Expression of p53, Ki67, EcPV2- and EcPV3 DNA, and viral genes in relation to metastasis and outcome in equine penile and preputial squamous cell carcinoma.

REASONS FOR PERFORMING STUDY: Equine penile and preputial squamous cell carcinoma (SCC) is a potentially lethal disease of which little is known regarding the relationship between tumour characteristics and prognosis. OBJECTIVES: To assess the relationship between tumour differentiation grade (tumour subtype), presence of papillomaviruses, expression of viral genes (E2, E6, L1), nuclear proteins p53 and Ki67 and metastasis in equine penile and preputial SCC and to assess the relationship of tumour subtype, presence of papillomavirus type 2, p53 and Ki67 with survival. STUDY DESIGN: Retrospective case-control study using archived material. METHODS: Samples (n = 103) from 87 horses with penile and/or preputial intraepithelial neoplasia (PIN), papilloma or SCC and corresponding case files were evaluated. Tumours were graded microscopically and p53 and Ki67 expression evaluated immunohistochemically. Equine papillomavirus (EcPV) types 2 and 3 DNA was detected by conventional PCR. Real-time PCR was used for quantification of E2, E6 and L1 mRNA. RESULTS: Equine papillomavirus type 2 DNA was detected in 89.4% and EcPV3 in 1.5% of horses. No differences in quantitative expression of E2, E6 and L1 oncogenes between subtypes were found. Expression of p53 and occurrence of metastasis were positively correlated to a less differentiated subtype (r = 0.429, P<0.001 and r = 0.769, P = 0.001, respectively). Differences in survival between subtypes were significant (log Rank P<0.001); horses with less differentiated tumours were more likely to die of the disease (papilloma 8.3%; G1 26.1%; G2 26.3%; G3 63.3%). CONCLUSIONS: In equine penile and preputial SCC, tumour grading is an important prognosticator for survival and a predictor for presence of metastases. Expression of p53 and Ki67 and presence or expression of EcPV2 and EcPV3 do not appear to be important prognosticators.

Expression of cyclo-oxygenases-1 and -2, and microsomal prostaglandin E synthase-1 in penile and preputial papillomas and squamous cell carcinomas in the horse.

REASONS FOR PERFORMING STUDY: Penile and preputial papilloma and squamous cell carcinoma (SCC) are commonly diagnosed in horses. Papillomas have the potential to progress to potentially lethal SCC. Knowledge of pathogenetic mechanisms may help in prevention and definition of treatment targets. STUDY DESIGN: Retrospective study using archived material. OBJECTIVES: To determine the expression of cyclo-oxygenase 1 (COX-1), cyclo-oxygenase 2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) in penile and preputial normal tissue, papilloma and SCC in horses, and whether expression of these enzymes is influenced by degree of inflammation and differentiation grade. METHODS: Tumour differentiation grade, degree of inflammation and COX-1, COX-2 and mPGES-1 expression in 75 formalin-fixed paraffin embedded samples of penile and preputial papilloma and SCC of 68 horses were investigated by histopathology and immunohistochemistry. RESULTS: Inflammation was more prominent in SCC compared with papilloma. No correlation between expression of COX-1 or COX-2 and inflammation was found. Expression of mPGES-1 was weakly correlated with inflammation. Expression of COX-1, COX-2 and mPGES-1 was found in 42.6%, 50.7% and 96.0% of lesions respectively, but less than 1% of cells were immunopositive for COX-1 and COX-2 in 59.4% and 84.2% of cases respectively. Expression of COX-1 was moderately negatively correlated with differentiation grade, COX-2 was not correlated and mPGES-1 was poorly negatively correlated. CONCLUSIONS: Expression of COX-1 and COX-2 in penile and preputial SCC in the horse is poor and COX inhibitors may thus be of little value for prevention or treatment. Microsomal PGES-1 is more prominently expressed in well-differentiated tissue compared with poorly differentiated tissue. Further research on the role of mPGES-1 in carcinogenesis is needed to assess its potential use as a treatment target. Knowledge of arachidonic pathway enzyme expression and their role in equine penile and preputial carcinogenesis may help in developing preventive and therapeutic strategies.

Treatment and long-term follow-up of distichiasis, with special reference to the Friesian horse: a case series.

