Evaluation of an indigenous ELISA for diagnosis of Johne’s disease and its comparison with commercial kits

Country lacks sensitive and indigenous diagnostic kits for the screening of goats and sheep against Johne’s disease. Therefore an indigenous ELISA kit was developed using protoplasmic antigen from native Mycobacterium avium subspecies paratuberculosis ‘Bison Type’ strain of goat origin (Kit 1). In the present study, kit 1 and two commercial kits (kit 2 and 3) were evaluated with respect to ‘Gold Standard’ fecal culture in 71 animals (55 goats and 16 sheep). Kit 1 using indigenous antigen (protoplasmic antigen) was sensitive at very low concentration (0.1 μgm / well) as compared to purified commercial protoplasmic antigen (4 μgm / well) used in kit 2, in the Type 1 reactors (strong positive as positive). Screening of 71 animals by fecal culture detected 38.0% animals (goats-40.0%, sheep-31.2%) as positive (clinical shedders of bacilli) from these farm animals. Of the farm animals located at Central Institute for Research on Goats, herds of goat were endemic whereas, sheep flocks were comparatively resistant to Johne’s disease. The 29.5 and 61.9, 15.4 and 57.7 and 4.2 and 14.0% animals (goats and sheep) were in the category of sero-reactors type 1 and 2 of the ELISA kits 1, 2 and 3, respectively. In the type 1 sero-reactors, sensitivity and specificity of kit 1, 2 and 3 was 53.7 and 86.0, 17.8 and 86.0 and 3.5 and 94.7%, respectively. Indigenous ELISA test (kit 1) was significantly superior for the screening of goatherds and sheep flocks against JD as compared to commercial ELISA kits (Kit 2 and 3). In comparison to kit 2 and 3, kit 1 had highest sensitivity, comparable specificity and substantial to nearly perfect proportional agreement (Kappa Scores) with respect to ‘Gold standard’ fecal culture in goats and sheep. Disease being endemic in herds and flocks screened using ELISA kits, Type I sero-rectors had better correlation with fecal culture in comparison to Type II sero-reactors therefore, used for estimation of sero-prevalence. Newly developed Indigenous ELISA kit was simple, inexpensive, sensitive and reliable for screening of goats and sheep population against Johne’s disease. The study reports high prevalence of Johne’s disease in farm goatherds and sheep flocks, using sensitive tests (fecal culture and ELISA kit). Results of Type 1 reaction in kit 1 were optimally correlated with culture and were good for estimating the sero-prevalence. For controlling Johne’s disease in endemic herds initial removal of the animals in strong positive category (Tyep 1 reactors), may help to remove heavy shedders.     

Experimental and Natural Infections of Goats with Severe Fever with Thrombocytopenia Syndrome Virus: Evidence for Ticks as Viral Vector

Background

Severe fever with thrombocytopenia syndrome virus (SFTSV), the causative agent for the fatal life-threatening infectious disease, severe fever with thrombocytopenia syndrome (SFTS), was first identified in the central and eastern regions of China. Although the viral RNA was detected in free-living and parasitic ticks, the vector for SFTSV remains unsettled.

Methodology/Principal Findings

Firstly, an experimental infection study in goats was conducted in a bio-safety level-2 (BSL-2) facility to investigate virus transmission between animals. The results showed that infected animals did not shed virus to the outside through respiratory or digestive tract route, and the control animals did not get infected. Then, a natural infection study was carried out in the SFTSV endemic region. A cohort of naïve goats was used as sentinel animals in the study site. A variety of daily samples including goat sera, ticks and mosquitoes were collected for viral RNA and antibody (from serum only) detection, and virus isolation. We detected viral RNA from free-living and parasitic ticks rather than mosquitoes, and from goats after ticks’ infestation. We also observed sero-conversion in all members of the animal cohort subsequently. The S segment sequences of the two recovered viral isolates from one infected goat and its parasitic ticks showed a 100% homology at the nucleic acid level.

