Polychronis Kostoulas

Isolation of Mycobacterium avium subspecies paratuberculosis from non-ruminant wildlife living in the sheds and on the pastures of Greek sheep and goats

This study aimed to: (1) investigate whether non-ruminant wildlife interfacing with dairy sheep and goats of four Greek flocks endemically infected with Mycobacterium avium subspecies paratuberculosis (MAP) harboured MAP and (2) genetically compare the strains isolated from the wildlife to those isolated from the small ruminants of these flocks. We cultured and screened, by polymerase chain reaction (PCR), pooled-tissue samples from 327 wild animals of 11 species for the MAP-specific IS900 insertion sequence. We also cultured faecal samples from 100 sheep or goats from each of the four flocks. MAP was detected in samples from 11 sheep, 12 goats, two mice, two rats, a hare and a fox. Only one rat had histopathological findings. Genetic typing categorized 21 isolates as cattle-type strains and two, from a house mouse and a goat respectively, as sheep-type strains; this is the first report of a rodent harbouring a sheep-type strain. The MAP types that were most frequently isolated amongst the sheep and goats of each flock were also the ones isolated from sympatric rodents; those isolated from the fox and hare also belonged to the predominant ruminant strains.

STARD-BLCM: Standards for the reporting of diagnostic accuracy studies that use Bayesian latent class models

The Standards for the Reporting of Diagnostic Accuracy (STARD) statement, which was recently updated to the STARD2015 statement, was developed to encourage complete and transparent reporting of test accuracy studies. Although STARD principles apply broadly, the checklist is limited to studies designed to evaluate the accuracy of tests when the disease status is determined from a perfect reference procedure or an imperfect one with known measures of test accuracy. However, a reference standard does not always exist, especially in the case of infectious diseases with a long latent period. In such cases, a valid alternative to classical test evaluation involves the use of latent class models that do not require a priori knowledge of disease status. Latent class models have been successfully implemented in a Bayesian framework for over 20 years. The objective of this work was to identify the STARD items that require modification and develop a modified version of STARD for studies that use Bayesian latent class analysis to estimate diagnostic test accuracy in the absence of a reference standard. Examples and elaborations for each of the modified items are provided. The new guidelines, termed STARD-BLCM (Standards for Reporting of Diagnostic accuracy studies that use Bayesian Latent Class Models), will facilitate improved quality of reporting on the design, conduct and results of diagnostic accuracy studies that use Bayesian latent class models.

Effect of Anticoagulant and Storage Conditions on Platelet Size and Clumping in Healthy Dogs

The potential impact of preanalytical factors, such as type of anticoagulant, storage temperature, and time, on the formation of macroplatelets and platelet aggregates (platelet clumping) in dogs is largely elusive. The objective of the current study was to assess the effect of anticoagulant, temperature, and blood storage time in the light microscopy-generated macroplatelet percentages and the frequency of visually inspected platelet aggregates in clinically healthy dogs. Giemsa-stained blood smears from 70 healthy dogs were reviewed after exposure to different anticoagulants (ethylenediamine tetra-acetic acid [EDTA] vs. citrate), temperatures (25°C vs. 4°C), and storage times (up to 24 hr postsampling). The mean percentage of macroplatelets (platelets with diameter or length ≥ μm) was higher (P = 0.0006) when EDTA was used as the anticoagulant. For either anticoagulant, the mean percentage of macroplatelets was higher (P < 0.0001) at 25°C than at 4°C. Platelet clumping was 1.9 times (P < 0.0001) more likely to occur when citrate- rather than EDTA-anticoagulated blood was examined; regardless of the anticoagulant used, clumping occurred 3 times (P < 0.0001) more often when samples were preserved at 4°C than when they were preserved at 25°C. Storage time did not significantly influence the macroplatelet percentages or the frequency of platelet clumping. The results of this study indicate that macroplatelet percentages in the canine blood should be interpreted in relation to anticoagulant- and temperature-specific reference intervals and that future studies are warranted in order to investigate the clinical relevance of this calculation. In addition, the significant association of citrate with the formation of platelet aggregates may preclude its use for platelet enumeration in the dog.

Strain-specific sensitivity estimates of Mycobacterium avium subsp. paratuberculosis culture in Greek sheep and goats.

