A serological investigation of Bovine enterovirus-1, Bovine herpesvirus-1, Bovine viral diarrhea virus, and Parainfluenza-3 infections in camelsin Western Turkey.

Camels (Camelus dromedarius) are bred in Western Turkey, particularly in the province of Aydin, for touristic, social and cultural purposes. Bovine enterovirus­1 (BEV­1), Bovine herpesvirus type­1 (BHV­1), Bovine viral diarrhea virus (BVDV), and Parainfluenza­3 (PI­3) virus infections are significant causes of health and/or economic concerns in several animal species. These agents have not been investigated in the camel population in Turkey. The objective of this study was to serologically investigate the presence and infection rates of these viruses in camels in Aydin province, Western Turkey. Ninety­two serum samples were taken from clinically healthy camels that were kept in private farms or brought to the local slaughterhouses. Serum neutralization test was performed to assess the presence and the titers of specific antibodies against BEV­1, BHV­1, BVDV, and PI­3 virus in camel sera. Of the 92 camels tested, 30 (32.61%), 2 (2.17%), 54 (58.7%), and 20 (21.74%) were seropositive for BEV­1, BHV­1, BVDV, and PI­3, respectively. These results suggest that, except for BHV­1, these viral infections are common among camels in Western Turkey. To our knowledge, this the first comprehensive, large­scale study investigating these viral infections in camels in Turkey.

Improvement of eradication program for infectious bovine rhinotracheitis in France inferred by serological monitoring of singleton reactors in certified BoHV1-free herds.

Within the framework of the national voluntary eradication program for Bovine alphaherpesvirus 1 (BoHV1) in France, the proportion of certified-free herds which experienced no more than two positive animals (termed singleton reactors) steadily increased to reach up to 95% in 2015. The aim of this study was to collate and evaluate serological data to gain insight into these epidemiological questionable BoHV1 seropositive animals. Preliminary evaluation of the performances of BoHV1 ELISA kits using a collection of 997 field sera with well-defined status revealed a relatively low specificity of the two gB blocking ELISAs most used in France for confirmatory testing (93.2% and 97.5% for gB-IDVet and gB-Idexx, respectively). In both ELISAs, the suboptimal specificity was associated with the presence of antibodies against BoHV2. Reassessment of the cut-offs led to a specificity and a sensitivity higher than 99.3%. Consequently, a comprehensive analysis of gB-positive sera from 2551 singleton reactors was performed by using gB ELISAs with optimized cut-offs, combined with viral neutralization test (campaign 2014-2015) or gE ELISA (campaign 2015-2016). Fifty percent of the 728 sera collected in 2014-2015 reacted below the optimized cut-offs in both gB ELISAs. Analysis of new blood samples collected at a minimum 6-week interval showed that these weak-positive reactions did not increase with time and could not be confirmed by confirmatory tests. Among the 1823 sera collected in 2015-2016, only 84 samples tested positive by gE ELISA, most of them corresponding to sera with reactivity above the optimized cut-offs in gB ELISAs. Screening for BoHV2 antibodies revealed a significantly increased prevalence among herds with singleton reactors, compared with the between-herd prevalence in French cattle herds. Altogether, these results provided suitable analytical strategies to limit the occurrence of false-positive BoHV1 reactions and inappropriate withdrawal of the BoHV1-free status, without alteration of diagnostic costs and reliability of eradication programs.

Bayesian estimation of herd-level prevalence and risk factors associated with BoHV-1 infection in cattle herds in the State of Paraíba, Brazil.

