Pathological findings in horses dying during an outbreak of the paralytic form of Equid herpesvirus type 1 (EHV-1) infection.

In 1988 an outbreak of the paralytic form of Equid herpesvirus type 1 (EHV-1) infection occurred on a stud farm and several animals died. This provided an opportunity to perform detailed pathological investigations to gain insights into the pathogenesis of this spontaneous disease. Two paretic mares, three foals, an aborted foetus and its non-paretic dam were examined. The endotheliotropism of the virus was clearly demonstrated by the use of an indirect immunoperoxidase (IP) stain. At autopsy, evidence of viral infection was widespread in the foetus and foals, but limited or absent in the mares, probably reflecting differences in their immune status. Vascular lesions were present in the central nervous system (CNS) of the foals as well as the adults; they resulted in minimal neural lesions in the foals. Severe changes in the upper and lower respiratory tracts were a particular feature in the foals, two of which exhibited extensive vasculitis and thrombosis in the lungs. The IP technique was of great value in locating antigen-containing cells in the CNS of one mare when virus isolation was negative. It also revealed the presence of virus in less well documented sites such as the pancreas, gut, thyroid, uveal tract and the skin of the nares.     

Equid herpesvirus (EHV-1) live vaccine strain C147: efficacy against respiratory diseases following EHV types 1 and 4 challenges

The temperature sensitive and host range mutant clone 147 of equine herpesvirus 1 (EHV-1) was assessed for its ability to protect conventional, susceptible adult horses against respiratory infection by EHV-1 and equine herpesvirus 4 (EHV-4).Intranasal (IN) vaccination with 5.2 log(10) TCID(50) did not cause adverse clinical reactions although a limited virus shedding and viraemia (leukocytes) was observed in 11 of 15 and 10 of 15 vaccinated horses respectively. All 15 vaccinated horses showed a significant seroresponse to both EHV-1 and EHV-4 for virus neutralising (VN) antibody. None of 14 control horses shed virus or became viraemic or seroconverted prior to challenge. EHV-1 challenge (dose 6.0 log(10)) 6 weeks after vaccination resulted in pyrexia in all eight control horses while eight vaccinated horses remained unaffected. Six control horses developed nasal discharge, five of which were mucopurulent nasal discharge (mean duration 3.2 days) which also occurred in four vaccinated horses for 1 day. All eight control horses shed challenge EHV-1 at a significantly higher level (group mean titre 2.6+/-0.4 log(10) TCID(50) per sample) and for much longer (mean duration 4.8+/-1.5 days) than that (group mean titre 1.4+/-0.8 log(10) TCID(50) per sample and mean duration 1.5+/-0.5 days) in six vaccinated horses. Furthermore, all eight control horses became viraemic (mean duration 2.9 days) but viraemia did not occur in eight vaccinated horses. Following EHV-1 challenge, all eight control horses showed a significant VN antibody rise to both EHV-1 and EHV-4 but this occurred in only one vaccinated horse and to EHV-4 only. In EHV-4 challenge (dose of 4.2 log(10) TCID(50)) of a separate pair of seven vaccinated and six control horses, 6 weeks after EHV-1 vaccination resulted in pyrexia (mean duration 2.3 days) and nasal discharge (mean duration 1.8 days) in three and five control horses respectively but the only reaction observed in the vaccinated group was nasal discharge for 1 day in one animal. All six control animals shed virus (mean titre 2.5+/-0.6 log(10) TCID(50) per sample and mean duration 2+/-0.6 days) compared to one vaccinated animal. Although EHV-4 viraemia is rare, 3 of 6 control horses became viraemic after EHV-4 challenge but this was not observed in vaccinated horses. After EHV-4 challenge 3 and 5 of 6 control horses seroconverted for VN antibody to EHV-1 and EHV-4 respectively; a non-responsive control horse had high level of pre-existing VN antibody to EHV-4. However, only 1 of 7 vaccinated horses showed a significant antibody rise and only to EHV-4.     

