Detection of Salmonella spp in fecal specimens by use of real-time polymerase chain reaction assay

OBJECTIVE: To use real-time polymerase chain reaction (PCR) technology to develop a highly sensitive and specific diagnostic assay for the detection of Salmonella spp in fecal specimens. SAMPLE POPULATION: 299 fecal specimens from cattle, horses, and dogs. PROCEDURE: Enrichment of fecal specimens was followed by genomic DNA extraction by use of commercially available isolation kits. Real-time PCR assay was performed to target a Salmonella spp-specific DNA segment. Results of real-time PCR assay were compared with bacterial culture results to determine relative sensitivity and specificity. RESULTS: Use of the spaQ primer-probe set resulted in a relative sensitivity of 100% and a specificity of 98.2%, compared with bacterial culture results when tested on 299 clinical fecal specimens. CONCLUSIONS AND CLINICAL RELEVANCE: A rapid, sensitive, and specific assay for the detection of Salmonella spp from enriched clinical fecal specimens was developed. This technique would be highly valuable in clinical settings to help avoid or mitigate the complications arising from an outbreak of salmonellosis in a herd or among patients of a veterinary hospital.     

Enzootic pneumonia of pigs--a diagnostic dilemma

In an enzootic pneumonia-free Australian pig herd, an outbreak of a severe respiratory disease in the grow-out herd was initially diagnosed as acute tracheitis and pneumonia precipitated by the dusty environment, with a superimposed mixed infection of Pasteurella multocida and Arcanobacterium pyogenes. Culture for Mycoplasma hyopneumoniae, Salmonella sp and fungi was negative. The outbreak persisted. Subsequently, gross lesions consistent with enzootic pneumonia occurred, but histological lesions were equivocal and definitive tests for M hyopneumoniae remained negative. Eighteen months after the initial outbreak, gross and histological lesions were consistent with enzootic pneumonia but serological tests were still negative. Almost 2 years later, one of four nasal swabs was positive by the polymerase chain reaction test for M hyopneumoniae, and then lung samples were sporadically positive. The pneumonic disease became endemic in the herd. Gross lesions consistent with enzootic pneumonia occurred in another herd belonging to the same company nearly 2 years after the initial outbreak. Again, results of laboratory tests were inconsistent. Despite sporadic positive polymerase chain reaction tests for M hyopneumoniae, the respiratory disease resolved within 4 months and there has been no clinical evidence of enzootic pneumonia during the subsequent 4 years. These cases raise important questions about the role of the diagnostic tests and their interpretation, and the ecology of M hyopneumoniae and its role in enzootic pneumonia.     

Spatial analysis of an anthrax outbreak in Saskatchewan, 2006

An outbreak of anthrax in Saskatchewan in 2006 affected more than 800 animals at 150 locations. The purpose of this study was to assess the spatial and temporal patterns among the cases to determine if there were any significant trends associated with this outbreak. Case and population data were first analyzed for each individual farm location and then again as aggregate data per rural municipality using spatial and spatiotemporal statistical methods such as Oden's Ipop, Cuzick-Edwards' test, spatial scan test, and other mapping techniques. East central Saskatchewan was identified as a primary high risk area, particularly during July 2006. The results of the study led to the conclusion that within this high-risk region, flooding in spring followed by hot and dry conditions could have been a factor in the development of the outbreak.     

Significant features of the epidemiology of equine influenza in Queensland, Australia, 2007

An outbreak of equine influenza (EI) caused by influenza A H3N8 subtype virus occurred in the Australian states of Queensland and New South Wales in August 2007. Infection in the Australian horse population was associated with the introduction of infection by horses from overseas. The first case of EI in Queensland was detected on 25 August 2007 at an equestrian sporting event. Infection subsequently spread locally and to other clusters through horse movements prior to the implementation of an official standstill. There were five main clusters of infected properties during this outbreak and several outliers, which were investigated to find the potential mechanism of disease spread. To contain the outbreak, Queensland was divided into infection status zones, with different movement controls applied to each zone. Vaccination was implemented strategically in infected areas and within horse subpopulations. Control and eventual eradication of EI from Queensland was achieved through a combination of quarantine, biosecurity measures, movement control, rapid diagnostic testing and vaccination.     

