intractable infection

Dear Sir:

The most commonly seen bacteria in infections such as those you are dealing with are Vibrio spp., and the antibiotics you have been using (ciprofloxacin and neomycin sulfate + triple sulfa) are generally good choices for combating vibriosis. However, there are many strains of Vibrio and not all of them will respond to the same antibiotics, so it would certainly be worth trying another antibiotic to resolve the problems your Pinto and Sunburst have been having.

Above all, don’t forget that reducing the water temperature is often the best thing you can do to help bring such infections under control, so what ever antibiotic you use, be sure to cool down the water in the treatment tank as discussed below.

Some of the other antibiotics that have proven to be effective for vibriosis are listed below. In terms of effectiveness, I would rate them roughly as follows:

Chloramphenicol (i.e., Chloromycetin) either as a bath or administered orally
streptomycin, flumequine, and polymyxin B (all good choices but very hard to obtain)
kanamycin as a bath (one of the few antibiotics that works well in saltwater and is absorbed readily through the skin and gills)
tetracyclene/oxytetracycline (can be effective if administered orally)

furazolidone

For example, here is a preliminary report from Olin Feuerbacher after examining a seahorse that died of a Vibrio infection. Olin is a marine biologist who is now a Molecular Biologist and a member of the research staff at the Arizona Genomics Institute, and who runs a small aquaculture business- clownfish, gobies, a bit of coral, and all sorts of odd food items- lots of pods, microalgae, etc. He is also a seahorse keeper and has done a lot of research in tropical diseases for quite a while he was a paramedic. He is also a grad student working on marine microbiology, mainly ocean borne human pathogens, and his specialty has been the Vibrio bacteria!

In short, Olin really knows his stuff when it comes to this sort of thing. He examined the deceased seahorse, took cultures and ran their sensitivities. This is his preliminary report:

Hi all, I have some early results:
The most common bacteria present were Vibrios, at least 2 species are present. There is at least one that was found inside muscle tissue, not just superficially, so this is likely to be the main pathogen. It is not id’d to species yet (last count there were over 400 spp of Vibrio). But the good news is that Chloramphenicol killed all the bacteria types present, and Kanamycin was over 95% effective. This is of all the myriad bacteria that arrived, not just the pathogens, so I would expect either to be a good treatment. I will post more later when I have a better idea of what the actual etiologic agent is.
Olin

And here are his thoughts on bacterial infections in seahorses (these are observations he shared based on his past experience but before he did the necropsy and cultures described above):

Quote:

They typically start as a secondary infection after either mechanical damage or parasites or cnidarian stings. Once established, they are difficult to control. This is due in part to the fact that they are typically normal flora in all tanks. They are generally benign until they get an opportunity to invade. The symptoms you describe make me think of a particularly nasty species, Vibrio vulnificus. Unlike most of the other vibrios which are gastrointestinal
pathogens or cause localized tissue damage, V. vulnificus starts as a local infection, but then destroys enough tissue that it moves into the bloodstream and causes a highly fatal form of septicemia. If this is the case, you might want to be careful about sticking your hands into the tank if you have any open cuts. It is also a rare but serious human pathogen. For some reason, it is especially serious for alcoholics in whom it is fatal in about 40% of septicemic cases. So your horses had better cut down on the boozin’. It sounds like the third treatment might be the best idea. Oxytetracycline also sometimes works, but it is a bacteriastatic agent, meaning that it’s growth is halted but it is not killed outright. Instead, the horse’s immune system is responsible for ultimate clearance, and by the time symptoms appear, it is likely too far gone for this to work. The antibiotics that seem to work well for this include streptomycin (in some cases), chloramphenicol (dangerous in 1/100,000 humans) and ciprofloxacin (just hard to get).

As for the importance of avoiding heat stress when it comes to bacterial infections (or the value of maintaining reduced temperatures when fighting a bacterial infection), this is what he has to say:

It is interesting that you mentioned the elevated
temperatures. I think this is a critical factor in a
number of ways. First, elevated temperatures can have
many adverse effects on the immune status of many
organisms. Many of the enzymes and proteins involved
in an immune response are very temperature sensitive.
When studying an outbreak of vibriosis in echinoderms
during an El Nino event in the Sea of Cortez, I found
that several defensive enzymes in the echinoderms were
inactivated by a rise of only a few degrees in water
temperature.

