Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.     

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples

AIMS: To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis.

METHODS: Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay.

RESULTS: The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R²?=?0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6?×?10⁴ and 3.3?×?10⁶ genomes per µL of blood.

CONCLUSIONS: All qPCR assays had improved specificity and sensitivity over existing conventional PCR assays for diagnosis of T. orientalis Ikeda. The burden of Ikeda DNA in blood was demonstrated using an Ikeda-specific qPCR assay with titrated synthetic gene target.

CLINICAL RELEVANCE: Adoption of high-throughput DNA extraction and qPCR reduced T. orientalis and Ikeda diagnosis times. The Ikeda-specific qPCR assay provides a specific diagnosis for Ikeda in animals with signs of infection with T. orientalis and can be used to monitor the parasite load of Ikeda in blood.     

A Real-Time Polymerase Chain Reaction Assay for Quantification of Edwardsiella ictaluri in Catfish Pond Water and Genetic Homogeneity of Diagnostic Case Isolates from Mississippi

Abstract

A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri–specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.

Received October 20, 2010; accepted June 13, 2011     

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     

Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers

Background: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts.

Objective: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum.

Methods: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used.

Results: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ～10⁰ parasite mL^?1. For the blood samples, 40/69 (58%; CI 95%; 46%–69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing.

Conclusion: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.     

Diagnosis of bovine spongiform encephalopathy: A review

Summary

Cows affected with bovine spongiform encephalopathy (BSE) display chronic neurological signs consisting of behavioural changes, abnormalities of posture and movement, and/or hyperaesthesia. At present, there are no laboratory test available to diagnose BSE in the live animal. In this article, we describe the post‐mortem diagnostic examination of brains from BSE‐suspected cattle as currently performed at ID‐Lelystad. The routine laboratory diagnosis of BSE consists of histopathological examination of the brain and detection of the modified prion protein, PrP^BSE, in brain tissue. These tests, however, have the disadvantage of being laborious and time consuming, so that results are available only after several days.

Recently, at ID‐Lelystad a new post‐mortem test has been developed that enables screening of larger volumes of brain samples for PrP^BSE within 1 day. This BSE test is especially suited for slaughterline monitoring. A preliminary validation study has shown that both sensitivity and specificity are 100% compared to the gold diagnostic standard of histopathology.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

Evidence for absence of equine arteritis virus in the horse population of New Zealand

AIM: To summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of New Zealand.

METHODS: Analysis was carried out on results from laboratory tests carried out at the Ministry for Primary Industries Animal Health Laboratory (AHL) for equine arteritis virus from horses tested prior to being imported or exported, testing of stallions as part of the New Zealand equine viral arteritis (EVA) control scheme and testing as part of transboundary animal disease (TAD) investigations for exclusion of EVA. Horse breeds were categorised as Thoroughbred, Standardbred or other.

RESULTS: A total of 7,157 EVA serological test records (from import and export testing, EVA control scheme testing and TAD investigations) were available for analysis between 2005 and 2011. For the three breed categories a seroprevalence of ≤1.6% at the 95% confidence level was determined for each category. Between 2001 and 2011, as part of the EVA control scheme, the EVA status of 465 stallions was determined to be negative. During 2005–2011 EVA was excluded from 84 TAD investigations.

CONCLUSIONS: There was no evidence of equine arteritis virus being present in the general horse population outside of carrier stallions managed under the EVA control scheme.

CLINICAL RELEVANCE: Equine arteritis virus is absent from the general horse population of New Zealand.     

Blood chemistry for the diagnosis of hepatobiliary disease in birds. A review

Summary

A review is presented of current knowledge with regard to blood chemistry for the diagnosis of hepatobiliary disease in birds. Straightforward conclusions are difficult, because research on this subject has been limited.

Jaundice caused by hyperbilirubinaemia occurs infrequently in birds, because the main bile pigment is biliverdin and not bilirubin. A yellow discolouration of avian plasma is often caused by the presence of carotenoids. The diagnostic value of plasma ALAT concentrations is controversial. Although ASAT and LD are not specific for the liver they appear to be sensitive enzymes to detect liver cell damage.

