Quality of bulk tank milk samples from Danish dairy herds based on real-time polymerase chain reaction identification of mastitis pathogens.

Results of a commercial real-time PCR analysis for 11 mastitis pathogens from bulk tank milk (BTM) samples from all 4,258 Danish dairy herds in November 2009 to January 2010 were compared with somatic cell count (SCC) and total bacteria count (TBC) estimates in BTM. For Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis, a low real-time PCR cycle threshold (Ct) value (corresponding to high bacterial DNA quantity) was correlated with higher SCC and higher TBC. For Staphylococcus aureus, low Ct values were correlated only with higher SCC. For the environmental mastitis pathogens Klebsiella spp., Enterococcus spp., and Escherichia coli, low Ct values had a correlation with higher TBC. Staphylococcus spp. were found in the BTM from all herds, Strep. uberis in 95%, Staph. aureus in 91%, and Strep. dysgalactiae in 86%, whereas E. coli, Klebsiella, and Strep. agalactiae were found in 61, 13, and 7% of the herds. It is concluded that the real-time PCR used provides results that are related to the milk quality in the herds. Real-time PCR can be used in the same way as culture for monitoring BTM samples, and is especially useful for bacteria with low prevalence (e.g., Strep. agalactiae).

Population studies of 16 bovine STR loci for forensic purposes.

As a consequence of the close integration of cattle into the food chain of humans, forensically relevant cases involving cattle (Bos taurus) DNA analysis are common. However, scientific publications reporting the information content of the commonly used bovine short tandem repeat (STR) loci remains scarce. Population studies were performed for 16 polymorphic STR loci (BM1818, BM1824, BM2113, CSRM60, CSSM66, ETH3, ETH10, ETH225, HAUT27, ILSTS006, INRA023, SPS115, TGLA53, TGLA122, TGLA126, and TGLA227) including 4,162 randomly selected cattle representing 20 distinct breeds. The power of parental exclusion, expected and observed heterozygosity, probability of identity, and non-amplifying ("null") allele frequencies were calculated. Major differences existed in the information content between different cattle breeds. The selection of 16 STR loci, partially recommended by International Society for Animal Genetics as the minimum standard needed for bovine STR typing, was sufficient for forensic analysis. Furthermore, the efficacy of the loci was assessed in assigning unknown individuals to the correct breed based on genotype data. The individual assignment tests provided excellent success in several breeds.

Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria.

Fast and reliable identification of the microorganisms causing mastitis is important for management of the disease and for targeting antimicrobial treatment. Methods based on PCR are being used increasingly in mastitis diagnostics. Comprehensive field comparisons of PCR and traditional milk bacteriology have not been available. The results of a PCR kit capable of detecting 11 important etiological agents of mastitis directly from milk in 4h were compared with those of conventional bacterial culture (48h). In total, 1,000 quarter milk samples were taken from cows with clinical or subclinical mastitis, or from clinically healthy quarters with low somatic cell count (SCC). Bacterial culture identified udder pathogens in 600/780 (77%) of the clinical samples, whereas PCR identified bacteria in 691/780 (89%) of the clinical samples. The PCR analysis detected major pathogens in a large number of clinical samples that were negative for the species in culture. These included 53 samples positive for Staphylococcus aureus by PCR, but negative by culture. A total of 137 samples from clinical mastitis, 5 samples from subclinical mastitis, and 1 sample from a healthy quarter were positive for 3 or more bacterial species in PCR, whereas culture identified 3 or more species in 60 samples from clinical mastitis. Culture identified a species not targeted by the PCR test in 44 samples from clinical mastitis and in 9 samples from subclinical mastitis. Low SCC samples provided a small number of positive results both in culture (4/93; 4.3%) and by PCR (7/93; 7.5%). In conclusion, the PCR kit provided several benefits over conventional culture, including speed, automated interpretation of results, and increased sensitivity. This kit holds much promise as a tool to complement traditional methods in identification of pathogens. In conventional mastitis bacteriology, a sample with 3 or more species is considered contaminated, and resampling of the cow is recommended. Further study is required to investigate how high sensitivity of PCR and its quantitative features can be applied to improve separation of relevant udder pathogens from likely contaminants in samples where multiple species are detected. Furthermore, increasing the number of species targeted by the PCR test would be advantageous.

Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing.

In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The "no-growth" samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10(3) to 10(7) bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-beta-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such milk samples. Low bacterial concentration is commonly speculated to explain the no-growth milk samples. This hypothesis is not supported by the results of the current study.

Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens.

Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal beta-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and beta-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.

Comparison of tests for detection of beta-lactamase-producing staphylococci.

Penicillin resistance identification tests are important in veterinary medicine. Six enzyme assays and a PCR test were compared for the detection of beta-lactamase production or the beta-lactamase gene in 175 staphylococcal isolates. We conclude that the PCR test and two nitrocefin-based assays can be recommended for routine clinical use.

Isolation by distance within a river system: genetic population structuring of Atlantic salmon, Salmo salar, in tributaries of the Varzuga River in northwest Russia.

An important issue for designing any conservation programme aimed at preserving genetic diversity is estimation of the scale at which genetic structuring occurs. Additional relevant factors include distinguishing whether or not population structuring is expected to be stable as predicted by the member-vagrant hypothesis, or alternatively, whether populations are more prone to local extinction-recolonization processes, as predicted by the metapopulation evolutionary model. In this study, the population genetic structure of Atlantic salmon from 11 locations within or nearby the Varzuga River tributary system was assessed using 17 microsatellites. Mantel tests and spatial autocorrelation analyses revealed a significant isolation-by-distance signal within the tributary system as well as a negative association between the level of genetic diversity and waterway distance from the river mouth, indicating that dispersal is less likely to occur to populations deep in the tributary system. Individual-level spatial autocorrelation analyses indicated that the majority of migration occurred between populations situated within 20 km of each other. The relatively high level of genetic structuring and significant isolation-by-distance signal observed in the Varzuga tributaries are concordant with the predictions of the member-vagrant evolutionary model. However, one subpopulation in particular revealed signs of instability which may be due to its location in the tidal zone, or due to the fact that it is more affected by human impacts. The results suggest that preservation of a number of spawning sites spaced throughout the tributary system is recommendable for ensuring sustainable fishing tourism in the river.

Identification of reproductively isolated lineages of Amur grayling (Thymallus grubii Dybowski 1869): concordance between phenotypic and genetic variation.

We analysed variation at maternally (mitochondrial DNA control region sequences) and bi-parentally (10 microsatellites) inherited genetic markers, as well as across 12 meristic characters in 7 populations of Amur grayling, Thymallus grubii, from eastern Siberia. All three data sets were concordant in supporting the existence of three diagnosable, reciprocally monophyletic, and most probably reproductively isolated, lineages of grayling within the Amur drainage. There was a significant correlation between genetic and phenotypic divergence, both within and among lineages. Two phenotypically distinct forms (with and without an orange spot on the posterior portion of the dorsal fin), found in sympatry in the lower Amur, most likely result from secondary contact, as they demonstrate 4.6% sequence divergence at the mitochondrial DNA control region. This divergence, together with the existence of at least one nearby population of orange spot grayling outside the Amur drainage (0.8% divergence) underscore the palaeo-hydrological complexity of the system, which presumably promoted genetic divergence in a shifting allopatric framework throughout the Pleistocene. Grayling from the upper Amur, corresponding to the type locality for the species, formed a sister group (1.4-1.6% divergent) to the orange spot lineage perhaps diverging in the early Pleistocene (1.4-1.6 Ma).

Individual assignment using microsatellite DNA reveals unambiguous breed identification in the domestic dog.

