Factors associated with epidemiology of Anaplasma platys in dogs in rural and urban areas of Minas Gerais State, Brazil.

This epidemiological survey of Anaplasma platys was carried out in rural and urban areas of three distinct regions of the State of Minas Gerais, Brazil. EDTA blood samples were collected during the dry season from dogs living on farms with an attempt to resample the same dogs in the subsequent rainy season. Samples were also taken from dogs in urban areas. DNA was extracted from blood samples for real time PCR. Risk factors, such as age, breed, sex, presence of ticks, and packed cell volume were analyzed. During the rainy season, the prevalence of infection by A. platys in dogs in the rural areas was significantly higher (13.9%) than that observed in dogs in the urban areas (5.1%). Dogs in the Nanuque region were 3.74 times (p=0.001) more likely to be real-time PCR positive than dogs in the other two studied regions. Dogs infested with ticks showed higher rates of positivity. The results showed that in rural areas of Minas Gerais A. platys infection is influenced by climatic conditions. In areas of higher temperature and higher humidity, transmission occurs during both the dry and rainy seasons, while in areas with lower temperature and humidity transmission occurs mainly during the dry season.

In vitro culture of a novel genotype of Ehrlichia sp. from Brazil.

Ehrlichiae are obligate intracytoplasmic Gram-negative, tick-borne bacteria belonging to the Anaplasmataceae family. Ehrlichioses are considered emerging diseases in both humans and animals. Several members of the genus Ehrlichia have been isolated and propagated in vitro. This study describes the continuous propagation of a Brazilian Ehrlichia sp. isolate in IDE8 tick cells, canine DH82 cells and bovine aorta cells. Initially, the organisms were isolated from the haemolymph of a Rhipicephalus (Boophilus) microplus tick into IDE8 cells. Infected IDE8 cells were brought from Brazil to Germany, where the organisms were continuously propagated in IDE8, DH82 and bovine aorta cells. Bovine aorta cells were infected and propagated for 3 months, corresponding to six subcultures, whereas the other two infected cell lines were kept for more than 1 year. During the cultivation period, 36 and 14 subcultures were carried out in IDE8 and DH82 cell cultures, respectively. Reinfection of IDE8 cells with organisms grown in DH82 cells was achieved. Sequence analysis made with a fragment of the 16S rRNA gene showed that this Ehrlicha sp. is closely related to Ehrlichia canis. However, the maximum likelihood phylogenetic tree shows that it falls in a separate phylogenetic clade from E. canis.

Cross-protection between geographically distinct Anaplasma marginale isolates appears to be constrained by limited antibody responses.

The rickettsia Anaplasma marginale causes the haemolytic disease bovine anaplasmosis, an economic problem in tropical and subtropical areas worldwide. The closely related but less pathogenic Anaplasma centrale is commonly used as a live vaccine to prevent anaplasmosis, but it can only be produced from infected blood. UFMG1 is a low pathogenic Brazilian strain of A. marginale, which has been shown to protect cattle against a high pathogenic Brazilian isolate. As UFMG1 can be grown in tick cells, the strain was proposed as a possible cell culture-derived vaccine. We have evaluated whether UFMG1 could protect cattle against a geographically distant heterologous strain, using A. centrale vaccination as a standard for comparison. Trial calves were infected with UFMG1, A. centrale or PBS. UFMG1-infected animals were more symptomatic than those infected with A. centrale, but none required treatment. All calves were then challenged with the Israeli A. marginale Gonen strain (one of the most prevalent strain in Israel). The A. centrale group had the mildest symptoms, while UFMG1 and control groups both had a more severe response. Nevertheless, the challenge did not cause life-threatening disease in any group. Animals infected with A. centrale had a significantly higher IgG response than UFMG1, when measured in an ELISA against initial bodies from their homologous strain or Gonen. The level of cross-reactivity of the response to initial infection correlated significantly with reduced symptoms after challenge. In conclusion, UFMG1 had limited effect in preventing disease by the geographically distant heterologous Gonen strain. While the low pathogenicity of the Gonen strain in this trial makes it impossible to conclusively state that UFMG1 would have given no protective effect against more serious disease, the comparatively low IgG response to UFMG1 suggests it would not have been as effective as A. centrale.

Use of a Real Time PCR for detecting subspecies of Babesia canis.

