BACKGROUND
besnoitia besnoiti, a cyst-forming apicomplexan protozoon, is the causative agent of bovine besnoitiosis.
bovine besnoitiosis has spread within europe in the past few years, with latest outbreaks in hungary  <cit> , switzerland  <cit> , italy  <cit> , and germany  <cit> .
in  <dig>  the disease has been classified as “emerging disease” by the european food safety authority  <cit> .
clinical signs of infected cattle in the early stage of the disease include fever, edema, enlarged lymph nodes, anorexia, weight loss and, in bulls, swollen and painful testes  <cit> .
in the chronic stage, parasitic cysts arise in various organs, including skin, vascular walls, scleral conjunctivae, and other non-intestinal mucous membranes  <cit> .
clinical signs, morphological changes, pathological lesions, parasitological examinations and diagnosis of bovine besnoitiosis have been in the focus of researchers  <cit> .
however, the effects of the parasitic infection on laboratory parameters during acute and chronic stages of the disease were not investigated.
b. caprae, a parasite very closely related to b. besnoiti  <cit> , causes similar clinical signs in goats  <cit>  and data on altered laboratory parameters in caprine besnoitiosis are already available.
in naturally acquired caprine besnoitiosis in iran, alterations of various hematological and biochemical parameters as well as enzyme activities have been observed  <cit> .
however, similar examinations regarding bovine besnoitiosis are missing, and metabolic disturbances caused by tachyzoites and bradyzoites are unknown.
we hypothesized that pathological alterations during the acute stage like hemorrhages, necroses or degenerative lesions in different organs, for example muscle and liver cells, and activation of lymphatic tissues  <cit>  lead to alterations of laboratory parameters connected to those lesions.
hemorrhages may affect erythrocyte parameters, muscle and liver necroses may lead to an increase in organ specific enzyme activities, and alterations of lymphatic tissues or multifocal inflammation may affect leukocyte parameters.
in the chronic stage, we hypothesized that emaciation and the huge number of tissue cysts partially surrounded by chronic granulomatous inflammation in different organs  <cit>  may negatively affect serum albumin concentrations  or elevate serum globulin concentrations .
the objective of the present study was to monitor laboratory parameters during acute, subacute and chronic naturally acquired bovine besnoitiosis and to connect those findings to already obtained clinical and serological findings in the same animals  <cit> .
in addition, we aimed to compare these findings with laboratory parameters of b. besnoiti seronegative cattle and altered parameters of a larger group of chronically infected seropositive limousin cattle from a german cow-calf-operation   <cit> .
methods
animals
chronology of acute, subacute, and chronic bovine besnoitiosis monitoring infected simmental and limousin cattle
animals were part of a longitudinal study focusing on the different stages and disease progression of naturally acquired bovine besnoitiosis  <cit> .
permission for this study was granted by the responsible authorities .
the study consisted of a 12-week cohabitation period .
five healthy german simmental heifers  and one healthy german simmental bull  were kept on a pasture with three chronically infected and b. besnoiti seropositive limousin cows.
six healthy simmental heifers  between  <dig> and 20 months of age were kept on a paddock 20 m away as a control group  <cit> .
prior to the trial, the simmentals tested negative for b. besnoiti and neospora caninum antibodies in immunoblots and ifat  <cit> .
on trial day   <dig> and td  <dig>  two limousin cows  of herd-bbger <dig> suspected to be in the acute stage of bovine besnoitiosis, were added to the pasture.
infection of sa  <dig>  sa  <dig>  sa  <dig>  and sa  <dig> was confirmed clinically, histologically, and serologically.
in the case of sa  <dig>  infection was confirmed serologically   <cit> .
although sa  <dig> had already seroconverted on the day of admission, this day was regarded as day of seroconversion to make inter-animal comparison possible for data analyses.table  <dig>
overview of
b
.
besnoiti
affected simmental  and limousin  cattle
animal id
sa 4
sa 6
sa 8
sa 20
sa 22
breed
s
s
s
l
l
sa = study animal, s = simmental, l = limousin, np = non pregnant, p = pregnant, td = trial day.
the acute stage was classified according to clinical criteria.
animals had to show fever  or at least two of the following clinical signs/diagnoses of acute besnoitiosis: depression, conjunctivitis, subcutaneous edema, lymphadenitis, lameness.
as soon as cysts were clinically observed in the scleral conjunctivae, the term ‘chronic stage’ was used .
the subacute stage was defined as stage between the end of the acute and the beginning of the chronic stage.
blood samples were collected twice a week during the whole trial period and daily for 21 days after animals showed signs of acute besnoitiosis.
on day  <dig>  sa  <dig>  sa  <dig>  sa  <dig>  sa  <dig>  and sa  <dig> were bled during a routine herd health status examination.
in total,  <dig> samples collected from the control heifers and sa  <dig>  sa  <dig>  and sa  <dig> were used to determine reference ranges for simmental hematology, serum chemistry and enzymes activities.
values outside of three times interquartile range from the 25th and 75th percentile were defined as outliers and eliminated.
thereafter, the reference ranges were determined non-parametrically as interval between the  <dig> th and  <dig> th percentile.
hematological and biochemical parameters of sa  <dig>  sa  <dig>  and sa  <dig> were compared with these reference ranges.
values of sa  <dig> and sa  <dig> were compared with those of b. besnoiti seronegative limousin cattle .
herd-bbger1: monitoring of b. besnoiti seropositive limousin cattle
in total,  <dig> samples were collected from  <dig> female limousin cattle  during routine herd health status examinations in the years  <dig>   <dig>  and  <dig>  the sampling of these animals was conducted according to international guidelines and national law concerning animal welfare.
limousin cattle were free of bhv- <dig> and bvd infection and did not show clinical signs of gastrointestinal helminthoses or lung worm disease.
all animals were seropositive for b. besnoiti antibodies in immunoblots and ifat  <cit>  and  <dig> animals displayed cysts in the scleral conjunctivae and/or vaginal vestibula.
only single animals showed large numbers of parasitic cysts in the vaginal vestibula and the scleral conjunctivae as well as visible and palpable lesions in the skin of the trunk, limbs and udder.
values outside of three times interquartile range from the 25th and 75th percentile were defined as outliers and eliminated.
after that, the  <dig> th and  <dig> th percentiles were determined non-parametrically.
herd-bbger1: monitoring of b. besnoiti negative limousin cattle
samples were collected from two groups of female adult cattle of herd-bbger <dig>  fifteen samples from  <dig> healthy female limousin cattle  were collected during routine herd health status examinations in  <dig>   <dig> and  <dig>  the sampling of these animals was conducted according to international guidelines and national law concerning animal welfare.
twenty-seven limousin cows  without a history of bovine besnoitiosis were introduced into herd-bbger <dig> in spring  <dig>  samples from these animals were collected as part of the quarantine health check.
limousin cattle from both groups were free of bhv- <dig> and bvd infection and did not show clinical signs of gastrointestinal helminthoses or lung worm disease and sera were negative for b. besnoiti antibodies in immunoblots and ifat as previously described  <cit> .
to determine limousin reference ranges, values outside of three times interquartile range from the 25th and 75th percentile were defined as outliers and eliminated.
after that, reference ranges were determined non-parametrically as interval between the  <dig> th and  <dig> th percentiles.
blood sample collection and processing
blood samples were collected from the tail or jugular veins.
