acute phase proteins ( apps ) are a group of blood proteins that show concentration increases ( positive app ) or decreases ( negative app ) after inflammation , infection , trauma , some neoplastic growth , or immunological disorders . early reaction of the host to infection or tissue damage leads to a release of pro - inflammatory cytokines . the main cytokines released by infected cells are primarily inteferons , especially ifn- , from mononuclear inflammatory cells , although tnf- and il-1 may also be involved . the positive apps are mainly c - reactive protein ( crp ) , hp , and saa which are released by the hepatocytes after cytokine stimulation . when severe cellular destruction is present , a full acute phase response ( apr ) can be observed . the apr is considered to be a non - specific immune mechanism involving systemic and metabolic changes against insult before specific immunity is achieved [ 4 , 5 ] . during the acute phase response , the serum concentration of the apps changes dramatically . the circulating concentrations of the apps are related to the severity of the disorder and the extent of tissue damage in the affected animals . app levels are not suitable for establishing a specific diagnosis , but quantification of their concentration can provide objective information about the extent of ongoing lesions in individual animals [ 6 , 7 ] . ruminant coccidiosis is a contagious protozoal disease that occurs especially in lambs and kids , with a worldwide distribution . clinical coccidiosis occurs mainly in 4 - 6-month old kids and lambs and stress factors , such as inclement weather , weaning , dietary changes , traveling , and regrouping have important roles in caprine coccidiosis . in goats , 16 species have been described , of which eimeria christenseni , eimeria arloingi , eimeria caprina , and eimeria ninakohlyakimovae are of major concern [ 10 - 12 ] . e. arloingi , a common coccidian species of goats worldwide , causes high mortality in kids and leads to economic loss from both subclinical and clinical infections , including low growth performance , decrease in productivity , and treatment cost [ 13 , 14 ] . the diagnosis of coccidiosis depends on the clinical findings ( diarrhea , dehydration , and progressive emaciation ) , the presence of large numbers of oocysts in the feces , and appropriate signalment and intestinal lesions at necropsy . apps may be useful for providing information about the stage of clinical and subclinical infections and also for prognosis of their severity . to our knowledge , there is no document describing the changes in serum concentrations of apps and inflammatory mediators in caprine coccidiosis . the purpose of this study was to evaluate the dependent changes in the serum concentrations of hp , saa , tnf- , and ifn- in kids experimentally infected by e. arloingi . a pure strain of e. arloingi was obtained from the parasitology department of shiraz university , shiraz , iran . the oocysts were stored at 4 in 2.5% potassium dichromate ( k2cr2o7 ) solution for about 2 weeks before their use in the experiment . before inoculation , the suspension containing oocysts was freed of the potassium dichromate by series washing and centrifugation at 750 g for 5 min . the sediment was mixed with 100 ml distilled water and the number of oocysts was calculated for each ml of suspension . eighteen iranian crossbred kids were separated from their dams immediately after birth , fed with a milk replacer and reared under coccidia - free conditions in closed rooms with restricted access . at 15 days of age , the kids were divided into 2 groups . the kids of groups a ( n=6 ) and b ( n=6 ) were infected orally with 110 ( low dose ) and 110 ( high dose ) sporulated oocysts of e. arloingi per animal , respectively . fresh fecal samples , approximately 3 - 5 g , were obtained directly from the rectum by stimulation of the anus and were examined for the presence of oocysts every day . fecal consistency was assessed on the basis of a scoring system ( 1 , normal ; 2 , semi liquid to liquid ; 3 , watery ; 4 , hemorrhagic and/or with tissue ) . blood samples for determination of serum hp , saa , ifn- , and tnf- were collected from the jugular vein of each kid in both groups before inoculation and at days 7 , 14 , 21 , 28 , 35 , and 42 pi . the sera were separated following centrifugation for 10 min at 750 g and were stored at -20 until analyzed . haptoglobin ( hp ) was measured based on prevention of the peroxidase activity of hemoglobin , which is directly proportional to the amount of hp . the analytical sensitivity of this test in serum has been determined as 0.0156 mg / ml for hp by the manufacturer ( tridelta development plc , wicklow , ireland ) . serum amyloid a ( saa ) was measured by a solid phase sandwich - elisa . the analytical sensitivity of this test in serum has been determined as 0.3 g / ml for saa by the manufacturer ( tridelta development plc ) . ifn- and tnf- were measured by a solid phase sandwich - elisa ( abc 606 and abc 607 , respectively ; votre fournisseur abcys s.a . , paris , france ) . for histopathological examinations , 2 kids were selected from each group , euthanized , and necropsied at day 42 pi . after systematic postmortem examination , appropriate samples of the visceral organs including small and large intestines , liver , spleen , pancreas , and mesenteric lymph nodes from 4 affected kids were fixed in 10% neutral buffered formalin . paraffin - embedded tissues were sectioned at 5 m thickness , stained with hematoxylin - eosin , and examined with a light microscope . descriptive statistics , including the mean , standard deviation , median , minimum , and maximum were calculated for all variables . the anova and tukey tests were used for comparison of different parameters . data were analyzed by spss software , version 16 , and p<0.05 was accepted as a statistically significant difference . a pure strain of e. arloingi was obtained from the parasitology department of shiraz university , shiraz , iran . the oocysts were stored at 4 in 2.5% potassium dichromate ( k2cr2o7 ) solution for about 2 weeks before their use in the experiment . before inoculation , the suspension containing oocysts was freed of the potassium dichromate by series washing and centrifugation at 750 g for 5 min . the sediment was mixed with 100 ml distilled water and the number of oocysts was calculated for each ml of suspension . eighteen iranian crossbred kids were separated from their dams immediately after birth , fed with a milk replacer and reared under coccidia - free conditions in closed rooms with restricted access . at 15 days of age , the kids were divided into 2 groups . the kids of groups a ( n=6 ) and b ( n=6 ) were infected orally with 110 ( low dose ) and 110 ( high dose ) sporulated oocysts of e. arloingi per animal , respectively . fresh fecal samples , approximately 3 - 5 g , were obtained directly from the rectum by stimulation of the anus and were examined for the presence of oocysts every day . fecal consistency was assessed on the basis of a scoring system ( 1 , normal ; 2 , semi liquid to liquid ; 3 , watery ; 4 , hemorrhagic and/or with tissue ) . blood samples for determination of serum hp , saa , ifn- , and tnf- were collected from the jugular vein of each kid in both groups before inoculation and at days 7 , 14 , 21 , 28 , 35 , and 42 pi . the sera were separated following centrifugation for 10 min at 750 g and were stored at -20 until analyzed . haptoglobin ( hp ) was measured based on prevention of the peroxidase activity of hemoglobin , which is directly proportional to the amount of hp . the analytical sensitivity of this test in serum has been determined as 0.0156 mg / ml for hp by the manufacturer ( tridelta development plc , wicklow , ireland ) . serum amyloid a ( saa ) was measured by a solid phase sandwich - elisa . the analytical sensitivity of this test in serum has been determined as 0.3 g / ml for saa by the manufacturer ( tridelta development plc ) . ifn- and tnf- were measured by a solid phase sandwich - elisa ( abc 606 and abc 607 , respectively ; votre fournisseur abcys s.a . , paris , france ) . for histopathological examinations , 2 kids were selected from each group , euthanized , and necropsied at day 42 pi . after systematic postmortem examination , appropriate samples of the visceral organs including small and large intestines , liver , spleen , pancreas , and mesenteric lymph nodes from 4 affected kids were fixed in 10% neutral buffered formalin . paraffin - embedded tissues were sectioned at 5 m thickness , stained with hematoxylin - eosin , and examined with a light microscope . descriptive statistics , including the mean , standard deviation , median , minimum , and maximum were calculated for all variables . the anova and tukey tests were used for comparison of different parameters . data were analyzed by spss software , version 16 , and p<0.05 was accepted as a statistically significant difference . the oocyst shedding was observed after the prepatent period , which varied between 16 to 18 days in groups a and b. all inoculated kids produced comparable numbers of oocysts ( fig . the clinical signs of the infected kids varied with the size of the inoculation dose . a , diarrhea started from day 20 pi , a kid with semiliquid and the others with liquid diarrhea ( score 2 ) . severe diarrhea with variable amounts of mucus ( score 3 ) occurred in group b from day 18 pi . other signs included dehydration , anorexia , depression , paleness of eye mucosa , and listlessness . g / l , and a significant increase was detected at day 7 pi ( fig . g / l ) was detected at days 14 and 21 pi in groups a and b , respectively . the median saa concentration prior to inoculation was 6.1 g / ml , and concentrations of this protein were significantly increased at day 7 pi ( fig . the maximum median saa concentration ( 27.8 and 90.4 g / ml ) was reached at days 35 and 21 after inoculation in groups a and b , respectively . the levels of this protein remained increased until the end of the experiment . the mean tnf- concentration before inoculation was 22.7 pg / dl , and a significant increase ( about 2.4 times ) was observed at day 7 pi ( fig . the highest median concentrations were 57.6 pg / dl in group a and 58.5 pg / dl in group b. the mean ifn- concentration before inoculation was 15.3 pg / dl , and a significant increase ( about 3 times ) was observed at day 7 pi ( fig . the highest median concentrations were 46.7 pg / dl and 45.8 pg / dl in groups a and b , respectively . 