Detection of Chlamydia and Chlamydia-like organisms in bovine placental tissue.

This study aimed to investigate the presence of Chlamydia spp. and Parachlamydia acanthamoebae in bovine placental tissue originating from abortion and non-abortion cases in Belgium. Placentas of 164 late term bovine abortions (last trimester of gestation) and 41 non-abortion (collected after calving) cases were analysed by PCR for Chlamydia spp., Chlamydia abortus, C. psittaci and P. acanthamoebae. Additionally, a subset of 101 (75 abortion and 26 non-abortion cases) of these placenta samples were also analysed by histopathology to detect possible Chlamydia-induced lesions. In 5.4% (11/205) of the cases, Chlamydia spp. were detected, and three of those cases were positive for C. psittaci. Parachlamydia acanthamoebae was detected in 36% (75/205) of the cases, being 44% (n = 72) in abortions and 7.3% (n = 3) in non-abortions cases (p < .001). None of the cases was positive for C. abortus. Purulent and/or necrotizing placentitis with or without vasculitis was observed in 18.8% (19/101) of the histopathologically analysed placenta samples. In 5.9% (6/101) of the cases, placentitis was observed along with vasculitis. In the abortion cases, 24% (18/75) of the samples showed purulent and/or necrotizing placentitis, while purulent and/or necrotizing placentitis was visible in 3.9% (1/26) of the non-abortion cases. Placental lesions of inflammation and/or necrosis were present in 44% (15/34) of the cases where P. acanthamoebae was detected, while inflammation and/or necrosis was present in 20.9% (14/67) of the negative cases (p < .05). The detection of Chlamydia spp. and especially P. acanthamoebae, in combination with correlated histological lesions such as purulent and/or necrotizing placentitis and/or vasculitis in placental tissue following abortion, suggests a potential role of this pathogen in cases of bovine abortion in Belgium. Further in-depth studies are necessary to unravel the role of these species as abortifacient agents in cattle and to include them in bovine abortion monitoring programmes.

Prevalence of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Rickettsia spp. and Babesia spp. in cattle serum and questing ticks from Belgium.

BACKGROUND: Anaplasmosis, borreliosis, rickettsiosis and babesiosis are tick-borne diseases of medical, veterinary and economic importance. In Belgium, little is known on the prevalence of these diseases in animals and previous screenings relate only to targeted geographic regions, clinical cases or a limited number of tested samples. We therefore performed the first nationwide seroprevalence study of Anaplasma spp., A. phagocytophilum, Borrelia spp., Rickettsia spp. and Babesia spp. in Belgian cattle. We also screened questing ticks for the aforementioned pathogens. METHODS: ELISAs and IFATs were performed on a representative sample set of cattle sera stratified proportionally to the number of cattle herds per province. Questing ticks were collected in areas where the highest prevalence for the forenamed pathogens in cattle serum were observed. Ticks were analyzed by quantitative PCR for A. phagocytophilum (n = 783), B. burgdorferi sensu lato (n = 783) and Rickettsia spp. (n = 715) and by PCR for Babesia spp. (n = 358). RESULTS: The ELISA screening for antibodies to Anaplasma spp. and Borrelia spp. in cattle sera showed an overall seroprevalence of 15.6% (53/339) and 12.9% (52/402), respectively. The IFAT screening for antibodies against A. phagocytophilum, Rickettsia spp. and Babesia spp. resulted in an overall seroprevalence of 34.2% (116/339), 31.2% (99/317) and 3.4% (14/412), respectively. At the provincial level, the provinces of Liege and Walloon Brabant harboured the highest seroprevalence of Anaplasma spp. (44.4% and 42.7% respectively) and A. phagocytophilum (55.6% and 71.4%). East Flanders and Luxembourg exhibited the highest seroprevalence of Borrelia spp. (32.4%) and Rickettsia spp. (54.8%) respectively. The province of Antwerp showed the highest seroprevalence of Babesia spp. (11%). The screening of field-collected ticks resulted in a prevalence of 13.8% for B. burgdorferi s.l., with B. afzelii and B. garinii being the most common genospecies (65.7% and 17.1%, respectively). Rickettsia spp. was detected in 7.1% of the tested ticks and the only identified species was R. helvetica. A low prevalence was found for A. phagocytophilum (0.5%) and no Babesia positive tick was detected. CONCLUSIONS: The seroprevalence data in cattle indicate hot spots for tick-borne pathogens in specific provinces and highlights the importance of veterinary surveillance in anticipating the emergence of diseases among humans. The detection of all pathogens, with the exception of Babesia spp. in questing ticks, underlines the need of raising awareness among public and professionals on other tick-borne diseases along with lyme borreliosis.