immediately after sampling, a blood smear was performed on a glass slide for differentiation count of cells.
all blood samples were stored at approximately 8°c for up to 6 hours until transferred to the laboratory.
sera for antibody detection and serum protein electrophoresis were frozen at −80°c until transferred to the friedrich-loeffler-institut  and vet med labor gmbh  for further examination.
samples for serum chemistry, hematology and enzyme activities were kept overnight at 8°c and were processed the next day.
biochemical analysis and hematological examination
urea, creatinine, total protein , albumin, total bilirubin, conjugated bilirubin, calcium, magnesium, phosphor, sodium, chloride and potassium concentrations, aspartate transaminase , glutamate dehydrogenase , gamma-glutamyl transpeptidase , creatine kinase  activities were analyzed in sera with a hitachi  <dig> chemistry analyzer  in the laboratory of the clinic for ruminants.
globulin, albumin/globulin  ratio and unconjugated bilirubin were determined arithmetically.
a complete blood cell count was performed using edta-anticoagulated blood, analyzed with a hematological analyzer .
air-dried slides for differential blood count were stained using a pappenheim stain .
mean corpuscular hemoglobin concentration , mean corpuscular volume  and mean corpuscular hemoglobin  were determined arithmetically.
serum protein gel electrophoresis
sera from sa  <dig>  sa  <dig>  and sa  <dig> were examined on the day of seroconversion, 2 days post seroconversion ,  <dig> dps and on day  <dig>  sera from sa  <dig> and sa  <dig> were examined likewise with the exception of serum from  <dig> dps.
in addition, three sera from sa  <dig> and two sera from sa  <dig> were analyzed during the febrile phase before seroconversion.
serum protein gel electrophoresis was performed at vet med labor gmbh  with a semi-automated agarose gel electrophoresis system .
reference ranges were calculated from the  <dig> samples of sa  <dig> to sa  <dig> from the beginning and the mid of the trial as described above.
in order to exclude possible effects of parasite circulation on the determined parameters in sa  <dig>  sa  <dig>  and sa  <dig>  only samples taken before the calculated incubation period of 13 days  <cit>  were used.
serological examinations
to detect b. besnoiti antibodies in serum, three tests were performed as previously described: one ifat and two immunoblots either based on b. besnoiti tachyzoite or bradyzoite antigen.
a reciprocal positive cut-off titer of  <dig> was used in ifat and recognition of at least four of ten bands in both immunoblots was regarded specific  <cit> .
animals were regarded as positive if two of the three serological tests yielded a positive result.
statistical analysis
hematological results, serum chemistry and enzyme activities of b. besnoiti seronegative and b. besnoiti seropositive limousin cattle were compared statistically.
additionally, the same parameters of b. besnoiti seropositive subclinically affected and b. besnoiti seropositive clinically affected animals were compared statistically.
to assess normality, d’agostino and pearson omnibus normality test was applied.
to compare the two groups, the non-parametric mann–whitney-test was performed.
p values below  <dig>  where considered significant.
data were analyzed using the software graphpad prism  <dig>  for windows.
RESULTS
chronology of hematologic alterations during acute, subacute, and chronic bovine besnoitiosis
shortly before seroconversion, infected animals sa  <dig>  sa  <dig>  and sa  <dig> showed a period of decline in white blood cell  concentration followed by an increase.
sa  <dig> and sa  <dig> showed only a consistent increase in wbc concentrations after seroconversion.
in sa  <dig> and sa  <dig>  the decline was below the simmental reference range  for four days shortly after entering the acute stage .
reduced wbc concentrations consisted of an equal reduction in the concentrations of neutrophils and lymphocytes.table  <dig>
mean, sd and percentiles for laboratory parameters of
b
.
besnoiti
negative simmental cattle
hematology
mean ± sd
percentiles
<dig> th
<dig> th
serum chemistry
enzymes
sd = standard deviation.
chronology of hematologic parameters of study animal   <dig>  sa  <dig>  sa  <dig>  sa  <dig>  and sa  <dig>  a concentration of white blood cells .
b concentration of red blood cells .
c hemoglobin concentration.
d hematocrit .
blue continuous vertical line: beginning of acute stage of sa  <dig>  red hashed vertical line: beginning of acute stage of sa  <dig>  black continuous vertical line: day of seroconversion.
hashed horizontal lines delimit simmental reference range.
continuous horizontal lines delimit limousin reference range .
a decline in red blood cell  concentration was observed in sa  <dig> lasting for 20 days .
rbc concentrations of sa  <dig> fluctuated below the lower limit of the limousin reference range  for the whole trial period.table  <dig>
mean, sd and percentiles of laboratory parameters of
b
.
besnoiti
seronegative and
b
.
besnoiti
seropositive limousin cattle
seronegative
seropositive
hematology
mean ± sd
percentiles
mean ± sd
percentiles
<dig> th
<dig> th
<dig> th
<dig> th
<dig>  ±  <dig>
a,b
<dig>  ±  <dig>
b
<dig>  ±  <dig>
b
serum chemistry
<dig>  ±  <dig>
b
<dig>  ±  <dig>
b
<dig>  ±  <dig>
a,b
<dig>  ±  <dig>
a,b
<dig>  ±  <dig>
b
<dig>  ±  <dig>
b
<dig>  ±  <dig>
b
<dig>  ±  <dig>
a,b
<dig>  ±  <dig>
b
enzymes
<dig>  ±  <dig>
a,b
<dig>  ±  <dig>
a,b
samples from b. besnoiti seronegative limousins comprise  <dig> samples from  <dig> animals, samples from b. besnoiti seropositive limousins comprise  <dig> samples  respectively  <dig> samples  from  <dig> animals.
sd = standard deviation, a = normality test not passed, b = significantly  different versus seronegative limousins  highlighted in bold.
sa  <dig> and sa  <dig> showed a decline in hemoglobin concentration and hct shortly before seroconversion.
sa  <dig> showed strong fluctuation below or around the lower limit of the reference range for the whole trial period .
mean corpuscular hemoglobin concentration  showed strong fluctuations above the lower limit of reference range in all infected animals .
mean corpuscular volume  and mean corpuscular hemoglobin  concentrations were within the reference range in all infected animals with few borderline values .
chronology of changes in serum chemistry during acute, subacute, and chronic bovine besnoitiosis
elevations of tp and globulin concentrations plus decline in a/g ratio were detectable shortly after seroconversion in sa  <dig>  sa  <dig>  sa  <dig>  and sa  <dig> .figure  <dig>
chronology of serum chemistry parameters of study animal   <dig>  sa  <dig>  sa  <dig>  sa  <dig>  and sa  <dig>  a total protein  and globulin  concentrations.
b albumin/globulin  ratio.
c total bilirubin concentration.
d calcium concentration.
blue continuous vertical line: beginning of acute stage of sa  <dig>  red hashed vertical line: beginning of acute stage of sa  <dig>  black continuous vertical line: day of seroconversion.
hashed horizontal lines delimit simmental reference range .
continuous horizontal lines delimit limousin reference range .
albumin concentration declined within reference range shortly after the beginning of the acute stage in sa  <dig>  sa  <dig> displayed fluctuating albumin concentrations around the lower limit of the reference range during the whole trial period.
sa  <dig> and sa  <dig> showed increasing albumin concentrations for few days after seroconversion.
