and parasites in polar bears are a useful indicator of changing transmission dynamics in Arctic ecosystems. We examined prevalence and risk factors associated with exposure to parasites and viral and bacterial pathogens in Chukchi Sea polar bears. Serum antibodies to six pathogens were detected and prevalence increased between 1987–1994 and 2008–2017 for five: Toxoplasma gondii, Neospora caninum, Francisella tularensis, Brucella abortus/suis, and canine distemper virus. Although bears have increased summer land use, this behavior was not associated with increased exposure. Higher prevalence of F. tularensis, Coxiella burnetii, and B. abortus/suis antibodies in females compared to males, however, could be associated with terrestrial denning. Exposure was related to diet for several pathogens indicating increased exposure in the food web. Elevated white blood cell counts suggest a possible immune response to some pathogens. Given that polar bears face multiple stressors in association with climate change and are a subsistence food, further work is warranted to screen for signs of disease. [ABSTRACT FROM AUTHOR]

Vector‐borne diseases pose a significant threat to human and animal health worldwide, with arthropods, including fleas and lice, acting as key vectors for transmitting various pathogens. In Uzbekistan, where millions of domestic dogs coexist with humans, the diversity of vector‐borne pathogens carried by ectoparasites remains largely unexplored. This study aimed to investigate the diversity and prevalence of lice and pathogens carried by fleas and lice collected from domestic dogs in Uzbekistan. Ectoparasites were collected from 77 dogs across five regions and subjected to morphological identification and molecular analysis. The 47 lice collected were identified as Trichodectes canis Fabricius, 1780 (Psocodea: Trichodectidae), Linognathus africanus Kellogg and Paine, 1911 (Psocodea: Linognathidae) and Bovicola bovis Linnaeus, 1758 (Phthiraptera: Trichodectidae), alongside with 197 fleas. Notably, L. africanus was detected for the first time on dogs worldwide. Molecular screening detected Rickettsia spp. in 27.9% fleas and 44.7% lice, Dipylidium caninum Linnaeus, 1758 (Cyclophyllidea: Dipylidiidae) in 1.5% of fleas and Bartonella spp. in 0.5% of fleas. Sequencing confirmed Rickettsia asembonensisMaina et al., 2016 (Rickettsiales: Rickettsiaceae), Rickettsia helvetica Beati et al., 1993 (Rickettsiales: Rickettsiaceae), Rickettsia monacensis Simser et al., 2019 (Rickettsiales: Rickettsiaceae), D. caninum and Bartonella clarridgeiae Lawson and Collins, 1996 (Hyphomicrobiales: Bartonellaceae). These findings highlight the potential role of dogs as reservoirs for vector‐borne pathogens and emphasises the need for further research to inform effective disease control strategies. [ABSTRACT FROM AUTHOR]

Carcasses of wild mammals provide convenient samples for wildlife disease investigators. Specifically, antibodies can persist in the blood and tissues of animals up to a few days after death and can provide information on exposure to specific pathogens, including microorganisms with zoonotic implications, such as Toxoplasma gondii, Trichinella spp., Brucella spp., and Mycobacterium spp. Although screening samples from decaying wildlife for antibodies is not a new method, an overview of the literature with clear recommendations on best practices is lacking. Here, we present results from a systematic review of 115 studies using enzyme‐linked immunosorbent assay (ELISA) to detect pathogen antibodies in wild mammals across 298 individual research projects. We discuss the range of mammalian hosts and the pathogens most represented in the literature and suggest improvements to consider when using samples from decaying animals for seroinvestigation. Our results indicate that the most examined mammals were artiodactyl ungulates and that antibodies against the bacteria Brucella spp. are those most examined to date, followed by the protozoan Toxoplasma gondii, malignant catarrhal fever virus, Schmallenberg orthobunyavirus, and the coccidian Neospora caninum. The effect of haemolysis in serum samples on ELISA results was considered in only 3.7% of the research projects we reviewed, sample size decisions were not outlined in 83% of research projects, and half of the projects did not examine seroprevalence in different age classes. Nevertheless, these details are important for replicability and accurate interpretation of results. We highlight the benefits and limitations of detecting antibodies against pathogens in carcasses from wild mammalian groups. Wild mammals are reservoirs for a wide range of pathogens including those with zoonotic potential and investigating their carcasses can help wildlife investigators to better understand the epidemiology of many diseases with implications for wildlife, livestock, and human health. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Endemic infectious diseases remain a major challenge for dairy producers worldwide. For effective disease control programs, up-to-date prevalence estimates are of utmost importance. The objective of this study was to estimate the herd-level prevalence of bovine leukemia virus (BLV), Salmonella enterica ssp. enterica serovar Dublin (Salmonella Dublin), and Neospora caninum in dairy herds in Alberta, Canada, using a serial cross-sectional study design. Bulk tank milk samples from all Alberta dairy farms were collected 4 times, in December 2021 (n = 489), April 2022 (n = 487), July 2022 (n = 487), and October 2022 (n = 480), and tested for antibodies against BLV, Salmonella Dublin, and N. caninum using ELISA. Herd-level apparent prevalence was calculated as positive herds divided by total tested herds at each time point. A mixed-effect modified Poisson regression model was employed to assess the association of prevalence with region, herd size, herd type, and type of milking system. Apparent prevalence of BLV was 89.4%, 88.7%, 86.9%, and 86.9% in December, April, July, and October, respectively, whereas for Salmonella Dublin apparent prevalence was 11.2%, 6.6%, 8.6%, and 8.5%, and for N. caninum apparent prevalence was 18.2%, 7.4%, 7.8%, and 15.0%. For BLV, Salmonella Dublin, and N. caninum , a total of 91.7%, 15.6%, and 28.1% of herds, respectively, were positive at least once, whereas 82.5%, 3.6%, and 3.0% of herds were ELISA positive at all 4 times. Compared with the north region, central Alberta had a high prevalence (prevalence ratio [PR] = 1.13) of BLV antibody-positive herds, whereas south Alberta had a high prevalence (PR = 2.56) of herds positive for Salmonella Dublin antibodies. Furthermore, central (PR = 0.52) and south regions (PR = 0.46) had low prevalence of N. caninum -positive herds compared with the north. Hutterite colony herds were more frequently BLV positive (PR = 1.13) but less frequently N. caninum -positive (PR = 0.47). Large herds (>7,200 L/d milk delivered ∼>250 cows) were 1.1 times more often BLV positive, whereas small herds (≤3,600 L/d milk delivered ∼≤125 cows) were 3.2 times more often N. caninum positive. For Salmonella Dublin, Hutterite colony herds were less frequently (PR = 0.07) positive than non-colony herds only in medium and large strata but not in small stratum. Moreover, larger herds were more frequently (PR = 2.20) Salmonella Dublin-positive than smaller herds only in non-colony stratum but not in colony stratum. Moreover, N. caninum prevalence was 1.6 times higher on farms with conventional milking systems compared with farms with an automated milking system. These results provide up-to-date information of the prevalence of these infections that will inform investigations of within-herd prevalence of these infections and help in devising evidence-based disease control strategies. [ABSTRACT FROM AUTHOR]

