Hemorrhagic pneumonia in neonatal minks in Greece concomitant with Leismania infantum detection.

In the present study a severe outbreak of hemorrhagic pneumonia (HP) in neonatal minks concomitant with Leismania infantum (L. infantum) detection is reported. The outbreak took place on a Greek mink farm and affected 1,362 mink kits, with 524 dying. Macroscopic lesions of 14 necropsied affected kits were confined to the respiratory system with dark red, consolidated lung lobes and to the small intestine with severe, acute, hemorrhagic and necrotic enteritis. Microscopic examination of lung sections revealed severe hemorrhagic pyogranulomatous pneumonia. Bacteria were obtained in pure culture from the lungs of all necropsied animals and were confirmed as Pseudomonas aeruginosa (P. aeruginosa). Three out of 14 (21.4%) animals were positive for the presence of L. infantum DNA. The outbreak was attributed to the infection of minks with P. aeruginosa, possibly as a consequence of being immuno-suppressed by L. infantum. Further research is necessary, especially on the pathogenesis of P. aeruginosa/L. infantum co-infection and the implications of this interaction on HP disease outcome.

Rabies outbreak in Greece during 2012-2014: use of Geographical Information System for analysis, risk assessment and control.

The objectives of this work were (i) geographical analysis of the 2012-2014 outbreak of rabies in Greece using GIS and (ii) comparative analysis of animal cases with data of potential human exposure to rabies together with environmental data, in order to provide information for risk assessment, effective monitoring and control. Most animal cases (40/48) involved red foxes, while domestic animals were also diagnosed with rabies. Overall, 80% of the cases were diagnosed in central northern Greece; 75% of the cases were diagnosed in low altitudes (<343·5 m), within a distance of 1 km from human settlements. Median distance from livestock farms was 201·25 m. Most people potentially exposed to rabies (889/1060) presented with dog bite injuries. Maximum entropy analysis revealed that distance from farms contributed the highest percentage in defining environmental niche profiles for rabid foxes. Oral vaccination programmes were implemented in 24 administrative units of the country during 2013 and 2014, covering a total surface area of ~60 000 km2. Rabies re-occurrence in Greece emphasizes the need for ongoing surveillance in cross-border areas and in areas with intense human activity.

First evidence of Leishmania infection in European brown hare (Lepus europaeus) in Greece: GIS analysis and phylogenetic position within the Leishmania spp.

Although the existence of a sylvatic transmission cycle of Leishmania spp., independent from the domestic cycle, has been proposed, data are scarce on Leishmania infection in wild mammals in Greece. In this study, we aimed to investigate the presence of Leishmania infection in the European brown hare in Greece, to infer the phylogenetic position of the Leishmania parasites detected in hares in Greece, and to identify any possible correlation between Leishmania infection in hares with environmental parameters, using the geographical information system (GIS). Spleen samples from 166 hares were tested by internal transcribed spacer-1 (ITS-1)-nested PCR for the detection of Leishmania DNA. Phylogenetic analysis was performed on Leishmania sequences from hares in Greece in conjunction with Leishmania sequences from dogs in Greece and 46 Leishmania sequences retrieved from GenBank. The Leishmania DNA prevalence in hares was found to be 23.49 % (95 % confidence interval (CI) 17.27-30.69). The phylogenetic analysis confirmed that the Leishmania sequences from hares in Greece belong in the Leishmania donovani complex. The widespread Leishmania infection in hares should be taken into consideration because under specific circumstances, this species can act as a reservoir host. This study suggests that the role of wild animals, including hares, in the epidemiology of Leishmania spp. in Greece deserves further elucidation.

Dissemination of intestinal pathogens between lambs and puppies in sheep farms.

