A Multiplex Polymerase Chain Reaction Assay for Direct Detection and Differentiation of β-Hemolytic Streptococci in Clinical Samples from Horses

Streptococcus equi subspecies equi, S equi subspecies zooepidemicus, and S dysgalactiae subspecies equisimilis are β-hemolytic Streptococci, often isolated from horses with respiratory or genital diseases. The aim of this study was (i) defining and validating a multiplex polymerase chain reaction (PCR) protocol for identifying these Streptococci in bacterial cultures and for detecting them directly in equine clinical specimens, and (ii) defining and validating a cheap DNA extraction protocol for clinical specimens. When respiratory and genital samples from symptomatic and asymptomatic horses were tested by bacterial culture and by multiplex PCR, all the 150 samples culture-positive for S equi, S zooepidemicus, or S equisimilis were also positive by PCR. Of 150 culture-negative samples, 143 were negative by PCR. Seven samples were positive by PCR but negative by bacteriology. The multiplex PCR protocol described in this study is proven suitable for a sensitive, specific, and rapid detection and identification of S equi, S zooepidemicus, and S equisimilis in cultured bacterial colonies, as well as in clinical specimens from symptomatic or asymptomatic horses. The inclusion of internal control primers in the PCR protocol excludes false-negative results. A cheap DNA extraction method has been also validated for swabs, tracheal aspirates, bronchoalveolar lavage, and guttural pouches lavage samples.     

Bacteriological and Molecular Detection of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Equines of Northern India

Present study was undertaken to study the prevalence of β-haemolytic streptococci in equine of northern temperate region of Jammu and Kashmir, India. One hundred and forty one samples were collected in duplicate from nasopharyngeal tract of diseased (53) and apparently healthy equine (88) for isolation and direct PCR. A total of 77 isolates of streptococci were recovered from 141 samples with an overall prevalence rate of 54.60%. Out of these 77 isolates, 52 were from diseased and 25 from apparently healthy animals. Of the 77 isolates, 4 were identified as Streptococcus equi subsp. equi, 56 as S. equi subsp. zooepidemicus and 17 as S. dysgalactiae subsp. equisimilis. Thus the overall prevalence of S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis was 2.83, 39.71 and 12.05% respectively. The sensitivity of the PCR for the detection of S. equi species was found higher when attempted from direct swab samples.     

PCR based differentiation between Streptococcus dysgalactiae subsp. equisimilis strains isolated from humans and horses

Streptococcus dysgalactiae subsp. equisimilis (SDSE) can be severely pathogenic in humans and is increasingly isolated from horses with respiratory, reproductive or other diseases, although it is often considered a commensal bacterium. Here a PCR protocol is described for identifying SDSE recovered from humans. A multiplex PCR targeting the 16S rRNA and the streptokinase precursor gene has been optimized for differentiating between SDSE strains isolated from humans and those isolated from horses. Previously, the sequence of the streptokinase precursor gene of SDSE recovered from horses has been found in two human cases of pneumonia in Japan.     

A survey of respiratory viruses in New Zealand horses

AIMS: To determine which viruses circulate among selected populations of New Zealand horses and whether or not viral infections were associated with development of respiratory disease.

METHODS: Nasal swabs were collected from 33 healthy horses and 52 horses with respiratory disease and tested by virus isolation and/or PCR for the presence of equine herpesviruses (EHV) and equine rhinitis viruses.

RESULTS: Herpesviruses were the only viruses detected in nasal swab samples. When both the results of nasal swab PCR and virus isolation were considered together, a total of 41/52 (79%) horses with respiratory disease and 2/32 (6%) healthy horses were positive for at least one virus. As such, rates of virus detection were significantly higher (p<0.001) in samples from horses with respiratory disease than from healthy horses. More than half of the virus-positive horses were infected with multiple viruses. Infection with EHV-5 was most common (28 horses), followed by EHV-2 (27 horses), EHV-4 (21 horses) and EHV-1 (3 horses).

CONCLUSIONS: Herpesviruses were more commonly detected in nasal swabs from horses with respiratory disease than from healthy horses suggesting their aetiological involvement in the development of clinical signs among sampled horses. Further investigation to elucidate the exact relationships between these viruses and respiratory disease in horses is warranted.

CLINICAL RELEVANCE: Equine respiratory disease has been recognised as an important cause of wastage for the equine industry worldwide. It is likely multifactorial, involving complex interactions between different microorganisms, the environment and the host. Ability to control, or minimise, the adverse effects of equine respiratory disease is critically dependent on our understanding of microbial agents involved in these interactions. The results of the present study update our knowledge on the equine respiratory viruses currently circulating among selected populations of horses in New Zealand.     