A plethora of infectious and non-infectious causes of bovine abortions and perinatal mortalities (APM) have been reported in literature. However, due to financial limitations or a potential zoonotic impact, many laboratories only offer a standard analytical panel, limited to a preestablished number of pathogens. To improve the cost-efficiency of laboratory diagnostics, it could be beneficial to design a targeted analytical approach for APM cases, based on maternal and environmental characteristics associated with the prevalence of specific abortifacient pathogens. The objective of this retrospective observational study was to implement a machine learning pipeline (MLP) to predict maternal and environmental factors associated with infectious APM. Our MLP based on a greedy ensemble approach incorporated a standard tuning grid of four models, applied on a dataset of 1590 APM cases with a positive diagnosis that was achieved by analyzing an extensive set of abortifacient pathogens. Production type (dairy/beef), gestation length, and season were successfully predicted by the greedy ensemble, with a modest prediction capacity which ranged between 63 and 73 %. Besides the predictive accuracy of individual variables, our MLP hierarchically identified predictor importance causes of associated environmental/maternal characteristics of APM. For instance, in APM cases that happened in beef cows, season at APM (spring/summer) was the most important predictor with a relative importance of 24 %. Furthermore, at the last trimester of gestation Trueperella pyogenes and Neospora caninum were the most important predictors of APM with a relative importance of 22 and 17 %, respectively. Interestingly, herd size came out as the most relevant predictor for APM in multiparous dams, with a relative importance of 12 %. Based on these and other mix of predicted environmental/maternal and pathogenic potential causes, it could be concluded that implementing our MLP may be beneficial to design a more cost-effective, case-specific diagnostic approach for bovine APM cases at the diagnostic laboratory level. • We implement a MLP to predict maternal and environmental factors associated with infectious bovine APM. • Our greedy ensemble approach incorporates a standard tuning grid of four machine learning models. • The MLP correctly predicted production type (beef vs. dairy), gestation length, and season at a range of 63 to 73 %. • Implementation of a MLP allows a cost-effective, case-specific diagnostic approach for bovine APM cases. [ABSTRACT FROM AUTHOR]

Background: Neospora caninum is a protozoan parasite in the Apicomplexa controlled by complex signaling pathways. Transcriptional control, an important way to regulate gene expression, has been almost absent in the N. caninum life process. However, to date, research on the transcriptional regulation of the AP2 family factors in N. caninum has been extremely limited. A prior study demonstrated that removing rhoptry protein 5 (ROP5), a significant virulence factor, resulted in abnormal expression levels of predicted NcAP2XII-4 in N. caninum, suggesting that the factor may regulate the function of ROP5. This study aimed to identify NcAP2XII-4 and its function in transcriptional regulation. Methods: The NcAP2XII-4 gene was identified by analyzing the N. caninum genome. A polyclonal antibody against the protein was prepared and purified, and its expression and localization in the parasite were detected using western blot (WB) and immunofluorescence assay (IFA). The ΔNcAP2XII-4 strain was constructed from the Nc1 strain using CRISPR/Cas9 to study its effect on the growth and development of N. caninum, and DAP-Seq and electrophoretic mobility shift assay (EMSA) were used to verify the transcriptional regulatory functions of the gene. Results: Bioinformatic analysis showed that NcAP2XII-4 consists of 11,976 bp and encodes 3991 amino acids, with a predicted molecular mass of 410 kDa. The protein has two AP2 domains, 1207aa-1251aa and 3453aa-3500aa, and is predicted to be located in the nucleus. The results of PCR, WB, and IFA were in accordance with the bioinformatics analysis. ΔNcAP2XII-4 was successfully constructed, but the strain could not be released and ultimately succumbed within parasitophorous vacuoles (PVs). Plaque assays demonstrated that parasites lacking this gene could not form plaques. One motif was successfully identified using DAP-Seq technique. Two prokaryotic expression vectors containing the AP2 domain of NcAP2XII-4 were successfully constructed, and two prokaryotic expression proteins, AP2-D1 and AP2-D2, and ROP5 biotinylated probes were prepared. Using EMSA, NcAP2XII-4 was shown to regulate ROP5 transcription by binding to its promoter. Conclusions: NcAP2XII-4 is an essential gene in N. caninum. This study provides a foundation for further research on transcriptional regulation in N. caninum and identifies a new candidate factor for the development of vaccines against N. caninum. [ABSTRACT FROM AUTHOR]