Antimicrobial susceptibility of Escherichia coli F4, Pasteurella multocida,and Streptococcus suis isolates from a diagnostic veterinary laboratory and recommendations for a surveillance system

Antimicrobial susceptibility data on Escherichia coli F4, Pasteurella multocida, and Streptococcus suis isolates from Ontario swine (January 1998 to October 2010) were acquired from a comprehensive diagnostic veterinary laboratory in Ontario, Canada. In relation to the possible development of a surveillance system for antimicrobial resistance, data were assessed for ease of management, completeness, consistency, and applicability for temporal and spatial statistical analyses. Limited farm location data precluded spatial analyses and missing demographic data limited their use as predictors within multivariable statistical models. Changes in the standard panel of antimicrobials used for susceptibility testing reduced the number of antimicrobials available for temporal analyses. Data consistency and quality could improve over time in this and similar diagnostic laboratory settings by encouraging complete reporting with sample submission and by modifying database systems to limit free-text data entry. These changes could make more statistical methods available for disease surveillance and cluster detection.     

Use of the scan statistic on disaggregated province-based data: foot-and-mouth disease in Iran

The spatial scan statistic was applied to density-smoothed data that approximated the spatial distribution within the area and reduced the potential bias produced when location data have been aggregated for large areas. The method is illustrated, using data on the location of foot-and-mouth disease (FMD) outbreaks in Iran. Data examined were 4477 FMD outbreaks reported on a per province basis between June 1996 and September 2003. A kernel density of the outbreak locations was estimated, using a fixed radius and the centroid of each province as the designated location of all cases reported for the province. The radius that produced a density map with the highest correlation with expert opinion was 4° (latitude/longitude). Livestock density was used as a proxy for the underlying population at risk of acquiring FMD. Livestock and outbreak density maps were overlain to obtain the number of outbreaks and livestock in each of 15,599 cells covering the mapped surface of the country. A spatial scan statistic was applied to the density-smoothed data assuming that the outbreaks had a Poisson distribution. Results were compared with those obtained using a spatial scan statistic on provincially aggregated data. Application of the spatial scan statistic on the density-smoothed data allowed identification of clusters (P < 0.01) related more to the actual geographic distribution of cases (expert opinion) and of animals at risk, than to the distribution of the provinces. Significant clusters of FMD were identified that coincided with roads, neighboring countries, and high-density population areas, suggesting that the region may represent a route for cross-continent transmission of FMD.     

An Outbreak of Highly Pathogenic Avian Influenza at a Public Animal Exhibit in Seoul,Korea, During 2008

This study describes the first recorded outbreak of HPAI in the city of Seoul, in captive birds held in an exhibition for public viewing at a local district office. The index cases were two pheasants, which had been introduced into the exhibit on 24 April, 4 days prior to death, from a store in a local market in Gyeonggi‐do. Ducks and chickens from an HPAI outbreak farm, subsequently confirmed on 4 May, had also been held in this store. This outbreak highlights the potential role of local markets in AIV transmission. This outbreak led to considerable public health concern in Korea, however, no human cases were reported. The non‐commercial poultry sector needs to be considered in national plans for preparedness and response.     

Epidemiological enquiries in two Q fever outbreaks in a community of Baden-Württemberg during 2008 and 2009

In 2008 and 2009, two consecutive outbreaks of Q fever in humans were recorded in the district of Freudenstadt, northern Black Forrest, Baden-Württemberg, Germany. In 2008, a total of 41 persons from a single local community fell ill and were found infected with Coxiella burnetii. Although comprehensive diagnostic and epidemiological outbreak investigations were conducted and control measures taken which included vaccination of ruminants at risk in three parts of the affected community, re-occurrence of the disease in 2009 with further 29 confirmed human Q fever cases could not be prevented. While the origin of infection of the first outbreak was probably a flock of 550 sheep moved in the surrounding of the affected villages, the source of infection for the consecutive outbreak in 2009 could not be identified. It seems possible that meadows contaminated with infectious placenta or birth fluids represented the sources of infection.     