Detection of Anaplasma phagocytophilum in fetal and placental tissue of bovine abortions and perinatal mortalities.

BACKGROUND: Anaplasma phagocytophilum is a tick-borne zoonotic bacterium that is the aetiologic pathogen of tick-borne fever (TBF) in ruminants. In clinical bovine cases of TBF, abortion and stillbirth may be observed. However, in this regard, the pathophysiology of TBF has not yet been completely elucidated, and no clear guidelines to diagnose A. phagocytophilum-related abortions and perinatal mortalities (APM) are available. METHODS: This exploratory study aimed to investigate the presence of A. phagocytophilum in bovine cases of APM and determine whether placental or fetal spleen tissue has the greatest sensitivity for A. phagocytophilum identification. The placenta and fetal spleen of 150 late-term bovine APM cases were analysed using real-time PCR to detect A. phagocytophilum. RESULTS: A total of 2.7% of sampled placentas were positive for A. phagocytophilum, while none of the fetal spleen samples was. LIMITATIONS: No histopathology to detect associated lesions was performed. Consequently, no evidence of causality between the detection of A. phagocytophilum and APM events could be achieved. CONCLUSION: The detection of A. phagocytophilum suggests a potential role of this pathogen in bovine APM, and placental tissue seems to be the most suitable tissue for its identification.

A Randomized Large-Scale Cross-Sectional Serological Survey of Hepatitis E Virus Infection in Belgian Pig Farms.

Hepatitis E virus (HEV) is the causative agent of hepatitis E disease in humans. While sporadic HEV infections, which occur in industrialised countries and are typically due to HEV genotypes 3 or 4, are asymptomatic and self-limiting, a chronic form of the disease can lead to liver cirrhosis in immunocompromised individuals. Pigs share HEV 3 and 4 genotypes and are thus considered a major animal reservoir for human infection. A subset of animals has been shown to carry HEV particles at the age of slaughter, rendering raw or undercooked pig products potential vectors for human infection. To provide an overview of the current dissemination of HEV in Belgian pig herds, this study was designed as a randomized, robust, large-scale, cross-sectional, serological survey. HEV genotypes and subtypes recently circulating in Belgium (2020-2021) were investigated. Sample stratification as well as epidemiological investigation through the available demographic data of the sampled herds showed that HEV widely circulated in the Belgian pig population during this time and that a change in the circulating HEV strains may have occurred in the last decade. Herd size and type were identified as risk factors for HEV herd-seropositivity. Identifying farms at risk of being HEV-positive is an important step in controlling HEV spread and human infection.

Phylogenomic analysis of Mycoplasma bovis from Belgian veal, dairy and beef herds.

M. bovis is one of the leading causes of respiratory disease and antimicrobial use in cattle. The pathogen is widespread in different cattle industries worldwide, but highest prevalence is found in the veal industry. Knowledge on M. bovis strain distribution over the dairy, beef and veal industries is crucial for the design of effective control and prevention programs, but currently undocumented. Therefore, the present study evaluated the molecular epidemiology and genetic relatedness of M. bovis isolates obtained from Belgian beef, dairy and veal farms, and how these relate to M. bovis strains obtained worldwide. Full genomes of one hundred Belgian M. bovis isolates collected over a 5-year period (2014-2019), obtained from 27 dairy, 38 beef and 29 veal farms, were sequenced by long-read nanopore sequencing. Consensus sequences were used to generate a phylogenetic tree in order to associate genetic clusters with cattle sector, geographical area and year of isolation. The phylogenetic analysis of the Belgian M. bovis isolates resulted in 5 major clusters and 1 outlier. No sector-specific M. bovis clustering was identified. On a world scale, Belgian isolates clustered with Israeli, European and American strains. Different M. bovis clusters circulated for at least 1.5 consecutive years throughout the country, affecting all observed industries. Therefore, the high prevalence in the veal industry is more likely the consequence of frequent purchase from the dairy and beef industry, than that a reservoir of veal specific strains on farm would exist. These results emphasize the importance of biosecurity in M. bovis control and prevention.

Pilot study assessing the possible benefits of a higher level of implementation of biosecurity measures on farm productivity and health status in Belgian cattle farms.