This paper reports the development and use of a Real Time PCR for detection of Babesia canis canis, B. canis rossi, and B. canis vogeli in endemic areas of Brazil. The sequences of the internal transcribed spacer (ITS) of several organisms were aligned and five primers and four probes were designed for amplification of a fragment (around 125 bp) which differentiates subspecies of B. canis. Blood samples collected from dogs living in farms in three distinct rural regions within the State of Minas Gerais (Lavras, Belo Horizonte and Nanuque) were tested. Blood samples had been collected during a dry season (Lavras, n=100; Belo Horizonte, n=50; Nanuque, n=102); the dogs were re-sampled in the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=29; Nanuque, n=66). From each sample, DNA was extracted and Giemsa stained smears were microscopically examined for direct detection of Babesia parasites. B. canis vogeli was the only subspecies found, with an overall prevalence of 9.9% during the dry season and 10.8% during the rainy season. Dogs living in Nanuque and Belo Horizonte showed significantly higher prevalence rates than those living in Lavras (13.7%, 12.0% and 5.0%, respectively). The Real Time PCR developed proved to be appropriate to detect B. canis subspecies in endemic areas.

Isolation of viable Toxoplasma gondii from feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctolagus cuniculus) from Brazil.

Toxoplasma gondii was isolated from a feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctologus cuniculus) from Brazil for the first time. Serum and brains from 10 guinea fowl and 21 rabbits from Brazil were examined for T. gondii infection. Antibodies to T. gondii were found in 2 of 10 fowl and 2 of 21 rabbits by the modified agglutination test (titer 1∶25 or higher). Viable T. gondii (designated TgNmBr1) was isolated from 1 of the 2 seropositive fowl by bioassay in mice but not from the 8 seronegative fowl by bioassay in cat. Viable T. gondii was isolated from both seropositive rabbits (designated TgRabbitBr1, TgRabbitBr2) by bioassay in mice from 1 and by bioassay in cat from the other. The TgRabbitBr1 strain was highly virulent for out-bred mice; mice fed 1 infective oocyst died of acute toxoplasmosis. The remaining 2 isolates were relatively avirulent for mice; lethal dose for mice was 10,000 oocysts. All 3 isolates were grown in cell culture, and tachyzoite-derived DNA were genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The TgNmBr1 was found to be clonal Type II, a rare finding in Brazil in any host. The rabbit isolates were atypical, similar to isolates from cats from Brazil (TgRabbitBr1 was identical to TgCatBr5, and TgRabbitBr2 was identical to TgCatBr1, a common genotype in Brazil denoted type BrII). This is the first genetic characterization of T. gondii isolates from the rabbits and guinea fowl in Brazil and the first host record for T. gondii in the guinea fowl.

PCR detection of Anaplasma phagocytophilum in goat flocks in an area endemic for tick-borne fever in Switzerland.

Central Switzerland is a highly endemic region for tick-borne fever (TBF) in cattle, however, little is known about A. phagocytophilum in goats. In the present study, 72 animals from six goat flocks (373 EDTA blood-samples) in Central Switzerland were analysed for A. phagocytophilum DNA. A real-time PCR targeting the msp2 gene of A. phagocytophilum was performed and in positive samples the partial 165 rRNA, groEL and msp4 gene were amplified for sequence analysis. Four DNA extracts were positive. Different sequence types on basis of the amplified genes were found. For comparison, sequences of A. phagocytophilum from 12 cattle (originating from Switzerland and Southern Germany) were analysed. The 165 rRNA gene sequences from cattle were all identical amongst each other, but the groEL and msp4 gene differed depending on the origin of the cattle samples and differed from the variants from goats. This study clearly provides molecular evidence for the presence of different types of A. phagocytophilum in goat flocks in Switzerland, a fact which deserves more thorough attention in clinical studies.

Protection in the absence of exclusion between two Brazilian isolates of Anaplasma marginale in experimentally infected calves.

This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an appendage (UFMG1) could protect calves from infection with a pathogenic A. marginale isolate (UFMG2). Two groups of five Friesian calves were each inoculated with UFMG1 by intravenous injections of either A. marginale-infected tick cell cultures (group 1) or blood stabilates (group 2); a third (control) group was injected with saline. All animals were inoculated with a blood stabilate containing a high pathogenicity A. marginale isolate (UFMG2) 75 days after the UFMG1 inoculation. After infection with UFMG2, animals in groups 1 and 2 presented low rickettsaemia, but no clinical signs and no reduction in packed cell volume (PCV). Control animals became sick, with high rickettsaemia (16% infected erythrocytes) and a reduction in PCV (71%), resulting in 60% deaths. Up to 2 weeks after the UFMG2 inoculation, msp1α UFMG1 sequences were detected in groups 1 and 2. Four weeks after UFMG2 inoculation, UFMG2 sequences were detected in these animals, along with a new msp1α genotype sequence, closely related to that of the UFMG2 isolate. Control animals had UFMG2 msp1α sequences up to 4weeks after inoculation with UFMG2 and the new msp1α genotype sequence could be detected on the sixth week. The origin of the new A. marginale genotype was unknown, but may represent the first example of MSP1a antigenic variation in infected cattle. The results confirmed the low pathogenicity of the UFMG1 isolate, which provided clinical protection against the highly pathogenic A. marginale UFMG2. Infection with UFMG1 did not prevent the establishment of a second isolate, suggesting protection without infection-exclusion among A. marginale isolates.

