Early pathogenesis of equine Streptococcus equi infection (strangles)

Reasons for performing study: Little is known about entry and subsequent multiplication of Streptococcus equi following exposure of a susceptible horse. This information would have value in design of intranasal vaccines and understanding of shedding and protective immune responses. Objectives: To determine entry points and sites of subsequent replication and dispersion of S. equi at different times after intranasal infection or commingling exposure. Methods: Previously unexposed horses and ponies were subjected to euthanasia 1, 3, 20 or 48 h following intranasal inoculation with biotin labelled or unlabelled S. equi CF32. Some ponies were inoculated with suspensions of equal numbers of CF32 and its mutants lacking capsule, S. equi M‐like protein or streptolysin S. Others were infected by commingling exposure and subjected to euthanasia after onset of fever. Tonsils and lymph nodes were cultured for S. equi and tissues sectioned for histopathological examination and fluorescent microscopy. Results: Tonsillar tissues of both the oro‐ and nasopharynx served as portals of entry. Entry was unexpectedly rapid but involved few bacteria. Small numbers of organisms were detected in tonsillar crypts, in adjacent subepithelial follicular tissue and draining lymph nodes 3 h after inoculation. By 48 h, clumps of S. equi were visible in the lamina propria. At onset of fever, tonsillar tissues and one or more mandibular and retropharyngeal lymph nodes were heavily infiltrated by neutrophils and long chains of extracellular S. equi. Mutant S. equi lacking virulence factors were not seen in draining lymph nodes. Conclusions: Although very small numbers of S. equi entered the lingual and nasopharyngeal tonsils, carriage to regional lymph nodes occurred within hours of inoculation. This observation, together with visual evidence of intracellular and extracellular multiplication of S. equi in tonsillar lymphoid tissue and lymph nodes over the following days, indicates involvement of potent antiphagocytic activity and failure of innate immune defences. Relevance: Future research should logically address the tonsillar immune mechanisms involved including identification of effector cell(s) and antigens.     

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.     

Control of strangles outbreaks by isolation of guttural pouch carriers identified using PCR and culture of Streptococcus equi

Previous use of repeated nasopharyngeal swabbing and culture of Streptococcus equi showed that healthy carriers developed in more than 50% of 'strangles' outbreaks. The guttural pouches were the only detectable site of S. equi colonisation on endoscopic examination of horses during one of these outbreaks and S. equi was sometimes not detected by culture of nasopharyngeal swabs from carriers for up to 2 or 3 months before nasal shedding resumed sporadically. A more sensitive way of detecting S. equi on swabs from established guttural pouch carriers was therefore required. Conveniently selected 'strangles' outbreaks were investigated in detail using endoscopy, in order to develop and assess a suitable polymerase chain reaction (PCR) test. We report here 3 protracted 'strangles' outbreaks on different kinds of establishments in which between 29 and 52% of sampled horses were infected as detected by culture and/or PCR. Of the infected horses, between 9 and 44% were identified as carrying S. equi after clinical signs had disappeared and the predominant site of carriage was the guttural pouch. Prolonged carriage of S. equi, which lasted up to 8 months, did not cease spontaneously before treatment was initiated to eliminate the infections. The detection and isolation of the carriers, in conjunction with strict hygiene measures, apparently resulted in the control of the outbreaks and allowed the premises to return to normal activity. Comparing PCR and culture, many more swabs were found to be positive using PCR (56 vs. 30% of 61 swabs). Similar results were obtained for guttural pouch samples from 12 established carriers (PCR 76% and culture 59%). These results from repeated samples from relatively few animals need confirming using more long-term carriers. PCR can also detect dead organisms and is, therefore, liable to yield false positive results. Despite this drawback, it is argued that PCR provides a potentially useful adjunct to culture of nasopharyngeal swabs in the detection of asymptomatic carriers of S. equi following outbreaks of 'strangles'.     

Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis

Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi.     

Subscapular lymph node abscessation as a result of metastatic Streptococcus equi subspecies equi infection: An atypical presentation of bastard strangles in a mare

The case reported here represents an atypical presentation of bastard strangles in an 18‐year‐old Arab mare. The horse initially presented for progressive neck pain characterised by reluctance to lateral and ventroflexion of the neck. Subsequent diagnostics revealed a subscapular abscess and aspirates of the mass cultured positive for Streptococcus equi ssp. equi. Surgical drainage and debridement of the abscess was performed under general anaesthesia. Six months post surgery, the mare had made a complete recovery.     

Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs.

