Differential diagnosis of Eimeria species in farmed Japanese quails (Coturnix japonica).

Similarly to poultry industry, coccidiosis may cause significant economic losses also in the commercial quail industry, an emerging sector undergoing uneven development around the world. Although scant and mostly dated, the available literature reports detailed morphological and morphometric features of both oocysts and sporocysts of the Eimeria species hitherto recognized in Japanese quails, i.e. E. tsunodai, E. uzura, E. bateri, and E. fluminensis. Mixed infections are very common in the field and require an accurate differential diagnosis of diverse species of coccidia, identifying the highly pathogenic ones, in particular E. tsunodai (localized in the caeca), and E. uzura (localized in both caeca and small intestine). This goal is hampered by time-consuming laboratory procedures involving highly qualified staff and facilities, and poorly compatible with routine management practices in farmed quails. A supplemental difficulty is represented by the lack of nucleotide sequences available in GenBank. To overcome these issues, copromicroscopic and molecular analyses (amplifying the 18S rRNA region, and the internal transcribed spacers regions ITS1-5.8rRNA-ITS2) were performed on oocysts populations separately isolated from pools of 12 caecal and 12 cloacal contents collected from 240 naturally infected laying Japanese quails. Data on morphological and morphometric features of 1,000 sporulated oocysts were statistically compared, demonstrating the presence of different Eimeria species colonizing the 2 intestinal tracts. This result was also confirmed by PCR and phylogenetic analysis of the 18S rRNA gene. Overall results allowed to hypothesize the presence of E. uzura in our Japanese quails. Although a certain identification at species level was not obtained, the present study demonstrates that reasonable turnaround times of monitoring procedures performed on Japanese quail farms, shedding light on the in vivo and post-mortem differential diagnosis of coccidiosis can be achieved, and provide obvious benefits in disease understanding and control.

Identification of Eimeria spp. in domestic chickens raised in alternative poultry production systems in the State of São Paulo, Brazil.

The objective of this study was to identify Eimeria spp. in alternative poultry production systems (APPS) in the State of São Paulo, Brazil. Fecal samples (168) and DNA extracted from fecal samples obtained in APPS located in different Municipalities in the State of São Paulo (93) were examined by microscopy or genera-specific PCR (ITS-1 locus). Samples positive for Eimeria spp. were examined using Eimeria lata, Eimeria nagambie, and Eimeria zaria species-specific PCR protocols (ITS-2 locus) and another E. lata-specific PCR (candidate IMP1 genomic locus) followed by molecular cloning (E. lata and E. zaria ITS-2 amplicons) and genetic sequencing. All positive DNA samples were also submitted to genera-specific nested PCR (18S rRNA gene) followed by next-generation sequencing to identify Eimeria spp. Eimeria nagambie, E. zaria, and Eimeria sp. were identified by ITS2-targeted species-specific PCRs and genetic sequencing. Next-generation sequencing identified, in order of prevalence: E. nagambie; Eimeria acervulina; Eimeria mivati; Eimeria praecox; Eimeria brunetti; Eimeria mitis; Eimeria sp.; Eimeria maxima; E. zaria, and Eimeria necatrix/tenella. Our results confirmed, for the first time in Brazil, the identification of E. nagambie, E. zaria, and Eimeria spp. ITS-2 and 18S rRNA gene sequences not yet described in Brazil.

Assessment of Neospora caninum infection in bulls using serological and molecular techniques.

Neospora caninum is a significant cause of abortion and economic losses in cattle worldwide. The main aim of the present work was to detect the prevalence of N. caninum infection in bulls in Hamedan (Iran) using serology and molecular techniques. All blood samples (n = 792) were screened for detecting the antibodies to N. caninum using enzyme-linked immunosorbent assay (ELISA). Then seropositive animals were rechecked using the immunofluorescent antibody test (IFAT). Also, blood, epididymis, and spinal cord samples were collected from animals for molecular analysis using nested PCR. In serology, using ELISA, 3.91% of animals were seropositive for N. caninum. Additionally, true prevalence based on the sensitivity and specificity of the ELISA was calculated 1.25% (95% CI: 0.48-2.02%). Neospora-infection in animals, calculated as the number of bulls seropositive and/or one sample positive to nested PCR, was 3.40%; and 19 bulls tested positive by both serology and molecular diagnostic methods. The overlaps between ELISA and molecular results were observed in 74.19% of whole blood samples, 80.64% of the epididymis, and 87.09% of the spinal cord. Using ELISA, the seroprevalence of N. caninum was detected 1.8% in ≤2 and 5.45% in >2 years old group of animals (p = 0.009, PR = 3.1). In addition, the seropositivity in Holstein and native breed animals was calculated 6.57% and 2.93%, respectively (p = 0.019, PR = 2.3). Seven sequences with 94.9-99.3% similarity were detected in multiple alignments of positive PCR products. Our work was the first comprehensive evaluation of Neospora-infection/neosporosis in Iranian bulls. We detected a low prevalence of infection in animals compared to previous reports. The ELISA is a sensitive serological technique for detecting the highest number of positive bulls in the present investigation and, the nested PCR is a reliable technique to identify Neospora-DNA.

