Reemergence of Dourine in Italy: Clinical Cases in Some Positive Horses

In 2011, Trypanosoma equiperdum reemerged in Italy, almost 10 years after its last appearance. A total of eight infected horses have been observed to date. Six horses were affected by natural outbreaks of the disease, whereas two were infected experimentally. The aim of this study was to offer a recent perspective on clinical cases of dourine in Europe. Investigation of the clinical aspects confirmed the three stages reported in the literature: stage 1 (genital lesions), stage 2 (cutaneous signs), and stage 3 (nervous signs). The most common signs in the horses under study were notable weight loss, edematous skin eruptions and oedemas of the abdomen, mammary glands and hind legs. Three animals presented neurological signs (lip ptosis of lower lip and ataxia). Infections were paucisymptomatic or asymptomatic in some animals. Hyperthermia was not reported in infected animals and considerable anemia was observed. High antibody titers did not always correspond to clinical signs. Positive polymerase chain reaction test results of blood or tissue (skin, eye swab) often correspond to an advanced stage of the disease. Dourine is a variable disease; owing to its low prevalence and chronic manifestation, it can be difficult to make a quick diagnosis when facing a Dourine-positive horse.     

First recovery of A/equine/Fontainebleau/1/79 influenza viruses in Italy

In Italy epizootics of equine influenza often occur, but no virus isolation has been reported since 1971. This paper describes the antigenic and biochemical characterization of two equine influenza viruses isolated in Italy from 1985 to 1989. The virus isolates were shown to differ antigenically from earlier strains of the same subtype, A/equine/Miami/1/63 (H3N8). Monoclonal antibody analysis showed that the haemagglutinins of these strains were serologically indistinguishable from A/equine/Fontainebleau/1/79, a variant of A/equine/Miami, never isolated in Italy before. One of the two virus isolates was obtained from a horse immunized with a bivalent inactivated influenza vaccine, not containing A/equine/Fontainebleau/79 antigens.

The vaccine failure underlines the importance of antigenic relatedness between currently circulating viruses and vaccine strains. Therefore, to improve the protection afforded by equine immunization, the vaccine composition should be decided according to the results of a virological surveillance activity, systematically conducted among horses.     

Equine disease surveillance, April to June 2010

Recent outbreaks of equine infectious anaemia and equine viral arteritis in the UK. Update on the equine infectious anaemia situation in Europe. West Nile virus reported in several Mediterranean countries. Current and future approaches to equine viral arteritis control in the UK. These are among matters discussed in the quarterly equine disease surveillance report for April to June 2010, prepared by Defra, the Animal Health Trust and the British Equine Veterinary Association.     

Phylogenetic and Molecular Characterization of the Equine Influenza Virus A (H3N8) Causing the 1997 and 2004 Outbreaks in Morocco

Reported here are the results of antigenic and genetic characterization of equine influenza strains causing local outbreaks reported in Morocco, respectively, in 1997 and 2004. The antigenic and genetic characterizations of the equine influenza virus H3N8 are reported here. The highest similarity between the HA1 nucleotide sequences of A/equine/Nador/1/1997 and those of A/equine/Rome/5/1991 and A/equine/Italy/1199/1992 demonstrate that A/equine/Nador/1/1997 belongs to the European lineage. On the other hand, A/equine/Essaouira/2/2004 and A/equine/Essaouira/3/2004 were classified in the predivergent lineage. The present work emphasizes the importance of a national influenza survey program, which requires a collaborative laboratory network to promote the collection and characterization (antigenic and genetic) of equine influenza viruses in real time.     

Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our understanding of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field.     

