Application of Methods to Assess Animal Welfare and Suffering Caused by Infectious Diseases in Cattle and Swine Populations.

Control of infectious diseases in livestock has often been motivated by food safety concerns and the economic impact on livestock production. However, diseases may also affect animal welfare. We present an approach to quantify the effect of five infectious diseases on animal welfare in cattle (three diseases) and pigs (two diseases). We grouped clinical manifestations that often occur together into lists of clinical entities for each disease based on literature reviews, and subsequently estimated "suffering scores" based on an aggregation of duration, frequency, and severity. The duration and severity were based on literature reviews and expert knowledge elicitation, while frequency was based mainly on estimates from the literature. The resulting suffering scores were compared to scores from common welfare hazards found under Danish conditions. Most notably, the suffering scores for cattle diseases were ranked as: bovine viral diarrhoea and infection with Mycobacterium avium subsp. paratuberculosis > infectious bovine rhinotracheitis, and for pigs as: porcine respiratory and reproductive syndrome > Aujeszky's disease. The approach has limitations due to the limited data available in literature and uncertainties associated with expert knowledge, but it can provide decision makers with a tool to quantify the impact of infections on animal welfare given these uncertainties.

Maternally contributed Nlrp9b expressed in human and mouse ovarian follicles contributes to early murine preimplantation development.

PURPOSE: The aim of the study is to investigate presence and role of the gene encoding the maternally contributed nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) in human and mouse ovaries, respectively, and in preimplantation mouse embryo development by knocking down Nlrp9b. METHODS: Expression levels of NLRP9 mRNA in human follicles were extracted from RNA sequencing data from previous studies. In this study, we performed a qPCR analysis of Nlpr9b mRNA in mouse oocytes and found it present. Intracellular ovarian distribution of NLRP9B protein was accomplished using immunohistochemistry. The distribution of NLRP9B was explored using a reporter gene approach, fusing NLRP9B to green fluorescent protein and microinjection of in vitro-generated mRNA. Nlrp9b mRNA function was knocked down by microinjection of short interference (si) RNA targeting Nlrp9b, into mouse pronuclear zygotes. Knockdown of the Nlrp9b mRNA transcript was confirmed by qPCR. RESULT: We found that the human NLRP9 gene and its corresponding protein are highly expressed in human primordial and primary follicles. The NLRP9B protein is localized to the cytoplasm in the blastomeres of a 2-cell embryo in mice. SiRNA-mediated knockdown of Nlrp9b caused rapid elimination of endogenous Nlrp9b mRNA and premature embryo arrest at the 2- to 4-cell stages compared with that of the siRNA-scrambled control group. CONCLUSIONS: These results suggest that mouse Nlrp9b, as a maternal effect gene, could contribute to mouse preimplantation embryo development. It remains to investigate whether NLRP9 have a crucial role in human preimplantation embryo and infertility.

Comparing the treatment effect of narrow spectrum antimicrobial, probiotic and fluid with amoxicillin in mink kits (Neovison vison) with pre-weaning diarrhea.

Pre-weaning diarrhea in mink kits (PWD), also known as "sticky kits" is a multifactorial syndrome of considerable concern in the mink production. Evidence based treatment protocols are not available, and treatment is therefore empirical and often based on the use of antimicrobials. The purpose of the study was to test the effect of 3 alternative treatments to a standard antibiotic treatment, to characterize the study groups microbiologically, and finally to compare the intestinal microbiota of the different treatment groups at the age of 42 days. In total, 226 one to three week old mink kits with PWD from 36 litters were treated with either 1) Lactobacillus reuteri, 2) benzylpenicillin, 3) Ringer lactate or 4) amoxicillin (controls). Effects of the treatments were measured as weight gain from day 0 to day 15 and mortality. Multivariable linear mixed model regression showed no significant difference in weight gain between probiotic-, penicillin or fluid-treated mink kits and the amoxicillin treated controls. There was also no significant difference in mortality risk between the treatment groups. Bacterial culture and next generation sequencing of the viral contents showed that the study groups were uniform with a high frequency of Staphylococcus intermedius group (SIG) bacteria, Escherichia coli, Enterococcus hirae, Mamastrovirus and Sapovirus which were representative for mink kits with PWD. 16S sequencing results of the bacterial microbiota, when the kits were 42 days old were dominated by clostridia in all groups and showed no clear differences in the bacterial composition between the different treatment groups.

