Immunological studies on the effects of toltrazuril and neem extract in broiler chickens suffering from coccidiosis.

Background: The prevalence of avian coccidiosis in the poultry industry has grown, resulting in substantial financial losses from high mortality, stunted growth, reduced productivity, and expensive medical expenses. Aim: The purpose of the current study was to assess the immunological effects of neem leaf extract and toltrazuril on broilers that had contracted coccidiosis. Methods: In this investigation, 100 one-day-old Cobb broiler chicks without sexes were employed. The chicks were divided into five equal groups, with 20 birds in each. On the 14th day of life, the birds in groups 2, 3, 4, and 5 received an oral inoculation with 1 × 105 sporulated oocysts of Eimeria tenella (E. tenella) (field isolate). The first group (Gp), which consists of 20 healthy broilers, served as a negative control. Gp (2) contains experimentally infected broilers and nontreated (served as a positive control). Gp (3) contains experimentally infected broilers treated with toltrazuril (1 ml/l drinking water) for two consecutive days. Gp (4) contains experimentally infected broilers treated with neem leaf extract 4% (50 ml/l drinking water) for 5 successive days, and Gp (5) contains experimentally infected broilers treated with toltrazuril (1 ml/l drinking water) and a half dose of neem leaves extract 4% (25 ml/l drinking water) for 5 successive days. For the purpose of estimating body weight growth and feed conversion ratio, each broiler was weighed separately at the start of the trial and again on the 1st and 10th day after treatment. In addition to obtaining intestinal samples for immunohistochemistry, blood samples were also obtained for immunological examination. Results: As compared to the negative control group, the experimentally infested broilers with E. tenella showed significant decreases in serum nitric oxide, lysosome, phagocytic percent, and phagocytic index, along with significant increases in white blood cells (WBCs), lymphocyte, heterophilis, eosinophilis, basophilis, monocyte, serum total protein, γ globulin, fibrinogen, and haptoglobin. When compared to the control positive group, experimentally infested broilers treated with either neem or toltrazuril alone or in combination demonstrated significant increases in serum total protein, nitric oxide, lysozyme, phagocytic percent, and phagocytic index, but significant decreases in WBCs, lymphocytes, heterophile, eosinophile, basophile, and monocyte. The intestinal peroxidase stain of broilers infected with E. tenella exhibited a significant positive expression for CD4, but the infected broilers treated with toltrazuril and half a dosage of neem displayed a negative expression for CD4, identical to the negative control. Conclusion: The broiler chickens infested with E. tenella may have a variety of negative impacts on their immune systems and immunohistopathological findings. Nonetheless, toltrazuril and neem extract, either separately or in combination, function as anticoccidial medications that may enhance the broiler chicks' immune state.

Efficacy of dietary supplements of Glycyrrhiza glabra (Licorice) and maduramicin alone or in combination with Eimeria tenella infected chicks: A clinical study and molecular docking.

Background: Coccidiosis is one of the most economically significant poultry diseases worldwide, caused by the pathogenic Eimeria species, and is characterized by decreased weight gain (WG) and failure to grow due to malabsorption, low feed conversion rate, bloody diarrhea, and dehydration. Aim: This study investigated the effectiveness of licorice root extract (LRE) in controlling cecal coccidiosis to determine whether its combination with maduramicin could help alleviate the pathological, biochemical, and histopathological effects of cecal coccidiosis in Sasso broiler chicks. Methods: A total of 125 one-day-old Sasso broiler chicks were categorized into five equal groups (n = 25), each consisting of five replicates (n = 5 per replicate). G1-LE received a basal diet supplemented with LRE (3 g/kg); G2-ME received a basal diet containing maduramycin (0.5 g/kg); and G3-LME received a basal diet containing LRE and maduramicin together with the same rates. G4-E (positive control) and G5-N (negative control) received no additives in their feed. Birds in groups (G1-4) were challenged on day 14 of the experiment by orally intercropping a 1 ml suspension of Eimeria tenella sporulated oocysts. Results: Groups of birds fed on LRE and maduramicin separately or together appeared to be in good condition where no deaths or clinical abnormalities were observed, based on the analysis of clinicopathological examination. Compared with the G4-E positive control, the dropping scoring and oocyst shedding of groups G1-LE, G2-ME, and G3-LME along the 10th-day post-challenge (dpc), as well as macroscopic and microscopic lesions scoring at the 7th dpc, was considerably lower. The dual supplementation use of LRE and maduramicin in G3-LME's reduced the harmful effects of coccidian, which appeared only as a mononuclear cellular infiltration and a small number of oocysts invading the intestinal glands. Molecular docking revealed that LRE and maduramicin interacted with E. tenella DNA polymerase, E. tenella apical membrane antigen 1, and microneme protein binding sites resulting in reduced E. tenella replication and invasion. Conclusion: The inclusion of LRE and maduramicin, individually or in combination, in the diet might effectively mitigate the detrimental effects of coccidiosis.

Protective effect of Inonotus obliquus polysaccharide on mice infected with Neospora caninum.