Investigation on Opisthorchis felineus occurrence and life cycle in Italy

Opisthorchiasis is a fish borne parasitic infection caused by helminths of the genus Opisthorchis (Digenea, Opisthorchiidae), affecting humans and other fish-eating mammals. Despite Opisthorchis felineus was first described in Italy in 1884, no cases of human opisthorchiasis were reported in this country until 2004; from then on, 4 outbreaks due to this species have been recorded in Central Italy. Following the more relevant of these outbreaks, involving 34 people in August 2007, snails, fishes and fecal samples collected from the Bolsena and Bracciano lakes (Central Italy) were analyzed in order to define the cycle of O. felineus in the area and investigate its prevalence in the different hosts. Pools of 20-40 snails each (4983 specimens altogether) of the genus Bithynia were analyzed by PCR for parasite DNA detection. Eight hundred and ninety-four fish belonging to 12 species were collected from the two lakes and tested for metacercariae both by muscle compression and digestion techniques. Eighty-seven fecal samples of 5 putative definitive host species were collected very close to the two lakes and tested for parasite eggs detection by formalin-ethyl acetate concentration technique. Identification at the species level of metacercariae and eggs, respectively, from fish and stool was confirmed by PCR analysis and sequencing. O. felineus DNA was detected in 0.08% (overall minimum infection rate) of snails of the genus Bithynia from the two lakes. The tench, Tinca tinca, was the only fish found infested in both lakes (prevalence 88.5%). O. felineus eggs were found only in cat feces (prevalence 46.4%). The tench represents the only threat for the human consumption in the study area while Coregonus sp., the most economically important species for the local fishery and frequently consumed raw marinated, resulted to be not infected. The high prevalence recorded both in fish and in definitive host suggests a widespread and massive presence of the parasite in the area. Further studies are needed to better investigate the possible role of some cyprinids species as intermediate hosts, in order to check their safety for human consumption.     

Design and Results of an Intensive Monitoring Programme for Avian Influenza in Meat‐Type Turkey Flocks During Four Epidemics in Northern Italy

Surveillance programmes for low pathogenicity (LPAI) and high pathogenicity avian influenza (HPAI) infections in poultry are compulsory for EU Member States; yet, these programmes have rarely been evaluated. In Italy, following a 1999 HPAI epidemic, control measures, including vaccination and monitoring, were implemented in the densely populated poultry area (DPPA) where all epidemics in Italy have been concentrated. We evaluated the monitoring system for its capacity to detect outbreaks rapidly in meat‐type turkey flocks. The evaluation was performed in vaccination areas and high‐risk areas in the DPPA, in 2000–2005, during which four epidemics occurred. Serum samples and cloacal swabs were taken from vaccinated birds and unvaccinated (sentinel) birds. We compared the detection rate of active, passive and targeted surveillance, by vaccination status, using multinomial logistic regression. A total of 13 275 samplings for serological testing and 4889 samplings for virological testing were performed; 6315 production cycles of different bird species were tested. The outbreaks detection rate in meat‐type turkeys was 61% for active surveillance (n = 222/363 outbreaks), 32% for passive surveillance and 7% for targeted surveillance. The maximum likelihood predicted values for the detection rates differed by vaccination status: in unvaccinated flocks, it was 50% for active surveillance, 40% for passive surveillance and 10% for targeted surveillance, compared to respectively 79%, 17% and 4% for vaccinated flocks. Active surveillance seems to be most effective in detecting infection, especially when a vaccination programme is in place. This is the first evaluation of the effectiveness of different types of surveillance in monitoring LPAI infections in vaccinated poultry using field data.     

Vesicular stomatitis and other vesicular, erosive, and ulcerative diseases of horses.

Physical trauma, dietary factors, certain toxins, immune mediated disorders, and vesicular stomatitis virus (VSV) infection are known causes of stomatitis in horses. There is evidence that some outbreaks of equine stomatitis are caused by as yet unidentified infectious agents. It remains to be determined whether stomatitis is an emerging equine infectious disease, or if the increase in reported cases is simply the result of greater public awareness as a consequence of widespread outbreaks of VSV in the southwestern United States in recent years. Focused laboratory and epidemiological studies are necessary to more adequately define non-VS related infectious and noninfectious causes of equine stomatitis.     

