Critical roles of Rickettsia parkeri outer membrane protein B (OmpB) in the tick host.

Rickettsia parkeri is a pathogen of public health concern and transmitted by the Gulf Coast tick, Amblyomma maculatum. Rickettsiae are obligate intracellular bacteria that enter and replicate in diverse host cells. Rickettsial outer membrane protein B (OmpB) functions in bacterial adhesion, invasion, and avoidance of cell-autonomous immunity in mammalian cell infection, but the function of OmpB in arthropod infection is unknown. In this study, the function of R. parkeri OmpB was evaluated in the tick host. R. parkeri wild-type and R. parkeri ompBSTOP::tn (non-functional OmpB) were capillary fed to naïve A. maculatum ticks to investigate dissemination in the tick and transmission to vertebrates. Ticks exposed to R. parkeri wild-type had greater rickettsial loads in all organs than ticks exposed to R. parkeri ompBSTOP::tn at 12 h post-capillary feeding and after 1 day of feeding on host. In rats that were exposed to R. parkeri ompBSTOP::tn-infected ticks, dermal inflammation at the bite site was less compared to R. parkeri wild-type-infected ticks. In vitro, R. parkeri ompBSTOP::tn cell attachment to tick cells was reduced, and host cell invasion of the mutant was initially reduced but eventually returned to the level of R. parkeri wild-type by 90 min post-infection. R. parkeri ompBSTOP::tn and R. parkeri wild-type had similar growth kinetics in the tick cells, suggesting that OmpB is not essential for R. parkeri replication in tick cells. These results indicate that R. parkeri OmpB functions in rickettsial attachment and internalization to tick cells and pathogenicity during tick infection.

Systemic Caryospora-like coccidiosis in a clutch of hatchling red-eared slider turtles (Trachemys scripta elegans).

Caryospora-like organisms (CLOs) form a clade of at least 11 genotypes of related coccidia that can cause epizootic mortality in marine turtles. The biology, transmission, host species range, and host cell tropism of these organisms are still largely unknown. The goal of this study was to characterize the host cell tropism, pathologic and ultrastructural features, and phylogeny associated with the first report of a mortality event due to CLO in the freshwater red-eared slider turtle (Trachemys scripta elegans). Sudden mortalities within a clutch of captive-raised red-eared slider hatchlings (n = 8) were recorded, and deceased animals had severe segmental to diffuse, transmural, fibrinonecrotic enterocolitis and multifocal to coalescing hepatic necrosis, among other lesions associated with numerous intracytoplasmic developing stages of intralesional coccidia. Among the different developmental stages, merozoites were ultrastructurally characterized by an apical complex. A pan-apicomplexan polymerase chain reaction (PCR) yielded a 347 bp-amplicon matching the Schellackia/Caryospora-like clade with 99.1% identity to the US3 strain from green sea turtles (Chelonia mydas) and 99.1% identity to Schellackia sp. Isolate OC116. Surviving hatchlings were treated with toltrazuril sulfone (ponazuril) but were subsequently euthanized due to the risk of spreading the parasite to other chelonids in the collection. The ponazuril-treated hatchlings (n = 4) had mild proliferative anterior enteritis, with few intraepithelial coccidia in one hatchling confirmed as CLO by PCR. This is the first report of Caryospora-like coccidiosis in non-cheloniid turtles, highlighting the relevance of this disease as an emerging highly pathogenic intestinal and extra-intestinal form of coccidiosis of turtles with potential cross-species infectivity.

An outbreak of systemic chlamydiosis in farmed American alligators (Alligator mississippiensis).

Chlamydia spp are reported to causes systemic disease in a variety of hosts worldwide including few reports in crocodilians. Disease presentations vary from asymptomatic to fulminant disease, some of which are zoonotic. The aim of this study was to describe the pathological, immunohistochemical, and molecular findings associated with the occurrence of a previously unreported Chlamydia sp infection causing a major mortality event in farmed American alligators (Alligator mississippiensis). The outbreak presented with sudden death in juvenile alligators mainly associated with necrotizing hepatitis and myocarditis, followed by the occurrence of conjunctivitis after the initial high mortality event. The widespread inflammatory lesions in multiple organs correlated with intralesional chlamydial organisms identified via immunohistochemistry and confirmed by 23S rRNA-specific real-time quantitative polymerase chain reaction (qPCR) for Chlamydiaceae bacteria. By sequencing and phylogenetic analysis of the OmpA gene, this uncultured Chlamydia sp grouped closely with Chlamydia poikilothermis recently described in snakes. This study highlights the significance of such outbreaks in farmed populations. Enhanced epidemiological monitoring is needed to gain further insight into the biology of Chlamydia sp in alligators, disease dynamics, risk factors, and role of carrier animals.