Isolation of Nicoletella semolina from Equine Tracheal Washes

The aim of the present study was to describe the prevalence of Nicoletella semolina in equine airways and its relationships with cytological evaluation of tracheal wash (TW). Samples were collected in the framework of routine bacteriological diagnostics of equine TW between May 2010 and June 2011. N semolina has been isolated, along with either common pathogens or contaminants, from 19 (1.8%) of the 1,054 TW samples. Median TW neutrophil counts (87.0%) in specimens from N semolina-positive horses were significantly different from those from N semolina-“negative” horses (52.0%). The data presented in this report may suggest considering such bacteria in horses clinically suffering from airway inflammation.     

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.     

Prevalence of equine herpesvirus-1 and equine herpesvirus-4 infections in equidae species in Turkey as determined by ELISA and multiplex nested PCR

In this report we examined the presence of specific antibodies against equine herpesvirus type 1 (EHV-1), and equine herpesvirus type 4 (EHV-4) in several equidae, including mules, donkeys, horses. The presence of EHV-1 and EHV-4 in respiratory diseases of equids, and ability of multiplex nested polymerase chain reaction (PCR) screening in simultaneous diagnosis of horses acutely infected by EHV-1 and EHV-4 were also investigated. Sera from 504 horses, mules and donkeys sampled were tested for the presence of EHV-1 and EHV-4 specific antibodies. Blood samples taken from 21 symptomatic horses and nasal swabs taken from 40 symptomatic horses were tested for the presence of EHV-1 and EHV-4 by a multiplex nested PCR. A total of 14.3% (3/21) of buffy coat samples and 32.5% (13/40) nasal swab samples were found to contain EHV-1 DNA, while 19% (4/21) buffy coat samples and 22.5% (9/40) nasal swab samples were found to be positive for EHV-4 DNA. By species, 14.5% of horses, 37.2% of mules and 24.2% of donkeys tested were EHV-1 seropositive. EHV-4 specific antibodies were detected in 237 (81.7%) of 290 horse sera tested. Results from this investigation demonstrate that EHV-1 and EHV-4 are prevalent throughout the equid population, and that donkeys and mules might also represent an important source of infection for other equids. We also showed that the multiplex nested PCR assay might be useful for diagnosis of mixed respiratory infections in horses due to EHV-1 and EHV-4.     

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.     

Prevalence of bacterial genotypes and outcome of bovine clinical mastitis due to Streptococcus dysgalactiae and Streptococcus uberis

Background

Streptococcus dysgalactiae and Streptococcus uberis are common causes of clinical mastitis (CM) in dairy cows. In the present study genotype variation of S. dysgalactiae and S. uberis was investigated, as well as the influence of bacterial species, or genotype within species, on the outcome of veterinary-treated CM (VTCM). Isolates of S. dysgalactiae (n = 132) and S. uberis (n = 97) were genotyped using pulsed-field gel electrophoresis. Identical banding patterns were called pulsotypes. Outcome measurements used were cow composite SCC, milk yield, additional registered VTCMs and culling rate during a four-month follow-up period.

Results

In total, 71 S. dysgalactiae pulsotypes were identified. Nineteen of the pulsotypes were isolated from more than one herd; the remaining pulsotypes were only found once each in the material. All S. uberis isolates were of different pulsotypes. During the follow-up period, the SCC of S. dysgalactiae-cows was significantly lower than the SCC of S. uberis-cows (P <0.05). Median SCC of S. dysgalactiae-cows was 71 500 cells/ml and of S. uberis-cows 108 000 cells/ml. No other differences in outcome parameters could be identified between species or genotypes.

Conclusions

Identical S. dysgalactiae genotypes were isolated from more than one herd, suggesting some spread of this pathogen between Swedish dairy herds. The genetic variation among S. uberis isolates was substantial, and we found no evidence of spread of this pathogen between herds. The milk SCC was lower during the follow-up period if S. dysgalactiae rather than S. uberis was isolated from the case, indicating differences in treatment response between bacterial species.

Electronic supplementary material

The online version of this article (doi:10.1186/s13028-014-0080-0) contains supplementary material, which is available to authorized users.     

Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR

Background: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
Objectives: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses.
Animals: Fifteen horses experimentally infected with EHV-1.
Methods: Experimental study : Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed.
Results: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI ( P < .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86–100) and 27% (95% CI: 20–35).
Conclusions and Clinical Importance: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C _T values are provided as well as justification of a minimum 10-day quarantine period.     

The first nosocomial outbreak of methicillin-resistant Staphylococcus aureus in horses in Sweden

Background

Methicillin-resistant Staphylococcus aureus (MRSA) in animals is a rare finding in Sweden. In horses, MRSA was first detected in a screening survey in 2007. In 2008, six clinical cases occurred in an equine hospital, indicating an outbreak.