Use of the rose bengal test in the diagnosis of brucellosis in hares

The diagnostic effectiveness of the Rose bengal test was verified in a set of 72 samples of the blood of hare shot in a focus of brucellosis; the blood was found to be serologically positive by the following methods: rapid agglutination with serum, surface fixation test, method of slow agglutination, and the complement fixation reaction. The Rose bengal test was found positive in 57 cases (79.17%), the surface agglutination test was positive in 60 cases (83.33%), rapid agglutination with serum in 41 cases (56.94%), slow agglutination in 55 cases (76.39%), and the complement-fixation reaction in 63 cases (87.50%). In nine cases (12.5%) a positive reaction was obtained only when the Rose bengal test was used. The results confirm that the Rose bengal test is a sensitive qualitative serological method of low technical and time requirements, suitable for routine diagnostic uses. In the field epizootological investigations in the foci of hare brucellosis, the Rose bengal test is a good screening serological method.     

Avian Influenza A(H5N1) Virus Outbreak Investigation: Application of the FAO‐OIE‐WHO Four‐way Linking Framework in Indonesia

WHO, FAO and OIE developed a ‘four‐way linking’ framework to enhance the cross‐sectoral sharing of epidemiological and virological information in responding to zoonotic disease outbreaks. In Indonesia, outbreak response challenges include completeness of data shared between human and animal health authorities. The four‐way linking framework (human health laboratory/epidemiology and animal health laboratory/epidemiology) was applied in the investigation of the 193rd human case of avian influenza A(H5N1) virus infection. As recommended by the framework, outbreak investigation and risk assessment findings were shared. On 18 June 2013, a hospital in West Java Province reported a suspect H5N1 case in a 2‐year‐old male. The case was laboratory‐confirmed that evening, and the information was immediately shared with the Ministry of Agriculture. The human health epidemiology/laboratory team investigated the outbreak and conducted an initial risk assessment on 19 June. The likelihood of secondary cases was deemed low as none of the case contacts were sick. By 3 July, no secondary cases associated with the outbreak were identified. The animal health epidemiology/laboratory investigation was conducted on 19–25 June and found that a live bird market visited by the case was positive for H5N1 virus. Once both human and market virus isolates were sequenced, a second risk assessment was conducted jointly by the human health and animal health epidemiology/laboratory teams. This assessment concluded that the likelihood of additional human cases associated with this outbreak was low but that future sporadic human infections could not be ruled out because of challenges in controlling H5N1 virus contamination in markets. Findings from the outbreak investigation and risk assessments were shared with stakeholders at both Ministries. The four‐way linking framework clarified the type of data to be shared. Both human health and animal health teams made ample data available, and there was cooperation to achieve risk assessment objectives.     

Laboratory experience during the classical swine fever virus epizootic in the Netherlands in 1997-1998

From February 1997 till May 1998 the national reference laboratory for classical swine fever (CSF) in the Netherlands was confronted with millions of samples taken from pigs during an outbreak of CSF in a pig dense region. In a limited period major logistic problems needed to be solved regarding the processing of samples and information at the laboratory facilities.In total over 2.3 million samples were examined by different CSF diagnostic methods. The majority (approximately 2.1 million) of these samples were blood samples which were tested for CSF serum antibody in a semi-automated ELISA. Approximately 166,000 samples were examined for the presence of CSF virus or viral antigen. Automated preparation and testing of blood samples for CSF serum antibody, the obligatory identification and registration system of pig holdings and the computerised laboratory management system made it possible to process the huge amount of samples and information presented in a limited period. The majority of the test results was sent to the veterinary authorities via e-mail or a computerised fax system.Of the 429 outbreaks 82% were detected via a direct immunofluorescence technique performed on cryostat sections of the tonsil. The sampling of clinically suspected pigs ('guided' sampling) for this diagnostic method provided rapid positive and negative results and thus played a paramount role during the eradication campaign. Serological surveys identified 13.5% of the infected pig holdings: such surveys proved very effective in the screening of holdings which were subjected to restrictions (protection or surveillance zones) for many months. Virus isolation performed on different types of samples detected 4. 5% of the infected pig holdings.In conclusion, analysis of data collected in the laboratory and epidemiological analysis should result in an improved eradication plan for the future control of outbreaks of CSF in the Netherlands supported by optimised CSF diagnostic methods.     