01 ) , saa with tnf-(r=0.635 , p<0.01 ) and saa with ifn-(r=0.627 , p<0.01 ) in both groups . significant correlation was observed for tnf- and ifn-(r=0.961 , p<0.01 ) . as shown in fig . 2a and 2b , hp and saa concentrations were significantly higher in group b compared with group a ( p<0.05 ) in all weeks . however , there were no significant changes in concentrations of tnf- and ifn- between group a and group b. during the experiment , in group c , the concentrations of hp , saa , tnf- , and ifn- were within the reference interval . there were differences between groups a and b with regards to gross and histopathological lesions . the most remarkable macroscopic lesion was white to grey , slightly to well - raised pin - point foci to relatively large nodules mostly on affected mucosa in the middle jejunum to the ileum ( fig . 3 ) . the kids in group a had milder lesions , including a few scattered whitish non - pedunculated nodules in the mucosa of the jejunum and ileum . the kids in group b showed advanced widespread lesions characterized by thickened mucosa due to mucosal hyperplasia and adenomatous - like changes in the mucosa and a cerebriform or gyrate pattern of projections visible from the serosa . the main histopathological lesion in both groups was proliferative enteritis associated with the presence of the developmental stages of parasite including mature schizonts , microgametocytes , macrogametocytes , and immature oocysts in the epithelial cells of villi and crypts of the jejunum , ileum , cecum , and proximal colon ( fig . 4 ) . also , mild lymphoid hyperplasia of the peyer 's patches , remarkable congestion , and edema were observed in some cases . generally , the kids in group b showed more prominent microscopic lesions than those in group a. caprine coccidiosis is an important disease resulting from complex interactions between parasites and host , with many factors influencing the severity of the disease . age , stress , genetic susceptibility , physical condition , and the degree of immunity are important in pathogenesis [ 10 , 16 , 17 ] . coccidiosis causes high morbidity and mortality in livestock , and diagnosis is limited to clinical findings , the presence of large numbers of oocysts in the feces , and histopathological findings . there are relatively few reports of app induction by parasite infections , although infections with plasmodium falciparum , babesia canis , leishmania infantum , and trypanosoma brucei might be accompanied by increased levels of apps [ 19 - 23 ] . haptoglobin and saa are major acute phase proteins in most species studied . in our experiment , we have shown that the app response is induced in kids following infection with e. arloingi . the exact mechanism however , the induction of apps supports the hypothesis that pro - inflammatory cytokines play an important role . the main pathway leading to app production involves the initial release of pro - inflammatory cytokines by macrophages and other inflammatory cells at the site of inflammation or infection . the most important inducers of apps are cytokines of the il-1 , tnf , and il-6 families . tnf- , il-1 , and ifn- are essential for induction of other cytokines ( il-6 and il-8 ) and agents , such as platelet activating factor , prostaglandins , leukotrienes , and nitric oxide . when these pro - inflammatory cytokines spill into the circulation , they induce the release of apps by the liver into the circulation . il-6 is the principal regulator of most apps and induces the production of hp , whereas il-1 and tnf- induce expression of saa and 1-acid glycoprotein and hp , depending on the mammalian species . as was demonstrated in this study , serum hp , saa , tnf- , and ifn- concentrations were significantly higher on day 7 after inoculation compared with their concentrations before inoculation , and the levels of these proteins remained increased until the end of the experiment . hp is a major app in ruminants , in which species it has a slight circulating level in normal animals , but increases over 100 folds on stimulation . the mean hp concentrations in groups a and b at day 7 pi were 3.2 and 6.3 times higher than the levels before inoculation . in both groups , significant correlations were observed for hp with saa , tnf- , and ifn-. petersen et al .   