Dose response of black American mink to Aleutian mink disease virus.

INTRODUCTION: Aleutian mink disease virus (AMDV) causes a serious health problem for mink globally. The disease has no cure nor an effective vaccine and selection for tolerance using antibody titer is adopted by many mink farmers. The objective of this study was to investigate the effects of various doses of a local AMDV isolate on the response of black American mink to infection with AMDV. METHODS: Eight black American mink were each inoculated intranasally with 0.5 mL of eight serial 10-fold dilutions (100 to 10-7 ) of a 10% spleen homogenate containing a local AMDV isolate. Blood samples were collected on days 0, 20, 35, 56, 84, 140, and 196 postinoculation (dpi). Anti-AMDV antibodies and viral DNA were tested by counter-immunoelectrophoresis (CIEP) and PCR, respectively. Animals that were PCR or CIEP positive at 196 dpi (n = 41) were killed at 218 dpi, and samples of blood and seven organs were tested by CIEP and PCR. RESULTS: Antibody production persisted in all seroconverted mink until the termination of the experiment, whereas 71.1% of the mink showed short-lived viremia. Significant associations were observed between inoculum dose and the incidence of viremia until 84 dpi which disappeared thereafter, whereas associations between inoculum dose and the incidence of seropositive mink were significant on all sampling occasions. Antibody titer at 218 dpi significantly decreased with decreasing inoculum dose. AMDV DNA was detected in the bone marrow, lymph nodes, and spleen samples of almost all mink inoculated at every dose but was not detected in other organs of some mink. CONCLUSIONS: CIEP is more accurate than PCR for detecting AMDV infection in mink. Using antibody titer in naturally infected mink may not be accurate for the identification of tolerant mink.

Prevalence of the Aleutian mink disease virus infection in Nova Scotia, Canada.

Despite many years of testing mink for serum antibodies against the Aleutian mink disease virus (AMDV) by counterimmunoelectrophoresis (CIEP) and elimination of reactors, this virus has remained the number one disease threat for the mink industry in Nova Scotia (NS). The objective of this study was to analyze CIEP test results to determine the success of the AMDV-control strategy in NS. A total of 2,964,920 CIEP test results from 82 ranches, spanning an eight-year period between 1998 and 2005, were analyzed. This survey included approximately 60% of the active ranchers in the province. The number of ranchers that tested their animals was 42 in 1998, gradually increased to 58 in 2003 and then showed some decline. The overall proportion of CIEP-positive mink was 3.34%, and varied between 5.22% in 1999 and 1.35% in 2005. The proportion of infected ranches ranged between 23.8% in 1998 and 70.7% in 2003. The overall trend was for a smaller proportion of infected animals but a larger proportion of infected ranches during this time period. Of the 82 ranches, 24 (29.3%) had negative CIEP in all tests, 15 (18.3%) had CIEP positive animals in every year tested, and 43 (52.4%) had positive and negative results in different years, indicating that AMDV infection was widespread in NS. There were 23 infected ranches with 8 years of uninterrupted testing. These ranchers performed 75.8% of the total samples tested (2,246,711), implying that they have diligently been trying to eradicate the virus. Infection persisted on three of these ranches for the entire 8 year period, and only two of the ranches remained CIEP negative for longer than four years. The average percentage of CIEP-positive mink on these ranches was 2.2, which was lower than 6.35% for the 33 infected ranches with occasional testing, and 73.6% and 82.4% for two ranches that had never used the CIEP test, showing that persistent test-and-removal strategy has been effective in reducing the prevalence of infected animals but has failed to eradicate the virus from most of the infected ranches.

Caprine pleuropneumonia in Beetal goats [corrected].

