Detection of specific antibodies in dogs infected with Angiostrongylus vasorum

Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment.     

Serologic detection of Angiostrongylus vasorum infection in dogs

Angiostrongylus vasorum, French Heartworm, is a metastrongylid nematode infecting the pulmonary arteries and right heart of wild and domestic canids in various regions of the world. Infection in dogs can result in fatal cardiopulmonary disease. A single endemic focus of A. vasorum in North America occurs in the southeastern portion of Newfoundland, Canada. Dogs are currently diagnosed by detection of first-stage larvae shed in feces using the Baermann technique or fecal flotation. However, these procedures may lack sensitivity due to intermittent fecal larval shedding. The potential for using detection of circulating worm antigen for diagnosis was investigated by developing a sandwich-ELISA using rabbit anti-whole adult worm antiserum. This test detected circulating antigen in sera from 22/24 Baermann positive dogs naturally infected with A. vasorum. Negative results (0/52) were obtained from sera collected from Baermann negative dogs from outside of the endemic region, and from sera (0/30) from dogs from non-endemic regions that were infected with Crenosoma vulpis, the fox lung worm. Receiver operating curve analysis gave a specificity of 100% and a sensitivity of 92% for the sandwich-ELISA at an optical density cut-off of 0.19. Subsequently, 239 dogs from Newfoundland displaying clinical signs of cardiopulmonary disease, were examined using both the Baermann fecal examination and the sandwich-ELISA. Larvae were detected in 10% (24/239) of these dogs by fecal examination, whereas the sandwich-ELISA detected circulating antigen of A. vasorum in serum from 18.8% (45/239) of the dogs. This suggests that fecal diagnostics may have missed approximately half of the A. vasorum infected dogs, and that the sandwich-ELISA may be a useful tool in the diagnosis of this parasite.     

Serological survey of Neospora caninum in dogs in the Czech Republic and a long-term study of dynamics of antibodies

The main aim of the present study was to establish the prevalence of antibodies against Neospora caninum dogs from the Czech Republic and to examine the dynamics of antibody titers during a long-term period. For this purpose, sera of 858 dogs were examined for the presence of anti-N. caninum antibodies using an indirect immunofluorescent antibody test (IFAT). Four groups of dogs of various origins were included in the survey: the first group (A, n=470) comprised dogs purchased by the Czech Army from the civilian sector throughout the Czech Republic, with 22 (4.7%) N. caninum-positive dogs, second group (B, n=115) represented police dogs with no seropositive animal, third group (C, n=195) were pet dog sera collected for veterinary clinic with 5 (2.6%) anti-N. caninum sera and the fourth group (D, n=78) of canine shelter dogs with the seroprevalence of 19.2%. The differences in seroprevalence were significant (P< or =0.01) between groups B and A, and between D and A. None of the serologically positive animals had clinical signs of neurological disorders. Coprological examination did not reveal any dog shedding N. caninum oocysts. The seropositivity rates for N. caninum were analyzed in relation to other data, such as age, breed and gender. Increased prevalence rates of anti-N. caninum antibodies were found in the older age strata of the dog population sample tested in the present study. We found significantly higher (P=0.02) prevalence in 3-3.5-year-old dogs (11.1% of 36), as compared to 1-1.5-years-old dogs (2% of 98). A longitudinal study of antibody dynamics was carried out in 19 initially seropositive dogs over a period of 4 years. The second and third examinations revealed that antibody titers decreased in majority of positive dogs (10, 52.6%), of which in seven cases (36.8%) the titers fell to levels that are currently considered as being seronegative (titer <1:50), or even became undetectable (titer <1:25).     

Serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test

An indirect fluorescent antibody test was used for detection of serum antibodies to the platelet-specific rickettsial organism that is the causative agent of infectious cyclic thrombocytopenia (ICT) in dogs. The test converted from negative to positive in 7 of 7 experimentally inoculated dogs. One of 2 attempts to recover the rickettsial agent of ICT from naturally occurring seropositive dogs, by blood inoculation of experimental dogs, was successful. Seemingly, the test did not detect antibodies to Ehrlichia canis, nor did a similar test, using E canis antigen slides, detect antibodies to the rickettsial agent of ICT. The rickettsial agent of ICT has been classified tentatively as E platys. When applied to sera from a group of healthy random-source dogs, the test revealed a relatively low (5%) occurrence of positive reactions. A higher occurrence of positive reactions (35%) was noticed in sera from a group of thrombocytopenic dogs from the University of Florida. A majority of these positive sera were also positive for antibodies to E canis. The highest occurrence of positive reactions was found (greater than 50%) in E canis-positive sera from dogs at the University of Florida, as well as from dogs from 9 other states.     

Assessment of the combination of spinosad and milbemycin oxime in preventing the development of canine Angiostrongylus vasorum infections

Angiostrongylus vasorum is an increasingly reported parasite in Europe that develops in dogs after ingestion of infective third stage larvae (L3) that reside in gastropod molluscs which are needed to complete the parasite's life-cycle. Infection can produce a diversity of clinical signs, determined by involvement of the respiratory, neurological, and/or coagulation system, with a likely fatal outcome in the absence of treatment. Few drugs have been shown to reliably prevent infection, and data on treatment of infections is limited. A controlled, randomized, partially blinded laboratory study was therefore executed to evaluate the efficacy and safety of a combination tablet of spinosad/milbemycin oxime in dogs inoculated with approximately 250 A. vasorum L3. Sixteen healthy nematode free adult dogs were randomly allocated to two study groups of 8 dogs each. Thirty days post inoculation (dpi) all dogs in the fed state were treated: dogs in group B were treated with spinosad and milbemycin oxime at the dose rates of 45–60 mg/kg and 0.75–1.0 mg/kg bodyweight, respectively, approximately the lower half portion of the expected full unit dose range; dogs in group A were treated with placebo tablets. All dogs were euthanized and necropsied 56–58 dpi. The heart and lungs were examined to determine the presence of A. vasorum. All placebo group dogs were infected at necropsy with counts ranging from 22 to 98 adult worms and a geometric mean worm count of 55.2. In contrast, the geometric mean worm count in the spinosad/milbemycin oxime group was 0.7 with worm numbers ranging from 0 to 8. The results of this study demonstrate that a single treatment with the tablet combination of spinosad and milbemycin oxime administered 30 dpi provided 98.8% preventive efficacy against development of adult A. vasorum infections. Monthly treatments with spinosad and milbemycin oxime have the potential to prevent the establishment of infections with A. vasorum in dogs.     

Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus

Corticosteroids have been reported to induce immunosuppression in fish exposed to many types of bacterial antigens. We document a similar phenomenon in fish exposed to infectious pancreatic necrosis virus (IPNV). Fingerling striped bass that were injected with the steroid triamcinolone acetonide (100 mg/kg body weight) 24 hours before receiving intraperitoneal inoculation with IPNV became viremic 3 days post inoculation (dpi) and virus was still detected in the buffy coat cells 14 dpi. In contrast, viremia could not be detected after 7 dpi in fish that received virus but not steroids. Circulating virus neutralizing antibodies were first detected in steroid treated fish at 10 dpi compared to 7 dpi for the virus injected fish and titers were consistently lower in the steroid group. Steroid treatment of chronic IPNV-carriers did not induce detectable viremia nor alter circulating antibody levels in chronic IPNV-carriers. None of the striped bass demonstrated clinical signs of viral disease.     

