Comparative Evaluation of Molecular Diagnostic Tests for Nucleospora salmonis and Prevalence in Migrating Juvenile Salmonids from the Snake River,USA

Abstract

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and found that both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.

Received September 14, 2010; accepted November 14, 2010     

Field Evaluation of Sensitivity and Specificity of a Polymerase Chain Reaction (PCR) for Detection of Nucleospora salmonis in Rainbow Trout

Abstract

We determined the sensitivity and specificity of a nested polymerase chain reaction (PCR) for detection of the microsporidian parasite Nucleospora salmonis in kidney tissue of rainbow trout Oncorhynchus mykiss. Kidney tissues were sampled on three dates from 162 juvenile rainbow trout obtained from a California State fish hatchery where the organism was endemic. Kidney tissues were used to prepare imprints stained with May–Grünwald Giemsa and for extraction of genomic DNA for a nested PCR test for N. salmonis. Positive PCR results for N. salmonis were obtained from 1 of 100, 2 of 32, and 27 of 30 kidneys collected on the first, second, and third sample dates, respectively. Kidney tissues from 3 of 27 trout in the third sample that tested positively by PCR also had microscopic evidence of parasites in stained kidney imprints. No parasites were detected in the remaining 159 kidney samples examined microscopically. Sensitivity and specificity of the PCR assay were estimated by using maximum likelihood estimation based on cross-classified test results. This method yielded estimates of sensitivity of 99.99% and specificity of 99.87%. This field evaluation supports experimental evidence that the nested PCR test will be a valuable diagnostic tool for prevention and control of N. salmonis as well as for risk assessment associated with fish movements.     

Comparative diagnosis of parasitological,serological, and molecular tests in dourine-suspected horses

Study on comparative sensitivity of parasitological, serological, and molecular tests on 237 horses originating from two dourine-suspected districts of Arsi-Bale highlands of Ethiopia was conducted to determine the prevalence of the disease and degree of agreement of the diagnostic tests. Accordingly, the prevalence of the disease was found to be 4.6%, 36.7%, and 47.6% by parasitological Woo test, RoTat 1.2 and 18S PCR tests, respectively. The seroprevalence of the disease was 27.6% in CATT/Trypanosoma evansi test. In Ethiopia, it was for the first time that trypanosomes from dourine suspected horses were demonstrated in 4.6% of the animals using Woo test. The findings of the present study disclosed that dourine is highly prevalent and one of the major diseases of horses in the area. There was no statistically significant difference (P > 0.05) in prevalence of the disease between districts, sexes, and age groups of the animals. However, there was a statistically significant difference (P < 0.05) in the prevalence of the disease between emaciated and animals with good body condition. Assessment of the degree of agreement of the diagnostic tests employed revealed low to fair ( k = 0.1 - 0.4 ) \left( {k = 0.{1} - 0.{4}} \right) with significantly higher sensitivity by PCR than other tests.     

Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs

Rapid identification of porcine Brachyspira species is required in order to differentiate pathogenic from non-pathogenic species. The aim of our study was to compare a recently described genetic method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), nox RFLP-PCR assay, and three species-specific PCRs described previously in the literature with a 16S rRNA gene RFLP-PCR discriminatory reference assay (16S RFLP-PCR) for the identification of Brachyspira spp. of swine origin. In this study, 20 porcine spirochaetal strains were identified and compared to 33 reference strains by 16S RFLP-PCR and nox RFLP-PCR and three species-specific PCRs. RFLP-PCR methods showed concordant results for 47 strains and discordances for 6 strains (2 differently identified and 4 not revealed by nox RFLP-PCR). In our hands species-specific PCRs showed concordant results with 16S and nox RFLP-PCR for 43 strains and discordances for 10 strains (2 differently identified and 8 not amplified). The same results observed testing the 20 field-isolated spirochaetes were obtained for the corresponding porcine faecal samples. The detection limit was 10(2) -10(3) cells/g of faeces for 16S rRNA gene PCR and 10(4) cells/g of faeces for nox PCR. In our experience nox RFLP-PCR appeared successful for the speciation of B. hyodysenteriae reserving 16S RFLP-PCR for all other pathogenic and non-pathogenic Brachyspira species. Among the species-specific PCR assays tested only that for B. pilosicoli was useful in our hands.     

A field evaluation of serological and cellular diagnostic tests for bovine tuberculosis.