Seroprevalence, clinical findings, and lesions of contagious caprine pleuropneumonia (CCPP) in Beetal goats were recorded during an outbreak. The overall seroprevalence of CCPP was 32.50%. Confirmation of Mycoplasma mycoides in serum was carried out using counter immunoelectrophoresis (CIE) technique. The highest CIE-positive cases were recorded in the older goats (51.72%) as compared to young ones. Nasal swabs collected from 39 goats showing respiratory signs were found positive for M. mycoides. The most consistent clinical findings were mild to severe cough, purulent nasal secretion, emaciation, dyspnea, increased respiration rate, and pyrexia. Mortality due to CCPP was 9.17%. Consolidation of lungs exhibited the highest frequency (100%), followed by alveolar exudation (90.90%) and pleural adhesion (72.72%). Among the microscopic lesions, septal peribronchiolar fibrosis exhibited the highest frequency (81.81%), followed by fibrinous pleuritis (63.63%) and peribronchiolar cuffing of mononuclear cells (54.54%) in lungs. From these results, it was concluded that CCPP under subtropical conditions has high prevalence in Beetal goats and leads to significant mortality.

Zoonotic infections of buffalopox in India.

Four outbreaks of buffalopox in domestic buffaloes, with considerable mortality with high case fatality rates in young buffalo calves and high morbidity with significant productivity loss in terms of reduction in milk yield in adult animals along with severe zoonotic infection in milk attendants were recorded at various places in India, during 2006-2008. In buffaloes, the pox lesions were confined to udder and teats of the majority of the affected animals, and in few animals the lesions were appeared on the hindquarters, indicating generalized infection. The overall disease morbidity, mortality and case fatality rate were 6.8%, 0.7% and 11.4% respectively. Milkers developed pox-like lesions on the hands, forearms and forehead accompanied by fever, axillary lymphadenopathy and general malaise. The causative agent of the outbreaks, buffalopox virus (BPXV), was confirmed upon virus isolation in cell culture, electron microscopy, A-type inclusion (ATI) and ankyrin repeat protein (C18L) gene-specific polymerase chain reactions (PCR). Further, sequence analysis of the BPXV isolates from human and buffalo showed more identity of ATI and C18L genes sequences with that of other orthopoxviruses at nucleotide and amino acid levels and confirmed a close relationship of BPXV with Vaccinia virus (VACV) or VACV-like viruses. Considering the zoonotic impact and productivity losses of buffalopox infection, the control measures are imperative in curtailing economic and public health impact of the disease.

Antibody seroprevalences against Peste des Petits Ruminants (PPR) virus in sheep and goats in Sudan.

Counter immnuo-electrophoresis (CIEP) and Competitive ELISA (C-ELISA) tests were employed for seroprevalence of Peste des Petits Ruminants (PPR) infection in Sudan. The result of both tests showed high prevalence of PPRV antibodies in sheep and goats sera collected from six different regions of Sudan. Of the 519 serum samples examined for the presence of PPRV antibodies 307(59.15%) were positive by CIEP while 263(50.67%) were positive by C-ELISA. CIEP technique was shown to be more sensitive than C-ELISA technique for detection of PPRV antibodies (Kappa statistics 0.259). C-ELISA allowed rapid, simple, specific, sensitive and differential sero-diagnosis of PPRV and RPV in sheep, goats and cattle. CIEP is, unlike competitive ELISA, is group-specific test and can not differentiate between PPR and RP infections. Despite its low specificity CIEP can be a useful indicative screening test for PPRV antibodies in flocks that neither been vaccinated nor otherwise exposed to PPR or RP virus. Results obtained suggest that CIEP, like the HI test, could be a useful screening test where it is not possible to use C-ELISA.

An outbreak of sheep pox on a sheep breeding farm in Jammu, India.

An outbreak of sheep pox occurred in December 2001 on a sheep breeding farm in Jammu, India. The farm maintains three exotic breeds of sheep, i.e. American Merino, Rambouillet and Australian cross. The disease agent was confirmed as sheep pox virus by clinical and post-mortem examination as well as laboratory testing. Typical pock lesions were dispersed over the body of the affected animals with nodular lesions observed in the lung tissue of the dead animals. Sheep pox virus antigen and antibody were detected in infected tissue and convalescent sera, respectively, with serological tests. Viral deoxyribonucleic acid was extracted from the infected tissue and amplified using a diagnostic polymerase chain reaction. Sheep of the Rambouillet breed were found to be most susceptible to infection with morbidity and mortality rates of 26.9% and 8.3%, respectively. Morbidity and mortality rates in the entire flock were 18.4% and 6.3%, respectively. The grazing and migration pattern indicates that the disease was probably introduced to the farm by local sheep.