The study aimed to explore the protective effects of Inonotus obliquus polysaccharide (IOP) on Neospora caninum (N. caninum) infection. Our data showed that the survival rate of the mice was the highest and the survival time was the longest when the IOP was 2 mg/10 g. In agreement with these observations, IOP alleviated the pathological damage in the various organs and tissues of the mice. Compared with that in the Neosporidium infection model group, the content of N. caninum in the heart, liver, spleen, lung, kidney and brain, determined through HE staining, was significantly lower. In addition, IOP inhibited the levels of immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2a) from the 21st to 42nd day of the administration group, whereas the levels of interleukin-12 (IL-12) and serum tumor necrosis factor alpha (TNF-α) were down-regulated at 7 d - 42 d. The production of CD4+ T lymphocytes was promoted, the number of CD8+ T lymphocytes were significantly lower and the CD4+/CD8+ ratio was significantly elevated. Furthermore, IOP effectively balanced the levels of hormones including gonadotropin-releasing hormone (GnRH), luteotropic hormone (LH) and testosterone (T) in male mice, and progesterone (PROG), estradiol (E2) and prolactin (PRL) in female mice. These findings demonstrate that IOP exerts protective effects against pathological damage caused by N. caninum infection in mice, and improve the immune function of the organism and regulate the secretion balance of sex hormones.

Effects of natural extract from medicinal herbs on broilers experimentally infected with Eimeria tenella.

This study aimed to evaluate the effects of natural extracts from nine medicinal herbs (SMA) on the growth performance, immunity, and intestinal integrity of broilers experimentally infected with Eimeria tenella. A total of 252 one-day-old broiler chicks were divided into 7 groups with 3 replicates per group and 12 broilers per cage. The groups were uninfected-untreated blank control group (BC), infected-untreated negative control group (NC), SMA treatment groups, Chinese medicine positive control group (CM), and chemical drug positive control group (CD). The SMA groups were infected and fed a basal diet supplemented with 0.6 (SMA-L), 0.8 (SMA-M), and 1.0 (SMA-H) g/kg SMA. The CM and CD groups were infected and fed a basal diet supplemented with 15 g/kg Jiqiuchong San and 0.2 g/kg Diclazuril, respectively. Results showed that feeding SMA could significantly reduce the number of oocysts in infected chickens, especially 1.0 g/kg SMA, which exhibited moderate anticoccidial efficacy. When infected with E. tenella, the supplementation of 1.0 g/kg SMA increased the renal index; restored the hepatic, splenic, and bursal indexes to BC levels; increased the levels of immunoglobulin A (IgA), IgM, and IgY; and reduced the contents of tumor necrosis factor (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), and IL-10 of the infected chickens. Moreover, treatment with 1.0 g/kg SMA alleviated the pathological changes in cecal tissue and increased the contents of zonula occludens-1 (ZO-1), occludin, claudin-1, and mucoprotein 2 (mucin-2) in cecal tissues of E. tenella-infected chickens. We found that 1.0 g/kg SMA reduced the number of oocysts, improved immunity, and alleviated intestinal barrier damage, which could improve the growth performance of infected chickens. Thus, SMA proved to be an effective natural extract against E. tenella and has the potential to be used as an efficient anticoccidial drug or additive.

Therapeutic efficacy of Citrus aurantifolia (lime) juice in experimental Eimeria tenella-infected broiler chickens.

The purpose of the present experiment was to explore the therapeutic effect of the crude fruit juice of Citrus aurantifolia (CAJ) on Eimeria tenella disorder in chickens. One hundred twenty 3-week-old Ross 308 broilers of equal sexes were assigned to six experimental groups of 20 birds each. Groups A, B, C, D, and E were experimentally infected with 20,000 sporulated Eimeria tenella oocysts. Broilers in groups A, B, and C were infected and allocated to three treatment-graded doses of C. aurantifolia fruit juice (20, 10, and 5 mL/kg body weight, respectively) which were administered orally for 7 consecutive days of the trial. Group D was infected and treated with a reference drug, Amprolium 1.5 g/L of drinking water (positive control), group E served as infected-untreated control, and group F was uninfected and non-treated (negative control). Oocysts per gram of feces were counted using the McMaster counting device, weight gain was calculated, and blood samples from each experimental group were collected on days 0, 7, 14, 21, and 28 post-infection for hematological evaluation. Results revealed that medication of broilers with C. aurantifolia fruit juice dose-dependently increased body weight, improved cecal lesion, decreased the E. tenella oocyst production rate, and significantly (P<0.05) increased the PCV of the infected broilers. C. aurantifolia provided valuable therapeutic effects against E. tenella-induced coccidiosis in broiler chickens. The plant fruit juice should be further validated to explore the vital compounds responsible for the anticoccidial efficacy.

Nigella sativa as an antibiotic alternative to promote growth and enhance health of broilers challenged with Eimeria maxima and Clostridium perfringens.

