Development and validation of an indirect Enzyme-linked immunosorbent assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants.

To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

Schmallenberg virus outbreak in the Netherlands: routine diagnostics and test results.

At the end of 2011, a new Orthobunyavirus was discovered in Germany and named Schmallenberg virus (SBV). In the Netherlands malformations in new-born ruminants were made notifiable from the 20th of December 2011. After a notification, malformed new-borns were necropsied and brain tissue was sampled for reverse transcription-polymerase chain reaction (RT-PCR). In addition, blood samples from mothers of affected new-borns were tested for antibodies in a virus neutralization test (VNT). The aim of this study was to summarize and evaluate the diagnostic data obtained and to gain insight into the possible regional differences. In total 2166 brains were tested: 800 from lambs, 1301 from calves and 65 from goat kids. Furthermore 1394 blood samples were tested: 458 from ewes, 899 from cows and 37 from goats. Results showed that 29% of the lamb brains, 14% of the calf brains, and 9% of the goat kid brains were RT-PCR positive. The number of malformed and RT-PCR positive lambs decreased over time while the number of malformed and RT-PCR positive calves increased. In the VNT 92% of the ewes, 96% of the cows and 43% of the goats tested positive. Combining RT-PCR and VNT results, 18% of all farms tested positive in both the RT-PCR and VNT. The relative sensitivity and specificity of the RT-PCR are 19% and 97% respectively, and of the VNT 99% and 6%. The results show a widespread exposure to SBV and the regional evaluation seems to indicate an introduction of SBV in the central/eastern part.

Detection of PRRSV circulation in herds without clinical signs of PRRS: comparison of five age groups to assess the preferred age group and sample size.

A cross-sectional study was conducted to find the most effective diagnostic approach to detect circulation of porcine reproductive and respiratory syndrome virus (PRRSV). The study was performed in 10 Dutch swine herds, with sows and fattening pigs or breeding stock. Herds did not experience clinical signs of PRRS during the last 6 months before sampling, but a PRRSV infection was confirmed at most 2 years before sampling. Blood samples were collected from 5 age groups: sows during early and late gestation, weaners at 9 weeks of age, fatteners or breeding stock at 16 and 22 weeks of age. For each category, 20 serum samples were examined; in total 100 serum samples per herd. Samples were analysed for PRRSV antibodies with ELISA (n=1002), and rt-PCR when ELISA S/P-ratios were above 1.5 (n=307) or below 0.4 (n=187; random selection from each age group). A logistic regression analysis was used to obtain factors associated with the probability of virus detection in a pig (PCR positive test result). Herd, ELISA-result, and age group were included as explanatory variables. Variables remained in the model when statistically significant. ELISA results showed that none of the herds could be considered to be free of PRRSV infection. Mean PRRSV seroprevalence in unvaccinated animals varied between 18% and 82%, and mean PRRS-virus prevalence varied between 0% and 41%. In only one of the 10 herds, no PRRS-virus could be detected. The odds of finding PRRS-virus in blood samples were 8.6 (95% CI, 5.3-13.9) in pigs of 9 weeks of age and 4.6 (95% CI, 3.0-7.0) in pigs of 16 weeks of age, compared with fatteners of 22 weeks of age. This result indicates that 9- to 16-week-old pigs are the preferred age group to detect PRRS-virus, in herds without clinical signs of PRRS. We concluded that PRRS-virus circulation could be detected in 8 out of 9 of the study-herds, with a relatively low number of blood samples. Testing 12 blood samples in both rt-PCR and ELISA, with 6 samples in pigs 9 weeks of age and 6 samples in pigs 16 weeks of age, will lead to a cost-efficient first evaluation of the PRRSV infection-status in herds without clinical signs of PRRS.

Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria.