on day  <dig>  albumin concentrations were within reference range in sa  <dig> and sa  <dig> and borderline in sa  <dig>  sa  <dig>  and sa  <dig>
total bilirubin concentrations were above the reference range in sa  <dig>  sa  <dig>  and sa  <dig> .
in sa  <dig> and sa  <dig>  this elevation was mainly caused by an elevated unconjugated bilirubin concentration .
urea concentrations were below the reference range for five days in sa  <dig> starting 4 days ante seroconversion  and for four days in sa  <dig> starting  <dig> dps.
lowest values were  <dig>  mmol/l for sa  <dig> and  <dig>  mmol/l for sa  <dig>  sa  <dig> displayed an increased urea concentration on  <dig> das .
sa  <dig> showed decreased urea concentration  for one day  <dig> dps .
creatinine concentrations were above the reference range in sa  <dig> for five days starting  <dig> das  and for three days starting  <dig> das in sa  <dig> .
on day  <dig>  total bilirubin, urea and creatinine concentrations were within the respective reference ranges in all animals except creatinine concentration in sa  <dig>  .
concentrations of magnesium , phosphate, potassium, sodium and chloride displayed strong fluctuations within the respective reference ranges in all infected animals, with single values lying outside the upper or lower limits .
sa  <dig> displayed a decline of magnesium concentrations starting  <dig> das for four days below reference range.
calcium concentrations declined in all three simmentals shortly before seroconversion and were below the reference range in sa  <dig> and sa  <dig> .
chronology of serum protein gel electrophoresis during acute, subacute, and chronic bovine besnoitiosis
during the acute stage, sa  <dig> displayed no alterations of serum protein fractions compared with the simmental reference range .
sa  <dig> displayed a slightly elevated α1-globulin-fraction  and a diminished α2-globulin-fraction   <dig> das.
alterations of serum protein fractions on the day of seroconversion and thereafter are depicted in figure 3a-d.table  <dig>
mean, sd and percentiles of serum gel electrophoresis of
b
.
besnoiti
negative simmental cattle  and comparison with results from the *2-year-group  of alberghina et al.
<cit>
data obtained in the present study
literature data*
serum gel electrophoresis
mean ± sd
percentiles
mean ± sd
<dig> th
<dig> th
sd = standard deviation.
chronology of absolute concentrations of serum globulins of study animal   <dig>  sa  <dig>  sa  <dig>  sa  <dig>  and sa  <dig>  a α1-globulins.
b α2-globulins.
c β-globulins.
d γ-globulins.
serum protein gel electrophoresis, horizontal lines delimit simmental reference range.
chronology of enzyme activities during acute, subacute, and chronic bovine besnoitiosis
ast activities are depicted in figure  <dig> figure  <dig>
chronology of aspartate transaminase  activities of study animal   <dig>  sa  <dig>  sa  <dig>  sa  <dig>  and sa  <dig>  the empty symbols depict the days on which creatine kinase activities were above the respective reference range.
blue continuous vertical line: beginning of acute stage of sa  <dig>  red hashed vertical line: beginning of acute stage of sa  <dig>  black continuous vertical line: day of seroconversion.
hashed horizontal lines delimit simmental reference range.
continuous horizontal lines delimit limousin reference range.
ck activities were elevated for one day  <dig> dps in sa  <dig>  and  <dig> das in sa  <dig> .
serum activities of ggt and gldh remained below the upper limit of the reference range in all infected animals.
on day  <dig>  activities of ggt, gldh, and ck remained below the upper limit of the reference range in all animals.
herd-bbger <dig> monitoring: significant differences in laboratory parameters between b. besnoiti seronegative and b. besnoiti seropositive limousin cattle
means, standard deviations and percentiles of hematology, serum chemistry and enzyme activities for b. besnoiti seronegative and b. besnoiti seropositive limousin cattle are shown in table  <dig>
the rbc concentration was significantly higher and mcv and mch were significantly lower in b. besnoiti seronegative limousins.
tp, albumin and globulin concentrations were significantly higher in b. besnoiti seropositive limousins.
ast and ck activities were significantly lower in b. besnoiti seronegative limousins .
there were no statistical significant differences between parameters of b. besnoiti seropositive subclinically affected and b. besnoiti seropositive clinically affected limousin cattle.
during chronic besnoitiosis, laboratory parameters of sa  <dig> and sa  <dig> were within the percentiles of the b. besnoiti seropositive limousin group, except rbc and albumin concentrations which were lower in sa  <dig> and globulin concentration which was higher in sa  <dig>
discussion
the results of this study present the chronology of laboratory parameters during a longitudinal clinical trial monitoring five cattle with naturally acquired acute, subacute, and chronic bovine besnoitiosis.
in addition, the laboratory parameters of b. besnoiti seronegative and b. besnoiti seropositive chronically infected limousin cattle from a herd naturally affected by bovine besnoitiosis are compared.
variations exist between reference ranges of different laboratories, cattle breeds, age groups, lactation and pregnancy status  <cit> .
thus, reference ranges for the evaluation of laboratory parameters of b. besnoiti infected simmental cattle were calculated from values of healthy b. besnoiti negative simmental study animals.
although larger animal cohorts are usually used for calculation, these reference ranges were regarded representative for this study, because housing, feeding and handling were similar and examination of cattle samples of the same breed were examined in the same laboratories.
for the same reason, laboratory parameters of sa  <dig> and sa  <dig> were compared with reference ranges calculated from healthy b. besnoiti seronegative female limousin cattle of herd-bbger <dig>  furthermore, we could not find appropriate up-to-date reference values for adult limousin cows in the literature and application of older data could have caused misinterpretations because reference values may change due to genetic and environmental factors  <cit> .
the effect of bovine besnoitiosis on hematological parameters
several hematological parameters were altered during acute bovine besnoitiosis.
in the acute stage, leukopenia was observed in sa  <dig>  sa  <dig>  and sa  <dig>  leukopenia is a common finding in cattle with viral, bacterial and protozoan infections  <cit>  and is due to the low storage neutrophil pool of adult cattle as well as increased margination and tissue emigration during acute inflammation.
for the low rbc concentrations in sa  <dig> and sa  <dig> there are two possible explanations: first, multifocal hemorrhages, which have been described in acute besnoitiosis  <cit>  and were also clinically observed in sa  <dig>  <cit>  and in multiple consecutive histological skin sections taken during acute besnoitiosis in these animals  <cit> .
however, total bilirubin concentrations - although of limited value as a confirmation of hemorrhages in sick cattle  - returned to the normal range after a few days, indicating that hemorrhages are not the only reason contributing to this finding.
second, anemia can also be a result of a chronic inflammatory disease  <cit> .
the constantly low rbc concentrations of sa  <dig> and the lower rbc concentrations in the seropositive limousin group are most likely caused by the chronic inflammatory state.
reticulocyte stains of blood smears to assess the origin of anemia were not performed and reticulocytes were not observed in conventionally stained smears.
similar significant differences in hematological parameters, namely increased mcv and mch and decreased rbc concentrations were also observed in iranian goats infected by b. caprae  <cit> .
the effect of bovine besnoitiosis on serum chemistry values
the initial hypothesis of increased globulin concentrations during chronic bovine besnoitiosis proved true.
however, the effect of besnoitiosis on albumin concentrations was different than expected.