Background: The aim of the present study was to describe the presence of co-infection by Toxoplasma gondii and Neospora caninum in goats reared in extensive systems from Mexico. Materials and Methods: A cross-sectional study was conducted to determine the frequency of T. gondii and N. caninum, by detecting antibodies to each parasite by mean commercial ELISA kits. A total of 176 blood samples were randomly collected from mature females reared in extensive system herds from 20 municipalities of state of Guanajuato, Mexico. Results: The general seroprevalence was 23.9 and 21.0% for T. gondii and N. caninum, respectively, while co-infection rate was 3.6%. For geographic and environmental variables, no differences were observed among T. gondii and coinfection; however, it was observed that altitude, annual precipitation, annual average temperature, and rainy period showed significant differences with N. caninum seropositive goats. Conclusion: The seroprevalence of both parasites was appreciated in most of the studied herds. The present study is the first report of T. gondii and N. caninum co-infection in goats from extensive herds in Mexico. [ABSTRACT FROM AUTHOR]

Background: Early diagnosis of neosporosis in dogs is challenging. Objectives: To evaluate the feasibility of a compound multimodal testing approach for diagnosing in dogs neuromuscular and combined forms of neosporosis. Animals: A total of 16 dogs diagnosed with solely neuromuscular neosporosis or with a combination of neuromuscular and central nervous system neosporosis. Methods: Retrospective review of clinical signs, laboratory findings, treatment, and outcome with focus on the diagnostic utility of different tests. Development of a chromogenic in situ hybridization (ISH) assay for the identification of Neospora caninum in paraffin‐embedded muscle samples. Results: 13/16 dogs had only neuromuscular signs of neosporosis, 3/16 had disease signs with concomitant central nervous system (CNS) involvement. Serology was performed in 15/16, with 10/15 showing titers >1 : 160 at admission. PCR on muscle samples detected N. caninum DNA in 11/16. Immunohistochemistry (IHC) detected N. caninum in 9/16 and ISH in 9/16. Histopathology revealed inflammatory myopathy in 10/16, necrotizing myopathy in 5/16, borderline changes in 1/16 and tachyzoites in 9/16. In 4 cases, N. caninum infection was confirmed with all 5 diagnostic methods, 3 cases with 4, 2 with 3, 6 with 2, and 1 animal with 1. Conclusions and Clinical Importance: Diagnosis of N. caninum infection should rely on a multimodal diagnostic approach and negativity of 1 single test should not allow for exclusion. Serology in combination with direct parasite identification via histopathology, DNA via PCR, or both modalities, appears a reliable diagnostic approach. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to detect Neospora caninum by nested PCR (nPCR) in aborted fetuses of cattle, sheep, and goats in Bangladesh. Materials and Methods: The head portion of each aborted fetus (111) was dissected at each sampling site and transferred to the laboratory in an ice box. Data on risk factors associated with N. caninum infection were simultaneously collected. Deoxyribonucleic acid was extracted from brain tissue to perform nPCR targeting the internal transcribed spacer 1 (ITS1) ribosomal DNA (rDNA) gene of N. caninum and sequencing was performed from the representative positive samples. Results: By nPCR, N. caninum was found in 16.0% of aborted fetuses of cattle, followed by sheep (14.81%) and goats (11.78%). The highest prevalence was found in aborted fetuses of animals during the second trimester (27.78%) of pregnancy aged 2 to 4 years (18.75%). Obtained sequences showed they were completely matched with N. caninum ITS1 rDNA gene deposited in GenBank. Univariate analysis demonstrated that pregnancy stages (trimesters), abortion history of the animals, and access to dogs in animal farms were significantly (p = 0.05) correlated with N. caninum infection. Conclusion: This study represents the first investigation into the molecular detection, phylogenetic characterization, and analysis of risk factors associated with N. caninum in livestock in Bangladesh. According to the research findings, N. caninum infection may have a role in abortion cases and the ensuing financial losses in the nation's livestock industry. [ABSTRACT FROM AUTHOR]

Simple Summary: Abortions in ruminants lead to economic losses to livestock-owning communities. The major causes of these abortions are infectious agents. Surveillance of the infectious agents that cause abortions is important to the overall improvement of livestock productivity. Most LMICs have scarce or inadequate surveillance platforms for these pathogens. In this review article, we have summarized the current status of the available surveillance platforms in place for the infectious agents that lead to abortions in ruminants, as well as the sero-epidemiology of these pathogens. Ruminant abortion events cause economic losses. Despite the importance of livestock production for food security and the livelihoods of millions of people in the world's poorest communities, very little is known about the scale, magnitude, or causes of these abortions in Africa and Asia. The aim of this review was to determine the current status of surveillance measures adopted for ruminant abortigenic pathogens in Africa and Asia and to explore feasible surveillance technologies. A systematic literature search was conducted using PRISMA guidelines for studies published between 1 January 1990 and 1 May 2024 that reported epidemiological surveys of abortigenic pathogens Africa and Asia. A meta-analysis was used to estimate the species-specific sero-prevalence of the abortigenic agents and the regions where they were detected. In the systematic literature search, 39 full-text manuscripts were included. The most prevalent abortigenic pathogens with sero-prevalence greater than 10% were BHV-1, Brucella, Chlamydia abortus, Neospora caninum, RVFV, and Waddlia chondrophila in cattle, BVDV in sheep, and RVFV and Toxoplasma gondii in goats in Africa. In Asia, Anaplasma, BHV-1, Bluetongue virus, Brucella, and BVDV were prevalent in cattle, whereas Mycoplasma was important in goats and sheep. [ABSTRACT FROM AUTHOR]

