Changes in Serum Antibody Levels after Vaccination for Strangles and after Intranasal Challenge with Streptococcus equi subsp. equi in Horses

In this study, to evaluate the influence of strangles vaccination on serological test results, we investigated the changes in strangles serum antibody levels in horses after vaccination and subsequent intranasal challenge with S. equi. The horses were vaccinated for strangles with either a component vaccine (Group C) or a live vaccine (Group L). We measured changes in strangles serum antibody levels weekly for 20 weeks after vaccinating horses twice for strangles over a 3-week interval, and for 7 weeks after intranasal challenge with S. equi in the same horses. Serum antibody responses to the proline-glutamic acid-proline-lysine (PEPK) antigen with five repetitions (PEPK-5R) were higher at all times (up to 2.4-fold) following vaccination in Group C than in Group L, and the value peaked at 2.9-fold above the initial value after the second vaccination in Group C horses. However, the value was lower than that in horses infected with S. equi, and it gradually decreased, reaching the initial (week 0) value by the 15th week. Serum antibody responses to PEPK-5R after challenge with S. equi increased in both groups of horses, but the value tended to be lower than that reported for unvaccinated horses. In addition, the average value in Group C was 2.6-fold higher than that of Group L. These results suggest the serum antibody responses of horses infected with S. equi varies according to the type of vaccine with which they have been vaccinated. Although the serological diagnostic test for strangles in which PEPK-5R is used as an antigen is effective for the investigation of serum antibodies to strangles in vaccinated horses, the present data suggest it is necessary to consider the vaccination history when interpreting the results.     

Evaluation of a nested PCR test and bacterial culture of swabs from the nasal passages and from abscesses in relation to diagnosis of Streptococcus equi infection (strangles)

REASONS FOR PERFORMING STUDY: Streptococcus equi is the cause of strangles in horses. To improve diagnostic sensitivity, development and evaluation of DNA-based methods are necessary. OBJECTIVES: To evaluate diagnostic methods and observe the pattern of bacterial shedding during natural outbreaks. METHODS: Two herds with natural outbreaks of strangles were visited over a period of 15 weeks and 323 samples originating from 35 horses investigated. The diagnostic use of a nested PCR test was evaluated using a collection of 165 isolates of Lancefield group C streptococci (species specificity) and swabs from nasal passages or from abscesses from horses infected with S. equi (diagnostic sensitivity). RESULTS: All 45 S. equi isolates tested positive in the nested PCR, whereas no amplicon was formed when testing the other 120 Lancefield group C isolates. A total of 43 samples were collected from 11 horses showing clinical signs of strangles during the study period. The diagnostic sensitivity for PCR test was 45% and 80% for samples from the nasal passages and abscesses, respectively; the corresponding diagnostic sensitivity for cultivation was 18% and 20%. The diagnostic sensitivity was significantly higher for PCR than for bacterial cultivation. Furthermore, the shedding of S. equi in 2 infected horse populations was evaluated. An intermittent shedding period of S. equi of up to 15 weeks was recorded in this part of the study. It was also shown that shedding of S. equi occurred both from horses with and without clinical signs. CONCLUSIONS AND POTENTIAL RELEVANCE: The nested PCR test represents a species-specific and -sensitive method for diagnosis of S. equi from clinical samples. It may, however, be desirable in future to develop detection methods with high diagnostic sensitivity and specificity without the potential problems inherent in nested PCR.     

Description of an epizootic and persistence of Streptococcus equi infections in horses

The age-specific attack rates of Streptococcus equi infections of the upper respiratory tract and lymph nodes (strangles) in horses for the different age groups were 17.6% for broodmares, 47.5% for 1-year-old horses, and 37.5% for foals. Streptococcus equi was isolated from nasal, pharyngeal, or lymph node specimens in 31 (60.8%) of 51 sick horses. A male 1-year-old horse, shipped from Kentucky to farm A, was considered to be the index case. Six (19.4%) of 31 horses with strangles remained as shedders of S equi after clinical signs of the disease had ended. Shedders of S equi were not identified from horses that were exposed to infected horses but never developed strangles.     