a decrease in albumin synthesis, albumin loss or hemodilution can be the cause of sa 20′s hypoalbuminemia  <cit> .
however, clinical examinations and the results of laboratory tests did not reveal evidence for albumin loss or hemodilution.
two effects probably caused the decreased albumin synthesis in sa 20: inflammation and a negative energy balance.
albumin is a negative acute phase protein and inflammatory hypoalbuminemia can develop after on-going inflammatory states and is usually expected to be mild  <cit> .
moreover, it is tempting to speculate that the massive development of numerous b. besnoiti cysts in the tissues of sa  <dig> led to an increased demand in nutrients and subsequently to a negative energy balance.
the hypocalcemia in this animal is very likely associated with hypoalbuminemia  <cit> , as clinical signs of hypocalcemia were not observed.
the increased globulin concentrations are most likely due to increased antibodies against b. besnoiti, since reciprocal ifat titers displayed a similar increase post seroconversion  <cit> .
this hypothesis is supported by the electrophoresis findings displaying increased γ-globulin fractions  <dig> dps in sa  <dig> and on day  <dig> in sa  <dig> and sa  <dig>
the increase in the α1-fraction  during the febrile phase of sa  <dig> and the increase in both α1- and α2-fractions  of sa  <dig>  sa  <dig>  sa  <dig>  and sa  <dig> shortly after seroconversion are most likely a response to the acute inflammation.
interestingly, the α-fraction was only elevated during the febrile phase in sa  <dig> on one day.
a reason for this may be the rather mild clinical course of the disease in sa  <dig> and sa  <dig>  the increase in the β-fraction of sa  <dig> and sa  <dig> on  <dig> dps is most likely due to an increase in igm-antibodies or complement proteins, because transferrin, a main part of the β-fraction is expected to be low during acute inflammation  <cit> .
sa 20′s elevated γ-globulin fraction  displayed a two-peak pattern  <dig> dps, and elevated γ-globulin fractions of the other days displayed only one peak.
this is indicative of a polyclonal hypergammaglobulinemia shortly after seroconversion changing into a monoclonal or oligoclonal gammoglobulinemia in the chronic stage.
the significant hyperproteinemia of b. besnoiti seropositive limousin cows of herd-bbger <dig> compared with b. besnoiti seronegative limousin cows is mainly caused by hyperglobulinemia.
these findings correlate with the results obtained from the trial animals.
elevation of γ-globulin concentration is common during chronic inflammatory diseases  <cit> , and similar results have also been reported in goats suffering from natural chronic caprine besnoitiosis  <cit> .
unexpectedly, albumin concentrations were higher in b. besnoiti seropositive limousins.
albumin tends to decrease during inflammatory states , and decreased albumin concentrations have also been found in cases of caprine besnoitiosis  <cit> .
maybe the higher albumin concentrations are due to a slight dehydration of these animals, which is also indicated by slightly higher urea concentrations of b. besnoiti seropositive limousins compared with b. besnoiti seronegative limousins.
severely infected limousins in this study had a tendency to have higher total protein and globulin concentrations as well as lower a/g ratios.
the former two observations were also made in goats naturally infected with b. caprae in iran  <cit> .
the hyperbilirubinemia of sa  <dig>  sa  <dig>  and sa  <dig> most likely has two main causes: a prehepatic icterus following hemorrhages , or anorexia and sickness, which are common reasons for hyperbilirubinemia in different species  and can include elevated unconjugated bilirubin concentrations  <cit> .
the decrease in urea concentrations in sa  <dig> and sa  <dig>  as well as low and borderline magnesium concentrations observed for  <dig> das in sa  <dig> can be associated with a period of anorexia in these animals, too.
the elevation of creatinine concentrations in sa  <dig> and sa  <dig> are most likely due to dehydration  <cit> .
sa  <dig> and sa  <dig> showed clinical dehydration on several days and the dehydration period coincides with the anorectic period  <cit> .
urea/creatinine ratio to further assess azotemia was not helpful in these cases, because urea concentrations were reduced.
electrolyte concentrations in blood are influenced by feed intake, hydration status or sweating and are subject to daily change, as it was observed in the trial animals.
these factors are also a plausible explanation for the occasional changes observed in the two limousin groups.
using our data to conclude that bovine besnoitiosis affects electrolyte concentrations seems inappropriate at this point and further studies should be conducted to assess the importance of these alterations.
the effect of bovine besnoitiosis on enzyme activities
the increased ast activities of sa  <dig> and sa  <dig> are most likely associated with a loss of muscle fiber integrity, because ck activities were elevated at the same time and gldh and ggt displayed no elevated activities.
the longer half-life of ast  <cit>  explains the relatively rapid decline of ck activities and the slow removal of ast from serum.
muscle fiber damage may be caused by prolonged recumbence, because b. besnoiti affected cattle show apathy and are more likely to lie down and rest  <cit> .
or elevated ast activities could be a result of mild muscular degeneration and necrosis, lesions which are described in cattle during acute besnoitiosis after experimental infection with high parasite doses  <cit> .
although lower ast and ck activities are of little clinical significance, it is surprising that ast and ck activities were lower in b. besnoiti seropositive limousins compared with b. besnoiti seronegative limousins.
tissue cysts surrounded by severe chronic inflammatory reaction can be found in the musculature of diseased cattle  <cit> , and loss of muscle fiber integrity due to cysts or inflammation seems likely.
maybe the effect of tissue cysts on muscle fiber integrity is too low to get noticed or leads to mild, gradual release of muscle enzymes, which cannot be detected due to the short half-life of ck.
in comparison with the results obtained from the trial animals, loss of muscle fiber integrity seems to play only a role during acute and subacute besnoitiosis.
CONCLUSIONS
this study provides detailed results of laboratory parameters obtained during the course of naturally acquired acute, subacute and chronic bovine besnoitiosis for the first time.
even though a low number of animals were examined in acute and subacute besnoitiosis, it could be shown that hematologic parameters were altered especially during acute and subacute besnoitiosis, leading to reduced red and white blood cell concentrations.
furthermore, during acute and subacute besnoitiosis, elevation of serum globulin fractions reflected the acute inflammatory state, clinical parameters like disturbed condition resulted in increased bilirubin concentrations and lesions like muscle necroses, which are described in the literature led to increased aspartate transaminase and creatine kinase activities.
chronic besnoitiosis led to reduced concentrations of red blood cells and to hyper--globulinemia most likely due to the chronic inflammatory condition caused by the parasite.
abbreviations
a/g ratioalbumin/globulin ratio
astaspartate transaminase
ckcreatine kinase
dasdays ante seroconversion
dpsdays post seroconversion
ggtgamma-glutamyl transpeptidase
gldhglutamate dehydrogenase
hcthematocrit
herd-bbger1first german cattle herd diagnosed with cases of bovine besnoitiosis
ifatimmunofluorescent antibody test
mchmean corpuscular hemoglobin
mchcmean corpuscular hemoglobin concentration
mcvmean corpuscular volume
rbcsred blood cells
sastudy animal
tdtrial day
wbcswhite blood cells
competing interests
the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
prionics ag, schlieren, switzerland funded parts of the study but had no influence on the design of the experiment, data collection and analysis and was not involved in writing the manuscript.
authors’ contributions
mcl collected the samples, performed the data management and analysis and wrote the manuscript.
jcs collected the samples and performed data management.
cs did the statistical workup and revised the manuscript.
gs designed and coordinated the serological investigations and performed the respective data analysis.
nsg was involved in sample collection, data analysis, study design and helped drafting the manuscript.
all authors read and approved the final manuscript.