Comparison of four techniques for the detection of Clostridium perfringens type D epsilon toxin in intestinal contents and other body fluids of sheep and goats.

Polyclonal capture enzyme-linked immunosorbent assay (PC-ELISA), monoclonal capture ELISA (MC-ELISA), mouse neutralization test (MNT), and counterimmunoelectrophoresis (CIEP), were compared for their ability to detect epsilon toxin in intestinal contents and body fluids of sheep and goats. When used to evaluate intestinal contents of sheep artificially spiked with epsilon prototoxin, PC-ELISA detected 0.075 mouse lethal dose (MLD)50/ml, whereas the MNT, MC-ELISA, and CIEP detected 6, 25, and 50 MLD50/ml, respectively. Amounts of epsilon toxin detected by PC-ELISA, MC-ELISA, MNT, and CIEP in sheep pericardial fluid artificially spiked with epsilon prototoxin were 0.075, 0.75, 6, and 200 MLD50/ml, respectively. For assaying epsilon toxin in aqueous humor, PC-ELISA and MC-ELISA detected 0.075 MLD50/ml, whereas CIEP detected 200 MLD50/ml (MNT was not evaluated). When 51 samples of intestinal contents of sheep and goats (32 positive and 19 negative to MNT) were analyzed by the other 3 techniques, the relative sensitivity of PC-ELISA, MC-ELISA, and CIEP was 93.75, 84.37, and 37.50%, respectively. The specificity of PC-ELISA, MC-ELISA, and CIEP was 31.57, 57.89, and 84.21%, respectively. The absolute sensitivity of PC-ELISA, MC-ELISA, CIEP, and MNT was 90.90, 69.69, 15.15, and 54.54%. The absolute specificity of the 4 techniques was 100%. These results show that there is a marked inconsistency among techniques routinely used to detect Clostridium perfringens epsilon toxin. Until more consistent results are achieved, the diagnosis of enterotoxemia should not only be based solely on epsilon toxin detection, but also on clinical and pathological data.

Effect of a valine residue at codon 352 of the VP2 capsid protein on in vivo replication and pathogenesis of Aleutian disease parvovirus in mink.

OBJECTIVE: To determine whether a group of 3 genetic differences in the nonstructural protein (NS1) or 1 genetic difference in the structural protein (VP2) of Aleutian disease parvovirus (ADV) is responsible for an increase in the in vivo replication and pathogenicity of G/U-8, a chimera of ADV-G (nonpathogenic) and ADV-Utah (pathogenic), compared with G/U-10. ANIMALS: 32 eight-month-old female sapphire mink (Mustela vison). PROCEDURE: Chimeric viruses were constructed, propagated in vitro, and used to inoculate mink. Antiviral antibody responses, presence of serum viral nucleic acid, and serum gamma globulin concentrations were monitored for 120 days following inoculation. Histologic examination of the liver, kidneys, spleen, and mesenteric lymph nodes was performed after necropsy. RESULTS: A chimera containing only the 3 amino acid substitutions in NS1 did not elicit measurable responses indicative of replication or pathogenicity in inoculated mink. Serum antiviral antibody responses, frequency of detection of viral nucleic acid in serum, gamma globulin response, and histologic changes in mink inoculated with chimeras containing a valine residue at codon 352 (352V) of VP2 capsid were increased, compared with values from mink inoculated with chimeric viruses that did not contain 352V. CONCLUSIONS AND CLINICAL RELEVANCE: A valine residue at codon 352 in the VP2 capsid protein of ADV affects in vivo viral replication and pathogenicity. This amino acid may be part of an incompletely defined pathogenic determinant of ADV. Further characterization of the pathogenic determinant may allow future development of focused preventive and therapeutic interventions for Aleutian disease of mink.

