The Effect of Different Species of Eimeria with Clostridium perfringens on Performance Parameters and Induction of Clinical Necrotic Enteritis in Broiler Chickens.

Necrotic enteritis (NE) is a common disease that causes great economic loss to the broiler industry due to mortality and reduced performance. Although Clostridium perfringens (CP) is a necessary component of this disease, coccidia species are a well-defined predisposing factor that exacerbates the condition. Different Eimeria species have been reported to influence NE to different degrees. In a pair of experiments, six different Eimeria species were evaluated in the presence and absence of C. perfringens. Male broiler chicks were housed in battery cages for the duration of both experiments. Feed conversion, body weight gain, and NE mortality were reported in both experiments. Experiment 1 challenged birds with E. maxima, E. acervulina, E. tenella, E. necatrix, and E. brunetti at day 13 and subsequently inoculated birds with CP on days 18, 19, and 20. In the second experiment, E. maxima, E. acervulina, E. tenella, and E. praecox were inoculated on day 15 and challenged with CP on days 17, 18, 19, 20, 21, and 22 of the experiment. In the first experiment, E. acervulina, E. brunetti, E. maxima, and E. necatrix with the addition of CP all stimulated necrotic enteritis mortality. In the second experiment, E. praecox had minimal impact on performance during the challenge (14-23 days) while E. maxima + CP decreased body weight gain and increased mortality compared to the CP alone control. Eimeria maxima had the highest mortality (21.9%) in this experiment followed by E. acervulina (6.3%). The remaining Eimeria with added CP in the second experiment did not induce NE mortality. While the challenge with CP alone did not induce mortality, feed conversion was increased compared to the unchallenged control group. When using isolated Eimeria species in these experiments, disturbances created by E. brunetti and E. maxima resulted in the most-severe challenges. These experiments highlight the NE risk of these species of Eimeria and give insight into how other species interact with the host in a controlled CP challenge model.

Artículo regular­Efecto de diferentes especies de Eimeria con Clostridium perfringens sobre los parámetros de rendimiento y la inducción de enteritis necrótica clínica en pollos de engorde. La enteritis necrótica (NE) es una enfermedad común que causa grandes pérdidas económicas a la industria del pollo de engorde debido a la mortalidad y a la reducción del rendimiento. Aunque Clostridium perfringens (CP) es un componente necesario de esta enfermedad, las especies de coccidia son un factor predisponente bien definido que agrava la enfermedad. Se ha informado que diferentes especies de Eimeria influyen en la enteritis necrótica en diferentes grados. En un par de experimentos, se evaluaron seis especies diferentes de Eimeria en presencia y ausencia de C. perfringens. Pollos de engorde machos se alojaron en jaulas en batería durante la duración de ambos experimentos. En ambos experimentos se analizaron la conversión alimenticia, el aumento de peso corporal y la mortalidad por enteritis necrótica. En el Experimento 1 se desafió a las aves con E. maxima, E. acervulina, E. tenella, E. necatrix y E. brunetti en el día 13 y posteriormente se inoculó a las aves con C. perfringens en los días 18, 19 y 20. En el segundo experimento, E. maxima, E. acervulina, E. tenella y E. praecox se inocularon en el día 15 y se desafiaron con C. perfringens en los días 17, 18, 19, 20, 21 y 22 del experimento. En el primer experimento, E. acervulina, E. brunetti, E. maxima y E. necatrix junto con C. perfringens estimularon la mortalidad por enteritis necrótica. En el segundo experimento, E. praecox tuvo un impacto mínimo en el rendimiento durante el desafío (14 a 23 días) mientras que el tratamiento de E. maxima + C. perfringens disminuyó el aumento de peso corporal y aumentó la mortalidad en comparación con el control con solamente C. perfringens. Eimeria maxima tuvo la mayor mortalidad (21.9%) en este experimento seguida por E. acervulina (6.3%). El resto de las especies de Eimeria junto con C. perfringens en el segundo experimento no indujeron mortalidad por enteritis necrótica. Si bien el desafío con C. perfringens no solo no indujo mortalidad, sino que la conversión alimenticia aumentó en comparación con el grupo de control no desafiado. Cuando se utilizaron especies de Eimeria aisladas en estos experimentos, los problemas creados por E. brunetti y E. maxima resultaron en los desafíos más severos. Estos experimentos destacan el riesgo por enteritis necrótica con estas especies de Eimeria y dan una idea de cómo otras especies interactúan con el hospedador en un modelo de desafío con C. perfringens controlado.