The requirements for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) may be related to the strain‐type [sheep (S)‐ or cattle (C)‐type] and not to the host. The objective of this paper was to estimate and compare strain‐ and biological sample (faeces or pooled‐tissue) – specific sensitivities (Ses) of two solid culture media, Herrold’s egg yolk medium (HEYM) and Lowenstein–Jensen (LJ) medium, for the isolation of Map from Greek dairy sheep and goats. From 400 faecal samples collected from sub‐clinically infected sheep and goats of four flocks and from 214 pooled‐tissue samples (142 from sheep and 72 from goats) collected, at the abattoir, from >1‐year‐old routinely slaughtered animals, with gross pathology suggestive of paratuberculosis, we isolated 34 Map strains. Of those, by the IS1311 PCR, 18 were categorized into the C‐type and nine into the S‐type; seven were not typed. We used a Bayesian approach to estimate the strain‐specific Ses. SeHEYM‐C‐faecal = 17% (95% credible interval: 7, 40) was higher than SeHEYM‐S‐faecal = 2% (0.3, 11). Also, SeHEYM‐C‐faecal was higher than SeLJ‐C‐faecal = 4% (1, 12). In pooled‐tissue samples, the strain‐specific Ses did not differ between the two media.

Reference values and repeatability of buccal mucosal bleeding time in healthy sedated cats

Bleeding time is a screening test for the evaluation of primary haemostasis. As there is currently limited information on the reference interval (RI) and repeatability of the test in the cat compared with the dog, the purpose of the study was to establish the RI of buccal mucosa bleeding time (BMBT) in healthy cats and to investigate the intra-observer repeatability of the test. Fifty-six cats were prospectively enrolled in the study. The animals were deemed to be healthy based on history, physical examination, complete blood count, serum biochemistry, and negative serological testing for feline leukaemia and immunodeficiency viruses. All cats were sedated with ketamine, dexmedetomidine and morphine, and the BMBT was sequentially measured in the left and right exposed buccal mucosa following a standardised incision made by a commercially available, disposable, bleeding time device. The mean BMBT was 58.6 s and the RIs ranged from 34 to 105 s (Bootstrap estimation). The intra-observer repeatability was up to 87 s (Bland–Altman plot). The results of this study imply that the combination of ketamine, dexmedetomidine and morphine is a safe and useful sedative protocol allowing for the reliable measurement of BMBT in the cat. The RI of feline BMBT may range from 34 to 105 s and the BMBT may differ by up to 87 s for any two consecutive readings for an individual cat.

Effect of toltrazuril treatment in nursing piglets naturally infected with Isospora suis

Isospora suis is an important parasitic infection in intensive pig production worldwide, responsible for significant economic losses. In this study the efficacy of toltrazuril treatment against isosporosis was evaluated, under field conditions and throughout the nursing period, in reducing (i) the mean time to onset of diarrhoea and oocyst excretion, (ii) the odds of diarrhoea and, (iii) the odds and level of oocyst excretion, adjusting for the heterogeneity of I. suis infection among litters and across time. In a 300-sow farrow-to-finish commercial operation, twenty-five litters were randomly allocated to receive toltrazuril (thirteen litters) or no treatment (twelve litters). The course of infection was followed in all piglets by coprological examination from day 6 after farrowing until weaning. Parametric shared frailty models, generalised linear mixed models and a two-part random effects model were used in the analyses. Treated piglets had longer mean time to onset of oocyst excretion, lower odds of excreting oocysts and lower mean amount of excreted oocysts on any day during the nursing period. Diarrhoea was less likely to occur in treated piglets. Variance partition coefficients revealed that almost half of the variation in the odds of oocyst excretion and diarrhoea was ascribed to unknown or unmeasured factors that operate at higher than the piglet levels of aggregation. Thus, beyond toltrazuril treatment, control of isosporosis in commercial pig farms can be improved by identification and quantification of these factors.

Bayesian validation of a serum and milk ELISA for antibodies against Mycobacterium avium subspecies paratuberculosis in Greek dairy goats across lactation

We validated a commercial (Idexx Pourquier, Montpellier, France) serum and milk indirect ELISA that detects antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in Greek dairy goats. Each goat was sampled 4 times, starting from kidding and covering early, mid, and late lactation. A total of 1,268 paired milk (or colostrum) and serum samples were collected during the 7-mo lactation period. Bayesian latent class models, which allow for the continuous interpretation of test results, were used to derive the distribution of the serum and milk ELISA response for healthy and MAP-infected individuals at each lactation stage. Both serum and milk ELISA, in all lactation stages, had average and similar overall discriminatory ability as measured by the area under the curve (AUC). For each test, the smallest overlap between the distribution of the healthy and MAP-infected does was in late lactation. At this stage, the AUC was 0.89 (95% credible interval: 0.70; 0.98) and 0.92 (0.74; 0.99) for the milk and serum ELISA, respectively. Both tests had comparable sensitivities and specificities at the recommended cutoffs across lactation. Lowering the cutoffs led to an increase in sensitivity without serious loss in specificity. In conclusion, the milk ELISA was as accurate as the serum ELISA. Therefore, it could serve as the diagnostic tool of choice, especially during the implementation of MAP control programs that require frequent testing, because milk sampling is a noninvasive, rapid, and easy process.

