The present study was conducted to determine whether Varroa jacobsoni can transmit American foulbrood (AFB), caused by the bacterium Paenibacillus larvae to healthy colonies by the surface transport of spores. Five two-storey Langstroth colonies of Apis mellifera ligustica were infested by placing a sealed brood comb, with 10% Varroa prevalence, between the central brood combs of each colony. Two months later the colonies were inoculated with P. larvae by adding brood comb pieces with clinical signs of AFB (45±5 scales per colony). After 60 days the brood area was completely uncapped by means of dissecting needles and tweezers, separating the Varroa mites from the larvae and the collected mites were introduced at a rate of 51 per colony into four recipient hives placed in an isolated apiary. Twenty female Varroa specimens were separated at random and observed by SEM. Paenibacillus larvae spores were found on the dorsal shield surface and on idiosomal setae. All colonies died after 4–5 months due to a high incidence of varroosis. No clinical AFB symptoms or P. larvae spores were observed in microscopic preparations. It is concluded that Varroa jacobsoni does not transmit AFB from infected to healthy colonies; it does, however transport P. larvae spores on its surface.Facultad de Ciencias Agrarias y Forestale

Albo, Graciela Noemí

Alippi, Adriana Mónica

Leniz, Daniel

Marcangeli, Jorge Augusto

Noriega, Alicia E.

