Interdisciplinary studies on Coxiella burnetii: From molecular to cellular, to host, to one health research.

The Q-GAPS (Q fever GermAn interdisciplinary Program for reSearch) consortium was launched in 2017 as a German consortium of more than 20 scientists with exceptional expertise, competence, and substantial knowledge in the field of the Q fever pathogen Coxiella (C.) burnetii. C. burnetii exemplifies as a zoonotic pathogen the challenges of zoonotic disease control and prophylaxis in human, animal, and environmental settings in a One Health approach. An interdisciplinary approach to studying the pathogen is essential to address unresolved questions about the epidemiology, immunology, pathogenesis, surveillance, and control of C. burnetii. In more than five years, Q-GAPS has provided new insights into pathogenicity and interaction with host defense mechanisms. The consortium has also investigated vaccine efficacy and application in animal reservoirs and identified expanded phenotypic and genotypic characteristics of C. burnetii and their epidemiological significance. In addition, conceptual principles for controlling, surveilling, and preventing zoonotic Q fever infections were developed and prepared for specific target groups. All findings have been continuously integrated into a Web-based, interactive, freely accessible knowledge and information platform (www.q-gaps.de), which also contains Q fever guidelines to support public health institutions in controlling and preventing Q fever. In this review, we will summarize our results and show an example of how an interdisciplinary consortium provides knowledge and better tools to control a zoonotic pathogen at the national level.

Holoprosencephalia, hypoplasia of corpus callosum and cerebral heterotopia in a male belted Galloway heifer with adipsia.

BACKGROUND: Specialized neurons in the diencephalon detect blood hypernatremia in dehydrated animals. These neurons are connected with the pituitary gland, subsequently producing antidiuretic hormone to reabsorb water from urine in the kidneys, and to the forebrain to generate thirst and trigger drinking behavior. CASE PRESENTATION: This is the first case report describing clinical findings, magnetic resonance imaging (MRI) and necropsy results of a Belted Galloway heifer with severe clinical signs of dehydration and hypernatremia, but concurrent adipsia and isosthenuria. Due to insufficient recovery with symptomatic treatment, owners elected euthanasia. Postmortem MRI and necropsy revealed a complex forebrain malformation: mild abnormal gyrification of the forebrain cortex, lobar holoprosencephaly, and corpus callosum hypoplasia. The affected brain structures are well known to be involved in osmoregulation and generation of thirst in dogs, humans and rodents. CONCLUSIONS: Complex forebrain malformation can be involved in the pathogenesis of hypernatremia and adipsia in bovines.

Calcinosis in a roe deer fawn (Capreolus capreolus) in northern Germany.

BACKGROUND: Calcinosis has been reported for a broad range of different animals. Causes for calcinosis include metabolic disorders due to kidney failure, intoxication with calcinogenic plants, or iatrogenic overdose of vitamin D. Especially young animals seem to be very susceptible to developing calcinosis. Currently, however, there is a lack of information on calcinosis in wildlife. CASE PRESENTATION: The following case report describes a roe deer fawn admitted to a clinic due to general weakness and myiasis. Plasma levels for creatinine, urea and phosphate were highly elevated, whereas the total calcium level was decreased. Necropsy revealed calcinosis due to calcification in many organs. The reason for calcinosis in this particular case might be kidney failure. Plasma samples from other hunted roe deer fawns also showed high phosphate levels. CONCLUSIONS: Roe deer fawns might be susceptible to calcinosis due to high plasma phosphate, which could be a result of kidney failure or different feed. Further research into calcium and phosphate homeostasis in roe deer is necessary.

Blood leukocyte composition and function in periparturient ewes kept on different dietary magnesium supply.

