Mycobacterium avium Subspecies paratuberculosis DNA and Antibodies in Dairy Goat Colostrum and Milk.

Mycobacterium avium subspecies paratuberculosis (MAP) is endemic in the Dutch dairy goat population causing economic loss, and negatively influencing welfare. Moreover, there are concerns about a potential zoonotic risk. Therefore the industry's objectives are to decrease MAP prevalence, limit economic losses as well as reduce the concentration of MAP in (bulk) milk. To diminish within-farm spread of infection, vaccination, age dependent group housing with separation of newborns from adults, as well as rearing on artificial or treated colostrum and milk replacers are implemented. However, the importance of MAP contaminated colostrum and milk as a route of infection in dairy goat herds is unknown. Therefore the aim of this study was to detect the presence of MAP DNA in colostrum and milk from dairy goats in infected herds. A convenience sample of 120 colostrum samples and 202 milk samples from MAP infected dairy goat herds were tested by IS900 real-time Polymerase Chain Reaction (PCR) for MAP DNA. Furthermore, 22 colostrum samples and 27 post mortem milk samples of goats with clinical signs consistent with paratuberculosis from known infected herds were tested. The majority of samples were from goats vaccinated against MAP. Positive or doubtful PCR results were obtained in none of the 120 and two of the 22 colostrum samples, and in eight of the 202 and four of the 27 milk samples Negative PCR results were obtained in the remaining 140 (99%) colostrum samples and 217 (95%) milk samples.

Effects of Age and Environment on Adaptive Immune Responses to Mycobacterium avium subsp. paratuberculosis (MAP) Vaccination in Dairy Goats in Relation to Paratuberculosis Control Strategies.

Paratuberculosis infection is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In the Netherlands, 75% herd level prevalence of caprine paratuberculosis has been estimated, and vaccination is the principal control strategy applied. Most goat dairy farms with endemic paratuberculosis systematically vaccinate goat kids in the first months of life with a commercially available whole cell MAP vaccine. We hypothesized that the development of adaptive immune responses in goats vaccinated at young age depends on the environment they are raised in, and this has implications for the application of immune diagnostic tests in vaccinated dairy goats. We evaluated the early immune response to vaccination in young goat kids sourced from a MAP unsuspected non-vaccinated herd and raised in a MAP-free environment. Subsequently we compared these with responses observed in birth year and vaccination matched adult goats raised on farms with endemic paratuberculosis. Results indicated that initial adaptive immune responses to vaccination are limited in a MAP-free environment. In addition, adult antibody positive vaccinated goats raised in a MAP endemic environment are less likely to be IS900 PCR-positive as compared to antibody negative herd mates. We conclude that test-and-cull strategies in a vaccinated herd are currently not feasible using available immune diagnostic tests.

Phylogeographic Distribution of Human and Hare Francisella Tularensis Subsp. Holarctica Strains in the Netherlands and Its Pathology in European Brown Hares (Lepus Europaeus).

Sequence-based typing of Francisella tularensis has led to insights in the evolutionary developments of tularemia. In Europe, two major basal clades of F. tularensis subsp. holarctica exist, with a distinct geographical distribution. Basal clade B.6 is primarily found in Western Europe, while basal clade B.12 occurs predominantly in the central and eastern parts of Europe. There are indications that tularemia is geographically expanding and that strains from the two clades might differ in pathogenicity, with basal clade B.6 strains being potentially more virulent than basal clade B.12. This study provides information on genotypes detected in the Netherlands during 2011-2017. Data are presented for seven autochthonous human cases and for 29 European brown hares (Lepus europaeus) with laboratory confirmed tularemia. Associated disease patterns are described for 25 European brown hares which underwent post-mortem examination. The basal clades B.6 and B.12 are present both in humans and in European brown hares in the Netherlands, with a patchy geographical distribution. For both genotypes the main pathological findings in hares associated with tularemia were severe (sub)acute necrotizing hepatitis and splenitis as well as necrotizing lesions and hemorrhages in several other organs. Pneumonia was significantly more common in the B.6 than in the B.12 cases. In conclusion, the two major basal clades present in different parts in Europe are both present in the Netherlands. In hares found dead, both genotypes were associated with severe acute disease affecting multiple organs. Hepatitis and splenitis were common pathological findings in hares infected with either genotype, but pneumonia occurred significantly more frequently in hares infected with the B.6 genotype compared to hares infected with the B.12 genotype.

BRUCELLA PINNIPEDIALIS IN GREY SEALS ( HALICHOERUS GRYPUS) AND HARBOR SEALS ( PHOCA VITULINA) IN THE NETHERLANDS.