Comparison of blind and endoscopic-guided orotracheal intubation on laryngeal and tracheal damage in domestic rabbits (Oryctolagus cuniculus).

OBJECTIVE: To compare blind and endoscopic-guided techniques for orotracheal intubation in rabbits and the number of intubation attempts with laryngeal/tracheal damage. STUDY DESIGN: Prospective, randomized experimental study. ANIMALS: A total of 24 healthy, intact female New Zealand White rabbits, weighing 2.2 ± 0.2 kg (mean ± standard deviation). METHODS: Rabbits were randomly assigned to blind (group B) or endoscopic-guided (group E) orotracheal intubation with a 2.0 mm internal diameter uncuffed tube. Intramuscular (IM) alfaxalone (7 mg kg-1), hydromorphone (0.1 mg kg-1) and dexmedetomidine (0.005 mg kg-1) were administered, and additional IM alfaxalone (3-5 mg kg-1) and dexmedetomidine (0.025 mg kg-1) were administered to rabbits with strong jaw tone. An intubation attempt was defined as the advancement of the endotracheal tube from the incisors to the laryngeal entrance. Tracheal intubation was confirmed via capnography and anesthesia was maintained with isoflurane for 2 hours. Following euthanasia, laryngeal and tracheal tissues were submitted for histopathology. Quality of anesthesia for orotracheal intubation, intubation procedure and tissue damage were numerically scored. Data were analyzed using Poisson regression, Spearman's correlation, t test, mixed anova, Mann-Whitney U test, Friedman and Chi square tests as appropriate. RESULTS: Median (range) intubation attempts were 2 (1-8) and 1 (1-3) for groups B and E, respectively. More rabbits in group E (91.6%) required additional alfaxalone and dexmedetomidine than in group B (16.7%). Median (range) cumulative histopathology scores were 6 (3-10) and 6 (2-9) for groups B and E, respectively. Scores were highest in the cranial trachea, but there was no difference between groups and no correlation between laryngeal/tracheal damage and the number of intubation attempts. CONCLUSIONS AND CLINICAL RELEVANCE: Both orotracheal intubation techniques were associated with laryngeal/tracheal damage. Although blind orotracheal intubation was associated with a higher number of attempts, the tissue damage was similar between groups.

Hepatocyte ploidy in cats with and without hepatocellular carcinoma.

BACKGROUND: Domestic cats rarely develop hepatocellular carcinoma. The reason for the low prevalence is unknown. Reductions in hepatocellular ploidy have been associated with hepatic carcinogenesis. Recent work in mice has shown that livers with more polyploid hepatocytes are protected against the development of hepatocellular carcinoma. Hepatocyte ploidy in the domestic cat has not been evaluated. We hypothesized that ploidy would be reduced in peri-tumoral and neoplastic hepatocytes compared to normal feline hepatocytes. Using integrated fluorescence microscopy, we quantified the spectra of ploidy in hepatocellular carcinoma and healthy control tissue from paraffin embedded tissue sections. RESULTS: Feline hepatocytes are predominantly mononuclear and the number of nuclei per hepatocyte did not differ significantly between groups. Normal cats have a greater number of tetraploid hepatocytes than cats with hepatocellular carcinoma. CONCLUSIONS: Total hepatocellular polyploidy in normal cat liver is consistent with values reported in humans, yet cellular ploidy (nuclei per cell) is greater in humans than in cats. Tetraploid cat hepatocytes are predominantly mononuclear.

Determining the efficacy of the bovine amniotic membrane homogenate during the healing process in rabbits' ex vivo corneas.