Surveillance of equine respiratory viruses in Ontario

The objective of this project was to develop and implement an active surveillance program for the early and rapid detection of equine influenza viruses in Ontario. For this purpose, from October 2003 to October 2005, nasopharyngeal swabs and acute and convalescent serum samples were collected from 115 client-owned horses in 23 outbreaks of respiratory disease in Ontario. Sera were paired and tested for antibody to equine influenza 1 (AE1-H7N7), equine influenza 2 (AE2-H3N8), equine herpesvirus 1 and 4 (EHV1 and EHV4), and equine rhinitis A and B (ERAV and ERBV). Overall, the cause-specific morbidity rate of equine influenza virus in the respiratory outbreaks was 56.5% as determined by the single radial hemolysis (SRH) test. The AE2-H3N8 was isolated from 15 horses in 5 outbreaks. A 4-fold increase in antibody levels or the presence of a high titer against ERAV or ERBV was observed in 10 out of 13 outbreaks in which AE2-H3N8 was diagnosed as the primary cause of disease. In conclusion, AE2-H3N8 was found to be an important contributor to equine respiratory viral disease. Equine rhinitis A and B (ERAV and ERBV) represented an important component in the equine respiratory disease of performing horses.     

Equine Infectious Anemia: Active Surveillance in Central Italy 2007-2009

Equine infectious anemia (EIA) is an infectious and potentially fatal viral disease of equids. EIA virus is usually transmitted from horse to horse by large biting insects, such as horseflies. The aim of this study was to evaluate the results of a national surveillance plan from 2007 to 2009 and evaluate the potential risk factors of EIA in horse populations in central Italy. In 2007, 18 of 6,773; in 2008, 30 of 7,940; and in 2009, 21 of 11,666 equines tested were seropositive for EIA. No statistical association was found between location or sex and the diagnosis of EIA. The seroprevalence rate (2007-2008-2009) was higher among older equids (older than 6 years) than among young (3 months to 5 years old) (P < .05). Likewise, the seroprevalence rate (2007-2008-2009) was higher among mules than among other horses (P < .05). Until 2007, the national equine register did not exist in Italy; therefore, it was difficult to measure the percentage of untested horses that presented a real but unquantified risk for continued EIA virus transmission. By introducing new laws governing the control and conducting active surveillance for EIA, it has been possible, in Italy, to develop a firm foundation of knowledge concerning the persistence and transmission of EIA and the risk factors and to better control the spread of this infection in horses.     

Molecular Characterization of Equine Rotavirus Group A Detected in Argentinean Foals During 2009–2014

Equine rotavirus group A (RVA) has been detected in several countries worldwide since its first detection in 1975. Currently, equine RVA is considered the major cause of dehydrating diarrhea in foals younger than 3 months, and the frequency of detection in clinical cases varies from 20% to 77%. The genotypes of epidemiologic relevance found in horses are G3P[12] and G14P[12]. In a survey conducted in Argentina from 1992 to 2008, equine RVA was detected in 21% and 39% of the fecal samples and outbreaks, respectively. Genotype distribution was 51% G3P[12] and 33% G14P[12]. In continuation with the surveillance, the aim of the present study was to characterize the equine RVA detected in Thoroughbred foals in Argentina from 2009 to 2014. A total of 436 stool samples (corresponding to 177 single diarrhea cases or outbreaks) were analyzed. Equine RVA was detected in 31% (135 of 436) of the samples, which corresponded to 42% (74 of 177) of outbreaks. From the positive cases, 42% (57 of 135) were genotyped. Of this, 63% were G3 (36 of 57) and 37% (21 of 57) were G14 genotype. Considering the whole data (1992–2014), equine RVA was detected in 25% (300 of 1,207) of the stool samples and 41% (119 of 293) of the diarrhea outbreaks. The results of this study also show a cyclic pattern of the G3 and G14 prevalence in the horse population with a change in G3:G14 frequencies from year to year. Furthermore, clustering in the phylogenetic tree suggests evolutionary and geographic relationships between the Argentinean strains compared with the strain circulating worldwide.     

