Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A (ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive (10 rDNA or ompA copies/microL DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism (RFLP) analysis using five Cp. psittaci field isolates (99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.     

Insulin resistance and abomasal motility disorders in cows detected by use of abomasoduodenal electromyography after surgical correction of left displaced abomasum

OBJECTIVE: To determine the correlation between insulin concentrations and myoelectrical activity of the abomasum in cows with a left displaced abomasum (LDA). ANIMALS: 14 dairy cows with an LDA at the onset of lactation. PROCEDURE: During surgical correction of an LDA, 3 pairs of electrodes were placed in the smooth muscle of the gastrointestinal tract (abomasal body, pars pylorica, and duodenum) of each cow. Electromyographic recordings were obtained once per day for 7 days. Samples were collected and tested to determine concentrations of insulin, glucagon, cortisol, glucose, beta-hydroxybutyrate, and nonesterified fatty acids. RESULTS: All 14 cattle had high glucose and insulin concentrations at the time of admission, independent of ketosis. Concentrations of glucose and insulin decreased slowly after surgical treatment and were associated with a progressive increase in abomasoduodenal myoelectric activity. The 14 cows were allocated into 2 groups (suspected insulin-resistant cattle, n = 7; suspected non-insulin-resistant cattle, 7) on the basis of persistent hyperinsulinemia during the postoperative period. Seven days after surgery, the abomasoduodenal myoelectric patterns were still significantly lower for the insulin-resistant cows, compared with patterns for the non-insulin-resistant cows. CONCLUSIONS AND CLINICAL RELEVANCE: Insulin resistance appears to be common in cows with an LDA. Analysis of results of this study reveals that abomasal atony in cows with an LDA depends on persistence of high serum concentrations of insulin. Results of this study could provide an explanation for a pathogenetic factor of LDAs and the frequent relapses of cattle affected by this condition.     

Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol

A real-time PCR assay was exploited for monitoring the Leishmania DNA load in different tissues from 18 naturally-infected dogs before and after treatment with a combination of meglumine antimoniate (100mg/kg/day, subcutaneously) and allopurinol (10mg/kg/day, orally) for 30 days. After the combined therapy, allopurinol was continued at the same dose until the end of the observation period. Whole blood samples, lymph node aspirates, and skin biopsies were collected at the time of diagnosis, 1 month after starting therapy, and every 3 months for 2 years. In six dogs parasite load assessments continued every 6 months for a further 3 years. At each assessment, the dogs were examined for signs of disease and a clinical score was recorded. At diagnosis, the highest Leishmania DNA load was detected in lymph node aspirates. From 1-6 months post-therapy a general improvement in clinical conditions was recorded in all dogs, which correlated with a decrease in the parasite DNA load in all tested tissues, even though it was less pronounced in lymph node aspirates. In the period from 9-24 months post-therapy, a re-increase in parasite load was observed in the tissues of some dogs, concomitant with a disease relapse. The results show that the combined therapy with meglumine antimoniate and allopurinol promoted a clinical improvement which was accompanied by a reduction in the parasitic load in the blood, skin and lymph nodes but, even after long period of allopurinol administration alone, Leishmania may persist in dog tissues.     

Epidemiological and genetic characterization of Toxoplasma gondii in urban pigeons from the area of Lisbon (Portugal)

Circumstantial evidence suggests that birds may be a good indicator of soil contamination, since they feed from the ground. Therefore a study was conducted to assess the prevalence of Toxoplasma gondii in urban pigeons (Columba livia) from different areas of the city of Lisbon and to genetically characterize T. gondii strains isolated from infected animals, in order to assess circulating genotypes. Sera from 695 pigeons captured at 33 different localities of the city of Lisbon were assayed by the direct agglutination test (DAT) showing a 4.6% prevalence. One third of flocks sampled revealed to be infected with T. gondii, with prevalence ranging between 5% and 62.5%. T. gondii DNA could be characterized directly from brain tissue from 12 of 23 seropositive pigeons. Microsatellite typing revealed that 9 strains belonged to type II, 2 were type III and one was type I. No recombinant or atypical genotypes were found. Attempts to recover viable T. gondii strains through bioassay in mice resulted in the isolation of 9 strains.     

