Roberta Perego

Conditioned Medium from Horse Amniotic Membrane-Derived Multipotent Progenitor Cells: Immunomodulatory Activity In Vitro and First Clinical Application in Tendon and Ligament Injuries In Vivo

We have recently demonstrated that heterologous transplantation of horse amniotic membrane-derived mesenchymal cells (AMCs) can be useful for cell therapy applications in tendon diseases, and hypothesized that these cells may promote tendon repair via paracrine-acting molecules targeting inflammatory processes. To test this hypothesis, here we examined the immunomodulatory characteristics of AMCs and of their conditioned medium (AMC-CM) in vitro, and studied the potential therapeutic effect of AMC-CM in thirteen different spontaneous horse tendon and ligament injuries in vivo. Our results demonstrate that AMCs are capable of inhibiting peripheral blood mononuclear cell (PBMC) proliferation after allogenic stimulation either when cocultured in cell-to-cell contact, or when the two cell types are physically separated by a transwell membrane, suggesting that soluble factors are implicated in this phenomenon. Our hypothesis is further supported by the demonstration that PBMC proliferation is inhibited by AMC-CM. In our in vivo studies, no significant adverse effects were observed in treated tendons, and clinical and ultrasonographical evaluation did not reveal evidence of inappropriate tissue or tumor formation. Clinical outcomes were favorable and the significantly lower rate (15.38%) of reinjuries observed compared to untreated animals, suggests that treatment with AMC-CM is very efficacious. In conclusion, this study identifies AMC-CM as a novel therapeutic biological cell-free product for treating horse tendon and ligament diseases.

Investigating the efficacy of amnion-derived compared with bone marrow–derived mesenchymal stromal cells in equine tendon and ligament injuries

BACKGROUND AIMS This is the first study to compare the treatment of horse tendon and ligament injuries with the use of mesenchymal stromal cells (MSCs) obtained from two different sources: amniotic membrane (AMSCs) and bone marrow (BM-MSCs). The objective was to prove the ability of AMSCs to exert beneficial effects in vivo. METHODS Five million allogeneic frozen-thawed AMSCs or autologous fresh BM-MSCs were injected intralesionally in horses belonging to group A (51 horses) and group B (44 horses). The interval lesion/implantation was of 6-15 days for the AMSCs and 16-35 days for the BM-MSCs. Healing was assessed clinically and ultrasonographically. Follow-up was monitored for 2 further years from return to full work. RESULTS No significant adverse effects after MSCs treatment were seen in any of the horses studied, independent of the type of stromal cell implanted. All animals belonging to group A resumed their activities between 4-5 months after treatment, whereas animals of group B resumed their activities after 4-12 months. The rate of re-injury in horses treated with AMSCs is lower (4.00%) compared with the average observed when horses were treated with BM-MSCs (23.08%). CONCLUSIONS The possibility to inject allogeneic AMSCs in real time, before any ultrasonographic change occurs within the injured tendon and ligament, together with the higher plasticity and proliferative capacity of these cells compared with BM-MSCs, represents the main features of interest for this novel approach for the treatment of equine tendon diseases. An obvious active proliferative healing in the area injected with AMSCs makes these cells more effective than BM-MSCs.

Seroprevalence of feline immunodeficiency virus, feline leukaemia virus and Toxoplasma gondii in stray cat colonies in northern Italy and correlation with clinical and laboratory data

Stray cat colonies in urban and rural areas of Lombardy, northern Italy, were surveyed for seroprevalence of feline immunodeficiency virus (FIV) antibodies, feline leukaemia virus (FeLV) antigen and Toxoplasma gondii IgG. Of 316 cats tested, 6.6% were positive for FIV and 3.8% were positive for FeLV infection; 203 cats were tested for T gondii IgG antibodies and a prevalence of 30.5% was detected. Statistical analysis tested the influence of provenience, age, gender, health status and laboratory results on seroprevalence and found male gender and adult age were risk factors for FIV infection. FIV-infected cats were more likely to have a decreased red blood cell count than FIV seronegative cats. No predictors were significantly associated with FeLV and T gondii seropositivity. Colony cats in this study posed a limited risk for retrovirus infection to pet cats allowed outdoors, whereas toxoplasmosis exposure was comparable with the worldwide data.

Prevalence of adverse food reactions in 130 dogs in Italy with dermatological signs: a retrospective study.