Apparent prevalence of dourine in the Khomas region of Namibia

A 15-year record of the results of horse sera from the Khomas region of Namibia tested by the complement fixation test for dourine at the Central Veterinary Laboratory in Windhoek before clearing the respective animals for export and competitive sport were subjected to statistical analysis. The range of percentage positive, taken as the apparent prevalence of dourine for the region, during the period of study, was 0-29.09%; the average regional level of apparent prevalence was 8.33%. These figures were thought to be lower than the real situation due to some bias in the sampling criteria. For more accurate results, the more reliable enzyme-linked immunosorbent assay techniques are recommended for use in sero-surveys for dourine in Khomas and other regions of Namibia to provide a basis for development of effective control strategies against the disease.     

Clinical and neuropathological features of West Nile virus equine encephalomyelitis in Italy

West Nile (WN) virus infection is a mosquito-borne flavivirosis endemic in Africa and Asia. Clinical disease is usually rare and mild and only in a few cases the infection causes encephalomyelitis in horses, fever and meningoencephalitis in man. We report here the clinical and pathological findings in an epidemic of the disease involving 14 horses from Tuscany, Italy. All cases were observed from August to October 1998. Affected horses showed ataxia, weakness paresis of the hindlimbs and, in 6 cases, there was paraparesis progressing to tetraplegia and recumbency within 2 to 9 days. Eight animals recovered without any important consequences. Serological investigations revealed positivity to WN virus in all the 14 horses and the agent was isolated from the cerebellum and spinal cord of an affected horse. Postmortem examination was carried out on 6 horses. The neuropathological pattern was that of a mild to moderate, nonsuppurative polioencephalomyelitis with constant involvement of the ventral horns of the thoracic and lumbar spinal cord, where focal gliosis and haemorrhage were also apparent in some cases. Differential diagnoses with other equine viral encephalomyelitides are discussed. Climatological and environmental characteristics of the geographic area in which the outbreaks occurred suggest the existence of suitable conditions for the development of the disease. This is the first report of WN virus equine encephalomyelitis in Italy.     

Contagious Equine Metritis: A Review

Contagious equine metritis is a highly contagious genital infection of mares, spread venereally, and was first described in 1977. Although most contagious equine metritis outbreaks involved Thoroughbreds, infection in other breeds has also occurred. The disease has been reported in Europe, Australia and the United States. In Canada, contagious equine metritis has been designated a reportable disease under the Animal Disease and Protection Act.Contagious equine metritis is characterized by an endometritis and infertility and infected mares show no signs of systemic infection. Clinical signs have not been observed in stallions. An asymptomatic carrier state exists in both mares and stallions.Infected mares respond clinically to the topical and parenteral administration of antibacterial drugs. However, a proportion of mares remain carriers of the contagious equine metritis organism. Treatment of stallions is successful. Haemophilus equigenitalis has been proposed as the species name of the Gram-negative, microaerophilic coccobacillus.Sample collection and laboratory methods for the diagnosis of contagious equine metritis are described.     

Assessment of the proportion of under-reporting during the 2007 equine influenza outbreak in New South Wales, Australia

During the 2007 equine influenza (EI) outbreak in Australia, there was no objective information about the possible under-reporting of cases by horse owners either so that they would avoid movement restrictions or because of their inability to detect infection. This investigation aimed to estimate the proportion of under-reporting during the outbreak based on the results of surveillance undertaken in conjunction with vaccination. The results provided improved estimates of morbidity during the outbreak and indicated the level of under-reporting likely to occur in future outbreaks of other infectious diseases in horses in Australia.     