The poultry industry has significant coccidiosis and necrotic enteritis (NE) challenges, leading to high mortality and unacceptable growth without antibiotic treatment. This research explored supplementing Nigella sativa (black cumin) seed oil in poultry feed to mitigate coccidiosis and prevent or lessen NE in broilers. In vivo studies consisted of 384 and 320 Cobb 500 male broiler chicks distributed in a randomized complete block experimental design for trials 1 and 2, respectively. The first trial compared 3 concentrations (1, 2, and 5 mL/kg) of black cumin seed oil (BCSO), and trial 2 compared 2 concentrations (2 and 5 mL/kg) BCSO, with birds challenged with Eimeria maxima and Clostridium perfringens (Cp) strains Cp#6 and Cp#4, respectively. Broiler live performance, NE disease outcomes, and Cp populations were measured for both trials. A commercially available BCSO oil product, determined in a preliminary in vitro study to have the highest anti-Cp activity, was selected for in vivo studies. Gas chromatography-mass spectrometry analysis indicated the major bioactive compounds p-cymene, thymoquinone, carvacrol, and thymol were present in the BCSO. In trial 1 with strain Cp#6, BCSO concentrations of 2 and 5 mL/kg reduced NE lesion score and mortality rate to 1.6% compared with 7.8% for positive control, with no adverse impact on live performance. In trial 2 with strain Cp#4, BCSO reduced NE lesion scores and mortality rate to 35.9% compared with 51.6% for positive control and also improved weight gain when there was a Cp infection in broiler chickens. The current study compared NE in broilers challenged with 2 different Cp strains producing different levels of NE. Following Cp infection, both the population of vegetative cells and spores of Cp in cecal contents decreased for all treatments in trial 2. In conclusion, BCSO at concentrations of 2 and 5 mL/kg enhanced broiler live performance and alleviated NE and has potential as a natural, non-medication antimicrobial nutritional supplement for use as a feed additive in chickens.

Pathogenic mechanism of Eimeria tenella autophagy activation of chicken embryo cecal epithelial cells induced by Eimeria tenella.

Eimeria tenella mainly invades and develops into cecal epithelial cells of chickens, resulting in cecal epithelial cell damage. Infectious intracellular pathogens possibly act by influencing the autophagy process after invading cells. The interaction between E. tenella and the autophagy of host cells was explored by infecting E. tenella with chick embryo cecal epithelial cells. Transmission electron microscopy, laser confocal microscopy, and Western blot analysis were used to demonstrate that E. tenella infection could induce autophagy in host cells. Results showed that infection with E. tenella induced the formation of autophagosomes in cells. The expression of ATG 5, Beclin-1, and LC3B-II proteins were significantly (P < 0.01) increased after E. tenella infected host cells. Expression of p62 protein levels were significantly (P < 0.01) decreased in host cells infected with E. tenella. Chloroquine (CQ) significantly (P < 0.01) increased the expression levels of LC3B-II and P62 in E. tenella-infected host cells. Rapamycin (RAPA) induced autophagy in host cells, thus reducing the intracellular infection of E. tenella. By contrast, the infection rate of E. tenella increased in cells treated with 3-Methyladenine (3-MA). Hence, E. tenella sporozoite infection could induce autophagy activation in chick embryo cecal epithelial cells, and enhanced autophagy could reduce the infection rate of E. tenella.

Dietary organic zinc and probiotic alleviate induced Eimeria tenella infection in Japanese quails model of coccidiosis.

The current study was conducted to evaluate the impact of organic zinc (OZn) and probiotic on growth performance, oocysts number, and histological features of cecum of quails following Eimeria tenella challenge. A total of 480 Japanese quails were distributed into six treatments as follows: untreated uninfected; untreated infected; E. tenella challenge + amprolium; E. tenella challenge + OZn; E. tenella challenge + probiotic; and E. tenella challenge + OZn + probiotic. Except untreated uninfected, all groups were orally gavaged at day 8 with 5 × 104 E. tenella sporulated oocysts. Supplementation of OZn + probiotic improved (P = 0.001) growth performance compared to the untreated infected group. Lesion score of intestine and mortality was lower (P < 0.01) in quails supplemented with OZn + probiotic. Moreover, oocysts per gram (OPG) and histological dimensions of cecum in challenged birds were alleviated in OZn + probiotic. The histological findings of quails supplemented with OZn + probiotic showed normal intestinal villi with gentle sloughed epithelium. We concluded that OZn + probiotic may be safely included in the diet of Japanese quails to control coccidiosis.

Effect of Monovalent Copper Oxide and Potentiated Zinc Oxide on Growth Performance and Gut Morphology of Broiler Chickens Challenged with Coccidiosis.