GLDH is liver specific in a number of animal species (including birds) and might prove to be a useful enzyme for diagnostic purposes. AP and GGT seem less useful. Little information is available on the diagnostic value of plasma bile acids and plasma dye clearance tests.     

Species-specificity of equine and porcine Lawsonia intracellularis isolates in laboratory animals

Lawsonia intracellularis infection causes proliferative enteropathy (PE) in many mammalian species, with porcine and equine proliferative enteropathy (PPE and EPE) known worldwide. Hamsters are a well-published animal model for PPE infection studies in pigs. There is no laboratory animal model for EPE infection studies and it is not known whether there is species-specificity for equine or porcine isolates of L. intracellularis in animal models. The objective of this study was to determine whether it is possible to generate typical EPE lesions in hamsters after inoculation with an equine strain of L. intracellularis (EPE strain) and whether it is comparatively possible to generate PPE lesions in rabbits after inoculation with a porcine strain of L. intracellularis (PPE strain). In 2 separate trials, 4-week-old and 3-week-old weanling golden Syrian hamsters were challenged with EPE strains and compared to uninfected (both trials) and PPE-infected controls (Trial 2 only). Concurrently, 6 female New Zealand white juvenile rabbits were infected with PPE strain and observed concomitantly to 8 similar rabbits infected with EPE strain for a different experiment. Hamsters and rabbits were observed for 21 to 24 days post-infection (DPI), depending on the experiment. Neither infected species developed clinical signs. The presence of disease was assessed with diagnostic techniques classically used for pigs and horses: immune-peroxidase monolayer assay on sera; quantitative polymerase chain reaction (qPCR) detection of molecular DNA in feces; and hematoxylin and eosin (H&E) stain and immunohistochemistry (IHC) on intestinal tissues. Our results showed that EPE-challenged hamsters do not develop infection when compared with PPE controls (IHC, P = 0.009; qPCR, P = 0.0003). Conversely, PPE-challenged rabbits do not develop typical intestinal lesions in comparison to EPE-challenged rabbits, with serological response at 14 DPI being significantly lower (P = 0.0023). In conclusion, PPE and EPE strains appear to have different host-specificities for hamsters and rabbits, respectively.     

Cyanides and their toxicity: A literature review

Summary

Cyanide is a potent and rapidly‐acting asphyxiant which prevents tissue utilization of oxygen by inhibition of the cellular respiratory enzyme, cytochrome oxidase. Inhalation or ingestion of cyanide produces reactions within a few seconds and death within minutes. Cyanide toxicity of dietary origin has been implicated in acute animal deaths and as major etiologic factors in toxic ataxic neuropathy in man and as a cause of vision failure in humans suffering from tobacco amblyopia and leber's hereditary optic atrophy. Diagnosis of cyanide toxicity may be confirmed by a variety of laboratory procedures, but accurate assay is essential for proper conclusions from analysis of animal tissues several hours after death or from human samples in instances of chronic dietary exposure. Biological detoxification of cyanide is available through several routes, and the application of sodium nitrite with sodium thiosulfate or administration of methylene blue are effective treatment procedure. The environmental availability of cyanide in its various forms necessitates an understanding of its pathophysiology and responsible management of hazardous situations.     

Serological patterns,antibody half-life and shedding in urine of Leptospira spp. in naturally exposed sheep

Abstract

AIMS: To determine within-farm prevalence, longitudinal pattern of exposure measured by serology, antibody titre longevity and point prevalence of shedding in urine of Leptospira borgpetersenii serovar Hardjo and L. interrogans serovar Pomona in naturally infected sheep on a sample of commercial farms in New Zealand.