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains.

Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two field strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identified as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a confirmatory test to identify serotype-specific capsular and somatic antigens.

Rocket immunoelectrophoresis in the diagnosis of infectious bursal disease.

The rocket immunoelectrophoresis (RIE) test was used for the qualitative detection and quantitative estimation of infectious bursal disease virus (IBDV) specific antigen in experimentally infected chickens and samples collected from suspected outbreaks. The IBDV specific antigen was detected in the bursae of experimentally inoculated chickens up to 5 days post infection (PI) by the agar gel precipitation (AGP) test and 7 days PI by the RIE test. The RIE detected IBDV specific antigen in a significantly greater number of samples collected from the field outbreaks than the conventional AGP test. Exudative bursae were found to have a higher antigen content than haemorrhagic bursae and are recommended as the material of choice for diagnosis of IBD. This test could also be used to quantify IBDV specific antigen in commercial killed vaccines.

Fasciola hepatica: partial characterization of circulating antigens.

Antigens present in sera from Fasciola hepatica-infected goats were partially characterized by means of polyacrylamide gel electrophoresis of immunoprecipitates obtained by counterimmunoelectrophoresis. Agar containing the 2 immunoprecipitin lines, agar containing no precipitin lines, a crude extract of F. hepatica, and normal rabbit IgG were assayed. The electrophoretic pattern of 1 precipitin line showed, apart from the light and heavy chains of IgG and other faint bands, 2 prominent polypeptides of about 70 and 85 kDa. The other precipitin line showed a similar pattern, but with the 70-kDa band absent.

Coproantigens: early detection and suitability of an immunodiagnostic method for echinococcosis in dogs.

In the present study immunodiagnostic tests for Echinococcus granulosus-specific coproantigens have been evaluated. The techniques are based on polyclonal antisera collected from dogs experimentally infected with E. granulosus protoscoleces. ELISA, Countercurrent immunoelectrophoresis (CCIEP) and Immunodot were evaluated for their application in coprodiagnosis. The level of coproantigens was detectable on day 7 post-infection and reached a maximum level on day 56 as detected by ELISA. Very high O.D values were obtained when faecal samples of 7-8 weeks post-infection were used. Besides this, immunodot and CCIEP were also tested and the 4th week post-infection samples gave significant reactions. It is evident from the results that ELISA was more sensitive and detected the coproantigens as early as the first week post-infection whereas immunodot and CCIEP detected coproantigens later. The latter methods are rapid, cost-effective and can be used to diagnose suspect cases of echinococcosis under field conditions. Thus, it is suggested that future studies should be aimed at early detection of echinococcosis by a rapid and cost-effective immunodot test.

Hydropericardium syndrome (HPS) in India: a preliminary study on the causative agent and control of the disease by inactivated autogenous vaccine.

Hydropericardium syndrome (HPS) in broiler birds of 3 to 6 weeks of age was recorded for the first time in the Haldwani area of Nainital district (UP) in India in November, 1994. The overall mortality in 6 poultry farms was 61.62 per cent. The disease was experimentally transmitted by bacteria free infected liver homogenate extract passed through membrane filters of 0.22 and 0.1 mu APD. The aetiological agent was inactivated by heat treatment at 56 degrees C for one hour and 80 degrees C for 10 min. A precipitin band was demonstrated in agar gel immunodiffusion and counter immunoelectrophoresis using infected liver homogenate extract as antigen and homologous antisera raised in the laboratory. The disease was effectively controlled by formalinised and heat inactivated autogenous vaccine prepared from the infected livers of birds which died of natural infection.

Comparative evaluation of agar gel precipitation, counterimmunoelectrophoresis and passive haemagglutination tests for the diagnosis of Dicrocoelium dendriticum infection in sheep and goats.