Propagation of a Brazilian isolate of Anaplasma marginale with appendage in a tick cell line (BME26) derived from Rhipicephalus (Boophilus) microplus.

Anaplasma marginale is a tick-borne pathogen of cattle responsible for the disease anaplasmosis. Data suggest that Rhipicephalus (Boophilus) microplus and R. annulatus may be the major tick vectors of A. marginale in tropical and subtropical regions of the world. In this work we demonstrated the first infection and propagation of a Brazilian isolate of A. marginale (UFMG1) in the BME26 cell line derived originally from embryos of R. (Boophilus) microplus. The establishment of A. marginale infection in a cell line derived from R. (Boophilus) microplus is relevant for studying the A. marginale/tick interface.

Canine babesiosis caused by Babesia canis vogeli in rural areas of the State of Minas Gerais, Brazil and factors associated with its seroprevalence.

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n=92; Belo Horizonte, n=50; Nanuque, n=102) and the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=28; Nanuque, n=66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.

Extração alcoólica de fenólicos e flavonóides totais de mirtilo Rabbiteye (Vaccinium ashei) e sua atividade antioxidante / Alcoholic extracts of total phenolics and flavonoids from rabbiteye blueberry (Vaccinium ashei) and their antioxidant activity

Uma infinidade de produtos de cosmetologia tem utilizado mirtilo, uma pequena fruta com inúmeras alegações de propriedades biológicas, na formulação de uma variedade de produtos como cremes hidratantes, esfoliantes, protetores da radiação ultravioleta, entre outros. Neste sentido, a composição química desta pequena fruta é associada a estas propriedades biológicas,no entanto, poucos relatos científicos são encontrados na literatura. Desta forma, o presente estudo avaliou os teores de compostos fenólicos e de flavonóides, bem como a relação destes compostos com a atividade antioxidante de extratos hidroalcoólicos preparados a partir de frutas de mirtilo cv. "Rabbiteye" (Vacciniumashei). Diferentes proporções de álcool (etanol ou metanol) em água deionizada (ou seja, 40:60, 60:40 e 80:20 (v/v)) foram avaliados quanto à capacidade de extração de fenólicos totais e de flavonóides de amostras de mirtilo em duas condições: frescas (frutas in natura)e secas (após secagem a 105ºC por 15 h). Todas as extrações alcoólicas foram realizadas à temperatura ambiente e sob agitação. Dentre as proporções utilizadas para a extração dos compostos fenólicos foi verificado que, em geral, a mistura metanol/água 80:20(v/v) e a mistura etanol/água 60:40 (v/v) foram os que apresentaram maiores teores de fenólicos e flavonoides totais, e que houve uma correlação positiva forte entre as concentrações de fenólicos e de flavonóides com a atividade antioxidante daqueles extratos hidroalcoólicos de mirtilo, segundo análise do coeficiente de correlação de Pearson.

A wide range of cosmetic products, including moisturizers, exfoliating scrubs and sunscreens, have been formulated with blueberry, a small fruit within numerable claims to biological properties. Although the chemical composition of these fruits has been associated with these biological properties, few studies can be found in the literature. In the present study, the phenolic and flavonoid contents of hydroalcoholic extracts of the rabbiteye blueberry (Vaccinium ashei)were analyzed and the correlation of these compounds with the antioxidant activity of the extracts investigated (by Pearson's correlation coefficient). Various mixtures of alcohol (ethanol or methanol) with deionized water (40:60, 60:40 and 80:20 (v/v)) were tested for their capacity to extract phenolics and flavonoids from blueberries in two conditions: fresh (as picked) and dried (at 105ºC for 15 h). All the extractions were carried out at room temperature with shaking. Out of all the solvent mixtures used for the extractions it was found that, in general, the 80:20 (v/v) methanol/water and 60:40 (v/v) ethanol/water mixtures showed the highest phenolic and flavonoid contents, respectively, with a strong positive correlation between the phenolic and flavonoid contents of the blueberry hydroalcoholic extracts and their antioxidant activity.