Transfer of amoxicillin to suckling mink (Neovison vison) kits via the milk from dams treated orally or intra-muscularly.

Treatment of mink kits with pre-weaning diarrhea (PWD) can be time-consuming and expensive for the farmer, and the efficacy of the treatment procedure may be questioned. Evidence-based treatment protocols for application on affected animals at farms with outbreaks of PWD are lacking. In Denmark, the dams are sometimes treated with amoxicillin, however, it is unknown if it is passed on to the mink kits via the milk. The aim of the present study was to investigate if amoxicillin is transferred via the milk to the kits after oral (PO) and intramuscular (IM) treatment, respectively, of the dam. Moreover, we estimated the concentrations of amoxicillin continuously in serum from the kits up to 8 h after administration. The concentration of amoxicillin was not affected by the route of administration (P = .64) and serum reached the highest level after 8 h (34 ng/mL, CI95% = [24.3-47.7]). The serum concentrations of amoxicillin in the mink kits achieved within 8 h were judged too low to exert antimicrobial impact on relevant bacterial species.

Dam characteristics associated with pre-weaning diarrhea in mink (Neovison vison).

BACKGROUND: Pre-weaning diarrhea (PWD) in mink, also known as "sticky kits", is a frequently occurring syndrome in suckling mink kits on commercial mink farms. Outbreaks of PWD result in weakened kits, increased mortality and reduced growth and welfare as well as considerable economic losses for the farmers. The syndrome is regarded as multifactorial with a complex etiology, and studies have focused on associations with environment, management and dam characteristics. The present study was conducted from May to June 2015 and included 70 dams with mink litters with and without PWD. The aims were to examine associations between PWD and mastitis (bacterial infection and histological signs of inflammation or other lesions in the mammary gland), and to examine associations between PWD and other dam-related characteristics (age, litter size, body mass index, and weight and number of active mammary glands of the dam). RESULTS: Using multivariable mixed logistic regression analyses with farm id as a random intercept, we found that the odds for PWD in the litter were significantly higher in 1 year old dams versus > 1 year old (OR = 13.3, CI 2.0-90.2, P = 0.01), higher if litter size observed after birth was > 5 kits versus ≤ 5 kits (OR = 16.5, CI 2.2-123.7, P = 0.01), higher if the number of active mammary glands per kit was ≤ 1.5 versus > 1.5 glands per kit (OR = 6.5, CI 1.2-36.0), P = 0.03), and higher in farms with high prevalence of PWD versus low prevalence (OR = 16.8, CI 2.9-97.6, P = 0.002). There were no significant associations between PWD and bacterial infection, histological signs of inflammation or other lesions of the mammary gland, body mass index or weight of mammary gland per kit. CONCLUSION: Pre-weaning diarrhea had a statistically significant association with age of the dam, litter size and the number of active mammary glands per kit. However, PWD was not associated with mastitis, body mass index and weight of mammary gland tissue per kit.

Investigation of the viral and bacterial microbiota in intestinal samples from mink (Neovison vison) with pre-weaning diarrhea syndrome using next generation sequencing.

Pre-weaning diarrhea (PWD) in mink kits is a common multifactorial syndrome on commercial mink farms. Several potential pathogens such as astroviruses, caliciviruses, Escherichia coli and Staphylococcus delphini have been studied, but the etiology of the syndrome seems complex. In pooled samples from 38 diarrheic and 42 non-diarrheic litters, each comprising of intestinal contents from 2-3 mink kits from the same litter, the bacterial populations were studied using Illumina Next Generation Sequencing technology and targeted 16S amplicon sequencing. In addition, we used deep sequencing to determine and compare the viral intestinal content in 31 healthy non-diarrheic and 30 diarrheic pooled samples (2-3 mink kits from the same litter per pool). The results showed high variations in composition of the bacterial species between the pools. Enterococci, staphylococci and streptococci dominated in both diarrheic and non-diarrheic pools. However, enterococci accounted for 70% of the reads in the diarrheic group compared to 50% in the non-diarrheic group and this increase was at the expense of staphylococci and streptococci which together accounted for 45% and 17% of the reads in the non-diarrheic and diarrheic group, respectively. Moreover, in the diarrheic pools there were more reads assigned to Clostridia, Escherichia-Shigella and Enterobacter compared to the non-diarrheic pools. The taxonomically categorized sequences from the virome showed that the most prevalent viruses in all pools were caliciviruses and mamastroviruses (almost exclusively type 10). However, the numbers of reads assigned to caliciviruses were almost 3 times higher in the diarrheic pools compared the non-diarrheic pools and Sapporo-like caliciviruses were more abundant than the Norwalk-like caliciviruses. The results from this study have contributed to the insight into the changes in the intestinal microbiota associated with the PWD syndrome of mink.