Objectives of the present work were (i) to confirm pathogens implicated in cases of diarrhoea in newborn and young lambs in sheep farms in Greece and (ii) to investigate a possible relation in dissemination of pathogens between lambs and dogs present in the farm. Work was carried out in 22 sheep farms, with (i) flock size over 150 animals, (ii) presence of clinical signs of diarrhoea in lambs in the flock and (iii) close and continuous contact and movement of shepherd dogs within the animal shed of each farm. Faecal sample collection from lambs was performed within 48 h of onset of clinical signs and prior to administration of any antimicrobial or antiparasitic medication to lambs. Faecal samples were also collected from puppies in the farm. In total, samples were collected from 126 lambs and 58 puppies. Samples were processed by using established techniques for isolation of bacteria, detection of viruses and observation of protozoan oocycts. Escherichia coli isolates obtained during the study, were tested for antimicrobial resistance against a variety of antimicrobial agents. In total, 236 bacterial isolates were recovered from faecal samples of lambs and 165 isolates from faecal samples of puppies. E. coli was the most frequently isolated microorganism: 104 isolates from lambs and 109 isolates from puppies were recovered. Other bacteria isolated were Enterobacter spp., Proteus spp., Klebsiella spp., (lambs and puppies), Clostridium perfringens, Citrobacter freundi, Salmonella enterica subsp. diarizonae (only lambs) and Streptococcus spp. (only puppies). Group A Rotavirus was detected in samples from lambs (2.5%) and Parvovirus in samples from puppies (5%). Cryptosporidium spp. oocysts were observed in samples from lambs and puppies. This is the first report of isolation of S. enterica subsp. diarizonae and of detection of Rotavirus from lambs in Greece. Rates of E. coli isolates from puppies resistant to antimicrobial agents were, in general, smaller than respective rates in isolates from lambs. Two pairs of isolates from the same farm (one from a lamb and one from a puppy) with identical patterns of resistance to antimicrobial agents were detected, which provides some evidence in support of a hypothesis that members of each pair might possibly have been spread from one animal species to the other.

Experiences from the 2014 outbreak of bluetongue in Greece.

Objective of this paper was to review relevant work and to present a general account of the bluetongue outbreak, which occurred in Greece in 2014. In total, 2895 outbreaks of the disease have been reported by the veterinary authorities of Greece; sheep, goats and cattle were affected with officially reported morbidity rates of 11.0%, 2.0% and 3.5%, respectively. No vaccinations were allowed and conservative measures were implemented to attempt to limit the disease, which at the end had expanded throughout the country. In field investigations, a significantly higher bluetongue morbidity rate (27.5%) in sheep has been reported. During that work, clinical anaemia was encountered, which was characterised as macrocytic, hypochromic, regenerative and non-haemolytic. Other investigations, which are reviewed in this paper, have described an outbreak of Citrobacter freundii-associated enteritis in newborn kids, offspring of goats subclinically infected with Bluetongue virus, increased rate of early embryonic deaths, reduced conception rates, increased incidence risk of mastitis and reduced milk yield in herds of subclinically-infected cattle and detection of the virus from hunter-harvested tissue samples of roe-deer. In 2015, vaccines against the disease have been licenced; vaccinations started in May 2015. Then, in 2015, only one outbreak of the disease was confirmed, which could have been the result of a combination of reasons acting concurrently to prevent further cases.

Epidemiology of human leishmaniasis in Greece, 1981-2011.

Leishmaniasis is endemic and mandatorily notifiable in Greece. Epidemiological surveillance data for leishmaniasis in Greece between 1981 and 2011 are presented. In 1998, the notification system began distinguishing between visceral and cutaneous leishmaniasis. The mean annual incidence of reported leishmaniasis cases between 1998 and 2011 was 0.36 per 100,000 population. Of a total 563 leishmaniasis cases reported after 1998, 523 (93%) were visceral leishmaniasis cases. Incidence of reported visceral leishmaniasis cases fluctuated during this period, generally decreasing after 2007, with a small re-increase in 2011. The mean annual incidence rate of reported visceral leishmaniasis cases was significantly higher in less than four year-olds (p <0.001). Leishmaniasis cases occurred both in the country mainland and islands. Between 1998 and 2011, Attica concentrated almost half of the reported visceral leishmaniasis cases, with incidence rates in western Attica and western Athens above 12.00 per 100,000 population. Compared to visceral leishmaniasis, cutaneous leishmaniasis had a rather sporadic distribution, with many prefectures appearing free of cases. From 2004, the notification also included risk factors and of 287 cases with known immune status, 44 (15%) were immunocompromised. Moreover having a dog at home was reported by 209 of 312 leishmaniasis cases (67%), whereas 229 of 307 cases (75%) reported the presence of stray dogs near their residence. Linking clinical surveillance data with laboratory data and improving collaboration with the veterinary public health sector are some of the future challenges for leishmaniasis surveillance in Greece.