A comparative study of serological tests used in the diagnosis of Toxoplasma gondii infection in small ruminants evidenced the importance of cross-reactions for harmonizing diagnostic performance.

Toxoplasma gondii is a major foodborne zoonotic pathogen that can be transmitted through the consumption of raw or undercooked meat of small ruminants, among others. Serology has been suggested as an epidemiological indicator and several tests are available nowadays. However, there is no comparative study with the most used ones. Therefore, the objective of this study was to develop and validate two in-house tests (Western blot -TgSALUVET WB- and ELISA -TgSALUVET ELISA 2.0-) and perform a comparative study including such tests and four commercial ELISA kits (IDScreen®, PrioCHECK®, Pigtype® and IDEXX). First, a specific pattern of recognition of immunodominant antigens by TgSALUVET WB was determined with serum panels of noninfected sheep and sheep infected with T. gondii or Neospora caninum. Next, TgSALUVET WB was used as a reference to preliminary validate TgSALUVET ELISA 2.0 using sera from sheep and goats naturally infected with T. gondii. Then, the abovementioned sheep serum panels were analyzed by all tests and subjected to TG-ROC analyses and agreement tests, and cross-reactivity with the anti-N. caninum IgGs was studied. All the techniques were accurate enough for the cutoff values initially suggested with all serum panels (Se and Sp ≥ 94%), except for PrioCHECK®, which showed 83% Sp. However, a cutoff readjustment improved their diagnostic performance. Additionally, cross-reactions between anti-N. caninum antibodies and T. gondii antigens were detected with all tests. Thus, a second cutoff readjustment was carried out and the use of both readjusted cutoff values is recommended to obtain comparable data and avoid false-positive results.

Identification of specific antigens between Toxoplasma gondii and Neospora caninum and application of potential diagnostic antigen TgGRA54.

Toxoplasma gondii is a zoonotic parasite that is very common in livestock. Meat products from livestock infected with T. gondii are one of the important transmission routes of toxoplasmosis. Rapid and reliable diagnosis is a prerequisite for the prevention and control of toxoplasmosis. Neospora caninum and T. gondii are similar in morphology and life history, and there are a large number of cross antigens between them, making clinical diagnosis of toxoplasmosis more difficult. In this study, immunoprecipitation-mass spectrometry (IP-MS) was used to screen for T. gondii-specific antigens, and the specific antigen was cloned and expressed in Escherichia coli. The specific antigen was then used to establish an indirect ELISA diagnostic method. A total of 241 specific antigens of T. gondii and 662 cross antigens between T. gondii and N. caninum were screened by IP-MS. Through bioinformatics analysis and homology comparison, seven proteins were selected for gene cloning and prokaryotic expression, and the most suitable antigen, TgGRA54, was selected to establish an indirect ELISA for T. gondii. Compared with the indirect immunofluorescent antibody test (IFAT), the positive coincidence rate of the ELISA based on rTgGRA54 was 100% (72/72) and the negative coincidence rate was 80.95% (17/21). The indirect ELISA method based on TgGRA54 recombinant protein was established to detect T. gondii antibodies in bovine sera, and the recombinant protein reacted well with T. gondii positive sera from sheep, mouse, and swine, indicating that the recombinant protein is a good diagnostic antigen for T. gondii.

Molecular detection and phylogenic characterization of Neospora caninum in naturally infected sheep in Alborz and Qazvin provinces, the north of the central region of Iran.