REASONS FOR PERFORMING STUDY: Distichiasis is very uncommon in horses and treatment options and outcome remain unknown. OBJECTIVES: To describe treatment and long-term follow-up of distichiasis in the horse. STUDY DESIGN: Retrospective clinical study. METHODS: Case records of horses admitted for diagnosis and treatment of uni- or bilateral distichiasis between 2003 and 2012 were reviewed. Long-term (>6 months after last treatment) follow-up was by telephone conversation with the owner or referring veterinarian. Data were analysed using a Chi-squared test. RESULTS: Eighteen cases were identified, of which 17 were Friesians. All horses had signs of corneal irritation or ulceration concurrent with distichiasis. In total, 29 eyes were affected with distichiasis of which 23 had clinical signs. All corneas were stained with fluorescein and 13 had fluorescein positive defects (45%). The lower eyelid was affected more often. Two different electrocautery devices were used for treatment. The complications were slight scarring of the eyelids (100%) and clinical recurrence (54%). Successful resolution of the clinical signs after one treatment occurred in 46% of the horses and in 77% of the horses satisfactory long-term results were found after a mean of 2 treatments (range: 1- 6 treatments). There was significant correlation between number of hairs and recurrence (P<0.002). CONCLUSIONS: There is a breed predisposition of distichiasis in the Friesian horse. In Friesian horses that present with uni- or bilateral corneal irritation or corneal ulceration, careful examination of the eyelid margins should be performed to diagnose distichiasis. Electrocautery is a valid and successful therapeutic modality which is minimally invasive, inexpensive, has a low complication rate and is easy to perform in the standing sedated horse. The recurrence rate is low in horses with less than 5 distichiae.

Penile and preputial tumours in the horse: literature review and proposal of a standardised approach.

Penile and preputial tumours are not uncommon in the horse, but can cause discomfort and lead to serious complications. Several types of tumour of the male external genitalia have been described. The most common type is the squamous cell carcinoma (SCC), which is found mainly in older horses. Reports of a breed predilection for penile tumour formation are equivocal, but castration, coat colour, poor hygiene and various infectious agents have all been suggested to predispose to the development of some types of tumour (e.g. SCC, papilloma and melanoma). Careful assessment of the primary tumour is an important first step in the design of an optimal treatment protocol. Invasiveness, differentiation grade, tumour size and presence of metastases are all relevant to the decision to pursue additional diagnostic procedures or specific treatment options. To date, no standard protocol has been reported for the approach to penile tumours in the horse and treatments range from minimally invasive therapies (e.g. topical use of 5-fluorouracil) to radical surgical interventions (e.g. en bloc penile and preputial resection with penile retroversion). Completeness of removal of the neoplasm and therefore risk of recurrence is highly dependent on the type of therapy chosen. However, the size and histopathological features of the primary tumour are also important factors with respect to the likelihood of recurrence. This review describes the most common penile and preputial neoplasms in the horse, and outlines a standard protocol aimed at arriving at a specific diagnosis and tailoring the therapeutic approach accordingly.

Penile and preputial tumours in the horse: a retrospective study of 114 affected horses.

REASONS FOR PERFORMING STUDY: Preputial and penile tumours are more common in horses than in other domestic animals, but no large surveys of male horses with tumours of the external genitalia are available. OBJECTIVE: To present a retrospective analysis of male horses with neoplasms of the external genitalia. METHODS: The penile and preputial tumours of 114 horses were evaluated. Data recorded included age, gelding or stallion and breed; type and site of lesion; involvement of regional lymph nodes; histopathology (including grading of squamous cell carcinoma); and results of radiographic examination of the thorax. RESULTS: Mean age of horses was 19.5 years with no apparent breed predilection. Common presenting clinical signs were irregularities (e.g. the presence of a mass and/or ulceration) on the integument of the penis and prepuce, and purulent or sanguineous discharge from preputial orifice. Squamous cell carcinoma (SCC) was the most prevalent neoplasm followed by papillomas and melanomas. A basal cell carcinoma, neurofibrosarcoma, adenocarcinoma or fibrosarcoma were each found on single horses. Squamous cell carcinomas with poor differentiation had a higher tendency to metastasise than did more differentiated tumours. CONCLUSIONS: Squamous cell carcinoma is the most common urogenital tumour of the male horse and occurs primarily in old horses. Horses with poorly differentiated SCCs tend to have a higher incidence of regional metastases. Pathology of lymph nodes, even when not palpably enlarged, is a valuable diagnostic exercise. Radiology of the thorax to detect lung metastases is of little value.