Research Note: The administration schedule of coccidia is a major determinant in broiler necrotic enteritis models.

A reliable and reproducible in vivo experimental model is an essential tool to study the pathogenesis of broiler necrotic enteritis and to evaluate control methods. Most current in vivo models use Eimeria as predisposing factor. Nevertheless, most models only result in a limited number of animals with intestinal necrosis. This research describes the necrotic enteritis incidence and severity using 2 previously described experimental models varying in the time point and frequency of Eimeria administration: single late and early repeated Eimeria administration models. In an in vivo model in which Clostridium perfringens is administered at 3 consecutive days between day 18 and 20 of age, birds belonging to the single late Eimeria administration regimen received a single administration of a tenfold dose of a live attenuated Eimeria vaccine on the second day of C. perfringens challenge. Broilers belonging to the early repeated administration regimen were inoculated with the same Eimeria vaccine 4 and 2 d before the start of the C. perfringens challenge. Early repeated coccidial administration resulted in a significant increase in average necrotic lesion score (value 3.26) as compared with a single late Eimeria administration regimen (value 1.2). In addition, the number of necrotic enteritis-positive animals was significantly higher in the group that received the early repeated coccidial administration. Single Eimeria administration during C. perfringens challenge resulted in a skewed distribution of lesion scoring with hardly any birds in the high score categories. A more centered distribution was obtained with the early repeated Eimeria administration regimen, having observations in every lesion score category. These findings allow better standardization of a subclinical necrotic enteritis model and reduction of the required numbers of experimental animals.

Pathogenic Pore Forming Proteins of Plasmodium Triggers the Necrosis of Endothelial Cells Attributed to Malaria Severity.

Severe malaria caused by Plasmodium falciparum poses a major global health problem with high morbidity and mortality. P. falciparum harbors a family of pore-forming proteins (PFPs), known as perforin like proteins (PLPs), which are structurally equivalent to prokaryotic PFPs. These PLPs are secreted from the parasites and, they contribute to disease pathogenesis by interacting with host cells. The severe malaria pathogenesis is associated with the dysfunction of various barrier cells, including endothelial cells (EC). Several factors, including PLPs secreted by parasites, contribute to the host cell dysfunction. Herein, we have tested the hypothesis that PLPs mediate dysfunction of barrier cells and might have a role in disease pathogenesis. We analyzed various dysfunctions in barrier cells following rPLP2 exposure and demonstrate that it causes an increase in intracellular Ca2+ levels. Additionally, rPLP2 exposed barrier cells displayed features of cell death, including Annexin/PI positivity, depolarized the mitochondrial membrane potential, and ROS generation. We have further performed the time-lapse video microscopy of barrier cells and found that the treatment of rPLP2 triggers their membrane blebbing. The cytoplasmic localization of HMGB1, a marker of necrosis, further confirmed the necrotic type of cell death. This study highlights the role of parasite factor PLP in endothelial dysfunction and provides a rationale for the design of adjunct therapies against severe malaria.

Comparison of the Pathogenicity of Five Clostridium perfringens Isolates Using an Eimeria maxima Coinfection Necrotic Enteritis Disease Model in Commercial Broiler Chickens.

Clostridium perfringens (CP) is the etiologic agent of necrotic enteritis (NE) in broiler chickens that is responsible for massive economic losses in the poultry industry in response to voluntary reduction and withdrawal of antibiotic growth promoters. Large variations exist in the CP isolates in inducing intestinal NE lesions. However, limited information is available on CP isolate genetics in inducing NE with other predisposing factors. This study investigated the ability of five CP isolates from different sources to influence NE pathogenesis by using an Eimeria maxima (EM) coinfection NE model: Str.13 (from soil), LLY_N11 (healthy chicken intestine), SM101 (food poisoning), Del1 (netB+tpeL-) and LLY_Tpel17 (netB+tpeL+) for NE-afflicted chickens. The 2-wk-old broiler chickens were preinfected with EM (5 × 103 oocysts) followed by CP infection (around 1 × 109 colony-forming units per chicken). The group of the LLY_Tpel17 isolate with EM coinfection had 25% mortality. No mortality was observed in the groups infected with EM alone, all CP alone, or dual infections of EM/other CP isolates. In this model of EM/CP coinfections, the relative percentages of body weight gain showed statistically significant decreases in all EM/CP groups except the EM/SM101 group when compared with the sham control group. Evident gut lesions were only observed in the three groups of EM/LLY_N11, EM/Del1, and EM/LLY_Tpel17, all of which possessed an essential NE pathogenesis locus in their genomes. Our studies indicate that LLY_Tpel17 is highly pathogenic to induce severe gut lesions and would be a good CP challenge strain for studies investigating pathogenesis and evaluating the protection efficacy for antibiotic alternative approaches.