OBJECTIVE: To evaluate the efficacy of bovine amniotic membrane homogenate (BAMH) on wounded ex vivo rabbit corneas. PROCEDURE: Eighteen corneas obtained from normal rabbit eyes were wounded equally using a 6 mm trephine and cultured into an air-liquid interface model. Corneas were treated with phosphate-buffered saline (PBS) (n = 6, control group), 0.2% ethylenediaminetetraacetic acid (EDTA; n = 6), or BAMH (n = 6). All treatments were applied topically 6 times/day. Each cornea was macrophotographed daily with and without fluorescein stain to assess epithelialization and haziness. After 7 days, corneal transparency was evaluated, and the tissues prepared for histologic analysis of viability, and total and epithelial thickness. RESULTS: The mean epithelialization time was 6.2 ± 0.82 days for the control group, 6.2 ± 0.75 days for the EDTA-treated group, and 5.1 ± 0.40 days for the BAMH-treated group, demonstrating a significant difference between the BAMH and the other groups. The corneas that received EDTA had better transparency compared with the other groups. Histologically, all corneas had adequate morphology and architecture after healing. Analysis of corneal and epithelial thickness revealed no significant difference among groups. CONCLUSION: Bovine amniotic membrane homogenate is an effective and promising treatment for stromal and epithelial ulcers.

Erythema Multiforme Major in a Dog Treated with Intravenous Human Immunoglobulin and Immunosuppressive Therapy.

An ∼12 yr old castrated mixed-breed dog was evaluated for a 7 wk progressive history of intermittent hyporexia, lethargy, and erosive dermatitis. Initial examination revealed disseminated papules and macules coalescing to irregularly shaped and serpiginous plaques with widespread erosion progressing to ulceration. Skin histopathology revealed transepidermal keratinocyte apoptosis with lymphocyte satellitosis and lymphocytic and histiocytic interface infiltrate. Histopathology combined with clinical signs and history were compatible with the diagnosis of erythema multiforme major. Treatment was initiated with multidrug immunosuppression. Following 36 hr with no improvement, intravenous human immunoglobulin (0.45 mg/kg IV) was administered resulting in notable improvement in the dog's attitude and appetite within 2 hr and the dog's skin lesions within 48 hr. Following discharge, the dog improved daily with near complete resolution of dermatologic disease achieved 1 mo postdischarge. All immunosuppressive medications were ultimately discontinued 5 mo following presentation. This is the first report of a dog with erythema multiforme major that has been successfully treated with a combination of intravenous immunoglobulin and immunosuppression.

Spotted Fever Group Rickettsia Infection and Transmission Dynamics in Amblyomma maculatum.

Tick vectors are capable of transmitting several rickettsial species to vertebrate hosts, resulting in various levels of disease. Studies have demonstrated the transmissibility of both rickettsial pathogens and novel Rickettsia species or strains with unknown pathogenicity to vertebrate hosts during tick blood meal acquisition; however, the quantitative nature of transmission remains unknown. We tested the hypothesis that if infection severity is a function of the rickettsial load delivered during tick transmission, then a more virulent spotted fever group (SFG) Rickettsia species is transmitted at higher levels during tick feeding. Using Amblyomma maculatum cohorts infected with Rickettsia parkeri or "Candidatus Rickettsia andeanae," a quantitative PCR (qPCR) assay was employed to quantify rickettsiae in tick salivary glands and saliva, as well as in the vertebrate hosts at the tick attachment site over the duration of tick feeding. Significantly greater numbers of R. parkeri than of "Ca Rickettsia andeanae" rickettsiae were present in tick saliva and salivary glands and in the vertebrate hosts at the feeding site during tick feeding. Microscopy demonstrated the presence of both rickettsial species in tick salivary glands, and immunohistochemical analysis of the attachment site identified localized R. parkeri, but not "Ca Rickettsia andeanae," in the vertebrate host. Lesions were also distinct and more severe in vertebrate hosts exposed to R. parkeri than in those exposed to "Ca Rickettsia andeanae." The specific factors that contribute to the generation of a sustained rickettsial infection and subsequent disease have yet to be elucidated, but the results of this study suggest that the rickettsial load in ticks and during transmission may be an important element.

Presumed post-traumatic ocular chondrosarcoma with intrathoracic metastases in a cat.