 besnoitia besnoiti, a cyst-forming apicomplexan protozoon, is the causative agent of bovine besnoitiosis. bovine besnoitiosis has spread within europe in the past few years, with latest outbreaks in hungary, switzerland, italy, and germany. in the disease has been classified as “emerging disease” by the european food safety authority. clinical signs of infected cattle in the early stage of the disease include fever, edema, enlarged lymph nodes, anorexia, weight loss and, in bulls, swollen and painful testes. in the chronic stage, parasitic cysts arise in various organs, including skin, vascular walls, scleral conjunctivae, and other non-intestinal mucous membranes. clinical signs, morphological changes, pathological lesions, parasitological examinations and diagnosis of bovine besnoitiosis have been in the focus of researchers. however, the effects of the parasitic infection on laboratory parameters during acute and chronic stages of the disease were not investigated. b. caprae, a parasite very closely related to b. besnoiti, causes similar clinical signs in goats and data on altered laboratory parameters in caprine besnoitiosis are already available. in naturally acquired caprine besnoitiosis in iran, alterations of various hematological and biochemical parameters as well as enzyme activities have been observed. however, similar examinations regarding bovine besnoitiosis are missing, and metabolic disturbances caused by tachyzoites and bradyzoites are unknown. we hypothesized that pathological alterations during the acute stage like hemorrhages, necroses or degenerative lesions in different organs, for example muscle and liver cells, and activation of lymphatic tissues lead to alterations of laboratory parameters connected to those lesions. hemorrhages may affect erythrocyte parameters, muscle and liver necroses may lead to an increase in organ specific enzyme activities, and alterations of lymphatic tissues or multifocal inflammation may affect leukocyte parameters. in the chronic stage, we hypothesized that emaciation and the huge number of tissue cysts partially surrounded by chronic granulomatous inflammation in different organs may negatively affect serum albumin concentrations or elevate serum globulin concentrations. the objective of the present study was to monitor laboratory parameters during acute, subacute and chronic naturally acquired bovine besnoitiosis and to connect those findings to already obtained clinical and serological findings in the same animals. in addition, we aimed to compare these findings with laboratory parameters of b. besnoiti seronegative cattle and altered parameters of a larger group of chronically infected seropositive limousin cattle from a german cow-calf-operation. methods animals chronology of acute, subacute, and chronic bovine besnoitiosis monitoring infected simmental and limousin cattle animals were part of a longitudinal study focusing on the different stages and disease progression of naturally acquired bovine besnoitiosis. permission for this study was granted by the responsible authorities. the study consisted of a 12-week cohabitation period. five healthy german simmental heifers and one healthy german simmental bull were kept on a pasture with three chronically infected and b. besnoiti seropositive limousin cows. six healthy simmental heifers between and 20 months of age were kept on a paddock 20 m away as a control group. prior to the trial, the simmentals tested negative for b. besnoiti and neospora caninum antibodies in immunoblots and ifat. on trial day and td two limousin cows of herd-bbger suspected to be in the acute stage of bovine besnoitiosis, were added to the pasture. infection of sa sa sa and sa was confirmed clinically, histologically, and serologically. in the case of sa infection was confirmed serologically. although sa had already seroconverted on the day of admission, this day was regarded as day of seroconversion to make inter-animal comparison possible for data analyses.table overview of b. besnoiti affected simmental and limousin cattle animal id sa 4 sa 6 sa 8 sa 20 sa 22 breed s s s l l sa = study animal, s = simmental, l = limousin, np = non pregnant, p = pregnant, td = trial day. the acute stage was classified according to clinical criteria. animals had to show fever or at least two of the following clinical signs/diagnoses of acute besnoitiosis: depression, conjunctivitis, subcutaneous edema, lymphadenitis, lameness. as soon as cysts were clinically observed in the scleral conjunctivae, the term ‘chronic stage’ was used. the subacute stage was defined as stage between the end of the acute and the beginning of the chronic stage. blood samples were collected twice a week during the whole trial period and daily for 21 days after animals showed signs of acute besnoitiosis. on day sa sa sa sa and sa were bled during a routine herd health status examination. in total, samples collected from the control heifers and sa sa and sa were used to determine reference ranges for simmental hematology, serum chemistry and enzymes activities. values outside of three times interquartile range from the 25th and 75th percentile were defined as outliers and eliminated. thereafter, the reference ranges were determined non-parametrically as interval between the th and th percentile. hematological and biochemical parameters of sa sa and sa were compared with these reference ranges. values of sa and sa were compared with those of b. besnoiti seronegative limousin cattle. herd-bbger1: monitoring of b. besnoiti seropositive limousin cattle in total, samples were collected from female limousin cattle during routine herd health status examinations in the years and the sampling of these animals was conducted according to international guidelines and national law concerning animal welfare. limousin cattle were free of bhv- and bvd infection and did not show clinical signs of gastrointestinal helminthoses or lung worm disease. all animals were seropositive for b. besnoiti antibodies in immunoblots and ifat and animals displayed cysts in the scleral conjunctivae and/or vaginal vestibula. only single animals showed large numbers of parasitic cysts in the vaginal vestibula and the scleral conjunctivae as well as visible and palpable lesions in the skin of the trunk, limbs and udder. values outside of three times interquartile range from the 25th and 75th percentile were defined as outliers and eliminated. after that, the th and th percentiles were determined non-parametrically. herd-bbger1: monitoring of b. besnoiti negative limousin cattle samples were collected from two groups of female adult cattle of herd-bbger fifteen samples from healthy female limousin cattle were collected during routine herd health status examinations in and the sampling of these animals was conducted according to international guidelines and national law concerning animal welfare. twenty-seven limousin cows without a history of bovine besnoitiosis were introduced into herd-bbger in spring samples from these animals were collected as part of the quarantine health check. limousin cattle from both groups were free of bhv- and bvd infection and did not show clinical signs of gastrointestinal helminthoses or lung worm disease and sera were negative for b. besnoiti antibodies in immunoblots and ifat as previously described. to determine limousin reference ranges, values outside of three times interquartile range from the 25th and 75th percentile were defined as outliers and eliminated. after that, reference ranges were determined non-parametrically as interval between the th and th percentiles. blood sample collection and processing blood samples were collected from the tail or jugular veins. immediately after sampling, a blood smear was performed on a glass slide for differentiation count of cells. all blood samples were stored at approximately 8°c for up to 6 hours until transferred to the laboratory. sera for antibody detection and serum protein electrophoresis were frozen at −80°c until transferred to the friedrich-loeffler-institut and vet med labor gmbh for further examination. samples for serum chemistry, hematology and enzyme activities were kept overnight at 8°c and were processed the next day. biochemical analysis and hematological examination urea, creatinine, total protein, albumin, total bilirubin, conjugated bilirubin, calcium, magnesium, phosphor, sodium, chloride and potassium concentrations, aspartate transaminase, glutamate dehydrogenase, gamma-glutamyl transpeptidase, creatine kinase activities were analyzed in sera with a hitachi chemistry analyzer in the laboratory of the clinic for ruminants. globulin, albumin/globulin ratio and unconjugated bilirubin were determined arithmetically. a complete blood cell count was performed using edta-anticoagulated blood, analyzed