METHODS: On eight commercial sheep farms, between September 2011 and January 2014, blood samples were collected from 115–217 ewe lambs on each farm, at intervals of 2–11 months. They were analysed by microscopic agglutination test (MAT) for antibodies to L. borgpetersenii serovar Hardjo and L. interrogans serovar Pomona, using a titre cut-point of 48. Urine from 98 animals was tested by quantitative PCR (qPCR). The half-life of antibodies was estimated in 185 sheep for serovar Hardjo and 21 for Pomona, and the seroprevalence and mean titre of animals lost to follow-up was compared with those remaining in the study.

RESULTS: Within-flock seroprevalence for serovar Hardjo reached a maximum at 17–22 months of age, ranging from 79 to 100%. Seroprevalence for serovar Pomona rose above 10% on three farms and increased to 21–54% by 4–14 months. Seroconversions occurred mainly from late autumn to early summer at 7–15 months of age. Seroprevalences ranging from 3 to 76% for serovar Hardjo and 0.5 to 15% for serovar Pomona were observed up to 3 months of age, likely due to maternally derived immunity. The half-life of antibody in response to infection was estimated to be 6.7 (95% CI=5.8–7.9) months for serovar Hardjo and 6.3 (95% CI=4.8–9.0) months for Pomona. The prevalence of sheep with urine positive for leptospires on qPCR on each farm ranged from 11 to 88%. All but one of the qPCR-positive animals were seropositive for serovar Hardjo. On two farms where Pomona exposure was observed, animals that were lost to follow-up had a higher geometric mean titre for serovar Pomona than those remaining in the study.

CONCLUSIONS: This study demonstrated seasonal exposure from autumn to early summer in young sheep, a wide range of within-flock serological and shedding prevalence, and gives an estimation of the half-life of MAT titres in sheep. More extensive data are needed to fully understand the epidemiology of leptospirosis in sheep flocks across New Zealand and, along with economic analysis, to justify and design cost-effective and efficient control measures to protect human and animal health.     

Validation of a point-of-care polymerase chain reaction assay for detection of Streptococcus equi subspecies equi in rostral nasal swabs from horses with suspected strangles

This study aimed to validate a point-of-care polymerase chain reaction (PCR) assay for detection of Streptococcus equi subsp. equi (S. equi) in rostral nasal swabs from horses with suspected acute strangles and to compare the results against the molecular gold standard of quantitative polymerase chain reaction (qPCR). Two hundred thirty-two individual swabs of rostral nasal passages were characterized by qPCR as S. equi positive, S. equi subsp. zooepidemicus (S. zooepidemicus) positive, or S. equi and S. zooepidemicus negative. The specificity and sensitivity of the point-of-care PCR assay were 89% and 84%, respectively. The limits of detection of the qPCR assay and the point-of-care PCR analyzer were 3 and 277 eqbE target genes of S. equi, respectively. Overall agreement and short turnaround time make the point-of-care PCR assay a potential molecular diagnostic platform that will enhance the capability of equine veterinarians to timely support a diagnosis of strangles and institute proper biosecurity protocols.     

Autonomic innervation of the airways

Summary

A review is given of the literature concerning the autonomic innervation of airway smooth muscle. The cholinergic, adrenergic and non‐cholinergic non‐adrenergic (NANC) systems in humans and several animal species are discussed. The diagnostic and therapeutic possibilities and limitations of new receptor specific agonists and antagonists are also discussed.     

Detection of Streptococcus equi subspecies equi using a triplex qPCR assay

Genome sequencing data for Streptococcus equi subspecies equi and zooepidemicus were used to develop a novel diagnostic triplex quantitative PCR (qPCR) assay targeting two genes specific to S. equi (eqbE and SEQ2190) and a unique 100 base pair control DNA sequence (SZIC) inserted into the SZO07770 pseudogene of S. zooepidemicus strain H70. This triplex strangles qPCR assay can provide results within 2 h of sample receipt, has an overall sensitivity of 93.9% and specificity of 96.6% relative to the eqbE singlex assay and detects S. equi at levels below the threshold of the culture assay, even in the presence of contaminating bacteria.     

