Customized Tracking Algorithm for Robust Cattle Detection and Tracking in Occlusion Environments.

Ensuring precise calving time prediction necessitates the adoption of an automatic and precisely accurate cattle tracking system. Nowadays, cattle tracking can be challenging due to the complexity of their environment and the potential for missed or false detections. Most existing deep-learning tracking algorithms face challenges when dealing with track-ID switch cases caused by cattle occlusion. To address these concerns, the proposed research endeavors to create an automatic cattle detection and tracking system by leveraging the remarkable capabilities of Detectron2 while embedding tailored modifications to make it even more effective and efficient for a variety of applications. Additionally, the study conducts a comprehensive comparison of eight distinct deep-learning tracking algorithms, with the objective of identifying the most optimal algorithm for achieving precise and efficient individual cattle tracking. This research focuses on tackling occlusion conditions and track-ID increment cases for miss detection. Through a comparison of various tracking algorithms, we discovered that Detectron2, coupled with our customized tracking algorithm (CTA), achieves 99% in detecting and tracking individual cows for handling occlusion challenges. Our algorithm stands out by successfully overcoming the challenges of miss detection and occlusion problems, making it highly reliable even during extended periods in a crowded calving pen.

An Absorbing Markov Chain Model to Predict Dairy Cow Calving Time.

Abnormal behavioral changes in the regular daily mobility routine of a pregnant dairy cow can be an indicator or early sign to recognize when a calving event is imminent. Image processing technology and statistical approaches can be effectively used to achieve a more accurate result in predicting the time of calving. We hypothesize that data collected using a 360-degree camera to monitor cows before and during calving can be used to establish the daily activities of individual pregnant cows and to detect changes in their routine. In this study, we develop an augmented Markov chain model to predict calving time and better understand associated behavior. The objective of this study is to determine the feasibility of this calving time prediction system by adapting a simple Markov model for use on a typical dairy cow dataset. This augmented absorbing Markov chain model is based on a behavior embedded transient Markov chain model for characterizing cow behavior patterns during the 48 h before calving and to predict the expected time of calving. In developing the model, we started with an embedded four-state Markov chain model, and then augmented that model by adding calving as both a transient state, and an absorbing state. Then, using this model, we derive (1) the probability of calving at 2 h intervals after a reference point, and (2) the expected time of calving, using their motions between the different transient states. Finally, we present some experimental results for the performance of this model on the dairy farm compared with other machine learning techniques, showing that the proposed method is promising.

Epidemiological study to investigate the incidence and prevalence of clinical mastitis, peracute mastitis, metabolic disorders and peripartum disorders, on a dairy farm in a temperate zone in Japan.

BACKGROUND: Our aim was to investigate the incidence and prevalence of clinical mastitis, peracute mastitis, metabolic disorders, and peripartum disorders, and to examine factors affecting the prevalence of each disease in cows raised on a large dairy farm in a temperate climate in Japan. The present study was performed on a large commercial dairy farm with approximately 2500 Holstein cows. Data were collected from 2014 to 2018, and involved 9663 calving records for 4256 cows. RESULTS: The incidence rate on the farm was 21.9% for clinical mastitis, 10.4% for peracute mastitis, 2.9% for metabolic disorders, and 3.2% for peripartum disorders. The prevalence rates for clinical mastitis, peracute mastitis, metabolic disorders, and peripartum disorders were 28.0, 13.3, 3.7, and 4.0%, respectively. In all four diseases, the probability of time to occurrence for each disease was associated with parity and calving season (P < 0.05). Regarding metabolic disorders and peripartum disorders, the probability of occurrence decreased during the first 10 days after calving. CONCLUSIONS: Our results showed that clinical mastitis occurred most often in this temperate zone, and that metabolic disorders and peripartum disorders occurred from calving to day 10 post-calving.

Technical note: Calving prediction in dairy cattle based on continuous measurements of ventral tail base skin temperature using supervised machine learning.