This paper describes the field evaluation of a serological test and a new in vitro assay for cell-mediated reactivity for the diagnosis of bovine tuberculosis. The use of a Mycobacterium bovis-specific antigen (MPB-70) in an ELISA to test the serological response to tuberculosis infection resulted in a specificity of 96.4% and a sensitivity of 18.1%. The most favourable results were obtained with the interferon gamma (IFN-gamma) assay which had a sensitivity of 81.8% and a specificity of 99.1%. Respective figures for the single intradermal tuberculin test were 68.1% and 96.7%. The use of MPB-70 as the antigen in the IFN-gamma assay reduced the sensitivity of this assay, without producing any useful increase in specificity. The IFN-gamma assay was also demonstrated to be a practical diagnostic test for use with large groups of cattle.     

Comparative diagnostic potential of three serological tests for abortive Q fever in goat herds

Performances of an ELISA, an immunofluorescence assay (IFA) and a complement fixation test (CFT) were assessed for detecting antibodies against Coxiella burnetii after Q fever abortions in naturally infected goats. The goal of the study was to provide information useful for veterinary serodiagnosis in regard to categories of goats either experiencing Q fever abortion or not, blood sampling times and recommended cut-offs. The study was conducted on eight goat herds with evidence of C. burnetii abortions. In each herd, at least 5 goats that had aborted and 10 goats prior to parturition or at term were monitored 15, 30 and 60 days (D15, D30, D60) after the onset of Q fever abortion. The overall CFT results distribution did not differ between the two groups of goats and showed poor agreement with the ELISA results. In contrast, the ELISA and IFA results revealed comparable significant differences, but overall the ELISA test was slightly more sensitive than the IFA test. Seroprevalence, according to ELISA and IFA respectively, was higher in the aborting (88% and 82%) than in the non-aborting group (60% and 50%). High levels of serum antibodies were detected in goats post-abortion with an average of 114 %OD using ELISA and a log10(titer) of 2.4 using IFA. Strongly positive ELISA (%OD>80) and positive IFA results (log10(titers)>1.9) were significantly associated with abortion. Sampling on D15 gave the best association with ORs of 10 for ELISA and 6 for IFA. The practical interest of these results is discussed.     

A Bayesian evaluation of six diagnostic tests for foot-and-mouth disease for vaccinated and non-vaccinated cattle

The sensitivity and specificity of six ELISA tests for foot-and-mouth disease (FMD) to discriminate between sero-converted (for non-structural FMD virus proteins) and non-sero-converted cattle were evaluated for vaccinated and unvaccinated cattle. Since none of the tests could be considered as a proper reference test and for about half of the tested sera the true status (sero-converted or not for non-structural proteins, i.e. presence of antibodies) of the animals was unknown, a Bayesian analysis employing a latent class model was used that did not rely on the use of a reference test or gold standard. Prior information about prevalence for subsets of the data and specificity of the tests was incorporated into the analysis. The specificity of the six tests for vaccinated and non-vaccinated cattle ranged from 96 to 99%. For vaccinated cattle, one test stood out with an estimated sensitivity of 94% (95% CI from 89.8 to 98.1%). Second best for vaccinated cattle were two tests with estimated sensitivities of 85% (95% CI from 78.9 to 89.7%) and 92% (95% CI from 86.2 to 95.6%). For non-vaccinated cattle, the sensitivities of these three tests were around 97%. The remaining three tests showed lower estimated sensitivity for vaccinated cattle, ranging from 57 to 79%.     

A Bayesian approach for the evaluation of six diagnostic assays and the estimation of Cryptosporidium prevalence in dairy calves

The prevalence of Cryptosporidium in calves and the test properties of six diagnostic assays (microscopy (ME), an immunofluorescence assay (IFA), two ELISA and two PCR assays) were estimated using Bayesian analysis. In a first Bayesian approach, the test results of the four conventional techniques were used: ME, IFA and two ELISA. This four-test approach estimated that the calf prevalence was 17% (95% Probability Interval (PI): 0.1-0.28) and that the specificity estimates of the IFA and ELISA were high compared to ME. A six-test Bayesian model was developed using the test results of the 4 conventional assays and 2 PCR assays, resulting in a higher calf prevalence estimate (58% with a 95% PI: 0.5-0.66) and in a different test evaluation: the sensitivity estimates of the conventional techniques decreased in the six-test approach, due to the inclusion of two PCR assays with a higher sensitivity compared to the conventional techniques. The specificity estimates of these conventional assays were comparable in the four-test and six-test approach. These results both illustrate the potential and the pitfalls of a Bayesian analysis in estimating prevalence and test characteristics, since posterior estimates are variables depending both on the data at hand and prior information included in the analysis. The need for sensitive diagnostic assays in epidemiological studies is demonstrated, especially for the identification of subclinically infected animals since the PCR assays identify these animals with reduced oocyst excretion, which the conventional techniques fail to identify.     