Re-emergence of animal rabies in northern Greece and subsequent human exposure, October 2012 - March 2013.

Greece has been rabies-free since 1987 with no human cases since 1970. During 2012 to 2013, rabies has re-emerged in wild and domestic animals in northern Greece. By end March 2013, rabies was diagnosed in 17 animals including 14 red foxes, two shepherd dogs and one cat; 104 subsequent human exposures required post-exposure prophylaxis according to the World Health Organization criteria. Human exposures occurred within 50 km radius of a confirmed rabies case in a wild or domestic animal, and most frequently stray dogs were involved.

Molecular detection and phylogenetic analysis of West Nile virus lineage 2 in sedentary wild birds (Eurasian magpie), Greece, 2010.

A West Nile virus (WNV) lineage 2 strain was molecularly identified and characterised in a Eurasian magpie hunted in Greece in 2010, during a WNV outbreak in humans. Phylogenetic analysis revealed the highest sequence similarity (>99%) with other WNV lineage 2 strains derived from birds of prey in Austria and Hungary (2004­2009). This first molecular detection of WNV in sedentary wild birds in Greece, which are possible reservoirs of the virus, is a public health concern.

Brucellosis underreporting in Greece: assessment based on aggregated laborato-ry data of culture-confirmed cases from public hospitals.

BACKGROUND: Brucella spp. isolation is one of the mainstays of brucellosis diagnosis. Simultaneously, the true brucellosis disease rate may be underrepresented in notification systems. This study aims at assessing the nosocomial capacity for Brucella spp. isolation and the underreporting rate of brucellosis cases in Greece. METHODS: Data for Brucella spp. culture capacity and the number of isolations were collected annually from public hospitals nationwide, during 2015-2018. The number of unreported cases was estimated after subtracting the National Mandatory Notification System cases from the survey-captured isolations, matched by hospital and year. RESULTS: Feedback was provided by 112 public hospitals (response rate: 97.4 %). Brucella spp. isolation capacity was completely absent in 27.7 % of hospitals; during the four years of the study, 11.3 %, 13.9 %, 20.0 %, and 25.2 % of the hospitals had isolation competence for one, two, three, or four years, respectively. Underreporting assessment was possible in hospitals that declared at least one Brucella spp. isolation (n =35) and unreported cases were identified in 19 (54 %). Α mean underreporting of 28.9 % of total cases was estimated for the whole period of the study ranging annually from 24.1 % to 35.0 %. The number of unreported cases per hospital ranged from one to 12 per year (median: 2, IQR: 5). CONCLUSIONS: Interventions for improving diagnosis and reporting of the disease are recommended. Assessment of brucellosis underreporting by comparing raw numerical data of survey-captured isolations and officially notified cases lacks the case by case specificity, however, keeping required data to a minimum achieves high feedback rate from hospitals and provides a tentative estimation of the notification deficit. HIPPOKRATIA 2019, 23(3): 106-110.

Study of potential environmental factors predisposing ewes to subclinical mastitis in Greece.

Objectives of the work presented herewith were to investigate association of prevalence of subclinical mastitis with environmental (climatic and topographic) factors and to identify factors potentially predisposing ewes to the disease. Milk samples were collected from 2198 sheep in 111 farms, in all 13 administrative regions of Greece, for bacteriological and cytological examination. Data on farm location were collected in the field using hand-held Global Positioning System Garmin units. The geo-references were resolved to specific farm level. Prevalence of subclinical mastitis was 0.260. Main aetiological agents were staphylococci (Staphylococcus aureus and coagulase-negative species), which accounted for 0.699 of all isolates recovered. In a multivariable mixed-effects analysis, the two environmental variables found to be associated with increased prevalence of subclinical mastitis were the minimum temperature of coldest month (coefficient: -0.084 ± 0.033, P = 0.014) and the mean temperature for 30 days prior to sampling date (coefficient: 0.031±0.014, P = 0.029).

Use of geographical information system and ecological niche modelling for predicting potential space distribution of subclinical mastitis in ewes.