In addition to the effects on the hosts, water
temperature may have very significant effects on the
pathogens as well. First, elevated temperature will
obviously increase the rate of microbial growth.
Perhaps more importantly, recent research has
implicated temperature as a major factor in the
regulation of virulence genes. When in the cooler
pelagic environment, a bacteria wants to conserve
energy, so virulence genes will not be expressed since
there is probably no host. However, in warmer temps,
these genes can be turned on resulting in pathogenesis.
This is especially true for bacteria such as the
Vibrios which exist both as normal aquatic flora and as
pathogens in many mammalian species with our nice warm
digestive tracts etc. One particularly interesting
study showed that the coral pathogen Vibrio strain AK1
was completely benign, despite heavy colonization, in
corals at one temp (I forget exactly what, I think it
was about 25C), but when temperature was raised by 3
degrees, all of the virulence genes in the Vibrio’s
pathogenicity island were turned on. This resulted in
severe infection and rapid death of the corals. Bad
news for aquarists, but I still think this kind of gene
regulation is really cool!
Olin

So raising or dropping the water temperature just a few degrees can make a huge difference. Likewise, here are Neil Garrick-Maidment’s observations on the importance of water temperature when treating a Vibrio infection:

[Quote]
I am not sure if it is of any help but I recently had a problem with vibriosis
in Hippocampus capensis coupled with a couple of gas bubbles in the end of the
tail. Having tried a number of treatments in the past that havn’t worked I took
a slightly more drastic approach this time and dropped the temperature from 23
down to 18 degrees C (64°F) having first isolated the infected animals into a separate
tank. I then left them like this for 4 weeks after which I increased the
temperature slowly up to 21 degrees C or 70°F (which it still is). After the second week
the vibriosis had gone completely (and has not returned) and the gas bubbles
were gone after the third week. In all the time the temperature was low the
animals reduced their feeding and it has now increased with the raising of the
temperature and they since gone on to have two broods of fry.

Best wishes,

Neil Garrick-Maidment
Seahorse Project Co-ordinator

Hippocampus erectus has an enormous range of the wild and can adapt to an equally wide range of temperatures, so your Sunburst (H. erectus) would be perfectly comfortable at the same temperatures Neil used to cure his H. capensis, if you can possibly drop the temperature in your treatment tank that far. If not, just dropping the temperature a few degrees from normal can often make a huge difference when treating bacterial infections.

Finally, here’s a research paper I thought you might be interested in since it documents a case of mass mortality among hippocampus kuda due to a Vibrio infection, and discusses the antibiotics that were found to be effective against that strain of Vibrio (note that chloramphenicol was again found to be the most effective medication):

Vibrio harveyi causes disease in seahorse, Hippocampus sp.
E Alcaide1, C Gil-Sanz1, E Sanjuµn1, D Esteve1, C Amaro1 and L Silveira2

A mass mortality among cultured seahorses,
Hippocampus kuda and Hippocampus sp., occurred
in spring 1998 in Tenerife, Spain. Seahorses were
held together with tropical shrimps, Stenopus
hispidus, in glass aquaria supplied with 1000 L of
sea water at 25 °C. The water supply was conducted
between different tanks that contained various
marine species, such as octopus, Octopus vulgaris,
star®sh, Asterias rubens, sea-urchin, Paracentrotus
lividu, greater weever, Trachinus draco, grouper,
Epinephelus guaz and Canarian shrimp, Lismata
amboiens. None of these species was affected,
including the shrimps that shared aquaria with the
seahorses. Mortalities of seahorses were very high
(more than 90%), and the fish died in 3-5 days
after the first clinical signs appeared. Moribund
seahorses were microbiologically analysed and subsequently,
chloramphenicol was used as a bath
(30 mg L)1) to control the outbreak. The mortality
decreased after a few days of antibiotic treatment.

Diseased seahorses presented clinical signs similar
to vibriosis: external haemorrhages, and haemorrhagic
liver and ascitic fluid accumulation in the
intestinal cavity. A bacterium identified as Vibrio
harveyi was obtained in pure culture from samples
of skin haemorrhages, mouth and liver of all
moribund seahorses. The aim of this study was
to characterize the V. harveyi strains isolated from
diseased seahorse, and to confirm its pathogenicity
by means of experimental infection.