Effects of Eimeria maxima and Clostridium perfringens infections on cecal microbial composition and the possible correlation with body weight gain in broiler chickens.

With the voluntary and regulatory withdrawal of antibiotic growth promoters from animal feed, coccidiosis and necrotic enteritis (NE) emerge as the top two enteric poultry infectious diseases responsible for major economic loss worldwide. The objective of this study was to investigate the correlation between the cecal microbiota compositions with the growth trait after coccidiosis and NE. In this study, the effects of Eimeria maxima and/or Clostridium perfringens infections on the microbial composition and potential correlation with the body weight gain were investigated in broiler chickens using 16S rRNA gene sequencing. E. maxima and C. perfringens coinfection successfully induced NE with its typical gut lesions and significant reductions in the percentage of relative body weight gain (RBWG%). The NE challenge model did not affect cecal microbial diversity, but influenced the cecal microbial composition. KEGG enzymes in microbiota were significantly altered in abundance following dual infections. Furthermore, significant correlations between cecal microbiota modules and RBWG% were identified in the sham control, E. maxima or C. perfringens infected groups. Understanding of host-microbiota interaction in NE would enhance the development of antibiotics-independent strategies to reduce the harmful effect of NE on the gut microbiota structure, and improve the gut health and poultry production.

Macrophages and T Lymphocytes in the Ovine Placenta After Experimental Infection With Toxoplasma gondii.

Early abortion in ovine toxoplasmosis has had limited investigation. This study evaluated the immune response in the placenta of sheep orally infected with Toxoplasma gondii and euthanized between 2 and 4 weeks postinfection. Toxoplasma infection of the placenta was only found at 4 weeks after infection. Parasitic debris in foci of necrosis were immunolabeled in the maternal caruncle, whereas well-preserved intracellular parasitic vacuole-like structures were found in trophoblasts of fetal cotyledon. Early abortions had increased macrophages in caruncular septa, whereas in later abortions the placentas containing the parasite had an increase of T lymphocytes and macrophages mainly in the fetal cotyledons. This study suggests that the immune response in both the fetal and maternal compartments of the placenta may contribute to the pathogenesis of ovine toxoplasmosis and that these responses differ between early and late presentations of the disease.

Reducing protein and supplementing crystalline amino acids, to alter dietary amino acid profiles in birds challenged for subclinical necrotic enteritis.

Necrotic enteritis (NE) is an infection of the gastrointestinal tract and is estimated to cost the global poultry industry billions of dollars annually. A study was conducted to examine whether reducing the crude protein might offset the severity of NE in broilers experimentally challenged with Eimeria spp. on day 9 and Clostridium perfringens on days 14 and 15. Furthermore, increasing the dietary amino acid (AA) density of the diet was also examined owing to identified benefits of improving performance compromised from low protein (LP) diets or NE. A 2 × 2 × 3 factorial arrangement of treatments at 6 replicates per treatment was used with 972 Ross 308 cockerels fed wheat-sorghum-soy-based diets to 35 D. Factors were NE challenge: no or yes; protein: standard (SP) or LP; and AA density: 100% AA, 115% with only essential AA (115% EAA) increased, and 115% AA with both essential and nonessential AA (115% AA) increased. The performance was measured in grower (days 7-21), finisher (days 21-35), and overall (day 7-35) periods. In addition, on day 16, intestinal lesion score and cecal short-chain fatty acids were measured. Only in nonchallenged birds fed LP diets, 115% AA increased grower feed intake (P < 0.01) and body weight gain (P < 0.05) compared to 115% EAA treatments. Challenge increased jejunal lesions (P < 0.001) with no difference between dietary treatments. Finisher body weight gain was greater in nonchallenged birds fed the 115% AA diets than in challenged birds (P < 0.05). Feeding diets with higher nonessential AA encouraged faster recovery from NE challenge. When fed the SP diets, NE challenge increased cecal butyric acid (P < 0.01) and total short-chain fatty acids (P < 0.05). The nutrient matrix used in LP diets does not favor beneficial butyric acid-producing bacteria. Using LP diets to mitigate NE severity does not offset the predisposing effect of E. spp. when attacking the gastrointestinal tract, and NE recovery is favored when feeding SP diets or additional AA.

