In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep

Oligodeoxynucleotides (ODN) containing cytosine-phosphate-guanosine (CpG) motifs have been shown to activate the innate immune system and protect mice and chicken from bacterial and viral infections. Unfortunately, similar studies in other veterinary species are lacking. In this study we assessed the in vivo immunostimulatory effects of CpG ODN 2007, an ODN with previously demonstrated in vitro biological activity. The in vivo effects of ODN 2007 were compared in two closely related outbred species, sheep and cattle, to determine if there were common biological responses. We demonstrated that subcutaneous (s.c.) injection of the CpG ODN induces an acute phase response in the form of a transient fever, a mild transient increase in circulating neutrophils and elevated serum haptoglobin in both sheep and cattle. Sheep injected with CpG ODN also exhibited increased serum 2'5'-oligoadenylate (2'5'-A) synthetase activity, but no increase in serum 2'5'-A synthetase was detected in cattle. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than phosphate buffer saline (PBS) alone. These in vivo data demonstrate for the first time that a CpG ODN induces acute phase immunostimulatory responses in sheep and cattle. However, CpG ODN-induced antiviral effector molecule 2'5'-A synthetase was detected only in sheep but not in cattle.     

Eimeria praecox infection ameliorates effects of Eimeria maxima infection in chickens

The effect of Eimeria praecox on concurrent Eimeria maxima infection was studied in susceptible chickens. Clinical signs of coccidiosis were assessed in single E. praecox or E. maxima infections and compared to dual infection with both Eimeria species. Groups infected solely with 10(4)E. maxima oocysts displayed weight gains that were 48% of weight gain in uninfected controls. Weight gain in chickens infected only with 10(4)E. praecox oocysts was 90% of uninfected controls. Average weight gain in chickens infected with both E. maxima and E. praecox was 79% of controls, and showed no significant difference (P>0.05) from weight gain in E. praecox-infected chickens. Feed utilization (feed conversion ratio, FCR) in chickens infected with both species showed no significant difference (P>0.05) from FCR in non-infected controls or chickens infected with E. praecox alone; all showing a significant difference (P<0.05) from FCR in chickens infected solely with E. maxima. Although E. praecox did not appear to have a negative effect on weight gain and FCR, it did cause a significant decrease in serum carotenoids. Analysis of oocysts excreted by chickens during dual infection showed little effect of E. praecox on E. maxima oocyst production.     

Protective effects of sugar cane extracts (SCE) on Eimeria tenella infection in chickens

The effects of oral administration of sugar cane extracts (SCE) on Eimeria tenella oocysts infection in chickens were studied with 2 different experiments. In Experiment 1, 3-week-old inbred chickens (MHC; H.B15) were inoculated into the crop with SCE (500 mg/kg/day) for 1 day or 3 consecutive days, and then challenged with E. tenella sporulated oocysts (2 x 10(4) cells/chicken). In Experiment 2, 1-week-old chickens were orally administered SCE at the same dose for 3 consecutive days, and then initially infected with E. tenella sporulated oocysts (2 x 10(3) cells/chicken). At 2 and 3 weeks of age, these chickens were immunized intravenously with the mixed antigens of sheep red blood cells (SRBC) and Brucella abortus (BA). At 4 weeks of age, chickens were challenged with E. tenella sporulated oocysts (1 x 10(5)/chicken). Challenged chickens with E. tenella oocysts showed markedly decreased body weight gain/day, severe hemorrhage and great number of shedding oocysts in feces and high lesion scores. Oral administration of SCE and initial infection with oocysts (2 x 10 (3)/chicken) resulted in a remarkable improvement in body weight gain/day, hemorrhage, the number of shedding oocysts and lesion score, compare to other infected groups. In addition, SCE-inoculated chickens with the initial infection showed a significant increase in antibody responses against SRBC and BA and also improvement in decreased relative proportions of Bu-1a(+) and CD4( )cells in cecal tonsil lymphocytes of E. tenella-challenged chickens. Cecal tissues of chickens administered SCE and initially infected with E. tenella oocysts showed lower numbers of schizonts, gametocytes and oocysts than those of infected control chickens. These results suggest that SCE have immunostimulating and protective effects against E. tenella infection in chickens.     