An experiment was conducted to evaluate the effect of copper oxide (Cu2O) and potentiated zinc oxide (ZnO) on performance, intestinal morphology, oocyst excretion, coccidial lesion scores, and antioxidant properties in broilers during an Eimeria spp. challenge. A total of 288 1-day-old male broiler chickens (Ross 308) were divided into 18 treatments. Treatments included three levels of Cu (0, 15, or 150 mg/kg) from Cu2O and three levels of Zn (0, 80, or 160 mg/kg) from potentiated ZnO which were added to the basal diet and fed to broilers with or without challenge, using a completely randomized design in a factorial arrangement for 42 days. Live body weight, feed intake, mortality, and the cause of death were recorded weekly and histomorphology of jejunum was measured at the end of the experiment. Results showed that birds fed Cu and Zn linearly decreased (P < 0.0001) oocyst shedding. The number of excreted oocysts was reduced eight times in broilers fed a diet containing 150 mg/kg copper from Cu2O and 160 mg/kg zinc from potentiated ZnO, compared to the infected group without Cu and Zn supplementation (P < 0.0001). Microscopic features of both non-challenged and challenged broiler jejunum revealed significant improvement along with increased Cu2O and potentiated ZnO doses. Supplementation of Cu2O and potentiated ZnO decreased the jejunum structure damages and intestinal lesion score (P < 0.002). Eimeria caused a decrease (P < 0.006) in total antioxidant capacity. Superoxide dismutase increased by dietary zinc supplementation (P < 0.05). Results suggested that a combination of Cu2O and potentiated ZnO could exhibit efficient anticoccidial activity.

Effect of dietary sophorolipids on growth performance and gastrointestinal functionality of broiler chickens infected with Eimeria maxima.

Two experiments were conducted to evaluate the effects of dietary sophorolipids (SLs) supplementation as antibiotic alternatives on growth performance and gut health of chickens infected with Eimeria maxima. In experiment 1, 336 (zero-day-old) male broilers were used. The chickens were weighed and randomly allocated to the following 6 treatments groups with 7 chickens/cage and 8 cages/treatment: control group that received a basal diet (NC), positive control group that received a basal diet and was challenged with E. maxima (PC), PC+C18:1 lactonic diacetyled SL (SL1), PC+C18:1 deacetyled SL (SL2), PC+C18:1 monoacetyled SL (SL3), and PC+C18:1 diacetyled SL (SL4). Each SL (200 mg/kg feed) was added to the corresponding treatment group. In experiment 2, 588 (zero-day-old) male broilers were used. The chickens were randomly allocated to the following experimental groups with 10 or 11 chickens/cage and 8 cages/treatment: NC, PC, PC+ monensin at 90 mg/kg feed (MO), PC+SL1 at 200 mg/kg feed (SL1 200), PC+SL1 at 500 mg/kg feed (SL1 500), PC+SL4 at 200 mg/kg feed (SL4 200), and PC+SL4 at 500 mg/kg of feed (SL4 500). The chickens and feed were weighed at 0, 7, 14, 20, and 22 d to determine growth performance. In both experiments, all chickens except the NC group were orally infected with E. maxima (10,000 oocysts/chicken) at d 14. One chicken per cage was euthanized at d 20 to sample jejunal tissue to measure lesion scores, cytokines, and tight junction (TJ) proteins. Excreta samples were collected daily between d 20 and 22 to measure oocyst numbers. Data were analyzed using Mixed Model (PROC MIXED) in SAS. In experiment 1, SLs did not affect the growth of broiler chickens, but SL4 decreased (P < 0.05) the lesion score and oocyst number compared to PC chickens. In terms of cytokines and TJ protein gene expression, SLs increased (P < 0.05) IL-1ß, IL-6, IL-17F, IL-4, IL-13, occludin, and ZO1 levels compared to PC chickens. In experiment 2, monensin increased (P < 0.05) body weight, and decreased (P < 0.05) the lesion score and oocyst number compared to the PC group. SL4 500 increased (P < 0.05) average daily gain and feed conversion ratio but decreased (P < 0.05) lesion score and fecal oocyst number. SL4 decreased (P < 0.05) IL-6, IL-17F, TNFSF-15, IL-2, and IL-10 levels but increased (P < 0.05) occludin and ZO-1 levels. Overall, dietary SL supplementation, especially SL4, improved growth and gastrointestinal functionality of young broiler chickens, demonstrating significant potential as an antibiotic alternative.

First report of Caryospora sp. infection in free-living green turtles (Chelonia mydas) in Northeastern Brazil.

Infection by Caryospora cheloniae has been reported to be responsible for green turtle strandings with high morbidity and mortality rates worldwide. Although studies have already shown the pathogenesis of these infections, many aspects of this protozoan are still poorly understood, including their life cycle and infection dynamics in free-living sea turtle populations. Due to the lack of information about the infection by this protozoan in sea turtles in Northeastern Brazil, our study aims to describe Caryospora sp. infection and its pathological findings in free-living Chelonia mydas found on the north coast of the Bahia state. Between 2018 and 2019, 64 specimens of green turtles were necropsied in partnership with Fundação Projeto Tamar; among these, 10 (1.56%) had oocysts morphologically compatible with Caryospora cheloniae in the evaluation of fecal samples and histopathological examination of intestinal samples. The infected animals were juvenile green turtles that were found stranded on the beaches of the north coast of Bahia. The pathological findings were restricted to the lower gastrointestinal tract, with different presentations and intensities. About 70% of the animals with coccidial infection exhibited erosive and ulcerative fibrinous enteritis. This is the first report of coccidiosis in green turtles on the north coast of Bahia.