A cross-sectional study was carried out to estimate the animal- and herd-level prevalence of bovine herpesvirus 1 (BoHV-1) infection in cattle in the State of Paraíba, and to identify risk factors associated with herd-level infection. The state was divided into three sampling strata, and for each stratum, the prevalence of herds infected with BoHV-1 was estimated through a two-stage sampling survey carried out from September 2012 to January 2013. In total, 2443 animals were sampled from 478 herds. A virus-neutralization test was used for BoHV-1 antibody detection. A Bayesian latent-class model was used to describe the data, taking into account imperfect diagnostic test characteristics and the non-independence of test results from animals within the same herd, and using a dynamic within-model risk factor selection method based on indicator variable selection. The adjusted herd-level prevalence was estimated to be 84% (95% CI: 80-88%) for the State of Paraíba, and the animal-level prevalence was estimated to be 73% (95% CI: 66-84%). Only five of the available risk factors were used by the model, with the three most influential being disposal of aborted foetuses (3.78, 95% CI: 1.11-13.85), sharing resources with other farms (3.0, 95% CI: 1.1-8,6), and a herd size of > 23 animals (2.5, 95% CI: 1.1-6.0). Our findings suggest that the animal- and herd-level seroprevalence of BoHV-1 infection in the State of Paraíba is high. While some risk factors such as herd size and sharing resources were identified as risk factors for BoHV-1 infection, these risk factors are initially likely to be of only minor relevance in a control programme due to the extremely high prevalence of infected farms. However, the results are relevant to the risk of reintroduction of disease on farms that have previously eradicated the disease.

Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1.

Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.

Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle.

Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.

A potentiometric biosensor for rapid on-site disease diagnostics.

Quantitative point-of-care (POC) devices are the next generation for serological disease diagnosis. Whilst pathogen serology is typically performed by centralized laboratories using Enzyme-Linked ImmunoSorbent Assay (ELISA), faster on-site diagnosis would infer improved disease management and treatment decisions. Using the model pathogen Bovine Herpes Virus-1 (BHV-1) this study employs an extended-gate field-effect transistor (FET) for direct potentiometric serological diagnosis. BHV-1 is a major viral pathogen of Bovine Respiratory Disease (BRD), the leading cause of economic loss ($2 billion annually in the US only) to the cattle and dairy industry. To demonstrate the sensor capabilities as a diagnostic tool, BHV-1 viral protein gE was expressed and immobilized on the sensor surface to serve as a capture antigen for a BHV-1-specific antibody (anti-gE), produced in cattle in response to viral infection. The gE-coated immunosensor was shown to be highly sensitive and selective to anti-gE present in commercially available anti-BHV-1 antiserum and in real serum samples from cattle with results being in excellent agreement with Surface Plasmon Resonance (SPR) and ELISA. The FET sensor is significantly faster than ELISA (<10 min), a crucial factor for successful disease intervention. This sensor technology is versatile, amenable to multiplexing, easily integrated to POC devices, and has the potential to impact a wide range of human and animal diseases.

Evaluation of total oxidative stress and total antioxidant status in cows with natural bovine herpesvirus-1 infection.

Viruses, including herpes viruses, can alter oxidative balance by either increasing the formation of free radicals or inhibiting synthesis of enzymes involved in oxidative defense within host cells. This study examined the occurrence of oxidative and antioxidative balance in cows naturally infected with bovine herpesvirus type 1 (BHV-1) under field conditions. Clinical history indicated that cows had been sick and showed mild to severe respiratory signs, characterized by dullness, coughing, and lacrimation, and a high febrile response. All samples obtained from the infected animals during clinical examination were confirmed as positive for bovine herpesvirus type 1 by PCR. Control cows showed no clinical abnormalities and PCR results were negative. Total antioxidative status, total oxidant status, oxidative stress index, and some biochemical parameters were measured. The level of total antioxidative status was significantly lower in infected animals, compared with the healthy control group (P = 0.025). However, there was no statistically significant difference between the 2 groups for total oxidant status and oxidative stress index levels. Furthermore, there was a significant decrease in the infected groups, with respect to concentrations of alkaline phosphatase, alanine transferase, γ glutamyl transferase, monocyte, and erythrocyte (P < 0.05). On the other hand, aspartate aminotransferase and creatinine kinase concentrations significantly increased in the cows infected with BHV-1. In conclusion, the data obtained hereby explained that animals with infected BHV-1 seemed to have more oxidative stress and low antioxidant defense. Moreover, future research conductance is needed on antioxidative and oxidative balance to understand pathophysiology of BHV-1 infections.