Utilizing technology to enhance laboratory–client interaction while encouraging best behavior

As client interactions with veterinary diagnostic laboratories have evolved, so have client expectations: faster results, enhanced accessibility to cases, and more seamless data transfer from the laboratory database; all of these factors have encouraged the evolution of diagnostic laboratory systems. This evolution started with 24-h access to laboratory results via the web, yet data quality remained at the mercy of the person filling out the form. If bad (incomplete) information was flowing in, then the data coming out was equally bad (incomplete or inconsistent). By designing a web-based system integrated into our existing reporting platform, the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) set out to improve the quality of submission data by including the premises identification number (PIN) and obtaining consistent location data, all while presenting to the client an easy-to-use interface. Efforts continued by incentivizing the use of this tool and client submission practices. As clients transitioned, data have become more complete, resulting in easier queries and an improved ability to leverage the diagnostic data. To further enhance the client experience, a streamlined daily reporting summary was designed to communicate laboratory results succinctly. The use of these web-based tools had a positive impact on the quality and consistency of the diagnostic data. As new ideas develop, the ISU-VDL strives to foster continuous improvement and positively impact the clients’ experience.     

Investigation of H7N2 avian influenza outbreaks in two broiler breeder flocks in Pennsylvania, 2001-02

An avian influenza (AI) outbreak occurred in meat-type chickens in central Pennsylvania from December 2001 to January 2002. Two broiler breeder flocks were initially infected almost simultaneously in early December. Avian influenza virus (AIV), H7N2 subtype, was isolated from the two premises in our laboratory. The H7N2 isolates were characterized as a low pathogenic strain at the National Veterinary Services Laboratories based on molecular sequencing of the virus hemagglutinin cleavage site and virus challenge studies in specific-pathogen-free leghorn chickens. However, clinical observations and pathologic findings indicated that this H7N2 virus appeared to be significantly pathogenic in meat-type chickens under field conditions. Follow-up investigation indicated that this H7N2 virus spread rapidly within each flock. Within 7 days of the recognized start of the outbreak, over 90% seroconversion was observed in the birds by the hemagglutination inhibition test. A diagnosis of AI was made within 24 hr of bird submission during this outbreak using a combination of virus detection by a same-day dot-enzyme-linked immunosorbent assay and virus isolation in embryonating chicken eggs. Follow-up investigation revealed that heavy virus shedding (90%-100% of birds shedding AIV) occurred between 4 and 7 days after disease onset, and a few birds (15%) continued to shed virus at 13 days post-disease onset, as detected by virus isolation on tracheal and cloacal swabs. AIV was not detected in or on eggs laid by the breeders during the testing phase of the outbreak. The two flocks were depopulated at 14 days after disease onset, and AIV was not detected on the two premises 23 days after depopulation.     

Development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to avian influenza virus

During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.     

Descriptive summary of an outbreak of porcine post-weaning multisystemic wasting syndrome (PMWS ) in New Zealand

CASE HISTORY: Investigations were conducted to determine the cause of an acute, multi-farm outbreak of porcine respiratory disease that included diarrhoea and subsequent loss of body condition in affected pigs. A definition for post-weaning multisystemic wasting syndrome (PMWS) including both clinical and pathological features, previously developed for the pig industry in New Zealand, was applied to the current outbreak. In addition to self-reporting by owners of affected farms, local veterinarians, disease and epidemiology consultants, and animal health officials from the Ministry of Agriculture and Forestry (MAF) were involved in conducting farm visits and submission of diagnostic specimens. CLINICAL FINDINGS AND DIAGNOSIS: Pathogens known to be endemic in the pig industry in New Zealand as well as likely exotic diseases were excluded as causative agents of the outbreak. Clinical signs including dyspnoea, diarrhoea, and rapid loss of body condition were consistent with the New Zealand case definition for PMWS. Interstitial pneumonia, pulmonary oedema, generalised lymph-node enlargement, and presence of porcine circovirus type 2 (PCV2) inclusion bodies were consistently identified in affected pigs. Classical swine fever virus (CSFv), Porcine reproductive and respiratory syndrome virus (PRRSv), and Influenza virus were ruled out, using molecular and traditional virological techniques. Spread of the disease between farms was hypothesised to be facilitated by locally migrating flocks of black-backed seagulls. The original source of the disease incursion was not identified. DIAGNOSIS: Based on the consistent presence of circovirus-associated lesions in lymphoid tissues in combination with generalised enlargement of lymph nodes, histiocytic interstitial pneumonia, clinical wasting, and poor response to antibiotic therapy, a diagnosis of PMWS was made. CLINICAL RELEVANCE: PMWS should be considered in the differential diagnoses of sudden onset of respiratory dyspnoea, diarrhoea, and rapid loss of body condition in young pigs in New Zealand pig herds.