Neospora caninum is a protozoan coccidian parasite that can act as a cause of abortion in sheep. The aim of this study was to investigate the presence of this parasitic agent and its role in causing abortion in sheep of Iran. Between June 2019 and February 2022, 100 samples [brain (n = 39), placenta (n = 8), embryonic membrane (n = 7), cotyledon (n = 7), umbilical cord (n = 2), homogenate mixture of tissues (heart, liver, spleen and digestive track) (n = 37)] that were collected following the necropsies of 39 aborted ovine fetuses from different parts of the Alborz and Qazvin provinces, the north of the central region of Iran were employed for DNA extraction. Nc-5 was selected as the target gene sequence for amplification of DNA by using four pairs of primers in two semi-nested PCR. Samples considered positive for the presence of the NC-5 gene were examined to further confirm the presence of the ITS1 gene. Sequence of NC-5 gene was detected from the 27 tissue samples of 23 aborted ovine fetuses. The ITS1 gene sequence was detected in all of the 27 tissue samples that were positive for the NC-5 gene analysis. Brain tissue was the most studied tissue, and the highest number of positive cases was observed in this tissue. The present study updated the situation of ovine neosporosis in the central region of Iran and confirmed the presence of the N. caninum among sheep flocks' abortion.

Molecular detection of Toxoplasma gondii, Neospora caninum and Sarcocystis spp in tissues of Sus scrofa slaughtered in southern Brazil.

The aim of this study was to determine the presence of deoxyribonucleic acid (DNA) from Toxoplasma gondii, Sarcocystis spp. and Neospora caninum, in tissues of wild boars slaughtered in southern Brazil. A total of 156 samples were collected from different organs of 25 wild boars, and DNA from at least one of the protozoa investigated was detected in 79 samples. To differentiate between infectious agents, restriction fragment length polymorphism was performed using the restriction enzymes DdeI and HpaII. For N. caninum, conventional PCR was performed with specific primers. The DNA of at least one of the studied pathogens was detected in each animal: 26.58% for T. gondii, 68.36% for Sarcocystis spp. and 5.06% for N. caninum. Coinfection between T. gondii and Sarcocystis spp. occurred in 14 animals, between T. gondii and N. caninum in only one male animal, between Sarcocystis spp. and N. caninum in a female, while co-infection with the three agents was equally observed in only one male animal. Considering the high frequency of detection and its zoonotic risk, especially T. gondii, it appears that wild boars can be potential sources of transmission of infectious agents and the adoption of monitoring measures in these populations should be prioritized.

Amplicon sequencing allows differential quantification of closely related parasite species: an example from rodent Coccidia (Eimeria).

BACKGROUND: Quantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended. METHODS: We here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population. RESULTS: We show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance. CONCLUSIONS: We conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body condition of mice in the natural environment.

Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen-capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria.

This study was conducted to develop an antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E which is present in all Eimeria species, using a set of 3-1E-specific mouse monoclonal antibodies (mAbs). Highly sensitive 3-1E-specific antigen-capture ELISA was established using compatible mAb pairs (#318 and #320) selected from 6 mAbs (#312, #317, #318, #319, #320, and #323) with high binding activity against recombinant 3-1E protein. These anti-3-1E mAbs specifically recognized E. tenella sporozoites and a higher level of 3-1E was detected in the lysate of sporozoites than in sporocysts. Immunofluorescence assay (IFA) using 2 mAbs (#318 and #320) showed specific staining around the membrane of E. tenella sporozoites. In order to measure the changes in the 3-1E level during in coccidiosis, serum, feces, jejunal, and cecal contents were individually collected daily for 7-days postinfection (dpi) with E. maxima and E. tenella. The new ELISA was sensitive and specific for 3-1E detection in all samples collected daily from E. maxima- and E. tenella-infected chickens for a week, and the detection sensitivity ranges were 2 to 5 ng/mL and 1 to 5 ng/mL in serum, 4 to 25 ng/mL and 4 to 30 ng/mL in feces, 1 to 3 ng/mL and 1 to 10 ng/mL in cecal contents, and 3 to 65 ng/mL and 4 to 22 ng/mL in jejunal contents. Following coccidiosis, the overall 3-1E levels started to increase from 4 dpi, and the highest production was shown on 5 dpi. Among the samples collected from Eimeria-infected chickens, the highest detection level was found in the jejunal contents of E. maxima-infected chickens. Furthermore, the level of IFN-γ in serum was significantly (P < 0.05) increased from 3 dpi and peaked on 5 dpi post E. maxima infection. Post E. tenella infection, the level of IFN-γ in serum gradually (P < 0.05) increased from 2 to 5 dpi and plateaued at 7 dpi. The level of TNF-α in serum was rapidly (P < 0.05) increased from 4 dpi and those levels were kept until 7 dpi post both Eimeria infections (E. maxima and E. tenella). More importantly, the daily changes in the 3-1E levels in different samples from E. maxima- and E. tenella-infected chickens were effectively monitored with this new antigen-capture ELISA. Therefore, this new immunoassay is a sensitive diagnostic tool to monitor coccidiosis in a large field population in the commercial poultry farms before clinical symptoms develop using serum, feces, and gut samples during the entire period of infection cycle starting from 1 d after infection.

