Development of two alternative Loop‐mediated isothermal amplification tools for detecting pathogenic pine wood nematodes

Loop‐mediated isothermal amplification (LAMP) detection tools have great potential for diagnosing the causal agents of infectious diseases in clinics and in agriculture. In this work, we developed two alternative LAMP protocols for detecting the pathogenic nematode Bursaphelenchus xylophilus, causal agent of pine wilt disease. We first identified a pectate lyase 3 gene as a biomarker for developing a LAMP Detection Kit, as there was no homologue in non‐pathogenic nematodes that live in pine timber or bark and show structural similarities to B. xylophilus. The first LAMP protocol used the Genie II equipment and an isothermal master mix containing Geobacillus sp. M 2.0 large fragment DNA polymerase showed approximately 10 times greater sensitivity with a shorter incubation period compared with that of the second LAMP protocol, which utilized a fluorescence metal indicator, calcein and an engineered Bacillus stearothermophilus DNA polymerase I, large fragment (Bst 2.0 DNA polymerase). However, the LAMP reactions with calcein and Bst 2.0 polymerase were the cost‐effective method because the reaction could be performed using a simple isothermal block and relatively inexpensive calcein as a fluorescence indicator visible to the naked eye.     

The effects of injection of an iodine counterirritant into the patellar ligaments of ponies: Application to stifle lameness

Patellar fixation is a common cause of lameness in horses and has been successfully treated by injection of the patellar ligaments with counterirritants. To investigate the effects of this treatment, the medial and middle patellar ligaments of 10 ponies were injected with an oil-based iodine counterirritant solution and then examined morphologically. Vacuoles of counterirritant were present in the paratenon and endotenon of the ligaments and caused severe necrosis and inflammation. At 1 day after injection, there was a marked neutrophilic infiltration which progressed to a lymphocytic infiltrate by 3 days. By 7 days, fibroplasia was present and became more organized with time. Disrupted collagen fibers and numerous, large, pale fibroblasts were present in the fasciculi, but neutrophilic infiltration was absent. The fibrous response in the fasciculi was similar to that present in the paratenon and endotenon. At 28 days, the drug was still present as lipid vacuoles in the paratenon and endotenon, and the damaged fasciculi had not regained their normal appearance. The clinical effect of this treatment was attributed to the change in size and shape of the patellar ligaments resulting from the inflammatory response.     

Peribiliary cysts associated with severe liver disease: a previously unrecognized tumor in a lion (Panthera leo).

A 13-year-old male lion (Panthera leo) from Dae Jeon Zoo, Republic of Korea, presented with clinical signs of lethargy and anorexia. Despite treatment with antibiotics and fluid therapy, the lion died 6 days after initial presentation. Postmortem examination revealed multiple masses measuring 5-10 cm in diameter and cysts throughout the liver. A diagnosis of spontaneous peribiliary cysts was made on the basis of microscopic lesions as well as special staining and immunohistochemical characteristics. Histologically, the neoplasm was surrounded and composed of compact collagenous tissue. The inner cystic single layer resembled biliary mucosa and was composed of cuboidal or flattened epithelial lining that was strongly immunopositive for cytokeratin AE1/AE3. This layer was surrounded by fibrous tissue that stained blue by Masson's trichrome staining. Given the presence of multiple organized cysts in the liver, the lesion was consistent with peribiliary cysts. To the authors' knowledge, this is the first report of peribiliary cysts in an animal.     

Experimental fecal transmission of human cryptosporidia to pigs, and attempted treatment with an ornithine decarboxylase inhibitor

Fecal material collected from an immunologically deficient man with persistent cryptosporidia infection was stored in potassium dichromate for two weeks and then fed (inoculated) to newborn pigs. The six inoculated newborn pigs shed the organism in their feces starting four to five days afer inoculation and continuing for as long as 22 days after inoculation. Pigs which were killed and necropsied while shedding had cryptosporidia infection of ileum, cecum, and colon. Infected pigs had atrophied ileal villi and flattened irregular cecal and colonic epithelium. Uninoculated littermate controls remained free to the infection and had histologically normal intestinal tracts at necropsy. Treatment of three of the six inoculated pigs with the ornithine decarboxylase inhibitor, DL-alpha-difluoromethylornithine, orally for ten days had no apparent effect on the infection.     

Age Dependent Resistance to Transmissible Gastroenteritis of Swine (TGE) I. Clinical Signs and Some Mucosal Dimensions in Small Intestine

Pigs were exposed to transmissible gastroenteritis (TGE) virus when three days old or when 21 days old. Diarrhea was earliest in onset, most frequent, most profuse and most prolonged in the youngest group. Pigs exposed when three days old also had a higher case fatality rate than those exposed when 21 days old. The histological response of both groups to exposure was atrophy of villi and hyperplasia of crypts in jejunum and ileum. However, from days three to seven post-exposure, when most fatalities occurred in the younger group, atrophy of villi was both more intensive and extensive in the younger group. Hyperplasia of crypts was also greater and more prolonged in the younger group. Regeneration of atrophic villi was more rapid in jejunum than ileum in both groups. Results were interpreted to indicate two populations, with different rates of regeneration, in the 21-day old group. Based on this interpretation, regeneration of villi was more rapid in one population from the 21-day old group than in the three-day old group.

The length of villi and depth of crypts in control pigs varied longitudinally (i.e. from site to site) in the intestine, within each age group. Length of villi and depth of crypts in control pigs also varied with age.

     

Studies on the carriage and transmission of porcine reproductive and respiratory syndrome virus by individual houseflies (Musca domestica)

The objectives of the study were to determine the site of porcine reproductive and respiratory syndrome virus (PRRSV) in individual houseflies, to assess whether an individual housefly could transmit PRRSV to a susceptible pig, and to compare the ability of PCR, virus isolation and a pig bioassay to detect PRRSV in houseflies. In the first experiment 26 houseflies were fed on a pig infected experimentally with PRRSV; 13 were processed as a whole fly homogenate, while an exterior surface wash and a gut homogenate were collected from the other 13. Infectious PRRSV was recovered from nine of the whole fly homogenates, 12 of the gut homogenates and one of the exterior surface washes. In the second experiment, two of 10 individual houseflies, which had fed on an infected pig, transmitted PRRSV to a susceptible pig in a controlled manual transmission protocol. In the third experiment, single flies or pools of 30 flies were immersed in different concentrations of a PRRSV inoculum, then tested by PCR, virus isolation and bioassay. The virus was detected at a concentration of 10(1) TCID50/ml by PCR, 10(2) TCID50/ml by the bioassay and 10(3) TCID50/ml by virus isolation.