In this study, we developed a calving prediction model based on continuous measurements of ventral tail base skin temperature (ST) with supervised machine learning and evaluated the predictive ability of the model in 2 dairy farms with distinct cattle management practices. The ST data were collected at 2- or 10-min intervals from 105 and 33 pregnant cattle (mean ± standard deviation: 2.2 ± 1.8 parities) reared in farms A (freestall barn, in a temperate climate) and B (tiestall barn, in a subarctic climate), respectively. After extracting maximum hourly ST, the change in values was expressed as residual ST (rST = actual hourly ST - mean ST for the same hour on the previous 3 d) and analyzed. In both farms, rST decreased in a biphasic manner before calving. Briefly, an ambient temperature-independent gradual decrease occurred from around 36 to 16 h before calving, and an ambient temperature-dependent sharp decrease occurred from around 6 h before until calving. To make a universal calving prediction model, training data were prepared from pregnant cattle under different ambient temperatures (10 data sets were randomly selected from each of the 3 ambient temperature groups: <15°C, ≥15°C to <25°C, and ≥25°C in farm A). An hourly calving prediction model was then constructed with the training data by support vector machine based on 15 features extracted from sensing data (indicative of pre-calving rST changes) and 1 feature from non-sensor-based data (days to expected calving date). When the prediction model was applied to the data that were not part of the training process, calving within the next 24 h was predicted with sensitivities and precisions of 85.3% and 71.9% in farm A (n = 75), and 81.8% and 67.5% in farm B (n = 33), respectively. No differences were observed in means and variances of intervals from the calving alerts to actual calving between farms (12.7 ± 5.8 and 13.0 ± 5.6 h in farms A and B, respectively). Above all, a calving prediction model based on continuous measurement of ST with supervised machine learning has the potential to achieve effective calving prediction, irrespective of the rearing condition in dairy cattle.

Body Condition Score Estimation Based on Regression Analysis Using a 3D Camera.

The Body Condition Score (BCS) for cows indicates their energy reserves, the scoring for which ranges from very thin to overweight. These measurements are especially useful during calving, as well as early lactation. Achieving a correct BCS helps avoid calving difficulties, losses and other health problems. Although BCS can be rated by experts, it is time-consuming and often inconsistent when performed by different experts. Therefore, the aim of our system is to develop a computerized system to reduce inconsistencies and to provide a time-saving solution. In our proposed system, the automatic body condition scoring system is introduced by using a 3D camera, image processing techniques and regression models. The experimental data were collected on a rotary parlor milking station on a large-scale dairy farm in Japan. The system includes an application platform for automatic image selection as a primary step, which was developed for smart monitoring of individual cows on large-scale farms. Moreover, two analytical models are proposed in two regions of interest (ROI) by extracting 3D surface roughness parameters. By applying the extracted parameters in mathematical equations, the BCS is automatically evaluated based on measurements of model accuracy, with one of the two models achieving a mean absolute percentage error (MAPE) of 3.9%, and a mean absolute error (MAE) of 0.13.

Exploration of factors determining milk production by Holstein cows raised on a dairy farm in a temperate climate area.

In this study, we examined factors that affected milk production by cows raised in a temperate climate area. We conducted this study on a large dairy farm containing approximately 2000 Holstein cows, located in a temperate climate area. We collected 7803 calving records for 4069 cows from 2012 to 2016. We then assessed the effect of hot weather on milk yield by examining three climate factors: season, maximum temperature (MAX), and the temperature and humidity index (THI). We found that increases in heat stress caused linear decreases in milk yield (P < 0.05). Additionally, the effects of the three climate factors on milk yield varied depending on cow parity and days open (P < 0.05). Thus, management procedures should consider cow parity and lactating stage to minimize the negative effects of heat stress on milk production. We also found that the lowest Akaike information criterion value was obtained in our model when using THI for 305-day milk yield. This suggests that THI is a more accurate variable for evaluating heat stress than MAX or season.

Paraoxonase-1 activity is related to Trichinella spiralis-induced hepatitis in rats.