A pilot study for the isolation of Eimeria spp. oocysts from environmental straw samples in comparison with individual faecal examination of fattening calves.

The diagnosis of eimeriosis in calves mainly relies on the presence of diarrhoea and the excretion of Eimeria oocysts in the faeces. Restraining the animals to collect rectal samples for diagnostic purposes is stressful and time-consuming. The aim of this study was to evaluate a method for the quantification of oocysts in environmental barn straw samples. To investigate the recovery rate of the method, straw and Eimeria negative faeces were spiked with Eimeria oocysts in plastic bags and mixed with water and 0.05% Tween 20 (v/v); the liquids were filtered twice through sieves (mesh size 300 and 52 µm), centrifuged and the number of oocysts in the sediment determined using a McMaster counting chamber. A recovery rate of 52.4% (95% confidence interval: 48.2-56.5%) was obtained. In the following, field straw (n = 156) and individual faecal samples (n = 195, also analysed by McMaster counting chambers) were collected on four different farms. Eimeria oocysts were present on all farms in faecal (84/195, 43.1%) and straw samples (119/156, 76.3%). In 37 (23.7%) straw samples, sporulated oocysts were observed, with a sporulation rate ranging from 0 to 40%. Despite high variability between farms and examination days, mean numbers of oocysts in the straw positively correlated with mean numbers of oocysts excreted in the faeces (ρSpearman = 0.60). The examination of environmental straw samples may represent an easy-to-perform, non-invasive, inexpensive preliminary diagnostic approach for surveillance of eimeriosis at group level, having the potential to assess the infection pressure.

Neospora caninum e toxoplasma gondii: revisão de literatura / Neospora caninum and toxoplasma gondii: review of literature / Neospora caninum y toxoplasma gondii: revisión de literatura

A Neospora caninum e a Toxoplasma gondii são os agentes etiológicos que causam a Neosporose e a Toxoplasmose, respectivamente. Estas duas doenças são consideradas de grande importância econômica e de distribuição mundial, que acometem tanto animais de produção quanto animais domésticos. Apresentam sinais clínicos inespecíficos, sendo a Neosporose frequentemente associada ao abortamento em fêmeas. Ambas enfermidades costumavam ser confundidas, dificultando o diagnóstico. São causadas por protozoários cosmopolitas de ciclos biológicos heteróxenos. O Toxoplasma gondii é responsável por doença clínica em cães e gatos, enquanto o Neospora caninum acomete somente cães. Além disso, não há, até o momento, relatos de Neosporose em humanos, diferente da Toxoplasmose. Ocasionalmente esta pode ocorrer em coiotes, suínos, ovinos, caprinos, equinos, cervídeos e bubalinos. Anticorpos contra Neospora tem sido descrito em raposas, camelos e felídeos. O objetivo da presente revisão, é elucidar a forma de transmissão, sinais clínicos, diagnóstico, tratamento e controle de ambas as doenças, mostrando suas semelhanças, afim de que se possa diagnosticá-las corretamente.(AU)

Neospora caninum and toxoplasma gondii are agents of great economic importance and worldwide distribution that affect production and domestic animals. They present nonspecific clinical signs, and neosporosis is a disease that frequently causes abortion in females, which is considered current, because both used to be confused, making diagnosis difficult. They are protozoan, cosmopolitan, of heterogeneous biological cycles. Toxoplasma gondii is responsible for clinical disease in dogs and cats, while Neospora caninum affects only dogs. Furthermore, there are no reports to date of neosporosis in humans, other than toxoplasmosis. Occasionally it may occur in coyotes, pigs, sheep, goats, horses, deer, and bubaline. Antibodies to Neospora have been described in foxes, camels, and felids. This review aims to elucidate the transmission, clinical signs, diagnosis, treatment, and control of both diseases, showing their similarities, so that they can be correctly diagnosed.(AU)