Isolation and characterization of BoHV-1 from seropositive cows after inducing artificial stress in West Bengal, India.

Infectious Bovine Rhinotracheitis (BoHV-1) is the most important emerging disease of cattle in India. With an aim to reactivate BoHV-1 from latently infected sero-positive cattle for molecular characteristics of the isolates prevalent in tropical and sub-tropical countries like India and further epidemiological investigations on IBR infections this study had been conducted. Artificial stress with dexamethasone at the dose rate of 0.1 mg kg(-1) body weight for 5 consecutive days was induced in BoHV-1 sero-positive cows. Then isolation from nasal swabs was attempted in Madin Darby Bovine Kidney (MDBK) cell line to find out the prevalent strain in India. The virus was isolated from all the three cows. All the three isolates were typed as BoHV-1.2 (Strain India 4, India 5 and India 6). The reactivation obtained in this study with dexamethasone suggests the usefulness of BoHV-1 cow latency model for epidemiological investigations on BoHV-1 infections in tropical and sub-tropical countries like India, Pakistan etc.

The effects of vaccination on serum hormone concentrations and conception rates in synchronized naive beef heifers.

Crossbred beef heifers (N = 59) were vaccinated at the time of synchronization/breeding with either a commercially available bovine herpesvirus type 1 modified live virus (MLV) (one dose) or inactivated virus vaccine (one or two doses). The estrus cycle was synchronized at vaccination and heifers were artificially inseminated 8 days (one dose) or 36 days (two dose) after initial vaccination. Pregnancy rates were greater for control heifers (90%; P = 0.02) and heifers given the inactivated virus vaccine (one dose: 86%; P = 0.08; or two: 90%; P < 0.01) than those given the MLV vaccine (48%). No control heifers experienced an abnormal estrous cycle, whereas only two (two dose; 2/21) and one (one dose; 1/7) heifers in the inactive virus groups had abnormal estrous cycles and were similar to control (P > 0.10). Heifers given the MLV vaccine had a greater (P = 0.02) percentage of abnormal estrous cycles (38%; 8/21) compared with the control and inactivated groups. Of the heifers with an abnormal estrous cycle, 100% of heifers given the inactivated vaccine (one or two dose) conceived at their return estrus, whereas only 38% of heifers given the MLV vaccine conceived at their return estrus (P > 0.10). During the synchronization period, concentrations of estrogen were greater (P < 0.01) in the control and the two-dose inactivated group compared with the MLV group. After AI, progesterone concentrations were greater (P < 0.01) in control heifers compared with the inactivated and MLV groups, but were similar (P ≥ 0.18) between the inactivated and MLV groups. Therefore, naïve heifers vaccinated with the inactivated vaccine were less likely to have an abnormal estrous cycle and had significantly higher pregnancy rates compared with heifers vaccinated with the MLV vaccine. In summary, vaccination of naïve heifers with an MLV vaccine at the start of a fixed-time AI protocol had a negative effect on pregnancy success.

Serological evidence of bovine herpesvirus-1 (BoHV-1) infection in yaks (Peophagus grunniens) from the National Research Centre on Yak, India.

Bovine herpesvirus-1 (BoHV-1) causes a variety of disease syndromes including, respiratory, nervous and reproductive disorders both in domestic as well as wild bovines and the disease is prevalent throughout the world including India. In this study, serum samples of yaks were screened for serological evidence of BoHV-1 in a yak farm with history of abortion and kerato-conjuctivitis by competition-ELISA. The result of seroprevalence of BoHV-1 infections in yaks (Poephagus grunniens) revealed that the overall seroprevalence was 60.1%. The sero-prevalence of BoHV-1 infections was highest in male calf (67.7%) followed by yak cows (62.6%), yak bulls (56.8%), and yak heifers (50.0%).