In vivo priming heterophil innate immune functions and increasing resistance to Salmonella enteritidis infection in neonatal chickens by immune stimulatory CpG oligodeoxynucleotides

Oligodeoxynucleotides (ODN) containing CpG dinucleotides (CpG-ODN) mimic bacterial DNA and stimulate the innate immune system of vertebrates. Here, we investigated the effects of intraperitoneal (ip) administered CpG-ODN on the innate immune functions of chicken heterophils. Our results demonstrated CpG-ODN-dependent priming of chicken heterophil degranulation and oxidative burst. Heterophils from chickens treated with CpG-ODN exhibited significantly higher (p<0.05) degranulation activity compared to PBS and control ODN (ODN containing no CpG motif) treated groups when stimulated with opsonized Salmonella enterica serovar enteritidis. Similarly, oxidative burst activity, which generates bactericidal reactive oxygen species, was significantly higher (p<0.05) in heterophils from the CpG-ODN treated group than from PBS and control ODN groups when stimulated with formalin-killed S. enteritidis. The priming effects of CpG-ODN on heterophil immune functions continued at least 4 days post-treatment. In the infection study, newly hatched chickens were treated with CpG-ODN, control ODN or PBS for 24h then challenged with oral inoculation of S. enteritidis. A significant reduction (p<0.05) in colonization by S. enteritidis was observed in chickens treated with CpG-ODN. Our study provides evidence that immunostimulatory CpG-ODN potentiates the innate immune responses of heterophils and enhances resistance to infectious pathogens in neonatal chickens.     

Factors influencing enhanced Salmonella typhimurium infection in Eimeria tenella-infected chickens

To investigate a possible mechanism involved in the enhancement of Salmonella typhimurium infection in chickens concurrently infected with Eimeria tenella, S typhimurium was given orally to chickens 7 days after E tenella inoculation. The number of viable S typhimurium decreased in the ceca of chickens not inoculated with E tenella, whereas the number gradually increased in the ceca of chickens inoculated with E tenella. Cecal contents were analyzed for pH value, oxidation-reduction potential, and amounts of short-chain fatty acids and bile acids. In the ceca of E tenella-inoculated chickens, the oxidation-reduction potential significantly (P less than 0.05) shifted to the oxidative phase, and the concentration of volatile fatty acids (acetic acid, propionic acid, and butyric acid) significantly (P less than 0.05) decreased. In both aerobic and anaerobic incubations, the number of viable S typhimurium in vitro decreased as the molar concentration of fatty acids increased. Experimental evidence indicated that multiplication of S typhimurium in the ceca of E tenella-inoculated chickens was associated with decreased concentrations of volatile fatty acids.     

Influence of reticuloendotheliosis on the severity of Eimeria tenella infection in broiler chickens

Broiler chickens free of maternal immunity to reticuloendotheliosis virus (REV) were used in the experiment. Two groups of 25 chickens were inoculated with REV at one day of age. One of these groups and another group of 25 chickens were inoculated with Eimeria tenella sporulated oocysts at 7 days of age. Chickens inoculated with E. tenella showed bloody diarrhoea from 12 to 14 days of age. Six out of 25 chickens died (P less than 0.05) at 13 and 14 days of age in the dual infected group. At 14 days of age, when chickens were killed, the lesion score in the combined infection group, was statistically different from that in the chickens inoculated with E. tenella alone. Also the weights of the bursa of Fabricius and thymus were lower in the two REV infected groups than in the controls. Although REV infection alone adversely affected the weight gain and feed conversion, with combined infection this effect was much greater. Following REV inoculation most of the chickens showed feathering defects and all the examined chickens were viraemic at 21 days of age. At the same age, all but one chicken failed to show precipitating antigenaemia and about one-half of these chickens showed a very low serum neutralisation titre. None of these chickens showed precipitating antibodies.     