Method

All MRSA isolates detected, 11 spa-type t011 and one t064 (n = 12), in infected horses (n = 10) and screening of horses (n = 2) in Sweden from December 2007 to March 2010 were retrospectively analysed with pulsed-field gel electrophoresis (PFGE) using Cfr9I and ApaI restriction enzymes, to study relationship between the isolates. Medical records of infected horses and outbreak investigation notes were scrutinised to monitor the clinical outcome and other aspects of the outbreak.

Results

Eight of the 10 infected horses were linked to one equine hospital and two to another hospital in the same region. The six horses infected with MRSA in 2008 underwent surgery during the period 22 May-7 July in one of the hospitals. Four more infections linked to the two hospitals were notified between 2009 and March 2010.Nine of the 11 spa-type t011 isolates had identical Cfr9I and ApaI PFGE pattern. All six infected horses from 2008 presented with this MRSA. Two t011 isolates differed in one and two bands, respectively, in PFGE.Nine horses suffered from surgical site infections (SSI). No antimicrobials were used following the MRSA diagnosis and the infections cleared. The time from surgery to MRSA diagnosis differed greatly between the horses (range 15-52 days).

Conclusions

Association in time and space of six horses infected with an identical MRSA strain of spa-type t011 confirmed an outbreak. Two isolates found in 2009 and 2010 in the outbreak hospital were closely related to the outbreak strain, indicating one circulating strain. Both spa-type t011 and t064 have been reported in horses in Europe prior to these findings. The observation that the infections cleared although antimicrobials were not used is encouraging for future prudent use of antimicrobials. The time from surgery to bacteriological diagnosis was not acceptable in most cases, as contagious spread was a risk. Sampling when symptoms of infection are noticed and accurate analysis are thus important.     

Evaluation of a real-time polymerase chain reaction assay for rapid identification of methicillin-resistant Staphylococcus aureus directly from nasal swabs in horses

Screening for nasal colonization is an important aspect of many methicillin-resistant Staphylococcus aureus (MRSA) control programs. Real-time polymerase chain reaction (RT-PCR) is an attractive alternative to standard culture techniques because of the considerably shorter turnaround time. An assay has been validated for diagnostic purposes in humans, however this methodology has not been evaluated in horses. The purpose of this study was to compare an RT-PCR assay for rapid identification of MRSA directly from nasal swabs in horses to standard culture techniques. Nasal swabs collected from 293 horses were processed using a commercial RT-PCR assay (IDI-MRSA, GeneOhm Sciences, San Diego, CA) according to the manufacturer's instructions. The swabs were also cultured and MRSA was identified according to standard protocols. Initially only 176/293 samples yielded valid PCR results. Two of 176 and 167/176 samples were positive and negative, respectively, by both PCR and culture. Seven of 176 samples were positive by PCR and negative by culture, whereas 0/176 samples were negative by PCR and positive by culture. The kappa statistic was 0.35, which represented poor agreement between the tests. Of the remaining 117 samples, 105 samples were initially reported as "unresolved". Following one freeze-thaw cycle of the lysates, the recommended technique to resolve such samples, 61/110 (55%) samples remained unresolved. In this study, the IDI-MRSA assay was not a clinically practical screening test for horses harbouring nasal MRSA. Its agreement with culture was poor and the high unresolved rate (37%) also significantly decreased the clinical utility of the test.     

Salmonella Antimicrobial Activity of Selected Strains of Enterolactobacillus Species Isolated from the Gastrointestinal Tract of the Horse

The gastric mucosa and the mucosa of the right and left dorsal colon were biopsied in each of the 15 horses, and a total of 45 samples were collected. Mucosal samples were cultured using a Lactobacillus enrichment broth. While numerous Lactobacillus strains were identified, Lactobacillus reuteri was the most common organism identified. Sixteen strains of Lactobacillus reuteri were selected for antimicrobial testing. Salmonella antimicrobial activity was identified in six out of 16 strains tested. Organisms with Salmonella antimicrobial activity were cultured from the stratified squamous epithelium of the stomach and the mucosa of the right and left dorsal colon.     

Use of pooled samples for the detection of Salmonella in feces by polymerase chain reaction.

