Histological Evaluation of Resected Tissue as a Predictor of Survival in Horses with Strangulating Small Intestinal Disease.

Strangulating small intestinal disease (SSID) in horses carries a poor prognosis for survival, especially following resection of ischaemic tissue. The margins of a resection are principally based on visual appraisal of the intestine during surgery. We hypothesized that histological evaluation of resected tissue may identify occult changes indicative of prognosis. Small intestinal samples from 18 horses undergoing resection for SSID and 9 horses euthanised for reasons unrelated to gastrointestinal pathology were utilised. Histological appearance was used to generate a 'total damage score' (TDS) for the control tissue, grossly normal tissue at oral and aboral extremities (sections OR1 and AB1) of the resected intestine, and oral and aboral extremities of visually abnormal tissue (sections OR2 and AB2) from SSID horses. The relationship between TDS and long-term post-operative survival was investigated. TDS was not different between control tissues and OR1 and AB1 sections. Five surgical cases were alive at follow-up, the longest follow-up time being 2561 days. Based on the median scores for SSID cases versus controls, cut-off values were generated to evaluate post-operative survival versus TDS. Only OR2 TDS was significantly associated with survival, with a higher (worse) score indicating longer survival. More severe tissue insult may expedite rapid progression to surgery, improving post-operative outcomes.

Toxoplasmosis in Captive Ring-Tailed Lemurs (Lemur catta).

Toxoplasma gondii is one of the most common protozoan parasites and is widely present in all warm-blooded animals. Although clinical disease is uncommon, some species, including ring-tailed lemurs (Lemur catta), have been found to develop acute and lethal toxoplasmosis. The aim of this study was to describe the pathologic, immunohistochemical, serological, and molecular findings of an outbreak of fatal toxoplasmosis in three captive ring-tailed lemurs in Central Italy in 2009. The animals died acutely within few days. The necropsy was immediately performed; necrotic lesions in the spleen, liver, and kidney, as well as interstitial pneumonia, were found histologically. All animals had high titers of anti-T. gondii-specific antibodies (1:1280 IgM and 1:640 IgG) according to a modified agglutination test (MAT) and immunohistochemistry showed scattered tachyzoites in the target organs. Diagnosis was confirmed by PCR and clonal type II was identified. In addition, the seven co-habiting lemurs were seronegative. This paper reports the first outbreak of acute disseminated toxoplasmosis in captive ring-tailed lemurs in Italy. These findings confirm the high susceptibility of this endangered species to toxoplasma infection, which may be considered a further threat to captive population viability.

Pulmonary bleeding in racehorses: A gross, histologic, and ultrastructural comparison of exercise-induced pulmonary hemorrhage and exercise-associated fatal pulmonary hemorrhage.

Exercise-induced pulmonary hemorrhage (EIPH) is a common condition of Thoroughbred racehorses that is usually responsible for reduced performance, while exercise-associated fatal pulmonary hemorrhage (EAFPH) is characterized by severe pulmonary bleeding of unknown pathogenesis resulting in sudden death during strenuous exercise. The aim of the study was to characterize and compare anamnestic data together with pulmonary gross, histologic, and ultrastructural findings in racehorses with EIPH (n = 10), EAFPH (n = 10), and control horses (n = 5). No differences in anamnesis were identified between the 3 groups. Grossly cranial lobe reddening and edema scores were significantly more prevalent and severe in the EAFPH group compared with the EIPH and control groups. Histologically, hemorrhage scores were higher in the EAFPH group, while hemosiderophages, iron encrustations of collagen and elastin fibers, and vascular remodeling scores were significantly higher in EIPH group compared with the EAFPH and control groups. In all groups, caudal lung locations exhibited a significantly higher score for vascular remodeling, hemosiderophage accumulation, iron encrustation, and type II pneumocyte hyperplasia when compared with cranial, dorsal, and ventral locations. Ultrastructural analysis of perivascular collagen showed fibrils with significantly larger diameters in the EAFPH group compared with the EIPH group but not compared with the control group. This study demonstrates that lungs of horses that experienced EAFPH show significantly less vascular remodeling and other long-term pulmonary abnormalities that characterize horses with EIPH.