Fast and reliable identification of the microorganisms causing mastitis is important for management of the disease and for targeting antimicrobial treatment. Methods based on PCR are being used increasingly in mastitis diagnostics. Comprehensive field comparisons of PCR and traditional milk bacteriology have not been available. The results of a PCR kit capable of detecting 11 important etiological agents of mastitis directly from milk in 4h were compared with those of conventional bacterial culture (48h). In total, 1,000 quarter milk samples were taken from cows with clinical or subclinical mastitis, or from clinically healthy quarters with low somatic cell count (SCC). Bacterial culture identified udder pathogens in 600/780 (77%) of the clinical samples, whereas PCR identified bacteria in 691/780 (89%) of the clinical samples. The PCR analysis detected major pathogens in a large number of clinical samples that were negative for the species in culture. These included 53 samples positive for Staphylococcus aureus by PCR, but negative by culture. A total of 137 samples from clinical mastitis, 5 samples from subclinical mastitis, and 1 sample from a healthy quarter were positive for 3 or more bacterial species in PCR, whereas culture identified 3 or more species in 60 samples from clinical mastitis. Culture identified a species not targeted by the PCR test in 44 samples from clinical mastitis and in 9 samples from subclinical mastitis. Low SCC samples provided a small number of positive results both in culture (4/93; 4.3%) and by PCR (7/93; 7.5%). In conclusion, the PCR kit provided several benefits over conventional culture, including speed, automated interpretation of results, and increased sensitivity. This kit holds much promise as a tool to complement traditional methods in identification of pathogens. In conventional mastitis bacteriology, a sample with 3 or more species is considered contaminated, and resampling of the cow is recommended. Further study is required to investigate how high sensitivity of PCR and its quantitative features can be applied to improve separation of relevant udder pathogens from likely contaminants in samples where multiple species are detected. Furthermore, increasing the number of species targeted by the PCR test would be advantageous.

Multiple strains of Mycobacterium avium subspecies hominissuis infections associated with aborted fetuses and wasting in pigs.

A herd of pigs being reared for breeding and fattening, in which there had been incidences of abortion and wasting, reduced growth rates and an increase in mortality for the past year, were tested for Mycobacterium infection by pathological examinations, skin test, serology and Mycobacterium culture. In one placenta, and also in the lung tissues of fetuses, Ziehl-Neelsen staining revealed acid-fast bacilli in combination with infiltrations of neutrophils, macrophages and multinucleated giant cells. Acid-fast bacilli were also found in the mesenteric lymph nodes, liver and/or spleen and jejunum of pigs with wasting and in slaughtered animals. The specimen cultures were identified as Mycobacterium avium subspecies hominissuis using IS1245-specific PCR and IS1245 restriction fragment length polymorphism (RFLP). IS1245 RFLP revealed that the herd was infected with multiple M avium subspecies hominissuis strains belonging to at least two different clades. It is suggested that this infection may have played a more important role in the economic losses of the pig farm than had been assumed previously.

A study on the severity and relevance of porcine circovirus type 2 infections in Dutch fattening pigs with respiratory diseases.

This study was set up to get more insights in the severity and relevance of porcine circovirus type 2 (PCV2) infections in Dutch fattening farms in an endemic PCV2-situation with no clinical signs of post-weaning multisystemic wasting syndrome (PMWS). In part A of the study, in total 29 commercial fattening farms with varying percentages of pneumonia and pleurisy at slaughter were examined. Blood samples were collected at random by cross-sectional sampling; 10 in the age of 10-12 weeks, 10 at the age of 16 weeks and 10 blood samples at the end of the finishing period (20-22 weeks of age). Serum samples were examined for the presence of PCV2 IgM and IgG antibodies and for antibodies against other porcine lung pathogens. In part B, 8 "high" and 8 "low" herds were selected. The 8 "high" herds were defined as herds having high percentages of lung lesions (pneumonia) at slaughter, and the 8 "low" herds had low percentages of pneumonia at slaughter. For both the "high" and "low" herds, 3 pigs showing signs of respiratory distress were selected for necropsy (n=48). Lung tissue samples were examined post-mortem for macroscopic and histopathological lesions, and for the presence of bacteria and viruses. The results of part A showed that, pigs at 16 weeks of age with IgM antibodies against PCV2 had a lower probability of having pleuritis at slaughter (OR 0.34, P<0.000). Pigs in the age category of 20-22 weeks, and with IgM antibodies against PCV2, also had a lower probability of having pneumonia at slaughter (OR 0.29, P=0.032). In part B lobus apicalis pneumonia, PCV2 in macroscopically unaffected lungs, Pasteurella multocida, Mycoplasma hyopneumoniae, and swine influenza viruses were all found significantly more often in "high" than in "low" pigs at autopsy. High PCV2 DNA loads (>10(4) PCV2 DNA copies/mg) were found in lungs of 14 (58%) "high", and in 7 (29%) of the "low" pigs (P=0.13). In 11 of the 19 affected lungs from "high" pigs, high PCV2 DNA loads were found in combination with one or more other lung pathogens, while this was found only in 5 of the 17 affected lungs from "low" pigs (P=0.02). This study confirms the hypothesis that PCV2 plays a role in pneumonia and pleurisy in 10-24 weeks old fattening pigs, not only in herds with a high prevalence of PMWS, but also in herds with no clinical signs of PMWS.