The effect of high pre‐slaughter environmental temperature on meat quality traits of Italian autochthonous pig Suino Nero Lucano

The aim of the present study was to evaluate the effect of high pre‐slaughter environmental temperature on meat (Longissimus lumborum muscle) quality traits of Italian autochthonous pigs Suino Nero Lucano (SNL). High pre‐slaughter environmental temperature influenced the meat quality of autochthonous pigs. In particular, the meat from SNL pigs exposed to heat stress (H) showed significantly lower pH values at 24 and 48 hr post mortem, haem iron content and redness (a*) value, and a significantly higher shear force, drip loss and lightness (L*) values, compared to meat from SNL pigs reared under comfortable environmental temperature (C). Moreover, H meat showed a significant reduction of vitamin A, vitamin E, creatine and carnosine content, compared to C meat. Despite this, no pale‐soft‐exudative pork was found in our study, and no significant difference was detected between two studied groups for intramuscular fat and protein content of meat. In addition, despite the meat from SNL pigs exposed to heat stress has suffered a qualitative decrease, it showed a good endogenous antioxidants content.     

Late onset and rapid progression of cerebellar abiotrophy in a domestic shorthair cat

A two-year-old male domestic shorthair cat was seen because of sudden onset of incoordination and tremors that had rapidly worsened over five days. Neurological examination revealed severe cerebellar ataxia, intention tremors and bilaterally decreased menace response. Blood work evaluation included a complete cell blood count (CBC), serum biochemistry profile, urinalysis, faecal flotation, cerebrospinal fluid (CSF) analysis and retroviral testing. Except for testing positive for feline immunodeficiency virus (FIV) antibodies, all other results were within the normal range. The patient was euthanased two days later because of progression of clinical signs, and a necropsy was performed. Histologically, lesions were limited to the cerebellum and consistent with cerebellar abiotrophy. No secondary diseases that could explain the rapid development of clinical signs were found. It was considered unlikely that cerebellar degeneration was related to FIV positivity, as virus invasion of the central nervous system (CNS) is mainly limited to the cerebral cortex. This case report is the first to describe late onset and rapid progression cerebellar abiotrophy in a cat.     

Seed transmission of Plectosphaerella cucumerina, causal agent of leaf spot of Diplotaxis tenuifolia in Italy

Plectosphaerella cucumerina has been described recently as the causal agent of a leaf spot on wild rocket (Diplotaxis tenuifolia). Eight seed samples of wild rocket obtained from commercial seed lots used for sowing by farms severely affected by P. cucumerina, were assayed for the presence of the pathogen. Isolations were carried out on subsamples of seeds (400) unwashed or disinfected in 1% sodium hypochloride. The pathogenicity of the isolates of P. cucumerina obtained was tested in two trials carried out on wild rocket; four out of eight samples of rocket seeds were contaminated by P. cucumerina. Eleven isolates of P. cucumerina were obtained from 7,200 not disinfected seeds tested, while none was isolated from an equal number of disinfected seeds. All isolates were pathogenic on wild rocket. The results obtained indicate that rocket seeds are a potential source of inoculum for P. cucumerina. The possibility of isolating the pathogen from seeds, albeit from a low percentage of them, supports the hypothesis that the rapid spread of this new disease of rocket recently observed in Italy is due to the use of infected propagation material. Measures for prevention and control of the disease are discussed.     

Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs

The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.     

Evaluation of assay procedures for prediction of passive transfer status in lambs

OBJECTIVE: To compare 4 assay procedures for prediction of passive transfer status in lambs. ANIMALS: Thirty-one 1-day-old Sardinian lambs. PROCEDURE: Serum IgG concentration was determined by use of single radial immunodiffusion. The following were determined: serum total protein concentration as measured by refractometry (ie, refractometry serum total protein concentration), serum total protein concentration as determined by the biuret method (ie, biuret method serum total protein concentration), serum gamma-globulin concentration as determined by serum protein electrophoresis, and serum gamma-glutamyltransferase (GGT) activity as measured by spectrophotometry. Accuracy of these assays for estimation of serum IgG concentration in 1-day-old lambs was established by use of linear regression analysis. RESULTS: Refractometry serum total protein concentration, biuret method serum total protein concentration, and serum gamma-globulin concentration were closely and linearly correlated with serum IgG concentration. The natural logarithm (ln) of serum GGT activity was closely and linearly correlated with serum IgG concentration (ln). Refractometry serum total protein concentration, biuret method serum total protein concentration, and gamma-globulin concentration accounted for approximately 85%, 91%, and 95% of the variation in serum IgG concentration, respectively. Serum GGT activity (ln) accounted for approximately 92% of the variation in serum IgG concentration (ln). CONCLUSIONS AND CLINICAL RELEVANCE: For prediction of passive transfer status in 1-day-old lambs, serum GGT activity or biuret method serum total protein concentration determination will allow for passive transfer monitoring program development. Immediate refractometry serum total protein concentration determination is beneficial in making timely management and treatment decisions. Serum gamma-globulin concentration determination can be used as a confirmatory test.