Molecular survey on the presence of arthropod-borne bacteria and protozoans in roe deer (Capreolus capreolus) and ticks from Central Italy.

Environmental changes, due to climatic emergency and to anthropogenic activities severely impact on the epidemiology of vector borne diseases, mostly when transmitted by ticks. The data about the distribution of microorganisms responsible for them in roe deer (Capreolus capreolus) population living in Italy are scanty and completely lacking in Tuscany, so a molecular survey was carried out to estimate the prevalence of some zoonotic tick-borne pathogens in roe deer, and ticks removed from them, living in areas of Central Italy with high risk of arthropod exposure. Spleen samples from 72 roe deer were tested by PCR for Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Francisella tularensis and piroplasms. Moreover, 345 ticks were removed from 65 roe deer, morphologically or molecularly identified and grouped into 162 pools that were submitted to PCR for detecting the same pathogens. Forty-six (63.88%) roe deer were positive for at least one investigated pathogen: 43 (59.72%) for A. phagocytophilum, 2 (2.78%) for Babesia capreoli, 1 (1.39%) for B. burgdorferi, and 1 (1.39%) for Babesia sp. No animals were PCR positive for F. tularensis.  All ticks were identified as Ixodes ricinus. Seventy-six (46.91%) tick pools showed DNA of one or more pathogens: 66 (40.74%) were positive for A. phagocytophilum, 22 (13.58%) for B. burgodorferi s.l., 6 (3.70%) for B. venatorum and 3 (1.85%) for B. capreoli. No pools were positive for F. tularensis. Two or three pathogens were detected in 23 (14.19%) pools.

Elephant Endotheliotropic Herpesvirus 4 and Clostridium perfringens Type C Fatal Co-Infection in an Adult Asian Elephant (Elephas maximus).

Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is an acute, often fatal, multisystemic hemorrhagic disease and one of the most significant causes of mortality of Asian elephants in captivity. Most fatal cases of EEHV-HD are associated with EEHV1A and EEHV1B in juveniles. This case report describes the clinical and pathological features of a fatal co-infection of Clostridium perfringens type C and EEHV-HD, caused by EEHV4, in an adult female Asian elephant. Although fatal clostridial enterotoxemia has been occasionally reported in elephants, this report highlights the importance of having both EEHV-HD and clostridial enterotoxemia as potential differential diagnoses in cases of widespread tissue necrosis and internal hemorrhage in elephants, regardless of the animal age group, due to their macroscopic similarities, frequent co-occurrence and cumulative morbid potential.

Leukocyte numbers and intestinal mucosal morphometrics in horses with no clinical intestinal disease.

Healthy horses and other animals have large numbers of resident leukocytes in the intestinal wall, but there is scant information regarding which and how many leukocytes are normally present in the equine intestinal wall. Our aim was to provide a reference range of leukocytes in the intestinal mucosal and submucosal propria of normal horses. We included in our study intestinal tissues from 22 Thoroughbred racehorses with no clinical intestinal disease, which had been euthanized because of catastrophic musculoskeletal injuries. Neutrophils, lymphocytes, eosinophils, macrophages, and plasma cells were counted in 5 random 17,600-µm2 areas of villus lamina propria of the duodenum, jejunum, and ileum, and deep lamina propria of the duodenum, jejunum, ileum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon, and small colon. Other features investigated in the same intestinal segments included villus height and width (small intestine), presence of ciliated protozoa, Paneth cells number, subcryptal leukocyte layers (number of leukocyte layers between the bottom of the crypts and the muscularis mucosae), and submucosal leukocytes. Lymphocytes were the most numerous cells in all segments analyzed, followed by plasma cells, eosinophils, macrophages, and neutrophils. Eosinophil numbers were significantly higher in both lamina propria and submucosa of the large intestine than in the small intestine. The duodenum had shorter and thinner villi than either jejunum or ileum. The data provided from our study will be useful for diagnosticians examining inflammatory processes in the intestinal tract of horses.

Ultrasound-guided erector spinae plane block in horses: a cadaver study.