Equine herpesvirus 1 (EHV-1) glycoprotein D DNA inoculation in horses with pre-existing EHV-1/EHV-4 antibody

We have shown previously that equine herpesvirus 1 (EHV-1) glycoprotein D (gD) DNA elicited protective immune responses against EHV-1 challenge in murine respiratory and abortion models of EHV-1 disease. In this study, 20 horses, all with pre-existing antibody to EHV-4 and two with pre-existing antibody to EHV-1, were inoculated intramuscularly with three doses each of 50, 200 or 500microg EHV-1 gD DNA or with 500microg vector DNA. In 8 of 15 horses, inoculation with EHV-1 gD DNA led to elevated gD-specific antibody and nine horses exhibited increased virus neutralising (VN) antibody titres compared to those present when first inoculated. A lack of increase in gC-specific antibody during the 66 weeks of the experiment showed that the increase in gD-specific antibodies was not due to a natural infection with either EHV-1 or EHV-4. The increase in EHV-1 gD-specific antibodies was predominantly an IgGa and IgGb antibody response, similar to the isotype profile reported following natural EHV-1 infection.     

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Equine herpesvirus type 1 (EHV-1) replication in primary murine neurons culture

Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. The occurrence of abortions caused by EHV-1 has repeatedly been confirmed in Poland, but neurological manifestations of the infection have not been described yet. Also it is unknown how the infection of neurons with non-neuropathogenic strains is regulated. To further understand the virus-neuron interaction we studied two strains of EHV-1 in murine primary neuron cell cultures. Both strains were isolated from aborted fetuses: Rac-H, a reference strain isolated by Woyciechowska in 1959 (Woyciechowska 1960) and Jan-E isolated by Bańbura et al. (Bańbura et al. 2000). Upon infection of primary murine neuronal cell cultures with Jan-E or Rac-H strains, a cytopathic effect was observed, manifested by a changed morphology and disintegration of the cell monolayer. Positive results of immunofluorescence, nPCR and real-time PCR tests indicated high virus concentration in neurons, meaning that both EHV-1 strains were likely to replicate in mouse neurons in vitro without the need for adaptation. Moreover, we demonstrated that some neurons may survive (limited) virus replication during primary infection, and these neurons (eight weeks p.i.) harbour EHV-1 and were still able to transmit infection to other cells.     

Equine herpesvirus type 1 (EHV-1) myeloencephalopathy: a case report

An outbreak of neurological disease occurred in a well-managed riding school. Ataxia and paresis were observed in several horses, five of which became recumbent and were euthanized. Post-mortem analysis revealed scattered haemorrhages along the spinal cord, that were reflected by multiple haemorrhagic foci on formalin-fixed sections, with the thoracic and lumbar segments being the most affected. Pathohistologically, perivascular mononuclear cuffing and axonal swelling, especially in the white matter, were evident. Parallel to the course of disease, alterations in myelin sheets and activation of astrocytes and microglial cells were also observed. Virological findings confirmed an acute equine herpesvirus type 1 infection and virus was isolated from the spinal cord of a 26-year-old mare.     

Characterization of equid herpesvirus 1 (EHV-1) related viruses from captive Grevy's zebra and blackbuck

Equid herpes virus 1 (EHV-1) related isolates from a captive blackbuck (strain Ro-1) and Grevy's zebra (strain T965) behaved similarly to EHV-1 and EHV-9 in respect to their host cell range. Restriction enzyme analysis and a phylogenetic tree confirmed that Ro-1 and T965 were identical and more closely related to EHV-1 than to EHV-9. Differences from EHV-1 became obvious firstly, by amino acid alignments revealing two unique substitutions in the gB protein of Ro-1 and T965. Secondly, an EHV-1 type-specific monoclonal antibody did not detect its antigen on Ro-1, T965 or EHV-9 infected cells by immunohistochemistry. The results support the view that Ro-1 and T965 isolates represent a distinct, previously unrecognized species of equid herpesviruses.     