Conclusions/Significance

In our natural infection study, close contact between goats does not appear to transmit SFTSV, however, the naïve animals were infected after ticks’ infestation and two viral isolates derived from an infected goat and its parasitic ticks shared 100% of sequence identity. These data demonstrate that the etiologic agent for goat cohort’s natural infection comes from environmental factors. Of these, ticks, especially the predominant species Haemaphysalis longicornis, probably act as vector for this pathogen. The findings in this study may help local health authorities formulate and focus preventive measures to contain this infection.     

Visna Virus Meningoencephalomyelitis in Goats

A progressive paresis was encountered in herds of Swedish goats. The symptoms developed during a period of weeks or months, and were initially often seen as a weakness of the hind limbs before the animals became paralytic. The development and the histopathological lesions of the disease in the GNS and the lungs were similar to those of visna in sheep. In vitro grown choroid plexus cells, prepared from affected goats, showed foci of polykaryocytes. Electron microscopy revealed the presence of particles morphologically similar to those of sheep visna virus (SVV). Goats experimentally infected with the goat visna virus (GVV) developed GNS lesions similar to those of visna in sheep and became seropositive to SVV. The results of complement fixation tests, carried out on sera from 11 goat herds, showed a coincidence between seropositiveness and the occurrence of disease in one and the same herd. Using the ELISA method, an average of 80 % of the goats in 5 herds were found to be seropositive to GVV.     

Experimental concurrent infection of Afar breed goats with Oestrus ovis (L1) and Haemonchus contortus (L3): interaction between parasite populations, changes in parasitological and basic haematological parameters

The study was conducted to examine the occurrence and interaction between Oestrus ovis and Haemonchus contortus in experimentally infected Ethiopian Afar breed of goats. Twenty goats were divided into four groups (O, OH, H, and C) of five animals each. Each animal of groups O and OH received weekly infections for 5 weeks with 66 first instar larvae (L₁) of O. ovis. Then animals of groups OH and H were infected with a single dose of 5000 third stage larvae (L₃) of H. contortus. Goats of group C were kept free of any infection as non-infected control. Faecal egg count (FEC), blood cell count, total serum protein level and body weight were recorded weekly throughout the study period. At necropsy worm burden, female worm length, fecundity and larval burden of O. ovis in the nasal-sinus cavities of infected animals were assessed. The results showed that the presence of H. contortus in the abomasum of goats of group OH had no influence on the development of O. ovis. On the contrary, a significant reduction (P < 0.05) in FEC, worm burden, fecundity and female worm length was revealed in group OH animals compared to the mono-infected animals (group H). This was associated with eosinophilia and reduced packed cell volume.     

Genome-wide association study for antibody response to Mycobacterium avium ssp. paratubeculosis in goats

Mycobacterium avium ssp. paratuberculosis (MAP), causes Johne's disease (JD), or paratuberculosis, a chronic enteritis of ruminants, which in goats is characterized by ileal lesions. The work described here is a case–control association study using the Illumina Caprine SNP50 BeadChip to unravel the genes involved in susceptibility of goats to JD. Goats in herds with a high occurrence of Johne's disease were classified as healthy or infected based on the level of serum antibodies against MAP, and 331 animals were selected for the association study. Goats belonged to the Jonica (157) and Siriana breeds (174). Whole-genome association analysis identified one region suggestive of significance associated with an antibody response to MAP on chromosome 7 (p-value = 1.23 × 10⁻⁵). These results provide evidence for genetic loci involved in the antibody response to MAP in goats.     

Evaluation of a positive conditioning technique for influencing big sagebrush (Artemisia tridentata subspp. wyomingensis) consumption by goats

Diets of angora goats (Capra hircus) positively conditioned to eat big sagebrush (Artemisia tridentata subspp. wyomingensis) were contrasted with control groups to assess the effects of positive conditioning. Goats were conditioned by including ever-increasing amounts of sagebrush in the daily ration, to a maximum of 25% by weight as fed. Conditioning effects were evaluated by comparing relative consumption of big sagebrush in a rangeland setting. Field trials were conducted at the Squaw Butte Experimental Range, a shrub steppe rangeland in eastern Oregon. Our results indicate that neither conditioned does nor kids had significantly different intake of sagebrush when compared with control animals. Young animals consumed shrub species sooner than adults and ate significantly more shrubs throughout all seasons until the second summer when diets did not differ between age groups.     