Animals in aging research: Requirements,pitfalls and a challenge for the laboratory animal specialist

Summary

The prime goal of aging research is to gain some insight into the basic mechanisms underlying the aging process. The long lifespan and frequent mobility of humans as well as the legal and ethical constraints on human experimentation make man unsuitable for studying the aging process. Therefore animals, particularly rodents, are used in aging research. In studying the aging process in animals, one hopes to find means for the prevention or amelioration of at least some of the disabilities of old age in man. Many intrinsic and extrinsic factors can influence the outcome of animal experiments; hence an extensive monitoring program is a prerequisite in aging research. An important role in controlling the quality of the animals is reserved for the laboratory animal specialist. Since he is faced with all aspects of laboratory animal science, aging research is a challenging area for such a specialist.     

Early Detection of Brucella Canis via Quantitative Polymerase Chain Reaction Analysis

Canine brucellosis is a reportable zoonotic disease that can lead to canine reproductive losses and human infection through contact with infected urine or other genitourinary secretions. Although many locations require testing and euthanasia of positive dogs, current diagnosis is limited by the time required for seroconversion, for example, presence of B. canis‐specific antibodies. The goal of this study was to determine the diagnostic ability of Brucella canis‐specific quantitative polymerase chain reaction (qPCR) assay to detect B. canis in field samples prior to serological positivity for faster diagnosis and prevention of transmission within kennels or in households. Two kennels, one of which was located in the owner's home, were sampled following observation of suggestive clinical signs and positive serology of at least one dog. Specimens obtained were comparatively analysed via serology and qPCR analysis. 107 dogs were analysed for B. canis infection via qPCR: 105 via whole‐blood samples, 65 via vaginal swab, six via urine and seven via genitourinary tract tissue taken at necropsy. Forty‐five dogs were found to be infected with canine brucellosis via qPCR, of which 22 (48.89%) were seropositive. A statistically significant number (P = 0.0228) of qPCR‐positive dogs, 5/25 (20.00%), seroconverted within a 30‐day interval after initial serologic testing. As compared to serology, qPCR analysis of DNA from vaginal swabs had a sensitivity of 92.31% and specificity of 51.92%, and qPCR analysis of DNA from whole‐blood samples had a sensitivity of 16.67% and specificity of 100%. B. canis outer membrane protein 25 DNA qPCR from non‐invasive vaginal swab and urine samples provided early detection of B. canis infection in dogs prior to detection of antibodies. This assay provides a critical tool to decrease zoonotic spread of canine brucellosis, its associated clinical presentation(s), and emotional and economic repercussions.     

The Gordon Memorial Lecture: novel approaches to controlling bacterial infections

ABSTRACT

1. There is huge emphasis in veterinary and agricultural science in understanding the basics of processes and exploiting them for benefits to the economy and human and animal welfare. It is always valuable to be able to step back from existing or favourite hypotheses and paradigms to look at an area of work or problem and see whether a different approach might be productive particularly by drawing parallels with other sometimes unrelated problems.

2. This approach has been used to explore (i) the use of live, attenuated Salmonella vaccines to generate a new form of competitive exclusion, (ii) gene expression technology for the design of improved inactivated vaccines (iii) use of cytokine therapy to reduce persistent carriage by Salmonella, (iv) using bacteriophages to reduce carcass contamination by food-borne pathogens and reduce carriage of antibiotic resistance plasmids.

3. The potential for extending virus therapy to parasite infections is also discussed.     

Endoscopy in birds

Summary

Endoscopic examination is described as a valuable diagnostic method in birds. In the introduction, reference is made to the literature on this subject. Apart from the classical approaches, alternative sites to enter the thoraco‐abdominal cavity in birds are discussed. The basic equipment for endoscopy in birds is described and a new biopsy forceps which makes a secundary puncture redundant is introduced.

After a discussion of the indications and complications, the value of the endoscopic examination is compared with other diagnostic methods for the diagnosis of avian tuberculosis in birds.