Bayesian mixture models for partially verified data: Age- and stage-specific discriminatory power of an antibody ELISA for paratuberculosis

Bayesian mixture models can be used to discriminate between the distributions of continuous test responses for different infection stages. These models are particularly useful in case of chronic infections with a long latent period, like Mycobacterium avium subsp. paratuberculosis (MAP) infection, where a perfect reference test does not exist. However, their discriminatory ability diminishes with increasing overlap of the distributions and with increasing number of latent infection stages to be discriminated. We provide a method that uses partially verified data, with known infection status for some individuals, in order to minimize this loss in the discriminatory power. The distribution of the continuous antibody response against MAP has been obtained for healthy, MAP-infected and MAP-infectious cows of different age groups. The overall power of the milk-ELISA to discriminate between healthy and MAP-infected cows was extremely poor but was high between healthy and MAP-infectious. The discriminatory ability increased with increasing age. The great overlap between the distributions of the different infection stages would have hampered our ability to discriminate between the different infection stages. Thus, the proposed method, which uses partially verified data on the true status for some individuals, is an intuitive extension to the standard non-gold standard methods, especially in the case of infections with a long latent period.

A Bayesian Weibull survival model for time to infection data measured with delay

Survival analysis methods can be used to identify factors associated with the time to induction of infection. In the absence of a perfect test, detection of infection is generally delayed and depends on the duration of the latent infection period. We assess, via simulations, the impact of ignoring the delayed detection of infection on estimated survival times and propose a Bayesian Weibull regression model, which adjusts for the delayed detection of infection. The presence of non-differential detection delay seriously biased the baseline hazard and the shape of the hazard function. For differential detection delay, the associated regression coefficients were also biased. The extent of bias largely depended on the longevity of the delay. In all considered simulation scenarios our model led to corrected estimates. We utilized the proposed model in order to assess the age at natural infection with Mycobacterium avium subsp. paratuberculosis (MAP) in Danish dairy cattle from the analysis of available time to milk-seropositivity data that detected infection with delay. The proposed model captured the inverse relationship between the incidence rate of infection and that of seroconversion with time: susceptibility to infection decreases with time (shape parameter under the proposed model was rho=0.56<1), while older animals had a higher probability of sero-converting (rho=2.67>1, under standard Weibull regression). Cows infected earlier in their lives were more likely to subsequently shed detectable levels of MAP and, hence, be a liability to herd-mates. Our approach can be particularly useful in the case of chronic infections with a long latent infection period, which, if ignored, severely affects survival estimates.

The effect of butorphanol on the incidence of dexmedetomidine-induced emesis in cats

OBJECTIVE Τo evaluate the antiemetic effect of butorphanol (BUT) when co-administered with dexmedetomidine (DEX) in cats. STUDY DESIGN Double-blind, randomized controlled cross-over experimental study. ANIMALS Fourteen purpose-bred healthy Domestic Short Hair cats, seven females and seven males, aged median (range) 14-84 (78) months and weighing 1.7-5.5 (4.0) kg. METHODS Each cat received five different treatment protocols intramuscularly (IM): (A) 25 μg kg(-1) DEX; (B) 20 μg kg(-1) DEX and 0.2 mg kg(-1) BUT; (C) 20 μg kg(-1) DEX and 0.1 mg kg(-1) BUT; (D) 25 μg kg(-1) DEX and 0.2 mg kg(-1) BUT; and (E) 20 μg kg(-1) DEX. Episodes of emesis, incidence and severity of nausea, and time to lateral recumbency were recorded for a period of 8 minutes after treatment administration, and the sedation was scored at the end of this period. The Friedman test and the Cochrans Q-test were used to analyse the data. Significance was evaluated at the 5% level. RESULTS The proportion of cats that vomited was significantly lower with the treatment protocols that included BUT (B, C and D) compared with the protocols that included only DEX (A and E). The proportion of cats that had nausea was significantly higher with the protocols that included only DEX (A and E) compared with protocols B and D. Time to lateral recumbency (p=0.09) and sedation score (p=0.07) was not statistically different between the treatment protocols. CONCLUSIONS AND CLINICAL RELEVANCE Butorphanol can be used to prevent emesis and reduce the incidence and the severity of nausea caused by DEX in cats. It seems that the combination of BUT and DEX is very useful not only when emesis could result in serious complications, but also to provide comfort and well-being in cats sedated for minor procedures.