An indoor-only, 5-year-old, spayed female domestic shorthair cat presented for an ophthalmic examination of the left eye. An intraocular tumor with secondary glaucoma and blindness was diagnosed; the globe was enucleated and sent for histopathological examination. Gross examination revealed a solid white mass filling the entire vitreous space and replacing the iris and ciliary body. The lens and retina appeared to be similarly replaced by the neoplasm. Histological examination revealed a complete loss of the internal ocular structures, with a ruptured capsule as the only remnant of the lens within an extensive malignant mesenchymal neoplastic cell proliferation. The cells were polygonal, with well-defined cytoplasmic borders and abundant weakly basophilic cytoplasm, embedded within the islands of chondroid matrix. No neoplastic invasion of the sclera was apparent. The animal died 6 months after the enucleation due to respiratory distress. Gross examination revealed numerous firm, white to tan nodular masses with smooth to mildly irregular surfaces dispersed throughout the parietal pleura, thoracic surface of the diaphragm, tracheobronchial and mediastinal lymph nodes, pericardium, and lungs. On cross-section, the neoplastic nodules were solid and variably translucent, resembling hyaline cartilage. Histologically, these nodules were similar to the neoplasm identified earlier in the left globe. Metastasis of post-traumatic ocular chondrosarcoma has not yet been described in cats. This is therefore believed to be the first report of metastases of this type of neoplasm in cats. This case adds to the limited set of data on the outcome of this type of tumor.

Long-term clinical control of feline pancreatic carcinoma with toceranib phosphate.

An 11-year-old, spayed female, domestic shorthair cat was presented with a non-resectable abdominal mass diagnosed as carcinomatosis of pancreatic origin. Treatment with toceranib phosphate was started. Abdominal ultrasound approximately 1 year after diagnosis revealed progressive disease. The cat was humanely euthanized approximately 792 days after initial presentation due to progressive clinical signs.

Contrôle clinique à long terme du carcinome pancréatique félin avec le tocéranib phosphate. Un chatte commune domestique stérilisée âgée de 11 ans a été présentée avec une masse abdominale non résécable diagnostiquée comme une carcinomatose d'origine pancréatique. Le tocéranib phosphate a été administré. L'échographie abdominale environ 1 an après le diagnostic a révélé une maladie progressive. La chatte a été euthanasiée sans cruauté environ 792 jours après la présentation initiale en raison de signes cliniques progressifs.(Traduit par Isabelle Vallières).

Tissue tropisms, infection kinetics, histologic lesions, and antibody response of the MR766 strain of Zika virus in a murine model.

BACKGROUND: The appearance of severe Zika virus (ZIKV) disease in the most recent outbreak has prompted researchers to respond through the development of tools to quickly characterize transmission and pathology. We describe here another such tool, a mouse model of ZIKV infection and pathogenesis using the MR766 strain of virus that adds to the growing body of knowledge regarding ZIKV kinetics in small animal models. METHODS: We infected mice with the MR766 strain of ZIKV to determine infection kinetics via serum viremia. We further evaluated infection-induced lesions via histopathology and visualized viral antigen via immunohistochemical labeling. We also investigated the antibody response of recovered animals to both the MR766 and a strain from the current outbreak (PRVABC59). RESULTS: We demonstrate that the IRF3/7 DKO mouse is a susceptible, mostly non-lethal model well suited for the study of infection kinetics, pathological progression, and antibody response. Infected mice presented lesions in tissues that have been associated with ZIKV infection in the human population, such as the eyes, male gonads, and central nervous system. In addition, we demonstrate that infection with the MR766 strain produces cross-neutralizing antibodies to the PRVABC59 strain of the Asian lineage. CONCLUSIONS: This model provides an additional tool for future studies into the transmission routes of ZIKV, as well as for the development of antivirals and other therapeutics, and should be included in the growing list of available tools for investigations of ZIKV infection and pathogenesis.

Ultrastructure and molecular phylogeny of Pleistophora hyphessobryconis (Microsporidia) infecting hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a Brazilian aquaculture facility.

A microsporidian infecting the skeletal muscle of hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a commercial aquaculture facility in Brazil is described. Affected fish exhibited massive infections in the skeletal muscle that were characterized by large opaque foci throughout the affected fillets. Histologically, skeletal muscle was replaced by inflammatory cells and masses of microsporidial developmental stages. Generally pyriform spores had a wrinkled bi-layer spore wall and measured 4·0 × 6·0 µm. Multinucleate meronts surrounded by a simple plasma membrane were observed. The polar filament had an external membrane and a central electron dense mass. The development of sporoblasts within a sporophorous vesicle appeared synchronized. Ultrastructural observations and molecular analysis of 16S rDNA sequences revealed that the microsporidian was Pleistophora hyphessobryconis. This study is the first report of a P. hyphessobryconis infection in a non-ornamental fish.

Retinal dysplasia in American pit bull terriers--phenotypic characterization and breeding study.

