Background: The Finnish dairy herd recording system maintains production and health records of cows and herds. Veterinarians and farmers register veterinary treatments in the system. Milk samples for microbiological analysis are routinely taken from mastitic cows. The laboratory of the largest dairy company in Finland, Valio Ltd., analyzes most samples using real-time PCR. This study addressed pathogen-specific microbiological data and treatment and culling records, in combination with cow and herd characteristics, from the Finnish dairy herd recording system during 2010-2012. Results: The data derived from 240,067 quarter milk samples from 93,529 dairy cows with mastitis; 238,235 cows from the same herds served as the control group. No target pathogen DNA was detected in 12% of the samples. In 49% of the positive samples, only one target species and in 19%, two species with one dominant species were present. The most common species in the samples with a single species only were coagulase-negative staphylococci (CNS) (43%), followed by Staphylococcus aureus (21%), Streptococcus uberis (9%), Streptococcus dysgalactiae (8%), Corynebacterium bovis (7%), and Escherichia coli (5%). On average, 36% of the study cows and 6% of the control cows had recorded mastitis treatments during lactation. The corresponding proportions were 16 and 6% at drying-off. For more than 75% of the treatments during lactation, diagnosis was acute clinical mastitis. In the milk samples from cows with a recorded mastitis treatment during lactation, CNS and S. aureus were most common, followed by streptococci. Altogether, 48% of the cows were culled during the study. Mastitis was reported as the most common reason to cull; 49% of study cows and 18% of control cows were culled because of mastitis. Culling was most likely if S. aureus was detected in the milk sample submitted during the culling year. Conclusions: The PCR test has proven to be an applicable method also for large-scale use in bacterial diagnostics. In the present study, microbiological diagnosis was unequivocal in the great majority of samples where a single species or two species with one dominating were detected. Coagulase-negative staphylococci and S. aureus were the most common species. S. aureus was also the most common pathogen among the culled cows, which emphasizes the importance of preventive measures.

Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser-induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high-throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.

Infections with the nine human herpesviruses (HHVs) are globally prevalent and characterized by lifelong persistence. Reactivations can potentially manifest as life-threatening conditions for which the demonstration of viral DNA is essential. In the present study, we developed HERQ-9, a pan-HHV quantitative PCR designed in triplex reactions to differentiate and quantify each of the HHV-DNAs: (i) herpes simplex viruses 1 and 2 and varicella-zoster virus; (ii) Epstein-Barr virus, human cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus; and (iii) HHV-6A, -6B, and -7. The method was validated with prequantified reference standards as well as with mucocutaneous swabs and cerebrospinal fluid, plasma, and tonsillar tissue samples. Our findings highlight the value of multiplexing in the diagnosis of many unsuspected, yet clinically relevant, herpesviruses. In addition, we report here frequent HHV-DNA co-occurrences in clinical samples, including some previously unknown. HERQ-9 exhibited high specificity and sensitivity (LOD95 of similar to 10 to similar to 17 copies/reaction), with a dynamic range of 10' to 10 6 copies/p.I. Moreover, it performed accurately in the coamplification of both high- and low-abundance targets in the same reaction. In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance to clinical management, the method is valuable for the assessment of hitherto-unexplored synergistic effects of herpesvirus coinfections. Furthermore, its high sensitivity enables studies on the human virome, often dealing with minute quantities of persisting HHVs. IMPORTANCE By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.

Voles (Arvicolinae, Rodentia) are known carriers of zoonotic bacteria such as Bartonella spp. and Francisella tularensis. However, apart from F. tularensis, the bacterial microbiome of voles has not previously been determined in Finland and rarely elsewhere. Therefore, we studied liver samples from 61 voles using 16S ribosomal RNA gene PCR analysis, followed by Sanger sequencing. Twenty-three of these samples were also studied with tag-encoded pyrosequencing. The samples originated from 21 field voles (Microtus agrestis), 37 tundra voles (Microtus oeconomus), and 3 bank voles (Myodes glareolus). With the more conventional 16S rDNA PCR analysis, 90 (33%) of the recovered 269 sequence types could be identified to genus level, including Bartonella, Francisella, Mycoplasma, Anaplasma, and Acinetobacter in 31, 15, 9, 9, and 9 sequences, respectively. Seventy-five (28%) matched best with sequences of uncultured bacteria, of which 40/75 could be classified to the order Clostridiales and, more specifically, to families Lachnospiraceae and Ruminococcaceae. Pyrosequencing from 23 samples revealed comparable and similar results: clinically relevant bacterial families such as Mycoplasmataceae, Bartonellaceae, Anaplasmataceae, and Francisellaceae were recognized. These analyses revealed significant bacterial diversity in vole livers, consisting of distinct and constant sequence patterns reflecting bacteria found in the intestinal gut, but including some known zoonotic pathogens as well. The molecular bacterial sequence types determined with the two different techniques shared major similarities and verified remarkable congruency between the methods.