Over the last few years, the interest of decision-makers and control agencies in biosecurity (BS), aiming at preventing and controlling the introduction and spread of infectious diseases, has considerably increased. Nevertheless, previous studies highlighted a low implementation level of biosecurity measures (BSM), especially in cattle farms; different reasons were identified such as perceived costs, utility, importance, increased workload and lack of knowledge. In order to convince cattle farmers to adopt BSM, it is necessary to gather more information and evidence on their cost-effectiveness and their importance or utility in terms of disease prevention and control. The objectives of this study were to determine whether the farm or farmers' profile correlated with the implementation level of BSM and if there was a positive correlation between the BSM implementation and the farm production and health parameters. Data were collected through face-to-face interviews conducted in 100 Belgian farms as part of a stratified and randomized survey. The Regional Animal Health Services provided the farm health status and production data. A general BS score and five sub-scores related to the five BS compartments (bio-exclusion, bio-compartmentation, bio-containment, bio-prevention and bio-preservation) were calculated for each farm based on the implementation level of different BSM grouped in 16 domains. The study highlighted a significant and negative correlation between the mortality rates in adult cattle (over 24 months of age) and young calves (aged 0-7 days) and different BS compartment scores. The study also demonstrated that the farms having a higher general BS score were indeed more likely to have a BVD-free status. These evidence-based findings are encouraging as they demonstrate the benefits of implementing BSM and could promote their adoption by farmers.

Biosecurity practices in Belgian veal calf farming: Level of implementation, attitudes, strengths, weaknesses and constraints.

The shift from cure toward prevention in veterinary medicine involves the implementation of biosecurity. In cattle farming, the application of biosecurity measures has been described to a limited degree, yet no data on biosecurity on veal farms is available. A high degree of commingling of veal calves from multiple farms causes frequent disease outbreaks, and thereby high antimicrobial usage and increased risk of antimicrobial resistance. Therefore, this study aimed to describe the current implementation of biosecurity on veal farms in Belgium. To this extent, a list of the most important calf diseases (n = 34) was created, and risk factors and related biosecurity measures for these diseases were determined and included in a questionnaire. Herd visits and face-to-face interviews were conducted on 20 randomly selected veal farms, comprising 8.3% of the target population. A categorical principal component and clustering analysis were performed to determine the influence of the veal companies on the farms' biosecurity level. Awareness of biosecurity was very low among the farmers. All farms used an "all-in, all-out" production system with calves originating from multiple farms without quarantine. On average, farms were filled in 11.4 days (range 2-52). The degree of commingling for these farms was 1.24, meaning that, on average, 124 calves originated from 100 farms. Veterinarians wore farm-specific boots on eight farms (40%) and farm-specific clothes on six farms (30%), while technical advisors wore farm-specific boots on six farms (30%) and farm-specific clothes on four farms (20%). Disinfection footbaths were only used in five farms (25%) despite being present in all farms in the sample. Concerning internal biosecurity, none of the farmers isolated sick animals; only one farmer (5%) had a physically separated hospital pen, and only 11 farmers (55%) both cleaned and disinfected the stables after each production cycle. In most farms, animals were of comparable age. Healthy calves generally remained in the same compartment during the entire production cycle, limiting the risks associated with the movement of animals. No influence of the integrations on the biosecurity level could be determined. It can be concluded that a few biosecurity measures, such as "'all-in, all-out" and compartmentation, are implemented relatively well, while other measures, such as good cleaning and disinfection and proper entrance measures for visitors and personnel can easily be improved. The improvement of measures regarding the introduction of animals of different origins will require more fundamental changes in the veal industry.

Classification of adult cattle infectious diseases: A first step towards prioritization of biosecurity measures.

An emphasis on biosecurity in the cattle industry was made over the years to improve animal and public health. Nevertheless, the level of implementation of biosecurity measures (BSM) remains largely insufficient due to certain constraints. It is therefore necessary to prioritize the different BSM to be applied in accordance with the individual context and the main infectious diseases affecting cattle. Previous prioritization exercises of infectious diseases were neither specific to Belgium nor based on an exhaustive list of diseases. This study aimed at classifying the most important infectious diseases affecting cattle in Belgium. A list of 74 cattle infectious diseases reported in Europe was compiled based on a literature review. Through an online survey, Belgian rural veterinary practitioners (RVP) were asked to assign a score to each disease according to their frequency (question 1), their trends estimated between 2013-15 (question 2), and finally to list the five most important diseases for adult cattle (question 3). Respectively, 107 and 93 RVP answered the first two questions and the last one. Results of the survey were used to classify the diseases based on their frequency, trends, and importance through an additional weighting system and a subsequent regression tree analysis. Belgian laboratory databases and previous disease prioritization exercises were also analysed and taken into account as additional data sources. For the most important diseases identified (those ranked as important by the three data sources), a literature review was performed in PubMed to identify their related risk factors and BSM. A total of 48 infectious diseases were classified as important in Belgium with six of them considered as important from the three data sources: bovine respiratory diseases (BRD), bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), Q fever, and salmonellosis. Their related BSM should be prioritized in terms of BSM implementation.