OBJECTIVES: To describe dye distribution and spinal nerve involvement after a simulated erector spinae plane (ESP) block performed on fresh equine cadavers. STUDY DESIGN: Experimental cadaver study. ANIMALS: A group of 11 adult equine cadavers. METHODS: The spinal region surrounding the sixteenth thoracic vertebra (Th16) of one cadaver was removed and underwent magnetic resonance imaging. In 10 adult equine cadavers [body weight, 549 ± 58 kg (mean ± standard deviation)], 0.2 mL kg-1 of a 50:1 2% lidocaine/dye solution was injected bilaterally (n = 20 injections) into the fascial plane between the transverse process of Th16 and the erector spinae muscles. An in-plane ultrasound-guided technique with a convex transducer was used to guide injection. Dissection was performed immediately following injection. The craniocaudal and lateral extent of dye distribution was measured (cm) and the number of vertebral bodies involved were counted (n = 20). Abdominal and thoracic cavities as well as the epidural space were also examined for presence of dye (yes/no) (n = 20). Further dissection was performed to evaluate if staining of the dorsal and ventral rami of the spinal nerves and sympathetic chain occurred (n = 14). RESULTS: The thoracolumbar fascia was stained in 17/20 (85%) injections and three injections terminated intramuscularly. Multisegmental staining of the dorsal rami was observed in the 14 injections in which staining was evaluated. Ventral rami staining was observed in 3/14 injections where staining was evaluated. Epidural migration was observed in 4/20 (20%) injections. No evidence of dye was found in the thoracic and abdominal cavities or on the sympathetic chain. CONCLUSIONS AND CLINICAL RELEVANCE: The ESP block may prove beneficial to desensitize structures innervated by the dorsal rami of the thoracic spinal nerves. Further investigation is necessary to evaluate complications caused by epidural contamination.

Haemoproteus spp. and Leucocytozoon californicus Coinfection in a Merlin (Falco colombarius).

The Leucocytozoon genus comprises numerous widely distributed parasites which have been less investigated than other avian hemoprotozoa. Their occurrence is common, with very variable prevalence values and pathogenicity degrees. Leucocytozoon species are characterized by a great taxonomic diversity, and infections are usually restricted to birds of the same family. In the present paper, a mixed hemosporidia infection by Leucocytozoon californicus and Haemoproteus sp. in an adult male merlin (Falco columbarius) which died during hospitalisation is reported, indicating, for the first time, a newly described avian host species. A molecular investigation was carried out through cytochrome b gene analysis, revealing a 100% match with L. californicus and Haemoproteus spp. A blood smear examination allowed us to detect Leucocytozoon fusiform mature gametocytes and different degrees of maturity of Haemoproteus gametocytes. Histopathology revealed foci of necrosis, hemorrhagic areas and extramedullary hematopoiesis in the liver, the presence of microthrombi in the heart and lung and scattered hemorrhages in the lung.

Detection of Neospora Caninum DNA in Wild Birds from Italy.

The role of avian species in the Neospora caninum life cycle has not been completely elucidated, and epidemiological data from Europe are scant. The aim of the present report was to evaluate the presence of N. caninum DNA in the tissues of 302 birds belonging to different avian species, along with IFAT titers. Forty-two out of the 302 birds (13.9%) showed low serological titers (1/50 and 1/100) against N. caninum. These data, positive for 31 animals (10.3%), were corroborated by PCR. Twenty-two hearts and eighteen brains scored positive, while nine subjects resulted had parasite DNA both in their hearts and brains. Serological data showed significantly higher results in waterfowl in respect to non-waterfowl avian species. This finding indicates a higher exposure of water birds to the parasite. These avian species, in fact, which feed directly from soil and/or water, are prone to ingest oocysts excreted by final canid hosts. The present study adds information to the state of art of N. caninum epidemiology in Italy, even if more investigations using bio-assays are needed to allow for a serological/parasitological follow-up to evaluate the real impact of the avian species in maintaining the parasite in main reservoirs.

Molecular survey on the occurrence of avian haemosporidia, Coxiella burnetii and Francisella tularensis in waterfowl from central Italy.