ABSTRACT Neospora caninum is one of the most common pathogens causing reproductive failure in ruminants (e.g., cattle and goats) worldwide. However, due to a poor understanding of the pathogenic mechanisms of N. caninum infection, no effective drugs and vaccines are currently available. Long non-coding RNAs (lncRNAs) have been reported to be important regulators involved in a great number of physiological and pathological processes. Our previous study found that N. caninum infection induced significantly aberrant expression of lncRNA profiles in caprine endometrial epithelial cells (EECs). In the present study, we found that N. caninum infection specifically suppressed the expression of a novel lncRNA, XR_001919077.1, and knockdown of XR_001919077.1 with small interfering RNA significantly promoted the propagation of N. caninum in caprine EECs. Rapid amplification of cDNA ends analysis generated six splice variants of XR_001919077.1, with lengths ranging from 592 to 694 nt. Transfection of the full length of each variant markedly inhibited the propagation of N. caninum in caprine EECs. Further study suggested that XR_001919077.1 acted as a sponge of Chi-miR-93-5p to promote the expression of sirt1, and the XR_001919077.1/Chi-miR-93-5p/sirt1 axis significantly delayed the in vitro growth of N. caninum in caprine EECs by regulating host cell mitochondrial function and autophagy. Our findings provide a novel insight to understand the interactions between N. caninum and host cells.IMPORTANCEThe uterus is an indispensable reproductive organ for embryo implantation and fetal growth. The endometrium is more vulnerable to infection by pathogenic microorganisms resulting in an increased risk of miscarriage. Neospora caninum is one of the most common pathogens causing miscarriage in ruminants and is able to naturally inhabit the uterus, with N. caninum tissue cysts found in the endometrium. Recent advances in N. caninum research have revealed aberrant expression of long non-coding RNA (lncRNA) profiles in infected caprine endometrial epithelial cells. In the present study, N. caninum, but not Toxoplasma gondii, which has similar morphological and biological features to N. caninum, specifically suppresses the expression of a host lncRNA, XR_ 001919077.1, to impair host’s defense through the competitive endogenous RNA mechanism to modulate the host cell mitochondrial function and autophagy to facilitate parasite propagation. The findings suggest a novel immune evasion strategy of N. caninum to facilitate intracellular propagation and provide an alternative path to develop control strategies against neosporosis.

ABSTRACT Background Neospora caninum (Apicomplexa, Sarcocystidae) is a protozoan parasite regarded as a major cause of reproductive failure in cattle. Swine are susceptible to N. caninum infection; however, the role of these animals in the circulation, maintenance, and transmission of N. caninum remains unclear. Therefore, we aimed to estimate the global seroprevalence of N. caninum in domestic pigs (Sus domesticus) and wild boars (Sus scrofa). Methods Online databases including PubMed, Web of Science, Scopus, and Google Scholar were systematically searched to identify relevant studies up to 30 April 2024. A random‐effects meta‐analysis model was used to estimate the overall and subgroup‐pooled prevalence of N. caninum across studies. The Cochrane test (Q) and the I2 index were employed to heterogeneity. Funnel plots and Egger's regression test were used to evaluate publication bias, year of publication, and sample size. Results A total of 11,124 animals from 26 eligible studies were included. The pooled seroprevalence of N. caninum in swine was 17% (95% CI: 7%–26%) with a prevalence of 17% in domestic pigs and 16% in wild boars. In the subgroup analysis, the highest prevalence was observed among female animals, with a pooled seroprevalence of 17% (95% CI: 7%–26%). The pooled seroprevalence of N. caninum was nearly equal in adult and sub‐adult swine (18% and 14%, respectively). The overall seroprevalence of N. caninum in swine, based on sample size and year of publication, showed no statistically significant correlations. Conclusion The present study showed that domestic swine are suitable intermediate hosts of N. caninum based on serological findings. However, further studies are needed to describe the epidemiology of the N. caninum and to isolate the parasite from swine to confirm this hypothesis.

Neospora caninum is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to N. caninum virulence will provide potential candidates for vaccine development against neosporosis. In the present study, N. caninum c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the N. caninum CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in N. caninum tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the N. caninum challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against N. caninum infection and vertical transmission.

Background: Neospora caninum is a protozoan parasite and a main cause of abortions in cattle worldwide. However, its role in abortions and decreased fertility in sheep is not completely understood, especially due to the complex, multifactorial etiology of abortions. This study aimed to perform a longitudinal field study to investigate the epidemiology of neosporosis and its effect on fertility in endemic sheep herds. Methods: Serological (IFAT) and clinical (outcome of pregnancy) data from 153 ewe-lambs was collected in four intensive management farms in Israel during three consecutive pregnancies. Results: The seroprevalence in ewe-lambs at different farms varied between 24% and 93%. The overall seroprevalence increased from 50% in ewe-lambs to 96.6% at the end of the third pregnancy. Horizontal infection was observed in all farms, with seroconversion in 59% of seronegative sheep. Abortion rates were lower (p = 0.004) in seropositive ewes in the first pregnancy and not significantly higher in seropositive sheep in consecutive pregnancies. Seropositivity or seroconversion were not associated with abortions or repeated abortions; however, many aborting ewes were removed from the flock. Conclusions: No direct short- or long-term association was found between Neopsora infection and abortions. The variations between flocks and pregnancies suggest a more complex etiology.

A new cyclic natural compound formed by succinic acid and two alanine amino acid units was isolated from the Tetragonisca angustula honey extract. The chemical structure of 1 was established based on spectroscopic data analysis, including one- (1H and 13C NMR) and two-dimensional NMR techniques (1H-1H-COSY, HSQC and HMBC). A primary culture model previously infected with Neospora caninum was used to evaluate 1 for two time intervals (24 and 72 h), showing a reduction (40-56%) of the number of tachyzoites in the first 24 h and until 72 h, a dose-dependent reduction in parasite proliferation (25-50%). Glial cells treated with 1 did not demonstrate toxicity at concentrations up to 25 ug/mL. Treated and infected cultures showed an increase in NO when compared to control cells in 24 h and 72 h. In silico studies suggest that the new compound may affect DNA synthesis and impair -protein production. [ABSTRACT FROM AUTHOR]