OBJECTIVES To determine the prevalence of adverse food reactions (AFRs) in dogs with dermatological signs presented to the referral dermatological clinic of the University of Milan. METHODS The medical records of dogs with dermatological signs were reviewed. Prevalence of AFRs was calculated. Owner and clinician pruritus scores were compared. Breed, sex and age predisposition were statistically tested, as was the association between AFR and selected clinical features. RESULTS The prevalence of AFRs in dogs with dermatological signs was 12% (16 of 130). AFR was diagnosed in 26% of dogs with allergic disease and 48% of those subjected to a dietary trial. There was a significant association between AFRs and early onset of clinical signs (< 1 year) (OR=3.8; P=0.0221, 95% CI=1.27 to 11.16). There was a significant association between AFRs and both otitis externa (OR=5.9; P=0.0015, 95% CI=2 to 17.9) and perianal fistula (OR=26.1; P=0.0058, 95% CI=2.52 to 269.4), although all dogs with perianal fistulas were German shepherd dogs. CLINICAL SIGNIFICANCE The prevalence of AFRs in the study population was higher than most reported values. Further studies are warranted to investigate the true prevalence of AFR and its possible association with perianal fistula and other potential markers.

Comparison of gel column agglutination with monoclonal antibodies and card agglutination methods for assessing the feline AB group system and a frequency study of feline blood types in northern Italy

BACKGROUND A new commercial gel column agglutination system is reported to have high sensitivity in detecting cats with blood type AB. OBJECTIVES The aims of this study were to compare gel column agglutination and card agglutination methods for feline blood-typing and to determine the frequency distribution of feline blood types in northern Italy. METHODS Blood-typing was performed on 120 cats using both a commercial gel column containing monoclonal antibodies (ID Gel-Test Micro Typing System) and a card agglutination method (RapidVet-H Feline). Results were confirmed with back-typing. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the 2 methods. A second group of 140 Domestic Shorthair (DSH) cats was blood-typed using the gel column technique to determine the frequency distribution of feline blood types in northern Italy. RESULTS The card agglutination method demonstrated poor sensitivity in identification of type-AB cats (61%) and was only 95% specific when identifying type-B cats. The gel column agglutination technique demonstrated 100% sensitivity and specificity for typing all 3 blood types (A, B, and AB). The frequency distribution study of 140 cats demonstrated that 127 (90.7%) cats were type A, 10 (7.1%) were type B, and 3 (2.1%) were type AB. CONCLUSION When blood-typing cats of breeds with a relatively high frequency of blood types B and AB, methods that use monoclonal antibodies for detection of blood types B and AB are recommended. Alternatively, blood type can be confirmed by more sensitive supplemental testing, such as back-typing. The high frequency of blood type A in DSH cats in northern Italy was comparable to previously reported frequencies in Italy and world-wide.

Relationship between Leishmania IFAT Titer and Clinicopathological Manifestations (Clinical Score) in Dogs

During canine leishmaniasis (CanL) due to Leishmania infantum, high levels of antibodies production are associated with the presence of various clinical signs, because of the deposition of soluble immune complexes in organs and tissues. The immunofluorescence antibody test (IFAT) is one of the most commonly used techniques for detection of anti-Leishmania antibodies. The purpose of this study was to assess whether there is a correlation between clinical signs and IFAT titers in dogs naturally infected with Leishmania. A retrospective study was performed on medical records of 49 dogs diagnosed with CanL. Information extracted from the medical records of each dog with CanL was clinical score, IFAT titer, serum total protein (TP), gamma globulin (IgG) and creatinine concentration, and protein creatinine ratio in urine sample (UP/UC) at each follow-up examination. Results show that dogs with highest IFAT titers recorded had higher mean clinical scores indicating a positive relationship (P < 0.0001) between anti-Leishmania antibodies (IgG) and clinical manifestations, which becomes more evident in severe clinical forms of canine leishmaniasis. Higher TP and IgG serum concentrations were recorded in dogs with higher clinical scores. Significant association was observed between UP/UC and the IFAT titer (P = 0.004).

Pituitary-dependent hyperadrenocorticism and generalised toxoplasmosis in a cat with neurological signs

A 12-year-old female neutered cat presented with acute onset unilateral vestibular syndrome, a spontaneous cutaneous wound, polyuria, polydipsia, and diabetes mellitus. Hyperadrenocorticism was demonstrated by means of hyper-responsiveness to adrenocorticotropic hormone stimulation, elevated urine cortisol-to-creatinine ratio, bilaterally enlarged adrenal glands on abdominal ultrasound, and pituitary enlargement on computed tomography imaging. The cat was euthanased and post-mortem histological examination revealed feline skin fragility syndrome; confirmed a pituitary cromophobe macroadenoma; and generalised toxoplasmosis with tachyzoites in the pancreas, bowel and brain. This report is the first to describe the concurrence of macroadenoma pituitary-dependent hyperadrenocorticism and generalised toxoplasmosis in a cat with central vestibular syndrome.