Use of a national identification database to determine the lifetime prognosis in cattle with necrotic laryngitis and the predictive value of venous pCO2.

BACKGROUND: Necrotic laryngitis, caused by Fusobacterium necrophorum, frequently requires surgical intervention (laryngostomy) in the chronic stage. HYPOTHESIS/OBJECTIVES: To determine survival until slaughter of cattle surgically treated for necrotic laryngitis and to identify predictors of mortality. ANIMALS: A total of 221 cattle diagnosed with necrotic laryngitis by laryngoscopy and surgically treated METHODS: Retrospective cohort study. Clinical records were matched with the national cattle identification, registration, and movement database. Information on possible predictors including clinical examination, biochemistry, and surgery was collected. A multivariable Cox proportional hazard model was used to identify predictors of mortality. RESULTS: The overall survival rate was 65.2% and 58.6% of the animals with a completed life cycle could be slaughtered. Animals <6 months old experienced significantly higher mortality risk (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.1-3.5). The venous partial pressure of carbon dioxide (pCO2 ; HR, 2.4; 95% CI, 1.4-4.2) at a 64.5 mm Hg cut-off was most significantly associated with mortality. Sensitivity and specificity of the final model consisting of age and pCO2 were 49.1 and 86.4%, respectively. Instead of pCO2 , total carbon dioxide (TCO2 ) could also be used, with similar diagnostic accuracy. CONCLUSIONS AND CLINICAL RELEVANCE: The lifetime prognosis for chronic necrotic laryngitis in cattle with surgical intervention appears fair. Age, venous pCO2 and TCO2 are easily accessible predictors of survival to support owners and veterinarians in their decision process of whether or not to operate and to identify high risk animals that require more intensive follow-up.

Serological and virological BVDV prevalence and risk factor analysis for herds to be BVDV seropositive in Belgian cattle herds.

Bovine viral diarrhoea virus (BVDV) is a worldwide spread virus that most commonly infects cattle and can cause considerable economic losses. To determine the prevalence of BVDV in Belgium, a cross-sectional study was performed between November 2009 and March 2010. Young stock aged between 6 and 12 months from 773 randomly selected Belgian cattle herds were tested for BVDV-specific antibodies and antigen. With a target and maximum of 10 animals per sampled herd, a total of 5246 animals were selected. Additionally a questionnaire including different herd management topics and questions about participation in animal health programmes, including BVDV, was sent to 1100 Belgian cattle herds, including the 773 herds for BVDV testing. This paper focuses on results regarding these 773 herds. The true prevalence of BVDV-specific antibodies and antigen at herd level was respectively 47.4% and 4.4%, while at animal level this was respectively 32.9% and 0.3%. In 44.4% of the herds where BVDV-specific antibodies were detected at least 60% of the sampled young stock was BVDV seropositive. Interestingly, 83.4% of these farmers stated not to have suffered from problems related to BVDV. Moreover, only 8.4% of all farmers who completed the questionnaire (n=895) reported problems possibly related to BVDV the past 3 years. This demonstrates that farmers are often unaware of the presence of BVDV in their herd. Risk factors for a herd to be BVDV seropositive were identified by means of a multivariable logistic regression model. Large herds were significantly more likely to be BVDV seropositive (OR=1.004, p<0.01). The interaction between "Antigen positive animal detected in this study" and "BVDV vaccination in 2009" was significant (p<0.01). In non-vaccinating herds, the detection of antigen positive animals was significantly associated with BVDV seropositive herds (OR=13.8, p<0.01). In herds with no antigen positive animals detected, vaccination resulted in a significant risk factor to be BVDV seropositive compared to non-vaccinating herds (OR=3.4, p<0.01). Herds reporting BVDV-related problems the past 3 years were more likely to be BVDV seropositive (OR=1.9, p<0.05). This relation became non-significant (OR=1.8, p=0.08) when only a subset of herds with no vaccination of animals <12 months was taken into account. The results of the current study suggest an active circulation of BVDV in a considerable number of Belgian cattle herds.

Association between herd exposure to BVDV-infection and bulk milk somatic cell count of Flemish dairy farms.