Mink (Neovison vison) kits with pre-weaning diarrhea have elevated serum amyloid A levels and intestinal pathomorphological similarities with New Neonatal Porcine Diarrhea Syndrome.

BACKGROUND: Pre-weaning diarrhea (PWD) is a syndrome affecting farm-raised neonatal mink kits. Apart from diarrhea it causes greasy skin exudation, dehydration, and distressed behavior and can ultimately lead to death. No specific causative agents have been identified and the syndrome is regarded as multifactorial. The aim of the present study was to investigate a possible inflammatory state in mink kits with PWD, as indicated by raised serum concentrations of the acute phase protein serum amyloid A (SAA) and by changes in intestinal pathomorphology and intestinal contents of bacteria. Samples collected from 20 diarrheic mink kits with PWD and 20 age-matched non-diarrheic control mink kits from two commercial Danish farms during the pre-weaning period (April-May) in 2016 were analyzed. RESULTS: Concentrations of SAA in serum samples from mink kits with PWD were significantly higher (up to 1000-fold) compared to non-diarrheic control mink kits. Significant features of enterocytic vacuolization, atrophy and fusion of villi in jejunum and mucosal atrophy of the colon of kits with PWD were found. Moreover, attachment of coccoid bacteria to enterocytes was more often found in kits suffering from PWD, while intra-cytoplasmic eosinophil bodies were more frequently observed in control kits. Cellular infiltrations with mononuclear and neutrophil leukocytes were not associated with disease status. Bacteria from the Staphylococcus intermedius group, such as Staphylococcus delphini, were more frequently cultivated from control mink kits, whereas Enterococcus spp. dominated in mink kits with PWD. Escherichia coli was cultivated from both control and mink kits with PWD, but with a higher frequency from mink kits with PWD. CONCLUSION: A significant increase in circulating concentrations of SAA was found in PWD affected mink kits from 6 to 23 days old compared to controls. The histopathological changes in PWD mink kits suggest that the type of diarrhea is secretory. Attachment of coccoid bacteria, therefore, might be responsible for an enterotoxic effect causing a loss of balance in movements of ions and water leading to the vacuolization and swelling of the enterocytes. The slight to moderate infiltrations of neutrophils irrespectively of diarrheic status and the attachment of coccoid bacteria to enterocytes are comparable to observations found in piglets suffering from New Neonatal Porcine Diarrhea Syndrome. Mechanisms behind the correlation between increased SAA levels and the observed pathological intestinal features remain obscure.

Risk factors associated with diarrhea in Danish commercial mink (Neovison vison) during the pre-weaning period.

BACKGROUND: Pre-weaning diarrhea in mink, also known as "sticky kits", is a syndrome and outbreaks occur every year on commercial mink farms in all mink producing countries. Morbidity and mortality can be considerable on a farm with huge economic consequences for the farmer as well as compromised welfare for the mink kits. Although efforts have been taken to identify etiologic agents involved in outbreaks, the syndrome is still regarded as multifactorial and recurring problems on the same farms draw attention to management and environmental risk factors. In the pre-weaning period from May to June 2015, a case control study was carried out on 30 Danish mink farms. Data concerning management, biosecurity, hygiene, feed consumption, antibacterial prescription and production efficiency were analyzed. RESULTS: The proportion of 1-year old females, farm size (total number of females), energy supply per female in the late gestation period, and dogs accessing the farm area were significantly associated with being a case farm. Case farms were prescribed almost twice the amount of antibacterials per gestational unit (female and litter) as in control farms. Farmers on case farms spent significantly more time nursing and treating the animals and experienced more females with mastitis compared to farmers on control farms. No significant differences in cleaning practices or hygienic measures between case and control farms were found and there were no differences in drinking water quality, bedding material, composition neither of color types nor in management regarding litter equalization. CONCLUSIONS: Results from this study showed an association between the occurrence of pre-weaning diarrhea on mink farms and parity profile, farm size and feeding intensity in the gestational period. The access of dogs to the farm area was a significant risk factor, but needs further clarification.