Serological response in cattle immunized with inactivated oil and Algel adjuvant vaccines against infectious bovine rhinotracheitis.

Infectious bovine rhinotracheitis (IBR) virus was grown in Madin Darby bovine kidney (MDBK) cell line using a roller culture system for its large-scale production. Optimum multiplicity of infection (MOI) of 1:750 was found to give consistent virus yield. To determine the appropriate payload, three batches of antigen with virus titres ranging from 10(8.37) to 10(6.37) TCID50 per ml were used to prepare experimental inactivated IBR oil adjuvant vaccine. Beta-propiolactone (BPL) was used as inactivant. The vaccine formulation using inactivated BHV-1 virus antigen with a pre-inactivation titer of 10(8.37) TCID50 per dose elicited better sero-conversion in cattle calves as evidenced from the mean log SN titre of 1.02. To choose the appropriate adjuvant, two batches of vaccine each containing aluminum hydroxide gel (Algel) and Montanide oil respectively were tested in calves. Two groups of 16 calves each were inoculated with Algel and oil adjuvant vaccine respectively twice at four weeks to test the immunogenicity. Adequate titres of vaccine induced anti BHV-1 antibodies could be demonstrated both by ELISA and MNT up to 180 days post vaccination in both the groups.

Serological survey of bovine herpesvirus type 1 infection in China.

To understand the nationwide seroprevalence of bovine herpesvirus type 1 (BoHV-1) infection of cows in China, 1344 sera of dairy cows from 29 provinces and 765 sera from 6 herds in Hubei province were collected with stratified random sampling. Another 483 sera from imported cows were included. The serum antibody was tested by BoHV-1 gG ELISA. The results demonstrated that the overall nationwide seroprevalence was 35.8% (481/1344), while the prevalence for individual province ranged from 12.1% to 77.8%. Although each province had positive samples, the prevalence was clustered in areas based on the cow population size. In Hubei Province, the overall seroprevalence was 22.2% (170/765) while the prevalence for individual farms varied greatly from 0.0% to 41.5%. The sera from imported cows had a moderate prevalence of 21.7% (105/483).

[Use of polymeric suspensions as a viral sorbent to detect cattle serum antibodies].

The paper shows it possible to use stained polymeric microspheres, 1.7 microm in diameter, that contain viruses onto the surface, in the latex agglutination test to detect antibodies to the bovine serum viruses of infective rhinotracheitis, parainfluenza-3, viral diarrhea, respiratory syncytial infection, and adenoviral infection.

Stochastic modelling as a tool for planning animal-health surveys and interpreting screening-test results.

Traditionally, the planning of surveys (in particular, sample-size calculations) has relied on assumptions including the assumption of perfect screening tests. This paper presents a novel approach that can be used for planning animal-health surveys and interpreting screening-test results in the context of these surveys. A stochastic simulation model developed to assess the properties of herd-level sampling schemes and surveys has been adapted for large surveys aimed at substantiating freedom from infection at a national or regional level. We use a Bayesian approach to derive the post-survey probability of freedom from infection from the pre-survey probability of freedom and the likelihood ratio that is associated with screening-test results. We applied the model to two consecutive surveys conducted in 1998 and 1999 in Switzerland to substantiate freedom from infectious bovine rhinotracheitis (IBR) in the cattle population of about 56000 herds (median herd size of 15 cattle > 2 yr of age in 1999). In 1998, serum samples were taken from five cattle > 2 yr in 4672 herds, and in 1999 from all cattle > 2 yr old in 648 herds; samples were analysed by ELISA. The survey of 1999 provided less evidence than that of 1998 to support a status of freedom from infection; also, the characteristics of both herd-level sampling schemes were similar. We argue that the rationale for survey planning depends on the pre-survey probability of freedom from infection (i.e. our level of confidence that the infection does not occur in the targeted animal population). In consequence, surveys should be tailored to individual populations in the respective countries or regions. The model has been developed in an Excel spreadsheet to allow flexibility of use, and adaptation to many other animal-health issues.