BACKGROUND: Transition period (TP) is characterised by physiological and metabolic changes contributing to immunodysregulation. Since knowledge about this period in sheep is scarce, we analysed changes in selected immune variables during the TP in ewes and whether dietary magnesium (Mg) supplementation could modulate these immune variables. Pregnant ewes (2nd and 3rd lactation) were divided into a control group (CONT, n = 9) and a Mg group (MAG, n = 10) supplemented with Mg oxide resulting in a daily Mg intake of approximately 0.30 and 0.38% (MAG) of dry matter during ante- (a.p.) and post-partum (p.p.) periods, respectively. Blood samples were collected between days (d) 30 a.p. and d 30 p.p.. Whole blood neutrophil phagocytic activity, monocyte subset (classical cM, intermediate intM, non-classical ncM) composition and the proliferative capacity of lymphocytes were determined flow cytometrically. At d 14 a.p., all ewes were vaccinated against Mycobacterium avium subsp. paratuberculosis (MAP). RESULTS: Both groups showed a sharp increase in the total leukocyte counts (TLC) and neutrophil counts (P < 0.0001), at d 1 p.p., while, monocytes and their subpopulations displayed the highest values at d 30 p.p. (P ≤ 0.05). At d 1 p.p. the neutrophil phagocytic activity was higher (P < 0.05) in MAG ewes. Throughout the TP, the proliferative response of CD4+ cells was significantly higher in the MAG group (P < 0.05). Ewes in both groups responded with an increase in the TLC, neutrophil numbers (P ≤ 0.05) and ncM (P < 0.001) 24 h post vaccination, whereas monocytes and cM dropped in numbers (P ≤ 0.05). Numbers of intM only increased in MAG ewes (P < 0.05), whereas lymphocyte numbers decreased (P < 0.01). Mg supplementation did not affect the significant increase in MAP-specific antibodies at d 7 and 21 post vaccination. Total Mg and Ca serum levels did not show any differences between the two groups. CONCLUSION: Whereas TP-associated fluctuations in blood leukocyte numbers are not influenced by Mg supplementation, neutrophil phagocytic activity, the proliferative capacity of CD4+ cells and the cellular response within 24 h after a vaccination are subject to modulation.

First report on nasal myiasis in an alpaca "Vicugna pacos" - a case report.

BACKGROUND: An infestation of bot fly larvae causes myiasis which is known to cause respiratory symptoms in ruminants. There are reports of bot fly larvae in llamas, but to our knowledge there are no previous reports of nasal myiasis due to bot flies in alpacas ("Vicugna pacos"). CASE PRESENTATION: The following case report describes a neutered male alpaca showing sneezing and mild nasal discharge. Endoscopic examination of the upper respiratory tract revealed bot fly larvae in one nostril. After treatment with doramectin, there was no evidence of living bot fly larvae visible in the nostril. CONCLUSION: Bot fly larvae should be considered as a potential cause of respiratory symptoms in alpacas. In the present case, a treatment with doramectin was successful.

Host-pathogen interplay at primary infection sites in pigs challenged with Actinobacillus pleuropneumoniae.

BACKGROUND: Actinobacillus (A.) pleuropneumoniae is the causative agent of porcine pleuropneumonia and causes significant losses in the pig industry worldwide. Early host immune response is crucial for further progression of the disease. A. pleuropneumoniae is either rapidly eliminated by the immune system or switches to a long-term persistent form. To gain insight into the host-pathogen interaction during the early stages of infection, pigs were inoculated intratracheally with A. pleuropneumoniae serotype 2 and humanely euthanized eight hours after infection. Gene expression studies of inflammatory cytokines and the acute phase proteins haptoglobin, serum amyloid A and C-reactive protein were carried out by RT-qPCR from the lung, liver, tonsils and salivary gland. In addition, the concentration of cytokines and acute phase proteins were measured by quantitative immunoassays in bronchoalveolar lavage fluid, serum and saliva. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. RESULTS: Significant cytokine and acute phase protein gene expression was detected in the lung and the salivary gland however this was not observed in the tonsils. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter investigations, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. The bacteria isolated from the upper and lower respiratory tract showed distinct IR spectral patterns reflecting the organ-specific acute phase response of the host. CONCLUSIONS: In summary, this study implies a metabolic adaptation of A. pleuropneumoniae to the porcine upper respiratory tract already during early infection, which might indicate a first step towards the persistence of A. pleuropneumoniae. Not only in lung, but also in the salivary gland an increased inflammatory gene expression was detectable during the acute stage of infection.