Objective was to recognise areas potentially of high risk for increased frequency of subclinical mastitis in ewes. Milk samples were collected, for bacteriological and cytological examination, from 2198 clinically healthy ewes in 111 farms in all administrative regions of Greece. Data on farms were located in the field using hand-held Global Positioning System Garmin units. Collected data were analysed by an Ecological Niche Model under the framework of a geographic information system. Two separate analyses were performed: one for subclinical mastitis independently of causal agent (prevalence in population sampled: 0.260) and one for subclinical mastitis caused specifically by slime-producing staphylococci (prevalence in population sampled: 0.153). A model was constructed in which sheep farms were divided into two clusters, according to prevalence of subclinical mastitis: farms in the upper three quartiles of prevalence were used as occurrence points for the Ecological niche modelling procedure ('infected farms'); farms in the lower quartile of prevalence within each category were (pseudo)negative points. Significant differences in environmental parametres prevailing in locations of farms into the study, were identified for up to 13 parametres between locations of farms according to management system applied in farms. When farms in each management system were considered separately, differences became evident between farms in each management system, as well as between the two infections. The factor with the highest relative contribution in the analyses was the distance from other sheep farms; other factors also of importance in the predictive models were the altitude, the maximum temperature of warmest month and the total precipitation of driest month. Verification of the model revealed that ≥ 0.760 of infected farms' were located in areas predicted as high risk for prevalence of subclinical mastitis or slime staphylococcal subclinical mastitis. The paper describes for the first time potential association of mastitis with environmental factors and presents predictive models for mastitis in ewes taking into account environmental parametres.

Atypical PrPsc distribution in goats naturally affected with scrapie.

The brain and spinal cord of 48 goats from two Greek herds in which scrapie had been reported were examined. All animals were symptomless at the time of euthanasia. Notably, no lesions were observed either at the level of the obex or at other regions of the brain and spinal cord. Immunohistochemical examination revealed PrPsc labelling of the linear and fine punctuate types, mainly in the cerebral cortices, of 36 goats. Twenty-seven of them were negative by ELISA (designed to detect proteinase-resistant PrP) at the level of the obex but positive in a pooled brain sample, and the majority carried PrP genotypes associated with scrapie susceptibility. Surprisingly, in 16 of the 27 animals, PrPsc deposits were detected only in the rostral parts of the brain. In addition, nine animals which were ELISA-positive at the level of the obex exhibited positive immunoreactivity, but not in the dorsal vagal nucleus. The findings indicate that this unusual scrapie type may have been underdiagnosed previously and may be of importance in scrapie surveillance programmes.

A clinical case of chicken infectious anemia disease and virus DNA detection in naturally infected broilers in Greece.

In this study, chicken infectious anemia virus (CIAV) DNA was detected from 12-day-old broilers. Clinical history showed that the clinical features were diarrhea, blue wing disease, depression, and death. Necropsy findings were pale liver, severe atrophy of bursa of Fabricius and thymus, and discoloration of the bone marrow as well as hemorrhages subcutaneously and a few in skeletal muscles. The majority of the necropsied broilers had developed gangrenous dermatitis. Histopathology showed hypoplasia of bone marrow and depletion of lymphocytes in spleen, bursa, and subcapsular thymic cortex. Karyorrhexis of lymphocytes was scattered in the thymic cortex and most pronounced in the bursal follicles. Eosinophilic intranuclear inclusion bodies were mainly located in lymphocytes of thymus, with a few in hemopoietic cells of bone marrow. CIAV DNA was detected by polymerase chain reaction from bursa, thymus, and bone marrow. A virus strain was detected and genetically characterized in 639 base pairs of VP1 gene. Phylogenetic analysis revealed that the Greek isolate was clustered together with isolates from Alabama, China, Slovenia, and Bangladesh.

Genetic variability of encephalomyocarditis virus (EMCV) isolates.