with a hematological analyzer. air-dried slides for differential blood count were stained using a pappenheim stain. mean corpuscular hemoglobin concentration, mean corpuscular volume and mean corpuscular hemoglobin were determined arithmetically. serum protein gel electrophoresis sera from sa sa and sa were examined on the day of seroconversion, 2 days post seroconversion, dps and on day sera from sa and sa were examined likewise with the exception of serum from dps. in addition, three sera from sa and two sera from sa were analyzed during the febrile phase before seroconversion. serum protein gel electrophoresis was performed at vet med labor gmbh with a semi-automated agarose gel electrophoresis system. reference ranges were calculated from the samples of sa to sa from the beginning and the mid of the trial as described above. in order to exclude possible effects of parasite circulation on the determined parameters in sa sa and sa only samples taken before the calculated incubation period of 13 days were used. serological examinations to detect b. besnoiti antibodies in serum, three tests were performed as previously described: one ifat and two immunoblots either based on b. besnoiti tachyzoite or bradyzoite antigen. a reciprocal positive cut-off titer of was used in ifat and recognition of at least four of ten bands in both immunoblots was regarded specific. animals were regarded as positive if two of the three serological tests yielded a positive result. statistical analysis hematological results, serum chemistry and enzyme activities of b. besnoiti seronegative and b. besnoiti seropositive limousin cattle were compared statistically. additionally, the same parameters of b. besnoiti seropositive subclinically affected and b. besnoiti seropositive clinically affected animals were compared statistically. to assess normality, d’agostino and pearson omnibus normality test was applied. to compare the two groups, the non-parametric mann–whitney-test was performed. p values below where considered significant. data were analyzed using the software graphpad prism for windows.  chronology of hematologic alterations during acute, subacute, and chronic bovine besnoitiosis shortly before seroconversion, infected animals sa sa and sa showed a period of decline in white blood cell concentration followed by an increase. sa and sa showed only a consistent increase in wbc concentrations after seroconversion. in sa and sa the decline was below the simmental reference range for four days shortly after entering the acute stage. reduced wbc concentrations consisted of an equal reduction in the concentrations of neutrophils and lymphocytes.table mean, sd and percentiles for laboratory parameters of b. besnoiti negative simmental cattle hematology mean ± sd percentiles th th serum chemistry enzymes sd = standard deviation. chronology of hematologic parameters of study animal sa sa sa and sa a concentration of white blood cells. b concentration of red blood cells. c hemoglobin concentration. d hematocrit. blue continuous vertical line: beginning of acute stage of sa red hashed vertical line: beginning of acute stage of sa black continuous vertical line: day of seroconversion. hashed horizontal lines delimit simmental reference range. continuous horizontal lines delimit limousin reference range. a decline in red blood cell concentration was observed in sa lasting for 20 days. rbc concentrations of sa fluctuated below the lower limit of the limousin reference range for the whole trial period.table mean, sd and percentiles of laboratory parameters of b. besnoiti seronegative and b. besnoiti seropositive limousin cattle seronegative seropositive hematology mean ± sd percentiles mean ± sd percentiles th th th th  ±  a,b  ±  b  ±  b serum chemistry  ±  b  ±  b  ±  a,b  ±  a,b  ±  b  ±  b  ±  b  ±  a,b  ±  b enzymes  ±  a,b  ±  a,b samples from b. besnoiti seronegative limousins comprise samples from animals, samples from b. besnoiti seropositive limousins comprise samples respectively samples from animals. sd = standard deviation, a = normality test not passed, b = significantly different versus seronegative limousins highlighted in bold. sa and sa showed a decline in hemoglobin concentration and hct shortly before seroconversion. sa showed strong fluctuation below or around the lower limit of the reference range for the whole trial period. mean corpuscular hemoglobin concentration showed strong fluctuations above the lower limit of reference range in all infected animals. mean corpuscular volume and mean corpuscular hemoglobin concentrations were within the reference range in all infected animals with few borderline values. chronology of changes in serum chemistry during acute, subacute, and chronic bovine besnoitiosis elevations of tp and globulin concentrations plus decline in a/g ratio were detectable shortly after seroconversion in sa sa sa and sa.figure chronology of serum chemistry parameters of study animal sa sa sa and sa a total protein and globulin concentrations. b albumin/globulin ratio. c total bilirubin concentration. d calcium concentration. blue continuous vertical line: beginning of acute stage of sa red hashed vertical line: beginning of acute stage of sa black continuous vertical line: day of seroconversion. hashed horizontal lines delimit simmental reference range. continuous horizontal lines delimit limousin reference range. albumin concentration declined within reference range shortly after the beginning of the acute stage in sa sa displayed fluctuating albumin concentrations around the lower limit of the reference range during the whole trial period. sa and sa showed increasing albumin concentrations for few days after seroconversion. on day albumin concentrations were within reference range in sa and sa and borderline in sa sa and sa total bilirubin concentrations were above the reference range in sa sa and sa. in sa and sa this elevation was mainly caused by an elevated unconjugated bilirubin concentration. urea concentrations were below the reference range for five days in sa starting 4 days ante seroconversion and for four days in sa starting dps. lowest values were  mmol/l for sa and  mmol/l for sa sa displayed an increased urea concentration on das. sa showed decreased urea concentration for one day dps. creatinine concentrations were above the reference range in sa for five days starting das and for three days starting das in sa. on day total bilirubin, urea and creatinine concentrations were within the respective reference ranges in all animals except creatinine concentration in sa. concentrations of magnesium, phosphate, potassium, sodium and chloride displayed strong fluctuations within the respective reference ranges in all infected animals, with single values lying outside the upper or lower limits. sa displayed a decline of magnesium concentrations starting das for four days below reference range. calcium concentrations declined in all three simmentals shortly before seroconversion and were below the reference range in sa and sa. chronology of serum protein gel electrophoresis during acute, subacute, and chronic bovine besnoitiosis during the acute stage, sa displayed no alterations of serum protein fractions compared with the simmental reference range. sa displayed a slightly elevated α1-globulin-fraction and a diminished α2-globulin-fraction das. alterations of serum protein fractions on the day of seroconversion and thereafter are depicted in figure 3a-d.table mean, sd and percentiles of serum gel electrophoresis of b. besnoiti negative simmental cattle and comparison with results from the *2-year-group of alberghina et al. data obtained in the present study literature data* serum gel electrophoresis mean ± sd percentiles mean ± sd th th sd = standard deviation. chronology of absolute concentrations of serum globulins of study animal sa sa sa and sa a α1-globulins. b α2-globulins. c β-globulins. d γ-globulins. serum protein gel electrophoresis, horizontal lines delimit simmental reference range. chronology of enzyme activities during acute, subacute, and chronic bovine besnoitiosis ast activities are depicted in figure  figure chronology of aspartate transaminase activities of study animal sa sa sa and sa the empty symbols depict the days on which creatine kinase activities were above the respective reference range. blue continuous vertical line: beginning of acute stage of sa red hashed vertical line: beginning of acute stage of sa black continuous vertical line: day of seroconversion. hashed horizontal lines delimit simmental reference range. continuous horizontal lines delimit limousin reference range. ck activities were elevated for one day dps in sa and das in sa. serum activities of ggt and gldh remained below the upper limit of the reference range in all infected animals. on day