Bovine neosporosis is a widespread parasitic disease associated with significant economic losses. Its effects on the reproductive performance of cows have resulted in losses that run into the hundreds of millions of US dollars in dairy industries in various countries (Reichel et al., Int J Parasitol 43:133–142, 2013). Due to outdated and scant information on the occurrence of Neospora caninum infection in South Africa, the study aimed to determine the seroprevalence and risk factors associated with infection in dairy cattle in South Africa. A total of 1401 blood samples were randomly collected from cattle on 48 dairy farms in seven of the nine provinces in South Africa. A close-ended questionnaire was used in a cross-sectional study to obtain farm-level and animal-level data. Serological testing was done using a commercial IDvet Screen® Neospora caninum Indirect ELISA. An overall seroprevalence, adjusted for test sensitivity and specificity, of 2.3% (95% CI, 1.3–4.1) was detected and 48% (23/48) of sampled farms had at least one animal testing positive. The highest seroprevalence of N. caninum was in the KwaZulu-Natal province with 7.5% (95% CI, 3.8–14.3), and the lowest in Western Cape with 0.1% (95% CI, 0–1.2). The highest within-farm seroprevalence of 25% was detected on a farm in the North West Province. In a multivariable logistic regression model, the odds of N. caninum seropositivity were higher in Holstein–Friesian cattle when compared to other breeds. Good hygiene was identified as a protective factor. Cattle left out on pasture had increased odds of testing positive for N. caninum compared to those that were penned. The odds of testing seropositive for N. caninum was higher on farms that practised segregation of cattle into different age groups. The purchase of replacement animals was a significant risk factor, as open herds had increased odds of N. caninum seropositivity. Cattle on farms that did not have a specific calving location were more likely to be seropositive. This is the first such study in South Africa and shows that N. caninum is widely distributed in the country at a low seroprevalence, but it may be a cause of concern on certain farms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background and Aim: In the Caribbean region of Colombia, the concomitance of endemic infectious agents is a common problem, and coinfections are possible, increasing the complexity of cattle herds' sanitary, reproductive, and productive problems. This study aimed to estimate the seroprevalence of bovine leukemia virus and its association with bovine infectious abortion in grazing Creole breeds from tropical herds in the Colombian Caribbean. Materials and Methods: For the determination of bovine leukemia virus (BLV), bovine viral diarrhea virus (BVDV), bovine herpes virus-1 (BoHV-1), and Neospora Caninum (NC), the enzyme-linked immunosorbent assay technique was used. Matrix analysis was performed to represent multiple seroprevalence in the same cow. To explore the association between the seroprevalence of BLV and bovine infectious abortion agents, a multivariate logistic regression model was used. Results: The seroprevalence was as follows: BLV 30.78%, BVDV 33.01%, BoHV-1 12.85%, and NC 8.96%. In the multivariate logistic regression model, seroprevalence of BVDV (OR 10.8; 95% CI: 7.5-15.6) and seroprevalence of BoHV-1 (OR 1.8; 95% CI: 1.1-3.0) were associated with the seroprevalence of BLV. Conclusion: Animals infected with BLV are more susceptible to coinfections with BVDV and BoHV-1. Implementing healthy measures against these two immunosuppressive infections could enhance the hygiene of numerous cattle herds. This study was designed as a retrospective cross-sectional study, which limits the ability to confirm that BLV is the primary infection. Further studies to confirm the primary infection of BLV with an active viral coinfection are necessary and the factors associated with these phenomena. [ABSTRACT FROM AUTHOR]

The objective of this study was to apply and preliminarily evaluate a High-Resolution Melting (HRM) analysis technique coupled with qPCR, that allows the simultaneous detection of 10 different ruminant abortogenic pathogens, for investigating abortions in sheep and goats throughout Greece. A total of 264 ovine and caprine vaginal swabs were obtained the week following the abortion from aborted females and analyzed using a commercially available kit (ID Gene™ Ruminant Abortion Multiplex HRM, Innovative Diagnostics). Results indicated a high prevalence of Coxiella burnetii and Chlamydophila spp., which were detected in 48.9% and 42.4% of the vaginal swabs, respectively. Results for these most commonly detected pathogens were compared with those of a well-established commercial qPCR kit, with near-perfect agreement. Toxoplasma gondii, Salmonella spp., Brucella spp., Anaplasma phagocytophilum, Campylobacter fetus, and Neospora caninum were also identified, the two latter reported for the first time in the country in small ruminants. Mixed infections occurred in 35.6% of the animals examined. This technique allows for the simultaneous detection of many abortogenic pathogens in an accurate and cost-effective assay. Detection of uncommon or not previously reported pathogens in various cases indicates that their role in ovine and caprine abortions may be underestimated. [ABSTRACT FROM AUTHOR]