Characterization of surfactant alterations in pigs infected with Actinobacillus pleuropneumoniae.

PURPOSE/AIM OF THE STUDY: Surfactant, a surface active complex of phospholipids and proteins located at the inner surface of alveoli and small conducting airways is necessary for breathing. Bacterial respiratory tract infections frequently lead to surfactant alterations and to an increase in surface tension. Pigs, often used in experimental lung research, could suffer from severe pleuropneumonia, a highly contagious disease often characterized by sudden onset, short clinical course, high morbidity, and high mortality. It is caused by the gram-negative bacterium Actinobacillus pleuropneumoniae (A.pp.). This study tests the hypothesis that also in the subacute stage pathomorphological lung alterations are accompanied with increased inactive surfactant components. Clinical lung scores, functional and ultrastructural analysis of porcine surfactant were performed in pigs before infection and in the subacute state of infection. Clinical signs were determined using inter alia different subscores. Surfactant was isolated from the BALF for functional and quantitative ultrastructural studies. RESULTS: In the subacute stage clinical, ultrosonographic and radiographic scores as well as the overall Respiratory Health Score showed significantly higher values than before infection. However, surfactant surprisingly contained more active surfactant subtypes and significantly less inactive subtypes such as unilamellar vesicles. The quantity of multilamellar vesicles with unclear function did not differ. The minimal surface tension of surfactant before and after infection was comparable. CONCLUSIONS: Thus, in spite of continued severe lung tissue alterations the surfactant system show signs of recovery. This may be the result of an effective adaption to inflammatory lung disorders caused by swine-specific pathogens.

Molecular analysis of Coxiella burnetii in Germany reveals evolution of unique clonal clusters.

The causative agent of Q fever, Coxiella burnetii, is a query agent occurring naturally all over the world. We studied 104 German Coxiella burnetii strains/DNA samples obtained between 1969 and 2011 using a 14 microsatellite marker Multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA) technique. We were able to divide our collection into 32 different genotypes clustered into four major groups (A-D). Two of these (A and C) formed predominant clonal complexes that covered 97% of all studied samples. Group C consisted exclusively of cattle-associated isolates/DNA specimens, while group A comprised all other affected species including all sheep-derived strains/DNA samples. Within this second cluster, two major genotypes (A1, A2) were identified. Genotype A2 occurred in strains isolated from ewes in northern and central Germany, whereas genotype A1 was found in most areas of Germany. MLVA analysis of C. burnetii strains from neighbouring countries revealed a close relationship to German strains. We thus hypothesize that there is a western and central European cluster of C. burnetii. We identified predominant genotypes related to relevant host species and geographic regions which is in line with findings of the Dutch Q fever outbreak (2007-2010). Furthermore three of our analyzed German strains are closely related to the Dutch outbreak clone. These findings support the theory of predominant genotypes in the context of regional outbreaks. Our results show that a combination of 8 MLVA markers provides the highest discriminatory power for attributing C. burnetii isolates to genotypes. For future epidemiological studies we propose the use of three MLVA markers for easy and rapid classification of C. burnetii into 4 main clusters.

Anaemia in South American camelids - an overview of clinical and laboratory diagnostics.