OBJECTIVE: The purpose of this study was to investigate the inheritance and phenotype of retinal dysplasia (RD) in the American pit bull terrier. ANIMALS STUDIED: A breeding colony established from a single female pure-bred American pit bull terrier dog with RD. PROCEDURES: A female pure-bred American pit bull terrier with RD was donated to the Veterinary Hospital of Federal University of Paraná, Curitiba, Brazil. A breeding colony was established and the phenotype and inheritance of the condition investigated. Regular ophthalmic examinations and fundus photography were performed on three generations of offspring from the founder animal. Some animals were additionally studied by optical coherence tomography. Ocular histopathology was performed on some animals from the colony. RESULTS: Fifty-seven offspring were produced in two generations from the affected founder female. Thirty-two were diagnosed with RD and showed a spectrum of severity of lesions including multifocal, and or geographic lesions and some developed retinal detachment. Histologic examination demonstrated retinal folds, rosettes, and areas of retinal detachment. The affected dogs were shorter in stature than the unaffected littermates. Breeding studies suggested the trait has an autosomal dominant mode of inheritance. DNA testing showed that the affected dogs were negative for the known gene mutations for canine dwarfism with RD. CONCLUSION: This is a report of a novel inherited form of RD that affects American pit bull terriers.

Sepsis and multi-organ dysfunction associated with postgrooming furunculosis in a dog.

BACKGROUND: Postgrooming furunculosis, as previously described in the dog, is a type of pyoderma that typically responds to routine antimicrobial therapy. Systemic clinical signs are common but are usually mild. ANIMAL: A 3-year-old spayed female great dane was presented with marked dorsal furunculosis of 24 h duration. The clinical signs and blood analyses were consistent with sepsis, systemic inflammatory response syndrome and disseminated intravascular coagulopathy (DIC). METHODS AND RESULTS: Skin culture revealed multi-drug resistant Pseudomonas aeruginosa infection. The dog did not respond to aggressive medical therapy including intravenous antibiotic therapy, fresh frozen plasma and haemodynamic support, and was humanely euthanized. Postmortem findings were consistent with postgrooming furunculosis with associated sepsis and DIC affecting multiple organs including the lungs and brain. CONCLUSIONS AND CLINICAL IMPORTANCE: Postgrooming furunculosis can progress to sepsis and multiple organ dysfunction in the dog and can lead to death.

Puberty-specific promotion of mammary tumorigenesis by a high animal fat diet.

INTRODUCTION: Increased animal fat consumption is associated with increased premenopausal breast cancer risk in normal weight, but not overweight, women. This agrees with our previous findings in obesity-resistant BALB/c mice, in which exposure to a high saturated animal fat diet (HFD) from peripuberty through adulthood promoted mammary tumorigenesis. Epidemiologic and animal studies support the importance of puberty as a life stage when diet and environmental exposures affect adult breast cancer risk. In this study, we identified the effects of peripubertal exposure to HFD and investigated its mechanism of enhancing tumorigenesis. METHODS: Three-week-old BALB/c mice fed a low-fat diet (LFD) or HFD were subjected to 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis. At 9 weeks of age, half the mice on LFD were switched to HFD (LFD-HFD group) and half the mice on HFD were switched to LFD (HFD-LFD group). Tumor gene expression was evaluated in association with diet and tumor latency. RESULTS: The peripubertal HFD reduced the latency of DMBA-induced mammary tumors and was associated with tumor characteristics similar to those in mice fed a continuous HFD. Notably, short-latency tumors in both groups shared gene expression characteristics and were more likely to have adenosquamous histology. Both HFD-LFD and continuous HFD tumors showed similar gene expression patterns and early latency. Adult switch from HFD to LFD did not reverse peripubertal HFD tumor promotion. Increased proliferation, hyperplasia, and macrophages were present in mammary glands before tumor development, implicating these as possible effectors of tumor promotion. Despite a significant interaction between pubertal diet and carcinogens in tumor promotion, peripubertal HFD by itself produced persistent macrophage recruitment to mammary glands. CONCLUSIONS: In obesity-resistant mice, peripubertal HFD is sufficient to irreversibly promote carcinogen-induced tumorigenesis. Increased macrophage recruitment is likely a contributing factor. These results underscore the importance of early life exposures to increased adult cancer risk and are consistent with findings that an HFD in normal weight premenopausal women leads to increased breast cancer risk. Notably, short-latency tumors occurring after peripubertal HFD had characteristics similar to human basal-like breast cancers that predominantly develop in younger women.