An undescribed species of Sarcocystis associated with necrotizing meningoencephalitis in naturally infected backyard chickens in the Midwest of Brazil.

Sarcocistys -associated menigoencephalitis is virtually an unrecognized cause of neurological disease in chickens. An undescribed species of Sarcocystis cause fatal infection in two backyard chickens in the Midwest of Brazil. Infected chickens presented anorexia, weight loss, incoordination, ataxia and opisthotonos. Yellow necrotic foci in the gray and white matter of the telencephalon were the main gross lesion. Microscopically, necrotizing granulomatous and heterophilic meningoencephalitis with intralesional Sarcocystis -like schizonts and mezoites were observed in the central nervous system. Molecular analysis of frozen brain samples of the two chickens was identical and the protozoan was named Sarcocystis sp. Chicken-2016-DF-BR. Complete nested PCR- sequence of Sarcocystis sp. Chicken-2016-DF-BR was equally similar to Sarcocystis anasi (EU553477) and Sarcocystis albifronsi (EU502868). This is the first report of Sarcocistys -associated meningoencephalitis with molecular characterization in backyard chickens.

Effect of Aspirin on the Intestinal Response to a Necrotic Enteritis Challenge.

The effect of aspirin on intestinal lesions was evaluated in birds undergoing an experimental challenge with Clostridium perfringens as part of a model for inducing subclinical necrotic enteritis (SNE). Broilers were raised on clean wood shavings and randomly assigned to three treatments: Uninfected (U), Infected (I), and Infected + Aspirin (I+A; 0.025% acetylsalicylic acid in drinking water during days 21-25). Birds in the I and I+A groups were gavaged with Eimeria maxima on day 18 and their feed was inoculated with C. perfringens (1 × 109 CFU/bird) during days 23-25. On day 26, birds were euthanatized, intestinal lesions were evaluated, and intestinal tissue was collected for qPCR assessment of genes thought to be involved in the immune response to SNE: IL-1ß, IL-10, MMP-2, and MMP-7. Birds in the I+A group had more-severe and numerous lesions compared to the I group. For all genes except MMP-2, expression was upregulated in the I group compared to the U group, but did not differ between the I and I+A groups. These results indicate that aspirin exacerbated the intestinal lesions associated with this disease. Aspirin could play a role in the development of a reliable and consistent model for the induction of necrotic enteritis under experimental settings.

Efecto de la aspirina en la respuesta intestinal a un desafío por enteritis necrótica. El efecto de la aspirina en las lesiones intestinales se evaluó en aves sometidas a un desafío experimental con Clostridium perfringens como parte de un modelo para inducir enteritis necrótica subclínica (SNE). Los pollos de engorde se criaron sobre viruta de madera limpia y se asignaron aleatoriamente a tres tratamientos: no infectado (U), infectado (I) e infectado + aspirina (I+A; ácido acetilsalicílico al 0.025% en el agua potable durante los días 21-25). A las aves de los grupos I e I+A se les administró por sonda gástrica Eimeria maxima en el día 18 y el alimento fue inoculado con C. perfringens (1×109 unidades formadoras de colonias/ ave) durante los días 23-25. En el día 26, las aves fueron sacrificadas, se evaluaron las lesiones intestinales y se recolectó tejido intestinal para la evaluación cuantitativa por PCR en tiempo real de los genes que se cree están involucrados en la respuesta inmune a la enteritis necrótica subclínica: IL-1ß, IL-10, MMP-2, y MMP-7. Las aves del grupo I+A tuvieron lesiones más graves y numerosas en comparación con el grupo I. Para todos los genes, excepto MMP-2, la expresión se regulaba positivamente en el grupo I en comparación con el grupo U pero no difería entre los grupos I e I+A. Estos resultados indican que la aspirina exacerbó las lesiones intestinales asociadas con esta enfermedad. La aspirina podría desempeñar un papel en el desarrollo de un modelo confiable y consistente para la inducción de enteritis necrótica bajo condiciones experimentales.