BACKGROUND: Little is known about the potential adverse effects of a chronic zoonotic nematode Trichinella spiralis infection on hepatic inflammation and its relationship to paraoxonase (PON)-1 and butyrylcholinesterase (BuChE) activities. Therefore, we aimed to examine the effects of T. spiralis infection on hepatic synthesis of PON1. METHODS: Wistar rats were infected with 2500 first-stage larvae (L1) of T. spiralis, and serum PON1 and BuChE activities were evaluated. Hepatic expression levels of PON1, BuChE and various cytokines and chemokines [interleukin (IL)-1, IL-4, IL-6, IL-10, tumour necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and transforming growth factor (TGF)-ß1] were evaluated for up to 9 weeks post-infection (p.i.). The effect of these changes on the degree of hepatic apoptosis was also investigated. RESULTS: Trichinella spiralis infection in rats induced significant decreases in serum PON1 activities from day 2 until week 7 p.i. and BuChE activity starting from day 4 until 2 weeks p.i. Moreover, T. spiralis infection increased serum pro-inflammatory cytokines IL-1, IL-6 and TNF-α as well as chemokines MCP-1, MIP-1α and TGF-ß1 during the enteral phase of the parasite life cycle. The anti-inflammatory cytokines IL-4 and IL-10 showed significant increases during the enteral phase for the former and the muscle phase for the latter. These were associated with hepatic inflammation and apoptosis. These events typically decreased hepatic PON1 and BuChE mRNA expression. CONCLUSIONS: Immune responses mounted against T. spiralis infection in rats were associated with hepatic inflammation and a subsequent decrease in serum PON1 and BuChE activities.

Strongyloides spp. infections of veterinary importance.

This paper reviews the occurrence and impact of threadworms, Strongyloides spp., in companion animals and large livestock, the potential zoonotic implications and future research. Strongyloides spp. infect a range of domestic animal species worldwide and clinical disease is most often encountered in young animals. Dogs are infected with Strongyloides stercoralis while cats are infected with different species according to geographical location (Strongyloides felis, Strongyloides tumefaciens, Strongyloides planiceps and perhaps S. stercoralis). In contrast to the other species, lactogenic transmission is not a primary means of infection in dogs, and S. stercoralis is the only species considered zoonotic. Strongyloides papillosus in calves has been linked to heavy fatalities under conditions of high stocking density. Strongyloides westeri and Strongyloides ransomi of horses and pigs, respectively, cause only sporadic clinical disease. In conclusion, these infections are generally of low relative importance in livestock and equines, most likely due to extensive use of macrocyclic lactone anthelmintics and/or improved hygiene. Future prevalence studies need to include molecular typing of Strongyloides species in relation to different hosts. More research is urgently needed on the potential zoonotic capacity of Strongyloides from dogs and cats based on molecular typing, information on risk factors and mapping of transmission routes.

Molecular and morphological variation of Paragonimus westermani in Vietnam with records of new second intermediate crab hosts and a new locality in a northern province.

Paragonimus westermani is one of the most medically important lung flukes and is widely distributed in Asia. It exhibits considerable variation in morphological, genetic and biological features. In central provinces of Vietnam, a high prevalence of metacercariae of this species has been reported from the crab intermediate host, Vietopotamon aluoiense. In this study, we detected P. westermani metacercariae in two additional crab hosts, Donopotamon haii in Quang Tri Province, central Vietnam and Indochinamon tannanti in Yen Bai Province in the north. The latter is a new locality for P. westermani in a northern region of Vietnam where P. heterotremus is the only species currently known to cause human paragonimiasis. Paragonimus westermani metacercariae found in Vietnam showed considerable morphological variation but slight genetic variation based on DNA sequences from the nuclear ribosomal ITS2 region and the mitochondrial 16S gene. Co-infection of the same individual crabs with P. westermani and P. heterotremus and/or some other Paragonimus species was found frequently, suggesting potential for co-infection in humans. The findings of the present study emphasize the need for highly specific molecular and immunodiagnostic methods to differentially diagnose between P. westermani and P. heterotremus infections.

Quantitative effects of a declaration of a state of emergency on foot-and-mouth disease.

OBJECTIVES: The law in Japan requires the declaration of a state of emergency and implementation of countermeasures for an epidemic of a new infectious disease. However, because a state of emergency has never been declared in Japan, its effects remain unknown. The required countermeasures are similar to those implemented in the foot-and-mouth disease epidemic in Miyazaki in 2010. This study aimed to quantitatively estimate the effect of the declaration in 2010 and investigate the nature of the epidemic based on the day on which the declaration took effect. METHODS: Only publicly available data were used. Data for farms in the most affected town were analyzed. A modified susceptible-infected-recovered model was used to estimate the effect and for the simulation. Another model was used to estimate the effective reproduction number. RESULTS: After the declaration, the intra-bovine transmission rate decreased by 18.1 %, and there were few days when the effective reproduction number was >1.0. A few weeks delay in the declaration significantly increased the possibility of epidemic, number of farms at peak, and final infection scale. CONCLUSIONS: Based on the substantial decrease in the transmission rate after the declaration of a state of emergency in 2010, a future declaration will have a similar effect for a new infectious disease even though a direct extrapolation is not valid. Although a declaration should be carefully considered owing to the potential socioeconomic effects, it is essential to prepare for the implementation given that a delay of only a few weeks should be acceptable.

