Fulvio Laus

Clinical investigation on Theileria equi and Babesia caballi infections in Italian donkeys

BackgroundInterest in the welfare and diseases of donkeys is constantly increasing in several countries. Despite this, clinical research into donkeys needs to be in continual development since they show different reactions compared to horses in many conditions, including infectious diseases, and need specific clinical and therapeutic approaches. No reports are currently available on clinical and clinical pathology data regarding donkeys with natural piroplasms infection.ResultsVenous blood samples were taken from one hundred and thirty eight donkeys and underwent indirect fluorescent antibody test (IFAT) to detect IgG antibodies against Theileria equi and Babesia caballi and real-time polimerase chain reaction (PCR) to detect Babesia spp. and Theileria spp. Clinical examinations, haematological analyses and serum bilirubin evaluation were also performed and compared with positive or negative status. A seroprevalence of 40.6% and 47.8% was found for T. equi and B. caballi, respectively; double positivity was detected in 19.6% of the animals. PCR results showed that 17.4% of the animals tested positive for T.equi and 3.6% for B. caballi with no double positivity. Twelve donkeys (8.7%) had clinical signs consistent with chronic forms of the disease and no acute forms were detected. Fifty-eight donkeys had haematological and serum bilirubin alterations and 56 (96.6%) of them were IFAT and/or PCR positive. Changes in erythrocyte number, packed cell volume, hemoglobin concentration, mean corpuscular hemoglobin, platelets number and total bilirubin were significantly associated with positive and symptomatic animals.ConclusionNonspecific clinical presentation seems to be very common in donkeys and several clinical pathology alterations persist after natural infection. Therefore, apparently healthy donkeys can have masked but severe clinical pathology alterations. Acute forms are very seldom observed in donkeys. Clinical monitoring of chronically infected donkeys is recommended since such animals represent a risk both for transmission to other animals and for their own health; furthermore, their production performances could be reduced. The study should also be intended as a contribution for veterinary practitioners because it describes the most usual clinical presentations and laboratory findings of equine piroplasmosis in naturally infected donkeys in endemic areas.

Detection of Streptococcus dysgalactiae subsp. equisimilis in equine nasopharyngeal swabs by PCR

Streptococcus (S.) dysgalactiae subsp. equisimilis is responsible for severe diseases in humans, including primary bacteraemia, pneumonia, endocarditis, and toxic shock syndrome. Infection in some animal species can also occur, although a few studies have looked into cross-species infectivity. In horses, S. equisimilis is generally considered infrequent or opportunistic, but has recently been isolated from cases of strangles-like disease. Rapid and sensitive diagnostic techniques could enable epidemiological studies and effective investigation of outbreaks involving these bacteria. In this study, PCR protocols previously described in cattle and in humans to detect the species S. dysgalactiae and the subspecies equisimilis were evaluated to detect specific sequences in equine samples. For this purpose, 99 monolateral nasal swabs were collected from horses from stud farms with a history of S. equisimilis infection and were tested blindly by bacteriological isolation and by single and duplex PCR. DNA for PCR was extracted both from the colonies grown on agar media and from enrichment broth aliquots after incubation with nasal swab samples. S. equisimilis was identified by bacteriological isolation in 23 out of 99 swab samples, and PCR assays on these colonies were fully concordant with bacteriological identification (kappa statistic = 1.00). In addition, PCR of the enrichment broth aliquots confirmed the bacteriological results and detected S. equisimilis in 6 samples more than the bacteriological examination (kappa statistic = 0.84). The PCR protocols appeared to be reliable for the rapid identification of S. equisimilis in equine nasal swab samples, and could be useful for microbiological diagnosis.

Characteristic clinical, haematological and histopathological findings in feline mastocytoma

M.T. Antognoni1*, A. Spaterna2, E. Lepri3, A. Fruganti2 and F. Laus2 1Department of Clinical Sciences, Section of Internal Medicine, Faculty of Veterinary Medicine, University of Perugia; 2Department of Veterinary Sciences, Clinical Section, Faculty of Veterinary Medicine, University of Camerino; 3Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Perugia, Italy *Correspondence: Department of Clinical Sciences, Section of Internal Medicine, Faculty of Veterinary Medicine, University of Perugia, 06126-Perugia, Italy E-mail: mta@unipg.it

Occurrence of Borrelia lusitaniae infection in horses.