reported that clinical signs of lameness , diarrhea , respiratory disease , and ear necrosis were reflected in a high serum hp concentration . many studies have reported the serum hp as a clinically useful parameter for measuring the occurrence and severity of inflammatory responses in cattle with enteritis , peritonitis , endometritis , mastitis , pneumonia , endocarditis , abscesses , and other natural or experimental infectious conditions [ 15,28 - 37 ] . the mean saa concentrations in groups a and b at day 7 pi were 4.2 and 13.7 times higher than the levels before inoculation . in both groups , significant correlations were observed for saa with hp , tnf- , and ifn-. saa is a remarkably moderate acute phase protein in cattle increasing around 2 - 5 times during an acute phase response [ 38 - 40 ] . because of the difficulty of measuring serum saa levels , the application of saa assays to veterinary diagnosis has not been as widespread as that of hp assays . saa has been suggested to be more useful in distinguishing acute and chronic inflammations than neutrophil counts and white blood cell counts   in our study , the magnitude and the duration of the hp and saa responses correlated well with the inoculation doses and severity of the clinical signs and diarrhea in individual animals . hp and saa concentrations were significantly higher in group b compared with group a ( p<0.05 ) . these results were consistent with histopathological investigations , which showed advanced widespread lesions in group b. in some papers , no correlation between hp and disease severity was observed . sickness behavior with decreased appetite or anorexia is mediated by the pro - inflammatory cytokines through induction of prostaglandins . the elevation in apps following e. arloingi infection very strongly indicated that pro - inflammatory cytokines were released into the peripheral circulation . significant increases in tnf- and ifn- concentrations ( about 2.4 and 3.0 times , respectively ) were observed at day 7 pi in both groups . significant correlations were observed for tnf- and ifn- with hp and saa in both groups . although serum concentrations of tnf- and ifn- increased after inoculation in both groups , significant correlations were not observed with the inoculation doses and severity of the clinical signs and diarrhea in individual animals . it is important to note that the app - response - inducing cytokines are small molecules with a very short half - life . therefore , cytokines are not very useful for most general diagnostic purposes , in contrast to the apps , which are stable proteins and have relatively long half - lives . in addition , assays for the individual pro - inflammatory cytokines , tnf- , il-1 , and il-6 are not commercially available for animals . according to our study , it seems that saa and hp are 2 major apps that significantly increase in caprine coccidiosis . considering the timing of the response and the correlation between the magnitude of the response and the severity of the infection , it can be established that parasites causing focal lesions can induce acute phase responses , most probably indirectly via parasite - mediated tissue perturbations . in conclusion , measuring hp and saa can be a suitable indicator of inflammatory reactions in caprine coccidiosis . in our experiment , therefore , apps could be used as a non - specific marker for disease as well as for monitoring the response to treatment in coccidiosis . measuring apps along with clinical signs and oocysts per gram ( opg ) can be useful for providing information about the stages of clinical and subclinical coccidiosis .
this experiment was conducted to assess the changing patterns and relative values of acute phase proteins and inflammatory cytokines in experimental caprine coccidiosis . 
 eighteen newborn kids were allocated to 3 equal groups . 
 two groups , a and b , were inoculated with a single dose of 1103 and1105 sporulated oocysts of eimeria arloingi , respectively . 
 the third group , c , received distilled water as the control . 
 blood samples were collected from the jugular vein of each kid in both groups before inoculation and at days 7 , 14 , 21 , 28 , 35 , and 42 post - inoculation ( pi ) , and the levels of haptoglobin ( hp ) , serum amyloid a ( saa ) , tnf- , and ifn- were measured . for histopathological examinations , 
 2 kids were selected from each group , euthanized , and necropsied on day 42 pi . 
 mean hp concentrations in groups a and b ( 0.34 and 0.68 
 g / l ) at day 7 pi were 3.2 and 6.3 times higher than the levels before inoculation . 
 the mean saa concentrations in groups a and b ( 25.6 and 83.5 g / ml ) at day 7 pi were 4.2 and 13.7 times higher than the levels before inoculation . the magnitude and duration of the hp and saa responses correlated well with the inoculation doses and the severity of the clinical signs and diarrhea in kids . 
 these results were consistent with the histopathological features , which showed advanced widespread lesions in group b. in both groups , significant correlations were observed for tnf- and ifn- with saa and hp , respectively . in conclusion , hp and saa can be useful non - specific diagnostic indicators in caprine coccidiosis .