Differential expression of microRNAs in the caecal content and faeces of broiler chickens experimentally infected with Eimeria.

Coccidiosis caused by Eimeria spp. incurs significant morbidity and mortality in chickens, and is thus of great economic importance. Post-mortem intestinal lesion scoring remains one of the most common means of diagnosis; therefore alternative, non-invasive methods of diagnosis and monitoring would be highly desirable. Micro-RNAs (miRNAs) have been shown to be stable in faeces of human and animal species with expression altered in gastrointestinal disease. We hypothesized that miRNA is stable in caecal content of chickens, and that differential miRNA expression patterns would be seen in Eimeria-infected versus uninfected individuals. Initially, RNA was extracted from Eimeria tenella-infected (n = 3; 7 days post infection) and uninfected (n = 3) chicken caecal content to demonstrate miRNA stability. Subsequently, next-generation miRNA sequencing was performed on caecal content from E. tenella-infected chickens with high (lesion score (LS) 3-4; n = 3) or low (LS1; n = 3) levels of pathology, and uninfected controls (n = 3). Comparative analysis identified 19 miRNAs that exhibited significantly altered expression in the caecal content of E. tenella, infected chickens versus uninfected chickens (t-test, False Discovery Rate (FDR) < 0.05). Eight of these miRNAs showed significant up-regulation in infection (fold change of 9.8-105, FDR <0.05). Quantitative PCR was performed using separate biological replicates to confirm differential regulation in eight of these miRNA candidates in caecal and faecal content. This work has identified a panel of miRNA candidates which may be appropriate for use as non-invasive faecal markers of active caecal coccidiosis without the need for culling. RESEARCH HIGHLIGHTSE. tenella induced differential miRNA expression in caecal content and faeces.

Comparison of macroscopy, histopathology and PCR for diagnosing Eimeria spp. in broiler chickens / Comparação da macroscopia, histopatologia e PCR para o diagnóstico de Eimeria spp. em frangos de corte

Coccidiosis is a disease of great importance in industrial poultry. The correct diagnosis directs the poultry industry to its best treatment and control. Thus, a survey of Eimeria spp. was carried out in intestines of 64 broiler flocks, with an average age of 29 days. Eight broilers from each flock were randomly removed from the slaughter line, in a total of 512 samples. Macroscopic and histopathological lesions in the intestine were classified into Scores 0 to 4. Polymerase chain reaction (PCR) was used to research the oocysts from the seven species of Eimeria spp. in the intestinal content. The macroscopic evaluations showed that 59.4% (38/64) of the flocks were positive for E. acervulina, 32.8% (21/64) for E. maxima, 29.7% (19/64) for E. tenella, and 34.4% (22/64) for E. brunetti. The histopathological evaluation showed that 87.5% (56/64) of the flocks had at least one broiler with parasitic structures compatible with Eimeria spp. in the duodenum, 70.3% (45/64) in the jejunum, 18.8% (12/64) in the ileum, 46.9% (30/64) in the cecum, and 4.7% (3/64) in the colon. In PCR, 21.9% (14/64) of the flocks were positive for E. acervulina, 12.5% (8/64) for E. maxima, 3.1% (2/64) for E. mitis, and 32.8% (21/64) for E. tenella. The Kappa Cohen test between macroscopy, histopathology, and PCR demonstrated concordance ranging from weak to moderate with the exception of histopathology and PCR of the cecum, which was strong. In the comparison between macroscopy and histopathology, there were significative differences between Scores 0 and 1 (apart from the cecum). For Score 3, there were significative differences in duodenum, jejunum and cecum (p<0.05). In conclusion, the macroscopic diagnosis and PCR can generate false-negative results, and the histopathological exam proved to be effective, making it essential to associate different techniques for the correct diagnosis of Eimeria spp. in broiler chickens.(AU)

A coccidiose é uma doença de grande importância na avicultura industrial. O diagnóstico correto direciona a indústria avícola ao seu melhor tratamento e controle. Desta forma, realizou-se a pesquisa de Eimeria spp. em intestinos de 64 lotes de frangos de corte, com idade média de 29 dias. Em cada lote foram retirados aleatoriamente oito frangos da linha de abate, totalizando 512. Os intestinos foram classificados na macroscopia e na histopatologia em Grau de 0 a 4. No conteúdo intestinal pesquisou-se por reação em cadeia da polimerase (PCR) oocistos das sete espécies de Eimeria. As avaliações macroscópicas demonstraram que 59,4% (38/64) dos lotes foram positivos para E. acervulina, 32,8% (21/64) para E. maxima, 29,7% (19/64) para E. tenella e 34,4% (22/64) para E. brunetti. Na avaliação histopatológica, 87,5% (56/64) dos lotes apresentaram pelo menos um frango com estruturas parasitárias compatíveis com Eimeria spp. no duodeno, 70,3% (45/64) no jejuno, 18,8% (12/64) no íleo, 46,9% (30/64) no ceco e 4,7% (3/64) no cólon. Na PCR 21,9% (14/64) dos lotes foram positivos para E. acervulina, 12,5% (8/64) para E. maxima, 3,1% (2/64) para E. mitis e 32,8% (21/64) para E. tenella. O teste de concordância de Kappa Cohen entre macroscopia, histopatologia e PCR demonstrou concordância variando de fraca a moderada com exceção da histopatologia e PCR do ceco que foi forte. Na comparação dos graus de macroscopia e histopatologia, foram encontradas diferenças significativas entre o Grau 0 e 1 (exceto no ceco) e no Grau 3 houve diferença para duodeno, jejuno e cecos (p<0,05). Conclui-se que o diagnóstico macroscópico e a PCR podem gerar resultados falsos negativos e que o exame histopatológico se demostrou eficaz, tornando fundamental a associação de diferentes técnicas para o correto diagnóstico de Eimeria spp. em frangos de corte.(AU)

