Sequelae and complications of Streptococcus equi subspecies equi infections in the horse

Streptococcus equi ssp. equi infection in the horse, or strangles, commonly results in abscessation of the submandibular, submaxillary or retropharyngeal lymph nodes. Although this classical presentation of strangles is associated with a low mortality rate, complications and sequlae may worsen the prognosis and increase mortality rates. This article reviews sequelae and complictions of S. equi ssp. equi infection in the horse, including guttural pouch empyema, bastard strangles and immune mediated diseases such as purpura haemorrhagica, myopathies and myocarditis.     

Lymph Drainage from the Ovine Tonsils: An Anatomical Study of the Tonsillar Lymph Vessels

Although the tonsils of sheep have gained much attention during the last decade, only few data are available on their lymph vessel architecture. Tonsillar lymph vessels are immunologically important as they form the efferent routes for locally activated immune cells to reach the draining lymph nodes. To gain insight into the tonsillar lymph drainage in the sheep, Indian ink and a casting polymer were injected into the interstitium of the five tonsils present in the heads of slaughtered sheep. This enabled us to determine the draining lymph node and to examine the microscopic organization of lymph vessels using light and scanning electron microscopy. No lymph vessels were observed within the tonsillar lymphoid follicles. The corrosion casts demonstrated that the lymphoid follicles are surrounded by numerous sacculated lymph sinuses that drain into a dense interfollicular lymph vessel network. From here, the lymph flows into single small lymph vessels that in turn drain into larger lymph vessels extending towards the medial retropharyngeal lymph node. The presented results can be valuable for immunological studies, for example during oral or intranasal vaccine development.     

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.     

Experimental Studies on the Pathogenesis of Corynebacterium equi Infection in Foals

Four month-old foals were infected orally with 75 mL of a suspension of 5.0 × 10⁸ Corynebacterium equi per mL. Two foals were killed after ten days and had scanty number of C. equi in the caeco-colic lymph nodes. No C. equi were recovered from the other two foals, killed 20 days after infection. No gross pathological change was detected in these four foals, although mild microscopic lesions were seen in the ileum of one foal. Results of lymphocyte blastogenesis using peripheral blood lymphocytes and C. equi antigens showed, however, that lymphocytes became sensitized to C. equi following this challenge.

In a second experiment four month-old foals were given orally the same dose of organisms but on five consecutive days. Two foals were killed ten days after infection and showed mild histological changes in the large bowel mucosa and C. equi could be recovered from all intestinal lymph nodes cultured. In one of these foals moderate numbers of C. equi were present in the bronchial lymph node. Of the other two foals, one died after 22 days with severe ulcerative enterocolitis and intestinal lymphadenitis. Only one small pulmonary abscess was detected despite large numbers of C. equi in the lungs. The other foal developed similar intestinal changes and was euthanized 25 days after infection. No C. equi were detected in the lungs or bronchial lymph node. Lymphocyte blastogenesis in these animals showed a rapid rise in response to C. equi antigens.

These studies suggest that C. equi pneumonia in foals does not always arise from an intestinal infection, that minor intestinal infection causes a cellular immune response and that massive exposure of the bowel over a sustained period is necessary to induce intestinal lesions.

     

CD21 B cell populations are altered following subcutaneous scrapie inoculation in sheep

In order to gain a better understanding of the pathogenesis of scrapie in sheep an experimental model was developed to characterise immune system cells in the minutes following inoculation with scrapie-brain homogenate. Four 1-year-old susceptible (ARQ/ARQ) sheep were inoculated via the subcutaneous route at four different peripheral lymph node (LNs) drainage sites, at specific time points, prior to euthanasia of the sheep. The LNs were removed post-mortem at 30, 90, 180 and 300 min after inoculation. Flow cytometric triple-labelling was carried out on the LN cells and indicated that inoculation of scrapie-brain homogenate adjacent to a lymph node may delay or even inhibit the number of host CD21⁺ B cells expressed within the first 5 h. Immunohistochemistry was used to attempt detection of the abnormal form of prion protein (PrP^sc) in draining LNs adjacent to inoculation sites, with negative results at those time points.     