Evaluation of the MGL method to detect Paragonimus eggs and its improvement.

Dog feces containing 500 Paragonimus westermani eggs per gram were examined by the Medical General Laboratory (MGL), the simple sedimentation (SS), and the Army Medical School III (AMS III) methods. The number of eggs per gram of feces (EPG) obtained by the MGL method was 17.2 and was significantly lower than those obtained by the SS method (324.0) and the AMS III method (505.6). When isolated P. westermani eggs were processed by the MGL method and four layers (ether, ether-fecal, formalin layers, and sediment) of the final centrifugation product were separately examined, almost 100% of eggs were found at the ether-fecal layer. Similarly, when fecal samples containing P. westermani, Paragonimus skrjabini miyazakii, Paragonimus ohirai, or Paragonimus harinasutai eggs were processed by the MGL method, more than 95% of the eggs were found in the supernatant layers. The formalin-ethyl acetate (FEA) method showed a similar tendency as the MGL method and over 90% of eggs remained in the supernatant layers. Contrary to Paragonimus eggs, 63 and 96% of Clonorchis and Metagonimus eggs were found in the sediment in the MGL method, respectively. When surfactant (Tween 80) was added to fecal solution, most of Paragonimus eggs spun down in the sediment in the MGL and FEA methods, suggesting that Paragonimus eggs have hydrophobic components on their surface. It is suggested that surfactant addition to the fecal solution should be considered when the MGL method is used for detection of Paragonimus eggs.

Immunomodulatory and antiparasitic effects of garlic extract on Eimeria vermiformis-infected mice.

We investigated the immunomodulatory and parasiticidal effects of garlic extract on coccidiosis caused by Eimeria vermiformis infection in male ICR mice. One group received garlic extract daily until the end of the experiment by the oral route from 10 days prior to oral infection with 300 sporulated E. vermiformis oocysts (infected-garlic(+)). The other group served as a control positive with E. vermiformis infection alone (infected-garlic(-)). In the infected-garlic(+) group, garlic extract treatment induced a significant reduction in fecal oocyst output when compared with the infected-garlic(-) group. Histopathological, immunohistochemical, and gene expression analysis for inflammatory cytokines in ileal tissues showed that the garlic extract treatment impaired intracellular development of E. vermiformis during the early stages by increasing the number of intraepithelial CD8(+) T cells and decreasing IL-10 expression. This induced cell cytotoxicity which was reflected by a decrease in oocyst numbers in the intestinal villi and the feces, indicating anticoccidial effects of the garlic extract. However, further studies to explore the precise mechanism of the observed effects of garlic treatment during Eimeria infection are needed to verify our results.

Comparative analysis of LTR and structural genes in an equine infectious anemia virus strain isolated from a feral horse in Japan.

Although equine infectious anemia virus (EIAV) poses a major threat to the equine industry worldwide, the molecular epidemiology of this virus is poorly understood. Recently, an EIAV strain (EIAVMiyazaki2011-A) representing a new monophyletic group was discovered in feral horses in southern Japan. In the present study, the EIAVMiyazaki2011-A proviral genome is compared with evolutionarily divergent EIAV isolates to investigate conservation of functional elements or motifs within the long terminal repeats (LTRs) and structural genes. This analysis represents a significant step forward in increasing understanding of the molecular conservation and variation between geographically distinct strains of this equine lentivirus.

Identification of a novel equine infectious anemia virus field strain isolated from feral horses in southern Japan.

Although equine infectious anemia (EIA) was described more than 150 years ago, complete genomic sequences have only been obtained from two field strains of EIA virus (EIAV), EIAV(Wyoming) and EIAV(Liaoning). In 2011, EIA was detected within the distinctive feral Misaki horse population that inhabits the Toi-Cape area of southern Japan. Complete proviral sequences comprising a novel field strain were amplified directly from peripheral blood of one of these EIAV-infected horses and characterized by nucleotide sequencing. The complete provirus of Miyazaki2011-A strain is 8208 bp in length with an overall genomic organization typical of EIAV. However, this field isolate possesses just 77.2 and 78.7 % nucleotide sequence identity with the EIAV(Wyoming) and EIAV(Liaoning) strains, respectively, while similarity plot analysis suggested all three strains arose independently. Furthermore, phylogenetic studies using sequences obtained from all EIAV-infected Misaki horses against known viral strains strongly suggests these Japanese isolates comprise a separate monophyletic group.

