Ovine keratoconjunctivitis experimentally induced by instillation of Mycoplasma conjunctivae

Five sheep, free from Mycoplasma conjunctivae and ocular Chlamydia infection, were experimentally inoculated with M. conjunctivae and five more sheep were exposed to the infection by contact. Keratoconjunctivitis developed in all ten sheep. As in natural outbreaks of infectious keratoconjunctivitis (IKC), clinical signs were generally moderate and transient, and recurred in some sheep. M. conjunctivae was detected throughout the 53-day observation period. Clinical diagnosis was confirmed by histopathologic examination of three sheep. Moraxella ovis, found in six of the ten sheep before the start of the experiment, appeared to play no etiologic role in the development of IKC.     

Ovine infectious keratoconjunctivitis: a microbiological study of clinically unaffected and affected sheep's eyes with special reference to Mycoplasma conjunctivae

In a field survey of ovine infectious keratoconjunctivitis, the microbiological flora of 240 clinically unaffected eyes from sheep in 10 flocks was compared with the flora of an equivalent number of clinically affected eyes from 12 natural outbreaks of the disease. Totals of 16 and 17 genera of bacteria were recovered from unaffected and affected eyes, respectively. Staphylococcus, bacillus and branhamella were isolated significantly more often than the other genera of bacteria, in both the unaffected and affected eyes (P less than 0.05). Branhamella ovis and Escherichia coli occurred more frequently in affected eyes, and Staphylococcus aureus occurred more frequently in severely than mildly affected eyes. The genera Mycoplasma and Acholeplasma were isolated from both groups, and Mycoplasma conjunctivae occurred in 92 affected eyes (38.3 per cent), and 27 unaffected eyes (11.3 per cent).     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.     

Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene.

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).     

Use of laparoscopy for diagnosing experimentally induced acute pancreatitis in dogs

Diagnosis of acute pancreatitis in dogs remains a significant challenge despite the development of advanced diagnostic methodologies. Visual inspection and pancreas biopsy using laparoscopy are generally considered to be procedures free of complications when conducted on healthy animals. However, the usefulness of laparoscopy for diagnosing acute pancreatitis has not been assessed. In the present study, the efficacy of laparoscopy for diagnosing acute pancreatitis in dogs was evaluated in animals with experimentally induced acute pancreatitis. Gross appearance of the pancreatic area was examined by laparoscopy to survey for the presence of edema, adhesions, effusion, pseudocysts, hemorrhage, and fat necrosis. Laparoscopic biopsy was performed and the histopathologic results were compared to those of pancreatic samples obtained during necropsy. The correlation between laparoscopy and histopathologic findings of the pancreas was evaluated. The presence of adhesions, effusion, and hemorrhage in the pancreatic area observed by laparoscopy significantly correlated with the histopathologic results (p < 0.05). There was no significant relationship between the histopathologic and laparoscopic biopsy findings. Results of this study suggested that laparoscopic evaluation of gross lesions has clinical significance although the laparoscopic biopsy technique has some limitations. This method combined with additional diagnostic tools can be effective for diagnosing acute pancreatitis in dogs.     

Isolation,proliferation and characterization of endometrial canine stem cells

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.     

Some characteristics of four attenuated vaccine virus strains and a virulent strain of Aujeszky's disease virus

Summary

In the present study four attenuated virus strains, used as vaccines, and a virulent strain of Aujeszky's disease virus (AD V) were compared with respect to their virulence in mice, their ability to induce virus‐specified thymidine kinase (TK) in infected cells, and their cleavage profiles of viral DNA's after treatment with the restriction endonuclease Kpnl.

The survival time of mice inoculated with the B‐KAL or the virulent NIA‐3 strain was comparable. whereas the Hanha and BUK strains required significantly loniser periods to kill mice. Mice were resistant to the MK‐25 strain of ADV.

The strains were assayed for TK phenotype by plaque autoradiography after ³H‐thymidine labelling of infected cells. MK‐25 proved to be the only strain defective in induction of TK in pig kidney cells. Restriction endonuclease analysis of viral DNA's revealed that each vaccine strain showed a characteristic fragment pattern that could easily be differentiated from that of other vaccine and field strains of ADV. The present results demonstrate that the mouse virulence lest and the TK assay detect differences in biological properties of ADV strains, but that restriction endonuclease analysis is required for unambiguous identification of vaccine and field strains of ADV.     