Effects of a herbal complex against Eimeria tenella infection in chickens

A liquid and a powder made from a herbal complex consisting of Uncariae Ramulus cum Uncis, Agrimoniae Herba, Sanguisorbae Radix, Eclipta Prostrate Herba, Pulsatillae Radix, Sophorae Flavescentis Radix, Rehmanniae Radix and Glycyrrhizae Radix were studied for their anticoccidial activities in chickens. Chickens were administered with herbal liquid, powder, diclazuril or without medication during the study and challenged with oocysts of Eimeria tenella. Results indicated that the birds medicated showed less bloody faeces than those without medication. The intestinal lesion was mild in the chicks medicated with herbal liquid without significantly different lesion score when compared with uninfected chicks. The birds with medication had significantly higher body weight gains than birds without medication. Therefore, the herbal complex used in this study was effective against E. tenella infection in chickens.     

Antioxidant status during the course of Eimeria tenella infection in broiler chickens

The antioxidant status of broiler chickens (Cobb 500 hybrids) infected with Eimeria tenella was monitored by determining blood plasma malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT). The results of the experiment showed an increase in MDA - a marker of radical-induced damage of E. tenella-infected birds, compared to healthy chickens (3.01 micro mol/L vs. 2.55 micro mol/L, P<0.05). Correspondingly, a decreased SOD activity was observed in infected birds compared to controls (2429.0 U/g Hb vs. 3044.6 U/g Hb, P<0.05). Furthermore, CAT activity in infected birds was higher than in healthy ones (2242.2 U/g Hb vs. 1367.0 U/g Hb, P<0.001). The observed enzyme changes suggest an impaired antioxidant status of chickens during the course of an E. tenella infection and the occurrence of oxidative stress following infection. Alterations in the caecum, oocyst production, weight gain and feed conversion ratio were indicative of a severe infection involving pathogenic oxidative stress and impaired ecological oxidative balance.     

Does Plasmodium gallinaceum induce relapse of Eimeria acervulina infection in chickens?

An experiment was carried out to test the hypothesis that chickens previously infected with Eimeria acervulina, but having ceased producing oocysts, recommence E acervulina oocyst production when infected with Plasmodium gallinaceum. No relapse of coccidiosis was caused by the malarial infection. Subsequent treatment with the immunosuppressant betamethasone of the control chicks infected with E acervulina only did not reveal any occult coccidial infection. The results are critically compared with previously published results which apparently supported the hypothesis.     

不同品种地方鸡对柔嫩艾美球虫的易感性分析

按每只鸡12×10~4个孢子化卵囊的剂量,分别感染20日龄大骨鸡、鹿苑鸡、油鸡和固始鸡,以存活率、相对增重率、血便扣分、病变扣分、卵囊扣分和抗球虫指数为评价指标,分析比较4个地方品种鸡对柔嫩艾美球虫的易感性。试验重复1次。结果显示,4个地方品种鸡对柔嫩艾美球虫的易感性不同。固始鸡对柔嫩艾美球虫易感性最高,表现为感染后的死亡率最高,病变扣分最多,抗球虫指数也最低。油鸡对柔嫩艾美球虫的易感性最低,表现为感染后的死亡率为零,病变扣分和血粪扣分最少,每鸡平均排卵囊数最少,抗球虫指数最高。大骨鸡和鹿苑鸡对柔嫩艾美球虫的易感性相近,位于固始鸡与油鸡之间。     

Immunization of young chickens by trickle infection with Eimeria tenella.

Immunization of chickens was attempted with low levels of Eimeria tenella oocysts (50 oocysts per day) over the first 1 or 2 weeks of life--the "trickle infection" (TI) method. When chickens were immunized by TI at 0-13 days of age, no cecal lesions and a reduced number of oocysts in ceca were observed after challenge at 17 days of age. TI at 0-6 days of age conferred better protection against challenge with E. tenella than did TI at 7-13 days. However, cecal lesions were observed in almost all of these chickens. These findings indicated that TI for 2 weeks (0-13 days of age) provided better immunity than TI for 1 week (0-6 or 7-13 days of age).     