Comparison of macroscopy, histopathology and PCR for diagnosing Eimeria spp. in broiler chickens / Comparação da macroscopia, histopatologia e PCR para o diagnóstico de Eimeria spp. em frangos de corte

Coccidiosis is a disease of great importance in industrial poultry. The correct diagnosis directs the poultry industry to its best treatment and control. Thus, a survey of Eimeria spp. was carried out in intestines of 64 broiler flocks, with an average age of 29 days. Eight broilers from each flock were randomly removed from the slaughter line, in a total of 512 samples. Macroscopic and histopathological lesions in the intestine were classified into Scores 0 to 4. Polymerase chain reaction (PCR) was used to research the oocysts from the seven species of Eimeria spp. in the intestinal content. The macroscopic evaluations showed that 59.4% (38/64) of the flocks were positive for E. acervulina, 32.8% (21/64) for E. maxima, 29.7% (19/64) for E. tenella, and 34.4% (22/64) for E. brunetti. The histopathological evaluation showed that 87.5% (56/64) of the flocks had at least one broiler with parasitic structures compatible with Eimeria spp. in the duodenum, 70.3% (45/64) in the jejunum, 18.8% (12/64) in the ileum, 46.9% (30/64) in the cecum, and 4.7% (3/64) in the colon. In PCR, 21.9% (14/64) of the flocks were positive for E. acervulina, 12.5% (8/64) for E. maxima, 3.1% (2/64) for E. mitis, and 32.8% (21/64) for E. tenella. The Kappa Cohen test between macroscopy, histopathology, and PCR demonstrated concordance ranging from weak to moderate with the exception of histopathology and PCR of the cecum, which was strong. In the comparison between macroscopy and histopathology, there were significative differences between Scores 0 and 1 (apart from the cecum). For Score 3, there were significative differences in duodenum, jejunum and cecum (p<0.05). In conclusion, the macroscopic diagnosis and PCR can generate false-negative results, and the histopathological exam proved to be effective, making it essential to associate different techniques for the correct diagnosis of Eimeria spp. in broiler chickens.

A coccidiose é uma doença de grande importância na avicultura industrial. O diagnóstico correto direciona a indústria avícola ao seu melhor tratamento e controle. Desta forma, realizou-se a pesquisa de Eimeria spp. em intestinos de 64 lotes de frangos de corte, com idade média de 29 dias. Em cada lote foram retirados aleatoriamente oito frangos da linha de abate, totalizando 512. Os intestinos foram classificados na macroscopia e na histopatologia em Grau de 0 a 4. No conteúdo intestinal pesquisou-se por reação em cadeia da polimerase (PCR) oocistos das sete espécies de Eimeria. As avaliações macroscópicas demonstraram que 59,4% (38/64) dos lotes foram positivos para E. acervulina, 32,8% (21/64) para E. maxima, 29,7% (19/64) para E. tenella e 34,4% (22/64) para E. brunetti. Na avaliação histopatológica, 87,5% (56/64) dos lotes apresentaram pelo menos um frango com estruturas parasitárias compatíveis com Eimeria spp. no duodeno, 70,3% (45/64) no jejuno, 18,8% (12/64) no íleo, 46,9% (30/64) no ceco e 4,7% (3/64) no cólon. Na PCR 21,9% (14/64) dos lotes foram positivos para E. acervulina, 12,5% (8/64) para E. maxima, 3,1% (2/64) para E. mitis e 32,8% (21/64) para E. tenella. O teste de concordância de Kappa Cohen entre macroscopia, histopatologia e PCR demonstrou concordância variando de fraca a moderada com exceção da histopatologia e PCR do ceco que foi forte. Na comparação dos graus de macroscopia e histopatologia, foram encontradas diferenças significativas entre o Grau 0 e 1 (exceto no ceco) e no Grau 3 houve diferença para duodeno, jejuno e cecos (p<0,05). Conclui-se que o diagnóstico macroscópico e a PCR podem gerar resultados falsos negativos e que o exame histopatológico se demostrou eficaz, tornando fundamental a associação de diferentes técnicas para o correto diagnóstico de Eimeria spp. em frangos de corte.

Kinetics of the Cellular and Transcriptomic Response to Eimeria maxima in Relatively Resistant and Susceptible Chicken Lines.