Hepatectomía derecha y hemicolectomía derecha por necrosis hepática y perforación cólica causadas por Entamoeba histolytica / Right hepatectomy and right hemicolectomy for liver necrosis and colonic perforation caused by Entamoeba histolytica

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Two different Clostridium perfringens strains produce different levels of necrotic enteritis in broiler chickens.

Subclinical necrotic enteritis (NE) is primarily caused by the gram-positive bacterium, Clostridium perfringens (Cp). The trend towards removal of in-feed antimicrobials and subsequent increased emergence of infection in poultry has resulted in a wide interest in better understanding of the mechanism behind this disease. The virulence of NE, to a large extent, depends on the virulence of Cp strains. Thus, this study was to assess how 2 different strains of Cp affect performance and gut characteristics of broiler chickens. Ross 308 male broilers (n = 468) were assigned to a 2 × 3 factorial arrangement of treatments with antibiotics (Salinomycin at 72 ppm and zinc bacitracin at 50 ppm -, or +) and challenge (non-challenge, Cp EHE-NE18, or Cp WER-NE36). Oral administration of Eimeria oocysts (day 9) followed by inoculation with 1 mL 108 CFU Cp strains (day 14 and 15) were used to induce NE. Broiler performance was analyzed at day 10, 24, and 35. On day 16, intestinal lesion score and intestinal pH were evaluated and samples of cecal content were analyzed for bacterial counts and short-chain fatty acid concentrations (SCFA). Birds in both challenged groups showed higher feed conversion ratio (FCR), lower weight gain (P < 0.001), increased lesion scores in the jejunum (P < 0.01), and reduced pH in the ileum and cecum (P < 0.01), compared to the non-challenged birds. They also showed decreased numbers of Bacillus spp. (P < 0.001), and Ruminococcus spp. (P < 0.01) in the cecal content. On day 35, the NE36 challenged birds had a lower weight gain (P < 0.001) and higher FCR (P < 0.001) compared to the NE18 challenged birds. Interestingly, cecal Lactobacillus and lactate were increased by the NE challenge (P < 0.001), and to a greater extent in birds challenged with NE36 compared to the NE18 strain (P < 0.001). This study suggests that Cp strains varying in virulence produce different levels of disease in broiler chickens through modulating the gut environment, intestinal microbiota, and SCFA profile to different extents.

Comparison of multiple methods for induction of necrotic enteritis in broilers: II. Impact on the growth curve.

Necrotic enteritis (NE) is a disease that has gained relevance in the poultry industry with both immediate and sustained effects on BW of broilers. The objective of the 3 experiments was to evaluate the impact of NE, induced by methods that reflect common broiler production systems, on the growth curve throughout the growth period. In addition, the impact of Eimeria maxima (EM) on NE, as well as the long-term impact of Clostridium perfringens (CP) on BW, were analyzed. In experiment 1, a dual infection model of EM and CP was compared to a non-challenged control, while experiment 2 evaluated 2 different strains of EM dual infection, as well as 6 CP-only groups. Similarly, experiment 3 tested dual infection and both high and low dose CP-only groups. Both NetB and non-NetB strains of CP were used to evaluate whether NetB toxin may potentially play a role in NE induction. In all 3 experiments, BW was measured immediately before infection on day 16, then weekly through the end of the test period. In all 3 experiments, a decrease (p < 0.05) in BW was observed immediately following the acute NE disease period of day 21 to day 23, with a negative impact also observed of BW gain during NE disease period (p < 0.05). A long-term effect on BW was most clearly detected in the EM + CP dual infection models, as well as when high levels of CP-only were administered. In these cases, BW was impacted long-term, with a requisite week or more to return to a BW similar to the non-challenged control. The separation in BW, though not significant, was nearly parallel with the non-challenged control throughout the growth period, indicating a shift in the growth curve. In addition to showing the long-term impact of various forms of NE on broiler growth, these shifts in the growth curve can be used to measure the effects of treatments on prevention and recovery of broilers impacted by NE.

Acanthocytosis and brain damage in area postrema and choroid plexus: Description of novel signs of Loxosceles apachea envenomation in rats.