Strangles in horse studs: Incidence, risk factors and effect of vaccination

A questionnaire survey of 179 horse studs in New South Wales was conducted to estimate the incidence of strangles during 1985 to 1988, to identify risk factors for strangles outbreaks and to assess the effect of strangles vaccination. Forty-nine of the studs (27.4%) had at least one strangles outbreak during this period and 62 studs (34.6%) had at least one case of strangles. The average incidence of strangles was 2.1 cases per 100 horses per year. The risk of strangles increased progressively with the total horse population and rose markedly when more than 100 mares had been served in the 1988-89 season. Certain types of feeders, fences and water sources were also significantly associated with outbreaks of strangles. Strangles vaccine was used on 63 studs (35.2%). Thirty-seven of these (58.7%) used the manufacturer's recommended vaccination regime. When other risk factors were taken into account, vaccination had no significant effect on the likelihood of a strangles outbreak.     

Clinical observations on an outbreak of strangles

An outbreak of strangles, which occurred during the spring, summer and fall of 1980 on a Standardbred stud farm in eastern Alberta is described. The infective organism, Streptococcus equi, may have been introduced by an outside mare that was brought to the stud for breeding. All of the groups of horses on the farm were affected. For the most part, the disease was allowed to run its natural course. Only severely affected individuals were treated. During the outbreak, the foals were prophylactically treated with penicillin to prevent them from contracting the disease. Ten horses died of complications of strangles infection.     

Proline-glutamic acid-proline-lysine repetition peptide as an antigen for the serological diagnosis of strangles

The reactivity of the proline-glutamic acid-proline-lysine (PEPK) repetition peptide antigen in 3176 serum samples was investigated to evaluate its utility as an antigen for the serological diagnosis of strangles. The reactivity of the sera of horses infected with Streptococcus equi subspecies equi was high when the peptide had several PEPK repetitions. However, as the number of PEPK repetitions increased, the reactivity of the antigen with the sera of horses infected with Streptococcus equi subspecies zooepidemicus also increased. In horses infected experimentally with S equi, the reactivity of the PEPK antigen with five repetitions increased one week after inoculation and continued to increase during the following four weeks. The optical density (OD) values of test sera from horses infected experimentally with S equi and sera from horses that had recovered from strangles were high. The od values of sera from horses that had recovered from an experimental infection with S zooepidemicus and of sera from healthy horses were comparatively low.     

Proline-glutamic acid-proline-lysine peptide set as a specific antigen for the serological diagnosis of strangles

The reactivity of synthesised peptide sets for the M-like proteins SeM and SzPSe with sera from horses infected with Streptococcus equi or Streptococcus zooepidemicus, or control horses, was investigated by an ELISA. Seventeen horses were infected experimentally with S equi or S zooepidemicus, convalescent sera were obtained from 25 horses and control sera were obtained from 1945 horses. The serum antibody responses of individual horses to the peptide sets were highly variable. Some of the peptide sets for SeM reacted strongly with the sera from the horses infected experimentally with S equi, but also reacted with sera from some of the horses infected experimentally with S zooepidemicus. However, the proline-glutamic acid-proline-lysine (PEPK) repeats peptide set, synthesised from the PEPK repeats areas of SzPSe, reacted most strongly with the sera from the horses infected experimentally with S equi and the horses convalescing from strangles, and reacted only minimally with the sera from the horses infected experimentally with S zooepidemicus and the control horses.     