Analyses of the results of different test systems in the 2005 global proficiency testing schemes for infectious bursal disease virus and Newcastle disease virus antibody detection in chicken serum.

The results of global proficiency testing schemes (PTS) for serological tests to detect antibodies against infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) in chicken serum, in which 125 and 120 laboratories, respectively, participated from Africa, Asia, Europe, Central and South America, were used to analyse the performances of different antibody test systems such as virus neutralization tests, haemagglutination inhibition tests, enzyme-linked immunosorbent assays and agar gel precipitation tests. All laboratories were asked to carry out their routine diagnostic tests for the detection of IBDV and NDV antibodies as usual. This global ring trial provided a large amount of data on variation within and between laboratories and test systems used worldwide. The data showed that the variation between the quantitative test results of different laboratories (R(between)) using the IBDV virus neutralization tests and the NDV haemagglutination inhibition test was higher (about double) compared with the variation within commercial enzyme-linked immunosorbent assay systems. Although both tests are often referred to and used as the "gold standard" in experimental and scientific studies, official procedures and for the validation of tests, this study shows that there is an urgent need for a global implementation of recommended test procedures and/or the inclusion of international reference sera in these studies.

[Review: diagnostic methods for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) infections]. / Review: diagnostische methodieken voor de detectie van porcine reproductive and respiratory syndrome virus (PRRSV) infecties.

This review describes methods that have been developed for the diagnose porcine reproductive and respiratory syndrome virus (PRRSV) infections. It summarizes the organs and tissues which should be sampled and the sampling times, and methods to detect viral RNA, viral antigens, and antibodies directed against PRRSV. The sensitivity, specificity, and limitations of the various tests are also described.

[Risk factors for clinical signs of PMWS and PDNS in pigs in The Netherlands: a case-control study]. / Bedrijfsrisicofactoren voor varkens met klinische verschijnselen van PMWS of PDNS in Nederland : een case-control onderzoek.

Potential risk factors for clinical signs of post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) in pigs in the Netherlands were investigated in a matched case-control study using a questionnaire (personal interview). Eighty-two pig farmers were questioned about management, hygiene, husbandry systems, disease history, and preventive health care. In this study, 30 pig herds with (cases) and 30 pig herds without (controls) characteristic clinical signs of PMWS were compared. For PDNS, 11 pig herds with (cases) and II pig herds without (controls) characteristic clinical signs of PDNS were compared. Univariate analysis (P < 0.10) showed that the following occurred relatively more often in the PMWS case herds than in the control herds: 1) clinical signs of PDNS, porcine reproductive and respiratory syndrome (PRRS), porcine parvovirus (PPV) infections, meningitis, coccidiosis, and pre-weaning diarrhoea observed by the farmer; 2) vaccination against PRRS and mycoplasma; 3) non-optimal climatic conditions in the nursery rooms, a large variation in weaning age, a high occurrence of cross-fostering of piglets, a large number of sows with lactation problems, poor colostrum intake by piglets; and 4) (historical) use of breeding stock (including semen for artificial insemination) of Anglo-Saxon origin. In the final multivariate statistical model, one variable remained significantly associated with PMWS case herds, namely, the presence of clinical signs of PRRS (and/or the associated use of vaccination against PRRS). It should be noted that in almost all cases animals were vaccinated against PRRS because of clinical signs of PRRS that appeared a few months after the first occurrence of clinical signs of PMWS. This excludes PRRS vaccination as a primary factor in causing PMWS. Analysis of the PDNS case-control data showed comparable results with those of the PMWS study. In the final statistical model, the presence of clinical signs of PRRS (and/or the associated use of vaccination against PRRS) was significantly associated with PDNS case herds.

Bovine herpesvirus 4-associated postpartum metritis in a Spanish dairy herd.