Experimental and Applied Acarology

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Expetqmental & AppliedAcarology, 19 (1995) 607-613 607 Short Communication The mite Varroa jacobsoni does not transmit American foulbrood from infected to healthy colonies Adriana  M. Alippi, Graciela  N. Albo,  Jorge  Marcangeli  a, Daniel  Leniz  and Alicia Noriega Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, ec 31, 1900 La Plata, Argentina aFacultad de Cieneias Exactas y Naturales, Universidad Naeional De Mar del Plata, Funes 3350, 7600 Mar del Plata, Argentina ABSTRACT The present study was conducted to determine whether Varroa jacobsoni can transmit American foulbrood (AFB), caused by the bacterium Paenibacillus larvae to healthy colonies by the surface transport of spores. Five two-storey Langstroth colonies of Apis mellifera ligustica were infested by placing a sealed brood comb, with 10% Varroa preva- lence, between the central brood combs of each colony. Two months later the colonies were inoculated with P. larvae by adding brood comb pieces with clinical signs of AFB (45 _+ 5 scales per colony). After 60 days the brood area was completely uncapped by means of dissecting needles and tweezers, separating the Varroa mites from the larvae and the collected mites were introduced at a rate of 51 per colony into four recipient hives placed in an isolated apiary. Twenty female Varroa specimens were separated at random and observed by SEM. Paenibacillus larvae spores were found on the dorsal shield surface and on idiosomal setae. All colonies died after 4-5 months due to a high incidence of varroosis. No clinical AFB symptoms or P. larvae spores were observed in microscopic preparations. It is concluded that Varroajacobsoni does not transmit AFB from infected to healthy colonies; it does, however transport P. larvae spores on its surface. Key words: American foulbrood, Paenibacillus larvae, Varroa jacobsoni, spores, varroosis, honey bees, Apis mellifera, SEM. T h e  mi te  Varroa jacobsoni  Oud.  (Acari :  V a r r o i d a e )  is an agen t  which t ransmits  several  pa thogen ic  m i c r o o r g a n i s m s  to Apis  mellifera L. It  has b e e n  d e m o n s t r a t e d  tha t  Varroa can  t r ansmi t  b o t h  sacb rood  virus (SBV) (Bailey,  1991) and  acu te  paralysis  virus  (A P V )  (Ball,  1985, 1988; Ball and  Al len ,  1988) by inocula t ing  virus par t ic les  in to  the  h a e m o l y m p h  o f  h o n e y  0168-8162 © 1995 Chapman & Hall 608 A.M. ALIPPI E T A L .  bees. By means of SEM studies, Puerta et al. (1990) found spores of Ascosphaera apis (Maasen ex Clausen) Olive and Spiltoir and Aspergillus flares (Link ex Fries), on the cuticle of adult female Varroa. The mite is also one of the vectors of Hafnia alvei M¢ller, which causes septicaemia (Strick and Madel, 1988) and of several other bacteria (Glinski and Jarosz, 1990a, 1992). On the other hand, when varroosis and American foulbrood (AFB) develop simultaneously, colony death is accelerated (Grobov, 1977). SEM and cultural studies have shown that V. jacobsoni carries viable spores of PaenibaciUus larvae (White) Ash, Priest and Collins (Ash et al., 1993) which causes AFB (Alippi, 1992). However, there is no information concerning the transmission of this disease from colonies with AFB to healthy colonies. The purpose of the present work was to determine whether V. jacobsoni can transmit AFB to healthy colonies by the surface transport of P. larvae spores. Five two-storey standard Langstroth colonies of Apis mellifera ligustica L. were used. The selected hives had four brood combs each of which were treated with Fluvalinate (Apistan ®, manufactured by Sandoz-Zbecon) 40 days before the experiment was started in order to ensure the absence of Varroa. At the beginning of the austral summer (23 December 1993), a sealed brood comb with 10% Varroa prevalence (approximately 150 mites) was placed in the centre of the four brood combs of each colony. Two months later, the five colonies were inoculated with P. larvae by adding brood combs with clinical signs of AFB. Four comb pieces of 126-144 cells each on both sides containing a total of 45 + 5 AFB scales were used (each colony received 40-50 scales). These were placed at the four corners of each central brood comb (A. M. Alippi, G. N. Albo, D. Leniz, I. Rivera, M. Zanelli and A. E. Roca, unpublished manuscript). The colonies were examined weekly to determine disease development. Clinical symptoms of AFB were observed after 35 days in the central comb and in both contiguous sides of the near combs. In mid-autumn (13 April 1994) the brood area was completely uncapped by means of dissecting needles and tweezers, separating Varroa mites from the larvae. In the cells with larvae which had advanced AFB symptoms, either there were no Varroa present, or those observed were dead. The mites collected were introduced, at a rate of 51 Varroa per colony, into four recipient colonies placed in an isolated apiary in La Plata area. These colonies had similar quantities of brood, adult bee population and food (pollen and honey). To ensure the absence of mites, the receptive colonies had been treated with fluvalinate 40 days before the beginning of TRANSMISSION OF AMERICAN FOULBROOD 609 the experiment. Honey samples taken from the brood chambers were analysed for P. larvae (Alippi, 1991; Hornitzky and Clark, 1991; A. M. Alippi, submitted). None had P. larvae spores, and there were no clinical symptoms of AFB disease. Before introducing the mites to the receptive colonies, 20 female Varroa (five from each _colony) were separated at random and e _xamined to determine, by SEM observations, if they had P. larvae spores on their surfaces. The mites were fixed in 2% glutaraldehyde in Sorensen buffer (pH 7.5) for 1 h at 4°C, then washed in the same buffer (three times, 15 min each) and dehydrated in a series of ethanol solutions of increasing concentration: 20, 30, 50 and 70% (10 min each), 96% (30 rain) and the last two in absolute ethanol (30 rain each). Mites were mounted on stubs with diphase tape, vacuum metallized with a 200 ,~ width gold layer and observed by a Jeol scanning microscope JSM-T 100, scanning the whole surface of each mite. Paenibacillus larvae spores were found on the dorsal shield surface (Figs 1 and 2) and on idiosomal setae (Fig. 3). Eighty per cent of the examined mites carried P. larvae spores, but only one mite Fig. 1. Scanning electron micrograph of P. larvae spores on the dorsal shield surface of a female V. jacobsoni. 610 A.M. ALIPPI E T A L .  