A preliminary study on the responses to experimental Haemonchus contortus infection in indigenous goat genotypes

A preliminary study on the responses of different goat genotypes to experimental haemonchosis was assessed in 48 castrated 6-months old Boer, Xhosa-Boer, Nguni and Xhosa goats randomly assigned to four balanced groups: non-infected and non-supplemented (NINS); infected, non-supplemented (INS); infected, supplemented (IS) and non-infected, supplemented (NIS). Each goat in the infected groups received a dose of 7200 L3 H. contortus larvae and kept for 90 days. Of the infected groups, the IS Xhosa goats had the lowest faecal larval counts (FLC) and maintained the highest PCV values throughout the study. The INS Xhosa and Nguni goats maintained body weight, but INS Boer goats lost body weight markedly. In all the treatments, the Xhosa goats also maintained high PCV values throughout the study. In conclusion, the Xhosa and Nguni genotypes performed better in terms of tolerance to haemonchosis. With protein supplementation, the Xhosa and Nguni genotypes also appeared to be much more capable of increasing PCV and eosinophil counts as worm counts increased, unlike the Boer and Xhosa-Boer genotypes.     

Home Range Use and Movement Patterns of Non-Native Feral Goats in a Tropical Island Montane Dry Landscape

Advances in wildlife telemetry and remote sensing technology facilitate studies of broad-scale movements of ungulates in relation to phenological shifts in vegetation. In tropical island dry landscapes, home range use and movements of non-native feral goats (Capra hircus) are largely unknown, yet this information is important to help guide the conservation and restoration of some of the world’s most critically endangered ecosystems. We hypothesized that feral goats would respond to resource pulses in vegetation by traveling to areas of recent green-up. To address this hypothesis, we fitted six male and seven female feral goats with Global Positioning System (GPS) collars equipped with an Argos satellite upload link to examine goat movements in relation to the plant phenology using the Normalized Difference Vegetation Index (NDVI). Movement patterns of 50% of males and 40% of females suggested conditional movement between non-overlapping home ranges throughout the year. A shift in NDVI values corresponded with movement between primary and secondary ranges of goats that exhibited long-distance movement, suggesting that vegetation phenology as captured by NDVI is a good indicator of the habitat and movement patterns of feral goats in tropical island dry landscapes. In the context of conservation and restoration of tropical island landscapes, the results of our study identify how non-native feral goats use resources across a broad landscape to sustain their populations and facilitate invasion of native plant communities.     

复合微量元素缓释丸对放牧反刍动物Cu、Co、Se营养价值的研究

从放牧生态环境大群的大别山黄牛和英山白山羊中选择年龄,体重相近的２０头牛和２０头羊,各随机分为两组,每组１０头,雌、雄均匀配,以复合微量元素缓释丸为试验材料,、每Ⅰ组投丸为试验组,每Ⅱ组不投丸为对照组,牛的两组每组以５头补料,５头不补料,试验期１３５ｄ。羊的两组均不补料,为全放牧方式,试验期６０ｄ,试验结束对羊Ⅰ、Ⅱ组各随机抽取３头屠宰,取样测定肝、肾、脾、肌肉、心、血和瘤胃内容物测定Ｃｕ、Ｃｏ和     

Genetic and phenotypic parameters of body weight in West African Dwarf goat and Djallonké sheep