Evidence for bovine parafilariosis in Italy: first isolation of Parafilaria bovicola (Tubangui, 1934) from autochthonous cattle

In May and July 2010 and in March 2011, the presence of multiple bleeding nodules at the level of the neck, shoulders, withers, back and rump in five cattle from three herds in north-central Italy were observed. A diagnosis of parafilariosis was made. Fragments of adult females of Parafilaria bovicola could be identified from the derma of three to five bioptic extirpates. Larvated eggs and free larvae were observed in serohemorrhagic exudates.This paper is the first report of the occurrence of parafilariosis in Italy and subsequent to the various outbreaks reported, it can now be said that bovine parafilariosis is also currently present in Italy.     

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay.cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healhty horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16–35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection.     

Immunity to equine influenza: relationship of vaccine-induced antibody in young Thoroughbred racehorses to protection against field infection with influenza A/equine-2 viruses (H3N8)

Field outbreaks of influenza that occurred in vaccinated Thoroughbred racehorses in Newmarket in 1995 and 1996 were investigated by nucleoprotein ELISA and serology. Investigations showed that serum levels of vaccine-induced single radial haemolysis (SRH) antibody correlated closely with protective immunity against equine influenza and were consistent with observations made in previous experimental studies using nebulised aerosol challenge. In the second part of this study, antibody levels stimulated by vaccination were investigated to examine probable protection in high risk groups, such as yearlings and horses in training. Results for yearlings correlated closely with experimentally derived antibody profiles described for several equine influenza vaccines. The horses in training had levels of antibody immediately prior to revaccination, which were higher than those measured in the yearlings. In conclusion, SRH antibody, used in the investigation of outbreaks and surveillance of post vaccination responses, was shown to correlate with and validate experimental vaccination and challenge models currently used in ponies in the licensing of modern vaccines. There may be benefit from serological monitoring of horses following vaccination through identification of susceptible periods to infection and demonstration of poor vaccine responders. This would allow appropriate and timely amendment of vaccination strategies to maximise protective immunity against influenza.     

Characterisation of three equine influenza A H3N8 viruses from Germany (2000 and 2002): evidence for frozen evolution

Reported here are the results of antigenic and genetic characterisation of equine influenza strains causing local outbreaks reported to the Equine Diagnostic Centre in Berlin, Germany. In 2000, equine influenza virus was detected in a nasal swab from a non-vaccinated horse using a rapid diagnostic kit, but was not successfully isolated. Partial direct sequencing of the haemagglutinin (HA1) gene, indicated that the virus was a European lineage H3N8 subtype strain representative of strains isolated in several European countries during 2000. In 2002, two equine influenza viruses were isolated from nasal swabs both taken from unvaccinated horses with acute respiratory symptoms housed at the same stables. Antigenic characterisation using a panel of ferret antisera suggested that these isolates also belonged to the European lineage of H3N8 viruses. Analysis of deduced HA1 amino acid sequences confirmed that the HA1 of both isolates were identical and belonged to the European lineage. However, from phylogenetic analysis, both strains appeared to be more closely related to viruses isolated between 1989 and 1995 than to viruses isolated more recently in Europe. These results suggested that viruses with fewer changes than those on the main evolutionary lineage may continue to circulate. The importance of expanding current equine influenza surveillance efforts is emphasised.     

Upper Respiratory Disease in Thoroughbred Horses: studies of its viral etiology in the Toronto area, 1960 to 1963

From outbreaks of upper respiratory infection of horses in the Toronto area between 1960 and 1963, several viruses have been isolated. The viruses, isolated in tissue cultures or eggs, include an equine strain of Myxovirus parainfluenzae 3; two strains of equine influenza virus, A/equi-1/Prague/56, and A/equi-2/Miami/63; equine rhinopneumonitis virus, and two newly recognized viruses of the horse, equine rhinoviruses. In addition serological evidence suggested a widespread infection with these viruses in the population under study. Because of the identical clinical picture seen and the complex etiology of the disease, immunization against upper respiratory disease of the horse does not appear to be completely feasible at this time.