Loxocelism is a neglected medical problem that depends on its severity, can cause a cutaneous or viscero-cutaneous syndrome. This syndrome is characterized by hemostatic effects and necrosis, and the severity of the loxoscelism depends on the amount of venom injected, the zone of inoculation, and the species. In the Chihuahuan desert, the most abundant species is L. apachea. Its venom and biological effects are understudied, including neurological effects. Thus, our aim is to explore the effect of this regional species of medical interest in the United States-Mexico border community, using rat blood and central nervous system (CNS), particularly, two brain structures involved in brain homeostasis, Area postrema (AP) and Choroid plexus (PC). L. apachea specimens were collected and venom was obtained. Different venom concentrations (0, 0.178 and 0.87 µg/g) were inoculated into Sprague-Dawley rats (intraperitoneal injection). Subsequently, blood was extracted and stained with Wright staining; coronal sections of AP were obtained and stained with Hematoxylin-Eosin (HE) staining and laminin γ immunolabelling, the same was done with CP sections. Blood, AP and CP were observed under the microscope and abnormalities in erythrocytes and fluctuation in leukocyte types were described and quantified in blood. Capillaries were also quantified in AP and damage was described in CP. L. apachea venom produced a segmented neutrophil increment (neutrophilia), lymphocyte diminishment (leukopenia) and erythrocytes presented membrane abnormalities (acanthocytosis). Extravasated erythrocytes were observed in HE stained sections from both, AP and CP, which suggest that near to this section a hemorrhage is present; through immunohistofluorescence, a diminishment of laminin γ was observed in AP endothelial cells and in CP ependymal cells when these structures were exposed to L. apachea venom. In conclusion, L. apachea venom produced leukopenia, netrophilia and acanthocytosis in rat peripheral blood, and also generated hemorrhages on AP and CP through degradation of laminin γ.

Case Series: Virulent hemosporidiosis infections in juvenile great horned owls (Bubo virginianus) from Louisiana and California, USA.

A total of eight juvenile great horned owls (Bubo virginianus) were found lethargic and on the ground in spring 2015, 2016, and 2017, including one fledgling from Louisiana, USA and seven nestlings from California, USA. One bird survived to release after rehabilitation; seven birds died or were euthanized due to poor prognosis and were necropsied. Necropsy findings were similar and included general pallor of all tissues, particularly the subcutis and lungs, and enlarged liver and spleen. Histopathology revealed multi-organ necrosis, abundant meronts containing merozoites, and intracytoplasmic pigmented haemosporidian parasites in blood cells in one bird. Leucocytozoon lineages lSTOCC16 and BUVIR06 were identified by polymerase chain reaction and genetic sequencing. The systemic Leucocytozoon infections were likely associated with morbidity and mortality in these owls. A second parasite, Haemoproteus lineage hSTVAR01, was also identified in an owl from Louisiana. This is the first identification of Leucocytozoon lineages that have been associated with mortality in young great horned owls.

A secondary wave of neutrophil infiltration causes necrosis and ulceration in lesions of experimental American cutaneous leishmaniasis.

We evaluated the importance of neutrophils in the development of chronic lesions caused by L. Viannia spp. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R2 = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p<0.001 each). In contrast, reduced neutrophil infiltration via cyclophosphamide-mediated depletion led to more benign lesions and lower parasite loads compared to controls (p<0.001 each). Neutrophils of the second wave expressed significantly lower GM-CSF, reactive oxygen species and nitric oxide than those of the first wave, suggesting that they had less efficient anti-leishmania activity. However, there was increased inflammatory cytokines and expression of neutrophil proteases (myeloperoxidase, cathepsin G and elastase) in lesions during the second wave of neutrophil infiltration compared with the levels reached during the first wave (6h p.i.). This suggests that augmented neutrophil proteases and inflammatory cytokines during the secondary wave of neutrophils could contribute to skin inflammation, ulceration and necrosis in ACL. The overall results indicate that neutrophils were unable to clear the infection in this model, and that the second wave of neutrophils played an important role in the severity of ACL.

Laser-mediated rupture of chlamydial inclusions triggers pathogen egress and host cell necrosis.

Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.

Prevalence and morphopathological characteristics of linguatulosis in one-humped camel (Camelus dromedarius) in Yazd, Iran.

