Zinc Deficiency-Like Syndrome in Fleckvieh Calves: Clinical and Pathological Findings and Differentiation from Bovine Hereditary Zinc Deficiency.

BACKGROUND: Zinc deficiency-like (ZDL) syndrome is an inherited defect of Fleckvieh calves, with striking similarity to bovine hereditary zinc deficiency (BHZD). However, the causative mutation in a phospholipase D4 encoding gene (PLD4) shows no connection to zinc metabolism. OBJECTIVES: To describe clinical signs, laboratory variables, and pathological findings of ZDL syndrome and their utility to differentiate ZDL from BHZD and infectious diseases with similar phenotype. ANIMALS: Nine hospitalized calves with crusting dermatitis and confirmed mutation in PLD4 and medical records from 25 calves with crusting dermatitis or suspected zinc deficiency. METHODS: Prospective and retrospective case series. RESULTS: The 9 calves (age: 5-53 weeks) displayed a moderate to severe crusting dermatitis mainly on the head, ventrum, and joints. Respiratory and digestive tract inflammations were frequently observed. Zinc supplementation did not lead to remission of clinical signs in 4 calves. Laboratory variables revealed slight anemia in 8 calves, hypoalbuminemia in 6 calves, but reduced serum zinc concentrations in only 3 calves. Mucosal erosions/ulcerations were present in 7 calves and thymus atrophy or reduced thymic weights in 8 calves. Histologically, skin lesions were indistinguishable from BHZD. Retrospective analysis of medical records revealed the presence of this phenotype since 1988 and pedigree analysis revealed a common ancestor of several affected calves. CONCLUSIONS AND CLINICAL IMPORTANCE: ZDL syndrome should be suspected in Fleckvieh calves with crusting dermatitis together with diarrhea or respiratory tract inflammations without response to oral zinc supplementation. Definite diagnosis requires molecular genetic confirmation of the PLD4 mutation.

Naturally acquired bovine besnoitiosis: Disease frequency, risk and outcome in an endemically infected beef herd.

The recent spread of bovine besnoitiosis warrants further epidemiological investigations to improve the knowledge on disease development. Thus, a 4-year longitudinal open cohort study was conducted in the first German cattle herd naturally infected with Besnoitia besnoiti. At seven herd-visits between 2008 and 2012, fourteen breeding bulls (>1.5 years) and 131 females (>1 year) were examined clinically and serologically. In females, clinical and serological prevalences, incidence and remission rates were determined. In addition, the association of age, antibody levels and number of visible parasitic cysts with clinical and serological outcome was investigated. The seroprevalence (89.4%-100%) and serological incidence rate (140.5 per 100 animal-years) were considerably higher than the clinical prevalence (23.5%-36.6%) and clinical incidence rate (16.7 per 100 animal-years). Of 33 new clinical and 12 new serological cases, only 6.7% (3/45) attracted attention with clinical signs of acute bovine besnoitiosis. The apparent serological remission rate (1.9 per 100 animal-years) was considerably lower than the clinical remission rate (37.3 per 100 animal-years). A median cyst score of <1 and mean immunofluorescent antibody test (IFAT) titre of ≤1,600 over the entire observation period was significantly associated with a negative clinical outcome at the end. Overall cyst score was not significantly associated with serological outcome and age had no significant influence on clinical and serological outcome. Within 4 years, there was a significant reduction in cyst scores and IFAT titres in the same animals, leading to eight clinically and serologically negative animals in the end. Two initially negative animals achieved clinical and apparent serological remission in about 2.5 years. In bulls, the time between herd entry and seroconversion was 7-30 months and the serological incidence rate was nearly identical to the rate in females (142.0 per 100 animal-years). This shows that a high B. besnoiti prevalence leads to infection of bulls within a short time period.

Naturally acquired bovine besnoitiosis: Differential distribution of parasites in the skin of chronically infected cattle.