Many epidemiological studies of Salmonella rely on conventional bacteriological culture methods to detect Salmonella in fecal samples. These culture-based methods are inefficient for epidemiological studies in populations with a low prevalence of Salmonella. The objective of this study was to optimize a protocol that uses pooled Salmonella enrichment broth cultures of bovine feces and polymerase chain reaction (PCR) for the detection of the invA gene of Salmonella in feces. In one field trial, 196 animals were sampled, and all samples were tested by culture, invA PCR on individual samples, invA PCR on pools of 5 samples, and BAX PCR on individual samples. All assays showed a high agreement on individual samples (kappa > or = 0.75). The invA PCR was run on each of 40 pools and detected 19 of 22 culture-positive pools. In another field trial, 152 samples were taken from 4 dairies, and the invA PCR was performed on pools of 5 samples in addition to bacteriological culture of individual samples. Salmonella was detected in 5 of the 32 pools (7 total positive samples) by both PCR and culture. One pool was PCR-positive but culture-negative. Pooling did not dramatically affect the performance of the invA PCR; most of the culture-positive samples were detected, including all of the samples when there were 4 or more Salmonella colonies on the agar plate. Based on these field trials, invA PCR on pooled samples appears to be an efficient method of Salmonella detection as long as Salmonella loads are not extremely low.     

Seroprevalence of Sarcocystis neurona and Its Association With Neurologic Disorders in Argentinean Horses

Equine protozoal myeloencephalitis (EPM) is generally caused by Sarcocystis neurona and can produce substantial economic losses on equine production in America. The aims of the present study were to evaluate the seroprevalence of S. neurona in the main horse-production area of Argentina and associate it with the occurrence of neurologic disorders. Serum samples were collected from 640 horses in nine Argentinean provinces. Most of the samples correspond to animals ≥1.5-year-old from different breeds (n = 628); 12 samples were from younger horses. Further seroprevalence comparison was conducted from the older animals grouped with (n = 148) or without neurologic signs (n = 480). Immunoblot: proteins from 2 × 10⁷S. neurona merozoites were used as antigen on each membrane. Reactivity to antigens with relative mobility of 7, 10, and 16 kDa was considered specific for antibodies against S. neurona; reactivity at 30 kDa was recorded separately. The overall seroprevalence for S. neurona was 26.1% (167/640), and all the provinces had positive horses. Seroprevalence of animals with neurologic signs was greater (P < .001) than what was observed in normal horses (39.2% vs. 22.1%), with an odds ratio of 2.27. Reactivity at 30 kDa was detected in 71% of all samples. This study identified a wide distribution of S. neurona–positive animals in Argentina and horses with neurologic signs having a greater seroprevalence than normal horses. Sarcocystis neurona infection should be considered for early differential diagnosis and treatment of animals with neurologic disorders to decrease the economic impact of EPM in Argentina.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Nasal bacterial microbiota during an outbreak of equine herpesvirus 1 at a farm in southern Ontario

The objective of this study was to investigate the nasal bacterial microbiota of healthy horses and horses infected with equine herpesvirus 1 (EHV-1). The nasal bacterial microbiota of 10 horses infected with EHV-1 and 11 control horses from a farm experiencing an outbreak was characterized using the Illumina MiSeq platform targeting the V4 region of the 16S ribosomal RNA gene. The nasal bacterial microbiota of healthy horses and EHV-1 horses was significantly different in community membership and structure. Horses shedding EHV-1 had lower bacterial richness (P = 0.002), evenness (P = 0.008), and diversity (P = 0.026) than healthy horses. Healthy horses had a higher relative abundance of Firmicutes and Bacteroidetes, but lower Proteobacteria than horses with EHV-1 (P < 0.05). This study provides the basis for generating hypotheses and investigations on the role of bacterial-viral interactions in the health and diseases of adult horses.     

Comparison of immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae 168 strain in pigs

The aim of this study was to evaluate the immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae (Mhp) 168 strain in the local respiratory tract in pigs. Twenty-four pigs were randomly divided into 4 groups: an intranasal immunization group, an intrapulmonary immunization group, an intramuscular immunization group and a control group. The levels of local respiratory tract cellular and humoral immune responses were investigated. The levels of interleukin (IL)-6 in the early stage of immunization (P<0.01), local specific secretory IgA (sIgA) in nasal swab samples (P<0.01); and IgA- and IgG-secreting cells in the nasal mucosa and trachea were higher after intranasal vaccination (P<0.01) than in the control group. Interestingly, intrapulmonary immunization induced much stronger immune responses than intranasal immunization. Intrapulmonary immunization also significantly increased the secretion of IL-6 and local specific sIgA and the numbers of IgA- and IgG-secreting cells. The levels of IL-10 and interferon-γ in the nasal swab samples and the numbers of CD4⁺ and CD8⁺ T lymphocytes in the lung and hilar lymph nodes were significantly increased by intrapulmonary immunization compared with those in the control group (P<0.01). These data suggest that intrapulmonary immunization with attenuated Mhp is effective in evoking local cellular and humoral immune responses in the respiratory tract. Intrapulmonary immunization with Mhp may be a promising route for defense against Mhp in pigs.