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes

Modulation of the immune system is known to be important for successful pregnancy but how immune function might differ between the lymph nodes draining the reproductive tract and peripheral lymph nodes is not well understood. Additionally, if immune system changes in response to the presence of an embryo during early pregnancy, and if this response differs in local versus peripheral immune tissue, has not been well characterized. To address these questions, we examined expression of genes important for immune function using NanoString technology in the ampulla and isthmus of the oviduct, endometrium, lymph nodes draining the reproductive tract (lumbo-aortic and medial iliac) as well as a peripheral lymph node (axillary), the spleen, and circulating immune cells from ewes on day 5 of the estrous cycle or pregnancy. Concentrations of estradiol and progesterone in plasma were also determined. Principal component analysis revealed separation of the local from the peripheral lymph nodes (MANOVA P = 3.245e-08, R² = 0.3) as well as separation of tissues from pregnant and nonpregnant animals [lymph nodes (MANOVA P = 2.337e-09, R² = 0.5), reproductive tissues (MANOVA P = 2.417e-14, R² = 0.47)]. Nine genes were differentially (FDR < 0.10) expressed between lymph node types, with clear difference in expression of these genes between the lumbo-aortic and axillary lymph nodes. Expression of these genes in the medial iliac lymph node was not consistently different to either the axillary or the lumbo-aortic lymph node. Expression of IL10RB was increased (FDR < 0.05) by 24% in the reproductive tissue of the pregnant animals compared to nonpregnant animals. Analysis of gene categories revealed that expression of genes of the T-cell receptor pathway in reproductive tract tissues was associated (P < 0.05) with pregnancy status. In conclusion, assessment of gene expression of reproductive and immune tissue provides evidence for a specialization of the local immune system around the reproductive tract potentially important for successful establishment of pregnancy. Additionally, differences in gene expression patterns in reproductive tissue from pregnant and nonpregnant animals could be discerned as early as day 5 of pregnancy. This was found to be associated with expression of genes important for T-cell function and thus highlights the important role of these cells in early pregnancy.     

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.     

Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens.

The proliferation responses of antigen-specific lymphocytes from various anatomical sites were studied in dairy goats locally immunized with heat-killed Staphylococcus aureus (HKS). Animals were inoculated three times subcutaneously in the right udder with HKS at 1 month intervals. One week following the last inoculation, prescapular, mesenteric and ipsilateral (draining) and contralateral (non-draining) suprammammary lymph nodes were collected and the cells assayed in 3- and 6-day cultures to determine the immune proliferative responses of antigen-specific lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). The cells from draining and non-draining supramammary lymph nodes responded to HKS in 3-day cultures. Peripheral lymph nodes, such as the prescapular, showed similar responses. In contrast, mesenteric lymph nodes responded optimally in 6-day cultures, notably to lower concentrations of the antigen. Cells from all lymph nodes tested showed increased responses to PHA in immunized animals, although non-draining lymph nodes demonstrated a greater response to the T cell mitogen than those of draining lymph nodes. These results suggest that unilateral introduction of Staphylococcus cell antigens to the supramammary region can induce an anamnestic response in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. Furthermore, these data indicate that cells from intestinal lymph nodes respond differently from those of peripheral lymph nodes, suggesting the presence of a unique gastrointestinal lymphoid cell circulation in goats. Concomitant peripheral responses may be attributed to memory cell migration or to antigen leakage and relocation to distant sites from the inoculated region. Analysis with PHA suggests a difference in general responsiveness and perhaps, immunocompetence, by lymphocyte populations in various lymphoid tissues of immunized animals.     

A note recording the occurrence of bovine viral vaginitis in New Zealand

Episodes of frenzy lasting approximately 30 minutes were observed among horses confined to enclosures surfaced with sand or soil. The probability of sighting these episodes increased by a factor of three when within 24 hours there was 0.2mm or more of rain, a maximum air temperature between 16.7 – 26.6°and a soil temperature of 16.3 – 23.9°C at 30 cm. High egg counts of Strongyloides westeri appeared in faeces four to five days later and persisted for several days.

Rhodococcus equi was recovered from all soil samples, and from the faeces of 76% of mares and 82% of foals. The youngest foal was five days old when the organism was isolated from rectal faeces. In contrast to the majority of reports, the lesions of R. equi in the foals were confined to limbs and peripheral lymph nodes.

It is proposed that the percutaneous invasion of these foals by third stage larvae of S. westeri facilitated invasion of R. equi, and ubiquitous saprophytic opportunist pathogen.⁽²⁾     

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.     