The Macavirus genus, Gammaherpesvirinae subfamily, Herpesviridae family, contains ovine gammaherpesvirus 2 (OvGHV2), the cause of sheep-associated malignant catarrhal fever (SA-MCF). Members of the Macavirus genus associated with the development of malignant catarrhal fever (MCF) in their respective hosts share the 15A antigenic epitope, are conserved within the DNA polymerase gene and are collectively referred to as the malignant catarrhal fever virus (MCFV) complex. The ability of MCFV and/or OvGHV2 to produce abortions in ruminants is currently unknown, with little documentation of infections by these agents in bovine fetuses. This report presents the findings observed due to the detection of OvGHV2 DNA and MCFV tissue antigens in aborted bovine fetuses from southern Brazil. Four aborted bovine fetuses from three farms, located in a geographical region of Paraná State with elevated immunohistochemical (IHC) prevalence of MCFV tissue antigens, with gestational ages varying between 78 to 208 days were investigated. Significant gross and histopathological alterations were not observed in any of these fetuses. An IHC assay using the 15A-monoclonal antibody (15A-MAb), which is based on the 15A antigenic epitope of Macavirus, identified MCFV tissue antigens in multiple organs from two fetuses (#1 and #4); however, positive immunoreactivity to the 15A-MAb IHC assay was not detected in Fetus #2 and #3. Molecular testing amplified OvGHV2 DNA only from the myocardium and lungs of Fetus #1 that had positive intracytoplasmic immunoreactivity to the 15A-MAb IHC assay in these tissues. Furthermore, infections by Leptospira spp. were confirmed by molecular assays in fetuses #1, #3, and #4, while PCR detected Neospora caninum in the myocardium of Fetus #2. Additionally, molecular assays to identify well-known fetopathy agents of cattle, including bovine viral diarrhea virus, bovine alphaherpesvirus 1, Histophilus somni, and Listeria monocytogenes, did not amplify the nucleic acids of these pathogens. PCR assays to identify bovine gammaherpesvirus 6 (BoGHV6), another Macavirus known to infect cattle in Brazil, were unsuccessful. These findings confirmed that the 15A-MAb IHC assay can be efficiently used to detect MCFV antigens in organs of aborted bovine fetuses. The identification of MCFV antigens with the simultaneous detection of OvGHV2 DNA confirmed that Fetus #1 was infected by OvGHV2 and added to the few descriptions of this infection in aborted fetuses of ruminants worldwide. Moreover, the IHC detection of MCFV in multiple organs of Fetus #4, without the molecular detection of OvGHV2 or BoGHV6, may suggest that this fetus was infected by a Macavirus that was not previously diagnosed in cattle herds from Brazil. These findings strongly suggest that OvGHV2 and MCFV can produce transplacental infections in cattle. [ABSTRACT FROM AUTHOR]

Objectives: Studies in humans have demonstrated the role of Toxoplasma gondii, a protozoan parasite, in epileptic seizures. This study aimed to investigate the serological correlation between T. gondii and N. caninum and epilepsy in dogs. Materials and Methods: The medical record database of the Veterinary Teaching Hospital, University of Perugia, was searched for dogs serologically tested by IFAT for T. gondii and N. caninum and following specific inclusion criteria. Dogs were stratified by having a clinical diagnosis of epilepsy or suffering different conditions. Results: One‐hundred and twenty‐eight dogs were included, 64 with epilepsy and 64 without clinical signs of epilepsy. Seventeen of the 64 epileptic dogs (26.6%; 95% CI: 15.7% to 37.4%) and twenty‐one of the 64 non‐epileptic dogs (32.8%; 95% CI: 21.3% to 44.3%) tested positive for T. gondii. Eight of the epileptic dogs (12.5%; 95% CI: 4.4% to 20.6%) and three of the non‐epileptic dogs (4.7%; 95% CI: 0% to 9.9%) tested positive for N. caninum. There was no statistically significant difference in the rate of T. gondii or N. caninum seroreactivity between epileptic and non‐epileptic dogs. Clinical Significance: The results obtained do not seem to support the role of T. gondii and N. caninum as causative agents of dog epilepsy. [ABSTRACT FROM AUTHOR]

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcDGRA7 and NcDROP40. For NcDGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcDROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfrts marker were integrated into the genomic DNA during gene editing by CRISPRCas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains. [ABSTRACT FROM AUTHOR]

Toxoplasma (T.) gondii and Neospora (N.) caninum are two significant abortifacient apicomplexan protozoa causing severe economic losses in livestock farming. This study was designed to investigate the serological status of T. gondii and N. caninum in sheep and goats across the Southeastern Anatolia Region of Türkiye, including some provinces bordering Syria and Iraq. Totally, 906 serum samples collected from female sheep (n=510) and goats (n=396) were investigated for specific antibodies against these protozoa by recombinant TgSAG2 and NcSAG1 protein-based indirect ELISA (iELISA). The individual seroprevalence of T. gondii and N. caninum in sheep was determined as 32.6 and 3.1%, respectively, while the seroprevalence of infection with both species was 33.1%. On the other hand, T. gondii and N. caninum seroprevalence in goats were 32.1 and 20.2%, respectively, while the total seroprevalence of infection with both species was 39.4%. The highest seroprevalence values for T. gondii (P<0.05) and N. caninum (P>0.05) were determined in sheep from Diyarbakır and Mardin provinces, respectively, while in goats, higher seroprevalence values for both protozoa were determined in Diyarbakır province compared to other provinces (P<0.05). Statistical analyses revealed that seroprevalence values varying between provinces were significant (P<0.05), except for ovine neosporosis (P>0.05) and when seroprevalence values between animal species were compared, caprine neosporosis was statistically significantly higher (P<0.05). The fact that 35.9% of the small ruminants enrolled in the study had been exposed to abortive apicomplexan protozoa during their lives is a serious concern, given the zoonotic aspects of these protozoa and the economic damage they can cause. [ABSTRACT FROM AUTHOR]

With a long history and a wide distribution across the globe, the population of buffaloes (Bubalus bubalis) increased by 1.3% annually between 2002 and 2017, suggesting rising interest in this species. The results of assisted reproduction technologies in buffaloes are not as fulfilling as in cows; for this purpose, any other possible inconvenience must be removed. Parasitic and infectious diseases represent the major cause that negatively impact biotechnologies, especially in embryo transfer, since, although the relocation of an embryo from a seropositive donor to a seronegative recipient seems to be disease-free, in 25% of cases abortion occurs. Neosporosis is a parasitosis with effects that interest mainly the female reproduction, being one of the most important abortigenic entities among buffaloes, with an average prevalence in Australia and America of approximately 88%, in Africa 68%, in Asia 54.7-66.7%, and in Europe varying from 9.1% (Czech Republic) to 68.5% (Romania). In.order.to.monitor and control neosporosis, it is imperative that all three existing categories involved in the embryo transfer process (donors, recipients, embryos) should be tested and proved to be free. [ABSTRACT FROM AUTHOR]

