Vitamin C and infections in animals

by Harri Hemilä

This text is based on pages 7-9 of Hemilä (2006).
This document has up to date links to documents that are available via the net.

Harri Hemilä
Department of Public Health
University of Helsinki, Helsinki, Finland
harri.hemila@helsinki.fi

Home: http://www.mv.helsinki.fi/home/hemila

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Version August 27, 2009

When you can measure what you are speaking about,
and express it in numbers, you know something about it;
but when you cannot measure it, when you cannot express it in numbers,
your knowledge of it is of a meager and unsatisfactory kind.

Lord Kelvin (1883)

Since primates and the guinea pig have lost their capability to synthesize vitamin C (Burns 1957; Pauling 1970b; Chatterjee 1973; Sato & Udenfriend 1978; Nishikimi & Yagi 1996; Nandi et al. 1997; Smirnoff 2001), these animals are of primary interest when considering the possible effects of low vitamin C intake on susceptibility to infection.

In the early studies on vitamin C deficiency in the guinea pig, bacteria were so often found in histological sections to indicate the infectious nature of scurvy to some authors. Hess (1920 pp 133-4) concluded that such results show merely that the tissues of scorbutic animals frequently harbor bacteria, but "There is no doubt that invasion of the bloodstream does occur readily in the course of scurvy, but this takes place generally after the disease has developed and must be regarded as a secondary phenomenon and therefore unessential from an etiological standpoint. Indeed one of the striking and important symptoms of scurvy is the marked susceptibility to infection" (p 32). Summarizing autopsy findings of experimental scurvy in the guinea pig, Hess also noted that "Pneumonia is met with very frequently and constitutes a common terminal infection" (p 123) and "The superficial lymphatic glands are frequently palpable in scurvy, especially those in the inguinal region… It has seemed to us attributable less to the nutritional condition than to the infections which so frequently complicate the disorder" (p 141).

A large number of studies have reported that guinea pigs fed a vitamin C deficient diet have low resistance to various infections (for review, see Höjer 1924; Clausen 1934; Robertson 1934; Perla & Marmorston 1937a 1937b; Birkhaug 1938; McCullough 1938; Scrimshaw et al. 1968; Hemilä 1997a, 1998). Most of the early animal studies did not examine the effect of pure vitamin C, the ‘vitamin C group’ being administered oranges or other fruit or vegetables containing vitamin C. Even though the positive findings in such studies are consistent with the notion that vitamin C intake may affect susceptibility to infection, the effect may be caused by other substances in fruit and vegetables as well (see Hemilä 1999). The studies in which pure vitamin C was administered to the ‘vitamin C group’ are thus presented separately (Appendix 2 of Hemilä 2006) from those in which the ‘vitamin C group’ was administered fruit or vegetables as the source of the vitamin (Appendix 3 of Hemilä 2006). Studies in which animals were administered diphtheria toxin, tetanus toxin, or endotoxin are included in the tables, since these toxins are essential components in the pathogenesis of the bacteria concerned. A meta-analytic summary of the animal studies with pure vitamin C is shown in Table 2.

The studies in Appendix 2 are ordered with respect to the importance of the animal model to human beings so that those with primates are first, followed by guinea pigs, and thereafter studies with animals producing vitamin C, first mammals and then birds. At the end of Appendix 2 of Hemilä 2006 there are studies with fish, which are incapable of synthesizing vitamin C, yet in evolutionary terms they are most distant from humans. In a series of studies with rhesus monkeys infected with poliovirus, Jungeblut (1937a, 1937b, 1939) found significant benefit from vitamin C in several settings, but there was considerable variation in the results. In a small study, Albert Sabin (1939) found no effect using vitamin C. The studies by Jungeblut and Sabin differ in the method of virus inoculation, and Sabin administered oranges to his control monkeys, whereas Jungeblut does not describe the basal diet; these are some examples of potential factors explaining the inconsistency in their findings. With marmosets, Murphy et al. (1974) found that vitamin C increased resistance against experimental parainfluenza infection. Several studies with guinea pigs found that vitamin C protected against M. tuberculosis and M. bovis, and against various other micro-organisms (Hemilä 2006 Appendix 2).

Rabbits, cats and mice synthesize vitamin C and thus cannot be used to study the effects of vitamin C deficiency, but the effects of supplementation can be studied in these species (Hemilä 2006 Appendix 2). Three studies with rabbits found protection against pneumococcus using vitamin C, and cats administered vitamin C recovered faster from rhinotracheitis. Several studies with mice found benefits from vitamin C supplementation.