The International Trypanotolerance Centre's small ruminant breeding programme was initiated in 1995. The aim was to increase the efficiency of meat production and the trypanotolerance of the animals (sheep and goat). To achieve that goal, selection was based on estimated breeding values for daily weight gain from 4 to 12 months of age measured on trypanotolerance challenge. The purpose of this study was to estimate genetic parameters for growth traits and to evaluate genetic trends in West African Dwarf goat and Djallonké sheep resulting from the breeding programme under a low input production environment. Data for West African Dwarf goat and Djallonké sheep included birth weight (BW), weaning weight (W120), yearling weight (W360), pre-weaning (GR0–4) and post-weaning (GR4–12) growth rate. The data were analysed using an animal model that accounted for fixed effects of sex, year of birth, season of birth, parity of the dam, type of birth and the interaction year by season of birth. Estimates of heritability for BW, W120, W360, GR0–4 and GR4–12 were 0.5, 0.43, 0.30, 0.32 and 0.11 for goats and 0.39, 0.54, 0.21, 0.54 and 0.23 for sheep, respectively. The genetic correlation between BW and W120 was high for goats (0.74) and moderate for sheep (0.47). Genetic correlations between W120 and GR4–12 were high (0.92) for goats and moderate (0.49) for sheep. Between GR0–4 and BW the correlation was positive but low for sheep (0.26) and moderate for goats (0.60). Positive trends were found in mean estimated breeding values for animals born in the period 1995–2002 which demonstrated the effectiveness of the implemented breeding programs.     

Mycoplasma agalactiae detected in the semen of goat bucks

Contagious agalactia (CA) is among the most significant diseases affecting small ruminant populations in Mediterranean countries. This study was designed to detect the excretion in semen of CA-causing mycoplasmas in goats (Capra hircus) reared in Spain, where the disease is considered endemic. Culture techniques and PCR were conducted on 147 semen samples collected from 113 goat bucks to detect mycoplasmas. No animal showed clinical symptoms of CA at the moment of the screening. M. agalactiae was identified using both diagnostic methods in three semen samples collected from three different bucks. These animals belonged to a group of animals in which semen had been analyzed twice and only the second sample proved positive, suggesting the possibility of intermittent excretion. This is the first report of the isolation of M. agalactiae from semen collected from naturally infected goats. Future studies should investigate whether semen could be a real source of CA infection by determining if the agent may be transmitted during natural service or when semen is used for artificial insemination.     

Efficacy study of novel diamidine compounds in a Trypanosoma evansi goat model

Three diamidines (DB 75, DB 867 and DB 1192) were selected and their ability to cure T. evansi experimentally infected goats was investigated. A toxicity assessment and pharmacokinetic analysis of these compounds were additionally carried out. Goats demonstrated no signs of acute toxicity, when treated with four doses of 1 mg/kg/day (total dose 4 mg/kg). Complete curative efficacy of experimentally infected goats was seen in the positive control group treated with diminazene at 5 mg/kg and in the DB 75 and DB 867 groups treated at 2.5 mg/kg. Drug treatment was administered once every second day for a total of seven days. Complete cure was also seen in the group of goats treated with DB 75 at 1.25 mg/kg. DB 1192 was incapable of curing goats at either four-times 2.5 mg/kg or 1.25 mg/kg. Pharmacokinetic analysis clearly demonstrated that the treatment failures of DB 1192 were due to sub-therapeutic compound levels in goat plasma, whilst compound levels for DB 75 and DB 867 remained well within the therapeutic window. In conclusion, two diamidine compounds (DB 75 and DB 867) presented comparable efficacy at lower doses than the standard drug diminazene and could be considered as potential clinical candidates against T. evansi infection.     

Diagnosis of caseous lymphadenitis by ELISA in naturally infected goats from Venezuela

Due to the absence of previous reports, the goal of this work was to detect caseous lymphadenitis (CLA) in goat flocks from Venezuela using an indirect immunoenzymatic assay (ELISA). Eighteen farms were randomly selected in Falcon State, North-Western Venezuela. Blood samples were taken from 259 goats, 65 of them with abscesses. Experimental inoculations were made to healthy kids with 0.5 mL inocula containing 4.7 × 10⁵ of Corynebacterium pseudotuberculosis to observe the kinetics of antibody response. Immunoenzymatic assays were carried out using exotoxin of C. pseudotuberculosis as antigen. Antibody response in experimentally inoculated animals was detected 2 weeks after infection. Of 259 field goat sera, 55.98% were positive by ELISA. Of 65 goats with abscesses, 67.69% had CLA demonstrated by bacteriological methods; from these, 72.73% showed antibodies by ELISA. Of the remaining goats negative to CLA, 47.62% had antibodies by ELISA. Sensitivity was calculated in 72.73% and specificity in 67.74%. The immunoenzymatic assay applied in this research could be useful to detect CLA in naturally infected goat flocks from Venezuela.     