Presence of Coxiella burnetii DNA in inflamed bovine cardiac valves.

BACKGROUND: Bacterial endocarditis is a recognised disease in humans and animals. In humans, infection with Coxiella burnetii can cause endocarditis, but this has not been investigated thoroughly in animals. Endocarditis in cattle is a common post-mortem finding in abattoirs and studies have identified Trueperella pyogenes as a major cause. Despite exposure of cattle to C. burnetii, the significance of this particular bacterium for development and progression of endocarditis has not been studied in detail. Cardiac valves of cattle affected with endocarditis (n = 100) were examined by histology, fluorescence in situ hybridization (FISH) and real time quantitative polymerase chain reaction (PCR). Serum was examined for anti-C. burnetii antibodies by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serology revealed that 70% of the cattle were positive for antibodies to C. burnetii, while PCR analysis identified 25% of endocarditis valve samples as being positive. C. burnetii was not detected by FISH, probably due to the low infection levels. Most cattle had chronic valvular vegetative endocarditis with lesions being characterised by a core of fibrous tissue covered by significant amounts of fibrin, sometimes with areas of liquefaction, and with a coagulum covering the surface. In a few cases, including the case with the highest infection level, lesions were characterized by extensive fibrosis and calcification. Histologically, bacteria other than C. burnetii were observed in most cases. CONCLUSIONS: The presence of C. burnetii DNA is relatively common in cattle affected with valvular endocarditis. The role of C. burnetii remains however unknown as lesions did not differ between C. burnetii infected and non-infected cattle and because T. pyogenes-like bacteria were present in the inflamed valves; a bacterium able to induce the observed lesions. Heart valves of normal cattle should be investigated to assess if C. burnetii may be present without preexisting lesions.

Gross and histopathological evaluation of human inflicted bruises in Danish slaughter pigs.

BACKGROUND: Human inflicted bruises in slaughter pigs are hampering animal welfare, are an infringement of the animal protection act, and are a focus of public attention. The aim of the present study was to evaluate the gross appearance of human inflicted bruises in slaughter pigs and to compare the inflammatory changes in two lesions as a basis for estimating the age of lesions in the same pig. Pigs with human inflicted bruises slaughtered at two major slaughterhouses in Denmark from November 2013 to May 2014 were evaluated. After slaughter, the bruises were examined grossly and skin and underlying muscle tissue from two similar but separate bruises (a and b) on each pig were sampled for histology. RESULTS: Skin and muscle tissue from 101 slaughter pigs were subjected to gross evaluation. Eighty-one of these were also subjected to histological evaluation. Most frequently (51 out of 101 pigs, 50 %), bruises had a tram-line pattern due to blunt trauma inflicted with long objects such as sticks. Other bruises reflected the use of tattoo-hammers, plastic paddles, double U profiles and chains. Histological evaluation of two bruises from a pig with multiple lesions was found insufficient to assess the overall age of the lesions as substantial variation in the inflammatory response between bruises was present. CONCLUSIONS: Grossly, the pattern of bruises often reflected the shape of the object used for inflicting the lesions. When determining the age of multiple bruises on a pig more than two lesions should be evaluated histologically.

Anatomical distribution and gross pathology of wounds in necropsied farmed mink (Neovison vison) from June and October.

BACKGROUND: Wounds are regarded as an indicator of reduced welfare in mink production; however, information on the occurrence and significance of wounds is sparse. To provide a basis for assessment and classification of wounds in farmed mink, the distribution pattern and characteristics of wounds in farmed mink in June and October, respectively, is described. A total of 791 and 660 mink from 6 to 12 Danish mink farms, respectively, were examined. The mink were either found dead or were euthanized due to injury or other disease. Mink included from June were kits in the pre-weaning and weaning period (1-2 months old). Mink included from October were juveniles in the late growth period (approximately 5-6 months old) or older. Macroscopic pathology and wound location was systematically recorded. RESULTS: There was considerable variation in morphology as well as location of wounds between June and October. Wounds were primarily located on the front parts of the body and in the head in June (1-2 month old kits) and mainly on the rear parts of the body and on the tail in October (5-6 month old kits and older). Moreover, there were significantly more females than males with wounds for most wound types, and significant differences in occurrence of ear and tail base wounds between certain colour types. CONCLUSIONS: Wounds varied significantly from June to October with respect to morphology and anatomical location. Wounds in June were primarily located on the front parts of the body and in the head, while wounds in October were mainly present on the hind parts of the body and on the tail. The majority of the wounds were found in specific well defined skin areas and could therefore be grouped into categories according to anatomical location.