Penile and preputial squamous cell carcinoma in the horse: a retrospective study of treatment of 77 affected horses.

REASONS FOR PERFORMING STUDY: The most common penile and preputial neoplasm in the horse is the squamous cell carcinoma (SCC), but no large surveys of treatment and effects of the grade of the tumour, based on the degree of differentiation, on outcome of affected horses are available. OBJECTIVES: Analysis of treatment of male horses affected with SCC of the external genitalia and long-term results of treatment. METHODS: Seventy-seven cases of SCC were evaluated. Data recorded included treatment, outcome, post operative histopathology and retrospective tumour grading. RESULTS: Treatments included: cryosurgery, excision, partial phallectomy, partial phallectomy and sheath ablation, and en bloc penile and preputial resection with penile retroversion and removal of inguinal lymph nodes. The incidence of recurrence after partial phallectomy was 25.6% (10/39) and following incomplete removal was 17.9% (7/39). The incidence of recurrence after en bloc resection with retroversion was 12.5% (1/8). In horses with confirmed inguinal lymph node metastasis, the incidence of recurrence was 25.0% (1/4). Poorly differentiated SCCs were more likely to metastasise than well differentiated SCCs, and there was a greater chance that the treatment would be unsuccessful. The success of treatment, complete removal and in preventing recurrence of the tumour, of male horses with SCC of the external genitalia was 55.7%. CONCLUSIONS: Horses that receive only partial phallectomy for treatment for SCC of the external genitalia have a high incidence of recurrence in contrast to horses that receive an en bloc resection. Tumour grading of SCC can help predict prognosis and guide selection of treatment.

Clinical efficacy of intravenous administration of marbofloxacin in a Staphylococcus aureus infection in tissue cages in ponies.

Tissue cages (TC), implanted subcutaneously in the neck in eight ponies, were inoculated with Staphylococcus aureus (S. aureus) to determine the clinical efficacy of marbofloxacin in the treatment of this infection. From 21 h after inoculation, marbofloxacin (6 mg/kg) was administered intravenously (i.v.) once daily for 7 days. Samples of the tissue cage fluid (TCF) were taken to determine marbofloxacin concentrations (days 1, 3 and 7), using high-pressure liquid chromatography, and numbers of viable bacteria [colony forming units (CFU)] (days 1, 3, 7, 14 and 21). Statistical analysis was used to compare CFU before and after treatment. Clinical signs and CFU were used to evaluate the efficacy of treatment. Although, there was a slight decrease in CFU in all TC initially, the infection was not eliminated by marbofloxacin treatment in any of the ponies and abscesses formed. As the MIC (0.25 microg/mL) did not change during treatment and the concentration of marbofloxacin during treatment (mean concentration in TCF was 0.89 microg/mL on day 1, 0.80 microg/mL on day 3 and 2.77 microg/mL on day 7) was above MIC, we consider that the treatment failure might be attributable to the formation of a biofilm by S. aureus. Based on the present results, i.v. administration of marbofloxacin alone is not suitable for the elimination of S. aureus infections from secluded sites.

Clinical efficacy of local administration of ceftiofur in a Staphylococcus aureus infection in tissue cages in ponies.

Ceftiofur concentrations in an infected and uninfected environment were compared and the efficacy of locally administered ceftiofur was evaluated in an experimental infection with Staphylococcus aureus in tissue cages. Eight ponies had tissue cages (TCs) implanted s.c. on each side of the neck. Into one of the cages 150 mg of ceftiofur was administered and fluid samples were taken to determine ceftiofur concentrations. After 1 week the other TC was infected with S. aureus and subsequently treated with 150 mg ceftiofur administered locally into the TC once daily for 21 days. Samples of fluid were taken to determine ceftiofur concentrations and for bacterial counts. Ceftiofur concentrations did not differ significantly in the infected and uninfected environments after single dose of 150 mg of ceftiofur. Concentrations were considerably in excess of the minimum inhibitory concentration (MIC) of the S. aureus strain used. A marked decrease of viable bacteria in tissue cage fluid (TCF) occurred. In five of seven ponies; however, the infection was not eliminated and abscess formation occurred. Therefore, local application of ceftiofur alone is not advisable for infections with S. aureus in secluded sites in horses, but should be used only with adjunctive therapy.