Experimental infection with equine herpesvirus type 1 (EHV-1) induces chorioretinal lesions

Equine herpesvirus myeloencephalitis (EHM) remains one of the most devastating manifestations of equine herpesvirus type 1 (EHV-1) infection but our understanding of its pathogenesis remains rudimentary, partly because of a lack of adequate experimental models. EHV-1 infection of the ocular vasculature may offer an alternative model as EHV-1-induced chorioretinopathy appears to occur in a significant number of horses, and the pathogenesis of EHM and ocular EHV-1 may be similar. To investigate the potential of ocular EHV-1 as a model for EHM, and to determine the frequency of ocular EHV-1, our goal was to study: (1) Dissemination of virus following acute infection, (2) Development and frequency of ocular lesions following infection, and (3) Utility of a GFP-expressing virus for localization of the virus in vivo. Viral antigen could be detected following acute infection in ocular tissues and the central nervous system (experiment 1). Furthermore, EHV-1 infection resulted in multifocal choroidal lesions in 90% (experiment 2) and 50% (experiment 3) of experimentally infected horses, however ocular lesions did not appear in vivo until between 3 weeks and 3 months post-infection. Taken together, the timing of the appearance of lesions and their ophthalmoscopic features suggest that their pathogenesis may involve ischemic injury to the chorioretina following viremic delivery of virus to the eye, mirroring the vascular events that result in EHM. In summary, we show that the frequency of ocular EHV-1 is 50-90% following experimental infection making this model attractive for testing future vaccines or therapeutics in an immunologically relevant age group.     

Polycystic kidney disease in goldfish (Carassius auratus) from Hamilton Harbour, Lake Ontario, Canada

Severe kidney enlargement was observed in 6.3% (5 of 80) of goldfish collected from a heavily polluted industrial basin. Externally the fish had generalized swelling and abdominal distension. The kidneys contained numerous large, clear, fluid-filled cysts (polycystic) that ranged in size from microscopic to 4 cm in diameter. Affected kidneys had a wide range of histological changes-including the presence of large multiple cysts that caused severe distension and compression of normal renal tissue, multifocal granulomas, and signs of early, proliferative glomerulonephritis. The histology of affected kidneys is compared with other goldfish from Hamilton Harbour, and with goldfish collected from an alternate site (a population apparently free of polycystic kidney enlargements). This afflication is rare in feral fish populations, and its occurrence in a deteriorated environment such as Hamilton Harbour may provide further evidence of a link between fish health and environmental quality.     

A review of insecticide poisonings among domestic livestock in southern Ontario, Canada, 1982-1989

From 1982 to 1989, inclusive, 20 poisonings were investigated by the Ontario Ministry of Agriculture and Food following ingestion by domestic livestock of granular insecticides including terbufos (13 poisonings), disulfoton (two poisonings), fonofos (two poisonings), phorate (two poisonings), and carbofuran (one poisoning); all are used for rootworm (Diabrotica spp.) control in corn. A further three poisonings of livestock occurred following the ingestion of the foliar insecticide, endosulfan (two poisonings), and the seed protectant insecticides diazinon plus lindane (one poisoning). There were six poisoning cases as a result of excessive topical applications of the three insecticides coumpahos, fenthion, and lindane as dusts or sprays to control external parasites. Together, these events caused the deaths of 258 domestic animals of which 200 were cattle, 23 were swine, and 35 were sheep. Not all deaths are reported to the Ministry and the cases reported here may only represent 30-50% of the actual deaths over the period. Based on total populations of livestock, the percent losses were very small but they represent serious losses to individual growers. The economic loss is estimated at $160,000 over the eight years, or $20,000 per annum, and this does not include veterinary costs.

Some of the poisoned animals died within as little as three to four hours of ingestion while others were sick but survived for several days. Lethal doses of insecticide were found in the rumen, abomasum, or stomach of dead animals. Signs typical of cholinesterase inhibition caused by organophosphorus poisoning were observed in most cases. Cholinesterase readings were found to be zero in dying animals. Necropsy findings were rarely more than pulmonary edema or myocardial hemorrhage. Where organochlorine insecticides were ingested, convulsions were the major manifestation.

Contamination of feed was most often accidental, and chemical analysis was most helpful in identifying both potent and minor sources, thus facilitating cleanup procedures.

     

Bacterial isolates from equine infections in western Canada (1998-2003)

All bacterial samples of equine origin submitted to the diagnostic laboratory at the Western College of Veterinary Medicine from January 1998 to December 2003 from either "in-clinic" or Field Service cases were accessed (1323 submissions). The most common bacterial isolates from specific presenting signs were identified, along with their in vitro antimicrobial susceptibility patterns. The most common site from which significant bacterial isolates were recovered was the respiratory tract, followed by wounds. Streptococcus zooepidemicus was the most common isolate from most infections, followed by Escherichia coli. Antimicrobial resistance was not common in the isolates and acquired antimicrobial resistance to multiple drugs was rare. The results are compared with previous published studies from other institutions and used to suggest appropriate antimicrobial treatments for equine infections in western Canada.     