Neospora caninum y Toxoplasma gondii son los agentes etiológicos que causan Neosporosis y Toxoplasmosis, respectivamente. Estas dos enfermedades se consideran de gran importancia económica y de distribución mundial, afectando tanto al ganado como a los animales domésticos. Presentan signos clínicos inespecíficos y la neosporosis se asocia con frecuencia al aborto en mujeres. Ambas dolencias solían ser erróneas, lo que hacía difícil el diagnóstico. Son causados por protozoos cosmopolitas de ciclos biológicos heterogéneos. Toxoplasma gondii es responsable de enfermedades clínicas en perros y gatos, mientras que Neospora caninum sólo ataca a perros. Además, no se han notificado casos de Neosporosis en humanos hasta el momento, diferente de Toxoplasmosis. Ocasionalmente esto puede ocurrir en coyotes, cerdos, ovejas, cabras, caballos, ciervos y bubalinos. Se han notificado anticuerpos contra la Neospora en zorros, camellos y felinos. El propósito de esta revisión es dilucidar la forma de transmisión, los signos clínicos, el diagnóstico, el tratamiento y el control de ambas enfermedades, mostrando sus similitudes, de manera que puedan ser diagnosticadas correctamente.(AU)

Use and comparison of serologic assays to detect anti-Neospora caninum antibodies in farmed red deer (Cervus elaphus).

Neospora caninum is a protozoan parasite that cause abortion in different ruminant species, including red deer ( Cervus elaphus). There are no validated assays to be performed with sera from red deer. At the present work, we evaluated the agreement among indirect fluorescent antibody test (IFAT), competitive inhibition ELISA based on a recombinant protein (ciELISA tSAG1) and immunoblot (IB) to detect anti- N. caninum antibodies in a red deer herd that presented reproductive losses due to N. caninum. In addition, we analyzed the relationship between the serologic results and 15 hinds were analyzed by IFAT, ciELISA tSAG1 and IB to detect anti- N. caninum antibodies. In the three assays, the cut-off established for cattle was used. Besides, sera were analyzed by IFAT to detect anti- Toxoplasma gondii antibodies. The hinds were monitored by ultrasound scanning during the gestational period to detect abortions. Gwet's agreement coefficient (AC1) and the percentage of agreement were used to estimate the agreement between pairs of assays. Chi-square test and odds ratio (OR) were used for the statistical association between abortion and seropositivity to N. caninum or to T. gondii. The N. caninum seropositivity rate was 53.9% (62/115), 57.4% (66/115) and 55.7% (64/115) for IFAT, ciELISA tSAG1 and IB, respectively. The AC1 and percentage of agreement were 0.760% and 87.8% for the pair ciELISA tSAG1 /IFAT, 0.793% and 89.6% for the pair IFAT/IB, and 0.966% and 98.3% for the pair IB/ciELISA tSAG1. The T. gondii seropositivity rate was 53.0% (61/115). Seropositive hinds to N. caninum were more likely to abort than seronegative hinds by the 3 assays. The OR for the association between N. caninum seropositivity and abortion was 72.70, 22.96 and 83.24 when ciELISA tSAG1, IFAT or IB assays were used, respectively. between T. gondii seropositivity and abortion. The three serologic assays were useful to detect N. caninum infected hinds. The validation of the assays for use in red deer would be an improvement for diagnosis of neosporosis in this species.

Automated enumeration of Eimeria oocysts in feces for rapid coccidiosis monitoring.