Pubertal high fat diet: effects on mammary cancer development.

INTRODUCTION: Epidemiological studies linking dietary fat intake and obesity to breast cancer risk have produced inconsistent results. This may be due to the difficulty of dissociating fat intake from obesity, and/or the lack of defined periods of exposure in these studies. The pubertal mammary gland is highly sensitive to cancer-causing agents. We assessed how high fat diet (HFD) affects inflammation, proliferative, and developmental events in the pubertal gland, since dysregulation of these can promote mammary tumorigenesis. To test the effect of HFD initiated during puberty on tumorigenesis, we utilized BALB/c mice, for which HFD neither induces obesity nor metabolic syndrome, allowing dissociation of HFD effects from other conditions associated with HFD. METHODS: Pubertal BALB/c mice were fed a low fat diet (12% kcal fat) or a HFD (60% kcal fat), and subjected to carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis. RESULTS: HFD elevated mammary gland expression of inflammatory and growth factor genes at 3 and 4 weeks of diet. Receptor activator of nuclear factor kappa-B ligand (RANKL), robustly induced at 4 weeks, has direct mitogenic activity in mammary epithelial cells and, as a potent inducer of NF-κB activity, may induce inflammatory genes. Three weeks of HFD induced a transient influx of eosinophils into the mammary gland, consistent with elevated inflammatory factors. At 10 weeks, prior to the appearance of palpable tumors, there were increased numbers of abnormal mammary epithelial lesions, enhanced cellular proliferation, increased growth factors, chemokines associated with immune-suppressive regulatory T cells, increased vascularization, and elevated M2 macrophages. HFD dramatically reduced tumor latency. Early developing tumors were more proliferative and were associated with increased levels of tumor-related growth factors, including increased plasma levels of HGF in tumor-bearing animals. Early HFD tumors also had increased vascularization, and more intra-tumor and stromal M2 macrophages. CONCLUSIONS: Taken together in this non-obesogenic context, HFD promotion of inflammatory processes, as well as local and systemically increased growth factor expression, are likely responsible for the enhanced tumorigenesis. It is noteworthy that although DMBA mutagenesis is virtually random in its targeting of genes in tumorigenesis, the short latency tumors arising in animals on HFD showed a unique gene expression profile, highlighting the potent overarching influence of HFD.

Sensitivity and specificity of in situ hybridization for diagnosis of cutaneous infection by Leishmania infantum in dogs.

An accurate diagnosis of infection by Leishmania infantum in dogs is fundamental for the control of zoonotic visceral leishmaniasis (VL). Histopathology (HP) and immunohistochemistry (IHC) are frequently used for the histological diagnosis of L. infantum in dogs but have shown limited accuracy. To improve the sensitivity and specificity of the histological diagnosis of VL, we evaluated automated in situ hybridization (ISH) using a generic probe for Leishmania and a specific probe for L. infantum in surgical skin biopsy specimens of dogs. The ISH results were compared with those of HP and IHC, using parasitological culture as the reference standard. Skin samples from 51 dogs with cutaneous L. infantum infection and 51 noninfected dogs were randomly selected from samples of dogs from various cities in Brazil where canine VL is endemic. These samples were processed for parasitological culture, HP, IHC, and ISH using both probes. The sensitivities of ISH using the specific probe, ISH using the generic probe, IHC, and HP were, respectively, 74.5%, 70.6%, 69.5%, and 57.6%. The specificity of both ISH probes tested was 100%, and there was no cross-hybridization of the generic and specific probes with selected pathogenic fungi and protozoa. The specific probe discriminated L. infantum from the other species of Leishmania that infect dogs in the New World. ISH is highly sensitive and specific for the diagnosis of L. infantum in histologic samples of skin from infected dogs and can be used on routine biopsy material to make a diagnosis of leishmaniasis.

The Oncolytic herpes simplex virus type-1 (HSV-1) vaccine strain VC2 causes intratumor infiltration of functionally active T cells and inhibition of tumor metastasis and pro-tumor genes VEGF and PDL1 expression in the 4T1/Balb/c mouse model of stage four breast cancer.