In more than 10 Spanish dairy cows, a bovine herpesvirus 4 (BHV4) associated postpartum metritis was confirmed by virus isolation, BHV4-glycoprotein B (gB) PCR and/or serology. In this study, 12 cows with, and, at the time of sampling, 3 cows without clinical signs of acute postpartum metritis from one large dairy herd in Spain were examined for bacterial and viral infections. Blood, placenta/caruncles and uterine contents were collected between day 1 and day 20 post-calving, and examined for the presence of bacteria and for viruses by virus isolation, BHV4 DNA by BHV4-gB PCR and/or BHV4 antibody titres. Bovine herpesvirus 4 was detected in 83% of the cases with clinical signs of acute postpartum metritis by virus isolation and/or BHV4-gB PCR. An increase of BHV4 antibodies was detected in all examined postpartum metritis cows and in the 3 cows without clinical metritis. Two of these 3 cows developed severe metritis a few dayss after collecting the first blood sample. A concurrent infections of BHV4 and bacteria, mainly Arcanobacterium pyogenes and Streptococcus sp., were detected in 73% of the examined uterine contents collected from postpartum metritis affected cows. This case-report study showed a clear association between BHV4 infections and acute postpartum metritis in dairy cows. In addition, the BHV4-associated postpartum metritis appeared to be an emerging syndrome in this Spanish herd.

Genome sequence analysis of 10 Dutch porcine circovirus type 2 (PCV-2) isolates from a PMWS case-control study.

The factors responsible for the emergence of post-weaning multisystemic wasting syndrome (PMWS) as an epidemic disease with significant impact upon the pig industry are not all known. Porcine circovirus type 2 (PCV-2) has been shown to be necessary but not sufficient for the expression of PMWS. Retrospective serological and molecular surveys have shown that PCV-2 was widespread and was maintained with only occasional reports of sporadic PMWS in the 30 year period prior to the recent emergence of the epidemic form of the syndrome. However, the recent spread of the disease in Europe and elsewhere has pointed to the transmission of a novel pathogen. One explanation to reconcile this paradox is that PWMS is caused by a unique PCV-2 variant that is being spread through pig populations. To test this hypothesis, complete genomes (1767 bp) of 10 Dutch PCV-2 isolates from 4 PMWS affected premises and 6 farms without PMWS were sequenced. Phylogenetic analysis showed that these sequences were grouped together although they differed on 77 nucleotide positions relative to each other (95.6-100% identity between the 10 isolates). None of these nucleotide changes identified impacted upon transcriptional elements or other important recognised features of the genome of the PCV-2. Amino acid changes were recorded on 4 positions in ORF1 and on 16 positions in ORF2 but, importantly, no consistent pattern was evident between PCV-2 isolates from affected and control pigs. These data provide further evidence to suggest that factor(s) in addition to PCV-2 are necessary in the development of PMWS.

The presence of co-infections in pigs with clinical signs of PMWS in The Netherlands: a case-control study.

In this study, 60 pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) from 20 different pig herds and 180 control pigs (without clinical signs of PMWS) were examined to get more insights into the frequencies of porcine circovirus 2 infections and the presence of co-infections in pigs with and without clinical signs of PMWS in the Netherlands. Porcine circovirus type 2 was detected in 100% of the pigs with clinical signs of PMWS by virus isolation and/or PCR and in 50% of the pigs from PMWS-free herds. There was an association between the levels of infectious PCV2 and/or PCV2 DNA load and the severity of clinical signs as described for PMWS. A high variation in PCV2 antibody titres was found in the clinically affected pigs, and 27% of these pigs did not mount PCV2 antibody titres higher than 1:200. A concurrent infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) was found in at least 83% of the pigs with clinical signs of PMWS and in 35% of the pigs from PMWS-free herds. Co-infections of European- and American-type PRRSV were detected only in PMWS herds and in one control herd with a history of PMWS clinical signs.

Excessive porcine circovirus type 2 antibody titres may trigger the development of porcine dermatitis and nephropathy syndrome: a case-control study.