The Limits of Test-Based Scrapie Eradication Programs in Goats

Small ruminant post-mortem testing programs were initially designed for monitoring the prevalence of prion disease. They are now considered as a potential alternative to genetic selection for eradicating/controlling classical scrapie at population level. If such policy should be implemented, its success would be crucially dependent on the efficiency of the surveillance system used to identify infected flocks. In this study, we first determined the performance of post-mortem classical scrapie detection in eight naturally affected goat herds (total n = 1961 animals) according to the age at culling. These results provided us with necessary parameters to estimate, through a Monte Carlo simulation model, the performance of scrapie detection in a commercial population. According to this model, whatever the number of tests performed, post mortem surveillance will have limited success in identifying infected herds. These data support the contention that scrapie eradication programs relying solely on post mortem testing in goats will probably fail. Considering the epidemiological and pathological similarities of scrapie in sheep and goats, the efficiency of scrapie surveillance in both species is likely to be similar.     

The effects of buck teasing on synchronization of estrus in goats after intravulvo-submucosal administration of cloprostenol

A study was conducted on 35 East African shorthorned female goats to determine if a combination of buck teasing and low doses of a prostaglandin (PGF(2) alpha) analogue, cloprostenol, given intravulvo-submucosally (i.v.s.m.) would be suitable for synchronization of estrus. Goats were allotted, with the onset of estrus, to seven groups (n = 5 goats per group). Five of the seven groups received varying doses of cloprostenol: Group 1 (125 mug cloprostenol i.m. per goat); Group 2 (62.5 mug cloprostenol i.v.s.m. per goat); Group 3 (62.5 mug cloprostenol i.v.s.m. per goat plus buck teasing); Group 4 (31.25 mug cloprostenol i.v.s.m. per goat); Group 5 (31.25 mug cloprostenol i.v.s.m. per goat plus buck teasing); Group 6 (buck teasing); Group 7, (2 ml physiological saline i.v.s.m. per goat, control group). Plasma progesterone concentration was measured on day of treatment and for 6 d thereafter. All goats in groups 1, 2, 3 and 5 exhibited estrus within 68 h. Thus, the number of goats receiving low doses of PG-cloprostenol intravulvo-submucosally observed in estrus increased (P < 0.05) with exposure to bucks. Exhibition of behavioral signs of estrus was maximal between 2 and 20 h after onset of signs of estrus. The exposure of females to males prior to intrauterine penetration was an advantage because copious mucus eased penetration.     

Profile of the Caprine arthritis-encephalitis virus (CAEV) in blood, semen from bucks naturally and experimentally infected in the semi-arid region of Brazil

The aim of this study was to report the chronology of Caprine arthritis-encephalitis virus elimination and compare the blood and semen viral profiles of animals naturally and experimentally infected by SRLV raised in the semi-arid region of Brazil. The experiment was carried out at the Brazilian Center for Goat Research (Embrapa). Nine bucks were selected, four naturally infected by CAEV and five animals proven negative that were inoculated with the goat lentivirus (CAEV-Cork strain). Every week the animals were submitted to semen collection using an artificial vagina. The blood was collected by puncturing the jugular vein with tubes containing EDTA, 7 days after inoculation (experimentally infected group) or at the start of the experiment (naturally infected group) and then at every 30 days. The genomic viral DNA was extracted from semen and blood and then Nested-PCR was applied. An agar gel microimmunodiffusion was performed to detect anti-CAEV antibodies. The results were described in percentage and analyzed by the Chi square test (P < 0.05). The presence of anti-CAEV antibodies was detected in the 16th week after inoculation that characterized the seroconversion from four of the five naturally infected goat bucks (80%). The fifth reproducer presented late seroconversion, totaling 32 weeks post-inoculation. A quantity was observed in the total of samples collected of 12.50 and 17.14% positive results in the blood and 10.98 and 11.25% positive results in the semen of the naturally and experimentally infected animals, respectively, and there was no statistical difference. No statistically significant differences were observed regarding the presence of proviral DNA in the blood and semen of the naturally and experimentally infected animals. A viral elimination pattern was not identified during the assessment period, but the presence of proviral DNA was shown at shorter intervals after the 18th week and the 22nd week, for the experimentally and naturally infected bucks, respectively. Therefore, recently infected goats in the period prior to seroconversion eliminated small ruminant lentivirus proviral DNA in the semen and are important sources of infection that should be considered in a control program of this lentivirus, and the Nested-PCR technique is a relevant tool to select virus-free ejaculates.     