巨型艾美尔球虫感染对雏鸡肠道乳酸杆菌的影响

取200只红羽肉杂雏鸡,饲喂至8日龄,随机分为3个球虫卵囊剂量感染组(0.5×105、1.0×105和2.0×105个/羽),并设对照组;于感染后1、3、5、7 d,各组分别处死雏鸡5只,测定小肠中段乳酸杆菌的定植数量。结果表明:与对照组比较3个试验组雏鸡肠道的乳酸杆菌定植数量均有降低。其中试验Ⅰ组,在感染后1、3 d,肠道乳酸杆菌定植数对数值分别为(5.46±0.14)和(5.57±0.14),与对照组比差异不显著(P>0.05);感染5、7 d后,分别为(5.71±0.45)和(5.80±0.52),达到差异显著水平(P<0.05)。试验Ⅱ、Ⅲ组,在感染后1 d,达到差异显著水平(P<0.05)。感染后3、5、7 d,肠道乳酸杆菌定植数量对数值与对照组比较达到极显著水平(P<0.01)。试验证明大剂量球虫感染可明显降低雏鸡小肠段乳酸杆菌的定植数量。     

Interaction of aflatoxin with Eimeria tenella infection and monensin in young broiler chickens.

Young broiler chicks inoculated with Eimeria tenella and given a diet containing 2.5 mug aflatoxin/g had significantly higher mortality than birds with aflatoxicosis or coccidiosis alone or uninoculated controls. This effect was seen even when a light coccidial infection alone did not increase mortality or cause weight depression. In addition this higher mortality with the combination began earleir and occurred at a higher rate than did mortality from aflatoxin or cecal coccidiosis alone. Dietary monensin sodium (99 umg/g) did not completely prevent mortality and weight depression when aflatoxin and E. tenella were in combination. Aflatoxin and E. tenella singly significantly depressed three-week body weights; however, the depression was most severe when the two were in combination. Both dietary aflatoxin and E. tenella significantly reduced hemoglobin, packed cell volume, and plasma pigmentation, and in combination resulted in more severely reduced hemoglobin, packed cell volume, and plasma pigmentation. Coccidial lesion scores were significantly less for the combination of E. tenella and aflatoxicosis than for coccidiosis alone. This atypical response of the ceca to E. tenella in the presence of dietary aflatoxin was characterized by less distended ceca, very little coagulated blood in the ceca, and apparently more profuse cecal hemorrhage.     

Eimeria brunetti and Eimeria necatrix in chickens of Argentina and confirmation of seven species of Eimeria

Ten poultry farms (broiler breeder pullets, layer pullets, and broilers) in the provinces of Entre Rios and Buenos Aires in Argentina were examined for presence of Eimeria spp. Litter samples obtained from flocks 7-11 wk old were taken to the laboratory for oocyst counting and sporulation, then concentrated for inoculation into coccidia-free chickens. Species were identified by prepatent period, oocyst size, location and appearance of lesions in the intestine, microscopic examination of mucosal smears, and histology (to confirm Eimeria brunetti). On this basis, Eimeria praecox was found in two samples, Eimeria mitis in two, Eimeria acervulina in nine, Eimeria maxima in seven, Eimeria necatrix in three, Eimeria tenella in seven, and E. brunetti in four. These results confirm the presence of all seven recognized species of Eimeria in chickens in the Republic of Argentina.     

Effect of Eimeria acervulina infection on nutrient retention with special reference to fat malabsorption in chickens.