Eimeria maxima is a common cause of coccidiosis in chickens, a disease that has a huge economic impact on poultry production. Knowledge of immunity to E. maxima and the specific mechanisms that contribute to differing levels of resistance observed between chicken breeds and between congenic lines derived from a single breed of chickens is required. This study aimed to define differences in the kinetics of the immune response of two inbred lines of White Leghorn chickens that exhibit differential resistance (line C.B12) or susceptibility (line 15I) to infection by E. maxima. Line C.B12 and 15I chickens were infected with E. maxima and transcriptome analysis of jejunal tissue was performed at 2, 4, 6 and 8 days post-infection (dpi). RNA-Seq analysis revealed differences in the rapidity and magnitude of cytokine transcription responses post-infection between the two lines. In particular, IFN-γ and IL-10 transcript expression increased in the jejunum earlier in line C.B12 (at 4 dpi) compared to line 15I (at 6 dpi). Line C.B12 chickens exhibited increases of IFNG and IL10 mRNA in the jejunum at 4 dpi, whereas in line 15I transcription was delayed but increased to a greater extent. RT-qPCR and ELISAs confirmed the results of the transcriptomic study. Higher serum IL-10 correlated strongly with higher E. maxima replication in line 15I compared to line C.B12 chickens. Overall, the findings suggest early induction of the IFN-γ and IL-10 responses, as well as immune-related genes including IL21 at 4 dpi identified by RNA-Seq, may be key to resistance to E. maxima.

Reproductive injury in male BALB/c mice infected with Neospora caninum.

BACKGROUND: Neospora caninum is one of the main causes of abortion in pregnant animals. However, N. caninum-induced reproductive injury in male mice is still unclear. METHODS: Male BALB/c mice were infected with a bovine isolate of N. caninum, and the organ coefficients of the testis and epididymis were measured. Lesions in the testis and epididymis were observed by light microscopy and transmission electron microscopy. Expression of the spermatogenic cell apoptosis-related proteins p53 and caspase-3 was detected by western blot. The expression of spermatogenesis-related genes in the testis was detected by reverse transcription-PCR. Sperm morphology and motility were observed. The levels of nitric oxide (NO) and antisperm antibody (AsAb) in the testicular homogenates and hormones in the serum were detected by enzyme-linked immunosorbent assay. The reproductive capacity of the male mice was detected using a reproduction test. RESULTS: The organ coefficients of the testis and epididymis of the experimental group were significantly downregulated. Light microscopy examination revealed that the spermatogenic cells of the testis were arranged in a disordered manner, and the number was reduced. The number of sperm in the epididymal lumen was significantly reduced, and the cytoplasm exhibited vacuolation and degeneration. Ultrastructural studies revealed that the cells of the testis and epididymis tissues showed varying degrees of disease. The level of p53 and caspase-3 expression in the testis was significantly upregulated. The expression of the testicular spermatogenesis-related genes Herc4, Ipo11 and Mrto4 were strongly downregulated. Observation of sperm by microscopic examination revealed significantly reduced sperm density and sperm motility, and the number of sperm deformities was significantly increased. The level of NO and AsAb was significantly increased. The levels of luteinizing hormone, follicle-stimulating hormone and gonadotropin-releasing hormone were significantly upregulated, whereas the levels of testosterone, thyrotropin-releasing hormone, thyroxine and thyroid-stimulating hormone were significantly downregulated. After challenge, the infected male mice and healthy female mice were caged together: the subsequent fetal death rate was increased, and the conception rate, litter size, number of live births and the birth weight were significantly reduced. CONCLUSIONS: Infection of male BALB/c mice with the bovine isolate of N. caninum induced varying degrees of injury to the testis, epididymis and sperm of the mice, destroyed spermatogenesis and affected the reproductive capacity.

Establishment of a model of Neospora caninum infection in pregnant mice.

The aim of this study was to establish an animal model of Neospora caninum infection in pregnant BALB/c mice infected with different doses of N. caninum tachyzoites. After infection, the female BALB/c mice were housed with male BALB/c mice. The aim of this study was to observe clinical signs and pathological changes, detect Nc5 gene expression in the main organs, and measure the wet weight and coefficient of the placenta of the pregnant mice. In addition, the level of cytokines and placental hormones in the serum was measured in pregnant mice. Our results showed that the optimal dose of the mice in the infected model was 105 tachyzoites. The infected pregnant mice presented with various clinical signs, including depression, ataxia, and variable mortality. Pathological observations of the brain, liver, and spleen in the mice exhibited hyperemia, bleeding, and swelling. Moreover, N. caninum tissue cysts or tachyzoites were observed in the brain, liver, and spleen tissues by hematoxylin-eosin (HE). The Nc5 gene was detected in the brain, liver, spleen, and placental tissues of the mice. With the increase in infection days, the weight of the placenta in the model mice increased, and the placenta ratio decreased gradually. Compared with the control group, the placenta weight and placental ratio were significantly different (P < 0.05). Furthermore, the levels of the placental hormones, corticotropin-releasing hormone (CRH), chorionic gonadotropin (CG), prolactin (PRL), and estriol (E3), and cytokines IFN-γ, IL-4, and TGF-ß were differentially expressed between the model and the control group (P < 0.05 or P < 0.01), which indicated that infection with N. caninum caused an imbalance in the regulatory function of the placental hormones and cytokines in pregnant mice. A pregnant mouse model of N. caninum infection was successfully established in this study, providing a foundation for the study of the pathogenic mechanisms of N. caninum.