Davelaar and Bos (1992) found substantial protection in chickens against infectious bronchitis virus (IBV). This is an interesting report because IBV belongs to the family of coronaviruses which includes the recently identified SARS (severe acute respiratory syndrome) coronavirus (Hemilä 2003b). Vitamin C also protected chickens against Salmonella and E. coli infections (Hemilä 2006 Appendix 2). Finally, a few studies found that vitamin C supplementation improved the resistance of fish to various infections.

Appendix 2 (Hemilä 2006) lists 148 separate animal studies with vitamin C, 86 of which found a statistically highly significant benefit, P ≤ 0.01, on at least one infectious disease outcome, while 58 studies reported benefit at a level of P ≤ 0.001 (Table 2). Of 100 studies with mammals, 58 found highly significant benefit, P ≤ 0.01, from vitamin C on some infectious disease outcome.

Although many of the studies in Appendix 2 are old, it seems unlikely that administering a fixed dose of vitamin C and evaluating mortality were meaningfully different in the older days compared with modern methods. Furthermore, 97 of the papers were published in the 1970s or later, and 56 of these newer papers reported a statistically highly significant effect, P ≤ 0.01, on at least one infectious disease outcome (Appendix 2 of Hemilä 2006). Furthermore, among the non-significant recent studies, Bourke et al. (1980) reported over 100% longer median survival time in vitamin C supplemented mice infected with malaria, but the number of animals was not reported, making any statistical interpretation impossible. In mice infected with pneumococcus, Esposito (1986) found no effect on mortality; nevertheless, vitamin C significantly enhanced the clearance of pneumococci from the lungs. Thus, there is no apparent pattern that the newer studies would have yielded less positive results than the older. In most of the studies in Appendix 2, statistical analyses of the results are missing or inappropriate in the original papers, and in this respect the statistical analyses in Appendix 2 are essentially all novel.

Matsumoto et al. (1992) examined the effect of 2-octadecylascorbic acid in parallel with ascorbic acid, but only the results with the latter are presented in Appendix 2 of Hemilä 2006. Nevertheless, assuming that the effect of vitamin C on infections is based on the antioxidant effect, the results with the derivative are also of interest. When 2-octadecylascorbic acid was administered subcutaneously in E. coli infected rats, only 2 of 8 rats produced renal scarring of the two highest scores (2 or 3), in contrast to all 8 control rats (mid-P = 0.002). Similarly with pyelonephritis caused in rats by Serratia marcensces, subcutaneous administration of the derivative resulted in none of 8 rats producing renal scarring of the two highest scores (2 or 3), in contrast to 7 of the 8 control rats (mid-P = 0.0004).

Nonaka et al. (1990) studied the effect of 2-octadecylascorbic acid on mouse endotoxemia. None of the mice administered the vitamin C derivative died (0/20) whereas 45% (9/20) of the control mice died (mid-P = 0.0003).

Appendix 3 lists studies in which vitamin C group was administered either fruit or vegetables as the source of vitamin C. Albert Sabin (1939) found that rhesus monkeys fed vitamin C deficient diet had a high mortality due to infections, in particular pneumonia. In a number of studies using guinea pigs, fruit or vegetables protected against various infections better than a deficient diet. Although vitamin C is an important substance in fruit and vegetables, it is also possible that the differences are caused by substances other than vitamin C (see Hemilä et al. 1999). In this respect, no specific conclusion can be drawn from Appendix 3 as to the importance of vitamin C per se, although these studies are consistent with the notion of benefits from higher levels of vitamin C intake.

In addition to the animal studies yielding quantitative data on the effect of vitamin C on infections (Appendices 2 and 3 in Hemilä 2006), a few studies reported qualitative findings on its role on infections. Russell et al. (1944) found that caseous foci caused by tuberculosis were considerably larger in vitamin C deficient guinea pigs than in animals administered lettuce as a source of vitamin C. Tuberculous lesions in the spleen were also substantially larger and less well demarcated in vitamin C deficient guinea pigs than in normal animals. Höjer (1924) infected guinea pigs with tuberculosis bacilli and, compared with normally nourished animals, he found that in animals that had scurvy, tuberculous foci in muscles, lymph-glands and the spleen were not surrounded or penetrated by any connective tissue, and the necrotic parts passed into non-tubercular tissue without such demarcation. Meyer and Meyer (1944) reported that abscesses caused by staphylococcal infection remained soft and undemarcated in vitamin C deficient guinea pigs, whereas they were hard and button-like in animals administered pure vitamin C. In an uncontrolled study, Hans Zinsser et al. (1931) found that the infection caused by Rickettsiae was very severe in vitamin C deficient guinea pigs, so that particularly high concentrations of Rickettsiae were found in pleural and peritoneal exudates. Witt et al. (1988) reported that an unexpected outbreak of pneumococcal infections in guinea pigs was associated with a diet unintentionally low in vitamin C. Nungester et al. (1951) did not find any difference in the clearance of streptococci from the lungs of rats and guinea pigs, or in the occurrence of pneumonia by vitamin C level administered.