Coccidiosis represents a major driver in the economic performance of poultry operations, as coccidia control is expensive, and infections can result in increased feed conversion ratios, uneven growth rates, increased co-morbidities with pathogens such as Salmonella, and mortality within flocks. Shifts in broiler production to antibiotic-free strategies, increased attention on pre-harvest food safety, and growing incidence of anti-coccidial drug resistance has created a need for increased understanding of interventional efficacy and methods of coccidia control. Conventional methods to quantify coccidia oocysts in fecal samples involve manual microscopy processes that are time and labor intensive and subject to operator error, limiting their use as a diagnostic and monitoring tool in animal parasite control. To address the need for a high-throughput, robust, and reliable method to enumerate coccidia oocysts from poultry fecal samples, a novel diagnostic tool was developed. Utilizing the PIPER instrument and MagDrive technology, the diagnostic eliminates the requirement for extensive training and manual counting which currently limits the application of conventional microscopic methods of oocysts per gram (OPG) measurement. Automated microscopy to identify and count oocysts and report OPG simplifies analysis and removes potential sources of operator error. Morphometric analysis on identified oocysts allows for the oocyst counts to be separated into 3 size categories, which were shown to discriminate the 3 most common Eimeria species in commercial broilers, E. acervulina, E. tenella, and E. maxima. For 75% of the samples tested, the counts obtained by the PIPER and hemocytometer methods were within 2-fold of each other. Additionally, the PIPER method showed less variability than the hemocytometer counting method when OPG levels were below 100,000. By automated identification and counting of oocysts from 12 individual fecal samples in less than one hour, this tool could enable routine, noninvasive diagnostic monitoring of coccidia in poultry operations. This approach can generate large, uniform, and accurate data sets that create new opportunities for understanding the epidemiology and economics of coccidia infections and interventional efficacy.

First autochthonous clinical case of Hepatozoon silvestris in a domestic cat in Italy with unusual presentation.

Hepatozoon spp. is the causative agent of a vector-borne parasitic disease in many animal species. In felids, Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris have been molecularly isolated. Hepatozoonosis usually causes asymptomatic infections in domestic cats, but clinical cases have recently been reported in Europe. We describe the first Italian case of hepatozoonosis in a cat with an unusual presentation. An 11-year-old neutered European shorthair cat was urgently hospitalized for intestinal intussusception. Hematology, biochemistry, FIV-FeLV tests, blood smears and molecular investigation targeting the 18S rRNA gene of Hepatozoon spp. were performed on blood samples; in addition, histological and molecular investigations were performed to analyze surgical samples to identify Hepatozoon infection. Hepatozoon gamonts were detected in granulocytes in the blood smear, and Hepatozoon spp. DNA was confirmed by PCR on blood. The intussusception was caused by a sessile endoluminal nodule that was surgically removed. Histologically, many elements referring to parasitic tissue forms were identified in the intestinal cells, and then the specimens were molecularly confirmed to harbor H. silvestris. This is the first description of symptomatic hepatozoonosis in a domestic cat in Italy. Hepatozoon silvestris has been described in wild felids, which are usually resilient to the infection, whereas the domestic cat seems to be more susceptible. Indeed, H. silvestris in cats usually presents tropism for skeletal muscle and myocardium with subsequent clinical manifestations. This is the first description of a domestic cat with H. silvestris localized in the intestinal epithelium and associated with intussusception.

Hepatozoonosis of Dogs and Cats.

Hepatozoon canis and Hepatozoon americanum are tick-borne infections of dogs transmitted by different tick species, with dissimilar geographic distributions, target organs, and clinical syndromes. H canis is transmitted mostly by the brown dog tick Rhipicephalus sanguineus sensu lato, affects hemolymphoid organs, is associated with anemia and other hematologic abnormalities, and is widely prevalent globally, whereas H americanum is transmitted by the Gulf Coast tick Amblyomma maculatum, causes severe myositis, and is an emerging parasite in the southern United States. Treatment of these 2 infections decreases the parasitic load without elimination. Domestic cats are infected with 3 Hepatozoon species.

Neospora caninum-associated abortions in cattle from Southern Brazil: Anatomopathological and molecular characterization.

This study aimed to evaluate the N. caninum associated abortions in cattle in the state of Santa Catarina, in the southern Brazil. Aborted bovine fetuses were necropsied, submitting organ samples for histopathological evaluation. Brain fragments were analyzed by polymerase chain reaction (PCR). The diagnosis of abortion due to N. caninum was established through histopathology and molecular analysis in 53.84% (28/52) of the cases, with PCR detection in 71.42% (20/28). The histopathological evaluation showed lesions in 75% of the cases, characterized by mononuclear necrotizing encephalitis, mononuclear myocarditis, mononuclear myositis, mixed placentitis, and mononuclear pneumonia. Neospora caninum was the primary etiological agent associated with causes of abortion in cattle in the present study.

Neospora-related abortions in sheep in Israel - A serological diagnostic challenge in an endemic area.