Linguatula serrata is a cosmopolitan zoonotic parasite. Its adult form inhabit the nasal and respiratory passages of canids as the definitive hosts while its immature stages localize in the mesenteric lymph nodes or in other organs of herbivorous intermediate hosts. We examined the liver, mesenteric, and mediastinal lymph nodes of 272 camels slaughtered at the slaughterhouse of Yazd, Iran. Forty-one out of 272 camels (15.1 %) were infected with nymphs of L. serrata. Twenty-four out of 166 males (14.45 %) and 17 out of 106 females (16 %) were positive. The livers of five camels, which also had nymphs in their lymph nodes, were infected with the larval stage of this parasite. The infection rate increased with age and was highly significant, while sex did not play a significant role in the prevalence of this parasitic infection. The infected lymph nodes were grossly enlarged, edematous, and consisted of hemorrhagic and necrotic lesions. Histopathologically, the architecture of the infected lymph nodes was degraded, necrotic, and sectioned migrating stages of L. serrata were clearly visible. In some lymph nodes, parasitic granulomatous lymphadenitis with necrosis and in some cases, degenerated parasite in central area was observed. High prevalence of infection in camels suggests possibility of similar high rate of infection in other animals of this region. In view of the consumption of raw or undercooked visceral organs of camel by humans of this region, the importance of careful inspection at slaughterhouse needs to be emphasized.

Heat Production and Energy Efficiency of Broilers Infected With Necrotic Enteritis.

Necrotic enteritis (NE) in poultry is the most important bacterial disease in terms of economic losses. The present study was conducted to evaluate the effect of an experimental challenge with necrotic enteritis on respiration and heat production in birds pretreated with dietary acylated starch or antibiotics (AB) zinc bacitracin (50 mg/kg) plus salinomycin (60 mg/kg). In total, 48 1-day-old Ross 308 male broilers were assigned to floor pens until day 10. On day 11, birds were randomly placed into 16 calorimetric chambers with four replicates of three birds per treatment. Treatments were: control, AB, acetylated high-amylose maize starch (SA), or butyrylated high-amylose maize starch (SB). Birds were NE challenged by inoculation with 5000 sporulated oocysts each of Eimeria maxima and Eimeria acervulina and 2500 sporulated oocysts of Eimeria brunetti on day 9 and Clostridium perfringens (3.8 × 10(8) colony-forming units) on day 14. The results showed that heat production (HP), respiratory quotient (RQ), heat increment, weight gain (WG), feed intake (FI), and livability (LV) of birds fed control, SA, and SB diets were lower than birds fed AB at 19 and 42 hr postinoculation (P < 0.05). At 65 hr postchallenge, increased FI and WG of birds were observed, indicating recovery from NE. During the entire period, from day 14 to day 17, birds fed control, SA, and SB had lower WG, FI, HP, RQ, metabolizable energy intake (MEI), and metabolizable energy (P < 0.01) than those fed AB. The data demonstrate that Eimeria sp. and C. perfringens challenge reduces growth performance, HP, RQ, metabolizable energy, and MEI of birds fed control, SA, and SB but not AB diets.

Physalis angulata induces death of promastigotes and amastigotes of Leishmania (Leishmania) amazonensis via the generation of reactive oxygen species.

Leishmaniasis are a neglected group of emerging diseases that have been found in 98 countries and are caused by protozoa of the genus Leishmania. The therapy for leishmaniasis causes several side effects and leads to drug-resistant strains. Natural products from plants have exhibited activities against Leishmania in various experimental models. Physalis angulata is a widely used plant in popular medicine, and in the literature it has well-documented leishmanicidal activity. However, its mechanism of action is still unknown. Thus, this study aims to evaluate the mechanism driving the leishmanicidal activity of an aqueous extract of P. angulata root (AEPa). AEPa was effective against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. This effect was mediated by an increase of reactive oxygen species (ROS), but not of nitric oxide (NO). The increased production of ROS induces cell death by phenotypes seems by apoptosis cell death in Leishmania, but not autophagy or necrosis. In addition, morphological analysis of macrophages showed that AEPa induced a high number of cytoplasmic projections, increased the volume of cytoplasm and number of vacuoles, caused cytoskeleton alterations and resulted in high spreading ability. AEPa also promoted superoxide anion (O2(-)) production in both uninfected macrophages and those infected with Leishmania. Therefore, these results revealed that AEPa causes cell death by phenotypes seems by apoptosis cell death in L. amazonensis and modulates macrophage activation through morphofunctional alterations and O2(-) generation to induce Leishmania death.