Bovine besnoitiosis is caused by Besnoitia besnoiti, an apicomplexan parasite closely related to Toxoplasma gondii and Neospora caninum. In the acute stage of besnoitiosis, cattle suffer from pyrexia, swollen lymph nodes, anorexia and subcutaneous edema. In the chronic stage, tissue cysts are formed in a variety of tissues including the skin. Knowledge about the distribution of tissue cysts of different parts of the skin of infected animals is scarce. Four chronically infected cattle were euthanized and skin samples were taken from a total of 77 standardized cutaneous locations per animal. Portions of the dermis were taken, from which DNA was extracted and examined by real-time PCR. Cycle of transition (Ct) values reflecting the amount of parasite DNA in the samples were determined. For statistical analysis, samples were attributed to 11 larger skin regions ('OuterHindlegDistal', 'Rump, ForelegMiddle', 'NoseFrontEars', 'CheekEye', 'SideLowerPart', 'ForelegDistal', 'SideUpperPart', 'LegsInner', 'VentralHeadNeck', 'DorsalNeckWithersBackTail'). While all samples revealed a positive result in three female cattle, only 63.6% (49/77) of the samples of a bull showed positive results. For statistical analysis, a Ct value of 45 was assumed for samples with a negative result. The dams showed median Ct values of 16.1, 17.5 and 19.4, while in skin samples of the bull a median Ct value of 37.6 was observed. To determine the differences in DNA concentrations between different locations of the skin of the animals, a relative Ct (relCt) was determined by subtracting for each animal indv the MedianCtindv from each sample Ct. Analyses of the relCt values showed that the highest relative parasite DNA concentrations were observed in the categories 'OuterHindlegDistal', 'Rump', 'ForelegMiddle' and 'NoseFrontEars'. The relCt values in these categories differed statistically significantly from those determined for the categories 'VentralHeadNeck' and 'DorsalNeckWithersBackTail'. The analysis showed clear differences in the distribution and the detectability of parasite DNA in the skin of cattle infected with B. besnoiti. In all four animals, samples from the 'Rump' region (Regio fermoris) showed high parasite DNA concentrations. Because this region is also easily accessible for veterinarians, this skin location appears to be optimal for taking skin biopsies for detection or isolation of B. besnoiti.

Besnoitia besnoiti infection in cattle and mice: ultrastructural pathology in acute and chronic besnoitiosis.

Current knowledge on bovine besnoitiosis, caused by the emerging apicomplexan pathogen Besnoitia besnoiti, is still fragmentary. So far, studies dealing with ultrastructural pathology focused mainly on the easily accessible chronic stage, whereas ultrastructural investigations of tachyzoites were confined to in vitro studies. In the study presented here, the ultrastructural pathology of naturally B. besnoiti-infected cattle in the acute and chronic disease stages and experimentally B. besnoiti-infected mice was monitored. Further, the ultrastructure of tachyzoites and bradyzoites was investigated. Skin samples of two adult Limousin cows and one adult Limousin bull naturally infected with B. besnoiti and liver and skin samples of gamma-interferon knockout mice infected with B. besnoiti were examined in semithin sections stained with toluidine blue and safranin and in ultrathin sections contrasted with uranyl acetate and lead citrate. Samples of vessel walls of the bull and nasal mucosa of one cow were examined by scanning electron microscopy. Few tachyzoites-like endozoites were detected for the first time in bovine skin, and large numbers of tachyzoites were detected in murine skin and liver. Within tissue cysts in bovine skin, numerous bradyzoites were observed displaying signs of degeneration. Tachyzoites had apicomplexan endozoite ultrastructure. B. besnoiti tachyzoites and bradyzoites differed in shape and the number of amylopectin granules. Transmission and scanning electron microscopy confirmed the presence of two different cyst wall layers, and the present results on cyst wall ultrastructure were in accordance with those previously obtained by histological sections.

Naturally acquired bovine besnoitiosis: histological and immunohistochemical findings in acute, subacute, and chronic disease.