First Report of Molecular Characterization of Argentine Isolates of Streptococcus equi subsp. equi by Pulsed-Field Gel Electrophoresis

Strangles is one of the most frequently diagnosed equine respiratory infectious diseases in the world. It is caused by Streptococcus equi subsp. equi (S. equi), and it is an acute infection characterized by pyrexia, nasal discharge, pharyngitis, and abscessation of lymph nodes. Frequently, healthy horses might continue to harbor S. equi after clinical recovery. Although the genetic distance between S. equi isolates is short, strains can be differentiated by pulsed-field gel electrophoresis (PFGE) and single locus sequence typing for epidemiological studies. The aim of this study was to characterize by PFGE Argentine isolates of S. equi obtained from horses with acute strangles and those that had recovered. Bacterial isolation and identification of 80 S. equi isolates by phenotypic and genotypic tests were performed using samples from 29 horses with acute strangles and 95 from healthy animals. Also, the isolates were characterized by PFGE using Bsp120I and SmaI. Visual comparison of macrorestriction patterns generated with both enzymes revealed three different DNA fragment profiles with variations of one or two bands. Interestingly, an identical profile was found in isolates from the same horse and from horses that were infected at the same time, and the horses recovered from strangles continue to carry the same strain. Some vaccinated horses have been mild infected for a different strain from that of carriers suggesting other source of infection. This is the first molecular characterization of Argentine isolates of S. equi, which shows the presence of three strains between 2010 and 2013 in Buenos Aires.     

In vivo pathogenicity and resistance to phagocytosis of Streptococcus equi strains with different levels of capsule expression.

The glossy non-encapsulated strain of Steptococcus equi, NCTC 9682, was compared with the matt strain Hidaka/95/2 which expresses a medium sized capsule and with the mucoid CF32 which expresses a large sized capsule in phagocytosis assays and for virulence in inoculated horses. The three strains, NCTC 9682, Hidaka /95/2 and CF32 produced 2.0, 3.1, and 5.3 mg/g wet cells respectively after 3 h incubation, but similar amounts of M-like proteins, cytotoxin and mitogen. NCTC 9682 showed no resistance to phagocytosis by equine neutrophils regardless of the presence of opsonin while strains Hidaka /95/2 and CF32 showed almost complete resistance to phagocytosis. Furthermore, NCTC 9682 produced no clinical disease although it infected the guttural pouch and caused seroconversion. Typical strangles with guttural pouch invasion was observed in all horses infected with encapsulated strains.     

Lessons learned from a strangles outbreak on a large Standardbred farm

Streptococcus equi subspecies (ssp.) equi infection (strangles) remains one of the most frequently diagnosed and costly infectious diseases of horses. Large breeding herds, where a disease outbreak competes for personnel and financial resources needed for foaling management, present a special challenge for equine practitioners. A 15‐month outbreak involving 62 clinical cases of strangles occurred on a large Standardbred breeding farm (average population of 1400 horses). Sixteen asymptomatic horses were found to be PCR (polymerase chain reaction)‐positive for S. equi ssp. equi. During the outbreak, serological samples from 48 clinically normal horses were found to be seropositive for S. equi ssp. equi, confirming herd‐wide exposure. After several clinical cases of strangles had been diagnosed, an intranasal S. equi ssp. equi vaccine was administered to clinically normal horses (n = 558) considered to be at risk of exposure. Strangles complications included 7 fatalities (none in vaccinated horses) and 6 cases of purpura haemorrhagica (4 in vaccinated horses). Midway through the outbreak, injectable, sustained release ceftiofur crystalline free acid (CCFA), given as an initial dose followed by a second dose 4 days later, was used exclusively for systemic antimicrobial treatment of clinically affected and PCR‐positive horses. This antimicrobial regimen coincided with a reduction in disease incidence and eventual resolution of the outbreak. Two horses with persistent guttural pouch infection were endoscopically confirmed as carrier horses. The herd history demonstrated that a strangles outbreak will often result in asymptomatic carrier horses and that identification and treatment of these horses are necessary to eliminate long‐term sources of infection. Ceftiofur crystalline free acid was found to be a suitable antimicrobial due to its activity against S. equi ssp. equi and the efficiencies associated with twice parenteral dosing during a 10‐day treatment period. Occurrence of purpura in 4 vaccinated horses suggests that vaccination should be reserved for healthy seronegative horses and avoided during an active outbreak.     