Analysis by enzyme-linked immunosorbent assay and 2-dimensional electrophoresis of haptoglobin in the high-density lipoprotein fraction in cows.

Haptoglobin (Hp) is a hemoglobin (Hb)-binding acute-phase protein. Besides its relevance in inflammation, Hp is involved in the regulation of lipid metabolism. In cattle, in addition to the lipoprotein-deficient fraction, Hp is distributed in high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL) fractions. The purpose of this study was to determine Hp concentrations in the lipoprotein fractions using an enzyme-linked immunosorbent assay (ELISA) based on the affinity with Hb, and also to detect structural differences of HDL Hp from that in the lipoprotein-deficient fraction using 2-dimensional electrophoresis. When purified Hp was used as the antigen for the ELISA, the detection limit was 7.4 ng/ml and linearity was obtained from 14.8 to 475 ng/ml. The correlation coefficient between the ELISA and single radial immunodiffusion was 0.884. The ELISA was shown to be applicable to evaluate Hp concentrations in the lipoprotein fractions. Hp concentrations in the lipoprotein fractions were in the range of 0.94 to 8.77 microg of Hp/ml (n = 4), and concentration ratios were 0.2 to 0.3% of whole serum Hp. Of the lipoprotein fractions, Hp was most abundant in HDL, moderate in VHDL and faint in chylomicrons, the very low-density lipoprotein fraction and low-density lipoprotein fraction. By 2-dimensional electrophoresis, alpha- and beta-chains of serum Hp were each separated into 5 spots, and their isoelectric point (pI) values were from 5.05 to 6.28 in the alpha-chain and from 5.92 to 6.95 in the beta-chain. The pI values of HDL Hp were indistinguishable from those of serum Hp. These results indicate that the ELISA based on the affinity with Hb is useful for evaluating Hp concentrations in lipoprotein fractions, and also suggest that HDL Hp is structurally similar to that in the lipoprotein-deficient fraction.

Prevalência de anticorpos neutralizantes contra o herpesvírus bovino tipo 1, decorrente de infecçäo natural, em rebanhos com distúrbios reprodutivos / Prevalence of neutralizing antibodies against bovine herpesvirus type 1, due natural infection, in herds with reproductive problems

A detecçäo de anticorpos anti-Herpesvírus Bovino tipo 1 (BHV-1) foi realizada, através da técnica de soroneutralizaçäo, em 1235 amostras de soro de bovinos adultos, näo vacinados contra Rinotraqueíte Infecciosa Bovina. As amostras de soro analisadas foram colhidas em 81 rebanhos, com histórico de problemas reprodutivos, incluindo animais com aptidäo para carne e leite, provenientes de 30 municípios do Estado do Paraná. Na amostragem proveniente de rebanhos leiteiros, 41,9 por cento (409/977) das amostras de soro e 90,5 por cento (57/63) dos rebanhos foram considerados positivos. Em bovinos de corte, o índice de soropositividade foi de 50,8 por cento (131/258) e 100 por cento (18/18) para amostras de soro e rebanhos, respectivamente. As frequências de 43,7 por cento (540/1235) de animais e 92,6 por cento (75/81) de rebanhos soropositivos demonstram que as infecçöes por BHV-1 apresentam-se amplamente disseminadas nas regiöes estudadas.

Prevalence of antibodies to IBR and BVD viruses in dairy cows with reproductive disorders.