Detection of the causative agent of furunculusis, Aeromonas salmonicida in salmonids of the Krka River

In this paper we describe the bacterial community associated with salmonids from the Krka River. Diversity analysis demonstrated that majority of the recovered bacteria were related to Aeromonadaceae group. Bacterial analysis also revealed the presence of Shigella spp. and Pseudomonas fluorescens. Isolation of Aeromonas salmonicida from trout, presents first isolation of this bacteria Croatian rivers.     

Estimation of sensitivity and specificity of diagnostic tests and disease prevalence when the true disease state is unknown

The performance of a new diagnostic test is frequently evaluated by comparison to a perfect reference test (i.e. a gold standard). In many instances, however, a reference test is less than perfect. In this paper, we review methods for estimation of the accuracy of a diagnostic test when an imperfect reference test with known classification errors is available. Furthermore, we focus our presentation on available methods of estimation of test characteristics when the sensitivity and specificity of both tests are unknown. We present some of the available statistical methods for estimation of the accuracy of diagnostic tests when a reference test does not exist (including maximum likelihood estimation and Bayesian inference). We illustrate the application of the described methods using data from an evaluation of a nested polymerase chain reaction and microscopic examination of kidney imprints for detection of Nucleospora salmonis in rainbow trout.     

Comparative evaluation of positive tests to Mycobacterium avium subsp. paratuberculosis in clinically healthy sheep and goats in south-west Greece using molecular techniques, serology, and culture

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis, which affects mainly ruminants although there is a growing concern about its possible implication in Crohn's disease in humans especially in connection with environmental spread and risks to the food chain. Retail cheese may represent a significant source of human exposure to MAP and the aim of this study was to assess MAP status in clinically healthy sheep and goats in Greece, comparing techniques routinely used in the positive diagnosis of the disease. From a total of 30 flocks, 632 sheep and goats had faecal, serum, and whole-blood samples examined by culture, complement fixation test (CFT), and polymerase chain reaction (PCR) targeted at IS900, IS1245, and IS6110. PCR produced positive results in 21% of the animals tested, with 5.6%, 3.9%, and 11.5% being identified as MAP, Mycobacterium avium subsp. avium, and Mycobacterium tuberculosis complex, respectively. CFT produced positive and suspicious results in 4.4% and 14.4% of the cases. Faecal cultures were negative in all but a single case that was identified as restriction fragment length polymorphism (RFLP)-type BC1. Agreement between results obtained by PCR and CFT was poor with isolated cases although an assessment of the MAP positive tests produced similar results for both methods. The findings indicate the need for additional measures of control, although the costs may be substantial if public health protection justifies elimination of MAP from livestock.     

国产碱式氯化铜与美国碱式氯化铜物相对比研究

通过对5个国产(深圳)的饲料级碱式氯化铜样品和2个美国微量营养元素公司生产的饲料级碱式氯化铜样品及其粉晶X射线衍射数据进行了分析鉴定及物相对比研究,5个国产的碱式氯化铜衍射谱图完全相同,鉴定结果为斜氯铜矿(Botallackite)型碱式氯化铜,不论5个样品的生产工艺有何差异,但产品的物相和纯度是一致的;2个美国产碱式氯化铜虽然样品的形态差异较大,但其衍射谱图也完全相同,鉴定结果主要为氯铜矿(Atacmite)型碱式氯化铜,含有微量的副氯铜矿(Parata-camite)型碱式氯化铜,二者之间为同质异构关系,为国产和美国饲料级碱式氯化铜的结构鉴定提供了依据。     

Bovine and Caprine Brucellosis in Bangladesh: Bayesian evaluation of four serological tests,true prevalence,and associated risk factors in household animals