Introduction: Oncolytic viruses (OVs) provide new modalities for cancer therapy either alone or in combination with synergistic immunotherapies and/or chemotherapeutics. Engineered Herpes Simplex Virus Type-1 (HSV-1) has shown strong promise for the treatment of various cancers in experimental animal models as well as in human patients, with some virus strains licensed to treat human melanoma and gliomas. In the present study we evaluated the efficacy of mutant HSV-1 (VC2) in a late stage, highly metastatic 4T1 murine syngeneic. Method: VC2 was constructed VC2 using double red recombination technology. For in-vivo efficacy we utilized a late stage 4T1 syngeneic and immunocompetent BALB/cJ mouse model breast cancer model which exhibits efficient metastasis to the lung and other organs. Results: VC2 replicated efficiently in 4T1 cells and in cell culture, achieving titers similar to those in African monkey kidney (Vero) cells. Intra-tumor treatment with VC2 did not appreciably reduce average primary tumor sizes but a significant reduction of lung metastasis was noted in mice treated intratumorally with VC2, but not with ultraviolet-inactivated VC2. This reduction of metastasis was associated with increased T cell infiltration comprised of CD4+ and CD4+CD8+ double-positive T cells. Characterization of purified tumor infiltrating T cells revealed a significant improvement in their proliferation ability compared to controls. In addition, significant T cell infiltration was observed in the metastatic nodules associated with reduction of pro-tumor PD-L1 and VEGF gene transcription. Conclusion: These results show that VC2 therapy can improve anti-tumor response associated with a better control of tumor metastasis. improve T cell responses and reduce pro-tumor biomarker gene transcription. VC2 holds promise for further development as an oncolytic and immunotherapeutic approach to treat breast and other cancers.

Supplementation with galacto-oligosaccharides increases the percentage of NK cells and reduces colitis severity in Smad3-deficient mice.

The gut microbiota plays an essential role in intestinal immunity. Prebiotics, including galacto-oligosaccharides (GOS), are fermentable fibers that beneficially affect the host by stimulating the growth of specific microbial populations. We investigated the effect of GOS on colitis development and on immune variables in Smad3-deficient mice treated with the pathogen Helicobacter hepaticus. Mice were supplemented daily with 5000 mg GOS/kg body weight 2 wk prior to infection and 4 wk postinfection, a time period during which colitis severity peaks in this model. Mice (n = 4-8/treatment at each time) were killed preinfection (0 d) and at 3, 7, and 28 d postinfection to evaluate immune variables in the spleen and in mesenteric lymph nodes (MsLN) by flow cytometry. Colon and cecum samples were collected for histopathologic analysis. Fecal pellets (n = 8-9/treatment) were collected prior to infection to measure relative changes in Bifidobacterium ssp. and Lactobacillum ssp. by real-time PCR. GOS significantly reduced colitis severity in response to H. hepaticus (P < 0.0001). This was associated with a significant increase in the percentage of NK cells in the spleen (P < 0.001) and in MsLN (P < 0.001) at 3 d postinfection and a 1.5-fold increase in fecal Bifidobacterium ssp. (P = 0.003). GOS stimulated NK expression of CCR9, a chemokine receptor involved in lymphocyte trafficking to the gut preinfection (0 d) in the blood (P = 0.02), spleen (P = 0.033), and MsLN (P = 0.017). In addition, GOS stimulated colonic IL-15 production 3 d postinfection (P < 0.001). These data suggest that GOS reduces colitis by modulating the function and trafficking of NK cells and may provide a novel therapeutic strategy for individuals with inflammatory bowel disease.

Bilateral uveal metastasis of a subcutaneous fibrosarcoma in a cat.

A 6-year-old neutered male domestic short-haired cat was presented to the Comparative Ophthalmology service at Michigan State University with a 3-week history of decreased appetite and redness of the left eye. The left forelimb had been removed 15 months previously because of the presence of a subcutaneous fibrosarcoma. In the left globe, a large iridal mass was associated with increased intraocular pressure and retinal detachment. A smaller mass involving the right iris was also present. Imaging revealed a 2-cm mass in the left caudodorsal lung lobe, and abdominal ultrasound showed multifocal bilateral renal masses. Aspirates of these masses were nondiagnostic. The left globe was removed for palliative reasons, and histopathology showed that fibrosarcoma was infiltrating the iris, choroid, and optic nerve. Despite systemic chemotherapy with doxorubicin, the animal died 4 months after initial presentation. Histopathology confirmed highly angioinvasive metastatic fibrosarcoma also in the right uveal tract, the lungs, and both kidneys.