In order to evaluate the variability of encephalomyocarditis virus (EMCV), field isolates originating from different European regions and inducing different clinical pictures in pigs have been molecularly characterised. The regions targeted were the poly(C) tract, a part of the 5'-UTR (360 nucleotides), the Leader gene (201 nucleotides), the complete capsid coding region (2502 nucleotides), the 2A gene (403 nucleotides), the end of the 3D polymerase gene (305 nucleotides) and the 3'-UTR (123 nucleotides). Analyses have also been performed on a virulent field isolate, which had been subjected to serial passages in vivo and in vitro resulting, in the case of the in vitro passaged virus, in attenuation, as demonstrated by animal experiments. The present study shows that different clinical pictures, such as acute fatal myocarditis or reproductive failure, may not only be caused by EMCV isolates which are genetically diverse but also by the same isolate. Thus no correlation could be demonstrated between genotype and clinical disease. However, the European isolate which showed the highest genetic divergence also gave rise to a more complex clinical picture. Despite EMCV having been isolated from cases of acute fatal myocarditis in pigs in certain areas of the world for many years, clinical disease, including a variety of clinical pictures and pathogenicity, has only been recognised in Europe since 1986 and thus it can be considered an emerging disease in this region. These findings, associated with the reported phenotype changes of the virus under environmental changes (passages), along with its wide distribution among vertebrate species (including higher primates), shows the validity of considering EMCV as a potential pathogen for recipients in xenotransplantation.

Application of a semi-dependent latent model in the Bayesian estimation of the sensitivity and specificity of two faecal culture methods for diagnosis of paratuberculosis in sub-clinically infected Greek dairy sheep and goats.

In this study, we compared the frequency of isolation of Mycobacterium avium subsp. paratuberculosis (MAP) from faecal samples grown on Herrold's egg-yolk medium (HEYM) or on Lowenstein-Jensen (LJ) medium and estimated the sensitivity (Se) and specificity (Sp) of the methods separately in sub-clinically infected Greek dairy sheep and goats, using latent-class models and Bayesian estimation procedures. Faecal and blood samples were collected from 400 animals > or =1 year old in April-May 2002. The HEYM supported growth of MAP better than the LJ method and their agreement was very poor (weighted kappa=0.062 (95% CI: -0.098, 0.222)). There was no evidence of dependence between the Ses whereas the Sps were positively correlated. Thus, a semi-dependent model that assumed independence of Ses and accounted for the dependence of Sps was adopted. Under this model, the parallel interpretation of the results of the two methods gave median estimates and 95% credible intervals (CrIs) for Se(par), Sp(par) of 15% (CrIs: 3, 45%), 96% (92, 98%) in sheep and 16% (6, 36%) and 97% (94, 99%) in goats.

Bayesian estimation of sensitivity and specificity of serum ELISA and faecal culture for diagnosis of paratuberculosis in Greek dairy sheep and goats.

Latent class models were used to estimate the sensitivity (Se) and the specificity (Sp) of a serum ELISA and a faecal culture (FC) method for the diagnosis of paratuberculosis separately, in sheep and goats. The estimates were obtained by a Bayesian method. Possible dependence of diagnostic errors was investigated by comparing models where independence was assumed to models allowing for conditional dependence given the true disease status. ROC analysis for the serum ELISA was also performed and optimized cut-off values based on the misclassification cost term were determined. No evidence of conditional dependence was found. Assuming independence, posterior medians and 95% credible intervals for the Se(ELISA), Sp(ELISA), Se(FC) and Sp(FC), were 63% (42, 93%), 95% (90, 98%), 8% (2, 17%) and 98% (95, 100%) in goats and 37% (10, 80%), 97% (93, 99%), 16% (2, 48%) and 97% (95, 99%) in sheep. AUC was calculated 0.702 for sheep and 0.847 for goats. For the serum ELISA, there is need of species- and purpose-specific cut-off selection. For instance, with 20% prevalence situation and assuming equal and five-fold cost of a false negative to a false positive test result, the optimal cut-off is 0.3 and 0.05 in sheep, respectively, while it is 0.6 and 0.1 in goats, respectively. Serum ELISA performed better in goats than in sheep. Lowering the cut-off, in relation to the one recommended by the manufacturer, improved Se(ELISA) without seriously compromising Sp(ELISA), in either species.

The association of sub-clinical paratuberculosis with the fertility of Greek dairy ewes and goats varies with parity.