Cytological and parasitological analysis of bronchoalveolar lavage fluid for the diagnosis of Angiostrongylus vasorum infection in dogs

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of many pulmonary diseases. The aims of this work were to perform this procedure in dogs in the acute and chronic phases of an Angiostrongylus vasorum infection for cytological analysis and to evaluate the potential of this technique as a diagnostic method for this lung-heart worm. The BAL procedure was performed through the use of an endotracheal tube on seven A. vasorum infected dogs and on five non-infected dogs lined as a control group. Sixty days post-infection (dpi) active and live larvae were retrieved from the bronchoalveolar fluid (BALF) of all infected dogs. Furthermore, in one animal it was possible to retrieve larvae in its BALF before the pre-patent period. This work reports that the A. vasorum infection resulted in an increase of relative neutrophils and eosinophils counts. In contrast, there was a significant decrease in the alveolar macrophage relative count in infected animals from 60 to 330 dpi. This study shows that the BAL is an accurate technique for the diagnosis of canine angiostrongylosis. Moreover, the technique allows us to retrieve cells and other elements that line the lung surface for cytological evaluation, which provides information about inflammatory diseases, and the diagnosis and prognosis of pulmonary parasites such as A. vasorum.     

Serodiagnosis of experimental and natural Babesia equi and B. caballi infections

The sensitivity and specificity of the complement fixation (CF) test for the diagnosis of Babesia infections in equines was assessed, using the indirect fluorescent antibody (IFA) test as a reference. Antibodies were first detected between 11 and 20 days post infection (dpi) in the CF test and between 7 and 14 dpi in the IFA test in ponies infected experimentally with B. equi (USDA strain). The CF test became negative in four of five ponies 63-174 dpi although B. equi was demonstrated microscopically in two of these four ponies up to 364 and 455 dpi. The IFA test remained positive up to 476 dpi (end of the examination period). Ponies infected experimentally with B. caballi (USDA strain) showed positive reactions in the CF test at first between 13 and 15 dpi and in the IFA test 10 or 11 dpi. The CF test became negative in two of three ponies 80 and 140 dpi, whereas the IFA test remained positive up to 190 dpi (end of the examination period). Cross-reactions of sera with heterologous antigens occurred at dilutions of 1/5 in the CF test and up to 1/20 in the IFA test. A total of 3944 CF tests was performed on 3765 horses from various European countries during 1980-1984. Sera that gave positive or trace CF reactions were retested in the IFA test. All 123 CF-positive sera were also IFA-positive and 26 of 31 sera (B. equi) and 11 of 32 sera (B. caballi) showing CF trace reactions were positive in the IFA test. Sera of two CF-negative horses were positive in the IFA test (B. equi); one of these horses was also positive upon microscopic examination. In seven of 21 horses repeatedly examined over longer periods the IFA titers (B. equi) persisted for up to 454 days longer than the CF titers. Sera of horses from highly endemic areas gave the following reactions: Sudan, 62 of 91 sera CF- and 86 of 91 IFA-positive; Zaire, 58 of 75 sera CF- and 72 of 75 IFA-positive; Columbia, 51 of 56 sera CF- and 56 of 56 IFA-positive; Brazil, 17 of 25 sera CF- and 21 of 25 IFA-positive. Only B. equi infections were demonstrated in Zaire. The combined use of the CF and IFA tests is recommended for safe identification of equine Babesia infections.     

Comparative evaluation of enzyme-linked immunosorbent assay based on crude and purified antigen in the diagnosis of canine visceral leishmaniasis in symptomatic and oligosymptomatic dogs

This study evaluated the performance of crude total antigen (CTA) and fucose-mannose ligand antigen (FML) in an enzyme-linked immunosorbent assay for diagnosis of canine visceral leishmaniasis (CVL). The assays used sera from known negative controls (n=30), clinically symptomatic (n=30) and oligosymptomatic (n=30) parasitologically proven infection (by microscopy). Aspirates of popliteal lymph node from infected canines were colleted to score parasitism and compared with the ELISA results. The study indicated that FML used in ELISA provided high sensitivity for detecting oligosymptomatic dogs (90%) and CTA showed greater sensitivity than FML for symptomatic canines (90%). In oligosymptomatic dogs, specificity was 100% for CTA-ELISA, but in symptomatic dogs, FML specificity was higher (96.7%) than CTA-ELISA (93.3%). A significant correlation was observed between the degree of parasitism and the results obtained in CTA-ELISA. Since no available antigen offers 100% specificity and sensitivity for CVL diagnosis, the choice of antigen used must depend on the aim of the investigation.     