The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly or difficult for routine diagnostic use. Several polymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nasal or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 microorganisms and did not cross-react with other mycoplasma species or with other microorganisms commonly found in the respiratory tract of pigs. This assay was better suited for detection of M. hyopneumoniae from nasal swabs than was conventional PCR. Nasal swab samples were taken at different time periods following experimental challenge of 10 susceptible pigs. Only 2 of the 55 swabs examined gave a positive result with conventional PCR, whereas 30 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak where M. hyopneumoniae had been diagnosed also gave a positive result with the nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae.     

Prevalence and serum protein values of strangles (Streptococcus equi) affected mules at Remount Depot,Sargodha (Pakistan)

The prevalence of Streptococcus equi serovar equi (S.equi) in nasal discharge and pus samples from sub‐mandibular lymph nodes in mules at the Remount Depot, Sargodha was examined and total serum proteins, serum albumin, serum globulin and fibrinogen measured. A total of 250 nasal swabs and pus samples were collected from mules and examined microbiologically: 99 (39.6%) were positive for S. equi. A higher occurrence of S. equi was recorded in foals as compared to adults. The concentrations of total serum protein, serum globulin and fibrinogen were significantly increased (P<0.05), while the concentration of serum albumin significantly decreased (P<0.05) in strangles‐affected mules. It was concluded that increased total serum proteins, serum globulin and fibrinogen along with decreased serum albumin were important indicators of infection by S. equi in mules.     

Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams

The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams.     

Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested PCR and culture methods

We examined nasal swab and lung homogenate samples collected from pigs experimentally and naturally infected with Mycoplasma hyopneumoniae for the detection of M. hyopneumoniae by the nested PCR (nPCR) and culture methods. In the 23 experimentally infected pigs, M. hyopneumoniae was commonly detected in nasal swabs by the nPCR and culture methods at 4 weeks after inoculation, and there was a significant correlation (P<0.01) between the titers of viable organisms in nasal swabs and in lung homogenates in the experimentally inoculated pigs. In the naturally infected pigs, on the other hand, discrepancies in detection were found between nasal swab and lung homogenate samples in 17 of 36 cases, although the presence of gross lung lesions correlated relatively well with the detection of organisms from the samples. Our results indicated that the diagnosis of mycoplasmal pneumonia by nPCR in individual pigs with nasal swabs is reliable under these experimental conditions. At present, nPCR with nasal swabs should only be used for monitoring the disease status at the herd level under field conditions.     

Field validation of a real-time PCR test for the detection of Mycoplasma hyopneumoniae in nasal swabs of live pigs

Enzootic pneumonia (EP) of pigs, caused by Mycoplasma hyopneumoniae has been a notifiable disease in Switzerland since May 2003. The diagnosis of EP has been based on multiple methods, including clinical, bacteriological and epidemiological findings as well as pathological examination of lungs (mosaic diagnosis). With the recent development of a real-time PCR (rtPCR) assay with 2 target sequences a new detection method for M. hyopneumoniae became available. This assay was tested for its applicability to nasal swab material from live animals. Pigs from 74 herds (average 10 pigs per herd) were tested. Using the mosaic diagnosis, 22 herds were classified as EP positive and 52 as EP negative. From the 730 collected swab samples we were able to demonstrate that the rtPCR test was 100% specific. In cases of cough the sensitivity on herd level of the rtPCR is 100%. On single animal level and in herds without cough the sensitivity was lower. In such cases, only a positive result would be proof for an infection with M. hyopneumoniae. Our study shows that the rtPCR on nasal swabs from live pigs allows a fast and accurate diagnosis in cases of suspected EP.     

Evaluation of a novel nested PCR for the routine diagnosis of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV)

Laboratory diagnosis of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) usually involves both viruses, as the clinical signs are similar and coinfection may occur. Serological methods may not represent an accurate diagnosis: maternal antibodies or cross-reactions may give false positive results to FIV, and false negative results may occur in latent FeLV status, or in certain FIV infection stages. A nested polymerase chain reaction (PCR) technique was designed to detect FeLV, FIV and feline endogenous retrovirus simultaneously. The detection of endogenous sequences was considered indicative of successful DNA extraction. The technique was used to diagnose FIV and FeLV in the blood cells of 179 cats. The kappa value with the serological data was 0.69 for FeLV and 0.87 for FIV. The joint detection of FeLV and FIV by this novel nested PCR is sensitive, specific, fast and convenient, and its applicability for clinical diagnosis is promising, as the direct evidence of the presence of the virus is more realistic than the indirect data provided by the serological detection.     