activities of ggt, gldh, and ck remained below the upper limit of the reference range in all animals. herd-bbger monitoring: significant differences in laboratory parameters between b. besnoiti seronegative and b. besnoiti seropositive limousin cattle means, standard deviations and percentiles of hematology, serum chemistry and enzyme activities for b. besnoiti seronegative and b. besnoiti seropositive limousin cattle are shown in table  the rbc concentration was significantly higher and mcv and mch were significantly lower in b. besnoiti seronegative limousins. tp, albumin and globulin concentrations were significantly higher in b. besnoiti seropositive limousins. ast and ck activities were significantly lower in b. besnoiti seronegative limousins. there were no statistical significant differences between parameters of b. besnoiti seropositive subclinically affected and b. besnoiti seropositive clinically affected limousin cattle. during chronic besnoitiosis, laboratory parameters of sa and sa were within the percentiles of the b. besnoiti seropositive limousin group, except rbc and albumin concentrations which were lower in sa and globulin concentration which was higher in sa discussion the results of this study present the chronology of laboratory parameters during a longitudinal clinical trial monitoring five cattle with naturally acquired acute, subacute, and chronic bovine besnoitiosis. in addition, the laboratory parameters of b. besnoiti seronegative and b. besnoiti seropositive chronically infected limousin cattle from a herd naturally affected by bovine besnoitiosis are compared. variations exist between reference ranges of different laboratories, cattle breeds, age groups, lactation and pregnancy status. thus, reference ranges for the evaluation of laboratory parameters of b. besnoiti infected simmental cattle were calculated from values of healthy b. besnoiti negative simmental study animals. although larger animal cohorts are usually used for calculation, these reference ranges were regarded representative for this study, because housing, feeding and handling were similar and examination of cattle samples of the same breed were examined in the same laboratories. for the same reason, laboratory parameters of sa and sa were compared with reference ranges calculated from healthy b. besnoiti seronegative female limousin cattle of herd-bbger furthermore, we could not find appropriate up-to-date reference values for adult limousin cows in the literature and application of older data could have caused misinterpretations because reference values may change due to genetic and environmental factors. the effect of bovine besnoitiosis on hematological parameters several hematological parameters were altered during acute bovine besnoitiosis. in the acute stage, leukopenia was observed in sa sa and sa leukopenia is a common finding in cattle with viral, bacterial and protozoan infections and is due to the low storage neutrophil pool of adult cattle as well as increased margination and tissue emigration during acute inflammation. for the low rbc concentrations in sa and sa there are two possible explanations: first, multifocal hemorrhages, which have been described in acute besnoitiosis and were also clinically observed in sa and in multiple consecutive histological skin sections taken during acute besnoitiosis in these animals. however, total bilirubin concentrations - although of limited value as a confirmation of hemorrhages in sick cattle - returned to the normal range after a few days, indicating that hemorrhages are not the only reason contributing to this finding. second, anemia can also be a result of a chronic inflammatory disease. the constantly low rbc concentrations of sa and the lower rbc concentrations in the seropositive limousin group are most likely caused by the chronic inflammatory state. reticulocyte stains of blood smears to assess the origin of anemia were not performed and reticulocytes were not observed in conventionally stained smears. similar significant differences in hematological parameters, namely increased mcv and mch and decreased rbc concentrations were also observed in iranian goats infected by b. caprae. the effect of bovine besnoitiosis on serum chemistry values the initial hypothesis of increased globulin concentrations during chronic bovine besnoitiosis proved true. however, the effect of besnoitiosis on albumin concentrations was different than expected. a decrease in albumin synthesis, albumin loss or hemodilution can be the cause of sa 20′s hypoalbuminemia. however, clinical examinations and the results of laboratory tests did not reveal evidence for albumin loss or hemodilution. two effects probably caused the decreased albumin synthesis in sa 20: inflammation and a negative energy balance. albumin is a negative acute phase protein and inflammatory hypoalbuminemia can develop after on-going inflammatory states and is usually expected to be mild. moreover, it is tempting to speculate that the massive development of numerous b. besnoiti cysts in the tissues of sa led to an increased demand in nutrients and subsequently to a negative energy balance. the hypocalcemia in this animal is very likely associated with hypoalbuminemia, as clinical signs of hypocalcemia were not observed. the increased globulin concentrations are most likely due to increased antibodies against b. besnoiti, since reciprocal ifat titers displayed a similar increase post seroconversion. this hypothesis is supported by the electrophoresis findings displaying increased γ-globulin fractions dps in sa and on day in sa and sa the increase in the α1-fraction during the febrile phase of sa and the increase in both α1- and α2-fractions of sa sa sa and sa shortly after seroconversion are most likely a response to the acute inflammation. interestingly, the α-fraction was only elevated during the febrile phase in sa on one day. a reason for this may be the rather mild clinical course of the disease in sa and sa the increase in the β-fraction of sa and sa on dps is most likely due to an increase in igm-antibodies or complement proteins, because transferrin, a main part of the β-fraction is expected to be low during acute inflammation. sa 20′s elevated γ-globulin fraction displayed a two-peak pattern dps, and elevated γ-globulin fractions of the other days displayed only one peak. this is indicative of a polyclonal hypergammaglobulinemia shortly after seroconversion changing into a monoclonal or oligoclonal gammoglobulinemia in the chronic stage. the significant hyperproteinemia of b. besnoiti seropositive limousin cows of herd-bbger compared with b. besnoiti seronegative limousin cows is mainly caused by hyperglobulinemia. these findings correlate with the results obtained from the trial animals. elevation of γ-globulin concentration is common during chronic inflammatory diseases, and similar results have also been reported in goats suffering from natural chronic caprine besnoitiosis. unexpectedly, albumin concentrations were higher in b. besnoiti seropositive limousins. albumin tends to decrease during inflammatory states, and decreased albumin concentrations have also been found in cases of caprine besnoitiosis. maybe the higher albumin concentrations are due to a slight dehydration of these animals, which is also indicated by slightly higher urea concentrations of b. besnoiti seropositive limousins compared with b. besnoiti seronegative limousins. severely infected limousins in this study had a tendency to have higher total protein and globulin concentrations as well as lower a/g ratios. the former two observations were also made in goats naturally infected with b. caprae in iran. the hyperbilirubinemia of sa sa and sa most likely has two main causes: a prehepatic icterus following hemorrhages, or anorexia and sickness, which are common reasons for hyperbilirubinemia in different species and can include elevated unconjugated bilirubin concentrations. the decrease in urea concentrations in sa and sa as well as low and borderline magnesium concentrations observed for das in sa can be associated with a period of anorexia in these animals, too. the elevation of creatinine concentrations in sa and sa are most likely due to dehydration. sa and sa showed clinical dehydration on several days and the dehydration period coincides with the anorectic period. urea/creatinine ratio to further assess azotemia was not helpful in these cases, because urea concentrations were reduced. electrolyte concentrations in blood are influenced by feed intake, hydration status or sweating and are subject to daily change, as it was observed in the trial animals. these factors are also a plausible explanation for the occasional changes observed in the two limousin groups. using our data to conclude that bovine besnoitiosis affects