The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results.     

Comparison of Resistance to Tomato Leaf Curl Virus (India) and Tomato Yellow Leaf Curl Virus (Israel) among Lycopersicon Wild Species, Breeding Lines and Hybrids

The objective of this study was to screen wild and domesticated tomatoes for resistance to Tomato yellow leaf curl virus, Israel (TYLCV-Is) and Tomato leaf curl virus from Bangalore isolate 4, India (ToLCV-[Ban4]) to find sources of resistance to both viruses. A total of 34 tomato genotypes resistant/tolerant to TYLCV-Is were screened for resistance to ToLCV-[Ban4] under glasshouse and field conditions at the University of Agricultural Sciences, Bangalore, India. Resistance was assessed by criteria like disease incidence, symptom severity and squash-blot hybridization. All the tomato genotypes inoculated with ToLCV-[Ban4] by the whitefly vector Bemisia tabaci (Gennadius) produced disease symptoms. In some plants of the lines 902 and 910, however, the virus was not detected by hybridization. The tomato genotypes susceptible to ToLCV-[Ban4] by whitefly-mediated inoculation were also found susceptible to the virus under field conditions. However, there were substantial differences between genotypes in disease incidence, spread, symptom severity and crop yield. Despite early disease incidence, many genotypes produced substantially higher yields than the local hybrid, Avinash-2. Sixteen tomato genotypes from India resistant/tolerant to ToLCV-[Ban4] were also tested for TYLCV-Is resistance at the Hebrew University of Jerusalem, Rehovot, Israel. Accessions of wild species, Lycopersicon hirsutum LA 1777 and PI 390659 were the best sources of resistance to both viruses. Lines 902 and 910, which were, resistant to TYLCV-Is were only tolerant to ToLCV-[Ban4] and accession Lycopersicon peruvianum CMV Sel. INRA, resistant to ToLCV-[Ban4], was only tolerant to TYLCV-Is. Implications of using the resistant lines in breeding programme is discussed.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.     

Identification of Chlamydophila pneumoniae in an emerald tree boa, Corallus caninus.

Tissues were evaluated from emerald tree boas, Corallus caninus, from a collection in which chlamydiosis was diagnosed. To determine the strain of chlamydia infecting these snakes, tissue samples from 5 frozen snakes were tested by a quantitative TaqMan polymerase chain reaction (PCR) test and a PCR sequence analysis test. Of the 22 samples tested, 9 were categorized as either positive or weakly positive with the TaqMan test, and 6 yielded an amplicon using a serial PCR test that amplified a portion of the 23S ribosomal RNA gene. A PCR product suitable for sequencing was obtained from the heart of one of the snakes. Sequence analysis showed that the snake had been infected with Chlamydophila pneumoniae. These findings show that C. pneumoniae can infect emerald tree boas, broadening the range of reptiles known to be infected by this primarily human pathogen.     

Investigation of a possible chain of Streptococcus suis infection in a pig breeding community using PCR-based genotyping

Streptococcus (S.) suis plays an important role in pig breeding causing invasive diseases such as meningitis and polyarthritis. Herd problems with Streptococcus suis are common in many pig farms and are frequently characterised by disease outbreaks. In this context, it is often important to identify chains of infection, e.g. between a farrow-to-wean and a grower farm. In the following case report a possible chain of infection among the different farms of a farrow-to-finish system was investigated. In two grower units herd problems with S. suis were present; the mortality was higher than 5 %. It appeared likely, that the streptococci causing these problems were transmitted from a single farrow-to-wean unit to the two different grower farms. In the respective farms swabs were taken from different healthy animals and, in the case of the grower farms, also from the infected animals. Genotypic profiling of strains by a multiplex-PCR and AFLP typing method revealed that two different S. suis pathotypes were responsible for the herd problems. Both pathotypes could not be detected in the farrow-to-wean farm.Thus, a chain of infection originating from the farrow-to-wean farm appeared unlikely. The multiplex PCR was in this case sufficient to elucidate the described problem.     

Hepatitis E virus in Cuba: A cross-sectional serological and virological study in pigs and people occupationally exposed to pigs

Surveillance of hepatitis E virus (HEV) in risk groups is an important strategy to monitor its circulation pattern and to timely detect changes thereof. The aims of this cross-sectional study were to estimate the prevalence of HEV infections in pigs and humans from different regions of the country, to identify risk factors for increasing anti-HEV IgG prevalence and to characterize HEV strains. The presence of anti-HEV antibodies was assessed by commercial ELISA in serum samples from the general population, farm and slaughterhouse employees, as well as pigs sampled in the three regions of Cuba from February to September 2016. Overall, individuals with occupational exposure to swine or swine products (70/248, 28.2%) were 4 times more likely to be seropositive compared to the general population (25/285, 8.7%; OR: 4.18; p < .001). Within the risk group, risk factors included age, number of years working in a professional activity with direct exposure to swine, geographic region and distance between residence and closest professional swine setting, while wearing gloves had a protective effect. Prevalence of total anti-HEV antibodies in swine was 88.2% (165/187) and HEV RNA was detected by real-time RT-PCR in 9.2% (16/173) swine stools. All HEV strains sequenced clustered within genotype 3. Some strains clearly belonged to subtype 3a, while another group of strains was related with subtypes 3b and 3 k but partial HEV sequences did not allow unequivocal subtype assignment. These findings suggest that the high HEV exposure in Cuban individuals with swine-related occupations could be due to enzootic HEV in certain regions, direct contact with infectious animals or their products as well as environmental contamination.