Modern individual clustering methods utilising hypervariable nuclear microsatellite DNA polymorphisms are being increasingly applied in the field of population genetics. This study explores the efficiency of the clustering methods in identifying the breeds of origin of 250 domestic dog (Canis familiaris) individuals based on 10 microsatellite loci. An allele sharing distance (DAS) matrix and the corresponding neighbour-joining tree of individuals revealed monophyletic assemblages that corresponded perfectly with the breeds of origin of the dogs. Individual assignment tests using a Bayesian statistical approach, an allele frequency based method, and a DCE genetic distance based method were all extremely powerful. Most strikingly, the Bayesian method provided 100% assignment success of individuals into their correct breeds of origin and 100% exclusion success of individuals from all alternate reference populations with a high level of statistical confidence (P < 0.0001). A Bayesian Markov Chain Monte Carlo clustering approach revealed clear distinction of individuals into groups according to their breeds of origin, with a near-zero level of 'genetic admixture' among breeds. The results demonstrate that an FST of 0.18, mean expected gene diversity of 0.6 across 10 loci, and approximately 50 individuals per reference population suffice to provide maximum individual assignment success in C. familiaris. This refutes the traditional view that DNA based dog breed identification is not feasible at the individual level of resolution.

Microsatellite data resolve phylogeographic patterns in European grayling, Thymallus thymallus, Salmonidae.

The phylogeography of an endangered salmonid, European grayling (Thymallus thymallus), was studied based on analysis of 17 nuclear microsatellite DNA loci. In agreement with earlier mitochondrial DNA (mtDNA) studies, phylogenetic relationships of the populations suggested that northern Europe was colonized from two distinct Pleistocene refugia. Furthermore, microsatellites revealed highly supported grouping of mainland Swedish, Norwegian, Danish, German and Slovenian populations, suggesting that grayling from the northwestern and central Europe have descended from their southern conspecifics. The level of divergence between populations was substantial, even across short geographical distances. Although this was in part due to postglacial colonization patterns and contemporary barriers for gene flow, the high divergence estimates between hydrologically connected sampling locations implied efficient interpopulation reproductive isolation. Microsatellites revealed that the populations exhibited, on average, only 3.5 (+/-2.2) alleles per locus, indicating that T. thymallus has strikingly low levels of intrapopulation genetic diversity as compared with other freshwater fish species. Accordingly, as indicated by analysis of molecular variance (AMOVA), only 49.1-58.0% of the total grayling microsatellite diversity resided within populations. A latitudinal genetic diversity gradient, potentially resulting from glaciation-mediated founder events, was not evident. Alternatively, it is possible that grayling display limited dispersal behaviour/capability, leading to low long-term effective population sizes and, consequently, depauperate intrapopulation polymorphism. These findings have implications for conservation of T. thymallus. Importantly, they exemplify that microsatellites can be highly informative for intraspecific phylogeography studies dealing with substantial divergence scales.

Assessment of the population structure of five Finnish dog breeds with microsatellites.

Genetic variabilities within and between Finnish populations of Golden Retrievers, German Shepherds, Wirehaired Dachshunds, Pembroke Welsh Corgis and Bedlington Terriers were quantified with microsatellite allele numbers, observed heterozygosities, expected heterozygosities, FIS estimates, FST estimates and DS distances. In a sample of 50 individuals from each, breed and ten polymorphic loci, the highest genetic diversity was exhibited in the Wirehaired Dachshunds and lowest in the Bedlington Terriers. Although statistically significant deviations from the Hardy-Weinberg (H-W) equilibrium were observed, they occurred at an unexpectedly low frequency. Most strikingly, the extremely small Bedlington Terrier population displayed genotypes in H-W proportions in all investigated loci. The H-W deviations always occurred with positive FIS estimates, which, on average, were not larger than values reported for free-living canids. Genetic differentiation between the breeds was very large. As a comparison, present estimates were, on average, over two times higher than previously observed between breeds of sheep, and over two times higher than the highest estimates reported between human populations. Moreover, the highest DS distances were only slightly lower than the lowest values inferred between humans and chimpanzees. Severe bottlenecks in the recent past of the examined breeds were not statistically supported. The presented data imply genetic isolation and intense artificial selection in the history of these breeds of dogs.