Samples from skin haemorrhages, mouth and liver
were analysed by streaking a piece of aseptically
obtained tissue onto tryptone-soy-agar supplemented
with 1% NaCl (TSA-1) and incubating at 25 °C
for 24±48 h. Pure cultures were obtained from all
samples. The isolated strains were Gram-negative
rods, motile, oxidase- and catalase-positive, sensitive
to the vibriostatic agent O129 at 150 lm and
fermentative. The isolates were first characterized
by API 20NE (BioMeÂrieux, S.A. France) strips,
which gave the same profile in all cases (7474445),
identi®ed by the database APILAB Plus
(BioMeÂrieux) supplied by the manufacturer as
V. vulni®cus, with a probability of 95.1%. Further
identification was achieved by colony hybridization
as previously described (Biosca, Amaro, Larsen &
Pedersen 1997), using an alkaline phosphataselabelled
oligonucleotide DNA probe (VVAP) specific
for V. vulni®cus, constructed from a portion of
the V. vulni®cus haemolysin±cytolysin (hlyA) gene
sequence (Wright, Miceli, Landry, Christy, Watkins
& Morris 1993). Positive and negative controls used
were V. vulni®cus ATCC 27562 and V. cholerae
ATCC 14035, respectively. All isolates were negative
in colony hybridization experiments, which indicated
that they were misidentified as V. vulni®cus.
Identification was continued by testing additional
biochemical characteristics as described by Biosca,
Oliver & Amaro (1996). On the basis of the results
obtained, the seahorse isolates were identified as
V. harveyi. They were almost identical to the type
strain of V. harveyi, except for growth at 12 °C and
luminescence (Table 1). Vibrio harveyi is a synonym
of V. carchariae (Pedersen, Verdonck, Austin, Austin,
Blanch, Grimont, Jofre, Koblavi, Larsen, Tiainen,
Vigneulle & Swings 1998), which is recognized as a
fish pathogen (Yii, Yang & Lee 1997). The present
strains differed from the type strain of V. carchariae in
swarming, production of urease, growth with 8%
NaCl and at 40 °C, and the utilization of sucrose,
arabinose, d-mannitol and l-citruline.

Cultures grown on TSA-1 were suspended in
sterile phosphate buffered saline (PBS) at pH 7.2 and
DO600 nmwas adjusted to 0.4. Aliquots of 0.1 mL
of this suspension were spread onto Mueller±Hinton
agar (Oxoid, Basingstoke, 2 UK), and antimicrobial
sensitivity tested using antimicrobial discs (Becton
3Dickinson, Pharmaceuticals, NJ, USA). The following
drugs were used: tetracycline (30 lg), flumequine
(30 lg), chloramphenicol (30 lg), oxolinic acid
(10 lg), trimethoprim-sulphametoxazol (25 lg),
nitrofurantoin (50 lg), oxytetracycline (30 lg),
erythromycin (15 lg), furazolidone (50 lg), gentamicin
(10 lg), kanamycin (30 lg) and polymyxin
B (300 U). Strains were sensitive to tetracycline,
flumequine, chloramphenicol, nitrofurantoin and polymyxin
B. Chloramphenicol and flumequine produced
the widest inhibition halos in the test plates.
The 50% lethal dose (LD50) test, with batches of
six seahorses per dose, were conducted by intraperitoneal
(i.p.) injection as previously described (Alcaide,
Amaro, TodolõÂ & Oltra 1999). Seahorse (mean
weight 4 g ®sh)1), were injected with 0.05 mL of a
bacterial suspension containing 107)102 cfu mL)1
(determined by plate counts on TSA-1), in PBS.
Sterile PBS was injected i.p. into seahorses as a
control. Mortalities were recorded daily for 14 days,
and were only considered positive if the injected
strain was recovered from assayed seahorses. The
LD50 as calculated by the method of Reed&Muench
(1938) was 4 ´ 103 cfu ®sh)1. Pure cultures of the
inoculated strains were re-isolated from liver and skin
haemorrhages of moribund seahorse. No mortality
was detected in the controls. Clinical signs appeared
12±24 h after i.p. injection and mortalities began
1±7 days post-challenge. The signs observed in
challenged seahorses reproduced those observed
during the outbreak. This result confirmed the role
of V. harveyi as the causative agent of the disease.
In the present work, an infectious disease affecting
seahorse, Hippocampus kuda and Hippocampus sp., is
described for the first time. The isolates from diseased
seahorse had the same morphological and biochemical
characteristics, and were identi®ed as V. harveyi
from comparison of their biochemical characteristics
with the type strain of the species. Vibrio harveyi is
a marine bacterium that causes luminous vibriosis
(Zhang & Austin 1999) and is an important
pathogen of cultured penaeid shrimp (Lavilla-Pitogo,
Baticados, de Cruz-Lacierda & de la PenÄa 1990;
Karunasagar, Pai, Malathi & Karunasagar 1994;
Liu, Lee, Yii, Kou & Chen 1996; Montero & Austin
1999). It has also been reported as an opportunistic
pathogen of common snook (Kraxberger-Beatty,
McGarey, Grier & Lim 1990), and has been isolated
from diseased marine fish such as Acanthopagrus
cuvieri (Saeed 1995), sea bream, Sparus aurata
(Balebona, MorinÄigo, Faris, Krovacek, MaÈnsson,
Bordas & Borrego 1995), and dentex, Dentex dentex,
cultured on the Mediterranean coast of Spain
(Company, SitjaÁ-Bobadilla, Pujalte, Garay, Alvarez-
Pellitero & PeÂrez-SaÂnchez 1999). Further studies are
in progress to characterize the virulence factors
involved in the pathogenicity of V. harveyi isolates.