The purpose of this study was to investigate the statistical association between herd bovine viral diarrhoea (BVD) status based on bulk milk antibody detection and monthly bulk milk somatic cell count (BMSCC) as a reflection of the udder health. A distinction was made between vaccinating and non-vaccinating herds via a questionnaire concerning BVD-vaccination. No significant difference in BMSCC was found between vaccinating (228,300 cells/ml; SD 180,019) and non-vaccinating (237,070 cells/ml; SD 77,900) herds. Non-vaccinating herds (n=243) were selected, and the relationship between a single BVDV-antibody titre and the BMSCC of each herd over a 12-month observation period evaluated. For this purpose, the non-vaccinating herds were divided into five groups depending on bulk milk BVDV-antibody titres. Overall, no significant relationship between the antibody titre and the BMSCC was found. Still, when comparing the category with the lowest S/P ratio (essentially BVDV-naïve herds; BMSCC=211,390 cells/ml) with the combined four other categories (BMSCC=242,790 cells/ml), a significant difference in BMSSC was observed (P=0.01).

Assessing biosecurity practices, movements and densities of poultry sites across Belgium, resulting in different farm risk-groups for infectious disease introduction and spread.

The existing diversity within poultry systems affects the potential risk of infectious disease introduction and spread. Population data on the level of biosecurity and between-farm contacts is scarce, despite its importance for identifying possible routes of disease transmission. A study was carried out in Belgium to investigate and differentiate professional and hobby poultry sites based on their biosecurity levels and farm movements. Questionnaire data from a total of 37 professional poultry farms, 19 hatcheries and 286 hobby poultry sites were analyzed using a combination of a linear scoring system, a Categorical principal component analysis (CATPCA) and a Two-Step cluster analysis (TSCA). In general, the level of biosecurity was lower in hobby poultry flocks, mainly due to the poor confinement against the outdoor environment and the poor infrastructural hygiene. Most Belgian professional poultry farms and hatcheries had an acceptable level of adoption of standard biosecurity practices, however less attention was given to the way transportation vehicles and employers were brought onto farms and professional visitors welcomed. Considerable variation in the movements and in the structure of the networks arising from these movements was found. Movement frequencies were higher at professional farms compared to hobby farms. Results showed that multiple category farming systems had the highest total movement frequencies. Monthly frequencies of professional visits often exceeded those of poultry and egg movements. Professional and hobby poultry sites were also connected, but movements of poultry and eggs were found only to occur from professional to hobby sites. However, hobby poultry keepers were personally purchasing the poultry and eggs on the professional poultry sites. Six groups of poultry sites were differentiated, which are interpreted as very low to very high risk groups, based on the potential of infectious disease introduction and spread.

Control of bovine virus diarrhoea at the herd level: reducing the risk of false negatives in the detection of persistently infected cattle.

The need to detect and eliminate cattle persistently infected (PI) with bovine viral diarrhoea virus (BVDV) is key to the control of BVD and has been shown to be very effective in eradicating BVDV from infected herds. However, because of pitfalls in the detection procedures, some PI animals can be missed and, as a result, are not identified and removal is delayed. The high prevalence of BVDV in cattle populations in some countries (such as Belgium and neighbouring countries) means there is a high risk of reinfection of a herd from which BVDV has been eradicated. Based on both practical experience and a literature study, this review considers those points that are critical to minimising the number of false negatives in the detection of PI cattle.

[Mental notes in tracking BVDV carriers]. / Aandachtspunten bij het opsporen van BVDV-dragers.

The detection and elimination of animals persistently infected with bovine viral diarrhoea virus (BVDV) is the key to BVD control. This method has proven to be very efficient in eradicating BVDV in a herd. Several pitfalls in the detection procedure can make that some persistently infected (PI) animals do not get identified or are removed too late, supporting the assumption that circulation of the virus could be possible in absence of PI animals. Furthermore the risk of reintroduction is high since the prevalence of BVD is high in the Netherlands and Belgium. Based on both practical experience and literature, here we review critical control points in order to minimise the risk of a false negative BVDV screening.

Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection.

The diagnostic properties of several assays on live animals were studied using data from different experiments. These experiments involved 128 classical swine fever virus (CSFV) infected pigs (weaner pigs, fatteners and sows). Since all pigs in the study were infected with CSFV, only the proportion of test positive results and the time until a test positive result is obtained were evaluated. The RT-nPCR detected the highest proportion of infected pigs (98.9%), whereas the Antigen ELISA gave the worst detection results (74.7%). Within the group of test positive animals, infection was detected earliest using the leukocyte count and latest using Antigen ELISA. Using the virus neutralisation test, antibodies against CSFV were detectable on average 7.6 days after the onset of viraemia in virus isolation in whole blood. Using survival analysis, the time until the first positive diagnosis and the proportion of detected animals were combined in one test. Results showed that RT-nPCR performed significantly better than either virus isolation in different blood fractions or antigen ELISA. It is concluded that the RT-nPCR technique is the best diagnostic tool available for early detection of a classical swine fever infection.