A cross-sectional study was carried out to estimate the true prevalence of Brucella spp. and identify allied risk factors/indicators associated with brucellosis in the Dinajpur and Mymensingh districts of Bangladesh. A total 320 stratified random blood samples were collected and tested in parallel for Brucella antibodies using Rose Bengal (RBT), slow agglutination (SAT), and indirect and competitive ELISA. In addition, a structured questionnaire was administered to each household herd owner to gather information regarding potential risk factors. Both univariate and multivariate logistic regression analyses were used to identify potential risk factors or indicators at animal level. A Bayesian approach was used to estimate the true prevalence of brucellosis along with the test performances (Se and Sp). The estimated animal level true prevalence in cattle was 9.70 % (95 % CPI 5.0–16 %) and in goat 6.3 % (95 % CPI 2.8–11.0 %). The highest sensitivity was achieved by SAT ranges from 69.6 to 78.9 %, and iELISA was found to be more specific (97.4 to 98.8 %) in comparison with other tests. On the other hand, a significant level of (P?<?0.05) Brucella seropositivity was found in cattle that breed naturally compared with those that undergo artificial insemination. In goats, exotic breeds were significantly associated (P?<?0.05) with Brucella seroprevalence compared with indigenous breeds. Goats with a previous records of abortion and/or retained placenta were also found to have significant levels (P?<?0.05). Cows with previous abortion records showed higher odds (18 times) of being seropositive. None of the evaluated tests can be recommended to apply alone for the diagnosis of bovine and caprine brucellosis.     

High prevalence and genotypes of Toxoplasma gondii isolated from organic pigs in northern USA

The ingestion of undercooked pork infected with Toxoplasma gondii is considered an important source of transmission of this parasite. While T. gondii infection in confinement raised market pigs (market pigs are typically used for fresh, unprocessed pork products) in the USA has decreased significantly over the last 20 years, infection levels in pigs with access to the outdoors can be quite high. An upsurge in consumer demand for 'organically raised', 'humanely raised' and 'free range' pork products has resulted in increasing numbers of hogs being raised in non-confinement systems. To determine T. gondii infection rate in these organic pigs, prevalence of T. gondii in organically raised pigs in two establishments (Farm 1, Farm 2) in Michigan was investigated. Serum and tissue samples from 33 pigs on the farm were available for T. gondii evaluation at slaughter. Serological testing was performed using both ELISA and the modified agglutination test (MAT). Antibodies to T. gondii were detected by both ELISA and MAT in 30 of 33 animals with MAT titers of 1:25 in three, 1:50 in six, 1:100 in seven, 1:200 in 13, and 1:400 in one. Hearts of all 33 pigs were bioassayed for T. gondii in mice; T. gondii was isolated from 17 pigs including one from a seronegative (both ELISA and MAT) pig. Genetic typing of 16 of the 17 T. gondii isolates using the SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico loci revealed clonal Type II from Farm 1 and clonal Type III on Farm 2. These results revealed very high prevalence of T. gondii in organic pigs for the first time in USA, indicating potentially increased health risk of consuming organic swine products.     

Comparison of the sensitivity of different diagnostic tests for pancreatitis in cats

The objective of this study was to compare the sensitivity of different diagnostic tests for pancreatitis in cats. Twenty-one cats with confirmed pancreatitis were evaluated at the Small Animal Clinic of the School of Veterinary Medicine in Hannover, Germany, between September 1997 and January 1999. Clinical signs of affected cats were nonspecific, with 95% of the cats showing anorexia and 86% lethargy. Also, hematologic and biochemical abnormalities of affected cats were nonspecific. Serum feline trypsin-like immunoreactivity (fTLI) in these 21 cats with pancreatitis was 127.5 +/- 109.5 microg/L (mean +/- SD; range, 24-500 microg/L). Fourteen of the 21 cats with pancreatitis had complicating conditions. Their serum fTLI was 153.9 +/- 124.3 microg/L (mean +/- SD; range, 29 500 microg/L). In this study, abdominal ultrasound showed a sensitivity for pancreatitis of 24%, and abdominal computed tomography had a sensitivity of 20%. Serum fTLI had a sensitivity between 86% when a cut-off value of 49 microg/L was used (upper limit of the control range) and 33% when a cut-off value of 100 microg/L was used. We conclude that in this group of cats with pancreatitis, measurement of serum fTLI was the most sensitive diagnostic test of those evaluated. Abdominal ultrasound, however, may be a valuable diagnostic tool in some cats with pancreatitis.     