Indirect enzyme-linked immunosorbent assay for the detection of antibody against Rift Valley fever virus in domestic and wild ruminant sera

An indirect enzyme-linked immunosorbent assay (I-ELISA) for the detection of specific IgG immunoglobulins against Rift Valley fever virus (RVFV) was validated in-house. A total of 3055 sera from sheep (n = 1159), goats (n = 636), cattle (n = 203), African buffalo (n = 928), and other wild ruminants (n = 129), including eland, kudu, and black wildebeest, was used. Sera from domestic ruminants were collected in West (n = 10), South (n = 1654) and East Africa (n = 334), and sera from wild ruminants (n = 1064) were collected in South Africa. In addition, 136 sera from eight experimentally RVFV-infected sheep, taken during a period of 28 days post infection (dpi), were used to study the kinetics of RVFV antibody production. Field sera were tested by the serum neutralization (VN) test and experimental sera by VN and haemagglutination-inhibition (HI) test. Based on VN test results, negative sera were regarded as reference controls from RVFV-free, and positive sera were regarded as reference controls from RVFV-infected subpopulations of animals. ELISA data were expressed as the percentage positivity (PP) of an internal high positive control. The two-graph receiver operating characteristics approach was used for the selection and optimization of I-ELISA cut-offs including the misclassification costs term and Youden index (J). In addition, cut-off values were determined as the mean plus two-fold standard deviation of the result observed with the RVFV-free subpopulations. Established optimal cut-offs were different for each of the data sets analyzed, and ranged from 1.65 PP (buffalo) to 9.1 PP (goats). At the cut-off giving the highest estimate of combined measure of diagnostic accuracy (highest J value), the I-ELISA test parameters were determined as follows: (1) Diagnostic sensitivity (%): cattle--84.31, buffalo--94.44, sheep--98.91, goats--99.18. (2) Diagnostic specificity (%): cattle--99.34, buffalo--98.28, sheep--99.16, goats--99.23 and other game ruminants--99.26. In the group of RVFV-experimentally infected sheep, seroconversion In all individuals was detected by VN on 4-6 dpi, by HI on 5-7 dpi, and by I-ELISA on 6-7 dpi. All tests showed the same kinetic pattern of immunological response. Antibody levels were low for a very short period before increasing to high titres, after which it was easily detectable by all tests. Compared to traditional tests, the lower sensitivity of I-ELISA in the detection of the earliest stage of immunological response may be practically insignificant, particularily when this assay is used in population-based, disease-surveillance programmes. The high sensitivity and specificity of I-ELISA established in this study, especially for the statistically more representative subpopulations of animals tested, seem to support this prediction. Test parameters determined in this study should, however, be regarded as in-house diagnostic decision limits, for which further updating is recommended, particularly for specimens from other countries, and preferably by applying a standardized method for sampling of new subpopulations of animals to be targeted by the assay.     

Detection of Sarcocystis antigens in the sera of experimentally-infected pigs and mice by an immunoenzymatic assay