Clinical and histopathological features and prognosis of gastrointestinal adenocarcinomas in Jack Russell Terriers

There has been an increase in the number of Jack Russell Terriers (JRTs) diagnosed with adenomas and adenocarcinomas of the gastrointestinal tract in Japan. This study retrospectively investigated the clinical and histopathological features and prognosis of adenocarcinomas arising in the gastrointestinal tract in JRT dogs. Seven JRTs and 39 dogs of other breeds diagnosed with gastrointestinal adenocarcinoma were included in the study. The most common sites of gastrointestinal adenocarcinoma in JRTs were the pylorus and rectum. On histopathological examination, these adenocarcinomas showed a papillary or tubular growth pattern, and the lesions were confined within the mucosal epithelium and poorly invasive. Among all dogs with gastric adenocarcinoma, the median survival time (MST) for five of the JRTs could not be determined because more than half of the cases remained alive, while the MST for nine non-JRT dogs was 34 days. Among all dogs with adenocarcinoma in the large intestine, the MST for three of the JRTs could not be determined, while the MST for nine non-JRT dogs was 1,973 days. The difference in MST between JRT and non-JRT dogs with gastric adenocarcinoma was significant (P=0.0220). Since gastrointestinal adenocarcinomas in JRTs show distinct characteristics with respect to their clinical features, treatment course, and prognosis, a different surgical and medical treatment plan should be considered compared to the management of gastrointestinal adenocarcinomas in other dog breeds.     

Retrospective analysis of factors affecting clinical outcome following CHOP‐based chemotherapy in dogs with primary nodal diffuse large B‐cell lymphoma

Numerous factors are known to affect the prognosis of dogs with chemotherapy‐treated lymphomas. However, prognostic factors for dogs with specific subtypes of lymphoma are less clearly defined. The objective of this study was to identify prognostic factors for dogs receiving CHOP‐based chemotherapy for primary nodal diffuse large B‐cell lymphoma (DLBCL). Medical records of dogs treated for DLBCL at the Purdue Veterinary Teaching Hospital (PUVTH) from 2006 to 2016 were reviewed. Factors potentially related to prognosis were analysed using multivariable statistical methods. Ninety‐eight dogs were included in the study. Best overall response to chemotherapy was complete remission in 80 dogs (81.6%) and partial remission in 18 dogs (18.4%). Median progression‐free survival (PFS) for the entire population was 252 days (range 19‐1068). Factors significantly associated with achieving partial (rather than complete) remission following CHOP included presence of thrombocytopenia at diagnosis (OR 6.88; 95% CI 1.98‐23.93; P = .002), baseline serum globulin concentration (OR 2.63; 95% CI 1.03‐6.75; P = .044), and age at diagnosis (OR 1.36; 95% CI 1.08‐1.71; P = .009). Factors significantly associated with PFS in the lowest quartile (≤93 days) included presence of thrombocytopenia at diagnosis (OR 8.72; 95% CI 1.54‐49.33; P = .014), age at diagnosis (OR 1.47; 95% CI 1.12‐1.94; P = .005), and baseline neutrophil count (OR 1.18; 95% CI 1.02‐1.37; P = .025). Presence of thrombocytopenia, greater age, higher neutrophil count, and higher serum globulin concentration all may be associated with a particularly poor outcome in dogs receiving CHOP‐based chemotherapy for DLBCL.     