Serum paraoxonase-1 as biomarker for improved diagnosis of fatty liver in dairy cows.

BACKGROUND: Fatty liver is a major metabolic disorder in dairy cows and is believed to result in major economic losses in dairy farming due to decreased health status, reproductive performance and fertility. Currently, the definitive means for diagnosing fatty liver is determining the fat content of hepatic tissue by liver biopsy, which is an invasive and costly procedure, making it poorly suited to dairy farms. Therefore, the key aim of this study was to investigate the measurement of serum paraoxonase-1 (PON1), an enzyme exclusively synthesized by the liver, as a sensitive noninvasive biomarker for diagnosis of fatty liver in dairy cows. RESULTS: A comparative cohort study using serum specimens from Holstein-Friesian dairy cows (46 healthy and 46 fatty liver cases) was conducted. Serum PON1 (paraoxonase, lactonase and arylesterase) activity and other biochemical and hematological parameters were measured. We found that serum PON1 activity was lower (P<0.001) in cows suffering from fatty liver. The area under the receiver operating characteristic curve (AUC-ROC) of PON1 activity for diagnosis of fatty liver was 0.973-0.989 [95% confidence interval (CI) 0.941, 1.000] which was higher than the AUC-ROC of aspartate aminotransferase (AST), lecithin-cholesterol acyltransferase (LCAT), alkaline phosphatase (ALP), non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL). We found that adding serum PON1 measurement to different batteries of serum diagnostic panels showed a combination of high sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (+LR), negative likelihood ratio (-LR), diagnostic odd ratio (DOR) and overall diagnostic accuracy in diagnosing fatty liver. CONCLUSIONS: The present results indicate that addition of serum PON1 activity measurement to the biochemical profile could improve the diagnosis of fatty liver in dairy cows, which would have a considerable clinical impact and lead to greater profitability in the dairy industry.

Presence of a temperature gradient among genital tract portions and the thermal changes within these portions over the estrous cycle in beef cows.

The aim of the present study was to describe the temperature of the different portions of the female genital tract and their relation to rectal temperature and to investigate the effect of steroid hormones profiles on these variables over the estrous cycle in cattle. Four nonpregnant Japanese Black cows were investigated daily over two successive estrous cycles using a digital thermometer with a long probe and rounded-end sensor to record the temperature of the rectum (RT), vagina (VT), cervix (CT), uterine body (UBT) and uterine horns (UHT). Blood samples were collected immediately before temperature recording to assay peripheral levels of progesterone (P(4)) and estradiol-17ß (E(2)). Moreover, transrectal ultrasonography was carried out after temperature recording to monitor the ovulatory follicle and track ovulation. During the experiment, the ambient temperature and relative humidity were recorded for further calculation of the temperature humidity index (THI). The temperature within the genital tracts in these cows progressively increased towards the uterine horns from the vagina. The VT, CT, UBT and UHTs were significantly higher in association with peripheral P(4) concentrations greater than 4 ng/ml (mid-luteal phase) when compared with lower peripheral P(4) concentrations. The VT was more significantly (P<0.01) correlated to the CT, UBT and UHTs than RT. In conclusion, a temperature gradient was present among the vagina, cervix and uterus over the estrous cycle, and changes in peripheral P(4) concentrations were associated with the thermal variations within these portions. The VT could be more beneficial than RT in monitoring temperature of deeper portions of the female genital tract in bovine.

Paragonimus and paragonimiasis in Vietnam: an update.