Nutritional balance studies were conducted to determine the effect of Eimeria acervulina infection on the retention of nutrients and the efficiency of utilization of protein and energy in the diet during the course of intestinal coccidiosis in chickens. Both light and electronmicroscopic studies were conducted to elucidate the pathogenesis of fat malabsorption in the infected chickens. The infection with E. acervulina during the acute phase of the disease (day 4 to day 8 postinfection) caused a reduction (P smaller than 0.01) in the retention of percent protein, percent ether extract and percent gross energy of the diet with a concomitant increase (P smaller than 0.01) in the concentration of ether extract and gross energy of the excreta. The metabolizable energy value of the diet was also depressed during this phase. During the recovery phase of the disease (day 9 to day 13 postinfection) infected chickens voided excreta containing a slightly greater concentration of ether extract when compared to the controls but differences in the retention of percent protein and percent gross energy were no longer observed. An increased retention of percent phosphorus also occured during the recovery phase. Studies involving the carcass composition revealed that the efficiency of utilization of protein and metabolizable energy of the diet for tissue deposition was reduced (P smaller than 0.05) during day 0 to day 8 postinfection but only the efficiency of metabolizable energy utilization was found to be depressed (P smaller than 0.05) when data for day 0 to day 14 postinfection were analyzed. Both light and electronmicroscopic studies of the duodenal villi of infected chickens during day 5 to day 6 postinfection revealed accumulation of large globules of fat in the villus epithelial cells parasitized by the gammonts of E. acervulina indicating a possible blackade of "fat exit" from these cells.     

Effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens introduced into chickens

The effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens (KGW 851) newly introduced into chickens was studied. Four groups of chickens consisted of: (1) Uninoculated controls; (2) inoculated with C perfringens (KGW 851); (3) inoculated with E tenella; and (4) inoculated with C perfringens (KGW 851) followed by E tenella. Five chickens in each group were necropsied on each of days 0, 3, 5, 7, 10 and 14 after the E tenella inoculation. The mean total C perfringens counts in the caecal contents increased at five days after E tenella inoculation and reached maximum counts at seven days after the inoculation. Also lincomycin-resistant C perfringens readily established itself at approximately one 10th of the total C perfringens population in the presence or absence of E tenella infection.     

In vitro and in vivo protective effects of three mycotoxin adsorbents against ochratoxin A in broiler chickens

1. The objective of this study was to investigate in vitro and in vivo (in broiler chickens) ochratoxin A (OTA) adsorption efficiency of three different adsorbents: inorganic (modified zeolite); organic (esterified glucomannans) and mixed (inorganic and organic components plus enzymes).

2. The aim of the study was to investigate which of these adsorbents provided the best protection against the presence of residues of OTA in the pectoral muscle and liver of broilers given an OTA-contaminated diet. In addition, it was important to test and compare the results of adsorbent efficiency using two different in vitro methods.

3. The results from classical in vitro investigations carried out in the artificial intestinal fluid, showed that the inorganic adsorbent (Mz), exhibited the highest adsorption, having adsorbed 80.86 ± 1.85% of OTA, whereas average in vitro adsorption abilities of organic (30.52 ± 3.50%) and mixed (32.00 ± 2.60%) adsorbents were significantly lower.

4. In the investigation of absorption in everted sacs of broiler duodenal segments (Everted Duodenal Sacs Procedure), higher OTA adsorption in gut was exhibited by organic adsorbent, 74.26 ± 4.48%. Furthermore, the mean adsorption efficiency of mixed and inorganic adsorbent was 65.26 ± 4.76% and 45.75 ± 7.14%, respectively.

5. In the in vivo investigation, broilers were fed for 21 d on diets containing 2 mg/kg of OTA and supplemented with inorganic (Mz), organic (Ms) or mixed adsorbent (Mf) at the recommended concentration of 2 g/kg of feed. All three adsorbents significantly decreased OTA residue concentrations in the pectoral muscle and livers, but the order of effectiveness was mixed > organic > inorganic. The most efficient was the mixed adsorbent which decreased residue concentration by 72.50% in pectoral muscle and 94.47% in livers.

6. The Everted Duodenal Sac in vitro method provided results similar to those obtained in the in vivo study. However, further studies are required to investigate the efficiencies of adsorbents against various mycotoxins using this method.