Marbofloxacin for the treatment of experimentally induced Mycoplasma haemofelis infection in cats

BACKGROUND: Administration of tetracyclines or fluoroquinolones is associated with improvement in clinical and laboratory abnormalities in cats infected with Mycoplasma haemofelis. No treatment protocol has consistently eliminated the organism, and antimicrobial susceptibility may vary among M. haemofelis isolates. Continued search for effective therapies is warranted. HYPOTHESIS: Marbofloxacin administered at the onset of clinical illness will be safe and effective for the treatment of M. haemofelis. ANIMALS: Fourteen young adult, laboratory-reared cats housed together in a specific pathogen-free facility. METHODS: Twelve cats were inoculated IV with 2.0 mL of blood from 2 M. haemofelis positive cats. Clinical parameters were assessed daily. CBC and hemoplasma polymerase chain reaction (PCR) assay were performed before inoculation, weekly for 1-3 weeks postinoculation (PI) and twice weekly 3-6 weeks PI. Treatment with marbofloxacin (2.75 mg/kg PO daily for 14 days) was initiated in 6 randomly selected cats when PCV was <30% or fever was >102.5 degrees F (39.2 degrees C). Cats that were PCR positive on day 7 of therapy were treated for 28 days. Cats that were PCR negative on day 42 PI were treated with 20 mg/kg methylprednisolone acetate IM on day 50 PI. RESULTS: Significant differences between groups on some days after inoculation included higher PCV and red blood cell counts, lower mean cell volume, and higher mean cell hemoglobin content in marbofloxacin-treated cats. No differences in PCR assay results were noted between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Marbofloxacin was safe and resulted in more rapid hematologic improvement in M. haemofelis-infected cats, but did not change clinical scores and did not consistently eliminate infection.     

Radiographic and computed tomographic evaluation of experimentally induced lung aspiration sites in dogs

This study was performed to radiographically examine the prevalence of aspiration sites and to evaluate their atomical correlation with the bronchial pattens. Ten healthy beagle dogs were repeatedly radiographed, at weekly intervals, in the left and right lateral, ventrodorsal (VD) and dorsoventral (DV) positions. Three mililiters of iohexol distilled with same volume of saline was infused into the tracheal inlet. Which lung lobe was aspirated was decided upon by the presence of a significant alveolar pattern due to the contrast medium. Alveolar patterns were identified at the left (100%) and right cranial lung lobes (77%) with the dogs in dependant lateral recumbency, at the right caudal lung lobe (71%) with the dogs in VD recumbency and at the right middle lung lobe (59%) with the dogs in DV recumbency, respectively. The anatomical correlation was evaluated by performing computed tomography. The right principal bronchus (165.8 ± 1.6°) was more straightly bifurcated than was the left principal bronchus (142.7 ± 1.8°, p < 0.01). In VD position, the right side lung had a greater opertunity to become aspirated. The ventrally positioned right middle lobar bronchial origin was more easily to be aspirated the other laterally positioned ones. We think that these anatomical characteristics can be one of the causes for aspiration pneumonia to occur more frequently in the right side lung.     

Randomized Phase III Trial of Piroxicam in Combination with Mitoxantrone or Carboplatin for First‐Line Treatment of Urogenital Tract Transitional Cell Carcinoma in Dogs

Background

Reported response rates of transitional cell carcinoma (TCC) in dogs to piroxicam in combination with either mitoxantrone or carboplatin are similar; however, it is unknown whether either drug might provide superior duration of response.

Hypothesis/Objectives

To determine if the progression‐free interval (PFI) of dogs with TCC treated with mitoxantrone and piroxicam was different than that of dogs receiving carboplatin and piroxicam. The hypothesis was that the efficacy of mitoxantrone is no different from carboplatin.

Animals

Fifty dogs with TCC without azotemia.

Methods

Prospective open‐label phase III randomized study. Either mitoxantrone or carboplatin was administered every 3 weeks concurrently with piroxicam with restaging at 6‐week intervals. Twenty‐four dogs received carboplatin and 26 received mitoxantrone.

Results

Response was not different between groups (P = .56). None of the dogs showed complete response. In the mitoxantrone group, there were 2 (8%) partial responses (PR) and 18 (69%) dogs with stable disease (SD). In the carboplatin group, there were 3 PR (13%) and 13 (54%) dogs with SD. The PFI was not significantly different between groups (mitoxantrone = 106 days; carboplatin = 73.5 days; P = .62; hazard ratio 0.86; 95% confidence interval 0.47–1.56). Dogs with prostatic involvement experienced a shorter survival (median, 109 days) compared to dogs with urethral, trigonal, or apically located tumors; this difference was significant (median 300, 190, and 645 days, respectively; P = .005).