Serological evidence of equine herpesvirus type 1 (EHV-1) activity in polo horses in Nigeria.

Serological evidence of Equine Herpes virus type 1 (EHV-1) activity in Polo horses in Nigeria is reported for the first time. Eighty-two percent of horses tested with known antigen had precipitating antibodies to EHV-1 while 43% of sera tested against antigen prepared from nasal discharges were positive suggesting that the virus was being excreted in the nasal discharges and probably acting as a source of infection for incontact animals as occurs in on-going acute infections. The result of this study indicates a high prevalence of EHV-1 activity among Polo horses in Nigeria and demonstrates the ubiquitous distribution of the virus in a country that has not been previously investigated.     

Serological study of an outbreak of paresis due to equid herpesvirus 1 (EHV-1).

Six cases of paresis occurred in a Swedish stud with 48 mares and a stallion. Complement-fixation tests revealed a recent infection with EHV-1 in most horses of the stud. Serumneutralisation tests showed rapid antibody-titre increases during the course of the disease. This type of antibody response was interpreted as induced by reinfection or, possibly, recurrent infection. Two diseased mares were sacrificed. No virus could be isolated from their central nervous system (CNS), liver or spleen, but there is a presumptive evidence for the presence of an antigen specific to EHV-1 in the CNS and liver. Neutralising antibodies to EHV-1 were demonstrated in the liver and kidneys following elution by acidification of the tissues. No such antibodies could be demonstrated in the brain and spinal cord. A possible reason for this failure is discussed.     

Low-dose ginseng (Panax quinquefolium) modulates the course and magnitude of the antibody response to vaccination against Equid herpesvirus 1 in horses

The purpose of this study was to determine if ginseng fed at low levels enhances a horse's antibody response to vaccination against Equid herpesvirus 1 (EHV-1). For 28 d, 5 horses received ground, powdered ginseng (35 mg/kg body weight, 1.7 mg/kg total ginsenosides) in molasses as a carrier, and 5 received molasses only. On day 14, each horse was vaccinated against EHV-1. The time course of the antibody response to vaccination was significantly altered in the horses receiving ginseng, a clinically relevant increase in antibody titer being observed by postvaccination day 2 compared with day 6 in the control horses. The horses receiving ginseng also had a significant decrease in serum levels of sodium and a significant increase in serum levels of potassium. No adverse effects of ginseng treatment were identified by hematologic and blood biochemistry profiles. Thus, low-dose dietary supplementation with ginseng in healthy horses may be a useful adjunct to vaccination.     

Observational study on antimicrobial resistance in Escherichia coli and Salmonella isolates from Ontario calf samples submitted to a diagnostic laboratory from 2007 to 2020

The objectives of this study were to i) describe Escherichia coli and Salmonella isolates; ii) investigate the temporal trends in antimicrobial resistance (AMR) profiles; and iii) evaluate the impact of season and age on these AMR profiles from diagnostic and post-mortem samples in Ontario calves ≤ 2-months-old submitted from 2007 to 2020 to the Animal Health Laboratory in Guelph, Ontario, Canada. Antimicrobial susceptibility testing results were measured by the Kirby-Bauer disk diffusion method. A total of 1291 isolates with AMR profiles were obtained from calves, with E. coli (n = 434) and Salmonella (n = 378) being the most common bacteria characterized for AMR. For E. coli, 79% of isolates tested showed a positive result in F5/K99, whereas for Salmonella isolates, S. Typhimurium (33%) and S. Dublin (22%) were the 2 most common serotypes identified. Multivariable logistic regression models were built to evaluate AMR profiles for E. coli (n = 414) and Salmonella (n = 357) to each antimicrobial tested. Most E. coli isolates (91%) and Salmonella isolates (97%) were resistant to at least one of the antimicrobials tested. In general, E. coli and Salmonella had higher odds of resistance in calves aged ≥ 2 wk compared to 1-week-old calves, and little difference was seen in the level of resistance over the years observed or between seasons in most of the antimicrobials tested. Prospective research should investigate potential risk factors for the development of AMR in calves examples being antimicrobial use and farm management practices.