Rawal et al. (1974) reported that survival of mice infected with Pseudomonas aeruginosa was increased by vitamin C supplementation in a dose-dependent fashion. Senatuite and Biziulevicius (1986) reported that in rats infected with Trichinella spiralis the average number of muscle larvae after 3 weeks was 40% lower in the vitamin C administered group. Chaiyotwittayakun et al. (2002) induced mastitis in cows using intramammary infusion of endotoxin, and vitamin C reduced the fall in milk production caused by endotoxin. Goldschmidt (1991) reported that in rhesus monkeys vitamin C administration dropped oral Actinomyces viscosus counts by six orders of magnitude.

Two case-series were reported describing the therapeutic benefit of vitamin C on dogs afflicted by the canine distemper virus. Belfield (1967) described a series of 10 dogs that appeared to benefit from 1 – 2 g/day of vitamin C injected i.v. over 3 consecutive days. Leveque (1969) had some ten years of veterinary practice and with the pessimistic observation that only 5-10% of dogs recovered from canine distemper with signs of central nervous system (CNS) disturbance, he became interested in Belfield’s case series. In a series of 16 dogs showing CNS disturbance that were treated with vitamin C, the proportion of dogs that recovered was 44% (95% CI: 20% to 70%; based on 7/16)(Leveque 1969).

Although the great majority of studies found benefits with higher vitamin C intake on various infections, a study on cryptosporidiosis in mice found more oocysts and a higher infection score in the vitamin C supplemented group (Leitch & He 1999).

Mortality and severity of infection in animals are hard outcomes, rather than surrogates such as various laboratory measures of the immune system. In this respect the animal studies with actual infections are more interesting from the human perspective than studies on laboratory measures of the human immune system. It is noteworthy that dramatic effects have been reported in numerous animal species ranging from rhesus monkey to fish, and with a great diversity of infecting organisms. Still, it is not clear to what degree the animal studies can be extrapolated to human subjects. It would seem unlikely that human beings qualitatively differ from the other animal species with respect to the general role of vitamin C on infections. Therefore, the fundamental question in human beings is not whether vitamin C affects susceptibility to and severity of infections, but rather the relevant questions seem to be focused on estimation: e.g., what the population groups that might get some benefit from higher vitamin C intakes are, what the dose-dependency relation between intake and the effects on infections is, and how great the proper levels of intake might be.

Table 2: Meta-analysis of the studies on animal infections: effect of vitamin C

	No. of studies	No. of studies	No. of studies
Category	Total in the category	Benefit in any infectious disease outcome with P[1-tail]<0.01	Benefit in any infectious disease outcome with P[1-tail]<0.001

All studies	148	86	58
Published >=1970	97	56	39
Mammals	100	58	40
Monkeys or guinea pigs	49	25	15

A ‘study’ is defined here operationally as the group of findings with a stated number of animals in Appendix 2 of Hemilä 2006.
Appendix 2 is based on extensive literature searches. Studies in which the number of events is very small are excluded, since they are uninformative. A few studies used different levels of vitamin C, the lowest being compared with the highest in most cases in the appendices, but in some cases different vitamin groups are combined and, when pertinent, different levels of intake are shown separately against the lowest level.
In some studies, different amounts of bacteria or different time-points, etc. were used and in these cases the selection of results shown in Appendix 2 involved subjective considerations, but the goal was to present the findings as objectively as possible given the complexity of some publications. A few reports stated in their text that vitamin C had no effect on a relevant outcome but no explicit data was mentioned preventing inclusion of those studies in the Appendix.

References

NOTE: All the links in the main text should be freely accessible at least as an abstract, but some links below require a permission from publisher for any access.

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Copyright: © 2006-2009 Harri Hemilä. This text is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Vitamin C and infections in animals by Harri Hemilä is licensed under a Creative Commons Attribution 1.0 Finland License.
Based on a work at www.mv.helsinki.fi/home/hemila/animals