The pathogenesis of bovine besnoitiosis, a disease of increasing concern within Europe, is still incompletely understood. In this study, disease progression after natural infection with the causative apicomplexan Besnoitia besnoiti was monitored in histological skin sections of 5 individual female cattle over time. High-frequency skin sampling of 2 cattle with mild and 2 with severe acute, subacute, and chronic besnoitiosis, as well as from 1 animal during subclinical disease, enabled documentation from the beginning of the disease. Skin sections were stained with hematoxylin and eosin and Giemsa, periodic acid-Schiff reaction, and anti-Besnoitia immunohistochemistry. In all 4 clinically affected animals, tachyzoite-like endozoites could be detected for the first time by immunohistochemistry, and tissue cyst evolution was monitored. Besnoitiosis-associated lesions were not detected in the animal showing the subclinical course. Because of the inconsistency of the nomenclature of Besnoitia tissue cyst layers in the literature, a new nomenclature for B. besnoiti cyst wall layers is proposed: tissue cysts consist of a hypertrophied host cell with enlarged nuclei, an intracytoplasmic parasitophorous vacuole with bradyzoites, a sometimes vacuolated inner cyst wall, and an outer cyst wall in more developed cysts. Inner and outer cyst walls can be readily distinguished by using special stains. In 1 animal, extracystic B. besnoiti zoites were immunohistochemically detected during the chronic stage. At necropsy, the 2 severely affected cows displayed large numbers of B. besnoiti cysts in a variety of tissues, including the corium of the claws, contributing mainly to the development of chronic laminitis in these 2 cases.

Sarcocystis sinensis is the most prevalent thick-walled Sarcocystis species in beef on sale for consumers in Germany.

Bovines are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta, and Sarcocystis hominis, which use canids, felids, or primates as definitive hosts, respectively. Cattle represent also intermediate hosts of Sarcocystis sinensis, but the definitive hosts of this parasite are not yet known. Sarcocystosis in cattle is frequently asymptomatic. The infection is characterized by the presence of thin-walled (S. cruzi) or thick-walled muscle cysts or sarcocysts (S. hominis, S. sinensis, and S. hirsuta). Recent reports suggest high prevalence of the zoonotic S. hominis in beef in Europe. We therefore aimed at differentiating Sarcocystis spp. in beef offered to consumers in Germany using molecular and microscopical methods, focusing on those species producing thick-walled sarcocysts. A total of 257 beef samples were obtained from different butcheries and supermarkets in Germany and processed by conventional and multiplex real-time PCR. In addition, 130 of these samples were processed by light microscopy and in 24.6% thick-walled cysts were detected. Transmission electron microscopical analysis of six of these samples revealed an ultrastructural cyst wall pattern compatible with S. sinensis in five samples and with S. hominis in one sample. PCR-amplified 18S ribosomal DNA (rDNA) fragments of 28 individual thick-walled cysts were sequenced, and sequence identities of ≥98% with S. sinensis (n = 22), S. hominis (n = 5) and S. hirsuta (n = 1) were observed. Moreover, nine Sarcocystis sp. 18S rDNA full length gene sequences were obtained, five of S. sinensis, three of S. hominis, and one of S. hirsuta. Out of all samples (n = 257), 174 (67.7%) tested positive by conventional PCR and 179 (69.6%) by multiplex real-time PCR for Sarcocystis spp. Regarding individual species, 134 (52%), 95 (37%), 17 (6.6%), and 16 (6.2%) were positive for S. cruzi, S. sinensis, S. hirsuta, and S. hominis, respectively. In conclusion, S. sinensis is the most prevalent thick-walled Sarcocystis species in beef offered for consumption in Germany. Further studies are needed to identify the final host of S. sinensis as well as the potential role of this protozoan as a differential diagnosis to the zoonotic species S. hominis.

Novel tools for the diagnosis and differentiation of acute and chronic bovine besnoitiosis.

Diagnosis of acute bovine besnoitiosis is a major diagnostic problem. We developed diagnostic tests to serologically diagnose and differentiate acute and chronic cases of bovine besnoitiosis using affinity purified antigens of Besnoitia besnoiti tachyzoites in immunoblots and in both, a conventional ELISA and an avidity ELISA. Sera of acutely and chronically infected cattle were investigated using these tests. Acutely infected cattle initially recognised an antigen of 74 kDa relative molecular mass, followed by reactions with increasing intensity against 81 and 28 kDa antigens. In addition, faint reactions against antigens with 36, 37, 39 and 42 kDa molecular mass started soon after seroconversion and increased over time. An antigen of 45 kDa molecular mass was transiently recognised early after infection but not or only weakly in the chronic stage. At least two antigens, the 39 and the 42 kDa antigens, seem to be located on the surface of B. besnoiti tachyzoites as determined by biotinylation. Affinity purified antigen was used to establish an APure-BbELISA which showed excellent sensitivity (100%) relative to a serological reference system in naturally, most likely chronically, infected cattle. Specificity was also high (99.8%) as determined in cattle from herds with Neospora caninum-associated abortions. The antibody levels in APure-BbELISA were correlated with the parasite load in the skin or the mucous membrane of the vestibulum vaginae as determined by real-time PCR. In acute cases of bovine besnoitiosis (confirmed by the detection of low avidity IgG in the APure-BbELISA) first specific antibodies were detected by ELISA in all animals except one, at the same time or earlier than in the serological reference system. The detection of parasite DNA in skin by real-time PCR was clearly superior to serological analysis in detecting infected cattle during acute besnoitiosis.