Clinical efficacy of prophylactic administration of trimethoprim/sulfadiazine in a Streptococcus equi subsp. zooepidemicus infection model in ponies.

Tissue chambers, implanted subcutaneously in the neck in six ponies, were inoculated with Streptococcus equi subsp. zooepidemicus in order to determine the clinical efficacy of prophylactic administration of trimethoprim/sulfadiazine (TMP/SDZ) against this infection. The TMP/SDZ treatment consisted of one intravenous (i.v.) injection of 5 mg/kg TMP and 25 mg/kg SDZ and the same dose of TMP/SDZ per os (p.o.), both given 3 h before inoculation. The oral dose was then repeated every 12 h for 5 days. TMP/SDZ concentrations in tissue chamber fluid (TCF) were above 10 times MIC at the moment of inoculation, and they were maintained at this level or higher throughout the duration of treatment. Trimethoprim/sulfadiazine treatment resulted in a marked reduction of viable bacteria in the tissue chamber but did not eliminate the infection, resulting in abscessation from day 19 onwards in all six ponies. This shows that, even when TCF is not yet purulent, TMP/SDZ is unable to eliminate the streptococci. Therefore, TMP/SDZ should not be the antimicrobial treatment of choice in infections in secluded sites in horses.

[A horse with a badly-healing wound? A case report]. / Een paard met een slecht genezende wond? Een case report.

A 14-year-old Haflinger mare was presented with a wound on the right metatarsus which it had sustained 3 years earlier. The wound had never completely healed but had only recently become a problem. Over a period of several months, the wound became larger, produced a lot of exudate, and the horse became lame on the affected limb. Clinical examination and radiographs failed to reveal the cause of the deterioration. Histological evaluation of tissue removed during debridement of the wound revealed squamous cell carcinoma. Because the tumour had already invaded the bone, the prognosis was unfavourable and the horse was euthanised. Necropsy showed the tumour to have metastasised to the right inguinal area and the adjacent mammary gland.

[Rectal tears in the horse: two case reports]. / Rectumbeschadigingen bij het paard: twee casus.

Rectal tears occur incidentally and often have a poor prognosis. Early recognition of the problem and adequate therapy determine the outcome. The history, clinical examination, therapy, and outcome of two horses with grade IIIb and grade IV rectal tears are discussed.

Diagnostic anaesthesia of the equine lower limb: a comparison of lidocaine and lidocaine with epinephrine.

The anaesthetic potency, onset of action, duration of action, and side effects of lidocaine and lidocaine plus epinephrine for proximal metacarpal block of the lateral and medial palmar nerves were determined. Ten horses were used and legs were injected using a cross-over model with three test solutions: 1) solvent and lidocaine (2%) plus epinephrine (SLE); 2) solvent and lidocaine (2%) without epinephrine (SL); and 3) solvent only (S). The contra-lateral leg was injected with saline (placebo; P). In both the SL and SLE groups, the onset of anaesthesia occurred between 5 and 15 minutes after injection. In the SL group the effect was short-lived, lasting 60 minutes, with a maximum effect at 15 minutes. SLE induced long-lasting anaesthesia in most animals moderate or total anesthesia lasted 6 hours, and in two horses more than 9 hours. The maximum effect was achieved at 60 and 90 minutes. Swelling, the main side effect, was seen halfway between the injection site and the fetlock joint. Swelling on day 1 was minimal in the legs injected with P (105.3%) but was progressively greater with S (129%), SL (136%), and SLE (172%). On day 1 and day 2 the differences between SLE and SL, and between SLE and S were significant (P < 0.05). The difference between S and SL was not significant. On day 3, the swelling had subsided and differences were no longer significant.