We determined the prevalence of antibodies to infectious bovine rhinotracheitis virus (IBRV) and bovine viral diarrhea virus (BVDV) in sera of dairy cows on 4 different farms in the Republic of Croatia. A high percentage (60.8%) of cows had various reproductive disorders. The results showed that seroprevalence of infectious bovine rhinotracheitis (IBR) was 85.8% and that of bovine viral diarrhea (BVD) was 79.2% in tested cows. Antibodies to both viruses were found in 80.8% of cows with reproductive disorders but in only 46.8% of cows without reproductive disorders. This difference was statistically significant (P<0.01), and indicated a connection between reproductive disorders and simultaneous infections with IBR and BVD viruses in dairy cows.

Standardization of enzyme-linked immunosorbent assays (ELISAs) for quantitative estimation of antibodies specific for infectious bovine rhinotracheitis virus, respiratory syncytial virus, parainfluenza-3 virus, and bovine viral diarrhea virus.

Commercial enzyme-linked immunosorbent assays (ELISAs) for detection of serum antibodies to bovine viral diarrhea virus (BVDV), parainfluenza-3 virus (PI3V), respiratory syncytial virus (RSV), and infectious bovine rhinotracheitis virus (IBRV) were standardized to give a quantitative result when testing was performed at a single optimum dilution. For each test, serum samples were titrated and their end point titers calculated by an algebraic method directly from a plot of each titration series and also from a regression line fitted to this plot. The corrected optical density (COD) of each sample when tested at dilutions of 1/25, 1/50, and 1/100 was expressed as a percentage of the COD of a positive reference serum included on each plate, this value was the sample/positive (S/P) ratio. For each test, the linear relationship between the S/P ratio obtained at a dilution of 1/25, 1/50, and 1/100 and the end point titer calculated by each method was determined. In each case, the best linear relationship existed when samples were tested at a dilution of 1/100 (r = 0.973 for BVDV, 0.962 for PI3V, 0.961 for RSV, 0.947 for IBRV). From the equation of these lines, an increase in the S/P ratio between acute and convalescent serum samples of 31%, 23%, 21%, and 35% would correspond to a 4-fold rise in ELISA titer to BVDV, PI3V, RSV, and IBRV, respectively. ELISA titers calculated from S/P ratios at 1/100 were significantly related to virus neutralization titers to BVDV, RSV, and IBRV and to hemagglutination inhibition titers to PI3V (P < < 0.001 in all cases). Samples with low S/P ratios had the greatest intraassay and interassay variation. Intraassay reproducibility ranged from 3.5% to 22.3% (coefficient of variation), with a median value of 9.5%. Interassay reproducibility was lower, ranging from 6.0% to 50.6%, with a median of 17.4%.

A comparison of different models for assessing variations in the sero-prevalence of infectious bovine rhinotracheitis by farm, area and district in Kenya.

The relative variability of the sero-prevalence of antibodies to infectious bovine rhinotracheitis (IBR) due to cow, farm, and agroecological area levels were investigated for three contrasting districts in Kenya: Samburu, an arid and pastoral area; Kiambu, a tropical highland area; and Kilifi, a typical tropical coastal area. Cattle were selected by two-stage cluster sampling and visited once between August 1991 and 1992. Data on animal, farm, and area factors were analyzed using Schall's algorithm and MLn (multi-level, n-level), two generalized mixed-model programs suitable for multi-level analysis. Most variation in IBR sero-prevalence was from farm-to-farm. This was reflected by the many farm-level fixed effects (farm size, disease control measures and type of breeding) significant in models both ignoring and accounting for single variance components (clustering) at farm, area, and district levels. Area-to-area and district-to-district variations were noted but the area and district variance components were one-third and one-fifth the size of the farm variance components for both methods. As farm-to-farm variation differed markedly by farm size and district, models in MLn were extended to allow for multiple farm-level variance components by these categories. For each, sero-prevalence of IBR increased with age and was significantly decreased on small-sized zero-grazing farms. These models, particularly the model with different farm variance components by districts, fit the data better and highlighted well that there was considerable farm-to-farm variation--differing by district--and that the available farm-level fixed effects did not predict IBR sero-prevalence well.