Manutenção in vitro de células IDE8 em dois tipos de soro bovino / In vitro maintenance of IDE8 cells using two types of bovine serum

The present study had the objective of defining the culture conditions, optimizing the maintenance and expansion of an IDE-8 cell line in Brazil, with the aim to propose its use as a model for in vitro infection and multiplication of Brazilian strains of rickettsia and other hemoparasites. The supplementation of IDE-8 cells with two distinct fetal bovine sera (a Brazilian and an imported) was evaluated. Culture media were changed weekly and subcultures were carried out every 15 days. The development of cultures and subcultures was evaluated by the percentage of viability and cellular morphology. The results indicate that the imported SFB can be replaced by the Brazilian SFB one, as no significant differences (P<0.05) were seen among culture viabilities.

Dopplerfluxometria das artérias renais: valores normais das velocidades sistólica e diastólica e do índice resistivo nas artérias renais principais / Renal arteries Dopplerfluxometry: normal systolic and diastolic flow velocities and resistive index values in the main renal arteries / Renal arteries Dopplerfluxometry: normal systolic and diastolic flow velocities and resistive index values in the main renal arteries

Dopplerfluxometry of renal arteries has been used to estimate renal perfusion in humans. The aim of this study was to use Dopplerfluxometry technique to calculate the resistive index of main renal arteries in dogs, measuring their systolic and diastolic blood flow velocities. Twenty (10 males, 10 females), adult mongrel dogs, were used in this study. The dogs were submitted to Doppler sonographic evaluation of left and right main renal arteries. The systolic and diastolic blood flow velocities, expressed (in centimeters per second) as mean and standard deviation were 79.96± 8.82 and 28.86± 5.11 in the right main renal artery and 80.22± 6.99 and 29.62± 4.14 in the left main renal artery. The value of resistive index expressed as mean ± standard deviation was 0.64± 0.04 for the right main renal artery and 0.63± 0.028 in the left main renal artery.

Retrospective study (1998-2001) on canine ehrlichiosis in Belo Horizonte, MG, Brazil

The present work describes a retrospective study of clinical cases of ehrlichiosis in dogs examined from March 1998 to September 2001. From the clinical records with laboratorial confirmation of Ehrlichia canis or E. platys infections, the following parameters were analyzed: demographic aspects (age, race, sex, period of the year and origin), clinical characteristics (body temperature, exposure to ticks and clinical signs), and hematological characteristics (blood cell counts and type of infected cell). A total of 194 clinical records were analyzed, from which 31 animals were infected with E. canis and 21 animals with E. platys. The number of cases of canine ehrlichiosis increased considerably from the year 2000 onwards, and 24.4 percent of the cases occurred in 13- to 24-month-old animals, in different urban and per-urban regions of the municipality of Belo Horizonte. The most frequent symptoms were fever, anorexia, apathy, abdominal pain, lymphadenopathy and dispnea. Regarding hematological alterations, 70.3 percent of the animals presented anemia, 50 percent presented thrombocytopenia and 30 percent leukopenia, and most E. canis morulae were seen in monocytes. The results point to the importance of canine ehrlichiosis, as 35.9 percent of the dogs with suspected hemoparasitic diseases were infected with Ehrlichia canis or E. platys

Uso de inóculo padronizado de Anaplasma marginale e da quimioprofilaxia no controle da anaplasmose bovina / Use of standardized inoculum of Anaplasma marginale and chemoprophylaxis to control bovine anaplasmosis

O uso de inóculo homólogo padronizado de Anaplasma marginale foi comparado à prática de quimioprofilaxia com diidrato de oxitetraciclina na reduçäo da riquetsemia e do volume globular (VG) causada pela anaplasmose bovina. Os animais que receberam o inóculo (10(7) hemácias com Anaplasma marginale)apresentaram, ao serem desafiados em campo, riquetsemia média de 1,2 por cento e reduçäo média de VG de 23,0 por cento, significativamente inferiores às do grupo-controle (P<0,05). No experimento de quimioprofilaxia os animais que receberam três doses de diidrato de oxitetraciclina (20mg/kg), com intervalos de 25 dias, apresentaram riquetsemia de 2,7 por cento e reduçäo de 36,3 por cento no VG, significativamente inferiores às do grupo-controle (P<0,05). Ambas as medidas preventivas testadas foram eficientes na reduçäo da intensidade da riquetsemia e na queda do VG

Infecçäo experimental de cäes com amostras de Babesia canis isoladas em Minas Gerais / Dog experimental infection of Babesia canis isolated in Minas Gerais State

The objective of the present study was to evaluate parasitemia and packed cell volume patterns of dogs experimentally inoculated with two isolates of Babesia canis: one from Belo Horizonte (BH) and the other from Lavras (Lv), Minas Gerais State, Brazil. Both isolates showed similar patterns, with the peak of parasitemia occurring three days post-infection. From the fourth day, parasitemia was detected in low levels (0.01 percent) with small periodical increases. The packed cell volume decreased after the parasitemia beginning, with oscillations during the experimental period. All dogs remained apparently normal, except one, which had been inoculated with the BH isolate and presented classical clinical signs of babesiosis (weakness, anemia, hemoglobinuria and depression). The results suggest that the studied isolates have low pathogenicity, and point to the need for further studies aiming to characterize the subspecies of Brazilian isolates

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle.