Evaluation of bioelectronics sensor compared to other diagnostic test in diagnosis of Johne's disease in goats

A bioelectronics sensor has been developed and it is evaluated for the diagnosis of paratuberculosis in goats. Initially hematite nanoparticles were prepared and using this nanoparticles as core, electrically active polyaniline coated magnetic (EAPM) nanoparticles are synthesized from aniline monomer (made electrically active by acid doping). These EAPM nanoparticles were fabricated with rabbit anti-goat IgG for the detection of goat antibodies on the capture pad. The protoplasmic antigen of Mycobacterium avium subspecies paratuberculosis (MAP) immobilized onto the capture pad will detect the antibody against MAP in the goat sera samples. This bound goat antibody will be detected by the anti-goat IgG previously bound to EAPM. Upon detection the EAPM nanoparticles bridges an electric circuit between the silver electrodes, flanking the capture membrane. The electrical conductance, caused by EAPM, was measured as direct charge transfer between the electrodes. Testing of the biosensor with known Johne's disease (JD) positive and negative serum samples gave significant difference in the electrical conductance value. Further the efficacy of this biosensor was compared with other serological tests like agar gel immunodiffusion (AGID) and absorbed ELISA using field sera. Out of 265 goat sera tested, positive results recorded were; AGID 36 (13.59%), bioelectronics sensor 49 (19.14%), and absorbed ELISA 51 (19.25%). This biosensor was also compared in live animals using intradermal Johnin test and nested PCR (detecting mycobacterial DNA in feces) in 65 animals. Of which, positive results recorded in animals were; Johnin test 21 (32%), biosensor 26 (40%) and fecal PCR detected mycobacterial DNA in 28 (43%) animals. Though the nanobioelectronics sensor was slightly less sensitive (not statistically significant) compared to absorbed ELISA and fecal nested PCR for mycobacterial DNA but it was simple to perform in field conditions and requires less time. The speed of detection and the equipment involved would support its application toward the various point-of-care opportunities aimed at control and management of Johne's disease in goats.     

Can Immune Response Mechanisms Explain the Fecal Shedding Patterns of Cattle Infected with Mycobacterium avium Subspecies paratuberculosis?

Johne’s disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns.     

Corynebacterium Pseudotuberculosis Infection in Goats V.

In 2 goat herds, one infected with Gorynebacterium pseudo-tuberculosis and one free from the infection,, goats were examined for superficial swellings on the shoulder and chest. All animals in this study had been vaccinated against paratuberculosis before the age of 4 weeks. The vaccine had been applied subcutaneously behind the shoulder. Twenty-two of 40 (55 %) and 31 of 45 (69 %) goats had such lesions in the infected and non-infected herds, respectively. The difference between the herds was not significant, P > 0.05. Swellings found behind the shoulder in 19 goat carcasses derived from 4 herds in which G. pseudotuberculosis infection occurred were examined bacteriologically. No bacteria could be isolated from such lesions in 15 animals, while G. pseudotuberculosis in pure culture was isolated from 3 carcasses, and a mixed bacterial flora from the re-maining carcass. Bacteria could not be isolated from lesions situated behind the shoulder in 7 carcasses from 3 herds free from G. pseudo-tuberculosis infection. It is concluded that most swellings on the shoulder and chest in goats were granulomas resulting from vaccination against paratuber-culosis.