The aim of the study was to investigate Borrelia burgdorferi sensu lato (s.l.) infection in horses exposed to heavy tick infestations. Blood samples of 98 healthy horses from 5 stud farms were examined by SNAP(®) 4D× and PCR to detect antibodies against B. burgdorferi s.l. and Borrelia DNA, respectively. Ten samples (15.3%) were antibody positive and 5 samples (5.1%) were both antibody and PCR positive. Sequence analysis showed the highest homology with the B. lusitaniae genospecies. No differences were found between sexes and stud farms, while age was significantly related to seropositivity (p<0.05). Our data confirms the presence of B. lusitaniae infection in horses, previously not clearly demonstrated.

Molecular and serological detection of tick-borne pathogens in donkeys (Equus asinus) in Italy

Donkeys, owing to the frequent outdoor activity, are exposed to a high risk of infection with tick-borne pathogens. This work aimed to detect exposure to Theileria equi, Babesia caballi, Anaplasma phagocytophilum and Borrelia burgdorferi s.l. of donkeys reared in Central Italy. For this purpose 122 adult donkeys were selected within 11 herds and submitted to blood collection. IgG antibodies to T. equi, B. caballi, A. phagocytophilum and B. burgdorferi s.l. were detected by IFAT. Conventional PCRs targeting the genes MSP2 and the flagellin were used for the detection of A. phagocytophilum and B. burgdorferi s.l. respectively and a Real Time PCR Sybr Green was used to detect Babesia/Theileria spp…. The species identity was determined by amplicons sequencing. Forty eight (39.3%) and 58 (47.5%) animals tested positive for T. equi and B. caballi antibodies, respectively; nine animals (7.4%) were found positive for antibodies against A. phagocytophilum whereas negative results were obtained for B. burgdorferi s.l. Twenty-six (21.3%) animals showed antibodies for both T. equi and B. caballi. Twenty-three (18.8%) donkeys were positive to Babesia/Theileria spp. PCR assay. Out of 21 sequenced amplicons, 20 were identified as T. equi, belonging to three main groups designated A, B and D and one as B. caballi group A. Neither A. phagocytophilum nor B. burgdorferi PCR results were positive. The study showed a high exposure of donkeys to tick-borne pathogens and provides information on the genetic identity of the T. equi strains circulating in Central Italy.

The Effects of Photobiomodulation of 808 nm Diode Laser Therapy at Higher Fluence on the in Vitro Osteogenic Differentiation of Bone Marrow Stromal Cells

The literature has supported the concept of mesenchymal stromal cells (MSCs) in bone regeneration as one of the most important applications in oro-maxillofacial reconstructions. However, the fate of the transplanted cells and their effects on the clinical outcome is still uncertain. Photobiomodulation (PBM) plays an important role in the acceleration of tissue regeneration and potential repair. The aim of this in vitro study is to evaluate the effectiveness of PBM with 808 nm diode laser therapy, using a flat-top hand-piece delivery system at a higher-fluence (64 J/cm2) irradiation (1 W, continuous-wave) on bone marrow stromal cells (BMSCs). The BMSCs of 3 old female Balb-c mice were analyzed. The cells were divided into two groups: irradiated group and control group. In the former the cells were irradiated every 24 h during 0 day (T0), 5 (T1), 10 (T2), and 15 (T3) days, whereas the control group was non-irradiated. The results have shown that the 64 J/cm2 laser irradiation has increased the Runt-related transcription factor 2 (Runx2). Runx2 is the most important early marker of osteoblast differentiation. The higher-fluence suppressed the synthesis of adipogenic transcription factor (PPARγ), the pivotal transcription factor in adipogenic differentiation. Also, the osteogenic markers such as Osterix (Osx) and alkaline phosphatase (ALP) were upregulated with an increase in the matrix mineralization. Furthermore, western blotting data demonstrated that the laser therapy has induced a statistically valid increase in the synthesis of transforming growth factor β1 (TGF-β1) but had no effects on the tumor necrosis factor α (TNFα) production. The data has statistically validated the down-regulation of the important pro-inflammatory cytokines such as interleukin IL-6, and IL-17 after 808 nm PBM exposition. An increase in anti-inflammatory cytokines such as IL-1rα and IL-10 was observed. These in vitro studies provide for first time the initial proof that the PBM of the 808 nm diode laser therapy with flat-top hand-piece delivery system at a higher-fluence irradiation of 64 J/cm2 (1 W/cm2) can modulate BMSCs differentiation in enhancing osteogenesis.