Associations between biosecurity and outbreaks of canine distemper on Danish mink farms in 2012-2013.

BACKGROUND: During 8 months from July 2012 to February 2013, a major outbreak of canine distemper involving 64 mink farms occurred on the Danish peninsula of Jutland. The canine distemper outbreak was associated with exposure of farmed mink to infected wild carnivores and could represent a deficit in biosecurity on the mink farms. The aim of this study was to investigate the extent and association of specific biosecurity measures with the outbreak. The study was carried out in an epidemiological case-control design. The case group consisted of the 61 farms, which had a confirmed outbreak of canine distemper from July 2012 to February 2013. The control group included 54 farms without an outbreak of canine distemper in 2012 or 2013, selected as the closest geographical neighbour to a case farm. RESULTS: The results showed that significantly more control than case farms had vaccinated their mink against canine distemper virus. Mortality was only assessed on the case farms, and there was a non-significantly lower mortality on vaccinated farms than on the non-vaccinated farms. Furthermore, the proportion of farms with observations of wild red foxes (Vulpes vulpes) inside the farm enclosures were larger for case farms, indicating that the control farms had a better biosecurity or were not equally exposed to canine distemper virus. Generally, all farms had very few specific precautions at the gate entrance in respect to human visitors as well as animals. The use of biosecurity measures was very variable in both case and control farms. Not using plastic boot covers, presence of dogs and cats, presence of demarcated area for changing clothes when entering and leaving the farm area and presence of hand washing facilities significantly lowered the odds of the farm having a canine distemper virus outbreak. CONCLUSIONS: The results of the study indicate that consistent use of correct vaccination strategies, implementation of biosecurity measures and limiting human and animal access to the mink farm can be important factors in reducing the risk for canine distemper outbreaks.

The apparent prevalence of skin lesions suspected to be human-inflicted in Danish finishing pigs at slaughter.

Skin lesions on pigs inflicted by humans compromise animal welfare and are the subject of increased public and political attention in Denmark. Systematic surveillance of such skin lesions was enforced in April 2010 at all Danish pig abattoirs, through the recording of meat inspection Code 904 for the presence of skin lesions suspected to be human inflicted. The objectives of the present study were to (a) estimate the apparent prevalence of Code 904s at the pig and herd owner level; (b) characterise the distribution of deliveries with pigs demonstrating a Code 904; (c) characterise the distribution of herd owners with repeated Code 904 recordings; and (d) determine the developments in Code 904 prevalence over time in Danish finishing pigs in the period from May 1, 2010 to September 30, 2013. Data from the 12 largest finishing pig abattoirs from Denmark were included and recordings were comprised from 65,504,021 pigs from 651,681 deliveries originating from 10,796 herd owners. Overall, 7200 (0.011%) of the pigs were recorded with a Code 904, and 21% of herd owners had a minimum of one Code 904 delivery with at least one pig with skin lesions inflicted by humans. On the pig-level, the apparent prevalence was 0.020% in 2010, which was reduced to 0.008% in 2013. In the first quarter of the study period, 17% of the herd owners had a Code 904 delivery, while 7% had one in the last quarter. Nine per cent of the herds had more than one Code 904 recording, with up to 16 Code 904 deliveries from one herd owner. Most deliveries included one single pig with a Code 904, but up to 102 Code 904 recordings were made in a single delivery. The apparent prevalence at the four smallest and four middle sized abattoirs decreased from the first to the second quarter, while the apparent prevalence decreased more substantially in the largest four abattoirs; with significant decreases from both the first to the second, and from the second to the third quarter. The study showed that recorded skin lesions suspected to be inflicted by humans are prevalent, but the apparent prevalence decreased from 2010 to 2012 and 2013. The development in Code 904 over time could be due to a real decrease or be due to other factors such as changes in the way the lesions were recorded, while both underestimation and overestimation appeared to be present.