Prevalence of osseous fragments in distal and proximal interphalangeal, metacarpo- and metatarsophalangeal and tarsocrural joints of Hanoverian Warmblood horses

The prevalence of osseous fragments in different limb joints was analysed in 3749 young Warmblood riding horses, which were pre-selected for sale at auction from 1991 to 1998 by the Association of Hanoverian Warmblood Breeders (Verband hannoverscher Warmblutzüchter e.V.) in Verden (Aller), Germany. The evaluation of these horses was based on a standardized radiological examination, including the distal interphalangeal (DIJ), the proximal interphalangeal (PIJ), the metacarpo- and metatarsophalangeal (MC/MTJ) and the tarsocrural joints (TCJ). In 32% of all horses at least one osseous fragment was found on these radiographs. TCJ were affected in 9.6% of the horses and MC/MTJ in 20.7%. The percentage of horses affected in the MTJ was significantly higher (13.7%) than that of horses affected in the MCJ (9.5%). The prevalences of osseous fragments were considerably lower in DIJ (4.5%) and PIJ (0.9%). Osseous fragments only occurred either in DIJ or PIJ or MC/MTJ or TCJ in 87% of the affected horses. Depending on the joint type, osseous fragments were found in analogous joints in the front and the hind limbs in up to 26% of the affected horses, whilst bilateral affection occurred in between 10% and 46% of the horses. Distribution of osseous fragments in the limb joints investigated here did not differ significantly between male and female horses. There was a significant increase in the percentage of horses with osseous fragments in MC/MTJ during the study period. The results of this study indicate the need to act against the high prevalences of osseous fragments in the limb joints of young Warmblood riding horses.     

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.     

Hematologic parameters of foals during a strangles epizootic

Hematologic parameters of 23 foals were studied at weeks 0, 2, 4, 6, and 10 following the onset of a strangles epizootic. The epizootic was initiated by group exposure to a foal experimentaUy infected with S, equi. The group consisted of 12 foals previously exposed to an S. equi epizootic 6 months prior, and 11 previously unexposed foals. Seventeen percent of the previously exposed foals and 91% of the unexposed foals developed clinical signs of strangles. Significant increases in mean white blood cell count, neutrophil cell count, fibrinogen concentration, and plasma protein concentration were seen in strangles cases, compared to foals not classified as cases, and were associated with clinical signs. Decreases in packed cell volume, hemoglobin concentration, and red blood cell count, although statistically insignificant, were observed in strangles cases compared to noncases during weeks 4, 6, and 10 and may have biological significance. Similar, but more pronounced, changes were observed in the hematologic parameters of the foal experimentally inoculated with S. equi. The effect of Streptococcus equi infection on the hematology of foals should be considered in their convalescent care.     

Serum bactericidal responses to Streptococcus equi of horses following infection or vaccination

An indirect test based on horse blood was used to study bactericidal responses of the horse to Streptococcus equi following infection or vaccination. Bactericidal antibody appeared in convalescent sera between two and four weeks and high titres were usually attained by eight weeks. Infection without clinical evidence of abscessation was also effective in eliciting strong bactericidal responses. Serum bactericidal activity of horses either recovered from strangles or immunised with commercial bacterin had declined eight months after vaccination. However, horses that developed strangles eight to 10 months after vaccination exhibited rapid and substantial increases in serum bactericidal activity. Groups of yearlings immunised with commercial S equi vaccines consisting either of M protein or bacterin developed clinical strangles within six months of vaccination although the majority of the animals had exhibited strong serum bactericidal activity a few weeks before occurrence of the disease. Similarly, a group of seven yearling ponies hyperimmunised with experimental vaccine, rich in M protein, were found to be highly susceptible to an intranasal challenge of 5 X 10(8) colony forming units of S equi, although their sera exhibited strong bactericidal activity at the time of challenge. These observations suggest that the role of serum bactericidal antibody in protection of the horse against strangles has been overrated.     