Immunoglobulins raised against Sarcocystis miescheriana and Sarcocystis muris cystozoite antigens were isolated from rabbit immune sera by affinity chromatography (using CNBr-activated Sepharose 4B for antigen immobilization). The specific immunoglobulins were incorporated into double-antibody sandwich immuno-enzymatic assays which were firstly quantitated and then used to detect soluble Sarcocystis antigens in the sera of experimentally-infected pigs and mice. Assays employing immunoglobulins attached to the solid-phase at concentrations of 80 micrograms/ml were capable of detecting homologous soluble cystozoite and sporozoite reference antigens at concentrations as low as 8 micrograms/ml. Circulating antigens were detected both in the presence and absence of acute clinical disease in pigs experimentally-infected with high and low doses of S. miescheriana sporocysts. The antigenemia detected was transitory (occurring from 3-20 days post-inoculation: dpi) and coincided well with the sporozoite and merozoite phases of parasite development. Circulating antigens were also detected during subclinical infections in mice (from 4-49 dpi) following their experimental infection with low doses of S. muris sporocysts. Specific immuno-enzymatic assays for circulating Sarcocystis antigens may therefore prove useful in the clinical diagnosis of acute sarcocystosis.     

Seroepidemiological survey of Rhodococcus equi infection in asymptomatic horses and donkeys from southeast Turkey

In order to assess the level of Rhodococcus equi infection in southeast Turkey, 679 sera from healthy foals and adult horses and 78 sera from donkeys were tested by indirect ELISA using a R. equi reference strain (ATCC 33701) as antigen. Eighty (11.7%) sera from horses and 9 (11.5%) sera from donkeys with titres >0.85 were positive. The prevalence of seropositive horses in Sanliurfa Province was higher than in Diyarbakir Province; 56 (13.9%) horses in Sanliurfa Province and 24 (8.7%) horses in Diyarbakir Province were defined as seropositive. In Sanliurfa Province 14.5% of female (n=343) and 10.1% of male (n = 59) horses tested were defined as seropositive, while in Diyarbakir Province more males (11.4%, n=114) were seropositive than females (6.7%, n=163). Horses 1 to 5 years of age were found to have the highest seropositivity rate in both provinces. A total of 78 sera from donkeys were investigated in Sanliurfa Province, of which 9 (11.5%) were positive by ELISA. Among the 9 positive sera, 6 (12.8%) were from donkeys 1-5 years old and 3 (13.6%) were from donkeys >5 years of age. No positive sera were found in donkeys less than 1 year old. Five (12.5%) sera of females and 4 (10.5%) sera of males tested were positive. These results indicate the existence of R. equi in the horse populations in Sanliurfa and Diyarbakir Provinces. Similar infection rates were found for donkeys in Sanliurfa. This suggests the importance of serological surveys to diagnose R. equi infection in the region and to prevent the zoonotic risk.     

Pulmonary lesions in guinea pigs experimentally infected with Actinobacillus pleuropneumoniae (A.p.) serovar 1

Pathological studies were carried out on the lungs of guinea pigs intratracheally inoculated with 4.6 x 10(6-8) colony forming units (CFU)/head of Actinobacillus pleuropneumoniae serovar 1. All animals in the highest dose group died within 24 hr post inoculation (hpi) and showed pulmonary lesions being hemorrhagic in nature while all animals in the lowest dose group were killed as scheduled at 11 days post inoculation (dpi) and showed only hyperplasia of peribronchial lymphoid tissues. In the middle dose group, two died within 24 hpi, two died at 9 dpi, and the remaining one was killed at 11 dpi. Two guinea pigs which died at 9 dpi showed fibrinonecrotic pleuropneumonia which is the most characteristic acute pulmonary lesion in swine, and has not yet been reproduced in laboratory animals up to the present time. This suggests that guinea pigs may be a useful laboratory animal for studying the pathogenesis of Actinobacillus pleuropneumoniae infection in swine.     