Increasing prevalence of Coxiella burnetii seropositive Danish dairy cattle herds.

A study based on bulk tank milk samples from 120 randomly selected dairy cattle herds was conducted to estimate the prevalence of Coxiella burnetii seropositive dairy herds, to describe the geographical distribution, and to identify risk factors. Using the CHEKIT Q-fever Antibody ELISA Test Kit (IDEXX), the study revealed a prevalence of 79.2% seropositive herds, 18.3% seronegative herds, and 2.5% serointermediate herds based on the instructions provided by the manufacturer. Multifactorial logistic regression showed statistically significant associations (P < 0.01) between C. burnetii seropositivity and increasing herd size (OR = 1.02 per cow increment) and increasing regional average number of cattle per dairy herd (OR = 1.02 per animal increment). Herds >150 cows had 17.9 times higher odds of testing positive compared to herds <80 cows. The regional average number of cattle herds per square kilometer was borderline significantly related to the occurrence of seropositive dairy herds (P = 0.06). The results indicate an increased prevalence of seropositive dairy herds since the previous survey in 2008 and an adverse impact of increasing herd size and cattle density on the risk of seropositivity.

Prevalence and risk factors of Coxiella burnetii seropositivity in Danish beef and dairy cattle at slaughter adjusted for test uncertainty.

Antibodies to Coxiella burnetii have been found in the Danish dairy cattle population with high levels of herd and within herd seroprevalences. However, the prevalence of antibodies to C. burnetii in Danish beef cattle remains unknown. The objectives of this study were to (1) estimate the prevalence and (2) identify risk factors associated with C. burnetii seropositivity in Danish beef and dairy cattle based on sampling at slaughter. Eight hundred blood samples from slaughtered cattle were collected from six Danish slaughter houses from August to October 2012 following a random sampling procedure. Blood samples were tested by a commercially available C. burnetii antibody ELISA kit. A sample was defined positive if the sample-to-positive ratio was greater than or equal to 40. Animal and herd information were extracted from the Danish Cattle Database. Apparent (AP) and true prevalences (TPs) specific for breed, breed groups, gender and herd type; and breed-specific true prevalences with a random effect of breed was estimated in a Bayesian framework. A Bayesian logistic regression model was used to identify risk factors of C. burnetii seropositivity. Test sensitivity and specificity estimates from a previous study involving Danish dairy cattle were used to generate prior information. The prevalence was significantly higher in dairy breeds (AP=9.11%; TP=9.45%) than in beef breeds (AP=4.32%; TP=3.54%), in females (AP=9.10%; TP=9.40%) than in males (AP=3.62%; TP=2.61%) and in dairy herds (AP=15.10%; TP=16.67%) compared to beef herds (AP=4.54%; TP=3.66%). The Bayesian logistic regression model identified breed group along with age, and number of movements as contributors for C. burnetii seropositivity. The risk of seropositivity increased with age and increasing number of movements between herds. Results indicate that seroprevalence of C. burnetii is lower in cattle sent for slaughter than in Danish dairy cows in production units. A greater proportion of this prevalence is attributed to slaughtered cattle of dairy breeds or cattle raised in dairy herds rather than beef breeds.

Risk factors for Coxiella burnetii antibodies in bulk tank milk from Danish dairy herds.

The aim was to identify risk factors associated with Coxiella burnetii antibody positivity in bulk tank milk (BTM) samples from 100 randomly selected Danish dairy cattle herds. Antibody levels were measured by an enzyme-linked immuno-sorbent assay. Before testing the herds, the farm managers were interviewed about hired labour, biosecurity, housing and herd health during the 12 months prior to the study. Variables considered important for C. burnetii antibody positivity in multivariable logistic regression analysis included the sharing of machines between farms (OR = 3.6), human contacts (OR = 4.2), artificial insemination by other people than artificial insemination technicians (OR = 7.7), routine herd health contract with the veterinarian (OR = 4.3) and hygiene precautions taken by veterinarians (OR = 5). In addition, herd size, hired labour, trading of cattle between farms, quarantine and use of calving and disease pens also showed significant association in univariable analysis. This study demonstrates that strict biosecurity is important for the prevention of infections with C. burnetii.