electrolyte concentrations seems inappropriate at this point and further studies should be conducted to assess the importance of these alterations. the effect of bovine besnoitiosis on enzyme activities the increased ast activities of sa and sa are most likely associated with a loss of muscle fiber integrity, because ck activities were elevated at the same time and gldh and ggt displayed no elevated activities. the longer half-life of ast explains the relatively rapid decline of ck activities and the slow removal of ast from serum. muscle fiber damage may be caused by prolonged recumbence, because b. besnoiti affected cattle show apathy and are more likely to lie down and rest. or elevated ast activities could be a result of mild muscular degeneration and necrosis, lesions which are described in cattle during acute besnoitiosis after experimental infection with high parasite doses. although lower ast and ck activities are of little clinical significance, it is surprising that ast and ck activities were lower in b. besnoiti seropositive limousins compared with b. besnoiti seronegative limousins. tissue cysts surrounded by severe chronic inflammatory reaction can be found in the musculature of diseased cattle, and loss of muscle fiber integrity due to cysts or inflammation seems likely. maybe the effect of tissue cysts on muscle fiber integrity is too low to get noticed or leads to mild, gradual release of muscle enzymes, which cannot be detected due to the short half-life of ck. in comparison with the results obtained from the trial animals, loss of muscle fiber integrity seems to play only a role during acute and subacute besnoitiosis.  this study provides detailed results of laboratory parameters obtained during the course of naturally acquired acute, subacute and chronic bovine besnoitiosis for the first time. even though a low number of animals were examined in acute and subacute besnoitiosis, it could be shown that hematologic parameters were altered especially during acute and subacute besnoitiosis, leading to reduced red and white blood cell concentrations. furthermore, during acute and subacute besnoitiosis, elevation of serum globulin fractions reflected the acute inflammatory state, clinical parameters like disturbed condition resulted in increased bilirubin concentrations and lesions like muscle necroses, which are described in the literature led to increased aspartate transaminase and creatine kinase activities. chronic besnoitiosis led to reduced concentrations of red blood cells and to hyper--globulinemia most likely due to the chronic inflammatory condition caused by the parasite. abbreviations a/g ratioalbumin/globulin ratio astaspartate transaminase ckcreatine kinase dasdays ante seroconversion dpsdays post seroconversion ggtgamma-glutamyl transpeptidase gldhglutamate dehydrogenase hcthematocrit herd-bbger1first german cattle herd diagnosed with cases of bovine besnoitiosis ifatimmunofluorescent antibody test mchmean corpuscular hemoglobin mchcmean corpuscular hemoglobin concentration mcvmean corpuscular volume rbcsred blood cells sastudy animal tdtrial day wbcswhite blood cells competing interests the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. prionics ag, schlieren, switzerland funded parts of the study but had no influence on the design of the experiment, data collection and analysis and was not involved in writing the manuscript. authors’ contributions mcl collected the samples, performed the data management and analysis and wrote the manuscript. jcs collected the samples and performed data management. cs did the statistical workup and revised the manuscript. gs designed and coordinated the serological investigations and performed the respective data analysis. nsg was involved in sample collection, data analysis, study design and helped drafting the manuscript. all authors read and approved the final manuscript. 
serum total protein, globulin, total bilirubin and creatinine concentrations were increased and aspartate transaminase and creatine kinase activities were elevated. in chronic besnoitiosis, erythrocyte parameters were statistically significantly lower, and total protein and globulin concentrations were significantly higher in b. besnoiti seropositive compared with b. besnoiti seronegative limousin cows. clinical signs of acute besnoitiosis are pyrexia, anorexia and subcutaneous edema. in subacute and chronic besnoitiosis parasitic cysts arise in a variety of tissues and affected cattle display skin lesions and weight loss. further, laboratory parameters of b. besnoiti seropositive limousin cows and healthy b. besnoiti seronegative limousin cows were compared. in this study, the impact of naturally acquired acute, subacute and chronic bovine besnoitiosis on hematologic parameters, serum chemistry, and enzyme activities was investigated. in this study, altered laboratory parameters during the course of naturally acquired acute, subacute and chronic bovine besnoitiosis are described for the first time. to determine aberrations of laboratory parameters, values were compared with reference ranges obtained from b. besnoiti negative simmentals and limousins. during acute and subacute besnoitiosis, a reduction of leukocyte and erythrocyte concentrations, hematocrit, serum albumin, urea, magnesium, and calcium concentrations were observed. only a few animals were examined in acute and subacute besnoitiosis, however the alterations of laboratory parameters during these stages reflected i) the acute inflammatory state, ii) clinical findings such as disturbed condition, and iii) lesions such as muscle necroses described in the literature. laboratory parameters of two simmental heifers and two limousin cows were monitored during acute, subacute and chronic besnoitiosis and in another simmental heifer during subclinical besnoitiosis. the emerging disease bovine besnoitiosis is caused by the apicomplexan parasite besnoitia besnoiti. in all stages of bovine besnoitiosis, lesions can be found in many organ systems and therefore presumably alter a variety of laboratory parameters. bovine besnoitiosisbesnoitia besnoitihematological and biochemical parameterscattleissue-copyright-statement© the author 2015 chronic besnoitiosis led to typical alterations of chronic inflammatory diseases like hyper--globulinemia or reduced erythrocyte concentrations.
 the emerging disease bovine besnoitiosis is caused by the apicomplexan parasite besnoitia besnoiti. clinical signs of acute besnoitiosis are pyrexia, anorexia and subcutaneous edema. in subacute and chronic besnoitiosis parasitic cysts arise in a variety of tissues and affected cattle display skin lesions and weight loss. in all stages of bovine besnoitiosis, lesions can be found in many organ systems and therefore presumably alter a variety of laboratory parameters. in this study, the impact of naturally acquired acute, subacute and chronic bovine besnoitiosis on hematologic parameters, serum chemistry, and enzyme activities was investigated. laboratory parameters of two simmental heifers and two limousin cows were monitored during acute, subacute and chronic besnoitiosis and in another simmental heifer during subclinical besnoitiosis. to determine aberrations of laboratory parameters, values were compared with reference ranges obtained from b. besnoiti negative simmentals and limousins. further, laboratory parameters of b. besnoiti seropositive limousin cows and healthy b. besnoiti seronegative limousin cows were compared.  during acute and subacute besnoitiosis, a reduction of leukocyte and erythrocyte concentrations, hematocrit, serum albumin, urea, magnesium, and calcium concentrations were observed. serum total protein, globulin, total bilirubin and creatinine concentrations were increased and aspartate transaminase and creatine kinase activities were elevated. in chronic besnoitiosis, erythrocyte parameters were statistically significantly lower, and total protein and globulin concentrations were significantly higher in b. besnoiti seropositive compared with b. besnoiti seronegative limousin cows.  in this study, altered laboratory parameters during the course of naturally acquired acute, subacute and chronic bovine besnoitiosis are described for the first time. only a few animals were examined in acute and subacute besnoitiosis, however the alterations of laboratory parameters during these stages reflected i) the acute inflammatory state, ii) clinical findings such as disturbed condition, and iii) lesions such as muscle necroses described in the literature. chronic besnoitiosis led to typical alterations of chronic inflammatory diseases like hyper--globulinemia or reduced erythrocyte concentrations. keywords bovine besnoitiosisbesnoitia besnoitihematological and biochemical parameterscattleissue-copyright-statement© the author 2015 