Morphological and molecular characterisation of Gyrodactylus salmonis (Platyhelminthes, Monogenea) isolates collected in Mexico from rainbow trout (Oncorhynchus mykiss Walbaum)

Gyrodactylus salmonis (Yin et Sproston, 1948) isolates collected from feral rainbow trout, Oncorhynchus mykiss (Walbaum) in Veracruz, southeastern Mexico are described. Morphological and molecular variation of these isolates to G. salmonis collected in Canada and the U.S.A. is characterised. Morphologically, the marginal hook sickles of Mexican isolates of G. salmonis closely resemble those of Canadian specimens - their shaft and hook regions align closely with one another; only features of the sickle base and a prominent bridge to the toe permit their separation. The 18S sequence determined from the Mexican specimens was identical to two variable regions of SSU rDNA obtained from a Canadian population of G. salmonis. Internal transcribed spacer (ITS) regions (spanning ITS1, 5.8S and ITS2) of Mexican isolates of G. salmonis are identical to ITS sequences of an American population of G. salmonis and to Gyrodactylus salvelini Kuusela, Zi?tara et Lumme, 2008 from Finland. Analyses of the ribosomal RNA gene of Mexican isolates of G. salmonis show 98-99% similarity to those of Gyrodactylus gobiensis Gl?ser, 1974, Gyrodactylus salaris Malmberg, 1957, and Gyrodactylus rutilensis Gl?ser, 1974. Mexican and American isolates of G. salmonis are 98% identical, as assessed by sequencing the mitochondrial cox1 gene. Oncorhynchus mykiss is one of the most widely-dispersed fish species in the world and has been shown to be an important vector for parasite/disease transmission. Considering that Mexican isolates of G. salmonis were collected well outside the native distribution range of all salmonid fish, we discuss the possibility that the parasites were translocated with their host through the aquacultural trade. In addition, this study includes a morphological review of Gyrodactylus species collected from rainbow trout and from other salmonid fish of the genus Oncorhynchus which occur throughout North America.     

Comparative evaluation of Rose Bengal plate agglutination test, mallein test, and some conventional serological tests for diagnosis of equine glanders.

The Rose Bengal plate agglutination test (RBT) was evaluated for the diagnosis of equine glanders, and its diagnostic efficiency was compared with that of mallein and other serological tests, including indirect hemagglutination test (IHAT), complement fixation test (CFT), and modified counter immunoelectrophoresis test (mCIET). Sera from 70 naturally infected culture-positive, 96 potentially exposed cohorts, and 110 healthy equines were tested. All tests but mCIET showed 100% specificity when testing the sera from glanders-negative equines. The calculated sensitivities of RBT, IHAT, CFT, mCIET, and mallein test when testing culture-positive equines were 90.0, 97.1, 91.4, 81.4, and 75.7%, respectively. The RBT was significantly (P < 0.05) more sensitive than the mallein test and mCIET. The positive and negative predictive values of each test (RBT, IHAT, CFT, mallein test, and mCIET) were as follows: 100 and 94, 100 and 98.2, 100 and 96.7, 100 and 86.6, and 90.5 and 88.6, respectively. On comparing glandered and nonglandered animals, the highest agreement (0.987) was found between RBT and CFT followed by RBT and IHAT (0.940), RBT and mallein test (0.871), and RBT and mCIET (0.852). Because the RBT is simpler and rapid to perform, the inclusion of the test as a supplementary test for the diagnosis of glanders in field conditions is recommended.     

Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests

We review the principles and practical application of receiver-operating characteristic (ROC) analysis for diagnostic tests. ROC analysis can be used for diagnostic tests with outcomes measured on ordinal, interval or ratio scales. The dependence of the diagnostic sensitivity and specificity on the selected cut-off value must be considered for a full test evaluation and for test comparison. All possible combinations of sensitivity and specificity that can be achieved by changing the test's cut-off value can be summarised using a single parameter; the area under the ROC curve. The ROC technique can also be used to optimise cut-off values with regard to a given prevalence in the target population and cost ratio of false-positive and false-negative results. However, plots of optimisation parameters against the selected cut-off value provide a more-direct method for cut-off selection. Candidates for such optimisation parameters are linear combinations of sensitivity and specificity (with weights selected to reflect the decision-making situation), odds ratio, chance-corrected measures of association (e. g. kappa) and likelihood ratios. We discuss some recent developments in ROC analysis, including meta-analysis of diagnostic tests, correlated ROC curves (paired-sample design) and chance- and prevalence-corrected ROC curves.