[High incidence of jaundice in young calves in Southern Germany]. / Häufung von Ikterus bei jungen Kälbern in Süddeutschland.

OBJECTIVE: Between September, 2010, and August, 2011, a series of cases of jaundice of unknown origin in young calves was detected in a number of farms in Southern Germany. This paper describes the syndrome on the basis of 57 cases, and the approach taken to discover the cause. MATERIAL AND METHODS: The clinical course of the disease is described in 19 patients. Using a case definition (calves aged 1-3 weeks, total serum bilirubin > 20 µmol/l and/or serum glutamate dehydrogenase [GLDH] activity >50U/l and/or autopsy findings with striking liver pathology [jaundice, liver dystrophy, cirrhosis]), 36 farms were included in an epidemiological survey. In a feeding trial, two batches of a dietary supplement feed, previously used in diseased calves on farms, were fed at the dosage recommendations of the manufacturer to four clinically healthy calves over 5days. Four other calves served as controls. The calves were clinically monitored daily, and blood samples were investigated using clinical chemistry and haematology. RESULTS: Clinical examination revealed behavioural alterations (weakness, tonic-clonic seizures and bawling just before death), recumbency, jaundice and discolouration of faeces. In less severe cases without clinical signs, there was an increase in serum bilirubin concentration and/or GLDH activity. In the epidemiological survey of affected farms, the feeding of a diet supplement feed was registered in 54 of 57 cases. The feeding of two batches of that diet supplement feed to four clinically healthy calves resulted in a significant (p<0.05) increase in bilirubin and lactate concentrations, as well as the GLDH activity in serum, but without serious impairment of the general condition, whereas in control calves, no comparable changes were observed. CONCLUSION: The results of the epidemiological survey and the feeding trial suggest a causal involvement of a dietary supplement feed. The toxic principle is unknown. CLINICAL RELEVANCE: Knowledge of the clinical picture and the probable feed-related context is important to detect this disease early. The suspected dietary supplement feed has been taken off the market, but with other products similar problems may arise.

Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal inherited disease in Caucasians and is caused by mutations in the CFTR gene. The disease is incurable and medical treatment is limited to the amelioration of symptoms or secondary complications. A comprehensive understanding of the disease mechanisms and the development of novel treatment options require appropriate animal models. Existing CF mouse models fail to reflect important aspects of human CF. We thus generated a CF pig model by inactivating the CFTR gene in primary porcine cells by sequential targeting using modified bacterial artificial chromosome vectors. These cells were then used to generate homozygous CFTR mutant piglets by somatic cell nuclear transfer. The homozygous CFTR mutants lack CFTR protein expression and display severe malformations in the intestine, respiratory tract, pancreas, liver, gallbladder, and male reproductive tract. These phenotypic abnormalities closely resemble both the human CF pathology as well as alterations observed in a recently published CF pig model which was generated by a different gene targeting strategy. Our new CF pig model underlines the value of the CFTR-deficient pig for gaining new insight into the disease mechanisms of CF and for the development and evaluation of new therapeutic strategies. This model will furthermore increase the availability of CF pigs to the scientific community.

Quantitative real time polymerase chain reaction assays for the sensitive detection of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely understood. Sensitive and quantitative DNA detection methods are needed to determine whether serologically positive animals are infectious and to examine the role of vectors (e.g. haematophagous insects) in the transmission of the parasite. To this end, we established two different 5'-nuclease quantitative assays to detect B. besnoiti infection in cattle and to estimate the parasite load in samples (BbRT1 and BbRT2). These PCRs are based on the sequence of the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene. Tests with serial dilutions of B. besnoiti genomic DNA in a buffer containing 100 ng/µl bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification was possible in samples containing ≥1 pg B. besnoiti genomic DNA with a coefficient of variation of ≤ 2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used, 90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings, were significantly correlated. The specificity of the PCRs was confirmed using genomic DNA from related parasites, including genomic DNA of Besnoitia spp., Neospora caninum, Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Isospora spp., Sarcocystis spp., Eimeria bovis, Cryptosporidium parvum, and Trypanosoma brucei brucei. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis.