The aim of the present study was to evaluate the occurrence of some avian Haemosporidia, Coxiella burnetii and Francisella tularensis in waterfowl from Tuscany wetlands. One-hundred and thirty-three samples of spleen were collected from regularly hunted wild birds belonging to 13 different waterfowl species. DNA extracted from each sample was submitted to PCR assays and sequencing to detect the pathogens. Thirty-three samples (24.81%) were positive with PCR for at least one pathogen: 23 (17.29%) for Leucocytozoon spp., 6 (4.51%) for Plasmodium spp., 4 (3%) for C. burnetii, 2 (1.5%) for Haemoproteus spp. No specific F. tularensis amplifications (0%) were detected. To the best of our knowledge, this study firstly reports data about haemosporidian and C. burnetii infections in waterfowl from Italy.

Multicentric Molecular and Pathologic Study On Canine Adenovirus Type 1 in Red Foxes (Vulpes vulpes) in Three European Countries.

Canine adenovirus type 1 (CAdV-1) is the agent of infectious canine hepatitis, a severe frequently fatal disease affecting primarily dogs (Canis lupus familiaris). The virus has been detected in many wild carnivore species. Our aim was to evaluate the prevalence and genetic and histopathologic features of CAdV-1 in wild red foxes (Vulpes vulpes). Kidney and liver samples were obtained from 86 subjects, coming from the UK (n=21), Italy (n=36), and Germany (n=29). We used PCR, targeting the viral E3 gene and flanked regions, to detect the presence of the virus; viral E3, fiber, and E4 genes were sequenced and their sequences were compared with published sequences. Kidneys and liver from foxes in Italy and Great Britain (n=57) were prepared for histologic and immunohistochemical examination for CAdV-1. Viral DNA was detected in 22% (19 of 86) kidney samples, with E3 and E4 genes showing reported and unreported single nucleotide changes. No pathologic changes or viral immunopositive signals were detected in the examined tissues. Our study suggests that red foxes could be considered potential shedders of CAdV-1, as they showed a relatively high prevalence without related pathologic changes in the organs examined.

Serological and Molecular Investigation on Toxoplasma gondii Infection in Wild Birds.

Toxoplasma gondii is an obligate apicomplexan zoonotic parasite that infects humans and other animals and is responsible for toxoplasmosis. This parasite causes one of the most common parasitic infections in humans worldwide. Toxoplasmosis meets the requirements for a One Health Disease due to its ability to affect the health of human beings as well as domestic and free ranging animals. Integrating human, domestic animal, and wildlife data could better assess the risk and devise methods of control. A first step of such an approach would be the knowledge of the prevalence of parasitosis in humans and animals in selected areas. Therefore, the aim of the present study was to evaluate the occurrence of Toxoplasma infection in 216 free ranging birds belonging to different genera/species by serology and molecular techniques. Twenty-five out of 216 animals (11.6%) were positive to the immunofluorescence antibody test (IFAT) with antibody titers ranging from 1/20 to 1/320, and 19 of them (8.8%) also showed a positive PCR for Toxoplasma DNA. The results confirmed the widespread occurrence of Toxoplasma infection in wild birds and serological data were corroborated by molecular results in birds that also had low antibody titers. The knowledge of the wide occurrence of the parasite in game and wild birds should enhance the accurate estimation of the risks in handling, managing, and eating these species with regard to domestic carnivores as well as the impact of viscera and offal in the environment.

Molecular survey on the occurrence of arthropod-borne pathogens in wild brown hares (Lepus europaeus) from Central Italy.

Data about the spreading of arthropod-borne pathogens among hare populations are very scant, so the aim of the present preliminary study was to investigate, through molecular analysis, the occurrence of Anaplasma phagocytophilum, Bartonella sp., Borrelia burgdorferi sensu lato, Coxiella burnetii, Francisella tularensis, Leishmania spp. and piroplasms DNA in blood of 51 wild hares (Lepus europaeus) living in protected areas in Tuscany. All hares resulted negative for A. phagocytophilum, Bartonella sp., B. burgdorferi s.l., C. burnetii and F. tularensis. Five animals (9.8%) were positive for Leishmania and one hare (1.9%) tested positive for piroplasms. Sequencing of this sample showed a piroplasm similar to one Babesia isolate from the same animal species in Turkey. Therefore, to the best of our knowledge, this is the first molecular report of piroplasms occurring in wild hares from Italy, and the second worldwide. The examined hares appeared to be in good health status, corroborating the hypothesis of a chronic carrier state of some vector borne agents for this animal species.