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.

Quimioprofilaxia da babesiose bovina com diidroxitetraciclina de longa duraçäo / Chemoprophylaxis of bovine babesiosis with long-acting diidroxytetracycline

A açäo da diidroxitetraciclina de longa duraçäo foi testada em bezerros experimentalmente inoculados com Babesia bigemina e em desafio natural em área endêmica. Em uma propriedade de gado de leite, 18 bezerros foram inoculados com 10 elevado a oitava potência hemácias parasitadas por B. bigemina. Dez animais foram tratados com 20 mg/kg de diidroxitetraciclina de longa açäo, sete dias após o inóculo, e oito foram deixados como controle. Todos os animais do grupo controle e apenas três dos tratados apresentaram parasitemia, sendo que dois já estavam infestados no dia do tratamento. Em um segundo experimento, dez bezerros receberam dois tratamentos de 20 mg/kg de diidroxitetraciclina de longa açäo aos 15 e 36 dias após serem liberados em piquetes e oito bezerros foram deixados como testemunhas. Os animais do grupo tratado apresentaram aumento significativo do período pré-patente e menor reduçäo do volume globular, em relaçäo aos do grupo controle

Prevalence of equine herpesvirus type 1 latency detected by polymerase chain reaction.

In this study, an improved polymerase chain reaction (PCR) was used for detection of DNA of latent EHV-1 strains from several sources. Three pairs of oligonucleotide primers spanning fragments of 333 bp, 226 bp and 268 bp of the thymidine kinase (tk) gene, and one primer pair spanning 225 bp of the glycoprotein C (gC) gene were used in specific amplifications. Primers for EHV-4 PCR were also designed. Restriction digests with TaqI confirmed the identity of tk PCR fragments from EHV-1. The sensitivity to detect PCR products was further improved by visualisation in silver-stained acrylamide gels. PCR assays were applied to 267 samples including pools of tissue, peripheral blood leukocytes (PBL) and nasal swabs of archived, farms and abattoir specimens from a total of 116 animals. The EHV-1 DNA was found in 88% of the analysed samples. The prevalence of the EHV-1 latent or persistent form in adult horses was similar to others reports but found higher than previously described in foetuses and young foals. EHV-4 latency was not detected in the Brazilian studied specimens.

Detecçäo de DNA de herpesvírus eqüino-1 em embriäo e em sêmen eqüino por meio de PCR / Detection of equine herpesvirus 1 DNA in a single embryo and in horses semen by polymerase chain reaction

O genoma das amostras de um embriäo e sêmen de três eqüinos, coletados em uma fazenda brasileira, foram testados para a presença de seqüências específicas dos genes da timidina kinase (TK) de herpesvírus eqüino-1 (HVE-1) e herpesvírus eqüino-4 (HVE-4) por meio de reaçäo em cadeia da polimerase (PCR). A PCR detectou sequências específicas do gene de TK de HVE-1 em todas as amostras testadas. O DNA template extraído de leucócitos periféricos da égua doadora do embriäo também foi amplificado pelos pares de primers designados para TK do HVE-1. Vírus infeccioso näo foi isolado desses espécimes. Os resultados indicaram que o PCR foi mais sensível do que o método de isolamento em cultura de células para a detecçäo do HVE-1 em sêmen de cavalos portadores

Development of a differential multiplex PCR assay for equine herpesvirus 1 and 4 as a diagnostic tool.

In this study, a multiplex polymerase chain reaction (PCR) procedure was developed for differentiation of strains and field isolates of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Specific oli-gonucleotide primers were combined to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4, which would yield fragments of different lengths for each virus in the same amplification reaction. The specificity of the largest PCR amplicon for EHV-4 was confirmed by restriction digestion with HindIII. The multiplex PCR proved to be a fast and sensitive method for typing EHV-1 and EHV-4 isolates and for detection and differentiation of both viruses in field samples in which infectious virus is no longer available. The sensitivity was improved by combining cycling optimization and visualization of PCR products in ethidium bromide and silver-stained acrylamide gels.