Molecular study on selected vector-borne infections in urban stray colony cats in northern Italy

Feline vector-borne diseases can be caused by a range of pathogens transmitted by arthropods. Many of these infections have zoonotic implications, and stray cats are potential sentinels for human and pet health. This study investigated the prevalence of selected vector-borne infections in stray colony cats in Milan. Blood samples from 260 stray cats were evaluated, using conventional polymerase chain reaction tests (cPCRs), for the presence of DNA associated with Rickettsia species, Anaplasma phagocytophilum and Ehrlichia species. Positive cPCR results occurred in 127/260 subjects (48.9%; 95% confidence interval [CI] = 40.7–58.1), with a prevalence of 31.9% (83/260, 95% CI = 25.4–39.6) for Rickettsia species, 17.7% (46/260, 95% CI= 13.0–23.6) for A phagocytophilum, and 5.4% (14/260, 95% CI = 2.9–9.0) for Ehrlichia species. There was no statistical association between a positive PCR test for vector-borne infections surveyed and colony location, age, gender, body condition score or complete blood count abnormalities, nor feline immunodeficiency virus, feline leukaemia virus or Toxoplasma gondii status. The only variable linked to positive PCR results was detection of signs of ocular infection and PCR positivity for Rickettsia species (P = 0.04, odds ratio [OR] = 2.2, 95% CI = 1.1–4.4, P = 0.02). There is a significant prevalence of vector-borne infections with zoonotic potential in urban stray cats in Milan. Thus, dogs and pet cats with outdoor access should be monitored and treated for ectoparasites on a regular basis to minimise risks of disease and the potential transmission of zoonotic agents to people.

Prevalence of haemoplasma infections in stray cats in northern Italy.

This study investigated the prevalence of feline haemoplasma infections in a number of stray cat colonies in Milan, Northern Italy. Blood samples from 260 stray cats were evaluated, with conventional PCR, for the presence of DNA associated with Mycoplasma haemofelis (Mhf) and “Candidatus Mycoplasma haemominutum” (CMhm). Odd ratios (OR) were calculated to identify risk factors for haemoplasma infections. PCR was positive in 86 out of 260 subjects (33.1%), with a prevalence of 10.8% (28/260 cats) for Mhf and 22.3% (58/260 cats) for CMhm. No coinfections were registered. There were significant associations between infections and season of sampling, that is, a negative association between winter sampling and a haemoplasma positive status (OR = 0.29, P = 0.001), or CMhm positive status (OR = 0.29, P = 0.01). Haemoplasma infections are common in stray cats in Milan. Thus, domestic cats with outdoor access should be routinely monitored and treated for ectoparasites to minimize risks of disease acquisition. Moreover, as these infections are transmitted via blood, feline blood donors from this area should be screened by PCR and preferably be drawn from a population of indoor cats regularly treated for fleas.

Clinical and haematological responses of feline blood donors anaesthetised with a tiletamine and zolazepam combination

This prospective study investigated the effect on clinical and haematological variables of the anaesthetic combination of tiletamine and zolazepam in feline blood donors. Blood (10 ml/kg bodyweight to a maximum volume of 60 ml) was collected from the jugular vein of 31 owned healthy cats anaesthetised with 2.5 mg/kg of tiletamine and 2.5 mg/kg of zolazepam intramuscularly. Rectal temperature (RT), systolic arterial pressure (SAP), mean arterial pressure (MAP), diastolic arterial pressure (DAP), heart rate (HR) and complete blood count (including red blood cells [RBC], haemoglobin [HB], haematocrit [HT], platelet [PLT] count, white blood cells [WBC], lymphocytes, neutrophils, eosinophils, monocytes and basophils) were evaluated pre- and postdonation. RT decreased significantly (P <0.01) after blood donation (mean change in RT −0.7°C). Significant increases in SAP (P = 0.03), MAP (P <0.01) and DAP (P <0.01) occurred after blood donation (mean increase 13 mmHg, 12 mmHg and 11 mmHg, respectively). Although RBC, HT, HB, WBC, PLT, neutrophil and monocyte counts decreased, and HR, and lymphocyte, eosinophil and basophil counts increased after blood donation this change was not statistically significant. Mean time from pre- to postdonation evaluation was 39 ± 11 mins (range 24–76 mins). None of the cats had evidence of pallor or collapse after recovery from anaesthesia. The collection of blood at 10 ml/kg bodyweight to a maximum volume of 60 ml in healthy cats using a low dose tiletamine and zolazepam anaesthetic appears to be well tolerated by feline blood donors.