Behavioral asymmetries in ticks - Lateralized questing of Ixodes ricinus to a mechatronic apparatus delivering host-borne cues.

Ticks are considered among the most dangerous arthropod vectors of disease agents to both humans and animals worldwide. Lateralization contributes to biological fitness in many animals, conferring important functional advantages, therefore studying its role in tick perception would critically improve our knowledge about their host-seeking behavior. In this research, we evaluated if Ixodes ricinus (L.) (Ixodiidae) ticks have a preference in using the right or the left foreleg to climb on a host. We developed a mechatronic device moving a tuft of fox skin with fur as host-mimicking combination of cues. This engineered approach allows to display a realistic combination of both visual and olfactory host-borne stimuli, which is prolonged over the time and standardized for each replicate. In the first experiment, the mechatronic apparatus delivered host-borne cues frontally, to evaluate the leg preference during questing as response to a symmetrical stimulus. In the second experiment, host-borne cues were provided laterally, in an equal proportion to the left and to the right of the tick, to investigate if the host direction affected the questing behavior. In both experiments, the large majority of the tested ticks showed individual-level left-biased questing acts, if compared to the ticks showing right-biased ones. Furthermore, population-level left-biased questing responses were observed post-exposure to host-mimicking cues provided frontally or laterally to the tick. Overall, this is the first report on behavioral asymmetries in ticks of medical and veterinary importance. Moreover, the mechatronic apparatus developed in this research can be exploited to evaluate the impact of repellents on tick questing in highly reproducible standardized conditions.

Stonebrood and chalkbrood in Apis mellifera causing fungi: in vitro sensitivity to some essential oils.

Aim of the present study was to evaluate the in vitro antimycotic activity of 17 chemically defined essential oils (EOs) both alone and as a mixture, against agents responsible for stonebrood caused by Aspergillus flavus, and chalkbrood caused by Ascosphaera apis in European honeybees. Cinnamomum zeylanicum yielded the lowest MIC value against A. flavus, but was not effective against A. apis, while Litsea cubeba and Pelargonium graveolens appeared to be effective against all checked fungi. Aspergillus niger showed the lower sensitivity. Two mixtures composed by L. cubeba, C. zeylanicum and Cymbopogon flexuosus (M1) and by L. cubeba, C. zeylanicum, P. graveolens and C. flexuosus (M2), respectively, were tested, both resulting effective. The components of M1 showed a synergistic effect. The use of mixtures allowed to decrease the total amount of EOs. The use of these products could be of interest for an alternative natural approach in honeybee disease management.

Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study.

BACKGROUND: The study was aimed to characterize tumor response after combined treatment employing electrochemotherapy with IL-12 gene electrotransfer in dogs with spontaneous mast cell tumors (MCT). MATERIALS AND METHODS: Eleven dogs with eleven MCTs were included in the study. Histological changes were investigated in biopsy specimens collected before the treatment (T0), and 4 (T1) and 8 weeks (T2) later. Cellular infiltrates were characterized immunohistochemically by using anti CD3, CD20, Foxp3 (Treg), CD68 and anti MHC-class II antibodies. Proliferation and anti-apoptotic activity of neoplastic cells were assessed using anti Ki-67 and Bcl-2 antibodies. Angiogenetic processes were investigated immunohistochemically by using anti Factor VIII and anti CD31 antibodies and micro vessel density quantification. RESULTS: Histopathological examination of samples at T0 confirmed the diagnosis and the presence of scanty infiltrates consisted mainly of T-lymphocytes and macrophages. At T1 and T2 neoplastic cells were drastically reduced in 7/11 cases, small clusters of neoplastic cells were detected in 3/11 cases and 1/11 cases neoplastic cells were still evident. Proliferation activity of neoplastic cells was significantly reduced at T1 and T2 and expression of anti-apoptotic protein at T1. Microvessel density was drastically reduced in all samples after treatment. The number of T-lymphocytes increased at T1, although not significant, while Treg were significant higher at T1 and macrophages at T2. CONCLUSIONS: The combined electrochemotherapy and IL-12 gene electrotransfer effectively induced a cellular response against neoplastic cells characterized mainly by the recruitment of T-lymphocytes and macrophages and a fibrotic proliferation with reduction of microvessels.