Genetic lineages and postglacial colonization of grayling (Thymallus thymallus, Salmonidae) in Europe, as revealed by mitochondrial DNA analyses.

In stark contrast to other species within the Salmonidae family, phylogeographic information on European grayling, Thymallus thymallus, is virtually nonexistent. In this paper, we utilized mitochondrial DNA polymerase chain reaction-restriction fragment length polymorphism (mtDNA PCR-RFLP) and sequence variation to infer the postglacial dispersal routes of T. thymallus into and within northern Europe, and to locate geographically, potential evolutionarily distinct populations. Mitochondrial analyses revealed a total of 27 T. thymallus haplotypes which clustered into three distinct lineages. Average pairwise interlineage divergence was four and nine times higher than average intralineage divergence for RFLP and sequence data, respectively. Two European grayling individuals from the easternmost sample in Russia exhibited haplotypes more genetically diverged from any T. thymallus haplotype than T. arcticus haplotype, and suggested that hybridization/introgression zone of these two sister species may extend much further west than previously thought. Geographic division of the lineages was generally very clear with northern Europe comprising of two genetically differentiated areas: (i) Finland, Estonia and north-western Russia; and (ii) central Germany, Poland and western Fennoscandia. Average interpopulation divergence in North European T. thymallus was 10 times higher than that observed in a recent mtDNA study of North American T. arcticus. We conclude that (i) North European T. thymallus populations have survived dramatic Pleistocene temperature oscillations and originate from ancient eastern and central European refugia; (ii) genetic divergence of population groups within northern Europe is substantial and geographically distinct; and (iii) the remainder of Europe harbours additional differentiated assemblages that likely descend from a Danubian refugium. These findings should provide useful information for developing appropriate conservation strategies for European grayling and exemplify a case with a clear need for multinational co-operation for managing and conserving biodiversity.

The one that did not get away: individual assignment using microsatellite data detects a case of fishing competition fraud.

Assignment of an individual to the population from which it most probably originated based on its multilocus genotype has been widely applied in recent years. In this study, individual assignment based on microsatellite data was used to identify a case of fishing competition fraud. Despite the fact that the true population of origin was most probably not among the reference populations, recent modifications of the assignment tests were used in confidently excluding (p < 0.0001) the possibility of a 5.5 kg salmon (Salmo salar) originating from the fishing competition location, Lake Saimaa (south-east Finland). In fact, the probability of the suspect salmon originating from one of the regions that supply most of Finland's fish markets was found to be over 600 times higher than it originating from Lake Saimaa. When presented with this evidence, the offender confessed to purchasing the salmon at a local fish shop and criminal charges were laid. This study emphasizes the potential practical application of the individual assignment procedure, in particular the usefulness of confidently excluding populations as the origin of an individual. A similar strategy could be also used, for example in suspected cases of illegal poaching, in order to assign or exclude individuals from originating from a claimed population.

A convenient and efficient microsatellite-based assay for resolving parentages in dogs.

We present a practical and efficient parentage analysis test for dogs. The method makes use of ten polymorphic microsatellite markers combined in three multiplex-PCR reactions. All three multiplexes can be performed under the same cycling conditions and using a multiple-dye detection system the PCR-products can be pooled for electrophoresis. Amplifications in four different thermal cyclers produced easily interpretable results throughout demonstrating the repeatability of the assay. In order to evaluate the method's efficiency a total of 100 dogs from four different breeds were typed. Assuming one known parent, exclusion probabilities ranging from 99.34% to 99.93% were attained.