Fig. 2. Scanning electron micrograph of P. larvae spores in the dorsal shield surface of a female V. jacobsoni. Fig. 3. Paenibacillus larvae spores on an idiosome seta of V. jacobsoni. TRANSMISSION OF AMERICAN FOULBROOD 611 showed a considerable number of spores (Fig. 2) while, the mean density of spores on the body surfaces of the rest were 3 + 1 per mite. The colonies were evaluated every 15 days to detect AFB clinical symptoms. Three colonies died 4 months after the mites had been intro- duced due to a high incidence of varroosis, while the remaining colony died after the fifth month. These colonies died because they were not able to bear the high parasitic mite levels. This phenomenon is more common in winter when the brood area is smaller and, consequently, most cells are parasitized. As a result of this, some bees had malformed wings and others were wingless. These symptoms, which are caused by V. jacobsoni have been previously observed in local 'Creole bees' (A. rn. mellifera × A. m. ligustica) (Marcangeli et al., 1992). Two to three cells with suspected symptoms of foulbrood were found in a brood area of approximately 209 average cells per comb side; they were examined by use of the modified hanging drop (Shimanuki and Knox, 1991) and nigrosin colouration methods (Gochnauer et al., 1979), applied to the material collected from inside the cells. Vegetative forms resem- bling Melissococcus pluton (White) Bailey, the causal agent of European foulbrood (EFB), were observed in nigrosin preparations, as well as spores of Paenibacillus alvei (Cheshire & Cheyne) Ash et al. and Bacillus lateros- porus Laubach, microorganisms commonly associated with this disease. Although no spores similar to those of P. larvae were found, isolations in J medium (Gordon et al., 1973) and in J medium supplemented with 6/zg ml-1 nalidixic acid were made following previously described techniques (Hornitzky and Karlovskis, 1989; Alippi, 1991; Hornitzky and Clark, 1991). Only P. alvei and B. laterosporus colonies were obtained on plates with J medium. Larvae with chalkbrood symptoms were also found in the colonies, with approximately five larvae per colony. The presence of EFB and chalk- brood might be due to weakening of the colonies caused by the high incidence of V. jacobsoni. This conclusion is based on the observation that at the beginning, no colony presented clinical symptoms of these diseases, which are endemic in the area where the experiment was carried out. Both these diseases are known to be related to the weakening of colonies. It is thus concluded that V. jacobsoni does not transmit AFB from infected to healthy colonies; however, it does transport P. larvae spores on their surfaces as observed by SEM (see also Alippi, 1992). The number of P. larvae spores that the mites carry on their surfaces and that can be introduced into receptive colonies is probably not suffi- cient to trigger AFB. Normally, the mode of transmission of P. larvae spores which develop in AFB infections is by ingestion of larval brood 612 A.M, ALIPPI E T A L ,  food. However, because Varroa is a blood-sucking ectoparasite of larvae and adults, the probability that the mite can contaminate the larval food by contact is very low. ACKNOWLEDGEMENTS The assistance of Ms P. Sarmiento and Mr R. Urrejola in the examination of samples with the scanning electron microscope and to SADA for the provision of the colonies used in this study is gratefully acknowledged. We also thank Drs M. Gilliam and D. De Jong for their critique of the manuscript. This work was supported by the UNLP (Universidad Nacional de La Plata), the CIC (Comisi6n de Investigaciones Cientificas de la Provincia. de Buenos Aries) and by the International Foundation for Science, Sweden (IFS grant no. B/1859-2 to A.M.A.). REFERENCES Alippi, A. M. 1991. A comparison of laboratory techniques for the detection of significant bacteria of the honey bee, Apis mellifera, in Argentina. J. Apicult. Res. 30: 75-80. Alippi, A. M. 1992. Transporte de esporas de Bacillus larvae por el ficaro Varroa jacobsoni. Rev. Facult. de Agronomi. de La Plata 68: 83-86. Ash,C, Priest, F.G. and Collins, M.D. 1993. Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek 64: 253-260. Bailey, L. 1991. Honey Bee Pathology, 2nd edn. Academic Press, London, New York. Ball, B. V. 1985. Acute paralysis virus isolates from honey bee colonies infested with Varroa jacobsoni. J. Apicult. Res 24:115-119 Ball, B. V. 1988. The impact of secondary infections in honey-bee colonies infested with the parasitic mite Varroa jacobsoni. In Africanized honey bees and bee mites, Needham, J.G. (ed.), Ellis Horwood, Cheshire. pp. 457-461. Bail, B.V. and Allen, M. F. 1988. The prevalence of pathogens in honey bee (Apis mellifera) colonies infested with the parasitic mite Varroa jacobsoni. Ann. Appl. Biol. 113: 237-224. Glinski, Z. F. and Jarosz, J. 1990a. Serratia marcescens, artificially contaminating brood and worker honeybees, contaminates the Varroajacobsoni mite. J. Apicult. Res. 29" 107-111. Glinski, Z. F. and Jarosz, J. 1990b. Micro-organisms associated fortuitously with Varroa jacobsoni. Microbios 62: 59-68. Glinski, Z. F. and Jarosz, J. 1992. Varroa jacobsoni as a carrier of bacterial infections to a recipient bee host. Apidologie 23: 25-31. Gochnauer, T. A., Furgala, B. and Shimanuki, H. 1979. Diseases and enemies of the honey bee. In The hive and the honey bee, Dadant & Sons, Hamilton, IL. (eds.) pp. 617-619. Gordon, R., Haynes, W.C. and Pang, C. H-N. 1973. The genus Bacillus. USDA Handbook 427. Grobov, O.F. 1977. Varroasis de las abejas. In La varroasis, enfermedad de ia abeja mellfera, Editorial Apimondia, Bucarest. pp. 52-92. Washington, D.C., USA. TRANSMISSION OF AMERICAN FOULBROOD 613 Hornitzky, M. A. Z. and Clark, S. 1991. Culture of Bacillus larvae from bulk honey samples for the detection of American Foulbrood. J. Apicult. Res. 32: 51-52. Hornitzky, M.A.Z. and Karlovskis, S. 1989. A culture technique for the detection of Bacillus larvae in honey bees. J. Apicult. Res. 28: 118-120. Marcangeli, J., Monetti, L. and Fernandez, N. 1992. Malformations produced by Varroa jacobsoni on Apis rnellifera in the province of Buenos Aires, Argentina. Apidologies 23: 399-402. Puerta, rz, ~ores, i ~ lirn,~n,~, a 1 n,,~,,~¢ wl ~nd P~rlill~ l~ 100/) ]3n~e~rmt~d~d~e secundarias a la parasitaci6n por varroa en Apis mellifera. Vida Aplcola 43: 54-59. Shimanuki, H. and Knox, D.A. 1991. Diagnosis of Honey Bee Diseases. US Department of Agriculture, Springfield, VA, USA. Strick, H. and Madel, G. 1988. Transmission of the pathogenic bacterium Hafnia alvei to honey bees by the ectoparasitic mite Varroa jacobsoni. In Africanized honey bees and bee mites, Needham, J.G. (ed.), pp. 462-466. Ellis Horwood, Cheshire. 

The mite Varroa jacobsoni does not transmit American foulbrood from infected to healthy colonies

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The mite Varroa jacobsoni does not transmit American foulbrood from infected to healthy colonies

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