South American camelids (SACs) play an increasing role in veterinary care in Europe. Many alpacas or llamas presented to veterinarians suffer from anaemia, regularly with a packed cell volume (PCV) below 0.10 l/l, which is a life-threatening condition for the animals. This review article presents clinical and laboratory diagnostic tools for the diagnosis of anaemia in SACs. Clinical identification of anaemic animals can be performed by assessing the FAMACHA© score and the Body Condition Score (BCS), since anaemia in alpacas and llamas correlates with pale mucous membranes and a lowered BCS. Haematological examination of a blood sample can provide a more differentiated diagnosis of anaemia in SACs. A common finding is regenerative anaemia with an increased number of reticulocytes that is often caused by blood loss due to Haemonchus contortus. Changes in a blood smear from an alpaca or llama with regenerative anaemia may include normoblasts (nucleated red blood cells), anisocytosis, poikilocytosis, polychromasia, Howell-Jolly bodies or basophilic stippling. Furthermore, non-regenerative anaemia, often caused by trace element deficiency or cachexia, can also occur.

Exposure of small ruminants to the Schmallenberg arbovirus in Germany from 2017 to 2018 - animal-specific and flock-management-related risk factors.

The Schmallenberg virus (SBV), an emerging Orthobunyavirus of mainly ruminant hosts, caused a substantial epidemic in European ruminant populations between 2011 and 2013. The pathogen is transmitted by arthropod vectors (Culicoides spp.) and can cause reproductive disorders and severe malformations of the offspring or stillbirth. The present study aimed to assess SBV seroprevalence among German sheep and goats a few years after the first virus detection in the country (November 2011). In addition, an extensive risk factor analysis including host-specific and husbandry-related factors was implemented. Seroprevalence was determined by examining serum samples from 2759 sheep and 446 goats out of a total of 70 flocks across five German federal states. The samples were withdrawn in the period between 2017 and 2018. Using a commercial competitive ELISA, antibodies against SBV were detected in all 70 investigated flocks. A percentage of 60.1 % (1657/2759) of the sheep and 40.4 % (180/446) of the goat sera contained SBV antibodies. Generalized linear mixed modeling revealed significant effects of host species (sheep > goats), age (old > young) and sex (female > male) on SBV seroprevalence. For both species, also the farming purpose, and for goats, ectoparasite treatment and the presence of cattle on the farm played a role in terms of risk for SBV exposure. The observations from this study still emphasize a wide distribution of the pathogen in Germany. Nevertheless, the observed seroprevalence might not be sufficient to achieve effective herd immunity. Pinpointing risk factors identified susceptible populations for targeted vaccination programs to reduce potential animal losses caused by SBV.

Body condition scoring in alpacas (Vicugna pacos) and llamas (Lama glama) - a scoping review.

Assessment of the body condition score (BCS) is an important tool to check the nutritional status of an animal, and thus can provide important information in herd management, but also in the veterinary examination of individual animals. In dairy farming, BCS has been recognized as an important parameter for a long time already, but over the years body condition scoring has also become more important for other species. Especially in South American camelids (SACs: alpacas and llamas), whose dense coat can hide a poor nutritional status for a long time, regular assessment of the BCS is recommended by different authors and organizations. To date, there are several instructions for the assessment of the BCS in SACs, which, however, differ significantly in some points. For a consistent approach, a total of 35 instructions for the BCS in SACs were compared in a scoping review. The spine, especially the lumbar vertebrae as well as the ribs were identified as the two main body sites recommended for assessing the BCS. Furthermore, the area between the front and rear leg and the pelvis were mentioned frequently; however, these body sites were discussed controversially. The paralumbar fossa, shoulder, and udder were only mentioned sporadically. Among the various scales, the 1-5 scale was mentioned most frequently, with the optimal BCS being 3. Nonetheless, physiological fluctuation due to breed, age, sex, pregnancy, and lactation have to be considered. The BCS should be assessed each time the animals are handled, but at least monthly. In contrast to body weight, the BCS reflects the nutritional status of an animal even after a single examination. The review showed that much of the available information on the assessment of BCS in SACs is based on practical experience rather than scientific evidence. Indeed, some of the assumptions made there might still have to be verified or modified.

Hematologic parameters in female alpacas during age progression: a retrospective study.