Common bean (Phaseolus vulgaris) is an annual grain legume that was domesticated in Mesoamerica (Central America) and the Andes. It is currently grown widely also on other continents including Africa. We surveyed seedborne viruses in new common bean varieties introduced to Nicaragua (Central America) and in landraces and improved varieties grown in Tanzania (eastern Africa). Bean seeds, harvested from Nicaragua and Tanzania, were grown in insect-controlled greenhouse or screenhouse, respectively, to obtain leaf material for virus testing. Equal amounts of total RNA from different samples were pooled (30-36 samples per pool), and small RNAs were deep-sequenced (Illumina). Assembly of the reads (21-24 nt) to contiguous sequences and searches for homologous viral sequences in data-bases revealed Phaseolus vulgaris endornavirus 1 (PvEV-1) and PvEV-2 in the bean varieties in Nicaragua and Tanzania. These viruses are not known to cause symptoms in common bean and are considered non-pathogenic. The small-RNA reads from each pool of samples were mapped to the previously characterized complete PvEV-1 and PvEV-2 sequences (genome lengths ca. 14 kb and 15 kb, respectively). Coverage of the viral genomes was 87.9-99.9%, depending on the pool. Coverage per nucleotide ranged from 5 to 471, confirming virus identification. PvEV-1 and PvEV-2 are known to occur in Phaseolus spp. in Central America, but there is little previous information about their occurrence in Nicaragua, and no information about occurrence in Africa. Aside from Cowpea mild mosaic virus detected in bean plants grown from been seeds harvested from one region in Tanzania, no other pathogenic seedborne viruses were detected. The low incidence of infections caused by pathogenic viruses transmitted via bean seeds may be attributable to new, virus-resistant CB varieties released by breeding programs in Nicaragua and Tanzania.

Background. Congenital cytomegalovirus (cCMV) infection is the most common congenital infection and causes significant morbidity. This study was undertaken to evaluate the benefits of screening newborns for cCMV and to understand the cCMV disease burden in Finland. Methods. Infants born in Helsinki area hospitals were screened for CMV by testing their saliva with a real-time polymerase chain reaction assay. The CMV-positive infants and matched controls were monitored to determine their neurodevelopmental, audiological, and ophthalmological outcomes at 18 months of age. Griffiths Mental Development Scales, otoacoustic emission and sound field audiometry, and ophthalmologic examination were performed. Results. Of the 19 868 infants screened, 40 had confirmed cCMV infection (prevalence, 2 in 1000 [95% confidence interval, 1.4-2.6 in 1000]). Four (10%) infants had symptomatic cCMV. Griffiths general quotients did not differ significantly between the CMV-positive (mean, 101.0) and control (mean, 101.6) infants (P = .557), nor did quotients for any of the Griffiths subscales (locomotion, personal-social, hearing and language, eye and hand, performance) (P = .173-.721). Four of 54 CMV-positive ears and 6 of 80 CMV-negative ears failed otoacoustic emission testing (P = 1.000). The mean minimal response levels over the frequencies 500 Hz to 4 kHz in the sound field audiometry did not differ between CMV-positive (mean, 34.31-dB hearing level) and control (mean, 32.73-dB hearing level) infants (P = .338). No CMV-related ophthalmologic findings were observed. Conclusions. The prevalence of cCMV was low, and outcomes at 18 months of age did not differ between the infected infants and healthy control infants. With such a low burden in Finland, universal newborn screening for cCMV seems unwarranted.