Influence of Eimeria spp. infection and dietary inclusion of arginine on intestine histological parameters, serum amino acid profile and ileal amino acids digestibility in broiler chicks.

Coccidiosis is considered to be one of the most important challenge in the poultry industry causes economic losses due to the destruction in the digestive tract of chicken. It disturbs amino acids profile and their digestibility, leading to weight lost and economic burden. Using dietary arginine may decrease the adverse effects of coccidiosis on chicken digestive tract. This study aimed to evaluate the effects of dietary inclusion of arginine on intestine histological parameters, serum amino acid concentration and ileal amino acid digestibility of broiler chicks infected with coccidiosis. A total number of 384 one-d-old broiler chicks (Ross 308) of mixed sex with initial weight of 42 ±â€¯2 g was allocated into 8 groups with 8 birds/pen from grower period. At 21 days of age, broiler chicks were infected with a mixture of Eimeria spp. Broiler chicks were divided into infected and un-infected groups and received arginine at recommended levels of 85, 100, 125 and 150 %. Intestinal morphology and lesions, serum amino acid concentration and ileal amino acid digestibility were evaluated. Broiler chicks infected with Eimeria spp. showed lower villus height and villus height: crypt depth ratio and also higher intestinal lesions (P < 0.05). Coccidia infection decreased the ileal amino acid digestibility for all studied amino acids and also reduced serum concentrations of amino acids, except lysine and isoleucine (P < 0.05). Dietary supplementation of arginine especially in higher levels significantly increased villus height and villus height:crypt depth ratio and decreased lesions (P < 0.05). Moreover, dietary supplementing of arginine increased the serum concentration of arginine (P < 0.05), but it did not have any significant effect on its digestibility (P > 0.05). In sum, coccidiosis decreases amino acid digestibility and serum amino acid concentration, but dietary inclusion of higher levels of arginine significantly improved histological parameters of broiler chicks infected with coccidiosis.

Disseminated Neospora caninum infection in a dog with severe colitis.

A 12-y-old spayed female Schipperke dog with a previous diagnosis of inflammatory bowel disease was presented with a 2-mo history of severe colitis. The patient's condition progressed to hepatopathy, pneumonia, and dermatitis following management with prednisolone and dexamethasone sodium phosphate. Colonic biopsies identified severe necrosuppurative colitis with free and intracellular parasitic zoites. Postmortem examination confirmed extensive chronic-active ulcerative colitis, severe acute necrotizing hepatitis and splenitis, interstitial pneumonia, ulcerative dermatitis, myelitis (bone marrow), and mild meningoencephalitis with variable numbers of intracellular and extracellular protozoal zoites. PCR on samples of fresh colon was positive for Neospora caninum. Immunohistochemistry identified N. caninum tachyzoites in sections of colon, and a single tissue cyst in sections of brain. Administration of immunosuppressive drugs may have allowed systemic dissemination of Neospora from the intestinal tract.

First record of clinical coccidiosis (Eimeria ovinoidalis) in adult sheep from northwestern Argentina.

Coccidiosis of sheep is an intestinal infection caused by protozoa of the genus Eimeria. An outbreak of the disease in adult sheep from Salta province, northwestern Argentina, was studied to establish its clinical, epidemiological, pathological and etiological aspects. The affected animals were part of a flock of 20 sheep brought from Formosa province about 10 days before. Most sheep (80% incidence) showed hemorrhagic diarrhea, dehydration and loss of body condition; six of them died and two that became permanently recumbent were euthanized. Three necropsied sheep showed mild mesenteric lymphadenomegaly, diffuse proliferative enteritis in the small and large intestines, and mucosal thickening. Histopathological studies exhibited diffuse proliferative enteritis and presence of structures compatible with intracellular coccidia at different stages of development. Parasitological studies (n = 12) resulted in an average of 16,636.6 (± 15,266.8) Eimeria oocysts per gram of feces (range 1680-46,400). Taxonomy of Eimeria species based on analysis of sporulated oocysts derived from 4 fecal samples (n = 100 oocyst per sample) showed, on average, a high prevalence of E. ovinoidalis (61.5%), followed by E. parva (27.2%), and lower proportions of E. crandallis (5.3%), E. ahsata (3.2%) and E. intricata (2.8%). Clinical and pathological findings confirmed the diagnosis of coccidiosis in the affected sheep; parasitological results showed that E. ovinoidalis was the main species responsible for the clinical signs. Clinical coccidiosis is considered unusual in adult sheep, but the present case shows that under favorable environmental and/or management conditions, this infection may be highly deleterious for adult sheep.