Clinical efficacy of trimethoprim/sulfadiazine and procaine penicillin G in a Streptococcus equi subsp. zooepidemicus infection model in ponies.

Tissue chambers, implanted subcutaneously on both sides of the neck in eight ponies, were inoculated with Streptococcus equi subsp. zooepidemicus in order to compare the clinical efficacy of trimethoprim/sulfadiazine (TMP/SDZ) and penicillin G treatment in a purulent infection. The TMP/SDZ treatment consisted of one intravenous (i.v.) injection of 5 mg/kg TMP and 25 mg/kg SDZ and the same dose of TMP/SDZ per os (p.o.), both given 20 h after inoculation. The oral dose was then repeated every 12 h for 21 days. The penicillin treatment consisted of one i.v. injection of 20 000 IU/kg sodium penicillin G and intramuscular (i.m.) injection of 20 000 IU/kg procaine penicillin G, both given 20 h after infection. The i.m. dose was then repeated every 24 h for 21 days. Eight ponies, each with two tissue chambers, were used in a cross over design; in the first experiment the left tissue chamber (TC) was infected and in the second experiment the right. TMP/SDZ treatment resulted in a limited reduction of viable bacteria in the TC but did not eliminate the infection, resulting in abscessation in 10-42 days in all eight ponies. However, penicillin treatment eliminated the streptococci in seven of eight ponies, and only one pony suffered abscessation on day 10. This constitutes a significantly better efficacy of the penicillin treatment in this model. The most probable cause of the failure of TMP/SDZ to eliminate the streptococci is inhibition of the action of TMP/SDZ in the purulent TCF. Therefore, TMP/SDZ should not be used to treat purulent infections in secluded sites in horses.

Brain abscesses as a metastatic manifestation of strangles: symptomatology and the use of magnetic resonance imaging as a diagnostic aid.

REASONS FOR PERFORMING STUDY: The occurrence of unexpectedly high numbers of horses with neurological signs during two outbreaks of strangles required prompt in-depth researching of these cases, including the exploration of magnetic resonance imaging (MRI) as a possible diagnostic technique. OBJECTIVES: To describe the case series and assess the usefulness of MRI as an imaging modality for cases suspected of space-occupying lesions in the cerebral cavity. METHODS: Four cases suspected of suffering from cerebral damage due to Streptococcus equi subsp. equi infection were examined clinically, pathologically, bacteriologically, by clinical chemistry (3 cases) and MRI (2 cases). In one case, MRI findings were compared to images acquired using computer tomography (CT). RESULTS: In all cases, cerebral abscesses positive for Streptococcus equi subsp. equi were found, which explained the clinical signs. Although the lesions could be visualised with CT, MRI images were superior in representing the exact anatomic reality of the soft tissue lesions. CONCLUSIONS: The diagnosis of bastard strangles characterised by metastatic brain abscesses was confirmed. MRI appeared to be an excellent tool for the imaging of cerebral lesions in the horse. POTENTIAL CLINICAL RELEVANCE: The high incidence of neurological complications could not be explained but possibly indicated a change in virulence of certain strains of Streptococcus equi subsp. equi. MRI images were very detailed, permitting visualisation of much smaller lesions than demonstrated in this study and this could allow prompt clinical intervention in less advanced cases with a better prognosis. Further, MRI could assist in the surgical treatment of brain abscesses, as has been described earlier for CT.

Intramuscular dosing strategy for ampicillin sodium in horses, based on its distribution into tissue chambers before and after induction of inflammation.

The Pharmacokinetics (PK) and distribution into tissue chamber fluid (TCF) of intramuscularly (i.m.) administered ampicillin sodium were examined in horses in order to design adequate dosing strategies. Concentration-time curves of ampicillin in plasma and TCF were determined in six horses following administration of 15 mg/kg ampicillin sodium, before and after the induction of local inflammation with 0.5% carrageenan. The calculated parameters were used to simulate various dosage-dosing interval combinations. Ampicillin was absorbed very rapidly following i.m. administration. Plasma concentrations were maximual between 18 and 21 min after administration. None of the plasma PK parameters were affected significantly by local (TC) inflammation. Penetration of ampicillin into and elimination from the TCF were affected significantly by inflammation and the half-life of elimination from the tissue fluid t1/2(d) was significantly shorter in inflammation. In the simulated dosage-dosing interval scenarios, only a dosage of 15 mg ampicillin/kg four times daily would successfully treat all ampicillin-susceptible bacterial isolates in well vascularized tissues. However a dosage as low as 10 mg/kg twice daily, would, in theory, treat all ampicillin-susceptible isolates in the inflamed poorly vascularized tissues. Decreasing the dosage results in loss of efficacy that cannot be completely compensated for by increasing the frequency of dosing.