Alpacas, like all camelids, have elliptical red blood cells (RBCs) in contrast to other mammals. This particular shape is important for increased osmotic resistance and stability. Age-related changes in the RBC count are known in other species, with alterations in both red and white blood cells being described. In alpacas, there are few data on age-related changes, and only a comparison of crias with adult animals. We characterized age-related hematologic changes in a study of 21 female alpacas from a research herd. A total of 87 records of clinically healthy alpacas of different ages were statistically analyzed retrospectively from the hematologic records over a nine-year period. Significant positive correlations of age with hemoglobin (Hb), HCT, MCV, MCH, neutrophils, platelet-to-lymphocyte ratio (PLR), and neutrophil-to-lymphocyte ratio (NLR) were found as well as significant negative correlations of age with lymphocytes in addition to lymphocyte-to-monocyte ratio (LMR). A paired comparison of eight older animals in the herd at three different ages also showed significant differences in the parameters Hb, HCT, MCV, MCH, MCHC, lymphocytes, eosinophils and neutrophils. Similar changes in hematologic parameters have been reported in other species and should be taken into account when interpreting hematologic results in alpacas.

Detection of Coxiella burnetii in the mammary gland of a dairy goat.

The zoonotic bacterium Coxiella (C.) burnetii can be excreted by infected goats through birth products and milk. The detection of C. burnetii DNA in the mammary gland tissue of infected dairy goats and intermittent milk shedders has been reported, but confirmation of C. burnetii bacteria in the udder remained pending. The pathogen caused abortions in a 152-head dairy goat herd, resulting in the vaccination against C. burnetii of the entire herd with annual boosters. To monitor the C. burnetii shedding at herd level, monthly bulk tank milk (BTM) samples were analyzed using PCR (IS1111). Despite vaccination, C. burnetii DNA was detected in BTM samples within the first 16 months of the study. Therefore, individual milk samples were tested on four different occasions several months apart to identify potential intermittent milk shedders. Only one goat (#67455) tested positive three times. This goat was necropsied to investigate the presence of C. burnetii in the udder and other organs. PCR detected C. burnetii DNA solely in both mammary glands and the left teat cistern. Immunohistological examination identified C. burnetii antigen in mammary gland tissue, confirmed by the detection of C. burnetii bacteria in the mammary epithelial cells using fluorescence in situ hybridization. The removal of goat #67455 led to negative BTM samples until the end of the study. The findings demonstrate the occurrence of C. burnetii in the mammary gland of a naturally infected and vaccinated goat. The presence possibly contributed to intermittent milk shedding of goat #67455, and the mammary gland tissue may serve as a replicative niche for C. burnetii.

Rhododendron poisoning in alpacas (Vicugna pacos) in Northern Germany.

Poisoning is often suspected to be the origin of disease in South American camelids (SACs) by owners, but only in a few cases this assumption can be confirmed. In small ruminants, rhododendron poisoning is a common emergency for livestock veterinarians. However, this condition has rarely been reported in SACs so far. This paper provides information regarding clinical findings, hematology, clinical chemistry, and treatment of four alpacas after presumed intake of rhododendron leaves including pathological findings of one of the animals. Rhododendron leaves contain grayanatoxins that lead to hyperpolarization of excitable cells. Clinical signs that were observed in the presented alpacas comprised: salivation, dehydration, decreased motility of compartment 1, uncoordinated regurgitation, and cardiac arrhythmia. Clinical chemistry revealed that rhododendron poisoning was associated with metabolic acidosis and azotaemia, hyponatremia and hyperkalemia. Most striking macroscopic and histopathological findings included gastric ulceration, and renal infarcts along with inflammatory changes. Leaves of Rhododendron spp. were identified in the forestomach content of this animal. Affected animals were treated symptomatically as there is no specific antidote in rhododendron poisoning. This included parenteral rehydration, treatment of metabolic acidosis (infusion of sodium bicarbonate solution), and oral administration of activated charcoal to bind potential toxins. In addition, antibiotic treatment might be necessary to prevent aspiration pneumonia in case of uncoordinated regurgitation. Of the four animals, the worst affected alpaca was euthanized, one had minimal signs and two responded to supportive care and recovered. In conclusion, rhododendron poisoning might be fatal for alpacas in individual cases and therefore rhododendron bushes should not be placed in the habitat of SACs.