Evaluation of a commercial ELISA for the specific detection of antibodies against Besnoitia besnoiti.

Bovine besnoitiosis is an economically important disease in cattle caused by the protozoan parasite Besnoitia besnoiti, which occurs endemically in many countries of Africa and Asia and is spreading in Europe. Serological identification of subclinically infected cattle is important to avoid the introduction of infected animals into naive herds. Here we determine the sensitivity and specificity of the PrioCHECK(®) Besnoitia Ab, a serological test recently introduced into the European market. Analytical specificity was examined using sera from animals experimentally infected with parasites related to B. besnoiti (n=27). Three animals experimentally infected with Neospora caninum or Toxoplasma gondii showed inconclusive reactions in the ELISA (percent positivity relative to the positive control [PP] 10% ≤ 20%) while all other sera reacted negative (PP<10%). An estimate of the diagnostic specificity was obtained by analysing field sera from bovine herds without besnoitiosis but with abortion problems associated to N. caninum (n=403). The analysis revealed a specificity of 94.3% or 96.8% depending on the applied cut-off (PP 10% or 20%, respectively). Sensitivity was assessed with sera from 110 animals of a herd in Germany where clinical bovine besnoitiosis was first diagnosed in September 2008. A positive serological reference standard was defined regarding sera from animals as reference positive, if these animals had tested positive in at least two of a panel of three other serological tests (two different B. besnoiti immunoblots and one immunofluorescence antibody test) on both of two sampling dates, November 2008 and April 2009. A diagnostic sensitivity of 91.8% or 75.5% was determined for sera collected in November 2008 and a sensitivity of 82.7% or 50% for sera collected in April 2009 (cut-off PP 10% or PP 20%, respectively). The marked drop in sensitivity from November 2008 to April 2009 was predominantly observed in reference-positive cattle without clinical signs. We conclude that PrioCHECK(®) Besnoitia Ab is a valuable diagnostic tool to detect clinically infected animals. Thus it may be used to support control measures, e.g., for the separation of infected animals from the remaining herd to avoid a further transmission of the infection within the herd.

Comparative evaluation of immunofluorescent antibody and new immunoblot tests for the specific detection of antibodies against Besnoitia besnoiti tachyzoites and bradyzoites in bovine sera.

Besnoitia besnoiti, an apicomplexan parasite causes economically important disease in cattle in many countries of Africa and Asia is re-emerging in Europe. Serological identification of infected cattle is important because introduction of these animals into naive herds seems to play a major role in the transmission of the parasite. We report new, simplified immunoblot-based serological tests for the detection of B. besnoiti-specific antibodies. Antigens were used under non-reducing conditions in the immunoblots, because reduction of the antigen with beta-mercaptoethanol diminished the antigenicity in both, tachyzoites and bradyzoites. Ten B. besnoiti tachyzoite and ten bradyzoite antigens of 15-45 kDa molecular weight were recognized by B. besnoiti infected cattle, but not or only weakly detected by cattle infected with related protozoan parasites, Neospora caninum, Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, or Sarcocystis hirsuta. The sensitivity and specificity of B. besnoiti immunoblots were determined with sera from 62 German cattle with clinically confirmed besnoitiosis and 404 sera from unexposed German cattle including 214 sera from animals with a N. caninum-specific antibody response. Using a new scoring system, the highest specificity (100%) and sensitivity (90%) of the immunoblots were observed when reactivity to at least four of the ten selected tachyzoite or bradyzoite antigens was considered as positive. When a cut-off based on this scoring system was applied to both the tachyzoite- and the bradyzoite-based immunoblots, there was an almost perfect agreement with the indirect fluorescent antibody test with a titre of 200 as the positive cut-off. We identified and partially characterized 10 tachyzoite and 10 bradyzoite B. besnoiti antigens which may help to develop new specific and sensitive serological tests based on individual antigens and in the identification of possible vaccine candidates.