Tissue protozoa play a significant role as a cause of economic losses in reproductive and productive aspects in camels beside the zoonotic importance. A Cross-sectional study was designed to estimate the seroprevalence and risk factors of Toxoplasma gondii and Neospora caninum in dromedary camels in Matrouh and Aswan border governorates of Egypt. One hundred and eighty-two serum samples from apparently healthy dromedary camels and tested serologically for neosporosis and toxoplasmosis. The prevalence of possible related risk factors was investigated from December 2020 to November 2021. The serological testing of 182 camel serum samples revealed a prevalence (15.93%, 29/182) for Neospora caninum, (58.24%, 106/182) for Toxoplasma gondii and (9.34%, 17/182) for both infections together. The total seroprevalence rate was (64.84%, 118/182). Camels in Matrouh governorate were at high risk (p < 0.05) of infection with tissue protozoa 3.74 times more than camels in Aswan governorate. Maghrabi camels were found more significant (p < 0.05) prevalent for T. gondii infection (62/78, 79.49%) by 5.28 times than Sudani camels, Otherwise, in Sudi camels, N. caninum is almost 2.70 times Maghrabi camels. The results showed that age and sex were mostly significant for N. caninum and T. gondii. This study revealed that camels are a possible source of infection for the studied tissue protozoa, some of which are significant for public health. Further research is needed to describe their true situation and epidemiology in dromedary camels. [ABSTRACT FROM AUTHOR]

Objectives: To describe the clinical signs and outcome of a large cohort of dogs presenting with neurological signs secondary to Neospora caninum infection. Materials and Methods: Retrospective review of cases presenting to two UK referral centres with neurological signs secondary to N. caninum infection between 2014 and 2023. Presenting signs, diagnostic test results, treatment, short‐ and long‐term outcome analysed. Results: A total of 1690 cases were assessed for eligibility. Forty‐four cases with a diagnosis of neosporosis were obtained. Three cases were then excluded due to non‐neurological presentations (two hepatitis and one myocarditis). A total of 41 cases were included in the study. Cerebello‐vestibular signs predominated; however, presenting clinical signs were varied and the neurolocalisation was often multifocal in nature (46.3%), making neosporosis an important differential diagnosis for meningoencephalitis of unknown origin. Complete clinical improvement was rare (5.6%), and relapses were common (27.8% cases with follow‐up). Clinical Significance: Neosporosis remains an important differential diagnosis for dogs at any age presenting with multifocal neurological signs. The outcome is considered poor and relapse rate is high. [ABSTRACT FROM AUTHOR]

Bovine neosporosis is an infection caused by the protozoan parasite Neospora caninum and has substantial veterinary hazards. Neosporosis cannot be controlled by vaccination or chemotherapy. Thus, accurate diagnosis followed by isolation and culling of infected animals is regarded as the most efficient method of control. In vivo diagnosis often relies on serologic testing of the animals, and milk represents a non-invasive and easy-to-collect sample matrix. However, indirect enzyme-linked immunosorbent assay (ELISA) specifically designed for antibody detection in milk are sometimes not easily available and it is tempting to use ELISA kits that are originally designed for use in serum in milk samples instead. Herein, we evaluated a widely used commercial ELISA (ID Screen® Neospora caninum competition Multispecies ELISA (ID. Vet, Grabels, France)), developed for detection of N. caninum antibodies in serum samples, for its performance on milk samples. Milk samples from dairy ruminants (cows, buffaloes, sheep, and goats; n = 149) were tested in parallel with the serum ELISA and a commercial milk ELISA as a standard test (Neospora caninum Milk Competitive ELISA, ID. Vet, Grabels, France). The detected prevalence values were 28.2% (42/149), 17.4% (26/149), and 17.4% (26/149) using milk ELISA, serum ELISA, and both ELISAs, respectively. Sensitivity, specificity, positive predictive value, and negative predictive value for the serum ELISA used with milk samples were 61.9%, 100%, 100%, and 87%, respectively. The agreement and kappa value between the two ELISAs were 89.3% and 0.70, respectively, suggesting substantial agreement. High values of Pearson correlation coefficient (0.904, p ≥ 0.0001) and area under the receiver operating characteristic (ROC) curve (0.789, p ≥ 0.0001) demonstrated the high diagnostic performance of the serum ELISA in milk samples. Also, a Bland–Altman Plot and histogram describing the frequency of distribution of ELISA optical densities confirmed the high agreement of both serum and milk ELISAs. The current results revealed the high specificity but moderate sensitivity of the serum ELISA used for milk samples compared with the milk ELISA. However, the excellent positive predictive value of the serum ELISA makes it an alternative option in case of the unavailability of milk ELISAs. With this study, we provided additional evidence that a widely used serum ELISA test kit may also be used for the detection of N. caninum antibodies in milk samples. [ABSTRACT FROM AUTHOR]

Neospora caninum is one of the most frequently diagnosed abortifacient pathogens in cattle. There is abundant genomic information about the parasite itself, but very little is known about the genetic variability of resistance in the most common intermediate host. The aim of this review was to compile all the available information about the genetic variability associated with the resistance to N. caninum both between and within cattle breeds. We systematically searched for published studies that investigated the influence of genetics of the host on the prevalence of N. caninum and risk of abortion. Beyond the potential confounding effects of feeding systems, management and animal density, some lines of evidence suggest that Holstein, the most popular breed for milk production, has a comparatively higher risk of abortion due to infections by N. caninum, whereas some beef breeds from Continental Europe seem to be more resistant. It is still not clear if different genetic mechanisms of resistance are involved in the two known routes of infection: postnatal ingestion of oocysts or transplacental transmission from the infected dam to the fetus. Genomic information associated with susceptibility to infection and risk of abortion in different cattle breeds is still scarce. The information reported here could be useful to identify new research alternatives and to define novel strategies to deal with this major problem of animal production. [ABSTRACT FROM AUTHOR]