Our cross-sectional study investigated the association of sub-clinical Mycobacterium avium subsp. paratuberculosis (MAP) infection with failing to produce a live offspring the season of lambing/kidding (November 2001 to January 2002) before testing (in April-May 2002), in four dairy-sheep and/or goat flocks in Greece (369 animals >or=1.5-year-old). From each selected animal 10 ml of blood and 10 g of feces from the rectum were obtained. The harvested sera were tested for antibodies to MAP with a commercial ELISA test kit; the feces were cultured on Herrold's egg-yolk medium supplemented with mycobactin J and antibiotics. An animal was considered sub-clinically infected when found either seropositive or culture positive. The true prevalence of sub-clinically infected animals, adjusted for the sensitivity and specificity of the parallel testing, was 14% (0.1-28%) and 35.9% (9.2-62.7%) in sheep and goats, respectively. The association of fertility of sheep and goats with sub-clinical paratuberculosis was investigated in random-effects logistic models. Sub-clinically infected animals (compared to uninfected) had OR for live offspring the previous year of 5.4 for parity <4, OR=0.05 for parity >6, and a non-significant OR for the middle parity category.

Pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in mice.

Mice (n=20) aged 8 weeks were infected, either by oronasal inoculation or by contact, with one of two different myocardial strains of encephalomyocarditis virus (EMCV), namely, the Greek strain 424/90 and the Belgian strain B279/95. The animals were killed at 18-59 days post-infection (dpi), except for two mice that died at 6 and 32 dpi, and samples of brain, heart, pancreas, kidney, Peyer's patches, spleen, lung and thymus were processed for virological, histopathological and immunohistochemical examination. Apart from the two deaths, the experimental infection was inapparent, but virus was invariably recovered from faeces and several organs. The main histopathological lesions were focal interstitial pancreatitis, depletion of thymus and Peyer's patches, and interstitial pneumonia. Additionally, in the two mice that died, multifocal interstitial myocarditis was observed. EMCV antigen was detected in the cytoplasm of pancreatic acinar cells and in macrophages of the lung and the thymus. Antigen was also detected in the cytoplasm of cardiac muscle cells from three animals, including the two that died. The results support the role of mice, in addition to rats, as reservoir hosts in the epidemiology of EMCV infections on pig farms.

Pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats.

Rats (n=40) aged 8 weeks were infected, either by oronasal inoculation or by contact, with one of two different myocardial strains of encephalomyocarditis virus (EMCV), namely, the Greek strain 424/90 and the Belgian strain B279/95. The animals were killed at 11-62 days post-infection (dpi) and samples of brain, heart, pancreas, kidney, Peyer's patches, spleen, lung and thymus were processed for virological, histopathological and immunohistochemical evaluation. This experimental infection was inapparent, but virus was isolated from faeces and several organs of all animals. The main histopathological changes were focal interstitial pancreatitis, degeneration and necrosis of pancreatic acinar cells, depletion of thymus and Peyer's patches, and interstitial pneumonia. EMCV antigen was detected in the cytoplasm of cardiac muscle cells, pancreatic acinar cells and hepatic epithelial cells, and in macrophages of the spleen, lung and thymus. In the heart (the target organ of EMCV in pigs), the presence of EMCV in cardiac muscle cells without lesions lends support to the hypothesis that the rat is a natural reservoir host species of EMCV. The persistence of virus in the macrophages of the thymus may represent a mechanism of perpetuation and reactivation, under immunosuppressive conditions, of the infection.

Histopathological and immunohistochemical features of natural goat scrapie.

Histopathological and immunohistochemical examinations were performed on the brain and spinal cord of 37 goats from two Greek herds in which scrapie had been reported. Of the 37 animals, 18 were from a herd consisting only of goats and 19 were from a herd of goats mixed with sheep. The goats studied were grouped on the basis of the presence or absence of clinical signs. Distinctive lesions and PrP(sc) (PrP, prion protein) deposition were found in the central nervous system (CNS) of eight clinically affected animals and six symptomless animals. The lesion profile and PrP(sc) distribution varied both between and within groups, variation being particularly pronounced in the symptomless goats. The results concerning the latter group suggested a poor correlation between the intensity of lesions, the amount of PrP(sc) in the CNS, and the manifestation of clinical signs. Immunohistochemical examination revealed 10 different PrP(sc) types, four of which are reported for the first time in goats. All scrapie-affected animals carried the VV(21)II(142)HH(143)RR(154) genotype, with the exception of two goats that carried the HR(143) dimorphism and had detectable PrP(sc) deposits. The results suggest that the histopathological and immunohistochemical profile of the natural disease in goats is influenced by the PrP genotype and age of the animals but may not be directly associated with the presence or otherwise of clinical signs.