Antithrombin III activity in horses with large colon torsion

A chromogenic peptide substrate assay was used to determine serially plasma antithrombin III (AT III) activity in 4 groups of horses. Group I consisted of healthy, mature horses in which AT III activity was determined twice daily for 7 consecutive days. Groups 2 and 3 contained healthy horses in which AT III activity was monitored for 7 days after controlled, but varying, conditions of general anesthesia and surgery (median celiotomy). Group 4 was made up of patients with a presurgical diagnosis of colonic torsion. In healthy awake horses (group I), there was no difference in AT III values over time. Postoperative AT III activity in the halothane-anesthetized horses (group 2) and in the sham-operated horses (group 3) was not significantly (P = 0.05) different from base-line values at any time. A significant decrease (P = 0.05) in AT III activity was observed on postoperative days 1 through 3 in the group of horses with large colon torsion, but returned to preoperative values by day 4 after surgery in the horses that survived. In those horses that did not survive, AT III activity remained below base-line values for the duration of observation. Seemingly, plasma AT III activity in horses was not significantly affected by halothane anesthesia or surgery. Serial evaluation of AT III activity may be useful for predicting survival in horses with large colon torsion.     

Prevalence of Ocular Disorders in an Indian Population of Horses

The objective of this clinical study was to report the prevalence of ocular disorders in a large population of horses from the Indian army. Both eyes of 500 horses were examined using a direct ophthalmoscope and by a transillumination procedure. Intraocular pressures were measured in several study horses. Fluorescein dye, Schirmer tear test, and menace testing were performed when indicated. Corneal culture swabs were collected from horses affected with corneal ulcers found on ophthalmoscopic examination. A total of 100 horses were found to be affected with some form of ocular disorder. The eye problems recorded were congenital eye lid disorders (2%) and included a subcutaneous cyst in the lower eye lid and slit in upper eye lid; conjunctivitis (12%); chronic interstitial keratitis (30%); a parasite in the anterior chamber (1%), which was however not removed for genus and species identification; phthisis bulbi (1%); uveitis (24%); glaucoma (13%); cataracts (3%); unilateral retinal hemorrhage (1%); luxation of cataractous lens (3%); posterior synechia (2%); and corneal ulceration (8%). Pseudomonas spp was the most common bacteria identified in horses affected with corneal ulceration. Vision-threatening ocular disorders are high in Indian army horses; this is a cause of major concern for the rider and the horse and, therefore, it is important to include ocular examination in horses as part of a routine and complete physical examination.     

Prevalence of Surra among camels and horses in Jordan

The prevalence of Trypanosoma evansi infection among camels and horses in Jordan was studied using thick blood smears and inoculation techniques with mice and rats. A total of 437 camels and 83 horses from four climatic zones were surveyed. In addition, 40 donkeys, 32 cattle and 35 goats in contact with infected camels and horses were also tested in the same way. Clinical disease was evident in 8.2% of the camels (36 out of 437) and in 9.6% of the horses (8 out of 83). Infection was limited only to the Sweama area on the Dead Sea (within the warm desert-climatic zone), with prevalence of 30.5% and 33.3%, respectively, for camels and horses. Donkeys, cattle and goats examined were all free from T. evansi. Clinically affected camels were positive by both, thick blood smear and mouse and rat inoculations. Rat and mouse inoculations revealed (X²=3.2, df=1, exact p=0.07) greater number of positive cases in horses than those revealed by thick blood smears. T. evansi-infected camels and horses showed all the clinical signs known for Surra. In addition, it was observed that 100% of infected camels stared at the sun.     

Pathologic findings in reintroduced Przewalski's horses (Equus caballus przewalskii) in southwestern Mongolia.