Distribution of orally administered trimethoprim and sulfadiazine into noninfected subcutaneous tissue chambers in adult ponies.

The distribution of trimethoprim (TMP) and sulfadiazine (SDZ) into subcutaneously implanted noninfected tissue chambers was studied in healthy adult ponies. Six ponies were given an oral TMP/SDZ paste formulation at a dose of 5 mg/kg TMP and 25 mg/kg SDZ at 12 h intervals for 2 days in order to reach steady-state concentrations. Plasma concentrations and tissue chamber fluid (TCF) concentrations of both drugs were measured at regular intervals during a period commencing 24 h after the last oral administration. The peak concentration of TMP (mean +/- SD) was 2.92 +/- 0.86 microg/mL for plasma and 1.09 +/- 0.25 microg/mL for TCF. For SDZ, the mean peak concentration was 40.20 +/- 14.74 microg/mL for plasma and 23.48 +/- 5.84 microg/mL for TCF. TMP peak concentrations in plasma were reached at 3.17 +/- 03.48 h and those in TCF at 7.33 +/- 03.72 h. SDZ peak concentrations in plasma were reached at 1.83 +/- 02.04 h and those in TCF at 8.00 +/- 03.10 h. Concentrations of TMP and SDZ in TCF remained above the generally accepted breakpoint for susceptibility (0.5/9.5 for the TMP/SDZ combination) for 12 h. Therefore, in ponies oral administration of TMP/SDZ at a dose rate of 30 mg/kg given twice daily in the form of a paste should be appropriate for effective treatment of infections caused by susceptible bacteria.

[Priapism in the stallion]. / Priapismus bij de hengst.

A review of the literature is given concerning the anatomy and pathophysiology of the equine penis with regard to priapism: a prolonged erection of the penis not associated with sexual arousal. Several treatment options, such as flushing of the corpus cavernosum penis with heparinized saline and the creation of shunt between the corpus cavernosum penis and the corpus spongiosum penis are discussed. Subsequently, a case of priapism in a stallion following the injection of acepromazine, is discussed. The priapism resolved after the corpus cavernosum penis was flushed with the stallion under general anaesthesia.

Factors influencing the occurrence of thrombophlebitis after post-surgical long-term intravenous catheterization of colic horses: a study of 38 cases.

Thrombophlebitis is a well-known complication of the use of long-term in-dwelling catheters. In humans, catheter material has been shown to strongly influence the occurrence of thrombophlebitis. In the horse, the influence of catheter material has been studied in healthy experimental animals, but information on the relative importance of this factor is lacking. To investigate which factors have most impact on the frequency of jugular vein thrombosis in post-surgical colic horses, a clinical study was performed on 38 animals. Horses were randomly divided into two groups. In one group a polytetrafluoroethylene catheter was used, in the other a polyurethane catheter. Both groups were clinically monitored and screened for signs of thrombophlebitis. Seven out of 38 horses developed thrombophlebitis. The type of catheter material used had no influence on thrombophlebitis development. Dwell time was significantly longer in horses that developed thrombophlebitis compared with those that did not. There was no relationship between the occurrence of thrombophlebitis and underlying disease or surgical treatment, suggesting that the general state of debilitation these horses experienced was the most important determinant for the development of thrombophlebiris. This was further stressed by the fact that seven horses developed thrombophlebitis of the contralateral vein that had been used for the induction of anaesthesia (this incidence is much higher than in horses anaesthetized for elective surgery). It is concluded that the state of debilitation is the most important determining factor for the occurrence of thrombophlebitis after the use of long-term in-dwelling intravenous catheters. This makes a substantial reduction of the incidence of thrombophlebitis difficult, but some progress can be made by consequently restricting dwell time.