Development of an enzyme-linked immunosorbant assay (ELISA) for the serodiagnosis of canine dermatophytosis caused by Microsporum canis

Abstract In dogs, dermatophytosis should be considered in any case of alopecic, papular or pustular lesion. The aim of this study was to develop an enzyme-linked immunosorbant assay (ELISA) as an aid in the diagnosis of canine dermatophytosis. The antigen used was a whole fungal extract obtained from an isolate of Microsporum canis cultured on a liquid medium from the parasitized hair of a cat with patches of alopecia. To assess the ELISA performances, sera from 18 dogs with dermatophytosis caused by M. canis (group A, n = 18), 20 dogs with skin diseases other than dermatophytosis and 22 healthy dogs (group B, n = 42) were tested. Four further animals were tested: three with dermatophytosis caused by M. gypseum and one by T. mentagrophytes. A significant difference (P < 0.01, Wilcoxon's test, w = 364) was found between IgG-specific levels of sera of recently M. canis-infected dogs (infection < 15 days) and controls (although three dogs had negative titres at this stage). A highly significant difference (P < 0.001, w = 462) was noted between controls and dogs with infection of longer duration (> 30 days). All dogs had positive titres at this stage. A highly significant correlation (P < 0.001, Spearman's test, rho = 0.86) between duration of infection and IgG concentration was noted. The test has good sensitivity (83.3%) and high specificity (95.2%) but some dogs retained positive titres after elimination of infection. The sensitivity is higher than that of direct microscopic hair examination and similar to that of fungal culture with DTM (dermatophyte test medium).     

Differentiation between canine cutaneous and visceral leishmaniasis by the detection of immunoglobulin G specific for Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens using flow cytometry

Flow cytometry employing Leishmania (L.) chagasi (Lc) and L. (Viannia) braziliensis (Lb) antigen was used to establish the differential diagnosis between visceral (VL) and cutaneous leishmaniasis (CL) in dogs. Flow cytometry permitted the detection of Leishmania-specific immunoglobulin G in sera from 19 dogs: nine with CL and 10 with VL. A significant difference in the percentage of positive staining was observed in sera from dogs with CL between the homologous antigen (69% for Lb) and the heterologous antigen (42% for Lc). However, this difference was not significant in sera from dogs with VL (61% for Lb and 73% for Lc). No significant staining was observed in control sera (0.6% for Lb and 0.4% for Lc) consisting of samples from healthy dogs, or in the group with sporotrichosis (1.8% for Lb and 1.5% for Lc), a differential diagnosis of CL. The results suggest that flow cytometry might be useful for the differentiation between CL and VL in dogs, with practical applications in areas where the two infections overlap.     

3种ELISA试剂盒检测不同亚型外源性鸡白血病病毒的比较

为建立一种稳定、快速从感染鸡体内检测或分离外源性鸡白血病病毒(ALV)的简易方法,作者使用A亚型(ALV-A)、C亚型(ALV-C)以及2株J亚型(ALV-J-PY和ALV-J-WS)鸡白血病病毒(ALV)按高、中、低(即100、10、1μL)3种接种量人工接种DF1细胞,在接种后不同时间用A、B、C 3种ELISA试剂盒检测ALV抗原(P27)。结果表明,对ALV-A和ALV-J-PY,高剂量接种时,用A试剂盒在接种后第3天即可检测出;低剂量接种时,第7天可检出。使用B试剂盒,2株病毒的检出时间延长(高剂量组分别为第7天和第5天;低剂量组分别为第15天和第13天);使用C试剂盒,所需检出时间最长(接种后第13天)。对ALV-C和ALV-J-WS毒株,A试剂盒对高剂量组,分别能够在接种后第5天和第9天检出,其它中、低接种量组15 d内均没有检出;而B、C2个试剂盒对3个接种剂量组均没有检测出。从以上结果看出,3种ELISA试剂盒对3种亚型ALV毒株的检出时间存在明显不同,A试剂盒灵敏度最高,能最早作出检测;B试剂盒灵敏度次之。同时,无论使用哪种试剂盒ALVs的检出时间与病毒接种量间存在很大的相关性。本研究结果为外源性ALV的检测及病毒分离研究提供一定的科学依据。     

Western blot analysis of the humoral response of dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866)

Seven cross-bred dogs were inoculated with Angiostrongylus vasorum and serum samples were analyzed using the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). ELISA detected specific antibodies anti-A. vasorum, from 14 to 28 days after inoculation (DAI) and persisted throughout the experiment. Using WB, the main antigens detected had molecular weight of approximately 115, 102, 86, 76, 69, 56, 41, 32, 28, 20-22 and 10kDa.     