Paragonimiasis is a food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. In Vietnam, research on Paragonimus and paragonimiasis has been conducted in northern and central regions of the country. Using a combination of morphological and molecular methods, 7 Paragonimus species, namely P. heterotremus, P. westermani, P. skrjabini, P. vietnamensis, P. proliferus, P. bangkokenis and P. harinasutai, have been identified in Vietnam. Of these, the first 3, P. heterotremus, P. westermani and P. skrjabini, are known to infect humans in other countries. However, in Vietnam, only P. heterotremus, found in some northern provinces, has been shown to infect humans. Even nowadays, local people in some northern provinces, such as Lai Chau and Yen Bai, are still suffering from P. heterotremus infection. In some provinces of central Vietnam, the prevalence and infection intensity of P. westermani metacercariae in freshwater crabs (the second intermediate hosts) are extremely high, but human cases have not been reported. Likewise, although P. skrjabini was found in Thanh Hoa Province, its pathogenicity to humans in Vietnam still remains uncertain. The results of molecular phylogenetic analyses of Vietnamese Paragonimus species provides new insights on the phylogeny and taxonomy of the genus Paragonimus. Comprehensive molecular epidemiological and geobiological studies on the genus in Vietnam and adjacent countries are needed to clarify the biodiversity and public health significance of the lung flukes.

Paragonimus paishuihoensis metacercariae in freshwater crabs, Potamon lipkei, in Vientiane Province, Lao PDR.

Among Paragonimus species, P. paishuihoensis is one of the most mysterious and poorly understood species. Metacercariae are characterized by having a unique dendritically branched excretory bladder. However, the morphology of the adult worm remains unknown. To date, metacercariae of this species have been reported only in China and Thailand. In this study, we first found P. paishuihoensis metacercariae in freshwater crabs, Potamon lipkei, in Hinheub District, Vientiane, Lao PDR, with a prevalence of 77.7% and the average intensity of 10.3 (range 1-28) metacercariae per crab. The molecular data based on ITS2 and CO1 markers indicated that P. paishuihoensis from Laos and Thailand were almost completely identical and were close to members of the Paragonimus bangkokensis/Paragonimus harinasutai complex. Attempts to infect experimental animals (cats, dogs, and rats) with P. paishuihoensis were unsuccessful, suggesting that these animals might be unsuitable definitive hosts for the species. Further studies are necessary to elucidate the taxonomic status and life cycle of P. paishuihoensis.

Cow detection and tracking system utilizing multi-feature tracking algorithm.

In modern cattle farm management systems, video-based monitoring has become important in analyzing the high-level behavior of cattle for monitoring their health and predicting calving for providing timely assistance. Conventionally, sensors have been used for detecting and tracking their activities. As the body-attached sensors cause stress, video cameras can be used as an alternative. However, identifying and tracking individual cattle can be difficult, especially for black and brown varieties that are so similar in appearance. Therefore, we propose a new method of using video cameras for recognizing cattle and tracking their whereabouts. In our approach, we applied a combination of deep learning and image processing techniques to build a robust system. The proposed system processes images in separate stages, namely data pre-processing, cow detection, and cow tracking. Cow detection is performed using a popular instance segmentation network. In the cow tracking stage, for successively associating each cow with the corresponding one in the next frame, we employed the following three features: cow location, appearance features, as well as recent features of the cow region. In doing so, we simply exploited the distance between two gravity center locations of the cow regions. As color and texture suitably define the appearance of an object, we analyze the most appropriate color space to extract color moment features and use a Co-occurrence Matrix (CM) for textural representation. Deep features are extracted from recent cow images using a Convolutional Neural Network (CNN features) and are also jointly applied in the tracking process to boost system performance. We also proposed a robust Multiple Object Tracking (MOT) algorithm for cow tracking by employing multiple features from the cow region. The experimental results proved that our proposed system could handle the problems of MOT and produce reliable performance.

Development of a nested PCR assay to detect equine infectious anemia proviral DNA from peripheral blood of naturally infected horses.

Equine infectious anemia (EIA) has posed a major challenge and caused significant losses to the equine industry worldwide. PCR detection methods have considerable potential as an adjunct to conventional serological diagnostic techniques. However, most published PCR methods, including that recommended by the OIE, were designed using laboratory-adapted virus strains and do not function with field isolates of EIA virus (EIAV). In the present study, a nested PCR assay for detection of EIAV proviral DNA in peripheral blood cells of naturally infected horses was developed. Primer sets were designed based on conserved 5' regions of the viral genome extending from the LTR to the tat gene. Preliminary studies demonstrated that the method has a detection limit of 10 genomic copies and, when applied to a naturally EIAV-infected feral horse population, shows 100 % correlation with conventional serological diagnostic techniques. This assay provides a powerful new tool in the control of EIAV.