Bayesian estimation of sensitivity and specificity of Coxiella burnetii antibody ELISA tests in bovine blood and milk.

Serological tests for Coxiella burnetii (the causative agent of Q fever) antibodies are usually based on enzyme linked immunosorbent assay (ELISA) although this method is not thoroughly evaluated. The objective of this study was to determine the sensitivity and specificity of an ELISA for detection of C. burnetii antibodies in milk and blood samples, using latent class models in a Bayesian analysis. Blood and milk samples of 568 lactating cows from 17 Danish dairy cattle herds collected in 2008 were used. The best combination of sensitivity and specificity estimates was revealed at a sample to positive (S/P) cut-off of 40 for both blood and milk ELISAs. At this cut-off, sensitivity of milk ELISA was 0.86 (95% posterior credibility interval [PCI] [0.76; 0.96]). This was slightly but insignificantly higher than sensitivity of blood ELISA (0.84; 95% PCI [0.75; 0.93]). The specificity estimates of the ELISA methods on milk and blood were equal at 0.99. No conditional dependence was observed between the specificity estimates of the two test methods. However, the sensitivity estimates of both tests were significantly reduced when conditional covariances ≥ 40 were used. Collection of milk samples from lactating cows is relatively easy, non-invasive and inexpensive and hence milk ELISA may be a better option for screening lactating cows. But, blood ELISA is an option for screening non-lactating cattle.

Factors associated with Coxiella burnetii antibody positivity in Danish dairy cows.

The aim of the study was to identify associations between the level of Coxiella burnetii (C. burnetii) antibodies in individual milk samples and cow and herd level factors in Danish dairy cows. The study, designed as a prospective cross sectional study with follow up, included 24 herds identified by a stratified random sampling procedure according to the level of C. burnetii antibodies in one bulk tank milk (BTM) sample at the beginning of the study. Ten herds were BTM positive, ten herds were BTM negative and four herds had an intermediate level. The samples were tested with an ELISA and results determined as S/P (sample to positive control) values. Three cross sectional studies of all lactating cows within each herd were then conducted during an 11 months follow up period with collection of a total of 5829 milk samples from 3116 cows. Each sample was tested with the same ELISA as used for BTM testing, and cows were considered test positive for S/P values ≥ 40, and otherwise negative. Individual cow information was extracted from the Danish Cattle Database and herd information was obtained from a telephone interview with each farmer. From multivariable logistic regression analysis accounting for hierarchical structures in the data it was concluded that odds for seropositivity increased with Danish Holstein breed, increasing number of parity and high milk protein contents, but decreased with increasing milk yield and high milk fat contents. Cows were at a higher risk during summer than other seasons. Among the herd level factors, herd size, tie stall housing system, quarantine of newly purchased animals and good hygienic precautions taken by the veterinarian before entering into the stable were also significantly associated with reduced odds of C. burnetii antibody positivity. The prevalence of test positive cows was almost constant during the study period in herds which were initially BTM positive and BTM intermediate, whilst the prevalence of positive cows in a few of the initial BTM negative herds changed from almost zero to higher than 60%. This indicates that herd infections last quite long and that test negative herds may convert to positive due to a few latently infected cows or due to transmissions from other herds.

Identification of biosecurity measures and spatial variables as potential risk factors for Aleutian disease in Danish mink farms.

Eradication of Aleutian disease was initiated in Denmark in 1976. The prevalence of positive farms has since then been reduced from 100% to only being continuously present in the region of Vendsyssel, Northern Denmark since 2004. In this study, we attempted to identify risk factors for the infection in this region based on logistic regression of spatial (environmental, neighbourhood) variables and biosecurity measures. Information on potential biosecurity (management) risk factors in the region was obtained from interviews in 342 registered farms in the region using a structured questionnaire. A total of 279 questionnaires were completed (response rate 82%). Additional spatial variables were included in the analysis. The study shows that farm size (the number of animals in the farm) and proportion of infected neighbouring farms were significant risk factors for infection with Aleutian Mink Disease Virus. These factors account for 35% of the variation of the infection status of mink farms located in Vendsyssel during 2009. These results indicate that only a coordinated effort from the farmers in the area will succeed in eradicating the disease from Denmark, because individual farms that have eradicated the disease will be at risk of re-infection from test-positive neighbours.