[Choanal atresia in an alpaca cria (Vicugna pacos)]. / Choanenatresie bei einem Alpakacria (Vicugna pacos).

After birth, an alpaca cria was noticed to exhibit weakness and respiratory distress, particularly when attempting to suckle milk from the dam's udder. Clinical findings indicated the presence of bilateral choanal atresia and the animal was subsequently euthanised. A computed tomography scan and a pathological examination were performed to describe the malformation in detail. Choanal atresia is a common malformation in neonatal South American camelids, surgical treatment is not recommended. This case report provides an overview of the clinical findings as well as the results of imaging and pathologic examinations and may help to raise awareness of this malformation for early recognition.

Control of Coxiella burnetii shedding in a dairy goat herd by annual offspring vaccination.

A Coxiella burnetii vaccination program, targeting only doelings, was introduced on a German goat farm to curb bacterial shedding. In 2018, adults were vaccinated with a C. burnetii Phase I vaccine at three-weeks apart following pathogen diagnosis, with a booster administered six months later due to sustained high shedding. From 2018 to 2021, doelings received two vaccine doses without any further boosters. To assess the program's efficacy, vaginal swabs from up to 40 animals per age group were collected during kidding seasons from 2019 to 2022. Bulk tank milk (BTM) samples were gathered monthly from January 2018 to October 2022 to monitor herd-level shedding. Real-time PCR analysis determined genome equivalents in all three sample types. Serum samples were taken before the initial immunization and during the post-kidding season from up to 40 goats per age group annually from 2018 to 2022. Phase-specific ELISAs determined IgG Phase I and Phase II antibodies. Additionally, two serum samples per age group from 2022 were analyzed using a neutralization assay. A few goats continued shedding small quantities during subsequent kidding seasons. Although positive BTM samples decreased, they displayed an undulating trend. Most age groups exhibited robust IgG Phase I responses and lower IgG Phase II levels post immunization. Mean IgG levels remained elevated until the study ended compared to pre-vaccination levels in most age groups. Additionally, neutralizing antibodies were present regardless of IgG response. Overall, double vaccination induced lasting antibody levels, but did not entirely prevent C. burnetii shedding. The resilience of the observed humoral immune activity requires further investigation.

Relationships between body condition score, body weight and body measurements in alpacas.

BACKGROUND: The nutritional status in alpacas is often masked by their dense fibre coat. Its assessment is commonly approached by different body condition scores (BCS) that rely on manual palpation of defined anatomical regions. However, BCS is an important diagnostic tool to aid recognition of diseased South American camelids (SACs) and low BCS has been associated with conditions like anaemia and neutrophilia. For dose-dependent veterinary treatment, body weight (BW), that should be as accurate as possible, is required. As on-site weighing with scales is often not possible, BW can mostly only be roughly estimated. To date, it remains unclear whether BCS in alpacas provides reliable information on BW or the ratios of BW to body length commonly known as Body Mass Index (BMI) or Ponderal Index (PI). Equations to estimate BW based on body measurements are available in the literature. Nonetheless, respective equations were developed in growing alpacas or adult llamas and BCS was not included. RESULTS: To compare six different BCS approaches and to examine the relationship between BCS and BW, body measurements and BCS scores were recorded in a herd of 105 alpacas. The examined BCS approaches showed significant (p < 0.05) but poor to moderate positive correlations to BW, BMI or PI. A solely visual inspection of BCS, in contrast, was not correlated with BW, BMI or PI. Equations previously developed in other studies provided an accurate estimation of BW. Multiple linear regression showed that the accuracy in predicting BW could be further increased by adding BCS data and sex. CONCLUSION: Our observations indicate that most selected BCS approaches are not only important measures of nutritional status but can also be used to create more accurate models for BW calculation in alpacas. The study also supports the claim that a purely visual inspection of alpacas is not an adequate method to evaluate the nutritional status of these animals.