The sensitivity and specificity of the agar gel precipitation test (AGPT), counter immunoelectrophoresis (CIEP) and passive haemagglutination test (PHT) were evaluated for the diagnosis of Dicrocoelium dendriticum infection in naturally infected sheep and goats. Two hundred and forty five sheep and goat sera samples were tested using phosphate buffered saline, pH 7.2 extracted adult fluke antigen. CIEP detected 69.8% of the infected animals and was found to be the most sensitive, followed by PHT which detected 50.0% of the infected sheep and goats. AGPT was found to be the least sensitive, detecting only 23.8% of the infected animals. The specificity of

Quantitative counter-immunoelectrophoresis for estimation of antibodies to infectious bursal disease virus.

Quantitative counter-immunoelectrophoresis was standardized to detect antibodies to the avian infectious bursal disease virus. This technique correlated well with the conventional quantitative agar gel precipitation test in estimating antibodies to IBDV. The use of blood dried on filter paper as an alternative to serum is discussed. QCIE is simple, easy to perform and faster than QAGP.

Duck hepatitis B virus (DHBV) infection in Indian domestic ducks: a pilot study.

One hundred and two apparently healthy Indian domestic ducks from the Poultry Research Station, Madras were screened for duck hepatitis B virus (DHBV) infection by; 1. screening for the duck hepatitis B virus surface antigen (DHBsAg) in their sera using hepatitis B virus (HBV) reagents, 2. screening for DHBsAg using specific duck hepatitis B virus (DHBV) reagents and 3. demonstration of DHBV DNA using DHBV DNA probe by dot blot hybridisation. While 5 ducks (4.9%) were consistently positive with HBV reagents, use of DHBV reagents showed a total of 4 ducks (including 3 of the above 5) to be positive for DHBsAg. DNA hybridisation showed 6 ducks to be positive for DHBV DNA. On clinical examination, 5 out of these 6 ducks did not reveal abnormalities, the other one showed hepatomegaly and ascites. Post-mortem studies showed the presence of nodules on the surface of the liver in all 5 which were positive with HBV reagents including the one with hepatomegaly. On histopathological evaluation, they were found to be hepatocellular carcinoma with or without bile duct carcinoma. The present study is a pilot report on the occurrence of DHBV infection in Indian domestic ducks and the possibility of antigenic cross reactivity between human HBV and duck hepatitis B virus antigens.

Evaluación de la contrainmunoelectroforesis y la prueba de ELISA en el diagnóstico de la fasiolasis experimental de ratones / Evaluation of the counterelectrophoresis-CEP and the enzyme-linked immunosorbent assay-ELISA in the diagnosis of fasiolasis in mice

Dos pruebas serológicas (contrainmunoelectroforesis-CEP y el ensayo inmunoenzimático-ELISA) fueron utilizadas para hacer un seguimiento longitudinal de 28 ratones infectados con Fasciolasis hepatica. Un grupo de 8 y 10 ratones fueron infectados con 1-2 metacercarias. Los anticuerpos séricos fueron detectados por ELISA a partir de la 1-2da. semana postinfección. Uno de los ratones recién alcanzó un valor de absorbancia diagnóstica a la 5ta. semana. Las precipitinas fueron demostradas por la CEP a la 2-3ra. semana de infección. Sólo en dos casos con diagnóstico comprobado de fasciolasis, no se detectaron precipitinas. Un tercer ratón, positivo mediante ELISA a la 5ta. semana, recién mostró reactividad a la 8va. semana. La prueba de ELISA fue ligeramente más sensible que la CEP para la detección temprana de la infección.

Seroepidemiological survey for yellow fever antibodies in domestic animals.

A total of 192 out of 300 serum samples from camel, cattle, sheep and goats tested for yellow fever virus antibody by the counterimmunoelectrophoresis were found positive. This test was confirmed by the single radial haemolysis and serum neutralization tests. Twenty-one and 36 sera were positive for specific yellow fever virus antibodies by the single radial haemolysis and serum neutralization tests respectively. The possible role of these animals in the epidemiology of yellow fever is discussed.