An immunohistochemical study of the tonsils in pigs with acute African swine fever virus infection

An immunohistochemical study of the tonsils was carried out to gain further insight in the pathogenesis of acute African swine fever (ASF). Twenty-one pigs were inoculated by intramuscular route with a highly virulent isolate of ASF virus and painlessly killed at 1-7dpi. Viral antigen was highly distributed in the tonsil from 3 to 4dpi and an increase in the number of monocyte-macrophages was very evident at the same days post inoculation. This phenomenon was observed together with an increase of the expression of proinflammatory cytokines (Tumour necrosis factor alpha and Interleukin-1 alpha) and the apoptosis of lymphocytes studied by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) technique and haemorrhages. With these results, we can conclude that the tonsil is suffering similar lesions than those observed in other lymphoid organs in acute African swine fever, even when the route of inoculation is the intramuscular and not oral-nasal.     

Angiostrongylus vasorum infection in 23 dogs (1999-2002)

Angiostrongylosis was diagnosed in 23 dogs presenting to the Queen Mother Hospital for Animals between June 1999 and August 2002. The animals' clinical records were reviewed retrospectively and certain risk factors were compared with a control population of 3407 dogs. Twenty-two of the 23 dogs were from south-east England and dogs from Surrey (n=8) were significantly overrepresented. There were also significantly more Cavalier King Charles spaniels (n=5) and Staffordshire bull terriers (n=5) among the affected dogs than in the control group. The median age of affected dogs was 10 months (range five to 90 months). The most common presenting signs were cough (65 per cent), dyspnoea (43 per cent), haemorrhagic diathesis (35 per cent) and collapse (26 per cent). Four dogs were thrombocytopenic and eight had significant prolongations in prothrombin time and/or activated partial thromboplastin time. Thoracic radiographs were abnormal in 18 of 19 dogs. A variety of changes were observed, the most typical being a patchy alveolar-interstitial pattern affecting the dorsocaudal lung fields. Angiostrongylus vasorum larvae were found in seven of 10 bronchoalveolar lavage specimens and 19 of 19 faecal samples. Three dogs died shortly after admission to the hospital. The remainder were successfully treated with fenbendazole at a dose of 50 mg/kg for five to 21 days. A vasorum should now be considered endemic to south-east England.     

Prevalence of antibodies against canine herpesvirus 1 in dogs in The Netherlands in 1997-1998

Canine herpesvirus (CHV1) is found in dogs all over the world and may spread by oronasal or sexual contact. We developed an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against CHV1 in dogs. The antigen used for this ELISA was prepared by purifying CHV1 virions from the medium of infected A72 cells. To investigate the prevalence of CHV1 in The Netherlands, a panel of 145 sera of dogs boarding at a kennel in Lelystad, The Netherlands, was screened using this ELISA. The dogs originated from all parts of The Netherlands and represented many different breeds. The sera were collected both at the start and at the end of the boarding period. Of the 145 paired sera 61 (42.1%) were positive, 79 (54.5%) were negative and 5 (3.4%) could not be attributed to either group. None of the negative dogs became seropositive during the boarding period, which lasted normally two to three weeks. We also tested 79 individual sera taken from dogs at various other places in The Netherlands and found that 27 (34.2%) were positive. Hence, in total 224 dog sera, collected from April 1997 to March 1998, were tested and 88 (39.3%) were found positive. We conclude that the prevalence of CHV1 seropositive dogs in The Netherlands in this period was about 40%, and that boarding at a dogs kennel did not contribute to the spread of CHV1. In addition, CHV1 has been isolated from two clinical cases of fatal haemorrhagic disease in The Netherlands.