Review of Corynebacterium (Rhodococcus) equi lung abscesses in foals: pathogenesis, diagnosis and treatment

Corynebacterium (Rhodococcus) equi is becoming increasingly significant as a cause of bronchopneumonia and lung abscessation in foals. The organism can survive within macrophages and may thus escape normal pulmonary defence mechanisms, particularly in immunocompromised animals. The disease has hitherto been associated with mortality rates as high as 80 per cent, partly as a result of inappropriate therapy. The selection of lipid-soluble antibiotics capable of intracellular penetration is critical for the successful treatment of C equi lung abscesses. A combination of two such antibiotics, erythromycin (25 mg/kg three times daily) and rifampicin (5 mg/kg twice daily) has been used on foals since 1981. Most of these animals had radiographic evidence of extensive lung abscessation, and in all cases the presence of C equi was confirmed on culture of tracheal aspirates. The duration of therapy ranged from four to nine weeks. Mild gastritis and diarrhoea were occasionally noted, but never such as to require termination of the therapy. No other adverse side effects were encountered. The success rate, as judged by a return to normal of chest radiographs and plasma fibrinogen concentrations, has exceeded 80 per cent.     

Antibody to equi factor(s) in the diagnosis of Corynebacterium equi pneumonia of foals.

Antibody to equi factor(s) in cases of Corynebacterium equi pneumonia in foals was detected using C. pseudotuberculosis exotoxin sensitized calf red blood cells. The test was standardized using antitoxin produced in rabbits by injection of equi factor(s). All sera from ten foals with culture-diagnosed C. equi pneumonia had antibodies to equi factor(s) (titre range 8-256, mean 74.0) and nine sera from 11 foals with suspected C. equi pneumonia also showed antibodies (titre range 4-512, mean 136.4). Two of five pneumonia foals with transtracheal aspirate cultures not yielding C. equi had such antibodies. Fifty-eight of 59 control horse sera had no antibodies; the one positive serum came from a foal on a farm where C. equi pneumonia was endemic. By contrast only five of 15 foals with experimentally-induced C. equi pneumonia had antibodies to equi factor(s), probably because the acute nature of the disease produced did not mimic the chronic course of the natural disease. Antibody to equi factor(s) can be used in the diagnosis of naturally-occurring corynebacterial pneumonia in foals.     

Enzyme-linked immunosorbent assay for diagnosis of Corynebacterium (Rhodococcus) equi infection in foals

An enzyme-linked immunosorbent assay (ELISA) was used to diagnose Corynebacterium (Rhodococcus) equi infection in foals. In tests done with different antigen-extraction procedures (sodium dodecyl sulfate, sodium deoxycholate, polyoxy-ethylene [9] p-tert-octylphenol, polyoxy-ethylene [9-10] p-tert-octylphenol, sonification, homogenization, and heat treatment at 121 C), Tween 20 was a satisfactory reactive antigen. Using hyperimmune rabbit sera or infected foal sera, we investigated the specificity and the sensitivity of the ELISA with the Tween 20 antigen of the different serotypes or of the isolates. Corynebacterium equi strain ATCC 6939 antigen had the best activity for detecting antibodies to C equi in foals. Sera from 218 healthy horses, 11 healthy foals, 17 healthy newborn foals, a foal with suspected C equi infection, and 5 infected foals were evaluated for antibodies to C equi, using ELISA. The optical density values of 206 healthy horses, 17 healthy newborn foals, and 9 healthy foals were less than 0.1. Infected foal sera, except from foal 3, and serum from a foal with suspected C equi infection had higher optical density values. Using ELISA, specific antibodies against C equi were detected in a naturally infected 6-week-old foal after the foal had a rapid increase in the number of bacteria in the feces and after the initial development of clinical signs of illness at 5 weeks of age. Therefore, ELISA was useful for the early diagnosis of C equi infection in foals.     

Utility of the Cryptosporidium oocyst wall protein (COWP) gene in a nested PCR approach for detection infection in cattle

A preliminary molecular epidemiological study was carried out to investigate the utility of the Cryptosporidium oocyst wall protein (COWP) gene in the detection of Cryptosporidium oocysts in fecal samples. A nested polymerase chain reaction (PCR) approach using COWP gene primers was adopted for this purpose. Fecal samples were spiked with each of 1, 10, and 100 oocysts of C. parvum, four samples for each number, and the DNA was extracted from each sample using a glassbead method. The presence of oocysts was determined using the nested PCR with COWP gene primers, and the limit of detection of oocysts by the PCR was determined. The limit of detection was 100 oocysts spiked in 1 ml of fecal material (50% sold material) (four positives/four samples tested). Seventy-five percent of DNA extracted samples spiked with 1 and 10 oocysts was positive by the PCR (three positives/four samples tested). Based on this, small sample size using the COWP gene primers with a nested PCR analysis could reliably identify infected animals rather conveniently and accurately.