Abortions in sheep flocks is a common multifactorial problem, which affects animal productivity and welfare. Israel is endemic to several infectious abortifacient pathogens, with neosporosis identified as a prominent cause. High seroprevalence of N. caninum is present in the general sheep population, making complicate to associate it as the causative agent of abortion in sheep. Here we describe two investigations of abortion storms in sheep flocks with high seroprevalence of neosporosis. In flock A, higher anti-Neospora antibody titers were demonstrated in aborting versus non-aborting ewes, suggesting that it may be the cause of abortions. In flock B, several infectious abortifacients were identified, but only the seroprevalence of border disease differed statistically between aborting and non-aborting ewes. These reports highlight the difficulty of diagnosing the cause of abortion in a multifactorial situation, and confirm the necessity to assess paired samples from aborting and non-aborting ewes, for reliable interpretation of the results.

RPA assay coupled with CRISPR/Cas12a system for the detection of seven Eimeria species in chicken fecal samples.

Chicken coccidiosis is one of the most common and economically important diseases in the global poultry industry, and it is caused by at least one of the seven Eimeria species. A simple and reliable way to distinguish Eimeria species in infected chicken is critical for the surveillance, control, and eradication of chicken coccidiosis. In this study, a recombinase polymerase amplification (RPA) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system (RPA-CRISPR/Cas12a) was developed for the detection of Eimeria species in chicken fecal samples. This assay is highly specific to the seven Eimeria species and it does not cross react between species. Assessment of analytical sensitivity revealed that a single copy of plasmid DNA could be detected. Comparative analysis revealed strong agreement between RPA-CRISPR/Cas12a assays and real-time qPCR to reliably detect all seven Eimeria species in fecal chicken samples. Importantly, the cleavage products could be visualized under a blue light instrument, making it possible for the rapid detection of Eimeria species for on-site testing. Collectively, our study demonstrated that RPA-CRISPR/Cas12a assays offer a simple and reliable diagnostic method for Eimeria species.

Seroprevalence, risk factors, and serological cross-reactivity for diagnosis of Toxoplasma gondii and Neospora caninum infections in goats in India.

Toxoplasma gondii and Neospora caninum are genetically related cyst-forming protozoan parasites that cause reproductive failures in ruminants. Given the limited information on the epidemiology of these infections in goats in India, the study aimed to estimate the seroprevalence, assess antibody cross-reactivity for diagnosis, and identify associated risk factors. A total of 695 sera were evaluated for antibodies to T. gondii and N. caninum infections using Modified Agglutination Test (MAT for Toxoplasma)/Neospora agglutination test (NAT), Enzyme-linked immunosorbent assay (ELISA), and Indirect Fluorescent Antibody Test (IFAT for tachyzoite and bradyzoite stages). The seroprevalence rate of T. gondii and N. caninum infections was 56.9% and 10.9%, respectively. Inter-rater agreement (kappa value - κ) was calculated to assess agreements between various diagnostic assays, using the IFAT as the gold standard (for detecting both infections), the agreements for MAT/NAT (κ = 0.97) and the ELISA (κ = 0.95) were excellent. The acute infection among seropositive goats were determined using serological (IgG avidity test - measures the binding strength between IgG antibodies and parasite antigens) and molecular diagnoses (PCR for repetitive DNA sequences - Toxoplasma B1 gene: 131 bp and Neospora NC5 gene: 328 bp). Among seropositive goats ≥80% had high IgG avidity and <10% of animals had low IgG avidity antibodies for both infections. Most low IgG avidity goats were PCR positive for the TgB1 gene (94.4%) or Nc5 gene (85.7%). In the serological assays, we used different dilutions of test serum to rule out the cross-reactivity owing to similar antigenic makeup between these two parasites. When the serological cross-reactivity was analyzed using invasion assay at a serum titer of ≥200, more than 90% T. gondii positive sera showed host cell invasion of N. caninum and vice versa. Largely, the serological results indicate that cut-off serum dilution of ≥1:200 for ELISA and IFAT and ≥1:25 for MAT/NAT avoids serological cross-reactivity between T. gondii and N. caninum. Further, the Univariate and multivariate analyses showed that adult animals (>2 years), reservoir hosts, and extensive rearing systems are common risk factors for these infections. However, the history of abortion was identified as a significant risk factor for T. gondii infection. This study revealed that T. gondii and N. caninum infections are highly prevalent in this region and the use of an appropriate cut-off serum dilution is necessary to avoid cross-reactivity between these closely related parasites.