Association of Streptococcus equi with equine monocytes

Streptococcus equi (Se), the cause of equine strangles, is highly resistant to phagocytosis by neutrophils and is usually classified as an extracellular pathogen. Large numbers of the organism in tonsillar tissues during the acute phase of the disease are completely eliminated during convalescence by mechanisms not yet understood. In this study we demonstrate in an opsono-bactericidal assay and by cytometry and confocal microscopy that Se is interiorized and killed by equine blood monocytes. This finding supports the hypotheses that adaptive immune clearance is mediated by tonsillar macrophages and that macrophages monocytes could serve as a vehicle for transport from the tonsil to local lymph nodes.     

Adherence of Streptococcus equi on tongue,cheek and nasal epithelial cells of ponies

Streptococcus equi was found to adhere to tongue, cheek and nasal epithelial cells of ponies, in vitro. Maximum adherence was observed at pH 7.5 after one hour of incubation of bacteria with epithelial cells. This adherence was more on epithelial cells from adult animals than foals. Streptococci exposed to heat (60°C for 10 min) or treated with pepsin or trypsin showed a reduced adherence, whereas an increase occurred on treatment with hyaluronidase. Antibodies against whole S. equi cells or M-like protein blocked the adherence, whereas antibodies against group-specific carbohydrate or lipoteichoic acids did not. Pretreatment of epithelial cells with either the M-like protein or crude extract of S. equi lowered the adherence, whereas an extract of S. zooepidemicus did not. Adherence of S. equi to the epithelial cells was considered to be mediated by structures specific to S. equi.     

EHV-1 gene63 is not essential for in vivo replication in horses and mice, nor does it affect reactivation in the horse: short communication

The aim of this study was to investigate the role of immediate early gene (gene63) in the pathogenesis of equine herpesvirus 1 (EHV-1) acute and latent infections in equine and murine models. EHV-1 gene63 mutant virus (g63mut) along with EHV-1 (Ab4) was used for intracerebral and intranasal infection of 3 and 17-day-old mice. Both viruses were recovered at the same frequency from tissues after infection. Two Welsh ponies were infected via the intranasal route with each of the viruses. Acute infection was monitored by virus isolation from nasal swabs and peripheral blood leukocytes. Six weeks post infection, peripheral blood leukocytes were taken from ponies and in vitro reactivation was positive for both viruses. At autopsy, both viruses were isolated by co-cultivation from bronchial and submandibular lymph nodes. These findings indicate that the mutation of EHV-1 gene63 does not play a role in the establishment and reactivation from latency.     

Characterization of a Streptococcus equi ssp. equi Isolate From a Strangles Outbreak in Thailand

Strangles, which is caused by Streptococcus equi ssp. equi, is one of the major infectious respiratory diseases in horses. Knowledge of isolates from different areas of the world is important for investigating the different strains of the disease. In contrast to many other countries, currently little is known about S. equi ssp. equi isolates in Thailand. In 2014, a farm in Thailand imported 20 horses from Europe. Approximately 1 month after arrival, 50% of the horses had developed pyrexia, mucopurulent nasal discharge, and abscesses of the mandibular lymph nodes. Nasal swabs of mucopurulent discharge were sent to a diagnostic laboratory, and two isolates of S. equi ssp. equi were identified. One of the isolates was further characterized using seM gene polymerase chain reaction and sequence analysis. The seM sequence was then compared to the database of PubMLST-seM. It was found to contain SeM allele 48, an allele isolated from horses in the United Kingdom in 2006 and 2010. This result demonstrates the usefulness of SeM allele identification as a tool for investigating the source of related strains and for the epidemiologic study of strangles. To the best of the authors' knowledge, this is the first report of the identification of an SeM allele of the S. equi ssp. equi isolate in Thailand.     

Strangles and its complications

Strangles, caused by the Gram‐positive bacteria Streptococcus equi subspecies equi (S. equi), is a highly contagious upper respiratory infection in horses. The infection is transmitted by inhalation or direct contact with mucopurulent discharge from an infected animal, resulting in fever, depression, and submandibular and retropharyngeal lymph node enlargement that can lead to respiratory distress. Complications include secondary cellulitis at external abscessation sites, guttural pouch empyema and its persistence into the carrier state, purpura haemorrhagica, metastatic abscessation, emergency tracheostomies and rarely secondary S. equi pneumonia or myositis. Control of outbreaks requires strict isolation protocols and hygiene measures. Detection methods of the index case and carrier state are constantly being refined to assist in the identification and prevention of disease perpetuation.     