All things considered, I would say that chloramphenicol (i.e. Chloromycetin) is the treatment of choice for most Vibrio infections. It is effective both as a bath for prolonged immersion or when administered orally. Your Sunburst is not eating, so administering the chloramphenicol to the treatment tank would be a good option for him. However, I would recommend administering the chloramphenicol to your Pinto orally, via bio-encapsulated shrimp, which would allow you to treat the Pinto in the main tank where it is most comfortable, without subjecting it to any additional stress.

The treatment protocol for Chloramphenicol or Chloromycetin is as follows:

Chloramphenicol can be used to treat Vibriosis at 40 mg/ litre of water (which comes out to about 150 milligrams per gallon) in a bath for 10-20 hours. It is important to watch the quality of the water, and if it starts to become turbid, the water must be changed. It is best to treat in a separate tank. In stubborn cases, a series of such baths may be necessary to resolve the problem, in which case a complete water change should be performed before the medication is redosed.

Chloramphenicol can also be used as an additive to the feed, if the fish are still eating (all to often in a major infection they will refuse to eat, but this treatment may be most useful in preventing the horizontal spread of the infection). When used as an addition to the feed use 500 mg per 100 gram of feed. (In the case of seahorses, the flake food medicated with chloramphenicol in this way would first be bio-encapsulated in live feeder shrimp, which would then in turn be fed to the seahorses.)

If you do obtain the chloramphenicol, be sure to be very careful when handling it. Remember, in a few rare individuals exposure to chloramphenicol can cause a potentially fatal side effect (aplastic anemia). These are rare cases and almost always involve patients who were being treated with the medication, but I would use gloves when handling it as a precaution and if you crush crush up tablets of chloramphenicol, be very careful not to inhale any of the power.

Because of this side effect, which affects one in 100,000 humans, chloramphenicol is no longer available as a medication for fishes and can therefore be difficult to obtain. If you find that is the case, you might have better luck switching to kanamycin instead. A fish medication that includes kanamycin sulfate as its primary ingredient should be available from any well-stocked LFS:

Kanamycin sulfate

This is a potent broad-spectrum, gram+/gram- aminogylcoside antibiotic. It is wonderfully effective for aquarium use because it is one of the few antibiotics that dissolves well in saltwater and that is readily absorbed through the skin of the fish. That makes it the treatment of choice for treating many bacterial infections in seahorses. Kanamycin can be combined safely with neomycin (as well as metronidazole) to further increase its efficacy. Like other gram-negative antibiotics, it will destroy your biofiltration and should be used in a hospital tank only.

If, for some reason, you have difficulty locating the kanamycin, you will surely be able to locate a fish medication based on tetracycline or oxytetracycline at your LFS. These medications are completely useless when added to the saltwater in a marine aquarium, since they bind with calcium and magnesium, but they have proven to be very effective in treating bacterial infections, including certain strains of Vibrio, when administered orally, as described below:

Tetracycline and oxytetracycline can also be used like Chloramphenicol as an addition to the feed, with the limitation as already mentioned that so often with Vibrio infections the fish will not eat. When attempting to use Tetracycline in the feed take 750 mg tetracycline HCL and mix same with 100 gram of feed. Use this mixture for at least one week, feeding twice daily in the morning and early afternoon. The dosages for oxytetracycline are exactly the same as for tetracycline. (Again, when treating seahorses seahorses, the flake food medicated with tetracycline/oxytetracycline in this way would first be bio-encapsulated in live feeder shrimp, which would then in turn be fed to the seahorses.)

Feeding your Pinto feeder shrimp that have been gut loaded with chloramphenicol or tetracycline/oxytetracycline may enable you to clear up his bloated condition once and for all.

Best of luck treating your ailing seahorses, sir!

Respectfully,
Pete Giwojna