The Przewalski's horse (Equus caballus przewalskii) was extinct in the wild by the mid 1960s. The species has survived because of captive breeding only. The Takhin Tal reintroduction project is run by the International Takhi Group; it is one of two projects reintroducing horses to the wild in Mongolia. In 1997 the first harem group was released. The first foals were successfully raised in the wild in 1999. Currently, 63 Przewalski's horses live in Takhin Tal. Little information exists on causes of mortality before the implementation of a disease-monitoring program in 1998. Since 1999, all dead horses recovered (n = 28) have been examined and samples collected and submitted for further investigation. Equine piroplasmosis, a tick-transmitted disease caused by Babesia caballi or Theileria equi, is endemic in Takhin Tal and was identified as the cause of death of four stallions and one stillborn foal. In December 2000, wolf predation was implicated in the loss of several Przewalski's horses. However, thorough clinical, pathologic, and bacteriologic investigations performed on dead and surviving horses of this group revealed lesions compatible with strangles. The extreme Mongolian winter of 2000-2001 is thought to have most probably weakened the horses, making them more susceptible to opportunistic infection and subsequent wolf predation. Other occasional causes of death since 1999 were trauma, exhaustion, wasting, urolithiasis, pneumonia, abortion, and stillbirth. The pathologic examination of the Przewalski's horses did not result in a definitive diagnosis in each case. Several disease factors were found to be important in the initial phase of the reintroduction, which could potentially jeopardize the establishment of a self-sustaining population.     

Treatment of maxillary cheek teeth apical infection caused by patent infundibula in six horses (2007–2013)

Six young horses presented with clinical signs of maxillary cheek teeth apical infection (bilateral in 2 horses) that included the presence of rostral maxillary swellings with discharging sinus tracts and were unexpectedly found to have no evidence of endodontic infection in 7 affected teeth. The apical infection of these teeth was attributed to infundibular dysplasia that allowed inoculation of the periapical tissue with contaminated oral contents. Treatment was by debridement and obturation of affected infundibula from their occlusal aspect using techniques normally used for endodontic therapy. Five out of 6 cases showed resolution of clinical signs over a mean follow‐up period of 19 months (range 6–48 months). These cases demonstrate that patency of the infundibular apex is a potential cause of cheek teeth apical infection in young horses, which can be successfully treated by debridement and subsequent infundibular restoration.     

Training-related factors associated with dorsometacarpal disease in young Thoroughbred racehorses in the UK

REASONS FOR PERFORMING STUDY: Dorsometacarpal disease (DMD) is common in young Thoroughbred (TB) racehorses, often interfering with their training programme. No reports exist on incidence rate or associated risk factors in the UK. OBJECTIVES: To estimate the incidence rate of DMD in TB horses in training for flat racing in the UK and identify associated risk factors, particularly in relation to training regimens. METHODS: Data were drawn from a large prospective study in which TB horses in 13 training yards were monitored for a period of up to 2 years, recording daily training information and details of musculoskeletal injury, including DMD. The incidence of DMD was calculated and survival analysis performed to determine features of the training regimen related to the risk of disease. RESULTS: A total of 335 horses provided 4235 months at risk of DMD. Seventy-nine first episodes were recorded, resulting in an incidence rate of 1.87/100 horse months (95% confidence interval 1.50-2.33). Increasing exercise distances at canter and high speed in short periods (up to 1 month) were associated with an increasing risk of DMD. However, increasing cumulative exercise distances since entering training were associated with a decreasing risk of the disease. CONCLUSIONS: The risk of DMD in young TBs decreases with accumulation of distances exercised at canter and high speed; this reflects the adaptive response of the third metacarpal bone to the loads placed upon it. However, increasing exercise distances in short periods (up to 1 month) increases the risk of DMD, probably as a consequence of microdamage and its associated remodelling response, and should therefore be avoided. POTENTIAL RELEVANCE: Training regimens for young TBs can be adjusted to prevent the occurrence of DMD. Early but gradual introduction of small amounts of high-speed exercise may be beneficial. Canter exercise should be kept minimal on high-speed work days and large amounts of both cantering and high-speed work per week, 2 weeks or per month should be avoided, particularly during the early stages of training, in order to minimise the risk of DMD.