In a case-control study, the role of porcine circovirus 2 (PCV2) and putative co-factors in the development of porcine dermatitis and nephropathy syndrome (PDNS) were investigated. Pigs with and without PDNS were examined for macroscopic lesions and histopathology. In addition, organs and tissues were collected at necropsy and examined for the presence of fibrinous deposits (immune complexes), CD8+ cells, and for the presence of bacterial and viral infections. Results from PDNS cases were compared with those of three control groups comprising pigs without clinical signs of PDNS and selected from; (1) the same compartment as PDNS cases, (2) another compartment but in the same PDNS herd, and (3) a control herd without any history of PDNS or post-weaning multisystemic wasting syndrome. Macroscopic and histopathological lesions found in PDNS cases were comparable to those previously documented for PDNS e.g. skin lesions and renal lesions representing glomerulonephritis associated with fibrinous deposits and to a lesser extent with interstitial nephritis. PCV2 was detected by PCR in 100% of the PDNS cases, mainly in lymph nodes and tonsils, and in 63% of the control pigs from PDNS free herds. Virus isolation did not reveal infectious PCV2 in all cases. In PDNS affected pigs the PCV2 serum antibody titres were consistently extremely high and the mean PCV2 antibody titre in PDNS pigs was significantly higher than the mean PCV2 antibody titres in pigs from all 3 control groups. Immunohistochemical investigation of kidneys from PDNS affected pigs revealed an increased accumulation of IgG1 + IgG2 and IgM, the complement factors C1q and C3, but also an increase of CD8+ cells. The amounts of IgA and the complement factor C5 in kidneys of PDNS pigs were only slightly increased as compared to control pigs. This study demonstrates that PCV2 infections can result in extremely high PCV2 antibody titres and that PCV2 is a candidate as primary agent in the development of PDNS. The causative physiological basis for PDNS may be the excessive levels of PCV2 antibodies.

Susceptibility of bovine umbilical cord endothelial cells to bovine herpesviruses and pseudocowpox virus.

The purpose of the study was to determine the susceptibility of bovine umbilical cord endothelial (BUE) cells to bovine herpesvirus (BHV) 1, BHV2, BHV4 and BHV5, and to pseudocowpox virus. The detection limits and growth curves of these viruses in BUE cells were compared with those in Vero, Madin-Darby bovine kidney (MDBK). or bovine fetal diploid lung (BFDL) cells. Detection limits were determined by inoculating cell cultures with serial 10-fold dilutions of these viruses, and growth curves by titration of virus, harvested at various times after infecting cells at a multiplicity of infection of 0.1. The detection limits of BHV2 and BHV4 were lower in BUE cells than in Vero or MDBK cells, and cytopathic effects were observed earlier in BUE cells. In addition, BHV2 and BHV4 grew to higher titres in BUE cells than in Vero or MDBK cells. BUE cells appeared to be equally susceptible to BHV5, but less susceptible to BHV1.1 and BHVI.2 than MDBK cells. The study showed that BUE cells are highly susceptible to BHV2 and BHV4. and that the use of BUE cells can improve the laboratory diagnosis of these viruses. The use of BUE cells could also improve the isolation and growth of pseudocowpox virus.

Viral infections and bovine mastitis: a review.

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.

[The role of virus infections in bovine mastitis]. / De rol van virusinfecties bij bovine mastitis.

Mastitis is an often occurring disease in dairy cattle with an enormous economic impact for milk producers worldwide. Despite intensive research, which is historically based on the detection of bacterial udder pathogens, still around 20-35% of clinical cases of bovine mastitis have an unknown aetiology. Due to the high number of unknown causes of clinical mastitis, studies were undertaken to gain more insight into the role of viruses in this important disease. This review deals with the role of viruses in the aetiology of bovine mastitis, including the results of the recently performed study on the role of bovine herpesvirus 4 (BHV4) in this aetiology. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore their importance and their economic impact needs further attention.

Presence of European bat lyssavirus RNas in apparently healthy Rousettus aegyptiacus bats.

Apparently healthy Rousettus aegyptiacus bats were randomly chosen from a Dutch colony naturally infected with European bat lyssavirus subgenotype 1a (EBL1a). These bats were euthanised three months after the first evidence of an EBL1a infection in the colony. EBL1a genomic and antigenomic RNAs of the nucleoprotein gene were detected by nested reverse transcriptase PCR in 75% of the examined Rousettus aegyptiacus bats. The EBL1a RNAs of the nucleoprotein gene were detected mainly in brain tissues, but also in other organs. EBL1a messenger RNAs of the nucleoprotein gene and the glycoprotein gene were detected in brain tissues. The standard fluorescent antibody test revealed the presence of lyssavirus antigens in brain tissues from 7 (17.5%) Rousettus aegyptiacus bats. Furthermore, EBL1a could not be detected by virus isolation on murine neuroblastoma cells or by intracerebral inoculation of suckling mice. Neutralising antibodies directed against EBL1 were detected in 11% of the examined bats. This study shows that at least 85% of the apparently healthy Rousettus aegyptiacus bats must have been infected with EBL1a, and that these bats may survive from an EBL1a infection. Furthermore, the study supports the possibility of a long-term maintenance of EBL1a genome in Rousettus aegyptiacus bats.