Cutaneous lesions due to Trichosporon jirovecii in a tortoise (Testudo hermanni).

Cutaneous mycoses have been rarely reported in Chelonians. A Testudo hermanni adult male showed a thick erosion of the dorsal neck covered by necrotic material. Trichosporon sp. was cultivated, while arthrospores and hyphae were observed in histological sections. The causative agent was identified as Trichosporon jirovecii by PCR. After a surgical intervention povidone iodine and Iruxol® ointment were daily administered through the drainage for 2 weeks, along with enrofloxacin 5 mg diluted in saline 0.5 ml via the intracelomatic route. After treatment the ulcer healed with residual scars. No relapse was registered after 12 months. T. jirovecii is considered as a rare yeast pathogen and the presented case is the first report of a dermatomycosis in tortoises caused by this yeast species.

Prevalence of the microsporidian Nosema ceranae in honeybee (Apis mellifera) apiaries in Central Italy.

Nosema ceranae and Nosema apis are microsporidia which play an important role in the epidemiology of honeybee microsporidiosis worldwide. Nosemiasis reduces honeybee population size and causes significant losses in honey production. To the best of our knowledge, limited information is available about the prevalence of nosemiasis in Italy. In this research, we determined the occurrence of Nosema infection in Central Italy. Thirty-eight seemingly healthy apiaries (2 to 4 hives each) were randomly selected and screened from April to September 2014 (n = 11) or from May to September 2015 (n = 27). The apiaries were located in six areas of Central Italy, including Lucca (n = 11), Massa Carrara (n = 9), Pisa (n = 9), Leghorn (n = 7), Florence (n = 1), and Prato (n = 1) provinces. Light microscopy was carried out according to current OIE recommendations to screen the presence of microsporidiosis in adult worker honeybees. Since the morphological characteristics of N. ceranae and N. apis spores are similar and can hardly be distinguished by optical microscopy, all samples were also screened by multiplex polymerase chain reaction (M-PCR) assay based on 16S rRNA-gene-targeted species-specific primers to differentiate N. ceranae from N. apis. Furthermore, PCR-positive samples were also sequenced to confirm the species of amplified Nosema DNA. Notably, Nosema spores were detected in samples from 24 out of 38 (63.2%, 95% CI: 47.8-78.5%) apiaries. Positivity rates in single provinces were 10/11, 8/9, 3/9, 1/7, or 1/1 (n = 2). A full agreement (Cohen's Kappa = 1) was assessed between microscopy and M-PCR. Based on M-PCR and DNA sequencing results, only N. ceranae was found. Overall, our results highlighted that N. ceranae infection occurs frequently in the cohort of honeybee populations that was examined despite the lack of clinical signs. These findings suggest that colony disease outbreaks might result from environmental factors that lead to higher susceptibility of honeybees to this microsporidian.

Molecular detection of tick-borne pathogens in wild red foxes (Vulpes vulpes) from Central Italy.

Spleen samples from 153 red foxes, shot during regular hunting season in the province of Pisa (Central Italy), were examined to detect DNA of Anaplasma phagocytophilum, Ehrlichia canis, Coxiella burnetii, Francisella tularensis, Hepatozoon canis and Babesia sp./Theileria sp. DNA of vector-borne pathogens was detected in 120 (78.43%; 95% CI: 71.06-84.66%) foxes. Specifically, 75 (49%; 95% CI: 40.86-57.22%) animals scored PCR-positive per H. canis, 68 (44.44%; 95% CI: 36.42-52.69%) for E. canis, 35 (22.88%; 95% CI: 16.48-30.35%) for piroplasms (Theileria annae), 3 (1.96%; 95% CI: 0.41-5.62%) for C. burnetii and 1 (0.65%; 95% CI: 0.02-3.59%) for A. phagocytophilum. No positive reaction was observed for F. tularensis. Fifty-six animals (36.6%; 95% CI: 28.97-44.76%) were positive for two or three pathogens. Red foxes result to be involved in the cycle of vector-borne pathogens that are associated to disease in dogs and humans.