First Evidence of Leishmania infantum Antibodies in Sheep (Ovis aries) from Southern Germany.

In Europe, Leishmania infantum is the most prevalent Leishmania species, and this protozoan is transmitted by phlebotomine sandflies. A recent publication has shown that sheep harbor L. infantum antibodies. This raises questions about the epidemiological role of small ruminants. Therefore, sera from small ruminants located in two southern German federal states, Baden-Wuerttemberg (BW) and Bavaria (BAV), were analyzed with an ELISA to determine the presence of L. infantum antibodies. The species, sex and age (gimmer vs. ewe) were recorded, and a univariate analysis was conducted to determine possible associations. In total, seven sheep flocks (274 sheep/10 goats) from BW and seven sheep flocks (277 sheep/78 goats) from BAV were examined. In BW, four sheep from three flocks tested positive for L. infantum antibodies. In BAV, the same number of positive sheep were detected but in four flocks. The total seropositivity rate in sheep was 1.45%. All goats tested negative. No significant association (p > 0.05) was detected between Leishmania seropositivity and the variables evaluated. Our study reveals the exposure of sheep to L. infantum in a non-endemic area. Further investigation is needed to determine whether sheep can be used as sentinels to identify new phlebotomine habitats and Leishmania risk areas.

[Diaphragmatic hernia in a female alpaca (Vicugna pacos)]. / Zwerchfellhernie bei einer Alpakastute (Vicugna pacos).

A 3-year-old pregnant female alpaca was noticed due to increased lying and decreased feed intake after shearing. Twelve days after shearing, she aborted an almost completely developed cria. After another two days, the alpaca was recumbent and subsequently died during transport to the clinic. A dissection of the carcass revealed a diaphragmatic hernia. Diaphragmatic hernias have been described in many different species, diagnosis can be made by radiography or ultrasound. Depending on the extent, surgical therapy may be successful.

Humoral and cellular immune responses in sheep following administration of different doses of an inactivated phase I vaccine against Coxiella burnetii.

An inactivated Coxiella burnetii Phase I (PhI) vaccine (Coxevac®) is licensed in several European countries for goats and cattle to prevent coxiellosis. The vaccine is also applied to sheep, although detailed information about the ovine immune response and vaccine dose is missing. Eighteen gimmers from a C. burnetii unsuspected flock were randomly divided into three groups of six. Group 1 (Cox1) and 2 (Cox2) were vaccinated twice with 1 ml and 2 ml Coxevac®, respectively, three weeks apart (primary vaccination). The same procedure was applied with Cox3 (2 ml sodium chloride, control group). A third injection (booster) was performed after nine months. Potential side effects were determined by measuring the rectal body temperature and skin thickness at the injection site. Blood samples were collected to detect phase-specific IgM and IgG antibodies and interferon-É£ (IFN-É£) release by immunofluorescence assay and ELISAs, respectively. Moreover, a cell infection neutralization assay determined the appearance of neutralizing sera. Body temperatures increased for one day post vaccination, and the skin swelled only slightly. Regardless of the vaccine volume, immunized sheep reacted first with an IgM and IgG PhII response. Ten weeks after the primary vaccination, IgG PhI antibodies predominated. Boosting eight months after primary vaccination resulted in a robust IgG PhI increase and strong IFN-É£ response. In the vaccinated animals, the neutralizing effect is more widespread after the administration of 1 ml than after the treatment with 2 ml. In summary, differences between 1 and 2 ml Coxevac® are minor, and a vaccine volume of 1 ml seems to be sufficient. A booster after the primary vaccination is apparently necessary to stimulate the cell-mediated immune response in naïve sheep.