Equine laminitis: Ultrastructural lesions detected in ponies following hyperinsulinaemia

Reasons for performing study: Anatomical changes in the hoof lamellar tissue induced by prolonged hyperinsulinaemia have not been described previously. Analysis of the induced lesions may promote understanding of hyperinsulinaemic laminitis pathogenesis and produce clinical benefit. Objectives: To use light and transmission electron microscopy (TEM) to document hoof lamellar lesions in ponies clinically lame after prolonged hyperinsulinaemia. Methods: Nine clinically normal, mature ponies were allocated randomly to either a treatment group (n = 5) or control group (n = 4). The treatment group received insulin via a modified, prolonged euglycaemic hyperinsulinaemic clamp technique (EHCT) and were subjected to euthanasia when clinical signs of Obel grade II laminitis occurred. The control group was sham treated with an equivalent volume of 0.9% saline and killed at 72 h. Lamellar tissues of the right front feet were harvested and processed for TEM. Results: Lamellae from insulin treated ponies were attenuated and elongated with many epidermal basal cells (EBC) in mitosis. Unlike carbohydrate induced laminitis in horses there was no global separation at the lamellar dermal/epidermal interface among ponies. Sporadic EBC basement membrane (BM) separation was associated with the proximity of infiltrating leucocytes. In 2 ponies, the lamellar BM was thickened. The number of hemidesmosomes/μm of BM was decreased in all insulin treated ponies. Conclusions: Prolonged hyperinsulinaemia causes unique lamellar lesions normally characteristic of acute and chronic laminitis. Lamellar proliferation may be an insulin effect through its mitogenic pathway. Aberrant lamellar mitosis may lengthen and weaken the lamellar, distal phalanx attachment apparatus and contribute to the clinical signs that developed. Potential relevance: The study shows that insulin alone, in higher than normal circulating concentrations, induces profound, changes in lamellar anatomy. Medical control of insulin resistance and hyperinsulinaemia may ameliorate lesions and produce clinical benefit.     

Granuloma development in cattle after intratonsilar inoculation with Mycobacterium bovis

OBJECTIVE: To examine the temporal development of tuberculous lesions in cattle inoculated with Mycobacterium bovis. ANIMALS: 15 mature crossbred cows obtained from a herd with no history of M bovis infection. PROCEDURE: Inoculation of cattle was done by intratonsilar instillation of 1.48 X 10(5) to 5.4 X 10(7) colony-forming units of M bovis strain 2045T. At 3 to 4 hours, 4 weeks, 6 weeks, and 8 weeks after inoculation, tissues were examined for gross and microscopic lesions and processed for isolation of M bovis. RESULTS: Retropharyngeal lymph nodes from cattle examined 4 weeks after inoculation contained microgranulomas consisting of aggregates of macrophages with few neutrophils. Retropharyngeal lymph nodes from all cattle examined 6 and 8 weeks after inoculation contained multiple, large, coalescing granulomas consisting of central areas of necrosis with mild fibrosis, numerous macrophages, lymphocytes, plasma cells, multinucleated giant cells, and neutrophils. Three of 8 cattle examined 6 or 8 weeks after inoculation had lesions in nonretropharyngeal sites with morphologic characteristics similar to that seen in retropharyngeal lymph node granulomas from cattle examined 4 weeks after inoculation. CONCLUSION: Granulomas can develop in draining lymph nodes of cattle in as little as 4 weeks after inoculation via intratonsilar instillation of M bovis. Intralesional morphologic changes between 4 and 6 weeks after inoculation indicate an increase in cellular chemotaxis and differentiation. Dissemination of bacteria to distant sites most likely was by lymphatic and hematogenous routes after establishment of the primary infection in retropharyngeal lymph nodes.     

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.     

Osteomyelitis of the scapulohumeral joint associated with Rhodococcus equi and unresponsive to antibiotherapy in a foal

This case report describes the treatment and outcome of a 3.5-month-old lame foal, with a history of pneumonia associated with Rhodococcus equi. Clinical examination identified osteomyelitis of the scapulohumeral joint. The unusual location of the Rhodococcus equi osteomyelitis unresponsive to prevention and adequate treatment led to euthanasia of the patient. Post-mortem findings confirmed bronchopneumonia due to Rhodococcus equi as well as the osteomyelitis of the scapulohumeral joint. This case highlights that Rhodococcus equi should be considered as a pathogen in extrapulmonary locations such as septic arthritis and osteomyelitis in foals with history of bronchopneumonia. Early and aggressive treatment should be considered.