Adenocarcinoma Involving the Tongue and the Epiglottis in a Horse

ABSTRACT Tumors involving the oral cavity of the horse are uncommon. No cases of equine adenocarcinoma on the dorsum of the tongue have been reported in the literature. We report a case of adenocarcinoma located on the dorsum of the posterior one-third of the tongue in a 29-year-old gelding with severe dysphagia. Endoscopy revealed an epiglottis involvement, and histology was consistent with adenocarcinoma arising from minor salivary glands, which was associated with a severe fungal colonization of affected tissues. The goals of this report are to present an uncommon case of dorsum of the tongue-associated neoplasia and to highlight the association with atypical fungal colonization, to review the literature and to discuss possible clinical approach and prognosis.

In vivo Biocompatibility of p(HPMAm-lac)-PEG Hydrogels Hybridized with Hyaluronan

The present study reports on the biocompatibility in vivo after intramuscular and subcutaneous administration in Balb/c mice of vinyl sulphone bearing p(HPMAm‐lac1–2)‐PEG‐p(HPMAm‐lac1–2)/thiolated hyaluronic acid hydrogels, designed as novel injectable biomaterials for potential application in the fields of tissue engineering and regenerative medicine. Ultrasonography, used as a method to study hydrogel gelation and residence time in vivo, showed that, upon injection, the biomaterial efficiently formed a hydrogel by simultaneous thermal gelation and Michael Addition cross‐linking forming a viscoelastic spherical depot at the injection site. The residence time in vivo (20 days) was found to be shorter than that observed in vitro (32 days), indicating that the injected hydrogel was resorbed not only by chemical hydrolysis but also by cellular metabolism and/or enzymatic activity. Systemic biocompatibility was tested by analysing routine haematological parameters at different time‐points (7, 14 and 21 days after administration) and histology of the main organs, including the haematopoietic system. No statistically significant difference between parameters of the saline‐treated group and those of the hydrogel‐treated group was found. Importantly, a time‐dependent decrease of important pro‐inflammatory cytokines (TREM1 (Triggering Receptor Expressed on Myeloid cells‐1), tumour necrosis factor‐α and interleukin‐1β) in cultured bone marrow cells extracted from hydrogel treated mice was observed, possibly correlated to the anti‐inflammatory effect of hyaluronic acid released in time as hydrogel degraded. Copyright

Development and application of a UHPLC‐MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11-deoxycortisol, 11-deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra-high-performance liquid chromatography-tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra-high-performance liquid chromatography-tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922-0.9986, and the limits of detection and limits of quantification were in the range of 0.002-2 and 0.0055-5.5 ng ml-1 , respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml-1 ) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml-1 free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25-51.26 ng ml-1 ) and cortisol (range 32.57-52.24 ng ml-1 ), followed by 17-OH-pregnenolone, dihydrotestosterone and pregnenolone. Copyright

Ultrasonographic biometry of the eyes of healthy adult donkeys

Sixty-two healthy adult donkeys were included in this study, giving a total of 124 eyes for examination. The weight of the donkeys was estimated and an ultrasonography of the eyes was performed using a curvilinear transducer. Ocular measurements were taken in a horizontal plane and included the following values: globe axial length (GAL), anterior chamber depth (ACD), vitreous body depth (VD), lens diameter (LDi) and lens depth (LDe). The mean and sds for each measurement are reported in mm: GAL 34.22±2.05; ACD 3.01±0.58; VD 20.20±1.63; LDi 17.96±1.66 LDe 11.06±0.71. Gender was not a variability factor for ocular biometry in donkeys, while the weight was directly related to the ultrasonographic ocular values. Lens dimensions represented an exception and further investigation should be carried out to verify a possible correlation with age rather than weight. This is the first paper reporting reference data for ocular biometry in donkeys. The ultrasonographic evaluation of the equine eye is a manageable procedure that is easy to perform and can provide information not always obtainable with direct ocular examination. It allows the imaging of intraocular and retrobulbar structures and the diagnosis of some important disorders involving these areas (Scotty and others 2004, Michau 2005, Dietrich 2007). Ocular ultrasound is also indicated where it is impossible to directly visualise (eg, with an ophthalmoscope) posterior structures of the globe in cases of corneal oedema or ulceration, cataract or ocular masses (Withcomb 2002). Ultrasound can be used to investigate enophthalmos, buphthalmos or exophthalmos in cases of ocular protrusion and suspicion of disparity in globe size (Withcomb 2002). The most common diseases that can be detected or confirmed with ultrasound are corneal diseases, cataract, lens luxation, intraocular cysts or masses, glaucoma and retinal detachment (Reef 1998, Withcomb 2002). Although horses and donkeys can be affected by …