Brucellosis is a zoonotic disease with terrestrial or marine wildlife animals as potential reservoirs for the disease in livestock and human populations. The primary aim of this study was to assess the presence of Brucella pinnipedialis in marine mammals living along the Dutch coast and to observe a possible correlation between the presence of B. pinnipedialis and accompanying pathology found in infected animals. The overall prevalence of Brucella spp. antibodies in sera from healthy wild grey seals ( Halichoerus grypus; n=11) and harbor seals ( Phoca vitulina; n=40), collected between 2007 and 2013 ranged from 25% to 43%. Additionally, tissue samples of harbor seals collected along the Dutch shores between 2009 and 2012, were tested for the presence of Brucella spp. In total, 77% (30/39) seals were found to be positive for Brucella by IS 711 real-time PCR in one or more tissue samples, including pulmonary nematodes. Viable Brucella was cultured from 40% (12/30) real-time PCR-positive seals, and was isolated from liver, lung, pulmonary lymph node, pulmonary nematode, or spleen, but not from any PCR-negative seals. Tissue samples from lung and pulmonary lymph nodes were the main source of viable Brucella bacteria. All isolates were typed as B. pinnipedialis by multiple-locus variable number of tandem repeats analysis-16 clustering and matrix-assisted laser desorption ionization-time of flight mass spectrometry, and of sequence type ST25 by multilocus sequence typing analysis. No correlation was observed between Brucella infection and pathology. This report displays the isolation and identification of B. pinnipedialis in marine mammals in the Dutch part of the Atlantic Ocean.

Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast.

The presence of Brucella (B.) spp. in harbour porpoises stranded between 2008 and 2011 along the Dutch coast was studied. A selection of 265 tissue samples from 112 animals was analysed using conventional and molecular methods. In total, 4.5% (5/112) of the animals corresponding with 2.3% (6/265) Brucella positive tissue samples were Brucella positive by culture and these were all confirmed by real-time polymerase chain reaction (real-time PCR) based on the insertion element 711 (IS711). In addition, two more Brucella-positive tissue samples from two animals collected in 2011 were identified using real-time PCR resulting in an overall Brucella prevalence of 6.3% (7/112 animals). Brucella spp. were obtained from lungs (n=3), pulmonary lymph node (n=3) and lungworms (n=2). Multi Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) typing based on the MLVA-16 showed that the Brucella isolates were B. ceti. Additional in silico Multi Locus Sequence typing (MLST) after whole genome sequencing of the 6 Brucella isolates confirmed B. ceti ST 23. According to the Brucella 2010 MLVA database, the isolated Brucella strains encountered were of five genotypes, in two distinct subclusters divided in two different time periods of harbour porpoises collection. This study is the first population based analyses for Brucella spp. infections in cetaceans stranded along the Dutch coast.

A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis.

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.

In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.

Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

Molecular epidemiology of Coxiella burnetii from ruminants in Q fever outbreak, the Netherlands.

Q fever is a zoonosis caused by the bacterium Coxiella burnetii. One of the largest reported outbreaks of Q fever in humans occurred in the Netherlands starting in 2007; epidemiologic investigations identified small ruminants as the source. To determine the genetic background of C. burnetii in domestic ruminants responsible for the human Q fever outbreak, we genotyped 126 C. burnetii-positive samples from ruminants by using a 10-loci multilocus variable-number tandem-repeat analyses panel and compared them with internationally known genotypes. One unique genotype predominated in dairy goat herds and 1 sheep herd in the human Q fever outbreak area in the south of the Netherlands. On the basis of 4 loci, this genotype is similar to a human genotype from the Netherlands. This finding strengthens the probability that this genotype of C. burnetii is responsible for the human Q fever epidemic in the Netherlands.

Interlaboratory comparison of real-time polymerase chain reaction methods to detect Coxiella burnetii, the causative agent of Q fever.

The bacterium Coxiella burnetii, which has a wide host range, causes Q fever. Infection with C. burnetii can cause abortions, stillbirth, and the delivery of weak offspring in ruminants. Coxiella burnetii infection is zoonotic, and in human beings it can cause chronic, potentially fatal disease. Real-time polymerase chain reaction (PCR) is increasingly being used to detect the organism and to aid in diagnosis both in human and animal cases. Many different real-time PCR methods, which target different genes, have been described. To assess the comparability of the C. burnetii real-time PCR assays in use in different European laboratories, a panel of nucleic acid extracts was dispatched to 7 separate testing centers. The testing centers included laboratories from both human and animal health agencies. Each laboratory tested the samples using their in-house real-time PCR methods. The results of this comparison show that the most common target gene for real-time PCR assays is the IS1111 repeat element that is present in multiple copies in the C. burnetii genome. Many laboratories also use additional real-time PCR tests that target single-copy genes. The results of the current study demonstrate that the assays in use in the different laboratories are comparable, with general agreement of results for the panel of samples.