Conclusions and Clinical Importance

This study did not detect a different in outcome in dogs with TCC treated with either mitoxantrone or carboplatin in combination with piroxicam.     

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.     

Molecular markers of prognosis in canine cortisol‐secreting adrenocortical tumours

Hypercortisolism is caused by a cortisol‐secreting adrenocortical tumour (ACT) in approximately 15%‐20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol‐secreting ACTs were included from 40 dogs, of which follow‐up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate‐high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT‐PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour‐transforming gene‐1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor‐1 (SF‐1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets.     

Correlating the immune response with the clinical-pathological course of persistent mastitis experimentally induced by Mycoplasma agalactiae in dairy goats

To correlate the clinical course of mycoplasma mastitis with its immune response, right mammary glands of 15 lactating goats were inoculating with 10¹⁰ colony-forming units (cfu) of Mycoplasma agalactiae (Ma). Before sacrificing the animals at 5, 15 or 45 days post-inoculation (dpi), blood Ma antibody titres and milk mycoplasma colony and somatic cell counts were monitored. Ma colonised the mammary gland and milk counts increased to over 10¹² cfu/ml within 5 dpi. During this period, an innate immune response involving neutrophils and macrophages was observed, and Ma antigen appeared in the degenerated acinar epithelium. From 7 dpi, a specific antibody response coincided with reduced viable mycoplasmas in milk. The humoral immune response was limited; by 37 dpi, all animals scored negative for anti-Ma antibodies, and around 10⁸ cfu/ml were shed. Results indicate an early immune response to Ma inoculation unable to control mycoplasmal invasion. An ensuing humoral response, despite reducing the mycoplasma burden, leads to chronic, persistent infection.     

C-reactive protein as an indicator of inflammatory responses to experimentally induced cystitis in dogs

The aim of this study was to demonstrate and assess C-reactive protein (CRP) changes in dogs with induced bacterial cystitis with or without antibiotics. We also evaluated availability of CRP levels to serve as an indicator for monitoring or diagnosing bacterial cystitis. Serial CRP concentrations in dogs with induced bacterial cystitis were higher than those of controls (p < 0.001). CRP concentrations peaked on day 7 and gradually decreased thereafter. In the treatment group, CRP concentrations decreased after medication compared to the untreated group (p = 0.032). CRP levels had a linear correlation with urine white blood cell counts among all groups (r = 0.837, p < 0.001, n = 140). Compared to the negative urine culture group, dogs with positive urine culture results had higher CRP concentrations (median 43.8 mg/L vs. 5.9 mg/L; p < 0.001). Area under the receiver operating characteristic curve was 0.955; when cut-off value was 12.2 mg/L, CRP measurements were found to have a sensitivity of 92.3% and specificity of 86.4%. This result indicates that rapid increases of CRP occurred after inducing bacterial cystitis and CRP may be a useful indicator for monitoring or diagnosing canine bacterial cystitis together with sediment urinalysis and urine bacterial culture.     

Radiation therapy for the treatment of tumours in small companion animals

Radiation is becoming widely available to treat tumours in veterinary patients. Orthovoltage machines capable of delivering low energy external beam radiation are less versatile than linear accelerators and cobalt-60 machines that deliver megavoltage radiation. In addition, electron beam capabilities that are available with some linear accelerators allow more targeted treatment in smaller patients. Acute effects of radiation are to be expected, but in nearly all cases such side effects resolve without limiting protocols. In contrast, late effects of radiation are dose limiting and are more likely with higher doses per treatment fraction. Protocols that use smaller doses per fraction have a lower risk of late effects thereby allowing higher total doses to be delivered which leads to higher tumour control rates. It is possible to provide long-term tumour control in cats and dogs using radiation therapy, particularly for mast cell tumours, soft tissue sarcomas, oral tumours and brain tumours in dogs and soft tissue sarcomas and skin tumours in cats. Individualization of treatments for tumours based on tumour staging and proliferative fraction should be considered, rather than making blanket assumptions about the behaviour of histologically determined tumour types.