Background: Neospora caninum is a protozoan parasite and a main cause of abortions in cattle worldwide. However, its role in abortions and decreased fertility in sheep is not completely understood, especially due to the complex, multifactorial etiology of abortions. This study aimed to perform a longitudinal field study to investigate the epidemiology of neosporosis and its effect on fertility in endemic sheep herds. Methods: Serological (IFAT) and clinical (outcome of pregnancy) data from 153 ewe-lambs was collected in four intensive management farms in Israel during three consecutive pregnancies. Results: The seroprevalence in ewe-lambs at different farms varied between 24% and 93%. The overall seroprevalence increased from 50% in ewe-lambs to 96.6% at the end of the third pregnancy. Horizontal infection was observed in all farms, with seroconversion in 59% of seronegative sheep. Abortion rates were lower (p = 0.004) in seropositive ewes in the first pregnancy and not significantly higher in seropositive sheep in consecutive pregnancies. Seropositivity or seroconversion were not associated with abortions or repeated abortions; however, many aborting ewes were removed from the flock. Conclusions: No direct short- or long-term association was found between Neopsora infection and abortions. The variations between flocks and pregnancies suggest a more complex etiology. [ABSTRACT FROM AUTHOR]

ABSTRACT Toxoplasma gondii is capable of being transmitted by nearly all warm-blooded animals, and rodents are a major source of parasite dissemination, yet mechanisms driving its broad host range are poorly understood. Although a phylogenetically close relative of T. gondii, Neospora caninum differs from T. gondii in that it does not infect mice and only infects a small number of ruminant and canine species. We recently showed that T. gondii and N. caninum grow similarly in mice during the first 24 h post-infection, but only N. caninum induces an IFNγ-driven response within hours that controls the infection. The goal of the present study was to understand the cellular basis of this rapid response to N. caninum. To do this, we compared immune cell populations at the site of infection 4 h after T. gondii or N. caninum infection in mice. We found that both parasites induced similar frequencies of peritoneal monocytes, while macrophages and dendritic cell populations were not increased compared to uninfected mice. Through a series of knockout mouse experiments, we show that B, T, and NKT cells are not required for immediate IFNγ production and ultimate control of N. caninum infection, suggesting that natural killer (NK) cells are the primary inducers of immediate IFNγ in response to N. caninum. N. caninum infections exhibited significantly more IFNγ+ NK cells in the peritoneum compared with T. gondii-infected and uninfected mice. Finally, we demonstrate that differences in early IFNγ production during N. caninum and T. gondii infections in mice are at least partly due to differences in soluble antigen(s) produced by tachyzoites.IMPORTANCEPathogen differences in host range are poorly understood at the molecular level even though even closely related pathogen species can have dramatically distinct host ranges. Here, we study two related parasite species that have a dramatic difference in their ability to infect mice. Here, we show that soluble proteins from these species determine one driver of this difference: induction of interferon gamma by cells of the innate immune system.

Abstract Background Toxoplasma gondii and Neospora caninum are closely related protozoan parasites that are considered important causes of abortion in livestock, causing huge economic losses. Hunan Province ranks 12th in the production of beef and mutton in China. However, limited data are available on the seroprevalence, risk factors and molecular characterization of T. gondii and N. caninum in beef cattle and goats in Hunan province, China. Methods Sera of 985 beef cattle and 1147 goats were examined for the presence of specific antibodies against T. gondii using indirect hemagglutination test (IHAT) and anti-N. caninum IgG using competitive-inhibition enzyme-linked immunoassay assay (cELISA). Statistical analysis of possible risk factors was performed using PASW Statistics. Muscle samples of 160 beef cattle and 160 goats were examined for the presence of T. gondii DNA (B1 gene) and N. caninum DNA (Nc-5 gene) by nested PCR. The B1 gene-positive samples were genotyped at 10 genetic markers using the multilocus nested PCR-RFLP (Mn-PCR-RFLP). Results Specific IgG against T. gondii were detected in 8.3% (82/985) and 13.3% (153/1147) and against N. caninum in 2.1% (21/985) and 2.0% (23/1147) of the beef cattle and goats, respectively. Based on statistical analysis, the presence of cats, semi-intensive management mode and gender were identified as significant risk factors for T. gondii infection in beef cattle. Age was a significant risk factor for T. gondii infection in goats (P 3 years was a significant risk factor for N. caninum infection in beef cattle (P

Objectives: The study aimed to detect Neospora caninum by nested PCR (nPCR) in aborted fetuses of cattle, sheep, and goats in Bangladesh. Materials and Methods: The head portion of each aborted fetus (111) was dissected at each sampling site and transferred to the laboratory in an ice box. Data on risk factors associated with N. caninum infection were simultaneously collected. Deoxyribonucleic acid was extracted from brain tissue to perform nPCR targeting the internal transcribed spacer 1 (ITS1) ribosomal DNA (rDNA) gene of N. caninum and sequencing was performed from the representative positive samples. Results: By nPCR, N. caninum was found in 16.0% of aborted fetuses of cattle, followed by sheep (14.81%) and goats (11.78%). The highest prevalence was found in aborted fetuses of animals during the second trimester (27.78%) of pregnancy aged 2 to 4 years (18.75%). Obtained sequences showed they were completely matched with N. caninum ITS1 rDNA gene deposited in GenBank. Univariate analysis demonstrated that pregnancy stages (trimesters), abortion history of the animals, and access to dogs in animal farms were significantly (p ≤ 0.05) correlated with N. caninum infection. Conclusion: This study represents the first investigation into the molecular detection, phylogenetic characterization, and analysis of risk factors associated with N. caninum in livestock in Bangladesh. According to the research findings, N. caninum infection may have a role in abortion cases and the ensuing financial losses in the nation's livestock industry. [J Adv Vet Anim Res 2024; 11(3.000): 618-626]

Abstract Background Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. Methods Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. Results The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 μM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. Conclusions In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis. Graphical Abstract